Made available in DSpace on 2016-02-26T12:59:20Z (GMT). No. of bitstreams: 1 FabioFernandoStefanello.pdf: 387128 bytes, checksum: ab00691ba985715c2d4d480a0eaf642d (MD5) Previous issue date: 2009-07-19 / The transgenic glyphosate-resistant soybean (soybean GR) has been widely cultivated in the world. Although of the benefits of this technology on weed control, currently observed at some regions that, after the application glyphosate, the soybean GR were with leaf chlorosis and could be caused by glyphosate-induced deficiency of Manganese. Thus, the objective of this study was to evaluate the post-emergence application of glyphosate and Mn on soybean GR at different phenological growth stages and your effect in nutrients uptake and soybean GR yield. The experiment was carried out at two farms (Lages de Pedra and São Manoel), both at city of Rio Brilhante-MS. The study was in a completely randomized block design with four (São Manoel) and six (Lages de Pedra) replicates. The treatments were analyzed as factorial 3x8 with three times of application (without glyphosate; glyphosate application (720 g e.a.) in phenological growth stages V2 + V4 (480 g e.a.) and application only V4 (1.200 g e.a.)) and eight foliar Mn application of Mn in different phenological growth stages (without Mn; Mn on V4; Mn on V4 + V8; Mn on V4 + R2; Mn on V4 + V8 + R2; Mn on V8; Mn on V8 + R2 e Mn on R2), completing 24 treatments. It was not observed at no moment of the development of the culture visual symptoms of plant toxicity effects caused by glyphosate application. As well, it was not observed effects of glyphosate application on soybean GR in uptake of Mn and nutrients as N, P, K, Fe, Zn e Cu, as well, in mass of 100 grains and yield culture. The foliar Mn application did not influence the mass of 100 grains and yield culture, however increased leaf Mn content when the application was carried at phenological growth stages V8 and R2 or inside these possible combinations. The leaf Fe content decreased with the foliar Mn application / A soja transgênica resistente ao glifosato tem sido amplamente cultivada no mundo. Apesar dos benefícios dessa tecnologia no controle de plantas daninhas, recentemente tem-sê observado em alguns locais que, após a aplicação de glifosato as plantas de soja RR encontravam-se amarelecidas, e isso poderia ser causado por uma deficiência induzida de Mn. Assim o objetivo deste trabalho foi avaliar a aplicação de glifosato em pós-emergencia em diferentes estádios fenológicos da soja RR e, seus efeitos nos teores foliares de nutrientes e produtividade de grãos, em condições de campo. O experimento foi desenvolvido na Faz. Lages de Pedra e Faz. São Manoel, ambas no município de Rio Brilhante MS. O delineamento experimental foi em blocos casualizados, com quatro e seis repetições nos diferentes locais. Os tratamentos foram dispostos em arranjo fatorial 3 x 8, e consistiram da combinação de três tratamentos com aplicação de glifosato (sem glifosato; 720 g i.a. de glifosato no estádio fenológico da soja V2 + 480 g i.a. em V4 e 1.200 g i.a. de glifosato no estádio V4 da soja RR) e oito tratamentos com aplicação de Mn (sem aplicação de Mn; Mn no estádio fenológico da soja V4; Mn em V4 + V8; Mn em V4 + R2; Mn em V4 + V8 + R2; Mn em V8; Mn em V8 + R2 e Mn no estádio R2 da soja) (em cada aplicação de Mn foi utilizado 332 g de Mn ha-1), totalizando 24 tratamentos. Não foi observado em nenhum momento do desenvolvimento da cultura sintomas visuais de fitotoxidade na soja RR oriundos da aplicação de glifosato. Neste estudo não foi observado efeito do glifosato sobre os teores foliares de Mn na soja RR, assim como dos nutrientes N, P, K, Fe, Zn e Cu, bem como na massa de 100 grãos e na produtividade de grãos da soja. A aplicação foliar com Mn não influenciou a massa de 100 grãos e a produtividade, entretanto aumentou os teores foliares de Mn quando a aplicação foi realizada de forma isolada ou nas possíveis combinações nos estádios fenológicos V8 e R2 da soja transgênica. O teor foliar de Fe diminuiu com a aplicação foliar de Mn na Faz. Lages de Pedra

The mixture of herbicides is a widely used technique in weed control in several crops, including maize. However, interactions that might potentially result from mixing herbicides are a matter of constant concern and research. Soils are ideal settings to study transport and behavior of herbicides along gradients of physicochemical properties. The aim of this study was to evaluate sorption-desorption, leaching mineralization, biodegradation, and microbial respiration of mesotrione applied alone and mixed with S-metolachlor + terbuthylazine in samples from Brazilian sites cultivated with maize. The sorption-desorption of 14C-labeled [cyclohexane-2-14C] mesotrione was evaluated using the batch method, leaching was in glass columns, mineralization and degradation experiments were conducted using biometric flask, and microbial respiration was established according to the soil microorganisms: carbon transformation test with 14C-glucose solution in biometric flasks as well. Sorption of mesotrione applied alone and mixed had Kd sorption coefficient) values ranging from 0.08 to 5.05 kg L-1 and from 0.09 to 5.20 kg L-1, respectively, with similar behavior across soils. Mesotrione sorption was influenced primarily by the clay mineral (CM) content and the soil pH. Leaching of mesotrione is relatively high in the tropical soils and correlates with the pH (R2 = -0.84) and CM content (R2 = 0.75) and may pose a potential groundwater contamination risk. From the 49 d laboratory incubation data, increased mineralization half-life of mesotrione were observed for the mixture of herbicides, ranging from a 4 d increase for the sandy loam soil to a 1 d increase in the sandy clay texture soils. Mesotrione degradation rate had a 2-fold increase in the sandy loam compared to the sandy clay soil. Two metabolites can be identified from mesotrione degradation, 4-methyl-sulfonyl-2-nitrobenzoic acid (MNBA) and 2-amino-4-methylsulfonyl benzoic acid (AMBA). For unamended soil - control (without herbicide), microbial activity followed similar behavior to amended soil with herbicides in total 14CO2 released and accumulated, ranging from 23 to 27%. The mode of application of mesotrione did not influence sorption, desorption or leaching. Mesotrione sorption was relatively low in soils, which creates the high potential for leaching in maize producing areas. Thus, recommendations for mesotrione application, without the prior knowledge of the soil physical and chemical properties can result in an inefficient weed control. Mesotrione biotransformation was relatively quick, indicating this herbicide has low persistence, and consequently, low residual effect on crops and weeds when present in similar soils to this present study. Microbial respiration for all treatments was slightly higher in the sandy clay compared with sandy loam soil; although soil samples with application of herbicides (alones and in a mixture) did not have decreased basal microbial respiration or mineralization rate of glucose / A mistura de herbicidas é uma técnica amplamente utilizada no controle de plantas daninhas em diversas culturas, incluindo o milho. No entanto, as interações que podem potencialmente resultar da mistura de herbicidas são uma questão de constante preocupação e pesquisa. Os solos são os locais ideais para estudar o transporte e o comportamento de herbicidas ao longo dos gradientes de propriedades físico-químicas. O objetivo deste estudo foi avaliar a sorção-dessorção, lixiviação, mineralização, biodegradação e respiração microbiana do mesotrione aplicado isolado e misturado com S-metolachlor + terbuthylazine em amostras de locais brasileiros cultivados com milho. A sorção-dessorção do mesotrione 14C-marcado [ciclohexano-2-14C] foi avaliada utilizando o método de lote, a lixiviação foi em colunas de vidro, experimentos de mineralização e de degradação foi realizados utilizando frascos biométricos e a respiração microbiana foi estabelecida de acordo com os microorganismos do solo: teste de transformação de carbono com uma solução de 14C-glicose em frascos biométricos também. A sorção do mesotrione aplicado isolado e misturado apresentou valores de Kd (coeficiente de sorção) varando de 0,08 à 5,05 kg L-1 e de 0,09 à 5,20 kg L-1, respectivamente, com comportamento semelhante nos solos. A sorção do mesotrione foi influenciada principalmente pelo teor de argila mineral (CM) e o pH do solo. A lixiviação do mesotrione é relativamente elevada nos solos tropicais e correlaciona-se com o pH (R2 = -0,84) e teor de CM (R2 = 0,75) e pode representar um potencial risco de contaminação das águas subterrâneas. A partir dos dados de laboratório de 49 dias de incubação, o aumento da meia-vida de mineralização do mesotrione foi observado para a mistura de herbicidas, variando de um aumento de 4 dias do solo franco-arenoso para um aumento de 1 dia nos solos de textura argilo-arenosa. A taxa de degradação do mesotrione apresentou um aumento de 2 vezes no franco-arenoso em relação ao solo de textura argilo-arenosa. Dois metabólitos podem ser identificados a partir de degradação do mesotrione, o ácido 4-metil-sulfonil-2-nitrobenzóico (MNBA) e 2-amino-4-metilsulfonil benzóico (AMBA). Para o solo sem alterações - controle (sem aplicação de herbicida), a atividade microbiana seguiu comportamento similar ao solo tratado com herbicidas no total de 14CO2 liberado e acumulado, variando de 23 a 27%. O modo de aplicação do mesotrione não influenciou a sorção, dessorção ou lixiviação. A sorção do mesotrione foi relativamente baixa nos solos, que gera alto potencial de lixiviação em áreas produtoras de milho. Assim, as recomendações para a aplicação do mesotrione, sem o conhecimento prévio das propriedades físicas e químicas do solo podem resultar em um ineficiente controle de plantas daninhas. A biotransformação do mesotrione foi relativamente rápida, indicando que este herbicida tem baixa persistência, e, consequentemente, baixo efeito residual sobre as culturas e plantas daninhas, quando presentes em solos semelhantes ao presente estudo. A respiração microbiana em todos os tratamentos foi ligeiramente superior no argilo-arenoso em comparação com o solo franco-arenoso; embora as amostras de solo com aplicação de herbicidas (isolados e em uma mistura) não têm diminuído a respiração microbiana basal ou a taxa de mineralização da glicose

Faz-se necessária a continuidade no processo de geração de informações provenientes da pesquisa dirigida, buscando-se caracterizar a situação atual do uso de glyphosate e outros herbicidas aplicados em pós-emergência, na cultura do milho Roundup Ready 2 (RR2) e Liberty Link (LL), isso por meio da obtenção de informações que possam fomentar o posicionamento mais seguro destas tecnologias. Sem dúvida, esse assunto exige novas pesquisas, visando proporcionar condições que favoreçam o uso sustentável desta ferramenta que vêm apresentando grande potencial de utilização pelos produtores brasileiros, propiciando benefícios relacionados ao controle de plantas daninhas. O presente trabalho teve como objetivo a avaliação das respostas do milho RR2, submetido à aplicação de manejos, formulações e doses do herbicida glyphosate e, também, as repostas do milho RR2/LL à aplicação de diferentes associações de herbicidas. Para isso, foram conduzidos experimentos testando dois manejos, duas formulações e cinco doses de glyphosate (fatorial triplo 2x2x5), em milho de primeira safra (verão) e segunda safra (safrinha), durante uma sequência de dois anos agrícolas (2012/13 e 2013/14), totalizando quatro experimentos com 20 tratamentos cada, que foram realizados na estação experimental da C.Vale - Cooperativa Agroindustrial (Palotina- PR). Além destes, conduziu-se um experimento complementar, com os mesmos tratamentos, numa propriedade agrícola em Marechal Cândido Rondon-PR, na primeira safra de 2012/13. Também foram realizados dois experimentos distintos com oito tratamentos cada, utilizando glyphosate, amônio-glufosinato e herbicidas aplicados normalmente no milho convencional (atrazine e nicosulfuron), estes foram conduzidos na primeira safra de 2012/13 e repetidos na primeira safra de 2013/14, ambos em uma propriedade agrícola em Marechal Cândido Rondon-PR. Para o aprimoramento dos dados, foram conduzidos mais seis experimentos em casa de vegetação (três em Piracicaba-SP e três em Palotina-PR), com os mesmos tratamentos dos experimentos realizados em campo, com o propósito de alcançar informações adicionais que permitissem melhor entendimento do comportamento da cultura após ser submetida à aplicação dos tratamentos. Foram analisadas diversas variáveis relacionadas ao desempenho agronômico da cultura (aferições de alturas, diâmetro de colmo, índices de clorofila, massa fresca e seca de parte aérea, massa seca da raiz, produtividade e massa de 100 grãos) e relacionadas a qualidade dos grãos produzidos (teste de germinação, teor de proteínas, teores de macro e micronutrientes). A partir dos resultados alcançados, demonstrou-se que mesmo variando a formulação ou manejo utilizado, altas doses de glyphosate podem ser prejudiciais ao desenvolvimento do milho RR2, e também que nicosulfuron e algumas associações triplas de herbicidas podem trazer danos ao milho apresentando tecnologias transgênicas que conferem tolerância a herbicidas. / It is necessary to continue the information generation process from directed research, trying to characterize the current situation of the use of glyphosate and another herbicides applied in post-emergence, in corn Roundup Ready (RR2) culture, this by obtaining informations that can foment safer positioning of this technology. No doubt, this topic demands new researches, aiming to provide conditions that favor the sustainable use of this tool that has been showing high potential of use by Brazilian producers, providing these benefits related to the weeds control. The present work has the objective of evaluation of RR2 Corn replies, submitted to application managements, formulations and dosages of glyphosate herbicide and, also the replies of Corn RR2 and RR2/LL, to the application of different associations of herbicides. For that it was conducted experiments testing two managements, two formulations and five dosages of glyphosate (triple factorial 2x2x5), in corn of first crop (summer) and second crop (winter corn crops), during one sequence of two agricultural years (2012/13 and 2013/14), totalizing four experiments of 20 treatments each, that were performed in the experimental station of C.Vale Cooperativa Agroindustrial (Palotina-PR). Besides these, it was conducted one complementary experiment, with the same treatments, in an agricultural property in Marechal Cândido Rondon-PR, in the first crop of 2012/13. It was also realized two different experiments with eight treatments each, using glyphosate, ammoniunglufosinate and herbicides usually applied in conventional maize (atrazine and nicosulfuron), these were conducted in the first crop of 2012/13, and repeated in the first crop of 2013/14, both in an agricultural property in Marechal Cândido Rondon- PR. For data improvement, it was conducted more six experiments in a greenhouse (three in Piracicaba-SP and three in Palotina-PR), with the same treatments of the experiments carried out in field, with the purpose to reach additional information that allowed a better understanding of the crop behavior after being submitted to the treatments application. Several variables were analyzed related to the agronomic development of the crop (culture measurements, stem diameter, chlorophyll index, fresh and dry matter of the air part, root dry mass, productivity and weight of 100 grains) and related to quality of produced grains (germination test, proteins content, macro content and micronutrients). From the results achieved, it was shown that even by varying the formulation or management used, high dosages of glyphosate can be harmful to the development of RR2 corn and that also nicosulfuron and some triple associations of herbicides can bring damage to corn showing transgenic technologies that confer tolerance to herbicides.

Atualmente existe uma grande preocupação com relação aos efeitos a curto e longo prazo que muitos herbicidas podem ter sobre a saúde pública e o meio ambiente. Estes podem causar sérios danos ao metabolismo celular, levando geralmente a inibição do crescimento em microrganismos. Em exposição a herbicidas, esses microrganismos aumentam significativamente a geração de EAOs (Espécies Ativas de Oxigênio), causando estado de estresse oxidativo. O objetivo desse trabalho foi estudar a relação entre o efeito do herbicida acetochlor e a resposta do sistema antioxidante. Três linhagens de Klebsiella oxytoca foram submetidas ao crescimento em meio nutritivo com este herbicida nas concentrações de 0, 62 e 620 mM. O perfil protéico foi avaliado em SDS-PAGE, o qual revelou diferenças de intensidade das bandas, bem como a presença e ausência das mesmas em meio com herbicida. O herbicida causou estresse oxidativo nas três linhagens, o qual foi avaliado através da peroxidação de lipídeos, observado principalmente na concentração de 620 mM. As atividades da Catalase (CAT) e Superóxido Dismutase (SOD) foram altas para as três linhagens na concentração de 62 mM seguida de queda em 620 mM. Para a atividade da Glutationa Redutase (GR) foi observado decréscimo gradativo da atividade com o respectivo aumento da concentração de herbicida para as três linhagens. Porém para a atividade da Glutationa-S-transferase (GST), as três linhagens apresentaram comportamento diferencial para os mesmos tratamentos. O estudo da resposta antioxidativa de microrganismos na presença de xenobióticos são importantes para a compreensão de mecanismos de tolerância, biodegradação bem como para aplicação na biorremediação de ambientes contaminados. / Currently there is a great concern with respect to short and long term effects that many herbicides may cause on public health and the environment. They may seriously damage the cellular metabolism, generally leading to inhibition of the microbial growth. With the exposure to herbicides, these microorganisms significantly increase the generation of EAOs (Active Oxygen Species), generating a state of oxidative stress. The aim of this work was to study the effect of acetochlor herbicide in the response of antioxidant system. Three strains of the bacteria Klebsiella oxytoca were grown in a nutritional media containing the herbicide in concentrations of 0, 62 and 620 mM. The protein profile was evaluated in SDS-PAGE, which showed differences in the intensity of the bands, as well as the presence and absence in the media with herbicide. The oxidative stress was evaluated through the lipid peroxidation, and herbicide caused stress in all the three strains, mainly at the concentration of 620 mM. The activities of Catalase (CAT) and Superoxide Dismutase (SOD) were higher for the three strains in the concentration of 62 mM followed by a decrease at the concentration of 620 mM. For Glutathione Reductase (GR) it was observed a gradual reduction in its activity with the increase of herbicide concentration for all the three strains. However, in the case of Glutathione-S-transferase (GST) activity, the three strains showed differential behavior for the same treatments. The study of the antioxidative response of microorganisms in the presence of xenobiotics is important for the understanding of tolerance mechanisms, biodegradation as well as for application in the bioremediation of contaminated environments.

O aumento da produção de espécies ativas de oxigênio (EAOs) é observado em resposta a uma variedade de agentes estressantes, incluindo herbicidas. Alguns estudos relacionam o sistema antioxidante com a proteção celular e a resistência aos agroquímicos. O s-metolachlor é um herbicida chloroacetanilida, de alta toxicidade e um dos agroquímicos mais freqüentemente detectados em estudos de contaminação ambiental. Esse trabalho objetivou estudar a relação entre a toxicidade do herbicida e a resposta do sistema antioxidante em três bactérias isoladas de solo agrícola. Os microrganismos foram cultivados em meio nutritivo com doses crescentes de herbicida (0, 34 e 340 mM). Na maior dose de s-metolachlor, foi observada alta taxa de peroxidação lipídica, a qual reflete os danos causados pelo estresse oxidativo. O perfil protéico foi diferente entre as espécies bacterianas em análise de SDS-PAGE, porém não foi observada nenhuma diferença significativa entre as doses de s-metolachlor. As enzimas superóxido dismutase e catalase apresentaram indução da atividade na presença do herbicida a 34 mM, o que pode estar relacionado ao aparecimento de uma nova isoforma de CAT em análise de PAGE nativo. Para a enzima glutationa-redutase (GR), houve o aparecimento de novas isoformas na presença do herbicida, principalmente na concentração de 340 mM em PAGE. Já a atividade da glutationa S-transferase e da GR, em espectrofotômetro, refletiram a toxicidade do herbicida, apresentando queda em meio com maior dose de s-metlachlor. A variedade de respostas encontradas entre as bactérias foi muito maior do que o fator que produziu a resposta, sendo que esta diversidade pode condicionar o encontro de uma espécie bacteriana com vantagens na resistência ao herbicida. Os resultados sugerem que em baixas doses (34 mM), o herbicida pode induzir a atividade de algumas enzimas antioxidantes, porém, em doses maiores (340 mM) ele é tóxico para as bactérias. / The increase of reactive oxygen species (ROS) production is observed in response to a variety of stressful agents, such as herbicides. Some studies can associate the antioxidant system with cell protection and resistance to agrochemicals. S-metolachlor is a chloroacetanilide herbicide of high toxicity and one of the most commonly detected agrochemicals in studies concerning environmental contamination. The objective of this work was to investigate the relation between the herbicide toxicity and the oxidative responses in three bacteria isolated from an agricultural soil. These microorganisms were cultivated in nutritive media in the presence of increasing herbicide concentrations (0 mM, 34 mM and 340 mM). At the high concentration of smetolachlor, 340mM, lipidic peroxidation was observed, reflecting the damage caused by the oxidative stress. The protein profile was different among bacterial species in SDS-PAGE analyze, but no significant difference occurred between the different s-metolachlor dosages. The superoxide dismutase and catalase enzymes showed activity induction in the presence of 34 mM of s-metolachlor, which can be related to the emergence of a new CAT isoform in native PAGE analyze. In the case of glutathione-reductase (GR) it was also observed the induction of new isoforms in the presence of the herbicide, mainly in the 340 mM PAGE concentration. The activity of glutathione S-transferase and GR in spectrophotometer reflected the herbicide toxicity, it decreased in the media with the 340 mM of s-metolachlor. The variety of responses showed among the bacterias was much higher than the factor that induced the response. This diversity can lead to found bacterial species with advantages in herbicide resistance. The results suggest that at low doses (34 mM), the herbicide may induce the activity of some antioxidant enzymes, but in larger doses (340 mM) it is toxic to the bacteria.

Concentrations of herbicides in our aquatic ecosystems increase more and more. Among these, the herbicide glyphosate is the most common one. This ecotoxicological study was performed in order to examine the toxic effect of the herbicide glyphosate on aquatic invertebrates. Odonata were selected as study organisms in order to serve as bio-indicators of environmental contamination. Two populations, each of two species (Erythromma najas and Libellula quadrimaculata) were collected from four different locations, to study inter-specific differences, as well as, differences among populations within a species, in response to herbicide exposure. The experiment was conducted for 15 days in a 2 x 4 factorial design with 4 replicates (n = 32). The most common brand of weed-killer ‘Roundup’ containing 7.2 gL-1 of glyphosate) was used as source of glyphosate. Glyphosate was applied at a concentration of 7.6 mgL-1 in the experiment equalling the high end of environmentally relevant concentrations of glyphosate present in contaminated shallow waters. Response variables measured were larval survival, growth and activity. The results showed that glyphosate exposure reduced the survival of the larvae, but the magnitude of the glyphosate effects depended on species identity of the larvae and varied also with population within species. This study clearly shows that herbicide effects on invertebrate fitness depends on species identity and may even vary within species from different populations, possibly due to evolved resistance of random genetic variations between populations or due to random genetic variation between populations.

The global human population is expected to exceed 9 billion individuals by 2050, putting greater strain on the natural resources needed to sustain such a population. To feed this many people, some expect agricultural production will have to double and agrochemical use will have to increase anywhere from two- to five-fold relative to the turn of the century. Although industrial agriculture has provided many benefits to society, it has caused declines in biodiversity, both directly (e.g., through conversion of habitat) and indirectly (e.g., through contamination of adjacent natural habitats). Agricultural activity has also been linked to increased prevalence and intensity of trematode infections in wildlife and humans - directly by increasing available aquatic habitat for the snail intermediate hosts of trematode parasites and indirectly by altering the biological composition of aquatic habitats in ways that increase snail density. While the effects of single agrochemical contaminants on aquatic communities and trematode disease risk have been examined, agrochemical pollution typically occurs as mixtures of multiple chemical types in surface waters and the effects of mixtures on aquatic communities have received less attention. Moreover, given the high number of chemicals approved for agricultural use, the number of potential combinations of agrochemicals renders testing all possible combinations implausible. Thus, there is a critical need to develop better risk assessment tools in the face of this complexity. I developed and tested a theoretical framework that posits that the net effects of agrochemical mixtures on aquatic communities can be predicted by integrating knowledge of each functional group's 1) sensitivity to the chemicals (direct effects), 2) reproductive rates (recovery rates), 3) interaction strength with other functional groups (indirect effects), and 4) links to ecosystem properties. I conducted a freshwater mesocosm experiment to quantify community- and ecosystem-level responses to pairwise mixtures of four major agrochemical types (fertilizer, herbicide, insecticide, and fungicide) and single chemical treatments. The responses of biodiversity and ecosystem properties to agrochemicals alone and in mixtures were indeed predictable. Moreover, these results show that community ecology theory holds promise for predicting the effects of contaminant mixtures and offer recommendations on which types of agrochemicals to apply together and separately to reduce their impacts on aquatic ecosystems. I extended this framework to test if the direct effects of pesticides can be predicted by chemical class and/or mode of action. I performed standard toxicity trials on two invertebrate predators of snails (crayfish and giant water bugs) exposed to six insecticides belonging to two chemical classes (organophosphates and pyrethroids) to determine if environmental risk can be generalized to either insecticide class or insecticide exposure. Survival analyses demonstrated that insecticide class accounted for 55.7% and 91.1% of explained variance in crayfish and water bug survival, respectively. Simulated environmental exposures using US EPA software suggested that organophosphate insecticides present relatively low risk (as defined by the US EPA) to both crayfish and water bugs, while pyrethroid insecticides present consistently high risk to crayfish but not to water bugs, where only λ-cyhalothrin produced consistently high-risk exposure scenarios. Thus, risk to non-target organisms is well predicted by pesticide class. Furthermore, identifying insecticides that pose low risk to aquatic macroarthropods might help meet increased demands for food while mitigating against potential negative effects on ecosystem functions. Because evidence from field data and manipulated experiments demonstrated both top-down and bottom-up effects of agrochemical pollution that increased snail densities and trematode infections in wildlife, I conducted an additional agrochemical mixture experiment with freshwater communities containing the snail hosts of schistosomiasis, which has also been linked to agriculture. As expected, top-down and bottom-up effects of insecticide, herbicide, and fertilizer exposure indirectly increased snail densities, individually and as mixtures. Agrochemical exposure and snail density together accounted for 88% of the variation in the density of infected snails. Thus, agrochemical pollution has great potential to increase human exposure to schistosome parasites, and underscores the importance of identifying low-risk alternative pesticides. A subsequent mesocosm experiment with the same six insecticides used previously in laboratory trials confirmed that insecticide exposure indirectly mediates the densities of snail hosts that can transmit schistosomiasis through the direct effects of insecticides on crayfish mortality. Importantly, crayfish mortality in semi-natural mesocosm trials closely matched mortality from controlled laboratory trials. Thus, standard laboratory toxicity tests can be a useful tool for identifying alternative insecticides that might pose lower environmental risks to important predators that regulate snail densities. Identifying practices or agrochemicals that minimize this risk is critical to sustainably improving human health in schistosome-endemic regions. The theoretical framework presented here demonstrates the feasibility of predicting the effects of contaminant mixtures and highlights consistent effects of major agrochemical types (e.g. fertilizers, insecticides, etc.) on freshwater aquatic community composition. Furthermore, the strong top-down effects of invertebrate snail predators highlight that managing for high snail predator densities in might be a particularly effective strategy for reducing the burden of schistosomiasis in tropical countries.

Palmer amaranth (Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.

Master of Science / Department of Agronomy / Randall S. Currie / Mithila Jugulam / Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot), one of the problem weeds of the US, evolved resistance to multiple herbicides including glyphosate due to selection in Arkansas (AR). Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and amplification of EPSPS gene, the molecular target of glyphosate confers resistance to this herbicide in several weed species, including Italian ryegrass from AR. The objective of this study was to determine the expression of EPSPS gene and protein as well as distribution of EPSPS copies on the genome of glyphosate-resistant Italian ryegrass (ARR) using a known susceptible Italian ryegrass (ARS) from AR. EPSPS gene copies and expression of ARR and ARS were determined using quantitative PCR with appropriate endogenous controls. EPSPS protein expression was determined using Western blot analysis. Fluorescence in situ hybridization (FISH) was performed on somatic metaphase chromosomes to determine the location of EPSPS copies. Based on qPCR analysis, ARR plants showed a wide range of 12 to 118 EPSPS copies compared to a single copy in ARS. EPSPS gene expression correlated with the gene copy number in both ARR and ARS. Individuals with high EPSPS copies showed high protein expression in Western blot analysis. FISH analysis showed presence of brighter EPSPS signals, distributed randomly throughout the genome of ARR individuals compared to a faint signal in ARS plants. Random distribution of EPSPS copies was previously reported in glyphosate-resistant Palmer amaranth. Overall, the results of this study will help understand the origin and mechanism of EPSPS gene amplification in Italian ryegrass.

Maize damage has been observed from time to time in many parts of South Africa where registered herbicides were applied. Differential cultivar tolerance to certain herbicides was identified as an important factor in many cases where herbicide selectivity was inadequate. A study was done to investigate the existence of differential tolerance of maize genotypes to selected herbicides. Several maize genotypes were screened for tolerance to selected herbicides, both in the greenhouse and in the field. Maize genotypes demonstrated significant differences in their tolerance to all herbicides. Some genotypes were severely injured by certain herbicides while others were not affected at all by the same herbicide. This suggests that maize genotypes should be screened for tolerance to herbicides in order to reduce crop injury by using only appropriate ones for specific genotypes. Generally, hybrids were more tolerant to herbicides than inbreds, indicating that screening may be most necessary in seed production, where inbreds are used, than in commercial production where only hybrids are used. Tolerance of maize to herbicides was more variable to metazachlor than to other herbicides, and metazachlor also injured more genotypes than other herbicides. This was probably due to differences in herbicide mechanism of action. Shoot or root dry mass reduction of some of the maize genotypes occurred without visual injury symptoms, thus suggesting that visual injury may not reliably indicate susceptibility or tolerance to herbicides. The degree of correspondence of herbicide effects on maize in the greenhouse and the field was determined. Comparison of results from the greenhouse and the field showed that there is positive correlation between herbicide effects in the greenhouse and in the field. There was generally good correspondence of major parameters, such as shoot dry mass and injury symptoms, in the greenhouse and in the field. Similarly these parameters were positively correlated with the grain yield obtained from the field. It appears that shoot dry mass and visual injury symptoms could be good predictors of the yield. This indicates that reliable data could be generated through quicker screening at greenhouse level. A total of four herbicides, metazachlor, dimethenamid, acetochlor and the combination atrazine / metolachlor / terbuthylazine, had significant correlations while only two, flufenacet and acetochlor + atrazine/sulcotrione, had no significant correlations for major parameters with the yield. This indicates that the correlation of data was herbicide-dependent. The influence of temperature on maize tolerance to alachlor, metazachlor and metolachlor was investigated. Results showed that low temperatures reduce the tolerance of maize to these herbicides. This could mean that low temperature may reduce the selectivity of these herbicides. Fluctuating temperature conditions of 10°C at night and 35°C during the day, which are found in some maize producing areas, did not affect maize tolerance to the herbicides. The possibility of improving metazachlor tolerance in maize was also investigated. Evidence provided for possible gene effects on the tolerance of metazachlor indicates that maize tolerance to the herbicide could be improved by crossing tolerant parents. The results suggest that it may be possible to improve metazachlor tolerance by crossing appropriate parent lines with dominant genes for tolerance to metazachlor. Ultrastructural changes in the maize seedling root and shoot cells caused by metazachlor were investigated. In susceptible genotypes root cell nucleoli were found to be abnormally large, empty and more abundant than those in untreated control plants. In susceptible plants the chromatids appeared disorganised in cell nucleoli, and both the nuclear and plasma membranes showed signs of disintegrating. There were more and larger vacuoles in the herbicide-susceptible plants. Leaf cells from the susceptible plants had more empty vacuoles and more chloroplasts with generally disorganised content. The bundle sheath chloroplast membranes were dilated in susceptible plants, and the orientation of the grana was disrupted. In the herbicide-tolerant plants, the ultrastructure was not different from that of all the untreated plants. The established differential tolerance of maize to herbicides necessitates the screening of all genotypes to all registered herbicides in order to recommend specific herbicides for certain maize genotypes. Due to the large number of genotypes that would require screening, techniques that yield reliable data quickly have obvious merit. Pot experiments under controlled conditions, which could be selected to promote herbicide bioactivity, are likely to provide data with which the best possible predictions on the risk of herbicide damage in the field could be made. Based on this requirement, environmental factors that should be considered for greenhouse work are: soil with low adsorptive capacity, soil water content close to the field capacity level, and cool temperatures. When screening for herbicide tolerance, the use of herbicide rates in excess of the recommended rate could obviate the need for special environmental conditions, since all the aforementioned factors basically promote the accumulation of higher than usual amounts of herbicide at the site of action in the plant. Therefore, the use of at least a 2X-herbicide rate in screening experiments is advised. / Thesis (PhD (Plant Production: Weed Science))--University of Pretoria, 2006. / Plant Production and Soil Science / unrestricted