Made available in DSpace on 2014-06-11T19:30:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-10Bitstream added on 2014-06-13T21:01:04Z : No. of bitstreams: 1 cavalieri_sd_dr_botfca.pdf: 541032 bytes, checksum: 35c98291dc7bcdb9b1ec86f4f88d72ba (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo da seletividade e dos efeitos secundários dos herbicidas nas culturas agrícolas é de extrema importância para o sucesso da agricultura. No presente trabalho, três experimentos foram realizados. O primeiro, conduzido em casa-de-vegetação localizada na Faculdade de Ciências Agronômicas, UNESP, Campus de Botucatu (SP), teve como objetivo avaliar o efeito de formulações comerciais de glyphosate em parâmetros nutricionais e acúmulo de matéria seca de duas cultivares de soja RR resistentes ao glyphosate. Os tratamentos avaliados resultaram do arranjo fatorial entre formulações de glyphosate (Roundup Original®, Roundup Ready®, Roundup Transorb®, Roundup WG®, Roundup Ultra® e Zapp Qi®), mais uma testemunha e cultivares de soja RR (CD 225 RR e V Max RR), conduzidos em delineamento de blocos completos casualizados com seis repetições. As aplicações dos herbicidas ocorreram quando as plantas de soja apresentavam-se no estádio V3 (25 dias após a emergência), na dosagem de 960 g e.a. ha-1. Transcorridos 15 dias após o tratamento, a parte aérea das plantas de soja foi colhida e seca em estufa. Depois de secas, obtiveram-se os dados de massa da matéria seca de hastes, folhas e parte aérea (hastes + folhas), sendo em seguida o material triturado e enviado para laboratório para análise dos teores de macronutrientes (N, P, K, Ca, Mg e S) e micronutrientes (Cu, Fe, Mn, Zn e B). Após isso, de posse dos resultados das análises laboratoriais, calculou-se o acúmulo de nutrientes na parte aérea de cada planta presente nos vasos. De forma geral, o acúmulo de macronutrientes, micronutrientes e matéria seca na parte aérea das plantas de soja sempre foi maior na cultivar V Max RR em relação à CD 225 RR. As formulações Roundup Ready® e Roundup Ultra®, não apresentaram problemas no que diz respeito ao acúmulo de nutrientes e matéria seca na parte aérea... / The study of selectivity and secondary effects of herbicides on crops is extremely important to the success of agriculture. In this study, three experiments were conducted. The first one, carried out in a greenhouse located at the College of Agronomic Sciences, UNESP, Botucatu (SP), aimed to evaluate the effect of commercial formulations of glyphosate on nutritional status and dry matter accumulations in two glyphosate-resistant soybeans cultivars (GR). The treatments were arranged in a factorial arrangement involving six commercial formulations of glyphosate Roundup Original®, Roundup Ready®, Roundup Transorb®, Roundup WG®, Roundup Ultra® and Zapp Qi® plus a control treatment, and two soybean cultivars (CD 225 RR and V Max RR), arranged in a randomized complete block design replicated six times. The herbicide applications occurred when the soybean plants were at V3 growth stage (25 days after emergence) using a dose of 960 g a.e. ha-1. After 15 days after application, the shoot of soybeans was harvested and dried in an oven. Once dried, we obtained data of dry matter of stems, leaves and shoots (stems + leaves), and then the plants were crushed and sent to the laboratory for analysis of macronutrients (N, P, K, Ca, Mg and S) and micronutrients (Cu, Fe, Mn, Zn and B). After that, ownership of the results of nutritional analysis, we calculated the accumulation of nutrients in the shoots in each plant of the pots. In general, the accumulation of macronutrients, micronutrients and dry matter in the shoot of soybean plants was always greater in V Max RR cultivar than CD 225 RR cultivar. The formulations Roundup Ready®, Roundup Ultra® and Zapp QI® showed no problems in regard to the accumulation of nutrients in shoots of cultivars. Furthermore, the Roundup Original®, Roundup Transorb® and Roundup WG® formulations caused the greatest damage to the nutrition provided the cultivars... (Complete abstract click electronic access below)

Submitted by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-27T13:30:37Z No. of bitstreams: 5 61.pdf: 450363 bytes, checksum: cd83469eae8b523d2f8e1536e42d83d6 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2015-02-27T19:40:11Z (GMT) No. of bitstreams: 5 61.pdf: 450363 bytes, checksum: cd83469eae8b523d2f8e1536e42d83d6 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) / Made available in DSpace on 2015-02-27T19:40:11Z (GMT). No. of bitstreams: 5 61.pdf: 450363 bytes, checksum: cd83469eae8b523d2f8e1536e42d83d6 (MD5) license_url: 52 bytes, checksum: 3d480ae6c91e310daba2020f8787d6f9 (MD5) license_text: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) license.txt: 2109 bytes, checksum: aa477231e840f304454a16eb85a9235f (MD5) Previous issue date: 2013 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (Capes) / Funda??o de Amparo ? Pesquisa do estado de Minas Gerais (FAPEMIG) / O herbicida glyphosate n?o ? seletivo e de largo espectro de controle de plantas daninhas, seu mecanismo de a??o ocorre com a inibi??o da enzima 5-enolpiruvilchiquimato-3-fosfato sintase (EPSPs), acontecendo o bloqueio da rota do ?cido chiqu?mico, precursor de amino?cidos arom?ticos e de outros metabolitos secund?rios. O objetivo deste trabalho foi avaliar as altera??es nas caracter?sticas fotossint?ticas e do uso eficiente da ?gua por plantas de cafeeiro submetidas ? aplica??o de glyphosate. O experimento foi conduzido em casa de vegeta??o utilizando-se tr?s cultivares de caf? (Coffea arabica): Acai? (MG-6851), Catuca? Amarelo (2 SL) e Top?zio (MG-1190) e tr?s subdoses do glyphosate (0,0; 115,2 e 460,8 g ha-1), em esquema fatorial 3x3, com 4 repeti??es. Os cultivares de caf? se diferiram quanto ? atividade fotossint?tica. Com o aumento das subdoses do herbicida, observou-se maiores consequ?ncias negativas sobre as vari?veis fotossint?ticas. Tais efeitos podem ser atribu?dos aos danos diretos na atividade fotossint?tica ou pelos indiretos, afetando o metabolismo da planta. Com a aplica??o do herbicida, as plantas de cafeeiro apresentaram redu??es de taxa transpirat?ria e condutividade estom?tica, por?m menor efici?ncia do uso da ?gua apenas aos 15 DAA na quarta folha. Os cultivares apresentaram efeitos negativos com a aplica??o das subdoses de glyphosate, quanto a transpira??o e condut?ncia estom?tica. Pode-se concluir que o cultivar Acai? apresentou-se mais tolerante, pois n?o mostrou efeitos prejudiciais na efici?ncia do uso da ?gua. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Produ??o Vegetal, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2011. / ABSTRACT The herbicide glyphosate is nonselective and of wide-spectrum weed control; its mechanism of action is the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), blocking shikimic acid route, a precursor of aromatic amino acids and other secondary metabolites. The objective of this study was to evaluate the changes in photosynthetic characteristics and of water use efficiency for coffee plants submitted to glyphosate application. The experiment was conducted in a greenhouse using three varieties of coffee (Coffea Arabica): Acai? (MG-6851), Catuca? Amarelo (2 SL) e Top?zio (MG-1190) and, three subdoses of glyphosate (0,0; 115,2 and 460,8 g ha-1), in a factorial 3x3, with four replicates. The coffee cultivars subjected to reduced rates of glyphosate to differ in terms of photosynthetic activity. With the increase in sub dosage of the herbicide, there have been observed more negative consequences on the photosynthetic variables. These effects can be attributed to the direct damages on photosynthetic activity or to the indirect ones, affecting the metabolism of the plant. With the herbicide application, coffee plants presented reduction of perspiration rate and stomatal conductivity, however less efficiency in water use only at 15 DAA, on the fourth leaf. The cultivars presented negative effects with the application of a sub dosage of glyphosate regarding perspiration and stomatal conductivity. It can be concluded that the cultivar Acai? showed to be more tolerant, as it did not show prejudicial effects in water use efficiency.

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.

The resistance of Palmer amaranth (PA) and the tolerance (natural resistance) of pitted morningglory (PM) to glyphosate have made these species among the most common and troublesome weeds in the southeastern U.S. since the adoption of glyphosate-resistant (GR) crops. Populations of GR PA (R1 and R2) were identified in Mississippi. The inheritance of glyphosate resistance was examined in reciprocal crosses (RC) between glyphosate-resistant (R) and -susceptible (S) parents (Female-S × Male-R, S/R, and Female-R × Male-S, R/S), and second reciprocal crosses (2RC) (Female-S/R × Male-S/R, S/R//S/R, and Female-R/S × Male-R/S, R/S//R/S). Dose-response assays resulted in 17- to 4old resistance to glyphosate compared with S. Population S accumulated 325- and 8-times more shikimate at the highest glyphosate dose than in R1 and R2, respectively. cDNA sequence analysis of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no target site mutation. Genomes of R1, R2, RC, and 2RC contained from 1- to 59old more copies of EPSPS gene than S; EPSPS was highly expressed in R1 and R/S, but was poorly expressed in S, S/R, and R2. EPSPS activity was lower in S and S/R than in R and R/S, glyphosate absent; all were inhibited by glyphosate. Western Blot analysis confirmed an increased EPSPS protein level to EPSPS copy number correlation. Thus, the level of resistance was decidedly influenced by the direction of the cross. R and S female plants were reproductively isolated and seed were still produced, suggesting that PA can produce seed both apomictically and sexually (facultative apomixis). This mode of reproduction determined the low copy number inheritance, as well as guaranteeing the GR trait stability in the R populations. Dose-response assays resulted in 2.6old variability in tolerance to glyphosate between the most tolerant (MT) and the least tolerant (LT) PM populations. The level of tolerance positively correlated with the time of exposure to GR-crop system. Less shikimate was recovered in MT as compared to LT. Levels of aminomethylphosphonic acid (AMPA) were not different between populations and sarcosine was not present in either populations. Consequently, metabolism of glyphosate to AMPA or sarcosine is not a common factor in explaining natural resistance levels.

No description available.

Este trabalho teve como objetivo, desenvolver um modelo molecular que possa descrever propriedades de equilíbrio, bem como as forças de interação envolvidas em um sistema enzima-inibidor, onde a enzima é a 5-Enolpiruvil-Shikimato-3-Fosfato Sintase (EPSPs) e seu inibidor o herbicida glifosato (GPJ). A participação do substrato Shikimato-3- Fosfato (S3P) também foi estudada. A estabilidade e a distribuição de cargas ao longo da superfície da enzima EPSPs foram analisadas para subsidiar o desenvolvimento de um nanobiossensor específico para a detecção do herbicida glifosato através da funcionalização da ponta de um microscópio de força atômica (AFM) com a enzima EPSPs. A parametrização de dois ângulos diedros para o herbicida GPJ foram obtidos através de cálculos mecânico- quânticos ao nível de Hartree Fock, utilizando como conjunto de bases o 6-31G*. Os parâmetros obtidos, relacionados aos coeficientes de Ryckaert-Bellemans, foram implementados no campo de força OPLS-AA, utilizado pelo programa GROMACS. Simulações de dinâmica molecular direcional (SMD), foram realizadas em três possíveis caminhos de retirada do herbicida do sítio ativo, aplicando-se forças harmônicas externas com constante de força equivalente à do cantiléver do AFM, com o objetivo de simular experimentos de detecção do herbicida por microscopia de força atômica. Uma expansão cumulante de segunda ordem da igualdade de Jarzynski foi utilizada para os cálculos do perfil de energia livre ao longo da coordenada de reação e, portanto, para se obter o Potencial de Força Média (PMF) das simulações de SMD. Os três caminhos escolhidos apresentaram diferentes barreiras energéticas relacionadas com o rompimento das interações do inibidor com a enzima. Estas barreiras energéticas calculadas pelas simulações estão na mesma ordem de magnitude das energias livres de ligação para sistemas semelhantes obtidas a partir das constantes de inibição experimentais. / This work aims to developed a molecular model that can describe equilibrium properties, as well as the interacting forces involved in an enzyme-inhibitor system, where the enzyme is the 5-Enolpyruvyl-Shikimate-3-Phosphate Synthase (EPSPs) and its inhibitor is the glyphosate herbicide (GPJ). The participation of the substrate Shikimate-3-Phosphate (S3P) was also studied. The stability and distribution of charges along the surface of the EPSPs enzyme were analyzed for the development of a specific nanobiosensor for glyphosate herbicide detection through the functionalization of the tip of an atomic force microscope (AFM) with the enzyme EPSPs. The parameterization of two dihedral angles for the GPJ herbicide was obtained through mechanical-quantum calculations at the Hartree Fock level using the 6-31G* base set. The parameters obtained, related to the Ryckaert-Bellemans coefficients, were implemented in the OPLS-AA force field, used by the GROMACS program. Steered Molecular Dynamics Simulations (SMD) were performed in three possible unbinding pathways, to remove the herbicide from the active site, applying external harmonic forces with a force constant equivalent to that of the AFM cantilever, in order to simulate experiments to detect the herbicide by atomic force microscopy. A second-order cumulant expansion of the Jarzynski’s equation was used to calculate the free energy profile along the reaction coordinate and to obtain the potential of mean force (PMF) of the SMD simulations. The three paths chosen presented different energetic barriers related to the disruption of inhibitor interactions with the enzyme. These energy barriers calculated by the simulations are in the same order of magnitude as the bond free energies for similar systems obtained from the experimental inhibition constants. / Tese (Doutorado)

A utilização ampla do glyphosate como herbicida na agricultura brasileira e mundial é conseqüência, dentre outros fatores, de seu custo relativamente baixo, alta eficácia no controle de plantas daninhas, amplo espectro de controle de espécies de plantas daninhas, baixa toxicidade, e curta persistência no ambiente. No entanto, o uso intensivo do glyphosate tem proporcionado a seleção de biótipos de plantas daninhas resistentes a este herbicida, como é o caso da buva (Conyza bonariensis). Apesar de estudos já terem sido conduzidos pela comunidade científica de diversos países, para elucidar o fenômeno sob diversos aspectos, no Brasil são poucos os estudos científicos que esclareçam os mecanismos de resistência de plantas daninhas ao glyphosate. Neste sentido, esta pesquisa teve por objetivo estudar comparativamente populações resistentes (R) e suscetíveis (S) de C. bonariensis ao herbicida glyphosate para caracterizar o nível de resistência das populações R; comparar os níveis de acúmulo de ácido shiquímico entre as populações R e S; elucidar os principais mecanismos de resistência dos biótipos R, através da análise da absorção e translocação do herbicida, determinar a expressão gênica da EPSPS, através de análises semi-quantitativas RTPCR e sugerir alternativas químicas de controle para buva em pomares cítricos do Estado de São Paulo. Os resultados obtidos através do estudo de ácido shiquímico indicaram que o mecanismo de resistência das populações R buva estudadas não está relacionado com insensibilidade da EPSPS ao glyphosate. Já os estudos de translocação diferencial evidenciaram ser esta uma das causas dos mecanismos envolvidos na resistência do biótipo R. Através da análise da expressão gênica observou-se alto grau de similaridade entre seqüências obtidas dos genes EPSPS de C. bonariensis e as seqüências de C. canadensis depositadas no GenBank. Além disso, é possível sugerir que há relação entre a expressão dos genes EPSPS em C. bonariensis e a condição de resistência à ação do herbicida glyphosate em alguns indivíduos dessa espécie de planta daninha. / The extensive use of glyphosate as herbicide in the Brazilian and worldwide agriculture is a consequence, among other factors, of the relatively low cost, high weed control efficacy; wide weed species control spectrum; low toxicity, and short persistence in the environment. However, the intensive use of glyphosate has imposed the selection of certain resistant weed biotypes to this herbicide, such as the case of the weed Conyza bonariensis. Despite the fact that some studies has been developed in some countries around the world, in order to elucidate the phenomenon in several aspects, in Brazil a few studies have been conducted scientifically in order to elucidate the mechanisms of weed resistance to glyphosate. Therefore, this research was developed with the objective of studying the populations of C. bonariensis to characterize the resistance level of populations to glyphosate; compare the levels of shikimic acid accumulation between resistant (R) and susceptible (S) plants; elucidate the main mechanisms of resistance of resistant biotypes to glyphosate, by the absorption and translocation of the herbicide, and determine the EPSPS gene expression, by the RT-PCR semi-quantitative analysis. The results obtained by the shikimic acid study indicated that the mechanism of resistance of the biotype of C. bonariensis studied is not related with insensitivity of the EPSPS to glyphosate. On the other hand, the differential translocation maybe considered as one of the mechanisms involved in the resistance of the biotype R of C. bonariensis. With regards to gene expression, it was observed high degree of similarity among the sequences obtained of the EPSPS gene of C. canadensis, and the sequences of C. canadensis deposited in the GenBank. Furthermore, it is possible to suggest that there is relation between the gene expression in C. bonariensis and the resistance condition to the action of the herbicide glyphosate in some individuals of this weed specie.

A utilização ampla do glyphosate como herbicida na agricultura brasileira e mundial é conseqüência, dentre outros fatores, de seu custo relativamente baixo, alta eficácia no controle de plantas daninhas, amplo espectro de controle de espécies de plantas daninhas, baixa toxicidade, e curta persistência no ambiente. No entanto, o uso intensivo do glyphosate tem proporcionado a seleção de biótipos de plantas daninhas resistentes a este herbicida, como é o caso da buva (Conyza bonariensis). Apesar de estudos já terem sido conduzidos pela comunidade científica de diversos países, para elucidar o fenômeno sob diversos aspectos, no Brasil são poucos os estudos científicos que esclareçam os mecanismos de resistência de plantas daninhas ao glyphosate. Neste sentido, esta pesquisa teve por objetivo estudar comparativamente populações resistentes (R) e suscetíveis (S) de C. bonariensis ao herbicida glyphosate para caracterizar o nível de resistência das populações R; comparar os níveis de acúmulo de ácido shiquímico entre as populações R e S; elucidar os principais mecanismos de resistência dos biótipos R, através da análise da absorção e translocação do herbicida, determinar a expressão gênica da EPSPS, através de análises semi-quantitativas RTPCR e sugerir alternativas químicas de controle para buva em pomares cítricos do Estado de São Paulo. Os resultados obtidos através do estudo de ácido shiquímico indicaram que o mecanismo de resistência das populações R buva estudadas não está relacionado com insensibilidade da EPSPS ao glyphosate. Já os estudos de translocação diferencial evidenciaram ser esta uma das causas dos mecanismos envolvidos na resistência do biótipo R. Através da análise da expressão gênica observou-se alto grau de similaridade entre seqüências obtidas dos genes EPSPS de C. bonariensis e as seqüências de C. canadensis depositadas no GenBank. Além disso, é possível sugerir que há relação entre a expressão dos genes EPSPS em C. bonariensis e a condição de resistência à ação do herbicida glyphosate em alguns indivíduos dessa espécie de planta daninha. / The extensive use of glyphosate as herbicide in the Brazilian and worldwide agriculture is a consequence, among other factors, of the relatively low cost, high weed control efficacy; wide weed species control spectrum; low toxicity, and short persistence in the environment. However, the intensive use of glyphosate has imposed the selection of certain resistant weed biotypes to this herbicide, such as the case of the weed Conyza bonariensis. Despite the fact that some studies has been developed in some countries around the world, in order to elucidate the phenomenon in several aspects, in Brazil a few studies have been conducted scientifically in order to elucidate the mechanisms of weed resistance to glyphosate. Therefore, this research was developed with the objective of studying the populations of C. bonariensis to characterize the resistance level of populations to glyphosate; compare the levels of shikimic acid accumulation between resistant (R) and susceptible (S) plants; elucidate the main mechanisms of resistance of resistant biotypes to glyphosate, by the absorption and translocation of the herbicide, and determine the EPSPS gene expression, by the RT-PCR semi-quantitative analysis. The results obtained by the shikimic acid study indicated that the mechanism of resistance of the biotype of C. bonariensis studied is not related with insensitivity of the EPSPS to glyphosate. On the other hand, the differential translocation maybe considered as one of the mechanisms involved in the resistance of the biotype R of C. bonariensis. With regards to gene expression, it was observed high degree of similarity among the sequences obtained of the EPSPS gene of C. canadensis, and the sequences of C. canadensis deposited in the GenBank. Furthermore, it is possible to suggest that there is relation between the gene expression in C. bonariensis and the resistance condition to the action of the herbicide glyphosate in some individuals of this weed specie.

Orientador: Edivaldo Domingues Velini / Banca: João Domingos Rodrigues / Banca: Robinson Antonio Pitelli / Banca: Robinson Osipe / Banca: Cleber Daniel de Goes Maciel / Resumo: O estudo da seletividade e dos efeitos secundários dos herbicidas nas culturas agrícolas é de extrema importância para o sucesso da agricultura. No presente trabalho, três experimentos foram realizados. O primeiro, conduzido em casa-de-vegetação localizada na Faculdade de Ciências Agronômicas, UNESP, Campus de Botucatu (SP), teve como objetivo avaliar o efeito de formulações comerciais de glyphosate em parâmetros nutricionais e acúmulo de matéria seca de duas cultivares de soja RR resistentes ao glyphosate. Os tratamentos avaliados resultaram do arranjo fatorial entre formulações de glyphosate (Roundup Original®, Roundup Ready®, Roundup Transorb®, Roundup WG®, Roundup Ultra® e Zapp Qi®), mais uma testemunha e cultivares de soja RR (CD 225 RR e V Max RR), conduzidos em delineamento de blocos completos casualizados com seis repetições. As aplicações dos herbicidas ocorreram quando as plantas de soja apresentavam-se no estádio V3 (25 dias após a emergência), na dosagem de 960 g e.a. ha-1. Transcorridos 15 dias após o tratamento, a parte aérea das plantas de soja foi colhida e seca em estufa. Depois de secas, obtiveram-se os dados de massa da matéria seca de hastes, folhas e parte aérea (hastes + folhas), sendo em seguida o material triturado e enviado para laboratório para análise dos teores de macronutrientes (N, P, K, Ca, Mg e S) e micronutrientes (Cu, Fe, Mn, Zn e B). Após isso, de posse dos resultados das análises laboratoriais, calculou-se o acúmulo de nutrientes na parte aérea de cada planta presente nos vasos. De forma geral, o acúmulo de macronutrientes, micronutrientes e matéria seca na parte aérea das plantas de soja sempre foi maior na cultivar V Max RR em relação à CD 225 RR. As formulações Roundup Ready® e Roundup Ultra®, não apresentaram problemas no que diz respeito ao acúmulo de nutrientes e matéria seca na parte aérea... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The study of selectivity and secondary effects of herbicides on crops is extremely important to the success of agriculture. In this study, three experiments were conducted. The first one, carried out in a greenhouse located at the College of Agronomic Sciences, UNESP, Botucatu (SP), aimed to evaluate the effect of commercial formulations of glyphosate on nutritional status and dry matter accumulations in two glyphosate-resistant soybeans cultivars (GR). The treatments were arranged in a factorial arrangement involving six commercial formulations of glyphosate Roundup Original®, Roundup Ready®, Roundup Transorb®, Roundup WG®, Roundup Ultra® and Zapp Qi® plus a control treatment, and two soybean cultivars (CD 225 RR and V Max RR), arranged in a randomized complete block design replicated six times. The herbicide applications occurred when the soybean plants were at V3 growth stage (25 days after emergence) using a dose of 960 g a.e. ha-1. After 15 days after application, the shoot of soybeans was harvested and dried in an oven. Once dried, we obtained data of dry matter of stems, leaves and shoots (stems + leaves), and then the plants were crushed and sent to the laboratory for analysis of macronutrients (N, P, K, Ca, Mg and S) and micronutrients (Cu, Fe, Mn, Zn and B). After that, ownership of the results of nutritional analysis, we calculated the accumulation of nutrients in the shoots in each plant of the pots. In general, the accumulation of macronutrients, micronutrients and dry matter in the shoot of soybean plants was always greater in V Max RR cultivar than CD 225 RR cultivar. The formulations Roundup Ready®, Roundup Ultra® and Zapp QI® showed no problems in regard to the accumulation of nutrients in shoots of cultivars. Furthermore, the Roundup Original®, Roundup Transorb® and Roundup WG® formulations caused the greatest damage to the nutrition provided the cultivars... (Complete abstract click electronic access below) / Doutor

Glyphosat ist eines der weltweit am häufigsten eingesetzten Herbizide. Sein Einsatz wird u.a. auf Grund einer möglichen karzinogenen Wirkung und eines möglichen negativen Einflusses auf die Biodiversität kritisch diskutiert. Um Aussagen über die Verbreitung von Glyphosat in der Umwelt treffen zu können, werden verlässliche Nachweissysteme benötigt. Das Ziel der vorliegenden Arbeit bestand darin, ein einfaches optisches System zum schnellen Nachweis von Glyphosat in wässrigen Proben, basierend auf einer Hydrophobin-funktionalisierten Oberfläche, die das Glyphosat Zielprotein präsentiert, zu entwickeln. Hierfür wurden verschiedene Fusionsproteine aus dem Glyphosat Zielprotein, der 5-Enolpyruvylshikimat-3-phosphatsynthase (EPSPS, hier aus dem Bakterium Escherichia coli (EcEPSPS)) und dem zur Selbstassemblierung an hydrophilen/hydrophoben Grenzflächen befähigten Hydrophobin Ccg2 aus Neurospora crassa erzeugt, welche für die Oberflächenfunktionalisierung eingesetzt wurden. Die Expression und Reinigung der Fusionsproteine und von Ccg2 in E. coli verlief erfolgreich. Nach initialen Kontaktwinkelmessungen zur Untersuchung der Funktionalität des Hydrophobins und Enzymaktivitätsmessungen für die Fusionsproteine, konnte deren Aktivität auch nach der Reinigung nachgewiesen werden. Dabei erwies sich das Fusionsprotein Ccg2_GS_EcEPSPS, aufgrund einer hohen enzymatischen Aktivität nach Immobilisierung, als am besten geeignet. Es wurden verschiedene Belegverhältnisse zwischen Hydrophobin und Fusionsprotein untersucht, um etwaige sterische Behinderungen zu minimieren. Hierbei erwies sich ein Belegverhältnis von 1 µM Ccg2_GS_EcEPSPS und 5 µM Ccg2 für die künftigen Messungen als gut geeignet. Auf Basis der so funktionalisierten Oberfläche wurden zwei Verfahren zum optischen Nachweis von Glyphosat entwickelt. Eines der Verfahren, der Malachitgrün-Assay, weist die enzymatische Aktivität der EPSPS auf der Oberfläche nach, genauer das entstehende anorganische Phosphat (Pi). Durch Glyphosathemmung entsteht weniger Pi, dies kann mittels Malachitgrün-Assay nachgewiesen werden. Unter Laborbedingungen konnte ein Detektionslimit von 50 nM erreicht werden. Des Weiteren zeigte der Assay keine nennenswerte Querempfindlichkeit und erwies sich damit als sehr spezifisch. Zusätzlich wurde der Einfluss unterschiedlicher Temperaturen und pH-Werte untersucht. Es zeigte sich, dass Schwankungen dieser Parameter den Assay beeinflussen. Auch ein Einfluss der Ionenstärke konnte festgestellt werden. Deshalb sind entsprechende Kontrollen unerlässlich. Der Einfluss nicht-reaktionsbedingten Phosphates konnte durch Vorinkubation der Oberfläche mit der Glyphosat-haltigen Analyselösung mit anschließender Entfernung der Selbigen und Durchführung des Malachitgrün-Assays minimiert werden. Das zweite Verfahren, der Hydrogelsonden (HGS)-Assay, weist direkt die Interaktion von Glyphosat und der immobilisierten EcEPSPS nach. Hierfür wurden verformbare, Glyphosat-dekorierte HGS aus Polyethylenglykol benötigt. Bei Abwesenheit von freiem Glyphosat liegen die Bindestellen der immobilisierten EPSPS frei vor, sodass sie für die Bindung des immobilisierten Glyphosats an den HGS zur Verfügung stehen. Zwischen den HGS und der Oberfläche entsteht auf diese Weise eine große Kontaktfläche, welche mittels Reflektionsinterferenzkontrastmikroksopie messbar ist. Freies Glyphosat in der Analyselösung reduziert die verfügbaren Bindestellen an der Oberfläche. Dies resultiert in einer kleineren Kontaktfläche. Auf diese Weise kann durch Ermittlung der Größe der Kontaktfläche zwischen HGS und funktionalisierter Oberfläche und der daraus berechneten Adhäsionsenergie, auf das Vorhandensein von Glyphosat in der Analyselösung geschlossen werden. Im Rahmen dieser Arbeit konnte nach Optimierung der Oberflächenbeschichtung, ein positiver Machbarkeitsbeweis für dieses Verfahren erbracht werden.