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.

Difficulties with stand establishment are a major factor limiting further agronomic use of native warm season grasses. One significant cause of stand failure is competition with rapidly growing annual weed species during the early development of the perennial native grass. Broad spectrum preemergent herbicides can provide the needed weed control, but only if tolerance exists in the desired grass. Herbicide safeners, synthetic compounds that protect crops from herbicide injury, applied as seed treatments offer a potential strategy to achieving the needed herbicide tolerance where it does not naturally occur. This study tested the efficacy of five herbicide safeners (benoxacor, fenclorim, fluxofenin, naphthalic anhydride, and oxabetrinil) in protecting three native warm season grass species (big bluestem, Andropogon gerardii Vitman; little bluestem, Schizachyrium scoparium (Michx.) Nash; indiangrass, Sorghastrum nutans (L.) Nash) from herbicidal injury caused by preemergent application of S-metolachlor and quantifies this establishment method’s impact on early stand performance.

The application of commercial herbicide restricts weed growth and significantly improves control over crop vitality and yield. Despite their utility in the agriculture sector, herbicides have the potential to contaminate local water sources. To minimize environmental impacts, the development of efficient separation processes to clean-up contaminated water bodies is necessary. However, complex speciation and conformational flexibility in the condensed phase poses a significant challenge. In this work, we investigate structure and speciation of three common organic herbicides (glyphosate, atrazine, and metolachlor) in aqueous solution. We employ the PBE-D3 density functional to perform ab initio molecular dynamics (MD) simulations in the canonical and isothermal-isobaric ensembles. We analyze MD trajectories to understand hydrogen bonding dynamics and lifetime as well as diffusional and vibrational characteristics. To enhance configurational sampling, we conduct metadynamics simulations to obtain the free energies of dissociation and intramolecular proton transfer of glyphosate.

Herbicide safeners (protectants, antidotes) are used to protect crop plants from herbicide injury. Currently our understanding of the mechanisms involved in the protection of plants by safeners is not well defined; therefore, investigations were conducted to elucidate the mechanism(s) of action of the oxime ether safeners. Molecular comparisons of selected herbicide-safener combinations using computer-aided molecular modeling revealed that the chemical structures of safeners and herbicides are very similar at the molecular level; and, indicate that these compounds could bind at the same active site of the target protein or they may serve as inducers of metabolic enzymes which detoxify herbicides. Metolachlor at 10 μM and seed-applied CGA-133205 had no effect on germination while treatment with seed-applied oxabetrinil significantly reduced germination of grain sorghum. Results from experiments on ¹⁴C-acetate incorporation into lipids indicate that metolachlor and the oxime ether safeners influence lipid metabolism causing a redistribution of carbon in the lipid fractions of germinating sorghum roots. Results from studies with acetyl-CoA carboxylase indicate that this enzyme is not a target site for either metolachlor or the oxime ether safeners. Metolachlor and the oxime ether safeners enhanced glutathione levels in grain sorghum seedlings at 12 to 48 hr after imbibition was initiated with oxabetrinil being more stimulatory than metolachlor or CGA-133205. Glutathione reductase activity was also stimulated in safener-treated grain sorghum seedlings. Both safeners slightly enhanced nonenzymatic and enzymatic conjugation of metolachlor with reduced glutathione. Oxabetrinil conjugated enzymatically or nonenzymatically with reduced glutathione at a slow rate, but CGA-133205 did not. These data suggest that during the early stages of seed germination and seedling development of grain sorghum, safeners can enhance the detoxication of metolachlor by enhancing glutathione levels and enzymatic and nonenzymatic conjugation of metolachlor with glutathione. It appears that oxabetrinil and CGA-133205 are conferring protection to grain sorghum by increasing the rate of metolachlor metabolism. / Ph. D.

Orientador: Dagoberto Martins / Banca: Robson Antonio Pitelli / Banca: Ana Catarina Cataneo / Banca: Pedro Luis da Costa Aguiar Alves / Banca: Mario Sérgio Tomazela / Resumo: Este trabalho teve o objetivo de avaliar a eficiência do safener fluxofenim no tratamento de sementes de sorgo (DKB510 e SCG340), trigo (Avante e Ônix) e arroz (Bonanza e Aimoré), para o aumento da seletividade a aplicação do herbicida Smetolachlor em pré-emergência e determinar a atividade da enzima de detoxificação glutationa S-transferase (GST). O trabalho foi dividido em duas etapas. A primeira etapa constou da avaliação em campo da eficiência do safener em reduzir sintomas visuais de fitointoxicação causados pelo herbicida. Esta etapa foi conduzida na Estação Experimental pertencente à Syngenta Seeds no município de Uberlândia, Minas Gerais. Na segunda etapa foi determinada a atividade da GST, no Laboratório de Xenobióticos, do Departamento de Química e Bioquímica do Instituto de Biociências, UNESP, Campus de Botucatu, Botucatu, São Paulo. Foram comparadas a suscetibilidade ao herbicida por meio da avaliação visual de injúrias aos 3, 7, 15 e 30 dias após a emergência (DAE), massa seca de raiz e parte aérea aos 10 DAE, além da determinação da atividade da GST. Os tratamentos utilizados foram: aplicação ou não do safener na dose de 40mL por 100 kg ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aimed to evaluate the effectiveness of fluxofenim used for seed treatment as safener for sorghum, wheat and rice treated with the herbicide S-metolachlor applied in preemergence. The study was divided in two steps. In the first step it was measured the fluxofenim safener potential to reduce visual symptoms of S-metolachlor injury in the field. This phase was conducted in Uberlandia city, Minas Gerais, at Syngenta Experimental Station, and treatments were followed by S-metolachlor at 1.440 and 2.880 mL i.a. ha-1, and fluxofenim at 0, and 40 mL per 100 kg of seeds, and a check without treatment. The second phase was set up at Chemistry and Biochemistry Departament in Botucatu, UNESP, São Paulo, to evaluated glutathione S-transferase activity (GST) in the crops that responded to field treatments with safener. It was used two varieties/hybrids per crop and it was measured herbicide phytotoxity as visual symptoms at 3, 7, 15 and 30 days after emergence, dry matter at 10 DAE, and gluthationa S-transferase activity. Fluxofenim increased S-metolachlor tolerance for both sorghum hybrids, but best results were observed at 1.440 mL i.a. ha-1. Wheat varieties showed low tolerance to S-metolachlor in both rates, and fluxofenim increased S-metolachlor selectivity to wheat but not sufficient, reducing plant population to a non acceptable level. Seed treatment with fluxofenim for both rice varieties had no effect, and both S-metolachlor rates increased killed all plants treated with or without fluxofenim. Gluthationa S-transferase activity for sorghum and wheat increased when seeds treated with fluxofenim were submitted to S-metolachlor at 1.440 mL a.i. ha-1. / Doutor

Repeated use of acetolactate synthase (ALS)-inhibiting herbicides in recent years has resulted in the selection of 89 weed species resistant to these herbicides. One management strategy that can eliminate or slow the development of resistance is applying mixtures of herbicides with different modes of action. This research involved the characterization of ALS-inhibiting herbicide-resistant smooth pigweed (<i>Amaranthus hybridus</i> L.), as well as investigations on weed management programs in corn (<i>Zea mays</i> L.) utilizing mesotrione, a triketone, in mixtures with other herbicides. ALS-inhibiting herbicide-resistant smooth pigweed biotypes were collected from fields in Virginia, Delaware, Maryland, and Pennsylvania to evaluate response to ALS-inhibiting herbicides and to determine the molecular mechanisms of resistance. Sequencing of the ALS genes from these biotypes revealed two amino acid substitutions known to confer resistance, Ala₁₂₂ to Thr and Ser₆₅₃ to Asn, and one that has not been previously reported in plants, Asp₃₇₆ to Glu. The smooth pigweed biotype with an Asp₃₇₆ substitution displayed resistance to four classes of ALS-inhibiting herbicides that included imidazolinone (IMI), sulfonylurea (SU), pyrimidinylthiobenzoate (PTB), and triazolopyrimidine sulfonanilide (TP) chemistries. Transformation of this smooth pigweed ALS gene into <i>Arabidopsis thaliana</i> confirmed that the Asp₃₇₆ substitution is responsible for the resistance. Other biotypes that had a substitution at Ala₁₂₂ exhibited resistance to an IMI herbicide, little to no resistance to SU herbicides, and increased sensitivity to a PTB and a TP herbicide, whereas, biotypes that had a substitution at Ser₆₅₃ exhibited high-level resistance to an IMI herbicide and lower resistance to PTB and SU herbicides. Experiments were also conducted to investigate the effectiveness of mesotrione in preemergence (PRE) and postemergence (POST) corn weed management programs in Virginia. Mesotrione applied PRE in mixtures with <i>S</i>-metolachlor and atrazine controlled common lambsquarters (<i>Chenopodium album</i> L.), smooth pigweed, common ragweed (<i>Ambrosia artemisiifolia</i> L.), and morningglory (<i>Ipomoea</i> spp.) species when a timely rainfall followed application. POST applications of mesotrione controlled common lambsquarters and smooth pigweed, but common ragweed and morningglory species were not always controlled. Common ragweed and morningglory species were controlled by mesotrione in a mixture with atrazine POST. Large crabgrass [<i>Digitaria sanguinalis</i> (L.) Scop.] and giant foxtail (<i>Setaria faberi</i> Herrm.) control was generally better when the ALS-inhibiting herbicides nicosulfuron plus rimsulfuron or rimsulfuron plus thifensulfuron plus atrazine were applied in a mixture with mesotrione. Mixtures of mesotrione with other POST herbicides in a total POST program produced corn yields comparable to standard PRE followed by POST weed management programs. / Ph. D.

Les pesticides de nouvelle génération sont le plus souvent épandus à de faibles doses et en mélange. Peu d’études se sont intéressées jusqu’à présent à l’effet de ces mélanges sur la biodégradation et la toxicité de chaque pesticide et/ou métabolite. Le but de ces travaux de thèse était d’étudier les voies de biotransformation de chacun des trois herbicides d’un mélange constitué de mésotrione (β-tricétone), nicosulfuron (sulfonylurée) et S-métolachlore (chloroacétanilide) utilisé sur les cultures de maïs, ainsi que la toxicité (test Microtox ® ) des herbicides et de leurs métabolites, seuls et en mélanges. L’identification des métabolites de la mésotrione chez la souche Bacillus megaterium Mes11 et une étude de protéomique différentielle ont suggéré l’implication de nitroréductases dans la première étape de la biotransformation de cet herbicide, rôle confirmé ensuite par la caractérisation structurelle et fonctionnelle de deux enzymes capables de transformer la mésotrione : les nitroréductases NfrA1 et NfrA2, appartenant à la sous-famille NfsA-FRP des Nitro-FMN réductases. La voie de biotransformation du nicosulfuron a, quant à elle, été étudiée chez la souche Pseudomonas fluorescens SG-1 isolée à partir de sol agricole, capable de transformer cet herbicide par co-métabolisme. Cette biotransformation conduit à la formation de deux métabolites majoritaires issus du clivage de la liaison sulfonylurée du nicosulfuron, l’un deux (l’ADMP, 2-amino-4,6-diméthoxypyrimidine) présentant une toxicité 20 fois supérieure à celle de la molécule mère. Nous avons également étudié qualitativement et quantitativement la biotransformation de la mésotrione et du nicosulfuron par la souche Mes11 séparément ou en mélange, et en présence ou non de S-métolachlore Les résultats ont montré un effet négatif de la mésotrione sur la biotransformation du nicosulfuron et un effet positif du S-métolachlore sur la biotransformation de la mésotrione. Tous les mélanges d’herbicides testés ont montré des effets synergiques pour la toxicité vis-à-vis de A. fischeri, tandis que les mélanges de métabolites (avec ou sans S-métolachlore) étaient majoritairement synergiques ou antagonistes. La dernière partie des travaux de thèse est focalisée sur le développement d’une technique préventive de traitement de la pollution par les pesticides d’origine agricole (bioprophylaxie). Nous avons fait la preuve de concept de cette technique par une étude en microcosmes de sol. L’épandage simultané de l’herbicide 2,4-D (acide 2,4-dichlorophénoxyacétique) et de la souche Cupriavidus necator JMP134 capable de le minéraliser a en effet permis de réduire le temps de demi-vie de ce composé d’un facteur 3, tout en conservant son activité herbicide. / The new-generation pesticides are often sprayed at low dosages and in mixtures. Up to now, a few studies focused on the effect of these mixtures on the biodegradation and the toxicity of each pesticide and/or metabolite. The aim of this Ph.D. work was to study the biotransformation of each herbicide of a mixture composed of mesotrione (β-triketone), nicosulfuron (sulfonylurea) and S-metolachlor (chloroacetanilide) applied on maize crops, as well as the toxicity (Microtox® test) of the herbicides/metabolites alone or in mixture. The identification of mesotrione metabolites by the strain Bacillus megaterium Mes11 and a differential proteomic approach suggested the role of nitroreductases in the first step of mesotrione biotransformation. This was confirmed by the structural and functional characterization of two enzymes able to biotransform mesotrione: the NfrA1 and NfrA2 nitroreductases, belonging to the NfsA-FRP sub-family of Nitro-FMN reductases. The biotransformation pathway of nicosulfuron has been elucidated for the strain Pseudomonas fluorescens SG-1 isolated from an agricultural soil and able to co-metabolically biotransform nicosulfuron. Two major metabolites resulting from the cleavage of the sulfonylurea bridge were identified, one of them (ADMP, 2-amino-4,6-dimethoxypyrimidine) presenting a 20-fold higher toxicity than the parent compound. The simultaneous biotransformation of mesotrione and nicosulfuron by the strain Mes11 was also qualitatively and quantitatively studied, showing a negative effect of mesotrione on nicosulfuron biotransformation, and a positive effect of S-metolachlor on mesotrione biotransformation. All parent compound mixtures tested resulted in synergistic effects towards A. fischeri, while metabolite mixtures (with or without S-metolachlor) were mostly synergistic or antagonistic. The last part of the PhD work was devoted to the development of a preventive technique for the treatment of pollutions caused by agricultural pesticides (bioprophylaxis). We made the proof of concept of this method by using a soil microcosm study. The simultaneous spreading of 2,4-D (2,4-dichlorophenoxyacetic acid) herbicide and the strain Cupriavidus necator JMP134 able to mineralize it allowed a 3-fold reduction of 2,4-D half-life in soil, while preserving its herbicide activity.