Made available in DSpace on 2014-07-29T15:10:33Z (GMT). No. of bitstreams: 1 Tese - Flavio Marques Lopes.pdf: 1637206 bytes, checksum: 95899c1fbc6b76141c229cf07f0d9d1b (MD5) Previous issue date: 2008-10-23 / In this research, open ground samples (Brazilian Savannah Cerrado) proceeding from treated cultures of soy with Opera® (epoxiconazol and piraclostrobin) had been used as source of resistant microorganisms to fungicide. The sample were collected in July 2006, in three points of planted area and two depths, 0 to 20 cm, and from 80 cm to 1m, next to the state highway GO-139, in the towns of São Miguel do Passa Quatro, in a producing farm model of soy seeds, where Opera® was used since its launching in 2002. The collection point was characterized as a risk place for ecological accidents, presenting area with extensive agriculture activity, intense use of pest management, soil with good level of infiltration and storage of water, and areas of declivity. The microorganism s selection was carried through using selective solid J.E. supplemented with 0.1% of Opera® with microorganisms collected in depth of 80cm. In a previous selection, 9 microorganisms were isolated, and from these, the one that showed a better growth within the selective J.E. liquid supplemented with 0.03% of Opera® (1805) was chosen for the development of this research. The analysis for coloration of Gram-negative showed that the isolated microorganism is one bacillococcus Gram-negative with poliphormic characteristics, being sensible to all the tested antibiotics. Biochemical identification made through the method of Bactray®, presented a correlaction of 99% with the genus Klebsiella. Through molecular identification by the gene 16S rDNA, it was possible to find a correlaction of 99% of similarity with the species K.pneumoniae and K. oxytoca. The proteins released by the Klebsiella in a supplemented environment with 0.03% of Opera had been precipitated with acetone, obtaining two proteinic fractions, a soluble (FS) and the other insoluble (FI) in a 0,1 mol L-1 phosphate buffer, pH 6.0. In the soluble solution (FS), it was found an oxirredutase from the peroxidases family, which uses hydrogen peroxide as an agent and several substrates as reducing agents, presenting greater activity for pyrogallol, TMB, o-dianisidine and catechol. In a chromatogram obtained through separation by molecular exclusion in Sephadex G- 100, two peaks with peroxidasic activities had been observed. Peak 1 presented two bands in SDS-PAGE, one with39.3 kDa and another one with 24.6 kDa. In the FI, it was possible to determine the presence of one hydrolysis with capacity to break the bonds between the ester molecule, from the family of carboxylesterases esterase B. The esterase B presented activity against casein substrates (9.3 UE), BApNA (1.31 UE), pNPP (0.01), presenting 5,4% activity of inhibition after incubation with E64, 6.5% activity of inhibition after incubation with protease inhibitors cocktail containing AEBSF and 7.5% of inhibition after incubation with TLCK. The electrophoreses of FI presented two protein bands with molecular mass of approximately 61.7 kDa for superior band and 60.7 kDa for the inferior band. The enzymatic characterization using pNPP showed high activity in pH 5.0 and pH 7.0. Esterase B kept 100% of activity after 120 minutes of incubation at 60oC. The presence of ZnCl2, EDTA, MgSO4 and CuSO4 had presented an inhibition effect of 16%, 16%, 23.2% and 40.8% respectively, within the enzyme activity. Esterase B slightly presented higher activity in the presence of DMSO and acetone, and a reduction of up to 87% comparing to isopropyl alcohol (lower polarity). The vale of Km using pNPP as substratum was of 3.4 mmol L-1, with a Vmax of 0.019 UE. When the selective J.E. supplemented with 0.03% Opera was treated with Klebsiella for 120 h, it had a reduction of 43.7% in the absorbance of the compounds with a peak of 200 nm, 55.7% - 220 nm and 28.7% - 260 nm. Using released enzymes by Klebsiella for treatment of the epoxiconazol standards in the reduction of the absorbance 200, 220 and 260 nm the observed results were 99.7%, 95.7% and 99.5% respectively. Whereas for the pyraclostrobin, the observed reduction were 95.5% and 80.2% for the wave lengths 200 and 260 nm respectively / Neste trabalho amostras de solo de cerrado provenientes de culturas de soja tratadas com Opera® (epoxiconazol e piraclostrobin) foram utilizadas como fonte de microrganismos resistentes ao fungicida. A coleta foi realizada no mês de julho de 2006, em três pontos de área plantada e em duas profundidades, 0 a 20 cm e de 80 cm a 1 m, próximo à rodovia estadual GO-139, no município de São Miguel do Passa Quatro, em uma fazenda modelo produtora de sementes, em que o Opera® foi utilizado desde o seu lançamento em 2002. O ponto de coleta foi caracterizado como um local de risco a acidentes ecológicos, apresentando área com atividade agrícola extensiva, uso intenso de defensivos agrícolas, solos com boa taxa de infiltração e armazenamento de água e áreas de declividade. A seleção dos microrganismos foi feita com meio seletivo J. E. sólido - suplementado com 0,1% de Opera®, com microrganismos coletados na profundidade de 80 cm. Na primeira etapa 9 microrganismos foram isolados e, destes, o microrganismo 1805 foi o que apresentou melhor crescimento em meio seletivo J. E. líquido - suplementado com 0,03% de Opera® sendo escolhido para o desenvolvimento deste trabalho. A análise por coloração de Gram mostrou que o microrganismo isolado é um bacilococco Gram-negativo com características polimórficas, sendo sensível a todos os antibióticos testados. A identificação bioquímica pelo método de Bactray® apresentou correlação a nível de 99% com o gênero Klebsiella. Através de identificação molecular pelo gene 16S rDNA foi possível encontrar uma correlação de 99% de similaridade com as espécies K. pneumoniae e K. oxytoca. As proteínas secretadas pela Klebsiella em meio suplementado com 0,03% de Opera® foram precipitadas com acetona, obtendo duas frações protéicas, uma solúvel (FS) e outra insolúvel (FI) em tampão fosfato 0,1 mol L-1, pH 6,0. Na FS foi encontrada uma oxirredutase da família das peroxidases, que utiliza o peróxido de hidrogênio como agente oxidante e vários substratos como agentes redutores, apresentando maior atividade para pirogalol, TMB, o- ianizidina e catecol. No cromatograma obtido na separação por exclusão molecular em Sephadex G-100 foram observados dois picos com atividade peroxidásica. O pico 1 apresentou duas bandas em SDS-PAGE, uma mais forte com 39,3 kDa e outra com 24,6 kDa. Na FI foi possível determinar a presença de uma hidrolase, com capacidade de degradação de ligações éster, da família das carboxilesterases esterase B. A esterase B apresentou atividade contra os substratos caseína (9,3 UE), BApNA (1,31 UE), pNPP (0,01 UE), apresentando 5,4% de inibição com E64, 6,5% de inibição após incubação com coquetel de inibidores contendo AEBSF e 7,5% de inibição após incubação com TLCK. A eletroforese da FI apresentou duas bandas de proteínas com massa molecular de aproximadamente 61,7 kDa para a banda superior e 60,7 kDa para a banda inferior. A caracterização enzimática utilizando pNPP mostrou atividade alta em pH 5,0 e em pH 7,0. A esterase B manteve 100% de atividade após 120 minutos de incubação a 60 ºC. A presença de ZnCl2 aumentou a atividade em 14,2%, FeCl3 e MnSO4 não tiveram efeito significativo sobre a atividade e os demais sais, CaCl2, EDTA, MgSO4 e CuSO4 apresentaram um efeito inibitório de 16%, 16%, 23,2% e 40,8% respectivamente, na atividade da enzima. A esterase B apresentou atividade ligeiramente mais alta na presença de DMSO e acetona, e uma redução de até 87% frente ao isopropanol (solvente com menor polaridade). O valor de Km encontrado utilizando pNPP como substrato foi de 3,4 mmol L-1, com uma Vmáx de 0,019 UE. Quando o meio seletivo J. E. suplementado com 0,03% de Opera foi tratado com a Klebsiella por 120 h, houve uma diminuição 43,7% na absorvância dos compostos com pico a 200 nm, 55,7% a 220 nm e 28,7% a 260 nm. Usando as enzimas secretadas pela Klebsiella para tratamento do padrão epoxiconazol a redução da absorvância observada em 200, 220 e 260 nm foi de 99,7%, 95,7% e 99,5% respectivamente. Para o piraclostrobin a redução observada foi de 95,5 e 80,2% nos comprimentos de onda 200 e 260 nm respectivamente

Nach den umweltpolitischen Zielsetzungen der Bundesregierung für einen integrierten Pflanzenschutz wird gefordert, Pflanzenschutzmittelapplikationen in Abhängigkeit von dem Befallsgeschehen umwelt- und ressourcenschonend durchzuführen. Der Praxis stehen bereits unterschiedliche Prognosemodelle zur Verfügung, welche das Befallsgeschehen verschiedenster Schaderreger simulieren und die dazu genutzt werden können eine Applikation optimal zu terminieren. Nach erfolgter Applikation sind Landwirte jedoch insbesondere auf ihre Erfahrung angewiesen, über die Notwendigkeit und den Zeitpunkt einer Folgeapplikation zu entscheiden. Das Ziel dieser Arbeit war deshalb die Entwicklung eines Modells zur Prognose der Wirkungsdauer von Fungiziden an Winterweizen gegen Zymoseptoria tritici. In Freiland- und Laborversuchen wurden die Wirkung und die Wirkungsdauer der Fungizide Bravo 500 (500 g/l Chlorthalonil), Epoxion (125 g/l Epoxiconazol) und Imbrex (62,5 g/l Fluxapyroxad) sowie der Mischung Epoxion+Imbrex mit wissenschaftlichen Methoden untersucht. Es wurden insgesamt neun Parzellenversuche in den Jahren 2012 bis 2014 im Freiland durchgeführt. An einem Standort erfolgte ab der Applikation zusätzlich zu den Bonituren wöchentlich eine Analyse der Wirkstoffgehalte in den Blättern. In Halbfreilandversuchen mit Winterweizen in Töpfen wurde außerdem die Wirkungsdauer der Fungizide in Abhängigkeit vom Niederschlag untersucht. Darüber hinaus wurde ein Halbfreilandversuch zur kurativen Wirkung von Epoxion und Imbrex in Abhängigkeit von der Latenzzeit von Z. tritici durchgeführt. Die Untersuchung der Einflüsse von Temperatur und Konzentration auf das Wachstum von Z. tritici und des Einflusses der Temperatur auf die Fungizide erfolgte mit Hilfe von Mikrotitertests im Labor. Für die Modellerstellung wurde eine Methode zur Berechnung der Fungizidwirkungsdauer entwickelt. Der Kernpunkt dieser Methode ist die Berechnung des Befallsanstiegs für die unbehandelte Kontrolle und die Fungizidvariante basierend auf den Ergebnissen der Parzellenversuche. Ist der Befallsanstieg beider Varianten identisch, wird die Wirkung als beendet betrachtet. Von diesem Zeitpunkt wird dann die dynamisch berechnete Latenzzeit von Z. tritici abgezogen. Auf diese Weise wird der Tag ermittelt, ab dem die Wirkung des Fungizids nicht mehr ausgereicht hat, um das Blatt vor einer Infektion zu schützen. Die berechnete Wirkungsdauer lag im Mittel je nach Fungizid zwischen 16,2 und 22,0 Tagen. Durch einen Vergleich der berechneten Wirkungsdauer mit der berechneten Infektionswahrscheinlichkeit für Z. tritici und den Wirkstoffgehalten in den Blättern zum berechneten Wirkungsende, konnte bestätigt werden, dass die mit dieser Methode berechnete Wirkungsdauer plausibel ist. Da die Methode auf allgemeinen epidemiologischen Grundlagen basiert, ist sie nicht nur für Z. tritici an Winterweizen, sondern auch auf jedes andere Pathosystem anwendbar. Die Modellierung der Fungizidwirkungsdauer erfolgte mit der berechneten Wirkungsdauer der Fungizide. Hierfür wurde die Methode der binären multiplen logistischen Regression angewendet. Mit dieser wurden die Temperatur, der Niederschlag und je nach Fungizid auch die relative Luftfeuchte als Wetterparameter mit signifikantem Einfluss auf die Wirkungsdauer identifiziert. In den Labor- beziehungsweise Halbfreilandversuchen konnte ein Einfluss von Temperatur und Niederschlag auf die Wirkungsdauer jedoch nicht nachgestellt werden. Es wurde deshalb geschlussfolgert, dass diese Faktoren in erster Linie den pflanzlichen Stoffwechsel und das Pflanzenwachstum beeinflussen. In zweiter Linie wird dadurch auch die Wirkungsdauer der Fungizide beeinflusst, wie zum Beispiel durch eine erhöhte Metabolisierung oder Verdünnung des Wirkstoffs. Insgesamt wurden drei verschiedene Modellfunktionen entwickelt. Jeweils eine für Fungizide mit “mittlerer”, “guter” und “ sehr guter” Wirkung. Damit ist es möglich das Modell nicht nur für die in dieser Arbeit untersuchten Fungizide zu nutzen, sondern für alle Fungizide und Fungizidmischungen, die sich in diese Gruppen einordnen lassen. Die Einordnung der Fungizide basiert auf den Fungizidbewertungstabellen der Pflanzenschutzdienste der Länder und wird jährlich aktualisiert. Somit müssen keine aufwendigen Versuche durchgeführt werden, um das Modell zu aktualisieren. Auch eine verringerte Wirkungsdauer, wie sie zum Beispiel durch Resistenzen entsteht, wird auf diese Weise direkt berücksichtigt. Das entwickelte Modell trägt den Namen OPTIFUNG und prognostiziert in Abhängigkeit der Wetterparameter Temperatur, Niederschlag und relativer Luftfeuchte die Wirkungsdauer von Funigziden. Das Modell OPTIFUNG soll mit den Modellen SIMONTO (Ontogenesemodell) und SEPTRI (Zymoseptoria tritici-Modell) verknüpft und auf der Internetplattform www.isip.de zugänglich gemacht werden. Somit kann der Nutzer zukünftig informiert werden, wenn entweder eine neue, nicht mit behandelte Blattetage infiziert wird oder die Wirkungsdauer abgelaufen ist und die Wahrscheinlichkeit für eine neue Infektion mit Z. tritici groß ist. In dieser Arbeit wurde am Beispiel von Z. tritici eine allgemeingültige Methode zur Berechnung der Wirkungsdauer von Fungiziden entwickelt. Das daraus entstandene Modell ist durch die Bildung von Wirkungsgruppen für weitere Fungizide anwendbar und bietet dem Nutzer in Zukunft eine objektive und dynamische Unterstützung bei der Planung der Fungizidapplikationen.

Ce travail de thèse a cherché à déterminer si la contamination résiduelle à long terme des sols agricoles par les pesticides induit le développement de mécanismes d’adaptation aux pesticides chez les vers de terre. Il a aussi visé à identifier les coûts potentiels de l’adaptation de l’échelle de l’individu à celle de la population, et les conséquences pour l’écosystème sol. Une contamination résiduelle du sol par les pesticides est mesurée et comparée dans trois champs cultivés en agriculture conventionnelle (classés en fonction de l’historique cultural comme « haut », « moyen » et « bas » niveaux d’intrants), un champ cultivé en agriculture biologique et une prairie permanente biologique, tous dans ce type de management agricole depuis plus de 20 ans. En utilisant une méthode d’extraction des pesticides en milieu aqueux (représentant la fraction « biodisponible » des pesticides), 6, 8 et 4 résidus de pesticides sont détectés dans les champs à « haut », « moyen », et « bas » niveaux d’applications, respectivement, et un seul pesticide dans le sol du champ biologique (un résidu d’atrazine potentiellement vieux de plus de 20 ans). Les deux espèces endogées Allolobophora chlorotica et Aporrectodea caliginosa , communnes dans les sols des 5 champs,- mis à part A. chlorotica qui est absente du champ cultivé en agriculture biologique-, ont servi de modèles biologiques d’étude. Les stratégies d’adaptation aux pesticides sont étudiées en comparant les réponses de ces populations de vers de terre sur le terrain et après des expositions aux pesticides en laboratoire. Les réponses mesurées s’étendent de l’échelle moléculaire (enzymes de biotransformation et du stress oxidatif), biochimique (ressources énergétiques), métabolique (taux de respiration, métabolomique) à l’échelle de l’individu (biomasse, longueur) et de la population (traits de vie des cocons et des juvéniles), et aux possibles conséquences pour l’écosystème sol en termes de bioturbation (creusement et ingestion de sol) et de dissipation des pesticides comme service ecosystémique. Une capacité de détoxification augmentée et un plus grand potentiel anti-oxidant sont observés le long du gradient de contamination du sol et en laboratoire après exposition des vers de terre des champs conventionnel (population « pré-exposée ») et biologique (« naïve) à des pesticides. Des demandes énergétiques et des réarrangements métaboliques différents sont observés dans les deux populations, et sont plus prononcés chez la population pré-exposée. Une adaptation physiologique est démontrée chez les animaux pré-exposés, qui est associée à une ’augmentation de la bioturbation, et en cascade à une dissipation du pesticide dans le sol. Les conséquences au niveau de la population sont étudiées en termes de traits d’histoire de vie des deux populations pré-exposées et naïves. Le management en conventionnel incluant l’utilisation de pesticides semble diminuer le poids des adultes au champ, et implique potentiellement la réallocation des ressources énergétiques, des mécanismes reproductifs vers les processus métaboliques. Ceci aboutit à une diminution de la fécondité et du pourcentage d’éclosion et pourrait être un facteur participant à la diminution des populations de vers de terre dans les champs cultivés avec utilisation de produits phytosanitaires. / This work investigated if long-term residual contamination of agricultural soils leads to adaptation of earthworm populations to pesticides. It also aimed at identifying the costs of adaptation from the individual to the population level, and the consequences for the ecosystem. Residual contamination by pesticides was assessed and compared in three fields under conventional management (classified after evaluation of pesticide applications as ''high-'', ''medium-'', and ''low-''pesticide input), one field under organic agriculture requirements and one organic permanent pasture, all in this type of management for more than 20 years. Using a water extraction method, as indicative of the amount of bioavailable pesticides 6, 8 and 4 residues of pesticides were recovered in the ''high-'', ''medium-'', and ''low-input'' fields, respectively, and almost no pesticides were detected in the soil of the organic field except for low levels of residual (possibly 20 years old) atrazine. The endogeic species Aporrectodea caliginosa and Allolobophora chlorotica were found in common to the five fields, -except A. chlorotica which was absent from the organic field-, and were used as biological models. Adaptation strategies were investigated by comparing the populations of these earthworms between the different fields according to several endpoints in field and laboratory assessments. The endpoints measured ranged from the molecular (biotransformation and anti-oxidant enzymes), biochemical (main energy resources), and metabolic (respiration rate, metabolomics) levels, to individual (weight, length) and population-related parameters (cocoon and juvenile life traits), and to the possible consequences for the ecosystem in terms of bioturbation (earthworm burrowing behaviour) and pesticide disappearance as an crucial ecosystem service. Enhanced detoxification and anti-oxidant potential was demonstrated along the gradient of contamination in the fields, and in particular comparing the response to an experimental pesticide exposure between the pre-exposed, thus possibly adapted earthworms from the ''high-input'' field-, and the naïve population from the organic field. Distinct energetic demands and metabolic rearrangements were observed between the populations, more pronounced in the pre-exposed earthworms. Physiological adaptation was demonstrated in pre-exposed animals, and this was associated with an increase in burrowing behaviour and pesticide disappearance in the soil. Population-level consequences were assessed in life traits of the two populations. The conventional farming including the use of pesticides decreased the weight of adult worms in the field and resulted in reallocation of energy resources, possibly from reproductive to metabolic function. This led to lower fecundity and hatching success and could partly explain lower earthworm densities in pesticide-impacted soils. / I dette arbejde blev der forsket i om langsigtet residualforurening af landbrugsjord fører til pesticidadaptation hos regnorme. Forskningen sigtede også efter at identificere adaptations-omkostningerne fra individ- til populationsniveau, og konsekvenserne for økosystemet. Pesticiders residualforurening blev vurderet og sammenlignet i; tre konventionelt styrede marker (klassificeret efter evaluering af pesticid-anvendelser som ”højt-”, ”medium-”, og ”lav-input”), en mark styret ud fra økologiske betingelser, og et økologisk permanent græsningsareal, alle havde været styret på denne måde i mere end 20 år. Ved hjælp af en vand-ekstraktionsmetode blev 6,8 og 4 pesticidresiduums udvundet i henholdsvis ”høj-”, ”medium-”, og ”lav-input” markerne, og næsten ingen pesticider blev detekteret i jorden fra den økologiske mark bortset fra lave niveauer af resterende (højst sandsynligt 20 år gammelt) atrazin. Regnorms-populationerne Aporrectodea caliginosa og Allolobophora chlorotica blev fundet i alle fem marker, bortset fra A. chlorotica som ikke var til stede i den økologiske mark. Adaptationsstrategier blev undersøgt ved at sammenligne populationerne af disse regnorme de forskellige marker imellem ifølge flere parametre i felt- og laboratoriebedømmelserne. De målte parametre rangerede fra molekylære- (biotransformation og antioxiderende enzymer), biokemiske- (primære energiressourcer), og metaboliske- (respirationsrate, metabolomics) niveauer, til individuelle- (vægt, længde) og populations-relaterede parametre (puppe og juvenile livskarakterer), og til de mulige konsekvenser for økosystemet med hensyn til bioturbation (regnormes grave-adfærd) og nedgang i koncentrationen af ekstraherbare pesticider. Forbedret afgiftnings- og antioxiderende potentiale blev demonstreret langs gradienten af pesticid forurening i felten. Forbedret afgiftning i den pre-eksponerede population var særdeles tydeliggjort ved eksponeringen til pesticider i laboratoriet sammenliget med den økologiske population. Der blev observeret distinkte energibehov og metabolisk omgruppering populationerne imellem, dette var mere udtalt hos de pre-eksponerede regnorme. Der blev detekteret fysiologiske adaptationer hos de pre-eksponerede dyr, og dette hang sammen med en kompensatorisk øgning i grave-adfærd og nedgang i koncentrationen af ekstraherbare pesticider i jorden. Konsekvenser, på populationsniveau, blev bedømt ud fra de to populationers livskarakterer. Brug af pesticider resulterede i vægtnedgang hos de voksne orme i felten og omfordeling af energiressourcer, formodentlig fra reproduktiv til metabolisk funktion. Dette førte til lavere frugtbarhed og udklæknings-succes og kunne til dels forklare de lavere regnorme-densiteter i den pesticid-påvirkede jord.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The soybean cultivars reaction to the causal agent of asian rust (Phakopsora pachyrhizi Syd. & P. Syd.) and mixing responsiveness of DeMethylation Inhibitor (DMI) and Quinone outsite Inhibitor (QoI) fungicides are important factors to be determined for the management of the pathogen. The activity of these fungicides sprayed on the leaves depends on the amount that it reaches the biochemical action site and the compound efficacy. Thus, the proportion of leaf deposit that it enters the leaf, the rate of entry and dissipation in the leaf tissue are determinant for the activity and diseases residual control after the occurrence of a wash. The chapter I objective was to evaluate the reaction of fifteen soybean cultivars to the causal agent of soybean rust and the responsiveness mixing Active Ingredients (AI) epoxiconazole (EPOX) + pyraclostrobin (PYR) and prothioconazole (PROT) + trifloxystrobin (TRIFL). The parameters evaluated were the latency period, residual control, progression and the Area Under the Disease Progress Curve (AUDPC). In the Chapter II, the EPOX and PYR persistence associated with mancozeb (Mz) was determined under greenhouse conditions in two commercial soybean cultivars. Artificial washes were used to determine the concentration of the remaining AI on the foliar tissues after the spraying. For this purpose, the leaves were washed with water at 0.17, 1, 2, 4, 48, 96, 192 h after the fungicide spraying and immediately collected. The EPOX and PYR concentrations were also determined on leaves at 12, 16 and 20 days after spraying. Through the high performance liquid chromatographic analysis and the experimental design, it was possible to measure the EPOX and PYR concentrations penetrated and it dissipated in whole-plant leaves, associated or not to mancozeb. The influence of the interaction between leaflets age and cultivars in the EPOX and PYR concentration on leaves after the spraying was also evaluated. Due to the scarcity of detailed studies on the performance of site-specific fungicides associated with multisite fungicides, in the Chapter III was investigated the asian rust severity and the residual control of the EPOX and PYR mixture associated mancozeb. In the chapter I, it was verified that the responsiveness of DMI and QoI mixtures to P. pachyrhizi is different among the cultivars. The greatest residual control of the pathogen in the different cultivars was through of the PROT + TRIFL spraying. In the chapter II, it was verified that mancozeb influences the persistence of DMI and QoI fungicides in soybean cultivars. The EPOX penetration rate in the cultivars was faster without the association of mancozeb to the fungicide (DMI + QoI). The association of mancozeb to the fungicide (DMI + QoI) reduced the penetration rate of PYR in DM 6563 RSF IPRO cultivar. The PYR concentration in leaves of the two cultivars at 48 h after spraying was similar by the association or not of the fungicide with mancozeb. The cultivars showed different AI penetration rates in leaves. It was observed a reduction of the AI concentrations in the foliar tissues after 48 h of the spraying, evidencing a dissipation process of the AI, after this time. The association of mancozeb to the fungicide (DMI + QoI) only influenced the persistence of PYR at 16 days after spraying in DM 6563 RSF IPRO cultivar. The dissipation rate of PYR is different between the leaves cultivars. The penetration of the AI is greater in younger leaves than in older leaves. The concentrations of PYR in soybean leaves were higher than EPOX at different sampling times, after the fungicide spraying. In the chapter III, it was verified that mancozeb associated with EPOX + PYR in spray solution increases the residual control of P. pachyrhizi and it reduces the disease severity. / A reação de cultivares de soja ao agente causal da ferrugem asiática (Phakopsora pachyrhizi Syd. & P. Syd.) e responsividade de mistura de fungicidas Inibidores da DesMetilação (IDM) e Inibidores da Quinona oxidase (IQo) são importantes fatores a serem determinados para o manejo do patógeno. A atividade desses fungicidas pulverizados na folha depende da quantidade que atinge o local de ação bioquímico e da eficácia do composto. Assim, a proporção do depósito foliar que entra na folha, a taxa de entrada e sua dissipação no tecido foliar são determinantes para a atividade e residual no controle das doenças após a ocorrência de uma lavagem. O capítulo I teve como objetivo avaliar a reação de quinze cultivares de soja ao agente causal da ferrugem asiática e responsividade da mistura dos ingredientes ativos (IA) epoxiconazol (EPOX) + piraclostrobina (PIR) e protioconazol (PROT) + trifloxistrobina (TRIFL). Os parâmetros avaliados foram o período de latência, residual de controle, progresso e a área abaixo da curva de progresso da doença (AACPD). No capítulo II, a persistência de EPOX e PIR associadas com mancozebe (Mz) em duas cultivares comerciais de soja foi determinada em condições de casa de vegetação. Lavagens artificiais foram utilizadas para determinar a concentração dos IA remanescentes nos tecidos foliares após a pulverização. Para isso, as folhas foram lavadas com água 0,17, 1, 2, 4, 48, 96, 192 h após a pulverização do fungicida e imediatamente coletadas. As concentrações de EPOX e PIR também foram determinadas em folhas aos 12, 16 e 20 dias após a pulverização. Através da análise cromatográfica liquida de alta eficiência e do desenho experimental foi possível mensurar as concentrações de EPOX e PIR penetradas e dissipadas em folhas de planta inteira, associadas ou não à mancozebe. A influência da interação de idade de trifólios e cultivares sobre as concentrações de EPOX e PIR após a pulverização também foi avaliada. Devido à escassez de estudos detalhados sobre o desempenho dos fungicidas sítio-específicos associados aos fungicidas multissítios, no capítulo III foi investigado a severidade de ferrugem asiática e o residual de controle da mistura de EPOX e PIR associada ao mancozebe. No capítulo I foi verificado que a responsividade de misturas de IDM e IQo à P. pachyrhizi é distinta entre as cultivares. O maior residual de controle do patógeno nas diferentes cultivares foi através da pulverização de PROT + TRIFL. No capítulo II foi verificado que mancozebe influencia a persistência de fungicidas IDM e IQo em cultivares de soja. A taxa de penetração de EPOX nas cultivares foi mais rápida sem a associação de mancozebe ao fungicida (IDM + IQo). A associação de mancozebe ao fungicida (IDM + IQo) reduziu a taxa de penetração de PIR na cultivar DM 6563 RSF IPRO. A concentração de PIR em folhas das duas cultivares às 48 h após a pulverização foi semelhante pela associação ou não do fungicida com mancozebe. As cultivares apresentaram diferentes taxas de penetração dos IA em folhas. Observou-se uma redução das concentrações dos IA nos tecidos foliares após 48 h da pulverização, evidenciando um processo de dissipação dos IA, após esse tempo. A associação de mancozebe ao fungicida (IDM + IQo) somente influenciou a persistência de PIR aos 16 dias após a pulverização na cultivar DM 6563 RSF IPRO. A taxa de dissipação de PIR em folhas é diferente entre as cultivares. A penetração dos IA é maior em folhas mais novas do que em folhas velhas. As concentrações de PIR em folhas de soja foram superiores que EPOX nos diferentes tempos de coletas, após a pulverização do fungicida. No capítulo III foi verificado que mancozebe associado com EPOX + PIR em calda de pulverização aumenta o residual de controle de P. pachyrhizi e reduz a severidade da doença.