Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is one of the most destructive foliar diseases on sugar beet. It severely affects productivity and profitability of the sugar beet industry. CLS is managed by using resistant cultivars, rotating with non-host crops, and applying effective fungicides in a timely manner. In North Dakota and Minnesota, site-specific fungicides, such as quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), had been widely and extensively applied to control C. beticola which has developed resistances to these fungicides. The mycelial growth, spore production, spore germination, and aggressiveness of QoI and/or DMI resistant isolates were compared to sensitive isolates in a laboratory and greenhouse study. Results indicated that the QoI and/or DMI resistant isolates had a relatively slower disease development on sugar beet leaves due to their fitness penalty in sporulation and mycelial growth but still caused high disease severities as sensitive isolates. Fungicides were evaluated to manage the QoI and/or DMI resistant C. beticola isolates in a sensitivity study and a greenhouse and field study. Copper-based multisite activity fungicides were evaluated for controlling fungicide resistant C. beticola. The mean EC50 values for nine copper-based chemicals ranged from 1 to 10 ug/ml using a spore germination assay. In a greenhouse study, Fertileader (a copper-based fertilizer) caused leaf injury and was not evaluated, but the other tested chemicals provided significantly better control of CLS compared to the control check with Badge X2, Champion, Cuprofix, COCS and Ridomil having significantly small AUDPC. Newer site specific fungicides with different modes of action that have never been used for CLS management in North Dakota and Minnesota were also tested. The mean EC50 values were 4.9, 33.1, 99.4, and 481.6 ug/ml using mycelial growth assay and 5.7, 4.1, 9.2, and 4.2 ug/ml using spore germination assay for cyprodinil, fluazinam, pydiflumetofen, and Chlorothalonil, respectively. In a two-year field study, all the fungicides resulted in significantly better disease control, significantly higher beet tonnage, and recovered sucrose compared to the nontreated check. However, none of tested fungicide treatments performed better than the industry?s standard fungicide, triphenyltin hydroxide.

La phototransformation des matières actives à la surface des plantes après pulvérisation a une influence considérable sur l’efficacité des traitements. Dans ce travail, l’ensemble des réactions photochimiques se produisant sur des supports modèles des cires végétales dans des conditions proches des conditions environnementales ont été étudiées dans le cas d’un fongicide (le chlorothalonil) et d’un herbicide (la cycloxydime). Nous avons considéré les réactions photochimiques induites par l’absorption de lumière par les composés eux-mêmes et celles faisant intervenir des sensibilisateurs comme les métabolites secondaires des plantes. Dans chacun des cas, des études mécanistiques détaillées ont été conduites. Nous montrons que le chlorothalonil possède la capacité remarquable de produire de l’oxygène singulet avec un rendement quantique proche de l’unité. En parallèle, la réactivité de la cycloxydime avec l’oxygène singulet a été montrée. De ce fait, la phototransformation de la cycloxydime sur film de cire est accélérée lorsque des sensibilisateurs naturels, tels que les phytoalexines sont ajoutés à la cire, ou lorsque des traces de chlorothalonil sont présents à la surface du modèle foliaire. Ce travail a permis 1) de comprendre le mécanisme de phototransformation par excitation directe et sensibilisées du chlorothalonil et de la cycloxydime, 2) de mesurer de nombreuses données physico-chimiques (constantes de vitesse de réaction, rendements quantiques, durées de vie, caractéristiques spectrales des transitoires) et 3) de mettre au point un protocole expérimental permettant d’étudier la photochimie de molécules à l’état solide incluses dans des films de cire. / The phototransformation of active ingredients on plant surfaces after spraying has a significant impact on the treatment efficiency. In this work, the photochemical reactions taking place on leaf models and environmental conditions close to real ones have been overviewed for two pesticides: chlorothalonil and cycloxydim. We considered the photochemical reactions induced by light absorption by the compounds themselves and the reactions involving natural sensitizers such as plants secondary metabolites. Detailed mechanistic studies were conducted. We show that chlorothalonil has a noteworthy capacity to produce singlet oxygen with a quantum yield close to unity. Besides, the reactivity of cycloxydim towards singlet oxygen was demonstrated. Hence, the phototransformation of cycloxydim deposited on wax films is accelerated when natural sensitizers, such as phytoalexins, are included in the wax, or when chlorothalonil traces are present on the leaf model. This work allowed 1) to understand the mechanism of phototransformation by direct excitation and sensitization of chlorothalonil and cycloxydim, 2) to measure many physicochemical data (reaction rate constants, quantum yields, lifetime, spectral characteristics oft ransients and 3) to put in work an experimental protocol for the photochemical study of organic compounds in solid state and included in wax films.

Amphibians are the most threatened taxon on the planet. Declines have been associated with over-exploitation, habitat loss, pollution, and pathogenic diseases, but of these factors, pollution and disease have been relatively under-studied. Here, I investigated: 1) the impacts of commonly used pesticides on aquatic communities, 2) the effect of these pesticides on amphibian susceptibility to the pathogenic chytrid fungus, Batrachochytrium dendrobatidis (Bd), and 3) whether there are non-amphibian hosts of Bd and 4) how to best quantify the survival of Bd through ontogeny of the host. In my first research chapter, I quantified the effects of environmentally relevant concentrations of the mot commonly used synthetic fungicide in the US, chlorothalonil, on 34 species-, 2 community- and 11 ecosystem-level responses in a multitrophic-level system. Chlorothalonil increased mortality of amphibians, gastropods, zooplankton, algae, and a macrophyte (reducing taxonomic richness), reduced decomposition and water clarity, and elevated dissolved oxygen and net primary productivity. These ecosystem effects were indirect but were predictable based on changes in taxonomic richness. A path analysis suggests that chlorothalonil-induced reductions in biodiversity and top-down and bottom-up effects facilitated algal blooms that shifted ecosystem functions. In my second chapter, I investigated how a wide range of ecologically relevant concentrations of chlorothalonil affected four species of amphibians (Osteopilus septentrionalis, Rana sphenocephala, Hyla squirella and H. cinerea). I also evaluated the effects of chlorothalonil on liver tissue, immune cell density, and the stress hormone, corticosterone. Chlorothalonil killed nearly every amphibian at the expected environmental concentration (EEC) and, at concentrations to which humans are commonly exposed (up to the EEC), it was associated with elevated corticosterone levels and changes in immune cells. Three species (O. septentrionalis, R. sphenocephala, and H. cinerea) showed a non-monotonic dose-response, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. Corticosterone exhibited a similar non-monotonic dose response and chlorothalonil concentration was inversely associated with liver tissue and immune cell densities. These studies on chlorothalonil emphasize the need to re-evaluate its safety and to further link anthropogenic-induced changes in biodiversity to altered ecosystem functions. In my third research chapter, I investigated the effects of chlorothalonil and atrazine, one of the most commonly used herbicides in the US, on amphibian susceptibility to Bd, a leading cause of amphibian extinctions. Relative to controls, atrazine monotonically reduced Bd growth in culture and on tadpoles. In contrast, chlorothalonil non-monotonically reduced Bd growth in culture and on tadpoles, with low and high concentrations causing significantly greater mortality than intermediate concentrations and controls. This study is one of only a handful of studies to document a non-monotonic dose response of an invertebrate (Bd) to a pesticide. Although both pesticides reduced Bd growth on tadpoles and in culture, neither eliminated Bd entirely, and because we know little about the long-term effects of the pesticides on hosts (e.g., immunosuppression), I do not recommend using these chemicals to control Bd. In my fourth research chapter, I investigated whether there are non-amphibian hosts for Bd. Non-amphibian hosts could explain how Bd is able to persist in the environment after amphibians are extirpated, and the extreme virulence and distribution of Bd. In laboratory and field studies, I found that crayfish, but not mosquitofish, were hosts for Bd. I found that crayfish could be infected with Bd, could maintain that infection long term (at least 3 months) and could transfer that infection to susceptible amphibians. I also revealed that exposure to water that previously held Bd caused significant crayfish mortality and gill recession, suggesting that Bd releases a chemical that can cause host pathology in the absence of infection. Most efforts to conserve and restore amphibian populations challenged by Bd have been unsuccessful, but managing alternative hosts offers a new and potentially more effective approach to managing Bd. Likewise, identifying the specific pathology-inducing chemical released by Bd might facilitate the development of new strategies to reduce the risk posed by this pathogen. The fifth and sixth research chapters are aimed to improve the quality and efficiency of Bd research. During amphibian development, Bd infections transition from the mouthparts of tadpoles to the skin of post-metamorphic frogs but this transition has never been quantified and thus researchers might be sampling the wrong parts of amphibian bodies to detect Bd. I showed that Bd abundance in O. septentrionalis mouthparts declined from Gosner stages 35-42 and increased on epidermis from Gosner stages 38-46. Assuming our findings are general across species, I recommend sampling mouthparts of amphibians less than Gosner stage 41 and hind limbs of amphibians greater than Gosner stage 41. This should provide researchers with guidance on where to sample to maximize detection of Bd. I also investigated whether Trypan blue dye could be used to determine the viability of Bd. I showed that the proportion of zoospores stained with Trypan blue dye matched the proportion of known dead zoospores added to cultures. In contrast, all of the zoosporangia stage (including known dead zoosporangia) of Bd stained blue. These results demonstrate that Trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. I recommend using Trypan blue to report the number of live zoospores to which hosts are exposed and to help determine whether factors have lethal or sublethal effects on Bd. My work demonstrates that managing exposure to contaminants and biological reservoirs for Bd might provide new hope for imperiled amphibians. Further exploring how pesticides and pathogens are contributing to amphibian declines will allow us to formulate crucial management and conservation plans to begin remediation.

La phototransformation des matières actives à la surface des plantes après pulvérisation a une influence considérable sur l'efficacité des traitements. Dans ce travail, l'ensemble des réactions photochimiques se produisant sur des supports modèles des cires végétales dans des conditions proches des conditions environnementales ont été étudiées dans le cas d'un fongicide (le chlorothalonil) et d'un herbicide (la cycloxydime). Nous avons considéré les réactions photochimiques induites par l'absorption de lumière par les composés eux-mêmes et celles faisant intervenir des sensibilisateurs comme les métabolites secondaires des plantes. Dans chacun des cas, des études mécanistiques détaillées ont été conduites. Nous montrons que le chlorothalonil possède la capacité remarquable de produire de l'oxygène singulet avec un rendement quantique proche de l'unité. En parallèle, la réactivité de la cycloxydime avec l'oxygène singulet a été montrée. De ce fait, la phototransformation de la cycloxydime sur film de cire est accélérée lorsque des sensibilisateurs naturels, tels que les phytoalexines sont ajoutés à la cire, ou lorsque des traces de chlorothalonil sont présents à la surface du modèle foliaire. Ce travail a permis 1) de comprendre le mécanisme de phototransformation par excitation directe et sensibilisées du chlorothalonil et de la cycloxydime, 2) de mesurer de nombreuses données physico-chimiques (constantes de vitesse de réaction, rendements quantiques, durées de vie, caractéristiques spectrales des transitoires) et 3) de mettre au point un protocole expérimental permettant d'étudier la photochimie de molécules à l'état solide incluses dans des films de cire.

Submitted by Adriana Martinez (amartinez@unoeste.br) on 2016-10-11T12:17:24Z No. of bitstreams: 1 Jaqueline Nascimento da Silva.pdf: 2368376 bytes, checksum: b34072cdd99331d11d5544ee10f94320 (MD5) / Made available in DSpace on 2016-10-11T12:17:24Z (GMT). No. of bitstreams: 1 Jaqueline Nascimento da Silva.pdf: 2368376 bytes, checksum: b34072cdd99331d11d5544ee10f94320 (MD5) Previous issue date: 2016-04-25 / Brazil produces approximately 1% of natural rubber derived from rubber tree (Hevea brasiliensis) in the world, and Sao Paulo state is the major producer. The plantation of rubber tree might be stricken by fungal diseases, specially Antracnose. This fact requires the necessity of agriculture defenders as Cerconil WP®, composed by thiophanate methyl and chlorothalonil, one of the most used. Facing the large exposure to these compounds of workers and population that live near plantations, it is extremely important evaluate its possible toxicity and health impacts. Once that, the present work had as objective identify potential teratogenic effects and maternal toxicity of exposure to Cerconil WP® fungicide in different doses, using rat as an experimental model. Moreover, the study also aimed to identify effects of this fungicide on the chemistry structure latex of rubber tree, via Raman Spectroscopy. Therefore, Winstar female rats, adults and pregnant were divided into 4 experimental groups, that received 0 (control), 400 (EA), 800 (EB) or 1200mg/kg/day (EC) of Cerconil WP®, via gavage, from 6º to 15º gestational day (GD). In GD20, pregnant rats were euthanized and submitted to laparotomy for collection of uterus and maternal organs. The fetuses were collect, processed to skeletal and visceral analysis. Rats that were exposure to the fungicide presented clinical signs of toxicity. Reduction in weight evidenced in all groups in comparison to the Control group in GD12. Weight of maternal liver was lower in group EC than Control and histopathologic analysis of hepatic tissue presented areas of focal necrosis, inflammatory infiltrate, picnotic nuclei in hepatocytes and microvesicular fat degeneration areas moderated in groups EB and EC. The percentage of fetuses with adequate weight for gestational age was lower in groups EA and EB, in relation to the Control and EC groups. Porcentage of little fetuses increased in group EA, in relation to the other experimental groups, and in group EB in comparison to Control. In addition, there was an increase of total incidence of skeletal anomalies in groups exposure in relation to the control. Skeletal alterations observed in higher frequency were: decrease in esternal centres, absence of xiphoid process and caudal vertebrae, malformation of suppra-occiptal and reduction on number of anterior and metatarsus phalanges. Measures of Raman Spectroscopy (laser island of 514,5 nm, 25 seconds of exposure and 3 to 5 accumulations) did not show significant chemistry alterations on the latex exposure in vitro to 2g/L of Cerconil WP®. Results demonstrated that the oral exposure of pregnant rats to Cerconil WP®, during organogenesis, might affect on the embrio-fetal development and cause low toxicity to general maternal health. However, the molecular structure of latex exposed to the fungicide was not affect on the experimental study conditions. / O Brasil produz aproximadamente 1% da borracha natural derivada de seringueira (Hevea brasiliensis) no mundo, sendo o estado de São Paulo o maior produtor. O plantio de seringueira pode ser acometido por doenças fúngicas, especialmente a Antracnose. Isto leva à necessidade do uso de defensivos agrícolas, sendo o Cerconil WP®, composto por tiofanato metílico e clorotalonil, um dos mais utilizados. Diante da ampla exposição de trabalhadores rurais e da população do entorno das plantações a estes compostos, torna-se extremamente importante avaliar a sua possível toxicidade e impactos à saúde. Assim, o presente trabalho teve como objetivo identificar os potenciais efeitos teratogênicos e de toxicidade materna da exposição ao fungicida Cerconil WP® em diferentes doses, utilizando o rato como modelo experimental. Além disso, o estudo também pretendeu identificar os efeitos deste composto sobre a estrutura química do látex de seringueira, via Espectroscopia de Espalhamento Raman. Para tanto, ratas Wistar adultas prenhes foram divididas em 4 grupos experimentais, que receberam 0 (controle), 400 (EA), 800 (EB) ou 1200mg/kg/dia (EC) de Cerconil WP®, via gavagem, do 6º ao 15º dia gestacional (DG). No DG20, as ratas prenhes foram mortas e submetidas à laparotomia para coleta do útero gravídico e órgãos maternos. Os fetos coletados foram processados para análise esquelética e visceral. Ratas expostas ao fungicida apresentaram sinais clínicos de toxicidade. Houve redução no peso das ratas no DG12 em todos os grupos expostos em relação ao grupo controle. O peso do fígado materno foi reduzido no grupo EC em relação ao controle e a análise histopatológica do tecido hepático apresentou áreas com necrose focal, infiltrado inflamatório, presença de hepatócitos com núcleo picnótico e área com formação de degeneração gordurosa microvesicular de moderada intensidade nos grupos EB e EC. A porcentagem de fetos com peso adequado para a idade gestacional foi menor nos grupos EA e EB, em relação aos grupos controle e EC. Além disso, a porcentagem de fetos pequenos aumentou no grupo EA, em relação aos demais grupos experimentais, e no grupo EB em relação ao controle. Foi observado um aumento na incidência total de anomalias esqueléticas nos grupos expostos em relação ao grupo controle. As alterações esqueléticas observadas em maior frequência foram: diminuição dos centros esternais, ausência de processo xifoide e vertebras caudais, malformação do supra-occipital e redução no número de falanges anteriores e metatarsos. As medidas de Espectroscopia de Espalhamento Raman (linha de laser de 514,5 nm, 25 segundos de exposição e 3 a 5 acumulações) não apresentaram alterações químicas significativas no látex exposto in vitro a 2g/L de Cerconil WP®. Os resultados demonstram que a exposição oral de ratas prenhes ao Cerconil WP®, durante a organogênese, pode afetar o desenvolvimento embrio-fetal e causar toxicidade leve sobre a saúde geral materna. No entanto, a estrutura molecular do látex exposto ao fungicida não foi afetada nas condições experimentais do estudo.

Sclerotinia homoeocarpa (F.T. Bennett) is one of the most economically important pathogens on high amenity cool-season turfgrasses where it causes dollar spot. Due to decades of over-reliance and repeated chemical treatments, S. homoeocarpa has developed resistance and insensitivity to multiple classes of fungicides. To understand the genetic mechanisms of fungicide resistance, the whole genomes of two strains with varying resistance levels to fungicides, were sequenced. In unpublished data (Sang et al.), a RNA-sequencing analysis revealed three CYP450s that were validated to play a functional role in S. homoeocarpa’s resistance against different fungicide classes. We also identified CYP450 metabolic action on the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been known to be persistent in soils. Yet, S. homoeocarpa resistance to chlorothalonil has not been reported in the field. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP450 overexpression strains when compared to the wild-type. We show by GC-MS that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloroisophthalonitrile is produced by CYP450s’ metabolism.

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.

<p>  </p> <p>Over the past two decades, emerging fungal pathogens have been reported in numerous groups ranging from mammals to key crop species across the globe. To combat the rise of these fungal pathogens in industrial agriculture, fungicides have been developed and subsequently applied en masse. Despite their growing usage, research examining the effects of these anthropogenic contaminants on natural systems is severely limited compared to other major classes of pesticides. Of particular concern are their impacts on aquatic systems, which can be especially at risk due to their proximity to agriculture. Herein I used a tiered approach to assess the impacts of this class of pesticides on aquatic ecosystems using amphibians as model system. In the first chapter, I assessed the acute toxicity of two widely applied fungicides, pyraclostrobin and chlorothalonil, to six different species of amphibians commonly found in the Midwest. My results showed that these fungicides are very acutely toxic to several species of amphibians at levels within the expected environmental concentration (EEC). In the second chapter, I examined the impacts of long-term low-dose exposure of pyraclostrobin on the growth, development, and activity levels of three species of amphibians. Despite the acute toxicity of pyraclostrobin, sublethal effects of the fungicide were rather limited with only minor effect on growth and activity. In the third chapter, I investigated the effects of fungicide exposure on host-parasite interactions using trematodes and American Bullfrog tadpoles. I found that in all treatments pyraclostrobin increased parasite loads from ~3 to 8 times compared to control tadpoles. Additionally, parasite loads were approximately 2 times higher in tadpoles with the continued fungicide exposures compared to those tadpoles that were moved to fresh water following initial fungicide exposure. Finally, my fourth chapter investigated the impact of pyraclostrobin and chlorothalonil application regime on community-level interactions under semi-natural conditions. While chlorothalonil had limited effects on the community, I found that pyraclostrobin was acutely toxic to Gray Treefrogs at environmentally relevant concentrations and these effects were stronger with more frequent applications. Collectively, my research has demonstrated that fungicides can be acutely toxic to amphibians at EECs under laboratory and semi-natural conditions. Additionally, they have the potential to alter disease dynamics by increasing infection risk. Given our increasing usage of fungicides to control emerging fungal pathogens, a greater emphasis on understanding the environmental implications of this management strategy is needed to inform ecological risk assessments. </p>

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Pesticides are widely used in agriculture and public health for pest control and prevention. The indiscriminate use of these compounds can trigger environmental contamination by pesticides and increase the risk of human exposure. Human exposure to pesticides can occur directly, through occupational activity, or indirectly, through the environment and food. Chronic exposure to pesticides may result in neurological, reproductive, teratogenic and immunological disorders. Mancozeb, chlorothalonil and thiophanate methyl are fungicides widely used in the world. Despite their characteristics of low acute toxicity and low persistence in the environment, in vitro and in vivo studies demonstrate the cytotoxic effects of these compounds. However, the immunological effects that these pesticides can trigger are unexplored. Therefore, the objective of this study was to evaluate in vitro the pro-inflammatory and oxidative effects of mancozeb, chlorothalonil and thiophanate methyl pesticides. RAW 264.7 (ATCC® TIB-71™) macrophages were exposed to different concentrations (0.1-100 μg/mL) of each pesticide for 72 hours, and maintained in 5% CO2 atmosphere at 37°C. The pesticides were dissolved in dimethylsulfoxide, which was used as negative control. Phytohemagglutinin was used as positive control for inflammatory activation. The evaluation of cell proliferation, oxidative metabolism and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ), anti-inflammatory cytokine (IL-10) and caspases (Casp1, Casp3, Casp8) levels were performed by fluorimetric and molecular tests. The results showed a significant pro-inflammatory effect of mancozeb, chlorothalonil and thiophanate methyl pesticides at respective concentrations of 1, 3 and 100 μg/mL, with increase in cell proliferation and pro-inflammatory cytokines and caspases levels. However, the oxidative effect was only observed in macrophages exposed to chlorothalonil at 3 μg/mL. Thus, in these analysis conditions, the studied pesticides acted by activation of the immune system. The results found contribute to better understanding of immunological effects caused by exposure to these pesticides. / Os pesticidas são amplamente utilizados na agricultura e na saúde pública para o controle e prevenção de pragas. O uso indiscriminado desses compostos pode desencadear a contaminação ambiental por agrotóxicos e aumentar o risco de exposição dos seres humanos. A exposição aos agrotóxicos pelos humanos pode ocorrer de forma direta, através de atividade ocupacional, ou de forma indireta, pelo ambiente e pela alimentação. A exposição crônica aos pesticidas pode resultar em distúrbios neurológicos, reprodutivos, teratogênicos e imunológicos. Os pesticidas mancozebe, clorotalonil e tiofanato metílico são fungicidas amplamente utilizados no mundo. Apesar de suas características de baixa toxicidade aguda e baixa persistência no ambiente, estudos in vitro e in vivo demonstram os efeitos citotóxicos desses compostos. Entretanto, os efeitos imunológicos que esses pesticidas podem desencadear são pouco explorados. Diante disso, o objetivo deste estudo foi avaliar in vitro o efeito pró-inflamatório e oxidativo dos pesticidas mancozebe, clorotalonil e tiofanato metílico. Os macrófagos RAW 264.7 (ATCC® TIB-71™) foram expostos a diferentes concentrações (0,1 – 100 μg/mL) de cada pesticida por 72 horas, sendo mantidos em atmosfera com 5% CO2 a 37oC. Os pesticidas foram dissolvidos em dimetilsulfóxido, o qual foi utilizado como controle negativo. Como controle positivo para a ativação inflamatória, utilizou-se a fitohemaglutinina. A avaliação da proliferação celular, do metabolismo oxidativo e dos níveis das citocinas pró-inflamatórias (IL-1β, IL-6, TNF-α e IFN-γ), da citocina anti-inflamatória (IL-10) e das caspases (Casp1, Casp3, Casp8) foi realizada por testes fluorimétricos e moleculares. Os resultados obtidos demonstraram efeito pró-inflamatório significativo dos pesticidas mancozebe, clorotalonil e tiofanato metílico nas respectivas concentrações de 1, 3 e 100 μg/mL, ocorrendo aumento da proliferação celular e dos níveis de citocinas pró-inflamatórias e caspases. Entretanto, o efeito oxidativo somente foi observado nos macrófagos expostos ao clorotalonil na concentração de 3 μg/mL. Assim, nessas condições de análise, os pesticidas estudados atuam ativando o sistema imune. Os resultados encontrados contribuem para a melhor compreensão dos efeitos imunológicos que a exposição a estes pesticidas pode desencadear.