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Autophagie ist ein Degradationsprozess der streng reguliert ist und in welchem eine eukaryotische Zelle zelleigene Organellen und Proteine bei Nährstoffmangel abbaut. Außerdem konnte gezeigt werden, dass dieser Prozess auch in verschiedene Entwicklungsprozesse involviert ist. Die molekulare Entschlüsselung der Autophagie wurde hauptsächlich in der Bäckerhefe S. cerevisiae vorgenommen. Allerdings ist Beteiligung der Autophagie an Entwicklungsprozessen in multizellulären filamentösen Ascomyceten weitestgehend unbekannt. Die Fruchtkörperentwicklung von Pilzen ist ein komplex gestalteter Differenzierungsprozess der von einem zwei-dimensionalem Pilzgeflecht ausgeht das sich zu einem dreidimensionalem Perithezium entwickelt. Die Fruchtkörperentwicklung erfordert spezifische Umgebungsbedingungen und wird durch viele entwicklungsassoziierten Genen reguliert. In dieser Studie diente der Modellorganismus Sordaria macrospora zur Untersuchung des Einflusses der Autophagie auf die Fruchtkörperentwicklung. Der coprophytische filamentöse Ascomycet S. macrospora pflanzt sich lediglich sexuell fort, was ihn ideal für die Fragestellung dieser Arbeit macht. Für diese Arbeit wurden eine Reihe konservierter Autophagie bezogener Gene auserwählt. Folgende Gene die homolog zu denen anderer Ascomyceten sind wurden isoliert: Smvps34, Smvps15, Smatg8, Smatg4, und Smjlb1. Durch die Deletion dieser Gene sollte geklärt werden wie Autophagie in die Fruchtkörperentwicklung involviert ist. Die Deletion des Phospolipidkinase Gens Smvps34 und des Proteinkinase Gens Smvps15 führte zur Lethalität von S. macrospora was durch eine Auskeimungsuntersuchung belegt wurde. Die Deletion des Gens Smatg8, welches eine autophagosomale Strukturkomponente kodiert und des Gens Smatg4, das eine Cystein-Protease kodiert, die SmATG8 prozessiert, beeinträchtigte ebenfalls die Fruchtkörperentwicklung und das vegetative Wachstum. Durch Fluoreszenzmikroskopie konnte gezeigt werden, daß SmATG8 in Autophagosomen lokalisiert und SmATG4 vorwiegend im Zytoplasma lokalisiert ist. Die Prozessierung von SmATG8 durch SmATG4 wurde ebenfalls durch Fluoreszenzmikroskopie und Western-blot Analyse bestätigt. Die heterologe Expression von Smatg8 und Smatg4 in S. cerevisiae und der Ape1 Reifungsuntersuchung zeigte, das die cDNA von Smatg8 und Smatg4 den Deletionsphenotyp der jeweiligen Hefedeletionsmutanten aufheben konnte. Somit konnte die Konservierung dieser beiden Gene innerhalb der Ascomyceten gezeigt werden. Die Blockade der Fruchtköperentwicklung wurde durch die Deletion des bZIP Transkriptionsfaktor Gens Smjlb1 verursacht genauso wie die Beeinträchtigung des vegetativen Wachstums. SmJLB1 ist im Kern lokalisiert und durch qRT-PCR Experimente wurde gezeigt, dass die Autophagiegene Smatg8 und Smatg4 durch Smjlb1 reguliert werden. Dies läßt vermuten, dass Smjlb1 in den Prozess der Autophagie involviert ist. Die Ergebnisse dieser Arbeit weisen darauf hin, dass Autophagie und Fruchtkörperentwicklung des filamentösen Pilzes S. macrospora streng miteinander verknüpft sind.

En raison des forçages anthropiques exercés sur les écosystèmes aquatiques et des effets des changements globaux présents et à venir, on s’attend à une augmentation de la fréquence et de l’intensité des proliférations cyanobactériennes lacustres. Une meilleure connaissance des facteurs biotiques, et notamment du parasitisme, impliqués dans le control des dynamiques cyanobactériennes semble essentielle. Il est désormais établi que les Chytridiomycota (chytrides) constituent les principaux pathogènes fongiques du phytoplancton. Ainsi, les travaux de recherche menés au cours de cette thèse ont eu pour objectif d’étudier le parasitisme fongique associé aux efflorescences cyanobactériennes, et plus précisément l’écologie des chytrides parasites de la cyanobactérie Anabaena macrospora, espèce filamenteuse présentant des proliférations massives et récurrentes dans le lac d’Aydat (France). Par la mise en place d’un schéma d’échantillonnage temporel et spatial à haute fréquence et prenant en compte deux années consécutives (2010 et 2011), nous avons pu (i) étudier les cycles de vie des deux espèces de parasites fongiques associées à A. macrospora, (ii) évaluer l’impact de ces parasites sur la dynamique de la population cyanobactérienne, et (iii) caractériser des facteurs contrôlant la dynamique des systèmes hôtes-parasites d’intérêt. De plus, (iv) afin de mettre en exergue les relations étroites existantes entre l’hôte et la production fongique associée, nous avons mis au point des protocoles méthodologiques permettant une analyse microscopique fine de l’hôte, des sporanges et de leur contenu en zoospores (fécondité des chytrides). Les résultats acquis mettent en évidence la coexistence de deux espèces de chytrides, Rhizosiphon crassum et R. akinetum, associées à A. macrospora. La reconstruction des cycles de vie par analyse d’images prises à partir d’échantillons naturels nous a permis de montrer que les deux chytrides présentaient une durée de leur cycle de vie similaire, et estimée à environ 3j. pour R. crassum. Cependant, ces deux espèces se différencient d’un point de vue fonctionnel en parasitant des types cellulaires distincts : R. crassum influence directement la biomasse active en parasitant les cellules végétatives, alors que R. akinetum parasite les cellules de résistances (akinètes) de A. macrospora. Cette dernière espèce peut donc avoir un impact sur le recrutement, la structure génétique et la variabilité interannuelle de la population hôte. Par ailleurs, outre leur impact direct, nous montrons que l’action des chytrides parasites peut aboutir à une fragmentation mécanique des filaments de A. macrospora, augmentant potentiellement la perte de biomasse cyanobactérienne par broutage. De plus, nous avons pu mettre en évidence que la production zoosporique des chytrides dépendait des ressources nutritives disponibles tant d’un point vue quantitatif (taille de l’hôte) que qualitatif (groupe phytoplanctonique parasité, métabolisme de l’hôte...). La réduction de la biomasse active de cyanobactéries, la fragmentation mécanique, ainsi que la production de zoospores dont la qualité nutritive pour le zooplancton a été démontrée, établissent les chytrides comme lien trophique entre les proliférations cyanobactériennes filamenteuses « inedible », considérée comme impasses trophiques, et les niveaux trophiques supérieurs. Enfin, l’ensemble des résultats acquis montre l’intérêt de prendre en compte, désormais, le rôle fonctionnel des champignons microscopiques parasites, notamment comme agents régulateurs direct et indirect du développement phytoplanctonique. Cette prise en compte permettrait, sans aucun doute, d’améliorer les modèles de transferts de matière et d’énergie qui transitent dans les écosystèmes aquatiques, dans le contexte général d’optimiser la gestion des plans d’eau. / Face to both the important anthropogenic input in nutrients and the global change, numerous authors predict that the cyanobacterial blooms will increase in relative abundance in aquatic ecosystems. An exhaustive knowledge of the driving biotic factors of the cyanobacterial dynamic is essential. In lakes, the most common fungal parasites of phytoplankton belong to the phylum Chytridyomycota (i.e. chytrids). The aim of the thesis was to investigate the fungal parasitism associated to the cyanobacterial blooms, particularly the ecology of chytrids parasitizing the filamentous cyanobacterial species Anabaena macrospora, in Lake Aydat (France). During two successive years (2010-2011), investigations on (i) the chytrid cycle of life of two chytrid species parasitizing A. macrospora, (ii) the impact of the fungal parasitism on the cyanobacterial bloom dynamic and (iii) driving factors of the host-parasite pairings dynamics have been led during two spatio-temporal surveys using high resolution sampling strategies. Moreover (iv) a double staining method based on a combination of CFW and SYTOX green for counting, identifying, and investigating the fecundity of phytoplankton fungal parasites and the putative relationships established between hosts and their fungal parasites has been developed. Results underlined the coexistence of two chytrids, Rhizosiphon crassum and R. akinetum, which have similar life cycles but differed in their infective regimes depending on the cellular niches offered by their host. R. crassum infected both vegetative cells and akinetes while R. akinetum infected only akinetes. A reconstruction of the developmental stages suggested that the life cycle of R.crassum was completed in about 3 days. By infecting akinetes, R. akinetum could reduce or modify the genetic structure of the cyanobacterial bloom of the following year. Furthermore, chytrids may reduce the length of filaments of Anabaena macrospora significantly by ‘‘mechanistic fragmentation’’ following infection. All these results suggest that chytrid parasitism is one of the driving factors involved in the decline of cyanobacterial blooms, by direct mortality of parasitized cells and indirectly by the mechanistic fragmentation, which could weaken the resistance of A. macrospora to grazing. Moreover, we underlined that the production of zoospore depends on the nutritional host quantity (host size) and quality (host phytoplanktonic group, host metabolism...). The decrease of the cyanobacterial active biomass, mechanistic fragmentation, and production of zoospores which exhibit a high nutritional quality for the zooplankton, established the chytrids as a real link between the inedible filamentous cyanobacteria, considered as trophic dead ends, and the higher trophic levels. Overall, we consider that the acquisition of our data places the chytrid parasitism as an important driving factor of the phytoplankton dynamic, allowing the inclusion of fungi and their main function (parasitism) in the energy and matter fluxes in the pelagic ecosystems.

Made available in DSpace on 2016-12-08T16:44:37Z (GMT). No. of bitstreams: 1 PGPV12MA063.pdf: 46944968 bytes, checksum: 6b8d53259f4109a052b1401a704602a0 (MD5) Previous issue date: 2012-02-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The macrospora spot, caused by the fungus Stenocarpella macrospora, has become one of the most important maize diseases in Brazil in recent years. The objectives of the study were: a) evaluate the resistance of maize hybrids macrospora spot b) determine the sensitivity of S. macrospora to fungicides by inhibition of mycelial growth and conidial germination c) evaluate the protective and curative control of the disease in young corn plants d) quantify the effect of fungicides on control the expansion macrospora spot. The resistance was evaluated on 52 hybrids and two isolates of the fungus. The inoculation was made at the V2 developmental stage, placing each plant in 0.2 mL of the suspension of 7x104 conidia mL-1. Disease severity was evaluated 14 days after inoculation. The sensitivity of S. macrospora in vitro were evaluated 12 fungicides, six concentrations and two isolates of the fungus. The percentage of inhibition of mycelial growth and conidial germination was calculated in comparison with control, estimation of 50% inhibitory concentration (IC50). The efficiency of fungicides to control protective and curative was determined in a greenhouse, with the isolated SC and the hybrid AS1565. We evaluated 16 fungicides, applied at V2. Preventive control the inoculation was made depositing 0.2 mL of the suspension of 7x104 conidia mL-1 48 hours after application. In the curative control the inoculation was made 48 hours before application. Disease severity was determined 21 days after inoculation. In control the expansion of macrospora spot seven fungicides were evaluated, applied directly to leaves of maize with leaf lesions with hybrid AS 1565. The lesions were measured every three days for a period of 27 days. In each lesion was also quantified the number of pycnidia cm2-1. There was significant difference in severity leaf among the hybrids. There was no significant effect of isolates of the fungus on the severity of the disease. Resistant hybrids were not found. The disease severity ranged from 8.88% for hybrid 30A30HX to 17.76% for the AO 1050. The fungicides tested were effective in inhibiting the growth of the mycelium, and the IC50 was less than 1 ppm for all fungicides. There was no difference between isolates. The strobilurins had higher fungitoxicity in inhibition of germination of the conidia, and the IC50 was between 0.0035 and 0.03 ppm, and the isolated SC showed a higher sensitivity to the fungicides. In preventive aplication, all fungicides differed from the control, reducing the average 85% the severity macrospora spot compared to control. In the curative control all fungicides differed from the control, but mixtures of triazole + strobilurin showed greater efficiency, reducing an average of 75% disease severity, whereas the isolated products as the strobirulins reduced 62%, benzimidazoles 55% and triazoles 38% . The fungicide tebuconazole, and mixtures of triazoles + strobilurins, reduced on average 68% expansion of the macrospora spot, and 58.7% the formation of pycnidia cm2-1. It although no resistant hybrid was found genetic variability exists among the hybrids tested and that the application of fungicides significantly reduced the severity of the macrospora spot / A mancha-de-macrospora, causada pelo fungo Stenocarpella macrospora, tem se tornado uma das doenças mais importantes na cultura do milho nos últimos anos no Brasil. Os objetivos do trabalho foram: a) avaliar a resistência de híbridos de milho a mancha-demacrospora; b) determinar a sensibilidade de S. macrospora a fungicidas pela inibição do crescimento do micélio e germinação de conídios; c) avaliar o controle protetor e curativo da doença em plantas jovens de milho; d) quantificar o efeito de fungicidas na expansão da mancha-de-macrospora. Quanto a resistência foram avaliados 52 híbridos e dois isolados do fungo. A inoculação foi feita no estádio fenológico V2, depositando em cada planta 0,2 mL da suspensão de 7x104 conídios mL-1. A severidade da doença foi avaliada aos 14 dias após a inoculação. Quanto à sensibilidade de S. macrospora in vitro foram avaliados 12 fungicidas, seis concentrações e dois isolados do fungo. A porcentagem de inibição do crescimento micelial e germinação de conídios foi calculada em relação à testemunha, estimando-se valores de concentração inibitória de 50% (CI50). A eficiência de fungicidas no controle protetor e curativo foi determinada em casa-de-vegetação, com o isolado de SC e o híbrido AS1565. Foram avaliados 16 fungicidas, aplicados no estádio V2. No controle preventivo a inoculação de 0,2 mL da suspensão de 7x104 conídios mL-1 foi realizada 48 horas depois da aplicação. No controle curativo a inoculação foi realizada 48 horas antes da aplicação. A severidade da doença foi determinada 21 dias após a inoculação. No controle da expansão da mancha-de-macrospora foram avaliados sete fungicidas aplicados diretamente em folhas de milho AS 1565 com lesões foliares. As lesões foram mensuradas a cada três dias por um período de 27 dias. Em cada lesão também foram quantificados o número de picnídios cm2-1. Houve diferença significativa na severidade entre os híbridos. Não houve efeito significativo dos isolados do fungo sobre a severidade da doença. Não foi encontrado nenhum híbrido resistente. A severidade da doença variou de 8,88% para o híbrido 30A30HX a 17,76% para o AO 1050. Foi constatado que os fungicidas testados foram eficazes na inibição do crescimento do micélio, sendo que a CI50 foi menor que 1 ppm para todos os fungicidas, não havendo diferença entre isolados. Na inibição da germinação de conídios as estrobilurinas apresentaram maior fungitoxicidade, pois a CI50 ficou entre 0,0035 e 0,03 ppm, sendo que o isolado de SC mostrou maior sensibilidade aos fungicidas. Na aplicação preventiva todos os fungicidas diferiram da testemunha, reduzindo em média 85% à severidade da mancha-demacrospora em relação à testemunha. No controle curativo todos os fungicidas diferiram da testemunha, contudo as misturas de triazóis + estrobilurinas mostraram maior eficácia, reduzindo em média 75% a severidade da doença, enquanto que os produtos isolados como as estrobilurinas reduziram 62%, benzimidazóis 55% e triazóis 38%. O fungicida tebuconazole e as misturas de triazóis + estrobilurinas, reduziram em média 68% a expansão da mancha-demacrospora, e 58,7% a formação de picnídio cm2 -1. Foi constatado que apesar de não haver híbridos resistentes existe variabilidade genética entre os híbridos testados e que a aplicação de fungicidas reduz significativamente a severidade da mancha-de-macrospora

Made available in DSpace on 2016-12-08T16:44:43Z (GMT). No. of bitstreams: 1 PGPV13MA115.pdf: 363959 bytes, checksum: 6e95b30ef3170c9a81eb55320f07a3ed (MD5) Previous issue date: 2013-02-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The importance of pathogens that infect corn crop is a major obstacle to the continued increase in crop yield. Genetic resistance is a major strategy for disease control, however, in Brazil there is no information maize cultivars resistant to macrospora spot caused by the fungus Stenocarpella macrospora. With the objective to evaluate the resistance of different cultivars to macrospora spot, the impact of their occurrence on yield and quality of grain, get an ideal concentration of conidial S. macrospora to beused for inoculation of corn and investigate whether there are differences in disease severity from isolates of the fungus were developed four experiments. The first experiment tested six xoncentration: 0,60, 120, 180, 240 and 300 000 conidia mL-1 and the second tested isolates drom the regions of Vacaria, Passo Fundo, São Valentim, Campinas do Sul, state of Rio Grande do Sul, and Quilombo e Lages, Santa Catarina state, and Pato Branco, Paraná state. The inoculum concentration data were subjected to regression analysis (P <0.05) while data from different isolates were submitted to Tukey test (P <0.05). the third study was conducted in a greenhouse, in 2011, with 92 cultivars and three isolates of the fungus and mountainous regions west of the State of Santa Catarina (OSC and SSC) and Campos de Cima da Serra of the State of Rio Grande do Sul (CCSRS). It was used a completely randomized designs with five replicates. It was evaluated the severity of the macrospora spot 21 days after inoculation. Data were analyzed by ANOVA and means were compared by the Scott Knott test (P <0.05) and genotype groups responses being analyzed by orthogonal contrast (P <0.05). the fourth experiment was conducted in the field in Lages, SC, 2011/2012 season, with eigth genotypes: CD 393, NBX 920YG, TORK TL, AS 1565, DKB 240YG, SG 6304YG, P30F53YG and SCS 155 CATARINA under inoculating conidia of S. macrospora in different stages (V10, V12 and tasseling) and a control. It was evaluated the incidence of white rot cob, percentage of rot grains, thousand grain weigth and grain yield. Data were analyzed by analysis of variance and then by Tukey test (P <0.005). Dunnett s test was conducted to compare the stages of inoculation with control for each hybrid. The results in these experiments have revealed that there was infection and expression of symptoms in all cultivars, differing in severity. The hybrids showed uo for the three most resistant isolates, indicating that the gratest genetic variability of VPA and HD did not guarantee greater resistance to macrospora spot. It was also verified differneces in aggressiveness among isolates from different regions, being isolated from the Quilombo showed that on average greater disease severity, for this isolated significant difference between transgenic and conventional cultivars. It can be confirmed that was not detected complete resistance to S. macrospora. From the regression analysis it was determined that with 180.000 conidia ml-1 was reached maximum severity. Of the eighjt hybrids tested, five had decreased productivity when inoculated with S. macrospora. For all hybrids increased incidence of white rot cob and rot grains compared stages of inoculation with the witness (check) and as the stadium came from tasseling inoculation increased the percentage of rot cob and rot grain. It was demonstrated in this study that there is genetic variation for resistance to white rot cob caused by S. macrospora with inoculum derived from leaf lesions / A importância dos patógenos que infectam a cultura do milho constitui um dos principais entraves para o contínuo aumento na produtividade da cultura. A resistência genética é uma das principais estratégias de controle de doenças foliares, no entanto, no Brasil não há informações de cultivares de milho resistente à mancha de macrospora causada pelo fungo Stenocarpella macrospora. Com o objetivo de avaliar a resistência de diferentes cultivares comerciais à mancha de macrospora, o impacto da sua ocorrência na produtividade e qualidade de grãos, obter uma concentração ideal de conídios de S. macrospora a ser utilizada na inoculação do milho e investigar se há diferença na severidade da doença a partir de isolados do fungo foram desenvolvidos quatro experimentos. O primeiro experimento testou seis concentrações de conídios: 0, 60, 120, 180, 240 e 300 mil conídios mL-1 e o segundo testou isolados oriundos das regiões de Vacaria, Passo Fundo, São Valentim, Campinas do Sul, Estado do Rio Grande do Sul, Lages e Quilombo, Estado de Santa Catarina, e Pato Branco, Estado do Paraná. Os dados de concentração de inóculo foram submetidos à análise de regressão (P<0,05) enquanto os dados dos diferentes isolados foram submetidos ao teste de Tukey (P<0,05). O terceiro trabalho foi conduzido em casa de vegetação, no ano de 2011, com 92 cultivares e três isolados do fungo das regiões Oeste e Serrana do Estado de Santa Catarina (OSC e SSC) e Campos de Cima da Serra do Estado do Rio Grande do Sul (CCSRS). Utilizou-se delineamento experimental inteiramente casualizado com cinco repetições. Avaliou-se a severidade da mancha de macrospora aos 21 dias após a inoculação. Os dados foram submetidos à ANOVA e as médias comparadas pelo teste de Scott Knott (P<0,05), respostas ente grupos de genótipos analisadas por contraste ortogonal (P<0,05). O quarto experimento foi realizado a campo no município de Lages, SC, safra 2011/2012, com oito genótipos: CD 393, NBX 920YG, TORK TL, AS 1565, DKB 240YG, SG 6304YG, P30F53YG e SCS 155 CATARINA, sob inoculação de conídios de S. macrospora em diferentes estádios de (V10, V12 e Pendoamento) e uma testemunha. Avaliaram-se incidência de podridão branca de espiga, porcentagem de grãos ardidos, massa de mil grãos e rendimento de grãos. Os dados foram analisados por meio de análise de variância e posteriormente por teste de Tukey (P<0,05). Foi realizado teste de Dunnett para comparação dos estádios de inoculação com a testemunha, para cada híbrido. Os resultados obtidos em tais experimentos permitem afirmar que houve infecção e expressão de sintomas em todas cultivares avaliadas, diferindo no nível de severidade. Os híbridos simples demonstraram-se mais resistentes para os três isolados, demonstrando que a maior variabilidade genética da VPA e do HD não garantiu maior resistência à mancha de macrospora. Foi verificado também diferenças de agressividade entre os isolados de regiões diferentes, sendo o isolado de Quilombo o que apresentou na média maior severidade da doença. Para este isolado houve diferença significativa entre cultivares transgênicas e convencionais. Pode-se confirmar que não foi detectada resistência completa à S. macrospora. A partir da análise de regressão determinouse que com 180 mil conídios mL-1 foi alcançada máxima severidade. Dos oito híbridos avaliados, cinco apresentaram queda de produtividade quando foram inoculados com S. macrospora. Para todos os híbridos houve aumento de podridão branca da espiga e grãos ardidos quando comparados os estádios de inoculação com a testemunha e conforme o estádio de inoculação se aproximou do pendoamento aumentou espigas doentes e grãos ardidos. Ficou evidenciado neste estudo que existe variabilidade genética para resistência a podridão da espiga causada por S. macrospora com inóculo oriundo das lesões foliares

Made available in DSpace on 2017-07-25T19:29:52Z (GMT). No. of bitstreams: 1 Lincom Koguishi.pdf: 1991536 bytes, checksum: 2ab0674dbbe3a078502f0ecb0a0b2a5b (MD5) Previous issue date: 2011-12-20 / The objective of this study was to evaluate the effects of application timing of fungicides epoxiconazole + pyraclostrobin and azoxystrobin + cyproconazole in corn in the control of Phaeosphaeria leaf spot (Phaeosphaeria maydis), rust (Puccinia sorghi), Cercospora leaf spot (Cercospora zea-maydis), stain Diplodia (Diplodia macrospora) and stem rot. Three experiments were installed in a randomized block design with four replications in plots measuring 3.2 x 5.0 meters. In experiment 1, we applied the fungicide epoxiconazole + pyraclostrobin at a dose of 99.75 + 37.5 g ai ha-1, more mineral oil (Assist) at a dose of 500 mL pc ha-1 and Experiments 2 and 3 was applied fungicide azoxystrobin + cyproconazole at a dose of 70 gy + 28 ha-1, more paraffinic mineral oil (Nimbus) at a dose of 600 mL pc ha-1. In experiment 1, applications were made at the phenological stages V8, V16, V8 and V16, V12, V15, VT and R2, and evaluated the severity of Phaeosphaeria leaf spot and Cercospora leaf spot in at R3, R4 and R5 and rot stalk the stage R6. With the data of severity, we calculated the area under the disease progress curve to Phaeosphaeria leaf spot. The lowest AUDPC occurred in treatments with a V16 application, with two applications in V8 and V16, with controls from 74 to 80%. In Experiments 2 and 3, applications of fungicides were made in V8, V16, R2, V8 and V16, V8 and R2, and R2 V16, V8, V16 and R2. Was evaluated in experiment 2 to Cercospora leaf spot, Phaeosphaeria leaf spot and stem rot. In the control of Cercospora leaf spot were the best treatment applications in developmental stage V8, which coincided with the occurrence of disease in the crop. To Phaeosphaeria leaf spot, the lowest values were in AUDPC with two applications in V8 and V16, V16 and R2 and the three applications in V8, V16 and R2, with control ranging from 25 to 40%. In Experiment 3, we assessed the common rust, leaf spot Phaeosphaeria, stain and Diplodia stalk rot. To common rust treatments containing application in V8 were the most effective with the percentage of controls over 80%. In relation to Phaeosphaeria leaf spot, the best treatment for AUDPC was the three applications (V8, V17 and R2) with 27% control. The treatments did not control the stain of Diplodia. In three experiments, was also evaluated damaged kernels, weight of thousand seeds and productivity, there being only in experiment 2, significant difference in the number and developmental stage of application for the variables weight of thousand seeds and productivity, highlighting the three applications in V8, V16 (17), R2 and applications V16 (17), R2, V8, V17. Keywords: Phaeosphaeria maydis, Puccinia sorghi, Cercospora zea / O objetivo deste trabalho foi avaliar os efeitos da época de aplicação dos fungicidas piraclostrobin + epoxiconazole e azoxystrobin + ciproconazole na cultura do milho no controle da mancha foliar de phaeosphaeria (Phaeosphaeria maydis), ferrugem comum (Puccinia sorghi), mancha foliar de cercospora (Cercospora zea-maydis), mancha de diplodia (Diplodia macrospora) e a podridão de colmo. Foram instalados três experimentos, no delineamento de blocos ao acaso com 4 repetições, em parcelas medindo 3,2 x 5,0 metros. No experimento 1, foi aplicado o fungicida piraclostrobin + epoxiconazole na dose de 99,75 + 37,5 g i.a. ha-1, mais óleo mineral (Assist) na dose de 500 mL p.c. ha-1 e nos experimentos 2 e 3 foi aplicado o fungicida azoxystrobin + ciproconazole na dose de 70 + 28 g.i.a. ha-1, mais óleo mineral parafínico (Nimbus) na dose de 600 mL p.c. ha-1. No experimento 1, as aplicações foram realizadas nos estádios fenológicos V8; V16; V8 e V16; V12; V15; VT e R2, sendo avaliada a severidade da mancha foliar de phaeosphaeria e mancha foliar de cercospora nos estádios R3, R4 e R5 e a podridão de colmo no estádio R6. Com os dados de severidade calculou-se a área abaixo da curva de progresso da doença para a mancha foliar de phaeosphaeria. Os menores valores de AACPD ocorreram nos tratamentos com uma aplicação em V16, com duas aplicações em V8 e V16, com controles de 74 a 80%. Nos experimentos 2 e 3, as aplicações dos fungicidas foram realizadas em V8; V16; R2; V8 e V16; V8 e R2; V16 e R2; V8, V16 e R2. Avaliou-se no experimento 2 a mancha foliar de cercospora, mancha foliar de phaeosphaeria e a podridão de colmo. No controle da mancha foliar de cercospora os melhores tratamentos foram as aplicações no estádio fenológico V8, os quais coincidiram com a ocorrência da doença na cultura. Para mancha foliar de phaeosphaeria, os menores valores na AACPD foram os com duas aplicações em V8 e V16, V16 e R2 e as três aplicações em V8, V16 e R2, com controle variando de 25 a 40%. No experimento 3, foram avaliadas a ferrugem comum, mancha foliar de phaeosphaeria, mancha de diplodia e a podridão de colmo. Para ferrugem comum os tratamentos que continham aplicação em V8 foram os mais eficientes com porcentagem de controles acima de 80%. Em relação a mancha foliar de phaeosphaeria, o melhor tratamento para AACPD foi o com três aplicações (V8, V17 e R2) com 27% de controle. Os tratamentos não controlaram a mancha de diplodia. Nos três experimentos avaliou-se também grãos ardidos, peso de mil sementes e produtividade, constatando-se somente no experimento 2, diferença significativa quanto ao número e estádio fenológico de aplicação, para as variáveis peso de mil sementes e produtividade, destacando as 3 aplicações em V8, V16 (17), R2 e as 2 aplicações em V16 (17), R2 e V8, V17.

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