Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-13T12:15:49Z No. of bitstreams: 1 arquuivototal.pdf: 3577415 bytes, checksum: 0e9d2a60e77718e50720d15b7cad6fb1 (MD5) / Made available in DSpace on 2017-09-13T12:15:49Z (GMT). No. of bitstreams: 1 arquuivototal.pdf: 3577415 bytes, checksum: 0e9d2a60e77718e50720d15b7cad6fb1 (MD5) Previous issue date: 2016-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In recent decades fungal infections have increased, and their high rates of morbidity and mortality have brought them much attention. Immunocompromised patients are more susceptible to microorganism infections; in particular, fungal species of the genus Aspergillus spp. Among the current infectious filamentous fungi they have a rather high incidence. Antifungal treatments have been subjected to numerous studies, and the increasing number of resistant fungal species has been highlighted. Thus, we see the importance of seeking new, more effective, and less toxic therapeutic sources. The aim of this study was to evaluate the in vitro antifungal activity and structure activity relationships of coumarin compounds against Aspergillus species. For this the MIC of 24 coumarin compounds was determined and subsequent SAR studies were performed with computer software. 12 of the 24 tested coumarin derivatives have antifungal activity, with 5 has shown excelent activity. Thus two derivatives, 7-hydroxy-6-nitrocoumarin (Cou-UNO2), and 4-acetoxycoumarin (Cou-UMB16), with better MIC values were evaluated via inhibition of mycelial growth and conidial germination, and by mechanism of action testing. The products were evaluated in combination with antifungals standards. The results showed that 12 of the 24 tested coumarin derivatives have antifungal activity, with MIC values ranging from 1024-16μg/ml. SAR studies have showed that the presence of a short aliphatic chain, and/or electronegative groups like ring substituents are favorable for antifungal activity. The coumarin derivatives with better MIC values (16μg/ml); Cou-UNO2 and Cou-UMB16 were capable of inhibiting both the mycelial growth and conidial germination of Aspergillus spp. Their activity is on the structure of the fungal cell wall. Cou-NO2, in a sub-inhibitory concentration, enhanced the in vitro effects of azoles, and in combination with azoles (voriconazole and itraconazole) there was an additive effect. Cou-UMB16 in combination with azoles (voriconazole and itraconazole) had synergistic or additive effects depending on the strain used. Thus this study concludes that coumarin derivatives have antifungal activity against the species A. flavus and A. fumigatus. The coumarin Cou-UMB16 and Cou-UNO2 have able to inhibit the mycelial growth and conidio germination and that this activity is due to its action on the fungal cell wall and the presence of electronegative groups as substituents of the benzopyrone ring favors this activity. / As infecções fúngicas, nas últimas décadas, têm se destacado devido ao seu crescente aumento e suas elevadas taxas de morbidade e mortalidade. Pacientes imunocomprometidos são os que possuem maior susceptibilidade a estes micro-organismos. Dentre estas infecções fúngicas, as espécies pertencentes ao gênero Aspergillus spp. possuem alta incidência entre os fungos filamentosos. A terapêutica antifúngica tem sido alvo de numerosos estudos e nestes tem sido destacado o crescente aumento de espécies fúngicas resistentes. Dessa forma, destaca-se a importância da busca de novas fontes terapêuticas que se apresentem mais eficazes e com menos tóxicas ao hospedeiro. Assim o objetivo deste estudo foi avaliar a atividade antifúngica in vitro e relação estrutura atividade (SAR) dos compostos cumarínicos frente às espécies do gênero Aspergillus. Para tal foi determinada a CIM de 24 compostos cumarínicos e posteriormente realizado estudos SAR com softwares computacionais. Dos 24 compostos ensaiados, 12 demonstraram possuir atividade antifúngica e 5 obtiveram forte atividade antifúngica. Destes derivados cumarínicos com forte atividade antifúngica, dois merecem destaque, 7-hidroxi-6-nitrocumarina (Cou-UNO2) e 4-acetóxicumarina (Cou-UMB16), com melhores valores de CIM, foram avaliados via testes de inibição do crescimento micelial, germinação dos conídios e testes de determinação do mecanismo de ação. Por fim os produtos foram avaliados em combinação com antifúngicos padrões. Os resultados demonstraram que 12 derivados cumarínicos dos 24 ensaiados possuem atividade antifúngica, com valores de CIMs variando entre 1024-16μg/mL. A SAR demonstra que a adição de grupos eletronegativos, como substituintes do anel benzopirona, favorece a atividade antifúngica destes compostos. Os derivados cumarínicos com melhores CIM (16μg/mL), 7-hidroxi-6-nitrocumarina (Cou-UNO2) e 4-acetóxicumarina (Cou-UMB16) foram capazes de inibir o crescimento micelial e a germinação dos conídios de Aspergillus spp. E estes possuem ação sobre a estrutura da parede celular fúngica. Em uma concentração subinibitória, Cou-UNO2 potencializou a ação in vitro dos derivados azólicos e em combinação com os derivados azólicos (voriconazol e itraconazol) observou-se um efeito aditivo. Já Cou-UMB16 em combinação com os derivados azólicos (voriconazol e itraconazol) obteve um efeito sinérgico a aditivo, dependendo da linhagem utilizada. Logo este estudo conclui que os derivados cumarínicos possuem atividade antifúngica contra as espécies de A. flavus e A. fumigatus. As cumarinas Cou-UNO2 e Cou-UMB16 foram capazes de inibir o crescimento micelial e a germinação dos conídios e que esta atividade seja devido a sua ação sobre a parede celular fúngica. E que esta atividade é favorecida pela presença de moléculas eletronegativas no anel básico da cumarina (1,2-benzopirona).

Les champignons du genre Aspergillus sont des moisissures banales de l'environnement. Elles sont présentes dans le sol et sur des végétaux en décomposition. Les Aspergillus se propagent par l'intermédiaire de spores microscopiques en suspension dans l'air. L'Homme et les animaux sont exposés en permanence aux spores aspergillaires mais les défenses immunes empêchent leur développement dans l'organisme. Lorsque ces défenses sont amoindries, une aspergillose est possible. Dans ce cas, Aspergillus fumigatus et A. flavus sont le plus souvent incriminés. Les oiseaux sont beaucoup plus sensibles que les mammifères et l'environnement représenté par les élevages aviaires est propice à la prolifération des moisissures du genre Aspergillus. L'objectif de ce travail de thèse a été de caractériser la diversité génétique et la sensibilité aux antifongiques d'isolats d'Aspergillus provenant d'élevages aviaires dans la province du Guangxi en Chine. La première partie de la thèse est une analyse bibliographique sur les champignons du genre Aspergillus, les aspergilloses et les caractéristiques de l'élevage aviaire en Chine. Une première enquête a été réalisée dans 3 élevages près de la ville de Nanning et dans un élevage (incluant un éclosoir) à proximité de la ville de Guilin. Des écouvillonnages pharyngés et des prélèvements d'air ont été réalisés pendant plusieurs semaines. Des prélèvements ont également été faits sur des œufs dans l'éclosoir. Cette enquête a montré que le niveau de contamination fongique dépendait du type d'élevage. De nombreux isolats fongiques ont pu être collectés : 188 isolats d'A. fumigatus et 159 isolats d'A. flavus. La seconde partie du travail expérimental a porté sur la caractérisation de la diversité génétique d'A. fumigatus et d'A. flavus. Pour cela, la technique MLVA (multiple locus VNTR analysis) a été utilisée. Pour A. flavus, 8 marqueurs VNTR (variable-number tandem-repeat) ont été sélectionnés et une réaction PCR multiplex a été mise au point. Au total, 91 isolats d'A. flavus, incluant 6 souches de référence, ont été caractérisées avec le panel des 8 marqueurs VNTR. Cette analyse a permis de définir 78 génotypes distincts et un index de discrimination de 0,993. L'analyse de 188 isolats d'A. fumigatus avec 10 marqueurs VNTR a permis de définir 142 génotypes distincts. Certains génotypes d'A. flavus ou d'A. fumigatus sont clairement regroupés dans le nuage de point généré par l'analyse MST (minimum spanning tree). La troisième partie du travail expérimental a porté sur la sensibilité aux antifongiques de 177 isolats d'A. fumigatus. Ces isolats ont été récupérés dans des élevages aviaires en Chine et en France. Les isolats de Chine sont pour la plupart sensibles avec des valeurs minimales inhibitrices (vis-à-vis de l'itraconazole) comprises entre 0,38 et 0,75 µg/mL. Les isolats de France sont pour la plupart sensibles avec des valeurs minimales inhibitrices (vis-à-vis de l'itraconazole) comprises entre 0.19 and 1 µg/mL. Quatre souches ont été considérées comme résistantes : 2 souches provenant de deux élevages en Chine et 2 souches provenant de deux élevages en France. Des mutations sur le gène Cyp51A ont été détectées pour 11 isolats (3 résistants et 8 sensibles). Vingt et une mutations nucléotidiques ont été identifiées. Onze de ces mutations sont silencieuses et 9 sont à l'origine d'un changement de la composition de la protéine. Sept substitutions ont déjà été décrites dans la littérature ; les mutations A116R, E130D et Q131H sont originales. / Fungi of the genus Aspergillus are moulds, which occur most frequently in soil, water and decaying vegetation. They sporulate abundantly and the spores are easily dispersed into the environment by air. As a result of this ubiquitous presence, animals and people are constantly exposed to Aspergillus spores. Aspergillus fumigatus and A. flavus are recognized as predominant causes of fungal diseases in humans and wide range of animals. Birds are much more sensitive that mammals and in avian farms, environmental conditions are favorable to the development of many fungal species, including Aspergillus spp. The objective of the present study was to assess the genetic diversity and antifungal susceptibility of Aspergillus isolates from avian farms in Guangxi, China. The first part of the experimental work related the evolution of fungal contamination in 3 avian farms near the city of Nanning and one farm (including a hatchery) near the city of Guilin. Pharyngeal swabs and air samples were collected during several weeks and 3 cycles of hatching were monitored. The average contamination level with Aspergillus spp. and Mucorales was significantly different according to the farms. The survey allowed to collect a total number of 188 A. fumigatus and 159 A. flavus isolates. The second part of the work was about the genetic diversity of A. fumigatus and A. flavus. For that purpose, the Multiple Locus Variable-number tandem-repeat (VNTR) Analysis was specifically developed and used. For A. flavus, 8 VNTR markers were selected and a multiplex reaction was designed. A total number of 91 A. flavus isolates, including 6 reference strains were typed with the panel of 8 VNTRs. This analysis yielded 78 different genotypes, which corresponds to a combined loci index of 0.993. Among all genotypes, 71 were only found once. The analysis of 188 A. fumigatus isolates using 10 VNTR markers led to the resolution of 142 distinct genotypes. Clusters of A. flavus or A. fumigatus isolates could be defined by using the graphing algorithm Minimum Spanning Tree. The third part of the experimental work was about the antifungal susceptibility of 177 A. fumigatus isolates collected in avian farms in China and France. Most of the isolates from China were susceptible to itraconazole with a Minimum Inhibitory Concentration (MIC) comprised between 0.38 and 0.75 µg/mL. Most of the isolates from birds and avian farms in France were susceptible to itraconazole with a MIC comprised between 0.19 and 1 µg/mL. MIC values of isolates collected in farms with antifungal chemoprophylaxis were not higher than those of isolates collected from birds (that never received antifungal drugs before the sampling). Susceptibility testings demonstrated that 4 isolates should be considered as resistant to itraconazole: (2 isolates from avian farms in Guangxi, China and 2 isolates from avian farms in France). A modification of the Cyp51A sequence was identified in 11 isolates (3 azole-resistant and 8 azole-susceptible isolates). Twenty-one nucleotidic mutations were detected. Eleven of these mutations were silent and 10 yielded to amino acid substitutions. Seven of these substitutions had already been described whereas mutations A116R, E130D and Q131H were original.

L’aflatoxine B1 (AFB1) est une mycotoxine contaminant de nombreux substrats agricoles, notamment les céréales, arachides, pistaches, graines de coton et autres fruits secs. Elle est génotoxique, agit comme un initiateur de la cancérogenèse et elle est classée par le CIRC (Centre International de Recherche sur le Cancer) dans le groupe I des molécules carcinogènes pour les hommes et les animaux. Cette toxine est produite par des espèces fongiques appartenant essentiellement au genre Aspergillus et à la section Flavi ; A. flavus étant l’espèce la plus préoccupante. Cette espèce peut se développer à la fois au champ ou lors du stockage des matières premières après récolte. Plusieurs études montrent qu’un grand nombre de denrées alimentaires destinées à l’alimentation humaine et animale sont contaminées par les aflatoxines mettant en évidence la faible efficacité ou l’insuffisance des stratégies actuelles utilisées pour la maîtrise de cette contamination. L’objectif principal de cette étude est le développement d’une stratégie alternative basée sur l’utilisation de produits naturels et visant à réduire la production de la toxine par les moisissures aflatoxinogènes. Grâce à l’élaboration d’un outil moléculaire regroupant un ensemble de gènes impliqués directement ou indirectement dans la biosynthèse de l’AFB1, ce travail permet de mieux comprendre le mécanisme d’action moléculaire de composés anti-aflatoxinogènes. Des extraits aqueux de plantes méditerranéennes ont été testés pour leur efficacité à inhiber l’aflatoxinogénèse. Ainsi, l’extrait aqueux d’hysope, Micomeria graeca, présente des propriétés anti-aflatoxinogènes importantes sans modification de la croissance fongique. Son effet inhibiteur sur plusieurs souches productrices d’aflatoxines ainsi que son mécanisme d’action ont été caractérisés. Ce dernier semble impliquer des gènes pivots dans la régulation des métabolismes fongiques primaire et secondaire ainsi que des voies impliquées dans la réponse cellulaire au stress oxydatif. Ces travaux permettent de valider l’utilisation d’extraits naturels comme stratégie alternative de lutte contre la contamination des aliments par les aflatoxines. / Aflatoxin B1 (AFB1) is a mycotoxin contaminating numerous agricultural commodities such as cereals, peanuts, pistachios, cottonseed and other dried fruits. It is a genotoxic initiating carcinogenesis and is classified by the IARC (International Agency for Research on Cancer) in the group I of molecules that are carcinogenic for humans and animals. This toxin is produced by fungal species mainly belonging to the Flavi section of the Aspergillus genus, A. flavus being the most preoccupying species. This species can develop both in the field and during storage of commodities following harvest. Several studies show that a significant number of food and feedstuffs are contaminated by aflatoxins, highlighting the feeble efficiency or the shortfall of the strategies currently used to prevent such contamination. The main objective of this study is the development of an alternative strategy based on the use of natural products and aiming the reduction of toxin production by aflatoxinogenic fungi. Through the elaboration of a molecular tool grouping a number of genes directly or indirectly linked to AFB1’s biosynthesis, this work allows a better understanding of the molecular mechanism of action of anti-aflatoxinogenic compounds. Aqueous extracts of Mediterranean plants were tested for their ability to inhibit aflatoxinogenesis. Thus, hyssop’s aqueous extract - Micomeria graeca – is shown to present significant anti-aflatoxinogenic properties without reducing fungal growth. Its inhibiting effect in other AFB1-producing species and its mechanism of action were both characterized. The latter seems to involve pivotal genes in fungal primary and secondary metabolisms as well as genes implicated in the fungal cellular response to oxidative stress. These works allocate the use of natural extracts as an alternative strategy to counteract aflatoxin contamination of food and feed.

<p>Maize is a major calorie source for people living in Sub-Sahara Africa. In this region, <i>Aspergillus flavus</i> causes ear rot diseases in maize, contributing to food insecurity due to aflatoxin contamination. The biological control principle of competitive exclusion has been applied in both the United States and Africa to effectively reduce aflatoxin levels in maize at harvest by introducing atoxigenic strains that out-compete toxigenic strains. The goal of this study was to determine if the efficacy of preharvest biocontrol treatments carry over into the drying period, which is often delayed in Sub-Sahara Africa by the complexities of postharvest drying practices and lack of modern drying machinery. Maize was collected from fields in Texas and North Carolina that were treated with commercial biocontrol, and control fields that were untreated. To simulate moisture conditions similar to those experienced by farmers during drying in Sub-Sahara Africa, we adjusted the grain to 20% moisture content and incubated it at 28 ℃ for 6 days. Although the initial number of infected kernels in most samples were high, less than 24% of kernels were infected with <i>Aspergillus flavus</i> and aflatoxin levels were low (<4ppb). Both toxigenic and atoxigenic strains increased and spread through the grain over the incubation period, and aflatoxin levels increased, even in samples from biocontrol-treated fields. Our molecular analysis suggests that applied biocontrol strains from treated fields migrate to untreated fields. The results also indicate that the population of toxigenic <i>A. flavus</i> in the harvested grain will grow and produce aflatoxin during the drying period when moisture is high. Therefore, any potential postharvest reduction in aflatoxin accumulation will depend on how effective the biocontrol strain was at displacing the toxigenic populations prior to harvest.</p>

Orientador: Vera Aparecida de Oliveira Tiera / Banca: Dayane Batista Tada / Banca: Eduardo Alves de Almeida / Resumo: O presente trabalho descreve a síntese, caracterização e estudo das propriedades antimicrobianas de derivados anfifílicos de quitosana contra o fungo Aspergillus flavus. A quitosana utilizada nas sínteses foi obtida a partir da reação de desacetilação heterogênea de quitosana comercial. O grau médio de desacetilação foi determinado por Ressonância Magnética Nuclear de Hidrogênio, cujo resultado foi de 97,3%. As massas moleculares das quitosanas comercial e desacetilada foram determinadas por cromatografia de permeação em gel e os resultados obtidos foram 338,46 kDa e 137,12 kDa, respectivamente. Os derivados anfifílicos foram sintetizados em um processo de duas etapas: inicialmente realizou-se a alquilação da quitosana desacetilada pela inserção de uma proporção fixa de grupos dodecila (4%) na cadeia polimérica com posterior redução da base de Schiff empregando-se borocianohidreto de sódio. Em seguida foram inseridas proporções crescentes do grupo quaternário pentiltrimetilamônio. Os graus de substituição com grupos dodecila (GSDD) e grupos pentiltrimetilamônio (GDPE) foram determinados por RMN de 1H. O GSDD obtido foi de 4,0%, enquanto que os GDPE foram de 4,8; 12,7 e 46,3%. Todos os derivados sintetizados foram testados in vitro contra o fungo Aspergillus flavus em concentrações crescentes. Os ensaios microbiológicos mostraram que os derivados modificados com grupos dodecila e pentiltrimetilamônio são mais eficientes em inibir o crescimento do fungo Aspergillus flavus quando comparado à quitosana desacetilada e que a presença de grupos hidrofóbicos na cadeia do polímero proporciona uma interação mais forte com a membrana celular. Os resultados dos estudos utilizando modelos de membranas corroboraram com os dos ensaios microbiológicos, evidenciando que a presença de grupos dodecila na cadeia da quitosana aumenta a interação com as vesículas, resultando em... / Abstract: In this work is described the synthesis, characterization and study of antimicrobial properties of amphiphilic derivatives of chitosan against Aspergillus flavus. Deacetylated chitosan was obtained from the heterogeneous deacetylation of the commercial chitosan. The degree of deacetylation was determined by Hydrogen Nuclear Magnetic Resonance and the result was 97.3%. Molecular weights of commercial and deacetylated chitosans were determined by gel permeation chromatography and the obtained values were 338.46 kDa and 137.12 kDa, respectively. The hydrophobic derivatives were synthesized by a reductive amination reaction with dodecylaldehyde followed by reduction with sodium cyanoborohydride. Next increasing proportions of the quaternary group pentyltrimethylammonium was inserted on hydrophobic derivative. The degrees of substitution by dodecyl (4.0%) and pentyltrimethylammonium groups (4.8, 12.7 and 46.3%) were determined by Hydrogen Nuclear Magnetic Resonance. All synthesized derivatives were tested in vitro against Aspergillus flavus at increasing concentrations. The results showed that the modified derivatives with pentyltrimethylamonnium and dodecyl groups are more effective in inhibiting the growth of the fungus Aspergillus flavus when compared to deacetylated chitosan and the presence of hydrophobic groups in the polymer chain provides a stronger interaction with the cell membrane. The results of studies with model membranes agreed with the results of microbiological tests, showing that the presence of hydrophobic groups in chitosan backbone increases the interaction with lipid vesicles resulting in an increase in vesicles diameter and zeta potential. At first, it can be postulated that the mechanism of chitosan action against fungi is favored by hydrophobic interactions between chitosan derivatives and membrane lipids and may involve the adsorption on the cell wall and cell membrane disruption... / Mestre

Made available in DSpace on 2015-04-11T13:38:46Z (GMT). No. of bitstreams: 1 Renata Maria Leite Castelloes Mesquita.pdf: 1252525 bytes, checksum: 9ec0087057f4329331531ec3014e3df7 (MD5) Previous issue date: 2012-09-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Brazil nuts are an important commercial product of northern Brazil, however this product has been rejected by the international market due to high levels of aflatoxin contamination. The fungus considered more important in the production of these mycotoxins is Aspergillus flavus. In order to provide data for the development of an efficient strategy for the detection of aflatoxigenic A. flavus, which will contribute greatly to the quality control of the Brazil nuts, this main scope of this work was to study the genetic diversity of the fungi A. flavus isolated from almonds produced by different regions of Amazon. For this we have used microsatellite markers (SSR) which have proven to be effective for the study of genetic diversity between individuals and populations. We have selected 100 isolates of Aspergillus sp., and species identification was performed by sequencing the rDNA ITS region. It was identified 32 Aspergillus nomius, 49 Aspergillus flavus, 16 Aspergillus tamarii and 1 Aspergillus fumigatus. In the study of genetic diversity was used 12 SSR markers developed specifically for the species A. flavus and selected from the literature. Two groups of isolates of A. flavus were formed according to the origin of Brazil nuts (Humaitá / AM and Acre), and a third group isolated from nuts previously acquired in trade fairs in Belém / PA were also included in the study, totaling 86 individuals. Ten of the twelve markers used were efficient in PCR amplification. According to the result all the 10 loci studied were polymorphic, and from the 86 individuals analyzed 118 alleles were identified ranging from 110-390 base pairs. The number of alleles per locus ranged from 8-16 with a mean of 11.7. The statistical mean value for genetic differentiation (Gst) was 0.099, which represents a low genetic variation between the groups. However, the genetic diversity within groups accounted for 85.9% of the total genetic diversity, indicating a greater variability within groups. The coefficient of similarity wasused to estimate the genetic distance between the 86 A. flavus, it ranged from 0.7 to 1.0 with an average of 0.96. The genetic distance was also calculated between populations and ranged from 0.562 to 0.7287. Thus it is concluded that the isolates showed high polymorphism among them, however a greater variability was found within the groups (85.5%), while diversity among the groups was only 14.5%. And according to the groups formed by genetic diversity analysis of A. flavus using SSRs markers, there was no correlation with the origin of the isolates / A Castanha-do-brasil é um importante produto comercial da região norte do Brasil, entretanto esta vem sofrendo embargos na exportação devido aos altos níveis de aflatoxinas presente em lotes comercializados. A espécie de fungo considerada mais importante na produção destas micotoxinas é o Aspergillus flavus. No intuito de fornecer subsídios para o desenvolvimento de uma estratégia eficiente para a detecção de A. flavus aflatoxigênicos, que irá contribuir extremamente para o controle da qualidade da Castanha-do-brasil, pretendeu-se neste trabalho estudar a diversidade genética de fungos A. flavus isolados de amêndoas provenientes de diferentes municípios do estado do Amazonas e Acre. Para isso foram utilizados marcadores moleculares microssatélites (SSR), os quais tem se mostrado eficientes no estudo de variabilidade genética entre indivíduos e entre populações. Foram selecionados 100 isolados do gênero Aspergillus, e para identificação da espécie foi realizado o sequenciamento e análise da região ITS do rDNA. Ao total foram identificados 32 Aspergillus nomius, 49 Aspergillus flavus, 16 Aspergillus tamarii e 1 Aspergillus fumigatus. No estudo da variabilidade genética foram utilizados 12 marcadores SSR selecionados da literatura desenvolvidos especificamente para a espécie de A. flavus. Dois grupos de isolados de A. flavus foram formados de acordo com a origem da castanha (Humaitá/AM e Acre), e um terceiro grupo previamente isolado de castanhas adquiridas em feiras na cidade de Belém/PA também foram incluídos no estudo, totalizando 86 indivíduos. Dez dos doze marcadores utilizados foram eficientes na amplificação da PCR. Com o resultado verificou-se que todos os 10 locus analisados foram polimórficos, e a partir dos 86 indivíduos analisados foram identificados 118 alelos com amplicons variando entre 110 a 390 pares de base. O número de alelos variou de 8-16 por locus, com a média de 11,7. Ao realizar a análise estatística o valor médio da diferenciação gênica (GST) foi de 0,099, representando uma baixa variação genética entre os grupos analisados. Já a diversidade gênica dentro dos grupos foi responsável por 85,9% da diversidade genética total, indicando maior variabilidade dentro dos grupos. O coeficiente de similaridade utilizado para calcular a distância genética entre os 86 isolados variou de 0,7 a 1,0 com uma média de 0,96. A distância genética também foi calculada entre as populações e variaram de 0,562 a 0,7287. Assim conclui-se que os isolados apresentaram grande polimorfismo entre eles, sendo que a maior variabilidade ocorreu dentro do grupo (85,5%), enquanto que a diversidade entre os grupos estudados foi de 14,5%. De acordo com os resultados obtidos os agrupamentos formados pela análise da variabilidade genética de A. flavus utilizando marcadores SSRs, não apresentou correlação com a origem dos isolados.

Lipoxygenase (LOX)-derived oxylipins are known to play critical roles in defense against herbivores and pathogens. The objective of this study was to determine the biochemical, molecular and physiological roles of a specific maize lipoxygenase gene, ZmLOX10, with special emphasis on LOX10-derived oxylipins in plant-insect and plant-pathogen interactions. To achieve this goal, independent mutant alleles were generated and genetically advanced to create near-isogenic mutant and wild-type lines suitable for functional analysis. Here we provide genetic evidence that LOX10 is the sole LOX isoform in maize required for the biosynthesis of green leafy volatiles (GLV) in leaves and show that LOX10- mediated GLVs play a significant role in direct and indirect defense responses to insects through their regulation of jasmonic acid and volatile organic compound production. Contrary to the defensive role of LOX10 in plant-insect interactions, tests for susceptibility to fungal pathogens suggest that LOX10-mediated GLVs may contribute to the development of disease symptoms to the economically important maize pathogens, Aspergillus flavus and Colletotrichum graminicola. Specifically, LOX10-derived GLVs may facilitate aflatoxin accumulation in response to A. flavus infection and may play a positive role in anthracnose leaf blight and stalk rot caused by C. graminicola. Collectively, our results suggest that metabolites derived from GLV-regulated pathways have a significant impact on molecular plant-herbivore and plant-pathogen interactions.

Thesis (Ph.D.) -- Mississippi State University. Department of Biochemistry and Molecular Biology. / Title from title screen. Includes bibliographical references.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-06-15T19:21:25Z No. of bitstreams: 1 CELEIDA QUEIROZ DE LIMA - DISSERTAÇÃO PPGEA 2009..pdf: 7013625 bytes, checksum: 9063ee4b1275012362d4e2737d1c4d9f (MD5) / Made available in DSpace on 2018-06-15T19:21:25Z (GMT). No. of bitstreams: 1 CELEIDA QUEIROZ DE LIMA - DISSERTAÇÃO PPGEA 2009..pdf: 7013625 bytes, checksum: 9063ee4b1275012362d4e2737d1c4d9f (MD5) Previous issue date: 2009-04 / E premente a necessidade de controle do Aspergillus flavus, fungo importante por deteriorar sementes e grãos armazenados e produzir toxinas prejudiciais a saúde humana e animal. Produtos "naturais" como óleos essenciais ou extratos vegetais, vêm sendo testados e até mesmo utilizados com propósitos de eliminar ou reduzir os efeitos negativos apresentados pelos fungicidas sintéticos. O presente estudo realizou-se com extratos de nim (Azadirachta indica) - comercial e obtido de folhas-, canela (Cinnamomum zeylanicum) e alho (Allium sativum L.), aplicados nas proporções 1,10, 25, 50 e 100%, com o objetivo de avaliar "in vitro" os seus efeitos no desenvolvimento de A. flavus e sobre a qualidade fisiológica de sementes de amendoim (Arachis hypogeae L.) tratadas com os referidos extratos. O trabalho foi desenvolvido no Laboratório de Fitopatologia do Centro de Ciências Agrárias da Universidade Federal da Paraíba. Os conídios do fungo foram imersos nas soluções de extratos e avaliados quanto a germinação. Também foram plantados em meio de cultura BDA mais extratos, conídios e fragmentos miceliais (2mm de diâmetro) do fungo para se observar, respectivamente, o número de unidades formadoras de colónias (UFC) e o crescimento micelial. As sementes de amendoim, cv. BR-1 , antes ou após a inoculação de A. flavus, foram imersas nas soluções de extratos e, após, avaliadas quanto a incidência do fungo e qualidade fisiológica (Germinação e índice de Velocidade de Germinação - IVG). A análise estatística realizou-se segundo o delineamento inteiramente casualizado em arranjos fatorial, definidos conforme a natureza do ensaio. Os extratos empregados apresentaram atividade fungicida e/ou fungistáticas com relação a germinação de esporos, número de UFC e crescimento micelial, porém de forma diferenciada quanto a planta de onde foram obtidos e a concentração empregada. Os conídios imersos em extrato de canela a 10, 25, 50 e 100% e em extrato de nim comercial a 1 e 10% não germinaram e os menores números de UFC foram nos extratos de canela a 10, 25, 50 e 100%. O crescimento micelial foi nulo nas concentrações 50 e 100% de todos os extratos. Quanto ao tratamento das sementes, os produtos testados em relação à incidência de A. flavus exerceram mais efeito erradicantes, sendo também essas sementes as que apresentaram os maiores valores de germinação e IVG. / The fungus Aspergillus flavus cause deterioration of seeds and grains in storage and produce toxins that affect the human and animal health. Natural products as essential oils and plant extracts have been tested or applied instead fungicides to eliminate or reduce the negative effects of synthetics fungicides. This work was realized employing extracts of neem (Azadirachta indica) leaves and a commercial product, cinnamon (Cinnamomum zeylanicum) and apian (Allium sativum), in the proportions of 1, 10, 25. 50 and 100% aiming to evaluate "'in vitro" effects on the development of A. flavus and physiological quality of treated peanut seeds. The study was carried out in the Laboratory of Phytopathology/Federal University of Paraíba, Areia-Paraiba State-Brazil. The fungi conidia were immersed in the extracts solutions and observed to germination evaluation. Suspensions of conidia and discs of mycelium (2mm diameter) were planted in PotatoDextrose-Agar (PDA) aditioned of extracts solution to verify respectively the numbers of originated colonies (UFC) and mycelium growth. Peanut seeds cv. before and after inoculated with A. flavus conidia suspensions were treated with extracts solutions and evaluated about fungai incidence. and physiological qualities (germination and index of germination velocity - IVG). The statistical analyses were realized as an entirely randomized factorial design defined according the assays characteristics. The extracts shown fungicides and, or fungistatic effects to spores germination. numbers of UFC and mycelia growth but with variations according the origin (plant) and proportion of extract. Conidia immersed into cinnamon extract at the proportions of 10, 25, 50 and 100% and in neem commercial extract at the proportions of 1 and 10% did not germinate. AIso the cinnamon extract at the proportions of 10, 25, 50 and 100% when eddied to PDA cultural media reduced the numbers of UFC. The mycelia growth do not verified on PDA cultural media with the extracts at the proportions of 50 and 100%. Used in seed treatment the extracts presented more eradication effects than protective effects. The peanut seeds treated after the fungai inoculation presented the higher germination and IVG

Orientador: João Cláudio Thoméo / Banca: Vanildo Luiz del Bianchi / Banca: Vera Aparecida de Oliveira Tiera / Banca: Cecilia Cristina Marques dos Santos / Banca: Danila Comelis Bertolin / Resumo: Este trabalho teve como objetivo geral avaliar a viabilidade técnica da aplicação de biofungicidas extraídos de plantas no processamento de amendoim (Arachis hypogaea L.), de modo a reduzir a infestação pelo fungo Aspergillus flavus L. e possível contaminação por aflatoxina. Foi realizado na primeira etapa o estudo da contaminação fúngica e de aflatoxina, nas Safras 2011/12, 2012/13 e 2013/14, nas etapas da colheita e pós-colheita das vagens de amendoim Runner IAC 886 na região de Jaboticabal - SP, e a proposta da Análise de Perigos e Pontos Críticos de Controle (APPCC). Os resultados da umidade das vagens mostraram uma redução, em média, de 65,65% base seca (b.s.) no arranquio, para 7,17% b.s. no armazenamento. A contaminação fúngica foi maior na Safra 2013/2014 e os índices de aflatoxinas estavam abaixo do exigido pela legislação vigente. O plano APPCC apresentou os pontos de controle (PC) nas etapas do arranquio, secagem ao sol, colheita mecânica e transportes; e os Pontos Críticos de Controle(PCC), nas etapas de recepção, secagem e armazenamento, concluindo-se que a melhor etapa para a aplicação do biofungicida é antes da secagem artificial. A segunda parte consistiu na obtenção de biofungicidas nas formas de extratos (aquoso e alcoólico) e de óleo essencial das plantas: canela (Cinnamomum zeylanicum), cravo (Syzygium aromaticum), cidreira (Cymbopogon citratus), oregano (Origanum vulgare) e manjericão (Ocimum basilicum), e na avaliação destes quanto à inibição do fungo Aspergillus flavus L. e sintetização de aflatoxina. A eficiência de inibição dos biofungicidas sobre o Aspergillus flavus L. foi avaliada em meio Agar Dextrose Batata (BDA), pela técnica de inibição do crescimento micelial (ICM), concentração inibitória mínima (CIM) e por aplicações dos óleos mais eficientes nas vagens de amendoim contaminadas e armazenadas em umidades... / Abstract: This study aims to evaluate the technical feasibility of the application of biofungicides extracted from plants in the processing of peanut (Arachis hypogaea L.), with the objective of reducing infestation by fungus Aspergillus flavus L., and consequent contamination by aflatoxin, a chemical hazard. Firstly, we studied the fungal and aflatoxin contamination for the 2011/12, 2012/13, and 2013/14 crops, during the Runner IAC 886 peanut pods' harvesting and post-harvesting stages, in the region of Jaboticabal, Sao Paulo, Brazil, and proposed the Hazard Analysis and Critical Control Points (HACCP). The results of the pods' humidity showed a decrease, on average, of 65.65% d.b. on uprooting, to 7.17% d.b. on storage, and the fungal contamination was higher for 2013/2014 crops; the aflatoxin levels were below the required by current legislation. HACCP showed the control points (CP) during uprooting, sun drying, mechanical harvesting, and transportation; and the PCCs during stages of reception, drying, and storage proved the best stage for biofungicide application is before artificial drying. The second stage consisted of obtaining biofungicides as aqueous, alcoholic, and essential oil extracts from the plants: cinnamon (Cinnamomum zeylanicum), cloves (Syzygium aromaticum), lemon grass (Cymbopogon citratus), oregano (Origanum vulgare), and basil (Ocimum basilicum); then, we evaluated those in inhibiting the fungus Aspergillus flavus L. and synthesizing aflatoxin. The biofungicides' inhibiting efficiency on Aspergillus flavus L. was evaluated in Potato Dextrose Agar medium (PDA), by mycelial growth inhibition technique (MGI), by minimum inhibitory concentration (MIC), and by applying the most efficient oils on the contaminated peanut pods, stored in controlled humidity and room temperature, thus determining colony-forming units (CFU/g) and aflatoxins. The extracts' MGI results showed that there was no... / Doutor