Made available in DSpace on 2016-12-23T14:37:39Z (GMT). No. of bitstreams: 1 Carolina Oliveira Bernardes.pdf: 507333 bytes, checksum: f1bdcef3de907c50ccb23b8a69a35e0a (MD5) Previous issue date: 2009-12-10 / A soja é considerada um dos produtos de maior importância para a economia brasileira e dentre as pragas mais importantes, que limitam a sua produção, destaca-se Anticarsia gemmatalis. Visando reduzir a utilização de inseticidas no controle de pragas, têm-se buscado alternativas de controle, dentre as quais a utilização do controle biológico. Entre os agentes de controle biológico se destacam a bactéria entomopatogênica Bacillus thuringiensis (Bt) e os parasitoides de ovos do gênero Trichogramma. Este estudo avalia a interação desses dois agentes de controle biológico utilizados no controle de A. gemmatalis. Inicialmente, foram feitos testes de seleção com onze linhagens de Trichogramma, sendo a linhagem T. pretiosum 12, a que apresentou melhor desempenho para esta praga. Testes de patogenicidade e virulência foram realizados com 23 isolados de Bt, além do produto comercial Dipel® (B. thuringiensis var. kurstaki), para selecionar os mais agressivos para o inseto. Entretanto, os isolados 80, 997, 1054, 716 e 633 e o Dipel® foram os mais promissores. Por fim, foram realizados dois experimentos de interação entre o T. pretiosum 12, os isolados 80, 997, 1054, 716 e 633 e Dipel®. Foi observado que para o experimento em que os isolados foram misturados ao alimento houve uma interação positiva, em que os isolados 633 e 1054 causaram aumento no parasitismo. Com relação ao segundo experimento, foi observado que a imersão das cartelas com ovos de A. gemmatalis nas suspensões, contendo os diferentes isolados de Bt 80, 633, 716, 993, 1054 e o Dipel® reduziu o número total de ovos parasitados devido à repelência do T. pretiosum 12 aos ovos imersos em suspensão com Bt. Estudos histopatológicos da interação do Bt com as células do intestino médio de Trichogramma são necessários para elucidar como o Bt atua no inseto adulto. Esses estudos, aliados à caracterização molecular das toxinas presentes nos isolados, contribuirão para elucidar a interação parasitoide x entomopatógeno x hospedeiro / Soybean is considered one of the most important products to the brazilian economy and among the most important pests, which make its production limited, Anticarsia gemmatalis is detached. Aiming to reduce the use of insecticides on the pests control, alternatives of control have been searched, among these the use of biological control. Among the biological control agents the entomopathogenic bacterium Bacillus thuringiensis (Bt) and the egg parasitoids from the genus Trichogramma are detached. This study evaluates the interaction between these two biological control agents used on A. gemmatalis control. Initially selection tests were made with eleven Trichogramma strains and the strain T. pretiosum 12 was the one which presented best performance for this pest. Patogenicity and virulence tests were carried out with 23 Bt isolates and the commercial product Dipel® (B. thuringiensis var. kurstaki) to select the most virulent ones for the insect. This way the isolates 80, 997, 1054, 716, 633 and Dipel® were the most promising. With these results, two interaction experiments were performed among T. pretiosum 12, the isolates 80, 997, 1054, 716, 633 and Dipel®. It was observed that for the experiment in which the isolates were mixed to the food there was a positive interaction, where the isolates 633 and 1054 caused an increase on parasitism. Concerning the second experiment it was observed that the immersion of the cards containing A. gemmatalis eggs in the different Bt isolates 80, 633, 716, 993, 1054 and Dipel® reduced the total number of parasitized eggs due to repellence of T. pretiosum to the immersed eggs in Bt suspensions. Histopathologic studies of Bt interaction with T. pretiosum midgut cells are necessary to elucidate how Bt acts on the adult insect. These studies allied to molecular characterization of the toxins presented on the isolates will contribute to elucidate the interaction parasitoid x entomopathogen x host

Le groupe Bacillus cereus est composé de huit espèces de bactéries à Gram positif sporulantes qui peuvent coloniser plusieurs niches écologiques. Les espèces les plus importantes sont B. cereus, une bactérie ubiquitaire du sol et un pathogène opportuniste; B. thuringiensis, un entomopathogène très utilisé comme biopesticide; et B. anthracis l’agent de la maladie du charbon. Bien que ces espèces présentent différents phénotypes, elles sont étroitement liées génétiquement et leurs facteurs de virulences principaux sont portés par des plasmides. Le cycle infectieux de B. thuringiensis dans la larve d’insecte est régulé par l’activation séquentielle de systèmes de quorum sensing de la famille RNPP. Parmi eux, les systèmes Rap-Phr, caractérisés chez B. subtilis, ont très peu été étudiés dans le groupe B. cereus. Ces systèmes régulent divers processus bactériens importants dont la sporulation. L’objectif de cette étude est d’analyser les systèmes Rap-Phr dans le groupe B. cereus, pour connaitre leur distribution, leur localisation et leur diversité afin d’obtenir une vue globale de ces systèmes chez ces bactéries. De plus, leur possible implication dans la régulation du processus de sporulation a été prédite sur la base de données structurales décrites chez RapH de B. subtilis. Les gènes rap, toujours associés à un gène phr, sont présents dans toutes les souches étudiées avec une moyenne de six gènes rap-phr par souche et avec 30% de ces systèmes qui sont portés par des plasmides. Les souches de B. thuringiensis portent six fois plus de systèmes Rap-Phr plasmidiques que les souches de B. cereus. Par ailleurs, les souches phylogénétiquement proches possèdent un profil de gènes rap-phr similaire. Un tiers des protéines Rap sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Cette prédiction a été partiellement validée par des tests de sporulation suggérant que les résidus impliqués dans cette activité chez B. subtilis sont conservés mais insuffisants pour prédire cette fonction. Le système Rap63-Phr63 porté par le plasmide pAW63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. La protéine Rap63 a un effet modéré sur la sporulation et retarde l’expression des gènes régulés par Spo0A. La Rap63 est inhibée par son peptide Phr63, dont la forme mature correspond à l’extrémité C-terminale du pro-peptide. Les résultats de sporulation dans l’insecte suggèrent une activité synergique des systèmes Rap63-Phr63 et Rap8-Phr8 (porté par le pHT8_1) dans la régulation de la sporulation. Malgré la similarité entre les Phr63 et Phr8 aucun cross-talk n’a pu être mis en évidence, ce qui confirme la spécificité de ces systèmes de communication cellulaire. L’ensemble de ces résultats démontre la grande diversité des systèmes Rap-Phr dans le groupe B. cereus et souligne l’impact des systèmes plasmidiques dans le développement de ces bactéries. Par conséquent, les plasmides sont des éléments importants pour l’adaptation et la survie de ces bactéries et particulièrement pour B. thuringiensis. / The Bacillus cereus group of Gram positive spore forming bacteria is comprised by eight species that are able to colonize several ecological niches. The most important species are B. cereus, a ubiquitous soil bacterium and an opportunistic pathogen; B. thuringiensis, an entomopathogen widely used as biopesticide; and B. anthracis, the causative agent of anthrax. Even if they present different phenotypes, they are genetic closely related and their main virulence factors are encoded on plasmids. The infectious cycle of B. thuringiensis in the insect larvae is regulated by the sequential activation of quorum sensing systems from the RNPP family. Among them, the Rap-Phr was extensively studied in B. subtilis but just punctually in B. cereus group species. The Rap-Phr systems were shown to regulate various bacterial processes, including the sporulation. The objective of this study was to analyze the Rap-Phr systems in the B. cereus group, regarding their distribution, location and diversity to achieve an overview of these systems in these bacteria. Moreover, their possible involvement in the control of the sporulation process was predicted based on structural data described for RapH in B. subtilis. The rap genes, always associated with a phr gene, were present in all 49 studied strains with an average of six rap-phr genes per strain and 30% were located on plasmids. Comparison among B. cereus and B. thuringiensis strains revealed that the last one harbors six-fold more plasmid rap-phr system then the former. Moreover, phylogenetic closer strains possess a similar profile of rap-phr genes. Interestingly, 32% of the Rap proteins were predicted to inhibit sporulation and these proteins were preferentially located on plasmids and therefore in B. thuringiensis strains. This prediction was partially validated by sporulation efficiency assays suggesting that residues identified in B. subtilis as involved in the phosphatase activity are conserved but not sufficient to predict the sporulation function. Then, the plasmid-borne Rap63-Phr63 system from pAW63 plasmid of B. thuringiensis HD73 strain was further studied. The Rap63 protein moderately inhibits the sporulation and delays the expression of Spo0A-regulated genes. Rap63 is counteracted by its cognate Phr63 peptide, which mature form corresponds to the C-terminal end of the pro-peptide. Sporulation assays in insect larvae suggest a synergistic activity of Rap63-Phr63 and Rap8-Phr8 (from pHT8_1 of B. thuringiensis HD73 strain) systems on sporulation efficiency. Despite the similarities of Phr63 and Phr8 no cross-talk was found between these two systems, confirming their specificity. Altogether, these results reveal the high diversity of the Rap-Phr systems in the B. cereus group and highlight the relevance of the plasmid-borne systems to cell development. Therefore, the results demonstrated the importance of the plasmids in the adaptation and the survival of these bacteria, especially for B. thuringiensis.

Bacillus thuringiensis (Bt) est une bactérie produisant des inclusions protéiques cristallines à pouvoir insecticide et elle est largement exploitée à l'échelle industrielle. Dans cette étude, des souches de Bt ont été isolées du sol libanais. Nous avons étudié en premier la présence des principaux gènes cry1A codant pour des δ-endotoxines actives sur les lépidoptères. Les souches possédant ces gènes ont été testées pour leur toxicité sur des larves d'Ephestia kuehniella (E. kuehniella). Une souche nommée Lip, étant quatre fois plus toxique sur ces larves que la référence mondiale Bt subsp. kurstaki HD1, fut sélectionnée pour une étude plus approfondie. Après clonage et séquençage, nous avons identifié une nouvelle toxine de type Cry1Aa : Cry1Aa22 et une nouvelle variante de la toxine Cry1Ac. Ces dernières se sont montrées plus toxiques sur des larves d'E. kuehniella, et plus stables en présence des protéases intestinales de ces larves que Cry1Aa et Cry1Ac de HD1 permettant d'expliquer la toxicité élevée de la souche sauvage. D'autre part, nous avons optimisé la construction d'un modèle de membrane biomimétique incluant la membrane de la bordure en brosse intestinale (BBM) des larves d'E. kuehniella. Ces membranes nous ont servi à l'étude de l'interaction des toxines Cry1Aa et Cry1Ac de Lip et celles de HD1. Les toxines de Lip ont interagit différemment et avec une plus grande affinité avec ces modèles que celles de HD1.Tous ces résultats montrent que Lip est une souche intéressante pour une exploitation industrielle et que le modèle de membrane biomimétique est une alternative permettant la prédiction de l'affinité des toxines Cry. / Bacillus thuringiensis (Bt) is a bacterium that synthesizes insecticidal proteic crystallin inclusions and is widely used at an industrial scale. In this study, Bt strains were isolated from Lebanese soil. We studied the presence of the main cry1A genes encoding for δ-endotoxins active on Lepidoptera. Strains harboring these genes were tested for their toxicity against Ephestia kuehniella (E. kuehniella) larvae. The strain named Lip, being four folds more toxic to the larvae than the reference strain Bt subsp. kurstaki HD1, was selected for further study. We identified a novel Cry1Aa toxin, Cry1Aa22, and a variety of the Cry1Ac toxin after cloning and sequencing of the corresponding genes. These toxins were more toxic to E. kuehniella larvae and more stable in the presence of these larvae's intestinal midgut juice than Cry1Aa and Cry1Ac of HD1. Moreover, we optimized the construction of a biomimetic membrane model based on the intestinal brush border membrane (BBM) of E. kuehniella larvae. These models were used to study the interaction of Cry1Aa and Cry1Ac of Lip and HD1. Toxins of Lip interacted differently and with a greater affinity with these model membranes than toxins of HD1.These results show that Lip is an interesting Bt strain that could be exploited at an industrial scale. On another hand, the biomimetic membrane constructed in this study could be an alternative allowing the prediction of the Cry toxin's affinity.

A utilização do glicerol proveniente da fabricação do biodiesel como substrato para a obtenção de produtos biotecnológicos é uma alternativa promissora como forma de disposição adequada deste subproduto. O bioinseticida formulado com toxinas de Bacillus thuringiensis tem sido utilizado para o controle de insetos veiculadores de doenças, como a dengue, que atingem milhões de pessoas em todo o mundo. Para que este bioinseticida seja competitivo com os inseticidas químicos é necessário que o custo de sua produção seja diminuído, o que pode ser feito com a utilização de fontes de carbono alternativas, de forma a diminuir o custo do meio de fermentação. Neste trabalho, empregou-se o glicerol proveniente da fabricação do biodiesel de sebo bovino como componente do meio de fermentação para a produção de bioinseticida por Bacillus thuringiensis var. israelensis. Foram avaliados diferentes tratamentos do resíduo contendo glicerol, baseados em acidificação, decantação e aquecimento, para remoção de impurezas. O ajuste do pH até 7 pela adição de ácido fosfórico, seguido de decantação, aquecimento para remoção de metanol e nova decantação, foi a forma de tratamento escolhida, uma vez que proporcionou a maior atividade tóxica do meio fermentado contra larvas de Aedes aegypti. O meio de fermentação foi formulado determinando-se as concentrações de glicerol, extrato de levedura, sulfato de amônio e cloreto de cálcio que proporcionaram a maior atividade larvicida do meio fermentado, de acordo com planejamento fatorial 24. A toxicidade foi avaliada pela CL50 (concentração do complexo esporo-cristal necessária para matar 50% da população de larvas). O melhor valor de CL50 foi obtido com a utilização de glicerol a 10 g/L, extrato de levedura a 12 g/L, cloreto de cálcio a 0,24 g/L e sem adição de sulfato de amônio. A análise estatística dos dados confirmou a significância das variáveis glicerol, extrato de levedura e cloreto de cálcio sobre a atividade larvicida do meio fermentado. Cloreto de cálcio não influenciou significantemente a produção de esporos pela bactéria, e a taxa de esporulação não foi influenciada por cloreto de cálcio e sulfato de amônio nas concentrações estudadas. Para a otimização da produção de endotoxinas pela bactéria, foi realizado um planejamento fatorial 22, variando-se apenas as concentrações de extrato de levedura e de cloreto de cálcio, fixando-se a concentração de glicerol em 10 g/L e excluindo-se o sulfato de amônio do meio de fermentação. O melhor valor de CL50 (0,295 mg/mL) foi obtido no ensaio com 15 g/L de extrato de levedura e 0,4 g/L de cloreto de cálcio. / The use of glycerol derived from biodiesel production as a substrate for the acquisition of biotechnology products is a promising alternative as an adequate disposal mean of this by-product. The bioinsecticide formulated with toxins from Bacillus thuringiensis has been used to control vectors insects of diseases, such as dengue, which affect millions of people around the world. For this bioinsecticide be competitive with chemical insecticides is necessary a lower cost of its production, which can be achieved by the use of alternative carbon sources in order to reduce the fermentation medium cost. In this work, it was used glycerol from beef tallow biodiesel production as a component of the fermentation medium for the production of bioinsecticide by Bacillus thuringiensis var. israelensis. Different treatments of the residue containing glycerol, based on acidification, sedimentation and heating, to remove impurities, were evaluated. The adjustment of pH to 7 by addition of phosphoric acid, followed by decantation, then heating for removal of methanol, and a new decantation, was the treatment selected, since it provided the highest toxic activity of the fermented broth against Aedes aegypti larvae. The fermentation medium was formulated by determining the concentration of glycerol, yeast extract, ammonium sulfate and calcium chloride that provided the greatest larvicidal activity of the fermented broth according to 24 factorial design. The toxicity was measured by the LC50 (concentration of the spore-crystal complex required to kill 50% of the larvae). The best value of LC50 was obtained using glycerol at 10 g/L, yeast extract at 12 g/L, calcium chloride at 0.24 g/L and without addition of ammonium sulfate. Statistical analysis confirmed the significance of the variables glycerol, yeast extract and calcium chloride on the larvicidal activity of fermented broth. Calcium chloride did not influence significantly the production of spores by the bacteria and the sporulation ratio was not affected by calcium chloride and ammonium sulfate at the studied concentrations. For the endotoxins production optimization, a 22 factorial design was carried out, varying only the concentrations of yeast extract and calcium chloride, maintaining the concentration of glycerol at 10 g/L and excluding ammonium sulfate of the fermentation medium. The best value of LC50 (0,295 mg/mL) was obtained in the assay with 15 g/L of yeast extract and 0.4 g/L of calcium chloride.

A soja (Glycine max (L.) Merrill) com o evento DAS-81419-2 (ConkestaTM technology) que expressa as proteínas de Bacillus thuringiensis (Bt) Cry1F e Cry1Ac protege a cultura de danos de alimentação de lepidópteros-praga primários e secundários. Experimentos de campo foram conduzidos de 2011 à 2016 na região sul e central do Brasil para caracterizar a eficácia da soja DAS-81419-2 infestada com Anticarsia gemmatalis (Lepidoptera: Erebidae), Chrysodeixis includens (Lepidoptera: Noctuidae), Chloridea virescens (Lepidoptera: Noctuidae), Spodoptera cosmioides (Lepidoptera: Noctuidae), Elasmopalpus lignosellus (Lepidoptera: Pyralidae), Agrotis ipsilon (Lepidoptera: Noctuidae) e Helicoverpa armigera (Lepidoptera: Noctuidae) durante os estágios vegetativo (V4) e reprodutivo (R2 e/ou R4) da cultura. A eficácia da soja DAS-81419-2 foi comparada com a variedade isogênica não-Bt manejada com ou sem inseticidas foliares comerciais para controle de lepidópteros. DAS-81419-2 reduziu significantemente o dano de alimentação causado por A. gemmatalis, C. includens, C. virescens, S. cosmioides e H. armigera durante os estágios vegetativo e reprodutivo, bem como, a mortalidade de plântulas causada por E. lignosellus. Entretanto, DAS-81419-2 apresentou moderada atividade em A. ipsilon. Além disto, um estudo de campo de três anos avaliou os potenciais efeitos adversos de DAS-81419-2 por meio da inspeção da diversidade e abundância de artrópodes não-alvos comparado com a isolinha não-Bt com ou sem aplicações convencionais de inseticidas. Estes estudos foram conduzidos em três regiões produtoras de soja onde os artrópodes não-alvos da parte aérea e da superfície do solo foram monitorados. Em geral, a abundância e diversidade total não foram afetadas signifativamente pela soja Bt. Desta maneira, DAS-81419-2 mostrou eficácia consistente ao longo dos anos, localidades e diferente estágios da cultura contra lepidópteros-pragas primários e secundários de importância econômica da cultura da soja e não afetou adversamente os artrópodes não-alvos que ocorrem nos campos de soja. / Soybean (Glycine max (L.) Merrill) event DAS-81419-2 (ConkestaTM technology) expresses Bacillus thuringiensis (Bt) Cry1F and Cry1Ac proteins to provide protection against primary and secondary lepidopteran pest feeding damage. Field experiments were conducted from 2011 to 2016 in southern and central Brazil to characterize the efficacy of DAS-81419-2 soybean infested with Anticarsia gemmatalis (Lepidoptera: Erebidae), Chrysodeixis includens (Lepidoptera: Noctuidae), Chloridea virescens (Lepidoptera: Noctuidae), Spodoptera cosmioides (Lepidoptera: Noctuidae), Elasmopalpus lignosellus (Lepidoptera: Pyralidae), Agrotis ipsilon (Lepidoptera: Noctuidae), and Helicoverpa armigera (Lepidoptera: Noctuidae) during vegetative (V4) and reproductive (R2 and/or R4) crop developmental stages. The efficacy of DAS-81419-2 was compared to a non-Bt isogenic variety managed with or without applications of commercial foliar insecticides for lepidopteran control. DAS-81419-2 significantly reduced the feeding damage caused by A. gemmatalis, C. includens, C. virescens, S. cosmioides, and H. armigera during both vegetative and reproductive crop growth stages as well as the seedling stage plant mortality caused by E. lignosellus. However, DAS-81419-2 showed moderate activity on A. ipsilon. In addition, a 3-yr in-field study evaluated the potential adverse effects of DAS-81419-2 by examining the diversity and abundance of non-target arthropods (NTAs) compared to the non-Bt near isoline with and without conventional insecticide applications. These studies were conducted in three Brazilian soybean producing regions where both soil surface and aerial NTA were monitored. Overall, the total abundance and diversity were not significantly affected by Bt soybean. Therefore, DAS-81419-2 showed consistent efficacy across years, locations, and crop stages against the most economically important primary and secondary lepidopteran pests of soybean and did not adversely affect non-target arthropods occurring in soybean field.

Bactérias endofíticas provavelmente desenvolveram uma relação com planta hospedeira através de processos coevolucionários, podendo influenciar de alguma forma na fisiologia da planta. Na literatura, há muitos relatos sobre espécies de Bacillus endofíticos, no entanto, poucos se referem a espécie B. thuringiensis (Bt). Dessa forma, o objetivo desse trabalho foi isolar, identificar e caracterizar Bt de mandioca. Todos os isolados identificados como Bt produziram cristais pontuais e apenas um isolado, IIRAC22, produziu cristais amorfos maiores. Esse isolado apresentou genes cry4A, cry4B e cry10 e toxicidade a larvas de Aedes aegypti e Culex quinquefasciatus, evidenciando ser Bt var. israelensis. Todos os isolados produziram quitinase, sugerindo que elas podem ter ação controladora sobre fungos fitopatogênicos e insetos. Houve também produção de pectinase, enzimas envolvidas na penetração de planta hospedeira e estabelecimento como endófito. Esses Bt endofíticos podem levar ao desenvolvimento de produtos biotecnológicos para controle de insetos. / Endophytic bacteria probably developed a relationship with their plant host through coevoltionary processes what could influence in the physiology of the plant. In the literature, there are many reports about endophytic Bacillus species, however, few reports cite endophytic B. thuringiensis (Bt). Thus, the aim of the present work was to isolate, identify and characterize Bt from cassava. All the isolates identified as Bt were able to produce punctual crystals, but only one isolate, IIRAC22, produced large amorphous crystals. This isolate presented cry4A, cry4B and cry10 genes and toxicity against larvae of Aedes aegypti and Culex quinquefasciatus, evidencing to be Bt var. israelensis. All the isolates produced quitinase, suggesting that they can control phytopathogeni fungi and insects. There was also pectinase production, enzyme involved in the penetration of plant hosts and establishment as endophytes. These endophytic Bt can take to the development of biotechnological products for the control of insect.

Bt é uma bactéria formadora da proteína Cry1Ac, tóxica a lepidópteros. Plantas geneticamente modificadas expressam essa toxina. O objetivo deste trabalho foi isolamento e identificação de bactérias do solo de algodão transgênico e convencional potencialmente biodegradadoras dessa proteína. Estudou-se a dinâmica de crescimento das bactérias em meios com a proteína Cry1Ac ou glucose, a biodegradação, os genes apr, npr e sub. Em solo de algodão convencional a contagem foi menor; para algodão transgênico foi maior. Seis bactérias foram identificadas. O crescimento da bactéria B. pumilus foi maior em meio com a proteína Cry1Ac que glucose e a bactéria G. rubripertincta em meio contendo glucose teve o crescimento maior do que com a proteína Cry1Ac. Foi verificada a biodegradação da proteína pelas bactérias Bp e Gr. Os genes apr e sub foram detectados em solo de planta transgênica e o gene npr não. / Bacillus thuringiensis is a bacterium that forms the Cry1Ac protein, toxic to lepidopteran. Genetically modify plants expressing this toxin. The objective of this work was the isolation and identification bacteria from soil cultivated with transgenic cotton and conventional with potential degradation of this protein. We studied the dynamics growth from bacteria in media with the Cry1Ac protein or glucose, the biodegradation genes apr, npr and sub. In soil of conventional cotton, count was lower, for Bt cotton were higher. Six strains were identified. The growth bacterium B. pumilus was higher in medium with the Cry1Ac protein than bacteria with glucose and G. rubripertincta in medium containing glucose had higher growth than the Cry1Ac protein. Were verified Cry1Ac protein degradation by the bacteria Bp and Gr. Genes apr and sub were detected in soil with transgenic plant and the gene npr were not.

Neste trabalho foi feito um estudo para obtenção de formulações multiparticuladas, formadas por micropartículas de polímeros naturais, solúveis em água, não tóxicos e biodegradáveis. Os polímeros utilizados foram: Caseína, Hidroxietilcelulose (HEC), Hidroxipropilmetilcelulose (HPMC), Alginato de sódio e Quitosana. As técnicas utilizadas para obtenção das micropartículas foram o spray drying e a atomização de alginato de sódio em uma solução de CaCl2, para gelificação das gotículas formadas, com uma complexação ou não com quitosana. O estudo de micropartículas secas de alginato de cálcio e alginato de cálcio recoberta com uma membrana de quitosana revelou que diferentes procedimentos e variáveis de processo influenciavam nas características das micropartículas obtidas. O diâmetro médio das micropartículas de alginato e alginato-quitosana variou de 60 a 553 µm. A superfície de micropartículas com quitosana se mostrou mais rugosa, com uma grande quantidade de poros menores que 1 µm. As micropartículas com diâmetro médio de 60 µm apresentaram uma boa esfericidade e uma distribuição de tamanho de partícula uniforme. O teor de cálcio apresentou variações, diminuindo em processos com quitosana. A maioria das micropartículas de alginato e alginato-quitosana eram estáveis em água, mas instáveis em soro fisiológico e tampão fosfato 0,1M. Após a realização do estudo das características destas micropartículas foram incorporados a elas materiais com atividade bioinseticida (Bacillus thuringiensis var. krusfaki (Btk) e de Baculovirus (Bv)). As micropartículas obtidas com alginato de cálcio formaram pós contendo sistemas matriciais capazes de microencapsular e reter microrganismos entomopatogênicos, inclusive após redispersão em água. Os processos de microencapsulação de desenvolvidos e avaliados demonstraram ser adequados para a manutenção da viabilidade de Btk e da integridade dos poliedros de Bv, assim como, a conservação da capacidade bioinseticida destes microrganismos. As micropartículas obtidas com as misturas poliméricas Caseína/HEC e Caseína/HPMC pelo processo de spray drying revelaram-se inadequadas na medida que se dissolviam rapidamente depois de dispersas em água e não poderiam assim proteger o material bioinseticida no meio ambiente. / The aim of this work was to develop powder formulations containing microparticles, to be used as multiparticulate delivery systems. Two different methods were investigated : 1) the preparation of microparticles by spray drying using casein, hydroxyethylcellulose (HEC) and hydroxypropilmetilcellulose (HPMC) and 2) the preparation of calcium alginate and chitosan-alginate microparticles by using an atomizer device. Different experimental procedures to prepare calcium alginate and chitosanalginate microparticles were evaluated and variables beHeved to be important for the membrane formation were examined. The mean particles diarneter ranged from 60 to 553 µm. When a comparison was made between the surface morphology of calcium alginate and alginate-chitosan microparticles, remarkable roughness and more porous structure was observed in the chitosan-alginate microparticles. Some properties of the microparticles depended on the method and the procedure conditions of forming the chitosan-alginate complex. Calcium alginate and chitosan-alginate microparticles containing two different bioinsecticides were also prepared: 1) a spore/δ-endotoxinscomplex of Bacillus thuringiensis var.kurstaki (Btk) and 2) polyhedra of Baculovirus anticarsia (Bv), a viral insecticide. The results shown that the encapsulation of suspensions of Btk containing spore/toxin complex or polyhedra of B. anticarcia in calcium alginate and chitosan-alginate microparticles did not decrease the larvicidal activity of these biopesticides against lepidopterous pests. The formulations developed in this study remained unchanged, did not swell, did not release the spores (Btk) or the polyhedra (Bv), when dispersed in water and could be useful to be applied by aqueous spray as bioinsecticides in agriculture. On the other hand, casein, HEC and HPMC microparticles prepared by spray drying were not suitable to encapsulate bioinsecticides because they dissolved fastly after dispersion in water.

Les propriétés insecticides du Bacillus thuringiensis, découvert par ShigentaneIshiwatari, ont été utilisées pendant des décennies comme biopesticides et cette utilisation a augmenté rapidement en raison de préoccupations au sujet des effets environnementaux négatifs des pesticides chimiques. Actuellement, la toxine Bt dans la forme de biopesticides et des plantes transgéniques Bt peut compléter ou remplacer les pesticides chimiques. Il y a peu d’indication que la toxine Bt a un effet nocif pour l'environnement ou la santé humaine. Néanmoins, il ya des préoccupations que les cultures transgéniques commerciales peuvent avoir des effets néfastes sur l'environnement. Après son introduction dans le sol l'exsudation racinaire et la dégradation des résidus végétaux, la toxine Bt interagit avec les particules de sol. Les interactions de la toxine Bt avec des particules de sol influencent sa mobilité, sa biodisponibilité, sa persistance et sa toxicité.Dans cette étude, nous visons à établir l'importance relative des facteurs biologiques et physico-chimiques dans la détermination de la dynamique des protéines Cry détectables dans les sols, de clarifier si la protéine adsorbée conserve ses propriétés insecticides et d'identifier les propriétés du sol qui déterminent le devenir des protéines Cry dans le sol. Les résultats montrent que les protéines Cry ont une forte affinité sur la surface du sol. Cependant, il y avait peu de relation entre l'affinité pour le sol ou le rendement d'extraction et les propriétés du sol, y compris la teneur en argile, teneur en carbone organique et le pH du sol. Il y avait peu de rapport entre l'affinité et le rendement d'extraction. Les protéines diffèrent à la fois dans leur affinité pour les sols et leurs rendements d'extraction.Une évaluation du rôle du sol et des facteurs environnementaux dans le sort des protéines Cry de la formulation de biopesticides commerciale a montré un déclin rapide de la protéine Cry détectable soumise aux rayons du soleil sous la condition de laboratoire, alors que peu d'effet a été observé dans des conditions de terrain. La demi-vie des protéines dans le sol dans des conditions naturelles était d'environ 1 semaine. Des effets de la température forts ont été observés, mais ils diffèrent pour les biopesticides et la protéine purifiée, indiquant différentes étapes limitantes. Pour le biopesticide, la baisse observée était ralenties par des facteurs biologiques, y compris éventuellement sporulation. En revanche pour des protéines purifiées, augmentation de la température améliorée des changements conformationnels de la protéine adsorbée du sol, conduisant à une fixation et, par conséquent diminué efficacité d'extraction qui a diminué avec le temps. En outre, l'étude de la persistance de diverses protéines Cry dans les sols contrastés a été réalisée par immuno-détection et dosage biologique a montré que la toxine extractible diminue avec incubation allant jusqu'à quatre semaines. L'activité insecticide était toujours maintenue à l'état adsorbé, mais a disparue après deux semaines d'incubation à 25°C. La baisse de la protéine extractible et la toxicité était beaucoup plus faible à 4°C à 25°C. La stérilisation du sol n'a pas eu d'effet significatif sur la persistance de la toxine Cry indiquant que le déclin observé était provoqué par la fixation en fonction du temps de la protéine adsorbée ce qui diminue la quantité de toxine Cry extractable, la dégradation de la protéine par l’activité microbienne jouant un rôle plus mineur.L’exposition des insectes aux protéines Cry sous la forme adsorbé pourrait avoir un impact significatif sur les insectes cibles et même les insectes non cibles, et devrait être plus étudiée afin de déterminer son impact potentiel. / The insecticidal properties of Bacillus thuringiensis, discovered by Shigentane Ishiwatari, have been used for decades as biopesticides and this use has been increasing rapidly because of concerns about the negative environmental effects of chemical pesticides. Currently, Bt toxin in the form of both biopesticides and Bt transgenic plantsmay supplement or replace chemical pesticide. There is little evidence to demonstrate that Bt toxin has any harmful effect to the environment or to human health. Nevertheless, there are concerns that commercial transgenic crops may have harmful impacts on the environment. After release into soil via root exudation and breakdown of plant residues, Bt toxin interacts with soil particles. The interactions of Bt toxin with soil particles influence its mobility, its bioavailability, its persistence and its toxicity. In this study, we aim to establish the relative importance of biological and physicochemical factors in the determination of the dynamics of detectable Cry proteins in soils, to clarify if adsorbed protein maintains its insecticidal properties and to identify the soil properties that determine the fate of Cry proteins in soil. The results show that Cry proteins have strong affinity on soil surface. However, there was little relationship between affinity for soil or the extraction yield and soil properties including clay content, organic carbon content and soil pH. There was little relationship between the affinity and the extraction yield. The proteins differ in both their affinity for soil and their extraction yields.An assessment of role of soil and environmental factors in the fate of Cry protein from commercial biopesticide formulation showed a rapid decline of detectable Cry protein subjected to direct sunlight under the laboratory condition, whereas, little effect was observed under field conditions. The half-life of proteins in soil under natural conditions was about one week. Strong temperature effects were observed, but theydiffered for biopesticide and purified protein, indicating different limiting steps. For biopesticide, the observed decline was due to biological factors, possibly including sporulation. In contrast for purified proteins, increased temperature enhanced conformationalchanges of the soil-adsorbed protein, leading to fixation and hence extraction efficiency decreased that decreased with time. Moreover, the study of persistence of various Cry proteins in contrasting soils was carried out by immuno-detection and bioassay showed that extractable toxin decreased with incubation of up to four weeks. Insecticidal activity was still retained in the adsorbed state, but lost after two weeks of incubation at 25°C. The decline in extractable protein and toxicity was much lower at 4°C than 25°C. There was no significant effect of soil sterilization to persistence of Cry toxin indicating that decrease in detectable Cry toxin in soil may be time-dependent fixation of adsorbed protein as well as decreasing solubilization in larva midgut, but not microbial breakdown.Exposition to Cry in the adsorbed form could have a significant impact on target and even non target insects and should be investigation to determine the potential impact.

Made available in DSpace on 2017-07-25T19:29:56Z (GMT). No. of bitstreams: 1 Paulo Gallo.pdf: 1634093 bytes, checksum: f84f8b79e7d1355234216b134966f01a (MD5) Previous issue date: 2012-11-13 / The presence of Diabrotica speciosa larvae causes damage to corn root system and reduction in grain yield, mainly in second season crops. Applying insecticide in the seed furrow can be effective, but this assumes a form of preventive control and implies higher production costs and environmental impact. We evaluated the efficacy of genotypes AG7000RW and DKB330RW (Cry3Bb1) and their isolines to reduce the damage caused by D. speciosa larvae to corn root system, the efficacy of the insecticide fipronil applied in the furrow, and the effect of the interaction of both treatments on the yield of maize hybrids, as well as the planting date when the most severe damage occurs. The efficacy experiment was carried out in Itaberá, SP, in no-till planting system after oat crop. The damage to the roots was evaluated 52 days after emergence, using a visual score according to its intensity. Stalk lodging was determined by counting the plants leaning more than 25º in relation to its own axis at harvest. Genotypes AG7000 and DKB330 with no insecticide application presented damage and the resulting scores were 1.62 and 1.85, whereas the scores of their Bt isolines were 0.25 and 0.24, respectively. Genotypes AG7000 and AG700RW had the highest percentage of matter and amount of dry root in relation to DKB330 and DKB330RW, regardless of insecticide application. Stalk lodging of 46.7% found in non-Bt genotype DKB330 with no insecticide application reflected negatively on the yield of this treatment. In contrast, with insecticide application, stalk lodging ranged from 0.2% to 2.2% between genotypes, not influencing their yield. Cry3Bb1 protein was effective in reducing the damage to the root regardless of the genotype tested. Genotypes with high yield potential and lower root mass may present higher yield response when protected by the toxin or insecticide in the presence of a large larval population. In the 2010/2011 season, we assessed the damage in non-Bt corn root at four planting dates in Itaberá (10/14, 11/3, 11/25, and 12/20) and three in Castro, PR (9/29, 10/21, and 11/11). In the following season, 2011/2012, we evaluated six planting dates in Itaberá (9/21, 10/18, 11/21, 12/28, 1/30, and 2/20) and four in Castro (9/20, 10/10, 10/30, and 11/20). We observed that, from the second planting date on, the root damage reached scores over 3.0 and remained above 4.0 in all other planting dates in Itaberá in both seasons. In Castro, only the seeding performed in 11/20/2011 reached a score above 3.0. The use of protection in corn root, by applying insecticide or toxin, presented a greater response in sowing carried out from October on in the region of Itaberá. / A presença de larva de Diabrotica speciosa danifica o sistema radicular do milho e causa redução na produtividade de grãos, principalmente nas lavouras de segunda safra. A aplicação de inseticida no sulco de semeadura pode ser eficaz, porém, esta forma de controle assume caráter preventivo e implica maior custo de produção e impacto ambiental. Avaliaram-se a eficácia dos genótipos AG7000RW e DKB330RW (Cry3Bb1) e de suas isolíneas na redução do dano da larva de D. speciosa na raiz do milho, a eficácia do inseticida fipronil aplicado no sulco de semeadura e o efeito da interação de ambos os tratamentos sobre a produtividade dos genóti os de milho, bem como a época de semeadura em que ocorre maior dano da larva. O experimento de eficácia foi instalado em Itaberá, SP, na safra 2011/2012, sob sistema de plantio direto na palha após cultura de aveia. Os danos nas raízes foram avaliados aos 52 dias após a emergência, atribuindo-se nota visual de acordo com sua intensidade. O acamamento foi determinado contando-se as plantas com inclinação maior que 25º em relação ao próprio eixo no momento da colheita. Os genótipos AG7000 e DKB330 sem inseticida apresentaram dano, e as notas resultantes foram 1,62 e 1,85, enquanto as notas de suas isolíneas Bt foram 0,25 e 0,24, respectivamente. Os genótipos AG7000 e AG700RW obtiveram os maiores percentuais de matéria e quantidade de massa seca na raiz em relação a DKB330 e DKB330RW, independentemente da aplicação do inseticida. O acamamento de 46,7% encontrado para o genótipo DKB330 sem aplicação de inseticida refletiu negativamente na produtividade deste tratamento. Por outro lado, com a aplicação de inseticida, o acamamento variou de 0,2% a 2,2% entre os genótipos, não influenciando sua produtividade. A proteína Cry3Bb1 foi eficaz na redução dos danos na raiz independentemente do genótipo testado. Genótipos com alto potencial produtivo e com menor massa de raiz podem apresentar maior resposta de produtividade quando protegidos com a toxina ou o inseticida na presença de alta população de larvas. Na safra 2010/2011, avaliaram-se os danos na raiz do milho não Bt em quatro épocas de semeadura em Itaberá (14/10, 3/11, 25/11 e 20/12) e três em Castro, PR (29/9, 21/10 e 11/11). Na safra seguinte, de 2011/2012, avaliaram-se seis épocas em Itaberá (21/9, 18/10, 21/11, 28/12, 30/1 e 20/2) e quatro em Castro (20/9, 10/10, 30/10 e 20/11). Constatou-se que, a partir da segunda época, os danos na raiz atingiram nota superior a 3,0 e permaneceram acima de 4,0 em todas as demais épocas de semeadura em Itaberá, nas duas safras avaliadas. Em Castro, apenas a semeadura de 20/11/2011 atingiu nota superior a 3,0. A utilização da proteção na raiz do milho, pela utilização de inseticida ou toxina, apresentou maior resposta nas semeaduras realizadas a partir do mês de outubro na região de Itaberá.