The major digestive enzymes of larval Diabrotica undecimpunctata howardi, the southern corn rcotworm (SCR), have been investigated. A number of proteases have been identified, the majority cysteine proteases, some aspartic acid proteases are also present. In vitro assays of cysteine proteases showed that almost all activity could be arrested by E64 or chicken egg-white cystatin. This activity was also affected by TLCK, CpTI and thaumatin. Two inhibitory activities were demonstrated in protein extracts from rice seed. The first, oryzacystatin-I, caused marked inhibition of both the insect cysteine proteases and papain. The second produced strong inhibition of insect cysteine proteases but left papain virtually unaffected. Amino acid sequence data for this novel inhibitor was obtained, and significant homology demonstrated to the rice allergenic proteins (Izumi et a].,1992; Adachi et al., 1993). Oryzacystatin-I was expressed as a fully functional fusion protein (Rozc) in Escherchia coli, isolated, characterised and employed in feeding trials with larval SCR, a significant rise in mortality was demonstrated. Other protease inhibitors were also assayed in vivo, but none exhibited the efficacy of Rozc. A single iso-form of a-araylase was identified and characterised. In vitro assays with amylase inhibitors demonstrated the effectiveness of WAAI and the weak effect of BAAI. WAAI, employed in bioassay, produced a significant decrease in survival. Five lectins were tested by bioassay. WGA and GNA caused marked alterations in larval development, GNA was most effective. Saporin caused little effect when incorporated into artificial diet. WAAI, CpTI and GNA were employed in combination bioassays. An enhanced level of effectiveness was demonstrated with the double and triple combinations assayed. While further work is necessary, especially assaying protein efficacy in planta and establishing mechanisms of action, this project has successfully demonstrated the clear potential of plant antimetabolic proteins for use in the enhancement of inherent resistance of crop plants to insects, and of employing a number of proteins in a multi-mechanistic defence.

No description available.

The environmental conditions created in the Southwestern deserts of the United States are conducive to the production of green microalgae biomass, for use as a feedstock intended for conversion to carbon neutral liquid biodiesel. One promising heat-tolerant, rapidly-growing, high lipid content species is the chlorophyte, Chlorella sorokiniana (Shihira and Krauss, 1965) (isolate DOE 1412), which has been selected for pilot-scale production as part of a larger algal biofuels project to assess its potential for long-term productivity in open, outdoor monoculture production systems. Molecular analysis exposed the presence of the pathogenic bacterium, Vampirovibrio chlorellavorus (Gromov & Mamkaeva, 1972) causing infection and death of DOE 1412, which occurred most rapidly at air temperatures exceeding 34 °C. This Gram-negative bacterium has been reported to attach to and utilize the cellular contents of several Chlorella species, leading to yellowing and flocculation of algal cells, and death of the host. A quantitative PCR assay was developed to monitor pathogen accumulation using the 16S ribosomal RNA gene, in addition to the algal 18S ribosomal RNA gene for normalization. The assay is highly sensitive, with limits of quantification for the 16S and 18S gene targets calculated to be 19 and 131 copies, respectively. The qPCR assay was used to monitor several outdoor reactors inoculated with the DOE1412, throughout the warm season growth-to-harvest cycle to understand the disease cycle and inform disease management decisions. Further, the bacterium was monitored in paddlewheel DOE 1412 cultures treated with benzalkonium chloride (BAC), a biocide tested for the ability V. chlorellavorus attack of DOE 1412. The treatment resulted in a reduced growth rate for DOE 1412, but prolonged the duration of the production cycle resulting in increased total harvestable yield, compared to untreated control cultures.

The field slug Deroceras reticulatum is a major pest in UK agriculture and amidst growing concern and regulatory pressures surrounding chemical molluscicides, innovation is required to advance the current repertoire of slug controls. This study set out to investigate the bacteria associated with D. reticulatum to assess their importance to the slug and potential as a target for biological control. Slug gut bacterial isolates identified using the phenotypical API system (BioMérieux) and 16S rRNA gene sequencing, were mainly soil-dwelling organisms of the phyla Proteobacteria and Bacteriodetes some of which may be important in human or plant disease. A ribosomal intergenic spacer analysis (RISA) was developed to study microbial communities in the slug gut. Slugs had an average species richness of 12 and comparing the bacterial communities in slugs from different locations yielded a mean similarity of 0.159 (Jaccard index) which was significantly lower than similarity indices of slugs collected within a single location (Mean Jaccard index 0.205, p<0.001, ANOVA). Cloning and sequencing of RISA bands common to slugs and slug eggs, but absent from the surrounding soil and plants identified bacteria for future investigation as potential beneficial symbionts. Bacteria extracted from the slug gut were tested for sensitivity to 16 antibiotics and greatest inhibition of growth was observed for chloramphenicol, gentamicin and tetracycline. These antibiotics administered to slugs by feeding and injection caused a reduction in gut-associated bacteria in plate counts, and in bacterial 16S rDNA quantities estimated by real-time quantitative PCR. Field collected D. reticulatum has a large transient gut bacterial population which is reduced upon starvation to a low background level. No significant detrimental effect of antibiotic treatment on the fitness and survival of the slugs was seen, in some instances control slugs suffered greater mortality than slugs that had been injected with antibiotic. Slugs that died during bioassays had a significantly greater amount of bacterial 16S rDNA in their gut than slugs that were sacrificed as healthy individuals suggesting the presence of a bacterial pathogen. This study has found little evidence that a bacterial symbiont may exist and be important for optimal fitness and survival of D. reticulatum, but insight into slug associated bacteria will be valuable in the direction of future studies in this field.

Made available in DSpace on 2014-08-20T14:33:02Z (GMT). No. of bitstreams: 1 dissertacao_volnei_knopp_zibetti.pdf: 3412606 bytes, checksum: a6c4bfae28f662171d613db6bd7e1608 (MD5) Previous issue date: 2013-02-27 / The earthworm breeding corresponds to the creation of earthworm for different purposes like producing arrays and cocoons and worms for the production of vermicompost. The vermicompost, widely used as a fertilizer in agriculture and family farming systems ecologically based, also has important role as fitoprotetor. The study aimed to produce vermicompost of Eisenia andrei Bouché from cattle manure (CM) and combinations of this with peanut husk (PH), as structural materials, and spent coffee grounds (SCG), nutritional qualities of the source, and its evaluation in liquid and aerated form in suppressiveness Sclerotium rolfsii Sacc. The work was divided into two stages. At first, vermicompost produced up to four treatments and six replications: H1 CM 100%); H2 CM 75% + PH 25%; H3 CM 75% + SCG 25%; H4 CM 50% + SCG 25% + PH 25%. We evaluated the biomass of earthworms, cocoon production, yield and vermicompost microbiological and chemical variables. Treatments H3 and H4 had higher biomass and cocoon production. When assessed the density of bacterial colonies and fungal treatments H2 and H4 registered the highest rates. In the second step, vermicompost tea aerated were prepared from different combinations of vermicompost, in order to test their antagonistic effect against S. rolfsii. The vermicompost were packed in sachets and immersed in water and aerated for a period of 24 hours. Were quantified bacterial and fungal colonies present in vermicompost tea (VT), in selective culture media. Representative samples of VT obtained from each of the four treatments were evaluated on mycelial growth of S. rolfsii from mycelium and sclerotic under two conditions: unfiltered and filtered. The VT filtered did not inhibit mycelial growth of S. rolfsii, as opposed to VT unfiltered. Colonies of microorganisms coming from the unfiltered treatments, once isolated and purified, were paired with discs of mycelium of S. rolfsii, to evaluate the effect of antagonistic fungi and bacteria present in vermicompost on the sclerotic. The F3 isolated, on VT on treatment H2, identified as belonging to the genus Trichoderma, was the one who showed the greatest inhibition to S. rolfsii, among other microorganisms matched. The production and use of vermicompost in family farming should be encouraged, because besides the already established benefits of fertilization on cropping systems can also aid in the control of plant diseases. / A minhocultura corresponde à criação de minhocas destinadas a diferentes fins, como produção de matrizes e casulos, e minhocas destinadas a produção de húmus. O húmus de minhoca, amplamente utilizado como fertilizante na agricultura familiar e sistemas agrícolas de base ecológica, também tem relevante papel como fitoprotetor. O estudo teve por objetivo produzir húmus de minhoca da espécie Eisenia andrei Bouché a partir de esterco bovino (EB) e combinações deste com cascas de amendoim (CA), como material estruturante, e borra de café (BC), na qualidade de fonte nutricional, e sua avaliação na forma líquida e aerada na supressividade de Sclerotium rolfsii Sacc. O trabalho foi dividido em duas etapas. Na primeira, produziu-se húmus em quatro tratamentos e seis repetições, sendo: H1 EB 100%; H2 EB 75% + CA 25%; H3 EB 75% + BC 25%; H4 EB 50% + BC 25% + CA 25%. Avaliou-se a biomassa de minhocas, produção de casulos, rendimento de húmus e variáveis microbiológicas e químicas. Os tratamentos H3 e H4 apresentaram maiores índices de biomassa e produção de casulos. Quando avaliadas as densidades de colônias bacterianas e fúngicas, os tratamentos H2 e H4 registraram os maiores índices. Na segunda etapa, foram preparados húmus líquidos aerados a partir das diferentes combinações de húmus, com o objetivo de testar seus efeitos sobre escleródios de S. rolfsii. Os húmus foram acondicionados em sachês e mergulhados em água, sendo aerados pelo período de 24 horas. Foram quantificadas as colônias bacterianas e fúngicas presentes nos húmus líquidos (HL), em meios de cultura seletivos. Amostras representativas de HL obtidos de cada um dos quatro tratamentos foram avaliadas no crescimento micelial de S. rolfsii, a partir de micélio e escleródio, em duas condições: filtradas e não filtradas. Os HL filtrados não inibiram o crescimento micelial de S. rolfsii, em oposição aos HL não filtrados. As colônias de microrganismos oriundas dos tratamentos não filtrados, uma vez isoladas e purificadas, foram pareadas com discos de micélio de S. rolfsii, objetivando avaliar o efeito antagônico de fungos e bactérias presentes nos húmus sobre o escleródio. O isolado F3, do HL oriundo do tratamento H2, identificado como pertencente ao gênero Trichoderma, foi quem apresentou a maior inibição ao S. rolfsii, dentre os demais microrganismos pareados. A produção e o uso de húmus de minhoca na agricultura familiar devem ser estimulados, pois além dos benefícios já consagrados acerca da fertilização em sistemas de cultivo também podem auxiliar no controle de doenças de plantas.

L’utilisation non raisonnée d’insecticides a des conséquences sur l’environnement et la santé humaine mais aussi sur le développement de résistances chez les insectes nuisibles. Dans ce contexte, ce travail de thèse porte sur le développement d'une technique de nanoencapsulation d'un agent synergisant chimique, la deltaméthrine (pyréthrinoide), associé un pro-insecticide l’indoxacarbe (oxadiazine). Sur la base d’études in vitro sur cellules neurosecrétrices de blattes (Periplaneta americana), une action synergique entre la deltaméthrine et le DCJW (métabolite actif de l’indoxacarbe) a été démontrée via un mécanisme intracellulaire original dépendant du calcium. Des études toxicologiques in vivo sur des blattes adultes ont permis de confirmer l’effet synergique entre la deltaméthrine et l’indoxacarbe. Pour optimiser la formulation de l’association de ces composés, des nanocapsules lipidiques contenant la deltaméthrine utilisée comme agent synergisant ont été produites. Les études de toxicité sur blattes ont permis de déterminer les doses effectives les plus faibles de l’association deltaméthrine nanoencapsulée/indoxacarbe et d’obtenir une optimisation de l’effet synergique avec la deltaméthrine nanoencapsulée. Cet effet synergique est plus important que celui du piperonyl butoxyde, composé utilisé dans les formulations d'insecticides classiques. Les résultats indiquent que la deltaméthrine, protégée des estérases par la nanoencapsulation, permet d’optimiser l’efficacité du traitement tout en réduisant les doses d’indoxacarbe. Cette nouvelle stratégie est une première étape dans le développement d'une formulation phytosanitaire efficace contre les insectes nuisibles. / The over-use of pesticides has represented a concern not only for its consequences against the environment but also for the increase in resistance mechanisms in pest insects. In this context, our research project is focused on the development of a nanoencapsulation technique of a deltamethrin (pyrethroid), used as synergistic agent, combined with a pro-insecticide indoxacarb (oxadiazine). Based on in vitro studies performed on cockroach (Periplaneta americana) neurosecretory cells, synergistic effect between deltamethrin and DCJW (active metabolite of indoxacarb) has been characterized, occurring through an original calcium-dependent intracellular mechanism. In vivo toxicological studies on adult cockroaches have confirmed these previous results. To optimize the formulation including the synergistic agent and indoxacarb, lipid nanocapsules (LNCs) containing deltamethrin have been developed. It has been possible to determine the lower effective doses of LNC-deltamethrin/indoxacarb mixture producing the synergistic effect in whole insects. The synergism obtained is more important than that of obtained with piperonil butoxide (PBO), the well-known synergist used in classical insecticide formulations. The results also indicate that LNC-deltamethrin, protected from esterase-induced detoxification enhances the toxicity of indoxacarb while reducing doses. This novel strategy is a first step for the development of a novel formulation more efficient against pest insects.

Tesis por compendio / [ES] Los péptidos antimicrobianos (AMP) son una alternativa prometedora para el desarrollo de nuevos antifúngicos que puedan sustituir a los fungicidas usados en agricultura. Sin embargo, el alto coste de la síntesis química y la dificultad para su producción a gran escala han limitado su aplicación. Las proteínas antifúngicas (AFP) son AMP naturales, pequeñas, catiónicas, secretadas y ricas en cisteína con gran potencial para el control de hongos fitopatógenos. Las AFPs se encuentran en hongos filamentosos, son estables y pueden producirse en grandes cantidades. Sin embargo, el papel biológico en su hongo productor no se conoce en profundidad. En esta tesis, se estudió la diversidad de AFPs en genomas de hongos ascomicetos y se propuso una nueva clasificación en tres clases (A, B y C). Penicillium digitatum es el principal patógeno postcosecha de cítricos y codifica solo una AFP en su genoma de clase B (AfpB), mientras que Penicillium expansum, el principal patógeno postcosecha de manzana, codifica una AFP de cada clase (AfpA, AfpB y AfpC). En este trabajo describimos la producción biotecnológica y la caracterización de estas cuatro AFPs. Se ha caracterizado el papel biológico del gen afpB en P. digitatum mediante estudios de expresión génica y generación de mutantes nulos y de expresión constitutiva. Los resultados indicaron que afpB es prescindible para la biología y el ciclo vital del hongo, aunque la expresión del gen afpB bajo el promotor constitutivo gpdA de Aspergillus nidulans es perjudicial para su crecimiento y virulencia. Sorprendentemente, ni la cepa parental ni las cepas constitutivas produjeron cantidades detectables de AfpB a pesar de la alta expresión del gen codificante. El modelado molecular y el diseño racional permitieron predecir la estructura terciaria de AfpB y diseñar péptidos sintéticos para mapear motivos antifúngicos en su secuencia primaria. Confirmamos que los bucles catiónicos L2 y L3 mostraron actividad antifúngica moderada y que pueden actuar sinergísticamente. Con el objetivo de producir AfpB mediante biotecnología, usamos un casete de expresión de AFPs basado en las regiones promotora y terminadora del gen paf de Penicillium chrysogenum, hongo que produce naturalmente grandes cantidades de su propia proteína PAF. Este casete funcionó en P. digitatum y permitió la producción homóloga de AfpB. Los datos también mostraron que las secuencias del péptido señal (SP) y el pro-péptido de la SP-Pro-AfpB no determinan la producción de proteína. También demostramos la estabilidad térmica y la resistencia a la proteólisis de AfpB, y aportamos datos que sugieren que la estructura terciaria no es necesaria para la actividad antifúngica. Similar a lo descrito en P. digitatum, ninguna de las tres AFPs se detectó en los sobrenadantes de cultivo en medio rico de P. expansum. Sin embargo, AfpA se produjo en grandes cantidades en cultivos de medio mínimo de P. expansum. Para completar el repertorio de AFPs, produjimos las tres AFPs de P. expansum (AfpA, AfpB y AfpC) en P. chrysogenum con el casete paf. Las tres proteínas de P. expansum se produjeron, purificaron y caracterizaron con éxito. Ninguna de las AFPs producidas en este trabajo fue citotóxica frente a eritrocitos de mamíferos. AfpA de P. expansum seguida de AfpB de P. digitatum fueron las AFPs más activas contra hongos filamentosos, incluyendo patógenos de plantas y humanos, productores de micotoxinas y sus propios hongos productores, una característica previamente no descrita en las AFPs. Además, la AfpA de P. expansum y la AfpB de P. digitatum protegieron frente a la infección causada por el hongo Botrytis cinerea en plantas de tomate, y AfpA de P. expansum protegió frente a P. digitatum en frutos de naranja. Estos resultados confirman nuestra hipótesis de que las AFPs son buenas candidatas para el desarrollo de nuevos antifúngicos en protección vegetal y conservación postcosecha, pero ta / [CA] Els pèptids antimicrobians (AMP) són una alternativa prometedora per al desenvolupament de nous antifúngics que puguen substituir als fungicides utilitzats en agricultura. No obstant això, l'alt cost de la síntesi química i la dificultat per a la producció biotecnològica a gran escala han limitat la seua aplicació. Les proteïnes antifúngiques (AFP) són AMPs naturals, xicotetes, catiòniques, secretades i riques en cisteína que oferixen un gran potencial per al control de fongs fitopatogens. Les AFPs estan presents de en fongs filamentosos, són molt estables i poden produir-se en grans quantitats. No obstant això, el paper biològic d'estes AFPs en el seu fong productor encara no està clar. En esta tesi es va estudiar la diversitat d'AFPs en genomes de fongs ascomicets i es va proposar una nova classificació en tres clases (A, B i C). Penicillium digitatum, el principal patogen postcollita de cítrics, codifica només una AFP en el seu genoma de classe B (AfpB). Penicillium expansum, el principal patogen postcollita de poma, codifica una AFP de cada classe (AfpA, AfpB i AfpC). En este treball presentem la producció biotecnològica i la caracterització d'estes quatre AFPs. Hem caracteritzat el paper biològic del gen afpB en P. digitatum mitjançant estudis d'expressió gènica i la generació de mutants nuls i d'expressió constitutiva. Els resultats van indicar que afpB és prescindible per a la biologia i el cicle de vida d'este fong, encara que l'expressió del gen afpB davall el promotor constitutiu gpdA d'Aspergillus nidulans és perjudicial per al seu creixement i virulència sobre fruits cítrics. Sorprenentment, ni el cep parental ni els ceps constitutius van produir quantitats detectables d'AfpB malgrat l'alta expressió del gen afpB. El modelatge molecular i el disseny racional van permetre predir l'estructura terciària d'AfpB i dissenyar pèptids sintètics per a identificar motius antifúngics dins de la seqüència primària. Confirmarem que les estructures catiòniques L2 i L3 mostren activitat antifúngica i que poden actuar de forma sinèrgica. Amb l'objectiu de la producció biotecnològica d'AfpB, utilitzarem un casset d'expressió d'AFPs basat en les regions promotora i terminadora del gen paf de Penicillium chrysogenum, el qual produïx naturalment grans quantitats de la seua pròpia proteïna PAF. Este casset va funcionar en P. digitatum i va permetre la producció homòloga d'AfpB. Les dades també van mostrar que les seqüències del pèptid señal (SP) i el propèptid de la SP-Pro-AfpB no determinaren la producció de proteïna. També demostrarem l'extrema estabilitat tèrmica i la resistència proteolítica d'AfpB, i proporcionem dades que suggerixen que l'estructura terciària no és necessària per a l'activitat antifúngica. Semblant a P. digitatum, cap de les tres AFPs es van detectar en els sobrenadants de medi de cultiu ric de P. expansum. Al contrari, AfpA es va produir en grans quantitats en cultius de P. expansum en medi mínim. Per a completar el repertori d'AFPs, vam produir les tres AFPs de P. expansum (AfpA, AfpB i AfpC) en P. chrysogenum mitjançant l'ús del casset paf. Així, les tres proteïnes de P. expansum es van produir, purificar i caracteritzar amb èxit. Cap de les AFPs produïdes en este treball va ser citotóxica front eritròcits de mamífer. AfpA de P. expansum seguida d'AfpB de P. digitatum van ser les AFPs més actives contra fongs filamentosos, incloent patògens de plantes i humans, productors de micotoxines i els seus propis productors, una característica prèviament no descrita per a les AFPs. A més, AfpA de P. expansum i AfpB de P. digitatum van protegir front la infecció causada pel fong Botrytis cinerea en plantes de tomaca, i l'AfpA de P. expansum va protegir front P. digitatum en fruits de taronja. Estos resultats confirmen la nostra hipòtesi anterior de que les AFPs són bones candidates per al desenvolupament d'antifúngics en protecció / [EN] Antimicrobial peptides (AMPs) are promising antifungal alternatives to the fungicides used in agriculture. However, the high cost of chemical synthesis and the difficulties of large-scale production have limited their application. Antifungal proteins (AFPs) are a group of natural, small, cationic, secreted, cysteine-rich AMPs that offer a great potential to develop new biomolecules for the control of phytopathogenic fungi. AFPs are naturally present in filamentous fungi, are very stable, and can be produced in large amounts. However, the biological role of these AFPs in their producer fungus is still unclear. In this thesis, we first studied the diversity of AFPs in ascomycetous genomes and proposed a new classification in three different classes (A, B and C). Penicillium digitatum is the main citrus postharvest pathogen and encodes only one AFP from class B in its genome (AfpB), while Penicillium expansum is the main pome postharvest pathogen and encodes one AFP from each class (AfpA, AfpB and AfpC). In this work, we report the identification, efficient biotechnological production and characterization of these four AFPs. We characterized the biological role of the afpB gene in P. digitatum by the study of its gene expression pattern and the generation of null and constitutive expression mutants. Results indicated that afpB is dispensable for the biology and life cycle of this fungus, although expression of the afpB gene under the constitutive Aspergillus nidulans gpdA promoter is detrimental to growth and virulence to citrus. Surprisingly, neither the wild type nor the constitutive strains produced detectable amounts of AfpB in spite of the high afpB gene expression. Molecular modeling and rational design allowed us to predict the AfpB tertiary structure and design synthetic peptides to map antifungal motifs within the AfpB primary sequence. We confirmed that the cationic exposed loops L2 and L3 showed moderate antifungal activity and that they can act synergistically. With the objective of the biotechnological production of AfpB, we used an AFP expression cassette based on the promoter and terminator regions of the well-studied paf gene from Penicillium chrysogenum, which naturally produces high amounts of its own protein PAF. This paf cassette worked efficiently in P. digitatum and allowed the homologous production of AfpB. Data also showed that the signal peptide (SP) and pro-peptide sequences of the translated SP-Pro-AfpB do not determine protein production. We also demonstrated the thermal stability and resistance to proteolytic cleavage of the P. digitatum AfpB, and provided data that suggest that tertiary structure is not required for antifungal activity. Similar to P. digitatum, none of the three AFPs were detected in supernatants of cultures of P. expansum in rich medium. By contrast, AfpA was produced with very high yields in P. expansum cultures in minimal medium. To complete the repertoire of AFPs from P. expansum we produced the three AFPs from P. expansum (AfpA, AfpB and AfpC) in P. chrysogenum with the use of the paf cassette. With this combined approach, the three P. expansum proteins were successfully produced, purified and characterized. None of the four AFPs produced in this work were cytotoxic against mammal erythrocytes. The P. expansum AfpA followed by the P. digitatum AfpB were the most active AFPs against filamentous fungi, including plant and human pathogens, mycotoxin-producer fungi, and their own producers, a feature that had not been previously described for AFPs. Moreover, AfpA from P. expansum and AfpB from P. digitatum protected against fungal infection caused by Botrytis cinerea in tomato plants, and additionally the P. expansum AfpA protected against P. digitatum in orange fruits. These results confirm our previous hypothesis that AFPs are good candidates for the development of antifungals in plant protection and postharvest conservation, but also in clinic or food preservation. / Garrigues Cubells, SM. (2018). Producción y utilización Biotecnológica de nuevas proteínas antifúngicas de hongos filamentosos [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113162 / Compendio

A teca (Tectona grandis) é uma das principais espécies madeireiras do mundo, com alto valor econômico, muito famosa por sua beleza, resistência e durabilidade. A espécie ocorre naturalmente na Índia, Mianmar, Tailândia, Laos e Indonésia, onde estudos de diversidade têm sido realizados no que tange à conservação de recursos genéticos. Entretanto, existe a necessidade de estudos de diversidade genética de teca no Brasil que poderiam ser utilizados, principalmente, para a proteção de cultivares e para o melhoramento genético. Visto isso, o objetivo deste trabalho foi caracterizar a diversidade genética de genótipos de teca utilizados nos plantios brasileiros. Para tanto foram testados 10 primers de microssatélites, obtidos na literatura, para a avaliação de 60 genótipos, 33 provenientes de sementes de plantios de teca em Cáceres, 14 correspondentes a clones obtidos em Cáceres e 13 genótipos referentes a clones de procedências fora do Brasil, sendo estas, Honduras, Malásia, Índia, Indonésia, Costa do Marfim e Ilhas Salomão. Os genótipos foram divididos em oito grupos, de acordo com sua procedência, para a análise da diversidade genética. Foram realizadas análises multivariadas pelo método bayesiano no programa STRUCTURE, análises de agrupamento e coordenadas principais. Dos 10 primers testados, nove se mostraram polimórficos, sendo então utilizados para as análises estatísticas. Elevada variabilidade genética para os genótipos de teca foi detectada, sendo o número médio de alelos por loco igual a 5,22. Os genótipos de Cáceres apresentaram 100% de polimorfismo, seguido pelos clones da Índia com 90% de polimorfismo. A heterozigosidade média observada ( o= 0,352) foi menor que a heterozigosidade média esperada ( e =0,443). Coerentemente com outros estudos em teca, a maior parte da variabilidade genética concentrou-se dentro dos grupos (Hs = 0,436). Com as análises do programa STRUCTURE foi possível definir a divisão dos genótipos em três grupos, sendo 73,4% dispostos em um único grupo (vermelho) representado pela maioria dos genótipos de Cáceres, 13,3% alocados no grupo verde compostos por alguns clones da Índia, Ilhas Salomão, um clone da Malásia, um de Honduras e os clones da Costa do Marfim e os 13,3% dos genótipos restantes possuíram uma mistura dos dois grupos (vermelho e verde). A análise de agrupamento, utilizando índice de Jaccard, indicou a separação dos genótipos em seis grupos distintos: grupo I pertencente ao clone da Indonésia, grupo II possuindo dois clones da Índia, grupo III com os genótipos de Cáceres e dois clones de fora (um da Índia e outro da Malásia), grupo IV possuindo os genótipos de Honduras e Malásia, grupo V com clones da Índia e grupo VI pertencente aos clones da Costa do Marfim e das Ilhas Salomão, sendo coerente com a análise de coordenadas principais. Através do agrupamento utilizando distância de Nei, foi possível inferir duas possíveis origens da teca implantada no Brasil: Malásia e Índia. Após a avaliação das divergências genéticas, sugestões são feitas no que tange a utilização de genótipos contrastantes para o uso como parentais em programas de melhoramento genético. / Teak (Tectona grandis) is one of the main timber species in the world with high economic value, famous for its beauty, strength and durability. The species occurs naturally in India, Myanmar, Thailand, Laos and Indonesia, where diversity studies have been conducted with regard to the conservation of genetic resources. However, there is a need for studies of genetic diversity of teak in Brazil that could be used mainly for the protection of plant varieties and for breeding. Therefore, the objective of this study was to characterize the genetic diversity of teak genotypes used in Brazilian plantations. We tested 10 microsatellite primers, obtained in the literature, to assess 60 teak genotypes, 33 genotypes from seeds of plantations in Caceres, 14 clones obtained in Caceres and 13 clones originated from Honduras, Malaysia, India, Indonesia, Ivory Coast and Solomon Islands. The genotypes were divided in eight groups, in accordance to its origin, for the genetic diversity analysis. Multivariate analysis were conducted using the Bayesian method implemented in the program STRUCTURE, as well as cluster and principal coordinates analysis. Of the 10 primers tested, 9 showed polymorphism, and were then used for statistical analysis. High genetic variability for the teak genotypes was detected, with the average number of alleles per locus equal to 5.22. Caceres genotypes showed 100% polymorphism, followed by the clones from India with 90% polymorphism. The average observed heterozygosity ( o = 0.352) was lower than the average expected heterozygosity ( e = 0.443). Consistent with other studies in teak, most of the genetic variability was concentrated within groups (Hs = 0.436). With the analysis of the STRUCTURE software it was possible to define the division of the genotypes into three groups, 73.4% placed in one group (red) represented the majority of the genotypes of Caceres, and 13.3% allocated in the green group composed of some clones from India, a clone from Solomon Islands, Malaysia and Honduras and the clones of the Ivory Coast. The 13.3% of the remaining genotypes possessed a mixture of the two groups (red and green). Cluster analysis using Jaccard index indicated the separation of the genotypes into six distinct groups: group I belonging to the clone from Indonesia, group II having two clones from India, group III with genotypes from Caceres and two clones from India and Malaysia, group IV having the Honduras and Malaysia genotypes, group V with clones from India and group VI with clones belonging to the Ivory Coast and the Solomon Islands. This result was consistent with the principal coordinate analysis. From the results described above, together with the cluster analysis using Neis distance, it was possible to infer two probable origins of teak implemented in Brazil: India and Malaysia. After assessing the genetic divergences, suggestions were made concerning the use of contrasting genotypes as parents in breeding programs.

L'utilisation de pesticides conventionnels, en particulier les pesticides chimiques de synthèse, a considérablement réduit les pertes de récoltes et a connu un succès commercial. Cependant, l'utilisation excessive de pesticides chimiques qui manquent de toxicité spécifique a provoqué une série de problèmes environnementaux et de santé publique. L'intérêt de la recherche vers de nouveaux biopesticides naturels avec de nouveaux modes d'actions vise à un meilleur équilibre entre l'efficacité des pesticides et la réduction des méfaits possibles pour l'environnement et les humains. Les plantes sont une source importante de biopesticides. Les acides chlorogéniques (CQA), isolés à partir de diverses plantes et présentent in vivo et in vitro un large spectre d'activités biologiques, ont attiré l'attention avec un potentiel comme biopesticides basé sur la toxicité brevetée de l'acide 3,5-di-O-caféoylquinique contre les larves de Myzus persicae. L'étude des propriété insecticides notamment la mode d'action et l'étude de certaines relations structure-activité pourrait bénéficier de la synthèse de différents dérivés et analogues de CQA. Des analogues des acides 4-désoxy-3,5-dicinnamoy quiniques et 3,4- et 4,5-dicinnamoylquiniques naturels ont été synthétisés. Sept analogues dans la série 4-désoxy ont été soumis à des essais insecticides et deux composés présentent une activité insecticide plus élevée que l'acide 3,5-dicaféoylquinique naturel. Comme perspectives à ce travail, confirmer l'activité des composés synthétisés sur d'autres espèces de pucerons d'importance agronomique pourrait être réalisé. De plus, d’autres collaborations avec des biologistes pourraient être établies afin d’évaluer d'autres activités des composés synthétisés ou les utiliser comme outils pour étudier des mécanismes de biosynthése. / The use of conventional pesticides, especially the synthetic chemical pesticides, has greatly reduced the crop losses and gained a commercial success. However, the excessive use of pesticides lacking toxic specificity has caused a series of environmental and public health problems. The research interest toward new naturally-occurring biopesticides with novel modes of actions aims at a better balance between the efficiency of pesticide and reducing possible harms to environment and humans. Botanicals are an important source of biopesticides. Cinnamoyl quinic acids (CQA), isolated from various plants and shown to exhibit in vivo and in vitro a wide spectrum of biological activities, have attracted the attention with potential as biopesticides based on the patented toxicity of 3,5-di-O-caffeoylquinic acid against the larvae of Myzus persicae. The investigation of the insecticidal activity of CQA including their mode of action and the study of some structure-activity relationships could benefit from the synthesis of different CQA derivatives and analogs. A series of natural CQA derivatives natural 3,4- and 4,5-dicinnmamoylquinic acid derivatives but also analogs like 4-deoxy-3,5-dicinnamoylquinic acids were so synthesized. Seven targeted 4-deoxy CQA analogs were subjected to insecticidal assays, and two compounds were found to exhibit higher insecticidal activities than natural 3,5-dicaffeoylquinic acid. As perspectives to this work, confirming the activity of the synthesized compounds on other aphid species of agronomic importance could be performed. Furthermore, other collaborations could be established with biologists dedicated to measure other bioactivities of the synthesized compounds or use them as tools to investigate various biological pathways.

A teca (Tectona grandis) é uma das principais espécies madeireiras do mundo, com alto valor econômico, muito famosa por sua beleza, resistência e durabilidade. A espécie ocorre naturalmente na Índia, Mianmar, Tailândia, Laos e Indonésia, onde estudos de diversidade têm sido realizados no que tange à conservação de recursos genéticos. Entretanto, existe a necessidade de estudos de diversidade genética de teca no Brasil que poderiam ser utilizados, principalmente, para a proteção de cultivares e para o melhoramento genético. Visto isso, o objetivo deste trabalho foi caracterizar a diversidade genética de genótipos de teca utilizados nos plantios brasileiros. Para tanto foram testados 10 primers de microssatélites, obtidos na literatura, para a avaliação de 60 genótipos, 33 provenientes de sementes de plantios de teca em Cáceres, 14 correspondentes a clones obtidos em Cáceres e 13 genótipos referentes a clones de procedências fora do Brasil, sendo estas, Honduras, Malásia, Índia, Indonésia, Costa do Marfim e Ilhas Salomão. Os genótipos foram divididos em oito grupos, de acordo com sua procedência, para a análise da diversidade genética. Foram realizadas análises multivariadas pelo método bayesiano no programa STRUCTURE, análises de agrupamento e coordenadas principais. Dos 10 primers testados, nove se mostraram polimórficos, sendo então utilizados para as análises estatísticas. Elevada variabilidade genética para os genótipos de teca foi detectada, sendo o número médio de alelos por loco igual a 5,22. Os genótipos de Cáceres apresentaram 100% de polimorfismo, seguido pelos clones da Índia com 90% de polimorfismo. A heterozigosidade média observada ( o= 0,352) foi menor que a heterozigosidade média esperada ( e =0,443). Coerentemente com outros estudos em teca, a maior parte da variabilidade genética concentrou-se dentro dos grupos (Hs = 0,436). Com as análises do programa STRUCTURE foi possível definir a divisão dos genótipos em três grupos, sendo 73,4% dispostos em um único grupo (vermelho) representado pela maioria dos genótipos de Cáceres, 13,3% alocados no grupo verde compostos por alguns clones da Índia, Ilhas Salomão, um clone da Malásia, um de Honduras e os clones da Costa do Marfim e os 13,3% dos genótipos restantes possuíram uma mistura dos dois grupos (vermelho e verde). A análise de agrupamento, utilizando índice de Jaccard, indicou a separação dos genótipos em seis grupos distintos: grupo I pertencente ao clone da Indonésia, grupo II possuindo dois clones da Índia, grupo III com os genótipos de Cáceres e dois clones de fora (um da Índia e outro da Malásia), grupo IV possuindo os genótipos de Honduras e Malásia, grupo V com clones da Índia e grupo VI pertencente aos clones da Costa do Marfim e das Ilhas Salomão, sendo coerente com a análise de coordenadas principais. Através do agrupamento utilizando distância de Nei, foi possível inferir duas possíveis origens da teca implantada no Brasil: Malásia e Índia. Após a avaliação das divergências genéticas, sugestões são feitas no que tange a utilização de genótipos contrastantes para o uso como parentais em programas de melhoramento genético. / Teak (Tectona grandis) is one of the main timber species in the world with high economic value, famous for its beauty, strength and durability. The species occurs naturally in India, Myanmar, Thailand, Laos and Indonesia, where diversity studies have been conducted with regard to the conservation of genetic resources. However, there is a need for studies of genetic diversity of teak in Brazil that could be used mainly for the protection of plant varieties and for breeding. Therefore, the objective of this study was to characterize the genetic diversity of teak genotypes used in Brazilian plantations. We tested 10 microsatellite primers, obtained in the literature, to assess 60 teak genotypes, 33 genotypes from seeds of plantations in Caceres, 14 clones obtained in Caceres and 13 clones originated from Honduras, Malaysia, India, Indonesia, Ivory Coast and Solomon Islands. The genotypes were divided in eight groups, in accordance to its origin, for the genetic diversity analysis. Multivariate analysis were conducted using the Bayesian method implemented in the program STRUCTURE, as well as cluster and principal coordinates analysis. Of the 10 primers tested, 9 showed polymorphism, and were then used for statistical analysis. High genetic variability for the teak genotypes was detected, with the average number of alleles per locus equal to 5.22. Caceres genotypes showed 100% polymorphism, followed by the clones from India with 90% polymorphism. The average observed heterozygosity ( o = 0.352) was lower than the average expected heterozygosity ( e = 0.443). Consistent with other studies in teak, most of the genetic variability was concentrated within groups (Hs = 0.436). With the analysis of the STRUCTURE software it was possible to define the division of the genotypes into three groups, 73.4% placed in one group (red) represented the majority of the genotypes of Caceres, and 13.3% allocated in the green group composed of some clones from India, a clone from Solomon Islands, Malaysia and Honduras and the clones of the Ivory Coast. The 13.3% of the remaining genotypes possessed a mixture of the two groups (red and green). Cluster analysis using Jaccard index indicated the separation of the genotypes into six distinct groups: group I belonging to the clone from Indonesia, group II having two clones from India, group III with genotypes from Caceres and two clones from India and Malaysia, group IV having the Honduras and Malaysia genotypes, group V with clones from India and group VI with clones belonging to the Ivory Coast and the Solomon Islands. This result was consistent with the principal coordinate analysis. From the results described above, together with the cluster analysis using Neis distance, it was possible to infer two probable origins of teak implemented in Brazil: India and Malaysia. After assessing the genetic divergences, suggestions were made concerning the use of contrasting genotypes as parents in breeding programs.