L’agroforesterie, et plus particulièrement les systèmes sylvo-arables, sont des systèmes peu étudiés qui présentent des intérêts agronomiques et environnementaux en systèmes tempérés. Les systèmes agroforestiers pourraient notamment fournir un microclimat favorable aux cultures maraîchères conduites en agriculture biologique en contexte pédoclimatique méditerranéen. Cependant, il existe encore peu de références scientifiques sur le sujet, et l’issue des interactions entre arbres et cultures sont encore me connues. En particulier, le microclimat généré par des arbres âgés est susceptible de modifier les relations entre cultures, bio agresseurs et pré dateurs généralistes, qui peuvent conditionner la performance d’une association agroforestière. Cette thèse avait donc comme objectifs :➢ D’évaluer l’impact de l’ouverture de la canopée sur la biodiversité et l’activité densite des coléoptères carabiques.➢ D’identifier comment le microclimat peut influencer les processus de régulation naturelle.Les systèmes agroforestiers se développent sur des temps longs (pluriannuels): l’étude s’est focalisée sur un système ayant déjà des arbres âgés de 20 ans pour en étudier l’impact sur le microclimat et la faune du sol. Pour faire varier ce microclimat, un gradient d’ouverture de la canopée a été réalisé. La température de l’air ambiant, l’hygrométrie et le pourcentage d’ouverture du milieu ont ensuite été mesurés.En premier lieu, la faune du sol a été échantillonnée durant 2 ans (non révolus), et les structures des communautés de Carabidae ont été analysées d’un point de vue taxonomique et fonctionnelle, au moyen de 5 traits écologiques. Les résultats montrent que la structure taxonomique est peu affectée, mais qu’à la fois l’activité-densité spécifique des espèces et les traits fonctionnels sont modifiés par le gradient d’ouverture du milieu. En particulier, la fermeture de la canopée du système agroforestier favorise les espèces ayant une affinité pour des milieux fermés et humides.L’activité journalière de deux prédateurs abondants a été caractérisée dans une période estivale chaude (juillet), et plus douce (septembre). Les résultats montrent qu’à la fois la saison mais également l’ouverture de la canopée peuvent modifier les rythmes journaliers de l’Arachnidae et du Carabidae les plus abondants à cette période (Pardosa hortensis, Pseudoophonus rufipes).Dans un troisième temps, le travail s’est focalisé sur le potentiel de prédation des prédateurs de la faune du sol, au moyen de cartes sentinelles de prédation à deux périodes estivales, juin et aout. Les résultats montrent que sur au moins une période, le potentiel de prédation sur larves de lépidoptères (Cydia pomonella) est différent entre les traitements. Ces différences sont probablement corrélées aux différences d’activité-densité et aux différences microclimatiques induites par les différences de couverts arborés.Dans un dernier temps, l’étude s’est focalisée sur une culture, la salade, et les dégâts causés par les limaces dans les différents traitements, sur les feuilles visibles. Plus de dégâts sous les arbres ont été constatés, malgré une activité-densité d’Arion lusitanicus et de Deroceras reticulatum non supérieures au témoin au mois de juin où les dégâts ont fortement augmenté. Les méthodes utilisées (planches, pots pièges neutres et attractifs) ont présenté des efficacités différentes, dont l’intérêt respectif est discuté. / Agroforestry, and more specifically sylvo-arable systems, are poorly studied systems with agronomic and environmental interests in temperate systems. They could provide a favorable microclimate for vegetable crops grown in organic agriculture in Mediterranean pedoclimatic context. However, there are still few scientific references on the subject, and the outcome of the interactions between trees and cultures are still unknown. The microclimate generated by old trees is likely to alter the relations between crops, pests and generalist predators, which can condition the performance of an agroforestry system.This thesis had two main objectives:• To evaluate the impact of canopy openness on ground beetle diversity and activity-density• Identify how the microclimate can influence natural regulation processes.Agroforestry systems develop over long periods (pluriannual): the study focused on a system already having 20-year-old trees to study the impact on microclimate and soil fauna. To modulate this microclimate, a canopy opening gradient has been realized. The ambient air temperature, the hygrometry and the percentage of canopy aperture were then measured.First, the soil fauna was sampled for 2 years and the structure of the Carabidae communities was analyzed from a taxonomic and functional point of view, using 5 ecological traits. The results show that the taxonomic structure is poorly affected, but that both species specific activity-density and functional traits are modified by the canopy openness, which favored species with affinity for closed and moist environments.The diel activity of two abundant predators was characterized in a hot summer period (July), and cooler (September). The results show that both the season and the opening of the canopy can modify the daily rhythms of the most abundant Arachnidae and Carabidae (Pardosa hortensis, Pseudoophonus rufipes).Thirdly, the work focused on the predation potential of predators of soil fauna, using sentinel preys method at two periods, (June and August). The results show that for at least one period, the predation potential on Lepidoptera larvae (Cydia pomonella) was different between treatments. These differences are probably correlated with differences in activity-density and microclimatic differences induced by the differences in canopy treatmentLastly, the study focused on lettuce crops and the damage caused by slugs in the different treatments, on the visible leaves. More damage under the trees was observed, despite a similar activity- density of Arion lusitanicus and Deroceras reticulatum compared to control in June, where the damage increased significantly. The methods used (boards, neutral traps and attractive traps) presented different efficiencies, whose respective interest is discussed.

Pour lutter contre les nuisibles des cultures (herbivores et pathogènes), de nouvelles voies ont été explorées et en particulier la lutte biologique. Utilisé la nature et ses organismes pour réguler les populations de pathogènes, tel est le but de la lutte biologique aussi appelé biocontrôle. Parmi les organismes couramment retrouvés dans le biocontrôle se trouve les micro-organismes, que cela soit des champignons ou des bactéries. Ces micro-organismes possèdent des capacités très intéressante pour des agriculteurs et agronomes. En effet, des études ont montré qu’ils étaient capables d’interagir avec les plantes pour permettre une meilleure croissance et santé de celle-ci. Alors, dans cette thèse, nous nous sommes intéressés aux bactéries pour trouver des méthodes de luttes alternatives à Fusarium graminearum, un pathogène des céréales responsables de fortes pertes de rendements, en particulier grâce à la production de mycotoxines qui va rendre les grains impropres à la consommation humaine et animales. Par l’intermédiaire d’une approche original consistant à garder des souches non-inhibitrice in vitro pour des tests in planta, nous avons pu montrer les limites de la sélection in vitro. Grâce à l’exploration métaboliques d’une souche particulièrement efficace, nous avons aussi pu mettre en évidence de potentiels nouvelles molécules antifongiques. Par l’intermédiaire d’une souche modèle, nous avons explorer les mécanismes de la mise en place d’une résistance systémique chez le blé induite par les bactéries. Et enfin, nous avons exploré le potentiel des combinaisons de bactéries dans la protection du blé qui semble représenter un vrai futur dans le monde du biocontrôle. Les travaux effectués s’inscrivent dans les besoins de nouvelles ressources pour limiter l’utilisation des pesticides, mais aussi dans un besoin de mieux comprendre les interactions tripartite entre blé, pathogène et bactéries bénéfiques / To combat pests of crops (herbivores and pathogens) new pathways have been explored, in particular biological control. Used the nature and its organisms to regulate the populations of pathogens, that is the goal of the biological fight also called biocontrol. Among the organisms commonly found in biocontrol are micro-organisms, be they fungi or bacteria. These microorganisms have very interesting capabilities for farmers and agronomists. Indeed, studies have shown that they are able to interact with plants to allow a better growth and health of it.Then, in this thesis, we were interested in bacteria to find alternative methods of struggle with Fusarium graminearum, a pathogen of cereals responsible for high yield losses, especially thanks to the production of mycotoxins that will make the seeds unfit for human and animal consumption.Through an original approach of keeping noninhibitory strains in vitro for in planta tests, we have been able to show the limitations of in vitro selection. Thanks to the metabolic exploration of a particularly efficient strain, we have also been able to highlight potential new antifungal molecules. Through a model strain, we explored the mechanisms of the establishment of systemic resistance in wheat induced by bacteria. And finally, we have explored the potential of bacterial combinations in wheat protection that seems to represent a real future in the world of biocontrol.The work done is in line with the needs of new resources to limit the use of pesticides, but also in a need to better understand the tripartite interactions between wheat, pathogen and beneficial bacteria

L’ochratoxine A (OTA) est une mycotoxine issue du métabolisme secondaire des champignons filamenteux appartenant aux genres Penicillium et Aspergillus. Cependant, Aspergillus carbonarius est le majeur producteur de l’OTA sur les raisins. L’OTA a été retrouvée dans différents types de denrées alimentaires ainsi que lesurs produits dérivés. Le profil toxicologique de l’OTA due aux effets néfastes qu’elle présente sur la santé humaine et animale (effets hépatotoxiques, immunotoxiques, génotoxiques, tératogènes et cancérogènes) a conféré à cette mycotoxine une attention majeure auprès des instances internationales afin de limiter son occurrence. Ce projet est dédié pour trouver un moyen de lutte biologique, pouvant réduire l’OTA produite par A. carbonarius d’une part, et détoxifier les matrices alimentaires non conformes aux normes d’une autre part. La première stratégie était d’employer des huiles essentielles (cardamome, céleri, cannelle, taramira, origan, feuille de laurier, cumin, fenugrec, mélisse, menthe, sauge, anis, camomille, fenouil, romarin, romarin et thym) ainsi que des composés phénoliques extraits de plantes médicinales (feuille de laurier, cumin, fenugrec, mélisse, menthe, sauge, anis, camomille, fenouil, romarin et thym) afin d’évaluer leur effet sur la production de l’OTA dans le milieu SGM. Cette approche a été complétée par une étude moléculaire dans le but d’évaluer l’expression des gènes de biosynthèse de l’OTA (acpks, acOTApks et acOTAnrps) ainsi que les gènes de régulation (veA et laeA) chez A. carbonarius. Les résultats ont décelé que les huiles essentielles ont une activité fongicide plus élevée que celle des extraits phénoliques. Effectivement, les huiles essentielles du thym, de l’origan, du taramira, et de la cannelle ont bloqué complètement la croissance d’A. carbonarius. Cependant, les huiles essentielles du fenouil, de la cardamome, de l’anise, de la camomille, du céleri et du romarin ont réduit l’OTA sans autant affecter la croissance fongique. Le mode d’action de ces dernières a été mis en évidence en suivant l’expression des gènes acpks, acOTApks, acOTAnrps, veA et laeA, impliqués dans la biosynthèse de l’OTA chez A. carbonarius. Le gène acpks a été réprimée le plus (99.2%) quand A. carbonarius a été mis en culture avec 5 µL/mL du fenouil, entrainant ainsi une réduction de 88.9% de l’OTA. La deuxième stratégie était de développer un moyen de lutte biologique pouvant détoxifier les matrices alimentaires contaminées. Cette méthodologie a été développée suite à l’utilisation de sept souches d’actinobactéries (AT10, AT8, SN7, MS1, ML5, G10 et PT1), en évaluant leur capacité à métaboliser l’OTA, adhérer cette toxine à leur paroi membranaire ainsi que leur effet sur l’expression des gènes impliqués dans la biosynthèse de l’OTA chez A. carbonarius (acpks, acOTApks, acOTAnrps, veA et laeA). Les résultats ont montré que toutes les souches possèdent la capacité d’adhérer l’OTA à leur surface, notamment la souche SN7 qui a réduit 33% de l’OTA après 60 minutes d’incubation dans une solution PBS (Phosphate Buffer Solution) non nutritive. Les souches AT10 et SN7 ont métabolisé 51.94 et 52.68% de l’OTA ajoutée au milieu ISP2 (International Streptomyces Project-2) après 5 jours de culture à 28 °C. Cependant, les souches MS1, ML5 et G10 étaient les seules à avoir un effet sur l’expression des gènes de biosynthèse de l’OTA chez A. carbonarius. Effectivement les gènes acpks, acOTApks et acOTAnrps ont été réprimé respectivement de 37.1, 23.9 et 21% par MS1, de 39, 23 et 11.1% par ML5 et de 39, 18.3 et 11.1% par la souche G10. Ce projet a mis en valeur la capacité des extraits naturels (composés phénoliques et huiles essentielles) et des actinobactéries à prévenir d’une part la production de l’OTA et d’autre part réduire ses taux, sans pourtant affecter l’équilibre naturel ni engendrer l’apparition des débris toxiques dans les aliments traités. / Ochratoxin A (OTA) is a mycotoxin derived from the secondary metabolism of filamentous fungi belonging to the Penicillium and Aspergillus genera. However, Aspergillus carbonarius is the major producer of OTA on grapes. OTA has been detected in different types of foodstuffs as well as several products derived from these commodities. The toxicological profile of OTA and its adverse effects on human and animal health (hepatotoxic, immunotoxic, genotoxic, teratogenic and carcinogenic effects) has given this mycotoxin a major attention of international committees in order to limit its occurrence. The aim of this project was to develop biological techniques that can reduce OTA produced by A. carbonarius and detoxify non-compliant food matrices. The first strategy was achieved by using essential oils (cardamom, celery, cinnamon, taramira, oregano, bay leaf, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary and thyme) and phenolic compounds extracted from medicinal plants (bay leaf, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary, and thyme) to evaluate their effect on OTA production in SGM medium. This approach was complemented by a molecular study to evaluate the expression of the OTA biosynthesis genes (acpks, acOTApks and acOTAnrps) as well as the regulatory genes (veA and laeA) in A. carbonarius. The results revealed that essential oils had more significant fungicidal activity than phenolic extracts. Indeed, the essential oils of thyme, oregano, taramira, and cinnamon completely blocked the growth of A. carbonarius. However, essential oils of fennel, cardamom, anise, chamomile, celery and rosemary reduced OTA without affecting fungal growth. The mode of action of these essential oils has been demonstrated by evaluating the expression of acpks, acOTApks, acOTAnrps, veA and laeA genes in A. carbonarius. The expression of acpks was repressed the most (up to 99.2%) when A. carbonarius was cultured with 5 L / mL of fennel essential oil, resulting in a 88.9% of reduction in the OTA produced by this fungus. The second strategy was developed in order to detoxify contaminated food matrices. This methodology was achieved by using seven strains of actinobacteria (AT10, AT8, SN7, MS1, ML5, G10 and PT1), and evaluating their ability to metabolize OTA, adhere this toxin to their membrane walls and their effect on the expression of the genes involved in the biosynthesis of OTA in A. carbonarius (acpks, acOTApks, acOTAnrps, veA and laeA). The results showed that all strains were able to bind OTA to their surfaces, specially the SN7 strain which reduced 33% of OTA after incubation for 60 minutes in PBS (Phosphate Buffer Solution). The strains AT10 and SN7 metabolized 51.94 and 52.68% of the OTA added to the ISP2 medium (International Streptomyces Project-2) after 5 days of culture at 28° C. However, MS1, ML5 and G10 were the only strains to have an effect on the expression of the OTA biosynthesis genes in A. carbonarius. Indeed, acpks, acOTApks and acOTAnrps genes were repressed respectively by 37.1, 23.9 and 21% by MS1, 39, 23 and 11.1% by ML5 and 39, 18.3 and 11.1% by the strain G10. This project highlighted the power of natural extracts (phenolic compounds and essential oils) as well as strains of actinobacteria to prevent OTA production on one hand and to detoxify contaminated commodities on the other hand without altering the natural microbial balance.

La protection des vignes contre les maladies cryptogamiques telles que le mildiou est assurée majoritairement par des fongicides de synthèse dont certains posent des problèmes environnementaux et sanitaires. L’induction de résistance de la vigne par des éliciteurs des réactions de défenses pourrait permettre de réduire leur utilisation. Toutefois, l’efficacité de cette stratégie est avérée en conditions de serres, mais reste plus variable au vignoble. L’efficacité des éliciteurs est conditionnée par le niveau de réponse défensif de la plante, et plus globalement, par son état physiologique. Dans ce contexte et dans le cadre de ce travail intégré au projet FUI IRIS+, l’objectif était de vérifier si des biostimulants, via leurs effets sur la physiologie de la plante, étaient susceptibles d’augmenter son niveau de réponse aux éliciteurs. Ce projet impliquait en amont le criblage d’éliciteurs et de biostimulants efficaces sur vigne. L’activation des défenses par des éliciteurs a un coût métabolique et énergétique que la plante doit assumer. Dans un premier temps, nous avons utilisé le modèle « suspensions cellulaires » afin de comparer l’impact de deux éliciteurs oligosaccharidiques sur le métabolisme primaire de la vigne. Des analyses enzymatiques et métaboliques ont permis de montrer que l’oligogalacturonide, qui a eu la plus forte capacité à activer le métabolisme secondaire associé à la défense, comparé à la laminarine, est également celui qui a eu l’impact le plus marqué sur le métabolisme primaire ; notamment sur certains glucides et acides aminés. Ainsi, l’élicitation mobilise des ressources, impacte le métabolisme primaire de la vigne et son efficacité pourrait être conditionnée par l’état physiologique global de la plante. Dans un second temps, un criblage a été réalisé afin d’identifier de nouveaux éliciteurs potentiels parmi sept fournis par le partenaire industriel. Des tests de protection contre Plasmopara viticola (agent du mildiou) réalisés sur boutures herbacées en conditions de serres, ont permis de retenir un extrait de plante codé SDN3. Les autres ont été écartés car déjà utilisés en laboratoire (l’un d’entre eux), insuffisants en terme d’efficacité, instables ou phytotoxiques. Des études in vitro et in planta ont révélé que l’activité biologique de SDN3 est liée à deux modes d’action : par activation des défenses et par effet direct contre l’agent pathogène. Dans un troisième temps, un criblage de cinq biostimulants potentiellement efficaces sur vigne était à réaliser mais aucune méthodologie de test n’était disponible au laboratoire. Ainsi, quatre dispositifs de complexités différentes ont dû être mis au point, permettant le suivi phénotypique des parties aériennes et/ou racinaires (et dans certains cas physiologique) : systèmes « godets », « rhizotron plan », « tubes » et un autre système appelé « X » (non décrit car lié à une protection industrielle). Seuls deux modèles ont montré un intérêt. En effet, le modèle « tube » a permis de montrer une augmentation du poids frais moyen par racine primaire en réponse à un apport par la voie racinaire de l’un des produits testés, codé BS3. Le modèle « X » a également mis en évidence une accélération de l’ouverture du bourgeon, et une augmentation du nombre de racines primaires en réponse à BS3 appliqué par voie racinaire. La mise au point d’applications d’éliciteurs et d’infection par P. viticola est en cours de finalisation afin de vérifier l’hypothèse de départ à l’aide de la combinaison de BS3 avec SDN3. / The protection of vineyards against cryptogamic diseases such as downy mildew is mainly ensured by synthetic fungicides, which cause serious environmental and health problems. The induction of resistance by elicitors could allow to reduce their use. However, even if their efficacy is demonstrated in greenhouse conditions, it remains quite variable in field conditions. Indeed, the efficacy of an elicitor depends on the plant’s ability to respond, or more generally, on the latter’s physiological status. In this context, part of the FUI project IRIS+, the aim of this present work is to evaluate whether biostimulants, through their effect on the plant’s physiology, would be able to increase their responsiveness to elicitors. The activation of defenses implies a metabolic and energetic cost that the plant must get in charge. First of all, we used a cell suspension model in order to compare two oligosaccharidic elicitors on the primary metabolism of grapevine. Enzymatic and metabolic analyses showed that the oligogalacturonide, which had a stronger impact on secondary metabolism related to defense, compared to laminarin, also showed a more notable impact on primary metabolism, particularly on some sugars and amino acids. The elicitation of grapevine defenses effectively turned out to require resources. Secondly, a screening was carried out to select the most effective elicitor among seven products that were provided by the company. Protection assays against Plasmopara viticola on herbaceous cuttings allowed us to identity SDN3 as the most interesting candidate. In vitro and in planta studies revealed that the mode of action of SDN3 relied on both the activation of defenses and a direct effect against the pathogen. Lastly, as no protocol, nor methodology were available in our laboratory to screen biostimulants, four systems were developed, in order to monitor phenotypic traits of aerial and/or root system: the “pot” model, the “rhizotron” model, the “tubes”, and another termed “X” (no description allowed because of an industrial protection). Only the “X” model showed potential interests, since it allowed to display biostimulating effects such as the acceleration of the bud opening, and the increase of the mean number of primary roots, in response to BS3, applied to the roots. The development of the protocol to apply the elicitor and to infect with P. viticola spores are currently in progress, in order to assess the initial hypothesis by using the combination of BS3 and SDN3.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-11-01T18:52:54Z No. of bitstreams: 2 Dissertação - Karina Roterdanny Araújo dos Santos - 2017.pdf: 1875951 bytes, checksum: b8afa2140cacad832c547e18a95b2b41 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-06T09:46:28Z (GMT) No. of bitstreams: 2 Dissertação - Karina Roterdanny Araújo dos Santos - 2017.pdf: 1875951 bytes, checksum: b8afa2140cacad832c547e18a95b2b41 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-06T09:46:28Z (GMT). No. of bitstreams: 2 Dissertação - Karina Roterdanny Araújo dos Santos - 2017.pdf: 1875951 bytes, checksum: b8afa2140cacad832c547e18a95b2b41 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-07-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The presence of heavy metals in the soil has increased, causing many problems of an agricultural nature. Trichoderma are a species which is among the most well know and used biocontrol agents wordwide, as well as possessing qualities such tha parasitism and antibiosis, they have great resistance to metals and can also absorb them. Therefore, it is necessary for more detailed studies of the resistance of this fungus to metals and to research further the processes involved. In this work, we aimed to better understand the tolerance of T. harzianum to aluminum chloride, though a series of experiments performed in BDA, MEX e MYG, as well Bradford tests for quantification of proteins. In addition, we evaluated the gene response to metal stress through the sequencing RNA samples obtained when the fungus was created in BDA medium and submitted to concentrations of 1.5 and 3.0 mg / mL of aluminum chloride, 1.5 mg / mL, the fungus showed significant inhibition. It was observed that T. harzianum showed higher mycelial growth when grown in BDA medium compared to MEX and MYG, presenting levels of protein secretion inversely proportional to increasing concentrations of aluminum. The amount of transcriptionspecific factors of the stress response was increased, as well as the induction of genes involved in G-mediated cell signaling and increase in genes involved in vacuolar transport and carrier proteins. A repression of genes encoding proteins associated with cellular processes important for the growth of T. harzianum was also observed. These results show that T. harzianum is resistant to aluminum since it is able to tolerate and absorb large concentrations of this metal, which is commonly harmful to agriculture. / A presença de metais pesados no solo tem sido cada vez mais frequente, aumentando dentre vários problemas, os de cunho agrícola. Espécies de Trichoderma estão entre os agentes de biocontrole mais conhecidos e empregados mundialmente, pois além de possuir ações como parasitismo e antibiose, possui grande resistência a metais, podendo também absorvê-los. Sendo assim, faz-se necessário, estudos mais aprofundados a cerca da resistência desse fungo a metais, envolvendo pesquisas que esclareçam melhor os processos envolvidos. Neste trabalho, objetivamos melhor compreensão a cerca da tolerância de Trichoderma harzianum ao alumínio, através do seu crescimento em diferentes meios de cultura com variadas concentrações do metal. Avaliamos também a secreção de proteínas quando o fungo crescia na presença de alumínio. Além disso, analisamos a resposta gênica ao estresse pelo metal através do sequenciamento de amostras de RNA obtidas quando o fungo foi crescido em meio BDA e submetido às concentrações de 1,5 e 3,0 mg/mL de cloreto de alumínio. Foi possível constatar que T. harzianum apresenta maior crescimento micelial quando crescido em meio BDA, comparado aos meios MEX e MYG, apresentando níveis de secreção de proteínas inversamente proporcionais às concentrações crescentes de alumínio. Foi observada alteração na expressão gênica através do aumento da quantidade de determinados fatores de transcrição de resposta a estresse, além da indução de genes envolvidos em sinalização celular mediada por proteína G e aumento em genes envolvidos com transporte vacuolar e proteínas transportadoras. A repressão de genes que codificam proteínas associadas a processos celulares importantes para o crescimento de T. harzianum também foi observada. No entanto, nossos resultados evidenciam que T. harzianum apresenta resistência significativa ao alumínio, uma vez que se mostra capaz de tolerar e absorver grandes concentrações deste metal, comumente prejudicial à agricultura.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-04-23T11:22:49Z No. of bitstreams: 2 Dissertação - Jacqueline Campos Borba de Carvalho - 2013.pdf: 2453380 bytes, checksum: 04cd0ef2d26879895e9dfc3e3f757f13 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-04-23T11:34:04Z (GMT) No. of bitstreams: 2 Dissertação - Jacqueline Campos Borba de Carvalho - 2013.pdf: 2453380 bytes, checksum: 04cd0ef2d26879895e9dfc3e3f757f13 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-04-23T11:34:04Z (GMT). No. of bitstreams: 2 Dissertação - Jacqueline Campos Borba de Carvalho - 2013.pdf: 2453380 bytes, checksum: 04cd0ef2d26879895e9dfc3e3f757f13 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-03-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Rice blast caused by the fungus Magnaporthe oryzae B. Couch [anamorfo - Pyricularia oryzae Cav.] occurs in all rice growing regions of the world. The sustainable agriculture requires the introduction of biological control as one of the components in the integrated disease management. The microorganisms associated to plants are capable of producing secondary metabolites such as alkaloids which may have a role in biological control. The objective of the present study consists, isolation and identification of secondary metabolites of the micorrhizal fungus Rhizoctonia sp. obtained from Epidendrum nocturnum and evaluate in vitro and in vivo antagonism to M. oryzae. Ten fungal isolates were used to test the antibiosis against M. oryzae. The isolate En07 of Rhizoctonia sp. exhibited a greater halo of inhibition and consequently was considered the best in vitro antagonist to M. oryzae. Crude, mycelial and lyophilized extracts of micorrhizal isolate were obtained. The analysis by CCD of these three extracts showed positive results in relation to Dragendorff, indicating the presence of phenolics. The analysis of RMN 1H and masses showed the presence of aromatic hydrogens and phenolics. Five concentrations of each extract were prepared and utilized in the studies on in vitro mycelial inhibition of M. oryzae and observed 77.86% of pathogen reduction by crude extract (700 μg/mL). Two crude extract treatments (520 μg/ml and 120 μg/ml) significantly reduced the radial growth of the pathogen compared to control. The crude extract showed best results for mycelia inhibition of the pathogen, followed by lyophilized and mycelial extracts. In two trials, the crude extract at 0.52 μg.μL-1 also reduced the formation of appressoria of M. oryzae by 100%. Two greenhouse experiments were conducted on leaf blast suppression with the cultivar Primavera, using completely randomized design with three replications. In both these trials, the mycelial extract (1860 μg/ml and M.o 3x105) showed marked reduction of leaf blast severity in relation to control by 59.27% and 77.58% respectively. In the second trial, the second treatment (1040 μg/mL and M.o3x105) of crude extract reduced AUDPC by 64.63% compared to control. The results showed that the metabolites of Rhizoctonia sp. posses great potential for biological control of rice blast. / A brusone ocorre em todas as áreas produtoras de arroz do mundo, e é causada pelo fungo Magnaporthe oryzae B. Couch [anamorfo - Pyricularia oryzae Cav.]. Seu controle, realizado pelo manejo que integra resistência genética, práticas culturais e controle químico, requer a inserção de agentes biológicos, além de assegurar uma agricultura mais sustentável. Os microrganismos associados às plantas são capazes de produzir metabólitos secundários como os alcaloides que podem atuar no controle biológico. O presente trabalho objetivou isolar os metabólitos secundários do fungo micorrízico Rhizoctonia sp. obtido de Epidendrum nocturnum, e avaliar o antagonismo in vitro e in vivo com M. oryzae. Todos os ensaios foram conduzidos em delineamento inteiramente casualizado. Um ensaio de antagonismo foi avaliado, pareando-se, 10 isolados fúngicos e M. oryzae, e identificou-se que o isolado En07 foi o que apresentou o maior halo de inibição e consequentemente o melhor antagonismo in vitro sobre M. oryzae. Foram obtidos três extratos do isolado micorrízico (bruto, micelial e liofilizado) que foram analisados por quatro métodos diferentes e complementares. A análise por Cromatografia em Camada Delgada dos três extratos mostrou-se positiva frente ao reativo de Dragendorf, sugerindo a presença de compostos fenólicos. Na análise dos espectros de Ressonância Magnética Nuclear e de massas verificou-se a presença de hidrogênios aromáticos e de compostos fenólicos. Cinco concentrações de cada extrato foram preparadas e utilizadas nos ensaios de inibição micelial de M. oryzae in vitro e observou-se 77,86% de redução do patógeno pelo extrato bruto (700 μg/mL). Dois tratamentos do extrato bruto (520 μg/ml e 120 μg/ml) reduziram o crescimento radial do patógeno de forma significativa quando comparado com a testemunha. Verificou-se que o extrato bruto apresentou os melhores resultados para a inibição micelial do patógeno, seguido dos extratos liofilizado e do micélio. Em dois ensaios o extrato bruto a 0,52 μg.μL-1 também reduziu o número de apressórios formados de M. oryzae em 100%. Foram conduzidos dois ensaios de supressão de brusone foliar em arroz, in vivo, com a cultivar Primavera em três repetições. Nos dois ensaios destacou-se o extrato micelial (1860 μg/ml e M.o 3x105) que proporcionou maiores reduções da severidade de brusone nas folhas em relação ao controle com 59,27% e 64,63%, respectivamente. No segundo ensaio o tratamento 2 (1040 μg/mL e M.o3x105) do extrato bruto reduziu a AACPD em 24,93% em relação à testemunha. Os resultados mostraram que o metabólito de Rhizoctonia sp. possui grande potencial para o controle biológico da brusone.

O guaraná, Paullinia cupana var. sorbilis, espécie nativa do Brasil é uma cultura de relevância para o país, principalmente nos estados da Bahia e Amazonas. O fruto contém substâncias estimulantes, como a cafeína, que conferem ao guaraná um grande potencial exploratório, visto que esta é uma das substâncias estimulantes mais consumidas no mundo. Apesar de sua importância, a cultura do guaraná é pouco estudada, portanto pouco se sabe sobre sua genética e as interações microbianas existentes, tanto em relação aos microrganismos endofíticos e epifíticos, quanto em relação aos patógenos. De tal forma que a cultura do guaraná na região Amazônica vem sendo afetada por condições fitossanitárias desfavoráveis, como a presença do fungo do gênero Colletotrichum, causador da antracnose, doença considerada uma ameaça à produção comercial do guaraná. Assim, visando compreender a dinâmica envolvida na interação planta - microrganismos e o possível controle da antracnose, o presente trabalho acessou a comunidade bacteriana associada às folhas com e sem sintomas de antracnose, de forma independente e dependente de cultivo; bem como, investigou o potencial biotecnológico e de biocontrole dos isolados bacterianos. A comunidade bacteriana acessada de forma dependente e independente de cultivo apresentaram similaridades, como maior valor de riqueza e menor diversidade bacteriana em folhas assintomáticas, quando comparadas com folhas sintomáticas, e com relação ao filo Proteobacteria, mais abundantemente acessado nas duas comunidades. Comparações realizadas com isolados bacterianos acessados epi e endofiticamente de folhas com e sem sintoma da antracnose apontaram que o aparecimento da doença antracnose na folha pareceu ser o fator que mais influenciou na estrutura da comunidade bacteriana. Com relação às diferenças entre tecidos sintomáticos e assintomáticos, foi possível identificar alguns gêneros abundantes mais frequentemente acessados em plantas sintomáticas, sendo que os gêneros Pseudomonas, Stenotrophomonas e Pantoea foram acessados em grande frequência tanto de forma dependente como independente de cultivo. Porém, de forma independente de cultivo foram acessados outros táxons abundantes que diferiram significativamente entre as plantas, sendo que folhas sintomáticas apresentaram Acinetobacter, Acidobacter_GP1 e Sphingobacteria, e em folhas assintomáticas foram acessados os táxons Methylobacterium, Beijerinckia, Bacilli e um grupo de Rhizobiales não classificados. Além disto, os microrganismos endofíticos e epifíticos cultivados foram testados com o fitopatógeno Colletotrichum sp. em ensaios de antagonismos, sendo que isolados do gênero Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, entre outros, apresentaram inibição do crescimento do fungo fitopatogênico. As bactérias cultivadas ainda foram testadas quanto ao potencial biotecnológico de produção de enzimas hidrolíticas e sideróforo. Muitos isolados apresentaram produção de protease e sideróforo. Foi possível também correlacionar a produção das enzimas amilase, lipase e poligalacturanase com os isolados provenientes de tecidos sintomáticos. / Guarana, Paullinia cupana var. sorbilis, is a native culture of Brazil that has relevance to the country, mainly in the states of Bahia and Amazonas. The fruit contains stimulants substances, such as caffeine, that gives a high exploration potential to the culture, since this is one of the most consumed stimulant substance in the world. Despite its importance, the culture of guarana hasnt gotten much of attention, and little is known about its genetics and microbial interactions with both epiphytic and endophytic microorganisms, and in relation to pathogens. So, the guarana culture in the Amazon region has been affected by bad phytosanitary conditions, such as the presence of the fungi Colletotrichum, which causes anthracnose disease thats considered a threat to guarana commercial production. Thus, to understand the dynamics involved in the interaction plant-microorganism and the possible control of anthracnose, this study accessed the bacterial community associated to leaves with and without anthracnose symptoms, combining culture-dependent and -independent methodologies and also investigated the biotechnology potential and biocontrol of culture-dependent bacteria. The bacterial community accessed by culture-dependent and -independent manner presented similarities, both presented higher bacterial richness but lower bacterial diversity in asymptomatic leaves when compared with symptomatic leaves, and both communities presented the Proteobacteria phylum as the most abundant one. Comparisons between the endophytic and epiphytic bacterial isolates associated with leaves with and without anthracnose symptoms showed that the onset of anthracnose disease on leaf seemed to be the most important factor that modified the bacterial community structure. Regarding the differences between symptomatic and asymptomatic tissue it was possible to identify some genera most frequently accessed in symptomatic plants such as Pseudomonas, Stenotrophomonas and Pantoea accessed in both culture-dependent and independent methodologies. However, the cultureindependent approach of bacterial accessed others abundants taxa that differed significantly between plants. Symptomatic leaves showed Acinetobacter, Acidobacter_GP1 and Sphingobacteria and asymptomatic leaves presented taxa Methylobacterium, Beijerinckia, group Bacilli and a group of unclassified Rhizobiales. In addition, endophytic and epiphytic isolates microorganisms were tested with the pathogen Colletotrichum sp. in antagonism assays, and isolates from the genus Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, and others, showed inhibition of growth of the fungus. The bacterial isolates were also tested for production of hydrolytic enzymes and siderophore. Many isolates showed protease and siderophore production. It was possible to correlate the production of amylase, lipase and poligalacturanase with isolates from symptomatic tissue.

Made available in DSpace on 2014-08-20T14:31:29Z (GMT). No. of bitstreams: 1 dissertacao_marina_cunha_sinott.pdf: 651389 bytes, checksum: a3911bf52b78e4b6458a51e537e40ea0 (MD5) Previous issue date: 2012-05-14 / Parasitic infections are the major cause of losses in livestock, particularly those involving sheep. Multidrug resistance makes the problem even more worrying because in addition to pathogens, parasites do not appear more susceptible to the active principles. Currently, for the progress to occur in sheep farming, there must be a need to seek alternatives that reduce the dependence on the success of the activity to the emergence of a molecule that makes a lack reduced, selective toxicity, and high degree of effectiveness in combating infestation. This context has led to studies in order to investigate the behavior of strains Bacillusspp, when in contact with eggs and young larvae of the forms tricostrongilídeo Haemonchuscontortus, which is undoubtedly the most pathogenic for sheep that are raised in the field. It was verified through in vitro tests, which B. circulans showed an 80.9% reduction in the number of larvae when compared to control groups. This reduction was also observed in the tests made in vivo, as well as the residual capacity of the agent through tests performed with Bacillus transformed and expressing the GFP gene. / As parasitoses que acometem ovinos são a maior fonte de prejuízos na pecuária. Devido aos tratamentos inadequados dessa enfermidade, os parasitos não se mostram mais suscetíveis aos princípios ativos químicos atuais. Para que ocorram progressos na pecuária ovina, há a necessidade de buscar alternativas que visem reduzir a dependência do sucesso da atividade ao surgimento de uma molécula que apresente carência reduzida, toxicidade seletiva com alto grau de eficácia no combate à infestação parasitária. O presente estudo avaliou a toxicidade de cepas de Bacillus spp. para ovos e formas jovens de larvas do tricostrongilídeo Haemonchus contortus. Através de testes in vitro utilizando uma suspensão esporotoxina contendo 2 x 108 UFC/mL foi possível verificar que os Bacillus thuringiensis var osvaldocruzi, Bacillus circulans, B. thuringiensis var israelensis, B. thuringiensis var kurstaki demonstraram ação larvicida com uma redução de 81; 80; 79 e 76% respectivamente no número de larvas. Nos testes in vivo, realizados com B. circulans em ovinos, observou-se atividade larvicida similar (80%) ao in vitro e esta atividade permaneceu por até sete dias após sua administração. Estes resultados sugerem que os Bacillus estudados são uma alternativa promissora no controle de helmintos em ovinos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Rio Grande do Sul (RS), the most important state of grape production in Brazil, harvests, approximately, 780 thousand tons annually. One of the biggest constraint factors to the obtainment of higher production numbers is the incidence of late season diseases in vineyards, being botrytis bunch rot, caused by Botrytis cinerea, one of the major contributors for field and post-harvest losses. Red grapes can show more resistance against this fungus, due to higher levels of phenolic compounds. The use of biological control agents (BCAs) emerges as a promising alternative to control botrytis disease. For this reason, the objectives of this study were to test isolates from this fungal pathogen, originated from the two main grape production regions in the state of RS, trough in vitro direct confront test against BCAs fungal isolates (Trichoderma spp. and Gliocladium sp.) and to test the same pathogen isolates on semi in vivo biological control, against the BCAs isolates which obtained the highest biological activity during the in vitro test, on the post-harvest storage of table grapes, on a red and a white cultivar. The B. cinerea isolates used were UFSM SG01, UFSM SG02, from serra (mountain range) region, UFSM CM01, and UFSM CM02, from campanha meridional (meridional pampas) region. The BCAs isolates used in this study were Tricoderma spp., UFSM T20, UFSM T17, UFSM T15.1 (obtained from soil), UFSM TSG, UFSM TCM (obtained from the same grape bunches where B. cinerea was isolated, representing each region), and Gliocladium sp., UFSM G4DB (obtained from soil). UFSM TSG, UFSM TCM and UFSM T15.1 showed the highest biocontrol activity (B. A.) in vitro, in general over 50% against all B. cinerea isolates. The same BCAs were selected to be used at the semi in vivo test with detached berries for three inoculation periods: B+T, pathogen and BCA inoculated at the same time; B+24hT, pathogen inoculated first and BCA 24 h later, T+24hB, BCA inoculated first and pathogen 24 h later. A higher control, considered as the lower damage level, on T+24hB period, showed the importance of preventive treatment. Cracks on berries played a more important role than the color of the skin for botrytis infection. / O Rio Grande do Sul, o estado produtor de uvas mais importante no Brasil, colhe em média 780 mil toneladas por ano. Um dos maiores entraves para a obtenção de maiores médias de produção é a incidência de doenças de final de ciclo (DFC), sendo Botrytis cinerea, agente causador da podridão-cinzenta, um dos maiores responsáveis por perdas de produção no campo e na pós-colheita. Uvas tintas podem apresentar maior resistência à podridão-cinzenta, devido à maior concentração de compostos fenólicos. O uso de agentes de controle biológico (BCAs) é uma alternativa promissora no controle da podridão de botrytis. Dessa maneira, os objetivos deste estudo foram testar isolados do patógeno coletados das duas principais regiões vitivinícolas do RS em confronto direto in vitro com isolados de agentes antagonistas (Trichoderma spp e Gliocladium sp.) e testar os mesmos isolados do patógeno em controle biológico semi in vivo com os isolados antagonistas que obtiveram as maiores médias de atividade de biocontrole no teste in vitro, na pós-colheita de uvas de mesa, em cultivares branca e tinta. Os isolados de B. cinerea empregados foram UFSM SG01, UFSM SG02, UFSM SG 03, oriundos da serra, e UFSM CM01 e UFSM CM02, oriundos da campanha meridional. Os isolados antagonistas empregados no teste in vitro foram UFSM T20, UFSM T17, UFSM T15.1 (oriundos de solo), UFSM TSG, UFSM TCM (oriundos de cachos de uva coletados nas mesmas regiões de coleta do patógeno), de Trichoderma spp., e UFSM G4DB, de Gliocladium sp.. Os isolados UFSM TSG, UFSM TCM e UFSM T15.1 foram os três que obtiveram as maiores médias de atividade de biocontrole, em geral acima de 50%, para todos os isolados de B. cinerea, testados em confronto direto in vitro. Esses isolados BCAs foram selecionados para o teste de controle biológico semi in vivo em bagas destacadas, em três períodos de inoculação: B+T, antagonista e patógeno aplicados ao mesmo tempo; B+24hT, patógeno inoculado primeiro e antagonista 24h após, T+24hB, antagonista aplicado primeiro e patógeno 24h após. O maior controle, assumido a partir do menor grau de dano, no período T+24hB, evidenciou a importância do tratamento preventivo. Rachaduras em bagas tiveram maior influência do que a coloração da casca na ocorrência da podridãocinzenta.

Cette thèse CIFRE porte sur la biologie, le comportement et le biocontrôle du frelonasiatique Vespa velutina, un prédateur invasif d’abeilles. Depuis son introduction en France, ce frelonétend maintenant son aire de répartition en Europe, impactant à la fois l’environnement etl’apiculture. L’objectif de ces travaux sera d’enrichir le savoir sur cette espèce pour perturber ledéveloppement des colonies de V. velutina à différents niveaux afin d’en limiter la prolifération. Lepremier axe porte sur la biologie des reproducteurs de V. velutina, afin d’empêcher la fondation decolonies en amont. Ce travail précise les données concernant la maturation sexuelle des mâles de V.velutina, compare certains traits liés à la fertilité des fondatrices avec celles du frelon européen, etmet en évidence une plus grande précocité et fertilité de V. velutina. Le deuxième axe porte sur labiologie des colonies, de la collecte à la distribution des ressources dans le nid. A l’aide de pucesélectroniques marquant des ouvrières (technique RFID), nous avons mesuré le rayon d’action et seslimites chez les ouvrières V. velutina. En marquant de la nourriture avec des métaux lourds, nousavons pu suivre l’évolution de sa distribution dans les colonies suivant leur structure. Le troisièmeaxe porte sur le biocontrôle de V. velutina avec des champignons entomopathogènes. Nous avonsévalué l’efficacité de différents isolats et de leur mode d’application contre V. velutina, puis décrit unchampignon naturellement parasitant V. velutina. Ces travaux ont permis de faire avancer lesconnaissances sur la biologie et la physiologie des frelons, mais également de proposer des pistes decontrôle durable de l’invasion européenne de V. velutina. / This CIFRE thesis deals with the biology, the behavior and the biological control of aninvasive predator of bees, the hornet Vespa velutina. Since its introduction in France, this hornet isnow invading most countries in occidental Europe, dealing damages both to the environment and thebeekeeping activity. In order to limit its proliferation, a good strategy could consist in disrupting itscolony development at different levels, explored in this work. The first axis deals with V. velutinareproductive biology, exploring the different paths to prevent colonies creation. First we describedthe sexual maturation of males in V. velutina, and second we compared different traits linked tofertility between foundresses of V. velutina and the European hornet, thus highlighting V. velutinahigher precocity and fertility potential. The second axis explored the biology of colonies, fromresource collection to resource distribution in the nest. Using RFID technic, we assessed the actionrange and its boundaries in V. velutina workers. We also labelled food and observed its distribution inV. velutina colonies in function of the colony size and structure. The third axis deals with V. velutinabiocontrol, using entomopathogenic fungi. We evaluated the efficiency of different isolates anddifferent application methods on V. velutina, and described a wild fungus found naturally parasitizingV. velutina. This work brought knowledge on biology behavior and physiology of this invasive hornet,and also proposed options that could be assayed for a durable control of V. velutina.