Tesis para optar al grado de Magíster en Ciencias Agropecuarias Mención Sanidad Vegetal / Botrytis cinerea produce importantes pérdidas en uva de mesa en Chile. En el control químico de este patógeno se utiliza entre otras moléculas a boscalid, carboxamida que actúa inhibiendo la enzima succinato deshidrogenasa. En aislados de Botrytis recolectados de cultivos sometidos a un uso intensivo del fungicida, se ha demostrado la asociación entre la pérdida de sensibilidad con mutaciones en el gen sdhB, en donde se han identificado las mutaciones P225F/L/T y N230I asociadas a aislados resistentes y en mayor frecuencia las mutaciones H272R/Y/L asociadas a aislados resistentes y moderadamente resistentes. Durante las últimas dos temporadas (2011-2012 y 2012- 2013), en la zona Central de Chile, se ha reportado una baja sensibilidad a boscalid. El objetivo de la presente investigación fue determinar la presencia de mutaciones en el gen sdhB en aislados chilenos de Botrytis cinerea de distinto nivel de sensibilidad a boscalid. Con este propósito, 50 aislados monoconidiales fueron caracterizados genética y fenotípicamente. Para la caracterización fenotípica se verificó la sensibilidad a boscalid mediante evaluación del comportamiento de germinación conidial, determinándose 4 categorías según valores EC50: Sensible (S) (>0,05-1,37μg.mL-1), Levemente Resistente (LR) (1,38-7,80μg.mL-1), Moderadamente Resistente (MR) (7,81-50μg.mL-1) y Resistente (R) (>50μg.mL-1). Aislados R y MR con mutaciones se compararon con sensibles, según parámetros como: crecimiento miceliar, esporulación, sensibilidad osmótica, capacidad formadora de esclerocios y virulencia. La detección de mutaciones se realizó mediante la técnica PCR-PIRA (Primer-Introduced Restriction Enzyme Analyses) y el uso de partidores específicos H272Y/R-fw y H272- rev, cuyos productos fueron digeridos con las enzimas de restricción EcoRV y HhaI respectivamente. Los aislados de Botrytis cinerea mostraron niveles de sensibilidad a boscalid variables entre 0,13 μg.mL-1 (S) y 1,1*109 μg.mL-1 (R). La utilización del PCR-PIRA, permitió identificar las mutaciones H272Y/R en el gen sdhB que resultaron ser inespecíficas de un determinado nivel de sensibilidad a boscalid. De acuerdo a los parámetros de adaptabilidad evaluados tales como crecimiento miceliar, capacidad formadora de esclerocios y esporulación de aislados resistentes con mutaciones H272Y/R respecto de los sensibles fueron significativamente diferentes (p<0,05), lo cual implicaría que las mutaciones detectadas y asociadas a la resistencia a boscalid generarían un costo metabólico en los aislados de Botrytis. / Botrytis cinerea produces serious losses in table grapes in Chile. In the chemical control of this pathogen is used among other molecules like boscalid, carboxamide that inhibits the succinate dehydrogenase enzyme. Botrytis isolates collected from different crops subjected to intensive fungicide application has shown an association between sensitivity loss and mutations in the sdhB gene, where P225F/L/T and N230I mutations have been identified in resistant isolates and most frequently mutations like H272R/Y/L on moderately resistant and resistant isolates. During the past two seasons (2011-2012 and 2012-2013) in the central Chile, has reported a low sensitivity to boscalid. The objective of this research was determine the mutations presence in the sdhB gene of Botrytis cinerea isolates with different sensitivity levels to boscalid. For this purpose, 50 monoconidial isolates were characterized genetically and phenotypically. For phenotypic characterization was verified the sensitivity to boscalid by conidial germination, were classified into four resistance phenotypes based on the EC50 values them: Sensitive (S) (>0.05-1.37 μg.mL-1), Low resistant (LR) (1.38-7.80 μg.mL-1), moderately resistant (MR) (7.81-50μg.mL-1) and resistant (R) (>50μg.mL-1). Isolate R and MR with mutations were compared with sensitive isolates, with parameters such as mycelial growth, sporulation, osmotic sensitivity, sclerotia production and virulence. Mutations detection was performed by PIRA-PCR (Primer- Introduced Restriction Enzyme Analyses) with specific primers H272-rev H272Y/R-fw whose products were digested with restriction enzymes EcoRV and HhaI respectively. Botrytis cinerea isolates showed different sensitivity levels between 0.13 μg.mL-1 boscalid (S) and 1.1*10-9 μg.mL-1 (R). The PIRA-PCR method has detected H272Y/R mutations, none were specific a sensitivity level to boscalid. According to adaptability parameters evaluated such as mycelial growth, sclerotia production and sporulation of resistant isolates with mutations H272Y/R compare with sensitive were significantly different (p <0.05), which imply that the mutations detected generate a metabolic cost in Botrytis cinerea isolates to boscalid.

Ureases, enzimas que catalisam a hidrólise da ureia formando amônia e dióxido de carbono, são sintetizadas por plantas, fungos e bactérias. A espécie leguminosa Canavalia ensiformis, conhecida em inglês como Jack Bean, produz diferentes isoformas de ureases, sendo a chamada Jack Bean Urease (JBU) a isoforma majoritária. Ureases possuem atividade inseticida já comprovada para diferentes espécies de insetos e um dos seus efeitos no animal é o bloqueio neuromuscular. O objetivo deste trabalho foi compreender o mecanismo de ação responsável pela atividade neurotóxica da JBU e pelo bloqueio neuromuscular em insetos. A injeção de JBU em baratas (Nauphoeta cinerea), apesar de não letal, induziu uma inibição da enzima acetilcolinesterase no SNC. A urease, assim como a acetilcolina ou o anticolinérgico neostigmina, também induziu um aumento no ritmo cardíaco das baratas. Insetos tratados com JBU tiveram um aumento no tempo de grooming, efeito comportamental este mimetizado com o tratamento por octopamina. Os resultados acima citados indicam um efeito da JBU no SNC do animal, interferindo com a neurotransmissão colinérgica e octopaminérgica e, possivelmente, também GABAérgica. A atividade de JBU sobre o sistema neuromuscular de insetos também foi estudada através de registros eletrofisiológicos do potencial de ação neural, muscular e de placa motora. Em preparação de músculo tarsal de gafanhoto (Locusta migratoria), a urease reduziu a amplitude do potencial de ação evocado; entretanto quando aplicada diretamente no músculo oviductal isolado, a JBU não alterou a resposta contrátil. Em preparações de placa motora de larvas da mosca-da-fruta Drosophila melanogaster, a urease aumentou a amplitude dos potenciais excitatórios pós-sinápticos (EJPs) e a frequência dos eventos miniaturas (mEJPs). O efeito da JBU sobre a junção neuromuscular de insetos foi reduzido conforme a concentração de cálcio no meio extracelular também foi reduzida e completamente revertido quando o cálcio foi removido do meio (presença de EGTA e cloreto de Co2+), indicando que a mobilidade de íons de cálcio possui um papel importante na toxicidade induzida por JBU no inseto. Experimentos de imagem de cálcio em gânglios de N. cinerea confirmaram um maior influxo de cálcio no neurônio na presença de JBU, indicando que mobilidade do cálcio através da membrana foi alterada. Os resultados obtidos confirmam que a urease induz neurotoxicidade em insetos tanto a nível central como periférico, atuando sobre os gânglios do cordão nervoso, sobre neurônios aferentes e sobre o terminal pré-sináptico de neurônios motores. A neurotoxicidade induzida por JBU aparenta ser dependente do influxo de íons de cálcio no neurônio. / Ureases, enzymes that catalyse the hydrolysis of urea into ammonia and carbon dioxide, are synthesized by plants, fungi and bacteria. The legume Canavalia ensiformis, known as jack bean, produces different isoforms of ureases, being the jack bean urease (JBU) its major isoform. Ureases have insecticidal activity against different insect species and one of effects induced by JBU is a neuromuscular blockage. The aim of this work is to further understand the mechanism of action behind this blockage. The injection of JBU into cockroaches (Nauphoeta cinerea), despite not lethal, caused inhibition of brain acetylcholinesterase enzyme. Urease, as well as acetylcholine or the anticholinesterase drug neostigmine, induced a positive cardiac chronotropism in cockroaches. Similar to octopamine, JBU induced an increase in the time spent on grooming behaviour. These results indicate that JBU may act on the central nervous system of the animal by interfering with neurotransmission, more specifically on the cholinergic, octopaminergic and, possibly, GABAergic pathways. The activity of JBU on the neuromuscular junction of insects was studied through electrophysiological recordings. On the tarsus muscle of locust (Locusta migratoria), JBU reduced the amplitude of evoked muscle potentials, but did not alter the contractile response when applied to the oviduct muscle isolated. When applied on the end-plate of flies (Drosophila melanogaster) JBU led to an increase in the amplitude of Excitatory Junctional Potentials (EJPs), as well as to higher frequency of miniature Excitatory Junctional Potentials (mEJPs). JBU’s effect on the neuromuscular junction was reduced when the extracellular calcium concentrations decreased, and it was completely reversed when in calcium free preparations (in the presence of EGTA and Cobalt chloride), indicating that calcium mobility may play an important role in JBU-induced toxicity. Calcium imaging of cockroach ganglions revealed increased calcium influx upon exposition to JBU. These results indicate that JBU induces a central and peripheral neurotoxicity in insects, by interfering on neurotransmitters release or signalling in the ventral nerve cord, in the afferent neurons and in the motor neurons. Moreover, JBU-induced neurotoxicity depends on the influx of calcium ions into the neurons.

Made available in DSpace on 2014-12-02T11:16:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-24Bitstream added on 2014-12-02T11:21:34Z : No. of bitstreams: 1 000800074.pdf: 1039235 bytes, checksum: 81d9e46f991603844eb8ba3b624a239b (MD5) / A incidência de radiação ultravioleta (UV 100 a 400 nm) na terra , em especial a radiação UV - B (280 - 320 nm), por ser filtrada exclusivamente pela camada de ozônio e apresentar grande efetividade biológica , quando comparada com os outros espectros da radiação UV , está sendo alterad a com as mudanças climáticas . Sendo a radiação solar um importante componente climático durante o desenvolvimento de um microrganismo no ambiente, se fez necessário avaliar a tolerância de fitopatógenos, bem como de agentes de biocont role à radiação UV - B . Assim , o presente trabalho teve como objetivo s estudar alguns aspectos d a interação morangueiro × Botrytis cinerea × Clonostachys rosea × radiação UV - B. Nos estudos foram observadas diferença s significativa s entre os 13 isolados de B. cinerea em relação a germinação de esporos e esporulação em discos de folhas de morango após irradiação com UV - B de 2, 9 a 8, 9 KJ m - 2 . A germinação relativa variou de 75% a 9 5% e a esporulação variou em mais do que 100% entre os isolados de B. cinerea após exposição à radiação UV - B de 6,4 KJ m - 2 . O isolado LQC - 150 de B. cinerea apresentou maior germinação e esporulação em discos de folhas após irradiação e foi selecionado como o mais tolerante. O isolado LQC - 150 de B. cinerea apresentou LD 50 de 6,2KJ m - 2 . A esporulação de ambos os fungos em discos de folhas de morangueiro , quando inoculados individualmente, foi inversamente proporcional ... / The incidence of ultraviolet (UV 100 to 400 nm) in the earth , especially UV - B radiation (280 - 320 nm) is being altered with climate change. The solar radiation is an import ant component for the development of microorganism in the environment, thus is important evaluate the tolerance of plant pathogens as well as the biocontrol agents to UV - B radiation. T he present study aimed to study the interaction of strawberry x Botrytis cinerea x Clonostachys rosea x UV - B radiation. There were significantly differences among the thirteen B. cinerea strains in relation to spore germination and sporulation on leaf disks after irradiation ranging from 2.9 to 8.9 KJ m - 2 . The relative germina tion ranged from 95 to 75% and the sporulation varied more than 100% among B. cinerea strains after exposure to 4 radiation of 6.4 KJ m - 2 . The LQC - 150 strain showed high germination and sporulation on leaf disk after irradiation and was selected as a toleran t strain. Survival curve of B. cinerea strain LQC - 150 showed lethal dose 50 (LD 50 ) of 6.2 KJ m - 2 . The sporulation of both fungi on leaf disks was inversely proportional to the dose of UV - B radiation, while inoculated alone. When confronted in the same leaf disk and not irradiated, C. rosea reduced the incidence of the pathogen and its sporulation in about 50% and 80%, respectively. However, the ability of C. rosea to control B. cinerea on leaf disks was gradually reduced with the increase of UV - B radiation, reaching 20% and 50%, respectively for pathogen incidence and sporulation, on higher UV - B doses. When the bioagent was applied in the morning, the development was lower than when applied afternoon. The effect of PABA in the induction of resistante in plan ts of Nicotiana benthamiana against B. cinerea was evaluated and it was found that plants treated with PABA were more resistant to the pathogen. The evaluations of size of plants and leaves ...

Ureases, enzimas que catalisam a hidrólise da ureia formando amônia e dióxido de carbono, são sintetizadas por plantas, fungos e bactérias. A espécie leguminosa Canavalia ensiformis, conhecida em inglês como Jack Bean, produz diferentes isoformas de ureases, sendo a chamada Jack Bean Urease (JBU) a isoforma majoritária. Ureases possuem atividade inseticida já comprovada para diferentes espécies de insetos e um dos seus efeitos no animal é o bloqueio neuromuscular. O objetivo deste trabalho foi compreender o mecanismo de ação responsável pela atividade neurotóxica da JBU e pelo bloqueio neuromuscular em insetos. A injeção de JBU em baratas (Nauphoeta cinerea), apesar de não letal, induziu uma inibição da enzima acetilcolinesterase no SNC. A urease, assim como a acetilcolina ou o anticolinérgico neostigmina, também induziu um aumento no ritmo cardíaco das baratas. Insetos tratados com JBU tiveram um aumento no tempo de grooming, efeito comportamental este mimetizado com o tratamento por octopamina. Os resultados acima citados indicam um efeito da JBU no SNC do animal, interferindo com a neurotransmissão colinérgica e octopaminérgica e, possivelmente, também GABAérgica. A atividade de JBU sobre o sistema neuromuscular de insetos também foi estudada através de registros eletrofisiológicos do potencial de ação neural, muscular e de placa motora. Em preparação de músculo tarsal de gafanhoto (Locusta migratoria), a urease reduziu a amplitude do potencial de ação evocado; entretanto quando aplicada diretamente no músculo oviductal isolado, a JBU não alterou a resposta contrátil. Em preparações de placa motora de larvas da mosca-da-fruta Drosophila melanogaster, a urease aumentou a amplitude dos potenciais excitatórios pós-sinápticos (EJPs) e a frequência dos eventos miniaturas (mEJPs). O efeito da JBU sobre a junção neuromuscular de insetos foi reduzido conforme a concentração de cálcio no meio extracelular também foi reduzida e completamente revertido quando o cálcio foi removido do meio (presença de EGTA e cloreto de Co2+), indicando que a mobilidade de íons de cálcio possui um papel importante na toxicidade induzida por JBU no inseto. Experimentos de imagem de cálcio em gânglios de N. cinerea confirmaram um maior influxo de cálcio no neurônio na presença de JBU, indicando que mobilidade do cálcio através da membrana foi alterada. Os resultados obtidos confirmam que a urease induz neurotoxicidade em insetos tanto a nível central como periférico, atuando sobre os gânglios do cordão nervoso, sobre neurônios aferentes e sobre o terminal pré-sináptico de neurônios motores. A neurotoxicidade induzida por JBU aparenta ser dependente do influxo de íons de cálcio no neurônio. / Ureases, enzymes that catalyse the hydrolysis of urea into ammonia and carbon dioxide, are synthesized by plants, fungi and bacteria. The legume Canavalia ensiformis, known as jack bean, produces different isoforms of ureases, being the jack bean urease (JBU) its major isoform. Ureases have insecticidal activity against different insect species and one of effects induced by JBU is a neuromuscular blockage. The aim of this work is to further understand the mechanism of action behind this blockage. The injection of JBU into cockroaches (Nauphoeta cinerea), despite not lethal, caused inhibition of brain acetylcholinesterase enzyme. Urease, as well as acetylcholine or the anticholinesterase drug neostigmine, induced a positive cardiac chronotropism in cockroaches. Similar to octopamine, JBU induced an increase in the time spent on grooming behaviour. These results indicate that JBU may act on the central nervous system of the animal by interfering with neurotransmission, more specifically on the cholinergic, octopaminergic and, possibly, GABAergic pathways. The activity of JBU on the neuromuscular junction of insects was studied through electrophysiological recordings. On the tarsus muscle of locust (Locusta migratoria), JBU reduced the amplitude of evoked muscle potentials, but did not alter the contractile response when applied to the oviduct muscle isolated. When applied on the end-plate of flies (Drosophila melanogaster) JBU led to an increase in the amplitude of Excitatory Junctional Potentials (EJPs), as well as to higher frequency of miniature Excitatory Junctional Potentials (mEJPs). JBU’s effect on the neuromuscular junction was reduced when the extracellular calcium concentrations decreased, and it was completely reversed when in calcium free preparations (in the presence of EGTA and Cobalt chloride), indicating that calcium mobility may play an important role in JBU-induced toxicity. Calcium imaging of cockroach ganglions revealed increased calcium influx upon exposition to JBU. These results indicate that JBU induces a central and peripheral neurotoxicity in insects, by interfering on neurotransmitters release or signalling in the ventral nerve cord, in the afferent neurons and in the motor neurons. Moreover, JBU-induced neurotoxicity depends on the influx of calcium ions into the neurons.

Botrytis cinerea is the principal member of the species complex that causes bunch rot of grapes and gray mold disease on other hosts including fruits and ornamental crops. It has developed resistance to many fungicides, and isolates from eastern US strawberry fields have regularly been identified with resistance to several modes of action. During the 2011-2015 growing seasons, 487 isolates were collected from Virginia wine grapes, strawberries, and ornamental crops and evaluated for sensitivity to eight different fungicides by a germ tube elongation method; for a subset of isolates, a 24-well plate mycelial growth assay was also used, and baseline sensitivity to polyoxin-D was evaluated. Resistance to benzimidazoles and quinone outside inhibitors, and low-level resistance to iprodione were common. Boscalid resistance was common in wine grapes and ornamentals. Resistance to the hydroxyanilide fenhexamid during germ tube elongation was found in only 5% of wine grape isolates, but in 33% of isolates from strawberries and ornamentals. All of the fenhexamid-resistant isolates were identified as B. cinerea carrying various mutations in the erg27 gene. An additional subset of isolates was identified with moderate resistance to fenhexamid during mycelial growth, but not germination and germ tube growth. These were identified as B. cinerea HydR2 isolates, which possess an unknown mechanism of resistance towards fenhexamid in mycelial growth. Moderate resistance to cyprodinil was common, but in grape inoculation tests, moderately resistant isolates were controlled by a field rate of cyprodinil. Diminished sensitivity to fludioxonil and fluopyram was rare. Polyoxin-D controlled most isolates in mycelial growth tests at 100 µg/ml. / Master of Science in Life Sciences

Des études récentes ont démontré l'existence d’un espace sensoriel typique des vins liquoreux de Bordeaux. Des composés volatils de familles chimiques différentes ont été identifiés et corrélés avec la typicité de ces vins. Cependant, les composés responsables des nuances aromatiques de « fruits confits », considérées comme des nuances « clés » de l’arôme des vins liquoreux de pourriture noble, n’avaient pas fait l’objet de recherches préalables. Les analyses classiques des extraits de vin par CPG-SM n'ont pas permis l’identification des composés responsables des nuances de « fruits confits » et plus précisément des notes d’ « orange confite ». Ainsi, une méthode d’étude alternative, reposant sur des approches sensorielles et instrumentales, a été mis en œuvre afin d’étudier les fractions obtenues par CLHP semi préparative. Des tests d’omission ont été réalisés par l’utilisation de la CPG préparative dans le but d’isoler une fraction contenant l’arôme d’ « orange confite ». Ces tests ont mis en évidence l’impact de cette fraction sur l’arôme des vins puisque son omission, au niveau de l’aromagramme, entraine la perte du caractère « fruit confit » dans les extraits. L’application de la CPG multidimensionnelle, il a permis d’identifier une nouvelle lactone spécifique des vins liquoreux, le 2-nonen-4-olide. Les expériences sensorielles de reconstitution et d’omission, menées à partir de fractions de vin ou en milieu modèle ont permis de souligner l’impact de composés clés - lactones, eugénol et 3-sulfanylhexanol - avec le 2-nonen-4-olide dans l’expression aromatique des nuances de « fruits confits », et plus particulièrement d’ « orange confite » au travers d’un phénomène d’accord aromatique. Par ailleurs, les corrélations entre le niveau de typicité de vins liquoreux de Bordeaux et les teneurs de ces composés ont été étudiées. Enfin, la genèse du 2-nonen-olide a été appréciée au cours de la botrytisation de raisins de Sémillon puis pendant l’élevage des vins en barrique. / Recent studies have demonstrated the existence of a typical sensory concept for Bordeaux dessert wines. Volatile compounds from several chemical families were identified and correlated with aromatic typicality in these wines. However, the compounds responsible for “key” aromas, particularly those recalling “overripe” fruits, had not previously been interpreted. Classical analysis of wine extracts by GC-MS was not successful in identifing the compounds responsible for these ripe fruity notes. An alternative strategy was developed in this research combining both analytical and sensory studies of wine fractions extracts obtained by semi-preparative HPLC. Omission tests were conducted using preparative GC with cryotrapping in order to evidence the fraction presenting overripe fruit nuances focusing on orange notes. These tests demonstrated the impact of this fraction on the wine aroma, as the overripe fruity note disappeared when this section was not trapped. Multidimensional CPG allowed the identification of a new lactone, 2-nonen-4-olide, typical to dessert wines. Reconstitution and omission tests carried out from wine HPLC fractions or in a model wine medium, highlighted the importance of specific compounds - lactones, eugenol and 3-sulfanylhexanol - with 2-nonen-4-olide, on the overripe fruit aromas expression and particularly on orange notes via perceptual blending. Furthermore, the correlations between the level of typicality of Bordeaux sweet wines and the levels of these compounds were studied. Finally, the evolution of 2-nonen-4-olide was evaluated during the botrytisation of Semillon grapes as well as in wines during aging in barrels.

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

Les champignons ont développé diverses interactions avec les végétaux. Ces interactions peuvent être bénéfiques pour la plante dans le cas des champignons établissant des symbioses mutualistes ou néfastes si le champignon est pathogène. Elles reposent sur des mécanismes moléculaires mal élucidés. Des études réalisées sur le champignon mutualiste Hebeloma cylindrosporum associé au pin Pinus pinaster ont permis de créer une collection de mutants affectés dans leur capacité à interagir avec les plantes et à former l’organe mixte de la symbiose, l’ectomycorhize. L’objectif de ma thèse a été d’étudier un mutant affecté dans le gène codant une alpha-tubuline Hctubα2. Les tubulines sont des protéines présentes chez tous les Eucaryotes et permettent la formation des microtubules, des éléments clés du cytosquelette. Chez les champignons, on trouve une ou deux alpha tubuline(s). H. cylindrosporum en possède deux. J’ai étudié l’expression de ces deux tubulines lors l’établissement de l’interaction avec les racines de l’hôte. Les résultats indiquent que ces deux gènes sont différentiellement exprimés lors de l’interaction. J’ai étudié au niveau protéomique l’impact de la mutation en comparant les protéomes intracellulaires des deux souches. On retrouve deux alpha-tubulines chez certains champignons phytopathogènes comme Botrytis cinerea. L’hypothèse de l’implication de l’alpha-tubuline 2 dans l’établissement de la pathogénie a été émise. J’ai donc construit des mutants de Botrytis cinerea dans lesquels ce gène a été inactivé. J’ai également tenté de localiser à l’aide de fusions traductionnelles chacune des alpha-tubulines chez le champignon mycorhizien et chez le pathogène / In all terrestrial ecosystems, plants live in close interaction with numerous fungi. The interaction has a negative or positive effect on host plant depending upon the pathogenic or symbiotic status of the fungus. The establishment of these interactions is based on a tightly regulated molecular dialog between symbiotic partners. Previous studies on the ectomycorrhizal fungi, Hebeloma cylindrosporum associated with maritime pine (Pinus pinaster), created a collection of mutants affected in their mycorrhizal abilitiy. The aim of my thesis was to characterize one of these mutants affected in a gene, Hctubα2, encoding an alpha tubulin. Tubulins are eukaryotic cytoskeletal proteins involved in microtubules formation. Fungi have one or two alpha-tubulin. For example, H.cylindrosporum has two alpha-tubulin. The site of mutagenic DNA insertion in fungal genome was characterized. I studied the expression of both alpha-tubulins during the establishement of mycorrhizal interaction. Results showed that the two genes are differentially expressed during the interaction with host plant. At proteomic level, I studied the impact of the mutation comparing the two strains using 2D gel electrophoresis and sequencing differentially accumulated spots. Pathogenic fungi also bear two alpha-tubulins, as Botrytis cinerea. The hypothesis of the involvement of the alpha-tubulin 2 in pathogenesis was investigated. I created Botrytis cinerea mutants deleted for this gene. I also created translational fusions in order to visualize both alpha-tubulins in Hebeloma cylindrosporum and in Botrytis cinerea

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

La perception et l’adaptation à l’environnement sont des processus indispensables pour la survie des organismes vivants. Le champignon phytopathogène Botrytis cinerea peut ainsi percevoir différents types de signaux qu’ils soient chimiques ou physiques. La voie de signalisation de la MAPK Sak1 est impliquée dans l’adaptation au stress osmotique, oxydatif et pariétal, mais aussi dans la sporulation et le pouvoir pathogène en régulant la pénétration de la plante et le développement des nécroses. Afin d’approfondir les connaissances existantes sur la voie de Sak1, nous avons réalisé des études globales basées sur des techniques de protéomique et phosphoprotéomique. L’analyse de protéomique comparative entre la souche sauvage et les mutants de signalisation ∆bos1 et ∆sak1 a notamment mis en évidence que la MAPK Sak1 régule l’abondance de protéines impliquées dans la voie des protéines G et la voie calcique. Cette connexion avec les protéines G a été confirmée par une baisse de la concentration en AMPc chez le mutant ∆sak1. L’utilisation du fludioxonil comme signal de l’activation de la MAPK Sak1 pour l’analyse par phosphoprotéomique a mis en évidence des modifications de l’état de phosphorylation de protéines. Parmi ces protéines différentiellement phosphorylées, la présence de PKAR (sous-unité régulatrice de la protéine kinase A) et du facteur de transcription CRZ1, indiquent respectivement une action sur la voie via protéines G et la voie calcique, validant les résultats obtenus par protéomique. Le phosphoprotéome a révélé une « phosducin-like protein », PhnA. Sa caractérisation fonctionnelle montre son rôle dans l’adaptation aux stress, la sporulation et la germination, ainsi que dans le pouvoir pathogène mettant ainsi en évidence un nouveau facteur de pathogénicité chez B. cinerea. Notre étude a permis de révéler des interactions entre Sak1 et d’autres voies de signalisation non suspectées, agissant aussi bien sur la production de certains composants (régulations transcriptionnelles et traductionnelles) que sur la phosphorylation (modifications post-traductionnelles). Nos résultats constitueront la base de nouvelles recherches pour compléter nos connaissances sur ces interactions impliquant l’adaptation au stress et la pathogénie de B. cinerea. / Perception and adaptation to the environment are essential processes for the survival of living organisms. The phytopathogenic fungus Botrytis cinerea can thus perceive different types of signals, whether they are chemical or physical. The signalling pathway of the Sak1 MAPK is involved in the adaptation to osmotic, oxidative and cell wall stress, but also in sporulation and pathogenicity by regulating plant penetration and necrosis development. In order to deepen existing knowledge of the Sak1 pathway, we have carried out global studies based on proteomics and phosphoproteomics techniques. A comparative proteomics analysis between the wild type and the signalling mutants ∆bos1 and ∆sak1 showed, among others, that Sak1 regulates the abundance of proteins involved in the G-protein pathway and calcium pathway. This connection with G-proteins was confirmed by a decrease in cAMP concentration in the ∆sak1 mutant. Using fludioxonil as signal for the activation of Sak1 for a phosphoproteomic analysis revealed changes in the state of protein phosphorylation. Among these differentially phosphorylated proteins, the presence of PKAR (regulatory subunit of protein kinase A) and the transcription factor CRZ1, indicates an action on the G-protein and calcium pathway respectively, validating the results obtained by proteomics. Phosphoproteomics revealed a phosducin-like protein, PhnA. Its functional characterization reveals its role in stress adaptation, sporulation and germination, as well as in pathogenicity, thus demonstrating a new pathogenicity factor in B. cinerea. Our study revealed interactions between Sak1 and other unsuspected signalling pathways, affecting both the production of certain components (transcriptional and translational regulations) and phosphorylation (post-translational modifications). Our results will create the basis for new research questions to complement our understanding of these interactions involving adaptation to stress and pathogenesis of B. cinerea.