Botrytis cinerea Pers., [forme imparfaite du Botryotinia fuckeliana (de Bary) Whetzel.] est le champignon ascomycète responsable de la pourriture grise (moisissure grise, pourriture de la grappe) chez des centaines de plantes hôtes au niveau mondial. Il est considéré comme un agent pathogène à haut risque de développement de résistance aux fongicides et s’attaque aux fruits, aux légumes mais, aussi aux plantes ornementales. Afin de lutter contre les maladies causées par B. cinerea, plusieurs familles de fongicides de synthèse sont homologuées au Canada. Un des fongicides récemment introduit et fréquemment utilisé est le boscalide. L’utilisation du boscalide combiné à la pression de sélection a mené au développement de la résistance dans les populations de B. cinerea. La génétique des mécanismes de résistance étant de plus en plus documentée, plusieurs mutations responsables de la résistance au boscalide ont été identifiées sur le gène de la sous-unité B de la succinate déshydrogénase (SdhB). Des substitutions d’acides aminés provoqués par des polymorphismes nucléotidiques (SNP) sont associées à cette résistance. Les mutations les plus fréquentes retrouvées chez les individus résistants sont la substitution d’une histidine par une tyrosine (H272Y), par une arginine (H272R), ou par une leucine (H272L) au codon 272 de la sous-unité SdhB. De plus, sur le codon 225, une proline est substituée par une phénylalanine (P225F) et sur le codon 230, une asparagine est substituée par une isoleucine (N230I). Il existe des outils moléculaires pour détecter les mutations H272Y, H272R, H272L, P225F et N230I, mais ils ne permettent pas d’analyses quantitatives et leur précision est limitée. Il devient donc important de développer une méthode efficace permettant de détecter et quantifier simultanément les mutations liées à la résistance au boscalide. L’objectif du travail était donc de développer un nouvel outil de détection et de quantification des mutations reliées à la résistance au boscalide chez B.cinerea. À l’aide d’une banque d’individus de B.cinerea déjà caractérisés pour la présence des cinq mutations (P225F, N230I, H272L, H272Y et H272R) et caractérisés pour la résistance au boscalide, un nouvel essai de pyroséquençage a été mis au point sur plateforme PyroMark Q24 (Qiagen). L’utilisation du PyroMark Q24 comme outil de détection et de quantification de cinq mutations reliées à la résistance au boscalide a permis de repousser les limites des techniques existantes. La technique est basée sur le séquençage par synthèse générant des données quantitatives avec une précision de 5%. Les résultats ont démontré une relation linéaire (R[indice supérieur 2]=0,99) entre les ratios (0% à 100%) de spores d'individus résistants et sensibles connus et prédit avec le PyroMark Q24. Le pyroséquençage est une technologie prometteuse puisqu’elle favorisera l’étude populationnelle en offrant la possibilité de combiner les échantillons et permettra ainsi d'analyser un grand nombre d’échantillons plus rapidement, avec une meilleure précision que les technologies de détection actuelles. La disponibilité d’informations quantitatives sur la proportion de chacune des mutations présentes dans une population permettra l’amélioration de la compréhension et de la gestion face à la résistance aux fongicides au sein de la communauté agricole.

Reproduction de : Thèse doctorat : Biologie et physiologie végétales : Reims : 2008. / Titre provenant de l'écran titre. Bibliogr. p.101-124.

Reproduction de : Thèse doctorat : Biologie et physiologie végétales : Reims : 2007. / Titre provenant de l'écran titre. Bibliogr. f. 123-154.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 115-135). Also available on the World Wide Web.

University, Diss., 2003--Bonn.

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.

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 ...

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.