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Made available in DSpace on 2015-03-26T13:51:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 952076 bytes, checksum: 7fbcb43414ecacd3439620826b6172cd (MD5) Previous issue date: 2012-07-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Colletotrichum lindemuthianum is the causal agent of common bean anthracnose. Genes that encode cell wall-degrading enzymes are essential for the development of this disease. The pectinases are characterized as the most important group of cell wall- degrading enzymes produced by phytopathogen fungi. The gene coding for pectate lyase, pecCl1, was previously identified in a suppressive subtractive library of bean infected with C. lindemuthianum. Isolation of the gene pecCl1 made it possible to obtain mutants and to analyze the regulation of this gene during development of anthracnose, determining whether the pectate lyase is a pathogenic factor. Thus, the aim of our study was structurally and functionally characterize the gene encoding pectate lyase in C. lindemuthianum. Initially, was performed the structural analysis of the gene pecCl1. The complete nucleotide sequence of the gene pecCl1 was deposited in Genbank with accession number JX270683. The analysis of the promoter region revealed some putative cis-elements and potential binding motifs of transcription factors involved in the regulation of pectate lyase gene expression. The deduced amino acid sequence of pecCl1 showed sequence identity with the pectate lyase F of Colletotrichum higginsianum and the pectate lyase C of Glomerella graminicola M1.001. Furthermore, it was found putative conserved domain pfam03211 of the pectate lyases superfamily. The gene pecCl1 is represented by a single copy in the C. lindemuthianum genome. However, into the genome of Colletotrichum graminicola, three sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, and into the genome of C. higginsianum seven sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, indicating that the C. lindemuthianum genome can possess other genes encoding pectate lyase. Phylogenetic analysis of pectate lyase amino acid sequences of filamentous fungi exhibited the formation of two distinct groups which are grouped on the basis of members of the pectate lyases multigene family. The Split-Marker technique was effective in C. lindemuthianum pecCl1 gene inactivation, allowing the study of pecCl1 function in a mutant by specific integrations and without ectopic integrations. The pecCl1 gene inactivation did not lead to complete loss of the pectate lyase activity, and consequently only decreased anthracnose symptoms in its host, which is consistent with the presence of other genes coding pectate lyase, allowing greater flexibility in pathogen aggressiveness. The analysis of differential expression of gene pecCl1 by qPCR was performed at different stages of bean infection and were observed expression levels of pecCl1 at all stages of development of the fungus in the plant, but a significant increase was observed five days after infection, in the onset of necrotrophic stage. At this stage, secondary hyphae cause extensive degradation of plant cell wall through the secretion of wide range of depolymerases, among these, the pectate lyase. Thus, the pectate lyase encoded by the gene pecCl1 is important to aggressiveness of C. lindemuthianum. The analysis of pectate lyases in C. lindemuthianum can not only assist in understanding the disease, but may also lead to discovery of one more target for disease control. / Colletotrichum lindemuthianum é o agente causal da antracnose do feijoeiro comum. Genes que codificam enzimas que degradam a parede celular são essenciais para o desenvolvimento dessa doença. As pectinases são caracterizadas como o grupo de enzimas que hidrolisam a parede celular mais importante produzidas por fungos fitopatogênicos. O gene pecCl1, que codifica pectato liase, foi previamente identificado em uma biblioteca subtrativa supressiva de feijoeiro infectado com C. lindemuthianum. O isolamento do gene tornou possível a obtenção de mutantes e análise da regulação deste gene durante o desenvolvimento da antracnose, visando determinar se a pectato liase é um fator de patogenicidade. Desta forma, o objetivo do nosso trabalho foi caracterizar estruturalmente e funcionalmente o gene que codifica pectato liase em C. lindemuthianum. Inicialmente, foi realizada a análise estrutural do gene pecCl1. A sequência completa de nucleotídeos do gene pecCl1 foi deposita no Genbank com número de acesso JX270683. A análise da região promotora revelou alguns possíveis cis-elementos e sítios de ligação a fatores de transcrição envolvidos na regulação da expressão gênica da pectato liase. A sequência de aminoácidos deduzida de pecCl1 apresentou identidade de sequências com a pectato liase F de Colletotrichum higginsianum e a pectato liase C de Glomerella graminicola M1.001. Além disso, detectou-se um possível domínio conservado pfam03211 da superfamília de pectato liases. O gene pecCl1 encontra-se representado por uma cópia única no genoma de C. lindemuthianum. No entanto, no genoma de Colletotrichum graminicola, três sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, e no genoma de C. higginsianum sete sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, indicando que o genoma de C. lindemuthianum pode possuir além do gene pecCl1 outros genes que codificam pectato liase. A análise filogenética de sequências de aminoácidos de pectato liases de fungos filamentosos mostrou a formação de dois grupos distintos, que se agruparam com base nos membros da família multigênica de pectato liases. A técnica de Split-Marker mostrou-se eficiente na inativação do gene pecCl1 de C. lindemuthianum, possibilitando o estudo da função do gene pecCl1, em um mutante com integração específica e livre de integrações ectópicas. A inativação do gene pecCl1 não levou a perda completa da atividade de pectato liase, e consequentemente, somente diminuiu os sintomas de antracnose em seu hospedeiro, o que é consistente com a presença de outros genes que codificam pectato liase no fungo, permitindo ao patógeno uma maior flexibilidade em sua agressividade. Foi realizada a análise da expressão diferencial do gene pecCl1 por qPCR nos diferentes estágios de infecção no feijoeiro e foram observados transcritos de pecCl1 em todas as fases de desenvolvimento do fungo na planta, mas houve um aumento significativo destes transcritos cinco dias após a infecção, no início da fase necrotrófica do fungo. Nesta fase, as hifas secundárias causam degradação extensiva da parede celular vegetal por meio da secreção de vasta gama de despolimerases, dentre estas, a pectato liase. Portanto, a pectato liase codificada pelo gene pecCl1 é importante para agressividade de C. lindemuthianum. A análise de pectato liases poderá não somente auxiliar na compreensão da antracnose em feijoeiro comum, mas também poderá levar a descoberta de mais um alvo para o controle dessa doença.

La réduction de la dépendance aux pesticides des systèmes de culture de laitue constitue un objectif à relativement court terme, partagé par les pouvoirs publics et les acteurs de la filière de production. Atteindre cet objectif suppose de disposer de moyens techniques permettant de substituer aux pesticides d’autres méthodes limitant les dégâts de bio-agresseurs de la laitue.Ces techniques peuvent être destinées à minimiser l’entrée d’inoculum dans les parcelles, à limiter la propagation des bio-agresseurs, à diminuer la sensibilité des plantes, ou à éradiquer les bio-agresseurs présents sur les cultures. L’évolution des systèmes de culture repose sur la démonstration de l’efficacité, en termes de protection des plantes, de ces techniques alternatives aux pesticides lorsqu’elles sont intégrées et combinées dans les itinéraires techniques. Elle dépend aussi de l’impact socio-économique et environnemental des itinéraires techniques alternatifs, dans un contexte commercial et réglementaire exigeant. L’objet de cette thèse a été de développer des stratégies alternatives de gestion des bio-agresseurs de la laitue d’hiver cultivée sous abri froid,plus économes en pesticides, et d’évaluer leurs performances agronomiques, économiques et environnementales,garantes de la durabilité des systèmes de production.Sur la base des techniques déjà disponibles, deux stratégies alternatives, nommées stratégie intermédiaire et stratégie bas-intrant ont été conçues, et testées dans deux exploitations agricoles et un domaine expérimental de l’INRA pendant deux hivers, en comparaison avec une stratégie conventionnelle, représentative des pratiques actuelles. Les stratégies intermédiaire et bas-intrant ont permis de réduire de 32% et 48% l’usage des pesticides, respectivement, et ont été suffisamment efficaces pour obtenir une qualité visuelle et des rendements équivalents à la stratégie conventionnelle. Le bénéfice environnemental de leur mise en oeuvre a également été démontré. La mise en place de ces stratégies a cependant entrainé un surcoût de production,essentiellement lié à la lutte biologique, d’environ 10 à 13%. Parallèlement, deux orientations techniques originales, dont l’efficacité n’était pas caractérisée, ont été explorées. Une première série d’essais expérimentaux a porté sur l’influence du génotype de l’hôte et de l’environnement de la plante sur sa sensibilité à deux champignons pathogènes d’importance majeure, Botrytis cinerea et Sclerotinia sclerotiorum. Il a été montré que le choix d’un génotype moins sensible couplé à une optimisation du rapport fructose : saccharose de la plante diminuait les symptômes observés après inoculation. Une deuxième démarche expérimentale, portant sur la lutte biologique contre le puceron Nasonovia ribisnigri, a révélé l’incapacité de parasitoïdes du genre Aphidius à contrôler les pucerons en fin de culture, lorsque la structure du couvert végétal devient trop complexe.En plus d’inscrire ces travaux de recherche dans un processus d’amélioration continue de l’efficacité et des performances des stratégies alternatives aux pesticides, la démarche scientifique utilisée, articulant approche analytique et approche intégrative, a permis d’étudier des techniques ayant potentiellement un impact sur plusieurs bio-agresseurs, telles que la réduction de la fertilisation azotée ou l’optimisation de l’espacement entre les laitues, qui pourraient permettre une meilleure gestion des pathogènes responsables de la pourriture du collet et des pucerons. / The reduction of pesticide reliance in lettuce cropping systems is a short term objective sharedby public authorities and by the stakeholders of lettuce market. Reaching this goal implies thesubstitution of pesticides by others techniques which may limit pest and pathogen damage. Thesetechniques can be intended to mitigate pest and pathogen invasion or propagation, to increaseplant defenses or to remove pests and pathogens from the crop. The improvement of currentcropping systems relies on i) the demonstration of the ability of alternative techniques to managediseases and pests when they are combined and integrated during the crop cycle; ii) theassessment of the socio-economic and environmental impacts of alternative strategies in a stringentcommercial and regulatory context. The objective of this thesis was to design alternativestrategies for pest and pathogen management of lettuce grown in winter under shelter, with fewerpesticide applications, and to assess their agronomic, economic and environmental performancesso as to ensure the sustainability of production.Based on currently available techniques, two alternative strategies, called intermediate andlow-input, were designed, and tested in two farms and an INRA experimental station duringtwo winters and compared to a conventional strategy representing current practices. The alternativestrategies enabled to reduce pesticide applications by 32% and 48% respectively. Theywere efficient enough to obtain similar yield and quality as compared to the conventional strategy.The environmental benefits of their implementation were also demonstrated. However, thesestrategies required a 10-13% extra production costs, almost exclusively due to biological controlproducts.In parallel, two original technical orientations, which efficacy had not been previously described,were examined. In a first set of experiments, the impact of lettuce genotype and growthconditions on plant susceptibility to two major pathogens, Botrytis cinerea and Sclerotinia sclerotiorum,was investigated. The use of a genotype displaying low susceptibility to these fungi,associated with an optimization of the ratio of fructose : sucrose in plants, appeared to reducethe symptoms after inoculation. A second experimental approach, dealing with biological controlagainst the aphid Nasonovia ribisnigri, highlighted the inability of Aphidius parasitoids tocontrol aphid populations at the end of the crop cycle, when the canopy structure becomes toocomplex.In addition to a contribution to cropping systems improvement, the scientific method used,combining analytical and integrative approaches enabled us to highlight the effect of techniquesacting on several pests and/or pathogens, such as the reduction of nitrogen fertilization or theoptimization of plant spacing, which could improve the management of fungi causing basal rotas well as aphids.

Stagonospora nodorum blotch (SNB), is a major foliar disease of wheat in the mid-Atlantic U.S., is caused by the necrotrophic fungus Parastagonospora nodorum. SNB is managed using cultural practices, resistant varieties, and foliar fungicides. There are increasing trends of severity and incidence of SNB in Virginia and the surrounding mid-Atlantic region, but it is not known if changes in the pathogen population are contributing to this trend. The overall goal of this research was to 1) determine the occurrence of quinone outside inhibitor (QoI) resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. For Objective 1, QoI resistant isolates of P. nodorum were identified from Virginia wheat fields, and this was the first report of QoI resistant P. nodorum in the United States. The G143A substitution in the cytochrome b gene of P. nodorum was associated with reduced QoI sensitivity, and in Objective 2, a state-wide, two-year survey of P. nodorum populations in Virginia determined that the G143A mutation was widespread in the state and among sampled fields the frequency ranged from 5-32% (mean = 19%). For Objective 3, P. nodorum was isolated from five different wheat cultivars across seven locations over two years in Virginia. SNB severity varied by cultivar but greater differences in disease severity were observed among locations and years suggesting environment plays an important role in SNB development. Among the necrotrophic effector (NE) genes examined, SnTox1 was predominant followed by SnTox3, and frequencies of NE genes did not vary by cultivar or location. P. nodorum populations in Virginia had high genetic diversity, but there was no genetic subdivision among locations or wheat cultivars from which individuals were isolated. Results also indicated that the P. nodorum population in Virginia undergoes a mixed mode of reproduction, but sexual reproduction made the greatest contribution to population structure. Overall, this work provides insights into the population biology of P. nodorum in Virginia and information on variability in fungicide sensitivity and cultivar susceptibility to SNB that has implications for the current and future efficacy of fungicides and host resistance for management of SNB. / Doctor of Philosophy / Wheat (Triticum aestivum L.) is one of the major cereal crops grown worldwide for food, feed, and other products. However, yields of this crop are often limited by fungal diseases including Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum. Increasing trends of severity and incidence of SNB may be due to reduced sensitivity of P. nodorum to fungicides or increased virulence of P. nodorum populations on commonly grown cultivars. Fungicides such as quinone outside inhibitors (QoIs) are one of the major classes of fungicides used for disease control and G143A substitution is the most common point mutation associated with complete resistance to QoIs. Therefore, the overall goal of this research was to better understand genotypic and phenotypic variation in Virginia populations of P. nodorum in the context of fungicide sensitivity and susceptibility of wheat cultivars to SNB. The specific objectives were to 1) determine the occurrence of quinone outside inhibitor (QoI) fungicide resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring QoI fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. Results from this research indicate that QoI fungicide resistance occurs in Virginia populations of P. nodorum due to a target site mutation (G143A substitution in the cytochrome b gene), and this mutation is widespread and relatively common in Virginia wheat fields. Based on a multi-year multilocation study, P. nodorum populations were genetically diverse, but there was no genetic subdivision among locations or wheat cultivars. SNB severity varied by location and cultivar, but disease severity was greatest at site-years with moderate springtime temperatures and high rainfall. Overall, this work contributes to a better understanding of P. nodorum populations including the current efficacy of fungicides and host resistance for management of SNB in the region.