Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens. The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation. The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oömycete pathogen, H. parasitica. Experiments with the DR5 / Ph. D.

Plants have evolved physical barriers and inducible defense responses to combat microbial pathogens. Inducible responses are mediated by R proteins, which recognize invading pathogens. R proteins must be precisely regulated to provide effective resistance, without inhibiting normal plant growth. However, little is known about R gene regulation under defense-inducing conditions. The interaction between the oomycete Hyaloperonospora parasitica and the model plant Arabidopsis thaliana provides an excellent model system to explore R gene regulation. My research focuses on RPP8, a CC-NBS-LRR gene, which provides resistance to the H. parasitica isolate Emco5. Previous work in the McDowell lab suggested that RPP8 is upregulated during defense responses. My research shows that RPP8 alleles from the Columbia and Landsberg erecta ecotypes are upregulated by H. parasitica and the defense signaling molecule salicylic acid, suggesting a potential feedback loop. RPP8-Ler is also systemically upregulated after infection of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Additionally, RPP8-Ler expression is increased during wounding and heat stress. I also examined the role of regulatory cis elements in the RPP8 promoter. Three W-boxes are essential for basal and inducible RPP8 expression, and are required for resistance to Emco5. The X-box, a unique cis element in the RPP8 promoter, is essential for strong basal expression and wound-induced upregulation, and affects spatial expression of RPP8-Ler. However, the X-box is not required for RPP8-Ler upregulation during pathogen or SA treatment. R genes may be induced as part of global defense responses, which could prime the host for more effective pathogen recognition. / Master of Science

Dans un contexte de réduction des intrants chimiques, l’utilisation des phytosanitaires naturels stimulant l’immunité végétale ouvre la porte vers une nouvelle approche de protection des plantes. Ces composés éliciteurs regroupés sous le terme de «Stimulateurs des Défenses naturelles des Plantes» (SDP) activent le système défensif de la plante la rendant plus résistante aux bio-agresseurs. Les SDP, de nature diverse, se présentent sous forme de composés uniques ou en mélange dans les extraits végétaux. Au cours de ma thèse, nous avons démontré l’activité SDP des extraits de marc de raisin. Les extraits issus de sous-produits de la vigne, marc de raisin rouge, marc de raisin blanc et pépins de raisin induisent diverses réactions de défense au sein de plantes modèles. Nous avons focalisé notre étude sur l’extrait de marc de raisin rouge (EMR) stimulant l’immunité chez le tabac. Infiltré sur feuilles, l’EMR induit la réponse de type HR caractérisée par l’apparition de lésions chlorotiques et accumulation de composés autofluorescents dans les tissus infiltrés. Ces réactions de défense locales ont été observées également chez l’arabette et la tomate. L’EMR déclenche les réponses LAR et SAR avec l’accumulation des transcrits des gènes de défense dans les feuilles de tabac et ce quelque soit son mode d’application (infiltration ou pulvérisation). Le mode d’action de l’EMR a été abordé sur cultures cellulaires de tabac BY-2. L’EMR induit une forte alcalinisation du milieu extracellulaire avec une mobilisation du calcium (Ca2+), l’expression des gènes de défense et la mort cellulaire. Une étude pharmacologique de la mort cellulaire suggère la mise en place de mort cellulaire programmée (PCD) dans les cellules de tabac. La caractérisation de la voie de signalisation activée par l’EMR a été étudiée avec le mutant NahG de tabac incapable d’accumuler l’acide salicylique (SA). Les réponses de défense (HR, LAR et SAR) sont faiblement induites par l’EMR chez le mutant nahG. L’EMR provoque une réponse de type HR fortement réduite avec une faible accumulation des composés autofluorescents et une diminution drastique de l’accumulation des transcrits des gènes PR suggérant l’intervention du SA dans l’induction des réactions de défense. Le degré de protection induit par l’EMR a été déterminé sur le pathosystème tabac/Phytophthora parasitica. Pulvérisé sur feuilles, l’EMR réduit de 45% les zones infectées par l’oomycète. Ce degré de protection semble être le résultat de l’activité antimicrobienne de l’EMR combinée à l’activité SDP. Aucune protection n’a été observée chez le mutant nahG confirmant l’implication de SA dans la résistance induite par l’EMR. Le fractionnement de l’EMR a permis de simplifier la formule active des extraits de raisin et d’identifier un mélange de molécules potentiellement capables d’induire l’activité SDP. Les composés actifs sont de nature polyphénolique et contiennent de la procyanidine B2 capable à elle seule d’induire la réponse de type HR et l’expression de l’antimicrobien PR1. Cependant, il semble que cette molécule agisse en association avec d’autres composés polyphénoliques pour stimuler le système défensif de la plante. / In order to reduce chemical inputs, the use of natural phytosanitary products stimulating plant immunity are emerging approaches in phytoprotection. These elicitor compounds known as "Plant Defense Inducers" (PDI) activate the plant defense system and improve their resistance to pests attack. PDI are single molecule or mixture of compounds extracted from plant. In my thesis, we demonstrated the PDI activity of different grape marc extracts. The winery byproducts, red grape marc extract, white grape marc extract and grape seed extract all induced various defense reactions in several plant models. We focused our study on the red grape marc extract (GME) which stimulates the immunity system in tobacco plants. When infiltrated into tobacco leaves, GME induced HR-like response characterized by the appearance of chlorotic lesions and accumulation of autofluorescent compounds in infiltrated tissues. Similar local defense reactions have been observed in Arabidopsis thaliana and tomato. GME also triggered LAR and SAR responses and induced defense gene transcript accumulation in tobacco leaves after infiltration or spraying. The GME mode of action was studied using the suspension-cultured cells of tobacco BY-2. GME induced rapid alkalinization of extracellular medium with calcium mobilization, expression of defense genes and cell death. A pharmacological approach of this defensive phenomenon suggests the establishment of programmed cell death (PCD) in tobacco cells. The characterization of the signaling pathway activated by GME was studied using tobacco nahG mutant unable to accumulate salicylic acid (SA). Defense responses (HR, LAR and SAR) induced by GME were impaired in the nahG mutant. GME drastically reduced HR-like response symptoms and PR transcript accumulation. These data suggest the implication of SA in the GME-induced plant defense reactions. The GME-induced protection was evaluated in the model pathosystem of compatible interaction between Nicotiana tabacum and Phytophthora parasitica var. nicotianae (Ppn). GME could reduce by 45% the infected areas induced by the oomycete on tobacco leaves. This level of protection was the result of the combined antimicrobial and PDI actions of GME. GME had no protecting effect against Ppn on NahG leaves evidencing the involvement of SA in the GME-induced resistance. GME fractionation led to identification of a bioactive molecule mixture capable of inducing the PDI activity. The active compounds are polyphenolics and involve procyanidin B2 which is by itself able to induce the HR-like response and PR1 transcript accumulation. This compound should act in combination with other polyphenolic molecules to stimulate the full plant defense reactions.

Les extensines sont des glycoprotéines pariétales appartenant à la famille des HRGPs (Hydroxyprolin-rich glycoproteins) impliquées dans plusieurs fonctions telles que la croissance, le développement et la défense des plantes contre les pathogènes. Toutefois, leur mode d’action dans la réponse immunitaire végétale n’est pas encore bien connu et reste à élucider. Les extensines interviennent dans le renforcement de la paroi, un des premiers remparts cellulaires contre les pathogènes, en se liant entre elles de manière intra- et intermoléculaire. Ce « cross-linking » est catalysé par des enzymes peroxydases spécifiques et nécessite une correcte conformation des extensines, laquelle est conférée par leur partie glycosylée. Dans ce projet de thèse, nous avons donc entrepris d’étudier l’impact de la glycosylation des extensines sur la défense racinaire et tenté de caractériser, de manière préliminaire, des peroxydases potentiellement impliquées dans le « cross-linking » chez Arabidopsis thaliana. Des techniques d’immunocytochimie réalisées sur une sélection de mutants affectés dans la glycosylation des extensines ont révélé une modulation de la distribution des extensines dans la racine d’A. thaliana en réponse à une élicitation avec un peptide bactérien, la flagelline 22. L’un des résultats majeurs de cette étude a été de montrer l’importance de l’arabinosylation des extensines dans la colonisation de la racine par l’oomycète pathogène Phytophthora parasitica. Ainsi, l’ensemble de ces résultats nous apermis d’élaborer un modèle proposant d’illustrer l’importance de l’arabinosylation des extensines dans l’organisation et l’architecture de la paroi, modulant ainsi l’adhésion du pathogène sur les cellules de la racine et influençant in fine la colonisation de cette dernière. / Extensins are cell wall glycoproteins involved in various biological processes including plantprotection. However, their mode of action in plant immunity response is not clearly established and remains to be elucidated. Extensins are able to strengthen the cell wall, one of the first cellular barriers against pathogens, through intra- and intermolecular cross-links. This cross-linking is catalysed by specific peroxidase enzymes and requires a correct conformation of extensins conferred by their glycan moiety. This PhD project aimed to investigate the impact of extensin glycosylation in root defence and to characterize, as a preliminary study, the peroxidases potentially involved in the extensin crosslinking in Arabidopsis thaliana. Through immunocytochemistry techniques on mutants impaired withextensin glycosylation, we have revealed that a modulation of extensin distribution occurs in A. thaliana root in response to elicitation with the bacterial peptide, flagellin 22. We have also showed that extensin arabinosylation plays a major role, although probably indirect, in the root colonization by the pathogen oomycete Phytophthora parasitica. We have therefore elaborated a model proposing to illustrate the importance of extensin arabinosylation in the cell wall organization and architecture,modulating pathogen adhesion on root cells and influencing in fine root colonization.

<p>Denna undersökning gjordes för att studera solitära steklars boplatsval speciellt med avseende på spatiala mönster. Aggregat av lämpliga bohål (<em>bibatterier</em>) tillverkades och gjordes tillgängliga förfrilevande steklar. Resultatet visade att steklar tenderar att anlägga bon i närheten av andra stekelbon. Ingen tendens fanns att anlägga bon i centralt belägna positioner. Steklarna verkar också föredra att anlägga bon till vänster om en tänkt mittllinje på bibatterierna. Aggregation kan vara ett sätt att undgå parasitering och predation vid boplatsen. Om detta orsakade aggregationstendensen var det dock något oväntat att inte också en central tendens kunde påvisas. Att steklarna föredrog bohål i bibatteriernas vänstra del är svårt att förklara.</p>

Denna undersökning gjordes för att studera solitära steklars boplatsval speciellt med avseende på spatiala mönster. Aggregat av lämpliga bohål (bibatterier) tillverkades och gjordes tillgängliga förfrilevande steklar. Resultatet visade att steklar tenderar att anlägga bon i närheten av andra stekelbon. Ingen tendens fanns att anlägga bon i centralt belägna positioner. Steklarna verkar också föredra att anlägga bon till vänster om en tänkt mittllinje på bibatterierna. Aggregation kan vara ett sätt att undgå parasitering och predation vid boplatsen. Om detta orsakade aggregationstendensen var det dock något oväntat att inte också en central tendens kunde påvisas. Att steklarna föredrog bohål i bibatteriernas vänstra del är svårt att förklara.

Several oomycete species of the genus Saprolegnia are recognized as devastating fish pathogens and are responsible for the loss of millions of fish annually for the aquaculture industry. Until recently, these pathogens were kept in check using malachite green; however, due to its toxicity, this chemical has now been banned from use. Saprolegnia parasitica is recognized as the major pathogen of aquaculture fish species. The industry is struggling to predict and control S. parasitica outbreaks in fish hatcheries and there is a need for new knowledge regarding the population genetic structure of this pathogen. Random amplified microsatellites were used to compare isolates of S. parasitica collected from a variety of hatchery locations during the period of November 2009 - August 2011, in order to determine the level of genetic variability and determine changes in genetic diversity over time. Allele frequencies of scored characters were graphically compared. Population genetic diversity was measured using Nei’s genetic distance, Shannon’s Information Index, number of polymorphic loci and phylogenetic trees. Due to the presence of Saprolegnia parasitica in the facilities tested, it appears to be ubiquitous in aquaculture facilities and treatment and prevention will be an ongoing concern in aquaculture management. Overall, genetic diversity of S. parasitica isolates was determined to be low with at least some sexual recombination occurring over time. There was a diversity of genotypes collected from the same hatchery on a single day, indicating there was not a single genotype present at a given time point. Genetic profiling, such as used here, could provide facility managers with a new approach to develop a series of best practices to control sporadic outbreaks of disease. Use of these genetic markers and close monitoring of S. parasitica genotypes will permit early detection and sanitation protocols. / Graduate / 2015-04-24 / 0476 / 0792 / 0369 / cren06@uvic.ca

Thesis (M.S.)--Mississippi State University. Department of Entomology and Plant Pathology. / Title from title screen. Includes bibliographical references.

Made available in DSpace on 2016-06-02T18:57:50Z (GMT). No. of bitstreams: 1 5940.pdf: 754678 bytes, checksum: d36d72758fb4a273690a2c5829a7ca95 (MD5) Previous issue date: 2014-05-29 / Financiadora de Estudos e Projetos / Among the phytosanitary problems faced by citrus sector it is highlighted the root rot caused by Phytophthora spp. which affects all stages of development of the citrus trees. Preventive measures associated with chemicals are used to control the disease, but the problems arising from the intensive use of fungicides have increased the interest in developing alternative techniques aimed at agricultural sustainability, among them the use of biofertilizers . Because of these facts, this study aimed to: (i) assess the effect of microbiota and metabolites present in biofertilizers produced from aerobic digestion (BSA) and anaerobic (BSAN) of swine manure on the mycelial growth of P. nicotianae; (ii) determining the effect of the products on inoculate of P. nicotinae in soil and roots of citrus tree, (iii) observing the effect on germination of citrus rootstocks . The results obtained in this study showed that the existing microorganisms and metabolites in biofertilizers affected the development of the colony of P. nicotianae; biofertilizer produced by aerobic digestion of swine manure (BSA) had the best efficiency in the elimination of inoculum of P. nicotianae ; BSA at concentrations of 5 and 10% and BSAN at concentration of 10% favored the germination of mandarin Sunki in substrate infested by the pathogen. Moreover BSA at concentration of 5% had a promising effect on seed germination of Rangpur lime under the same conditions. / Dentre os problemas fitossanitários enfrentados pelo setor citrícola, destacam-se doenças ocasionadas por Phytophthora spp. que afetam todos os estádios de desenvolvimento da planta cítrica. Para o controle dessas doenças são utilizadas medidas preventivas associadas com produtos químicos, porém, os problemas advindos do uso intensivo de fungicidas têm aumentado o interesse em desenvolver técnicas alternativas, visando a sustentabilidade agrícola e, dentre essas o uso de biofertilizante. Diante do exposto, o presente trabalho teve por objetivos: (i) avaliar o efeito da microbiota e dos metabólitos presentes em biofertilizantes, produzidos pela digestão aeróbica (BSA) e anaeróbica (BSAN) de esterco suíno, sobre o desenvolvimento micelial de P. nicotianae; (ii) verificar o efeito dos compostos sobre propágulos de P. nicotinae no solo e nas raízes de citros e, finalmente, (iii) observar o efeito na germinação de sementes de porta-enxertos de citros em substrato inoculado. Os resultados obtidos nesta pesquisa mostraram que os microrganismos e os metabólitos existentes nos biofertilizantes afetaram o desenvolvimento da colônia de P. nicotianae; o biofertilizante produzido pela digestão aeróbica de esterco suíno (BSA) foi o que apresentou melhor eficiência quanto à supressão de inóculo de P. nicotianae; BSA nas concentrações de 05 e 10% e BSAN a 10% favoreceram a germinação das sementes de tangerina Sunki, em substrato infestado pelo fitopatógeno; enquanto que, para o limão cravo, apenas BSA a 5% teve um efeito promissor na germinação das sementes nas mesmas condições.

Made available in DSpace on 2016-06-02T18:57:50Z (GMT). No. of bitstreams: 1 5948.pdf: 2099671 bytes, checksum: a2a82b16af89dab4eb5f364794eebc2f (MD5) Previous issue date: 2014-05-29 / Financiadora de Estudos e Projetos / The species of fungi belonging to the genus Trichoderma can be found in many environments, especially in the rhizosphere, as colonizers of roots and plant growth promoters, for their ability in facilitating the absorption of nutrients and protecting them against attack by pathogens. Thus, this study aimed to test isolates of Trichoderma spp. for application as growth promoting agents and inducers of resistance to Phytophthora nicotianae in citrus rootstocks. First, eighteen isolates of Trichoderma were tested in vitro for their ability in inhibiting mycelial growth of P. nicotianae, in terms of: production of volatiles or termostable antifungical substances and cell free compounds. The growth promotion test was done with three rootstocks: Sunki tangerine, Swingle citrumelo and Rangpur lime, whose height and number of leaves were measured at 30, 60, 90, 120, 150 and 180 days after treatment with the antagonistic; and in the end of the test, the measuring of dry weight for shoots and roots of plants was measured. The seeds of the rootstocks were treated with the Trichoderma spp isolates and, 180 days later, was tested their capacity of resistance induction through pathogen inoculation on stem, assessed by lesion length. The Phytophthora control assay was carried out by seeds microbiolization of tangerine Sunki and Rangpur lime. By the results, it was found that all Trichoderma spp. isolates provided more than 50% inhibition of the pathogen colony, however, no one produced volatile metabolites. Five isolates produced cell free metabolites and, only the ACB-38 produced termostable antifungal substances, capable of inhibiting the pathogen growth. Trichoderma spp. has the potential for rootstocks growth promoting and resistence inducing to P. nicotianae, but the responses vary according to the plant genotype and the type of isolate antagonist applied. Most isolates of Trichoderma spp. tested were able to control P. nicotianae in Sunki tangerine and Rangpur lime plants. / As espécies de fungos pertencentes ao gênero Trichoderma podem ser encontradas em muitos ambientes, principalmente na rizosfera, como colonizadores de raízes de plantas e promotores de crescimento das mesmas, por facilitarem a absorção de nutrientes, além de protegê-las contra o ataque de fitopatógenos. Sendo assim, esse trabalho teve por objetivo selecionar isolados de Trichoderma spp. com potencial de aplicação como agentes promotores de crescimento e indutores de resistência à Phytophthora nicotianae em porta-enxertos de citros. Primeiramente, avaliou-se in vitro a produção de metabólitos voláteis, termoestáveis e livres de células de dezoito isolados de Trichoderma spp., assim como, a atividade antagônica sobre o crescimento micelial do patógeno. Os testes in vivo compreenderam estudos de promoção de crescimento de três porta-enxertos: tangerina Sunki, citrumelo Swingle e limão Cravo, avaliando-se a altura e o número de folhas aos 30, 60, 90, 120, 150 e 180 dias após o tratamento com o antagônico e, a produção de massa seca da parte aérea e raiz das plantas. Microbiolizou-se as sementes dos porta-enxertos e testou-se a indução de resistência pela inoculação no caule, 180 dias após o tratamento, avaliada pelo comprimento de lesão. O teste de controle de Phytophthora foi realizado pela microbiolização de sementes de tangeriana Sunki e limão Cravo. Pelos resultados dos testes in vitro, verificouse que, todos os isolados de Trichoderma spp. proporcionaram mais de 50 % de inibição na colônia do fitopatógeno, porém, nenhum produziu metabólitos voláteis. Cinco isolados produziram metabólitos livres de células e, somente o ACB-38 produziu substâncias antifúngicas termoestáveis, com capacidade para inibir o desenvolvimento do patógeno. Trichoderma spp. têm potencial para promover crescimento de porta-enxertos cítricos, bem como induzir a resistência à P. nicotianae, porém, as respostas variam de acordo com o genótipo da planta e com o isolado do antagonista utilizado. A maioria dos isolados de Trichoderma spp. testada foi capaz de controlar P. nicotianae em plantas de tangerina Sunki e limão Cravo.