Quantitative resistance is controlled by multiple genes and has been shown to be a durable form of resistance to pathogens affecting cultivated crops including soybeans (Glycine max L. Merr). Root rot of soybean caused by Phytophthora sojae ranks among the most damaging soybean diseases. Quantitative resistance has proven durable in soybean against P. sojae, however the molecular mechanisms underlying this form of resistance are still unknown. The objective of this project is to gain insight into molecular basis of quantitative resistance in the soybean-P. sojae pathosystem. The approach was to use global transcriptional profiling based on microarray technology to identify genes that were differentially expressed in four cultivars of soybeans with varying levels of quantitative resistance at different time points during infection by P. sojae. Our results provide a better understanding of the potential regulatory factors that may contribute to quantitative resistance during early hours of P. sojae infection. / Ph. D.

Doctor of Philosophy / Department of Plant Pathology / Jan E. Leach / Because it is predicted to be durable and broad spectrum, quantitative trait loci (QTL)-based resistance is an important option for rice disease control. However, manipulation of this type of resistance requires knowledge of the contributing genes. This study demonstrates the contribution of two of three defense response (DR) genes to QTL-governed resistance, and identifies a third gene that negatively regulates resistance. The contribution to QTL-governed resistance of one of nine rice OsPAL genes, which encode phenylalanine ammonia-lyase, was determined using reverse genetics. Mutant ospal4 contains a 750 bp deletion in OsPAL4 and was identified using a PCR-pooling strategy. OsPAL4 underlies a QTL on chromosome 2, and is located in cluster with three other OsPAL members. Rice lines mutated in OsPAL4 are more susceptible to a virulent strain of Xanthomonas oryzae pv. oryzae (Xoo) than lines with the wild type allele. RNAi suppression was used to evaluate the contributions of genes encoding oxalate oxidase-like proteins (OsOXL) and a 14-3-3 protein (GF14-e) to disease resistance. Silencing of 12 OsOXL genes clustered on chromosome 8, varied from suppression of a few gene members to silencing of all expressed family members. Screening of transgenic lines by challenge with Magnaporthe grisea (Mg), the rice blast pathogen, revealed that the more chromosome 8 OsOXL genes suppressed, the more susceptible the plants were to Mg. GF14-e co-localizes with a disease resistance QTL on chromosome 2. Specific suppression of GF14-e by RNAi silencing did not result in enhanced susceptibility to Mg. Instead, the lines exhibited spontaneous HR-type lesions. The presence of this lesion mimic phenotype correlated with enhanced resistance to a virulent strain of Xoo, suggesting that the GF14-e encoded 14-3-3 protein functions as a negative regulator of plant cell death and bacterial resistance in rice. This study supports the hypothesis that DR genes, such as OsOXL and OsPAL4 contribute to disease resistance governed by QTL. The role of GF14-e is less clear, however its down regulation may contribute to QTL-governed resistance. Thus, incorporation of regions harboring the effective DR gene alleles into rice will enhance broad spectrum and durable resistance.

Parmi les Prunus, genre botanique d’intérêt économique important, l’abricotier (Prunusarmeniaca L.) est une culture emblématique du Bassin Méditerranéen. Il y est soumis à des contraintes biotiques importantes, parmi lesquelles le dépérissement bactérien, causé par Pseudomonas syringae (Psy), peut mener à des phénomènes de mortalité en verger au niveau des régions à hivers froids et humides. La mise en évidence de différences variétales en verger offre potentiellement des perspectives de contrôle de la maladie à travers le levier génétique. Aussi, ce travail de thèse avait pour principaux objectifs (i) d’identifier chez la plante des régions génomiques liées à la résistance partielle à la bactérie et (ii) d’étudier un plan factoriel d’interaction entre les diversités de la plante et de la bactérie (GxG) afin d’apprécier la généricité de la résistance et sa durabilité. Afin de répondre au premier objectif, deux approches complémentaires ont été mobilisées : une cartographie de QRLs (Quantitative Resistance Loci) sur quatre populations biparentales dont trois sont issues du croisement avec un géniteur commun ainsi qu’une analyse d’association sur une core-collection. Les données phénotypiques mobilisées correspondent à des symptômes issus d’inoculations contrôlées ainsi que des notes de mortalité obtenues suite à infection naturelle en verger. Ces deux approches (analyse de liaison et d’association) ont permis de mettre en évidence 22 QRLs de résistance, parmi lesquels seuls 2 QRLs sur les chromosomes 6 et 7 colocalisent entre les deux approches. Deux régions majeures détectées en étude d’association sur les chromosomes 5 et 6 se sont révélées être en déséquilibre de liaison et contrôlent près de 26 et 43% de la variation des symptômes. Deux mécanismes complémentaires reposant sur le blocage de l’infection de Psy et sur la limitation de la progression locale de la bactérie dans les tissus ont été mis en évidence à travers la détection de QRLs sur les chromosomes 3, 6, 8 d’une part et 1,4et 6 d’autre part. Le second objectif a été abordé grâce à une étude d’un plan factoriel d’interaction entre 20 accessions d’abricotier et 9 souches de Psy, échantillonnées d’après la connaissance de l’épidémiologie de la maladie en verger. L’analyse statistique de ce dispositif mis en œuvre à la fois en verger et en laboratoire a démontré la prédominance de l’effet du facteur souche dans la variabilité des symptômes étudiés et la très faible importance du facteur d’interaction GxG, indiquant une potentielle généricité des facteurs de résistance et des perspectives favorables à leur durabilité en verger.Les résultats issus de cette thèse contribuent à offrir une meilleure compréhension des mécanismes de résistance partielle au dépérissement bactérien de l’abricotier et fournissent des marqueurs et haplotypes, potentiellement mobilisables dans le cadre de programmes d’innovation variétale. / Within the genus Prunus, that contains highly valuable species, apricot (Prunusarmeniaca L.) is an emblematic Mediterranean crop. But apricot cultivation is constrainedby many biotic stresses, among which bacterial canker caused by Pseudomonas syringae(Psy) is particularly severe and can lead to the death of the trees in regions with humidand cold winters. Differences of susceptibilities have been observed between cultivars inorchards and create opportunities for disease management through genetic improvement.This thesis aimed to (i) identify genetic determinants linked to partial resistance to thebacterium and to (ii) study a factorial interaction design between both diversities of theplant and the pathogen (GxG interaction) in order to assess resistance genericity anddurability. With regard to the first objective, two complementary approaches were used :QRL (Quantitative Resistance Loci) mapping over four biparental progenies, amongwhich three were obtained with a cross involving a common genitor, and a genome-wideassociation study on a core-collection. The phenotypic data mobilized in this work rely onsymptoms issued from controlled inoculations and on mortality notations followingnatural infections in the orchard. These approaches led to the detection of 22 QRLs amongwhich only 2 QRLs, located on chromosomes 6 and 7, co-localized between the twomethods. Two main regions detected in the association study, over the chromosomes 5and 6, appeared to be in linkage disequilibrium and controlled 26 and 43% of the variationof the symptoms. A complementarity between two mechanisms, one that involves blockingthe infection of Psy and the other that limits bacterial mobility in the tissues has beenrevealed through the detection of QRLs over chromosomes 3, 6, 8 for one mechanism and1,4, 6 for the other, respectively. The second objective was fulfilled with a study of afactorial interaction design between 20 apricot accessions and 9 Psy strains, which weresampled according to the previous knowledge of the disease epidemiology in the orchard.Statistical analyses of phenotypic data obtained both from the orchard and a laboratorytest showed a clear predominance of the strain effect on symptom variability and a weakimportance of the GxG interaction factor. This last result highlighted a potentialgenericity of the resistance factors and favorable perspectives of durability in the orchard.The results issued from this thesis contribute to a better understanding of the mechanismsunderlying partial resistance of apricot to bacterial canker. Moreover, it provide markersand haplotypes of interest which could be mobilized in breeding programs.

La maladie de taches noires (TN ; champignon Leptosphaeria lindquistii) est une maladie importante en France. L'étude présentée dans cette thèse fournit des informations utiles sur l’épidémiologie de TN et sur la résistance quantitative du tournesol contre TN. Des expérimentations ont été menées sur petites parcelles au champ, plantes adultes (serre), et plantules (phytotron) pour (1) caractériser la dynamique spatiotemporelle de TN, (2) identifier les traits morphologiques affectant TN via des processus d’esquive de la maladie et en utilisant une procédure standardisée d'évaluation de la maladie, et (3) identifier des prédicteurs de résistance quantitative à TN. Cette étude suggère que (1) TN est associée à des épidémies monocycliques dans le sud ouest de la France, (2) les niveaux faibles de TN sont associés à des plantes ayant un grand nombre de feuilles vertes et de de grande taille, et (3) des prédicteurs de résistance quantitative à BS peuvent être identifiés expérimentalement. / Phoma black stem (BS) is caused by the fungus Leptosphaeria lindquistii, and is an important disease in France. The study presented in this dissertation provides useful information on BS epidemiology and sunflower quantitative resistance against the disease. Experiments were conducted on plants grown in small plots (field), adult plants (greenhouse), and seedlings (growth chamber) in order to (1) characterize the spatiotemporal dynamics of BS, (2) identify morphological traits affecting BS through disease escape processes and utilizing a standardised disease assessment procedure, and (3) identify predictors of quantitative resistance to BS. This study suggests that (1) BS is primarily associated to monocyclic epidemics in south west France, (2) low BS levels are associated with sunflower plants characterized by a large number of green leaves and large height, and (3) predictors of quantitative resistance to BS can be experimentally identified.

Brachypodium distachyon è stato recentemente proposto come pianta modello per le Triticeae che includono frumento e orzo. L’obbiettivo del presente studio è stato quello di identificare regioni genomiche associate con la resistenza quantitativa alla ruggine fogliare in Brachypodium. Le malattie causate dalle ruggini fogliari causano ingenti perdite in termini di produzione delle specie cerealicole. Una popolazione di 110 individui F2 è stata sviluppata incrociando due linee inbred di Brachypodium e una mappa di linkage di marcatori AFLP è stata create. La mappa di linkage consiste di 192 loci AFLP in dieci gruppi di linkage, e copre una lunghezza pari a 1,231 Kosambi cM. Allo scopo di identificare loci coinvolti nella resistenza quantitativa sulla mappa, i 110 individui F2 sono stati valutati per la loro reazione alla ruggine fogliare allo stadio di plantula e a quello adulto. Per confermare i risultati delle piante F2, le rispettive famiglie F3 sono state studiate per la loro resistenza alla ruggine fogliare in due esperimenti indipendenti. Due loci genomici sembrano essere maggiormente coinvolti nella resistenza. / Brachypodium distachyon has been proposed as a model species for the tribe of the Triticeae, which includes wheat and barley. The objective of our study was to identify the genomic regions associated with quantitative resistance to leaf rust in Brachypodium. Leaf rust diseases cause significant reductions annually in yield of cereal crops worldwide. An F2 mapping population of 110 individuals was generated between two Brachypodium inbred lines and a AFLP-based linkage map was developed. The linkage map consists of 192 AFLP loci in ten linkage groups, and spans a total genetic length of 1,231 Kosambi cM. To locate quantitative resistance loci on the map, the 110 F2 plants were evaluated for their reaction to the leaf rust at both seedling and adult plant stages. To improve QTL identification, F2-derived F3 families were studied for resistance to leaf rust in two independent experiments. Two major genomic regions involved in resistance to leaf rust were detected.

Leptosphaeria maculans est un champignon pathogène, responsable de l’une des principales maladies du colza (Brassica napus), la nécrose du collet. Le cycle de vie infectieux de L. maculans est particulièrement complexe. Après l’infection primaire des feuilles et des cotylédons, le champignon développe une longue phase de vie endophytique dans la tige. Cette phase de vie, qui est entièrement asymptomatique, dure plusieurs mois, avant que la nécrose ne se développe à la base de la tige, préjudiciable à l'élaboration du rendement. Durant cette phase, le colza peut présenter une « résistance adulte » limitant l'apparition et la gravité des symptômes. Alors que les gènes fongiques exprimés au cours de l’infection primaire du colza sont largement étudiés, très peu de connaissances étaient disponibles concernant la phase de colonisation systémique. Pour expliquer la capacité du champignon à coloniser la tige sans induire de symptômes, nous avons donc émis l’hypothèse que L. maculans exprimait à ce stade des effecteurs, c'est-à-dire des petites protéines sécrétées, interférant avec le système de défense de la plante. L’objectif de ma thèse était d'identifier de tels effecteurs et de les caractériser afin de mieux comprendre la colonisation systémique du colza par le champignon. Un des enjeux sous-jacents de cette thèse était aussi d'identifier de nouvelles résistances permettant la reconnaissance spécifique de ces effecteurs, qui pourraient expliquer, au moins en partie, la résistance adulte observée dans certaines variétés.Par une approche transcriptomique, j'ai pu identifier 307 effecteurs candidats "tardifs", spécifiquement exprimés lors de la colonisation de la tige et 107 effecteurs "précoces", spécifiquement exprimés lors de la colonisation des cotylédons. J’ai confirmé que les gènes codant des effecteurs précoces de L. maculans sont spécifiquement localisés dans les régions pauvres en gènes et riches en éléments répétés du génome fongique. A l'inverse les gènes codant des effecteurs candidats tardifs sont absents de ces régions et sont localisés dans les régions riches en gènes du génome. Les effecteurs de L. maculans ont donc une localisation génomique distincte en fonction de leur profil d'expression.Une analyse approfondie de cinq de ces effecteurs tardifs a permis de montrer leur conservation dans les populations naturelles de L. maculans et leur implication dans la suppression de la mort cellulaire végétale. Ces résultats associés à l'analyse de leur profil d'expression dans des échantillons de tige issus du champ au cours d'une saison culturale ont permis de proposer le modèle suivant: L. maculans coloniserait la tige de colza en sécrétant des effecteurs supprimant la mort cellulaire et donc interférant avec les défenses de la plante. A la fin de la saison culturale, la diminution de l'expression de ces effecteurs permettrait au champignon de passer d'un stade de vie biotrophe à un stade de vie nécrotrophe et d’induire la nécrose au collet. Cette transition entre les deux modes de vie serait donc basée sur un équilibre entre effecteurs supprimant la mort cellulaire et effecteurs induisant la mort cellulaire.Dans le but d'identifier de nouvelles sources de résistance spécifiques et/ou faciliter l’identification de résistances quantitatives dans le matériel végétal, j'ai créé des souches fongiques sur-exprimant précocement des effecteurs tardifs. Avec ces souches transformées j'ai évalué par test cotylédonnaire une grande collection de génotypes de colza pour identifier de potentielles relations de type gène-pour-gène. Une variété présentant une réponse hypersensible à un effecteur tardif a ainsi pu être identifiée, le contrôle monogénique de cette réponse a été validé et sa cartographie génétique effectuée dans deux descendances. Cette approche permet donc effectivement d'identifier de nouvelles sources de résistances pour lutter efficacement contre la nécrose du collet. / Leptosphaeria maculans is a pathogenic fungus, responsible for one of the main diseases of oilseed rape (Brassica napus), the stem canker disease. The infectious life cycle of L. maculans is especially complex. After the primary infection of leaves and cotyledons, the fungus develops a long endophytic stage in the stem. This infection stage, which is entirely asymptomatic, lasts several months before necrosis develops at the stem base, responsible of yield loss. At this stage, the oilseed rape may exhibit "adult resistance" limiting the onset and severity of symptoms. While the fungal genes expressed during the primary infection are extensively studied, very little knowledge was available concerning the systemic colonization. To explain the ability of the fungus to colonize the stem without inducing symptom, we have therefore hypothesized that L. maculans expressed effectors, i.e. small secreted proteins, interfering with the plant defense system.The objective of my thesis was to identify such effectors and to characterize them to better understand the systemic colonization of oilseed rape by L. maculans. One of the underlying challenges of this thesis was also to identify new resistances allowing the specific recognition of these effectors expressed during stem colonization, and which may explain, at least in part, the adult resistance observed in some varieties.Using a transcriptomic approach, I was able to identify 307 "late" effector candidates specifically expressed during stem colonization and 107 "early" effector candidates specifically expressed during cotyledon colonization. I confirmed that the genes encoding early effectors of L. maculans are specifically localized in gene-poor regions and rich in repeated elements of the fungal genome. Conversely, late candidate effectors are absent from these regions and are located in regions rich in genes of the genome. L. maculans effectors have thus a distinct genomic localization based on their expression profile.A detailed analysis of five of these late effectors showed their conservation in the natural populations of L. maculans and their involvement in the suppression of plant cell death. These results, associated with the analysis of their expression profile in stem samples from the fields during a growing season, allowed us to propose the following model: L. maculans would colonize systemically the oilseed rape stem by secreting effectors suppressing cell death and thus interfering with plant defenses. At the end of the growing season, the decreased expression of these effectors would allow the fungus to switch from a biotrophic to a necrotrophic lifestyle and to induce stem canker. This transition between the two ways of life would therefore be based on a balance between effectors suppressing and effectors inducing cell death.In order to identify new specific sources of resistance and / or to facilitate the identification of quantitative resistances in plant material, I created fungal strains over-expressing late effectors during cotyledon colonization. With these transformed strains I evaluated by cotyledonary test a large collection of oilseed rape genotypes to identify potential gene-for-gene. A variety with a hypersensitive response to a late effector was thus identified, the monogenic control of this response was validated and its genetic mapping carried out in two progenies. This approach therefore effectively enables the identification of new sources of resistance for effective control of L. maculans.

No description available.

Fusarium head blight (FHB), caused by Fusarium graminearum Schwabe, is a devastating fungal disease that affects barley production worldwide through damaged kernels, low yields, and production of mycotoxins. The most effective means of control is through the use of FHB resistant cultivars that are developed through gene pyramiding and incorporation of various sources of resistance. Resistance identified in winter barley cultivar Eve has been identified in Virginia Tech's Small Grains Breeding Program. The objectives of this study are to validate resistance and identify additional resistance QTL, and identify tightly linked and diagnostic markers for use in MAS programs. A population of F5:7 RILs derived from the cross Eve/Doyce were evaluated for FHB resistance. FHB incidence (Inc), FHB severity (Sev), Fusarium damaged kernels (FDK), and deoxynivalenol (DON) were assessed over 2014-15 and 2015-16 at Blacksburg, VA, Mount Holly, VA, Kinston, NC, and Lexington, KY. A QTL on chromosome 6H associated with FHB Sev, FDK, and DON was identified. The QTL accounted for as high as 14.5% of the phenotypic variation for DON accumulation, 8.3% for FHB Sev, and 11.87% to 17.63% for FDK. The QTL marker region spans 56.5 to 66.6 cM with flanking markers SCRI_RS_147342 and Bmag0613. QTL for morphological traits, heading date and height, were found in the same region as the FHB resistance traits. Diagnostic SNP makers associated with the FHB resistance QTL identified can be used for FHB resistance identification in MAS breeding programs to incorporate and pyramid resistance QTL and genes into other plant material. / Master of Science

L'enjeu de la thèse est de proposer une stratégie de gestion durable de la résistance génétique à la rouille brune du blé, basée sur des sources de résistance quantitative. Nous proposons d'identifier des résistances se traduisant par une diminution des performances du pathogène sur les différentes phases du cycle infectieux. Ainsi, l'exercice de contraintes diversifiées sur le pathogène devraient ralentir son adaptation et augmenter la durabilité de la résistance. La confrontation d'un ensemble de génotypes de blé à trois isolats de rouille brune a permis de mesurer le niveau de résistance pour cinq composantes en serre (efficacité d'infection, période de latence, taille de lésion, sporulation par lésion, sporulation par unité de surface sporulante), et à différentes étapes de l'épidémie au champ. Nous avons mis en évidence une grande diversité des composantes affectées, et une variabilité importante pour toutes les composantes. Le développement d'un modèle statistique a permis d'établir que l'ensemble des composantes intervient dans la détermination du niveau de résistance à l'échelle épidémique, mais l'efficacité d'infection et la latence sont les composantes qui jouent le rôle le plus important pour déterminer le niveau de résistance au champ. L'impact d'une composante sur le niveau global de résistance change selon les étapes de l'épidémie. Les trois isolats utilisés ont exprimé un profil d'agressivité contrasté vis-à-vis des différentes composantes. La cartographie des QTLs associés aux différentes composantes de résistance a permis d'établir que la diversité phénotypique observée est liée à une diversité génotypique. / The issue of this thesis is to propose a durable management of genetic resistance to wheat leaf rust, based on quantitative resistance. We propose to identify resistance factors reducing pathogen development across the different stages of the infectious cycle. Diversifying constraints exerted by host resistance on the pathogen development should slow down the pathogen adaptation, and increase resistance durability. A set of wheat genotypes was confronted to three leaf rust isolates, and resistance level was measured for five components in the greenhouse (infection efficiency, latent period, lesion size, spore production per lesion, spore production per unit of sporulating tissue), as well as at different stages of field epidemics. Across the germplasm investigated, the resistance components involved were diversified, and their resistance level varied. Developing a statistical model, we established that all the components are involved in the resistance level observed in field epidemics, the most important components being infection efficiency and latent period. The incidence of a component on the field resistance level varied across epidemic stages. The three pathogen isolates used displayed contrasted aggressiveness profiles, according to the different resistance components. QTL mapping of resistance associated to the different components showed that phenotypic diversity corresponded to genotypic diversity.

To improve understanding of the effectiveness of host resistance against Leptosphaeria spp., three aspects of effectiveness of resistance were investigated. With focus on the major Rlm-mediated resistance against L. maculans, changes in effectiveness of Rlm7-mediated resistance to prevent initiation of disease epidemics at the leaf spot stage were investigated in winter oilseed rape field experiments at five sites in the UK over the period with the cropping seasons 2009/2010 - 2013/2014. L. maculans isolates virulent against Rlm7 were identified in the UK. This may be associated with observed changes in lesion phenotypes on the Rlm7 cultivars in field conditions. However, despite increased severity of phoma leaf spotting on Rlm7 cultivars, there was no associated increase in phoma stem canker severity at the end of the cropping seasons. The effectiveness of winter oilseed rape cultivars for control of phoma stem canker (caused by L. maculans or L. biglobosa) was affected by the coexistence of the two Leptosphaeria species in oilseed rape crops. Weather conditions influenced ascospore release of both species and favoured L. biglobosa ascospore release in 2011, resulting in subsequent increased L. biglobosa phoma leaf spotting and stem canker severity. However, coexistence of Leptosphaeria spp. on oilseed rape crops was affected by the cultivar resistance against L. maculans. CE experiments showed that there were interactions between the two Leptosphaeria spp. in planta. Their coexistence on B. napus was influenced by the different host responses that they trigger during host colonisation. Effects of increased temperature on effectiveness of resistance against L. maculans and on severity of symptoms by Leptosphaeria spp. on B. napus were investigated. Increased temperature affected both Rlm4- and Rlm7-mediated resistance, when assessed by phenotypic and molecular techniques. Increased temperature was associated with increased symptom severity, for both L. maculans and L. biglobosa lesions on plants. Cultivar quantitative resistance background increased effectiveness of resistance against phoma stem canker pathogens at increased temperature and should be deployed in in strategies for adaptation to climate change to avoid increased phoma stem canker epidemics in the future.