Plants can interact with many different organisms on many trophic levels in their life. In this thesis I investigated how three biotic inertactions, powdery mildew infection, caused by Podosphaera ferruginea, predispersal seed predation by Dusky Large Blue (Phengaris nausithous) and Scarce Large Blue (P. teleius) and deer grazing affect life cycle and population biology of the Great Burnet (Sanguisorba officinalis L.). I also analysed their preferences of environmental conditions. Most of studied plant populations growth rates (λ) were lower than 1 and population decreased, but quite surprisingly for more populations vegetative reproduction was more important than reproduction by seeds. All intraction mentioned above affects more generative reproduction, and thus their influence on Great Burnet population dynamics is low, even with modelled maximal possible effect. Environment conditions analysis showed that powdery mildew occurs on sites with more flowering individuals, where spreading is very easy, butterflies preferred high, well noticeable flower heads grown high above other vegetation and host ant presence. For host ants' presence model locality was only significant variable.

[ES] Las cucurbitáceas son la segunda familia de hortícolas más importante a nivel mundial, solo por detrás de las solanáceas. Tradicionalmente su cultivo se ha llevado a cabo en las zonas templadas del planeta. Sin embargo, las condiciones de cambio climático, el comercio internacional y los modelos de agricultura intensiva favorecen la aparición de nuevas virosis y enfermedades fúngicas en zonas donde antes no estaban presentes. En este sentido, resulta esencial el monitoreo periódico de las principales zonas productoras, para así poder detectar los virus y hongos emergentes en cada territorio y adaptar los programas de mejora a los objetivos específicos de cada zona. En el caso concreto del melón (Cucumis melo) existe una gran variabilidad intraespecífica que puede servir como fuente de alelos de resistencia frente a estos patógenos. Sin embargo, las fuentes de resistencia suelen encontrarse dentro del germoplasma silvestre, normalmente originario de África o Asia, y en el que el nivel de domesticación es reducido. Para un mejor aprovechamiento de las accesiones resistentes, resulta necesario un estudio del control genético de los caracteres de interés, que permita localizar las regiones asociadas a la resistencia y diseñar marcadores moleculares asociadas a las mismas. Esto facilita los programas de mejora orientados a la introgresión de las resistencias manteniendo el fondo genético de las variedades de interés En la presente tesis doctoral, durante las campañas de verano de 2019 y 2020, se ha llevado a cabo un estudio de la incidencia y diversidad genética de 9 especies virales potencialmente limitantes para el cultivo de cucurbitáceas en el sur este español. Se ha podido observar que los virus transmitidos por pulgones son prevalentes frente a los transmitidos por mosca blanca. Dentro del primer grupo destacó la presencia de watermelon mosaic virus (WMV), cucurbits aphid borne yellows virus (CABYV) y cucumber mosaic virus (CMV), ya que fueron detectados en todas las zonas y cultivos estudiados, apareciendo frecuentemente en infecciones mixtas. Moroccan watermelon mosaic virus (MWMV) y tomato leaf curl New Delhi virus (ToLCNDV) también fueron detectados en algunas zonas, pero con porcentajes de infección más bajos y normalmente en infecciones mixtas con WMV. Los análisis filogenéticos de los distintos aislados encontrados ha permitido la identificación de 7 nuevos perfiles moleculares de WMV y de aislados recombinantes de CMV, lo que es consistente con los resultados obtenidos en otros países y pone de manifiesto la gran variabilidad de estos patógenos. Las accesiones silvestres de melón recogidas en distintos bancos de germoplasma son un valioso recurso para los programas de mejora genética frente a estreses bióticos. La accesión africana TGR-1551 ha sido descrita previamente como resistente a WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV y el hongo Podosphaera xanthii (Px, razas 1, 2 y 5) agente causal del oídio en melón. Además, es tolerante a la mosca blanca (Bemisia tabaci) y portadora del gen Vat (virus aphid transmission), el cual limita la transmisión de virus por pulgón. Por lo tanto, esta accesión constituye una buena fuente de alelos de resistencia y, al poder utilizar un único parental donante, su uso acortaría los programas de mejora. En el marco de la presente tesis doctoral, mediante el desarrollo de poblaciones segregantes de mapeo y el aprovechamiento de las tecnologías de genotipado masivo se han podido cartografiar los QTLs asociados a la resistencia a CYSDV derivados de esta entrada. En el caso de la resistencia a CYSDV, se han detectado dos QTL en el cromosoma 5. El primero de ellos es de efecto mayor y herencia dominante, estando asociado al desarrollo de síntomas. El segundo QTL, de efecto menor y también de herencia dominante, no confiere resistencia por sí mismo y está asociado a la carga viral durante la infección. Siguiendo una estrategia similar se han podido cartografiar y estrecha / [CA] Les cucurbitàcies són la segona família d'hortícoles més important a nivell mundial, només per darrere de les solanàcies. Tradicionalment el seu cultiu s'ha dut a terme a les zones temperades del planeta. No obstant això, les condicions de canvi climàtic, el comerç internacional i els models d'agricultura intensiva afavoreixen l'aparició de noves virosis i malalties fúngiques en zones on abans no estaven presents. En aquest sentit, resulta essencial el monitoratge periòdic de les principals zones productores, per a d'aquesta manera, poder detectar els virus i fongs emergents en cada territori i adaptar els programes de millora als objectius específics de cada zona. En el cas concret del meló (Cucumis melo) existeix una gran variabilitat intraespecífica que pot servir com a font d'al·lels de resistència enfront d'aquests patògens. No obstant això, les fonts de resistència solen trobar-se dins del germoplasma silvestre, normalment originari d'Àfrica o Àsia, i en el qual el nivell de domesticació és reduït. Per a un millor aprofitament de les accessions resistents, resulta necessari un estudi del control genètic dels caràcters d'interés, que permeta localitzar les regions associades a la resistència i dissenyar marcadors moleculars associats a aquestes. Això facilita els programes de millora orientats a la introgressió de les resistències mantenint el fons genètic de les varietats d'interés. En la present tesi doctoral, durant les campanyes d'estiu de 2019 i 2020, s'ha dut a terme un estudi de la incidència i diversitat genètica de nou espècies virals potencialment limitants per al cultiu de cucurbitàcies en el sud-est espanyol. S'ha pogut observar que els virus transmesos per pugons són prevalents enfront dels transmesos per mosca blanca. Dins del primer grup va destacar la presència de watermelon mosaic virus (WMV), cucurbits aphid born yellows virus (CABYV) i cucumber mosaic virus (CMV), ja que van ser detectats en totes les zones i cultius estudiats, apareixent sovint en infeccions mixtes. Moroccan watermelon mosaic virus (MWMV) i tomatoleaf curl New Delhi virus (ToLCNDV) també van ser detectats en algunes zones, però amb percentatges d'infecció més baixos i normalment en infeccions mixtes amb WMV. Les anàlisis filogenètiques dels diferents aïllats trobats ha permés la identificació de set nous perfils moleculars de WMV i d'aïllats recombinants de CMV, la qual cosa és consistent amb els resultats obtinguts en altres països i posa de manifest la gran variabilitat d'aquests patògens. Les accessions silvestres de meló recollides en diferents bancs de germoplasma són un valuós recurs per als programes de millora genètica enfront d'estressos biòtics. L'accessió africana *TGR-1551 ha sigut descrita prèviament com a resistent a WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV i el fong Podosphaera xanthii (Px, races 1, 2 i 5) agent causal de l'oïdi en meló. A més, és tolerant a la mosca blanca (Bemisia tabaci) i portadora del gen Vat (virus aphid transmission), el qual limita la transmissió de virus per pugó. Per tant, aquesta accessió constitueix una bona font d'al·lels de resistència i, en poder utilitzar un únic parental donant, el seu ús acurtaria els programes de millora. En el marc de la present tesi doctoral, mitjançant el desenvolupament de poblacions segregants de mapatge i l'aprofitament de les tecnologies de genotipat massiu s'ha pogut cartografiar els QTLs associats a la resistència a CYSDV derivats d'aquesta entrada. En el cas de la resistència a CYSDV, s'han detectat dues QTL en el cromosoma cinc. El primer d'ells és d'efecte major i herència dominant, estant associat al desenvolupament de símptomes. El segon QTL, d'efecte menor i també d'herència dominant, no confereix resistència per si mateix i està associat a la càrrega viral durant la infecció. Seguint una estratègia similar s'han pogut cartografiar i estrényer els *QTLs de resistència enfront de Px. En aquest cas es tracta d'una epistàsia dominant-re / [EN] Cucurbits represent the second most important horticultural family worldwide, second only the Solanaceae family. Traditionally, their cultivation has been concentrated in temperate regions across the globe. However, climate change conditions, international trade, and intensive agricultural practices are contributing to the emergence of new viral and fungal diseases in regions where they were previously absent. In this regard, it is crucial to regularly monitor major production areas to detect emerging viruses and fungi specific to each region. This monitoring allows for the adaptation of breeding programs to the unique goals of each area. In the case of melon (Cucumis melo), it exists significant intraspecific variability that can serve as a source of resistance alleles against these pathogens. However, sources of resistance are often found within wild germplasm, typically originating from Africa or Asia, and characterized by limited domestication. To better utilize these resistant accessions, a study of the genetic control of desirable traits is necessary. This study aims to locate regions associated with resistance and design molecular markers linked to these regions. Such an approach streamlines breeding programs focused on introgressing resistance traits while preserving the genetic background of the desired varieties. During the summer campaigns of 2019 and 2020, this doctoral thesis conducted a study on the incidence and genetic diversity of nine viral species potentially affecting cucurbit cultivation in southeastern Spain. It was observed that viruses transmitted by aphids were more prevalent than those transmitted by whiteflies. Within the first group, the presence of watermelon mosaic virus (WMV), cucurbits aphid borne yellows virus (CABYV), and cucumber mosaic virus (CMV) stood out, as they were detected in all the studied areas and crops, often in mixed infections. Moroccan watermelon mosaic virus (MWMV) and tomato leaf curl New Delhi virus (ToLCNDV) were also detected in some areas but with lower infection percentages, typically in mixed infections with WMV. Phylogenetic analyses of the found isolates have identified seven new molecular profiles of WMV and recombinant CMV isolates, which is consistent with results from other countries, highlighting the extensive variability of these pathogens. Wild melon accessions preserved in various germplasm banks represent a valuable resource for breeding programs against biotic stresses. The African accession TGR-1551 has been previously described as resistant to WMV, CYSDV (cucurbit yellow stunting disorder virus), CABYV, and the fungus Podosphaera xanthii (Px, races 1, 2, and 5), which causes powdery mildew in melons. Additionally, it is tolerant to whiteflies (Bemisia tabaci) and carries the Vat gene (Virus Aphid Transmission), limiting virus transmission by aphids. Therefore, this accession constitutes as an excellent source of resistance alleles, and its use, as a single donor parent, can expedite breeding programs. Within the scope of this doctoral thesis, through the development of segregating mapping populations and the utilization of high-throughput genotyping technologies, the QTLs associated with CYSDV resistance from this accession have been mapped. In the case of CYSDV resistance, two QTLs have been detected on chromosome 5. The first of these, with major effects and dominant inheritance, is associated with symptom development. The second QTL, with minor effects and also dominant inheritance, does not confer resistance by itself and is linked to viral load during infection. A similar strategy was employed to map and narrow down the QTLs for resistance against Px. In this case, it involves a dominant-recessive epistasis, with the recessive gene located on chromosome 12 and the dominant gene on chromosome 5, specifically in the same region where the major CYSDV resistance QTL is located. Regarding resistance against WMV, previous studies conducted by the research / This research was funded by the Spanish Ministerio de Ciencia e Innovación (MCIN/AEI/10.13039/501100011033), grant number PID2020-116055RB (C21 and C22), and by the Conselleria d’Educació, Investigació, Cultura i Esports de la Generalitat Valenciana, grant number PROMETEO/2021/072 (to promote excellence groups, cofinanced with FEDER funds). M.L. is a recipient of a predoctoral fellowship (PRE2018-083466) of the Spanish Ministerio de Ciencia, Innovación y Universidades co-financed with FSE funds. / López Martín, M. (2023). Breeding Melons for Resistance to Viral and Fungal Diseases. Exploiting the Multi-Resistant Accession TGR-1551 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202060

[ES] El pimiento (C. annuum) ocupa un importante lugar en la economía agrícola del mundo y en particular para España. La agricultura intensiva empleada en las últimas décadas está demostrando no ser sostenible a largo plazo, esto, y la aceleración del cambio climático, hace necesario la búsqueda de plantas más adaptadas a las nuevas presiones de estresores tanto bióticos como abióticos. Adicional al uso de variedades mejoradas para este tipo de estresores, es importante tomar en cuenta las interacciones que ocurren en el suelo e introducirlas en los programas de mejora genética vegetal. Por ello, esta tesis se ha centrado, por un lado, en la búsqueda de fuentes de resistencia frente a oídio (L. taurica), enfermedad que cada vez está resultando más problemática en pimiento, especialmente en agricultura ecológica. Por otro lado, y teniendo en cuenta la importancia de la sanidad y fertilidad del suelo para la resiliencia de los cultivos, se estudia la interacción del sistema radicular del pimiento con la zona de la rizosfera y la microbiota de la raíz. Como principales resultados obtenidos en la presente tesis se pueden destacar la identificación de fuentes de resistencia a oídio en las accesiones de C. annuum var. glabriusculum, lo que puede facilitar la introducción de genes de resistencia en variedades comerciales de pimientos. Además, se ha demostrado cómo las propiedades de la rizosfera (actividad enzimática, movilización de nitrógeno, respiración) y su microbioma es altamente dependiente de las condiciones de cultivo y del genotipo cultivado. Así se observó que la parcela de cultivo ecológico mostró mayores recuentos microbianos, actividades enzimáticas y movilización de nitrógeno que la convencional además de poseer más diversidad microbiana y mayor recuento de microorganismos. Por último, se observó también una gran especificidad de la interacción suelo-planta, observándose cómo ciertos microorganismos beneficiosos colonizaban preferentemente ciertos genotipos. Esta especificidad debería de ser considerada para futuros programas de mejoramiento para una agricultura más sostenible y adaptada. / [CA] El pebrot (C. annuum) ocupa un lloc important en l'economia agrícola del món i en particular per a Espanya. L'agricultura intensiva emprada en les últimes dècades està demostrant no ser sostenible a llarg termini, açò, i l'acceleració del canvi climàtic, fa necessari la recerca de plantes més adaptades a les noves pressions d'estressors tant biòtics com a abiòtics. Addicional a l'ús de varietats millorades per a este tipus d'estressors, és important tindre en compte les interaccions que ocorren en el sòl i introduir-les en els programes de millora genètica vegetal. Per açò, aquesta tesi s'ha centrat, d'una banda, en la recerca de fonts de resistència front a oídi (L. taurica), malaltia que cada vegada està resultant més problemàtica en pebrot, especialment en agricultura ecològica. D'altra banda, i tenint en compte la importància de la salut i fertilitat del sòl per a la resiliència dels cultius, s'estudia la interacció del sistema radicular del pebrot amb la zona de la rizosfera i la microbiota de l'arrel. Com a principals resultats obtinguts en la present tesi es poden destacar la identificació de fonts de resistència a oídi en les accessions de C. annuum var. glabriusculum, la qual cosa pot facilitar la introducció de gens de resistència en varietats comercials de pebrots. A més, s'ha demostrat com les propietats de la rizosfera (activitat enzimàtica, mobilització de nitrogen, respiració) i el seu microbioma és altament dependent de les condicions de cultiu i del genotip cultivat. Així es va observar que la parcel·la de cultiu ecològic va mostrar majors recomptes microbians, activitats enzimàtiques i mobilització de nitrogen que la convencional, a més de posseir més diversitat microbiana i major recompte de microorganismes. Finalment, es va observar també una gran especificitat de la interacció sòl-planta, observant-se com certs microorganismes beneficiosos colonitzaven preferentment certs genotips. Aquesta especificitat hauria de ser considerada per a futurs programes de millorament per a una agricultura més sostenible i adaptada. / [EN] The pepper (C. annuum) holds an important place in the global agricultural economy and particularly for Spain. Intensive agriculture used in recent decades is proving not to be sustainable in the long term, and, coupled with the acceleration of climate change, it necessitates the search for plants more adapted to new pressures from both biotic and abiotic stressors. In addition to using improved varieties for these types of stressors, it is important to consider the interactions that occur in the soil and incorporate them into plant genetic improvement programs. Therefore, this thesis has focused on one hand, on the search for sources of resistance against powdery mildew (L. taurica), a disease that is increasingly problematic in pepper, especially in organic agriculture. On the other hand, considering the importance of soil health and fertility for crop resilience, the interaction of the pepper root system with the rhizosphere zone and root microbiota is studied. The main results obtained in this thesis include the identification of sources of resistance to powdery mildew in the accessions of C. annuum var. glabriusculum, which can facilitate the introduction of resistance genes in commercial pepper varieties. Furthermore, it has been shown how the properties of the rhizosphere (enzymatic activity, nitrogen mobilization, respiration) and its microbiome are highly dependent on the cultivation conditions and the cultivated genotype. It was observed that the organic farming plot showed higher microbial counts, enzymatic activities, and nitrogen mobilization than the conventional one, in addition to possessing greater microbial diversity and higher counts of microorganisms. Lastly, a high specificity of the soil-plant interaction was also observed, with certain beneficial microorganisms preferentially colonizing specific genotypes. This specificity should be considered for future breeding programs for more sustainable and adapted agriculture. / El presente trabajo no podría haberse llevado a cabo sin el apoyo financiero de la beca predoctoral otorgada por CONACYT-CONCYTEP con el número identificador 47274 por parte del gobierno de México. / Morales Manzo, II. (2023). Mejora genética del chile (Capsicum annuum L.) para estrés biótico: Oídio (Leveillula taurica (Lév.) Arn.) y fertilidad del suelo: retos de la agricultura ecológica y búsqueda de fuentes de variación de interés y factores genéticos relacionados [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/194538

Maîtriser l’impact des maladies sur les cultures est un défi majeur de l’agriculture moderne. Cette préoccupation est un aspect important de l’optimisation de la productivité, notamment en viticulture. En France, le mildiou de la vigne causé par Plasmopara viticola est une des maladies cryptogamiques responsable des épidémies les plus dévastatrices et les plus redoutées. Les traitements reposent sur l’utilisation préventive, systématique et onéreuse de composés chimiques antifongiques dont l’utilisation massive constitue un risque à la fois pour l’homme et l’environnement. La réduction de l’utilisation de fongicide implique le développement d’outils de diagnostic au champ, qui requiert la compréhension des interactions entre la plante et les agents pathogènes. Les travaux de cette thèse pluridisciplinaire ont porté sur le pathosystème Plasmopara viticola - Vitis vinifera, notamment pour répondre à l’intérêt croissant pour un outil de diagnostic en temps réel de la maladie utilisable au vignoble. Les stilbènes sont des phytoalexines impliqués dans la défense de certaines plantes supérieures vis-à-vis de stress biotiques et abiotiques. L’autofluorescence de ces composés phénoliques, dont la biosynthèse est induite dans les feuilles de vigne par P. viticola, en fait un potentiel marqueur naturel de l’infection. En effet, la faible autofluorescence bleu-verte des feuilles de vigne saines est considérablement renforcée par l’autofluorescence violet-bleue des stilbènes à la surface de feuilles de vigne infectée par P. viticola. Cette étude a montré que quelque soit le niveau de résistance du génotype, l’autofluorescence violet-bleue des stilbènes induit par l’infection est présente au niveau des parois des cellules de l’épiderme. En dehors de la concentration, la viscosité s’est révélé être la principale variable physico-chimique influençant l’intensité de l’autofluorescence des stilbènes dans les différents compartiments cellulaires des feuilles de vigne. Ceci explique la fluorescence intense des parois, particulièrement rigides, des cellules de garde (stomates) des feuilles infectées. Le suivi cinétique journalier a révélé la nature transitoire de l’autofluorescence des stilbènes lors de l’infection. La robustesse et l’intérêt de ce signal a également été validée par la mesure à différentes échelles (de la cellule à la feuille entière) et avec différentes méthodes fluorimétriques. Les résultats de ce travail ont permis des avancées sur la connaissance du rôle de composés phénoliques induits et constitutifs dans la défense contre P. viticola. En plus de la localisation de l’autofluorescence des stilbènes en surface des feuilles, la microscopie confocale couplée à la microspectrofluorimetrie a révélé différentes localisations de ces phytoalexines dans la profondeur des tissus en corrélation avec le niveau de résistance des génotypes. L’utilisation de l’autofluorescence des stilbènes comme marqueur de l’infection a permis de mettre en évidence : 1) le fait que les flavonols constitutifs des feuilles de V. vinifera retardent le développement de l’infection par P. viticola; et 2) le fait que les acides hydroxycinnamiques constitutifs ne semble pas participer à la défense contre P. viticola. Enfin, une nouvelle méthode de diagnostic non-destructive du mildiou sur feuille basée sur l’autofluorescence des stilbènes a été développée. Elle a montré une détection pré-symptomatique du mildiou sur les feuilles de vigne entières dès le premier jour après l’infection sur la face abaxiale et le troisième jour sur la face adaxiale. Cette méthode de diagnostic du mildiou a été validée au laboratoire notamment grâce à un prototype de capteur proximal développé en collaboration avec la société Force-A. La validation de la méthode au vignoble dans le cadre d’infection naturelle est la prochaine étape pour une utilisation de ce capteur optique dans le cadre de l’agriculture durable et de la sélection variétale. / Controlling the impact of diseases on crops is a major challenge of modern agriculture. This concern is an important aspect of optimizing productivity, notably in viticulture. In France, downy mildew caused by Plasmopara viticola is a fungal disease responsible for the most devastating epidemics. The preventive and systematic treatments are expensive, while the massive use of antifungal chemicals is a risk to both humans and the environment. Reducing the use of fungicide involves the development of diagnostic tools in the field, which requires understanding the interactions between plants and pathogens. The work of this multidisciplinary thesis focused on the pathosystem Plasmopara viticola - Vitis vinifera, especially to meet the growing interest in a real-time diagnostic tool of disease applicable in the vineyard. Stilbenes are phytoalexins involved in the defense of certain higher plants against biotic and abiotic stresses. The autofluorescence of these phenolic compounds, whose biosynthesis is induced in grapevine leaves by P. viticola, makes it a potential marker of natural infection. Indeed, the low blue-green autofluorescence of grapevine leaves is greatly enhanced by the violet-blue autofluorescence of stilbenes on the surface of leaves infected by P. viticola. This study showed that whatever the level of resistance in various genotypes, violet-blue autofluorescence induced by stilbene is present in the walls of epidermal cells. In addition to their concentration, viscosity proved the main physico-chemical variable affecting the intensity of the autofluorescence of stilbenes in different compartments of vine leaves. This explains the intense fluorescence of the walls, particularly rigid, of guard cells (stomata) of infected leaves. Daily monitoring revealed a kinetic with a transient rise of the autofluorescence of stilbenes during infection. The robustness and value of this signal was also validated by measuring at different levels (cellular to whole leaf) and with various fluorimetric methods (imaging, spectroscopy, proximal sensing). These results advance our understanding of the role of constitutive and induced phenolic compounds in plant defence against P. viticola. In addition to a common location of the autofluorescence of stilbenes on the leaf surface, confocal microscopy coupled with microspectrofluorometry revealed distinctive localizations of these phytoalexins in the deep tissue correlated with the level of resistance in genotypes. This aspect no doubt needs broader testing. The use of autofluorescence of stilbene as a marker of infection allowed us to ascertain that: 1) constitutive flavonols of the leaves of V. vinifera retard the development of infection by P. viticola and 2) the constitutive hydroxycinnamic acids do not seem to participate in the defence against P. viticola. Finally, a new method for the non-destructive diagnosis of leaf infection based on the autofluorescence of stilbenes has been developed. We have demonstrated a pre-symptomatic detection of downy mildew on whole grape leaves from the first day after infection on the abaxial surface and from the third day on the adaxial surface. This method of diagnosis has been validated in the laboratory thanks to a proximal sensor prototype developed in collaboration with the company Force-A. The validation of the method in the vineyard in a context of natural infections is the next step for use of this optical sensor as a tool for sustainable agriculture and for genetic screening.

No presente trabalho, a metodologia NBS-profiling foi utilizada para o desenvolvimento de marcadores RGA (Resistance Genes Analogs) em duas populações de Phaseolus vulgaris, sendo uma derivada do cruzamento entre Bat 93 e Jalo EEP558 (BJ) e a outra derivada do cruzamento entre Carioca e Flor de Mayo (CFM). Uma vez identificados, foram mapeados 32 marcadores RGA na população BJ e 40 na população CFM. Nove dos marcadores alocados no mapa de ligação da população BJ se localizaram em proximidade a clusters de resistência, já identificados por outros pesquisadores. Foi realizado o sequenciamento de 32 dos RGA detectados, sendo 16 da cada população. Cinco sequências oriundas da população BJ e três sequências da população CFM apresentaram similaridade com proteínas de resistência identificadas em P. vulgaris, Glycine max e Medicago truncaluta. O mapa de ligação aqui gerado para a população CFM foi utilizado para o posicionamento de QTL (Quantitative Trait Loci) de resistência à mancha-angular (Pseudocercospora griseola (Sacc.) Crous & Braun) e ao oídio (Erysiphe polygoni DC.). Um total de 12 QTL foi mapeado, cinco associados à resposta à mancha angular e sete à resposta ao oídio. / In the presenty study, the NBS-profiling method was used for the development of RGA (Resistance Gene Analogs) markers in two populations of Phaseolus vulgaris, one derived from a cross between \'Bat 93\' and \'Jalo EEP558\' (BJ) and the other derived from a cross between \'Carioca\' and \'Flor de Mayo (CFM). After their identification, 32 RGA markers were mapped on the BJ population and 40 on the CFM population. Nine of the markers assigned to the linkage map of the BJ population were located in the proximity to clusters of resistance already identified by other researchers. We carried out the sequencing of 32 out of the RGA detected, being 16 from each population. Five sequences derived from the BJ population and three sequences from the CFM population showed similarity to resistance proteins identified in P. vulgaris, Glycine max and Medicago truncaluta. The linkage map here generated for the CFM population was used for the positioning of QTL (Quantitative Trait Loci) for resistance to angular leaf spot (Pseudocercospora griseola (Sacc.) Crous & Braun) and powdery mildew (Erysiphe polygoni DC.). Twelve QTL were mapped, five associated to the response to the angular leaf spot and seven to the powdery mildew.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

No presente trabalho, a metodologia NBS-profiling foi utilizada para o desenvolvimento de marcadores RGA (Resistance Genes Analogs) em duas populações de Phaseolus vulgaris, sendo uma derivada do cruzamento entre Bat 93 e Jalo EEP558 (BJ) e a outra derivada do cruzamento entre Carioca e Flor de Mayo (CFM). Uma vez identificados, foram mapeados 32 marcadores RGA na população BJ e 40 na população CFM. Nove dos marcadores alocados no mapa de ligação da população BJ se localizaram em proximidade a clusters de resistência, já identificados por outros pesquisadores. Foi realizado o sequenciamento de 32 dos RGA detectados, sendo 16 da cada população. Cinco sequências oriundas da população BJ e três sequências da população CFM apresentaram similaridade com proteínas de resistência identificadas em P. vulgaris, Glycine max e Medicago truncaluta. O mapa de ligação aqui gerado para a população CFM foi utilizado para o posicionamento de QTL (Quantitative Trait Loci) de resistência à mancha-angular (Pseudocercospora griseola (Sacc.) Crous & Braun) e ao oídio (Erysiphe polygoni DC.). Um total de 12 QTL foi mapeado, cinco associados à resposta à mancha angular e sete à resposta ao oídio. / In the presenty study, the NBS-profiling method was used for the development of RGA (Resistance Gene Analogs) markers in two populations of Phaseolus vulgaris, one derived from a cross between \'Bat 93\' and \'Jalo EEP558\' (BJ) and the other derived from a cross between \'Carioca\' and \'Flor de Mayo (CFM). After their identification, 32 RGA markers were mapped on the BJ population and 40 on the CFM population. Nine of the markers assigned to the linkage map of the BJ population were located in the proximity to clusters of resistance already identified by other researchers. We carried out the sequencing of 32 out of the RGA detected, being 16 from each population. Five sequences derived from the BJ population and three sequences from the CFM population showed similarity to resistance proteins identified in P. vulgaris, Glycine max and Medicago truncaluta. The linkage map here generated for the CFM population was used for the positioning of QTL (Quantitative Trait Loci) for resistance to angular leaf spot (Pseudocercospora griseola (Sacc.) Crous & Braun) and powdery mildew (Erysiphe polygoni DC.). Twelve QTL were mapped, five associated to the response to the angular leaf spot and seven to the powdery mildew.

Maîtriser l'impact des maladies sur les cultures est un défi majeur de l'agriculture moderne. Cette préoccupation est un aspect important de l'optimisation de la productivité, notamment en viticulture. En France, le mildiou de la vigne causé par Plasmopara viticola est une des maladies cryptogamiques responsable des épidémies les plus dévastatrices et les plus redoutées. Les traitements reposent sur l'utilisation préventive, systématique et onéreuse de composés chimiques antifongiques dont l'utilisation massive constitue un risque à la fois pour l'homme et l'environnement. La réduction de l'utilisation de fongicide implique le développement d'outils de diagnostic au champ, qui requiert la compréhension des interactions entre la plante et les agents pathogènes. Les travaux de cette thèse pluridisciplinaire ont porté sur le pathosystème Plasmopara viticola - Vitis vinifera, notamment pour répondre à l'intérêt croissant pour un outil de diagnostic en temps réel de la maladie utilisable au vignoble. Les stilbènes sont des phytoalexines impliqués dans la défense de certaines plantes supérieures vis-à-vis de stress biotiques et abiotiques. L'autofluorescence de ces composés phénoliques, dont la biosynthèse est induite dans les feuilles de vigne par P. viticola, en fait un potentiel marqueur naturel de l'infection. En effet, la faible autofluorescence bleu-verte des feuilles de vigne saines est considérablement renforcée par l'autofluorescence violet-bleue des stilbènes à la surface de feuilles de vigne infectée par P. viticola. Cette étude a montré que quelque soit le niveau de résistance du génotype, l'autofluorescence violet-bleue des stilbènes induit par l'infection est présente au niveau des parois des cellules de l'épiderme. En dehors de la concentration, la viscosité s'est révélé être la principale variable physico-chimique influençant l'intensité de l'autofluorescence des stilbènes dans les différents compartiments cellulaires des feuilles de vigne. Ceci explique la fluorescence intense des parois, particulièrement rigides, des cellules de garde (stomates) des feuilles infectées. Le suivi cinétique journalier a révélé la nature transitoire de l'autofluorescence des stilbènes lors de l'infection. La robustesse et l'intérêt de ce signal a également été validée par la mesure à différentes échelles (de la cellule à la feuille entière) et avec différentes méthodes fluorimétriques. Les résultats de ce travail ont permis des avancées sur la connaissance du rôle de composés phénoliques induits et constitutifs dans la défense contre P. viticola. En plus de la localisation de l'autofluorescence des stilbènes en surface des feuilles, la microscopie confocale couplée à la microspectrofluorimetrie a révélé différentes localisations de ces phytoalexines dans la profondeur des tissus en corrélation avec le niveau de résistance des génotypes. L'utilisation de l'autofluorescence des stilbènes comme marqueur de l'infection a permis de mettre en évidence : 1) le fait que les flavonols constitutifs des feuilles de V. vinifera retardent le développement de l'infection par P. viticola; et 2) le fait que les acides hydroxycinnamiques constitutifs ne semble pas participer à la défense contre P. viticola. Enfin, une nouvelle méthode de diagnostic non-destructive du mildiou sur feuille basée sur l'autofluorescence des stilbènes a été développée. Elle a montré une détection pré-symptomatique du mildiou sur les feuilles de vigne entières dès le premier jour après l'infection sur la face abaxiale et le troisième jour sur la face adaxiale. Cette méthode de diagnostic du mildiou a été validée au laboratoire notamment grâce à un prototype de capteur proximal développé en collaboration avec la société Force-A. La validation de la méthode au vignoble dans le cadre d'infection naturelle est la prochaine étape pour une utilisation de ce capteur optique dans le cadre de l'agriculture durable et de la sélection variétale.

Plant immune system is entirely based on the immunities of the individual cells in which systemic signals originate from the infection sites. Powdery mildew disease is one of the agents causing these infection sites, resulting in significant yield losses, if disease develops. Understanding the molecular basis of plant-pathogen interactions is the new trend for fighting against plant pathogens, since classical methods used in selection of resistant plants are becoming less and less efficient nowadays. Thus, finding out the genes which are responsible in plant&rsquo / s resistance is becoming very important. In this thesis, effect of &lsquo / General Control Nondepressible-2&rsquo / (GCN2) homolog protein in barley defense mechanism was aimed to be studied. The GCN2 of yeast was v previously identified in our laboratory as an interacting protein when the yeast cDNA library was screened with a putative yellow rust R gene (Yr10) fragment. There are reports available in the literature for the function of GCN2 protein, which makes it a good candidate for a role in disease resistance. Thus, the barley homologue of GCN2 might have a role in the R protein mediated early disease response of which may be proceeding via Programmed Cell Death (PCD). In order to observe such function of HvGCN2 in barley, silencing of its expression via Virus Induced Gene Silencing (VIGS) was investigated. Therefore, the GCN2 homologue was found to function as dampening the severity of the disease. The silencing with triple technical replicates was observed in 5 of the 6 samples, at an average of 43.2% by qRT-PCR analysis. The pathogen growth levels at different time points were analyzed under light microscope on the silenced and the control samples by measuring the primary and secondary hyphae lengths. The total of 24 seedlings and 292 individual spores were analyzed, and then the level of disease formation was quantitated with 603 primary hyphae and 106 secondary hyphae measurements. Up to 25% hyphae growth rate differences between the control and silenced groups were observed with a probability value less than 0.05 on t-test.

Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research Quality protein maize (QPM) has high nutritional value, but production is threatened by downy mildew (DM) and maize streak virus disease (MSVD) among other constraints. There are few studies of DM and MSVD resistance in QPM cultivars. The objective of this study was to improve resistance to DM and MSVD in three QPM populations. This was realized through ascertaining farmers’ key production constraints and special preferences for cultivars; determining the utility of recurrent selection method for improvement of three QPM populations (SussumaS2, ZM521Q and Pop62SRQ); and determining grain yield potential. The study was conducted in Mozambique for DM and in Zimbabwe for MSV, during 2003 to 2006. Surveys were conducted in Manica and Angonia districts in Mozambique to ascertain farmers’ perceptions and preferences for maize varieties, especially QPM. Participatory rural appraisal tools that included semi-structured questionnaires and focus group discussions were used to collect data. Results showed that farmers predominantly grew open pollinated varieties and fewer normal maize hybrids (non-QPM), and grain yield was estimated to be very low (0.2 to 0.6 t ha-1). Results showed that drought and insect pests were the dominant constraints to maize productivity in Mozambique, while diseases were ranked third. Downy mildew disease and MSVD were considered to be the most important diseases reducing maize productivity. Farmers also showed high preference for high yielding and early maturity cultivars in all areas. Predominantly, farmers were still using their local landraces because of sweet taste, particularly for home consumption and flint grain for storage. Farmers’ access to improved cultivars was limited due to high seed prices on the local market. Research priorities as perceived by the farmers included breeding for resistance to drought, grain weevils and diseases and sweetness. Generally, farmers showed little knowledge of QPM varieties and the importance of this trait, but they observed that the few QPM varieties they knew had some weaknesses such as poor storability and susceptibility to DM and MSVD which required improvement. These results should be considered in breeding new cultivars, both normal and QPM. To improve DM and MSV disease resistance in QPM varieties, S1 recurrent selection was conducted in three QPM populations, Sussuma, ZM521Q and Pop62SRQ at Umbeluzi Research Station in Mozambique and at CIMMYT-Harare Research. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.