Pseudoperonospora cubensis (Bert. et Curt) Rost. is the causal agent of cucurbit downy mildew (CDM). It is the most damaging cucumber pathogen on the Eastern Shore of Virginia and eastern parts of the United States. Pseudoperonospora cubensis is an obligate oomycete pathogen, infecting crops within the Cucurbitaceae family. The disease is characterized by angular chlorotic lesions and a downy or felt-like appearance on the abaxial side of the leaf. Control of this pathogen includes use of resistant cucumber cultivars and costly fungicide programs. Continuous use has led to resistance to commonly used fungicides. This has become a major concern and in response, seed companies have developed cucumber cultivars which claim downy mildew resistance.  This study evaluates different cucumber cultivars and assesses their level of resistance to CDM. The results indicate that an integrated management approach of reduced fungicide application and the use of resistant cultivars can suppress levels of CDM and yield a cucumber crop. Additionally, a molecular study was conducted, comparing the relative expression of genes encoding a basic PR-1 protein, a cytosolic ascorbate peroxidase protein and three resistance (R) gene proteins, in nineteen cultivars. All of the selected genes were analyzed using real-time PCR. The relative expression levels of the R-genes varied between cultivars. The basic PR-1 protein decreased expression in the majority of the cultivars, suggesting no involvement in the first twenty-four hours. Cytosolic ascorbate peroxidase relative expression levels suggest an increase in susceptible cultivars and a decrease in tolerant cultivars. / Master of Science

[ES] En este trabajo, se detectó por primera vez el virus de la clorosis amarilla de las cucurbitáceas (CCYV, cucurbit chlorotic yellows virus) infectando pepino y calabacín en Argelia. El CCYV (género Crinivirus, familia Closteroviridae) forma parte de un complejo de virus transmitidos por la mosca blanca Bemisia tabaci, que causan enfermedades de amarillamiento en las cucurbitáceas. Se obtuvo la secuencia completa del gen que codifica la proteína de la cápside (CP) y secuencias parciales de los genes RdRp y Hsp70 de un aislado argelino de CCYV y se compararon con las secuencias presentes en las bases de datos. ToLCNDV (género Begomovirus, familia Geminiviridae) también se detectó por primera vez infectando plantas de cucurbitáceas en Argelia. ToLCNDV es un begomovirus bipartito que causa importantes epidemias en cultivos económicamente valiosos de las familias Solanaceae y Cucurbitaceae. Se obtuvo la secuencia genómica completa de un aislado de ToLCNDV de pepino de Argelia (ToLCNDV-Biskra). La alineación de las secuencias de nucleótidos de los segmentos de ADN-A y ADN-B reveló identidades del 98,7% y 97,6%, respectivamente, con los segmentos de los aislados de la cuenca del Mediterráneo, mientras que la identidad con los aislados de Asia fue de aproximadamente del 90% y 81%. Los aislados presentes en la cuenca mediterránea son monofiléticos y forman un único grupo, mientras que los aislados de la cepa asiática presentan una mayor variabilidad genética y forman varios grupos. Todos los aislados de Argelia mostraron una variación de nucleótidos muy baja. Solo se observó una duplicación de 17 nucleótidos en el ADNB de algunos aislados que daría lugar a una proteína de movimiento 53 aminoácidos más larga, aunque la funcionalidad de esta supuesta proteína es desconocida. A pesar de esa duplicación, no se detectaron eventos de recombinación entre los aislados secuenciados. La baja variación genética podría dificultar la detección de recombinantes. / [CA] En aquest treball, es va detectar per primera vegada el virus de la clorosi groga de les cucurbitàcies (CCYV, cucurbit chlorotic yellows virus) infectant cogombres i carabassetes a Algèria. El CCYV (gènere Crinivirus, família Closteroviridae) forma part d'un complex de virus transmesos per mosca blanca que causen malalties d'esgrogueïment en les cucurbitàcies. Es va realitzar la determinació de la seqüència completa de la proteïna de la càpsida (CP) i seqüències parcials dels gens RdRp i Hsp70 d'un aïllat algerià de CCYV i es van comparar amb les seqüències publicades en les bases de dades. ToLCNDV (gènere Begomovirus, família Geminiviridae) també va ser detectat per primera vegada infectant plantes de cucurbitàcies a Algèria. ToLCNDV és un begomovirus bipartit que causa importants epidèmies en cultius econòmicament valuosos de les famílies Solanaceae i Cucurbitaceae. Es va obtindre la seqüència genòmica completa d'un aïllat de ToLCNDV de cogombre d'Algèria (ToLCNDV-Biskra). La alineació de les seqüències de nucleòtids del segments d'ADN-A i ADN-B van mostrar identitats del 98.7% i 97.6%, respectivament, amb els segments corresponents dels aïllats de la conca del Mediterrani, mentre que la identitat amb els aïllats d'Àsia va ser d'aproximadament el 90% i 81%. Els aïllats presents a la conca del Mediterrani són monofilètics i formen un únic grup, mentre que els aïllats de la soca asiàtica presenten una major variabilitat genètica i s'agrupen en diversos grups. Tots els aïllats algerians van mostrar una variació de nucleòtids molt baixa. Només es va observar una duplicació de 17 nucleòtids en d'ADNB d'alguns aïllats que lonaria lloc a una proteïna de moviment 53 aminoàcids més llarga, encara que la funcionalitat d¿aquesta suposada proteïnaés desconeguda. A més, malgrat la duplicació, no es van detectar esdeveniments de recombinació entre els aïllats seqüenciats. La baixa variació genètica podria dificultar la detecció de recombinants. / [EN] In this work, cucurbit chlorotic yellows virus (CCYV) was first detected infecting cucumber and zucchini in Algeria. CCYV (genus Crinivirus, family Closteroviridae) is part of a complex of whitefly-transmitted viruses that cause yellowing disease in cucurbits. Determination of the complete CP, and partial RdRp and Hsp70 sequences of an Algerian CCYV isolate was conducted to unveil the evolutionary relationships with the published isolates in databases. The phylogenetic analysis showed that the Algerian isolate clustered into group I together with the majority of the reported CCYV isolates. ToLCNDV (genus Begomovirus, family Geminiviridae) was also detected for the first time infecting cucurbit plants in Algeria. ToLCNDV is a bipartite begomovirus that causes major epidemics in economically valuable crops. epidemics in economically valuable crops of the Solanaceae and Cucurbitaceae families. Cucurbitaceae. The complete genome sequence of a ToLCNDV isolate from Algerian cucumber (ToLCNDV from Algeria (ToLCNDV-Biskra) was obtained. Alignment of the nucleotide sequences of the A-DNA and B-DNA segments revealed the identity of the DNA-A and DNA-B segments revealed identities of 98.7% and 97.6%, respectively, with the segments of the isolates from the Algerian segments of the Mediterranean basin isolates, while the identity with the Asian isolates was approximately 90% and 81%, respectively. was approximately 90% and 81%. The isolates present in the Mediterranean Basin are monophyletic and form a single monophyletic and form a single cluster, whereas isolates from the Asian strain have a higher genetic variability and form several clusters. genetic variability and form several clusters. All isolates from Algeria showed very low nucleotide variation. very low nucleotide variation. Only a duplication of 17 nucleotides was observed in the B-DNA of some isolates which would result in a longer 53 amino acid movement protein, although the functionality of this putative protein is unknown. Despite this duplication, no recombination events recombination events were detected among the sequenced isolates. The low genetic variation could hinder the detection of recombinants. / This work was supported by the following grants: Programa de Ayudas de Investigación y Desarrollo (PAID-11-21) of the Universitat Politècnica de València; PID2021-125787OR-C33 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”; and PROMETEO project for excellence groups 2021/072 Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana). / Kheireddine, A. (2023). First report of Cucurbit Chlorotic Yellows Virus (CCYV) and Tomato Leaf Curl New Delhi Virus (ToLCNDV) in Algeria and Lack of Evidence for Seed Transmission of ToLCNDV in Melon and Pumpkin [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196840

O meloeiro é uma cultura de relevância econômica no Brasil, principalmente na região Nordeste, onde grande parte da produção é exportada. Entre os fatores limitantes da cultura está o oídio, que pode ser controlado através do uso de variedades resistentes. No entanto, para que isto seja possível, é necessário que se tenha conhecimento sobre qual é o agente causal em ocorrência na região de produção e quais são as fontes de inóculo desta doença. O presente trabalho teve como objetivos o levantamento das espécies e raças de oídio com ênfase na região Nordeste, a verificação da patogenicidade de isolados provenientes de outras cucurbitáceas em relação ao meloeiro, a avaliação de germoplasma de meloeiro para resistência a P. xanthii raça 2 (francesa), a proposição de um sistema de cultivares de meloeiro para caracterização de raças de P. xanthii e a otimização de um protocolo de extração de DNA de P. xanthii. No total de 65 isolados analisados, observou-se que Podosphaera xanthii foi a única espécie encontrada entre as amostras. Dentre estas, as raças 1 e 2 foram as prevalentes. Também foram observadas as raças 0, 3, 4 e 5 em menor freqüência. As culturas de abóbora, abobrinha, melancia e pepino apresentaram-se como fontes de inóculo de oídio para o meloeiro, uma vez que todos os isolados provenientes destas culturas colonizaram discos foliares de meloeiro. Em geral, os genótipos de melão cultivados comercialmente no Brasil apresentaram-se resistentes a raça 2 (francesa) de P. xanthii. A partir da observação das raças ocorrentes no país e de dados da literatura, propôs-se um sistema de cultivares de meloeiro diferenciadoras para raças de P. xanthii a ser empregado em monitoramentos do patógeno. Um protocolo para extração de DNA de P. xanthii também foi obtido com a finalidade de facilitar estudos genéticos sobre o patógeno com base em marcadores moleculares. / Melon is an economically important crop in Brazil, mainly in the north-east region, where most of the production is exported. Among the limiting factors of the crop is powdery mildew, which can be controled by using resistant varieties. However, to make it possible, it is necessary to know the causal agent in occurrence in the production region and what are the inoculum sources of this disease. The present work had as objectives the survey of the powdery mildew species and races with emphasis in the north-east region; the verification of the strains pathogenicity deriving from others cucurbits in melon; the evaluation of melon germplasm to Podosphaera xanthii race 2 (french) resistance; the proposition of a system of melon cultivars to characterize the P. xanthii races and the optimization of a protocol to P. xanthii DNA extraction. Over all of the 65 strains analysed, it was observed that P. xanthii was the unique specie encountered between the samples. Within that, the races 1 and 2 were prevalents. Also were observed the races 0, 3, 4 and 5 at low frequency. Squash, zucchini, watermelon and cucumber crops showed up like inoculum sources of powdery mildew to melon. Usually, the melon genotypes commercially cultivated in Brazil showed up resistant to P. xanthii race 2 (french). After the observation of races in occurrence and datas from literature, a system of melon cultivars was proposed to differentiate P. xanthii races to be used in surveys of the pathogen. A protocol to extraction P. xanthii DNA also was proposed with the purpose to facility genetics studies about the pathogen basis on molecular markers.

The recently discovered cucurbit[n]uril are a range of macrocyclic hosts which have enormous potential in industrial, medical and academic applications. Cucurbit[n]uril have a rigid repeating structure of methylene bridged glycouril, which give cucurbit[n]uril their gourd like shape of a cavity with two carbonyl fringed portals. In this thesis the host-guest binding abilities of three cucurbit[n]uril (n = 6, 7, 8) have been examined for a range of potential guests. These guests ranged from simple alkyl amines through globular alkyl and carboranyl amines to bipyridyl systems. In total 45 guest molecules where examined. Most of the guests examined where either cationically charged, capable of hydrogen binding, contained a substantial molecular dipole, or a combination of these. Furthermore, all of the potential guests examined had some solubility in an acidified aqueous sodium sulfate solution within which the host-guest properties were examined. It was generally found that the larger guests did have selectivity for the larger hosts. However, when the host became too large weaker complexes would form and for the range of materials examined here cucurbit[7]uril was found to be the 'best' host system. In one example, p-xylene diamine, a 2:1 complex with cucurbit[8]uril was observed. While not the focus of this work a new rapid purification method was developed for the cucurbit[n]uril using different metal ions to either solubilise or precipitate the different cucurbit[n]uril. In the second part of this work these same guest molecules where used as potential templates in the synthesis of cucurbit[n]uril. Surprisingly the guests that bound strongly to an individual host did not seem to template the cucurbit[n]uril synthesis at all. Rather these strong binders inhibited the reaction such that little or no cucurbit[n]uril formed under the reaction conditions studied. However, several examples provided excellent template results. Indeed the results indicate that guests which bound with intermediate rates of exchange are the best templates and using templates under these conditions we have been able to produce cucurbit[7]uril as 46% by mass of the total cucurbit[n]uril product. This is the highest yield ever recorded for cucurbit[7]uril and it is the first example of cucurbit[7]uril being the major product of this condensation reaction. In an another example cucurbit[8]uril formed 18% of the product an increase of 150% over the standard reaction conditions. While studying both the template reactions and the host-guest binding properties of the cucurbit[n]uril a new supramolecular form, an 'inverse rotaxane' was discovered. Inverse rotaxanes are not held in place by large blocking groups, rather the molecular structure encapsulated by the cucurbit[n]uril host prevents decomplexation of the axle.

<p>Molecular recognition is arguably the most elementary physical process essential for life that arises at the molecular scale. Molecular recognition drives events across virtually all length scales, from the folding of proteins and binding of ligands, to the organization of membranes and the function of muscles. Understanding such events at the molecular level is massively complicated by the unique medium in which life occurs: water. In contrast to recognition in non-aqueous solvents, which are driven largely by attractive interactions between binding partners, binding reactions in water are driven in large measure by the properties of the medium itself. Aqueous binding involves the loss of solute-solvent interactions (desolvation) and the concomitant formation of solute-solute interactions. Despite decades of research, aqueous binding remains poorly understood, a deficit that profoundly limits our ability to design effective pharmaceuticals and new enzymes. Particularly problematic is understanding the energetic consequences of aqueous desolvation, an area the Toone and Beratan groups have considered for many years.</p><p> In this dissertation, we embark on a quest to shed new light on aqueous desolvation from two perspectives. In one component of this research, we improve current computational tools to study aqueous desolvation, employing quantum mechanics (QM), molecular dynamics (MD) and Monte Carlo (MC) simulations to better understand the behavior of water near molecular surfaces. In the other, we use a synthetic host, cucurbit[7]uril (CB[7]), in conjunction with a de novo series of ligands to study the structure and thermodynamics of aqueous desolvation in the context of ligand binding with atomic precision, a feat hitherto impossible. A simple and rigid macrocycle, CB[7] alleviates the drawbacks of protein systems for the study of aqueous ligand binding, that arise from conformational heterogeneity and prohibitive computational costs to model.</p><p> </p><p> We first constructed a novel host-guest system that facilitates internalization of the trimethylammonium (methonium) group from bulk water to the hydrophobic cavity of CB[7] with precise (atomic-scale) control over the position of the ligand with respect to the cavity. The process of internalization was investigated energetically using isothermal titration microcalorimetry and structurally by nuclear magnetic resonance (NMR) spectroscopy. We show that the transfer of methonium from bulk water to the CB[7] cavity is accompanied by an unfavorable desolvation enthalpy of just 0.49±0.27 kcal*mol-1, a value significantly less endothermic than those values suggested from previous gas-phase model studies. Our results offer a rationale for the wide distribution of methonium in biology and demonstrate important limitations to computational estimates of binding affinities based on simple solvent-accessible surface area approaches.</p><p> To better understand our experimental results, we developed a two-dimensional lattice model of water based on random cluster structures that successfully reproduces the temperature-density anomaly of water with minimum computational cost. Using reported well-characterized ligands of CB[7], we probed water structure within the CB[7] cavity and identified an energetically perturbed cluster of water. We offer both experimental and computational evidence that this unstable water cluster provides a significant portion of the driving force for encapsulation of hydrophobic guests.</p><p> The studies reported herein shed important light on the thermodynamic and structural nature of aqueous desolvation, and bring our previous understanding of the hydrophobic effect based on ordered water and buried surface area into question. Our approach provides new tools to quantify the thermodynamics of functional group desolvation in the context of ligand binding, which will be of tremendous value for future research on ligand/drug design.</p> / Dissertation

O meloeiro é uma cultura de relevância econômica no Brasil, principalmente na região Nordeste, onde grande parte da produção é exportada. Entre os fatores limitantes da cultura está o oídio, que pode ser controlado através do uso de variedades resistentes. No entanto, para que isto seja possível, é necessário que se tenha conhecimento sobre qual é o agente causal em ocorrência na região de produção e quais são as fontes de inóculo desta doença. O presente trabalho teve como objetivos o levantamento das espécies e raças de oídio com ênfase na região Nordeste, a verificação da patogenicidade de isolados provenientes de outras cucurbitáceas em relação ao meloeiro, a avaliação de germoplasma de meloeiro para resistência a P. xanthii raça 2 (francesa), a proposição de um sistema de cultivares de meloeiro para caracterização de raças de P. xanthii e a otimização de um protocolo de extração de DNA de P. xanthii. No total de 65 isolados analisados, observou-se que Podosphaera xanthii foi a única espécie encontrada entre as amostras. Dentre estas, as raças 1 e 2 foram as prevalentes. Também foram observadas as raças 0, 3, 4 e 5 em menor freqüência. As culturas de abóbora, abobrinha, melancia e pepino apresentaram-se como fontes de inóculo de oídio para o meloeiro, uma vez que todos os isolados provenientes destas culturas colonizaram discos foliares de meloeiro. Em geral, os genótipos de melão cultivados comercialmente no Brasil apresentaram-se resistentes a raça 2 (francesa) de P. xanthii. A partir da observação das raças ocorrentes no país e de dados da literatura, propôs-se um sistema de cultivares de meloeiro diferenciadoras para raças de P. xanthii a ser empregado em monitoramentos do patógeno. Um protocolo para extração de DNA de P. xanthii também foi obtido com a finalidade de facilitar estudos genéticos sobre o patógeno com base em marcadores moleculares. / Melon is an economically important crop in Brazil, mainly in the north-east region, where most of the production is exported. Among the limiting factors of the crop is powdery mildew, which can be controled by using resistant varieties. However, to make it possible, it is necessary to know the causal agent in occurrence in the production region and what are the inoculum sources of this disease. The present work had as objectives the survey of the powdery mildew species and races with emphasis in the north-east region; the verification of the strains pathogenicity deriving from others cucurbits in melon; the evaluation of melon germplasm to Podosphaera xanthii race 2 (french) resistance; the proposition of a system of melon cultivars to characterize the P. xanthii races and the optimization of a protocol to P. xanthii DNA extraction. Over all of the 65 strains analysed, it was observed that P. xanthii was the unique specie encountered between the samples. Within that, the races 1 and 2 were prevalents. Also were observed the races 0, 3, 4 and 5 at low frequency. Squash, zucchini, watermelon and cucumber crops showed up like inoculum sources of powdery mildew to melon. Usually, the melon genotypes commercially cultivated in Brazil showed up resistant to P. xanthii race 2 (french). After the observation of races in occurrence and datas from literature, a system of melon cultivars was proposed to differentiate P. xanthii races to be used in surveys of the pathogen. A protocol to extraction P. xanthii DNA also was proposed with the purpose to facility genetics studies about the pathogen basis on molecular markers.

Desde el comienzo de su modernización a finales del siglo XVIII, la Química se ha desarrollado continua y rápidamente a través de la teoría atómico-molecular; sin embargo, esta química del enlace covalente ha sido explotada hasta sus límites conceptuales, de forma que incluso el mejor químico sintético de hoy día no puede fabricar sistemas moleculares complicados usando sólo el repertorio de herramientas disponibles para crear enlaces covalentes. Puesto que el siguiente paso al aumentar el nivel de complejidad hacia los sistemas polimoleculares organizados presentes en los sistemas vivos se encuentra unido a las interacciones no covalentes, es el momento de que los químicos miren más allá de la síntesis clásica hacia la Química Supramolecular, una rama relativamente joven de la ciencia que se preocupa del estudio de las características básicas de estas interacciones no covalentes. En los últimos 25 años, una enorme parte de la investigación química se ha detenido en el campo de los dispositivos moleculares fotoactivos, observando que los resultados que pueden ser obtenidos de la interacción de la luz con la materia dependen del grado de organización de la misma; en este sentido, las entidades supramoleculares que contienen este tipo de componentes pueden desarrollar propiedades nuevas moduladas por la disposición de sus unidades constitutivas, dando lugar a procesos fotoquímicos distintivos de estas especies. A través de esta ruta es posible disponer componentes moleculares prefabricados que lleven a cabo una determinada propiedad relacionada con la luz, siendo posible diseñar sistemas organizados y funcionalmente integrados capaces de elaborar la señal proporcionada por los fotones para desarrollar funciones complejas. / Montes Navajas, PM. (2009). Sistemas supramoleculares basados en la encapsulación en cucurbit(n)uriles [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/6281

[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

<p dir="ltr">The striped cucumber beetle (<i>Acalymma vittatum</i>) is a leading pest across a variety of cucurbit crops in eastern North America. These beetles can rapidly infest and damage a field, leading to frequent insecticide applications to manage them. Chemical control is effective at reducing beetle populations, but it also causes detrimental non-target effects on pollinators, which are essential for cucurbit yield. Developing a holistic IPM (integrated pest management) approach is necessary to manage pests while avoiding non-target effects in cucurbit production systems. </p><p dir="ltr">A unique and well-characterized behavior of striped cucumber beetles is their olfactory attraction to both plant volatiles and their species-specific aggregation pheromone, vittatalactone. The interacting effects of combining these olfactory stimuli for cucumber beetle attraction have not previously been tested. I expected when presented with plant volatiles and pheromone together, the striped cucumber beetles will show synergistic attraction, resulting in enhanced attraction stronger than the sums of the separate effects. Along with targeting striped cucumber beetles, I expected to elicit cross-attraction of related cucurbit pests (e.g., spotted cucumber beetle) to these same signals based on previous findings indicating cross-attraction. The expected cross attraction may be due to the reliance on olfactory cues to inform the related insects on preferrable host plants and nutrition. </p><p dir="ltr">Cucurbit systems are also highly reliant on pollination services for high-quality fruit; thus, the feasibility of these semiochemical tools depends on their influence on pollinator behavior. I expect pollinators to be attracted to floral volatiles but not vittatalactone or individual plant volatiles without the full complement of floral scent (e.g., indole, leaf volatiles). Altogether, this research aims to develop a targeted management tool for striped cucumber beetles and other cucurbit pests, while avoiding pollinator distraction or other detrimental effects.</p><p dir="ltr">To measure the efficacy of using olfactory signals as attractants, clear sticky cards were deployed in the field with combinations of pheromone paired with volatiles (floral and/or leaf), as well as the individual components, to quantify pest responses. Simultaneously, a pan trap sampling method was implemented to measure pollinator responses to the same semiochemical combinations. The results of the study demonstrate that striped cucumber beetles are strongly attracted to volatile lures containing pheromones, floral volatiles, and combinations of the two, although the combined pheromone and floral volatile treatments did not synergize beetle attraction. </p><p dir="ltr">Though combined lures did not synergize attraction, this data demonstrates additive effects on beetle behavior. The findings also highlight the importance of understanding seasonal disparities between the behavior of early and late generations of striped cucumber beetles. Temporal variation in attraction demonstrated by the pests is crucial to understand when to implement lure-based management strategies. Two years of testing floral volatile and herbivory-induced plant volatile (HIPV) lure treatments on key cucurbit pollinators showed varying levels of attraction. There was no attraction of focal cucurbit bees to the striped cucumber beetle pheromone, but there were differences in the response of pollinators to plant volatile components. </p><p dir="ltr">Cucurbits require high pollination activity for successful fruit, though the most important bees in pollinating this system are bees belonging to the genera, <i>Apis</i> and <i>Eucera</i>. Along with these bee groups, other bees such as those belonging to <i>Melissodes</i>, <i>Lasioglossum</i>, and <i>Bombus</i> are also known important pollinators. <i>Lasioglossum</i> bees showed a strong attraction to the full-floral blend, TIC (1,2,4-trimethoxybenzene, indole, (E)-cinnamaldehyde), but no preference for indole or HIPVs. <i>Melissodes</i> bees were most attracted to methyl salicylate and TIC, whereas <i>Eucera</i> bees exhibited strong attraction to ocimene and TIC. These findings suggest that different pollinator taxa have unique preferences for plant volatiles, highlighting the importance of optimizing lure combinations to avoid disrupting pollination activities in cucurbit production.</p><p dir="ltr">Additionally, this study revealed that other key cucurbit pests, such as spotted cucumber beetles and western corn rootworms, also showed strong attraction to the tested volatile components. These findings suggest that the selected volatiles may have broader implications for pest management beyond striped cucumber beetles. Further research is needed to fully understand the efficacy and refine formulations of these volatile lures to implement in IPM.</p>

[Formulae and special characters can only be approximated here. Please see the pdf version of the Abstract for an accurate reproduction.] Several inert platinum complexes were synthesised: [(en)Pt([special character]-dpzm)2Pt(en)]4+, [{Pt(dien)}2[special character]-dpzm]4+, [{Pt(dien)}2[special character]-H2N-(CH2)6-NH2]4+, cis-[(NH3)2Pt([special character]--dpzm)2Pt(NH3)2]4+, trans-[Pt(NH3)2([special character]-dpzm)2]2+. Three active complexes, all with chloro ligands, were also synthesised: trans-[{Pt(NH3)Cl2}2[special character]-dpzm)], trans-[{Pt(NH3)2Cl}2[special character]-dpzm]2+ (di-Pt) and trans-[trans-{Pt(NH3)2Cl}2{trans-[Pt(NH3)2([special character]-dpzm)2]}]4+ (tri-Pt). 1H NMR established that multi-nuclear platinum complexes will preferentially associate in the DNA minor groove with a preference for A/T sequences, and with a binding constant [special character]-105 M-1, regardless of the charge, linking ligand, length or shape. Using [(en)Pt([special character]-dpzm)2Pt(en)]4+ and the oligonucleotide d(GC)5 it was determined that the metal complex binds G/C rich sequences also in the minor groove, but with a much reduced binding constant, 103 M-1. CD studies showed [(en)Pt([special character]-dpzm)2Pt(en)]4+ was able to induce a DNA conformation change from B-type to what appeared to be a partial Z-type. Transcription assays showed that even though the metal complex does not bind DNA covalently, it is still able to inhibit DNA transcription at particular sites. The complexes di-Pt, tri-Pt, [{Pt(dien)}2[special character]-dpzm]4+ and trans-[Pt(NH3)2([special character]-dpzm)2]2+ were tested for anti-cancer activity in the L1210 murine leukaemia cell line, and gave values of 3.8, 2.5, [special character]200 and 64 [special character]M respectively. In the cisplatin resistant line (L1210/DDP), trans-[Pt(NH3)2([special character]-dpzm)2]2+ showed an increase in activity with a drop to 32 [special character]M, while both di-Pt and tri-Pt showed decreases in activity to values of 8.8 and 3.6 [special character]M. In the human ovarian carcinoma 2008 cell line and its cisplatin resistant derivative C13[special character]5, both complexes showed good activity with values of 2.5 and 20.9 [special character]M respectively, but again both showed decreases in activity in the resistant line with values of 17.8 and 37.7 [special character]M respectively. To help explain the difference between activity of these complexes and the complexes BBR3464 and BBR3005, cell uptake and DNA interstrand cross-linking experiments were performed. The cell uptake studies showed that both di-Pt and tri-Pt are taken up by cells at very high levels, when administered at 100 [special character]M, thus indicating that the difference is unlikely to be due to large differences in cell uptake. The DNA interstrand cross-linking studies showed both complexes readily form interstrand adducts (50% interstrand cross-linking at 12 nM and 22 nM respectively, c.f cisplatin 3 [special character]M). These results suggest that the rigid nature of the dpzm linker may be affecting the DNA adducts formed, with more interstrand links being formed than BBR3464. Possibly, it is this that causes the large differences in cytotoxicity. The DNA binding of di-Pt and tri-Pt was examined with the nucleosides adenosine and guanosine and the dinucleotide d(GpG). Both complexes bound at the N7 of guanosine, but 2-fold slower than cisplatin. In addition, di-Pt bound at the N7 and either the N1 or N3 of adenosine, 7-fold slower than guanosine. Di-Pt forms a large variety of cross-links between two d(GpG) molecules, however it could not be established whether the 1,2-intrastrand adduct could be formed. Di-Pt, however, forms a 1,2-GG interstrand adduct with the oligonucleotide d(ATGCAT)2 resulting in a conformation change away from B-type DNA. The sugar pucker of the G3 nucleoside changes from 2[special character]-endo towards 3[special character]-endo, and the position of the nucleotide relative to the sugar changes from anti to syn. The ability of multi-nuclear platinum complexes to form covalent adducts in the DNA minor groove remains unclear. It appears that di-Pt can form up to 33% minor groove adducts with the oligonucleotide d(AT)5, but when added to the oligonucleotide d(GCCAAATTTCCG)2 no definite minor groove adducts are seen and the major adduct appears to be a 1,2-interstrand cross-link between the two A6's or between the G1 and G11. Finally, a study of the encapsulation of platinum complexes within cucurbit[7]uril (Q7) as a means of reducing drug toxicity was made. For complex A and di-Pt, encapsulation of the linker ligand occurred. The effect of Q7 on the rate of hydrolysis of di-Pt was at least a 3-fold reduction as compared to free di-Pt with guanosine. Studies with [{Pt(dien)}2[special character]-dpzm]4+/Q7 and the oligonucleotide d(CGCGAATTCGCG)2 showed that the metal complex could dissociate from the Q7 and associate with the oligonucleotide, where an equilibrium is achieved with 15 % of the metal complex bound to the oligonucleotide and 75 % encapsulated in Q7. Tests in the L1210 and L1210/DDP cancer cell lines showed that di-Pt/Q7 has almost the same activity compared to free di-Pt.