No description available.

La maladie du court-noué est principalement causée en Europe par le Grapevine fanleaf virus (GFLV) et l’Arabis mosaic virus (ArMV). Le principal moyen de lutte contre sa dispersion consiste à certifier l’absence de ces virus dans les vignes commercialisées par des méthodes sérologiques tels que le DAS-ELISA. De par leurs propriétés biophysique et structurale exceptionnelles, les Nanobodies (Nb) issus des domaines variables d’immunoglobulines (Ig) composées uniquement de chaînes lourdes, se distinguent avantageusement des Ig conventionnelles. L’objectif majeur de ma thèse était d’établir un test de diagnostic du court-noué à base de Nb. À partir de deux collections de Nb contre le GFLV et l’ArMV, j’ai identifié des Nb reconnaissant un large spectre d’isolats viraux. Leur fusion à une protéine fluorescente ou à la phosphatase alcaline a conduit à l’obtention de réactifs de détection performants du GFLV et de l’ArMV par DAS-ELISA. La structure atomique d’un complexe Nb/GFLV résolue à 2.8 Å par cryomicroscopie électronique a permis de cartographier l’épitope en surface du virus et a révélé une couverture maximale de la particule virale par le Nb. La comparaison des tests Nb à des réactifs sérologiques commerciaux a révélé leur supériorité en terme de sensibilité et de spécificité, ouvrant ainsi la voie à la commercialisation d’un nouveau test de diagnostic des virus du court-noué de la vigne. / The grapevine fanleaf disease is mainly caused in Europe by the Grapevine fanleaf virus (GFLV) and the Arabis mosaic virus (ArMV). The principal mean to limit their spread, is to certify their absence in marketed grapevines by serological methods such as DAS-ELISA. Their unique biophysical and structural properties make the variable domains of heavy chain-only immunoglobulin, called Nanobodies (Nb) a real asset for the development of a diagnostic test against fanleaf disease viruses. I identified Nb able to detect a broad spectrum of viral isolates from two Nb collections against GFLV and ArMV. Their fusion to a fluorescent protein or to a bacterial alkaline phosphatase resulted in the production of efficient DAS-ELISA detection reagents. The atomic structure of a Nb/GFLV complex was solved at 2.8 Å by cryoelectron microscopy, allowing the precise mapping of the viral epitope. This result showed a maximum coverage of the viral particle by the Nb, leading to a maximal signal in DAS-ELISA. The full Nb tests against GFLV and ArMV were compared to commercial reagents and showed the superiority of the former in both sensitivity and specificity, opening the way for the development and commercialization of a new type of serological kits for the detection of grapevine viruses.

This thesis deals with viral pathogens of pea and its seed transmission. It describes characteristic of pea seed-borne mosaic virus (PSbMV) and Pea enation mosaic virus (PEMV), mechanism of seed transmission, methods for detection viruses in seeds and pea resistence to both of viruses. In the experimental part was tested different stages of development of three breeding on new varietes (SM 247, 1019/12 and 1011/12) for presence PSbMV by DAS-ELISA. The aim of the experiment was to determine the developmental stage of pea relevant to determine the transmission PSbMV on pea plants. After the evaluation, it was concluded that this developmental stage is the embryo or one week plant. Furthermore, the selected lines of pea were tested for resistance to PEMV. On the basis of this experiment were obtained pea populations resistant to this virus.

White clover mosaic virus (WClMV) virions were purified using a modified method of Wetter (1960). Antisera were produced by rabbit immunisation and virus-specific polyclonal antibodies were obtained. Suitable conditions were optimised for serological detection of virus by DAS-ELISA and compared with a commercial kit.

No description available.

Thesis (MSc (Genetics))--Stellenbosch University, 2008. / South Africa is one of the top ten wine producing countries in the world. The South African wine industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product with 42.8% of wine being exported. To compete with the top wine producing countries and to ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and canes and is responsible for significant economic losses by reducing crop yields by as much as 80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The Breede River Valley contributes approximately 30% of the total production of the local wine industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act states that all propagation material sold must be tested for GFLV by a reputable scientific technique. The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linked Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to the South African viticulture industry. The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by using recombinant DNA technology to produce antibodies against bacterially expressed viral coat protein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein (CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase (GST) partner to the viral coat protein enhancing solubility and protein purification. Insufficient amounts of the soluble protein were expressed and purified, preventing the production of antibodies and thus the development of the DAS-ELISA. An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone- tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStrip tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability between the isolates as well as the variability between the South African isolates and GFLV sequences available in Genbank. Sequence identities between clones from different GFLV isolates from South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was found between geographical origin and symptoms, nor between geographical origin and sequence variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17 with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the most reliable technique for GFLV detection. Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was not successful, an alternative PCR based diagnostic system was developed, and proved to be sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.

Tesis por compendio / [ES] El pepino dulce (Solanum muricatum) y el tomate de árbol (S. betaceum) pertenecen al grupo de cultivos de la familia Solanaceae. Estos dos cultivos son originarios de América del Sur y actualmente se cultivan en varios países con climas tropicales, subtropicales y mediterráneos. Han sido infrautilizados durante mucho tiempo y han cobrado relevancia solo en los últimos años debido a su alta calidad nutricional. El pepino dulce exhibe niveles significativos de potasio, vitamina C y carotenoides y se informa que presenta propiedades antioxidantes, antidiabéticas, antiinflamatorias y antitumorales. Sus frutos se pueden consumir tanto como postre o en ensaladas. El tomate de árbol también destaca por su alto contenido en compuestos bioactivos como carotenoides, antocianinas, flavonoides y vitaminas. Varios productos como jugos, mermeladas, salsas y productos farmacéuticos son elaborados a partir de sus frutos. Debido a que estos cultivos se han introducido en nuevas regiones, donde pueden estar expuestos a estreses bióticos y abióticos que pueden amenazar su producción, y dado que el pepino dulce se ve especialmente afectado por la escasez de agua, fue necesario realizar un estudio para determinar la respuesta de siete cultivares de pepino dulce a parámetros fisiológicos y bioquímicos al estrés por sequía. Este trabajo puede ayudar a desarrollar programas de selección y mejoramiento que permitan generar nuevas variedades más tolerantes a la sequía. Por otro lado, en los países de clima mediterráneo, el pepino dulce se cultiva como cultivo protegido, aplicando las mismas técnicas agrícolas que otras solanáceas como el tomate y el pimiento. Estos sistemas agrícolas también brindan condiciones óptimas para el desarrollo de enfermedades como Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaico del pepino (PepMV) y virus del mosaico del tomate (ToMV), que potencialmente podrían causar grandes daños a los cultivos de pepino dulce. Por tal motivo, se realizó un estudio para evaluar la respuesta de una colección de pepino dulce y sus parientes silvestres contra estas cuatro enfermedades, y encontrar fuentes de resistencia/tolerancia a estos patógenos. Aunque el tomate de árbol es un cultivo frutal importante debido a su valor nutricional y efectos beneficiosos para la salud, actualmente no hay información genómica y transcriptómica disponible públicamente. Por lo tanto, fue fundamental secuenciar el transcriptoma de dos cultivares de tomate de árbol con frutos morados (A21) y frutos anaranjados (A23). Estos dos cultivares han sido ampliamente utilizados y cultivados comercialmente en países de la región andina como Ecuador y Colombia. La obtención del primer transcriptoma de tomate de árbol ha permitido realizar un estudio comparativo entre el tomate de árbol y sus especies cercanas, tomate y patata, identificar genes implicados en la ruta de biosíntesis de carotenoides y desarrollar marcadores de polimorfismo de nucleótido único (SNP). En general, esta Tesis Doctoral aporta información relevante sobre la respuesta del pepino a diversos estreses ambientales, que puede ser utilizada para el desarrollo de nuevas variedades de pepino resistentes a múltiples estreses. Mientras que en tomate de árbol, el desarrollo de herramientas genómicas acelerará los programas de mejoramiento. / [CA] El cogombre dolç (Solanum muricatum) i tomata d'arbre (S. betaceum) pertanyen al grup de cultius de la família Solanaceae. Aquests dos cultius són originaris d'Amèrica del Sud i actualment es cultiven en diversos països amb climes tropicals, subtropicals i mediterranis. Han sigut infrautilitzats durant molt de temps i han cobrat rellevància només en els últims anys a causa de la seua alta qualitat nutricional. El cogombre dolç exhibeix nivells significatius de potassi, vitamina C i carotenoides i s'informa que presenta propietats antioxidants, antidiabètiques, antiinflamatòries i antitumorals. Els seus fruits es poden consumir tant com postres o en ensalades. La tomaca d'arbre també destaca pel seu alt contingut en compostos bioactivos com carotenoides, antocianinas, flavonoides i vitamines. Dels seus fruits s'elaboren diversos productes com a sucs, melmelades, salses i productes farmacèutics. Pel fet que aquests cultius s'han introduït en noves regions on poden estar exposats a estressos biòtics i abiòtics que poden amenaçar la seua producció, atés que el cogombre es veu especialment afectat per l'escassetat d'aigua, va ser necessari realitzar un estudi per a determinar la resposta de set cultivars de cogombre dolç a paràmetres fisiològics i bioquímicos a l'estrés per sequera. Aquest treball pot ajudar a desenvolupar programes de selecció i millorament que permeten generar noves varietats més tolerants a la sequera. D'altra banda, als països de clima mediterrani, el cogombre dolç es cultiva com a cultiu protegit, aplicant les mateixes tècniques agrícoles que unes altres solanáceas com la tomaca i el pimentó. Aquests sistemes agrícoles també brinden condicions òptimes per al desenvolupament de malalties com Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), virus del mosaic del cogombre (PepMV) i virus del mosaic de la tomaca (ToMV), que potencialment podrien causar grans danys als cultius de cogombre dolç. Per tal motiu, es va realitzar un estudi per a avaluar la resposta d'una col·lecció de cogombre dolç i els seus parents silvestres contra aquestes quatre malalties, i trobar fonts de resistència/tolerància a aquests patògens. Encara que la tomaca d'arbre és un cultiu fruiter important a causa del seu valor nutricional i efectes beneficiosos per a la salut, actualment no hi ha informació genòmica i transcriptómica disponible públicament. Per tant, va ser fonamental seqüenciar el transcriptoma de dues cultivars de tomaca d'arbre amb fruits morats (A21) i fruits ataronjats (A23). Aquestes dues cultivars han sigut àmpliament utilitzats i cultivats comercialment en països de la regió andina com l'Equador i Colòmbia. L'obtenció del primer transcriptoma de tomaca d'arbre ha permés realitzar un estudi comparatiu entre la tomaca d'arbre i les seues espècies pròximes, tomaca i creïlla, identificar gens implicats en la ruta de biosíntesi de carotenoides i desenvolupar marcadors de polimorfisme de nucleòtid únic (SNP). En general, aquesta Tesi Doctoral aporta informació rellevant sobre la resposta del cogombre a diversos estressos ambientals, que pot ser utilitzada per al desenvolupament de noves varietats de cogombre resistents a múltiples estressos. Mentre que en tomaca d'arbre, el desenvolupament d'eines genòmiques accelerarà els programes de millorament. / [EN] Pepino (Solanum muricatum) and tree tomato (S. betaceum) belong to the group of crops of the Solanaceae family. These two crops are native to South America and currently are grown in various countries with tropical, subtropical and Mediterranean climates. They have been underutilized for a long time and have become relevant only in recent years due to their high nutritional quality. Pepino exhibit significant levels of potassium, vitamin C and carotenoids and it is reported to present antioxidant, antidiabetic, anti-inflammatory and antitumor properties. Its fruits can be consumed both as a dessert or in salads. Tree tomato also highlights high content of bioactive compounds such as carotenoids, anthocyanins, flavonoids and vitamins. Severals products such as juices, jams, sauces and pharmaceutical products are made from its fruits. Due to these crops have been introduced into new regions, where they may be exposed to biotic and abiotic stresses that can threaten their production, and since pepino is specially affected by water scarcity, a study was needed to determine the response of seven pepino cultivars to physiological and biochemical parameters to drought stress. This work can help develop selection and improvement programs that allow the generation of new varieties that are more tolerant to drought. On the other hand, in countries with a Mediterranean climate, pepino is grown as a protected crop, applying the same agricultural techniques as other solanaceous plants such as tomato and pepper. These agricultural systems also provide optimal conditions for the development of diseases such as Fusarium oxysporum f. sp. lycopersici (FOL), Verticillium dahliae (VE), pepino mosaic virus (PepMV) and tomato mosaic virus (ToMV), which could potentially cause great damage to pepino crops. For this reason, a study was performed to evaluate the response of a collection of pepino and their wild relatives against these four diseases, and find sources of resistance/tolerance to those pathogens. Although tree tomato is an important fruit crop due to its nutritional value and beneficial health effects, there is currently no publicly available genomic and transcriptomic information. Therefore, it was essential to sequence the transcriptome of two tree tomato cultivars with purple fruits (A21) and orange fruits (A23). These two cultivars have been widely used and cultivated commercially in countries of the Andean region such as Ecuador and Colombia. Obtaining the first tree tomato transcriptome has made it possible to perform a comparative study between tree tomato and its close species, tomato and potato, identify genes involved in the carotenoid biosynthesis pathway, and develop single nucleotide polymorphism (SNP) markers. In general, this Doctoral Thesis provides relevant information on the response of pepino to various environmental stresses, which can be used for the development of new varieties of pepino resistant to multiple stresses. While in tree tomato, the development of genomic tools will accelerating up breeding programs. / Pacheco Toabanda, JE. (2022). Development of Biotechnological Tools for the Genetic Improvement of Pepino (Solanum Muricatum) and Tree Tomato (S.betaceum) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/189205 / Compendio