<p><br></p> <p>Thrips, Thysanoptera, represent an ancient (~407 m.y.a.) order of ~6000 tiny insects from 9 families. Despite the small size of the order, thrips have a diversity of life histories, diets, and survival strategies. Thrips represent a challenge to fieldworkers and axonomists alike due to the morphological similarity between species and the lack of homologies between families. Recent </p> <p>molecular evidence has reopened debate over the phylogenetic relationships of the families of Thysanoptera.</p> <p>In this thesis we use genomic approaches to elucidate and clarify the early nodes in order to answer evolutionary questions about the Thysanoptera, their mitochondrion symbiotes, and their </p> <p>coevolutionary interactions with a group of economically important viruses; tospoviruses. Our results support previous ordinal hypotheses and show families in both sub-orders radiating </p> <p>around the emergence of the angiosperms ~120 m.y.a. We show that all thrips lineages likely have highly rearranged mitochondrial genomes, even on an intraspecies level, and that this rearrangement phenomena occurs very quickly in evolutionary time. We provide comment on the caution that must be taken with mitochondrial loci in any phylogenetic analysis with this new </p> <p>evidence and argue for the impact of among-site-rate-heterogeneity to be further investigated within thrips hylogenetics. We show that much more data is needed before thrips and tospovirus relationships can be fully elucidated but that two dueling hypotheses are emergent from our studies: either 5 very new separate vector/virus relationships, or one very old relationship that has been lost by the vast amount of thrips. We call for targeted taxa selection and show how new genomic methods can target certain taxa based upon the identification of </p> <p>assembled proteins from illumine shotgun read data.</p>

The primary objectives of this research are to determine effective biological and alternative control strategies of insect and disease pests in order to reduce harsh chemical use during greenhouse crop production and transport s. This research includes two separate studies: 1) testing the practical viability of rearing and storing four species of entomopathogenic nematode (EPN), Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica; and, 2) the efficacy of UV-C radiation applied, pre-transport, as a preventative disease control strategy against Botrytis cinerea. A study was conducted testing EPN infectious juvenile (IJ) rearing production counts and IJ viability after a six-day storage period. When all four species are compared, S. feltiae had a greater number of infectious juveniles emerge from the wax moth cadavers and S. carpocasae had the least. All four species survived the six day storage period but EPN infectious juvenile counts were significantly different among species. Our second study tested the efficacy of UV-C radiation as an alternative control to traditional fungicides to deactivate B. cinerea in vitro and to determine plant tolerance to UV-C. The crops tested were poinsettia (Euphorbia pulcherrima) and primula (Primula vulgaris). All the UV-C doses, 1.0, 2.8, 3.7 or 4 W/m2, significantly decreased B. cinerea conidial germination in vitro and resulted in zero percent damage on poinsettia bracts. However, all UV-C doses during both replications caused minor damage, 15% or less, to primula flowers. / Master of Science in Life Sciences / Entomopathogenic nematodes (EPN) shows promise in being non-chemical and environmentally friendly solution for greenhouse pest and disease control. These can also be referred to as Biological Controls (Biocontrols). Entomopathogenic nematodes are used widely to control multiple greenhouse plant pests which include both Lycoriella spp., Fungus Gnats, and Frankliniella spp., Western Flower Thrips. However, there are challenges with EPN viability and storage from the manufacture to the greenhouse producer. We studied four EPN species, Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora, Heterorhabditis indica, which were reared and stored to determine differences in production viability between species. Results show that the EPN species do not respond the same to storage and produce different amounts of infectious juveniles during rearing when conditions are the same. Separate from, but just as concerning as greenhouses plant pests are plant diseases. Ultraviolet radiation in the C spectra is known to be germicidal due to its narrow wavelengths. Because of this, UV-C has been shown to deactivate many different plant pathogens on contact and is being considered as a possible Biocontrol alternative to harsh traditional fungicides and bactericides. One disease that is known to contribute to the highest volume of annual crop losses is Botrytis cinerea. Botrytis cinerea is a plant disease that impacts floricultural crops to vegetables during propagation through the production supply chain to shipping and storage. We evaluated UV-C radiation at different doses, to determine if it could be used to replace a traditional fungicide before plants are shipped to reduce B. cinerea infection during transport. We found that UV-C successfully deactivated B. cinerea in vitro, but the viability of the application to plant tissue before transport has yet to be proven successful as a practical method of reducing B. cinerea during transport.

Tesis por compendio / [EN] Tomato spotted wilt virus is one of the most widespread and economically important viruses worldwide. It infects a large number of plant species, being the tomato and pepper the most affected. TSWV is transmitted from one plant to another by various species of thrips in a circulative and propagative manner, being Flankliniella occidentalis the main vector. The best strategy for disease control in tomato and pepper has been breeding resistant cultivars, but only tomato with the gene Sw-5 and pepper with gene Tsw have been effective against a wide spectrum of TSWV isolates, but resistance-breaking isolates often arise. To obtain a more effective and durable control is necessary: A) the genetic and biological characterization of conventional and resistance-breaking isolates of TSWV; B) the understanding the evolutionary and epidemiological factors involved in the emergence and dispersion of resistance-breaking isolates; C) the development of tools for viral detection and quantification; and D) evaluation of new sources of resistance (total or partial, estimated as the difficulty to viral infection or/and accumulation) or tolerance (total or partial, estimated as the difficulty to symptoms development and damage without affecting viral infection). In this work, TSWV isolates from Spanish tomato and pepper crops have been biologically and molecularly characterized. The complete genome of three TSWV isolates with different biotypes were sequenced: N (unable to infect Sw-5 resistant tomato and Tsw resistant pepper), T (tomato Sw-5 resistance-breaking), and P (pepper Tsw resistance-breaking). No correlation between genetic variation and the ability of overcoming resistance was found. These nucleotide sequences and others retrieved from the GenBank database were used to develop RT-qPCR, with high sensitivity and dynamic range. Primers and one TaqMan MGB were designed from conserved sequence stretches with the aim of detecting and quantifying any TSWV isolate. It was not possible to develop a molecular method to differentiate between conventional and resistance-breaking isolates since there is no correlation between genotype and biotype. Instead, two TaqMan MGB probes were designed to quantify the two main genotypes (according to the M segment) in mixed infections. RT-qPCR was used to evaluate TSWV accumulation in non-resistant tomato, non-resistant pepper and Datura stramonium (an important reservoir), as well as in the main vector F. occidentalis, which was considered to evaluate transmission efficiency. The results showed that resistance breakdown was not associated to a fitness cost (tradeoff) in the infectivity of susceptible hosts or transmissibility by thrips and that the resistance-breaking isolates have the same potential for dispersion in field as the conventional isolates. Finally, the information obtained from the previous studies and the RT-qPCR technique were used to evaluate the resistance and tolerance to TSWV of a new accession of Capsicum baccatum. In addition to considering the genetic and biological variation of TSWV, a new approach was used based on analysis of longitudinal data (those measured in the same individuals over time). The resistance level was estimated with two variables: A) absolute fitness, calculated from the variation in time of the viral titer, quantified by RT-qPCR; and B) infectition survival, the median time in which the virus was detected in a plant by ELISA. The tolerance level was estimated as symptom survival, the median time in which a plant developed severe symptoms. This analysis showed that this new accession is partially resistant and totally tolerant to TSWV conventional and resistance-breaking isolates and therefore is a good candidate for a pepper breeding program / [ES] Tomato spotted wilt virus (TSWV) es uno de los virus más extendidos y de mayor importancia económica del mundo. Infecta a un gran número de especies vegetales, siendo los cultivos de tomate y pimiento los más afectados. Este virus se transmite de una planta a otra por trips en una manera propagativa y circulativa, siendo Flankliniella occidentalis el más eficaz. El cultivo de variedades resistentes de tomate y pimiento permitió el control de la enfermedad, ya que estos genes inducen una respuesta hipersensible impidiendo la infección sistémica del virus. Sin embargo, con frecuencia aparecen aislados del virus capaces de superar estas resistencias. Para obtener un control de la enfermedad más eficaz y duradero es necesario: A) la caracterización genética y biológica de los aislados del TSWV que superan y no superan las resistencias; B) el estudio de los factores evolutivos y epidemiológicos implicados en la aparición y establecimiento de los aislados que superan las resistencias; C) el desarrollo de herramientas que permitan la detección y cuantificación de estos aislados virales; y D) la evaluación de nuevas fuentes de resistencia o tolerancia. En este trabajo se han caracterizado biológicamente y molecularmente diferentes aislados del TSWV procedentes de cultivos de tomate y pimiento de España. Se determinó la secuencia nucleotídica del genoma completo de tres aislados españoles correspondientes a tres biotipos: N (incapaz de infectar variedades resistentes de tomate o pimiento); T (supera la resistencia Sw-5 de tomate); y P (supera la resistencia Tsw de pimiento). No se encontró correlación entre la variación genética y la capacidad de superar la resistencia. Estas secuencias nucleotídicas y otras obtenidas de la base de datos Genbank se utilizaron para desarrollar una técnica basada en la RT-PCR cuantitativa (RT-qPCR) que permite detectar el virus con un alto grado de sensibilidad y cuantificar en un amplio rango dinámico. Se diseñaron los iniciadores y una sonda TaqMan MGB a partir de segmentos de secuencia conservados para que fueran válidos para todos los aislados del TSWV. También se desarrollaron dos sondas Taqman que permitían cuantificar diferencialmente variantes genéticas del virus en infecciones mixtas. La RT-qPCR se utilizó para evaluar la acumulación de varios aislados del TSWV en tomate sin Sw-5, pimiento sin Tsw y Datura stramonium, así como, en su principal vector F. occidentalis que se usó para evaluar la eficiencia de la transmisión. Se observó que la superación de la resistencia no suponía un coste en la eficacia biológica (fitness) tanto en la multiplicación del virus en estas plantas como en la transmisión por trips. Por tanto, los aislados que superan las resistencias tienen la misma capacidad de dispersión en campo que los aislados convencionales. Por último, se utilizó la RT-qPCR y la información obtenida para evaluar la capacidad de resistencia y tolerancia al TSWV de una accesión de Capsicum baccatum. El nivel de resistencia se estimó a partir de dos variables: A) la eficacia absoluta, calculada a partir de la variación en el tiempo del título viral, cuantificado por RT-qPCR; y B) la infectividad, como la mediana del tiempo que tarda el virus en ser detectado en una planta por ELISA. El nivel de tolerancia se estimó como la mediana del tiempo que tarda una planta en mostrar síntomas graves. Esta nueva accesión es parcialmente resistente y totalmente tolerante tanto para aislados del TSWV convencionales como para aquellos capaces de infectar e inducir síntomas severos en variedades de pimiento con el gen Tsw. Por tanto, esta nueva variedad de C. baccatum es un buen candidato para usarlo en programas de mejora genética de pimiento. / [CA] Tomato spotted wilt virus (TSWV) és un dels virus més estesos i de major importància econòmica del món. Infecta a un gran nombre d'espècies vegetals i els cultius de tomaca i pebrot són alguns dels més afectats. Aquest virus es transmet d'una planta a una altra, per trips de manera propagativa i circulativa, i Flankliniella occidentalis és el més eficaç. El cultiu de varietats resistents de tomaca i pebrot (en els quals s'han introduït per millora genètica els gens Sw-5 i Tsw, respectivament) va permetre el control de la malaltia, ja que aquests gens indueixen una resposta hipersensible i impedeixen la infecció sistèmica del virus (resistència total). No obstant açò, amb freqüència apareixen aïllats del virus que són capaços de superar aquestes resistències. Per a obtenir un control de la malaltia més eficaç i durador és necessari: A) la caracterització genètica i biològica dels aïllats del TSWV que superen i no superen les resistències; B) l'estudi dels factors evolutius i epidemiològics implicats en l'aparició i establiment dels aïllats que superen les resistències; C) el desenvolupament d'eines que permeten la detecció i quantificació d'aquests aïllats virals; i D) l'avaluació de noves fonts de resistència (total o parcial, que dificulten la infecció i multiplicació viral) o tolerància (total o parcial, que dificulten l'aparició de símptomes i danys encara que no tinguen un efecte en la infecció viral). En aquest treball s'han caracteritzat biològicament i molecularment diferents aïllats del TSWV procedents de cultius de tomaca i pebrot d'Espanya. Es va determinar la seqüència nucleotídica del genoma complet de tres aïllats espanyols corresponents a tres biotips: N (incapaç d'infectar varietats resistents de tomaca o pebrot); T (supera la resistència Sw-5 de tomaca); i P (supera la resistència Tsw de pebrot). Els resultats van mostrar que no hi havia una correlació entre la variació genètica i la capacitat de superar la resistència. Aquestes seqüències nucleotídiques i altres obtingudes de la base de dades Genbank es van utilitzar per a desenvolupar una tècnica basada en la RT-qPCR que permet detectar el virus amb un alt grau de sensibilitat i quantificar en un ampli rang dinàmic. Es van dissenyar els iniciadors i una sonda TaqMan MGB a partir de segments de seqüència conservats perquè foren vàlids per a tots els aïllats del TSWV. Tambe es van desenvolupar dos sondes Taqman que permetien quantificar diferencialment variants genètiques del virus en infeccions mixtes. La RT-qPCR es va utilitzar per a avaluar l'acumulació de diversos aïllats del TSWV en tomaca sense Sw-5, pebrot sense Tsw i Datura stramonium així com, en el seu principal vector F. occidentalis que es va usar per a avaluar l'eficiència de la transmissió. Es va observar que la superació de la resistència no suposava un cost en l'eficàcia biològica (fitness) tant en la multiplicació del virus en aquestes plantes com en la transmissió per trips. Per tant, els aïllats que superen les resistències tenen la mateixa capacitat de dispersió en camp que els aïllats convencionals. Finalment, es va utilitzar la RT-qPCR i la informació obtinguda per a avaluar la capacitat de resistència i tolerància d'una accessió de Capsicum baccatum al TSWV. El nivell de resistència es va estimar a partir de dues variables: A) l'eficàcia absoluta, calculada a partir de la variació en el temps del títol viral, quantificat per RT-qPCR; i B) la infectivitat, com la mitjana del temps que es tarda a detectar el virus en una planta per mitjèr d'ELISA. El nivell de tolerància es va estimar com la mitjana del temps que tarda una planta a mostrar símptomes greus. Aquesta nova accessió és parcialment resistent i totalment tolerant tant per a aïllats del TSWV convencionals com per a aquells capaços d'infectar i induir símptomes severs en varietats amb el gen Tsw. Per tant, aquesta nova varietat d / Debreczeni, DE. (2015). Caracterización de aislados del virus del bronceado del tomate (TSWV) que superan las resistencias de los genes Sw-5 en tomate y Tsw en pimiento. Identificación de una fuente de tolerancia en pimiento [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51460 / Compendio

vegetable crops. Micro-Tom is a model tomato cultivar used for research due to its small size and short time to fruiting. This project evaluated I. walleriana and Micro-Tom transformed with Agrobacterium. The construct contained GFP (green fluorescent protein) and hygromycin antibiotic-resistant selectable markers, and the antisense sequence of open reading frame of INSV nucleocapsid protein (N). The N gene is expected to confer INSV resistance by RNA interference or gene silencing. The presence of transgenes was confirmed by PCR. Transgenic Impatiens was selfed for two generations. Transgenic Micro-Tom was selfed for 4 generations. Spinach was used as an INSV reservoir. Impatiens, spinach and Micro-Tom were mechanically inoculated with INSV and evaluated visually, with assay tests, ELISA testing, and PCR. Spinach was successfully infected with INSV six times of seven attempts. Impatiens and Micro-Tom had no successful inoculations of three and five attempts, respectively. / Master of Science / Impatiens walleriana, also known as impatiens or ‘Bizzy Lizzy,’ is a popular ornamental plant. It has a wide variety of flower colors and grows well in shade. Impatiens necrotic spot virus (INSV) is an incurable virus that causes disfiguring dead spots on plants. Micro-Tom is miniature tomato used for research due to its small size and short time to fruiting. This project tested impatiens and Micro-Tom transgenic plants that had been genetically modified using bacteria. The bacteria had been modified to contain ‘markers’ which allow researchers to confirm the modifications were successful. It also contained a small piece of genetic material from the virus, which was expected to make the plants resistant to the virus by interfering with virus movement and reproduction. These transgenic plants were self-pollinated for multiple generations and tested to confirm the transgene was present. “Wild-type” (not genetically modified) spinach was infected with the virus by hand and infected spinach leaves were used to try to infect impatiens and Micro-Tom. The plants were inspected visually and leaves were tested for presence of the virus. Spinach was successfully infected with INSV six times of seven attempts. Impatiens and Micro-Tom had no successful inoculations of three and five attempts, respectively.