A non-persistently transmitted-virus induces a pull?push strategy inits aphid vector to optimize transmission and spread

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly,generally adapting to each type of virus?vector relationship in a way that enhances transmissionefficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant,cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypiiGlover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferousaphids changed their alighting, settling and probing behaviour activities over time when exposed toCMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate fromCMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), butshowed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min afterrelease. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected overmock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage andaphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph(EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of shortsuperficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (sec-ond hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much lesstime spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour includingan early increase in the number of short superficial probes and intracellular punctures followed by aphloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in aNP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting,settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spreadof the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NPmanner.

Integration of physical, chemical and biological tactics against insect pests and virus diseases in horticultural crops

Actualmente, la gestión de sistemas de Manejo Integrado de Plagas (MIP) en cultivos hortícolas tiene por objetivo priorizar los métodos de control no químicos en detrimento del consumo de plaguicidas, según recoge la directiva europea 2009/128/CE ‘Uso Sostenible de Plaguicidas’ (OJEC, 2009). El uso de agentes de biocontrol como alternativa a la aplicación de insecticidas es un elemento clave de los sistemas MIP por sus innegables ventajas ambientales que se utiliza ampliamente en nuestro país (Jacas y Urbaneja, 2008). En la región de Almería, donde se concentra el 65% de cultivo en invernadero de nuestro país (47.367 ha), MIP es la principal estrategia en pimiento (MAGRAMA, 2014), y comienza a serlo en otros cultivos como tomate o pepino. El cultivo de pepino, con 8.902 ha (MAGRAMA, 2013), tiene un protocolo semejante al pimiento (Robledo et al., 2009), donde la única especie de pulgón importante es Aphis gossypii Glover. Sin embargo, pese al continuo incremento de la superficie de cultivo agrícola bajo sistemas MIP, los daños originados por virosis siguen siendo notables. Algunos de los insectos presentes en los cultivos de hortícolas son importantes vectores de virus, como los pulgones, las moscas blancas o los trips, cuyo control resulta problemático debido a su elevada capacidad para transmitir virus vegetales incluso a una baja densidad de plaga (Holt et al., 2008; Jacas y Urbaneja, 2008). Las relaciones que se establecen entre los distintos agentes de un ecosistema son complejas y muy específicas. Se ha comprobado que, pese a que los enemigos naturales reducen de manera beneficiosa los niveles de plaga, su incorporación en los sistemas planta-insecto-virus puede desencadenar complicadas interacciones con efectos no deseables (Dicke y van Loon, 2000; Jeger et al., 2011). Así, los agentes de biocontrol también pueden inducir a que los insectos vectores modifiquen su comportamiento como respuesta al ataque y, con ello, el grado de dispersión y los patrones de distribución de las virosis que transmiten (Bailey et al., 1995; Weber et al., 1996; Hodge y Powell, 2008a; Hodge et al., 2011). Además, en ocasiones el control biológico por sí solo no es suficiente para controlar determinadas plagas (Medina et al., 2008). Entre los métodos que se pueden aplicar bajo sistemas MIP están las barreras físicas que limitan la entrada de plagas al interior de los invernaderos o interfieren con su movimiento, como pueden ser las mallas anti-insecto (Álvarez et al., 2014), las mallas fotoselectivas (Raviv y Antignus, 2004; Weintraub y Berlinger, 2004; Díaz y Fereres, 2007) y las mallas impregnadas en insecticida (Licciardi et al., 2008; Martin et al., 2014). Las mallas fotoselectivas reducen o bloquean casi por completo la transmisión de radiación UV, lo que interfiere con la visión de los insectos y dificulta o impide la localización del cultivo y su establecimiento en el mismo (Raviv y Antignus, 2004; Weintraub, 2009). Se ha comprobado cómo su uso puede controlar los pulgones y las virosis en cultivo de lechuga (Díaz et al., 2006; Legarrea et al., 2012a), así como la mosca blanca, los trips y los ácaros, y los virus que estos transmiten en otros cultivos (Costa y Robb, 1999; Antignus et al., 2001; Kumar y Poehling, 2006; Doukas y Payne, 2007a; Legarrea et al., 2010). Sin embargo, no se conoce perfectamente el modo de acción de estas barreras, puesto que existe un efecto directo sobre la plaga y otro indirecto mediado por la planta, cuya fisiología cambia al desarrollarse en ambientes con falta de radiación UV, y que podría afectar al ciclo biológico de los insectos fitófagos (Vänninen et al., 2010; Johansen et al., 2011). Del mismo modo, es necesario estudiar la compatibilidad de esta estrategia con los enemigos naturales de las plagas. Hasta la fecha, los estudios han evidenciado que los agentes de biocontrol pueden realizar su actividad bajo ambientes pobres en radiación UV (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007b; Legarrea et al., 2012c). Otro método basado en barreras físicas son las mallas impregnadas con insecticidas, que se han usado tradicionalmente en la prevención de enfermedades humanas transmitidas por mosquitos (Martin et al., 2006). Su aplicación se ha ensayado en agricultura en ciertos cultivos al aire libre (Martin et al., 2010; Díaz et al., 2004), pero su utilidad en cultivos protegidos para prevenir la entrada de insectos vectores en invernadero todavía no ha sido investigada. Los aditivos se incorporan al tejido durante el proceso de extrusión de la fibra y se liberan lentamente actuando por contacto en el momento en que el insecto aterriza sobre la malla, con lo cual el riesgo medioambiental y para la salud humana es muy limitado. Los plaguicidas que se emplean habitualmente suelen ser piretroides (deltametrina o bifentrín), aunque también se ha ensayado dicofol (Martin et al., 2010) y alfa-cipermetrina (Martin et al., 2014). Un factor que resulta de vital importancia en este tipo de mallas es el tamaño del poro para facilitar una buena ventilación del cultivo, al tiempo que se evita la entrada de insectos de pequeño tamaño como las moscas blancas (Bethke y Paine, 1991; Muñoz et al., 1999). Asimismo, se plantea la necesidad de estudiar la compatibilidad de estas mallas con los enemigos naturales. Es por ello que en esta Tesis Doctoral se plantea la necesidad de evaluar nuevas mallas impregnadas que impidan el paso de insectos de pequeño tamaño al interior de los invernaderos, pero que a su vez mantengan un buen intercambio y circulación de aire a través del poro de la malla. Así, en la presente Tesis Doctoral, se han planteado los siguientes objetivos generales a desarrollar: 1. Estudiar el impacto de la presencia de parasitoides sobre el grado de dispersión y los patrones de distribución de pulgones y las virosis que éstos transmiten. 2. Conocer el efecto directo de ambientes pobres en radiación UV sobre el comportamiento de vuelo de plagas clave de hortícolas y sus enemigos naturales. 3. Evaluar el efecto directo de la radiación UV-A sobre el crecimiento poblacional de pulgones y mosca blanca, y sobre la fisiología de sus plantas hospederas, así como el efecto indirecto de la radiación UV-A en ambas plagas mediado por el crecimiento de dichas planta hospederas. 4. Caracterización de diversas mallas impregnadas en deltametrina y bifentrín con diferentes propiedades y selección de las óptimas para el control de pulgones, mosca blanca y sus virosis asociadas en condiciones de campo. Estudio de su compatibilidad con parasitoides. ABSTRACT Insect vectors of plant viruses are the main agents causing major economic losses in vegetable crops grown under protected environments. This Thesis focuses on the implementation of new alternatives to chemical control of insect vectors under Integrated Pest Management programs. In Spain, biological control is the main pest control strategy used in a large part of greenhouses where horticultural crops are grown. The first study aimed to increase our knowledge on how the presence of natural enemies such as Aphidius colemani Viereck may alter the dispersal of the aphid vector Aphis gossypii Glover (Chapter 4). In addition, it was investigated if the presence of this parasitoid affected the spread of aphid-transmitted viruses Cucumber mosaic virus (CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infecting cucumber (Cucumis sativus L). SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersal in the short term, which enhanced CMV spread, though consequences of parasitism suggested potential benefits for disease control in the long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV in the long term. The flight activity of pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria rueppellii (Weidemann) under UV-deficient environments was studied under field conditions (Chapter 5). One-chamber tunnels were covered with cladding materials with different UV transmittance properties. Inside each tunnel, insects were released from tubes placed in a platform suspended from the ceiling. Specific targets were located at different distances from the platform. The ability of aphids and whiteflies to reach their targets was diminished under UV-absorbing barriers, suggesting a reduction of vector activity under this type of nets. Fewer aphids reached distant traps under UV-absorbing nets, and significantly more aphids could fly to the end of the tunnels covered with non-UV blocking materials. Unlike aphids, differences in B. tabaci captures were mainly found in the closest targets. The oviposition of lepidopteran T. absoluta was also negatively affected by a UV-absorbing cover. The photoselective barriers were compatible with parasitism and oviposition of biocontrol agents. Apart from the direct response of insects to UV radiation, plant-mediated effects influencing insect performance were investigated (Chapter 6). The impact of UV-A radiation on the performance of aphid M. persicae and whitefly B. tabaci, and growth and leaf physiology of host plants pepper and eggplant was studied under glasshouse conditions. Plants were grown inside cages covered by transparent and UV-A-opaque plastic films. Plant growth and insect fitness were monitored. Leaves were harvested for chemical analysis. Pepper plants responded directly to UV-A by producing shorter stems whilst UV-A did not affect the leaf area of either species. UV-A-treated peppers had higher content of secondary metabolites, soluble carbohydrates, free amino acids and proteins. Such changes in tissue chemistry indirectly promoted aphid performance. For eggplants, chlorophyll and carotenoid levels decreased with supplemental UVA but phenolics were not affected. Exposure to supplemental UV-A had a detrimental effect on whitefly development, fecundity and fertility presumably not mediated by plant cues, as compounds implied in pest nutrition were unaltered. Lastly, the efficacy of a wide range of Long Lasting Insecticide Treated Nets (LLITNs) was studied under laboratory and field conditions. This strategy aimed to prevent aphids and whiteflies to enter the greenhouse by determining the optimum mesh size (Chapter 7). This new approach is based on slow release deltamethrin- and bifenthrin-treated nets with large hole sizes that allow improved ventilation of greenhouses. All LLITNs produced high mortality of M. persicae and A. gossypii although their efficacy decreased over time with sun exposure. It was necessary a net with hole size of 0.29 mm2 to exclude B. tabaci under laboratory conditions. The feasibility of two selected nets was studied in the field under a high insect infestation pressure in the presence of CMV- and CABYV-infected cucumber plants. Besides, the compatibility of parasitoid A. colemani with bifenthrin-treated nets was studied in parallel field experiments. Both nets effectively blocked the invasion of aphids and reduced the incidence of both viruses, however they failed to exclude whiteflies. We found that our LLITNs were compatible with parasitoid A. colemani. As shown, the role of natural enemies has to be taken into account regarding the dispersal of insect vectors and subsequent spread of plant viruses. The additional benefits of novel physicochemical barriers, such as photoselective and insecticide-impregnated nets, need to be considered in Integrated Pest Management programs of vegetable crops grown under protected environments.

Influence of new integrated pest management strategies on aphidophagous predators in horticultural crops

El 1 de enero de 2014 entró en vigor la Directiva Europea 2009/128/CE sobre uso sostenible de plaguicidas y el Real Decreto 1311/2012 por el cual se traspone dicha normativa comunitaria al ámbito nacional. Estos reglamentos establecen el marco legal por el que las explotaciones agrícolas deben cumplir los principios generales de la Gestión Integrada de Plagas (GIP). Los principios de la GIP dan preferencia a aquellos métodos de control que sean sostenibles y respetuosos con el medio ambiente, dando prioridad al control biológico, al físico y a otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos con los enemigos naturales es necesario en ocasiones para el adecuado manejo de las plagas en cultivos hortícolas. Por ello, el objetivo general de esta Tesis ha sido aportar conocimientos para la mejora del control de plagas en cultivos hortícolas, mediante la integración de estrategias de lucha biológica, física y química. La primera de las líneas de investigación de esta Tesis se centró en el estudio del efecto de la presencia dos depredadores, larvas Chrysoperla carnea y adultos de Adalia bipunctata, en la dispersión del virus de transmisión no persistente Cucumber mosaic virus (CMV) y del virus de transmisión persistente Cucurbit aphid-borne yellows virus (CABYV), transmitidos por el pulgón Aphis gosypii en cultivo de pepino. La tasa de transmisión de CMV fue baja para los dos tiempos de evaluación ensayados (1 y 5 días), debido al limitado movimiento de su vector A. gossypii. Las plantas que resultaron infectadas se localizaron próximas a la fuente de inóculo central y la presencia de ambos enemigos naturales no incrementó significativamente el porcentaje de plantas ocupadas por pulgones ni la tasa de transmisión de CMV. Los patrones de distribución de A. gossypii y de CMV tan solo fueron coincidentes en las proximidades de la planta central infectada en la que se liberaron los insectos. En los ensayos con CABYV, la presencia de C. carnea y de A. bipunctata respectivamente provocó un incremento significativo de la dispersión de A. gossypii tras 14 días, pero no tras 7 días desde la liberación de los insectos. La reducción en el número inicial de pulgones en la planta central infectada con CABYV fue siempre mayor tras la liberación de C. carnea en comparación con A. bipunctata. Sin embargo, la tasa de transmisión de CABYV y su distribución espacial no se vieron significativamente modificadas por la presencia de ninguno de los depredadores, ni tras 7 días ni tras 14 días desde el inicio de los ensayos. Al igual que se estudió el efecto de la presencia de enemigos naturales en el comportamiento de las plagas y en la epidemiología de las virosis que transmiten, en una segunda línea de investigación se evaluó el posible efecto del consumo de pulgones portadores de virus por parte de los enemigos naturales. Este trabajo se llevó a cabo en el Laboratorio de Ecotoxicología del Departamento de Entomología de la Universidade Federal de Lavras (UFLA) (Brasil). En él se evaluó la influencia en los parámetros biológicos del enemigo natural Chrysoperla externa al alimentarse de Myzus persicae contaminados con el virus de transmisión persistente Potato leafroll virus (PLRV). El consumo de M. persicae contaminados con PLRV incrementó significativamente la duración de la fase larvaria, reduciendo también la supervivencia en comparación a otras dos dietas a base de M. persicae no contaminados con el virus y huevos del lepidóptero Ephestia kuehniella. La duración de la fase de pupa de C. externa no difirió significativamente entre las dietas a base de pulgones contaminados con PLRV y pulgones no contaminados, pero ambas fueron menores que con la dieta con huevos de E. kuehniella. Sin embargo, ni la supervivencia en la fase de pupa ni los parámetros reproductivos de los adultos emergidos mostraron diferencias significativas entre las dietas evaluadas. Por el contrario, la supervivencia de los adultos durante los 30 primeros días desde su emergencia sí se vio significativamente afectada por la dieta, siendo al término de este periodo del 54% para aquellos adultos de C. externa que durante su fase larvaria consumieron pulgones con PLRV. Dentro de la GIP, una de las estrategias de carácter físico que se emplean para el control de plagas y enfermedades en cultivos hortícolas protegidos es el uso de plásticos con propiedades fotoselectivas de absorción de la radiación ultravioleta (UV). Por ello, la tercera línea de investigación de la Tesis se centró en el estudio de los efectos directos e indirectos (mediados por la planta) de condiciones especiales de baja radiación UV sobre el crecimiento poblacional del pulgón A. gossypii y los parámetros biológicos del enemigo natural C. carnea, así como sobre las plantas de pepino en las que se liberaron los insectos. Los ensayos se realizaron en jaulones dentro de invernadero, utilizándose en el primero de ellos plantas de pepino sanas, mientras que en el segundo las plantas de pepino fueron previamente infectadas con CABYV para estudiar de qué manera afectaba la incidencia del virus en las mismas condiciones. Las condiciones de baja radiación UV (bajo plástico Térmico Antivirus®) ejercieron un efecto directo en las fases iniciales del cultivo de pepino, promoviendo su crecimiento, mientras que en fases más avanzadas del cultivo indujeron un aumento en el contenido en nitrógeno de las plantas. Las plantas de pepino que fueron sometidas a mayor intensidad de radiación UV (bajo plástico Térmico Blanco®) al inicio del cultivo mostraron un engrosamiento significativo de las paredes de las células epidérmicas del haz de las hojas, así como de la cutícula. El uso del plástico Térmico Antivirus®, utilizado como barrera fotoselectiva para crear condiciones de baja radiación UV, no alteró con respecto al plástico Térmico Blanco® (utilizado como control) el desarrollo poblacional del pulgón A. gossypii ni los parámetros biológicos evaluados en el depredador C. carnea. En el segundo experimento, realizado con plantas infectadas con CABYV, la incidencia de la virosis enmascaró las diferencias encontradas en experimento con plantas sanas, reduciendo aparentemente la influencia de las distintas condiciones de radiación UV. Por último, para el desarrollo de las estrategias de GIP es importante estudiar los posibles efectos secundarios que los plaguicidas pueden tener en los enemigos naturales de las plagas. Es por ello que en la Tesis se evaluaron la toxicidad y los efectos subletales (fecundidad y fertilidad) de flonicamida, flubendiamida, metaflumizona, spirotetramat, sulfoxaflor y deltametrina en los enemigos naturales C. carnea y A. bipunctata. Los efectos secundarios fueron evaluados por contacto residual tanto para larvas como para adultos de ambos enemigos naturales en condiciones de laboratorio. Flonicamida, flubendiamida, metaflumizona y spirotetramat fueron inocuos para larvas de último estadio y adultos de C. carnea y A. bipunctata. Por este motivo, estos insecticidas se presentan como buenos candidatos para ser incorporados dentro de programas de GIP en combinación con estos enemigos naturales para el control de plagas de cultivos hortícolas. Sulfoxaflor fue ligeramente tóxico para adultos de C. carnea y altamente tóxico para larvas de último estadio de A. bipunctata. Para A. bipunctata, sulfoxaflor y deltametrina fueron los compuestos más dañinos. Deltametrina fue también el compuesto más tóxico para larvas y adultos de C. carnea. Por tanto, el uso de deltametrina y sulfoxaflor en programas de GIP debería tomarse en consideración cuando se liberasen cualquiera de estos dos enemigos naturales debido al comportamiento tóxico que mostraron en condiciones de laboratorio. ABSTRACT On 1 January 2014 came into effect the Directive 2009/128/EC of the European Parliament about sustainable use of pesticides and the Royal Decree 1311/2012 that transposes the regulation to the Spanish level. These regulations establish the legal framework that agricultural holdings must adhere to in order to accomplish the general principles of Integrated Pest Management (IPM). The guidelines of IPM give priority to sustainable and eco-friendly pest control techniques, such as biological and physical measures. Nevertheless, the use of pesticides that are selective to natural enemies is sometimes a necessary strategy to implement accurate pest management programs in horticultural protected crops. Therefore, the general objective of this Thesis was to contribute to the improvement of pest management strategies in horticultural crops, by means of the integration of biological, physical and chemical techniques. The first research line of this Thesis was focused on the evaluation of the effects of two aphidophagous predators, Chrysoperla carnea larvae and Adalia bipunctata adults, on the spread of the non-persistently transmitted Cucumber mosaic virus (CMV, Cucumovirus) and the persistently transmitted Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), by the aphid vector Aphis gossypii in a cucumber crop under greenhouse conditions. The CMV transmission rate was generally low, both after 1 and 5 days, due to the limited movement of its aphid vector A. gossypii. Infected plants were mainly located around the central virusinfected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant where insects were released. In the CABYV experiments, the presence of C. carnea larvae and A. bipunctata adults induced significant A. gossypii dispersal after 14 days but not after 7 days. The reduction in the initial aphid population established in the central plant was always higher for C. carnea than for A. bipunctata. Nevertheless, CABYV spread was not significantly modified by the presence of each predator either in the short term (7 days) or in the long term (14 days). Furthermore, the percentage of CABYV-infected plants did not significantly differ when each natural enemy was present in any evaluation period. It is important to evaluate the influence that natural enemies have on pest dynamics and on the spread of viral diseases, but it should be also taken into account the possible effect on the performance of natural enemies when they feed on preys that act as vectors of viruses. Thus, in a second research line developed in the Laboratory of Ecotoxicology, Department of Entomology, of the Universidade Federal de Lavras (UFLA) (Brazil), it was evaluated the performance of Chrysoperla externa under the condition of consuming Myzus persicae acting as vector of Potato leafroll virus (PLRV). The diet composed of PLRV-infected M. persicae significantly increased the length and reduced the survival rate, of the larval period in regard to the other two diets, composed of non-infected M. persicae and Ephestia kuehniella eggs. The lengths of the pupal stage were not significantly different between the aphid diets, but both were significantly shorter than that of E. kuehniella eggs. Neither pupal survival nor reproductive parameters revealed significant differences among the diets. Nevertheless, the adult survival curves during the first 30 days after emergence showed significant differences, reaching at the end of this interval a value of 54% for those C. externa adults fed on PLRVinfected aphids during their larval period. According to the IPM guidelines, one of the physical strategies for the control of pests and diseases in horticultural protected crops is the use of plastic films with photoselective properties that act as ultraviolet (UV) radiation blocking barriers. In this sense, the third research line of the Thesis dealt with the study of the direct and plant-mediated influence of low UV radiation conditions on the performance of the aphid A. gossypii and on the biological parameters of the natural enemy C. carnea, as well as on the cucumber plants where insects were released. The experiments were conducted inside cages under greenhouse conditions, using for the first one healthy cucumber plants, while for the second experiment the cucumber plants were previously infected with CABYV in order to assess the influence of the virus in the same conditions. The low UV radiation conditions (under Térmico Antivirus® plastic film) seemed to exert a direct effect in the early stages of cucumber plants, enhancing their growth, and in an increasing nitrogen content at further developmental stages. The higher UV radiation exposure (under Térmico Blanco® plastic film) in the early stages of the cucumber crop induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves. The use of Térmico Antivirus® plastic film as a photoselective barrier to induce low UV radiation conditions did not modify, in regard to Térmico Blanco® plastic film (used as control), neither the population development of A. gossypii nor the studied biological parameters of the predator C. carnea. In the second experiment, done with CABYV-infected cucumber plants, the incidence of the virus seemed to mask the direct and plant-mediated influence of the different UV radiation conditions. In last term, for the development of IPM strategies it is important to study the potential side effects that pesticides might have on natural enemies. For this reason, in the Thesis were tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide, metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies C. carnea and A. bipunctata. The side effects of the active ingredients of the insecticides were evaluated with residual contact tests for the larvae and adults of these predators under laboratory conditions. Flonicamid, flubendiamide, metaflumizone and spirotetramat were innocuous to last instar larvae and adults of C. carnea and A. bipunctata. Therefore, these pesticides are promising candidates for being incorporated into IPM programs in combination with these natural enemies for the control of particular greenhouse pests. In contrast, sulfoxaflor was slightly toxic to adults of C. carnea and was highly toxic to last instar larvae of A. bipunctata. For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging compounds. Deltamethrin was also the most toxic compound to larvae and adults of C. carnea. In accordance with this fact, the use of sulfoxaflor and deltamethrin in IPM strategies should be taken into consideration when releasing either of these biological control agents, due to the toxic behavior observed under laboratory conditions.

Efecto de la infección por virus persistentes y no persistentes sobre el comportamiento alimenticio, la preferencia, tasa de aterrizaje y asentamiento y la eficacia biológica de Aphis gossypii Glover

En el complejo de plagas que atacan a los principales cultivos hortícolas protegidos, destacan principalmente los Hemípteros, y dentro de estos los pulgones, dada su importancia como vectores de virus que provocan considerables daños y pérdidas económicas. Debido a que la dispersión de la mayoría de los virus de plantas puede ser eficaz con densidades bajas de vectores y su control es muy complicado al no existir métodos curativos para su control, es necesario generar nuevos conocimientos sobre las interacciones virus-vector con el fin de desarrollar nuevas y eficaces estrategias de control. Por ello, el objetivo general de esta Tesis ha sido conocer el efecto de la infección viral (directo-mediado por la presencia del virus en el vector- e indirecto-mediado por las alteraciones físico-químicas que se originan en la planta como consecuencia de la infección viral-) sobre el comportamiento y eficacia biológica del vector Aphis gossypii Glover y sus posibles repercusiones en la epidemiología de virosis de transmisión no persistente (Cucumber mosaic virus, CMV, Cucumovirus) y persistente (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). El primer objetivo de esta Tesis Doctoral, se centró en el estudio del efecto indirecto del virus de transmisión no persistente CMV sobre el comportamiento alimenticio y la preferencia del pulgón A. gossypii en el cultivo de pepino. Los ensayos de despegue y aterrizaje mostraron que los pulgones que fueron liberados en las plantas de pepino infectadas con CMV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (60, 120 y 180 minutos después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CMV). El estudio de preferencia y asentamiento mostró que el vector A. gossypii prefiere asentarse en plantas infectadas con CMV en una etapa temprana de evaluación (30 minutos después de la liberación). Sin embargo, este comportamiento se revirtió en una etapa posterior (4 y 48 horas después de la liberación), donde los pulgones se asentaron más en las plantas no infectadas. A través de la técnica de Gráficos de Penetración Eléctrica (EPG) se observó un efecto indirecto del virus CMV, revelado por un cambio brusco en el comportamiento de prueba del pulgón a lo largo del tiempo, cuando éstos fueron expuestos a las plantas infectadas con CMV. Los primeros 15 minutos de registro EPG mostraron que los pulgones hicieron un número mayor de punciones intracelulares (potencial drops - pds) y pruebas en las plantas infectadas con CMV que en las plantas no infectadas. Por otra parte, la duración de la primera prueba fue más corta y la duración total de las pds por insecto fue mucho más larga en las plantas infectadas con CMV. Se observaron diferencias significativas en el tiempo transcurrido desde el final de la última pd hasta el final de la prueba, siendo ese tiempo más corto para los pulgones que estaban alimentándose en plantas infectadas con CMV. En la segunda hora de registro los pulgones rechazaron las plantas infectadas con CMV como fuente de alimento, permaneciendo menos tiempo en las fases de prueba en floema (fase de salivación – E1 y fase de ingestión del floema – E2). El comportamiento alimenticio observado sobre las plantas infectadas con CMV favorece la adquisición y posterior transmisión de los virus de transmisión no persistente, los cuales son adquiridos e inoculados durante la realización de pruebas intracelulares en las primeras pruebas de corta duración. En el segundo objetivo de la Tesis se evaluó el efecto directo e indirecto del virus de transmisión persistente CABYV en el comportamiento alimenticio y preferencia del pulgón A. gossypii en cultivo de pepino, especie susceptible al virus, y algodón, especie inmune al virus. No se observó un efecto directo del virus relevante en el comportamiento alimenticio del vector, ya que los resultados obtenidos a nivel floemático en plantas de pepino no se observaron en plantas de algodón, inmune al virus CABYV. Esto sugiere que los resultados obtenidos en pepino, pueden deberse a un “posible efecto indirecto” originado por la infección de las plantas susceptibles al virus durante la realización del ensayo, lo que indirectamente puede modificar el comportamiento del pulgón durante la fase de evaluación. Sin embargo, el virus CABYV modificó indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada. Los pulgones tardaron menos tiempo en llegar al floema, realizaron un mayor número de pruebas floemáticas y permanecieron durante más tiempo en actividades floemáticas en plantas infectadas con CABYV. El comportamiento observado sobre las plantas infectadas con CABYV favorece la adquisición de virus persistentes, los cuales son adquiridos durante la alimentación sostenida en floema. El estudio de preferencia y asentamiento de A. gossypii mostró que los pulgones virulíferos prefieren asentarse en plantas no infectadas a corto y largo plazo de evaluación (2, 4 y 48 horas después de la liberación). Los ensayos de despegue y aterrizaje mostraron que los pulgones virulíferos que fueron liberados en las plantas de pepino infectadas con CABYV tuvieron una mayor propensión en migrar hacia las plantas no infectadas (3, 6, 24 y 48 horas después de la liberación) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CABYV). Sin embargo, los pulgones no virulíferos no mostraron preferencia por plantas de pepino no infectadas o infectadas con CABYV en ninguno de los ensayos (preferencia o despegue) o periodos evaluados (corto y largo plazo). Los resultados indican que el virus CABYV es capaz de modificar indirectamente el comportamiento alimenticio de su vector a través de cambios en la planta infectada, favoreciendo su adquisición por su principal vector, A. gossypii. Una vez que los pulgones tienen capacidad de transmitir el virus (virulíferos) se produce un cambio en su comportamiento prefiriendo asentarse sobre plantas no infectadas optimizándose así la dispersión viral. El tercer objetivo de la Tesis, fue evaluar los efectos directos e indirectos del virus CABYV así como los efectos indirectos del virus CMV en la eficacia biológica del vector A. gossypii. Los resultados obtenidos en los ensayos realizados con el virus persistente CABYV indican que el virus parece no modificar directamente ni indirectamente la eficacia biológica del vector en plantas de pepino o algodón, no observándose diferencias estadísticas en ninguno de los parámetros poblacionales evaluados (tiempo de desarrollo, tasa intrínseca de crecimiento, tiempo generacional medio, tasa media de crecimiento relativo y ninfas totales). En cuanto a los ensayos realizados con el virus no persistente, CMV, los resultados muestran un efecto indirecto del virus sobre la biología del vector. Así resultó que tanto la tasa intrínseca de crecimiento natural (rm) como la tasa media de crecimiento relativo (RGR) fueron más altas para pulgones crecidos sobre plantas infectadas con CMV que sobre plantas no infectadas, favoreciendo la reproducción y crecimiento poblacional del vector sobre plantas infectadas con CMV. Los resultados obtenidos en la presente Tesis, ofrecen un ejemplo de como los virus de plantas pueden manipular directa e indirectamente a su vector, maximizando así su dispersión entre las plantas. Esos nuevos conocimientos generados tienen implicaciones importantes en la transmisión, dispersión y en la epidemiología de los virus y deben ser considerados para diseñar o ajustar los modelos de simulación existentes y patrones de dispersión que describen las epidemias de estos virus. ABSTRACT The main objective of this Thesis has been to understand the effect of the viral infection (direct-mediated by the presence of the virus in the vector and indirect mediated by the chemical and physical changes originated in the plant as a consequence of the viral infection) on the behaviour and biological efficacy of the vector Aphis gossypii Glover and its consequences in the epidemiology of two viral diseases, one with non-persistent transmission (Cucumber mosaic virus, CMV, Cucumovirus) and another with persistent transmission (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). The first objective of this Thesis was the study of the indirect effect of the nonpersistent virus CMV on the feeding behaviour and preference of the aphid A. gossypii in cucumber plants. The results of the alighting and settling behaviour studies showed that aphids exhibited no preference to migrate from CMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), but showed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min after release. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected over mockinoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage and aphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph (EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of short superficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (second hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much less time spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour including an early increase in the number of short superficial probes and intracellular punctures followed by a phloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in a NP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting, settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spread of the virus. The second objective of this work was to evaluate the effects that the persistently aphid transmitted Cucurbit aphid-borne yellows virus (CABYV) can induce directly and indirectly on the alighting, settling and probing behaviour activities of the cotton aphid A. gossypii. Only minor direct changes on aphid feeding behaviour was observed due to CABYV when viruliferous aphids fed on mock-inoculated plants. However, the feeding behaviour of non-viruliferous aphids was very different on CABYV-infected than on mockinoculated plants. Non-viruliferous aphids spent longer time feeding from the phloem when plants were infected by CABYV than on mock-inoculated plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. The vector alighting and settling preference was compared between nonviruliferous and viruliferous aphids. Viruliferous aphids showed a clear preference for mockinoculated over CABYV-infected plants at short and long time, while such behaviour was not observed for non-viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimize the transmission and spread of the virus. The third objective was to evaluate the direct and indirect effects of CABYV and indirect effects of the CMV on the A. gossypii fitness. Obtained results for the persistent virus CABYV showed that the virus did not modify the vector fitness in cucumber or cotton plants. None of the evaluated variables was statistically significant (development time (d), intrinsic growth rate (rm), mean relative growth rate (RGR) and total number of nymphs). On the other hand, data obtained for the non-persistent virus (CMV) showed an indirect effect of the virus on the vector fitness. Thus, the rm and RGR were higher for aphids grown on CMV-infected plants compared to aphids grown on mock-inoculated plants. Overall, the obtained results are clear examples of how plant viruses could manipulate directly and indirectly vector behaviour to optimize its own dispersion. These results are important for a better understanding of transmission, dispersion and epidemiology of plant viruses transmitted by vectors. This information could be also considered to design or adjust simulation models and dispersion patterns that describe plant virus epidemics.

Spatio-temporal dynamics of viruses are differentially affected by parasitoids depending on the mode of transmission by their insect vectors.

Relationships between agents in multitrophic systems are complex and very specific. Insect-transmitted plant viruses are completely dependent on the behaviour and distribution patterns of their vectors. The presence of natural enemies may directly affect aphid behaviour and spread of plant viruses, as the escape response of aphids might cause a potential risk for virus dispersal. The spatio-temporal dynamics of Cucumber mosaic virus (CMV) and Cucurbit aphid-borne yellows virus (CABYV), transmitted by Aphis gossypii in a non-persistent and persistent manner, respectively, were evaluated at short and long term in the presence and absence of the aphid parasitoid, Aphidius colemani. SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersion at short term, which enhanced CMV spread, though consequences of parasitism suggest potential benefits for disease control at long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV at long term. The impact of aphid parasitoids on the dispersal of plant viruses with different transmission modes is discussed.

Control of insect vectors and plant viruses in protected crops by novel pyrethroid-treated nets

Long-lasting insecticide-treated nets (LLITNs) constitute a novel alternative that combines physical and chemical tactics to prevent insect access and the spread of insect-transmitted plant viruses in protected enclosures. This approach is based on a slow-release insecticide-treated net with large hole sizes that allow improved ventilation of greenhouses. The efficacy of a wide range of LLITNs was tested under laboratory conditions against Myzus persicae, Aphis gossypii and Bemisia tabaci. Two nets were selected for field tests under a high insect infestation pressure in the presence of plants infected with Cucumber mosaic virus and Cucurbit aphid-borne yellows virus. The efficacy of Aphidius colemani, a parasitoid commonly used for biological control of aphids, was studied in parallel field experiments.

Ecological and genetic determinants of Pepino mosaic virus emergence

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether Pepino mosaic virus (PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the Solanaceae, multiplying efficiently in most assayed Solanum species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild Solanum species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different Solanum species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other Solanum species, would isolate tomato populations from those in other hosts.

Effects of a bifenthrin-treated net on the natural enemies Aphidius colemani (Haliday) and Adalia bipunctata L. in a cucumber crop in Central Spain. Semi-field experiments

Pest management practices that rely on pesticides are growing increasingly less effective and environmentally inappropriate in many cases and the search of alternatives is under focus nowadays. Exclusion of pests from the crop by means of pesticide-treated screens can be an eco-friendly method to protect crops, especially if pests are vectors of important diseases. The mesh size of nets is crucial to determine if insects can eventually cross the barrier or exclude them because there is a great variation in insect size depending on the species. Long-lasting insecticide-treated (LLITN) nets, factory pre-treated, have been used since years to fight against mosquitoes vector of malaria and are able to retain their biological efficacy under field for 3 years. In agriculture, treated nets with different insecticides have shown efficacy in controlling some insects and mites, so they seem to be a good tool in helping to solve some pest problems. However, treated nets must be carefully evaluated because can diminish air flow, increase temperature and humidity and decrease light transmission, which may affect plant growth, pests and natural enemies. As biological control is considered a key factor in IPM nowadays, the potential negative effects of treated nets on natural enemies need to be studied carefully. In this work, the effects of a bifentrhin-treated net (3 g/Kg) (supplied by the company Intelligent Insect Control, IIC) on natural enemies of aphids were tested on a cucumber crop in Central Spain in autumn 2011. The crop was sown in 8x6.5 m tunnels divided in 2 sealed compartments with control or treated nets, which were simple yellow netting with 25 mesh (10 x 10 threads/cm2; 1 x 1 mm hole size). Pieces of 2 m high of the treated-net were placed along the lateral sides of one of the two tunnel compartments in each of the 3 available tunnels (replicates); the rest was covered by a commercial untreated net of a similar mesh. The pest, Aphis gossypii Glover (Aphidae), the parasitoid Aphidius colemani (Haliday) (Braconidae) and the predator Adalia bipunctata L. (Coccinellidae) were artificially introduced in the crop. Weekly sampling was done determining the presence or absence of the pest and the natural enemies (NE) in the 42 plants/compartment as well as the number of insects in 11 marked plants. Environmental conditions (temperature, relative humidity, UV and PAR radiation) were recorded. Results show that when aphids were artificially released inside the tunnels, neither its number/plant nor their distribution was affected by the treated net. A lack of negative effect of the insecticide-treated net on natural enemies was also observed. Adalia bipunctata did not establish in the crop and only a short term control of aphids was observed one week after release. On the other hand, A. colemani did establish in the crop and a more long-term effect on the numbers of aphids/plant was detected irrespective of the type of net. KEY WORDS: bifenthrin-treated net, Adalia bipunctata, Aphidius colemani, Aphis gossypii, semi-field

Use of tomato and cucumber horticulture by-products in goat diets: effects o nrumen fermentation and microbial communities in batch and continuous cultures

Two in vitro experiments were conducted to analyse the effects of replacing dietary barley grain with wastes of tomato and cucumber fruits and a 1 : 1 tomato : cucumber mixture on rumen fermentation characteristics and microbial abundance. The control (CON) substrate contained 250 g/kg of barley grain on a dry matter (DM) basis, and another 15 substrates were formulated by replacing 50, 100, 150, 200 or 250 g of barley grain/kg with the same amount (DM basis) of tomato or cucumber fruits or 1 : 1 tomato : cucumber mixture. In Expt 1, all substrates were incubated in batch cultures with rumen micro-organisms from goats for 24 h. Increasing amounts of tomato, cucumber and the mixture of both fruits in the substrate increased final pH and gas production, without changes in final ammonia-nitrogen (NH3-N) concentrations, substrate degradability and total volatile fatty acid (VFA) production, indicating that there were no detrimental effects of any waste fruits on rumen fermentation. Therefore, in Expt 2 the substrates including 250 g of waste fruits (T250, C250 and M250 for tomato, cucumber and the mixture of both fruits, respectively) and the CON substrate were incubated in single-flow continuous-culture fermenters for 8 days. Total VFA production did not differ among substrates, but there were differences in VFA profile. Molar proportions of propionate, isobutyrate and isovalerate were lower and acetate : propionate ratio was greater for T250 compared with CON substrate. Fermentation of substrates containing cucumber (C250 and M250) resulted in lower proportions of acetate, isobutyrate and isovalerate and acetate : propionate ratio, but greater butyrate proportions than the CON substrate. Carbohydrate degradability and microbial N synthesis tended to be lower for substrates containing cucumber than for the CON substrate, but there were no differences between CON and T250 substrates. Abundance of total bacteria, Fibrobacter succinogenes and Ruminococcus flavefaciens, fungi, methanogenic archaea and protozoa were similar in fermenters fed T250 and CON substrates, but fermenters fed C250 and M250 substrates had lower abundances of R. flavefaciens, fungi and protozoa than those fed the CON substrate. Results indicated that tomato fruits could replace dietary barley grain up to 250 g/kg of substrate DM without noticeable effects on rumen fermentation and microbial populations, but the inclusion of cucumber fruits at 250 g/kg of substrate DM negatively affected some microbial populations as it tended to reduce microbial N synthesis and changed the VFA profile. More studies are needed to identify the dietary inclusion level of cucumber which produces no detrimental effects on rumen fermentation and microbial growth.

El Pisito 2011

Analyzing residential architecture in Spain today signifies tackling a complex, apparently fragmentary situation, one that makes comment or criticism quite difficult. In the last few years authority over housing and city planning has been transferred to the regions,1 and each has developed its own framework of laws, its own regulations and requirements, and has shaped urban and regional development on the basis of its own criteria. In parallel, schools of architecture have multiplied, reaching many of the zones previously regarded as ?peripheral.? It is easy to understand how the old administrative centrality has given way to a more pluralistic situation closer to the specific problems of each city and region. As a result cultural differences have been accentuated, even compromising the possibility of speaking of an ?architecture Spanish? or reducing it to a mere summation or mosaic of local circumstances. Part of the debate of recent years has been focused on underlining these differences. This essay takes the opposite approach. The premise from which it starts out is that analysis of the phenomenon of ?residential architecture in Spain? makes it possible to acknowledge the survival of a cultural unity that explains the present and the recent past and that does not conflict with the diversity of local experiences.

Characterization of vineyard's canopy through fuzzy clustering and svm over color images

In this work we propose an image acquisition and processing methodology (framework) developed for performance in-field grapes and leaves detection and quantification, based on a six step methodology: 1) image segmentation through Fuzzy C-Means with Gustafson Kessel (FCM-GK) clustering; 2) obtaining of FCM-GK outputs (centroids) for acting as seeding for K-Means clustering; 3) Identification of the clusters generated by K-Means using a Support Vector Machine (SVM) classifier. 4) Performance of morphological operations over the grapes and leaves clusters in order to fill holes and to eliminate small pixels clusters; 5)Creation of a mosaic image by Scale-Invariant Feature Transform (SIFT) in order to avoid overlapping between images; 6) Calculation of the areas of leaves and grapes and finding of the centroids in the grape bunches. Image data are collected using a colour camera fixed to a mobile platform. This platform was developed to give a stabilized surface to guarantee that the images were acquired parallel to de vineyard rows. In this way, the platform avoids the distortion of the images that lead to poor estimation of the areas. Our preliminary results are promissory, although they still have shown that it is necessary to implement a camera stabilization system to avoid undesired camera movements, and also a parallel processing procedure in order to speed up the mosaicking process.

Tomas Lopez's Geographic Atlas of Spain in the Peninsular War: A methodology for determining errors

During the Peninsular War, Napoleon's and Wellington's armies were aware of the lack of precision in the maps of Spain and its provinces that appeared in Tomas Lopez \s Geographical Atlas of Spain. The errors were due to the non-topographical surveying method he used which he had learned from his teacher Jean Baptiste Bourguignon D 'Anville. To map all of the Spanish provinces, Tomas Lopez divided them into circles of three leagues in diameter (16,718 m), taking a particular town as the centre. He asked the town's priest to draw a map of the territory and to complete a questionnaire that Tomas Lopez sent to him. The priest was to return the two documents after he had completed them. Subsequently, at his desk, Tomas Lopez used the maps and reports as well as other graphic and written sources from various locations to make an outline of each map. Next, he made a mosaic that served as a pattern for drawing the final provincial map. We will see the way that this method was applied in two concrete cases: the villages ofChavaler and Monteagudo, situated in the Spanish province of Soria, and verify their degree of accuracy. We will use the maps drawn by the priests in 1767, the final map of the province which was published in 1804 by Tomás López, and a current map of the province showing the angular and linear errors in Lopez \s map.

El injerto en pepino corto tipo español (Cucumis sativus L.) recomencaciones para su empleo en la zona central española

El injerto en hortalizas es uno de los temas de más actualidad en el panorama hortícola, no solo español, sino occidental, y recalcamos occidental, pues en muchos países que no corresponden a ese ámbito, sobre todo asiáticos: Japón, Corea, China, Filipinas, etc., esta es una técnica que cuenta con una gran difusión desde hace décadas, siendo, por ejemplo en Japón, la mayoría de sus cultivos de cucurbitáceas y solanáceas realizados con planta injertada. A finales de los noventa quedó claro que el empleo de bromuro de metilo tenía una fecha de caducidad y que las zonas que tenían una fuerte dependencia de este desinfectante de suelo debían de buscar alternativas a un plazo lo más corto posible, con un punto añadido sobre etapas anteriores, debían ser alternativas lo más respetuosas posible con el medio ambiente y que no incrementaran, de forma importante, los costes de producción. En la zona centro y concretamente en los invernaderos de la Comunidad de Madrid y zonas cercanas de Toledo y Guadalajara el pepino era y es el cultivo predominante, los horticultores empleaban el bromuro de metilo de forma sistemática para desinfectar sus suelos y la desaparición de este producto les planteaba una gran incertidumbre, lo que llevó a que desde diferentes instancias se buscaran diferentes alternativas. Tras analizar las posibilidades que se podían implementar y conocido el buen resultado que había dado el injerto en sandía en Almería, se decidió acometer los trabajos que conforman esta Tesis Doctoral, planteando en la zona, diferentes ensayos con la idea de conocer, si el injerto en pepino, con los cultivares empleados habitualmente, podía ser una alternativa real para los horticultores, tanto de Madrid, como los de las zonas cercanas de Toledo y Guadalajara. Se pretendía conocer sobre todo las repercusiones agronómicas y si esta técnica podría emplearse en solitario o era necesario complementarla con otras alternativas: desinfectantes químicos, solarización, biofumigación e incluso desinfección con vapor de agua. Los ensayos fueron realizados de forma secuencial entre el año 1999 y el 2011, comprobándose en primer lugar que el empleo de portainjertos híbridos de calabaza era posible con los cultivares de pepino corto tipo español, mayoritariamente empleados en los últimos años del siglo XX y primeros del XXI, fundamentalmente: Serena. Tras los primeros ensayos, Shintoza parecía el portainjerto híbrido de calabaza (Cucurbita maxima x C. moschata) con mejores perspectivas de empleo, pues presentaba la ventaja adicional de ser bien conocido por los semilleros que producen planta injertada al ser, en esos momentos, el portainjerto más empleado en sandía, lo que garantizaba por su lado, su empleo en pepino, y que los horticultores pudiesen disponer de planta injertada. Más adelante los trabajos se encaminaron hacia la determinación de la densidad y tipo de poda más adecuado para la planta injertada, realizándose múltiples ensayos en esta dirección, que culminaron con la conclusión de que el extravigor que los portainjertos conferían a las plantas permitía conducir estas a dos o más brazos (se suelen emplear dos, por mejor adaptación a los trabajos de manejo de la planta por parte de los agricultores), con lo que se podría disminuir la densidad de planta y por tanto ahorrar en este capítulo, cosa que preocupaba y preocupa a los agricultores. Se llegó a determinar que es posible reducir la densidad de plantación en alrededor de un 25%, estando la densidad de brazos más adecuada entre 3 y 3.5 br•m-2. Tras las primeras decisiones tomadas sobre el portainjerto y la densidad más adecuada, se continuó con el estudio de adaptación de estas propuestas a los nuevos cultivares que las empresas de semillas iban proponiendo y los agricultores adoptando. Estas acciones se complementaron con la introducción de nuevos portainjertos susceptibles de sustituir a Shintoza o rotar con él para cambiar de sistema radicular, lo que es conveniente cuando se emplean, como es el caso, portainjertos que no son resistentes a nematodos, principalmente de la especie Meloidogyne incognita, el mayor problema en la zona, debido al suelo. Cultivares como Trópico, en un primer momento, y Urano y Motril más recientemente, se adaptaron muy bien a esta técnica. Entre los portainjertos que mostraron buena adaptación a la técnica de injerto y suficientemente buena compatibilidad con la mayoría de los cultivares ensayados destacan: RS-841, Strongtosa y Camel. Azman también mostró un comportamiento relevante, pero este portainjerto no podrá ser empleado, al ser recientemente retirado del mercado por la empresa que lo obtuvo y comercializó Aunque no era el objetivo principal de esta Tesis Doctoral, se ha comprobado que puede ser interesante combinar el empleo del injerto con otras técnicas alternativas al bromuro de metilo para superar los problemas debidos a enfermedades del suelo o nematodos, pero debe seguirse trabajando pues este es un tema en continua evolución, tanto si se trata de desinfectantes, a la mayoría de los cuales les está siendo retirado el permiso para su comercialización, como de otros métodos como la biofumigación o el empleo de vapor de agua. Queda muy claro que el injerto puede considerarse entre los métodos respetuosos con el medio ambiente, si no el que más, en lo que alternativas al bromuro de metilo se refiere. También en otro momento, se comprobó que con plantas injertadas es posible reducir el aporte de nutrientes, sobre todo nitrógeno, lo que además de un ahorro supone una mejora más desde el punto de vista medioambiental. En definitiva, queda demostrado que es factible el empleo del injerto en pepino corto tipo español, que las selecciones de los híbridos entre Cucurbita maxima y C. moschata que habitualmente se están empleando en sandía son también de aplicación en estos pepinos y que su empleo puede llevarnos a producciones suficientemente remuneradoras, alargándose en muchos casos el ciclo y no modificando, de forma apreciable, la calidad. Queda también demostrado que aunque los portainjertos no sean resistentes a nematodos, su extravigor les hace desarrollarse, desde el punto de vista productivo, suficientemente, llegando por tanto, a “convivir” con ese problema. Al no ser resistentes los portainjertos, y permanecer e incluso agravarse el problema de nematodos es conveniente poder contar con diferentes portainjertos que nos permitan rotar entre ellos y utilizar diferentes sistemas radiculares que harán menos fácil el parasitismo de los nematodos, como recomiendan los nematólogos que se haga. ABSTRACT Vegetable grafting is one of the most current practices in horticulture, not only in Spain, but also in other Western and Asian countries, such as Japan, South Korea, China, the Philippines, etc. This is a decades-old, widespread technique: In fact, most cucurbit and solanaceous crops in Japan and Korea are grafted. At the end of the 1990s, it was clear that methyl bromide had an expiry date. Consequently, the areas strongly dependant on this soil disinfectant had to look for alternatives as quickly as possible. Besides, these had to be as environmentally friendly as possible and should not increase production costs significantly. The cucumber has been and still is the most important crop in greenhouses of the Comunidad de Madrid and in areas near Toledo and Guadalajara. Cucumber growers used methyl bromide systematically to disinfect the soil. The banning of this chemical product brought about uncertainty, which encouraged the search for different alternatives. After analyzing the different possibilities and taking into account the good results of watermelon grafting in Almería, it was decided to carry out the works that make up this doctoral thesis. Different trials were made in order to know if the cultivars used in cucumber grafting might be a real alternative for farmers, not only in Madrid, but also in the areas near Toledo and Guadalajara. The main aim was to assess the agronomic repercussions and whether that technique could be used alone, or if other complementary alternatives, such as chemical disinfectants, solarisation, biofumigation, or even steam disinfection, were necessary. Trials were carried out sequentially from 1999 to 2011. It was observed that the use of pumpkin hybrid rootstocks could be applied to cultivars of Spanish short cucumbers, mainly grown in the late 20th and early 21st centuries eg Serena. After the early trials, Shintoza (Cucurbita maxima x C. moschata), a pumpkin hybrid rootstock, seemed to be the best option, as it had the additional advantage of being well known by nurseries growing grafting plants. Bearing this in mind, Shintoza was then the hybrid rootstock to be used in cucumbers and consequently growers could have grafted plants at their disposal. Later on, research was focused on density and the most adequate type of pruning, by carrying out several trials. These experiments showed that, the extra vigour the rootstocks gave to the plants, allowed them to have two or three stems, (normally nurserymen use two, as it is easier for them to manage the plants). These findings would lead to the lessening the density of the plant and thus reduce costs, something which worried and still worries farmers. It was stated that it would be possible to reduce the density of the plants by about 25%, the optimum density of the stems ranging from 3 to 3.5 stem-m-2. Once decisions were taken both on the rootstock and the appropriate density, we went on to study how to apply these proposals to the new cultivars which the seed companies were proposing and the farmers were applying. These measures were complemented with the introduction of new rootstocks capable of replacing Shintoza, or rotating with it in order to change the root system. This is particularly necessary when rootstocks, non-resistant to nematodes, mainly of the species Meloidogyne incognita, are used. This is the main problem due to the soil of that area. Cultivars such as Trópico, at first, and Urano and Motril, more recently, adapted quite well to this technique. Among the rootstocks which adapted well to grafting and which were compatible with most tested cultivars, were, in particular, RS-841 Strongtosa and Camel. The behaviour of Azman was worth studying, but this rootstock was removed from the market by the company which had bought and commercialized it. Although not the main purpose of the research, it was observed that combining grafting with other alternatives to methyl bromide in order to overcome problems due to soil diseases or nematodes may be worthwhile. However, further research is needed, as this topic is in constant evolution, not only when we come to disinfectants, most of which are being affected by the removal of the permit for commercialization, but also when we refer to other techniques such as biofumigation or the use of steam. Results also showed that grafted plants may reduce the amount of fertilizers, particularly nitrogen, used: This means both saving money and the protection of the environment. We may conclude by saying that grafting Spanish short cucumbers is feasible, that the selections of the hybrids between Cucurbita maxima and C. moschata, habitually used in watermelon grafting, can also be applied to these cucumbers. It can also be concluded that the use of these grafting techniques may lead to profitable yields, by lengthening the growing cycle in many cases and by maintaining the quality to a large extent. Although these rootstocks are not resistant to nematodes, the results showed that their extra vigour enables them to develop in terms of production, and thus they cope with this problem. Since these rootstocks are not resistant to nematodes and the problem with these nematodes may even worsen, it is recommended that different types of rootstocks should be available to enable both the rotation and the use of different root systems, which will encourage the parasitism of nematodes.

Coevolución virus-planta: impacto de las infecciones virales en poblaciones silvestres de Chiltepín (capsicum annuum var. Aviculare (dierbach) d’arcy & eshbaugh) en México.

El impacto negativo que tienen los virus en las plantas hace que estos puedan ejercer un papel ecológico como moduladores de la dinámica espacio-temporal de las poblaciones de sus huéspedes. Entender cuáles son los mecanismos genéticos y los factores ambientales que determinan tanto la epidemiología como la estructura genética de las poblaciones de virus puede resultar de gran ayuda para la comprensión del papel ecológico de las infecciones virales. Sin embargo, existen pocos trabajos experimentales que hayan abordado esta cuestión. En esta tesis, se analiza el efecto de la heterogeneidad del paisaje sobre la incidencia de los virus y la estructura genética de sus poblaciones. Asimismo, se explora como dichos factores ambientales influyen en la importancia relativa que los principales mecanismos de generación de variabilidad genética (mutación, recombinación y migración) tienen en la evolución de los virus. Para ello se ha usado como sistema los begomovirus que infectan poblaciones de chiltepín (Capsicum annuum var. aviculare (Dierbach) D´Arcy & Eshbaugh) en México. Se analizó la incidencia de diferentes virus en poblaciones de chiltepín distribuidas a lo largo de seis provincias biogeográficas, representando el área de distribución de la especie en México, y localizadas en hábitats con diferente grado de intervención humana: poblaciones sin intervención humana (silvestres); poblaciones toleradas (lindes y pastizales), y poblaciones manejadas por el hombre (monocultivos y huertos familiares). Entre los virus analizados, los begomovirus mostraron la mayor incidencia, detectándose en todas las poblaciones y años de muestreo. Las únicas dos especies de begomovirus que se encontraron infectando al chiltepín fueron: el virus del mosaico dorado del chile (Pepper golden mosaic virus, PepGMV) y el virus huasteco del amarilleo de venas del chile (Pepper huasteco yellow vein virus, PHYVV). Por ello, todos los análisis realizados en esta tesis se centran en estas dos especies de virus. La incidencia de PepGMV y PHYVV, tanto en infecciones simples como mixtas, aumento cuanto mayor fue el nivel de intervención humana en las poblaciones de chiltepín, lo que a su vez se asoció con una menor biodiversidad y una mayor densidad de plantas. Además, la incidencia de infecciones mixtas, altamente relacionada con la presencia de síntomas, fue también mayor en las poblaciones cultivadas. La incidencia de estos dos virus también varió en función de la población de chiltepín y de la provincia biogeográfica. Por tanto, estos resultados apoyan una de las hipótesis XVI clásicas de la Patología Vegetal según la cual la simplificación de los ecosistemas naturales debida a la intervención humana conduce a un mayor riesgo de enfermedad de las plantas, e ilustran sobre la importancia de la heterogeneidad del paisaje a diferentes escalas en la determinación de patrones epidemiológicos. La heterogeneidad del paisaje no solo afectó a la epidemiología de PepGMV y PHYVV, sino también a la estructura genética de sus poblaciones. En ambos virus, el nivel de diferenciación genética mayor fue la población, probablemente asociado a la capacidad de migración de su vector Bemisia tabaci; y en segundo lugar la provincia biogeográfica, lo que podría estar relacionado con el papel del ser humano como agente dispersor de PepGMV y PHYVV. La estima de las tasas de sustitución nucleotídica de las poblaciones de PepGMV y PHYVV mostró una rápida dinámica evolutiva. Los árboles filogenéticos de ambos virus presentaron una topología en estrella, lo que sugiere una expansión reciente en las poblaciones de chiltepín. La reconstrucción de los patrones de migración de ambos virus indicó que ésta expansión parece haberse producido desde la zona central de México siguiendo un patrón radial, y en los últimos 30 años. Es importante tener en cuenta que el patrón espacial de la diversidad genética de las poblaciones de PepGMV y PHYVV es similar al descrito previamente para el chiltepín lo que podría dar lugar a la congruencia de las genealogías del huésped y la de los virus. Dicha congruencia se encontró cuando se tuvieron en cuenta únicamente las poblaciones de hábitats silvestres y tolerados, lo que probablemente se debe a una codivergencia en el espacio pero no en el tiempo, dado que la evolución de virus y huésped han ocurrido a escalas temporales muy diferentes. Finalmente, el análisis de la frecuencia de recombinación en PepGMV y PHYVV indicó que esta juega un papel importante en la evolución de ambos virus, dependiendo su importancia del nivel de intervención humana de la población de chiltepín. Este factor afectó también a la intensidad de la selección a la que se ven sometidos los genomas de PepGMV y PHYVV. Los resultados de esta tesis ponen de manifiesto la importancia que la reducción de la biodiversidad asociada al nivel de intervención humana de las poblaciones de plantas y la heterogeneidad del paisaje tiene en la emergencia de nuevas enfermedades virales. Por tanto, es necesario considerar estos factores ambientales a la hora de comprender la epidemiologia y la evolución de los virus de plantas.XVII SUMMARY Plant viruses play a key role as modulators of the spatio-temporal dynamics of their host populations, due to their negative impact in plant fitness. Knowledge on the genetic and environmental factors that determine the epidemiology and the genetic structure of virus populations may help to understand the ecological role of viral infections. However, few experimental works have addressed this issue. This thesis analyses the effect of landscape heterogeneity in the prevalence of viruses and the genetic structure of their populations. Also, how these environmental factors influence the relative importance of the main mechanisms for generating genetic variability (mutation, recombination and migration) during virus evolution is explored. To do so, the begomoviruses infecting chiltepin (Capsicum annuum var. aviculare (Dierbach) D'Arcy & Eshbaugh) populations in Mexico were used. Incidence of different viruses in chiltepin populations of six biogeographical provinces representing the species distribution in Mexico was determined. Populations belonged to different habitats according to the level of human management: populations with no human intervention (Wild); populations naturally dispersed and tolerated in managed habitats (let-standing), and human managed populations (cultivated). Among the analyzed viruses, the begomoviruses showed the highest prevalence, being detected in all populations and sampling years. Only two begomovirus species infected chiltepin: Pepper golden mosaic virus, PepGMV and Pepper huasteco yellow vein virus, PHYVV. Therefore, all the analyses presented in this thesis are focused in these two viruses. The prevalence of PepGMV and PHYVV, in single and mixed infections, increased with higher levels of human management of the host population, which was associated with decreased biodiversity and increased plant density. Furthermore, cultivated populations showed higher prevalence of mixed infections and symptomatic plants. The prevalence of the two viruses also varied depending on the chiltepin population and on the biogeographical province. Therefore, these results support a classical hypothesis of Plant Pathology stating that simplification of natural ecosystems due to human management leads to an increased disease risk, and illustrate on the importance of landscape heterogeneity in determining epidemiological patterns. Landscape heterogeneity not only affected the epidemiology of PepGMV and PHYVV, but also the genetic structure of their populations. Both viruses had the highest level of genetic differentiation at the population scale, probably associated with the XVIII migration patterns of its vector Bemisia tabaci, and a second level at the biogeographical province scale, which could be related to the role of humans as dispersal agents of PepGMV and PHYVV. The estimates of nucleotide substitution rates of the virus populations indicated rapid evolutionary dynamics. Accordingly, phylogenetic trees of both viruses showed a star topology, suggesting a recent diversification in the chiltepin populations. Reconstruction of PepGMV and PHYVV migration patterns indicated that they expanded from central Mexico following a radial pattern during the last 30 years. Importantly, the spatial genetic structures of the virus populations were similar to that described previously for the chiltepin, which may result in the congruence of the host and virus genealogies. Such congruence was found only in wild and let-standing populations. This is probably due to a co-divergence in space but not in time, given the different evolutionary time scales of the host and virus populations. Finally, the frequency of recombination detected in the PepGMV and PHYVV populations indicated that this mechanism plays an important role in the evolution of both viruses at the intra-specific scale. The level of human management had a minor effect on the frequency of recombination, but influenced the strength of negative selective pressures in the viral genomes. The results of this thesis highlight the importance of decreased biodiversity in plant populations associated with the level of human management and of landscape heterogeneity on the emergence of new viral diseases. Therefore it is necessary to consider these environmental factors in order to fully understand the epidemiology and evolution of plant viruses.

Discovery of ferromanganese hydrocarbon-related nodules associated with the Meknes mud volcano (Western Moroccan margin)

A suite of ferromanganese nodules were sampled during the MVSEIS-2008 cruise aboard of the R/V Hespérides in the flanks of Meknes mud volcano (Moroccan margin, NE Central Atlantic). The nodules were collected at water depths between 750-850 m within a seabed area characterized by high acoustic backscatter values. Debris of cold water corals and hydrocarbon-derived authigenic carbonate crusts were sampled at same time. The nodules show tabular morphology, up to 20 cm in maximum diameter and 2 kg of weight, brown-reddish external color and they are internally composed by a concentric to complex arrangement of laminae. The results of X-ray diffraction analysis show that these ferromanganese nodules are essentially composed of goethite and lepidocrocite, being Mn-oxides, silicates (quartz and clay minerals) and carbonates (calcite, dolomite and siderite) accessory to occasional minerals. All the samples display micritic to micro-sparitic mosaic under the petrographic microscope which forms massive, laminated or dendritic-mottled textures. The nodules show a high abundance of Fe, minor Mn and low contents of trace metals and REEs. Mature hydrocarbons, as n-alkanes derived from marine bacterial activity, and phenanthrene have been detected in all the ferromanganese nodules analyzed. These nodules display analogous characteristics (textural, mineralogical and geochemical) to the nodules studied by González et al (2009) in the carbonate mud-mounds in the Gulf of Cadiz, offshore Iberian margin. In this way, the same preliminary genetic model proposed for these nodules might be applicable to those find in the Meknes mud volcano. Therefore, the anaerobic oxidation of hydrocarbon-rich fluids within the mud-breccia sediments in the flanks of Meknes mud volcano would induce the formation of early diagenetic Fe-(Mn) carbonate nodules. Thus, the nodules were later exhumed by the erosive action of sea bottom currents generating the replacement of ferromanganese carbonates by Fe-Mn oxy-hydroxides. Thus, the hydrocarbon-rich fluid venting from deep seated reservoirs and erosive action of bottom currents must have been essential actors, as mineralization controls, for ferromanganese nodules generation and evolution. These findings imply that this type of nodules must be considered as new product as derived from the anaerobic/aerobic oxidation of hydrocarbons in areas of active seepages.