New insights into the regulation of NADPH oxidase dependent reactive oxygen species signaling during the plant immune response

Las NADPH oxidasas de plantas, denominadas “respiratory burst oxidase homologues” (RBOHs), producen especies reactivas del oxígeno (ROS) que median un amplio rango de funciones. En la célula vegetal, el ajuste preciso de la producción de ROS aporta la especificidad de señal para generar una respuesta apropiada ante las amenazas ambientales. RbohD y RbohF, dos de los diez genes Rboh de Arabidopsis, son pleiotrópicos y median diversos procesos fisiológicos en respuesta a patógenos. El control espacio-temporal de la expresión de los genes RbohD y RbohF podría ser un aspecto crítico para determinar la multiplicidad de funciones de estas oxidasas. Por ello, generamos líneas transgénicas de Arabidopsis con fusiones de los promoters de RbohD y RbohF a los genes delatores de la B-glucuronidasa y la luciferasa. Estas líneas fueron empleadas para revelar el patrón de expresión diferencial de RbohD y RbohF durante la respuesta inmune de Arabidopsis a la bacteria patógena Pseudomonas syringae pv. tomato DC3000, el hongo necrótrofo Plectosphaerella cucumerina y en respuesta a señales relacionadas con la respuesta inmune. Nuestros experimentos revelan un patrón de expresión diferencial de los promotores de RbohD y RbohF durante el desarrollo de la planta y en la respuesta inmune de Arabidopsis. Además hemos puesto de manifiesto que existe una correlación entre el nivel de actividad de los promotores de RbohD y RbohF con la acumulación de ROS y el nivel de muerte celular en respuesta a patógenos. La expression de RbohD y RbohF también es modulada de manera diferencial en respuesta a patrones moleculares asociados a patógenos (PAMPs) y por ácido abscísico (ABA). Cabe destacar que, mediante una estrategia de intercambio de promotores, hemos revelado que la región promotora de RbohD, es necesaria para dirigir la producción de ROS en respuesta a P. cucumerina. Adicionalmente, la activación del promotor de RbohD en respuesta al aislado de P. cucumerina no adaptado a Arabidopsis 2127, nos llevó a realizar ensayos de susceptibilidad con el doble mutante rbohD rbohF que han revelado un papel desconocido de estas oxidasas en resistencia no-huesped. La interacción entre la señalización dependiente de las RBOHs y otros componentes de la respuesta inmune de plantas podría explicar también las distintas funciones que median estas oxidasas en relación con la respuesta inmune. Entre la gran cantidad de señales coordinadas con la actividad de las RBOHs, existen evidencias genéticas y farmacológicas que indican que las proteínas G heterotriméricas están implicadas en algunas de las rutas de señalización mediadas por ROS derivadas de los RBOHs en respuesta a señales ambientales. Por ello hemos estudiado la relación entre estas RBOH-NADPH oxidasas y AGB1, la subunidad β de las proteínas G heterotriméricas en la respuesta inmune de Arabidopsis. Análisis de epistasis indican que las proteínas G heterotriméricas están implicadas en distintas rutas de señalización en defensa mediadas por las RBOHs. Nuestros resultados ilustran la relación compleja entre la señalización mediada por las RBOHs y las proteínas G heterotriméricas, que varía en función de la interacción planta-patógeno analizada. Además, hemos explorado la posible asociación entre AGB1 con RBOHD y RBOHF en eventos tempranos de la respuesta immune. Cabe señalar que experimentos de coímmunoprecipitación apuntan a una posible asociación entre AGB1 y la kinasa citoplasmática reguladora de RBOHD, BIK1. Esto indica un posible mecanismo de control de la función de esta NADPH oxidase por AGB1. En conjunto, estos datos aportan nuevas perspectivas sobre cómo, a través del control transcripcional o mediante la interacción con las proteínas G heterotriméricas, las NADPH oxidases de plantas median la producción de ROS y la señalización por ROS en la respuesta inmune. Nuestro trabajo ejemplifica cómo la regulación diferencial de dos miembros de una familia multigénica, les permite realizar distintas funciones fisiológicas especializadas usando un mismo mecanismo enzimático. ABSTRACT The plant NADPH oxidases, termed respiratory burst oxidase homologues (RBOHs), produce reactive oxygen species (ROS) which mediate a wide range of functions. Fine tuning this ROS production provides the signaling specificity to the plant cell to produce the appropriate response to environmental threats. RbohD and RbohF, two of the ten Rboh genes present in Arabidopsis, are pleiotropic and mediate diverse physiological processes in response to pathogens. One aspect that may prove critical to determine the multiplicity of functions of RbohD and RbohF is the spatio-temporal control of their gene expression. Thus, we generated Arabidopsis transgenic lines with RbohD- and RbohF-promoter fusions to the β-glucuronidase and the luciferase reporter genes. These transgenics were employed to reveal RbohD and RbohF promoter activity during Arabidopsis immune response to the pathogenic bacterium Pseudomonas syringae pv tomato DC3000, the necrotrophic fungus Plectosphaerella cucumerina and in response to immunity-related cues. Our experiments revealed a differential expression pattern of RbohD and RbohF throughout plant development and during Arabidopsis immune response. Moreover, we observed a correlation between the level of RbohD and RbohF promoter activity, the accumulation of ROS and the amount of cell death in response to pathogens. RbohD and RbohF gene expression was also differentially modulated by pathogen associated molecular patterns and abscisic acid. Interestingly, a promoter-swap strategy revealed the requirement for the promoter region of RbohD to drive the production of ROS in response to P. cucumerina. Additionally, since the RbohD promoter was activated during Arabidopsis interaction with a non-adapted P. cucumerina isolate 2127, we performed susceptibility tests to this fungal isolate that uncovered a new role of these oxidases on non-host resistance. The interplay between RBOH-dependent signaling with other components of the plant immune response might also explain the different immunity-related functions mediated by these oxidases. Among the plethora of signals coordinated with RBOH activity, pharmacological and genetic evidence indicates that heterotrimeric G proteins are involved in some of the signaling pathways mediated by RBOH–derived ROS in response to environmental cues. Therefore, we analysed the interplay between these RBOH-NADPH oxidases and AGB1, the Arabidopsis β-subunit of heterotrimeric G proteins during Arabidopsis immune response. We carried out epistasis studies that allowed us to test the implication of AGB1 in different RBOH-mediated defense signaling pathways. Our results illustrate the complex relationship between RBOH and heterotrimeric G proteins signaling, that varies depending on the type of plant-pathogen interaction. Furthermore, we tested the potential association between AGB1 with RBOHD and RBOHF during early immunity. Interestingly, our co-immunoprecipitation experiments point towards an association of AGB1 and the RBOHD regulatory kinase BIK1, thus providing a putative mechanism in the control of the NADPH oxidase function by AGB1. Taken all together, these studies provide further insights into the role that transcriptional control or the interaction with heterotrimeric G-proteins have on RBOH-NADPH oxidase-dependent ROS production and signaling in immunity. Our work exemplifies how, through a differential regulation, two members of a multigenic family achieve specialized physiological functions using a common enzymatic mechanism.

Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi.

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.

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.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias está aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la población, pero las causas de este aumento aún no están claras. Encontrar el origen del mecanismo por el cual una proteína inofensiva se convierte en capaz de inducir una respuesta alérgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterización de alérgenos relevantes ha ayudado a mejorar el manejo clínico y a aclarar los mecanismos básicos de las reacciones alérgicas, todavía queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (proteínas de transferencia de lípidos –LTPs- y taumatinas –TLPs-) y estudiando los mecanismos que median la sensibilización y la reactividad cruzada para mejorar tanto el diagnóstico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panalérgenos; y estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas. Para estudiar la reactividad cruzada entre miembros de la misma familia de proteínas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilización a alérgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional más relevante de origen alérgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con alérgenos y analizando los resultados por la teoría de grafos. En relación al estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas, se llevaron a cabo estudios sobre la interacción de los alérgenos alimentarios con células del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de moléculas co-transportadas con los alérgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y alérgeno principal del melocotón) se selección como modelo para llevarlo a cabo. Por otra parte, se analizó el papel de moléculas activadoras del sistema inmune producidas por patógenos en la inducción de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, alérgeno mayor de dicho hongo, en la sensibilización a Act d 2, alérgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigación innovadora aportando resultados de gran utilidad tanto para la mejora del diagnóstico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins –LTPs-, and Thaumatin-like Proteins –TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Transcriptional changes and oxidative stress associated with the synergistic interaction between potato virus X and potato virus Y and their relationship with symptom expression.

Many virus diseases of economic importance to agriculture result from mixtures of different pathogens invading the host at a given time. This contrasts with the relatively scarce studies available on the molecular events associated with virus---host interactions in mixed infections. Compared with single infections, co-infection of Nicotiana benthamiana with Potato virus X (PVX) and Potato virus Y (PVY) resulted in increased systemic symptoms (synergism) that led to necrosis of the newly emerging leaves and death of the plant. A comparative transcriptional analysis was undertaken to identify quantitative and qualitative differences in gene expression during this synergistic infection and correlate these changes with the severe symptoms it caused. Global transcription profiles of doubly infected leaves were compared with those from singly infected leaves using gene ontology enrichment analysis and metabolic pathway annotator software. Functional gene categories altered by the double infection comprise suites of genes regulated coordinately, which are associated with chloroplast functions (downregulated), protein synthesis and degradation (upregulated), carbohydrate metabolism (upregulated), and response to biotic stimulus and stress (upregulated). The expressions of reactive oxygen species?generating enzymes as well as several mitogen-activated protein kinases were also significantly induced. Accordingly, synergistic infection induced a severe oxidative stress in N. benthamiana leaves, as judged by increases in lipid peroxidation and by the generation of superoxide radicals in chloroplasts, which correlated with the misregulation of antioxidative genes in microarray data. Interestingly, expression of genes encoding oxylipin biosynthesis was uniquely upregulated by the synergistic infection. Virus-induced gene silencing of ?-dioxygenase1 delayed cell death during PVX?PVY infection.

Purification and Characterization of AsES Protein A Subtilisin Secreted by Acremonium Strictum is a Novel Plant Defense Elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

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.

Combinatorial interaction between AtbZIP44 and other bZIP transcription factors in the regulation of Arabidopsis thaliana seed maturation and germination

El gen AtbZIP44 se expresa en el endospermo micropilar y en la radícula de semillas en germinación. El mRNA del gen AtbZIP9 se localiza también en la radícula. Se realizan experimentos de interacción proteína-proteína entre los correspondientes factores transcripcionales.

Integrated plant monitoring to improve plant operation strategy and results

The more and more demanding conditions in the power generation sector requires to apply all the available technologies to optimize processes and reduce costs. An integrated asset management strategy, combining technical analysis and operation and maintenance management can help to improve plant performance, flexibility and reliability. In order to deploy such a model it is necessary to combine plant data and specific equipment condition information, with different systems devoted to analyze performance and equipment condition, and take advantage of the results to support operation and maintenance decisions. This model that has been dealt in certain detail for electricity transmission and distribution networks, is not yet broadly extended in the power generation sector, as proposed in this study for the case of a combined power plant. Its application would turn in direct benefit for the operation and maintenance and for the interaction to the energy market

3D Maize Plant Reconstruction Based on Georeferenced Overlapping LiDAR Point Clouds

3D crop reconstruction with a high temporal resolution and by the use of non-destructive measuring technologies can support the automation of plant phenotyping processes. Thereby, the availability of such 3D data can give valuable information about the plant development and the interaction of the plant genotype with the environment. This article presents a new methodology for georeferenced 3D reconstruction of maize plant structure. For this purpose a total station, an IMU, and several 2D LiDARs with different orientations were mounted on an autonomous vehicle. By the multistep methodology presented, based on the application of the ICP algorithm for point cloud fusion, it was possible to perform the georeferenced point clouds overlapping. The overlapping point cloud algorithm showed that the aerial points (corresponding mainly to plant parts) were reduced to 1.5%–9% of the total registered data. The remaining were redundant or ground points. Through the inclusion of different LiDAR point of views of the scene, a more realistic representation of the surrounding is obtained by the incorporation of new useful information but also of noise. The use of georeferenced 3D maize plant reconstruction at different growth stages, combined with the total station accuracy could be highly useful when performing precision agriculture at the crop plant level.