The role of secretion systems and small molecules in soft-rot enterobacteriaceae pathogenicity

Soft-rot Enterobacteriaceae (SRE), which belong to the genera Pectobacterium and Dickeya, consist mainly of broad host-range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants. They are found in plants, insects, soil, and water in agricultural regions worldwide. SRE encode all six known protein secretion systems present in gram-negative bacteria, and these systems are involved in attacking host plants and competing bacteria. They also produce and detect multiple types of small molecules to coordinate pathogenesis, modify the plant environment, attack competing microbes, and perhaps to attract insect vectors. This review integrates new information about the role protein secretion and detection and production of ions and small molecules play in soft-rot pathogenicity.

News in engineering education in Spain effective from 2010 in presence of external changes and mixed crisis, looking mostly to agro and civil engineers.

The engineering careers models were diverse in Europe, and are adopting now in Spain the Bolonia process for European Universities. Separated from older Universities, that are in part technically active, Civil Engineering (Caminos, Canales y Puertos) started at end of 18th century in Spain adopting the French models of Upper Schools for state civil servants with exam at entry. After 1800 intense wars, to conserve forest regions Ingenieros de Montes appeared as Upper School, and in 1855 also the Ingenieros Agrónomos to push up related techniques and practices. Other Engineers appeared as Upper Schools but more towards private factories. These ES got all adapted Lower Schools of Ingeniero Tecnico. Recently both grew much in number and evolved, linked also to recognized Professions. Spanish society, into European Community, evolved across year 2000, in part highly well, but with severe discordances, that caused severe youth unemployment with 2008-2011 crisis. With Bolonia process high formal changes step in from 2010-11, accepted with intense adaptation. The Lower Schools are changing towards the Upper Schools, and both that have shifted since 2010-11 various 4-years careers (Grado), some included into the precedent Professions, and diverse Masters. Acceptation of them to get students has started relatively well, and will evolve, and acceptation of new grades for employment in Spain, Europe or outside will be essential. Each Grado has now quite rigid curricula and programs, MOODLE was introduced to connect pupils, some specific uses of Personal Computers are taught in each subject. Escuela de Agronomos centre, reorganized with its old name in its precedent buildings at entrance of Campus Moncloa, offers Grados of Agronomic Engineering and Science for various public and private activities for agriculture, Alimentary Engineering for alimentary activities and control, Agro-Environmental Engineering more related to environment activities, and in part Biotechnology also in laboratories in Campus Monte-Gancedo for Biotechnology of Plants and Computational Biotechnology. Curricula include Basics, Engineering, Practices, Visits, English, ?project of end of career?, Stays. Some masters will conduce to specific professional diploma, list includes now Agro-Engineering, Agro-Forestal Biotechnology, Agro and Natural Resources Economy, Complex Physical Systems, Gardening and Landscaping, Rural Genie, Phytogenetic Resources, Plant Genetic Resources, Environmental Technology for Sustainable Agriculture, Technology for Human Development and Cooperation.

Habitat management for conservation of pollinators in agro-ecosystems of Central Spain

La gestión de hábitat orientada a la conservación de polinizadores en los agro-ecosistemas requiere una selección de especies vegetales atendiendo fundamentalmente a dos criterios: i) el potencial atractivo de sus flores a los polinizadores; y ii) la simplicidad en su manejo agronómico. Además de estas premisas, es necesario considerar la capacidad invasora de estas especies vegetales, debido a que algunas de las más atractivas pueden resultar invasoras en determinados agro-ecosistemas. Por lo tanto, es preciso determinar qué especies vegetales son las más indicadas para ser implementadas en cada agro-ecosistema. En la presente tesis doctoral se plantea la búsqueda de las especies vegetales adecuadas para atraer polinizadores en los agro-ecosistemas del centro de España. En una primera aproximación, se ha evaluado la atracción y expansión espacial (potencial invasivo) de seis plantas perennes de la familia Lamiaceae (aromáticas), elegidas por ser nativas de la región mediterránea. La elección de las especies vegetales se ha llevado a cabo con el fin de crear márgenes funcionales basados en la mezcla de especies vegetales con distintos periodos de floración, de modo que prolonguen la disponibilidad de recursos florales en el tiempo. Tras un primer año dedicado al establecimiento de las especies aromáticas, en los dos años siguientes se ha estudiado la atracción individual y combinada de las especies vegetales sobre los polinizadores, y como ésta se ve afectada por la densidad y la morfología floral, utilizando para ello un diseño experimental en bloques al azar. Los resultados de este estudio han puesto de manifiesto que la morfología floral no tuvo influencia sobre la atracción de las especies vegetales, pero si la densidad floral, puesto que las especies vegetales con mayor densidad de flores (Nepeta tuberosa e Hyssopus officinalis) han mostrado mayor atracción a polinizadores. Cabe destacar que de las seis especies consideradas, dos especies de verano (Melissa officinalis y Thymbra capitata) no han contribuido de forma efectiva a la atracción de la mezcla hacia los polinizadores, mostrando una reducción significativa de este parámetro respecto a las otras especies aromáticas a lo largo del verano. Se ha observado que ninguna de las especies aromáticas evaluadas ha mostrado tendencia invasora a lo largo del estudio. En base a estos resultados, se puede concluir que entre las especies aromáticas estudiadas, N. tuberosa, H. officinalis y Salvia verbenaca son las que ofrecen mayor potencial para ser utilizadas en la conservación de polinizadores. De forma similar al caso de las plantas aromáticas, se ha llevado a cabo una segunda experimentación que incluía doce plantas anuales con floración de primavera, en la que se evaluó la atracción a polinizadores y su comportamiento agronómico. Este estudio con especies herbáceas se ha prolongado durante dos años, utilizando un diseño experimental de bloques aleatorios. Las variables analizadas fueron: el atractivo de las distintas especies vegetales a los polinizadores, su eficiencia de atracción (calculada como una combinación de la duración de la floración y las visitas de insectos), su respuesta a dos tipos de manejo agronómico (cultivo en mezcla frente a monocultivo; laboreo frente a no-laboreo) y su potencial invasivo. Los resultados de esta segunda experimentación han mostrado que las flores de Borago officinalis, Echium plantagineum, Phacelia tanacetifolia y Diplotaxis tenuifolia son atractivas a las abejas, mientras que las flores de Calendula arvensis, Coriandrum sativum, D. tenuifolia y Lobularia maritima son atractivas a los sírfidos. Con independencia del tipo de polinizadores atraídos por cada especie vegetal, se ha observado una mayor eficiencia de atracción en parcelas con monocultivo de D. tenuifolia respecto a las parcelas donde se cultivó una mezcla de especies herbáceas, si bien en estas últimas se observó mayor eficiencia de atracción que en la mayoría de parcelas mono-específicas. Respecto al potencial invasivo de las especies herbáceas, a pesar de que algunas de las más atractivas a polinizadores (P. tanacetifolia and C. arvensis) mostraron tendencia a un comportamiento invasor, su capacidad de auto-reproducción se vio reducida con el laboreo. En resumen, D. tenuifolia es la única especie que presentó una alta eficiencia de atracción a distintos tipos de polinizadores, conjuntamente con una alta capacidad de auto-reproducción pero sin mostrar carácter invasor. Comparando el atractivo de las especies vegetales utilizadas en este estudio sobre los polinizadores, D. tenuifolia es la especie más recomendable para su cultivo orientado a la atracción de polinizadores en agro-ecosistemas en el centro de España. Esta especie herbácea, conocida como rúcula, tiene la ventaja añadida de ser una especie comercializada para el consumo humano. Además de su atractivo a polinizadores, deben considerarse otros aspectos relacionados con la fisiología y el comportamiento de esta especie vegetal en los agro-ecosistemas antes de recomendar su cultivo. Dado que el cultivo en un campo agrícola de una nueva especie vegetal implica unos costes de producción, por ejemplo debidos a la utilización de agua de riego, es necesario evaluar el incremento en dichos costes en función de demanda hídrica específica de esa especie vegetal. Esta variable es especialmente importante en zonas dónde se presentan sequías recurrentes como es el caso del centro y sur-este de la península Ibérica. Este razonamiento ha motivado un estudio sobre los efectos del estrés hídrico por sequía y el estrés por déficit moderado y severo de riego sobre el crecimiento y floración de la especie D. tenuifolia, así como sobre la atracción a polinizadores. Los resultados muestran que tanto el crecimiento y floración de D. tenuifolia como su atracción a polinizadores no se ven afectados si la falta de riego se produce durante un máximo de 4 días. Sin embargo, si la falta de riego se extiende a lo largo de 8 días o más, se observa una reducción significativa en el crecimiento vegetativo, el número de flores abiertas, el área total y el diámetro de dichas flores, así como en el diámetro y longitud del tubo de la corola. Por otro lado, el estudio pone de manifiesto que un déficit hídrico regulado permite una gestión eficiente del agua, la cual, dependiendo del objetivo final del cultivo de D. tenuifolia (para consumo o solo para atracción de polinizadores), puede reducir su consumo entre un 40 y un 70% sin afectar al crecimiento vegetativo y desarrollo floral, y sin reducir significativamente el atractivo a los polinizadores. Finalmente, esta tesis aborda un estudio para determinar cómo afecta el manejo de hábitat a la producción de los cultivos. En concreto, se ha planteado una experimentación que incluye márgenes mono-específicos y márgenes con una mezcla de especies atractivas a polinizadores, con el fin de determinar su efecto sobre la producción del cultivo de cilantro (C. sativum). La elección del cultivo de cilantro se debe a que requiere la polinización de insectos para su reproducción (aunque, en menor medida, puede polinizarse también por el viento), además de la facilidad para estimar su producción en condiciones semi-controladas de campo. El diseño experimental consistía en la siembra de márgenes mono-específicos de D. tenuifolia y márgenes con mezcla de seis especies anuales situados junto al cultivo de cilantro. Estos cultivos con márgenes florales fueron comparados con controles sin margen floral. Además, un segundo grupo de plantas de cilantro situadas junto a todos los tratamientos, cuyas flores fueron cubiertas para evitar su polinización, sirvió como control para evaluar la influencia de los polinizadores en la producción del cultivo. Los resultados muestran que la presencia de cualquiera de los dos tipos de margen floral mejora el peso y el porcentaje de germinación de las semillas de cilantro frente al control sin margen. Si se comparan los dos tipos de margen, se ha observado un mayor número de semillas de cilantro junto al margen con mezcla de especies florales respecto al margen mono-específico, probablemente debido al mayor número visitas de polinizadores. Puesto que el experimento se realizó en condiciones de campo semi-controladas, esto sugiere que las visitas de polinizadores fueron el factor determinante en los resultados. Por otro lado, los resultados apuntan a que la presencia de un margen floral (ya sea mono-especifico o de mezcla) en cultivos de pequeña escala puede aumentar la producción de cilantro en más de un 200%, al tiempo que contribuyen a la conservación de los polinizadores. ABSTRACT Habitat management, aimed to conserve pollinators in agro-ecosystems, requires selection of the most suitable plant species in terms of their attractiveness to pollinators and simplicity of agronomic management. However, since all flowers are not equally attractive to pollinators and many plant species can be weedy or invasive in the particular habitat, it is important to test which plant species are the most appropriate to be implemented in specific agro-ecosystems. For that reason, this PhD dissertation has been focused on determination of the most appropriate aromatic and herbaceous plants for conservation of pollinators in agro-ecosystems of Central Spain. Therefore, in a first approximation, spatial expansion (i.e. potential weediness) and attractiveness to pollinators of six aromatic perennial plants from the Lamiaceae family, native and frequent in the Mediterranean region, were evaluated. Preliminary plant selection was based on designing a functional mixed margins consisting of plants attractive to pollinators and with different blooming periods, in order to extend the availability of floral resources in the field. After a year of vegetative growth, the next two years the plant species were studied in a randomized block design experiment in order to estimate their attractiveness to pollinators in Central Spain and to investigate whether floral morphology and density affect attractiveness to pollinators. The final aim of the study was to evaluate how their phenology and attractiveness to pollinators can affect the functionality of a flowering mixture of these plants. In addition, the spatial expansion, i.e. potential weediness, of the selected plant species was estimated under field conditions, as the final purpose of the studied plants is to be implemented within agro-ecosystems. The results of the experiment showed that floral morphology did not affect the attractiveness of plants to pollinators, but floral density did, as plant species with higher floral density (i.e. Nepeta tuberosa and Hyssopus officinalis) showed significantly higher attractiveness to pollinators. In addition, of six plant species, two summer species (Melissa officinalis and Thymbra capitata) did not efficiently contribute to the attractiveness of the mixture to pollinators, which reduced its attractiveness during the summer period. Finally, as none of the plants showed weedy behaviour under field conditions, the attractive plant species, i.e. N. tuberosa, H. officinalis and the early spring flowering Salvia verbenaca, showed good potential to conserve the pollinators. Similarly, in a second approximation, the attractiveness to pollinators and agronomic behaviour of twelve herbaceous plants blooming in spring were studied. This experiment was conducted over two years in a randomized block design in order to evaluate attractiveness of preselected plant species to pollinators, as well as their attractiveness efficiency (a combination of duration of flowering and insect visitation), their response to two different agronomic management practices (growing in mixed vs. mono-specific plots; tillage vs. no-tillage), and their potential weediness. The results of this experiment showed that the flowers of Borago officinalis, Echium plantagineum, Phacelia tanacetifolia and Diplotaxis tenuifolia were attractive to bees, while Calendula arvensis, Coriandrum sativum, D. tenuifolia and Lobularia maritima were attractive to hoverflies. In addition, floral mixture resulted in lower attractiveness efficiency to pollinators than mono-specific D. tenuifolia, but higher than most of the mono-specific stands. On the other hand, although some of the most attractive plant species (e.g. P. tanacetifolia and C. arvensis) showed potential weediness, their self-seeding was reduced by tillage. After comparing attractiveness efficiency of various herbaceous species to pollinators and their potential weediness, the results indicated that D. tenuifolia showed the highest attractiveness efficiency to pollinators and efficient self-reproduction, making it highly recommended to attract bees and hoverflies in agro-ecosystems of Central Spain. In addition, this plant, commonly known as wild rocket, has a supplementary economic value as a commercialized crop. The implementation of a new floral margin in agro-ecosystems means increased production costs, especially in regions with frequent and long droughts (as it is Central and South-East area of Iberian Peninsula), where the principal agricultural cost is irrigation. Therefore, before recommending D. tenuifolia for sustainable habitat management within agro-ecosystems, it is necessary to study the effect of drought stress and moderate and severe deficit irrigation on its growth, flower development and attractiveness to pollinators. The results of this experiment showed that in greenhouse conditions, potted D. tenuifolia could be without irrigation for 4 days without affecting its growth, flowering and attractiveness to pollinators. However, lack of irrigation for 8 days or longer significantly reduced the vegetative growth, number of open flowers, total floral area, flower diameter, corolla tube diameter and corolla tube length of D. tenuifolia. This study showed that regulated deficit irrigation can improve water use efficiency, and depending on the purpose of growing D. tenuifolia, as a crop or as a beneficial plant to attract pollinators, it can reduce water consumption by 40% to 70% without affecting its vegetative and floral development and without reducing its attractiveness to pollinators. Finally, the following experiment was developed in order to understand how habitat management can influence on the agricultural production. For this purpose, it was evaluated if the vicinity of mixed and mono-specific field margins, preselected to conserve pollinators within agro-ecosystems, can improve seed production in coriander (C. sativum). The selection of this plant species for the experiment was based on its necessity for insect pollination for production of seeds (even though some pollen can be transmitted from one flower to another by wind) and the fact that under semi-controlled field conditions established in the field it is possible to estimate its total seed production. Since D. tenuifolia is attractive for both bees and hoverflies in Central Spain, the main objective of this experiment was to estimate the impact of two different types of field margins, i.e. mono-specific margin with D. tenuifolia and mixed margin with six herbaceous species, on the seed production of potted coriander. For that reason, it was tested: i) if open pollination (control without proximate field margin and treatments with nearby mono-specific and mixed margin) increases the seed production of coriander when compared with no-pollination (covered inflorescences of coriander) under field conditions; ii) if frequency of pollinator visitation to the flowers of coriander was higher in the presence of field margins than in the control without field margin; and iii) if seed production was higher in the presence of field margins than in control plants of coriander without field margin. The results showed that the proximity of both types of floral margins (mixed and mono-specific) improved the seed quality of coriander plants, as seed weight and germination rate were higher than in control plants without field margin. Furthermore, the number of seeds produced was significantly higher in coriander plants grown near mixed margins than near mono-specific margin, probably due to an increase in pollinator visits. Since the experiment was conducted under semi-controlled field conditions, it can be concluded that pollinator visits was the main factor that biased the results, and that presence of both mixed or mono-specific (D. tenuifolia) margins can improve the production of coriander for more than 200% in small-scale gardens and, in addition, conserve the local pollinators.

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process.

Plant β-diversity i in human-altered forest ecosystems: the importance of the structural, spatial, and topographical characteristics of stands in patterning plant species assemblages

An understanding of spatial patterns of plant species diversity and the factors that drive those patterns is critical for the development of appropriate biodiversity management in forest ecosystems. We studied the spatial organization of plants species in human- modified and managed oak forests (primarily, Quercus faginea) in the Central Pre- Pyrenees, Spain. To test whether plant community assemblages varied non-randomly across the spatial scales, we used multiplicative diversity partitioning based on a nested hierarchical design of three increasingly coarser spatial scales (transect, stand, region). To quantify the importance of the structural, spatial, and topographical characteristics of stands in patterning plant species assemblages and identify the determinants of plant diversity patterns, we used canonical ordination. We observed a high contribution of ˟-diversity to total -diversity and found ˟-diversity to be higher and ˞-diversity to be lower than expected by random distributions of individuals at different spatial scales. Results, however, partly depended on the weighting of rare and abundant species. Variables expressing the historical management intensities of the stand such as mean stand age, the abundance of the dominant tree species (Q. faginea), age structure of the stand, and stand size were the main factors that explained the compositional variation in plant communities. The results indicate that (1) the structural, spatial, and topographical characteristics of the forest stands have the greatest effect on diversity patterns, (2) forests in landscapes that have different land use histories are environmentally heterogeneous and, therefore, can experience high levels of compositional differentiation, even at local scales (e.g., within the same stand). Maintaining habitat heterogeneity at multiple spatial scales should be considered in the development of management plans for enhancing plant diversity and related functions in human-altered forests

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

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.

Functional response of Ulmus minor Mill. to drought, flooding and dutch elm disease

Ulmus minor es una especie arbórea originaria de Europa cuyas poblaciones han sido diezmadas por el hongo patógeno causante de la enfermedad de la grafiosis. La conservación de los olmos exige plantearse su propagación a través de plantaciones y conocer mejor su ecología y biología. Ulmus minor es un árbol de ribera, pero frecuentemente se encuentra alejado del cauce de arroyos y ríos, donde la capa freática sufre fuertes oscilaciones. Por ello, nuestra hipótesis general es que esta especie es moderadamente resistente tanto a la inundación como a la sequía. El principal objetivo de esta tesis doctoral es entender desde un punto de vista funcional la respuesta de U. minor a la inundación, la sequía y la infección por O. novo-ulmi; los factores que posiblemente más influyen en la distribución actual de U. minor. Con este objetivo se persigue dar continuidad a los esfuerzos de conservación de esta especie que desde hace años se dedican en varios centros de investigación a nivel mundial, ya que, entender mejor los mecanismos que contribuyen a la resistencia de U. minor ante la inoculación con O. novo-ulmi y factores de estrés abiótico ayudará en la selección y propagación de genotipos resistentes a la grafiosis. Se han planteado tres experimentos en este sentido. Primero, se ha comparado la tolerancia de brinzales de U. minor y U. laevis – otro olmo ibérico – a una inmersión controlada con el fin de evaluar su tolerancia a la inundación y comprender los mecanismos de aclimatación. Segundo, se ha comparado la tolerancia de brinzales de U. minor y Quercus ilex – una especie típica de ambientes Mediterránea secos – a la falta de agua en el suelo con el fin de evaluar el grado de tolerancia y los mecanismos de aclimatación a la sequía. El hecho de comparar dos especies contrastadas responde al interés en entender mejor cuales son los procesos que conducen a la muerte de una planta en condiciones de sequía – asunto sobre el que hay una interesante discusión desde hace algunos años. En tercer lugar, con el fin de entender mejor la resistencia de algunos genotipos de U. minor a la grafiosis, se han estudiado las diferencias fisiológicas y químicas constitutivas e inducidas por O. novo-ulmi entre clones de U. minor seleccionados a priori por su variable grado de resistencia a esta enfermedad. En el primer experimento se observó que los brinzales de U. minor sobrevivieron 60 días inmersos en una piscina con agua no estancada hasta una altura de 2-3 cm por encima del cuello de la raíz. A los 60 días, los brinzales de U. laevis se sacaron de la piscina y, a lo largo de las siguientes semanas, fueron capaces de recuperar las funciones fisiológicas que habían sido alteradas anteriormente. La conductividad hidráulica de las raíces y la tasa de asimilación de CO2 neta disminuyeron en ambas especies. Por el contrario, la tasa de respiración de hojas, tallos y raíces aumentó en las primeras semanas de la inundación, posiblemente en relación al aumento de energía necesario para desarrollar mecanismos de aclimatación a la inundación, como la hipertrofia de las lenticelas que se observó en ambas especies. Por ello, el desequilibrio del balance de carbono de la planta podría ser un factor relevante en la mortalidad de las plantas ante inundaciones prolongadas. Las plantas de U. minor (cultivadas en envases de 16 litros a media sombra) sobrevivieron por un prolongado periodo de tiempo en verano sin riego; la mitad de las plantas murieron tras 90 días sin riego. El cierre de los estomas y la pérdida de hojas contribuyeron a ralentizar las pérdidas de agua y tolerar la sequía en U. minor. Las obvias diferencias en tolerancia a la sequía con respecto a Q. ilex se reflejaron en la distinta capacidad para ralentizar la aparición del estrés hídrico tras dejar de regar y para transportar agua en condiciones de elevada tensión en el xilema. Más relevante es que las plantas con evidentes síntomas de decaimiento previo a su muerte exhibieron pérdidas de conductividad hidráulica en las raíces del 80% en ambas especies, mientras que las reservas de carbohidratos apenas variaron y lo hicieron de forma desigual en ambas especies. Árboles de U. minor de 5 y 6 años de edad (plantados en eras con riego mantenido) exhibieron una respuesta a la inoculación con O. novo-ulmi consistente con ensayos previos de resistencia. La conductividad hidráulica del tallo, el potencial hídrico foliar y la tasa de asimilación de CO2 neta disminuyeron significativamente en relación a árboles inoculados con agua, pero solo en los clones susceptibles. Este hecho enlaza con el perfil químico “más defensivo” de los clones resistentes, es decir, con los mayores niveles de suberina, ácidos grasos y compuestos fenólicos en estos clones que en los susceptibles. Ello podría restringir la propagación del hongo en el árbol y preservar el comportamiento fisiológico de los clones resistentes al inocularlos con el patógeno. Los datos indican una respuesta fisiológica común de U. minor a la inundación, la sequía y la infección por O. novo-ulmi: pérdida de conductividad hidráulica, estrés hídrico y pérdida de ganancia neta de carbono. Pese a ello, U. minor desarrolla varios mecanismos que le confieren una capacidad moderada para vivir en suelos temporalmente anegados o secos. Por otro lado, el perfil químico es un factor relevante en la resistencia de ciertos genotipos a la grafiosis. Futuros estudios deberían examinar como este perfil químico y la resistencia a la grafiosis se ven alteradas por el estrés abiótico. ABSTRACT Ulmus minor is a native European elm species whose populations have been decimated by the Dutch elm disease (DED). An active conservation of this species requires large-scale plantations and a better understanding of its biology and ecology. U. minor generally grows close to water channels. However, of the Iberian riparian tree species, U. minor is the one that spread farther away from rivers and streams. For these reasons, we hypothesize that this species is moderately tolerant to both flooding and drought stresses. The main aim of the present PhD thesis is to better understand the functional response of U. minor to the abiotic stresses – flooding and drought – and the biotic stress – DED – that can be most influential on its distribution. The overarching goal is to aid in the conservation of this emblematic species through a better understanding of the mechanisms that contribute to resistance to abiotic and biotic stresses; an information that can help in the selection of resistant genotypes and their expansion in large-scale plantations. To this end, three experiments were set up. First, we compared the tolerance to experimental immersion between seedlings of U. minor and U. laevis – another European riparian elm species – in order to assess their degree of tolerance and understand the mechanisms of acclimation to this stress. Second, we investigated the tolerance to drought of U. minor seedlings in comparison with Quercus ilex (an oak species typical of dry Mediterranean habitats). Besides assessing and understanding U. minor tolerance to drought at the seedling stage, the aim was to shed light into the functional alterations that trigger drought-induced plant mortality – a matter of controversy in the last years. Third, we studied constitutive and induced physiological and biochemical differences among clones of variable DED resistance, before and following inoculation with Ophiostoma novo-ulmi. The goal is to shed light into the factors of DED resistance that is evident in some genotypes of U. minor, but not others. Potted seedlings of U. minor survived for 60 days immersed in a pool with running water to approximately 2-3 cm above the stem collar. By this time, U. minor seedlings died, whereas U. laevis seedlings moved out of the pool were able to recover most physiological functions that had been altered by flooding. For example, root hydraulic conductivity and leaf photosynthetic CO2 uptake decreased in both species; while respiration initially increased with flooding in leaves, stems and roots possibly to respond to energy demands associated to mechanisms of acclimation to soil oxygen deficiency; as example, a remarkable hypertrophy of lenticels was soon observed in flooded seedlings of both species. Therefore, the inability to maintain a positive carbon balance somehow compromises seedling survival under flooding, earlier in U. minor than U. laevis, partly explaining their differential habitats. Potted seedlings of U. minor survived for a remarkable long time without irrigation – half of plants dying only after 90 days of no irrigation in conditions of high vapour pressure deficit typical of summer. Some mechanisms that contributed to tolerate drought were leaf shedding and stomata closure, which reduced water loss and the risk of xylem cavitation. Obviously, U. minor was less tolerant to drought than Q. ilex, differences in drought tolerance resulting mostly from the distinct capacity to postpone water stress and conduct water under high xylem tension among species. More relevant was that plants of both species exhibited similar symptoms of root hydraulic failure (i.e. approximately 80% loss of hydraulic conductivity), but a slight and variable depletion of non-structural carbohydrate reserves preceding dieback. Five- and six-year-old trees of U. minor (planted in the field with supplementary watering) belonging to clones of contrasted susceptibility to DED exhibited a different physiological response to inoculation with O. novo-ulmi. Stem hydraulic conductivity, leaf water potential and photosynthetic CO2 uptake decreased significantly relative to control trees inoculated with water only in DED susceptible clones. This is consistent with the “more defensive” chemical profile observed in resistant clones, i.e. with higher levels of saturated hydrocarbons (suberin and fatty acids) and phenolic compounds than in susceptible clones. These compounds could restrict the spread of O. novo-ulmi and contribute to preserving the near-normal physiological function of resistant trees when exposed to the pathogen. These results evidence common physiological responses of U. minor to flooding, drought and pathogen infection leading to xylem water disruption, leaf water stress and reduced net carbon gain. Still, seedlings of U. minor develop various mechanisms of acclimation to abiotic stresses that can play a role in surviving moderate periods of flood and drought. The chemical profile appears to be an important factor for the resistance of some genotypes of U. minor to DED. How abiotic stresses such as flooding and drought affect the capacity of resistant U. minor clones to face O. novo-ulmi is a key question that must be contemplated in future research.

Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica crops

Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin.

A role for jasmonate in pathogen defense of Arabidopsis

To investigate the role of jasmonate in the defense of plants against fungal pathogens, we have studied a mutant of Arabidopsis, fad3–2 fad7–2 fad8, that cannot accumulate jasmonate. Mutant plants were extremely susceptible to root rot caused by the fungal root pathogen Pythium mastophorum (Drechs.), even though neighboring wild-type plants were largely unaffected by this fungus. Application of exogenous methyl jasmonate substantially protected mutant plants, reducing the incidence of disease to a level close to that of wild-type controls. A similar treatment with methyl jasmonate did not protect the jasmonate-insensitive mutant coi1 from infection, showing that protective action of applied jasmonate against P. mastophorum was mediated by the induction of plant defense mechanisms rather than by a direct antifungal action. Transcripts of three jasmonate-responsive defense genes are induced by Pythium challenge in the wild-type but not in the jasmonate-deficient mutant. Pythium species are ubiquitous in soil and root habitats world-wide, but most (including P. mastophorum) are considered to be minor pathogens. Our results indicate that jasmonate is essential for plant defense against Pythium and, because of the high exposure of plant roots to Pythium inoculum in soil, may well be fundamental to survival of plants in nature. Our results further indicate that the fad3–2 fad7–2 fad8 mutant is an appropriate genetic model for studying the role of this important signaling molecule in pathogen defense.

East Gondwana ancestry of the sunflower alliance of families

The sunflower alliance of families comprises nearly 10% of all flowering plant species and includes the largest of all plant families, the sunflower family Asteraceae, which has 23,000 species, and the bellflower family Campanulaceae. Both are worldwide in distribution, but the majority of their species occur in the northern hemisphere. Recently it has been shown that a number of small, woody families from the Australian–Southwest Pacific area also belong in this relationship. Here we add yet another such family and present phylogenetic, biogeographic, and chronological analyses elucidating the origin of this large group of plants. We show that the ancestral lineages are confined to Malesia, Australia, New Guinea, and New Zealand and that the sunflower and bellflower families represent phylogenetically derived lineages within a larger group with a Cretaceous and southern-hemisphere, presumably East Gondwana, ancestry. Their highly derived position in the flowering plant phylogeny makes this significant for understanding the evolution of flowering plants in general.

The role of Wolbachia bacteria in reproductive incompatibilities and hybrid zones of Diabrotica beetles and Gryllus crickets

A rickettsial bacterium in the genus Wolbachia is the cause of a unidirectional reproductive incompatibility observed between two major beetle pests of maize, the western corn rootworm, Diabrotica virgifera virgifera, and the Mexican corn rootworm, D. v. zeae. These subspecies are allopatric except for two known regions of sympatry in Texas and Mexico. We demonstrate that populations of D. v. virgifera, with the exception of two populations in southern Arizona, are infected with a strain of Wolbachia. Populations of D. v. zeae are not infected. Treatment of D. v. virgifera with tetracycline eliminated the Wolbachia and removed the reproductive incompatibility. Similar patterns of reproductive incompatibility exist among taxa of the cricket genus Gryllus. Gryllus assimilis, G. integer, G. ovisopis, G. pennsylvanicus, and G. rubens are infected with Wolbachia whereas G. firmus is usually not. Populations of G. rubens and G. ovisopis carry the same Wolbachia strain, which is distinct from that of G. integer. G. pennsylvanicus is infected with two Wolbachia strains, that found in G. rubens and one unique to G. pennsylvanicus. Moreover, a proportion of G. pennsylvanicus individuals harbors both strains. Wolbachia may have influenced speciation in some members of the genus Gryllus by affecting the degree of hybridization between species. Given that Wolbachia infections are relatively common in insects, it is likely that other insect hybrid zones may be influenced by infections with Wolbachia.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the “oxidative burst,” they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C15 intermediate xanthoxin and C25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9′-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.