Molecular tools to improve chestnut management: El Bierzo as a case study

The European chestnut (Castanea sativa Mill.) is a multipurpose species that has been widely cultivated around the Mediterranean basin since ancient times. New varieties were brought to the Iberian Peninsula during the Roman Empire, which coexist since then with native populations that survived the last glaciation. The relevance of chestnut cultivation has being steadily growing since the Middle Ages, until the rural decline of the past century put a stop to this trend. Forest fires and diseases were also major factors. Chestnut cultivation is gaining momentum again due to its economic (wood, fruits) and ecologic relevance, and represents currently an important asset in many rural areas of Europe. In this Thesis we apply different molecular tools to help improve current management strategies. For this study we have chosen El Bierzo (Castile and Leon, NW Spain), which has a centenary tradition of chestnut cultivation and management, and also presents several unique features from a genetic perspective (next paragraph). Moreover, its nuts are widely appreciated in Spain and abroad for their organoleptic properties. We have focused our experimental work on two major problems faced by breeders and the industry: the lack of a fine-grained genetic characterization and the need for new strategies to control blight disease. To characterize with sufficient detail the genetic diversity and structure of El Bierzo orchards, we analyzed DNA from 169 trees grafted for nut production covering the entire region. We also analyzed 62 nuts from all traditional varieties. El Bierzo constitutes an outstanding scenario to study chestnut genetics and the influence of human management because: (i) it is located at one extreme of the distribution area; (ii) it is a major glacial refuge for the native species; (iii) it has a long tradition of human management (since Roman times, at least); and (iv) its geographical setting ensures an unusual degree of genetic isolation. Thirteen microsatellite markers provided enough informativeness and discrimination power to genotype at the individual level. Together with an unexpected level of genetic variability, we found evidence of genetic structure, with three major gene pools giving rise to the current population. High levels of genetic differentiation between groups supported this organization. Interestingly, genetic structure does not match with spatial boundaries, suggesting that the exchange of material and cultivation practices have strongly influenced natural gene flow. The microsatellite markers selected for this study were also used to classify a set of 62 samples belonging to all traditional varieties. We identified several cases of synonymies and homonymies, evidencing the need to substitute traditional classification systems with new tools for genetic profiling. Management and conservation strategies should also benefit from these tools. The avenue of high-throughput sequencing technologies, combined with the development of bioinformatics tools, have paved the way to study transcriptomes without the need for a reference genome. We took advantage of RNA sequencing and de novo assembly tools to determine the transcriptional landscape of chestnut in response to blight disease. In addition, we have selected a set of candidate genes with high potential for developing resistant varieties via genetic engineering. Our results evidenced a deep transcriptional reprogramming upon fungal infection. The plant hormones ET and JA appear to orchestrate the defensive response. Interestingly, our results also suggest a role for auxins in modulating such response. Many transcription factors were identified in this work that interact with promoters of genes involved in disease resistance. Among these genes, we have conducted a functional characterization of a two major thaumatin-like proteins (TLP) that belongs to the PR5 family. Two genes encoding chestnut cotyledon TLPs have been previously characterized, termed CsTL1 and CsTL2. We substantiate here their protective role against blight disease for the first time, including in silico, in vitro and in vivo evidence. The synergy between TLPs and other antifungal proteins, particularly endo-p-1,3-glucanases, bolsters their interest for future control strategies based on biotechnological approaches.

Polyolefin fibre-reinforced concrete : from material behaviour to numerical and design considerations

La presente Tesis proporciona una gran cantidad de información con respecto al uso de un nuevo y avanzado material polimérico (con base de poliolefina) especialmente adecuada para ser usada en forma de fibras como adición en el hormigón. Se han empleado fibras de aproximadamente 1 mm de diámetro, longitudes entre 48 y 60 mm y una superficie corrugada. Las prometedoras propiedades de este material (baja densidad, bajo coste, buen comportamiento resistente y gran estabilidad química) justifican el interés en desarrollar el esfuerzo de investigación requerido para demostrar las ventajas de su uso en aplicaciones prácticas. La mayor parte de la investigación se ha realizado usando hormigón autocompactante como matriz, ya que este material es óptimo para el relleno de los encofrados del hormigón, aunque también se ha empleado hormigón normal vibrado con el fin de comparar algunas propiedades. Además, el importante desarrollo del hormigón reforzado con fibras en los últimos años, tanto en investigación como en aplicaciones prácticas, también es muestra del gran interés que los resultados y consideraciones de diseño que esta Tesis pueden tener. El material compuesto resultante, Hormigón Reforzado con Fibras de Poliolefina (HRFP o PFRC por sus siglas inglesas) ha sido exhaustivamente ensayado y estudiado en muchos aspectos. Los resultados permiten establecer cómo conseguidos los objetivos buscados: -Se han cuantificado las propiedades mecánicas del PFRC con el fin de demostrar su buen comportamiento en la fase fisurada de elementos estructurales sometidos a tensiones de tracción. -Contrastar los resultados obtenidos con las bases propuestas en la normativa existente y evaluar las posibilidades para el uso del PFRC con fin estructural para sustituir el armado tradicional con barras de acero corrugado para determinadas aplicaciones. -Se han desarrollado herramientas de cálculo con el fin de evaluar la capacidad del PFRC para sustituir al hormigón armado con las barras habituales de acero. -En base a la gran cantidad de ensayos experimentales y a alguna aplicación real en la construcción, se han podido establecer recomendaciones y consejos de diseño para que elementos de este material puedan ser proyectados y construidos con total fiabilidad. Se presentan, además, resultados prometedores en una nueva línea de trabajo en el campo del hormigón reforzado con fibras combinando dos tipologías de fibras. Se combinaron fibras de poliolefina con fibras de acero como refuerzo del mismo hormigón autocompactante detectándose sinergias que podrían ser la base del uso futuro de esta tecnología de hormigón. This thesis provides a significant amount of information on the use of a new advanced polymer (polyolefin-based) especially suitable in the form of fibres to be added to concrete. At the time of writing, there is a noteworthy lack of research and knowledge about use as a randomly distributed element to reinforce concrete. Fibres with an approximate 1 mm diameter, length of 48-60 mm, an embossed surface and improved mechanical properties are employed. The promising properties of the polyolefin material (low density, inexpensive, and with good strength behaviour and high chemical stability) justify the research effort involved and demonstrate the advantages for practical purposes. While most of the research has used self-compacting concrete, given that this type of matrix material is optimum in filling the concrete formwork, for comparison purposes standard vibration compacted mixes have also been used. In addition, the interest in fibre-reinforced concrete technology, in both research and application, support the significant interest in the results and considerations provided by the thesis. The resulting composite material, polyolefin fibre reinforced concrete (PFRC) has been extensively tested and studied. The results have allowed the following objectives to be met: -Assessment of the mechanical properties of PFRC in order to demonstrate the good performance in the post-cracking strength for structural elements subjected to tensile stresses. -- Assessment of the results in contrast with the existing structural codes, regulations and test methods. The evaluation of the potential of PFRC to meet the requirements and replace traditional steel-bar reinforcement applications. -Development of numerical tools designed to evaluate the capability of PFRC to substitute, either partially or totally, standard steel reinforcing bars either alone or in conjunction with steel fibres. -Provision, based on the large amount of experimental work and real applications, of a series of guidelines and recommendations for the practical and reliable design and use of PFRC. Furthermore, the thesis also reports promising results about an innovative line in the field of fibre-reinforced concrete: the design of a fibre cocktail to reinforce the concrete by using two types of fibres simultaneously. Polyolefin fibres were combined with steel fibres in self-compacting concrete, identifying synergies that could serve as the base in the future use of fibre-reinforced concrete technology.

Influencia de un recubrimiento de vinilo en la tensión de fractura del vidrio en ensayos de anillos concéntricos con gran superficie

El comportamiento post-rotura de los vidrios laminados es uno de los temas que están siendo investigados para explicar la capacidad de carga remanente tras la rotura de la primera lámina. En investigaciones previas se ha observado que en el caso de impacto humano en vidrios recocidos se llega a una capacidad hasta 3 veces superior, sin explicación clara del comportamiento estructural del conjunto. Para realizar un acercamiento a la resistencia a la rotura del vidrio laminado se ha planificado una campaña de ensayos de rotura con anillos concéntricos de grandes superficies en vidrio recocido, termoendurecido y templado, con dos series adicionales de vidrio recocido y termoendurecido con una capa de butiral adherida justo después del proceso de fabricación. Para realizar la comparación de las distribuciones de Weibull de las distintas tensiones de rotura se utiliza un proceso iterativo basado en la distribución real de tensiones obtenida con un modelo de elementos finitos ajustado con datos experimentales. Las comparaciones finales muestran un aumento apreciable de la resistencia (45%) en el caso de vidrios recocidos, y menor en el de los termoendurecidos (25%).The post-fracture behavior of the laminated glasses is one of the research topics that are being studied to explain the load capacity after the break of the first sheet. Previous experimental work have shown, that in case of human impact in annealed glasses, the capacity of bearing load it can be up to 3 times higher without clear explanation of the structural behavior of the plate. To make an approximation to the post-fracture resistance, a experimental program to test annealed, heat-tempered and toughened glass plates has been prepared. Two additional series of annealed and heattempered, with a layer of polyvinyl butyral adhered just after the manufacturing process, have also been incorporated. Coaxial Double Ring with large test surface areas Coaxial Double Ring with large test surface areas is the standard that has been followed. To make the comparison of Weibull's distributions of the different fracture stress, an iterative process based on the actual stress distribution obtained with a finite elements model updated with experimental results has been used. Final comparisons show a great stress improvement for the annealed glass plates (45 %), and a minor increment for the heat-tempered (25 %).

Contribute to the Study of Vertical Distribution of Conventional No-Till Seeders and Spatial Variability of Seed Depth Placement of Maize in Alentejo, Portugal

Entre os vários fatores que contribuem para a produção de uma cultura de milho, a distribuição vertical dos semeadores avaliada através da localização da semente em profundidade é um fator-chave, especialmente na técnica de sementeira direta. Simultaneamente, dada a complexidade dos ecossistemas naturais e agrícolas em sistemas de agricultura de conservação, a gestão diferenciada e localizada das parcelas assume um importante papel na análise e gestão da variabilidade das propriedades do solo e estabelecimento das culturas, nomeadamente utilizando informação geo referenciada e tecnologia expedita. Assim, o principal objetivo desta Tese foi a avaliação em culturas de milho da variabilidade espacial da localização de semente em profundidade e estabelecimento da cultura em sementeira direta usando sistemas convencionais de controlo de profundidade, tendo-se comparado com diferentes sistemas de mobilização e recorrendo a tecnologias de agricultura de precisão. Os ensaios decorreram na região Mediterrânea do Alentejo, em propriedades agrícolas no decorrer das campanhas de 2010, 2011, 2012 e 2015 em 6 diferentes campos experimentais. O trabalho experimental consistiu em ensaios com avaliações in loco do solo e cultura, consumo de combustível das operações e deteção remota. Os resultados obtidos indicam que não só o sistema de mobilização afetou a localização da semente em profundidade, como em sementeira direta a profundidade de sementeira foi afetada pelo teor de humidade do solo, resistência do solo à profundidade e velocidade da operação de sementeira. Adicionalmente observaram-se condições heterogéneas de emergência e estabelecimento da cultura afetadas por condições físicas de compactação do solo. Comparando os diferentes sistemas de mobilização, obteve-se uma significativa redução de combustível para a técnica de sementeira direta, apesar de se terem observado diferenças estatísticas significativas considerando diferentes calibrações de profundidade de sementeira Do trabalho realizado nesta Tese ressalva-se a importância que as tecnologias de agricultura de precisão podem ter no acompanhamento e avaliação de culturas em sementeira direta, bem como a necessidade de melhores procedimentos no controlo de profundidade dos semeadores pelo respetivos operadores ou ao invés, a adoção de semeadores com mecanismos ativos de controlo de profundidade. ABSTRACT Among the various factors that contribute towards producing a successful maize crop, seeders vertical distribution evaluated through seed depth placement is a key determinant, especially under a no-tillage technique. At the same time in conservation agriculture systems due to the complexity of natural and agricultural ecosystems site specific management became an important approach to understand and manage the variability of soil properties and crop establishment, especially when using geo spatial information and affording readily technology Thus, the main objective of this Thesis was to evaluate the spatial variability of seed depth placement and crop establishment in maize crops under no-tillage conditions compared to different tillage systems, using conventional seed depth control no till seeders and precision farming technologies. Trials were carried out in the Mediterranean region of Alentejo, in private farms along the sowing operations season over the years 2010, 2011, 2012 and 2015 in 6 different experimental fields. Experimental work covered field tests with in loco soil and crop evaluations, fuel operation evaluations and aerial sensing. The results obtained indicate that not only tillage system affected seed depth placement but under no till conditions seed depth was affected by soil moisture content, soil resistance to penetration and seeders forward speed. In addition uneven crop seedling and establishment depended on seed depth placement and could be affected by physical problems of compaction layers. Significant reduction in fuel consumption was observed for no till operations although significant differences observed according to different setting calibrations of seed depth control. According to the results, precision agriculture is an important tool to evaluate crops under no till conditions and seed depth mechanisms should be more accurate by the operators or is determinant the adoption of new active depth control technology to improve seeders performance.