Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 x J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the use of 0.40 mM phloroglucinol during shoot multiplication, 0.20 mM previous root induction, and 6.81 mg/L Fe3+ (FeEDDHA). Moreover, the addition of 83.2 µM glucose during the root expression phase significantly improved plant survival during acclimatization. Phloroglucinol promoted microshoot elongation but inhibited rooting, especially at concentrations above 0.40 mM. Replacing FeEDTA by FeEDDHA diminished chlorotic symptoms and improved rooting, with up to 90% microshoots developing viable roots. Likewise, glucose was more efficient than sucrose or fructose in promoting plant survival. At the proposed working concentrations, neither glucose nor FeEDDHA caused any noticeable deleterious effect on walnut micropropagation. Microscopic analysis revealed the physical continuity between adventitious roots and stem pericycles. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5,800 ramets) have been established by applying this protocol.

Use of calculus of variations to determine the shape of hovering rotors of minimum power and its application to micro air vehicles

In this paper, calculus of variations and combined blade element and momentum theory (BEMT) are used to demonstrate that, in hover, when neither root nor tip losses are considered; the rotor, which minimizes the total power (MPR), generates an induced velocity that varies linearly along the blade span. The angle of attack of every blade element is constant and equal to its optimum value. The traditional ideal twist (ITR) and optimum (OR) rotors are revisited in the context of this variational framework. Two more optimum rotors are obtained considering root and tip losses, the ORL, and the MPRL. A comparison between these five rotors is presented and discussed. The MPR and MPRL present a remarkable saving of power for low values of both thrust coefficient and maximum aerodynamic efficiency. The result obtained can be exploited to improve the aerodynamic behaviour of rotary wing micro air vehicles (MAV). A comparison with experimental results obtained from the literature is presented.

Exceptionally well-preserved vegetal remains from the Upper Cretaceous of "Lo Hueco", Cuenca, Spain

Vegetal remains are considered labile structures that quickly become decayed in ecosystems. However, certain lignified tissues (woody plants) can largely resist decomposition, becoming sometimes exceptionally well preserved. At the Upper Cretaceous site of ?Lo Hueco? (Cuenca, Spain), those woody remains (trunks and branches) with resinous material in the inner tracheids and parenchyma cells that were buried rapidly under anoxic conditions experienced a low degree of maturation, becoming exceptionally well preserved. Those woody remains deposited under oxic conditions (subaerial or sub-aquatic exposure) were more intensely biodegraded and subsequently carbonified, partially or completely mineralized in gypsum and covered by a ferruginous crust. These two modes of preservation are scarce, with silicification or carbonification processes much more common, and both can be considered as ?exceptional preservation?. Other vegetal remains, such as carbonified leaves, stems and roots, were collected in the site. The different modes of preservation depend directly on: depositional micro-environment (sandy distributary channel, muddy flood plain); and type (trunk, branch, stem, leave, root) and state (presence or absence of resinous material) of the material. The great abundance and diversity of fossils in ?Lo Hueco? identify it as Konzentrat-Lagersta¿tten, sequentially formed by alternated events of flooding and drying depositional events, but the exceptional quality and rarity of determinate vegetal macroremains preservation suggest that certain deposits of this site can be considered as conservation deposits.

Flujos de gases de efecto invernadero en suelos forestales y agroforestales del centro de la Península Ibérica=Greenhouse gas flows in forest and agroforestry soils in the center of the Iberian Peninsula

Debido a la complejidad de los procesos que controlan el intercambio de gases de carbono (C) y nitrógeno (N) entre el suelo y la atmósfera, en los sistemas forestales y agroforestales, son comprensibles las incógnitas existentes respecto a la estimación de los flujos de los gases de efecto invernadero (GEI) y la capacidad como reservorios de carbono de los suelos, bajo diferentes formas de uso y regímenes de alteración a escala regional y global. Esta escasez de información justifica la necesidad de caracterizar la dinámica de intercambio de GEI en los ecosistemas Mediterráneos, en especial en el contexto actual de cambio climático, y el incremento asociado de temperatura y periodos de sequía, alteración de los patrones de precipitación, y el riesgo de incendios forestales; cuyas consecuencias afectarán tanto a los compartimentos de C y de N del suelo como a la capacidad de secuestro de C de estos ecosistemas. Dentro de este contexto se enmarca la presente tesis doctoral cuyo objetivo ha sido cuantificar y caracterizar los flujos de dióxido de carbono (CO2), de oxido nitroso (N2O) y de metano (CH4), junto con los stocks de C y N, en suelos forestales de Quercus ilex, Quercus pyrenaica y Pinus sylvestris afectados por incendios forestales; así como el estudiar el efecto de la gestión y la cubierta arbórea en la respiración del suelo y los stocks de C y N en una dehesa situada en el centro de la Península Ibérica. De manera que los flujos de CO2, N2O y CH4; y los parámetros físico-químicos y biológicos del suelo fueron estudiados en los diferentes tratamientos y ecosistemas a lo largo del trabajo que se presenta. Los resultados obtenidos muestran la existencia de variaciones temporales y espaciales de la respiración del suelo dentro de una escala geográfica pequeña, controladas principalmente por la temperatura y la humedad del suelo; y por los contenidos de C y N del suelo en un bosque de Pinus sylvestris en la vertiente norte de la Sierra de Guadarrama , en España. El análisis de los efectos de los incendios forestales a largo plazo (6-8 años) revela que las pérdidas anuales de C a través de la respiración del suelo en las zonas quemadas de Quercus ilex, Quercus pyrenaica y Pinus sylvestris fueron 450 gCm-2yr-1, 790 gCm-2yr-1 y 1220 gCm-2yr-1, respectivamente; lo que representa una reducción del 43%, 22% y 11% en comparación con las zonas no quemadas de dichas especies, debido a la destrucción de la masa arbórea. El efecto del fuego también alteró los flujos N2O y CH4 del suelo, de una forma diferente en los distintos ecosistemas y estacionalidades estudiadas. De tal modo, que los suelos quemados mostraron una mayor oxidación del CH4 en las masas de Q. ilex, y una menor oxidación en las de P. sylvestris; además de una disminución de los flujos de N2O en Q. pyrenaica. Los incendios también afectaron los parámetros microclimáticos de los suelos forestales, observándose un incremento de la temperatura del suelo y una disminución de la humedad en los emplazamientos quemados que en los no quemados. Los cationes intercambiables, el pH, el cociente C/N, el contenido en raicillas y la biomasa microbiana también disminuyeron en las zonas quemadas. Aunque el C orgánico del suelo no se alteró de manera significativa, si lo hizo la calidad de la materia orgánica, disminuyendo el carbono lábil y aumentando las formas recalcitrantes lo que se tradujo en menor sensibilidad de la respiración del suelo a la temperatura (valores de Q10) en las zonas quemadas. Los resultados del estudio realizado en la Dehesa muestran que las actividades silvopastorales estudiadas afectaron levemente y de forma no constante a la respiración del suelo y las condiciones microclimáticas del suelo. Se observó una reducción 12% de la respiración del suelo por efecto del pastoreo no intensivo. Sin embargo, se observaron incrementos de 3Mg/ha en los stocks de C y de 0.3 Mg/ha en los stocks de N en los suelos pastoreados en comparación con los no pastoreados. Aunque, no se observó un claro efecto de la labranza sobre la respiración del suelo en nuestro experimento, sin embargo si se observó una disminución de 3.5 Mg/ha en las reservas de C y de 0.3 Mg/ ha en las de N en los suelos labrados comparados con los no labrados. La copa del arbolado influyó de forma positiva tanto en la respiración del suelo, como en los stocks de C y N de los suelos. La humedad del suelo jugó un papel relevante en la sensibilidad de la respiración a la temperatura del suelo. Nuestros resultados ponen de manifiesto la sensibilidad de la respiración del suelo a cambios en la humedad y los parámetros edáficos, y sugieren que la aplicación de modelos estándar para estimar la respiración del suelo en áreas geográficas pequeñas puede no ser adecuada a menos que otros factores sean considerados en combinación con la temperatura del suelo. Además, las diferentes respuestas de los flujos de gases de efecto invernadero a los cambios, años después de la ocurrencia de incendios forestales, destaca la necesidad de incluir estos cambios en las futuras investigaciones de la dinámica del carbono en los ecosistemas mediterráneos. Por otra parte, las respuestas divergentes en los valores de respiración del suelo y en los contenidos de C y N del suelo observados en la dehesa, además de la contribución de la copa de los árboles en los nutrientes del suelo ilustran la importancia de mantener la gestión tradicional aplicada en beneficio de la capacidad de almacenar C en la dehesa estudiada. La información obtenida en este trabajo pretende contribuir a la mejora del conocimiento de la dinámica y el balance de C en los sistemas mediterráneos, además de ayudar a predecir el impacto del cambio climático en el intercambio de C entre los ecosistemas forestales y agroforestales y la atmósfera. ABSTRACT Due to the complexity of the processes that control the exchange of carbon (C) and nitrogen (N) gasses between soils and the atmosphere in forest and agroforestry ecosystems, understandable uncertainties exist as regards the estimation of greenhouse gas (GHG) fluxes and the soil sink capacity at regional and global scale under different forms of land use and disturbance regimes. These uncertainties justify the need to characterize the exchange dynamics of GHG between the atmosphere and soils in Mediterranean terrestrial ecosystems, particularly in the current context of climate change and the associated increase in temperature, drought periods, heavy rainfall events, and increased risk of wildfires, which affect not only the C and N pools but also the soil C sink capacity of these ecosystems. Within this context, the aims of the present thesis were, firstly, to quantify and characterize the fluxes of carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4) as well as the C and N stocks in Quercus ilex, Quercus pyrenaica and Pinus sylvestris stands affected by wildfires, and secondly, to study the effects of Quercus ilex canopy and management on both soil respiration and C and N pools in dehesa systems in the center of Iberian Peninsula. Soil CO2, N2O and CH4 fluxes, and soil physical-chemical and biological parameters were studied under the different treatments and ecosystems considered in this study. The results showed seasonal and spatial variations in soil respiration within small geographic areas, mainly controlled by soil temperature and moisture in addition to soil carbon and nitrogen stocks in mixed pine–oak forest ecosystems on the north facing slopes of the Sierra de Guadarrama in Spain. The analysis of long term effects of wildfires (6–8 years) revealed that annual carbon losses through soil respiration from burned sites in Quercus ilex, Quercus pyrenaica and Pinus sylvestris stands were 450 gCm-2yr-1, 790 gCm-2yr-1 and 1220 gCm-2yr-1, respectively; with burned sites emitting 43%, 22% and 11% less in burned as opposed to non-burned sites due the loss of trees. Fire may alter both N2O and CH4 fluxes although the magnitude of such variation depends on the site, soil characteristics and seasonal climatic conditions. The burned sites showed higher CH4 oxidation in Q.ilex stands, and lower oxidation rates in P. sylvestris stands. A reduction in N2O fluxes in Q. pyrenaica stands was detected at burned sites along with changes in soil microclimate; higher soil temperature and lower soil moisture content. Exchangeable cations, the C/N ratio, pH, fine root and microbial biomass were also found to decrease at burned sites. Although the soil organic carbon was not significantly altered, the quality of the organic matter changed, displaying a decrease in labile carbon and a relative increase in refractory forms, leading to lower sensitivity of soil respiration to temperature (Q10 values) at burned sites. The results from the dehesa study show that light grazing and superficial tilling practices used in the studied dehesa system in Spain had a slight but non-consistent impact on soil respiration and soil microclimate over the study period. The reduction in soil respiration in the dehesa system due to the effects of grazing was around 12 %. However, increments of 3Mg/ha in C stocks and 0.3 Mg/ha in N stocks in grazed soils were observed. Although no clear effect of tilling on soil respiration was found, a decrease of 3.5 Mg/ha in C stocks and 0.3 Mg/ha in N stocks was detected for tilled soils. The presence of a tree canopy induced increases in soil respiration, soil C and N stocks, while soil moisture was found to play an important role in soil respiration temperature response. Our results suggest that the use of standard models to estimate soil respiration in small geographical areas may not be adequate unless other factors are considered in addition to soil temperature. Furthermore, the different responses of GHG flux to climatic shifts, many years after the occurrence of wildfire, highlight the need to include these shifts in C dynamics in future research undertaken in Mediterranean ecosystems. Furthermore, divergent responses in soil respiration and soil C and N stocks to grazing or tilling practices in Dehesa systems, and the influence of tree canopy on soil respiration and soil nutrient content, illustrate the importance of maintaining beneficial management practices. Moreover, the carbon sequestration capacity of the Dehesa system studied may be enhanced through improvements in the management applied. It is hoped that the information obtained through this research will contribute towards improving our understanding of the dynamics and balance of C in Mediterranean systems, and help predict the impact of climate change on the exchange of C between forest and agroforestry ecosystems and the atmosphere.

Comparison of Airborne Laser Scanning Methods for Estimating Forest Structure Indicators Based on Lorenz Curves

The purpose of this study was to compare a number of state-of-the-art methods in airborne laser scan- ning (ALS) remote sensing with regards to their capacity to describe tree size inequality and other indi- cators related to forest structure. The indicators chosen were based on the analysis of the Lorenz curve: Gini coefficient ( GC ), Lorenz asymmetry ( LA ), the proportions of basal area ( BALM ) and stem density ( NSLM ) stocked above the mean quadratic diameter. Each method belonged to one of these estimation strategies: (A) estimating indicators directly; (B) estimating the whole Lorenz curve; or (C) estimating a complete tree list. Across these strategies, the most popular statistical methods for area-based approach (ABA) were used: regression, random forest (RF), and nearest neighbour imputation. The latter included distance metrics based on either RF (NN–RF) or most similar neighbour (MSN). In the case of tree list esti- mation, methods based on individual tree detection (ITD) and semi-ITD, both combined with MSN impu- tation, were also studied. The most accurate method was direct estimation by best subset regression, which obtained the lowest cross-validated coefficients of variation of their root mean squared error CV(RMSE) for most indicators: GC (16.80%), LA (8.76%), BALM (8.80%) and NSLM (14.60%). Similar figures [CV(RMSE) 16.09%, 10.49%, 10.93% and 14.07%, respectively] were obtained by MSN imputation of tree lists by ABA, a method that also showed a number of additional advantages, such as better distributing the residual variance along the predictive range. In light of our results, ITD approaches may be clearly inferior to ABA with regards to describing the structural properties related to tree size inequality in for- ested areas.

Prototype emitter for use in subsurface drip irrigation: Manufacturing, hydraulic evaluation and experimental analyses

The current research aims to analyse theoretically and evaluate a self-manufactured simple design for subsurface drip irrigation (SDI) emitter to avoid root and soil intrusion. It was composed of three concentric cylindrical elements: an elastic silicone membrane; a polyethylene tube with two holes drilled on its wall for water discharge; and a vinyl polychloride protector system to wrap the other elements. The discharge of the emitter depends on the change in the membrane diameter when it is deformed by the water pressure. The study of the operation of this emitter is a new approach that considers mechanical and hydraulic principles. Thus, the estimation on the membrane deformation was based on classical mechanical stress theories in composite cylinders. The hydraulic principles considered the solid deformation due to force based on water pressure and the general Darcy–Weisbach head-loss equation. Twenty emitter units, with the selected design, were handcrafted in a lathe and were used in this study. The measured pressure/discharge relationship for the emitters showed good agreement with that calculated by the theoretical approach. The variation coefficient of the handcrafted emitters was high compared to commercial emitters. Results from field evaluations showed variable values for the relative flow variation, water emission uniformity and relative flow rate coefficients, but no emitter was obstructed. Therefore, the current emitter design could be suitable for SDI following further studies to develop a final prototype.

High thermal neutron flux effects on structural and macroscopic properties of alkali-borosilicate glasses used as neutron guide substrate

The behaviour of four alkali-borosilicate glasses under homogeneous thermal neutron irradiation has been studied. These materials are used for the manufacturing of neutron guides which are installed in most facilities as devices to transport neutrons from intense sources such as nuclear reactors or spallation sources up to scientific instruments. Several experimental techniques such as Raman, NMR, SANS and STEM have been employed in order to understand the rather different macroscopic behaviour under irradiation of materials that belong to a same glass family. The results have shown that the remarkable glass shrinking observed for neutron doses below 0.5 · 10 18 n/cm 2 critically depends upon the presence of domains where silicate and borate network do not mix.

Unearthing the roots of degradation of Quercus pyrenaica coppices: an integrative perspective from clonal structure to carbon budgets

Quercus pyrenaica es una especie rebrotadora de raíz intensa e históricamente aprovechada en monte bajo para la obtención de leñas, carbón y pastos. Debido al éxodo rural y a la aparición de nuevas fuentes energéticas, este aprovechamiento fue abandonado en la década de 1970. Desde entonces, las bajas producciones de madera y bellota y el puntisecado de los pies evidencian el generalizado estancamiento de estas masas. Uno de los mayores retos actuales de la selvicultura en el ámbito mediterráneo es encontrar usos alternativos para estos montes abandonados, siendo la conversión a monte alto una de las alternativas preferidas. Se han realizado resalveos de conversión, sin embrago, éstos se aplican sin un conocimiento integral de las causas de la degradación. En esta tesis doctoral, estudiamos un hipotético desequilibrio entre la parte radical y la parte aérea (R:S) de las cepas de rebollo como causa subyacente de su decaimiento. En una parcela experimental, aprovechada al menos desde el siglo XII, se realizaron análisis genéticos a priori para elucidar la estructura genética del rodal, y así estudiar la influencia del tamaño clonal en el funcionamiento de las cepas. Las cepas de mayor tamaño presentaron un menor crecimiento diametral de sus pies, así como mayores tasas de respiración radical, estimadas a partir de flujos internos de CO2 a través del xilema (FT) y de los flujos de CO2 del suelo. Estos resultados sugieren que el desequilibrio R:S aumenta con el tamaño clonal, dado que la eliminación periódica de órganos aéreos, al mismo tiempo que las raíces permanecen intactas, da lugar a un gran desarrollo del sistema radical que consume gran parte de los carbohidratos no estructurales (NSC) en respiración de mantenimiento, comprometiendo así el desarrollo de órganos aéreos. Se excavaron y pesaron dos cepas compuestas por cuatro y ocho pies, las cuales mostraron ratios R:S (0.5 y 1, respectivamente) superiores a los registrados en pies de origen sexual. Al igual que en otras especies rebrotadoras de raíz, se observaron altas concentraciones de NSC en las raíces (> 20% en primavera) y una gran proporción de albura en el sistema radical (52%) que alberga una notable reserva de NSC (87 kg en la cepa de mayor tamaño). En el sistema radical de dicha cepa, estimada mediante dataciones radiocarbónicas en 550 años de edad, se contaron 248 uniones radicales. La persistencia de sistemas radicales grandes, viejos, y altamente interconectados sugiere que la gran cantidad de recursos almacenados y consumidos en las raíces compensan un pobre desarrollo aéreo con una alta resiliencia vegetativa. Para un mejor entendimiento de los balances de carbono y del agotamiento de NSC en las cepas de rebollo, se midieron los flujos internos y externos de CO2 en troncos y los flujos de CO2 del suelo, y se estimó la respiración de órganos aéreos (RS) y subterráneos (RR). Estacionalmente, RS y RR reflejaron las dinámicas de flujo de savia y de crecimiento del tronco, y estuvieron determinadas principalmente por los flujos externos de CO2, dada la escasa contribución de FT a RS y RR (< 10% y < 2%, respectivamente). En una escala circadiana, la contribución de FT a RS aumentó hasta un 25% en momentos de alta transpiración. Las bajas concentraciones de CO2 en el xilema ([CO2] hasta un 0.11%) determinaron comparativamente unos bajos FT, probablemente causados por una limitada respiración del xilema y una baja resistencia a la difusión radial del CO2 impuestos por la sequía estival. Los pulsos de [CO2] observados tras las primeras lluvias de otoño apoyan esta idea. A lo largo del periodo vegetativo, el flujo medio de CO2 procedente del suelo (39 mol CO2 day-1) fue el mayor flujo respiratorio, tres y cuatro veces superior a RS (12 mol CO2 day-1) y RR (8-9 mol CO2 day-1), respectivamente. Ratios RR/RS menores que la unidad evidencian un importante peso de la respiración aérea como sumidero de carbono adicional. Finalmente, se ensayó el zanjado de raíces y el anillamiento de troncos como tratamientos selvícolas alternativos con el objetivo de aumentar las reservas de NSC en los troncos de las cepas. Los resultados preliminares desaconsejan el zanjado de raíces por el alto coste derivado posiblemente de la cicatrización de las heridas. El anillado de troncos imposibilitó el transporte de NSC a las raíces y aumentó la concentración de almidón por encima de la zona anillada, mientras que sistema radical se mantiene por los pies no anillados de la cepa. Son necesarias más mediciones y datos adicionales para comprobar el mantenimiento de esta respuesta positiva a largo plazo. Para concluir, destacamos la necesidad de estudios multidisciplinares que permitan una comprensión integral de la degradación de los rebollares ibéricos para poder aplicar a posteriori una gestión adecuada en estos montes bajos abandonados. ABSTRACT Quercus pyrenaica is a vigorous root-resprouting species intensively and historically coppiced for firewood, charcoal and woody pastures. Due to the rural exodus and the appearance of new energy sources, coppicing was abandoned towards 1970. Since then, tree overaging has resulted in stand stagnation displayed by slow stem growth, branch dieback, and scarce acorn production. The urgent need to find new alternative uses for abandoned coppices is recognized as one of the biggest challenges which currently faces Mediterranean silviculture; conversion into high forest by thinning is one of the preferred alternatives. For this aim, thinning has been broadly applied and seldom tested, although without a comprehensive understanding of the causes of stand stagnation. In this PhD study, we test the hypothesis of an imbalance between above- and below-ground organs, result of long term coppicing, as the underlying cause of Q. pyrenaica decay. In an experimental plot coppiced since at least the 12th century, genetic analyses were performed a priori to elucidate inconspicuous clonal structure of Q. pyrenaica to evaluate how clonal size affects the functioning of these multi-stemmed trees. Clonal size negatively affected diametric stem growth, whereas root respiration rates, measured by internal fluxes of CO2 through xylem (FT) and soil CO2 efflux, increased with clonal size. These results suggest root-to-shoot (R:S) imbalance intensifying with clonal size: periodic removal of aboveground organs whilst belowground organs remain undisturbed may have led to massive root systems which consume a great proportion of non-structural carbohydrates (NSC) for maintenance respiration, thus constraining aboveground performance. Furthermore, excavation of two multi-stemmed trees, composed by four and eight stems, revealed R:S ratios (0.5 and 1, respectively) greater than those reported for sexually regenerated trees. Moreover, as similarly observed in several root-resprouting species, NSC allocation to roots was favored ([NSC] > 20% in spring): a large proportion of sapwood maintained throughout the root system (52%) stored a remarkable NSC pool of 87 kg in the case of the largest clone. In this root system of the eight-stemmed tree, 248 root connections were counted and, by radiocarbon dating, its age was estimated to be 550-years-old. Persistence of massive, old and highly interconnected root systems suggests that enhanced belowground NSC storage and consumption reflects a trade-off between vegetative resilience and aboveground development. For a better understanding of tree carbon budget and the potential role of carbon starvation in Q. pyrenaica decay, internal and external stem CO2 fluxes and soil CO2 effluxes were monitored to evaluate respiratory costs above- and below-ground. On a seasonal scale, stem and root respiration (RS and RR) mirrored sap flow and stem growth dynamics. Respiration was determined to the greatest extent by external fluxes of CO2 to the atmosphere or soil, since FT accounted for a low proportion of RS and RR (< 10% and < 2%, respectively). On a diel scale, the contribution of FT to RS increased up to 25% at high transpiration rates. Comparatively low FT was determined by the low concentration of xylem CO2 registered ([CO2] as low as 0.11%), likely as a consequence of constrained xylem respiration and reduced resistance to CO2 radial diffusion imposed by summer drought. Xylem [CO2] pulses following first autumn rains support this idea. Averaged over the growing season, soil CO2 efflux was the greatest respiratory flux (39 mol CO2 day-1), three and four times greater than RS (12 mol CO2 day-1) and RR (8-9 mol CO2 day-1), respectively. Ratios of RR/RS below one evidence an additional and important weight of aboveground respiration as a tree carbon sink. Finally, root trenching and stem girdling were tested as complimentary treatments to thinning as a means to improve carbon reserves in stems of clonal trees. Preliminary results discouraged root trenching due to the high cost likely incurred for wound closure. Stem girdling successfully blocked NSC translocation downward, increasing starch concentrations above the girdled zone whilst the root system is fed by non-girdled stems within the clone. Further measurements and ancillary data are necessary to verify that this positive effect hold over time. To conclude, the need of multidisciplinary approaches for an integrative understanding on the functioning of abandoned Q pyrenaica coppices is highlighted for an appropriate management of these stands.

Natural regeneration in mediterranean mixed forests: effect of environmental conditions on the physiological optimum niche of the species = Regeneración natural en masas mixtas mediterráneas: efecto de las condiciones ambientales en el nicho óptimo fisiológico de las especies

Los montes Mediterráneos han experimentado múltiples cambios en las últimas décadas (tanto en clima como en usos), lo que ha conducido a variaciones en la distribución de especies. El aumento previsto de las temperaturas medias junto con la mayor variabilidad intra e inter anual en cuanto a la ocurrencia de eventos extremos o disturbios naturales (como periodos prolongados de sequía, olas de frío o calor, incendios forestales o vendavales) pueden dañar significativamente al regenerado, llevándolo hasta la muerte, y jugando un papel decisivo en la composición de especies y en la dinámica del monte. La amplitud ecológica de muchas especies forestales puede verse afectada, de forma que se esperan cambios en sus nichos actuales de regeneración. Sin embargo, la migración latitudinal de las especies en busca de mejores condiciones, podría ser una explicación demasiado simplista de un proceso mucho más complejo de interacción entre la temperatura y la precipitación, que afectaría a cada especie de un modo distinto. En este sentido tanto la capacidad de adaptación al estrés ambiental de una determinada especie, así como su habilidad para competir por los recursos limitados, podría significar variaciones dentro de una comunidad. Las características fisiológicas y morfológicas propias de cada especie se encuentran fuertemente relacionadas con el lugar donde cada una puede surgir, qué especies pueden convivir y como éstas responden a las condiciones ambientales. En este sentido, el conocimiento sobre las distintas respuestas ecofisiológicas observadas ante cambios ambientales puede ser fundamentales para la predicción de variaciones en la distribución de especies, composición de la comunidad y productividad del monte ante el cambio global. En esta tesis investigamos el grado de tolerancia y sensibilidad que cada una de las tres especies de estudio, coexistentes en el interior peninsular ibérico (Pinus pinea, Quercus ilex y Juniperus oxycedrus), muestra ante los factores abióticos de estrés típicos de la región Mediterránea. Nuestro trabajo se ha basado en la definición del nicho óptimo fisiológico para el regenerado de cada especie a través de la investigación en profundidad del efecto de la sequía, la temperatura y el ambiente lumínico. Para ello, hemos desarrollado un modelo de predicción de la tasa de asimilación de carbono que nos ha permitido identificar las condiciones óptimas ambientales donde el regenerado de cada especie podría establecerse con mayor facilidad. En apoyo a este trabajo y con la idea de estudiar el efecto de la sequía a nivel de toda la planta hemos desarrollado un experimento paralelo en invernadero. Aquí se han aplicado dos regímenes hídricos para estudiar las características fisiológicas y morfológicas de cada especie, sobre todo a nivel de raíz y crecimiento del tallo, y relacionarlas con las diferentes estrategias en el uso del agua de las especies. Por último, hemos estudiado los patrones de aclimatación y desaclimatación al frio de cada especie, identificando los periodos de sensibilidad a heladas, así como cuellos de botella donde la competencia entre especies podría surgir. A pesar de que el pino piñonero ha sido la especie objeto de la gestión de estas masas durante siglos, actualmente se encuentra en la posición más desfavorable para combatir el cambio global, presentado el nicho fisiológico más estrecho de las tres especies. La encina sin embargo, ha resultado ser la especie mejor cualificada para afrontar este cambio, seguida muy de cerca por el enebro. Nuestros resultados sugieren una posible expansión en el rango de distribución de la encina, un aumento en la presencia del enebro y una disminución progresiva del pino piñonero a medio plazo en estas masas. ABSTRACT Mediterranean forests have undergone multiple changes over the last decades (in both climate and land use), which have lead to variations in the distribution of species. The expected increase in mean annual temperature together with the greater inter and intra-annual variability in extreme events and disturbances occurrence (such as prolonged drought periods, cold or heat waves, wildfires or strong winds) can significantly damage natural regeneration, up to causing death, playing a decisive role on species composition and forest dynamics. The ecological amplitude for adaptation of many species can be affected in such a way that changes in the current regeneration niches of many species are expected. However, the forecasted poleward migration of species seeking better conditions could be an oversimplification of what is a more complex phenomenon of interactions among temperature and precipitation, that would affect different species in different ways. In this regard, either the ability to adapt to environmental stresses or to compete for limited resources of a single species in a mixed forest could lead to variations within a community. The ecophysiological and morphological traits specific to each species are strongly related to the place where each species can emerge, which species can coexist, and how they respond to environmental conditions. In this regard, the understanding of the ecophysiological responses observed against changes in environmental conditions can be essential for predicting variations in species distribution, community composition, and forest productivity in the context of global change. In this thesis we investigated the degree of tolerance and sensitivity that each of the three studied species, co-occurring in central of the Iberian Peninsula (Pinus pinea, Quercus ilex and Juniperus oxycedrus), show against the typical abiotic stress factors in the Mediterranean region. Our work is based on the optimal physiological niche for regeneration of each species through in-depth research on the effect of drought, temperature and light environment. For this purpose, we developed a model to predict the carbon assimilation rate which allows us to identify the optimal environmental conditions where regeneration from each species could establish itself more easily. To obtain a better understanding about the effect of low temperature on regeneration, we studied the acclimation and deacclimation patterns to cold of each species, identifying period of frost sensitivity, as well as bottlenecks where competition between species can arise. Finally, to support our results about the effect of water availabilty, we conducted a greenhouse experiment with a view of studying the drought effect at the whole plant level. Here, two watering regimes were applied in order to study the physiological and morphological traits of each species, mainly at the level of the root system and stem growth, and so relate them to the different water use strategies of the species. Despite the fact that stone pine has been the target species for centuries, nowadays this species is in the most unfavorable position to cope with climate change. Holm oak, however, resulted the species that is best adapted to tolerate the predicted changes, followed closely by prickly juniper. Our results suggest a feasible expansion of the distribution range in holm oak, an increase in the prickly juniper presence and a progressive decreasing of stone pine presence in the medium term in these stone pine-holm oak-prickly juniper mixed forests.

Mannans and endo-beta-mannanases (MAN) in Brachypodium distachyon: expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24–30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36–42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.

Optimización del Abonado Nitrogenado en el Melón (Cucumis Melo L.) Tipo Piel de Sapo

Spain is the fifth-largest producer of melon (Cucumis melo L.) and the second exporter in the world. To a national level, Castilla-La Mancha emphasize and, specifically, Ciudad Real, where is cultivated 27% of national area dedicated to this crop and 30% of melon national production. Melon crop is cultivating majority in Ciudad Real and it is mainly located in the Alto Guadiana, where the major aquifers of the region are located, the aquifer 23 or Mancha Occidental and the aquifer 24 or Campo de Montiel, both declared overexploited and vulnerable zones to nitrate pollution from agricultural sources. The problem is exacerbated because in this area, groundwater is the basic resource of supply to populations, and even often the only one. Given the importance of melon in the area, recent research has focused on the irrigation of melon crop. Unfortunately, scant information has been forthcoming on the effect of N fertilizer on melon piel de sapo crop, so it is very important to tackle in a serious study that lead to know the N requirements on the melon crop melon by reducing the risks of contamination by nitrate leaching without affecting productivity and crop quality. In fact, the recommended dose is often subjective and practice is a N overdose. In this situation, the taking of urgent measures to optimize the use of N fertilization is required. To do it, the effect of N in a melon crop, fertirrigated and on plastic mulch, was studied. The treatments consisted in different rates of N supply, considering N fertilizer and N content in irrigation water, so the treatment applied were: 30 (N30), 85 (N85), 112 (N112) and 139 (N139) Kg N ha-1 in 2005; 93 (N93), 243 (N243) and 393 (N393) kg ha-1 in 2006; and 11 (N11), 61 (N61), 95 (N95) and 148 (N148) kg ha-1 in 2007. A randomized complete-block design was used and each treatment was replicated four times. The results showed a significant effect of N on dry biomass and two patterns of growth were observed. On the one hand, a gradual increase in vegetative biomass of the plant, leaves and stem, with increasing N, and on the other hand, an increase of fruit biomass also with increasing N up to a maximum of biomass corresponding to the optimal dose determined in 90 kg ha-1 of N applied, corresponding to 160 kg ha-1 of N available for melon crop, since this optimum dose, the fruit biomass suffers a decline. A significant effect was observed in concentration and N uptake in leaf, steam, fruit and whole plant, increasing in all of them with increasing of N doses. Fast N uptake occurred from 30-35 to 70-80 days after transplanting, coinciding with the fruit development. The N had a clear influence on the melon yield, its components, skin thickness and flesh ratio. The melon yield increased, as the mean fruit weight and number of fruits per m2 with increasing N until achieve an above 95% of the maximum yield when the N applied is 90 kg ha-1 or 160 kg ha-1 of N available. When N exceeds the optimal amount, there is a decline in yield, reducing the mean fruit weight and number of fruits per square meter, and was also observed a decrease in fruit quality by increasing the skin thickness and decrease the flesh ratio, which means an increase in fruit hollowed with excessive N doses. There was a trend for all indexes of N use efficiency (NUE) to decline with increasing N rate. We observed two different behaviours in the calculation result of the NUE; on the one hand, all the efficiency indexes calculated with N applied and N available had an exponential trend, and on the other hand, all the efficiency indexes calculated with N uptake has a linear trend. The linear regression cuts the exponential curve, delimiting a range within which lies the optimum quantity of N. The N leaching as nitrates increased exponentially with the amount of N. The increase of N doses was affected on the N mineralization. There was a negative exponential effect of N available on the mineralization of this element that occurs in the soil during the growing season, calculated from the balances of this element. The study of N leaching for each N rate used, allowed to us to establish several environmental indices related to environmental risk that causes the use of such doses, a simple way for them to be included in the code of Best Management Practices.

Rhizoctonia solani as causative agent of damping off of Swiss chard in Europe

During September 2011, post-emergence damping off of Swiss chard (Beta vulgaris subsp. cicla L.) was observed in a greenhouse in Villa del Prado (Spain). About 20% of the seedlings showed damping off symptoms. Lesions were initially water soaked, dark brown necrosis of crown tissue, irregular in shape and sunken in appearance on large plants, causing the infected seedlings to collapse and eventually die. Rhizoctonia solani was isolated consistently from symptomatic plants. After morphological and molecular identification of the isolates, pathogenicity was tested by placing agar plugs of four isolates adjacent to the stem at the three or four true leaf stage. In inoculated plants, brown crown and stem necrosis occurred while control plants did not show disease symptoms. Pathogenicity using non-germinated seeds was also tested. All four isolates produced extensive damping off when inoculated on non-germinated seeds. To our knowledge, this is the first report of damping off of Swiss chard caused by R. solani in Europe.

LiDAR remote sensing applied to forest resources assessment

Disponer de información precisa y actualizada de inventario forestal es una pieza clave para mejorar la gestión forestal sostenible y para proponer y evaluar políticas de conservación de bosques que permitan la reducción de emisiones de carbono debidas a la deforestación y degradación forestal (REDD). En este sentido, la tecnología LiDAR ha demostrado ser una herramienta perfecta para caracterizar y estimar de forma continua y en áreas extensas la estructura del bosque y las principales variables de inventario forestal. Variables como la biomasa, el número de pies, el volumen de madera, la altura dominante, el diámetro o la altura media son estimadas con una calidad comparable a los inventarios tradicionales de campo. La presente tesis se centra en analizar la aplicación de los denominados métodos de masa de inventario forestal con datos LIDAR bajo diferentes condiciones y características de masa forestal (bosque templados puros y mixtos) y utilizando diferentes bases de datos LiDAR (información proveniente de vuelo nacionales e información capturada de forma específica). Como consecuencia de lo anterior, se profundiza en la generación de inventarios forestales continuos con LiDAR en grandes áreas. Los métodos de masa se basan en la búsqueda de relaciones estadísticas entre variables predictoras derivadas de la nube de puntos LiDAR y las variables de inventario forestal medidas en campo con el objeto de generar una cartografía continua de inventario forestal. El rápido desarrollo de esta tecnología en los últimos años ha llevado a muchos países a implantar programas nacionales de captura de información LiDAR aerotransportada. Estos vuelos nacionales no están pensados ni diseñados para fines forestales por lo que es necesaria la evaluación de la validez de esta información LiDAR para la descripción de la estructura del bosque y la medición de variables forestales. Esta información podría suponer una drástica reducción de costes en la generación de información continua de alta resolución de inventario forestal. En el capítulo 2 se evalúa la estimación de variables forestales a partir de la información LiDAR capturada en el marco del Plan Nacional de Ortofotografía Aérea (PNOA-LiDAR) en España. Para ello se compara un vuelo específico diseñado para inventario forestal con la información de la misma zona capturada dentro del PNOA-LiDAR. El caso de estudio muestra cómo el ángulo de escaneo, la pendiente y orientación del terreno afectan de forma estadísticamente significativa, aunque con pequeñas diferencias, a la estimación de biomasa y variables de estructura forestal derivadas del LiDAR. La cobertura de copas resultó más afectada por estos factores que los percentiles de alturas. Considerando toda la zona de estudio, la estimación de la biomasa con ambas bases de datos no presentó diferencias estadísticamente significativas. Las simulaciones realizadas muestran que las diferencias medias en la estimación de biomasa entre un vuelo específico y el vuelo nacional podrán superar el 4% en áreas abruptas, con ángulos de escaneo altos y cuando la pendiente de la ladera no esté orientada hacia la línea de escaneo. En el capítulo 3 se desarrolla un estudio en masas mixtas y puras de pino silvestre y haya, con un enfoque multi-fuente empleando toda la información disponible (vuelos LiDAR nacionales de baja densidad de puntos, imágenes satelitales Landsat y parcelas permanentes del inventario forestal nacional español). Se concluye que este enfoque multi-fuente es adecuado para realizar inventarios forestales continuos de alta resolución en grandes superficies. Los errores obtenidos en la fase de ajuste y de validación de los modelos de área basimétrica y volumen son similares a los registrados por otros autores (usando un vuelo específico y parcelas de campo específicas). Se observan errores mayores en la variable número de pies que los encontrados en la literatura, que pueden ser explicados por la influencia de la metodología de parcelas de radio variable en esta variable. En los capítulos 4 y 5 se evalúan los métodos de masa para estimar biomasa y densidad de carbono en bosques tropicales. Para ello se trabaja con datos del Parque Nacional Volcán Poás (Costa Rica) en dos situaciones diferentes: i) se dispone de una cobertura completa LiDAR del área de estudio (capitulo 4) y ii) la cobertura LiDAR completa no es técnica o económicamente posible y se combina una cobertura incompleta de LiDAR con imágenes Landsat e información auxiliar para la estimación de biomasa y carbono (capitulo 5). En el capítulo 4 se valida un modelo LiDAR general de estimación de biomasa aérea en bosques tropicales y se compara con los resultados obtenidos con un modelo ajustado de forma específica para el área de estudio. Ambos modelos están basados en la variable altura media de copas (TCH por sus siglas en inglés) derivada del modelo digital LiDAR de altura de la vegetación. Los resultados en el área de estudio muestran que el modelo general es una alternativa fiable al ajuste de modelos específicos y que la biomasa aérea puede ser estimada en una nueva zona midiendo en campo únicamente la variable área basimétrica (BA). Para mejorar la aplicación de esta metodología es necesario definir en futuros trabajos procedimientos adecuados de medición de la variable área basimétrica en campo (localización, tamaño y forma de las parcelas de campo). La relación entre la altura media de copas del LiDAR y el área basimétrica (Coeficiente de Stock) obtenida en el área de estudio varía localmente. Por tanto es necesario contar con más información de campo para caracterizar la variabilidad del Coeficiente de Stock entre zonas de vida y si estrategias como la estratificación pueden reducir los errores en la estimación de biomasa y carbono en bosques tropicales. En el capítulo 5 se concluye que la combinación de una muestra sistemática de información LiDAR con una cobertura completa de imagen satelital de moderada resolución (e información auxiliar) es una alternativa efectiva para la realización de inventarios continuos en bosques tropicales. Esta metodología permite estimar altura de la vegetación, biomasa y carbono en grandes zonas donde la captura de una cobertura completa de LiDAR y la realización de un gran volumen de trabajo de campo es económica o/y técnicamente inviable. Las alternativas examinadas para la predicción de biomasa a partir de imágenes Landsat muestran una ligera disminución del coeficiente de determinación y un pequeño aumento del RMSE cuando la cobertura de LiDAR es reducida de forma considerable. Los resultados indican que la altura de la vegetación, la biomasa y la densidad de carbono pueden ser estimadas en bosques tropicales de forma adecuada usando coberturas de LIDAR bajas (entre el 5% y el 20% del área de estudio). ABSTRACT The availability of accurate and updated forest data is essential for improving sustainable forest management, promoting forest conservation policies and reducing carbon emissions from deforestation and forest degradation (REDD). In this sense, LiDAR technology proves to be a clear-cut tool for characterizing forest structure in large areas and assessing main forest-stand variables. Forest variables such as biomass, stem volume, basal area, mean diameter, mean height, dominant height, and stem number can be thus predicted with better or comparable quality than with costly traditional field inventories. In this thesis, it is analysed the potential of LiDAR technology for the estimation of plot-level forest variables under a range of conditions (conifer & broadleaf temperate forests and tropical forests) and different LiDAR capture characteristics (nationwide LiDAR information vs. specific forest LiDAR data). This study evaluates the application of LiDAR-based plot-level methods in large areas. These methods are based on statistical relationships between predictor variables (derived from airborne data) and field-measured variables to generate wall to wall forest inventories. The fast development of this technology in recent years has led to an increasing availability of national LiDAR datasets, usually developed for multiple purposes throughout an expanding number of countries and regions. The evaluation of the validity of nationwide LiDAR databases (not designed specifically for forest purposes) is needed and presents a great opportunity for substantially reducing the costs of forest inventories. In chapter 2, the suitability of Spanish nationwide LiDAR flight (PNOA) to estimate forest variables is analyzed and compared to a specifically forest designed LiDAR flight. This study case shows that scan angle, terrain slope and aspect significantly affect the assessment of most of the LiDAR-derived forest variables and biomass estimation. Especially, the estimation of canopy cover is more affected than height percentiles. Considering the entire study area, biomass estimations from both databases do not show significant differences. Simulations show that differences in biomass could be larger (more than 4%) only in particular situations, such as steep areas when the slopes are non-oriented towards the scan lines and the scan angles are larger than 15º. In chapter 3, a multi-source approach is developed, integrating available databases such as nationwide LiDAR flights, Landsat imagery and permanent field plots from SNFI, with good resultos in the generation of wall to wall forest inventories. Volume and basal area errors are similar to those obtained by other authors (using specific LiDAR flights and field plots) for the same species. Errors in the estimation of stem number are larger than literature values as a consequence of the great influence that variable-radius plots, as used in SNFI, have on this variable. In chapters 4 and 5 wall to wall plot-level methodologies to estimate aboveground biomass and carbon density in tropical forest are evaluated. The study area is located in the Poas Volcano National Park (Costa Rica) and two different situations are analyzed: i) available complete LiDAR coverage (chapter 4) and ii) a complete LiDAR coverage is not available and wall to wall estimation is carried out combining LiDAR, Landsat and ancillary data (chapter 5). In chapter 4, a general aboveground biomass plot-level LiDAR model for tropical forest (Asner & Mascaro, 2014) is validated and a specific model for the study area is fitted. Both LiDAR plot-level models are based on the top-of-canopy height (TCH) variable that is derived from the LiDAR digital canopy model. Results show that the pantropical plot-level LiDAR methodology is a reliable alternative to the development of specific models for tropical forests and thus, aboveground biomass in a new study area could be estimated by only measuring basal area (BA). Applying this methodology, the definition of precise BA field measurement procedures (e.g. location, size and shape of the field plots) is decisive to achieve reliable results in future studies. The relation between BA and TCH (Stocking Coefficient) obtained in our study area in Costa Rica varied locally. Therefore, more field work is needed for assessing Stocking Coefficient variations between different life zones and the influence of the stratification of the study areas in tropical forests on the reduction of uncertainty. In chapter 5, the combination of systematic LiDAR information sampling and full coverage Landsat imagery (and ancillary data) prove to be an effective alternative for forest inventories in tropical areas. This methodology allows estimating wall to wall vegetation height, biomass and carbon density in large areas where full LiDAR coverage and traditional field work are technically and/or economically unfeasible. Carbon density prediction using Landsat imaginery shows a slight decrease in the determination coefficient and an increase in RMSE when harshly decreasing LiDAR coverage area. Results indicate that feasible estimates of vegetation height, biomass and carbon density can be accomplished using low LiDAR coverage areas (between 5% and 20% of the total area) in tropical locations.

Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also investigated. Analysis of initial spatial IAA distribution in the cuttings revealed that approximately 40 and 10% of the total IAA pool was present in the leaves and the stem base as rooting zone, respectively. A negative correlation existed between leaf size and IAA concentration. After excision of cuttings, IAA showed an early increase in the stem base with two peaks at 2 and 24h post excision and, thereafter, a decline to low levels. This was mirrored by the expression pattern of the auxin-responsive GH3 gene. NPA treatment completely suppressed the 24-h peak of IAA and severely inhibited root formation. It also reduced activities of cell wall and vacuolar invertases in the early phase of AR formation and inhibited the rise of activities of glucose-6-phosphate dehydrogenase and phosphofructokinase during later stages. We propose a model in which spontaneous AR formation in Petunia cuttings is dependent on PAT and on the resulting 24-h peak of IAA in the rooting zone, where it induces early cellular events and also stimulates sink establishment. Subsequent root development stimulates glycolysis and the pentosephosphate pathway