Estructura histológica de la piel de tomate (Lycopersicon esculentum) en relación con su resistencia mecánica.

La resistencia de la piel es un factor determinante de la resistencia mecánica de los frutos de tomate. Los ensayos de punción se han mostrado muy útiles para el conocimiento de las diferencias entre las variedades en ese aspecto. El estudio histológico de las zonas epidérmica e hipodérmica de este mismo material muestra la importancia de la cutícula y de la forma de las células epidérmicas en relación a la resistencia a la punción.

Fungal microbiota from rain water and pathogenicity of Fusarium species isolated from atmospheric dust and rainfall dust

In order to determine the presence of Fusarium spp. in atmospheric dust and rainfall dust, samples were collected during September 2007, and July, August, and October 2008. The results reveal the prevalence of airborne Fusarium species coming from the atmosphere of the South East coast of Spain. Five different Fusarium species were isolated from the settling dust: Fusarium oxysporum, F. solani, F. equiseti, F. dimerum, and F. proliferatum. Moreover, rainwater samples were obtained during significant rainfall events in January and February 2009. Using the dilution-plate method, 12 fungal genera were identified from these rainwater samples. Specific analyses of the rainwater revealed the presence of three species of Fusarium: F. oxysporum, F. proliferatum and F. equiseti. A total of 57 isolates of Fusarium spp. obtained from both rainwater and atmospheric rainfall dust sampling were inoculated onto melon (Cucumis melo L.) cv. Piñonet and tomato (Lycopersicon esculentum Mill.) cv. San Pedro. These species were chosen because they are the main herbaceous crops in Almeria province. The results presented in this work indicate strongly that spores or propagules of Fusarium are able to cross the continental barrier carried by winds from the Sahara (Africa) to crop or coastal lands in Europe. Results show differences in the pathogenicity of the isolates tested. Both hosts showed root rot when inoculated with different species of Fusarium, although fresh weight measurements did not bring any information about the pathogenicity. The findings presented above are strong indications that long-distance transmission of Fusarium propagules may occur. Diseases caused by species of Fusarium are common in these areas. They were in the past, and are still today, a problem for greenhouses crops in Almería, and many species have been listed as pathogens on agricultural crops in this region. Saharan air masses dominate the Mediterranean regions. The evidence of long distance dispersal of Fusarium spp. by atmospheric dust and rainwater together with their proved pathogenicity must be taken into account in epidemiological studies.

The use of wind pumps for greenhouse microirrigation: a case study for tomato in Cuba.

Crop irrigation is a major consumer of energy. Only a few countries are self-sufficient in conventional non-renewable energy sources. Fortunately, there are renewable ones, such as wind, which has experienced recent developments in the area of power generation. Wind pumps can play a vital role in irrigation projects in remote farms. A methodology based on daily estimation balance between water needs and water availability was used to evaluate the feasibility of the most economic windmill irrigation system. For this purpose, several factors were included: three-hourly wind velocity (W3 h, m/s), flow supplied by the wind pump as a function of the elevation height (H, m) and daily greenhouse evapotranspiration as a function of crop planting date. Monthly volumes of water required for irrigation (Dr, m3/ha) and in the water tank (Vd, m3), as well as the monthly irrigable area (Ar, ha), were estimated by cumulative deficit water budgeting taking in account these factors. An example is given illustrating the use of this methodology on tomato crop (Lycopersicon esculentum Mill.) under greenhouse at Ciego de Ávila, Cuba. In this case two different W3 h series (average and low wind year), three different H values and five tomato crop planting dates were considered. The results show that the optimum period of wind-pump driven irrigation is with crop plating in November, recommending a 5 m3 volume tank for cultivated areas around 0.2 ha when using wind pumps operating at 15 m of height elevation.

Wind pumps for irrigating greenhouse crops: comparison in different socio-economical frameworks

Wind power can play an interesting role in irrigation projects in different areas. A methodology can determine the feasibility of the technology and the levels of daily irrigation demand satisfied by windmills at different levels of risk, using tomato (Lycopersicon esculentum Mill) as greenhouse crop. The present work compared the feasibility of the technology and the critical factors involved in three different countries: Cuba, Spain and Pakistan. The study considered as factors the wind speed level, the energy cost, the tomato prices, the reliability and distance to the electrical grid, and the crop development dates, determining the economic feasibility for each combination of factors in each country.

Wind pumps for irrigating greenhouse crops

Agriculture is a major consumer of energy in many countries of the world. Only a few of these countries are self-sufficient in conventional energy sources, which are also exhaustible. Fortunately, there are other sources of energy, such as wind, which has experienced recent developments in the area of wind power generation. From irrigation projects to power supply in remote farms, wind power generation can play a vital role. A simple methodology for technical evaluation of windmills for irrigation water pumping has been developed in this study to determine the feasibility per unit amount of water supplied and the levels of daily irrigation demand satisfied by windmill irrigation system at various levels of risk (probability of failure). For this purpose, a series of three hourly wind-speed data over a period of 38 years at Ciego de Ávila, Cuba, were analyzed to compute the diurnal wind pump discharge at varying levels of risk. The sizes of reservoirs required to modulate fluctuating discharge and to satisfy the levels of irrigation demand, on function of crop development dates, cultivated area and water elevation height, were computed by cumulative deficit water budgeting. An example is given illustrating the use of the methodology on tomato crop Licopersicon esculentum Mill) under greenhouse.

Caracterización de la madera de Castanea sativa Mill. de procedencia española

La madera de castaño de procedencia española no está presente como material estructural en la norma europea EN 1912, y por lo tanto no está asignada a ninguna clase resistente como sí lo están otras especies españolas. Por ello, en el presente trabajo se realizó una caracterización de madera aserrada de castaño de Asturias con fines estructurales, realizando los ensayos según la norma UNE EN 408:2004, y calculando sus valores característicos según la norma UNE EN 384:2010. Los valores obtenidos permitieron asignar una clase resistente de las establecidas en la norma UNE EN 338:2010. Fueron evaluadas un total de 260 probetas de dos secciones (40x100 y 40x150 mm) siendo asignada una clase resistente D24, resultando la densidad y la resistencia a flexión los parámetros limitantes y observándose un valor de módulo de elasticidad superior al correspondiente a dicha clase. La relación entre los módulo de elasticidad longitudinal y transversal obtenidos experimentalmente fue de 10 aproximadamente, mientras que la normativa establece, como valor genérico, 16. Se observó que la singularidad de la madera que provocó un mayor porcentaje de rotura fue la presencia de nudos, presentando estas probetas un valor de resistencia significativamente menor. Chestnut timber from Spain is not included as a structural timber in the European standard EN 1912 nor is it assigned to any strength class like other Spanish species. Therefore, a characterization of structural chestnut timber from Asturias was made according to the UNE EN 408:2004, and the characteristic values were calculated according to the UNE EN 384:2010. The values obtained allowed the assignment of a strength class according to UNE EN 338:2010. 260 samples of two sections (40x100 and 40x150 mm) were tested and a D24 strength class was assinged. Density and bending strength were the limiting parameters, and the value of modulus of elasticity was higher than the values asssigned to D24. The relationship between modulus of elasticity and the shear modulus obtained was approximately equal to 10, while this value in the UNE EN 384:2010 is set at 16. The characteristic of the wood which caused a higher percentage of failure was the presence of knots, resulting in significantly lower resistance values.

An empirical mixed model to quantify climate influence on the growth of Pinus halepensis Mill. stands in South-Eastern Spain

The influence of climate on forest stand composition, development and growth is undeniable. Many studies have tried to quantify the effect of climatic variables on forest growth and yield. These works become especially important because there is a need to predict the effects of climate change on the development of forest ecosystems. One of the ways of facing this problem is the inclusion of climatic variables into the classic empirical growth models. The work has a double objective: (i) to identify the indicators which best describe the effect of climate on Pinus halepensis growth and (ii) to quantify such effect in several scenarios of rainfall decrease which are likely to occur in the Mediterranean area. A growth mixed model for P. halepensis including climatic variables is presented in this work. Growth estimates are based on data from the Spanish National Forest Inventory (SNFI). The best results are obtained for the indices including rainfall, or rainfall and temperature together, with annual precipitation, precipitation effectiveness, Emberger?s index or free bioclimatic intensity standing out among them. The final model includes Emberger?s index, free bioclimatic intensity and interactions between competition and climate indices. The results obtained show that a rainfall decrease about 5% leads to a decrease in volume growth of 5.5?7.5% depending on site quality.

A new method for the identification of old-growth trees in National Forest Inventories: Application to Pinus halepensis Mill. stands in Spain

Old-growth trees play a very important role in the maintenance of biodiversity in forests. However, no clear definition is yet available to help identify them since tree age is usually not recorded in National Forest Inventories. To develop and test a new method to identify old-growth trees using a species-specific threshold for tree diameter in National Forest Inventories. Different nonlinear mixed models for diameter ? age were generated using data from the Spanish Forest Inventory in order to identify the most appropriate one for Aleppo pine in its South-western distribution area. The asymptote of the optimal model indicates the threshold diameter for defining an old-growth tree. Additionally, five site index curves were examined to analyze the influence of site quality on these models.

A fuzzy MCDA framework for safety assessment in the remediation of a uranium mill tailings site in Ukraine

The Pridneprovsky Chemical Plant was one of the largest uranium processing enterprises in the former USSR, producing a huge amount of uranium residues. The Zapadnoe tailings site contains most of these residues. We propose a theoretical framework based on multicriteria decision analysis and fuzzy logic to analyze different remediation alternatives for the Zapadnoe tailings, which simultaneously accounts for potentially conflicting economic, social and environmental objectives. We build an objective hierarchy that includes all the relevant aspects. Fuzzy rather than precise values are proposed for use to evaluate remediation alternatives against the different criteria and to quantify preferences, such as the weights representing the relative importance of criteria identified in the objective hierarchy. Finally, we suggest that remediation alternatives should be evaluated by means of a fuzzy additive multi-attribute utility function and ranked on the basis of the respective trapezoidal fuzzy number representing their overall utility.

Variation throughout the tree stem in the physical-mechanical properties of the wood of Abies alba Mill. from the Spanish Pyrenees

This study analyses the variation of main physical-mechanical properties of wood along the longitudinal and radial directions of the tree for Abies alba Mill. growing in the Spanish Pyrenees. Small clear specimens were used to study the properties of volumetric shrinkage (VS), density (?), hardness (H), bending strength (MOR), modulus of elasticity (MOE), maximum compressive strength parallel to the grain (MCS) and impact strength (K). Several models of properties variation in the longitudinal and radial directions were analyzed. Main trends of variation of properties throughout the tree stem were identified although none of them could be fitted to predictive statistical models. Along the longitudinal direction, the properties studied followed a downward trend from the base to the crown, which was not significant in all cases, indicating that no differences in quality existed. Throughout the radial direction the trend is upward for the first 40-50 growth rings, after which it slopes downwards, more gently at first until rings 70-75 and then more steeply. This behaviour is related to variation in wood structure from the pith to the bark, depending on whether the wood is juvenile, sapwood or heartwood, and to wood maturity and microfibril angle. Authors encourage carrying further studies on other populations of A. alba in the Spanish Pyrenees to check if the trends found in this study apply to other provenances.

Distribution of the coal flow in the mill-duct system of the As Pontes Power Plant using CFD modeling

The efficiency of a Power Plant is affected by the distribution of the pulverized coal within the furnace. The coal, which is pulverized in the mills, is transported and distributed by the primary gas through the mill-ducts to the interior of the furnace. This is done with a double function: dry and enter the coal by different levels for optimizing the combustion in the sense that a complete combustion occurs with homogeneous heat fluxes to the walls. The mill-duct systems of a real Power Plant are very complex and they are not yet well understood. In particular, experimental data concerning the mass flows of coal to the different levels are very difficult to measure. CFD modeling can help to determine them. An Eulerian/Lagrangian approach is used due to the low solid–gas volume ratio.

Mapping Genetic Diversity of Cherimoya (Annona cherimola Mill.): Application of Spatial Analysis for Conservation and Use of Plant Genetic Resources.

There is a growing call for inventories that evaluate geographic patterns in diversity of plant genetic resources maintained on farm and in species' natural populations in order to enhance their use and conservation. Such evaluations are relevant for useful tropical and subtropical tree species, as many of these species are still undomesticated, or in incipient stages of domestication and local populations can offer yet-unknown traits of high value to further domestication. For many outcrossing species, such as most trees, inbreeding depression can be an issue, and genetic diversity is important to sustain local production. Diversity is also crucial for species to adapt to environmental changes. This paper explores the possibilities of incorporating molecular marker data into Geographic Information Systems (GIS) to allow visualization and better understanding of spatial patterns of genetic diversity as a key input to optimize conservation and use of plant genetic resources, based on a case study of cherimoya (Annona cherimola Mill.), a Neotropical fruit tree species. We present spatial analyses to (1) improve the understanding of spatial distribution of genetic diversity of cherimoya natural stands and cultivated trees in Ecuador, Bolivia and Peru based on microsatellite molecular markers (SSRs); and (2) formulate optimal conservation strategies by revealing priority areas for in situ conservation, and identifying existing diversity gaps in ex situ collections. We found high levels of allelic richness, locally common alleles and expected heterozygosity in cherimoya's putative centre of origin, southern Ecuador and northern Peru, whereas levels of diversity in southern Peru and especially in Bolivia were significantly lower. The application of GIS on a large microsatellite dataset allows a more detailed prioritization of areas for in situ conservation and targeted collection across the Andean distribution range of cherimoya than previous studies could do, i.e. at province and department level in Ecuador and Peru, respectively.

Genetic characterization of chestnut (Castanea sativa Mill.) orchards and traditional nut varieties in El Bierzo, a glacial refuge and major cultivation site in Northwestern Spain

This paper presents a detailed genetic study of Castanea sativa in El Bierzo, a major nut production region with interesting features. It is located within a glacial refuge at one extreme of the distribution area (northwest Spain); it has a centenary tradition of chestnut management; and more importantly, it shows an unusual degree of genetic isolation. Seven nuclear microsatellite markers were selected to analyze the genetic variability and structure of 169 local trees grafted for nut production. We analyzed in the same manner 62 local nuts. The selected loci were highly discriminant for the genotypes studied, giving a combined probability of identity of 6.1 × 10−6. An unprecedented density of trees was sampled for this project over the entire region, and nuts were collected representing 18 cultivars marketed by local producers. Several instances of misclassification by local growers were detected. Fixation index estimates and analysis of molecular variance (AMOVA) data are supportive of an unexpectedly high level of genetic differentiation in El Bierzo, larger than that estimated in a previous study with broader geographical scope but based on limited local sampling (Pereira-Lorenzo et al., Tree Genet Genomes 6: 701–715, 2010a). Likewise, we have determined that clonality due to grafting had been previously overestimated. In line with these observations, no significant spatial structure was found using both a model-based Bayesian procedure and Mantel’s tests. Taken together, our results evidence the need for more fine-scale genetic studies if conservation strategies are to be efficiently improved.

Caracterización y selección de cultivares tradicionales de castaño (Castanea Sativa Mill.) en Galicia

Se han prospectado 41 de los 65 ayuntamientos de los que se disponía información sobre la existencia de cultivares de castaño en la comunidad gallega (FERNANDEZ y PEREIRA, 1981) . Se marcaron 350 clones correspondientes a 82 denominaciones varietales de castaño (Castanea sativa) de un total de 143 conocidas. Se introdujeron en Colección 195 clones (de los 350 marcados) correspondientes a un total de 75 denominaciones varietales. El rango de altitudes de selección fue desde los 140 a los 1.060 m sobre el nivel del mar. El 60,1 % de los árboles se han encontrado entre los 500 y 800 m.

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.