Procyanidins are potent inhibitors of LOX-1: a new player in the French Paradox

Lectin-like oxidized LDL receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL (oxLDL) and plays multiple roles in the development of cardiovascular diseases. We screened more than 400 foodstuff extracts for identifying materials that inhibit oxLDL binding to LOX-1. Results showed that 52 extracts inhibited LOX-1 by more than 70% in cell-free assays. Subsequent cell-based assays revealed that a variety of foodstuffs known to be rich in procyanidins such as grape seed extracts and apple polyphenols, potently inhibited oxLDL uptake in Chinese hamster ovary (CHO) cells expressing LOX-1. Indeed, purified procyanidins significantly inhibited oxLDL binding to LOX-1 while other ingredients of apple polyphenols did not. Moreover, chronic administration of oligomeric procyanidins suppressed lipid accumulation in vascular wall in hypertensive rats fed with high fat diet. These results suggest that procyanidins are LOX-1 inhibitors and LOX-1 inhibition might be a possible underlying mechanism of the well-known vascular protective effects of red wine, the French Paradox

Vasorelaxant and Antioxidant Activities of Spilanthes acmella Murr.

This study reports the effect of Spilanthes acmella Murr. extracts on phenylephrine-induced contraction of rat thoracic aorta as well as their antioxidant activity. Results show that the extracts exert maximal vasorelaxations in a dose-dependent manner, but their effects are less than acetylcholine-induced nitric oxide (NO) vasorelaxation. Significant reduction of vasorelaxations is observed in both NG-nitro-l-arginine methyl ester (l-NAME) and indomethacin (INDO). In the presence of l-NAME plus INDO, synergistic effects are observed, leading to loss of vasorelaxation of both acetylcholine and the extracts. Similarly, the vasorelaxations of the extracts are completely abolished upon the removal of endothelial cells. This demonstrates that the extracts exhibit vasorelaxation via partially endothelium-induced NO and prostacyclin in a dose-dependent manner. Significantly, the ethyl acetate extract exerts immediate vasorelaxation (ED50 76.1 ng/mL) and is the most potent antioxidant (DPPH assay). The chloroform extract shows the highest vasorelaxation and antioxidation (SOD assay). These reveal a potential source of vasodilators and antioxidants

The Influence of the Degree of Forest Management on Methylmercury and the Composition of Microbial Communities in the Sediments of Boreal Drainage Ditches

Funding: This work was supported by the Latvian Council of Science project no. lzp-2018/1-0434 “Interaction of microbial diversity with methane turnover and mercury methylation in organic soils”.Peer reviewedPublisher PD

The Influence of the Degree of Forest Management on Methylmercury and the Composition of Microbial Communities in the Sediments of Boreal Drainage Ditches

Inorganic mercury (Hg) can be methylated to the highly toxic and bioavailable methylmercury (MeHg) by microorganisms in anaerobic environments. The Hg methylation rate may be affected by forest management activities, which can influence the catchment soils, water, and sediments. Here, we investigate the influence of forest management in the form of ditch cleaning and beaver dam removal, as well as the seasonal variations, on sediment chemistry and microbiota. The relationships between MeHg concentrations in sediment samples and archaeal and bacterial communities assessed by 16S rRNA gene amplicon sequencing were investigated to determine the microbial conditions that facilitated the formation of MeHg. Concentrations of MeHg were highest in undisturbed catchments compared to disturbed or slightly disturbed sites. The undisturbed sites also had the highest microbial diversity, which may have facilitated the formation of MeHg. Low MeHg concentrations and microbial diversity were observed in disturbed sites, which may be due to the removal of organic sediment layers during ditch cleaning and beaver dam removal, resulting in more homogenous, mineral-rich environments with less microbial activity. MeHg concentrations were higher in summer and autumn compared to winter and spring, but the temporal variation in the composition and diversity of the microbial community was less than the spatial variation between sites. Beta diversity was more affected by the environment than alpha diversity. The MeHg concentrations in the sediment were positively correlated to several taxa, including Cyanobacteria, Proteobacteria, Desulfobacterota, Chloroflexi, and Bacteroidota, which could represent either Hg-methylating microbes or the growth substrates of Hg-methylating microbes

The Role of Microalgae in the Biogeochemical Cycling of Methylmercury (MeHg) in Aquatic Environments

Methylmercury (MeHg) is the most important and the most abundant organic Hg pollutant in the aquatic ecosystem that can affect human health through biomagnification. It is the most toxic organic Hg form, which occurs naturally and by human-induced contamination in water and is further biomagnified in the aquatic food web. MeHg is the only Hg form that accumulates in living organisms and is able to cross the blood–brain barrier, presenting an enormous health risk. Anthropogenic activity increases eutrophication of coastal waters worldwide, which promotes algae blooms. Microalgae, as primary producers, are especially sensitive to MeHg exposure in water and are an important entrance point for MeHg into the aquatic food web. MeHg assimilated by microalgae is further transferred to fish, wildlife and, eventually, humans as final consumers. MeHg biomagnifies and bioaccumulates in living organisms and has serious negative health effects on humans, especially newborns and children. Knowledge of the microalgae–MeHg interaction at the bottom of the food web provides key insights into the control and prevention of MeHg exposure in humans and wildlife. This review aims to summarize recent findings in the literature on the microalgae–MeHg interaction, which can be used to predict MeHg transfer and toxicity in the aquatic food webThis research was funded by the Spanish Ministry of Economic Transformation, Industry, Knowledge and Universities; by the European Regional Development Fund (FEDER) within the framework of the FEDER program of Andalusia (Spain) 2014–2020, grant number UHU–202065; and by Grant P20-00930 from the Andalusian Plan for Research, Development and Innovation, within the frame of the operational program “FEDER Andalucía 2014–2020” The authors wish to thank Erik Björn from Department of Chemistry, Umeå University, Sweden, for his constructive comments on the paper’s content. We wish to thank personnel from LICAH (Laboratorio de Investigación y Control Agroalimentario), University of Huelva, for their collaboration and cooperation under FEDER 2014–2020 UHU–202065 project. We also want to thank colleagues from BITAL (Algae Biotechnology Group), University of Huelva, for their kind assistance in the lab and for creating a productive working environmen

Comparison of Mercury Dynamics in High Arctic Lakes

Arctic lakes and their watersheds are being simultaneously subjected to the deposition of atmospheric pollutants such as mercury (Hg), and warming. Once Hg enters an ecosystem, it may become methylated, greatly increasing its toxicity and reducing organisms’ ability to eliminate it. Mercury is bioaccumulative and thus found at high concentrations in land-locked Arctic char (Salvelinus alpinus) and other top predators. In sediment, Hg methylation rate is temperature-dependent, and [Hg] in Arctic predatory fish has been correlated with trends in air temperature. Despite reductions in Hg emissions in North America and Europe, [Hg] continues to rise in some Arctic species. The purpose of this study was to better understand how climate change may influence Hg flow through Arctic lake food webs. The effect of temperature differences on Hg methylation and dynamics were examined in laboratory-based temperature manipulation experiments and by studying natural variation in temperature between shallow and deep lakes. Additionally, time-series of [Hg] in Arctic char were characterized and relationships between these time-series and climate trends were examined. The sediments of the shallow, warmer lakes demonstrate higher Hg methylation potentials than those of the cooler, deeper lakes, but differences between lakes were small, possibly due to the ultraoligotrophic nature of the sediments. Additionally, the midge larvae (Diptera: Chironomidae; which represent the bulk of the invertebrate biomass and the bulk of Arctic char diet) and Arctic Char) of the two shallow lakes exhibited lower methyl-Hg (MeHg) bioaccumulation factors than larvae and Arctic Char of the two deep lakes . The results of the analysis of time-series of [Hg] trends in Arctic char indicate that differences between the shallow and deep lakes Arctic char populations were sustained over time. Considered together, these findings indicate that while Hg methylation and MeHg demethylation influence concentrations of MeHg in sediment, differences in the MeHg bioaccumulation appear to account for differences in [Hg] between chironomid and Arctic char in the lakes, with deeper lakes exhibiting higher bioaccumulation factors. The higher bioaccumulation ratios observed in colder lakes is likely the result of less primary production on surface sediments leading to less secondary production (chironomid biomass) and therefore less food and greater retention of MeHg by both chironomids and Arctic char. The analysis of the time-series of Arctic char [Hg] in relation to climate variables failed to reveal any single climate variable that significantly influenced all six populations. However, for three of the populations, trends were positively related to sea ice duration. and in the lake whose Arctic char had the highest [Hg], there was a positive correlation with snowfall. This is consistent with prior research demonstrating the importance of snow pack and snow melt to influx of Hg to polar desert lakes. Three of the six Arctic char populations exhibited significant declines in [Hg] over time, consistent with reduced bioaccumulation factors associated with lake warming, but in one of the lakes, interpretation of this trend is confounded by recovery from historical waste-water inputs. Taken together, the results indicate that a warming Arctic should result slightly increased accumulation of MeHg in sediments, but, paradoxically, less Hg in biota because temperature-dependent faster growth rates result in biomass increases that exceed the increased rates of Hg methylation in sediments. However, the complex biogeochemistry of Hg prevents any further interpretation or accurate prediction of future effects

Os efeitos morfológicos, fisiológicos e bioquímicos causados pelo alagamento de plantas jovens de Guazuma ulmifolia Lam. oriundas da Amazônia e do Cerrado

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2018.O alagamento é um importante fator estressor que prejudica o desenvolvimento e a sobrevivência de muitas espécies de plantas. Entretanto, algumas espécies conseguem tolerar longos períodos de exposição a esse estressor, mostrando-se interessantes modelos de estudo. O Painel Intergovernamental de Mudança Climática (IPCC) alertou que as mudanças climáticas intensificarão a frequência de fortes precipitações e a atividade de ciclones tropicais. Isso propiciará um aumento da frequência de eventos de inundação, o que fará com que as plantas sejam mais comumente expostas ao estresse de alagamento. Nota-se, deste modo, a urgência no estudo da tolerância a esse estresse abiótico. O alagamento provoca a baixa oferta de oxigênio para as plantas e, para lidar com essa situação, o indivíduo precisa ajustar seu metabolismo, fisiologia e morfologia. Neste trabalho, objetivou-se avaliar respostas morfológicas, fisiológicas e metabólicas de Guazuma ulmifolia exposta ao alagamento parcial por 32 dias, seguida por 17 dias de recuperação pós-estresse. Além disso, objetivou-se comparar essas respostas em plantas provenientes de duas populações: a população amazônica, usualmente exposta a estresse de alagamento, e a população do Cerrado, adaptada ao ambiente seco. Para isso, utilizou-se 160 plantas jovens de G. ulmifolia organizadas em quatro tratamentos; Controle Amazônia (CoA), Alagado Amazônia (AlA), Controle Cerrado (CoC) e Alagado Cerrado (AlC). AlA e AlC foram submetidos a alagamento parcial e recuperação, enquanto CoA e CoC foram mantidos com regas diárias. Os parâmetros morfológicos mensurados foram o incremento da parte aérea, fração de massa radicular, número de folhas, diâmetro do caule e o aparecimento de lenticelas e raízes adventícias. Os dados fisiológicos avaliados foram fotossíntese máxima (Amax), condutância estomática (Gs), transpiração (E), carbono interno (Ci) e alocação de carbono e nitrogênio. Já os parâmetros metabólicos observados foram a porcentagem de amido e o perfil metabólico das raízes e folhas. Observou-se que as plantas do grupo AlA apresentaram maior Amax em relação a CoA do 7º até o 35º dia de experimento e as do grupo AlC exibiram maior Amax do 14º dia até o 35º em relação a CoC. Até o 7º dia de alagamento, os indivíduos AlA apresentaram maior Amax que os AlC. Indivíduos do grupo AlA apresentaram maior Gs que CoA do 7º ao 21º dia de alagamento. Já as plantas AlC mostraram menor Gs às 24h e 48h de alagamento em comparação com CoC, mas apresentaram maior Gs no 21º dia. Portanto, sob alagamento parcial, G. ulmifolia parece fazer mais fotossíntese para manter o metabolismo anaeróbico radicular. A avaliação do metabolismo mostra aumento de alanina, GABA, succinato e citrato em plantas alagadas. 8 No 7º dia de alagamento AlC apresentou aumento em glicina, serina, treonina, cisteína e prolina, indicando ajuste metabólico frente ao estresse. O perfil metabólico foliar apresentou muito menos diferenças que o perfil radicular, sugerindo que as alterações metabólicas ficam restritas à raiz, que é diretamente afetada pelo alagamento parcial. A partir dos dados fisiológicos e bioquímicos, sugere-se que as respostas ao alagamento desenvolvidas pelas plantas amazônicas são iniciadas precocemente e com maior intensidade em relação às do Cerrado, o que indica uma capacidade de aclimatação ao alagamento mais eficiente da população amazônica, ainda que as plantas de ambas as populações tenham mostrado alto grau de tolerância a esse estresse.Fundação de Apoio à Pesquisa do Distrito Federal (FAP-DF) e Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Flooding is an important stressor that impairs the development and survival of many plant species. However, some species can tolerate long periods of exposure to this stress and hence represent interesting models for study. The Intergovernmental Panel on Climate Change (IPCC) has warned that climate change will intensify the frequency of heavy rainfall and tropical cyclone activity. This will increase the frequency of flooding events and also the exposure of plants to flooding stress. Flooding causes a reduced supply of oxygen to plants, forcing them to adjust their metabolism, physiology and morphology in order to cope with this situation. The aim of this study was to evaluate the morphological, physiological and metabolic responses of Guazuma ulmifolia exposed to waterlogging for 32 days, followed by 17 days of recovery from stress. In addition, it we aimed to compare these responses in plants from two populations: the Amazon population, frequently exposed to flooding stress, and the Cerrado population, adapted to the dry environment. For this purpose, 160 young plants of G. ulmifolia were organized in four groups: Amazonian Control (CoA), Amazonian Flooded (AlA), Cerrado Control (CoC) and Cerrado Flooded (AlC). AlA and AlC were submitted to partial flooding and recovery, while CoA and CoC were maintained with daily watering. The morphological parameters assessed were shoot relative growth, root percentage, leaf number, stem diameter and emrgence of lenticels and adventitious roots. The physiological data evaluated were maximum photosynthesis (Amax), stomatal conductance (Gs), transpiration (E), internal carbon (Ci) and carbon and nitrogen allocation. The metabolic parameters observed were the leaf and root starch percentage and their metabolic profile. We observed that the plants of the AlA group presented higher Amax in relation to CoA from the 7th to the 35th day of experiment. Those of the AlC group showed higher Amax from the 14th day to the 35th in relation to CoC. Up until the 7th day of flooding, the AlA individuals presented a higher Amax than the AlC. Plants from the AlA group presented higher Gs than CoA from the 7th to the 21st day of flooding. AlC plants showed lower Gs at 24h and 48h of flooding compared to CoC, but showed higher Gs at day 21. Therefore, under partial flooding, G. ulmifolia seems to carry out more photosynthesis in order to maintain root anaerobic metabolism. The root metabolic profiling showed an increase of alanine, GABA, succinate and citrate in flooded plants. On the 7th day of waterlogging, AlC showed an increase in glycine, serine, threonine, cysteine and proline, indicating metabolic adjustment against stress. The leaf metabolic profile presented fewer alterations than the root profile, suggesting that the metabolic changes are restricted to the root, the part directly affected by waterlogging. Based on the physiological and biochemical data, it is suggested that the Amazonian plants initiated flooding responses earlier and with greater intensity in relation to the Cerrado plants. This indicates that the Amazonian population presents a more efficient capacity to acclimatize to waterlogging stress, although plants of both populations revealed great capacity to tolerate this stress

Efeito do sistema de produção nas características físico-químicas e no perfil lipídico de três músculos de suínos alentejanos

O presente trabalho tem como objetivo testar o efeito do sistema de produção com dieta rica em ácido oleico nas características zootécnicas, de carcaça, físico-químicas e no perfil lipídico dos músculos Longissimus dorsi (LD), Biceps femoris (BF) e Semimembranosus (SM) de suínos de raça Alentejana. Foram utilizados 15 animais, divididos em dois grupos, o grupo IN (n=9) foi mantido em parques individuais abertos ao exterior com 3 m2 e o grupo OUT (n=6) foi mantido numa área de 3 ha com acesso a pastagem. O grupo OUT obteve melhores performances zootécnicas. O rendimento de carcaça foi igual para ambos os grupos, e o rendimento comercial e proporção de peças nobres foi superior nos OUT. Estes obtiveram menores teores de lípidos intramusculares mas maiores teores de PUFA (ácidos gordos polinsaturados) e melhores valores para os rácios PUFA/SFA (ácido gordos saturados) e n-6/n-3. O SM foi menos afetado pelo sistema de produção; Abstract: Effect of the rearing system in the physical-chemical characteristics and the lipid profile of three muscles from Alentejano pigs The present study aims to test the effect of rearing system with oleic acid rich diet on performance, carcass and physical-chemical characteristics, and lipid profile of the muscles Longissimus dorsi (LD), Biceps femoris (BF) and Semimembranosus (SM) from Alentejano pigs. The study used 15 animals, divided into two groups. The Group IN (n = 9) was kept in individual parks open to the exterior with 3 m2, and the OUT (n = 6) was held in an area of 3 ha with access to pasture. The OUT group obtained the best performances. The carcass yield was the same for both groups, meat yield and the proportion of noble parts was superior in the OUT. These obtained lower levels of intramuscular fat but larger PUFA levels (polyunsaturated fatty acids) and best values for the ratios PUFA/SFA (saturated fatty acids) and n-6/n-3. The SM was the least affected by the rearing syste

Detección presintomática y no destructiva de enfermedades causadas por patógenos de suelo en maíz (Marchitez tardía) y en girasol (Jopo) mediante medidas térmicas y de fluorescencia multicolor

Algunas enfermedades de cultivos originadas por patógenos de suelo se caracterizan por la tardía aparición de síntomas. Este es el caso de la Marchitez tardía del maíz (causada por el hongo Harpophora maydis) y del Jopo del girasol (causado por la planta parásita de raíz Orobanche cumana). Harpophora maydis se refirió por vez primera en la Península Ibérica en 2010 y durante los últimos años se ha convertido en una preocupación importante para los productores de maíz. Por el contrario, los ataques de O. cumana son frecuentes en los cultivos españoles de girasol desde los años 80. Esta parásita es actualmente el principal limitante biótico de la producción de aceite de girasol en el mundo. Tanto H. maydis como O. cumana infectan la planta hospedante durante las primeras semanas después de la siembra, aunque los síntomas no se hacen visibles hasta la floración del cultivo o poco después de que ésta tenga lugar. También ambas enfermedades se controlan mediante la incorporación de genes de resistencia en las variedades cultivadas. El progreso y el éxito de los programas de mejora para resistencia a enfermedades dependen enormemente de un acertado y eficaz diagnóstico de la reacción de la planta hospedante al patógeno. Por otro lado, en la actualidad son frecuentes las técnicas basadas en el uso de sensores lejanos y/o de proximidad utilizadas en agronomía. Como alternativa a la inspección visual y al análisis de ADN destructivo, y debido a su sensibilidad a desórdenes fisiológicos en las plantas asociados al ataque de patógenos, estas técnicas pueden ser eficaces herramientas de detección en fitopatología. En el caso de proximidad, la monitorización indirecta de las plantas se efectúa principalmente mediante termometría, termografía,medidas de fluorescencia y técnicas espectrales. En el Capítulo 1 de esta Tesis Doctoral se desarrolla el estado del arte de la Marchitez tardía del maíz y del Jopo del girasol. También se presenta información científica actualizada sobre las técnicas de sensores lejanos y/o de proximidad utilizadas más comúnmente en fitopatología. Los objetivos se presentan en el Capítulo 2. En primer lugar se estudió la distribución de H. maydis en la Península Ibérica, se caracterizó su patogenicidad y también se determinaron otras especies de hongos presentes en maíz afectado por marchitez. Además, se evaluó el potencial de la termometría infrarroja para detectar las infecciones por H. maydis. En segundo lugar se detectó la presencia de O. cumana en girasol durante las fases de crecimiento subterráneo de la planta parásita utilizando para ello imágenes de fluorescencia multicolor (FMC). También se consideraron las posibles alteraciones fisiológicas en el girasol como consecuencia de la infección por O. cumana. En el Capítulo 3 se estudió la distribución geográfica de H. maydis en las principales zonas de cultivo de maíz en España y el sur de Portugal, prospectando 59 campos entre 2009 y 2013. La identidad de 14 de entre todos los aislados de H. maydis obtenidos se confirmó mediante amplificación ITS y estos mismos aislados se caracterizaron por su agresividad mediante inoculación y crecimiento de maíz susceptible crecido en condiciones de umbráculo durante todo el ciclo del cultivo. Uno de los aislados del hongo fue muy agresivo, causando síntomas severos en las plantas y reducciones significativas de peso de sus raíces y partes aéreas. Los aislados moderadamente agresivos causaron valores de enfermedad significativos, pero no todos ellos se asociaron a reducciones de peso de las plantas. En 2012 y 2013 se monitorizó la infección por H. maydis en maceta al aire libre y mediante medidas de temperatura de cubierta y del índice de estrés hídrico del cultivo en plantas control y en plantas inoculadas con el aislado más agresivo. Ambos índices respondieron a la infección por el hongo en los dos años, pudiendo detectarse dicha infección hasta 17 días antes de que los síntomas fueran visibles. Este estudio ha revelado la amplia distribución de H. maydis, que se localiza en los valles de todos los ríos de la Península Ibérica excepto el del Ebro y pone de relieve la importancia de la resistencia genética para controlar este patógeno en el sur de Europa. Además, la detección térmica de la infección previa al desarrollo de síntomas podría resultar en aplicaciones útiles para el diagnóstico presintomático y no destructivo de la enfermedad. En el Capítulo 4 se determinaron las especies de hongos asociadas a H. maydis como agente causal de marchitez de maíz. Para ello, se muestrearon 19 campos con síntomas de marchitez en las principales zonas de cultivo de la Península Ibérica entre 2011 y 2012. En el 47% de ellos no se identificó H. maydis sino otras especies: Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani y Trichoderma harzianum. En los campos restantes, junto a H. maydis se identificaron otros hongos de suelo en porcentajes apreciables: F. verticillioides (19%), F. proliferatum (19%), F. equiseti (9%), F. oxysporum (9%) y Pythium oligandrum (9%). El crecimiento vascular de H. maydis y de otras especies de hongos en maíz se confirmó analizando plantas con marchitez procedentes de tres campos diferentes. Tanto H. maydis como F. graminearum, F. equiseti, F. proliferatum y T. harzianum se recuperaron de la inserción entre la raíz y tallo y a 10 cm de altura en el tallo de las plantas. El efecto de la infección por H. maydis sobre la producción de las plantas de maíz se cuantificó en macetas y condiciones seminaturales en 2011. El peso de las mazorcas de plantas inoculadas se redujo en un 54%. Estas plantas también tuvieron pesos de raíz y de parte aérea (tallo y hojas) significativamente menores que los de las plantas control. Estos resultados apuntan al gran impacto que puede tener la Marchitez tardía sobre la producción de maíz en campo. Además, y aunque la patogenicidad de los hongos de suelo identificados en maíz debería ser confirmada, los resultados de este trabajo sugieren que la Marchitez tardía del maíz puede tener una etiología compleja. En cuanto al Capítulo 5, en él se analizó por primera vez la fluorescencia emitida por la clorofila de girasol en las bandas espectrales con máximos en el rojo (F680) y en el rojo lejano (F740). Se incubaron plantas sanas de girasol en macetas y condiciones de invernadero y, entre la segunda y la quinta semana de crecimiento se compararon los patrones de emisión de fluorescencia de los cuatro primeros pares de hojas (PHs) tanto en la superficie de la hoja como entre PHs. Los PHs de plantas sanas de girasol presentaron similares patrones de fluorescencia, tanto en el rojo como en el rojo lejano, que variaron dependiendo del grado de desarrollo de la hoja. La utilidad de F680 y F740 como indicadores de la infección de girasol por O. cumana durante las fases de desarrollo subterráneo de la plana parásita se evaluó en condiciones experimentales similares. En plantas infectadas por O. cumana se detectaron aumentos tempranos de F680 y F740, así como reducciones del ratio F680/F7403. Por otro lado, la significación de las diferencias de fluorescencia emitida por plantas control y plantas inoculadas dependió del PH que se considerara en cada momento. Las medidas de contenido clorofílico y de contenido de clorofila total apoyaron los resultados de la FMC, aunque fueron menos sensibles en la discriminación de plantas control y plantas inoculadas. Al final del experimento se confirmó la infección del girasol por la presencia de nódulos en las raíces de las plantas. Este trabajo revela el potencial de la fluorescencia en las regiones del rojo y el rojo-lejano para detectar de forma temprana la infección de girasol por O. cumana, lo que podría ser especialmente interesante para llevar a cabo un fenotipado temprano de material de programas de mejora. Más aún, y hasta donde hemos podido conocer, este es el primer trabajo donde se analiza el efecto de una planta parásita sobre su hospedante utilizando imágenes de fluorescencia en el rojo y en el rojo lejano. En el Capítulo 6 se analizó la emisión de fluorescencia azul y verde (FAV) en hojas de plántulas sanas de girasol. Además, se aplicaron tanto la FAV como la técnica de termografía para detectar la infección del girasol por O. cumana durante el desarrollo subterráneo de la planta parásita. En ambos experimentos se incubaron las plantas de girasol en macetas en invernadero y las medidas se tomaron tras el traslado temporal a cámara de condiciones controladas. En el primer experimento se observó que la FAV emitida por hojas de girasol sano aumentaba a lo largo de su desarrollo. En el caso de girasol parasitado, las hojas presentaban emisiones de FAV menores, y esta diferencia respecto a las hojas de las plantas control fue consistente a lo largo de todo su desarrollo. Al final del experimento se obtuvieron menores concentraciones de pigmentos, lo que sugiere que en las hojas de girasol ocurre un descenso de metabolitos secundarios tras la infección por O. cumana. Por otro lado, a lo largo de todo el experimento se detectaron mayores temperaturas de hoja en girasol inoculado con O. cumana en comparación con la temperatura de hojas de plantas control. Esto podría indicar que el ataque de la planta parásita induce un cierre estomático y una reducción de la transpiración del girasol. En el trabajo de nuevo se ha demostrado que es posible efectuar una monitorización no destructiva de la infección de girasol por O. cumana, en este caso utilizando FAV y termografía, y que dicha monitorización podría aplicarse al fenotipado rápido de girasol. Además, ambas técnicas se han revelado como útiles aproximaciones para estudiar los procesos mediante los cuales O. cumana altera la fisiología de su hospedante (metabolismo secundario y fotosíntesis). Por último, la discusión general de todos los resultados obtenidos en esta Tesis Doctoral y las conclusiones derivadas se presentan en los Capítulos 7 y 8 respectivamente.Some crop diseases caused by soilborne pathogens are characterised by very late symptoms appearance. This is the case of Late wilt of maize (caused by the fungus Harpophora maydis) and that of Broomrape of sunflower (caused by the root parasitic plant Orobanche cumana). Harpophora maydis was first reported in the Iberian Peninsula in 2010, and during the last years it has become a major concern to maize growers. On the contrary, attacks of O. cumana are frequent in sunflower growing areas of Spain since the 1980’s. Currently, the parasite is the first biotic constraint to sunflower oil production worldwide. Both H. maydis and O. cumana infect the host plant during the first weeks after sowing but symptoms are not observed until flowering or shortly after it. Also, they are controlled through the incorporation of genes of resistance into the crop varieties. The advancement and success of breeding programmes is highly dependent on an accurate and fast screening of the reaction of the host plant to the pathogen. On the other hand, techniques based on the use of remote and/or proximal sensors are frequently used with agronomical purposes. As an alternative to visual inspection and to destructive analyses of DNA, and because its sensitivity to physiological disorders in plants associated with pathogen attack, these techniques can constitute efficient detection tools in phytopathology. In the case of near distance, indirect monitoring of plants is majorly conducted by means of thermometry, thermography, fluorescence measurements and spectral techniques. In Chapter 1 of this Ph.D. Thesis, the state of the art of maize late wilt and sunflower broomrape is presented, as well as updated scientific information about the remote and proximal sensing techniques that are most commonly used in plant pathology. The objectives are presented in Chapter 2. First, the distribution of H. maydis in the Iberian Peninsula and its pathogenic characterization were addressed as well as the identification of other fungal species found in symptomatic maize. Also, the utility of infrared thermometry on the detection of maize infections by H. maydis was assessed. Second, the presence of O. cumana in sunflower was analysed during underground development stages by means of multicolour fluorescence (MCF). Possible physiological disorders in sunflower as a consequence of O. cumana infection were also considered. In Chapter 3 the geographical distribution of H. maydis in the main maize growing areas in the South of Portugal and Spain was determined by prospecting 59 fields from 2009 to 2013. Fourteen out of all the isolates of H. maydis were molecularly confirmed by ITS amplification, and their aggressiveness was analysed by inoculation and growth of susceptible maize under shadehouse conditions for the whole growing season. One of the isolates was highly aggressive, causing severe symptoms as well as significant weight reductions of both aboveground parts and roots of the inoculated plants. Moderately aggressive isolates caused significantly high symptoms severity, but not all of them were related to reductions in plant weight. In 2012 and 2013, the infection by H. maydis was monitored outdoors by means of measurements of canopy temperature and crop water stress index of potted control plants and plants inoculated with the most aggressive isolate. Both indices responded to the presence of fungal infection in both years, this infection being detected up to 17 days before symptoms in the plants were visible. This study shows the distribution of H. maydis in all the river valleys of the Iberian Peninsula, except that of the Ebro River, and highlights the importance of genetic resistance for controlling the pathogen in southern Europe. In addition, the thermal detection of the infection prior to symptoms development was possible, what might be further applied to the non-destructive pre-symptomatic diagnosis of Late wilt of maize. The species of fungi that are associated to H. maydis as the causal agent of maize wilt were identified in Chapter 4. Surveys were conducted in 2011 and 2012 in 19 fields where symptomatic plants were collected. The fields were located in the main maize growing areas of the Iberian Peninsula. In 47% of them the fungus infecting diseased plants was not H. maydis but Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani and/or Trichoderma harzianum. In the remaining fields H. maydis was identified together with other soilborne fungi that were also frequently isolated from diseased plants: F. verticillioides (19%), F. proliferatum (19%), F. equiseti (9%), F. oxysporum (9%) and Pythium oligandrum (9%). The vascular growth of H. maydis and other fungi into the host was confirmed by means of tissue analyses of diseased plants collected at three different locations. Harpophora maydis, as well as F. graminearum, F. equiseti, F. proliferatum and T. harzianum were recovered from the root-stem insertion, and from stem tissues up to 10 cm high. The effect of the infection by H. maydis on maize yield was assessed in inoculated potted plants that were grown in shadehouse in 2011. Cob production was reduced in 54% upon fungal infection. In addition, significantly low weights of roots and aboveground parts (stems and leaves) were obtained. These results point to the great economic impact that Late wilt can have on the yield of maize under field conditions. Likewise, this work suggests that it can be a disease of a complex etiology. Further work should address the pathogenicity of fungal species other than H. maydis on maize, so that the role they may play on disease incidence and on symptoms severity can be determined. Concerning Chapter 5, the fluorescence emitted by chlorophyll (Chl) of sunflower leaves in the spectral bands with peaks near red (F680) and far-red (F740) was analysed for the first time. Healthy sunflowers were grown in pots under greenhouse conditions. Fluorescence emission patterns across the leaf surface and throughout the plant were compared for the first four leaf pairs (LPs) and between the second and fifth weeks of growth. Similar fluorescence patterns, with a delay of three or four days between them, were obtained for LPs of healthy sunflower, showing that red and far-red fluorescence varied with the developmental stage of the leaves. The use of F680 and F740 as indicators of the infection of sunflower by O. cumana during underground development stages of the parasite was also evaluated under similar experimental conditions. Early increases in F680 and F740 as well as decreases in F680/F740 were detected upon infection, significant differences between inoculated and control plants being dependent on the LP that was considered at any time. Measurements of Chl contents and final total Chl content supported the results of MCFI, although they were less sensitive in differentiating healthy from inoculated plants. The infection of sunflowers was confirmed by the presence of broomrape nodules in the roots at the end of the experiment. This work revealed the potential of MCFI in the red and far-red regions for early detecting O. cumana in sunflower, what might be particularly interesting for early phenotyping in sunflower breeding programmes. Furthermore, and to the best of our knowledge, this is the first time that the effect of a parasitic plant in its host is analysed by means of MCFI. In Chapter 6 we analysed the blue-green fluorescence (BGF) emission of leaves of healthy sunflower plantlets, and we implemented BGF and thermal imaging in the detection of the infection by O. cumana during underground parasite development. In both experiments sunflowers were grown in pots and under greenhouse conditions and measurements were made after temporary movement to chamber of controlled conditions. Increases in BGF emission were observed in leaf pairs of healthy sunflowers during their development. Besides, lower BGF emission was consistently detected in parasitised plants throughout leaf expansion, and low pigment concentration was obtained at final time, supporting the interpretation of a decrease in secondary metabolites upon parasite infection. Also, parasite-induced stomatal closure and transpiration reduction were suggested by warmer leaves of inoculated sunflowers throughout the experiment. Techniques of BGF and thermal imaging allowed the non-destructive monitoring of sunflower broomrape, and they could be implemented for fast screening of sunflower genotypes. Additionally, these techniques were shown as valuable approaches to assess the processes by which O. cumana alters physiology (secondary metabolism and photosynthesis) of sunflower. Finally, the general discussion of all the results from the work included in this Ph.D. Thesis and the conclusions drawn from them are presented in Chapters 7 and 8 respectively

La Escuela de Farmacología de Madrid: de D. Teófilo Hernando a Instituto de I+D del medicamento de la Universidad Autónoma de Madrid

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología y Terapéutica. Fecha de lectura: 25 de Octubre de 201