Estudio de la dinámica de inóculo de los agentes de biocontrol de aflatoxinas en frutos secos, resistencia varietal al patógeno y caracterización de la población de Aspergillus spp. sección Flavi en España

Almond and pistachio nuts can be occasionally colonized with the fungal species Aspergillus flavus and A. parasiticus and, concomitantly, contaminated with aflatoxins, potent carcinogenic mycotoxins for humans. As reviewed in chapter I, the most effective pre-harvest management strategy for limiting aflatoxin contamination is the massive release of native atoxigenic strains of A. flavus for the competitive displacement (or competitive exclusion) of wild toxigenic isolates from the agroecosystem. Many farmers from the U.S.A., Africa, and Italy and diverse crop industries benefit from using this technology. Thereafter, in temperate and tropical regions, where the biocontrol strategy is still not implemented, the search for native atoxigenic A. flavus strains is a necessity. In California, for example, the US Environmental Protection Agency granted registration of the atoxigenic strain AF36 of A. flavus for use in pistachio and almond in 2012 and 2017, respectively. This strain is applied using sorghum grains as the AF36 spores carrier with the commercial name AF36 Prevail®. The use of AF36 Prevail® is a clear competitive advantage for U.S. farmers since commercial biocontrol agents to reduce aflatoxin contamination are not available in other nut-growing regions such as Australia, Spain, or Turkey. Although AF36 Prevail® was primarily developed for applying to row crops (e.g. maize, cotton), it has also been effective in limiting aflatoxin contamination in nut trees in California. Even so, AF36 Prevail® often fails in nut orchards because of differential characteristics between row and tree crops. Then, we evaluated (chapter II) the sporulation of the biocontrol strain AF36 in pistachio orchards and advised farmers to spread the AF36 Prevail® product in the moist soil area but avoid the site where the irrigation drops fall. Our study about the dynamics of A. flavus’ spores suggested that AF36 Prevail® could be applied every two rows obtaining an overlapping effect on the non-treated row whether the distance between tree rows is ≤ 10 m. Furthermore, we detected that tree debris in the canopy act as an inoculum source for Aspergillus species included in section Nigri, ochratoxins producers, and biocontrol strategies may act parallelly to protect against both mycotoxins. It is essential to monitor how the atoxigenic AF36 strain survives and competes with aflatoxin-producing species populations in the target agroecosystem to understand how it can displace wild isolates of Aspergillus spp. Traditionally, biocontrol strains of A. flavus have been monitored through vegetative compatibility assays (VCA), but these are tedious and time-consuming. Thus, we tackled this concern by developing and validating a mismatch-qPCR assay to quantify the proportion of AF36 vs. toxigenic genotypes of A. flavus and A. parasiticus from diverse soil and plant samples. Our mismatch-qPCR efficiently quantifies AF36 proportions in the Aspergillus population. To overcome the disadvantages (loss of grains, poor sporulation, etcetera) of applying the strain AF36 using sorghum grains as carriers, we studied (chapter II) the pistachio male inflorescences as an inoculum source of atoxigenic strains. Male inflorescences are an abundant and free substrate, regularly distributed in the orchard. In our trials, the density of AF36 spores on the pistachio canopy of the inflorescence-treated trees was similar (P > 0.05) to this of Prevail®-treated trees. Furthermore, our results indicated that in pistachio orchards, where biocontrol practices are not conducted, eliminating this critical source of toxigenic Aspergillus inoculum is recommended. In chapter III, we characterized the resistance of various almond cultivars against A. flavus and A. parasiticus colonization and aflatoxin contamination. Remarkably, we found high variability in response to aflatoxin contamination of almond cultivars caused by both Aspergillus species. In addition, the shells were an insurmountable barrier to the pathogen, regardless of their type of shell (hard, semihard, or paper shell). However, natural-opening shells often occur in paper shell almond cultivars in the field. Our results also pointed out the importance of peach for introgressing resistance to the pathogen in almond breeding programs. Finally, we presented the possibility of combining both cultivar resistance and biocontrol, which offers a particularly promising aflatoxin control strategy. In a final chapter IV, we surveyed two leading Spanish almond- and pistachio-producing regions, Andalusia and Castilla La Mancha. In these surveys, we isolated 78 strains of Aspergillus section Flavi. Remarkably, four A. flavus were identified as atoxigenic (i.e., no-aflatoxin and no-cyclopiazonic acid producers) and, to our knowledge, this is the first report of atoxigenic strains of A. flavus native to Spain. Besides, six A. tamarii strains resulted, for the first time, described as slightly aflatoxigenic. With the work advocated in this Ph. D Thesis, we have contributed definitely to the optimized use of this biological control strain in Californian tree nut crops. In addition, we are closer to offering a safe product option to be used infield shortly by the Spanish almond and pistachio producers.Las almendras y los pistachos son colonizados ocasionalmente por las especies fúngicas Aspergillus flavus y A. parasiticus y, por consiguiente, pueden contaminarse con aflatoxinas, potentes micotoxinas cancerígenas para los humanos. En el capítulo I de la presente Tesis Doctoral, revisamos una de las estrategias de control más efectiva para limitar la contaminación por aflatoxinas en campo: la liberación masiva de cepas atoxigénicas (no productoras de micotoxinas) nativas de A. flavus para el desplazamiento competitivo (o exclusión competitiva) de los aislados toxigénicos del agroecosistema. Muchos agricultores de EE. UU., África e Italia tienen acceso comercial a este tipo de agentes de biocontrol. En cambio, en las regiones templadas y tropicales donde aún no se implementa esta estrategia de biocontrol es necesaria la búsqueda de cepas atoxigénicas de A. flavus. En California, la Agencia de Protección Ambiental (EPA) de EE. UU. otorgó el registro de la cepa atoxigénica AF36 de A. flavus para uso en pistachero y almendro en 2012 y 2017, respectivamente. La cepa AF36 se aplica en campo utilizando granos de sorgo recubiertos de esporas con el nombre comercial AF36 Prevail®. El uso de AF36 Prevail® supone una ventaja competitiva para los agricultores estadounidenses ya que, en otras regiones productoras de frutos secos, como Australia, España o Turquía, no hay agentes de control biológico comerciales disponibles. El producto AF36 Prevail® se desarrolló para su uso en cultivos extensivos (ej. maíz y algodón), aunque también se ha mostrado eficaz disminuyendo la contaminación por aflatoxinas en frutos secos. Aun así, el control biológico de aflatoxinas en frutos secos mediante el uso de AF36 Prevail® fracasa con frecuencia debido a características agronomicas propias de este tipo de cultivos arbóreos. En el capítulo II, por lo tanto, evaluamos la esporulación y dispersión del producto AF36 Prevail® en campos de pistachero y recomendamos a los agricultores aplicar el producto en el área de suelo irrigada por los microaspersores aunque, evitando la zona donde impactan las gotas del agua de riego ya que afecta negativamente a su esporulación. Según nuestro estudio sobre la dispersión de las esporas de A. flavus, las esporas de AF36 fácilmente alcanzan la copa de los pistacheros próximos al punto de aplicación aunque disminuye marcadamente con la distancia a la fuente de inóculo y la altura, ajustándose a distintas ecuaciones de difusión. Nuestros datos apuntan a que AF36 Prevail® podría aplicarse en filas alternas de pistacheros obteniéndose un efecto de protección (densidad de esporas por árbol) similar en el conjunto de los árboles si la distancia entre filas es ≤ 10 m. Además, detectamos que los restos de tejido que quedan en la copa de los pistacheros actúan como fuente de inóculo para las especies de Aspergillus de la sección Nigri, productores de ocratoxinas, por lo tanto, la estrategia de biocontrol puede actuar de forma paralela contra ambas micotoxinas. Para comprender cómo la cepa atoxigénica AF36 sobrevive, compite y desplaza a las cepas silvestres de Aspergillus spp. productoras de aflatoxinas, es esencial la monitorización en los campos donde ha sido liberada. Tradicionalmente, las cepas de A. flavus se han monitoreado mediante ensayos de compatibilidad vegetativa (VCA), pero son tediosos y requieren varias semanas para su ejecución. Por lo tanto, desarrollamos y validamos (capítulo II) un protocolo de qPCR basado en un Mismatch para cuantificar la proporción de AF36 frente a los genotipos toxigénicos de A. flavus y la población general de A. parasiticus. Nuestra qPCR-Mismatch cuantifica de manera eficiente las proporciones de AF36 respecto a la población de Aspergillus en el suelo y la planta. Debido a los problemas derivados de aplicar la cepa AF36 en granos de sorgo (pérdida de granos, mala esporulación, etcétera), estudiamos (capítulo II) la posibilidad de utilizar las inflorescencias masculinas de pistachero como fuente de inóculo para las cepas atoxigénicas. Las inflorescencias masculinas constituyen un sustrato abundante, gratuito, y que se distribuye regularmente en la plantación. En nuestros ensayos, la densidad de esporas de AF36 en la copa de los pistacheros con inflorescencias del suelo inoculadas con AF36 fue similar (P > 0.05) a la de los árboles tratados con AF36 Prevail®. Estos resultados apuntan indirectamente a que, en los campos de pistacheros donde no se llevan a cabo prácticas de biocontrol, es recomendable eliminar las inflorescencias masculinas en el suelo al constituir una importante fuente de inóculo. En el capítulo III, caracterizamos la resistencia de cultivares y selecciones avanzadas de almendro a la colonización por A. flavus y A. parasiticus y, la subsiguiente, contaminación por aflatoxinas. Sorprendentemente, encontramos una alta variabilidad en la resistencia/susceptibilidad de los genotipos de almendro a la colonización por ambas especies. Además, la cáscara (endocarpo) intacta resultó ser una barrera infranqueable para el patógeno, independientemente del tipo (dura, semidura o de papel). Sin embargo, las aperturas de la cáscara, que naturalmente pueden aparecer en cultivares de almendro de cáscara de papel, constituyen un punto de entrada para las esporas del patógeno. En este capítulo, además, destacamos la importancia del melocotonero para la introgresión de genes de resistencia al patógeno en los programas de mejora del almendro. Finalmente, presentamos la posibilidad de combinar tanto la resistencia del cultivar como el biocontrol, lo que ofrece una estrategia de control de aflatoxina particularmente prometedora. En un capítulo final (IV), caracterizamos la población de Aspergillus spp. en dos de las principales regiones españolas productoras de almendras y pistachos, Andalucía y Castilla La Mancha. Durante las prospecciones realizadas, aislamos 78 cepas de Aspergillus sección Flavi y seis cepas de A. tamarii que sorprendentemente fueron caracterizadas como ligeramente aflatoxigénicas. Cabe destacar, que identificamos cuatro cepas de A. flavus como atoxigénicas (es decir, no productoras de aflatoxinas ni ácido ciclopiazónico) lo que constituye la primera descripción de cepas atoxigénicas de A. flavus españolas. En la presente Tesis Doctoral, hemos contribuido notablemente a la optimización del control biológico de aflatoxinas en los cultivos de frutos secos de California. Además, estamos más cerca de ofrecer agentes de control biológicos para reducir la contaminación por aflatoxinas que puedan emplear los productores españoles

«Por la palabra»: Exploración de una mínima poética de Jorge Guillén.

Sin resume

An ASIC channel for acid chemotransduction

Producción CientíficaSpontaneous breathing requires feedback controls in which detection of blood gas and pH are critical. While O2 detection is performed by pheripheral chemoreceptors, CO2/pH-sensitive chemoreceptors are in the carotid bodies (CBs), but major sites are also within the brain (the central chemoreceptors [CCR]). CO2/pH signals are related to the acid-base status of the blood and reflect the adequacy of breathing to metabolism. Small changes in CO2/pH can affect breathing, so that a rise in Pco2 as small as 1 mm Hg produces an evident change in ventilation.1 Such a high CO2 sensitivity relies in the inherent properties of CO2/pH-sensing molecules present both in CB and CCR cells, as shown in several recent studies on CO2/pH sensing ion channels and receptors.2,3 Functional properties of most proteins can be regulated by changes in pH, as this would only require 1 or a few titratable residues of the molecule, whose protonation can lead to conformation changes that translate into changes in activity. However, to define whether those molecules have a relevant role in CO2/pH chemoreception, some more criteria should be met, including their range of pH sensitivity, their location in chemoreceptor cells, and their functional contribution to the integrated chemoreceptor response. In this regard, the molecular characterization of pH-sensitive channels and transporters has progressed considerably within the last years, but conclusive evidences of their contribution to acid chemotransduction are not so well established for many of them. The main reason for this delay is the fact that the identity of the primary sensory cells constituting CCRs and of their neuronal networks remains elusive. In vitro, neurons from many brain locations are excited or inhibited by CO2/pH changes, but it has been difficult to link this neuronal chemosensitivity to chemoreception in vivo. Moreover, in addition to chemosensitive neurons, CCRs sites may also contain neurons with other integrative functions that are not clearly distinct from chemosensitive ones in their morphological or functional properties, making their experimental study difficult.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grants R006/009 and PI041044)Ministerio de Ciencia, Innovación y Universidades (grant BFU2004-05551)Junta de Castilla y León (grant VA011C05

Are Kv channels the essence of O2 sensing?

Producción CientíficaThis report represents a relevant contribution to the study of oxygen sensing for two main reasons. First, it shows an approach adequate for identification of a putative O2-sensitive K+ channel, by moving from the modulation by hypoxia of a recombinant channel back to its physiological context, the role of this channel in the response to low Po2 of the native cells. Second, it demonstrates that hypoxic inhibition of the recombinant Kv3.1 channels is retained in excised membrane patches, pointing to a membrane-delimited mechanism as the origin of hypoxic responses. The importance of this latter finding deserves additional comment

Functional Analysis of Lactic Acid Bacteria and Bifidobacteria and Their Effects on Human Health

Many lactic acid bacteria (LAB) and Bifidobacteria are beneficial components of human, animal, foods, and beverage microbiota [...]This research was funded by the Spanish Ministry of Science, Innovation and Universities, grant number RTI2018-097114-B-I00

Sector restaurantero: situación y oportunidades de mejora en la región Xalapa, Veracruz, periodo 2008-2009

En Xalapa el turismo se ha convertido en una de las actividades clave para apuntalar el crecimiento económico y la generación de empleos, por lo que el gobierno estatal promueve diversos programas para su desarrollo, en específico en el sector restaurantero, el cual en los últimos años se ha visto afectado por una serie de situaciones y dificultades para mantenerse competitivo y subsistir. Estos aspectos impulsaron el desarrollo de un proyecto de investigación para identificar y describir el estado actual de dicho sector, así como las áreas de oportunidad, con la finalidad de reconocer y proponer estrategias para enfrentar con ventajas los retos y aprovechar las oportunidades; de tal manera que estén en condiciones de permanecer y crecer competitivamente en este sector.In Xalapa, tourism has become one of the key activities to underpin economic growth and employment generation, so that the state government promotes several programs for development, specifically the restaurant industry, as in recent years affected by a number of situations and challenges to stay competitive and survival. Aspects behind the development of a research project to identify and explain the current state of the sector and the area of opportunity, in order to identify and propose strategies to deal with the challenges and benefits opportunities, in such so they are able to stay competitive and grow in this sector

“Zombie attack” a new way to teach Chemistry

The Higher Education requires new models which allow training people able to adapt and survive in changing environments. It is based on the use of technologies and the adaptation of knowledge to people. It is about an education according to circumstances, which is adapted to context and virtual behaviour of people. One of the main difficulties that lecturers find in the classroom is how to maintain students' attention and interest in their subject, especially when students also think that the subject is not important for their training. In order to motivate these students, innovation in educational techniques and methodologies, such as experiential learning, are progressively being imposed to and/or coordinated with the traditional ones. Escape Room is a very modern concept in education, based on the development of mental skills for the solution of enigmas and problems. It is a tool to develop the cooperative, cognitive, deductive and logical reasoning skills of the students. In this work, an educational gamification experience based on the escape room concept is presented. The students have 1 hour and 30 minutes to carry out this activity. They will have to solve four puzzles and enigmas that will give them the key to open a treasure chest and finally let them escape from the classroom. Logic, ingenuity and teamwork will allow participants to develop not only chemical competence, but also other basic skills. The story that is told throughout the escape room is a zombie attack: the city has been infected (with a virus) and only the occupants of the room where the activity takes place have not been infected. In addition, they can all protect themselves if they are able to open the chest where the antidote is located. The aim of this activity is to enhance the knowledge acquired throughout the semester as well as the development of skills.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Kvβ1.2 Subunit Coexpression in HEK293 Cells Confers O2 Sensitivity to Kv4.2 but not to Shaker Channels

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane α subunits and cytoplasmic modulatory β subunits. The differential expression and association of α and β subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvβ1.2 coexpression on the properties of Shaker and Kv4.2 KV channel α subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvβ1.2 functionally associates with these two α subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvβ1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvβ1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2′-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+β channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+β currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that β subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4.2+β channels to respond to hypoxia

A neuroimaging study of pleasant and unpleasant olfactory perceptions of virgin olive oil

Functional magnetic resonance imaging (fMRI) has been used to collect information from neurons that receive direct input from olfactory bulbs when subjects smell virgin olive oil. The pleasant aroma of three extra virgin olive oils (var. Royal, Arbequina and Picual) and three virgin olive oils with sensory defects (rancid, fusty and winey/vinegary) were presented to 14 subjects while a fMRI scan acquired data from the brain activity. Data were subjected to a two-sample t test analysis, which allows a better interpretation of results particularly when data are studied across different subjects. Most of the activations, which were located in the frontal lobe, are related to the olfactory task regardless of the hedonic component of perception (e.g. Brodmann areas 10, 11). Comparing the samples with pleasant and unpleasant aromas, differences were found at the anterior cingu�late gyrus (Brodmann area 32), at the temporal lobe (Brodmann area 38), and inferior frontal gyrus (Brodmann area 47), while intense aromas activated Brodmann area 6. The actual perceptions described by the subjects and the concentration of the odorant compounds in the samples were considered in the interpretation of the results.Estudio mediante neuroimagen de percepciones olfativas agradables y desagradables de aceites de oliva virgen. La imagen por resonancia magnética funcional (fMRI) ha sido empleada para estudiar la información de la respuesta cerebral producida al estimular las neuronas que participan en el proceso olfatorio tras percibir el aroma del aceite de oliva virgen (AOV). Se utilizó fMRI para la adquisición de los datos de la actividad cerebral de 14 sujetos a los que se presentaron tres aceites de oliva vírgenes de aroma agradable (var. Royal, Arbequina and Picual) y tres aceites de oliva vírgenes con defectos sensoriales (rancio, atrojado, avinado/avinagrado). Los datos se sometieron a una prueba t para observar diferencias entre dos grupos, la cual permite una mejor interpretación de los resultados, particularmente cuando los datos se estudian a través de diferentes sujetos. La mayoría de las activaciones, que se localizaron en lóbulos frontales, se relacionaron con la tarea olfatoria independientemente de la componente hedónica de la percepción (por ejemplo, áreas Brodmann 10, 11). Al comparar las muestras con aromas agradables y desagradables, se encontraron diferencias significativas en el giro cingulado anterior (área Brodmann 32), el lóbulo temporal (área Brodmann 38) y el giro frontal inferior (área Brodmann 47), mientras que los aromas más intensos activaron el área Brodmann 6. En la interpretación de los resultados se tuvo en cuenta tanto la percepción descrita por los sujetos como las concentraciones de los compuestos volátiles en las muestras.Ramón y Cajal Program AGL2008-01411, AGL2011- 30371-C02-01/02

Standardizing Process-Data Exploitation by Means of a Process-Instance Metamodel

The analysis of data produced by the enterprises during pro cesses execution is key to know how these processes are working and how they can be improved. These data may be consumed to make different types of analysis, for example, data could be used as input for process dis covery, decision-making and even process querying tools. However, each type of analysis needs data in different formats because they use different techniques and tackle the problem from a different point of view. Fortu nately, if we look at the data exploitation problem from a higher level of abstraction perspective, we can realize that all the points of view share a common ground: the business process model and its instantiation are in the kernel of all of them. In this paper, we propose the use of a Busi ness Process Instance Metamodel, which serves as a common interface to make independent the applications producing business process data from those applications that consume and exploit it. A tool has been implemented as a proof of concept to facilitate the matching between data from different data sources and the metamodel.Ministerio de Ciencia y Tecnología TIN2015-63502-C3-2-RMinisterio de Ciencia y Tecnología TIN2016-75394-