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Sequences of the forward and reverse primers and, probes used for TaqMan® RT-PCR. (DOCX 12 kb)

SSR profiles of the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc and those regenerated through somatic embryogenesis. (DOCX 17 kb)

Virus status and microsatellites (SSRs) profile of mother plants and plantlets from somatic embryos of cultivars Tempranillo and Pinot Blanc plants. (DOCX 15 kb)

Plant development measured using a shoot development index (DI) and a rooting index (RI), after 20 and 40 day of culture. As starting explants, 20 somatic embryos from direct embryogenesis events were sown in individual tubes. (DOCX 13 kb)

In this work we present a full-passive flexible multigas sensing tag for the determination of oxygen, carbon dioxide, ammonia, and relative humidity readable by a smartphone. This tag is based on near field communication (NFC) technology for energy harvesting and data transmission to a smartphone. The gas sensors show an optic response that is read through high-resolution digital color detectors. A white LED is used as the common optical excitation source for all the sensors. Only a reduced electronics with very low power consumption is required for the reading of the optical responses and data transmission to a remote user. An application for the Android operating system has been developed for the power supplying and data reception from the tag. The responses of the sensors have been calibrated and fitted to simple functions, allowing a fast prediction of the gases concentration. Cross-sensitivity has also been evaluated, finding that in most of the cases it is negligible or easily correctable using the rest of the readings. The election of the target gases has been due to their importance in the monitoring of modified atmosphere packaging. The resolutions and limits of detection measured are suitable for such kinds of applications., This work was supported by project CTQ2013-44545-R from the Ministry of Economy and Competitiveness (Spain) and Junta de Andalucía (Proyecto de Excelencia P10- FQM-5974). These projects were partially supported by European Regional Development Funds (ERDF). P. Escobedo wants to thank the Spanish Ministry of Education, Culture and Sport (MECD) for a pre-doctoral grant (FPU13/05032).

A test performance study (TPS) involving 26 European laboratories was conducted to evaluate the robustness and reliability of loop mediated isothermal amplification (LAMP) assays. These assays were for the detection of the quarantine listed species Liriomyza huidobrensis (a leaf mining insect) and Clavibacter michiganensis subsp. sepedonicus (CMS, a bacterial potato pathogen). The L. huidobrensis assay performed well with the main challenge being the interpretation of the results from non-target genera and DNA extraction from small insect parts. Many participants had problems with contamination of the CMS LAMP assay, and a decrease in the limit of detection for the assay was found. Some users found the correct interpretation of LAMP results difficult, and variation was seen upon the use of different amplification platforms. The TPS has highlighted that thorough training prior to implementation of a new diagnostic method would be beneficial, even in the hands of experienced diagnostic laboratories. Useful information has been gathered around the requirements for user instructions/protocols and the formulation of TPS material. Overall the feedback from the participants was positive and LAMP was viewed as a useful new tool, with the speed of testing and simple DNA extraction protocols particularly attractive to laboratories., Scientific report of the Euphresco funded project 'Euphresco: test performance study of LAMP assays' (QDetect+)

This work presents the design, fabrication, and characterization of a passive printed radiofrequency identification tag in the ultra-high-frequency band with multiple optical sensing capabilities. This tag includes five photodiodes to cover a wide spectral range from near-infrared to visible and ultraviolet spectral regions. The tag antenna and circuit connections have been screen-printed on a flexible polymeric substrate. An ultra-low-power microcontroller-based switch has been included to measure the five magnitudes issuing from the optical sensors, providing a spectral fingerprint of the incident electromagnetic radiation from ultraviolet to infrared, without requiring energy from a battery. The normalization procedure has been designed applying illuminants, and the entire system was tested by measuring cards from a colour chart and sensing fruit ripening.

International audience

Viruses cause significant damage in agricultural crops worldwide. Disease management requires sensitive and specific tools for virus detection and identification. Also, detection techniques need to be rapid to keep pace with the continuous emergence of new viral diseases. The genus Fabavirus is composed of five viruses infecting many economically important crops worldwide. This research describes the development of a procedure based on flow-through hybridisation (FTH), which is faster than and as sensitive as conventional hybridisation for virus detection in tissue-prints from infected plants. Six digoxigenin-labelled RNA probes were synthesised with two levels of specificity: (a) five specific for each viral species within this genus, and (b) a genus-specific probe that hybridises with a nucleotide sequence signature only found in the 5-untranslated region of the genus Fabavirus, which is the first of this type reported for plant viruses. The new procedure developed is useful for rapid detection and discrimination of the five fabaviruses identified so far and opens the possibility of discovering new species of this genus.

La embriogénesis somática es una técnica de cultivo in vitro que permite el saneamiento de cultivares de vid (Vitis vinifera L.). Se ha analizado la presencia de los virus ArMvV, GELY, GFkV, GLRaV-1 y GLRaV-3 mediante real-time multiplex RT-PCR en hojas de plantas adultas de los cultivares “Valencí Blanc” y “Botó de Gall”. Además, se ha establecido un protocolo de embriogénesis somática para ambas variedades. La adición de 2 mgL-1 de 2,4D en el medio de cultivo incrementa el porcentaje de embriones cultivados así como el número de embriones por callos.

National audience; Pour être adoptée en routine, une technique de diagnostic de virus doit idéalement présenter les caractéristiques suivantes : techniquement robuste, reproductible et répétable, diagnostic sensible et spécifique, facilité de mise en œuvre, y compris pour des utilisateurs peu formés ou inexpérimentés, automatisable, rapide, économiquement compatible avec les besoins de la filière et basée sur des standards industriels généralisés [1]. L’Elisa (Enzyme Linked ImmunoSorbent Assay), apparue dans les années 1970, et la PCR en temps réel, apparue à la fin des années 1990, possèdent la plupart de ces caractéristiques et sont ainsi devenues les deux standards utilisés de manière intensive pour le diagnostic des viroses, en particulier dans le domaine végétal. Pour chacune de ces techniques, une dizaine d’années ont été nécessaires entre les premiers développements et leur utilisation par les laboratoires de diagnostic de routine. Ces technologiques ont ainsi amélioré de manière significative notre capacité à diagnostiquer des infections virales. Cependant, leur utilité est limitée par la nécessité de déterminer a priori les virus ciblés par le test et par la disponibilité d’anticorps ou de séquences nucléotidiques spécifiques. Elles ne permettent donc pas le diagnostic de virus inconnus ou de souches virales divergentes peu ou mal caractérisées. L’indexage complet, c’est-à-dire l’identification de tous les virus présents dans un échantillon, était donc un objectif inatteignable jusqu’à tout récemment.

The Arabis mosaic virus (ArMV) is one of the causative agent of the grapevine fanleaf disease, one of the most widespread and damaging viral diseases of grapevine. Recently, the ArMV has been detected in Spanish vineyards, and its determination and molecular characterization was undertaken. To this aim, the nucleotide sequence of the genomic RNAs 1 and 2 of the first isolate of ArMV infecting grapevine detected in Spain (ArMV-DU13) has been determined. The ArMV-DU13 genomic sequences were compared to the corresponding sequences of other isolates of ArMV, or nepoviruses. The most divergent genes among ArMV isolates were the X1 and VPg genes on the RNA 1, and the 2A gene on the RNA 2, with identity levels at the amino acid level of 78% (X1 and VPg) or 69% (2A) between the most distant isolates. Interestingly, the VPg genes were identical between the two grapevine isolates ArMV-Du13 and -NW, suggesting a possible implication of the host. The phylogenetic analysis of the RNA 2 showed that the Spanish isolate was close to Grapevine fanleaf virus isolates. The analysis of the full length RNA 2 suggests a recombination event between ArMV-DU13 and GFLV-GHu isolates between nucleotides 54 and 586 in the ArMV-DU13 isolate. Altogether, these results confirm the high variability between isolates of ArMV, and will be helpful to design more appropriate and reliable molecular diagnostic techniques for the control of this emerging virus in Spain.

Las flores tropicales han tenido un auge importante desde los años ochenta, cuando los botánicos especializados en estudiar este tipo de flor selvática formaron en 1985 la Sociedad Internacional de Heliconias, organización creada con el ánimo de estudiar, recolectar, entender y conservar las heliconias del mundo. Con el tiempo han vinculado a personas interesadas en el tema, institucionalizando las actividades de investigación y divulgación. El género heliconia presenta entre 225 y 250 especies de plantas descritas hasta el momento en el mundo, siendo Colombia el país que mayor número tiene , originarias de Suramérica, Centroamérica, las islas del Pacífico e Indonesia. Se les llama platanillo por sus hojas o ave del paraíso y muela de langosta por las coloridas brácteas que envuelven. Administrador Ambiental y de los Recursos Naturales Pregrado

Esta monografía se realizó en las instalaciones y en las unidades prestadoras de servicios de la empresa TRANSGUÍAS S.A., con el objetivo de diseñar un plan de mejoramiento que permita controlar que permita reducir las cancelaciones de los servicios de transporte e incrementar la calidad del servicio hacia el cliente en la empresa, detallando claramente que es lo que se debe hacer, como se debe hacer, quien lo debe hacer, cuanto es el costo y cuáles son los beneficios que lograría la empresa con su implementación. Esta monografía está conformada por cuatro capítulos, los cuales comprenden los tópicos que se describen a continuación: El primer capítulo hace referencia al marco teórico en el cual se mencionan las bases conceptuales dentro de las cuales se encuentra enmarcado este proyecto y principales bases a tener en cuenta para darle solución al mismo En el segundo capítulo se mencionan aspectos generales relacionados con la empresa TRANSGUÍAS S.A., se detalla el proceso de prestación de servicio que efectúa la empresa y además se analiza el macro y micro ambiente en el cual esta se está desarrollando. En el tercer capítulo un diagnostico situacional, el cual muestra evidencias de los problemas que presenta la empresa TRANSGUÍAS S.A., con el fin de realizar una priorización que permita identificar cuál es el problema al cual se debe enfocar el plan de mejoramiento. Incluye bibliografía

Número de publicación: ES2335176 B1. Número de solicitud: 200802654., Dicho sistema comprende componentes de instrumentación (hardware), componentes lógicos de programación (software) y un conjunto de elementos de detección formados por un número de fotodetectores colocados en la suela del zapato deportivo. El funcionamiento se basa en el registro de los cambios en las señales de los fotodetectores sobre la base de una referencia temporal común a todos estos últimos., The invention relates to a system for measuring the temporal parameters of walking, running and jumping by means of photodetectors. Said system comprises instrumentation components (hardware), logical programming components (software) and a set of detection elements formed by a number of photodetectors placed on the sole of the sports shoe. Operation is based on recording the changes in the signals from the photodetectors on the basis of a temporal reference common to all of the latter., Universidad de Cádiz, Universidad de Granada

Resumen tomado de la publicación En este trabajo se describe una guía de trabajo autónomo, así como su valoración por parte del alumnado, en la asignatura Diseño de Receptores de Radio de la titulación de Ingeniería de Telecomunicaciones. Tras analizar los resultados, podemos ver cómo en torno al 70 por ciento de los encuestados, prefieren esta metodología para el aprendizaje práctico. Navarra Universidad Pública de Navarra. Biblioteca Universitaria; Campus de Arrosadia; 31006 Pamplona; Tel. +34948169060; Fax +34948169069; bupna@enavarra.es ESP

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for large-scale analyses. [Int Microbiol 2009; 12(1): 1-6]

Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future.

A nucleic acid sequence-based amplification method coupled with flow-through hybridisation (NASBA-FH) was developed for Plum pox virus (PPV) detection. The detection limit of the NASBA-FH was established at two copies of control transcripts, resulting 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 253 stone-fruit trees were collected during winter and analysed. The samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. PPV diagnosis by ELISA and NASBA-FH coincided in 90.5%, while ELISA and PCR-based methods coincided in the diagnosis of 91.3% of the trees and PCR-based methods with NASBA-FH agreed in 95.2%. Results support that NASBA-FH is a suitable molecular method for routine PPV detection in the winter period.

El diseño de protocolos de diagnóstico y detección de virus de especies leñosas implica no sólo la producción de reactivos (anticuerpos, sondas, cebadores...) específicos y la puesta a punto de técnicas sensibles. El establecimiento de la época, periodo vegetativo y órganos ideales a muestrear, es esencial para lograr detecciones y diagnósticos fiables. Algunos métodos de diagnóstico han permitido popularizar la detección de virus. Su aplicación sistemática para realizar prospecciones, controles de la producción, selección sanitaria, erradicación y estudios epidemiológicos, han sido definitivos para el control de algunas virosis graves y para ayudar al productor en la toma de decisiones. En el presente artículo se analizan las repercusiones de la existencia de protocolos fiables y de datos surgidos de estudios epidemiológicos, en el control del virus de la sharka de los frutales de hueso y del virus de la tristeza de los cítricos en España. Se discute la importancia y repercusión de los métodos de detección de calidad con dos ejemplos: el programa de erradicación del virus de la sharka agresivo en melocotonero, tipo M, y la reconversión citrícola provocada por la enfermedad de la tristeza.

Optimisation dynamique d'un procédé discontinu de séchage du riz. SIMO 2002 Système d'Information, Modélisation, Optimisation, Contrôle, Commande en Génie des Procédés

Phytophthora nicotianae Breda de Haan is one of the most important soil-borne plant pathogens. The identification of this pathogen based on morphological or physiological characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Based on this concept, conventional and single closed tube nested-PCRs were developed for the specific and sensitive detection of P. nicotianae. Two new specific primers, designed from the spacer regions ITS1 and ITS2, internal to the nucleotide sequence flanked by universal primers ITS4 and ITS6, were used. To evaluate the specificity of the method, 36 morphologically characterized isolates were tested. A positive reaction, characterized by an amplification product of 737 bp, was shown by all P. nicotianae isolates and two P. nicotianae/cactorum hybrids. No amplification product was observed when other Phytophthora species and genera were assayed. The sensitivity of this method was analysed by serial dilutions of a defined amount of fungal DNA in a healthy root extract. Nested-PCR was at least 1000 times more sensitive than conventional PCR. In addition, samples from different infection sites, origins and crops, samples from nutrient solution, water and the rockwool used in hydroponic cultures, were analysed to validate this method.