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Lo que se pretende en el presente TFM es la realización de una programación didáctica de la unidad dedicada a la industria quesera mediante la gamificación, utilizando diferentes técnicas de gamificación en la que los alumnos puedan aprender los conocimientos relacionados con la unidad de una manera más amena, Departamento de Ingeniería Agrícola y Forestal, Máster en Profesor de Educación Secundaria Obligatoria y Bachillerato, Formación Profesional y Enseñanzas de Idiomas

Verticillium wilt of olive is best managed by resistant cultivars, but those currently available show incomplete resistance to the defoliating (D) Verticillium dahliae pathotype. Moreover, these cultivars do not satisfy consumers' demand for high yields and oil quality. Highly resistant rootstocks would be of paramount importance for production of agronomically adapted and commercially desirable olive cultivars in D V. dahliae-infested soils. In this work, resistance to D V. dahliae in wild olive clones Ac-13, Ac-18, OutVert and StopVert was assessed by quantifying the fungal DNA along the stem using a highly sensitive real-time quantitative polymerase chain reaction (qPCR) protocol and a stem colonization index (SCI) based on isolation of V. dahliae following artificial inoculations under conditions highly conducive for verticillium wilt. Ac-13, Ac-18, OutVert and StopVert showed a symptomless reaction to D V. dahliae. The mean amount of D V. dahliaeDNA quantified in stems of the four clones ranged from 3.64 to 28.89 pg/100 ng olive DNA, which was 249 to 1537 times lower than that in susceptible Picual olive. The reduction in the quantitative stem colonization of wild olive clones by D V. dahliae was also indicated by a sharp decrease in the SCI. Overall, there was a pattern of decreasing SCI in acropetal progression along the plant axis, as well as correlation between positive reisolation and quantification of pathogen DNA. The results of this research show that wild olive clones Ac-13, Ac-18, OutVert and StopVert have a valuable potential as rootstocks for the management of verticillium wilt in olive.

Symptomless host and nonhost responses of Paulownia spp. to olive-defoliating (D) Verticillium dahliae is reported for the first time. Two paulownia clones, Paulownia elongata ‘PC-2’ and P. elongata × P. fortunei ‘PC-3’, were inoculated with a V. dahliae isolate representative of the D pathotype by either root dip or stem injection with a conidial suspension, repeated transplanting to a V. dahliae-infested soil mixture, or root dip in the conidial suspension followed by transplanting to the infested soil mixture. ‘Picual’ olive and ‘Sugar Baby’ watermelon were included in all experiments as susceptible standards to show that the inoculation procedures and incubation conditions were successful. Plants were incubated under conditions optimal for Verticillium wilt that caused severe disease in ‘Picual’ olive and ‘Sugar Baby’ watermelon in the growth chamber, shade house, and field microplots for 30 to 57 weeks in three independent experiments. No foliar symptoms developed on paulownia, whose stems were found free of V. dahliae both by isolation on semiselective NP-10 medium as well as by a nested-polymerase chain reaction assay using total genomic DNA from inoculated plants that effectively detected D V. dahliae in olive stems. V. dahliae was isolated to a limited extent from roots of PC-3 paulownia plants after 30 weeks of growth in the infested soil mixture but not from those that were root-dip inoculated or from PC-2 plants regardless the method of inoculation. The symptomless host and nonhost responses of Paulownia spp. to D V. dahliae may have practical applications in the use of fertile soils in southern Spain, particularly in those that are highly infested with the highly virulent D pathotype, as well as a replacement crop for Verticillium wilt-affected olive orchards in that region.

Paulownia (Paulownia spp.) es una dicotiledónea leñosa de origen asiático que ha generado interés en el sector forestal por su rápido crecimiento y eficiencia en la producción de biomasa. El uso de paulownia puede ser comprometido por la susceptibilidad a patógenos residentes en suelos agrícolas, entre los que Verticillium dahliae es particularmente relevante en Andalucía por la devastación que causa el patotipo defoliante (D) en extensas áreas olivareras. V. dahliae es uno de los hongos de más amplio espectro de plantas huésped, con más de 400 especies cultivadas y arvenses susceptibles, a las que hay que sumar aquéllas en que ocurren infecciones endofíticas. Sin embargo, la reacción de paulownia a V. dahliae es desconocida. Los resultados de este trabajo indican que los clones PC-2 de P. elongata y PC-3 (P. elongata x P. fortunei) de Plantas Continental S.A. son verdaderos no-huésped de V. dahliae D. Plantas de ambos clones crecieron asintomáticas durante más de 1 año tras su inoculación con una suspensión de conidias del hongo por inyección en el tallo o inmersión del sistema radical, mediante trasplante a suelo infestado, o conjuntamente por estos dos últimos métodos, con densidades de inóculo de V. dahliae D y condiciones de incubación en cámaras de crecimiento, umbráculo y microparcelas que determinaron enfermedad severa en olivo \’Picual\’ y sandía \’Sugar Baby\’ en tres experimentos independientes. Además, V. dahliae D no estableció infecciones en el tallo de las paulownias inoculadas, según los resultados negativos de ensayos de aislamiento en medio selectivo y \’nested\’ PCR\’ específica de dicho patotipo utilizando ADN total extraído de tallos de plantas inoculadas, que fueron positivos con tallos de olivos sintomáticos. V. dahliae se aisló en limitada extensión de raíces de dichas plantas 30 semanas después de la inoculación por trasplante a suelo infestado, pero no de aquéllas que se inocularon solo por inmersión radical. En consecuencia, paulownia pare

El uso de portainjertos resistentes al patotipo defoliante (D) de Verticillium dahliae sería de gran interés para el control integrado de la Verticilosis del olivo y la producción de cultivares de olivo susceptibles de interés comercial en áreas donde prevalece dicho patotipo. En investigaciones previas identificamos dos clones de acebuche (Ac-13, Ac-18) con fenotipo resistente a V. dahliae D con potencial de ser utilizados como portainjertos de olivos susceptibles. En este trabajo se ha confirmado y caracterizado la reacción resistente de dichos clones a V. dahliae D, y se ha identificado un nuevo clon resistente (Ac-4), en una serie de bioensayos utilizando protocolos de inoculación y condiciones de incubación óptimas para el desarrollo de la Verticilosis en olivo susceptible. La reacción de los clones se evaluó por la severidad e incidencia de síntomas foliares, el aislamiento del patógeno a lo largo del tallo, y la cuantificación del DNA del hongo en el tejido caulinar mediante un protocolo de qPCR con un límite de detección de 18 fg de V. dahliae DNA en tejidos infectados asintomáticos. Los clones AC13 and AC18 mostraron una reacción altamente resistente en inoculaciones por inmersión radical con V. dahliae 138I. La concentración de V. dahliae DNA por 100 ng de DNA de tallo fue 10,9 y 86,7 pg en plantas de \’AC13\’ y \’AC18\’, respectivamente, comparado con 42,9 pg y 16,6 ng en olivos \’Frantoio\’ y \’Picual\’, respectivamente. Bioensayos adicionales en condiciones óptimas para la infección por el patógeno demostraron que: i) la naturaleza de la reacción resistente de dichos clones no es modificada por la infección conjunta de su sistema radical por Glomus intraradices o Meloidogyne javanica, y ii) el injerto de olivo \’Arbequina\’ o \’Picual\’ sobre portainjertos de \’Ac-13\’ o \’Ac-18 protege satisfactoriamente a dichos cultivares de la enfermedad en condiciones que determinan la muerte de plantones autoenraizados de ellos.

[Background] Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris, a main threat to global chickpea production, is managed mainly by resistant cultivars whose efficiency is curtailed by Fusarium oxysporum f. sp. ciceris races., [Methodology] We characterized compatible and incompatible interactions by assessing the spatial-temporal pattern of infection and colonization of chickpea cvs. P-2245, JG-62 and WR-315 by Fusarium oxysporum f. sp. ciceris races 0 and 5 labeled with ZsGreen fluorescent protein using confocal laser scanning microscopy., [Findings] The two races colonized the host root surface in both interactions with preferential colonization of the root apex and subapical root zone. In compatible interactions, the pathogen grew intercellularly in the root cortex, reached the xylem, and progressed upwards in the stem xylem, being the rate and intensity of stem colonization directly related with the degree of compatibility among Fusarium oxysporum f. sp. ciceris races and chickpea cultivars. In incompatible interactions, race 0 invaded and colonized 'JG-62' xylem vessels of root and stem but in 'WR-315', it remained in the intercellular spaces of the root cortex failing to reach the xylem, whereas race 5 progressed up to the hypocotyl. However, all incompatible interactions were asymptomatic., [Conclusions] The differential patterns of colonization of chickpea cultivars by Fusarium oxysporum f. sp. ciceris races may be related to the operation of multiple resistance mechanisms. © 2013 Jiménez-Fernández et al.

Beauveria bassiana strain 04/01-Tip obtained from larvae of the opium poppy stem gall Iraella luteipes endophytically colonizes opium poppy plants and protect it against this pest. Development of a specific, rapid and sensitive technique that allows accurately determining the process and factors leading to the establishment of this strain in opium poppy plants would be essential to achieve its efficient control in a large field scale. For that purpose in the present study, species-specific primers that can be used in conventional or quantitative PCR protocols were developed for specifically identification and detection of B. bassiana in plant tissues. The combination of the designed BB.fw/BB.rv primer set with the universal ITS1-F/ITS4 primer set in a two-step nested-PCR approach, has allowed the amplification of up to 10. fg of B. bassiana. This represented an increase in sensitivity of 10000- and 1000-fold of detection than when using the BB.fw/BB.rv primers in a single or single-tube semi-nested PCR approaches, respectively. The BB.fw and BB.rv primer set were subsequently optimized to be used in real time quantitative PCR assays and allowed to accurately quantify B. bassiana DNA in different plant DNA backgrounds (leaves and seeds) without losing accuracy and efficiency. The qPCR protocol was used to monitor the endophytic colonization of opium poppy leaves by. B. bassiana after inoculation with the strain EABb 04/01-Tip, detecting as low as 26. fg of target DNA in leaves and a decrease in fungal biomass over time. PCR quantification data were supported in parallel with CLMS by the monitoring of spatial and temporal patterns of leaf and stem colonization using a GFP-tagged transformant of the B. bassiana EABb 04/01-Tip strain, which enabled to demonstrate that B. bassiana effectively colonizes aerial tissues of opium poppy plants mainly through intercellular spaces and even leaf trichomes. A decline in endophytic colonization was also observed by the last sampli

La presente invención proporciona un método, basado en PCR convencional o cuantitativa en tiempo real (qPCR), fiable, rápido, exacto y reproducible para la detección y cuantificación de cualesquiera de las ocho razas patogénicas del hongo Fusarium oxysporum f. sp. ciceris (0, 1A, 1B/C, 2, 3, 4, 5 ó 6) en una muestra biológica aislada. Dicho método permite la detección y cuantificación de, como mínimo, 1 pg de ADN del patógeno en muestras biológicas complejas incluyendo diferentes tejidos de la planta de garbanzo y suelos infestados, sin que su exactitud y eficacia se vea alterada. La invención también se refiere a dos parejas de cebadores útiles para llevar a cabo el método de la invención.

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infestedwith several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.© 2011 The American Phytopathological Society.

The proper identification and quantification of F. oxysporum populations inhabiting soil and plant rhizosphere niches are of importance for soil microbial ecology and plant pathology. In this study, we report the improvement of a PCR protocol for the specific identification of the F. oxysporum species complex and its conversion into a real-time qPCR assay for the quantification up to 1. pg of the fungus DNA in soil and different plant tissues. The amplification efficiency, sensibility and reproducibility of qPCR assays were not influenced by presence of non-target DNA from either plant or soil. The applicability of the newly developed qPCR protocol for F. oxysporum population studies was demonstrated using the technique for quantifying the fungus in different complex environmental samples. The use of the qPCR protocol allowed to accurately quantify up to 25. pg of F. oxysporum/g of naturally infested field soil, as well as to identify significant differences in the amount of F. oxysporum DNA in roots of different chickpea cultivars grown in a field soil infested with diverse pathogenic and nonpathogenic F. oxysporum populations. This qPCR protocol may be especially important for studies on soil microbial ecology and plant pathology since it provides a new opportunity for analyzing F. oxysporum populations and their interactions with the soil microflora, environment and plant host genotypes. © 2010 Elsevier B.V.