Estudio de la variabilidad genética y organización cromosómica en el hongo fitopatógeno "Botrytis Cinerea"

La presente memoria de Tesis Doctoral consta de 4 objetivos. En el primero de ellos se determinó las distintas poblaciones de hongos filamentosos presentes en los viñedos del marco vitivinícola de Jerez se estudió la influencia de la inoculación de una cepa de laboratorio de Botritis cinerea, obtenida tras el aislamiento sucesivo de conidios producidos in vitro a partir de un aislado de campo, sobre la micobiota propia de la vid. Los géneros de hongos filamentosos aislados en la viña fueron Aspergillus, Botrytis, Cladosporium y Penicillium, siendo Aspergillus y Cladospoium los mayoritarios y con comportamientos diferentes en cada año de muestreo, mientras que botrytis y Penicillium fueron más regulares y minoritarios. La inoculación de la viña modificó el desarrollo de la micobiota propia de la vid, así como el desarrollo de las cepas silvestres de Botrytis. Como segundo objetivo analizar la patogenicidad de B, cinerea asi como estudiar el control genético de este carácter en el cruzamiento entre dos cepas sexualmente compatibles, con el fin de contribuir al control racional de la enfermedad causada por este patógeno, denominada Podredumbre gris. El cruzamiento establecido ha dado lugar a cepas con niveles de virulencia muy diferentes, los cuales pueden ser empleados en retrocruzamientos e incluso en estudios comparativos que aporten nuevos datos sobre los mecanismos de patogenicidad de este hongo. En el tercer objetivo se realizó la caracterización molecular de numerosas cepas de B, cinerea y se evaluó al efecto que tien la reproducción sexual en la variabilidad genética del patógeno. El análisi de cariotipo electroforético puso de manifiesto que la reproducción sexual contriubuye considerablemente a la variabilidad de la especie y que es un proceso que tiene lugar en la naturaleza con mayor frecuencia de lo observado hasta el momento. Por último se realizó la estimación del número de cromosomas del patógeno así como el estudio de su variabilidad genética mediante la aplicación de la técnica denominada "telomeri fingerprinting". La aplicación de esta técnica y el análisis de cluster reveló un alto grado de variabilidad en B, cinerea y estableción entre 8 y 11 el número de cromosomas presentes en esta especie

Proteomic approach to Botrytis cinerea survival structures

Comunicaciones a congreso

Applying proteomic technology to dissect molecular aspect of phytopathogenic fungi, a Botrytis cinerea approach

Comunicaciones a congreso

Influence of the total concentration and the profile of volatile fatty acids on polyhydroxyalkanoates (PHA) production by mixed microbial cultures

Polyhydroxyalkanoates (PHAs) production from lignocellulosic biomass using mixed microbial cultures (MMC) is a potential cheap alternative for reducing the use of petroleum-based plastics. In this study, an MMC adapted to acidogenic effluent from dark fermentation (DF) of exhausted sugar beet cossettes (ESBC) has been tested in order to determine its capability to produce PHAs from nine different synthetic mixtures of volatile fatty acids (VFAs). The tests consisted of mixtures of acetic, propionic, butyric, and valeric acids in the range of 1.5–9.0 g/L of total acidity and with three different valeric:butyric ratios (10:1, 1:1, and 1:10). Experimental results have shown a consistent preference of the MMC for the butyric and valeric acids as carbon source instead other shorter acids (propionic or acetic) in terms of PHA production yield (estimated in dry cell weight basis), with a maximum value of 23% w/w. Additionally, valeric-rich mixtures have demonstrated to carry out a fast degradation process but with poor final PHA production compared with high butyric mixtures. Finally, high initial butyric and valeric concentrations (1.1 g/L and 4.1 g/L) have demonstrated to be counterproductive to PHA production.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was supported by the project CTM2016-79071-R (Spanish Ministry of Economy, Industry and Competitiveness) and financed by the Spanish State Research Agency ("Agencia Estatal de Investigacion" (AEI)) and by the European Regional Development Fund (ERDF) and by grant from the Program for the Promotion and Impulse of Research and Transfer of the University of Cadiz (Ref: IRTP04_UCA)

Development of Proteomics-Based Fungicides: New Strategies for Environmentally Friendly Control of Fungal Plant Diseases

Proteomics has become one of the most relevant high-throughput technologies. Several approaches have been used for studying, for example, tumor development, biomarker discovery, or microbiology. In this “post-genomic” era, the relevance of these studies has been highlighted as the phenotypes determined by the proteins and not by the genotypes encoding them that is responsible for the final phenotypes. One of the most interesting outcomes of these technologies is the design of new drugs, due to the discovery of new disease factors that may be candidates for new therapeutic targets. To our knowledge, no commercial fungicides have been developed from targeted molecular research, this review will shed some light on future prospects. We will summarize previous research efforts and discuss future innovations, focused on the fight against one of the main agents causing a devastating crops disease, fungal phytopathogens

4-Year in-situ assessment of a photocatalytic TiO2/SiO2 antifouling treatment for historic mortar in a coastal city

This manuscript presents a 4 years in-situ evaluation of TiO2/SiO2-based product applied to a mortar seating belonging to the bleachers of the Cadiz Roman Theatre, affected by biofouling and organic deposits. Antifouling performance was studied by colorimetric measurements and ATP analyses. Surface sampling was performed for biofouling identification, being remarkable the presence of filamentous fungi and cyanobacteria. Additionally, antifouling performance was checked during a 4 years in-situ monitoring study. SEM observations, carried out 4 years after the product application, demonstrate that the TiO2 integration in a SiO2 matrix produces a durable coating which allows for prolongation of the antifouling performance over time. According to the results obtained, 4 years after the application of the treatment, the treated area still maintains its antifouling efficacy, exhibiting a 40% reduction in organic deposit and 61% inhibition of biological growth compared to the untreated area. The results of this study show that the application of the SiO2/TiO2-based product may potentially reduce the costs of cleaning and maintenance works. At the same time, it is considered that the antifouling efficacy is more pronounced during the first two years and could be taken into account in the maintenance and re-application actions.This work has been supported by the Spanish Government MAT2017-84228-R (MINECO/AEI//FEDER, UE) and PID2020- 115843RB-I00/AEI/10.13039/501100011033. This work has been co-financed by the European Union under the 2014–2020 ERDF Operational Programme and by the Department of Economic Transformation, Industry, Knowledge, and Universities of the Regional Government of Andalusia. Project reference: FEDER-UCA18-106613. Giada M.C. Gemelli would also like to thank the Spanish Government for her pre-doctoral grants (PRE2018-085799). M. Luna would also like to thank the Spanish Government for his Margarita Salas grant (2021-067/PN/MS-RECUAL/CD) supported by the European Union-NextGenerationEU.Peer reviewe

Studying the influence of surface properties on the cell attachment and anti-fouling capacity of Ag/SiO2 superhydrophobic coatings for building materials

Anti-fouling coatings are a common solution for the protection of porous building materials from the effects of microbial colonization over their functionality and durability. Usually, this is achieved through the incorporation of biocides or the passive control by reducing bioreceptivity. Superhydrophobic surfaces are considered a promising strategy due to their reported capacity for reducing cell adhesion, but their affinity to non-polar substances may decrease their effectiveness under the right circumstances (e.g. organic contamination, cell walls with hydrophobic domains). The combination of these surfaces with active biocides may compensate these drawbacks, however, a close contact with the microorganisms is necessary to promote their effect. This work studies the factors that determine the anti-fouling capacity of a coating, tested on porous building materials, that combines superhydrophobic surface with a nanostructured Ag/SiO2 biocide agent. Special attention is paid to understanding to which extent the cell-surface interactions modulate the initial cell attachment to the surface and the biocidal effect. To this end, the electrostatic forces and surface energy balance were considered using different reference bacteria and a yeast. The results indicate that the hydrophobic character of the surface favors the cell attachment and the biocide agent may be unable to fully compensate this effect for all microorganisms. In addition, changes in micro and nano roughness seem to play an equally significant role. Overall, this study aims to provide a theoretical and experimental insight to assist in the future design of anti-fouling coatings tailored to the organisms responsible of fouling processes.This work has been financed by the Spanish State Research Agency (MCIN/AEI/ 10.13039/501100011033) R&D programs 2020–2021 (Project references: PID2020-115843RB-I00 and PDC2021-121652-I00); This work has been co-financed by the European Union under the 2014–2020 ERDF Operational Programme and by the Department of Economic Transformation, Industry, Knowledge, and Universities of the Regional Government of Andalusia (Project reference: FEDER- UCA18-106613)Peer reviewe

In Vitro Studies of Endophytic Bacteria Isolated from Ginger (<i>Zingiber officinale</i>) as Potential Plant-Growth-Promoting and Biocontrol Agents against <i>Botrytis cinerea</i> and <i>Colletotrichum acutatum</i>

Agriculture currently confronts a multitude of challenges arising from the excessive utilization of chemical pesticides and the proliferation of phytopathogenic fungi strains that exhibit resistance to commonly employed active compounds in the field. Botrytis cinerea and Colletotrichum acutatum are phytopathogenic fungi that inflict substantial economic losses within agriculture and food due to their high impacts on crops both pre- and post-harvest. Furthermore, the emergence of fungal strains that are resistant to commercial fungicides has exacerbated this problem. To explore more environmentally sustainable alternatives for the control of these pathogens, an investigation into the endophytic bacteria associated with ginger (Zingiber officinale Rosc.) was conducted. The primary focus of this study involved evaluating their inhibitory efficacy against the fungi and assessing their potential for promoting plant growth. The endophytic bacteria genera Lelliottia, Lysinibacillus, Kocuria, Agrococcus, Acinetobacter, Agrobacterium, Zymobacter, and Mycolicibacterium were identified. All isolates showed remarkable in vitro antagonistic ability against B. cinerea (>94%) and C. acutatum (>74%). Notably, the Lelliottia amnigena J29 strain exhibited a notable proficiency in producing extracellular enzymes and indole compounds (IAA), solubilizing phosphate and potassium, and forming biofilm. Furthermore, the Lysinibacillus capsici J26, Agrococcus citreus J28, and Mycolicibacterium sp. J5 strains displayed the capacity for atmospheric nitrogen fixation and siderophore production. These findings underscore the agricultural and biotechnological potential of endophytic bacteria derived from ginger plants and suggest the feasibility of developing alternative approaches to manage these two phytopathogenic fungi

Endophytic Microorganisms as an Alternative for the Biocontrol of <em>Phytophthora</em> spp.

The genus Phytophthora with more than 100 described species and 58 officially recognized, phylogenetically distributed in ten clades, are important pathogenic oomycete chromists that cause important diseases in agricultural crops, trees and forests worldwide. This genus is known as "The Plant Destroyer” which causes great economic losses with costs between 2 and 7 billion dollars per year in agricultural systems and unquantifiable losses in natural ecosystems. The host plants of the genus Phytophthora can vary from a wide range in some species to only one host, however, the host plants of the new species are still being determined and therefore the range continues to expand, that makes control exceedingly difficult. Plant damage can range from alterations in roots, fruits, trunks, stems, foliage and crown to invasive processes in highly susceptible species. Considering the wide range of hosts and organs that can be affected by Phytophthora, the use of endophytic microorganisms for the biocontrol of this phytopathogen can be an alternative to avoid losses of both crops and forests worldwide. Endophytes are microorganisms that live inside plant tissues without causing disease under any circumstances. The fact that endophytic microorganisms are able to colonize an ecological niche similar to that of some plant pathogens qualifies them as potential biocontrol agents. This chapter describes the endophytic bacteria and fungi isolated from different plant species that have shown antagonistic activity against different species of Phytophthora, as well as the metabolites isolated from these microorganisms that have shown fungicide activity and other biocontrol strategies (enzyme production, siderophores, substrate competition, among others) against Phytophthora