http://hdl.handle.net/10366/4028 2026-06-04T16:44:16Z http://hdl.handle.net/10366/171615 [EN]To enhance the framework of the mechanisms of action used by Trichoderma gamsiiT6085 for the control of Fusarium head blight (FHB), this work investigated its abil-ity to modulate the expression of defence-related genes of wheat (Triticum aestivum‘Apogee’) in response to endophytic colonization of plant tissues. Changes in relativeexpression of pal1, pr1, pgip2 and lox1 genes were assessed over time in wheat roots,in spikes colonized by T6085 alone and both T6085 and Fusarium graminearum, and inleaves from wheat seedlings root-inoculated with T6085. Results indicate the ability ofT6085 to induce local and systemic defence responses in wheat plants in the presenceof one of the causal agents of FHB. There was a general significant up-regulation ofthe plant defence-related genes analysed, especially in the first days after the applica-tion of T6085. According to these results, modulation of plant defence genes could beincluded within the arsenal of mechanisms used by T6085 when applied to wheat, anadditional feature of interest in the management of FHB. To evaluate the effect of theplant genotype on the ability of T6085 to endophytically colonize roots, root coloniza-tion was assessed on four cultivars of T. aestivum and two cultivars of T. durum. Datashowed that roots of only two T. aestivum cultivars were endophytically colonized byT6085, similar to cv. Apogee used here as control, thus demonstrating an effect of thehost genotype on the endophytic ability of T6085. 2023-01-01T00:00:00Z http://hdl.handle.net/10366/171614 [EN]We explore the combined use of two beneficial fungal isolates, Trichoderma gamsii T6085 (Tg) and Clonostachys rosea IK726 (Cr), to enhance Fusarium head blight (FHB) management by biological control. We found no evidence for mycoparasitism or inhibition via diffusible metabolites, but Tg volatiles inhibited Cr growth slightly. Although Cr reduced Tg spore germination and mycelial growth in liquid culture, this effect seemed absent in planta. The BCAs differently modulated defence-related (DR) genes when colonizing roots or spikes. At seven days post-inoculation (dpi), root-applied Cr, alone and co-inoculated, induced a minor upregulation of PR1. In leaves, a systemic signalling response by root inoculation was detected. In spikes, Pal1, PR1, and Lox1 were upregulated by Cr alone and co-inoculated at 96 hours post-inoculation (hpi). However, Lox1 activation was enhanced by co-inoculation. On spikes inoculated with Fg, the BCAs revealed different patterns of DR gene modulation indicating involvement of different biocontrol mechanisms. In detail, Pgip2 was primarily upregulated at 24 hpi in co-inoculated spikes whereas at 72 hpi activation of DR genes was observed only with Tg. Notably, the disease incidence was reduced by 93 % by co-inoculation. In addition, the inoculum potential of F. graminearum on straw was reduced by all BCAs treatments, with ≥ 96 % reduction of perithecia after six months incubation. Our results show the potential of combining Tg and Cr as a more effective and stable FHB management strategy, than by treatments with the individual strains. 2025-07-01T00:00:00Z http://hdl.handle.net/10366/171613 [EN]Fusarium graminearum (Fg) is one of the most virulent causal agents of Fusarium head blight (FHB) in Central Europe. The disease is also caused by other Fusarium species within the FHB Species Complex (FHBSC). Some Fusarium species secrete killer proteins (KPs) during host plant infection. Fg produces KP4-L killer toxins (FgKP4L), of which the clustered Fgkp4l-1, -2, -3 genes and Fgkp4l-4 (encoding for the heterodimeric KP4L-4 protein) are expressed in competitive interactions against the biocontrol agent Trichoderma gamsii T6085. We investigated the involvement of the four Fgkp4l genes in the competition with other plant-pathogenic Fusarium species either lacking KP4L proteins or carrying different combinations of them. Fusarium sporotrichioides (Fs) and Fusarium langsethiae (Fl) belonging to the FHBSC, and the outgroup Fusarium verticillioides (Fv), were used for both in vitro and in vivo tests. To monitor mycotoxin production, relative expression of Tri4 gene included within the trichothecene biosynthetic pathway was also evaluated. In dual cultures, modulation of KP4L-encoding genes was tailored according to the facing species and the distance between fungi. Against Fs, the three clustered Fgkp4l genes were up-regulated but no changes on gene expression occurred with Fl. Fgkp4l-2 was up-regulated facing Fv during the two contact independent stages (Early sensing and Sensing) of the interaction. The Tri4 gene was expressed only during the in vitro interaction with Fv, but not on spikes. Fgkp4l-2 and -3 were up-regulated at 3 days post-inoculation on wheat heads inoculated with a mix of the four Fusarium species. Competition against the other Fusarium did not influence Fg growth rate on spikes. 2025-01-01T00:00:00Z http://hdl.handle.net/10366/171612 [EN]Citrus fruits, widely consumed around the world, can be negatively affected by pests and fungal infections during their cultivation, handling, transportation, and storage, thus resulting in substantial yield losses and food waste. The use of natural preservatives like chitosan (CHT) and essential oils (EOs) is a promising approach for reducing chemical inputs to preserve food products. This study investigated the effects of CHT, extracted from crab shells and the fungus Pleorotus ostreatus, alone and in combination with mandarin (Citrus × reticulata Blanco, Rutaceae) essential oil (MEO), as an oviposition deterrent towards Ceratitis capitata (Diptera: Tephritidae), the Mediterranean fruit fly, and growth inhibitor of Penicillium (P. expansum, P. digitatum, and P. italicum) spp. fungi, the causal agents of apple and citrus rot. A solution of 1.0% CHT of both origins (from crab shells and P. ostreatus) added with MEO resulted as the best combination to significantly reduce the oviposition percentage of C. capitata as well as mycelial growth and spore germination of Penicillium isolates and their pathogenic activity on Citrus japonica Thunb. (kumquats) fruits. According to results here collected, CHT added with MEO represents a valid combination to be used as an edible film and coating as part of an integrated control strategy to improve the shelf-life of fresh citrus fruits. Furthermore, fungal CHT, here used for the first time in combination with MEO, can be an excellent alternative to reply to the eating habits and necessities of the final consumers. 2024-07-13T00:00:00Z http://hdl.handle.net/10366/171611 [EN]Plants constantly face the environment that surrounds them and fight for survival against biotic and abiotic stress factors. To deal with harmful conditions, plants have developed a multilayer defence system, making them capable of recognising threats and promptly recovering from them. This phenomenon, which takes advantage of the “memory effect”, is referred to as bio-priming and represents a new frontier in terms of crop protection. Here, we investigated the “indirect” protective mechanisms of a new yeast extract formulate in Vitis vinifera cv. Sangiovese plants at both the biochemical and genic levels. The formulate was applied once a week for three consecutive weeks, and grapevine leaves were sampled from the first to the fifth day after treatment (dat) at every week of the experiment. Increased levels of jasmonic acid (every week at 2 dat; +70% as average) and abscisic acid (at 1 dat of the first week, more than 1.7-fold higher than the control) and the underproduction of salicylic acid (from 2 dat; −18%) confirmed that these signalling molecules/”specialised compounds” are actively involved in the early activation of defence pathways in treated vines. In addition, pr2 and chit1b, two genes involved in regulating hormonal crosstalk, were significantly up-regulated (both in the first and second week of the trial) and were also found to underlie upstream molecular activation. The results obtained by this investigation confirm the use of this new product to prime and protect grapevines from a wide range of fungal and fungal-like plant pathogens through the induction of defence responses. 2024-06-01T00:00:00Z http://hdl.handle.net/10366/170354 [ES] Las RASopatías asociadas a alteraciones en los genes NF1 y SPRED1 presentan una marcada heterogeneidad clínica y molecular, donde el principal obstáculo tras el diagnóstico genético es la interpretación funcional de las variantes identificadas. Con el objetivo de superar este desafío, la presente tesis doctoral ha creado un flujo de trabajo experimental multidisciplinar que combina el análisis de variantes en NF1 y SPRED1 reportadas en bases de datos públicas y de pacientes de la DIERCyL, el modelado estructural y de estabilidad proteica in silico, la señalización celular, proliferación, y morfometría en las líneas celulares HEK293T y NIH3T3. Los análisis de las bases de datos genéticas identificaron hotspots estructurales críticos en la proteína neurofibromina (CSRD, GRD y Armadillo1), mientras que las variantes en SPRED1 mostraron una distribución homogénea, sugiriendo mecanismos patogénicos distintos. En el caso de las variantes en NF1 y SPRED1 de los pacientes, el análisis energético reveló un espectro continuo en neurofibromina que va desde haploinsuficiencia hasta dominancia negativa en truncantes tardías, mientras que en SPRED1 predominó la pérdida de función. Los ensayos celulares in vitro, mostraron dinámicas de señalización celular diferentes según el tipo de variante analizada en los genes NF1 y SPRED1. En particular, la hiperactivación de ERK emergió como el evento molecular más determinante para la patogenicidad de las variantes. La coactivación de p38 identificó variantes de mayor severidad clínica, mientras que la relación inversa p- ERK/p-AKT(Ser473) reveló un circuito de retroalimentación negativa entre las rutas de señalización MAPK y PI3K/AKT. Los ensayos de proliferación celular y el análisis cuantitativo del tamaño celular revelaron fenotipos consistentes con el grado de desregulación de MAPK, proporcionando biomarcadores fenotípicos adicionales para diferenciar entre variantes patogénicas y benignas en ambos genes. La relevancia tisular de HEK293T (contextos epiteliales/neuroectodérmicos) y NIH3T3 (mesenquimales) permitió reproducir la heterogeneidad clínica observada en pacientes. Los resultados de esta tesis doctoral han permitido reclasificar variantes de significado incierto en ambos genes e identificar la hiperactivación de ERK como diana terapéutica prioritaria, apoyando el uso de inhibidores de MEK en contextos compatibles con esta desregulación. En conjunto, este trabajo establece un marco integral para la interpretación funcional de variantes en RASopatías y para el desarrollo de futuras estrategias de medicina personalizada.; [EN] RASopathies associated with alterations in the NF1 and SPRED1 genes exhibit marked clinical and molecular heterogeneity, where the main challenge after genetic diagnosis lies in the functional interpretation of the identified variants. To address this issue, this doctoral thesis established a multidisciplinary experimental workflow that integrates the analysis of NF1 and SPRED1 variants reported in public databases and in patients from the DIERCyL cohort, together with in silico structural and protein-stability modeling, cellular signaling assays, proliferation studies, and morphometric analyses in HEK293T and NIH3T3 cell lines. Database analyses identified critical structural hotspots in neurofibromin (CSRD, GRD, and Armadillo1), whereas SPRED1 variants showed a more homogeneous distribution, suggesting distinct pathogenic mechanisms. In patient-derived NF1 and SPRED1 variants, energy-based analyses revealed a continuous spectrum in neurofibromin, ranging from haploinsufficiency to dominant-negative effects in late truncating variants, while SPRED1 alterations predominantly showed loss-of-function behavior. The in vitro cellular assays revealed distinct signaling dynamics depending on the type of variant analyzed in NF1 and SPRED1. Notably, ERK hyperactivation emerged as the most determinant molecular event for pathogenicity. Co-activation of p38 identified variants associated with more severe clinical outcomes, whereas the inverse p-ERK/p- AKT(Ser473) relationship revealed a negative feedback circuit between the MAPK and PI3K/AKT pathways. Cell-proliferation assays and quantitative cell-size analyses uncovered phenotypes consistent with the degree of MAPK deregulation, providing additional phenotypic biomarkers to discriminate between pathogenic and benign variants in both genes. The tissue relevance of HEK293T (epithelial/neuroectodermal contexts) and NIH3T3 (mesenchymal) cells allowed us to recapitulate the clinical heterogeneity observed in patients. The results of this doctoral thesis enabled the reclassification of variants of uncertain significance in both genes and identified ERK hyperactivation as a priority therapeutic target, supporting the use of MEK inhibitors in contexts consistent with this dysregulation. Taken together, this work establishes a comprehensive framework for the functional interpretation of variants in RASopathies and for the development of future personalizedmedicine strategies. 2025-01-01T00:00:00Z http://hdl.handle.net/10366/169986 [EN]Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing 2021-01-01T00:00:00Z http://hdl.handle.net/10366/169937 [EN]Colletotrichum is a fungal genus (Ascomycota, Sordariomycetes, Glomerellaceae) that includes many economically important plant pathogens that cause devastating diseases of a wide range of plants. In this work, using a combination of long- and short-read sequencing technologies, we sequenced the genome of Colletotrichum lupini RB221, isolated from white lupin (Lupinus albus) in France during a survey in 2014. The genome was assembled into 11 nuclear chromosomes and a mitochondrial genome with a total assembly size of 63.41 Mb and 36.55 kb, respectively. In total, 18,324 protein-encoding genes have been predicted, of which only 39 are specific to C. lupini. This resource will provide insight into pathogenicity factors and will help provide a better understanding of the evolution and genome structure of this important plant pathogen 2021-12-01T00:00:00Z http://hdl.handle.net/10366/169930 [EN]KP4 killer toxins are secreted proteins that inhibit cell growth and induce cell death in target organisms. In Fusarium graminearum, KP4-like (KP4L) proteins contribute to fungal virulence in wheat seedling rot and are expressed during Fusarium head blight development. However, fungal KP4L proteins are also hypothesized to support fungal antagonism by permeabilizing cell walls of competing fungi to enable penetration of toxic compounds. Here, we report the differential expression patterns of F. graminearum KP4L genes (Fgkp4l-1, -2, -3 and -4) in a competitive interaction, using Trichoderma gamsii as the antagonist. The results from dual cultures indicate that Fgkp4l-3 and Fgkp4l-4 could participate in the recognition at the distance of the antagonist, while all Fgkp4l genes were highly activated in the pathogen during the physical interaction of both fungi. Only Fgkp4l-4 was up-regulated during the interaction with T. gamsii in wheat spikes. This suggests the KP4L proteins could participate in supporting F. graminearum interspecific interactions, even in living plant tissues. The distribution of KP4L orthologous within the genus Fusarium revealed they are more represented in species with broad host-plant range than in host-specific species. Phylogeny inferred provides evidence that KP4L genes evolved through gene duplications, gene loss and sequence diversification in the genus Fusarium 2022-09-16T00:00:00Z http://hdl.handle.net/10366/169920 [EN]Species of fungal genus Trichoderma are characterized by a versatile lifestyle, high adaptability to the changing environmental conditions and the ability to establish sophisticated interactions with other organisms. Due to their ability to antagonize plant pathogens and to elicit the plant defence responses against biotic/abiotic stresses, Trichoderma spp. are commonly used as commercially biopesticides and biofertilizers. The Trichoderma success in the rhizosphere is supported by a wide arsenal of specialised metabolites (SMs) providing morphological and physiological autoregulation, self-protection and facilitating fungal communication. This review aims to explore the roles of SMs in the biology of fungi, with special emphasis on the genus Trichoderma and on how divergence in the SMs genetic structure determine Trichoderma lifestyles. Trichoderma genomes are endowed with a high number of SMs biosynthetic genes, and understanding the genetic basis of their biosynthesis is crucial for determining the role of these metabolites in Trichoderma ecophysiology and for expanding their application in crop protection. Recent advances on the characterization of the Trichoderma SMs genetic inventory driven by computational biology are discussed 2022-01-01T00:00:00Z http://hdl.handle.net/10366/169919 [EN]Trichoderma is a fungal genus comprising species used as biocontrol agents in crop plant protection and with high value for industry. The beneficial efects of these species are supported by the secondary metabolites they produce. Terpenoid compounds are key players in the interaction of Trichoderma spp. with the environment and with their fungal and plant hosts; however, most of the terpene synthase (TS) genes involved in their biosynthesis have yet not been characterized. Here, we combined comparative genomics of TSs of 21 strains belonging to 17 Trichoderma spp., and gene expression studies on TSs using T. gamsii T6085 as a model. An overview of the diversity within the TS-gene family and the regulation of TS genes is provided. We identified 15 groups of TSs, and the presence of clade-specific enzymes revealed a variety of terpenoid chemotypes evolved to cover diferent ecological demands. We propose that functional diferentiation of gene family members is the driver for the high number of TS genes found in the genomes of Trichoderma. Expression studies provide a picture in which diferent TS genes are regulated in many ways, which is a strong indication of diferent biological functions. 2020-10-01T00:00:00Z http://hdl.handle.net/10366/169918 [EN]Fungal pathogens are the main factors responsible for the most severe diseases affecting plants, leading to significant reduction in yield and crop quality and causing enormous economic losses worldwide. It is estimated that around 30% of the emerging diseases are caused by fungi thus requiring new strategies to improve their management. Biological control approach, frequently referred to the use of non-pathogenic microbial antagonists or products derived from their metabolism, represents a valid and promising alternative under a more ecological perspective to reduce the activities and to control populations of target pathogens. However, although the use of antagonists belonging to species different from that of the pathogen has been successfully reported, the use of competitors belonging to the same species of the pathogen is not widespread. A biocontrol strategy based on competition for space and nutrients and/or the induction of plant defenses against virulent pathogens performed by attenuated or avirulent pathogens could, therefore, be considered a valid alternative. 2019-02-15T00:00:00Z http://hdl.handle.net/10366/169917 [EN]Fusarium graminearum está entre los principales agentes causales del tizón de la espiga por Fusarium (FHB) o escabiosis en trigo y otros cereales, enfermedad presente en todo el mundo y producida por un complejo de especies de Fusarium. Además de causar pérdidas económicas importantes en rendimiento y calidad de los cultivos, F. graminearum representa una amenaza grave para la salud animal y humana. En este trabajo presentamos la primera secuencia genómica preliminar (borrador) del genoma completo de la cepa micotoxigénica Fusarium graminearum ITEM 124, proporcionando además información útil para estudios de genómica comparativa. 2017-11-09T00:00:00Z http://hdl.handle.net/10366/169884 [EN]The gene Bcin04g03490 has been shown to control development and pathogenicity in the plant necrotroph Botrytis cinerea. We have identified the orthologous gene in two formae speciales of the Fusarium oxysporum species complex; it maps to chromosome 7 in the core genome of the lycopersici forma specialis. It encodes a protein with two distinct domains, a GAL4-like Zn(II)2Cys6 binuclear cluster DNA-binding domain and an acetyltransferase domain, as previously shown in B. cinerea. Null mutants were generated in both formae speciales (phaseoli and lycopersici), and the phenotypes exhibited during in culture growth and plant colonization were analyzed. The results obtained demonstrate that this virulence factor is required for complete colonization of the plant host stem vascular tissue and full virulence in F. oxysporum but not for the initial stages of root colonization. In addition, null mutants showed a severe alteration in the formation of aerial mycelia and a drastic reduction of sporulation. These observations are consistent with a master role of FoG4MAT in the gene regulation of different processes crucial for normal development and full disease induction in the plant host. 2025-07-01T00:00:00Z http://hdl.handle.net/10366/169877 [EN]In planta gene expression analysis and GFP-based confocal microscopy are two powerful techniques that may be coupled to assess the extent and dynamics of plant colonization by a fungal pathogen. Here we describe methods to prepare common bean plants for inoculation with a highly virulent strain of Fusarium oxysporum f. sp. phaseoli, quantify the extent of colonization by RT-qPCR, and visualize the colonized tissues by confocal microscopy. 2022-01-01T00:00:00Z http://hdl.handle.net/10366/169874 [EN]Natural populations provide valuable information and resources for addressing the genetic characterization of biological systems. Botrytis cinerea is a necrotrophic fungus that exhibits complex responses to light. Physiological analysis of B. cinerea populations from vineyards in Castilla y León (Spain) allowed for the identification of isolate Bc116. This field isolate shows a reduced pathogenicity that is conditioned by the light regime. Light also delays germination and accentuates the negative effect it exerts on the vegetative growth of B. cinerea. Bc116 also displays a marked hyperconidiation phenotype and a characteristic sclerotia production pattern. Genetic analysis demonstrates that the alternative phenotypes regarding pathogenicity, conidiation, and pattern of sclerotia production co-segregate in the progeny of crosses between isolate Bc116 and a wild-type field isolate, Bc448, showing that they are under the control of a single locus. By applying a strategy based on bulked segregant analysis, the mutation in Bc116 has been mapped to a 200 kb region on Chr14 and the analysis of this region identifies a 2 kb deletion affecting the bcltf1 gene, encoding the B. cinerea Light Responsive Transcription Factor 1 described in the reference isolate B05.10. Transformation of Bc116 with the B05.10 bcltf1 allele restored the wild-type phenotypes, providing functional evidence that the natural mutant Bc116 is altered in gene bcltf1. This study offers additional information, derived from the analysis of the genetic background of a natural mutant, on the physiological processes regulated by BcLTF1 and supports the key role of this TF in the pathogenicity and photobiology of B. cinerea. 2025-04-08T00:00:00Z