Taxonomía multifásica de hongos endófitos del orden hypocreales en rubiaceae de bosque natural de Costa Rica
Fecha
2023
Tipo
tesis doctoral
Autores
Escudero Leyva, Efraín
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Editor
Resumen
Los problemas fitosanitarios relacionados al café en Latinoamérica y otras áreas en el mundo, han registrado un aumento en la frecuencia y la severidad de las enfermedades producidas por hongos como Hemileia vastatrix ó Mycena citricolor. Los hongos endófitos son importantes simbiontes de plantas, conocidos por tener un amplio rango de papeles ecológicos, habitando los tejidos internos sin causar efectos negativos aparentes o inmediatos en la salud del hospedero. Basados en la hipótesis de que los hongos endófitos aislados de bosques prístinos pueden conferir protección a las plagas a través de diferentes mecanismos, el presente trabajo comprende el aislamiento de hongos endófitos de plantas silvestres de Rubiaceae de áreas naturales de Costa Rica para probarlos contra patógenos que afectan al café. Se presentó un protocolo para analizar datos ITS fúngicos, desde el preprocesamiento de calidad de secuencias hasta la clasificación taxonómica, asignación de rasgos funcionales y análisis filogenético, clasificando 1400 aislamientos de hongos endófitos de Rubiaceae silvestre en aproximadamente 800 especies. Para comenzar a generar una alternativa verde para el control de M. citricolor, se seleccionaron los hongos endófitos Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12 y Xylaria multiplex GU14T. Los aislamientos de Trichoderma spp. produjeron los mayores porcentajes de inhibición del crecimiento in vitro. Posteriormente, se verificó la colonización endofítica, seguida de ensayos de promoción y antagonismo del crecimiento in planta, demostrando que Trichoderma CT5 y G1C tienen potencial para promover el crecimiento de las plantas y antagonizar contra M. citricolor, reduciendo la incidencia, la severidad y previniendo la mortalidad de las plantas. Estos dos aislamientos posteriormente mostraron tolerancia a fungicidas, obteniendo que CT5 tiene capacidad de remoción de hasta 89% para clorotalonil, 46,4% para ciproconazol y 33,1% para trifloxistrobina. En el caso de la azoxistrobina, la mayor remoción (82,2%) se produjo por adsorción a la biomasa fúngica, al mismo tiempo que detoxifica la matriz acuosa. Posteriormente, se compararon los perfiles volátiles de GU11N con y sin la presencia del patógeno M. citricolor mediante ensayos sin contacto y de cultivo dual, utilizando métodos de GC-MS-HS-SPME y UHPLC-HRMS/MS para identificar compuestos no volátiles de extractos orgánicos de los micelios involucrados en la interacción. Los resultados mostraron más compuestos volátiles usando HS-SPME (39 componentes) que con la técnica HS (13 componentes), compartiendo solo 12 compuestos. Las pruebas estadísticas sugirieron que D. eschscholtzii inhibe el crecimiento de M. citricolor a través de la liberación de COVs que contienen una combinación de 1,8-dimetoxinaftaleno y compuestos terpénicos afectando las estructuras reproductivas del patógeno, por lo que se corroboraron los efectos nocivos del 1,8-dimetoxinaftaleno en una prueba in vitro confirmando el daño estructural con fotografías SEM. Los datos de UHPLC-HRMS/MS, revelaron un predominio de derivados de ácidos grasos entre los compuestos identificados. Como parte del estudio de las plantas de Rubiaceae de donde provenían los endófitos, se desarrolló un flujo de trabajo de alto rendimiento para identificar diversos fitoquímicos. El estudio de metabolitos secundarios se realizó en hojas de una variedad comercial de café y dos especies silvestres de Rubiaceae, siendo la cafeína, la trigonelina y la teobromina muy abundantes en las hojas de café, mientras que las moléculas relacionadas con la defensa química, como los fenilpropanoides, el ácido giberélico y el monolignol sinaldehído se encontraron más abundantemente en las hojas silvestres de Rubiaceae. Finalmente, se colaboró integrando datos taxonómicos y metabolómicos a la plataforma MicrobeMASST, una herramienta de búsqueda de espectrometría de masas con información taxonómica altamente curada para la anotación de metabolitos microbianos en experimentos de metabolómica no dirigidos. Esta plataforma contiene 60 000 monocultivos microbianos, permitiendo buscar espectros de MS/MS conocidos y desconocidos y vincularlos con sus respectivos productores microbianos a través de patrones de fragmentación de MS/MS.
The phytosanitary problems related to the coffee in Latin America and other producer regions around the globe, have experienced an increase in the frequency and severity of the diseases produced by fungi like Hemileia vastatrix or Mycena citricolor, in part, because of the climate change. The endophytic fungi are important plant symbionts, known because of their wide ecological guilds, inhabiting the internal tissues without causing immediate negative damages in their hosts. Based in the hypothesis that, endophytic fungi isolated from pristine areas can confer protection against pathogens through different mechanisms, the current work, includes the endophytic fungi isolation from wild Rubiaceae plants in natural areas in Costa Rica to test them against coffee pathogens. A protocol to analyze fungal ITS data is presented, including the steps from the pre-processing of raw sequences up to the taxonomical classification, ecological guilds and phylogenetic analyses. With this, 1400 endophytic isolates from wild Rubiaceae plants were classified in nearly 800 species. Later, to propose a green alternative against M. citricolor, the following fungi were selected Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12 and Xylaria multiplex GU14T. From this, the isolates belonging to Trichoderma spp. obtained the better inhibition values on in vitro conditions. Later, thee endophytic colonization was confirmed and Trichoderma CT5 and G1C resulted with growth promotion and antagonistic capacities against M. citricolor, reducing incidence, severity and preventing mortality, in tests with coffee plantlets. These two isolates also resulted with tolerance to fungicides, where CT5 had a remotion capacity of 89% for clorotalonil, 46.4% for ciproconazol and 33.1% for trifloxistobine, according to available biomass. For azoxistrobine, the remotion (82.2%) occurred as a result of absorption to the fungal biomass, while detoxification of the matrix was a simultaneous process. In the third part of the results, the volatile profiling of GU11N with and in the absence of M. citricolor was done through contactless and in coculture experiments, using GC-MS-HS-SPME and UHPLC-HRMS/MS to identify the nonvolatile compounds from the organic extractions from mycelia recovered from the interaction zone. Results showed more volatile compounds using HS-SPME (39 components) rather than HS (13 components), sharing only 12 compounds. The statistical tests suggested that D. eschscholtzii was inhibiting the growth of M. citricolor trough the release of VOCs containing a combination of 1,8- dimethoxynaphtalene and terpenic compounds, affecting the reproductive structures of the pathogen, this was confirmed with an-in vitro test, obtaining SEM photographs with evidence of the structural damage in the pathogen. The UHPLC-HRMS/MS data revealed many fatty acids among the determined compounds. As part of the study related to the Rubiaceae plants from where the endophytes were retrieved, a pipeline for identification and quantification of polyphenols was done. The workflow was optimized to extract over 40 families of phytochemicals and the developed metabolomic platform was able of identification and quantification of 184 polyphenols in 15 minutes. The study of secondary metabolites was performed in leaves of coffee and two wild species of Rubiaceae. Caffeine, trigonelline and theobromine were abundant in the coffee leaves, while compounds related to the chemical defense, like phenylpropanoids, gibberellic acid and monolignol sinaldehyde were found abundantly in the wild Rubiaceae. Finally, as part of the collaboration with the MicrobeMASST platform, the taxonomic and metabolomic data related to the endophytes was integrated. This is a tool for the search of mass spectrometry with highly curated taxonomic data for the annotation of microbial metabolites in non-targeted metabolomic experiments. This platform contains 60 000 microbial monocultures, allowing to search for known and unknown MS/MS spectra and link them with their current microbial producer’s trough the MS/MS fragmentation patterns.
The phytosanitary problems related to the coffee in Latin America and other producer regions around the globe, have experienced an increase in the frequency and severity of the diseases produced by fungi like Hemileia vastatrix or Mycena citricolor, in part, because of the climate change. The endophytic fungi are important plant symbionts, known because of their wide ecological guilds, inhabiting the internal tissues without causing immediate negative damages in their hosts. Based in the hypothesis that, endophytic fungi isolated from pristine areas can confer protection against pathogens through different mechanisms, the current work, includes the endophytic fungi isolation from wild Rubiaceae plants in natural areas in Costa Rica to test them against coffee pathogens. A protocol to analyze fungal ITS data is presented, including the steps from the pre-processing of raw sequences up to the taxonomical classification, ecological guilds and phylogenetic analyses. With this, 1400 endophytic isolates from wild Rubiaceae plants were classified in nearly 800 species. Later, to propose a green alternative against M. citricolor, the following fungi were selected Daldinia eschscholzii GU11N, Nectria pseudotrichia GUHN1, Purpureocillium aff. lilacinum CT24, Sarocladium aff. kiliense CT25, Trichoderma rifaii CT5, T. aff crassum G1C, T. aff. atroviride G7T, T. aff. strigosellum GU12 and Xylaria multiplex GU14T. From this, the isolates belonging to Trichoderma spp. obtained the better inhibition values on in vitro conditions. Later, thee endophytic colonization was confirmed and Trichoderma CT5 and G1C resulted with growth promotion and antagonistic capacities against M. citricolor, reducing incidence, severity and preventing mortality, in tests with coffee plantlets. These two isolates also resulted with tolerance to fungicides, where CT5 had a remotion capacity of 89% for clorotalonil, 46.4% for ciproconazol and 33.1% for trifloxistobine, according to available biomass. For azoxistrobine, the remotion (82.2%) occurred as a result of absorption to the fungal biomass, while detoxification of the matrix was a simultaneous process. In the third part of the results, the volatile profiling of GU11N with and in the absence of M. citricolor was done through contactless and in coculture experiments, using GC-MS-HS-SPME and UHPLC-HRMS/MS to identify the nonvolatile compounds from the organic extractions from mycelia recovered from the interaction zone. Results showed more volatile compounds using HS-SPME (39 components) rather than HS (13 components), sharing only 12 compounds. The statistical tests suggested that D. eschscholtzii was inhibiting the growth of M. citricolor trough the release of VOCs containing a combination of 1,8- dimethoxynaphtalene and terpenic compounds, affecting the reproductive structures of the pathogen, this was confirmed with an-in vitro test, obtaining SEM photographs with evidence of the structural damage in the pathogen. The UHPLC-HRMS/MS data revealed many fatty acids among the determined compounds. As part of the study related to the Rubiaceae plants from where the endophytes were retrieved, a pipeline for identification and quantification of polyphenols was done. The workflow was optimized to extract over 40 families of phytochemicals and the developed metabolomic platform was able of identification and quantification of 184 polyphenols in 15 minutes. The study of secondary metabolites was performed in leaves of coffee and two wild species of Rubiaceae. Caffeine, trigonelline and theobromine were abundant in the coffee leaves, while compounds related to the chemical defense, like phenylpropanoids, gibberellic acid and monolignol sinaldehyde were found abundantly in the wild Rubiaceae. Finally, as part of the collaboration with the MicrobeMASST platform, the taxonomic and metabolomic data related to the endophytes was integrated. This is a tool for the search of mass spectrometry with highly curated taxonomic data for the annotation of microbial metabolites in non-targeted metabolomic experiments. This platform contains 60 000 microbial monocultures, allowing to search for known and unknown MS/MS spectra and link them with their current microbial producer’s trough the MS/MS fragmentation patterns.
Descripción
Palabras clave
FUNGI, TYPOLOGY, TROPICAL FORESTS, COSTA RICA