Caracterización de los metabolitos secundarios producidos por la cepa IBUN- 2755, involucrados en la actividad antimicrobiana y antifúngica contra patógenos de arroz

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dc.contributor.advisorUribe-Velez, Daniel
dc.contributor.advisorCastellanos Hernandez, Leonardo
dc.contributor.authorMiranda Martinez, Yessica Lorena
dc.contributor.researchgroupEstudio y Aprovechamiento de Productos Naturales Marinos y Frutas de Colombiaspa
dc.contributor.researchgroupMicrobiologia Agricolaspa
dc.date.accessioned2023-02-07T16:45:55Z
dc.date.available2023-02-07T16:45:55Z
dc.date.issued2022
dc.descriptionilustraciones, fotografías a colorspa
dc.description.abstractEl uso de organismos biocontroladores como estrategia que permita disminuir el uso de agroquímicos en los cultivos, es una práctica que está en aumento. Particularmente se hace relevante el desarrollo de productos de origen biológico que permitan el control de patógenos de arroz de manera específica y eficaz disminuyendo las tasas de contaminación y la reincidencia de enfermedades por la aparición de patógenos resistentes. Sin embargo, para llegar al desarrollo de un producto de origen biológico que tenga las características necesarias para ser efectivo en el control de enfermados fitosanitarias, es necesario tener un conocimiento amplio del organismo productor de los principios activos que harán parte de dicho producto. Bajo este contexto, el estudio de la cepa Bacillus velezensis IBUN 2755 se hace relevante ya que ha demostrado tener una fuerte actividad antibacteriana y antifúngica in vitro, contra tres patógenos que han sido reportados como los causantes de grandes pérdidas en cultivos de arroz y por tanto esta cepa se perfila como un potencial microorganismo biocontrolador. El estudio extensivo de la cepa incluye la caracterización de los posibles metabolitos secundarios bioactivos que produzca la cepa, que estén involucrados de manera directa en la actividad antibacterial y antifúngica conocida, asi como el efecto que tienen diferentes factores bióticos y abióticos sobre su producción. Este es precisamente el objetivo principal de este trabajo de investigación. El primer paso para llegar a este objetivo fue describir el efecto que tiene el crecimiento de la cepa IBUN 2755 en diferentes medios de cultivo, sobre la actividad antagónica de la cepa contra los patógenos Burkholderia. glumae, Rhizoctonia solani y Gaeumannomyces graminis. Para esto, se incubó la cepa IBUN 2755 en los medios de cultivo Landy, MES, MES modificado y MOLP P.E y MOLP P.U, que se caracterizan por tener condiciones nutricionales diferenciales. Se recuperaron los sobrenadantes libres de células de cada medio posterior al crecimiento de la cepa y se evaluó la actividad antagónica de los mismos sobre los tres patógenos antes mencionados. Se pudo concluir, que la composición nutricional de los medios tiene un efecto directo sobre la actividad antagónica de la cepa IBUN 2755, siendo el medio MES el que presentó sobrenadantes menos activos contra los tres patógenos y el medio MOLP P.U el que rindió los sobrenadantes más activos. Posteriormente con el fin de evaluar los metabolitos bioactivos que se estaban expresando en estos dos medios de cultivo que mostraron tener una actividad antagónica diferencial (MOLP.U y MES), se decidió hacer la extracción de estos usando dos metodologías de extracción: la partición liquido - liquido usando acetato de etilo y butanol y la precipitación ácida. Asi mismo estos procesos de extracción se realizaron sobre dos volúmenes diferentes de cada medio: 25 y 200 ml. De esta forma, al finalizar esta sección se tendrían tres parámetros a evaluar: primero, el efecto de la composición nutricional del medio de cultivo sobre la producción de metabolitos secundarios, segundo el efecto de la metodología de extracción sobre los metabolitos bioactivos recuperados de cada medio y tercero el efecto del volumen del medio de cultivo sobre la producción de metabolitos secundarios bioactivos por parte de la cepa IBUN 2755. La evaluación de la actividad biológica de los extractos de acetato de etilo, butanol y precipitación ácida obtenidos a partir de los sobrenadantes recuperados de los medios MOLP P.U y MES de 200 y 25 ml, sobre los tres patógenos, mostró que el extracto por precipitación acida del medio MOLP P.U de 25 ml, era el que demostraba una actividad antagónica consistente contra los tres patógenos. Asi mismo, se caracterizó el perfil metabolómico de cada uno estos extractos usando LC-MS/MS la construcción de redes moleculares y la derreplicación de compuestos, con esto se pudo establecer que la expresión de metabolitos secundarios por parte de la cepa IBUN 2755, era mayor en los medios de 25ml para el caso del medio MOLP P.U, además se evidencio que los extractos por precipitación acida y de acetato de etilo eran los más enriquecidos en compuestos con respecto a los extractos butanólicos y finalmente, se logró establecer que si bien la cepa IBUN 2755, es capaz de producir las mismas familias de compuestos, en los dos medios de cultivo (MOLP P.U y MES) esto no implica que se produzcan los mismos compuestos y en la misma proporción. Así mismo se evidencio la expresión de moléculas de interés como surfactinas, fengicinas e Iturinas. Una vez establecido el efecto de diferentes factores sobre la producción de metabolitos secundarios por parte de la cepa IBUN 2755, el siguiente paso fue identificar los compuestos responsables de la actividad antagónica de la cepa contra los patógenos B. glumae, R. solani y G. graminis, para esto se fracciono el extracto por precipitación con ácido del medio MOLP P.U y se evaluó la actividad de cada una de las fracciones, las fracciones activas fueron evaluadas por RMN y LC-MS/MS. Las fracciones F4 y F5 mostraron ser las más activas contra los tres patógenos y se logró determinar que los compuestos activos mayoritarios de estas fracciones eran la Surfactina C14 ß-OH ácido graso-MeO-Glu-Leu-Leu-Val-Asp-Leu-Leu y la surfactina C14 ß-OH ácido graso-Glu-Leu-Leu-Val-Asp-Leu-Leu. Finalmente, se decidió caracterizar de manera descriptiva haciendo uso de redes moleculares, el perfil metabolómico de la cepa IBUN 2755 bajo condiciones de cocoltivo con cada uno de los patógenos evaluados en este proyecto, con el fin de evaluar el efecto del crecimiento de la cepa en conjunto con otro microorganismo, sobre la producción de metabolitos secundarios potencialmente bioactivos. Este análisis metabolómico incluyo datos de LC-MS/MS tanto de los monocultivos de cada microorganismo, como de los cocultivos entre la cepa biocontroladora y cada una de los patógenos. Los resultados mostraron que en efecto cuando la cepa se encuentra bajo condiciones de estrés, como lo es enfrentarse a otro microorganismo, la expresión de metabolitos secundarios bioactivos se eleva drásticamente y se difunden hacia el área de crecimiento del patógeno, esto es especialmente cierto para los cocultivos de la cepa IBUN 2755 - B. glumae y IBUN 2755 - G. graminis. (Texto tomado de la fuente)spa
dc.description.abstractThe use of biocontrol organisms as a strategy to reduce the use of agrochemicals in crops is a practice that is on the rise. Particularly relevant is the development of products of biological origin that allow the control of rice pathogens in a specific and effective way, reducing contamination rates and the recurrence of diseases due to the appearance of resistant pathogens. However, to develop a product of biological origin that has the necessary characteristics to be effective in controlling phytosanitary diseases, it is necessary to have extensive knowledge of the organism that produces the active ingredients that will be part of said product. In this context, the study of the Bacillus velezensis IBUN 2755 strain becomes relevant since it has been shown to have a strong antibacterial and antifungal activity in vitro, against three pathogens that have been reported as the cause of great losses in rice crops and therefore this strain is outlined as a potential biocontrol microorganism The extensive study of the strain includes the characterization of the possible bioactive secondary metabolites produced by the strain, which are directly involved in the known antibacterial and antifungal activity, as well as the effect that different biotic and abiotic factors have on its production. This is precisely the main objective of this research work. The first step to reaching this goal was to describe the effect of the growth of the IBUN 2755 strain in different culture media on the antagonistic activity of the strain against the pathogens B. glumae, R. solani and G. graminis. For this, the IBUN 2755 strain was incubated in Landy, MES, modified MES and MOLP P.E, and MOLP P.U culture media, which are characterized by having differential nutritional conditions. Cell-free supernatants were recovered from each medium after the growth of the strain and their antagonistic activity on the three aforementioned pathogens was evaluated. It was concluded that the nutritional composition of the media has a direct effect on the antagonistic activity of the IBUN 2755 strain, with the MES medium being the one that presented less active supernatants against the three pathogens and the MOLP P.U medium the one that yielded the most active supernatants. assets. Subsequently, to evaluate the bioactive metabolites that were being expressed in these two culture media that showed a differential antagonistic activity (MOLP.U and MES), it was decided to extract them using two extraction methodologies: liquid partition - liquid using ethyl acetate and butanol and acid precipitation. Likewise, these extraction processes were carried out on two different volumes of each medium: 25 and 200 ml. In this way, at the end of this section there would be three parameters to evaluate: first, the effect of the nutritional composition of the culture medium on the expression of secondary metabolites, second, the effect of the extraction methodology on the bioactive metabolites recovered from each medium and third, the effect of the volume of the culture medium on the production of bioactive secondary metabolites by the IBUN 2755 strain The evaluation of the biological activity of the extracts of ethyl acetate, butanol, and acid precipitation obtained from the supernatants recovered from the MOLP P.U and MES media of 200 and 25 ml, on the three pathogens, showed that the extract by acid precipitation of the 25ml MOLP P.U medium was the one that demonstrated consistent antagonistic activity against the three pathogens. Likewise, the metabolic profile of each of these extracts was characterized using LC-MS/MS, the construction of molecular networks, and the dereplication of compounds, with this it was possible to establish that the expression of secondary metabolites by the IBUN 2755 strain was higher in the 25ml media for the case of the MOLP P.U medium, it was also evidenced that the extracts by acid precipitation and ethyl acetate were the most enriched in compounds concerning the butanol extracts and finally, it was possible to establish that although the IBUN 2755 strain, is capable of expressing the same families of compounds, in the two culture media (MOLP P.U and MES) this does not imply that the same compounds are expressed and in the same proportion. Likewise, the expression of molecules of interest such as surfactins, fengycins and Iturins was evidenced. Once the effect of different factors on the production of secondary etabolites by the IBUN 2755 strain had been established, the next step was to identify the compounds responsible for the antagonistic activity of the strain against the pathogens B. glumae, R. solani and G. graminis, for this, the extract was fractionated by acid precipitation of the MOLP P.U medium and the activity of each of the fractions was evaluated, the active fractions were evaluated by NMR and LC-MS/MS. Fractions F4 and F5 showed to be the most active against the three pathogens and it was possible to determine that the main active compounds of these fractions were Surfactin C14 ß-OH fatty acid-MeO-Glu-Leu-Leu-Val-Asp-Leu- Leu and surfactin C14 ß-OH fatty acid-Glu-Leu-Leu-Val-Asp-Leu-Leu. Finally, it was decided to characterize descriptively, using molecular networks, the metabolic profile of the IBUN 2755 strain under co-culture conditions with each of the pathogens evaluated in this project, to evaluate the effect of the growth of the strain on together with another microorganism, on the expression of potentially bioactive secondary metabolites. This metabolomic analysis included LC-MS/MS data from both the monocultures of each microorganism and the co-cultures between the biocontrol strain and each of the pathogens. The results showed that, in effect, when the strain is under stress conditions, such as facing another microorganism, the expression of bioactive secondary metabolites rises drastically and diffuses towards the area of growth of the pathogen, this is especially true for microorganisms. cocultures of strain IBUN 2755 - B. glumae and IBUN 2755 - G. graminis.eng
dc.description.degreelevelMaestríaspa
dc.description.degreenameMagíster en Ciencias - Microbiologíaspa
dc.description.researchareaMetabolomicaspa
dc.format.extentxxix, 260 páginasspa
dc.format.mimetypeapplication/pdfspa
dc.identifier.instnameUniversidad Nacional de Colombiaspa
dc.identifier.reponameRepositorio Institucional Universidad Nacional de Colombiaspa
dc.identifier.repourlhttps://repositorio.unal.edu.co/spa
dc.identifier.urihttps://repositorio.unal.edu.co/handle/unal/83355
dc.language.isospaspa
dc.publisherUniversidad Nacional de Colombiaspa
dc.publisher.branchUniversidad Nacional de Colombia - Sede Bogotáspa
dc.publisher.facultyFacultad de Cienciasspa
dc.publisher.placeBogotá - Colombiaspa
dc.publisher.programBogotá - Ciencias - Maestría en Ciencias - Microbiologíaspa
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dc.rights.accessrightsinfo:eu-repo/semantics/openAccessspa
dc.rights.licenseAtribución-NoComercial-SinDerivadas 4.0 Internacionalspa
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/spa
dc.subject.ddc540 - Química y ciencias afines::547 - Química orgánicaspa
dc.subject.lembCultivos (Biología)spa
dc.subject.lembCultures (Biology)eng
dc.subject.lembTécnicas de cultivo (Biología)spa
dc.subject.lembCulture techniques(Biology)eng
dc.subject.proposalMetabolitos secundariosspa
dc.subject.proposalMedios de cultivospa
dc.subject.proposalBiocontrolspa
dc.subject.proposalRedes molecularesspa
dc.subject.proposalderreplicaciónspa
dc.subject.proposalEspectrometría de masasspa
dc.subject.proposalcocultivosspa
dc.subject.proposalSecondary metaboliteseng
dc.subject.proposalCulture mediaeng
dc.subject.proposalBiocontroleng
dc.subject.proposalMolecular networkingeng
dc.subject.proposaldereplicationeng
dc.subject.proposalCocultureeng
dc.subject.proposalMass spectrometryeng
dc.titleCaracterización de los metabolitos secundarios producidos por la cepa IBUN- 2755, involucrados en la actividad antimicrobiana y antifúngica contra patógenos de arrozspa
dc.title.translatedCharacterization of the secondary metabolites produced by strain IBUN-2755, involved in antimicrobial and antifungal activity against rice pathogenseng
dc.title.translatedCharacterization of the secondary metabolites produced by strain IBUN-2755, involved in antimicrobial and antifungal activity against rice pathogenseng
dc.typeTrabajo de grado - Maestríaspa
dc.type.coarhttp://purl.org/coar/resource_type/c_bdccspa
dc.type.coarversionhttp://purl.org/coar/version/c_ab4af688f83e57aaspa
dc.type.contentDataPaperspa
dc.type.contentWorkflowspa
dc.type.driverinfo:eu-repo/semantics/masterThesisspa
dc.type.redcolhttp://purl.org/redcol/resource_type/TMspa
dc.type.versioninfo:eu-repo/semantics/acceptedVersionspa
dcterms.audience.professionaldevelopmentEstudiantesspa
dcterms.audience.professionaldevelopmentInvestigadoresspa
dcterms.audience.professionaldevelopmentMaestrosspa
dcterms.audience.professionaldevelopmentMedios de comunicaciónspa
dcterms.audience.professionaldevelopmentPersonal de apoyo escolarspa
dcterms.audience.professionaldevelopmentProveedores de ayuda financiera para estudiantesspa
dcterms.audience.professionaldevelopmentPúblico generalspa
oaire.accessrightshttp://purl.org/coar/access_right/c_abf2spa

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