Bioprospection of marine-derived bacteria with biocontrol activity against Fusarium oxysporum f. sp. lycopersici

dc.contributorRamos Rodriguez, Freddy Alejandro
dc.contributorSuárez Moreno, Zulma Rocío
dc.contributorEstudio y aprovechamiento de Productos Naturales Marinos y Frutas de Colombia
dc.creatorVinchira Villarraga, Diana Marcela
dc.date.accessioned2021-01-22T16:11:19Z
dc.date.available2021-01-22T16:11:19Z
dc.date.created2021-01-22T16:11:19Z
dc.date.issued2020-11-24
dc.identifierVinchira-Villarraga Diana Marcela (2020). Bioprospección de bacterias aisladas de ambientes marinos con actividad biocontroladora frente a Fusarium oxysporum f. sp. lycopersici. Tesis de doctorado. Universidad Nacional de Colombia.
dc.identifierhttps://repositorio.unal.edu.co/handle/unal/78875
dc.description.abstractla marchitez vascular, causada por el hongo fitopatógeno Fusarium oxysporum f. sp. lycopersici (FOL), es una limitante importante en la productividad de los cultivos de tomate (Solanum lycopersicum). Las bacterias derivadas de ambientes marinos producen metabolitos activos estructuralmente diversos. Estos compuestos han sido usados para el control de fitopatógenos, y recientemente, se ha propuesto evaluarlos junto con las bacterias que los producen como potenciales agentes de control biológico. En la presente investigación, se propuso usar bacterias derivadas de ambientes marinos como posibles biocontroladores de FOL. Para ello, este estudio se dividió en tres fases: (I) La selección y caracterización de bacterias con actividad antifúngica frente a FOL, (II) El análisis de competencia rizosférica y la evaluación de actividad antifúngica in vivo de las bacterias activas, y, (III) El análisis metabolómico no dirigido de la interacción tomate-Paenibacillus sp.-FOL59 bajo condiciones de invernadero y la interacción Paenibacillus sp.-FOL59 en co-cultivo. En la fase I, se llevó a cabo el tamizaje primario de actividad antifúngica con una colección de bacterias obtenidas de ambientes marinos compuesta por 152 aislamientos. De estas, 28 bacterias fueron seleccionadas con base en su actividad antifúngica in vitro contra nueve aislamientos de F. oxysporum y el fitopatógeno control FOL59. La caracterización fenotípica y la identificación molecular de estas bacterias se obtuvo con el fin de priorizar las cepas que deberían ser avaluadas en los ensayos de la fase II. Este proceso condujo a la selección de nueve aislamientos de bacterias pertenecientes al orden Bacillales, que presentaron la mayor actividad antifúngica contra FOL. A partir de los resultados obtenidos en la fase II, la bacteria Paenibacillus sp. PNM200 se seleccionó como el candidato más apropiado para el control de FOL. Paenibacillus PNM200 colonizó el sistema radicular de dos variedades de tomate (Milano y Santa Cruz Kada) bajo condiciones de invernadero, y generó un efecto positivo sobre el crecimiento de las plantas tratadas (Santa Cruz Kada) a los 30 días post inoculación. Este efecto fue correlacionado con la capacidad de Paenibacillus sp. PNM200 de producir Ácido indolacético y solubilizar fosfatos. En un esfuerzo por caracterizar la interacción tripartita tomate-Paenibacillus PNM200-FOL59, en la fase III se llevó a cabo un perfilado metabólico por LCMS bajo condiciones controladas. En los experimentos de perfilado metabólico de la interacción tomate-FOL59, se observó la acumulación de los glicoalcaloides esteroidales α-tomatina, hidroxitomatina, tomatidina tetrahexósido, y la aglicona tomatidina en las raíces de la planta. Sin embargo, este efecto no se observó en la interacción tripartita tomate-Paenibacillus sp. PNM200-FOL59, ni en el perfil metabólico de las raíces de plantas de tomate tratadas solo con Paenibacillus sp. PNM200. En ensayos metabolómicos adicionales, que pretendían caracterizar la interacción Paenibacillus sp. PNM200, se observó la producción diferencial de metabolitos en ambos microorganismos en mono y co-cultivo. Se evidenció también que la adición de tejido vascular de tomate genera cambios significativos en el perfil metabólico de ambos microorganismos. Al ser cultivado en un medio suplementado con tejido de tomate, el fitopatógeno FOL59 induce la síntesis de ácido fusárico, beauvericina J, y al menos cinco depsipéptidos estructuralmente relacionados a la beauvericina. Estos metabolitos son bien conocidos por su rol en la patogenicidad de Fusarium en plantas de tomate. Sin embargo, en co-cultivo con Paenibacillus sp. PNM200, se observó una reducción en la producción de estos metabolitos. Esta respuesta podría estar correlacionada con la disminución en la patogenicidad de FOL59 contra tomate en la interacción tripartita. Por otra parte, los cambios más relevantes en el perfil metabólico de Paenibacillus sp. PNM200 en su interacción con FOL59 se asociaron a la producción de una familia de nueve péptidos, con pesos moleculares entre 1100-1600 Da, que se relacionan estructuralmente con los lipopéptidos Pelgipeptina B y Paneipaptina C. Tres de los péptidos se produjeron exclusivamente en el co-cultivo en medio suplementado con tomate. Los demás péptidos detectados en el co-cultivo presentaron un incremento de entre 2 y 56 veces en su abundancia relativa en comparación con el monocultivo. Los extractos etanólicos enriquecidos en esta familia de péptidos mostraron actividad antifúngica contra la cepa tipo FOL CBS 164.85, con un valor de IC50 de 125 μg·mL-1, confirmando su rol como antibióticos frente a FOL. Ninguno de estos péptidos fue identificado por derreplicación usando bases de datos y fuentes bibliográficas, lo que permite sugerir que pueden tratarse de metabolitos nuevos. En este contexto, se propone que la actividad antifúngica in vivo de Paenibacillus sp. PNM200 está asociada a la síntesis de esta familia de péptidos, y que su producción es modificada (inducida o sobre-expresada) en respuesta a la presencia de FOL59. Esta hipótesis debe ser confirmada con nuevos experimentos dirigidos a evaluar la producción de los péptidos de interés bajo condiciones in vivo.
dc.description.abstractVascular wilt (VW), caused by the phytopathogenic fungus Fusarium oxysporum f. sp. lycopersici (FOL), is a significant disease that limits the yield in tomato crop (Solanum lycopersicum). Marine-derived bacteria produce structurally diverse bioactive metabolites. These compounds have been used for phytopathogens control, and recently, alongside marine-derived bacteria proposed as biological control candidates. In the present research, the use of marine-derived bacteria, isolated from the Colombian Caribbean Sea as biocontrol agents for FOL was proposed. The study was developed in three phases: (I) the selection and characterization of bacteria with antifungal activity against FOL, (II) the analysis of rhizospheric competence and in vivo antifungal activity of the active bacteria, and (III) the untargeted-metabolomic analysis of the interaction between tomato – Paenibacillus sp. - FOL under controlled greenhouse conditions and Paenibacillus sp. PNM200 - FOL by co-cultivation. In phase I, primary screening was carried out with a marine -derived bacterial collection composed of 152 isolates. Twenty-eight bacterial strains were selected based on in vitro antifungal activity test against nine F. oxysporum isolates and one control pathogen FOL59. The phenotypical characterization and molecular identification of these isolates were done aiming to prioritize the better candidates to be tested on phase II. These processes lead to the selection of nine bacteria belonging to the Bacillales order, which presented the higher antifungal activity against FOL. From phase II, isolate Paenibacillus sp. PNM200 was selected as a potential biocontrol strain. Paenibacillus sp. PNM200 colonized the root system of the tomato cultivars Milano and Santa Cruz Kada under greenhouse conditions and generated a positive effect on the growth of the treated plants after 30 days inoculation (Santa Cruz Kada tomato cultivar). This effect was correlated with the ability of Paenibacillus sp. PNM200 to produce IAA (Indoleacetic acid) and solubilize phosphates. These results are one of the first evidence of marine-derived bacteria’s rhizospheric competence of for its use as biocontrol agents. As an effort to characterize the tripartite tomato plant-Paenibacillus sp. PNM200-FOL59 interaction, a LCMS metabolic profiling approach was developed as part of phase III. A significant accumulation of the steroidal glycoalkaloids (SGA) α-tomatine, hydroxytomatine, tomatidine tetrahexoside, and the aglycone tomatidine on the plant's root of was observed in the metabolic profiling experiments to characterize the tomato-FOL59 interaction. However, this effect was not detected in the tripartite tomato-Paenibacillus sp. PNM200-FOL59 interaction, nor in the metabolic profiles of the tomato root system treated with Paenibacillus sp. PNM200. In other metabolomic experiments aimed at characterizing Paenibacillus sp. PNM200-FOL59 interaction, differential production of metabolites could be observed in the mono- and co-cultivation metabolic profiles of both microorganisms. Moreover, the addition of tomato vascular tissue also generated significant changes in the metabolic profile of both microorganisms. The pathogenic strain FOL59, when cultured in a growth medium supplemented with tomato plant tissue, induced the synthesis of fusaric acid, beauvericin J, and at least five additional depsipeptides structurally related to beauvericin. These are well-known metabolites related to the pathogenic effect of Fusarium against plants. However, in co-cultivation with Paenibacillus sp. PNM200, a reduction in the production of these metabolites was observed. This response could be related to the decrease of FOL59 pathogenicity against the tomato. On the other hand, the most relevant changes in the metabolic profiles of Paenibacillus sp. PNM200 were associated to the production of a family of nine peptides, with molecular weights between 1100-1600 Da, and structurally related to the peptide pelgipeptine B and paenipeptin C. Three of these peptides were produced exclusively in the co-culture when the growth medium was supplemented with tomato plant tissue. The other peptides detected in the co-culture showed a significant increase in their abundance compared to the monoculture (2 to 56-fold change), suggesting their role in the biocontrol of FOL59. The ethanolic extracts enriched in this family of peptides showed antifungal activity against FOL CBS 164.85, with an IC50 of 125 μg·mL-1, confirming the role mentioned above as antibiotics against FOL. None of these peptides could be identified by dereplication using databases and literature sources, suggesting that they may be new metabolites. In this context, it is proposed that the in vivo antifungal activity of Paenibacillus sp. PNM200 is associated with the synthesis of this family of peptides, and its production is modified (triggered and enhanced) in response to the presence of FOL59. This hypothesis must be confirmed by new experiments aimed at evaluating the peptides' production under in vivo conditions.
dc.languageeng
dc.publisherBogotá - Ciencias - Doctorado en Biotecnología
dc.publisherUniversidad Nacional de Colombia - Sede Bogotá
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dc.rightsAtribución-NoComercial-SinDerivadas 4.0 Internacional
dc.rightsAcceso abierto
dc.rightshttp://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rightsinfo:eu-repo/semantics/openAccess
dc.rightsDerechos reservados - Universidad Nacional de Colombia
dc.titleBioprospection of marine-derived bacteria with biocontrol activity against Fusarium oxysporum f. sp. lycopersici
dc.typeOtro


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