Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)

Autores
Pérez, María Laura
Año de publicación
2016
Idioma
español castellano
Tipo de recurso
tesis doctoral
Estado
versión aceptada
Colaborador/a o director/a de tesis
Mroginski, Luis Amado
Galdeano, Ernestina
Descripción
Argentina is the world's largest yerba mate producer; the producing provinces are Misiones and Corrientes. This crop has several drawbacks such as low genetic and physiological seed quality, slow, even and low percentage germination and heterogeneity of plants grown from seed, which led to the necessity of developing micropropagation systems. One of the major difficulties in the yerba mate micropropagation is in vitro contamination by fungi and bacteria. Among the various factors that can influence the contamination of in vitro culture are the time of year, type of explant and the presence of surface and/or endophytic plant-associated microorganisms. Since endophytes live within plant tissues, they are able to survive surface disinfection making them harder to control. The aim of this work was to characterize the microorganisms contaminant of yerba mate micropropagation systems and establish their source. The composition and diversity of the endophytic community associated with aerial organs of yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungal and bacterial endophytes were isolated from young and adult leaves and branches of three yerba mate clones growing under field condition in Corrientes province, Argentina. Besides, in order to establish if most of the contaminants of yerba mate in vitro culture corresponded to endophytes from field or greenhouse growing plants, contaminant microorganisms were isolated from in vitro micropropagated, and greenhouse plants. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer (ITS) rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. A total of 156 fungal and 193 bacterial isolates were obtained. Bacteria predominated in stem samples while leaf samples retrieved similar amount of bacterial and fungal isolates. No significant differences were found in isolate quantity nor diversity among seasons or between young and older leaves and stems. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Other 12 fungus genera were identified including Aspergillus, Cercospora, Clonostachys (Bionectria), Curvularia, Guignardia, Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe), and Phyllosticta. Actinobacteria represented 61% of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. They were isolated from all the organs assayed, 4 showing that they are ubiquitous endophytes. Other bacteria frequently found throughout the year were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. The predominant fungi and bacteria detected in yerba mate were similar to those reported in other species in which the same type of technique was used. Fungal genera Curvularia, Fusarium, Penicillium, Setophoma, and bacteria Pantoea and Staphylococcus were isolated as contaminants of in vitro culture and as endophytes in plants grown under greenhouse and field conditions. Alternaria was t isolated as a contaminant of in vitro culture and as endophyte in plants grown in the field, as well as the bacteria Janibacter. Fusarium was the most numerous fungal genus. 8 Fusarium species were identified, and pathogenicity on yerba mate was assayed in three of them (F. oxysporum, F. pseudocircinatum y F. subglutinans), showing that they behave as endophytes when plants are grown under field conditions but are pathogenic to in vitro plants (vitropathogens). The results obtained in this thesis showed that most of the yerba mate in vitro culture contaminants are endophytic microorganisms that occur also in field or greenhouse grown plants without showing any negative effect on ex vitro plants. We have also determined that the number and diversity of fungal and bacterial isolates are affected by the season, being higher during fall and summer, as well as the organ type and age.
Argentina es el primer productor mundial de yerba mate, las provincias productoras son Misiones y Corrientes. Este cultivo presenta varios inconvenientes, como la baja calidad genética y fisiológica de las semillas, la lenta y desuniforme germinación con un bajo porcentaje y la heterogeneidad de las plantas obtenidas a partir de semillas, que llevaron a la micropropagación. Una de las mayores dificultades en la micropropagación de yerba mate es la contaminación in vitro por hongos y bacterias. Entre los diversos factores que pueden influir en la contaminación de cultivo in vitro están la época del año, el tipo de explante y la presencia de microorganismos superficiales y/o endófitos asociados a las plantas. Los microorganismos endofíticos fueron encontrados en todas las plantas estudiadas en diferentes órganos vegetales, si bien su relación con las plantas no son comprendidas en su totalidad, se encontraron que producen efectos benéficos sobre sus hospedantes, cumpliendo algún rol importante para la fisiología de la planta, sin embargo cuando éstos se encuentran en explantes cultivados in vitro son considerados contaminantes. Su control se hace dificultoso debido a que al habitar los tejidos vegetales internos sobreviven a la desinfección superficial. El objetivo de esta tesis fue caracterizar a los microorganismos contaminantes de sistemas de micropropagación de yerba mate (Ilex paraguariensis) con el fin de determinar su origen. Existen muy pocos antecedentes sobre microorganismos endofíticos en la yerba mate, para ello en este trabajo se realizaron muestreos de hojas y tallos de plantas crecidas en el campo en diferentes fechas. Además, para confirmar que los contaminantes del cultivo in vitro de yerba mate son en su mayoría endófitos de plantas crecidas en el campo y/o invernadero, se aislaron contaminantes en la micropropagación de yerba mate y se realizaron muestreos de hojas y tallos de plantas crecidas en invernadero. Para la identificación de microorganismos se usaron la técnica morfológica y molecular amplificando por PCR y secuenciando el ITS para hongos y 16S para bacterias. De los endófitos aislados de plantas crecidas en el campo se identificaron 14 géneros de hongos Aspergillus, Cercospora, Colletotrichum, Curvularia, Fusarium, Gliocladium (Bionectria), Guignardia (Phyllosticta), Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe) y Trichoderma, y 20 géneros de bacterias, Aeromicrobium, Bacillus, Brevundimonas, 2 Curtobacterium, Herbiconiux, Kineococcus, Leucobacter, Lysinibacillus, Methylobacterium, Microbacterium, Micrococcus, Mycobacterium, Paenibacillus, Pantoea, Phenylobacterium, Rhizobium, Roseomonas, Sphingomonas, Staphylococcus y Xylophilus. Los géneros de hongos Curvularia, Fusarium, Penicillium, Setophoma, y de bacterias Pantoea, Staphylococcus fueron aislados como contaminantes de cultivo in vitro y como endófitos de plantas crecidas en invernadero y a campo. Alternaria fue el género de hongo que se aisló como contaminante de cultivo in vitro y como endófito de plantas crecidas a campo al igual que las bacterias Janibacter. También se determinó que el género de hongo que se encontró en mayor número fue Fusarium, se identificaron 8 especies y se probó la patogenicidad de 3 de ella en cultivo in vitro de yerba mate (F. oxysporum, F. pseudocircinatum y F. subglutinans) y se determinó que pueden ser endófitos en plantas crecidas en el campo y patógenos en el cultivo in vitro (vitropatógenos). Con los resultados de este trabajo se pudo confirmar que la mayoría de los contaminantes en cultivo in vitro son microorganismos endofíticos presentes en plantas crecidas a campo o en invernáculo que no afectan negativamente el crecimiento en condiciones ex vitro. Además, se determinó que el número de aislamientos y diversidad de hongos y bacterias están influenciados por la época de muestreo, presentándose los mayores valores en otoño y verano, además están influenciados por el tipo de órgano vegetal y su edad.
Fil: Pérez, María Emilia. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.
Fil: Pérez, María Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Fil: Mroginski, Luis Amado. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Fil: Galdeano, Ernestina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Materia
Yerba mate
Micropropagación
Microorganismos contaminantes
Nivel de accesibilidad
acceso abierto
Condiciones de uso
http://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
Institución
Universidad Nacional del Nordeste
OAI Identificador
oai:repositorio.unne.edu.ar:123456789/458

id RIUNNE_dcd4d0fb771f4aa0368133c421712f06
oai_identifier_str oai:repositorio.unne.edu.ar:123456789/458
network_acronym_str RIUNNE
repository_id_str 4871
network_name_str Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
spelling Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)Pérez, María LauraYerba mateMicropropagaciónMicroorganismos contaminantesArgentina is the world's largest yerba mate producer; the producing provinces are Misiones and Corrientes. This crop has several drawbacks such as low genetic and physiological seed quality, slow, even and low percentage germination and heterogeneity of plants grown from seed, which led to the necessity of developing micropropagation systems. One of the major difficulties in the yerba mate micropropagation is in vitro contamination by fungi and bacteria. Among the various factors that can influence the contamination of in vitro culture are the time of year, type of explant and the presence of surface and/or endophytic plant-associated microorganisms. Since endophytes live within plant tissues, they are able to survive surface disinfection making them harder to control. The aim of this work was to characterize the microorganisms contaminant of yerba mate micropropagation systems and establish their source. The composition and diversity of the endophytic community associated with aerial organs of yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungal and bacterial endophytes were isolated from young and adult leaves and branches of three yerba mate clones growing under field condition in Corrientes province, Argentina. Besides, in order to establish if most of the contaminants of yerba mate in vitro culture corresponded to endophytes from field or greenhouse growing plants, contaminant microorganisms were isolated from in vitro micropropagated, and greenhouse plants. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer (ITS) rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. A total of 156 fungal and 193 bacterial isolates were obtained. Bacteria predominated in stem samples while leaf samples retrieved similar amount of bacterial and fungal isolates. No significant differences were found in isolate quantity nor diversity among seasons or between young and older leaves and stems. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Other 12 fungus genera were identified including Aspergillus, Cercospora, Clonostachys (Bionectria), Curvularia, Guignardia, Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe), and Phyllosticta. Actinobacteria represented 61% of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. They were isolated from all the organs assayed, 4 showing that they are ubiquitous endophytes. Other bacteria frequently found throughout the year were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. The predominant fungi and bacteria detected in yerba mate were similar to those reported in other species in which the same type of technique was used. Fungal genera Curvularia, Fusarium, Penicillium, Setophoma, and bacteria Pantoea and Staphylococcus were isolated as contaminants of in vitro culture and as endophytes in plants grown under greenhouse and field conditions. Alternaria was t isolated as a contaminant of in vitro culture and as endophyte in plants grown in the field, as well as the bacteria Janibacter. Fusarium was the most numerous fungal genus. 8 Fusarium species were identified, and pathogenicity on yerba mate was assayed in three of them (F. oxysporum, F. pseudocircinatum y F. subglutinans), showing that they behave as endophytes when plants are grown under field conditions but are pathogenic to in vitro plants (vitropathogens). The results obtained in this thesis showed that most of the yerba mate in vitro culture contaminants are endophytic microorganisms that occur also in field or greenhouse grown plants without showing any negative effect on ex vitro plants. We have also determined that the number and diversity of fungal and bacterial isolates are affected by the season, being higher during fall and summer, as well as the organ type and age.Argentina es el primer productor mundial de yerba mate, las provincias productoras son Misiones y Corrientes. Este cultivo presenta varios inconvenientes, como la baja calidad genética y fisiológica de las semillas, la lenta y desuniforme germinación con un bajo porcentaje y la heterogeneidad de las plantas obtenidas a partir de semillas, que llevaron a la micropropagación. Una de las mayores dificultades en la micropropagación de yerba mate es la contaminación in vitro por hongos y bacterias. Entre los diversos factores que pueden influir en la contaminación de cultivo in vitro están la época del año, el tipo de explante y la presencia de microorganismos superficiales y/o endófitos asociados a las plantas. Los microorganismos endofíticos fueron encontrados en todas las plantas estudiadas en diferentes órganos vegetales, si bien su relación con las plantas no son comprendidas en su totalidad, se encontraron que producen efectos benéficos sobre sus hospedantes, cumpliendo algún rol importante para la fisiología de la planta, sin embargo cuando éstos se encuentran en explantes cultivados in vitro son considerados contaminantes. Su control se hace dificultoso debido a que al habitar los tejidos vegetales internos sobreviven a la desinfección superficial. El objetivo de esta tesis fue caracterizar a los microorganismos contaminantes de sistemas de micropropagación de yerba mate (Ilex paraguariensis) con el fin de determinar su origen. Existen muy pocos antecedentes sobre microorganismos endofíticos en la yerba mate, para ello en este trabajo se realizaron muestreos de hojas y tallos de plantas crecidas en el campo en diferentes fechas. Además, para confirmar que los contaminantes del cultivo in vitro de yerba mate son en su mayoría endófitos de plantas crecidas en el campo y/o invernadero, se aislaron contaminantes en la micropropagación de yerba mate y se realizaron muestreos de hojas y tallos de plantas crecidas en invernadero. Para la identificación de microorganismos se usaron la técnica morfológica y molecular amplificando por PCR y secuenciando el ITS para hongos y 16S para bacterias. De los endófitos aislados de plantas crecidas en el campo se identificaron 14 géneros de hongos Aspergillus, Cercospora, Colletotrichum, Curvularia, Fusarium, Gliocladium (Bionectria), Guignardia (Phyllosticta), Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe) y Trichoderma, y 20 géneros de bacterias, Aeromicrobium, Bacillus, Brevundimonas, 2 Curtobacterium, Herbiconiux, Kineococcus, Leucobacter, Lysinibacillus, Methylobacterium, Microbacterium, Micrococcus, Mycobacterium, Paenibacillus, Pantoea, Phenylobacterium, Rhizobium, Roseomonas, Sphingomonas, Staphylococcus y Xylophilus. Los géneros de hongos Curvularia, Fusarium, Penicillium, Setophoma, y de bacterias Pantoea, Staphylococcus fueron aislados como contaminantes de cultivo in vitro y como endófitos de plantas crecidas en invernadero y a campo. Alternaria fue el género de hongo que se aisló como contaminante de cultivo in vitro y como endófito de plantas crecidas a campo al igual que las bacterias Janibacter. También se determinó que el género de hongo que se encontró en mayor número fue Fusarium, se identificaron 8 especies y se probó la patogenicidad de 3 de ella en cultivo in vitro de yerba mate (F. oxysporum, F. pseudocircinatum y F. subglutinans) y se determinó que pueden ser endófitos en plantas crecidas en el campo y patógenos en el cultivo in vitro (vitropatógenos). Con los resultados de este trabajo se pudo confirmar que la mayoría de los contaminantes en cultivo in vitro son microorganismos endofíticos presentes en plantas crecidas a campo o en invernáculo que no afectan negativamente el crecimiento en condiciones ex vitro. Además, se determinó que el número de aislamientos y diversidad de hongos y bacterias están influenciados por la época de muestreo, presentándose los mayores valores en otoño y verano, además están influenciados por el tipo de órgano vegetal y su edad.Fil: Pérez, María Emilia. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.Fil: Pérez, María Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.Fil: Mroginski, Luis Amado. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.Fil: Galdeano, Ernestina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.Universidad Nacional del Nordeste. Facultad de Ciencias AgrariasMroginski, Luis AmadoGaldeano, Ernestina2016info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/acceptedVersionhttp://purl.org/coar/resource_type/c_db06info:ar-repo/semantics/tesisDoctoralapplication/ pdf110 p.application/pdfPérez, María Laura, 2016. Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis). Tesis doctoral. Corrientes: Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias.http://repositorio.unne.edu.ar/handle/123456789/458spainfo:eu-repo/semantics/openAccesshttp://creativecommons.org/licenses/by-nc-nd/2.5/ar/Atribución-NoComercial-SinDerivadas 2.5 Argentinareponame:Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)instname:Universidad Nacional del Nordeste2025-09-04T11:14:16Zoai:repositorio.unne.edu.ar:123456789/458instacron:UNNEInstitucionalhttp://repositorio.unne.edu.ar/Universidad públicaNo correspondehttp://repositorio.unne.edu.ar/oaiososa@bib.unne.edu.ar;sergio.alegria@unne.edu.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:48712025-09-04 11:14:16.856Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE) - Universidad Nacional del Nordestefalse
dc.title.none.fl_str_mv Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
title Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
spellingShingle Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
Pérez, María Laura
Yerba mate
Micropropagación
Microorganismos contaminantes
title_short Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
title_full Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
title_fullStr Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
title_full_unstemmed Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
title_sort Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis)
dc.creator.none.fl_str_mv Pérez, María Laura
author Pérez, María Laura
author_facet Pérez, María Laura
author_role author
dc.contributor.none.fl_str_mv Mroginski, Luis Amado
Galdeano, Ernestina
dc.subject.none.fl_str_mv Yerba mate
Micropropagación
Microorganismos contaminantes
topic Yerba mate
Micropropagación
Microorganismos contaminantes
dc.description.none.fl_txt_mv Argentina is the world's largest yerba mate producer; the producing provinces are Misiones and Corrientes. This crop has several drawbacks such as low genetic and physiological seed quality, slow, even and low percentage germination and heterogeneity of plants grown from seed, which led to the necessity of developing micropropagation systems. One of the major difficulties in the yerba mate micropropagation is in vitro contamination by fungi and bacteria. Among the various factors that can influence the contamination of in vitro culture are the time of year, type of explant and the presence of surface and/or endophytic plant-associated microorganisms. Since endophytes live within plant tissues, they are able to survive surface disinfection making them harder to control. The aim of this work was to characterize the microorganisms contaminant of yerba mate micropropagation systems and establish their source. The composition and diversity of the endophytic community associated with aerial organs of yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungal and bacterial endophytes were isolated from young and adult leaves and branches of three yerba mate clones growing under field condition in Corrientes province, Argentina. Besides, in order to establish if most of the contaminants of yerba mate in vitro culture corresponded to endophytes from field or greenhouse growing plants, contaminant microorganisms were isolated from in vitro micropropagated, and greenhouse plants. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer (ITS) rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. A total of 156 fungal and 193 bacterial isolates were obtained. Bacteria predominated in stem samples while leaf samples retrieved similar amount of bacterial and fungal isolates. No significant differences were found in isolate quantity nor diversity among seasons or between young and older leaves and stems. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Other 12 fungus genera were identified including Aspergillus, Cercospora, Clonostachys (Bionectria), Curvularia, Guignardia, Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe), and Phyllosticta. Actinobacteria represented 61% of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. They were isolated from all the organs assayed, 4 showing that they are ubiquitous endophytes. Other bacteria frequently found throughout the year were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. The predominant fungi and bacteria detected in yerba mate were similar to those reported in other species in which the same type of technique was used. Fungal genera Curvularia, Fusarium, Penicillium, Setophoma, and bacteria Pantoea and Staphylococcus were isolated as contaminants of in vitro culture and as endophytes in plants grown under greenhouse and field conditions. Alternaria was t isolated as a contaminant of in vitro culture and as endophyte in plants grown in the field, as well as the bacteria Janibacter. Fusarium was the most numerous fungal genus. 8 Fusarium species were identified, and pathogenicity on yerba mate was assayed in three of them (F. oxysporum, F. pseudocircinatum y F. subglutinans), showing that they behave as endophytes when plants are grown under field conditions but are pathogenic to in vitro plants (vitropathogens). The results obtained in this thesis showed that most of the yerba mate in vitro culture contaminants are endophytic microorganisms that occur also in field or greenhouse grown plants without showing any negative effect on ex vitro plants. We have also determined that the number and diversity of fungal and bacterial isolates are affected by the season, being higher during fall and summer, as well as the organ type and age.
Argentina es el primer productor mundial de yerba mate, las provincias productoras son Misiones y Corrientes. Este cultivo presenta varios inconvenientes, como la baja calidad genética y fisiológica de las semillas, la lenta y desuniforme germinación con un bajo porcentaje y la heterogeneidad de las plantas obtenidas a partir de semillas, que llevaron a la micropropagación. Una de las mayores dificultades en la micropropagación de yerba mate es la contaminación in vitro por hongos y bacterias. Entre los diversos factores que pueden influir en la contaminación de cultivo in vitro están la época del año, el tipo de explante y la presencia de microorganismos superficiales y/o endófitos asociados a las plantas. Los microorganismos endofíticos fueron encontrados en todas las plantas estudiadas en diferentes órganos vegetales, si bien su relación con las plantas no son comprendidas en su totalidad, se encontraron que producen efectos benéficos sobre sus hospedantes, cumpliendo algún rol importante para la fisiología de la planta, sin embargo cuando éstos se encuentran en explantes cultivados in vitro son considerados contaminantes. Su control se hace dificultoso debido a que al habitar los tejidos vegetales internos sobreviven a la desinfección superficial. El objetivo de esta tesis fue caracterizar a los microorganismos contaminantes de sistemas de micropropagación de yerba mate (Ilex paraguariensis) con el fin de determinar su origen. Existen muy pocos antecedentes sobre microorganismos endofíticos en la yerba mate, para ello en este trabajo se realizaron muestreos de hojas y tallos de plantas crecidas en el campo en diferentes fechas. Además, para confirmar que los contaminantes del cultivo in vitro de yerba mate son en su mayoría endófitos de plantas crecidas en el campo y/o invernadero, se aislaron contaminantes en la micropropagación de yerba mate y se realizaron muestreos de hojas y tallos de plantas crecidas en invernadero. Para la identificación de microorganismos se usaron la técnica morfológica y molecular amplificando por PCR y secuenciando el ITS para hongos y 16S para bacterias. De los endófitos aislados de plantas crecidas en el campo se identificaron 14 géneros de hongos Aspergillus, Cercospora, Colletotrichum, Curvularia, Fusarium, Gliocladium (Bionectria), Guignardia (Phyllosticta), Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe) y Trichoderma, y 20 géneros de bacterias, Aeromicrobium, Bacillus, Brevundimonas, 2 Curtobacterium, Herbiconiux, Kineococcus, Leucobacter, Lysinibacillus, Methylobacterium, Microbacterium, Micrococcus, Mycobacterium, Paenibacillus, Pantoea, Phenylobacterium, Rhizobium, Roseomonas, Sphingomonas, Staphylococcus y Xylophilus. Los géneros de hongos Curvularia, Fusarium, Penicillium, Setophoma, y de bacterias Pantoea, Staphylococcus fueron aislados como contaminantes de cultivo in vitro y como endófitos de plantas crecidas en invernadero y a campo. Alternaria fue el género de hongo que se aisló como contaminante de cultivo in vitro y como endófito de plantas crecidas a campo al igual que las bacterias Janibacter. También se determinó que el género de hongo que se encontró en mayor número fue Fusarium, se identificaron 8 especies y se probó la patogenicidad de 3 de ella en cultivo in vitro de yerba mate (F. oxysporum, F. pseudocircinatum y F. subglutinans) y se determinó que pueden ser endófitos en plantas crecidas en el campo y patógenos en el cultivo in vitro (vitropatógenos). Con los resultados de este trabajo se pudo confirmar que la mayoría de los contaminantes en cultivo in vitro son microorganismos endofíticos presentes en plantas crecidas a campo o en invernáculo que no afectan negativamente el crecimiento en condiciones ex vitro. Además, se determinó que el número de aislamientos y diversidad de hongos y bacterias están influenciados por la época de muestreo, presentándose los mayores valores en otoño y verano, además están influenciados por el tipo de órgano vegetal y su edad.
Fil: Pérez, María Emilia. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.
Fil: Pérez, María Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Fil: Mroginski, Luis Amado. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Fil: Galdeano, Ernestina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
description Argentina is the world's largest yerba mate producer; the producing provinces are Misiones and Corrientes. This crop has several drawbacks such as low genetic and physiological seed quality, slow, even and low percentage germination and heterogeneity of plants grown from seed, which led to the necessity of developing micropropagation systems. One of the major difficulties in the yerba mate micropropagation is in vitro contamination by fungi and bacteria. Among the various factors that can influence the contamination of in vitro culture are the time of year, type of explant and the presence of surface and/or endophytic plant-associated microorganisms. Since endophytes live within plant tissues, they are able to survive surface disinfection making them harder to control. The aim of this work was to characterize the microorganisms contaminant of yerba mate micropropagation systems and establish their source. The composition and diversity of the endophytic community associated with aerial organs of yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungal and bacterial endophytes were isolated from young and adult leaves and branches of three yerba mate clones growing under field condition in Corrientes province, Argentina. Besides, in order to establish if most of the contaminants of yerba mate in vitro culture corresponded to endophytes from field or greenhouse growing plants, contaminant microorganisms were isolated from in vitro micropropagated, and greenhouse plants. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer (ITS) rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. A total of 156 fungal and 193 bacterial isolates were obtained. Bacteria predominated in stem samples while leaf samples retrieved similar amount of bacterial and fungal isolates. No significant differences were found in isolate quantity nor diversity among seasons or between young and older leaves and stems. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Other 12 fungus genera were identified including Aspergillus, Cercospora, Clonostachys (Bionectria), Curvularia, Guignardia, Microsphaeropsis, Neofusicoccum, Nigrospora, Paecilomyces, Penicillium, Phomopsis (Diaporthe), and Phyllosticta. Actinobacteria represented 61% of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. They were isolated from all the organs assayed, 4 showing that they are ubiquitous endophytes. Other bacteria frequently found throughout the year were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. The predominant fungi and bacteria detected in yerba mate were similar to those reported in other species in which the same type of technique was used. Fungal genera Curvularia, Fusarium, Penicillium, Setophoma, and bacteria Pantoea and Staphylococcus were isolated as contaminants of in vitro culture and as endophytes in plants grown under greenhouse and field conditions. Alternaria was t isolated as a contaminant of in vitro culture and as endophyte in plants grown in the field, as well as the bacteria Janibacter. Fusarium was the most numerous fungal genus. 8 Fusarium species were identified, and pathogenicity on yerba mate was assayed in three of them (F. oxysporum, F. pseudocircinatum y F. subglutinans), showing that they behave as endophytes when plants are grown under field conditions but are pathogenic to in vitro plants (vitropathogens). The results obtained in this thesis showed that most of the yerba mate in vitro culture contaminants are endophytic microorganisms that occur also in field or greenhouse grown plants without showing any negative effect on ex vitro plants. We have also determined that the number and diversity of fungal and bacterial isolates are affected by the season, being higher during fall and summer, as well as the organ type and age.
publishDate 2016
dc.date.none.fl_str_mv 2016
dc.type.none.fl_str_mv info:eu-repo/semantics/doctoralThesis
info:eu-repo/semantics/acceptedVersion
http://purl.org/coar/resource_type/c_db06
info:ar-repo/semantics/tesisDoctoral
format doctoralThesis
status_str acceptedVersion
dc.identifier.none.fl_str_mv Pérez, María Laura, 2016. Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis). Tesis doctoral. Corrientes: Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias.
http://repositorio.unne.edu.ar/handle/123456789/458
identifier_str_mv Pérez, María Laura, 2016. Caracterización de microorganismos contaminantes en sistemas de micropropagación de yerba mate (Ilex paraguariensis). Tesis doctoral. Corrientes: Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias.
url http://repositorio.unne.edu.ar/handle/123456789/458
dc.language.none.fl_str_mv spa
language spa
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Atribución-NoComercial-SinDerivadas 2.5 Argentina
eu_rights_str_mv openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Atribución-NoComercial-SinDerivadas 2.5 Argentina
dc.format.none.fl_str_mv application/ pdf
110 p.
application/pdf
dc.publisher.none.fl_str_mv Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias
publisher.none.fl_str_mv Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias
dc.source.none.fl_str_mv reponame:Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
instname:Universidad Nacional del Nordeste
reponame_str Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
collection Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
instname_str Universidad Nacional del Nordeste
repository.name.fl_str_mv Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE) - Universidad Nacional del Nordeste
repository.mail.fl_str_mv ososa@bib.unne.edu.ar;sergio.alegria@unne.edu.ar
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