Caracterización Molecular de trips (Thysanoptera: Thripidae) procedentes de cultivos comerciales de aguacate (Persea americana Mill) del oriente antioqueño y estudio de la diversidad microbiana asociada

dc.contributorArango Isaza, Rafael Eduardo
dc.contributorMoreno Herrera, Claudia Ximena
dc.contributorSaldamando Benjumea, Clara Inés
dc.contributorBIOTECNOLOGÍA VEGETAL UNALMED - CIB y MiCROBIODIVERSIDAD Y BIOPROSPECCIÓN
dc.creatorCano-Calle, Daniela
dc.date.accessioned2020-11-06T14:57:11Z
dc.date.available2020-11-06T14:57:11Z
dc.date.created2020-11-06T14:57:11Z
dc.date.issued2020-10-06
dc.identifierhttps://repositorio.unal.edu.co/handle/unal/78594
dc.description.abstractThrips are considered insect pests that cause great economic losses in avocado, causing deformity in fruits, making them unfeasible for export. Some species of thrips are considered quarantine pests in the United States. On the other hand, the bacterial communities present in insects are known to play an important role in many crucial aspects of the life of their hosts, such as fitness, nutrition, development, protection against pathogens and survival in hostile environments. . In general, little is known about avocado thrips and the microbiota present in them. Therefore, the objective of this thesis was to study the molecular phylogeny and associated microbiota of thrips species (Thysanoptera: Thripidae), in commercial avocado (Persea americana Mill.) crops from eastern Antioquia. The thrips species present in the avocado were identified by morphological and molecular analysis based on partial sequences of data obtained from molecular DNA markers. The Cytochrome Oxidase I (COI) gene and the internal transcribed region (ITS). 2-parameter Kimura distances, haplotype networks and an approximation of the phylogeny of the thrips found in the avocado were performed with other sequences of thrips obtained from the sequence bank. 7 species of thrips are reported in avocado, Frankliniella gardeniae, F. gossypiana, F. panamensis, Microcephalothrips abdominalis, Thrips palmi, Scirtothrips hansoni and Liothrips perseae (Tubulifera). Two species of thrips T. simplex and T. trehernei were also found in the dandelion weed plant that is frequently found in avocado crops. The COI gene and the ITS region were useful tools to identify mostly thrips species except for F. gardeniae and F. gossypiana, which at the molecular level turned out to be the same genetic unit. Additionally, the microbiota present in avocado thrips in Antioquia was evaluated using dependent and independent culture methods. Bacterial isolates of individual and poles thrips were identified from macro and microscopic and molecular characterization by analysis of the inter-ribosomal region (ITS) and sequencing of the 16S RNAr and gyrase genes. For culture-independent methods Three thrips morphotypes were selected, DNA was extracted and microbial diversity present was analyzed by different molecular techniques, including amplification of the 16S rRNA gene region followed by time temperature gradient gel electrophoresis analysis (PCR-TTGE) and Next Generation Sequencing (NGS) using Miseq-Illumina technology. The culture-dependent results showed the presence of the Proteobacteria and Firmicutes phyla. The genera Bacillus, Serratia, Pantoea, Sphingomonas and Moraxella predominate. These results were corroborated by the results obtained by culture-independent methods. The TTGE revealed the presence of the phyla Proteobacteria, Firmicutes and Actinobacteria. Furthermore, a principal coordinate analysis showed differences in the microbiota between the three morphotypes of thrips: Scirtothrips hansoni (brown morphotype), Frankliniella (pale morphotype) y F. panamensis (dark morphotype). Likewise, a total of 641 unique OTUs were assigned, the richness showed to be greater in the brown morphotype, the data obtained suggest that there is small diversity of bacterial species in thrips species. The results indicated that the microbiota is composed of 6 phyla with a predominance of Proteobacteria (99%) followed by Cyanobacteria (37.81%), Firmicutes (7.48%), Deinococcus-Thermus (4.42%), Actinobacteria (3.23%) and Bacteroidetes (2.11 %). Significant bacterial genera were detected in the brown morphotype, such as the presence of the Wolbachia endosymbiont, and differences in the microbiota were observed between the morphotypes. In the brown morphotype associated with S. hansoni, Wolbachia prevailed (65-98%), for the pale morphotype of Frankliniella two genera Enterobacter (96.35%) and Rickettsia (46.04%) stood out, and in the dark morphotype of F. panamensis, Ehrlichia (96.13%) predominated. Principal Coordinate Analysis (PCoA) and Constrained Analysis of Principal coordinates (CAP) showed that the morphotype explains differences between the thrips microbiota 49.7% but did not show significant differences between the bacterial communities between the three morphotypes. (P> 0.05). On the other hand, in this investigation the natural infection and molecular identification of Wolbachia were detected in two morphotypes of natural thrips populations. The results showed 34.55% infection, and the presence of two Wolbachia supergroups A and B in thrips. Supergroup A present in the pale morphotype of Frankliniella and B in the brown morphotype of S. hansoni. The results suggest the presence of two new Wolbachia sequences in the two populations of thrips evaluated. In conclusion, these results allow us to elucidate the cultivable and non-cultivable microbiota of thrips, as well as the species of thrips present in avocado from eastern Antioquia. This will provide a baseline with information for future studies to clarify and complement the associated microorganisms, their interactions and the transmission dynamics of pathogens in the agronomic field and those bacteria with biotechnological activity.
dc.description.abstractLos trips se consideran insectos plagas que causan grandes pérdidas económicas en el aguacate, causando deformidad en las frutas, lo que las hace inviables para la exportación. Algunas especies de trips se consideran plagas cuarentenarias en los Estados Unidos. Por otro lado, se sabe que las comunidades bacterianas presentes en los insectos juegan un papel importante en muchos aspectos cruciales de la vida de sus hospederos, como el fitness, la nutrición, el desarrollo, la protección contra los patógenos y la supervivencia en entornos hostiles. En general, se sabe poco de los trips del aguacate y de la microbiota presente en ellos. Por lo tanto, como objetivo de esta tesis se planteo estudiar la filogenia molecular y microbiota asociada de las especies de trips (Thysanoptera: Thripidae), en cultivos comerciales de aguacate (Persea americana Mill.) del Oriente antioqueño. Se identificaron las especies de trips presentes en el aguacate mediante un análisis morfológico y molecular basado en secuencias parciales de datos obtenidos de marcadores moleculares de ADN. El gen Citocromo Oxidasa I (COI) y la región interna transcrita (ITS). Se realizaron distancias de Kimura 2-parametros, redes haplotipicas y una aproximación de la filogenia de los trips encontrados en el aguacate con otras secuencias de trips obtenidos del banco de secuencias. Se reportan 7 especies de trips en el aguacate, Frankliniella gardeniae, F. gossypiana, F. panamensis, Microcephalothrips abdominalis, Thrips palmi, Scirtothrips hansoni y Liothrips perseae (Tubulifera). También se encontró dos especies de trips T. simplex y T. trehernei en el arvense Diente de León planta que se encuentra con frecuencia en los cultivos de aguacate. El gen COI y la región ITS fueron herramientas útiles para identificar en su mayoría a las especies de trips excepto por F. gardeniae y F. gossypiana las cuales a nivel molecular resultaron ser las mismas unidad genética. Adicionalmente, se evaluó la microbiota presente en los trips de aguacate en Antioquia usando métodos dependientes e independientes de cultivo. Se identificaron aislados bacterianos de trips individuales y en pooles, a partir de la caracterización macro y microsopica y molecularmente por el análisis de la región inter-ribosomal (ITS) y la secuenciación de los genes RNAr 16S y girasa.Para los métodos cultivo-independiente se seleccionaron tres morfotipos de trips, se extrajo el DNA y se analizó la diversidad microbiana presente mediante diferentes técnicas moleculares, incluida la amplificación de la región del gen del RNAr 16S seguido por análisis de electroforesis en gel de gradiente de temperatura temporal (PCR-TTGE) y secuenciación de próxima generación (NGS) utilizando la tecnología Miseq-Illumina. Los resultados cultivo-dependiente mostraron la presencia de los filos Proteobacterias y Firmicutes. Con predominio de los géneros Bacillus, Serratia, Pantoea, Sphingomonas y Moraxella. Estos resultados se corroboraron por los resultados obtenidos por métodos cultivo-independeinte. El TTGE reveló la presencia de los filos Proteobacteria, Firmicutes y Actinobacteria. Además, un análisis de coordenadas principales mostró diferencias en la microbiota entre Scirtothrips hansoni (morfotipo café), Frankliniella (morfotipo pálido) y Frankliniella (morfotipo oscuro). Asimismo, se asignaron un total de 641 OTU únicas, la riqueza mostró ser mayor en S. hansoni (morfotipo café), los datos obtenidos sugieren que existe poca diversidad de especies de bacterias en las especies de trips. Los resultados indicaron que la microbiota está compuesta por 6 filos con predominio de Proteobacteria (99%) seguido de Cianobacteria (37.81%), Firmicutes (7.48%), Deinococcus-Thermus (4.42%), Actinobacteria (3.23%) y Bacteroidetes (2.11%). Se detectaron géneros bacterianos de importancia en el morfotipo café como la presencia del endosimbionte Wolbachia y se observó diferencias de la microbiota entre los morfotipos. En el morfotipo café asociado a S. hansoni prevaleció Wolbachia (65-98%), para el morfotipo pálido de Frankliniella sobresalió dos generos Enterobacter (96.35%) y Rickettsia (46.04%) y en el morfotipo oscuro de Frankliniella predominó Ehrlichia (96.13%). El análisis de coordenadas principales (PCoA) y de un análisis restringido de coordenadas principales (CAP, siglas en inglés) mostró que el morfotipo explica diferencias entre la microbiota de trips el 49.7% pero no presentó diferencias significativas entre las comunidades bacterianas entre los tres morfotipos (P > 0.05). Por otro lado, en esta investigación se detectó la infección natural e identificación molecular de Wolbachia en dos morfotipos poblaciones naturales de trips. Los resultados mostraron 34.55% de infección, y la presencia de dos supergrupos A y B de Wolbachia en trips. El supergrupo A presente en el morfotipo pálido de Frankliniella y el B en el morfotipo café de S. hansoni. Los resultados sugieren la presencia de dos nuevas secuencias de Wolbachia en las dos poblaciones evaluadas de trips. En conclusión, estos resultados permiten dilucidar la microbiota cultivable y no cultivable de trips así como también las especies de trips presentes en el aguacate del Oriente antioqueño. Lo cual suministrará una línea base con información para que en estudios futuros se logre esclarecer y complementar, los microrganismos asociados, sus interacciones y la dinámica de transmisión de agentes patógenos en el ambito agronómico y aquellas bacterias con actividad biotecnológica. Información que es clave en la implementación y desarrollo de nuevos productos biológicos que permitan generar planes de control biológico de trips.
dc.languagespa
dc.publisherMedellín - Ciencias - Doctorado en Biotecnología
dc.publisherEscuela de biociencias
dc.publisherUniversidad Nacional de Colombia - Sede Medellín
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dc.rightsAtribución-NoComercial-SinDerivadas 4.0 Internacional
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.titleCaracterización Molecular de trips (Thysanoptera: Thripidae) procedentes de cultivos comerciales de aguacate (Persea americana Mill) del oriente antioqueño y estudio de la diversidad microbiana asociada
dc.typeOtro


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