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Molecular Characterization and Genetic Diversity Of Molecular Characterization and Genetic Diversity of Entomopathogenic Nematodes, Rhabditida: (Steinernematidae and Heterorhabditidae), in Valle del Cauca as a Source for Biological Control Jorge Mario Londoño Caicedo Universidad Nacional de Colombia Facultad de Ciencias Agropecuarias Sede Palmira Palmira Valle del Cauca, Colombia 2017 Molecular Characterization and Genetic Diversity of Entomopathogenic Nematodes, Rhabditida: (Steinernematidae and Heterorhabditidae), in Valle del Cauca as a Source for Biological Control Jorge Mario Londoño Caicedo Tesis de grado presentada como requisito parcial para optar al título de: Magister en Ciencias Biológicas Director: PhD. Jaime Eduardo Muñoz Flórez Línea de Investigación: Biotecnología vegetal Grupo de Investigación: Diversidad Biológica Universidad Nacional de Colombia Facultad de Ciencias Agropecuarias Sede Palmira Palmira Valle del Cauca, Colombia 2017 2 Acknowledgements My sincere thanks to my parents for their daily support and to encourage me keep going forward in my career. Angélica Buitrago for her invaluable help in different fields; for some of the good friends from Diversidad Biológica research team for their company and support in our daily activities. Also special thanks to Fondo de Ciencia, Tecnología e investigación del Sistema General de Regalías FCTelSGR through the project “Desarrollo de Tecnologías Innovadoras para el Manejo Integrado de Plagas y Enfermedades Limitantes de Plátano y Banano en el Valle del Cauca” directed by Universidad Nacional de Colombia Sede Palmira. To professor Jaime Eduardo Muñoz for the opportunity to be part of the research group Diversidad Biológica. 3 Abstract and Resumen Abstract Banana and plantain are edible fruit crops widespread in tropical and subtropical regions, with high importance as a source of energy, nutrients and incomes for lots of families in developing countries. The Musa genus comprises twenty-eight species where M. acuminata and M. balbisiana are the most important species for commercial purposes. However several diseases and pests affect the productivity and fruit quality generating considerable losses every year. Banana weevil complex species are one of the main responsible of such losses worldwide; nevertheless several attempts have been carried out to control banana weevil populations. Cosmopolites sordidus is the most frequent species reported worldwide and with more interest for population control. In the present study four banana weevil species (C. sordidus, Metamasius hemipterus, M. hebetatus, Polytus mellerborgii) were found associated to banana and plantain stands. Genetic analysis on banana weevil species, from mtDNA and nr DNA Loci, indicated a widespread of few haplotypes across 12 municipalities of Valle del Cauca, also a low number of alleles were found indicating a possible bottleneck mediated by human activities. Additionally a survey for entomopathogenic nematodes (EPN) associated to plantain and banana stands was made, were Steinernematids and Heterorhabditids species were found, however molecular characterization indicates a low genetic diversity in EPN isolates. Two nuclear Loci (ITS and LSU) were sequenced and employed for species identification, indicating that from 14 isolates obtained, 10 of them belonged to Steinernema carpocapsae and four to Heterorhabditis spp. For the latter there was contrasting results when either ITS or LSU region was employed for species identification, nevertheless a concatenated analysis showed the presence of two species, Heterorhabditis bacteriophora and Heterorhabditis sp. These indigenous EPN isolates could be promising for biological control of weevil banana complex species, since this isolates were found associated to plantain and banana stands and, together with the low genetic diversity found in weevil species, it is expected to develop strategies for pests management using EPN isolates obtained in the present study. Molecular Characterization and Genetic Diversity of Entomopathogenic Nematodes, Rhabditida: (Steinernematidae and Heterorhabditidae), in Valle del Cauca as a Source for Biological Control Keywords: Banana weevil, Haplotype, Genetic Diversity, Cytochrome Oxidase I, Ribosomal DNA. Abstract and Resumen Resumen El plátano y el banano son frutos comestibles cultivados en las regiones tropicales y subtropicales, presentan importancia como fuente de energía, nutrientes e ingresos económicos para muchas familias de países en desarrollo. El género Musa comprende 28 especies siendo M. acuminata y M. balbisiana las especies mas importantes para propósitos comerciales. Sin embargo diversas enfermedades y pestes afectan la productividad y calidad del fruto generando pérdidas considerables cada año. El complejo de especies del picudo del banano son uno de los principales responsables de tales pérdidas a nivel mundial. No obstante varios intentos se han llevado a cabo para controlar las poblaciones del picudo del banano. Cosmopolites sordidus es la especie mas frecuente reportada a nivel mundial y en la cual mayor interés se ha llevado a cabo para su control. En el presente estudio cuatro especies de picudo del banano (C. sordidus, Metamasius hemipterus, M. hebetatus, Polytus mellerborgii) se encontraron asociadas a cultivos de plátano y banano. Análisis genéticos en especies de picudos, a partir de Loci mitocondrial y nuclear, indicaron un amplia distribución de pocos haplotipos en 12 municipios evaluados en el Valle del Cauca, también un bajo numero de alelos fue identificado indicando un posible efecto cuello de botella mediado por las actividades humanas. Adicionalmente se realizaron evaluaciones para obtención de nematodos entomopatógenos (NEP) asociados a plantaciones de plátano y banano, donde se encontraron especies de Steinernematidos y Heterorhabditidos, sin embargo la caracterización molecular mostró baja diversidad genética en los aislamientos. Dos Loci nucleares (ITS y LSU) fueron secuenciados y empleados para la identificación de especies, indicando que a partir de 14 aislamientos obtenidos 10 pertenecieron a Steinernema carpocapsae y cuatro a Heterorhabditis spp. Para este último hubo resultados contrastantes respecto a las regiones ITS o LSU y la asignación de especies, no obstante un análisis concatenado mostró la presencia de dos especies, Heterorhabditis bacteriophora y Heterorhabditis sp. Estos asilamientos nativos podrían ser promisorios para el control biológico de las especies del complejo de picudos, ya que fueron hallados en plantaciones de plátano y banano y, junto a la baja diversidad genética identificada en las especies del complejo de picudos, se espera desarrollar estrategias para el control de pestes usando los aislamientos de NEP obtenidos en este estudio. Palabras clave: Picudo del Banano, Haplotipo, Diversidad Genética, Citocromo Oxidasa I, ADN Ribosomal. 7 Summary Acknowledgements ....................................................................................................................... 3 Summary ...................................................................................................................................... 8 List of figures .............................................................................................................................. 10 List of tables ................................................................................................................................ 11 Introduction ................................................................................................................................. 12 1. Chapter 1 ............................................................................................................................. 14 Genetic diversity and haplotype distribution of banana weevil species complex (Curculionidae; Dryophthorinae). ......................................................................................................................... 14 1.1 Introduction .................................................................................................................... 15 1.2 Methodology .................................................................................................................. 19 1.2.1 Sampling area ......................................................................................................... 19 1.2.2 Taxon sampling and DNA extraction ...................................................................... 19 1.2.3 PCR amplification for mtDNA and rDNA Loci ......................................................... 21 1.2.4 DNA sequencing and Genetic analysis ................................................................... 21 1.3 Results ........................................................................................................................... 24 1.3.1 Genetic analysis in Weevil species through 28S Ribosomal RNA .......................... 24 1.3.1.1 Haplotype diversity ............................................................................................... 24 1.3.1.2 Haplotype Network ............................................................................................... 26 1.3.1.3 Genetic dissimilarity in weevil species ................................................................. 29 1.3.1.4 Principal Coordinate Analysis (PCoA) ................................................................. 30 1.3.1.5 Maximum Likelihood analysis in Weevils ............................................................. 35 1.3.2 Genetic analysis in Weevil species
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