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CHARACTERIZATION of Xenorhabdus Spp. Xptb1 and Xptc1 TOXIN GENES and EFFECT of the BACTERIA on Chilo Partellus and Busseola Fusca

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CHARACTERIZATION of Xenorhabdus Spp. Xptb1 and Xptc1 TOXIN GENES and EFFECT of the BACTERIA on Chilo Partellus and Busseola Fusca CHARACTERIZATION OF Xenorhabdus spp. XptB1 AND XptC1 TOXIN GENES AND EFFECT OF THE BACTERIA ON Chilo partellus AND Busseola fusca. By Nyongesa Lorine Nanjala (B.Sc.) I56/60441/2013. A research thesis submitted in partial fulfilment for the degree of Master of Science in Genetics at the School of Biological Sciences. UNIVERSITY OF NAIROBI September, 2016. DECLARATION This thesis is my original work and has not been presented for a degree award in any other university. Nyongesa Lorine Nanjala Signature…………………………… I56/60441/2013 Date………………………………… SUPERVISORS We confirm that the work reported in this thesis was carried out by the candidate under our supervision. Dr. Nelson Amugune Signature……………………………. School of Biological Sciences Date…………………………… ……. University of Nairobi Dr. Daniel Masiga International Centre of Insect Physiology and Ecology Molecular Biology and Bioinformatics Unit Signature……………………………. Date………………………... ……… ii DEDICATION To my family: mom Margaret and dad Moses, my siblings Kevin, Ivone, Bronvone, Angeline and nephew Peter. Thank you for your support and prayers. To my friends: Peter, Simon and Levi who helped sharpen my skills; Ryan and team Trek Science Kenya who have kept the dream alive; Susan, Davina, Rose, Mary, Caroline, Marjorie for believing in me. iii ACKNOWLEDGEMENTS I wish to acknowledge my supervisors Dr. Daniel Masiga and Dr. Nelson Amugune for their guidance and supervisory roles throughout my period of study. I am grateful that they availed the chance for me to study among professionals in the field as well as fellow students from various backgrounds. I am especially thankful for their patience. I also convey my appreciation to the International Centre of Insect Physiology and Ecology (icipe) Duduville Campus, Kasarani, for providing the facilities which made this study possible. I also appreciate Dr. Charles Waturu and Kenya Agricultural and Livestock Research Organization (KALRO) Horticultural Research Institute, Thika, for providing specimens used in this study. I thank the World Federation of Scientist (WFS) for awarding me a scholarship, tenable for one year through icipe’s Dissertation Research Internship Program (DRIP) that facilitated my study. I also thank the National Commission for Science Technology and Innovation (NACOSTI) for partially funding this work. I thank my friends, my classmates, and the entire team at the Molecular Biology and Bioinformatics Unit (MBBU) and the Animal Rearing Unit (ARU) - icipe for their support and for making this learning experience memorable. I also thank my friends and colleagues at Trek Science (Kenya) for their support. To many others who have not been mentioned, and were of great help directly or indirectly, friends and family, my sincerest thanks. Finally, my deepest thanks to the Lord Almighty, whose grace has been, and still is, sufficient. iv TABLE OF CONTENTS DECLARATION ......................................................................................................................... ii DEDICATION ........................................................................................................................... iii ACKNOWLEDGEMENTS ....................................................................................................... iv LIST OF FIGURES .................................................................................................................. viii LIST OF TABLES ..................................................................................................................... ix LIST OF APPENDICES ............................................................................................................. x LIST OF ABBREVIATIONS AND SYMBOLS ....................................................................... xi ABSTRACT ............................................................................................................................. xiii CHAPTER ONE .......................................................................................................................... 1 1.0 INTRODUCTION ................................................................................................................. 1 1.1 Research Problem and Justification ................................................................................... 4 1.2 Research Question ............................................................................................................. 5 1.3 Research Hypothesis ......................................................................................................... 5 1.4 General Objective .............................................................................................................. 5 1.5 Specific Objectives ............................................................................................................ 5 CHAPTER TWO ......................................................................................................................... 6 2.0 LITERATURE REVIEW ...................................................................................................... 6 2.1 The entomopathogenic enterobacteria ............................................................................... 6 2.1.1 Ecology and biology of Xenorhabdus bacteria........................................................... 6 2.1.2 Pathogenicity of Xenorhabdus spp. ............................................................................ 8 2.1.3 Xenorhabdus spp. insecticidal toxin complex .......................................................... 10 2.1.4 Molecular characterization of Xenorhabdus bacteria ............................................... 14 2.2 Maize production in Africa.............................................................................................. 17 2.3 The lepidopteran stem borers .......................................................................................... 17 2.3.1 Diversity of stem borers ........................................................................................... 17 2.3.2 Chilo partellus and Busseola fusca: distribution, ecology and pest status ............... 18 2.3.3 Biology of Chilo partellus ........................................................................................ 19 2.3.4 Biology of Busseola fusca ........................................................................................ 20 2.3.5 Damage caused by C. partellus and B. fusca. .......................................................... 21 2.3.6 Control measures ...................................................................................................... 22 CHAPTER THREE ................................................................................................................... 26 v 3.0 MATERIALS AND METHODS ........................................................................................ 26 3.1 Materials .......................................................................................................................... 26 3.2 Isolation and identification of Xenorhabdus spp. ............................................................ 26 3.2.1 Bacterial isolation from Steinernema nematodes ..................................................... 26 3.2.2 Morphological identification of bacteria .................................................................. 27 3.2.3 Short and long term bacterial storage ....................................................................... 27 3.2.4 Molecular identification of bacteria ......................................................................... 28 3.3 Molecular characterization of XptB1 and XptC1 genes ................................................... 30 3.3.1 Gene specific primer design and synthesis ............................................................... 30 3.3.2 Degenerate primer design and synthesis .................................................................. 31 3.3.3 PCR amplification of XptB1 and XptC1 genes ......................................................... 34 3.3.4 Gel electrophoresis and extraction of PCR products ................................................ 35 3.3.5 XptB1 and XptC1 gene sequencing ......................................................................... 36 3.4 Protein purification of XptB1 and XptC1 toxins ............................................................. 36 3.4.1. Determination of suitable cell lysis procedure and volumes ................................... 36 3.4.2. Determining the void volume and equilibrating the gel filtration column .............. 38 3.4.3 Gel filtration of Xenorhabdus sp. cell lysate ............................................................ 39 3.4.4 Ammonium sulphate protein concentration ............................................................. 39 3.4.5 Dialysis of concentrated proteins ............................................................................. 40 3.5 Insect bioassay of Xenorhabdus spp. against C. Partellus and B. fusca larvae............... 40 3.5.1 Proliferation of bacteria ............................................................................................ 40 3.5.2 Incorporation of bacteria into artificial diet .............................................................. 40 3.5.3 Introduction of C. partellus larvae into diet ............................................................. 41 3.5.4 Direct injection of Xenorhabdus sp. into C. partellus and B. fusca larvae .............. 41 3.6 Bioinformatics sequence analysis and
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