UNIVERSITY OF EMBU PHILEMON ORWA MASTERS THESIS 2020 DIVERSITY AND BIOLOGICAL CONTROL POTENTIAL OF HALOALKALIPHILIC FUNGI FROM LAKE MAGADI, KENYA PHILEMON ORWA A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF MASTER OF SCIENCE IN APPLIED MICROBIOLOGY IN THE UNIVERSITY OF EMBU JANUARY, 2020 DECLARATION This thesis is my original work and has not been presented for a degree in any other University Signature………………………. Date…………………………… Philemon Orwa Department of Biological Sciences B526/1125/2017 This thesis has been submitted for examination with our approval as the University Supervisors Signature…………………….. Date…………………............. Dr. Romano Mwirichia, Department of Biological Sciences, University of Embu. Signature …………………….. Date………………………… Dr. Vitalis Wekesa, Flamingo Horticulture (K) Ltd (Dudutech Division), Naivasha Signature……………………… Date…………………………. Dr. George Mugambi, Meru University of Science and Technology. ii DEDICATION I dedicate this work to Almighty God, my family and my mentor Dr. Romano Mwirichia. iii ACKNOWLEDGEMENT I acknowledge University of Embu for awarding me a scholarship to cover the tuition fees. Special thanks to my first supervisor Dr. Romano Mwirichia who was always there to oversee all my lab work and provided me with funds throughout my study. I also thank my other two supervisors, Dr. Vitalis Wekesa and Dr. George Mugambi for their guidance and advices. iv TABLE OF CONTENTS DECLARATION .............................................................................................................. ii DEDICATION ................................................................................................................. iii ACKNOWLEDGEMENT ................................................................................................ iv TABLE OF CONTENTS ................................................................................................... v LIST OF TABLES ............................................................................................................ ix LIST OF FIGURES ........................................................................................................... x LIST OF PLATES ............................................................................................................ xi LIST OF ABBREVIATIONS AND ACRONYMS ........................................................ xii ABSTRACT ................................................................................................................... xiii CHAPTER ONE .............................................................................................................. 1 INTRODUCTION ............................................................................................................ 1 1.1 Background information ........................................................................................... 1 1.2 Statement of the problem ......................................................................................... 4 1.3 Justification .............................................................................................................. 4 1.4 Hypothesis ................................................................................................................ 5 1.5 Objectives ................................................................................................................. 5 1.5.1 General objective ............................................................................................... 5 1.5.2 Specific objectives ............................................................................................. 5 CHAPTER TWO ............................................................................................................. 6 LITERATURE REVIEW ................................................................................................ 6 2.1 Soda lake environments ............................................................................................ 6 2.2 Microbial biodiversity and productivity of soda lakes ............................................. 6 2.3 Fungal diversity in extreme ecosystems ................................................................... 7 v 2.4 Fungal adaptation to extreme salinity ...................................................................... 8 2.5 Production of secondary metabolites by haloalkaliphilic/halotolerant fungi ........... 9 2.6 Fungi as biological control agents .......................................................................... 11 CHAPTER THREE ....................................................................................................... 13 MATERIALS AND METHODS .................................................................................. 13 3.1 Study site ................................................................................................................ 13 3.2 Sample collection ................................................................................................... 14 3.3 Media preparation ................................................................................................... 14 3.4 Fungal isolation ...................................................................................................... 15 3.4.1 Dilution plate technique ................................................................................... 15 3.5 Morphological characterization of the isolates....................................................... 15 3.5.1 Colonial morphology ....................................................................................... 15 3.5.2 Cellular morphology ........................................................................................ 15 3.6 Physiological characterization of the isolates......................................................... 15 3.6.1 Tolerance to different salt concentration ......................................................... 16 3.6.2 Tolerance to different pH ................................................................................. 16 3.6.3 Tolerance to different temperatures ................................................................. 16 3.7 Biochemical characterization of the isolates .......................................................... 16 3.7.1 Determination of esterase and lipase activity .................................................. 18 3.7.2 Determination of proteinase activity................................................................ 18 3.7.3 Determination of pectinase activity ................................................................. 18 3.7.4 Determination of Amylolytic activity .............................................................. 18 3.7.5 Determination of the cellulolytic activity ........................................................ 18 3.7.6 Determination of peroxidase/laccase activity .................................................. 19 3.7.7 Determination of chitinase activity .................................................................. 19 vi 3.8 Molecular characterization ..................................................................................... 19 3.8.1 DNA Extraction ............................................................................................... 19 3.8.2 PCR amplification of 18S rRNA ..................................................................... 20 3.8.3 Purification of PCR products and sequencing ................................................. 21 3.8.4 Phylogenetic data analysis ............................................................................... 21 3.9 Screening for antimicrobial activity ....................................................................... 21 3.9.1 Primary antimicrobial screening ...................................................................... 21 3.9.2 Secondary antimicrobial screening .................................................................. 22 CHAPTER FOUR .......................................................................................................... 24 RESULTS ....................................................................................................................... 24 4.1 Sampling ................................................................................................................. 24 4.1.1 Physicochemical characteristics ...................................................................... 24 ...................................................................................................................................... 24 4.2 Fungal isolation ...................................................................................................... 25 4.3 Morphological characterization of the isolated fungi ............................................. 25 4.3.1 Colony morphology ......................................................................................... 25 4.3.2 Cell morphology .............................................................................................. 25 4.4 Physiological characterization of the isolates......................................................... 30 4.4.1 Salt Tolerance .................................................................................................. 30 4.4.2 pH Tolerance.................................................................................................... 31 4.4.3 Temperature Tolerance .................................................................................... 31 4.5 Biochemical Characterization