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Isolation and Characterization of Actinobacteria with Potential for Control of Fusarium Spp and Colletotrichum Kahawae

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Isolation and Characterization of Actinobacteria with Potential for Control of Fusarium Spp and Colletotrichum Kahawae ISOLATION AND CHARACTERIZATION OF ACTINOBACTERIA WITH POTENTIAL FOR CONTROL OF FUSARIUM SPP AND COLLETOTRICHUM KAHAWAE JAMES OLUOCH NONOH A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Microbiology of Jomo Kenyatta University of Agriculture and Technology 2007 Declaration This thesis is my original work and has not been presented for a degree in any other university. Signature………………………… Date……………………….. James Oluoch Nonoh This thesis has been submitted for examination with our approval as supervisors Signature………………………… Date……………………….. Prof. Hamadi Boga JKUAT, Kenya Signature………………………… Date………………………. Dr. Bernard A. Nyende JKUAT, Kenya Signature………………………… Date……………………….. Dr. Wilber Lwande ICIPE, Kenya Signature………………………… Date……………………….. Dr. Dan Masiga ICIPE, Kenya ii Dedication This work is dedicated to my late parents, Mr. and Mrs. Nonoh. Thank you for all the support you gave me. You laid in me a good foundation that has seen me through to this level of education. iii Acknowledgements I am greatly indebted to Du Pont Agricultural Enterprise (USA) for the research grant without which it would not have been possible to do this work. I would like to thank the International Center of Insect Physiology and Ecology (ICIPE), through the Director of Training and Capacity Development, Dr. J.P.R Ochieng Odero for awarding me the Dissertation Research Internship Program (DRIP) fellowship and for availing to me the use of the institutions facilities. I am pleased to express my acknowledgement to Dr. Wilber Lwande, Head of Bioprospecting Programme (ICIPE), for all the necessary support he gave me throughout my study period. I sincerely thank Kenya Wildlife Service (KWS) for giving me permission to collect samples from protected National Parks. I would like to thank the department of Botany of Jomo Kenyatta University of Agriculture and Technology (JKUAT) for the assistance provided throughout my study period. I am deeply indebted to all my supervisors Prof. Hamadi Boga, Dr. Bernard Nyende, Dr. Wilber Lwande and Dr. Dan Masiga for the guidance, encouragement and correction that they provided throughout the study period. With pleasure, I wish to express my sincere gratitude to my wife Lillian for the endless love and moral support accorded during this study. Lastly, I thank all my colleagues in Applied Bioprospecting Program and Molecular Biology and Biotechnology Department for all their assistance and for encouraging a friendly working environment in the laboratory during this work. In particular, I thank Dr. Matilda Okech, Emma, Bwire, Priscillar, Rose, Moreka, David, Paul, Vincent, Harrison, Aman, Wainaina, Kabii, Kimondo and others. iv Table of contents Declaration.......................................................................................................................... ii Dedication..........................................................................................................................iii Acknowledgements............................................................................................................ iv Table of contents................................................................................................................. v List of tables....................................................................................................................... ix List of figures...................................................................................................................... x List of appendices ............................................................................................................. xii Abbreviations...................................................................................................................xiii Abstract............................................................................................................................ xiv 1.0 CHAPTER 1: INTRODUCTION................................................................................. 1 1.1 Background............................................................................................................... 1 1.2 Control of crop pathogens......................................................................................... 1 1.3 Actinobacteria........................................................................................................... 3 1.3.1 Diversity of actinomycetes................................................................................. 4 1.3.2 Actinobacteria as sources of antibiotics and other secondary metabolites ........ 5 1.3.3 Activity of actinobacteria in degradation of waste ............................................ 5 1.3.4 Use of actinobacteria in farming........................................................................ 6 1.3.5 Characterization of actinobacteria ..................................................................... 7 1.4 Justification...............................................................................................................9 1.5 Objectives ............................................................................................................... 10 1.5.1 General objectives............................................................................................ 10 1.5.2 Specific objectives ........................................................................................... 11 v 1.6 Hypothesis .............................................................................................................. 11 2.0 CHAPTER 2: LITERATURE REVIEW.................................................................... 12 2.1 Biological factors affecting agricultural productivity............................................. 12 2.2 Plant fungal pathogens............................................................................................ 15 2.2.1 Fusarium oxysporum ....................................................................................... 16 2.2.2 Colletotrichum kahawae .................................................................................. 19 2.3 Biological control of crop pathogens...................................................................... 21 2.4 Metabolites from microbial sources ....................................................................... 22 2.5 Biological control agents ........................................................................................ 23 2.6 Screening for bioactive natural products ................................................................ 24 2.7 Advantages of biological control............................................................................ 26 2.8 Genetic diversity among biological control agents................................................. 28 3.0 CHAPTER 3: MATERIALS AND METHODS ........................................................ 30 3.1 Plant pathogens....................................................................................................... 30 3.2 Soil sampling sites .................................................................................................. 30 3.3 Isolation and maintenance of actinobacterial cultures............................................ 32 3.4 Growth in broth cultures and extraction of secondary metabolites ........................ 32 3.5 Anti-fungal screening assays .................................................................................. 33 3.6 Characterization of isolates..................................................................................... 34 3.6.1 Morphological characterization ........................................................................... 34 3.6.1.1 Sporophore studies........................................................................................ 34 3.6.2 Biochemical characterization............................................................................... 35 3.6.2.1 Carbohydrate fermentation ........................................................................... 35 vi 3.6.2.2 Glucose oxidation fermentation (O-F) test ................................................... 36 3.6.2.3 Methyl Red-Voges- Proskauer (MR-VP) test............................................... 36 3.6.2.4 Starch hydrolysis........................................................................................... 36 3.6.2.5 Gelatin liquefaction....................................................................................... 37 3.6.2.6 Indole production and Hydrogen sulfide production.................................... 37 3.6.2.7 Catalase test .................................................................................................. 37 3.6.2.8 Oxidase test................................................................................................... 38 3.6.2.9 Citrate utilization .......................................................................................... 38 3.6.2.10 Nitrate reduction test................................................................................... 38 3.6.2.11 Urease test................................................................................................... 38 3.6.3 Molecular characterization of the isolates ........................................................... 39 3.6.3.1 DNA extraction from actinobacterial cells ................................................... 39 3.6.3.2 PCR amplification of actinobacterial 16S rDNA.......................................... 40 3.6.3.3 Purification
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