UNIVERSITY OF EMBU EDWARD NDERITU KARANJA PhD 2020 MICROBIAL COMMUNITY DIVERSITY AND STRUCTURE WITHIN ORGANIC AND CONVENTIONAL FARMING SYSTEMS IN CENTRAL HIGHLANDS OF KENYA EDWARD NDERITU KARANJA (MSc) A THESIS SUBMITTED IN PARTIAL FULFILLMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN APPLIED MICROBIOLOGY IN THE UNIVERSITY OF EMBU NOVEMBER, 2020 DECLARATION This thesis is my original work and has not been presented for a degree in any other University Signature……………………………. Date………….……….. Edward Nderitu Karanja Department of Biological Science B801/147/2015 This thesis has been submitted for examination with our approval as the University Supervisors Signature……………………………. Date………….………. Prof. Romano Mwirichia Department of Biological science University of Embu (UoEm), Kenya Signature………. …………………………. Date………….…………. Dr. Andreas Fliessbach Department of Soil Science Research Institute of Organic Agriculture - FIBL, Switzerland i DEDICATION I dedicated to my family; my wife Anne Kelly Kambura, my children; Shawn Karanja, Melissa Wangithi, Joseph Munyuithia, Shayne Koome and Ann Wanjiku, my parents; Mr. Samuel Karanja and Mrs. Agnes Wangithi, my siblings, Ruth Wairimu, Juliet Muthoni, Alex Ngochi, James Karuma and Nelly Njoki. I appreciate the support you have accorded me during my studies. Your inspiration and backing in this journey made it easier to manage all challenges encountered. ii ACKNOWLEDGEMENT I express gratitude toward Almighty God for his mercies from the beginning of this long and thought-provoking journey. This was conducted in the framework of long-term systems comparison program, with financial support from Biovision Foundation, Coop Sustainability Fund, Liechtenstein Development Service (LED) and the Swiss Agency for Development and Cooperation (SDC). I acknowledge icipe core funding for the kind contribution provided by UK-Aid from UK Government, Swedish International Development Cooperation Agency, Swiss Agency for Development and Cooperation, Federal Democratic Republic of Ethiopia and the Kenyan Government. I recognize icipe (executing agency of the project in Kenya) for the opportunity to carry out my PhD study within the project. This work could not have been accomplished without the wise counsel, specialized intellect and research mentorship of Prof. Romano Mwirichia and Dr. Andreas Fliessbach on farming systems microbiome studies. I deeply appreciate my supervisors for their patience and for having confidence in me. Thank you for standing by me. My most sincere gratitude goes to Dr. Noah Adamtey, Dr. Komi Fiaboe, Dr. Chrysantus Tanga and Dr. Martha Musyoka for the help they accorded me as I assumed training and attended to my work obligations at icipe. Your presence made this research promising and the challenges encountered functional. In addition, no adjectives can define the appreciation I have for the support offered by the SysCom project team; the late Mr. Peter Owuor, Mr. Felix Matheri, Mr. James Karanja, Ms. Jane Makena and Ms. Felistus Mutua during field activities, field maintenance, data collection and soil sampling. I appreciate their intellectual ability as they ensured exact sampling and sample handling techniques were achieved for this research. I am greatly indebted to them. Particular recognition goes to Crop nutrition laboratory services and KALRO NARL Soil physics laboratory staff led by Mr. Edward Komba and Mr. John Mwangi for their support and during soil samples handling, extraction and analysis process. iii I would like to index incomparable indebtedness to my loving wife for her unending encouragement, patience and personal sacrifices through the process of accomplishing this achievement. I cannot thank her enough for being there for me. iv TABLE OF CONTENTS DECLARATION ......................................................................................................................... i DEDICATION ........................................................................................................................... ii ACKNOWLEDGEMENTS ...................................................................................................... iii TABLE OF CONTENTS ........................................................................................................... v LIST OF TABLES .................................................................................................................... ix LIST OF FIGURES .................................................................................................................... x LIST OF APPENDICES ......................................................................................................... xiv LIST OF ABBREVIATIONS AND ACRONYMS ................................................................. xv ABSTRACT ........................................................................................................................... xvii CHAPTER ONE: INTRODUCTION ..................................................................................... 1 1.1 General Introduction ............................................................................................................. 1 1.2 Statement of the problem ...................................................................................................... 2 1.3 Justification ........................................................................................................................... 3 1.4 Hypothesis ............................................................................................................................ 4 1.5 Objectives ............................................................................................................................. 4 1.5.1 General Objective .............................................................................................................. 4 1.5.2 Specific Objectives ............................................................................................................ 4 CHAPTER TWO: REVIEW OF LITERATURE ................................................................. 5 2.1 General background of farming systems .............................................................................. 5 2.2 Sustainable and conventional agricultural systems .............................................................. 6 2.3 Organic resources used in agro-ecosystems ......................................................................... 7 v 2.4 Long-term farming system comparison in the tropics .......................................................... 8 2.5 Soil microbial ecology .......................................................................................................... 9 2.6 Prokaryotic organisms in soil ecosystems .......................................................................... 11 2.7 Microbial ecology in farming systems ............................................................................... 13 2.8 Fungal communities in soil habitats ................................................................................... 14 2.9 Effect of land use on microbial diversity ........................................................................... 15 2.10 Use of molecular techniques in microbial ecology ……………………………………..15 CHAPTER THREE: MATERIALS AND METHODS ...................................................... 18 3.1 Study sites ........................................................................................................................... 18 3.2 Farming systems ................................................................................................................. 19 3.3 Soil sampling ...................................................................................................................... 25 3.4 Microbial community analysis ........................................................................................... 26 3.4.1 Total DNA extraction ...................................................................................................... 26 3.4.2 Total RNA Extraction ...................................................................................................... 27 3.4.3 Synthesis of cDNA from 16S rRNA ............................................................................... 27 3.4.4 16S rRNA amplicon library preparation and sequencing ................................................ 28 3.4.5 Prokaryotic bioinformatic sequence processing, taxonomic identification and statistical analysis………………………………………………………………………………….28 3.4.6 Fungal amplicon DNA library preparation and sequencing ............................................ 30 3.4.7 Fungal Sequence analysis ................................................................................................ 29 3.4.8 cDNA synthesis from mRNA, library construction and sequencing .............................. 31 3.4.9 Transcriptome sequence analysis .................................................................................... 32 vi CHAPTER FOUR: RESULTS .............................................................................................. 34 4.1 Diversity and structure of prokaryotic communities within organic and conventional farming systems in central highlands of Kenya…......……………………………………34 4.1.1 General sequence analysis .............................................................................................. 34 4.1.2 Diversity indices of soil prokaryotic communities ......................................................... 42 4.1.3 Soil