GENETIC POLYMORPHISM OF PREVALENT ROOT NODULES BACTERIAL STRAIN FROM BAMBARA NUT (INDIGENOUS AFRICAN LEGUME) OAADEDAYO orcid.org/0000-0002-2151-6474 Dissertation submitted in fulfilment of the requirements for the degree Magister in Biology at Mafikeng Campus of the North-West University Supervisor: Prof. Olubukola 0 . Babalola Graduation October 2017 Student number: 27048187 http://dspace.nwu.ac.za/ NORTH-WEST UNIVERSITY ® 11111 YUNIBESITI YA BOKONE-BOPHIRIMA ...., NOORDWES·UNIVERSITEIT It all starts here ™ " GENETIC POLYMORPHISM OF PREVALENT ROOT NODULES BACTERIAL STRAIN FROM BAMBARA GROUNDNUT (INDIGENOUS AFRICAN LEGUME) BY OLALEKAN AYODELE ADEDAYO A Dissertation Submitted in Fulfilment of the requirements fo r the degree MASTER OF SCIENCE (BIOLOGY) DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF SCIENCE, AGRICULTURE AND TECHNOLOGY, NORTH-WEST UNIVERSITY, MAFIKENG CAMPUS, SOUTH AFRICA Supervisor: Professor Olubukola O. Babalola 2016 DECLARATION I, the undersigned, declare that this disse1iation submitted to the North-West University for the degree of Masters of Science in Biology in the Faculty of Science, Agriculture and Technology, School of Environmental and Health Sciences, and the work contained herein is my original work with exception of the citations and that this work has not been submitted at any other University in part or entirety for the award of any degree. STUDENT NAME Olalekan Ayodele ADEDAYO SIGNATURE ................................. DATE ....................................... SUPERVISOR'S NAME Professor Olubukola BABALOLA SIGNATURE ................................. DATE ....................................... 2 DEDICATION This dissertation is dedicated to the Almighty God who is the beginning and ending, the custodian of wisdom, knowledge and understanding, and for sparing my li fe to achieve this task to Him alone be praised. 3 ACKNOWLEDGEMENTS I would like to express my gratitude to the following people for their assistance. Firstly, my supervisor Prof. Olubukola Oluranti Babalola for her support, care, guidance and timely advice to ensure that this work is brought to completion. May you continue to soar higher like an eagle I will extend my acknowledgement to North-West University for offering me North­ West Postgraduate and North-West Institutional bursary awards to pursue this MSc degree. Heartfelt gratitude also goes to Prof. E. Mukwevho, the head of the Department of Biological Sciences for his valuable advice and encouragement throughout my studies. Many thanks also go to all academic and support staff of the Department of Biological Sciences for their love and assistance during this work, as well as to all faculty and staff members of the School of Environmental and Health Sciences, North-West University, for their valuable co­ operation. My sincere appreciation goes to Dr. B.R. Aremu, Dr. M.F. Adegboye, Mrs G. 0 . Onipede, Mrs C.F. Ajilogba, and Mr A.E. Ayangbero for the help rendered in the techniques or part of my research at one stage or the other. I can never thank you enough for all your efforts and patience. I would also li ke to thank all my colleagues in the Microbial Biotechnology Research Group for the good working relationship in the laboratory and their help. I appreciate the time spent with you all. My special thanks go to the co-executives of the Redeemed Campus Fellowship (RCF) of North-West University. I am also indebted to all my friends including Mrs. M.O. 4 Fashola, Ms. A.R. Adebayo, Mrs. A. Akindolire, Mr. and Dr. E.O. Fayemi, Dr. and Mrs. A. Ayeleso, Mr. and Mrs. Omotayo, for their invaluable support. I appreciate all the members of Redeemed Christian Church of God, Mafikeng and Deeper Life Campus Fellowship for all their love, assistance and prayers. I am very grateful to my parents Mr Titus Olanrewaju Adedayo and Late Mrs Felicia Moyosola Adedayo for nurturing in the way of the Lord. I am also indebted to all my spiritual parents; Pastor and Pastor (Mrs) Oduaran, Prophet Ojedele, Pastor Victor Essang, Pastor Joseph Adesina, Pastor Tope Fayemi, Pastor and Deaconess Salawu for all their prayers and encouragement I would also like to thank my father-in- law, mother-in-law, siblings, brother and sister-in-law, niece, nephew and cousins. They have given me their unequivocal support. Thanks to my wife, Mrs Oluwafunke Adedayo for her spiritual , financial and moral support. She always stood by me through the good and bad times with great patience at all times. Finally, I thank all those who have helped me directly or indirectly in the successful completion of this project. Anyone missed in this acknowledgement is also thanked. Above all, I give all the glory to God Almighty the creator of heaven and earth, the all sufficient God for His provisions, mercy, grace and Favour. To Him be all glory and honour forevermore. Amen 5 GENERAL ABSTRACT Bambara groundnut is an underutilized crop of good microbial resources in green manure. The knowledge of the true microbial diversity in rhizopheric bacteria of Bambara groundnut is fundamental to effective exploitation of these bacteria. This study employed morphological, biochemical as well as molecular approach in the isolation, identification and characterization of these bacteria. Morphological and biochemical analysis revealed only 8 out of 30 rhizopheric bacteria isolated from the Bambara root nodules as potential nitrogen fixing bacteria. These were further analyzed using the 16S rDNA and PCR-RFLP. The 16S rDNA sequences utilized in phylogenetic tree showed that 4 of the strains NWU A3 (Enterococcus durans), NWU A8 (Bacillus pumilus) NWU A 7 (Acinetobacter johnsonii) and NWU A 10 (Acinetobacter johnsonii) possessed 99% similarity index with the reference strains while 4 strains did not cluster with any of the reference strain due to their distinct nucleotide signature from the existing one. These strains are: NWU A2 (Fictibacillus nanhaiensis), NWU AS (Bacillus pumilus), NWU A6 (Fictibacillus arsenicus), and NWU A9 (Acinetobacter johnsonii). The PCR-RFLP expressed fingerprints of the strains generated by the ribosomal genes in three distinguished patterns of different combinations, representing three distinct l 6S rRNA genotypes among all the strains. Gene responsible for nodulation and nitrogen fixation in these strains are borne on their plasmid, which is an indication of probable horizontal gene transfer. In a nutshell, the rhizopheric bacteria found in the root nodules of Bambara groundnut can be used as biofertilizer in the future as main microbial resource, considering its rich bacterial diversity. This biological nitrogen fixation technology could be a right substitute to commercially available N fertilizer which are less environmentally friendly. In future research, three diverse group of rhizobia expressed from the PCR-RFLP point of view can further be analyzed and characterized through high throughput molecular technique to choose the ideal strain of rhizobia for Bambara. NWU· · 1 6 !LIBRARY Keywords: Bambara groundnut, bacteria, molecular, PCR-RFLP, rhizopheric 7 LIST OF PUBLICATIONS Chapter 2: Impact of Bambara groundnut symbiosis with rhizobium for sustainable crop yield improvement. This chapter has been formatted for publication in World Journal of Microbiology & Biotechnology. Authors: Candidate's Contributions: designed the study, managed the literature searches, and wrote the first draft of the manuscript. Chapter 3: Genetic diversity of root nodule bacteria associated with Bambara groundnut. This chapter has been formatted for publication in Turkish Journal ofBiology . Authors: Candidate's Contributions: designed the study, managed the literature searches, and wrote the first draft of the manuscript. Chapter 4: Morphological and molecular characterization of prevalent root nodule bacterial strain from African Bambara groundnut (Indigenous African Legume). This chapter has been formatted for publication in Archives ofMicrobiology. Authors: Candidate's Contributions: managed the literature searches, carried out all the wet laboratory bench work, performed all the analyses, interpreted the results and wrote the first draft of the manuscript. Chapter 5: Determination of genetic diversity and phylogeny of rhizobia isolated from root nodule bacterial of Bambara groundnut using PCR-RFLP. This chapter has been formatted for publication in Biology Bulletin Authors: 8 Candidate's Contributions: managed the literature searches, carried out all the wet laboratory bench work, performed all the analyses, interpreted the results and wrote the first draft of the manuscript. 9 CHAPTER ONE GENERAL INTRODUCTION 1.1 Background and Rationale Polymorphism refers to different phenotypes that exist in the same population of a species. The term is used by molecular biologists to describe certain point mutations in the genotype polymorphism which is common in nature. It is related to biodiversity, genetic variation and adaptation. It usually functions to retain variety of form in a population living in a varied environment (Adeparusi, 2001). In polymorphism that is genetically related, the genetic make-up determines the morphs. The extent of polymorphism is such that, except for identical twins, no two humans share the same genetic composition. According to Conrad et al. (2010), any two individual genomes taken from nature, in any species, will have dozens to hundreds of differences in their total number of functional genes. As Copy-Number Variants (CNVs) are due to both duplications and deletions, these differences will be due to newly