CHARACTERIZATION OF Pseudocercospora griseola, IDENTIFICATION OF LOCAL SOURCES OF RESISTANCE AND EFFECTIVENESS OF GENE PYRAMIDING IN CONTROLLING ANGULAR LEAF SPOT IN COMMON BEAN BY DDAMULIRA GABRIEL BSc. AGRIC, MSc. SOIL SCIENCE (MAK) REG. NO: 2008/HD02/14640U A THESIS SUBMITTED TO THE DIRECTORATE OF RESEARCH AND GRADUATE TRAINING FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN PLANT BREEDING AND BIOTECHNOLOGY OF MAKERERE UNIVERSITY OCTOBER, 2016 i DECLARATION The work presented in this thesis has not been submitted to any other institution of learning for any academic award. Signed……………………………............ Date…………………………………… Gabriel Ddamulira Department of Agricultural Production Makerere University, Kampala ii APPROVAL This thesis has been submitted to the Directorate of Research and Graduate Training, Makerere University with approval of the following supervisors: …………………………......................... Date……………………………............. Dr. Mildred Ochwo-Ssemakula Plant Pathologist, Department of Agricultural Production Makerere University, Kampala …………………………........................ Date……………………………… Dr. Clare Mukankusi Mugisha Plant Breeder, International Centre for Tropical Agriculture (CIAT) Kawanda, Uganda iii ACKNOWLEDGEMENTS I am very grateful to The Regional Universities Forum for Capacity Building in Agriculture (RUFORUM), Carnegie Corporation, Borlaug Leadership Enhancement in Agriculture Program (LEAP), and the International Centre for Tropical Agriculture (CIAT) for funding this research. My doctoral committee including supervisors: Drs. Mildred Ochwo-Ssemakula, Clare Mukankusi Mugisha, Richard Edema, Peter Sseruwagi, Denis Mpairwe and Mentors, Prof. Paul Gepts and Prof. Paul Gibson for the unreserved efforts towards getting this work accomplished. The Director General, National Agricultural Research Organisation (NARO) and the Director, National Crops Resources Research Institute, (NaCRRI) who recommended me for this scholarship. The laboratory staff of CIAT and the National Agricultural Research Laboratories (NARL), Kawanda for the assistance rendered to me during the execution of this research. My family, particularly my wife Harriet, children Gabriella and Timothy who endured my absence during the course of carrying out this research. My sincere and heartfelt thanks go to my mother (Maria Nalwoga), brothers Vincent (RIP), John Bosco, Edward, Joseph and sisters (Sylvia, Gorreth, Harriet, Margaret and Maurice) for the moral, educational and spiritual support rendered to me in my long education journey ever since our father‟s death. My colleagues especially the pioneer Plant Breeding & Biotechnology PhD class at Makerere University, who continuously comforted and supported me especially during the hard moments that came along while conducting this research. Almighty God, who has given me life despite the many friends that I have lost he has reserved me for a purpose. iv TABLE OF CONTENTS DECLARATION ....................................................................................................................... i APPROVAL ............................................................................................................................. ii ACKNOWLEDGEMENTS ...................................................................................................... iii TABLE OF CONTENTS .......................................................................................................... iv LIST OF TABLES ................................................................................................................... vii LIST OF FIGURES ................................................................................................................. ix ABSTRACT ............................................................................................................................. xi CHAPTER ONE: INTRODUCTION ....................................................................................... 1 1.1 Importance and uses of common bean ............................................................................. 1 1.2 Production and limitations to productivity of common bean ........................................... 2 1.3 Problem Statement ............................................................................................................ 2 1.4 Justification of the study .................................................................................................. 3 1.5 Objectives of the study ..................................................................................................... 5 1.6 Hypotheses ....................................................................................................................... 5 CHAPTER TWO: LITERATURE REVIEW ........................................................................ 6 2.1 Taxonomy of beans ....................................................................................................... 6 2.2 Origin, domestication and diversity of common bean .................................................. 6 2.3 Common bean production constraints in Africa .......................................................... 7 2.4 Angular leaf spot .......................................................................................................... 9 2.4.1 Taxonomy, etiology and occurrence ............................................................................ 9 2.4.2 Variability of Pseudocercospora griseola .................................................................... 9 2.4.3 Symptoms and epidemiology of angular leaf spot ................................................... 10 2.4.4 Factors affecting angular leaf spot occurrence and severity ...................................... 12 2.4.5 Angular leaf spot disease management .................................................................... 13 2.4.6 Use of angular leaf spot resistant sources ............................................................... 14 2.4.7 Tools used to characterise Pseudocercospora griseola ............................................ 15 2.4.7.1 Differential bean cultivars .......................................................................................... 15 2.4.7.2 Molecular markers .................................................................................................... 16 2.5 Genetic improvement of common bean .................................................................... 17 2.5.1 The history and progress of common bean breeding in Uganda .............................. 17 2.6 Genetics of angular leaf spot resistance .................................................................... 18 2.7 Gene-pyramiding to improve disease resistance ...................................................... 19 2.8 Diallel mating design ............................................................................................... 19 v 2.9 Marker-assisted gene pyramiding .............................................................................. 21 2.10 Summary ................................................................................................................... 21 CHAPTER THREE: DISTRIBUTION AND VARIABILITY OF Pseudocercospora griseola IN UGANDA ........................................................................................................... 23 3.1 Introduction ............................................................................................................... 22 3.2 Materials and methods ............................................................................................. 24 3.2.1 Incidence and severity of angular leaf spot .............................................................. 24 3.2.2 Fungal isolation and inoculum preparation ............................................................... 25 3.2.3 Inoculation and pathotype determination ................................................................. 27 3.2.4 Statistical data analysis of phenotypic data .............................................................. 28 3.2.5 Molecular characterisation of Pseudocercospora griseola ........................................ 28 3.2.6 Statistical analysis of molecular data ....................................................................... 29 3.4 Results ..................................................................................................................... 30 3.4.1 Incidence of angular leaf spot in the major bean growing areas of Uganda ......... 30 3.4.2 Severity of angular leaf spot in the major bean growing areas of Uganda .............. 30 3.4.3 Angular leaf spot under different bean crop stand ................................................... 32 3.4.4 Race characterisation of Pseudocercospora griseola based on pathogenicity levels on angular leaf spot differential set ......................................................................................... 32 3.4.5 Molecular characterisation of Pseudocercospora griseola ...................................... 33 3.5 Discussion ................................................................................................................. 35 3.6 Conclusion ................................................................................................................ 40 CHAPTER FOUR: IDENTIFICATION OF NEW SOURCES OF RESISTANCE TO ALS AMONG UGANDAN COMMON BEAN LANDRACES ............................................ 41 4.1 Introduction ..............................................................................................................