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Identification of Citrus (Citrus Sinensis) Postharvest Pathogens from Ethiopia and Their Control

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Identification of Citrus (Citrus Sinensis) Postharvest Pathogens from Ethiopia and Their Control University of Pretoria etd – Mekbib, S B (2007) Identification of citrus (Citrus sinensis) Postharvest Pathogens from Ethiopia and their Control By Sissay Bekele Submitted in partial fulfilment of the requirements for the degree of Ph.D. in Plant Pathology to the Faculty of Natural and Agricultural Sciences University of Pretoria 2006 University of Pretoria etd – Mekbib, S B (2007) DECLARATION I, the under signed hereby declare that the work reported herein is the result of my original research findings. Name: Sissay Bekele Signature & date: ____________________ II University of Pretoria etd – Mekbib, S B (2007) ACKNOWLEDGMENTS I sincerely wish to express my deepest gratitude and appreciation to my supervisor Prof. Lise Korsten, and co- supervisor Dr. Thierry Regnier without whose academic support and continued guidance throughout this work would not have made this study possible. I am very much grateful to all the staff members and students of Plant Pathology Laboratories, Department of Microbiology and Plant Pathology, University of Pretoria for encouragement, support and assistance. In particular, I am indebted to Mrs Amelita Lombard and Karin Zeeman who have given me the confidence and training to work in the ISO IEC 17075 accredited laboratories. I am also grateful to Mrs Daleen Muller for her unchanged smile and respect in due course of repeated enquiry in the process of learning. Special thanks must go to Colonel Teshome Ayalew and W/o Yigardu G/micheal family for their unreserved support and family care throughout my studies abroad. I would like to express my greatest appreciation to Dr. Seifu G/mariam from Melkassa Agricultural Research Institute, Nazareth for his guidance and sharing of ideas during my field survey. People who are too many to mention were key informants while collecting medicinal plants from and around citrus farms of Ethiopia are greatly acknowledged. I am deeply grateful to all the institutions who were directly or indirectly involved in this work. To mention: Upper Awash Agro Industry Enterprise, Horticulture Development Enterprise, Ethiopian III University of Pretoria etd – Mekbib, S B (2007) Fruit and Vegetables Marketing Corporation (Ethfruit), Ethiopian Coffee and Tea Authority library Addis Ababa, Hursso Cadet Training Center citrus farm, Toni citrus farm (Alemaya University), Tisabalima Amhara Regional Estate Development Farm, Methahara Sugar Estate citrus farm, Ethiopia. Faculty of Agriculture and Biotechnology Institute, Pretoria University, Letaba Estate fruit farms, Johan Kellerman (Crock Valley citrus packhouse) and Kieth Lesar (Citrus Research Institute, Nelspruit) South Africa are greatly acknowledged. I would like to thank the Agricultural Research Training Programme, Alemaya University for granting me a study fellowship through the World Bank Fund. I would like to extend my thanks to the Department of Microbiology and Plant Pathology, University of Pretoria for a kiss of life while doing my final research. Above all, I thank God for all power He gave me day and night to work. …. Oh God, you are my lamp who turns my darkness into light. You armed me with strength and make my way perfect and enable me to stand on the height! Samuel 22: 15 Vs 3 IV University of Pretoria etd – Mekbib, S B (2007) DEDICATION This thesis is dedicated to my wife W/o Tsigreda Gebremicheal, my children: Friezer, Bisirat, Biruktawit and the lovely young Yabsira Sissay for their enormous love, commitment and courage while I was away during my study. I don’t have a substitute to express my feelings for what you mean to me. V University of Pretoria etd – Mekbib, S B (2007) CONTENT CHAPTER ONE GENERAL INTRODUCTION 1 Literature cited 4 CHAPTER TWO LITERATURE REVIEW 8 Literature cited 23 CHAPTER THREE CITRUS (CITRUS SINENSIS ) DISEASE SURVEY: KNOWLEDGE, ATTITUDE AND MANAGEMENT PRACTICES IN ETHIOPIA 38 Abstract 38 Introduction 39 Material and Methods 39 Results and Discussion 41 Literature cited 56 CHAPTER FOUR DEVELOPING ETHIOPIAN MICROBIAL BIOCONTROL AGENT(S) FOR CITRUS POSTHARVEST DISEASE CONTROL 59 Abstract 59 Introduction 60 Material and Methods 61 Results 68 Discussion 87 Literature cited 89 VI University of Pretoria etd – Mekbib, S B (2007) CHAPTER FIVE IN VITRO ANTIMICROBIAL ASSAY OF SOME MEDICINAL PLANTS FROM ETHIOPIA AGAINST PLANT AND FOOD BORNE PATHOGENS 94 Abstracts 94 Introduction 95 Material and Methods 96 Results 101 Discussion 122 Literature cited 124 CHAPTER SIX CONTROL OF PENICILLIUM DIGITATUM GROWTH ON CITRUS FRUIT USING TWO PLANT EXTRACTS AND THEIR MODE OF ACTION 128 Abstracts 128 Introduction 129 Material and Methods 130 Results 134 Discussion 143 Literature cited 145 CHAPTER SEVEN SEMI COMMERCIAL EVALUATION OF PLANT EXTRACTS ON QUALITY RETENTION IN CITRUS SINENSIS 150 Abstract 150 Introduction 165 Material and Methods 152 Results 156 Discussion 162 Literature cited 163 VII University of Pretoria etd – Mekbib, S B (2007) CHAPTER EIGHT GENERAL DISCUSSION AND CONCLUSION 169 Literature cited 173 RESUME 174 APPENDIX I 176 APPENDIX II 195 APPENDIX 11I 209 VIII University of Pretoria etd – Mekbib, S B (2007) Identification of citrus (Citrus sinensis) Postharvest Pathogens from Ethiopia and their Control By Sissay Bekele Promoter: Prof. Lise Korsten Co-promoter: Dr. Thierry Regnier Department: Microbiology and Plant Pathology Faculty: Natural and Agricultural Sciences Degree: Ph.D. (Plant Pathology) SUMMARY From a world prospective, the continuous application of chemical pesticides has serious long- term effects on human health and environmental pollution, and can result in resistant pathogen strains. However, postharvest diseases cause major losses on the markets and need to be controlled effectively. The search for biopesticides using microbial antagonists and natural plant products has subsequently become more important as viable alternatives to control postharvest diseases. Currently, little information exists in terms of citrus production practices, disease management measures and postharvest losses in Ethiopia. The aim of this study was therefore to determine what the current situation in the country is in terms of production, disease management and postharvest disease incidence, disease management practices in Ethiopia and to develop an effective and safe disease control strategy for the industry. Citrus production in Ethiopia is mainly done by Government enterprises with little technical expertise. Disease control strategies are ineffective with postharvest losses exceeding 46%. The most important postharvest pathogen identified was Penicillium digitatum. In development of biopesticides, three yeast antagonists [Cryptococcus laurentii (strain MeJtw 10-2 and strain TiL 4-3) and Candida sake (TiL 4-2)] and plant leaf extracts of Acacia seyal and Withania somnifera were found to have some potential to 180 University of Pretoria etd – Mekbib, S B (2007) control Penicillium in in vitro and in vivo trials and ensure fruit quality. The modes of action of the yeast antagonists were not based on antibiosis. Instead, it involved competitive colonization where the antagonists inhibited P. digitatum spore germination and reduced mycelial growth by 75-100%. Extracts from the two plant species showed broad-spectrum antimicrobial activity against a range of several fungal and bacterial pathogens. The semi-commercial application of the antagonists and plant extracts improve fruit quality and the integration of these biopesticides were found effective in semi commercial trials and may provide a commercial solution for the citrus industry. 181 University of Pretoria etd – Mekbib, S B (2007) CHAPTER ONE GENERAL INTRODUCTION Citrus (Citrus sinesis L.) is one of the major commercial fruit crops that is widely consumed both as fresh fruit or juice. Its global demand is attributed to its high vitamin C content and its antioxidant potential (Gorinstein et al., 2001). Citrus is mainly cultivated in the subtropical and tropical regions of the world between 40 o north and south latitude in over 137 countries on six continents (Ismail and Zhang, 2004). Brazil is the largest producer followed by the United States of America (USA), China and Mexico. Spain, USA and South Africa are the largest exporter countries followed by Turkey and Morocco (Citrus Commodity Notes, 2005). Citrus is an important fruit crop in international trade next to grapes requiring excellent quality and shelf life attributes. Unfortunately, citrus is attacked by several plant pathogens that affect its fruit quality. In developing countries, where protection and proper handling of fresh fruit is inadequate, losses during transit and storage can represent in excess of 50% of the harvested crop (Eckert and Ogawa, 1985; Wisniewski and Wilson, 1992). Major postharvest losses have been recorded on the export markets associated with a range of pathogens. These include green and blue mould caused by Penicillium spp., gray mould caused by Botrytis cinerea Pers ex Fr (Agrios, 1997), Alternaria rot caused by Alternaria citri Elli and Pierce (Whiteside et al., 1988), anthracnose caused by Colletotrichum gloeosporioides Penz (Davies and Albrigo, 1994), Aspergillus rot caused by Aspergillus niger Van Tiegh, brown rot caused by Phytophthora parasitica Dast. (syn. P. nicotianae Breda de Haan), Diplodia stem-end rot caused by Diplodia natalensis Evans (Brown, 1994), sour rot caused by Geotrichum candidum Link ex Pers (Howard, 1936) and Trichoderma rot caused by Trichoderma viride Pos ex Gray (Whiteside et al., 1988). However, it is often reported that the importance
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