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Maize Ear Rot and Associated Mycotoxins in Western

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Maize Ear Rot and Associated Mycotoxins in Western MAIZE EAR ROT AND ASSOCIATED MYCOTOXINS IN WESTERN KENYA SAMMY AJANGA A thesis submitted in partial fulfilment of the requirements of the university of Greenwich for the Degree of Doctor of Philosophy December 1999 The research reported in this thesis was funded by the united Kingdom department forInternational development (DFID) forthe benefitof developing countries. The views expressed are not necessarily those ofDFID. [Cropprotection programme (R6582)] I certifythat this work has not been accepted in substance forany degree, and is not concurrently submitted for any degree other than of Doctor of Philosophy (PhD) of the university of Greenwich. I also declare that this work is the result of my own investigations except where otherwise stated. ll Dedicated to my family and parents Acknowledgements I would like to express my sincere gratitude to my supervisor, Dr. Rory Hillocks for his excellent supervision, guidance, kindness and encouragement through this study and preparation of this manuscript. I wish to thank Mr. Martin Nagler for his great support, supervision and guidance. His suggestions, comments and constructive criticism and assistance with the laboratory experimental know how is sincerely appreciated. I express my thanks to Dr. Angela Julian for her invaluable suggestions and contributions at the beginning of this study. I would like to register my thanks to the technical staff at KARI-Kakamega, Mr. G. Ambani and J. Awino for their tireless field visits and assistance in data collection. I am also grateful for the teamwork spirit, assistance, co-operation and friendliness accorded to me at all times by the KARI staff and the staff from the Ministry of Agriculture in Bungoma and Nandi Districts. My deep appreciation goes to the farmers in Tongaren and Kapsabet Divisions for their enthusiasm and participation in this project. I wish to extend my gratitude to the Centre Directors of KARI at Kakamega and Kitale on behalf of Director of KARI for their support to this project. Finally I would like to acknowledge and express my gratitude to the department for International development (DFID) for financing this study. IV Abstract Maize was established as the most important crop and cob rot as the most important crop protection problem in Western Kenya. The mean percentage rotten maize grain resulting from cob rot was 19% based on the PRA survey in Kapsabet and Tongaren Divisions and on-farm evaluation of the Kenyan hybrids in Tongaren Division in 1998 and 1999 seasons. The two important factors affecting the incidence of cob rot were the weather conditions at the period of maturation to harvest and stalk borer. Cob rot incidence was found to be strongly correlated with percentage borer damage (r = 0.87). Fusarium moniliforme was the most frequently isolated fungus in cobs that had stalk borer damage, occurring in 97% of the cobs that had rot and visible borer damage. F. moniliforme was found in 80%, F. graminearum in 56% and S. maydis in 49% of maize samples collected on the farms and markets in Western Kenya. Deoxynivalenol, zearalenone, fumonisin and aflatoxin were detected in samples collected from the farm and markets in the region. T-2 toxin was absent in all samples collected from the region. The levels of deoxynivalenol and zearalenone detected in the samples ranged from 0-1100 ng/g and 0-550 ng/g respectively and were mainly detected from rotten maize. The presence of zearalenone and deoxynivalenol is reported for the first time in maize in Western Kenya. Fumonisin and aflatoxin levels ranged from 0-2348 ng/g and 0-10 ng/g respectively. All the rotten maize harvested in the region was utilized as livestock feed and for brewing. Awareness of the potential risks associated with mycotoxins was low among the farmers and the extension workers. The Kenyan hybrids H627 and H622 were susceptible to Fusarium graminearum. The varieties did not show any difference in reaction to F. moniliforme and S. maydis. This fact was thought to be due to high disease pressure in the field and the genetic background of the hybrids. Abbreviations AFB2 Aflatoxin B2 AOAC Association of Official Analytical Chemistry AR Analytical reagent Av Average CAN Calcium ammonium nitrate CW Cold and wet DAP Di-ammonium phosphate DON Deoxynivalenol FBi FumonisinBi ELEM Equineleucoencephalomalacia ELISA Enzyme linked immuno-assay Ewt Effective weight GLS Gray leaf spot GPR General purpose reagent HH Hot and humid HPLC High performance liquid chromatography HPTLC High performance thin layer chromatography IAC Immuno assay chromatography IARC International agency for research on cancer KARI Kenya Agricultural Research Institute LH2 Lower highland zone 2 MAP Mono-ammonium phosphate Masl Metres above sea level ng Nanogram vi NRI Natural Resources Institute OA Ochratoxin A PDA Potato dextrose sugar PRA Participatory rural appraisal ppb Parts per billion ppm Parts per million SPE Solid phase extraction column TLC Thin layer chromatography TSP Tripple super phosphate UM1 Upper midland zone 1 UM3 Upper midland zone 3 UM4 Upper midland zone 4 uv Ultra violet ZEN Zearalenone Hg Microgram ul Microlitre Vll TABLE OF CONTENTS Statement of certification ii Dedication iii Acknowledgements iv Abstract v Abbreviations vi CHAPTER 1: INTRODUCTION 1 1.1 Maize production and loss to pests and diseases 1 1.2 Background to the present study 2 1.3 Objectives 3 CHAPTER 2: LITERATURE REVIEW 7 2.1 Distribution and importance of maize ear rots 7 2.2 Common fungi associated with maize ear rots 7 2.2.1 Fusarium moniliforme 8 2.2.2 Fusarium graminearum 11 2.2.3 Stenocarpella maydis 12 2.2.4 Aspergillus spp. 13 2.3 Mode of infection of ear rot fungi 14 2.4 Factors favouring maize ear rots and mycotoxin contamination 16 2.5 Strategy to minimize maize ear rots 20 2.5.1 Cultural control methods 21 2.5.2 Resistance breeding 26 2.6 Mycotoxins associated with maize ear rot fungi 29 Vlll 2.6.1 Trichothecenes 30 2.6.2 Zearalenone 30 2.6.3 Moniliformin 31 2.6.4 Fumonisin 32 2.6.5 Aflatoxins 32 2.6.6 OchratoxinA 33 2.7 Diseases associated with mycotoxins 34 2.7.1 Human mycotoxicoses 34 2.7.1.1 Aflatoxins 34 2.7.1.2 Fumonisins 36 2.7.1.3 Trichothecenes 37 2.7.2 Mycotoxicoses in livestock 37 2.7.2.1 Diplodiatoxin 37 2.7.2.2 Zearalenone 38 2.7.2.3 Trichothecenes 38 2.7.2.4 Fumonisins 38 2.7.2.5 Aflatoxin 39 2.7.2.6 OchratoxinA 40 2.8 Strategies to reduce mycotoxin contamination 43 2.8.1 Physical methods 43 2.8.1.1 Cooking and processing 43 2.8.1.2 Sieving and dehulling 45 2.8.2.3 Removal of broken grains and foreign matter 45 2.9.2 Chemical methods 46 IX 2.10 Detection of mycotoxins 46 CHAPTER 3: SURVEY OF FARMERS PERCEPTIONS AND 50 IN RELATION TO FARMER PRACTICES 3.1 Background information of surveyed areas 50 3.2 Methodology 52 3.3 Results 53 3.3.1 Crop enterprises 53 3.3.2 Livestock enterprises 56 3.3.3 Land use 56 3.3.4 Yield and area under maize 57 3.5 Production practices 59 3.4.1 Varieties 59 3.4.2 Source of seed 60 3.4.3 Land preparation 61 3.4.4 Input and fertilizer use 61 3.4.5 Planting 63 3.4.6 Weeding 64 3.4.7 Harvesting 64 3.4.8 Storage 64 3.4.9 Drying methods 73 3.5 Uses of maize stovers 73 3.6 Production constraints 76 3.7 Common pests 78 3.8 Common diseases 78 3.9 Yield loss due to ear rot 80 3.10 Strategies to minimize ear rot 82 3.11 Uses of rotten maize 83 3.12 Discussion 86 3.13 Conclusion 91 CHAPTER 4: EAR ROT INCIDENCE IN MAIZE PLOTS IN 93 IN WESTERN KENYA 4.1 Background 93 4.2 Methodology 94 4.3 Results 95 4.3.1 Survey results 95 4.3.2 Results of mycological analysis 105 4.4 Discussions 115 4.5 Conclusions 117 CHAPTER 5: IDENTIFICATION OF EAR ROT FUNGI 118 IN SAMPLES COLLECTED FROM MARKETS AND FARMS 5.1 Background 118 5.2 Methodology 118 5.3 Results 119 5.4 Discussion 124 5.5 Conclusion 125 XI CHAPTER 6: SURVEY OF MYCOTOXINS ASSOCIATED WITH 127 MAIZE EAR ROT IN WESTERN KENYA 6.1 Background 127 6.2 Mycotoxin survey 128 6.2.1 Design of mycotoxin survey 128 6.2.2 Sample collection 129 6.2.3 Sampling at farm level 129 6.2.4 Sampling at market level 130 6.2.5 Transportation of samples 131 6.2.6 Sample drying 131 6.2.7 Sample storage 131 6.2.8 Sample division 132 6.2.9 Preparation of mycotoxin laboratory sample 132 6.3 Mycotoxin analysis of maize samples 133 6.3.1 Estimation of aflatoxin in maize samples using Steiner method 133 6.3.1.1 Extraction 134 6.3.1.2 Clean-up and quantification 134 6.3.2 Aflatoxin analysis of samples using HPLC method 136 6.3.2.1 Extraction 136 6.3.2.2 Clean-up and quantification 136 6.3.3 Determination of T-2 toxin and deoxynivalenol (DON) by TLC 137 method solid phase clean up cartridges (Romer # 225) 6.3.31 Extraction 137 6.3.3.1 Quantification by3LC 137 6.3.3.3 Quantification of T-2 138 Xll 6.3.4 Determination of T-2 toxin and deoxynivalenol (DON) by HPTLC 13 8 method using solid clean up cartridges (Romer #225) 6.3.4.1 Extraction 138 6.3.4.2 Quantification by HPTLC 138 6.3.4.3 Quantification of T-2 139 6.3.4.4 Quantification of DON 139 6.3.5 Determination of Fumonisin in maize samples by HPLC 139 6.3.5.1 Extraction 139 6.3.5.2 Dilution and filtration 140 6.3.5.3 Fumonisin test affinity chromatography 140 6.3.6 Determination of zearalenone in maize samples by by base clean up 140 and HPTLC method 6.3.6.1 Extraction 140 6.3.6.2 Clean-up 141 6.3.6.3 Quantification 141 6.3.7 Analysis of zearalenone using Zearala-test affinity chromatography 142 6.3.7.1 Extraction 143 6.3.7.2 Dilution and filtration 143 6.3.7.3 Quantification 143 6.4 Results of mycotoxin analysis 143 6.4.1 Deoxynivalenol and T-2 levels in maize samples 143 6.4.2 Aflatoxin levels in maize samples 145 6.4.3 Zearalenone levels in maize samples 147 6.4.4 Fumonisin BI levels in maize samples 150 6.5 Discussion 151 Xlll 6.6 Conclusion 159 CHAPTER?: FIELD EVALUATION OF MAIZE VARIETAL 161 SUSCEPTIBLITY TO EAR ROT PATHOGENS 7.1 Background 161 7.2 Methodology 162 7.2.1 On-station evaluations 162 7.2.1.1 Inoculum preparation and inoculation 163 7.2.2 On-farm evaluations 164 7.3 Results 165 7.3.1 On-station experiments 165 7.3.
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