PHYTOCHEMICAL COMPOSITION AND INSECTICIDAL ACTIVITIES OF SOME BOTANICALS ON THREE STORED PRODUCT PESTS BY NEGBENEBOR, HELEN EHIMEMEN B.Sc. (Hons) Zoology, Ahmadu Bello University, Zaria. Post graduate Diploma in Education, Ahmadu Bello University, Zaria. M.Sc.Zoology, University of Ibadan, Ibadan. A THESIS SUBMITTED TO THE POSTGRADUATE SCHOOL, UNIVERSITY OF LAGOS, AKOKA, LAGOS, NIGERIA. FOR THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) DEGREE IN ZOOLOGY NOVEMBER, 2012 1 DECLARATION We hereby declare that this thesis titled ―Phytochemical composition and insecticidal activities of some botanicals on three stored product pests‖ is a record of an original research work carried out by Negbenebor Helen Ehimemen in the Department of Zoology, University of Lagos, Nigeria. Student Name: Negbenebor H. E. Signatures: …………………………… Supervisors Name: Professor (Mrs.) W. A. Makanjuola (Principal Supervisor) Signature: ……………………………………. Name: Dr. Adebayo Otitoloju (Co-Supervisor) Signature: …………………………………….. 2 SCHOOL OF POSTGRADUATE STUDIES UNIVERSITY OF LAGOS CERTIFICATION This is to certify that the Thesis: “Phytochemical Composition and Insecticidal Activities of Some Botanicals on Three Stored Product Pests” Submitted to the School of Postgraduate Studies University of Lagos For the award of the degree of DOCTOR OF PHILOSOPHY (Ph. D) is a record of original research carried out By: NEGBENEBOR, HELEN EHIMEMEN in the Department of Zoology ____________________ _______________ _______________ AUTHOR’S NAME Signature Date _____________________ ______________ _______________ 1st Supervisor’s Name Signature Date _____________________ _______________ _______________ 2ndSupervisor’s Name Signature Date ______________________ _______________ _______________ 2nd Internal Examiner’s Name Signature Date ______________________ _______________ _______________ Ist Internal Examiner’s Name Signature Date ______________________ __________________ _______________ External Examiner’s Name Signature Date ____________________ ______________ _______________ SPGS Representative Signature Date 3 DEDICATION I dedicate this work to the Almighty God who started the thesis and gave me the grace to complete the work despite all odds and also honoured his word in my life and family as well as for his provision and sustenance. 4 ACKNOWLEDGEMENTS My deep appreciation goes to my supervisors, Professor (Mrs.) Winifred Makanjuola for patiently supervising and seeing me through this work and my second supervisor Dr. Adebayo Otitoloju for his encouragement. I appreciate all the PG lecturers in the Department, particularly Prof. (Mrs.) Rosemary Egonmwan (Head of Department), Prof. (Mrs.) 0. A. Otubanjo, Dr. (Mrs.) Grace Adeoye, Dr. J. K. Saliu and Dr. J. C. Anikwe for their contributions and support at various times. I also sincerely thank Professor Titi Hassan for his support, encouragement and for believing in me. My sincere thanks go to Professor N.E.S. Lale who personally gave me materials for this study, Dr. B. Denloye for his support and intellectual contributions and Dr. Silva for his guidance and support during my defense of title. I wish to acknowledge the contribution of the International Institute for Tropical Agriculture (IITA), Ibadan for use of their Library facilities. I also acknowledge the use of laboratory facilities of the Biochemistry Department, Idi-araba for extraction and phytochemical analysis of the test plant materials. My appreciation goes to Mrs. Olafimihan, Mr. Mike and Mr. Daniel of Chemistry Department in University of Lagos for the gas chromatography and mass spectrometer analysis I acknowledge all the Entomology Laboratory staff of Nigerian Institute for Stored Product Research (NISPR), Abule Oja, Lagos, for the initial supply of insect cultures used for the bioassays and their management for the use of their laboratory. My special appreciation goes to the herbarium staff of the Botany Department, University of Lagos for the identification of test plant species and the voucher numbers of the plants. B 5 I will like to appreciate the late Professor Kio Don-Pedro who was my second supervisor for his immense input into my work and my mother and siblings for their prayer and support. My thanks go to Miss Irese Agbonzeke for hosting me and supporting with prayers. I thank all those who have contributed in one way or the other to the successful completion of this work but have escaped mention in this piece. Finally, I would like to thank my husband Raymond for his love, patience, financial support and encouragement. To my lovely children, Peter and Eric, I say thank you and God bless you. 6 TABLE OF CONTENTS CONTENTS PAGES Title i Declaration ii Certification iii Dedication iv Acknowledgement v Table of Contents vii List of Figures xi List of Plates xii List of Tables xiii Abstract xv CHAPTER ONE: INTRODUCTION 1.1. Background to study 1 1.2. Statement of the problem. 4 1.3. Significance of study 5 1.4. Purpose of study 5 1.5. Objectives of the study 5 1.6 Research questions 6 1.7. Definition of terms 7 CHAPTER TWO: LITERATURE REVIEW 2.1. Importance of Maize (Zea mays) 9 2.1.2. Cowpea (Vigna unguiculata) 10 2.1.3. The need for storage 11 2.2 Damage by stored product insects 13 2.3. Test Insect species 16 2.3.1. Sitophilus zeamais Motsch. (Coleoptera, Curculionidae) 16 2.3.2. Rhizopertha dominica (Coleoptera, Bostrichidae) 18 2.3.3. Callosobruchus maculatus F. (Coleoptera; Bruchidae) 20 2.4. Control measures and their limitations 22 2.4.1 Non–Chemical 22 7 2.4.1.1 Physical/ Mechanical methods 22 2.4.1.2 Biological control 23 2.4.1.3. Pheromones 25 2.4.1.4. Host resistance 26 2.5. Chemical control method 27 2.6. Botanicals 33 2.6.1. Mode of action of botanicals 37 2.7. Selected test plant species used in the study. 39 CHAPTER THREE: MATERIALS AND METHODS 3.0 Materials and methods 47 3.1. Selection of medicinal and agricultural plant materials 47 3. 1.2. Identification of test materials 47 3.2. Test synthetic insecticide 49 3.3. Experimental sites 49 3.4. Preparation of formulations of test plants 49 3.4.1 Dusts 49 3.4.2 Aqueous Extracts 50 3.4.3 Ethanol Extracts 50 3.4.4 Oil 51 3.5. Disinfestations of cowpea and maize seeds. 51 3.6. Experimental insects 51 3.6.1 Sources of test insects 51 3.6.2 Culture of test insects 52 3.6.2.1 Callosobruchus maculatus F. (Coleoptera; Bruchidae) 52 3.6.2.2 Sitophilus zeamais (Motsch) and Rhizopertha Dominica 52 3.7. General bioassay techniques 53 3.7.1. Application of plant materials 53 3.7.1.1. Admixture procedures 53 3.7.1.2. Dipping technique 53 3.8. Screening to detect bioactivity in test plant materials 53 3.8. 1. Preliminary screening of test plants materials 53 3.8.1.1 Dusts 53 3.8.1.2 Aqueous extract 54 8 3.8.1.3 Ethanol extract 54 3.8.2. Relative acute toxicity of test plant extracts 55 3.8.3. Evaluation of the rate of loss of bioactivity (persistence) of test ethanolic plants and oil against C. maculatus on cowpea seeds. 57 3.8.4. Effects of ethanolic test plant extracts on oviposition and progeny development of C. maculatus on cowpea 58 3.8.5. Effects of selected ethanolic test plant extracts on daily rate of oviposition of C. maculatus on grains 59 3.8.6. Effect of ethanolic extract of test plants on cowpea and maize under hermetic conditions for a period of six months. 59 3. 9. 0. Phytochemical analyses of selected test plants 60 3. 9. 1. Preparation of extract for quantitative determination of chemical constituents test plants 60 3.9.2. Quantitative determination of chemical constituents 60 3. 9. 3. Gas chromatography and mass spectrometer of plants 60 3.8. Data analyses 60 CHAPTER FOUR: RESULTS 4.0 Results 63 4.1. Preliminary screening of various formulations of test plants on adult insect species. 63 4.1.1. Effect of various formulations on adult S. zeamais 63 4.1.2 Effect of various formulations on adult R. dominica 68 4.1.3 Effect of various formulations on adult C. maculatus 70 4.2 Acute toxicity of test plant materials against test insect species 84 4.2.1. Sitophilus zeamais 84 4.2.2. Rhizopetha dominica 89 4.2.3. Callosobruchus maculatus 94 4.3 Rate of loss of insecticidal activity of test plant against adult C. maculatus on cowpea 99 4.4. Oviposition and progeny development of C. maculatus on cowpea treated with various test plant formulation 101 4.4.1. Daily rate of oviposition of C. maculatus on grains treated with various extracts 103 9 4.5. Effect of selected plant extracts on long term storage of maize and cowpea 105 4.6. Phytochemistry of the test plants 108 4.6.1 Gas Chromatography and Mass Spectrometer (GCMS) results 110 CHAPTER FIVE: DISCUSSION 5.0 Discussion 118 Conclusion 128 Summary of findings 130 Contribution to knowledge 133 REFERENCES 134 APPENDICES 169 LIST OF FIGURES 10 Figures Page Figure 1: Loss of bioactivity of test plants 87 Figure 2: Daily rate of oviposition of C. maculatus at 92 different concentrations on test plant materials Figure 3: Damage of maize grains protected test plant extracts 97 Figure 4: Damage of cowpea grains protected test plant extracts 100 Figure 5: Gas Chromatography-Mass Spectrometry of P. zeylanica 104 Figure 6: Gas Chromatography-Mass Spectrometry of J. curcas 106 Figure 7: Gas Chromatography-Mass Spectrometry of U. chamae 107 Figure 8: Gas chromatography-mass spectrometry of P. zeylanica 112 Figure 9: Gas chromatography-mass spectrometry of J. curcas 114 Figure 10: Gas chromatography-mass spectrometry of U.chamae 116 LIST OF PLATES 11 Plates Page 1.1 Adult of Sitophilus zeamais 17 1.2. Adult of Rhizopertha dominica 19 1. 3. Adult of C. maculatus 21 12 LIST OF TABLES Table Page Table 1: Plant species selected for evaluation of bioactivity during market survey.