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Evaluation of Different Media Formulations on Spore Production and Toxicity of Bacillus Thuringiensis Subsp

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Evaluation of Different Media Formulations on Spore Production and Toxicity of Bacillus Thuringiensis Subsp Evaluation of different media formulations on spore production and toxicity of Bacillus thuringiensis subsp. aizawai T.P Sehaole 20348819 Dissertation submitted in fulfilment of the requirements for the degree Master of Science in Environmental Science at the Potchefstroom Campus of the North-West University Supervisor: Dr. S. Claassens Co-Supervisor: Dr. J.J. Bezuidenhout March 2012 This work is dedicated to my parents, Rosy and Lucas Moloto and my siblings Karabo & Moeder. I thank them for their patience, encouragement and support throughout my entire studies. Thank you for this wonderful opportunity. May God Almighty bless you. i To God be the Glory. “For with men these things are impossible, but with God all things are possible. Luke 1:37” ii Table of contents PREFACE .................................................................................................................................................... v ACKNOWLEDGEMENTS......................................................................................................................... vi SUMMARY................................................................................................................................................. vii LIST OF FIGURES .................................................................................................................................... ix LIST OF TABLES ...................................................................................................................................... xi LIST OF ABBREVIATIONS ..................................................................................................................... xii CHAPTER 1- INTRODUCTION ................................................................................................................ 1 1.1 Background ....................................................................................................................................... 2 1.2 Problem statement ........................................................................................................................... 2 1.3 Aims and objectives ......................................................................................................................... 4 1.4 Chapter layout .................................................................................................................................. 4 CHAPTER 2 - LITERATURE REVIEW ................................................................................................... 5 2.1 The role of honey bees in agriculture & industry ......................................................................... 6 2.2 The wax moth (Galleria mellonella) ............................................................................................... 7 2.3 Galleria mellonella life cycle ........................................................................................................... 7 2.4 The effect of the wax moth on honeycomb .................................................................................. 8 2.5 Bacillus thuringiensis as a biopesticide ........................................................................................ 8 2.6 Specificity of Bacillus thuringiensis towards the wax moth ...................................................... 14 2.7 Culturing and enumeration of Bacillus thuringiensis spores ................................................... 16 2.8 Spore yield and toxicity ................................................................................................................. 18 CHAPTER 3 – MATERIALS AND METHODS ..................................................................................... 20 3.1 Organism ......................................................................................................................................... 21 3.2 Resuscitation and staining of organism ...................................................................................... 21 3.3 Media preparation and screening of media formulations ......................................................... 22 3.4 Bioassay for determination of toxicity ......................................................................................... 25 3.5 Toxin estimation ............................................................................................................................. 27 3.6 Morphology ..................................................................................................................................... 28 3.7 Statistical Analyses ........................................................................................................................ 29 CHAPTER 4 – RESULTS AND DISCUSSION .................................................................................... 30 4.1 Gram staining ................................................................................................................................. 31 iii 4.2 Endospore staining ........................................................................................................................ 31 4.3 Endospore count ............................................................................................................................ 32 4.4 Vegetative cell count ..................................................................................................................... 35 4.5 Spore percentage ........................................................................................................................... 37 4.6 Re-evaluation of 12 raw material formulations .......................................................................... 41 4.6.1 Endospore count ..................................................................................................................... 41 4.6.2 Vegetative cells ....................................................................................................................... 43 4.6.3 Spore percentage ................................................................................................................... 44 4.7 Cost analysis ................................................................................................................................... 46 4.8 Bioassay test ................................................................................................................................... 46 4.9 Morphology ..................................................................................................................................... 49 4.10 Protein analyses ........................................................................................................................... 52 CHAPTER 5 – CONCLUSIONS AND RECOMMENDATIONS ......................................................... 55 5.1 Conclusions ..................................................................................................................................... 56 5.2 Recommendations ......................................................................................................................... 57 REFERENCES .......................................................................................................................................... 58 APPENDIX A - Mortality rate graphs for Galleria mellonella using different raw material formulations ......................................................................................................................................... 69 APPENDIX B - Standard curve for protein analysis ............................................................................ 72 iv PREFACE The experimental work discussed in this dissertation was conducted during the period of February 2010 to March 2012 in the School of Environmental Sciences and Development, Microbiology, North-West University, Potchefstroom Campus, Potchefstroom, South Africa. The study was conducted under the supervision and co-supervision of Dr. S. Claassens and Dr. J.J. Bezuidenhout. I hereby declare that this is my own original, unaided work and has not been presented for any other degree, examination or research purpose. The reference style used in this dissertation is Harvard style. Perseverance Sehaole March 2012 v ACKNOWLEDGEMENTS I wish to express my sincere appreciation to the following persons and institutions for their contributions to the successful completion of this study; Dr. Sarina Claassens, School of Environmental Sciences and Development, Microbiology, North-West University, Potchefstroom Campus, for her supervision, patience, guidance and support throughout this study; Dr. Jaco Bezuidenhout, School of Environmental Sciences and Development, Microbiology, North-West University, Potchefstroom Campus, for his supervision and all his help and patience in many aspects of this study; Prof. Leon van Rensburg, Unit of Environmental Sciences and Management, North-West University, Potchefstroom Campus, for the financial assistance I received during this study; God the Father, the Son & the Holy Spirit, for wisdom, understanding, comfort, encouragement, love and everything He gave me; My family, for their unfailing love, support and patience; My friends and Thabiso, for always being there, for the advice and for all the talks we had; My Fellowship (PSCF), for encouragement, love, support, advice and any form of help. vi SUMMARY One of the major constrains in beekeeping is damage caused by the greater wax moth (Galleria mellonella). Due to problems and concerns regarding the
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