Entomopathogenic fungi as a biological control for the Greater melon fly Dacus frontalis (Becker) (Diptera: Tephritidae) Esam Omar Elghadi A thesis presented for the degree of Doctor of Philosophy School of Biology Faculty of Science, Agricultural and Engineering Newcastle University United Kingdom June-2016 i ii Abstract The Greater melon fly Dacus frontalis is one of the most economically damaging pests of cucurbit fruits in Africa. The aim of this study was to evaluate entomopathogenic fungi for use against D. frontalis and develop better strategies in using these biological agents for integrated fly management. Laboratory experiments were conducted to evaluate the pathogenicity of some commercial biopesticides based on several strains of entomopathogenic fungi against different life stages of the fly. Met52 Granular biopesticide (MET52) based on Metarhizium anisopliae caused the greatest pathogenicity to the adults. Therefore, MET52 was selected for further investigation. Pupal age and an increasing rate of MET52 had no effect on pupal mortality. However, post- emergence mortality increased when the fungus was applied on young pupae. Prior application of MET52 in a granule form caused considerable mortality to larval-pupal stages compared with a drench application. A sublethal effect of MET52 infection, reducing adult reproduction, was found. The fungus was more effective when applied as a granule in soil against emerging adults than as a direct spray against adults. MET52 fungus was able to persist in soil, reducing the adult emergence and subsequent fly population for more than two months after a single application, and also inducing new infections among the fly population. Percentage mortality of untreated females mating with inoculated males was greater than that of untreated males mating with inoculated females. The infection reduced the adult’s reproduction resulting in a considerable reduction in pupae recovered in the fungal treatments. Alone or in combination with MET52 neem had a dose-dependent effect on larval mortality and induced various effects on soil stages and adults of the fly. The efficacy of the fungus increased considerably at low concentration of neem showing the shortest LT50. In field cage trials, baited McPhail traps with yeast hydrolysate enzymatic had the greatest number of D. frontalis. MET52 gave lower adult mortality than insecticide. In conclusion, soil application of MET52 can be a promising effective control of D. frontalis, and can be combined with other control agents providing a possible effective strategy for integrated fly management. iii Dedication To my parents, wife and sons for their unlimited support iv Acknowledgements Deepest appreciation to my supervisor Dr Gordon Port for his support, guidance helpful and consideration over my PhD and at the difficult times that I faced during the course. I would like to thank very much to the Dean of Postgraduate Studies of the Faculty of Science, Agriculture and Engineer for his consideration during Libyan revolution and war later. Great thank to Dr Gordon Beakes and Prof Angharad Gatehouse for their useful comments on my annual reports. I acknowledge the Libyan government for financial support. Also, thanks to my supervisor in Libyan cultural affairs in the UK Mr Nuaman Alzarrok for his help during my study period. I would like greatly to thank the following people: Shokri Ashreef (Biotechnology Research Centre, Libya) for providing a culture of the Greater melon fly, Dacus frontalis, Alan Craig (Newcastle University) for providing some experimental equipment, Martha Koukidou and Camilla Beech (Oxitec Ltd, Oxford, UK) for providing a culture of Medfly, Ceratitis capitata, Dr N. Murugesan (T.Stanes and Company limited, India), Dr Adrian Jackson (Belchim Crop Protection Ltd, Cambridgeshire, UK), Dr Paul Sopp (Fargro Ltd. UK) for supplying some commercial products based on several species of entomopathogenic fungi and Dr David M Williams (Chairman/CEO Neem Biotech Ltd, UK) for providing neem oil and garlic extracts. I am very grateful to Ros Brown and Miriam Earnshaw, for their technical consultancies and laboratory assistance. I would like to express my gratitude to the Biotechnology Research Centre in Libya for providing facilities for insect culture and preparing experimental materials such as some traps for field trials conducted in Libya 2013. Thanks are extended to Fraj Abodheema (a researcher in Biotechnology Research Centre, Libya) for his assistance in finding a suitable cucurbit field and collecting infested cucurbit fruits in difficult times, Mohammed Alamaeei (a researcher in Biotechnology Research Centre, Libya) for providing some lab equipment (balance, plastic cups, gloves and Petri dishes) for preparing biopesticide and chemical pesticide and Dr Ali Kafu who provided some yellow traps during the field trials. My thanks to Dr Tariq Butt and Dr Ansari Minshad (Swansea University, Swansea, UK) for a useful short laboratory training and field practise works of using entomopathogenic fungi during my short stay in Swansea University in October 2009 before starting my PhD study. Finally, I sincerely thank all university staffs, my PhD colleagues at Newcastle University and my friends in Libya and the UK for their encouragement, support, advice and good relationship during the PhD study. v vi Table of Contents Abstract....................................................................................................................................iii Dedication.................................................................................................................................iv Acknowledgment.......................................................................................................................v List of Tables..........................................................................................................................xiii List of Figures..........................................................................................................................xv Chapter 1. General introduction.............................................................................................1 1.1 Background.....................................................................................................................1 1.2 Cucurbit crops................................................................................................................1 1.3 Fruit flies.........................................................................................................................2 1.4 The Greater melon fly, Dacus frontalis (Becker).........................................................3 1.4.1 Taxonomy history.....................................................................................................3 1.4.2 Global distribution...................................................................................................5 1.4.3 Economic important and host plants.......................................................................5 1.4.4 Life cycle and infestation symptoms........................................................................6 1.4.5 Dacus frontalis Control strategy.............................................................................9 1.4.6 Current control in Libya..........................................................................................9 1.5 Review of integrated fruit fly management...............................................................10 1.5.1 Cultural practices..................................................................................................11 1.5.2 Attract and kill technique.......................................................................................11 1.5.3 Sterile insect technique..........................................................................................12 vii 1.5.4 Chemical pesticides................................................................................................12 1.5.5 Biological control...................................................................................................13 1.6 Entomopathogenic fungi.............................................................................................16 1.6.1 Overview................................................................................................................16 1.6.2 Pathogenicity mechanism and life cycle................................................................16 1.6.3 Entomopathogenic fungi for insect control............................................................17 1.6.4 Application strategy...............................................................................................17 1.6.5 Factors influencing fungal pathogenicity..............................................................18 1.6.6 Combined use with other control agents................................................................18 1.7 Aims of thesis................................................................................................................19 Chapter 2. Potential use of entomopathogenic fungi as a biological control against the Greater melon fly Dacus frontalis (Becker) (Diptera: Tephritidae)......................................22 Abstract...............................................................................................................................22 2.1 Introduction..................................................................................................................22