FACULTY OF SCIENCES DEPARTMENT OF BIOLOFY Entomopathogenic nematodes as potential control agents of termites in citrus in Benin Lionel D. V. Zadji Promotor Prof. Dr. Wilfrida Decraemer (UGhent) Co-promotors Prof. Dr. Maurice Moens (UGhent) Dr Hugues Baimey (UNIPAR-Benin) Thesis submitted in the fulfilment of the requirements of the degree of Doctor (Ph.D.) in Sciences, Biology Proefschrift voorgelegd tot het behalen van de graad van Doctor in de Wetenschappen, Biologie September 2014 This work was supported by a grant of the Flemish Interuniversity Council – University Development Cooperation (VLIR-UOS) Dit werk werd mogelijk gemaak t door een beurs van het De Vlaamse Interuniversitaire Raad- Universitaire Ontwikkelingssamenwerking (VLIR-UOS) - 2 - Reading committee Prof. Dr. Ralf-Udo Ehlers (e-nema, Schwentinental, Germany) Prof. Dr. Roland N. Perry (University of Hertfordshire, UK) Prof. Dr. Patrick De Clercq (Ghent University, Belgium) Dr. Hugues Baimey (University of Parakou, Benin) Examination committee Prof. Dr. Ralf-Udo Ehlers (e-nema, Schwentinental, Germany) Prof. Dr. Roland N. Perry (University of Hertfordshire, UK) Prof. Dr. Patrick De Clercq (Ghent University, Belgium) Dr. Hugues Baimey (Co-promotor, University of Parakou, Benin) Prof. Dr. Maurice Moens (Co-promotor, Ghent University, Belgium) Prof. Dr. Wilfrida Decraemer (Secretary, promoter, Ghent University, Belgium) Prof. Dr. Wim Bert (Chairmain, Ghent University, Belgium) - 3 - Acknowledgements The research described in this thesis was funded by the Belgian Government and the Flemish Interuniversity Council – University Development Cooperation (VLIR-USO) in the framework of “Ecological Sustainable Citrus Production in Benin (ESCiP-Benin)” project which is a co-operation between Ghent University, Belgium and University of Parakou, Benin. I am deeply grateful to all those who have contributed to the realization of the present thesis. I would like to start out by expressing my very heartfelt thanking and gratitude to my promoters Prof. Wilfrida Decraemer, Prof. Maurice Moens and Dr. Hugues Baimey. It would not have been possible for me to do this work without your support and guidance and probably most importantly patience. I appreciate Prof. Dr. Wilfrida Decraemer’s countless e- mails to discuss the progress and direction of the research. I appreciate your intellectual support, encouragement, and enthusiasm. My infinity gratitude to Prof. Maurice Moens, your invaluable contributions, critical comments in correcting both my stylistic and scientific errors were very stimulating. I am very much indebted to you, Dear Dr. Hugues Baimey for believing in my capacity to achieve this thesis, and for your continue technical assistance throughout the different phases of my thesis works. I would like to express my deep gratitude to Dr. Leonard Afouda, head of the Multidisciplinary Laboratory of Faculty of Agronomy / University of Parakou, Benin, for providing me with the freedom to develop my research topic and conduct the studies. My special thanks are dedicated to the ESCiP-Benin project team, Olive Batcho, Nondichao Raimatou, Juldith Agbalenon, Raoul Balogoun, Brice Sinhouenon and all colleague-students, for sharing successes and frustrations bringing this project to reality. It has been a pleasure to work with you. I would like to express my sincere appreciation to the Institute of Agricultural and Fisheries Research (ILVO)-Unit Plant Crop Protection, Merelbeke, Belgium for allowing me to experience a professional, effective and efficient working environment. I wish to express my sincere thanks to all the staff members of the laboratory for their kind assistance. I am greatly - 4 - indebted to Lieven Waeyenberge, for his assistance and help with molecular work. I would also like to give my thanks to his wife, Nancy de Sutter, for accepting to teaching me the first necessary techniques used in nematology. I am especially grateful to Ms. Inge Dehennin for her friendship, continuous care and support during this long period. I am truly grateful to the members of the examination commission, Prof. Dr. Roland N. Perry, Prof. Dr. Ralf-Udo Ehlers, Prof. Dr. Patrick De Clercq and Prof. Dr. Wim Bert for their time and efforts in reading this work. The invaluable remarks and advice truly improved the quality of this thesis. I would like to thank my lab mates Francoise Kanga, Fouad Moukrini, Negin Ebrahimi, Yirina Valdes, Gideon Alake, and all colleague-students, for all the laughs and candid conversations and their sincere help in providing technical information one way or another. I am also thankful to my Beninese friends Prof. Dr. Achille Assogbadjo and his wife Dr. Flora Chadare, Mrs Carole Djidohopkin and her husband, Gladys Houssou, Chimene David, Anique Godjo and Herve Sossou for the short but sweet time spent together in Belgium. It has been a pleasure to have nice little dinners full of chatting. I would like to thank Mrs Marjolein Couvreur from Department of Biology, Faculty of Sciences, Ghent University, for the scanning electron micrographs. I feel unlimited gratitude to my wife, Rosaline Ekon, who did her best to support me to go to the end of this study. To my daughters, Carole and Maeva, and my son Teddy, who had to overcome being separated from their father by thousands of kilometers for a long time. It is your unconditional love and patience that has given me motivation and energy to complete this work. Last but not least, I wish to dedicate this dissertation to my late mother Leocadie Houngbadji. May her spirit continue to provide me with the motivation to keep working hard and pushing forward. Thanks to my father Martin Zadji, my sisters Estelle, Mireille, Emeline and Nina and my brothers Bellarmin and Franck for their love and supports throughout. - 5 - There are so many nice people I have met who helped me on one or the other way on this journey. I thank all of them, it is impossible to mention everyone here, but I am sure they know they have their own special place in my heart. - 6 - Contents Chapter 1 General introduction 8 Chapter 2 First record on the distribution of entomopathogenic 41 nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in Southern Benin Chapter 3 Characterization of biocontrol traits of heterorhabditid isolates 71 from South Benin targeting the termite pest Macrotermes bellicosus Chapter 4 Effectiveness of Heterorhabditis isolates from Southern Benin 94 for the biocontrol of the subterranean termite, Macrotermes bellicosus (Isoptera: Macrotermitinae), in laboratory trials Chapter 5 Comparative susceptibility of Macrotermes bellicosus and 116 Trinervitermes occidentalis (Isoptera: Termitidae) to EPN from Benin Chapter 6 Influence of pesticides, soil temperature and moisture on 134 entomopathogenic nematodes from southern Benin and their potential to control underground termite nest population Chapter 7 General discussion 161 Summary 178 Samenvatting 181 Curriculum Vitae 185 - 7 - 1 General introduction - 8 - General introduction 1 1.1 BACKGROUND 2 Termites are a large and diverse group of insects classified in an order of their own, the 3 Isoptera. They were first presented at the Royal Society of London in 1781 by Henry 4 Smeathman (Howse, 1970). There are approximately 3,500 species of termites (Engel 2011). 5 Africa is by far the richest continent in termite diversity (Eggleton, 2000). The most 6 economically important termite genera in agricultural and forest areas in Africa are 7 Macrotermes, Allodontermes, Amitermes, Pseudacanthotermes, Odontotermes, 8 Ancistrotermes, Trinervitermes, Hodotermes and Microtermes (Kumar & Pardeshi, 2011). 9 They dig underground tunnels with a small connection to the aboveground environment 10 (Noirot & Darlington, 2000) through which they reach and attack host plants. Since the major 11 constituent of the diet of termites is cellulose, the insects tunnel through stems and weaken 12 them by consuming the carbohydrates. Eventually the insects cause their collapse or give 13 access to fungi ( Guignardia citricarpa Kiely) and other disease causing agents (Tristeza 14 virus, the citrus bud mite Aceria sheldoni , the citrus nematode Tylenchulus semipenetrans 15 Cobb, or the burrowing nematode Radopholus similis Cobb ) (Kumar & Pardeshi, 2011). In 16 Uganda, losses of crops and tree stands ranging from 50 to 100% have been attributed to 17 termite attack (Sekamatte et al ., 2001). In the semi-arid savannah of Kenya termites were 18 reported to destroy 800-1500 kg/ha of pasture per year (Lepage, 1981). In the countries of 19 Southern Africa, harvester termites are a serious pest of rangeland, removing 60% or more of 20 the standing grass bio-mass during dry years (Coaston, 1958). 21 In Benin, citrus ( Citrus sinensis L.) is the second major fruit crop produced and exported after 22 pineapple (FAO, 2001). Regions of high production are located in the southern part of the 23 country, mainly in the departments of Zou, Collines, Oueme, Plateau, Mono, Couffo and 24 Atlantique. Orange trees are the most planted covering an average land of 2.38 ha per citrus 25 grower), followed by mandarin trees (1.73 ha per citrus grower), lemon trees (1.68 ha per 26 citrus grower) and grapefruit trees (0.12 ha per citrus grower). The importance of the 27 production of orange compared to other citrus fruits is due to its importance in the diet of 28 Muslims, especially during Lent of Muslim and it is being exported to Nigeria, a - 9 - Chapter 1 29 neighbouring country. The average annual income per producer from citrus is estimated to 30 1,437,293 CFA francs (2191 euro) for oranges, 143,070 CFA francs (218 euro) for lemon and 31 57,500 CFA francs (88 euro) for mandarin (ESCiP-Benin, 2012). Citrus harvested areas have 32 increased over the years from 1,470 ha in 1961 to 5,000 ha in 2012. However, in spite of the 33 adoption of new improved varieties, yields were lower than expected, decreasing from 72,624 34 hg/ha in 1961 to 28,000 hg/ha in 2012 (FAOSTAT, 2014). Citrus growers in different places 35 complain about damages caused to citrus trees by fruit flies, biting butterflies, caterpillars and 36 termites.