UiN :IVERSI'TV OF KWA.ZULU-NATAL Sugarcsne Stem Borers in Ethiopia: Ecology and Phylogeography Yoseph Assefa University ofKwaZulu-Natal Sugarcane Stem Borers in Ethiopia:.Ecology and Phylogeography by Yoseph Assefa BSc. Arid Land Crop Production (Asmara University, Eritrea) MSc. Crop Protection (Alemaya University, Ethiopia) A Thesis submitted to the School ofBiological and Conservation Sciences in partial fulfilment ofa PhD Degree in Entomology University ofKwaZulu-Natal Pietermaritzburg South Africa 2006 DECLARATION This thesis is the result ofthe author's original work except where acknowledged. I hereby certify that this research work has never been submitted for any degree or examination at any University or similar academic institution. Sign~8.#.it..... Dak Yoseph Assefa Signed'~~7"'Date -~r/t-?:-h~---- Dr. Desmond Edward Conlong (Supervisor) 11 ACKNOWLEDGEMENTS I would like to express my most sincere gratitude to my supervisors, Dr Des Conlong and Dr Andrew Mitchell, for their excellent supervision, patience, commitment and encouragement throughout my study period. Dr Brooo Le Rii for supplying photographs of stem borers and insect specimens for molecular studies. I am also indebted to Dr Pascal Moyal, IRD France, Dr David Barraclough, School of Biological and Conservation Sciences, UKZN, Durban and staff of the Biosystematics Division of the ARC-PPRI in Pretoria for their assistance in identifying insects. Special thanks are due to Professor Denis Brothers and Mrs Jane Flockhart, School of Biological and Conservation Sciences, UKZN, Pietermaritzburg for their contribution in facilitating an excellent working environment. I would like to express my special thanks to my Ethiopian, Eritrean and South African friends at UKZN, University of Pretoria, University of Cape Town and South African Sugarcane Association Research Institute (SASRI) for their assistance and encouragement throughout the study period. I am grateful to my friend Solomon for teaching me Arc View program, to Gudrun Dittrich for her assistances, to Alistair Barker and Wedi Qeshi (Negasi) for the transport and Black Label for the power. Grateful acknowledgement is made to SASRI for providing facilities for the study. Special thanks are due to staff of the Ethiopian sugarcane estates and staff of Crop Protection Section, Department of Plant Sciences, Alemaya University for their help in the course of sample collection from Ethiopia. The many institutes in Africa that sent specimens of Eldana saccharina and Cotesia jlavipes for analysis are gratefully acknowledged. The project was funded by Agricultural Research Training Project (ARTP), Alemaya University, Ethiopia 111 LIST OF PAPERS RESULTING FROM THIS THESIS THAT HAVE BEEN SUBMITTED TO OR ACCEPTED BY PEER REVIEWED JOURNALS Assefa Y., Conlong D.E., Mitchell A. 2005. Mitochondrial DNA variation among populations ofsugarcane borer Eldana saccharina Walker (Lepidoptera: Pyralidae). Proceedings of the South African Sugarcane Technologists' Association 79: 382­ 385. Assefa Y., Conlong D.E., Mitchell A. 2006. First record of the stem borer complex (Lepidoptera: Noctuidae; Crambidae; Pyralidae) in sugarcane estates of Ethiopia, their host plants and natural enemies. Proceedings ofthe South African Sugarcane Technologists' Association 80: 202-213. Assefa Y., Conlong, D.E., Mitchell A. 2006. Inter-population fertility in sugarcane stem borer Eldana saccharina Walker (Lepidoptera: Pyralidae). Proceedings ofthe South African Sugarcane Technologists' Association 80: 218-225. Assefa Y., Conlong, D.E., Mitchell A. 2006. Differences in mitochondrial DNA and fertility of crosses between populations of Eldana saccharina (Lepidoptera: Pyralidae) from Kenya and South Africa: possible evidence for cryptic species? Sugar Cane International 24: 11-14, 19. Assefa Y., Mitchell A., Conlong, D.E. 2006. Phylogeography of Eldana saccharina Walker (Lepidoptera: Pyralidae). Annales de la Societe entomologique de France (in press). Assefa Y., Conlong, D.E., Mitchell A. 2006. Status of Eldana saccharina Walker (Lepidoptera: Pyralidae), its host plants and natural enemies in Ethiopia. Bulletin of Entomological Research 96 (5): 497-504. IV Assefa Y., Conlong, D.E., Mitchell A. 2006. Busseola species complex (Lepidoptera: Noctuidae) in peasant sugarcane farms of Ethiopia. African Entomology (submitted). Assefa Y., Conlong D.E., Mitchell A. 2006. Chilo partellus Swinhoe (Lepidoptera: Crambidae) and its natural enemies in peasant sugarcane farms of Ethiopia. Biocontrol (submitted). Assefa Y., Mitchell A., Conlong D.E., Muirhead K. 2006. Evidence· for the Establishment ofCotesia jlavipes (Hymenoptera: Braconidae) in sugarcane fields of Ethiopia and the origin of the founding population. Journal of Economic Entomology (submitted). Assefa Y., Mitchell A., Conlong, D.E., Moyal P. 2006. Identification ofBusseola species complex (Lepidoptera: Noctuidae) in Ethiopian sugarcane based on Mitochondrial DNA data. African Entomology (submitted). v ABSTRACT Eldana saccharina Walker (Lepidoptera: Pyralidae) is an indigenous insect widely distributed throughout Sub-Saharan Africa that is a major pest of sugarcane in southern Africa. Studies have shown that populations from West Africa have distinct behavioural differences compared to populations from eastern and southern Africa. In addition, the parasitoid guilds attacking populations in these regions are markedly different. This marked behavioural and parasitoid guild variation evoked a hypothesis of genetic diversification. To evaluate this hypothesis a project on the phylogeography of E. saccharina was initiated. The project was planned to include sampling ofas many regions as possible in its known range in Africa, to obtain specimens of E. saccharina for genetic analysis. When these surveys were initiated in Ethiopia, it was found that there was no published literature available on the occurrence of stem borers in Ethiopian sugarcane. It was thus clear that no stalk borer/parasitoid surveys had been completed in either sugarcane or any large grass and! sedge indigenous hosts in Ethiopia. The study was thus expanded beyond the investigation of only the genetic diversity of E. saccharina, to include area-wide surveys to determine ecological aspects of the borer complex in suspected host plants, including sugarcane, in Ethiopia. In this way the host plant range and distribution of E. saccharina and other sugarcane borers in Ethiopia in particular could be determined, samples for a larger phylogeography project could be collected, and the insect's impact on sugarcane could be assessed. VI Quantified area-wide surveys of the sugarcane estates and small-scale farmer fields of Ethiopia were conducted between December 2003 and February 2004. The surveys verified the presence of four lepidopteran stem borer species on Ethiopian sugarcane. These were Chilo partellus Swinhoe (Lepidoptera: Crambidae), Sesamia calamistis Hampson (Lepidoptera: Noctuidae), Busseola fusca Fuller (Lepidoptera: Noctuidae) and Busseola phaia Bowden (Lepidoptera: Noctuidae). The surveys indicated that Busseola species are the major and most widely distributed sugarcane stem borers in sugarcane farms ofEthiopia. Over 70% ofthe peasant sugarcane fields visited were infested by these borers, with the highest levels of infestation (35% and 50%) being in the northern and western part ofthe country, respectively. Busseola was also the predominant stem borer of sugarcane in two of the three estates (Wonji and Finchawa). Chilo partellus and S. calamistis were recovered in very low numbers at all the commercial estates and from peasant farms in the western part ofEthiopia. However, C. partellus was the predominant sugarcane stem borer in lowland areas of northern, southern and eastern parts of the country. Eldana saccharina was recovered from large sedges in waterways of Metehara and Wonji sugar estates in the central part ofthe country, and sedges growing around lakes in northern and southern Ethiopia, but not from sugarcane anywhere in Ethiopia. The phylogeographic study conducted on E. saccharina populations from eleven countries of Africa clearly showed the population structure of the insect within the continent. Five hundred and two base pairs of the mitochondrial DNA (mtDNA), corresponding to the Cytochrome Oxidase subunit I (COl) region, were sequenced to clarify phylogenetic relationships between geographically distant populations from eastern, northern, southern and western Africa. Results revealed that E. saccharina is separated into four major populations corresponding to their geographical location, i.e. West African, Rift Valley vu and two southern African populations. Sequence divergence between the four populations ranged from 1% to 4.98%. The molecular data are congruent with an isolation by distance pattern except for some of the specimens from eastern and southern Africa where geographically close populations are genetically distant from each other. Geographical features such as the Rift Valley and large water bodies in the continent seem to have a considerable impact on the genetic diversity in E. saccharina. Identification of field-collected stem borer specimens was done using classical taxonomic techniques, except for Busseola spp. where DNA barcoding was used. As field-collected larval material of Busseola died before reaching the adult stage, identification of species using adult morphology was not possible. 'Sequence divergence in the COl gene was used as a tool to identify the species of Busseola attacking Ethiopian