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Pathogen Diversity and Host Resistance in Dieback Disease of Cocoa Caused by Fusarium Decemcellulare and Lasiodiplodia Theobromae

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Pathogen Diversity and Host Resistance in Dieback Disease of Cocoa Caused by Fusarium Decemcellulare and Lasiodiplodia Theobromae PATHOGEN DIVERSITY AND HOST RESISTANCE IN DIEBACK DISEASE OF COCOA CAUSED BY FUSARIUM DECEMCELLULARE AND LASIODIPLODIA THEOBROMAE Richard Kwame Adu-Acheampong, B. Sc. Agriculture, M. Phil. Entomology March 2009 Thesis presented for the degree of Doctor of Philosophy at Imperial College of Science, Technology and Medicine, and for the Diploma of Imperial College (DIC) Division of Biology, Imperial College London Silwood Park Campus, Ascot, Berkshire, SL5 7PY, UK 1 Abstract Dieback disease caused by Fusarium and Lasiodiplodia species is a major threat to cocoa production in Ghana and elsewhere in West Africa. Current recommendations involve insecticide application to control mirid bugs whose feeding punctures provide entry points for these fungi. Little is known about the true identity of the causal pathogens of this disease. Earlier work implicated F. decemcellulare as the causal agent and more rarely L. theobromae (Cotterell, 1927; Crowdy, 1947). A total of 117 single spore fungal cultures was established from diseased cocoa stems imported from Ghana. On morphological grounds cultures could be designated as either Fusarium or Lasiodiplodia spp. The Fusarium cultures exhibited inter-isolate variability with respect to macroscopic appearance and macro-conidium morphology, suggesting the presence of more than a single species. The isolates were further characterised by PCR amplification and sequencing of the ITS region of rDNA and comparison with authentic reference cultures. Thirty-seven Fusarium isolates were identified to twenty F. chlamydosporum, nine F. solani and four isolates each of F. oxysporum and F. proliferatum. The thirty-six Lasiodiplodia isolates were identified to two species, twenty-seven L. pseudotheobromae and nine L. theobromae. In pathogenicity tests, F. chlamydosporum, F. oxysporum, F. proliferatum, F. solani and L. pseudotheobromae, previously unknown as pathogens of either cocoa or any member of the Malvaceae, caused significant wilting and dieback in Amelonado seedlings similar to that observed in the field. All isolates exhibited optimal growth at 30 ºC on PDA. Disease incidence in 29 and 15 cocoa germplasm lines in the laboratory and greenhouse, respectively, showed reproducible differences in their reaction to necrotic lesion and dieback infection. LCTEEN 37/F was one of the most susceptible genotypes. CATIE 1000, T85/799 and MXC 67 were the most tolerant and could be used in cocoa breeding programmes for resistance to dieback. 2 DECLARATION OF OWN WORK I confirm that this thesis: Pathogen diversity and host resistance in dieback disease of cocoa caused by Fusarium decemcellulare and Lasiodiplodia theobromae is entirely my own work, conducted under the supervision of Dr Simon Archer and Dr Simon Leather. Where any material could be construed as the work of others, it is fully cited and referenced, and or with appropriate acknowledgement. No part of this research has been submitted in the past, or is being submitted, for a degree or examination elsewhere. The input of my supervisors to the research and to the thesis was consistent with normal supervisory practice. I grant copyright of this thesis to Imperial College London. March 2009 Name of student: Richard Kwame Adu-Acheampong Name of supervisor: Dr Simon Archer 3 Acknowledgements It is a pleasure to take a few moments and remember all of the support and assistance that I received as I worked through this thesis. Friends, family, supervisors, colleagues, and many others have sustained me throughout the process. I was fortunate to have been offered a scholarship by the Commonwealth Scholarships Commission of the United Kingdom, to whom many thanks are due. I am grateful to the late Dr M. R. Appiah, former Executive Director of the Cocoa Research of Ghana, for granting me permission to carry out the study. Two excellent advisors, the late Dr Beatrice Padi and Dr F. K. Oppong, encouraged and challenged me every step of the way. I wish also to thank Drs Y. Adu-Ampomah, F. K. Amoah, J. B. Ackonor, I. Y. Opoku, A. Y. Akrofi, Boamah-Adomako, Michael Assuah and the entire staff of the Entomology Division at CRIG for their stimulating support. Dr Andrew Daymond of University of Reading is acknowledged for the provision of budwood of cocoa clonal materials, likewise Dr Gary J. Sammuels (USDA) for the supply of Fusarium decemcellulare reference isolates and Rob Lockwood for useful references. I am indebted to my supervisors Dr Simon Archer and Dr Simon Leather for their support, continued interest and advice, and stimulating suggestions and encouragement that helped me during my entire stay at Silwood Park. For all of their support, I thank the following technicians, Paul Nicholas, Jim Culverhouse, Frank Wright, Paul Beasley and Glen Chalker. I am particularly grateful to the following persons from the Biology Division, Sandy Whitehead, Sue Lomas, Nabeil Salama, Biswanath Das and Hanno Schäfer for their help even by email and telephone and often at short notice. I am specially obliged to Dr Robert Coles Brian (formerly at Jealott’s Hill Research Station [I.C.I. Ltd, UK] and Ms Janet McLennan (of Sunningdale Baptist Church, and formerly Deputy Head of Coworth Park School, UK) who critically read the text. Coincidentally, Prof. Stephen Henry Crowdy who first investigated this disease problem in Ghana (then Gold Coast) worked alongside Dr Brian at Jealott’s Hill for 30 years. Janet looked closely at the final version of the thesis for English style and grammar, made corrections, and offered suggestions for improvement. Finally I would like to give my special thanks to my wife Beatrice whose patient love enabled me to complete this work. This work was conducted under the Department for Environment, Food and Rural Affairs (DEFRA) Plant Health Licence No. PHL 189B/5316(03/2006). 4 Table of contents Abstract..........................................................................................................................2 DECLARATION OF OWN WORK .............................................................................3 Acknowledgements........................................................................................................4 Table of contents............................................................................................................5 List of Figures................................................................................................................7 List of Tables ...............................................................................................................12 Abbreviations...............................................................................................................14 1. Chapter 1..............................................................................................................15 1.1. Literature review..........................................................................................15 1.1.1. Origin, history, geographic distribution and diseases of cocoa ...........15 1.1.2. Biology and ecology of cocoa..............................................................16 1.1.3. Pollinators ............................................................................................20 1.1.4. Taxonomy of cocoa..............................................................................20 1.1.5. Cultivation............................................................................................23 1.1.6. World cocoa production.......................................................................26 1.1.7. Major diseases of cocoa.......................................................................28 1.1.8. Insects associated with cocoa dieback disease and their management 39 1.1.9. Prospects for the use of host plant resistance for the control of mirid- related dieback of cocoa.......................................................................48 1.2. Overall thesis objective:...............................................................................49 1.2.1. Thesis outline.......................................................................................50 2. Chapter 2..............................................................................................................51 2.1. Characterization and pathogenicity of isolates of Fusarium and Lasiodiplodia species from dieback lesions of cocoa in Ghana .............................................51 2.2. Introduction..................................................................................................51 2.3. Materials and methods .................................................................................52 2.3.1. Comparative isolation rates of different fungi from infected cocoa stems on different culture media..........................................................52 2.3.2. Pure cultures from single spore isolates...............................................53 2.3.3. Morphological Studies.........................................................................54 2.3.4. Greenhouse pathogenicity tests............................................................55 2.4. Results..........................................................................................................63 2.4.1. Pathogen isolation and description ......................................................63 2.4.2. Growth rate studies..............................................................................69 2.4.3. The longevity of conidia in culture......................................................70
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