Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana Dennis M.W. Ochieno Thesis committee Thesis supervisors Prof. dr. Marcel Dicke Professor of Entomology Wageningen University Prof. dr. ir. Arnold van Huis Personal Chair at the Laboratory of Entomology Wageningen University Thesis co-supervisor Dr. Thomas Dubois Biocontrol Specialist International Institute of Tropical Agriculture Other members Prof. dr. TWM Kuijper, Wageningen University Prof. dr. RA Sikora, University of Bonn, Germany Dr. JM Raaijmakers, Wageningen University Dr. MNEJ Smit, IPM specialist, (private consultant) This research was conducted under the auspices of the C. T. de Wit Graduate School of Production Ecology and Resource Conservation Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana Dennis M.W. Ochieno Thesis submitted in partial fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Monday 1 November 2010 at 11 a.m. in the Aula Dennis M.W. Ochieno Endophytic control of Cosmopolites sordidus and Radopholus similis using Fusarium oxysporum V5w2 in tissue culture banana Thesis, Wageningen University, Wageningen, NL (2010) With references, with summaries in Dutch and English ISBN 978-90-8585-637-5 Table of contents Acknowledgements vii Abstract ix Chapter 1 The application of endophytes for the control of pests and diseases in crops and in particular banana 1 Chapter 2 Outline of this thesis 21 Chapter 3 Effects of endophytic Fusarium oxysporum V5w2 as influenced by host nutrition, and the artificial endophyte Beauveria bassiana G41, on the banana weevil Cosmopolites sordidus in tissue culture plants 33 Chapter 4 Comparative studies on effects of NPK nutrient deficiencies and soil sterility on banana plants and Radopholus similis infection 59 Chapter 5 Effects of soil sterilization on interactions between endophytic Fusarium oxysporum V5w2 and the root burrowing nematode Radopholus similis in tissue culture banana plants 87 Chapter 6 Interactions between Radopholus similis and Fusarium oxysporum V5w2 in tissue culture banana plants under nitrogen-starvation and total nutrient starvation as affected by soil sterility 105 Chapter 7 Effects of endophytic Fusarium oxysporum V5w2 on the root burrowing nematode Radopholus similis in v Table of contents tissue culture banana plants under phosphorus and potassium deficiencies as affected by soil sterility 125 Chapter 8 Effects of endophytic non-pathogenic Fusarium oxysporum V5w2 and mulching on field-grown tissue culture banana plants and root infection by Radopholus similis 145 Chapter 9 Summarizing discussion 161 References 177 Summary 195 Samenvatting 199 Personal history 205 PE&RC Education statement 207 Funding Acknowledgement 209 vi Acknowledgements I thank Prof. Arnold van Huis, Prof. Marcel Dicke (WUR), Dr. Thomas Dubois, Dr. Piet van Asten and Dr. Danny Coyne (IITA) for their strong commitment in nurturing me into the world of science. I acknowledge Prof. Joop van Lenteren (WUR), Dr. Cliff Gold (formally of IITA), Dr. Ellie Osir, Dr. Fritz Schulthess (formally of ICIPE), Prof. Micheni Ntiba and Prof. Lucy Irungu (University of Nairobi) for their efforts that led to my recruitment into this great study opportunity. I recognize the technical support that was provided by Abubakar Ezale, Agusi Franco, Arthur Wasukira, Cissy Nabulime, Dennis Nteza, Elvis Mbiru, Fred Kato, Fred Kimuli, Jane Luyiga, Jhamna Castillo, John Kibalama, Joshua Okonya, Juliana Nakintu, Juliet Akello, Margaret Nakawunde, Moses Kibirango, Moses Nyine, Patrick Emudong, Patrick Mayanja, Peter Semakula, Phillip Abidrabo, Ronald Kateriga, Rose Khainza, Stephen Segawa, Sinnia Kapindu, Stephen Ssebaggala, Markstuart Kibirango and Victoria Naluyange among other staff at IITA in Uganda. Fellow students and staff at the Laboratory of Entomology (WUR) provided comments and technical support that greatly improved this thesis. Thanks to Joost van Itterbeeck for the Dutch translation of the summary. I acknowledge the Kenyan and other student associations for creating social and academic occasions. I appreciate the efforts of Judith Naliaka Barasa, Charles Wekulo Barasa, Ashton Okiya, the Baziras, Solomy Kiwanuka, Dr. Edward Muge and Everlyne Kirwa in creating conducive work environment and neighbourhood during my PhD studies. Your prayers and advice played a great role towards the success of my studies. vii Acknowledgements To my dad Jackson J. Ochieno, mum Elizabeth N. Ochieno, my son Alvin Jonah Ochieno, all family members and friends, you gave me extra attention during my long absence from home while pursuing studies. Dad and Mum, you sacrificed a lot to ensure that I succeed in this challenging academic journey. Thanks a lot! viii Abstract Banana plants are being inoculated with Fusarium oxysporum V5w2 and Beauveria bassiana G41 for endophytic control of pests. The effects of F. oxysporum V5w2 and B. bassiana G41, soil sterility, fertilizer, and mulching, on Cosmopolites sordidus and Radopholus similis in banana plants, are investigated. Cosmopolites sordidus has low preference for plants inoculated with the two endophytes; corm damage is low on F. oxysporum V5w2-treated plants. High root damage and growth suppression are evident in R. similis-treated plants. Under N-deficiency, R. similis-treated plants are larger than those without the nematode. Compared to plants treated with complete nutrient solution (CNS), those under P-deficiency have higher root damage, but lower under K-deficiency, and not different under N-deficiency and when only water was applied. Plants under CNS have lower R. similis density than those under N-deficiency and those treated with water only. Also, C. sordidus larvae from plants under CNS are smaller than those given only water. Under N-deficiency and supply of only water, potted plants in non-sterile soil are smaller than those from sterile soil, but are larger under CNS, P or K-deficiencies when N is present. Radopholus similis densities are lower in roots from non-sterile soil, compared to those from sterile soil. Mulched plants are larger with bigger bunches than those without mulch, but are more prone to toppling when R. similis is present. Plants treated with F. oxysporum V5w2 have lower R. similis density under N-deficiency but higher under P-deficiency, and are smaller in size under K- deficiency, than endophyte-free ones. Fusarium oxysporum V5w2-treated plants are small and take short time to harvest. With mulch, R. similis- induced toppling is less in F. oxysporum V5w2-treated plants, possibly due ix Abstract to their smaller size during growth. In conclusion, data on the effect of nutrients, soil microorganisms and mulching do not support the transfer of F. oxysporum V5w2-treated banana plants to farmers, because the plants suffer from reduced performance. Understanding endophytic mechanisms of action and establishing successful inoculation is necessary for drawing a final valid conclusion. x Chapter 1 The application of endophytes for the control of pests and diseases in crops and in particular banana Endophytes growing from root pieces in media Dennis M.W. Ochieno · Marcel Dicke · Thomas Dubois · Arnold van Huis Chapter 1 Abstract Endophytes have become important for plant growth promotion. The technology has been made ready for crops like banana that are seriously constrained by pests and diseases. The current chapter reviews the literature on endophytes with emphasis on the application of non-pathogenic Fusarium oxysporum for the control of banana pests and diseases. A toppled banana plant 2 The application of endophytes for the control of pests and…………..…….. Pest problems facing banana production in Africa Banana and plantain are grown in over 100 tropical and subtropical countries worldwide (Sharrock and Frison 1999). In Africa, especially in the eastern and central regions of the continent, highland cooking banana cultivars (Musa spp., genome group AAA-EA) are a staple food crop for the livelihood of many people (Pillay et al. 2001). Uganda has been the leading African banana producer, with an annual production of 9.8 million tonnes (Sharrock and Frison 1999), and a consumption rate of 373 kg per capita per year (Smithson et al. 2001). In Rwanda, the annual banana production is 2 million tonnes (39% of total raw food production, with a consumption rate of 197 kg per capita per year) (Nsabimana and van Staden 2007). a b Fig. 1.1. The banana weevil Cosmopolites sordidus with eggs (a), and the root burrowing nematode Radopholus similis coiled inside a root cell (b). Corm damage by the weevil results in snapping while root burrowing by the nematode causes toppling of plants. Images byprovided provided by Moses Nyine and Phillip Abidrabo, respectively. 3 Chapter 1 However, banana production in Africa is threatened by a wide range of pests and diseases. The banana weevil, Cosmopolites sordidus (Coleoptera: Curculionidae) (Figure 1.1) is among the most damaging insect pests that feed on the corms resulting in snapping of plants (Gold et al. 2001). The root burrowing nematode, Radopholus similis (Tylenchida: Pratylenchidae) (Figure 1.1), is among the most damaging species of plant parasitic nematodes worldwide that had been given quarantine status (O'Bannon 1977; EPPO/CABI