Characterization of new Muscodor padawan and Muscodor sarawak, isolated from Sarawak, Malaysia: evaluation of their potential as a biological control agent for Ganoderma boninense, a pathogenic fungus of Elaeis guineensis By Noreha Mahidi A thesis presented in fulfilment of the requirements for the degree of Doctor of Philosophy at Swinburne University of Technology 2015 Abstract The aim of this thesis is to isolate endophytic Muscodor-like fungi that produces anti-Ganoderma volatile chemicals, from the rich biodiversity resources of Sarawak. These fungi were then examined for their potential to be developed as biological control agents to control Ganoderma boninense, a pathogenic fungus that causes basal stem rot disease in oil palm, Elaeis guineensis. Ten new isolates of endophytic Muscodor-like fungi were successfully obtained from leaves of different plants of Cinnamomum javanicum collected from the Padawan forest in Kuching, Sarawak, Malaysia, using a co-culture technique with Muscodor albus as the selection organism. Two isolates, Muscodor padawan and Muscodor sarawak were selected for further investigation. Muscodor padawan, when grown on potato dextrose agar, exhibits poor production of aerial mycelia, a yellowish colour, with 20 to 28mm colony diameter after 10 days of incubation at 250C. Muscodor sarawak forms whitish colony with a diameter of 23 to 30mm after 10 days of incubation at 250C and produces moderate aerial mycelia on potato dextrose agar. Scanning electron micrograph of the aerial mycelia of M. padawan showed hyphal formed coiled-like structures, spider mat-like attachments on the surface of hyphae and occasionally the presence of chlamydospores and clumps of hyphae. Formation of new hyphae at lateral main hyphae, chlamydospores at intermediate hyphae, half coiled hyphae at the tip and a strip of hyphae attached by lateral hyphae that formed short bridge-like structure were found in M. sarawak. Analysis on volatiles chemicals produced by both strains using Micro Extraction Gas Chromatography/ Mass Spectrograph showed Bicyclo [3.3.1] nona-2, 6-diene as a major compound in M. padawan. M. sarawak produces a musty odour and a major compound identified as (-) delta- Panasinsine. The ITS-5.8S rDNA sequence of both strains showed 96 to 99% similarities to Muscodor equiseti, indicating that both strains are representative members of Muscodor group. Phylogenetic analysis based on ITS-5.8S sequence showed that M. sarawak is clustered with M. vitigenus, M. sutura and M. equiseti but M. padawan clustered as an independent cluster. The anti-Ganoderma volatile chemicals produced by M. padawan and M. sarawak were affected by physicochemical conditions. Ganoderma boninense was completely killed by volatile chemicals produced by 5-day-old M. padawan and 7-day-old M. sarawak. Volatile chemicals produced by M. sarawak grown on all tested media were capable of killing G. boninense, but only volatile chemicals produced by M. padawan grown on oat extract agar and potato dextrose agar were able to kill G. boninense. At 250C and 300C, M. sarawak produced i volatile chemicals that kill G. boninense but M. padawan was only effective at 250C. At pH 5 and 9, M. padawan effectively killed G. boninense but M. sarawak showed the capability to kill G. boninense at all range of tested pH. In a pot assay system, volatile and non-volatile chemicals produced by M. sarawak did not show destructive impact to the growth rate (height, leaflet production, disease symptom and viability) of oil palm seedlings, although M. padawan showed suppressive impact towards the growth rate of the seedlings. M. padawan and M. sarawak showed success in killing G. boninense and also indirectly suppressed the growth rate of the air borne fungus, Trichoderma spp. This thesis discusses the isolation, characteristics and bioactivities of M. padawan and M. sarawak. The findings from this study suggest potential for the new endophytic fungi, M. sarawak, to be used as an alternative remedy to control the infection of G. boninense at the nursery stage, as well as to control the spread of basal stem rot disease in new or replanted area of oil palm plantations. The application of this newly discovered biofumigant agent could be expanded to other pre and post plant disease problems in the horticultural and agricultural industry. The isolated Muscodor strains described in this thesis may hold a lot of potential in the field of fungal biocontrol and this thesis can serve as a useful reference to the oil palm industries, researchers, and marketers. ii Acknowledgements I would like to thank the following departments and individuals that directly or indirectly contributed to my PhD project: • Dr. Rita Manurung, Ex-Chief Executive Officer (CEO) of Sarawak Biodiversity Centre who have always motivated me to achieve the goals in my study, • Dr. Yeo Tiong Chia, Chief Executive Officer (CEO) of Sarawak Biodiversity Centre and also as my co-supervisor in this project. He has always supported me to drive this project till completion, • Jabatan Ketua Menteri (Chief Minister’s Office) Sarawak, for funding the operational cost of this project, • Swinburne University of Technology Sarawak , for the fee waiver to support my study, • Sarawak Forest Corporation (SFC) and Sarawak Forest Department (SFD) for the information of targeted plants in Sarawak, • My supervisor Assoc. Prof. Dr. Peter Morin Nissom and ex-supervisor Prof. Clem Kuek for their guidance and support throughout my studies, • My Co-supervisor Dr. Moritz Mueller, who always supported and his trust in me on the direction that I selected, • My beloved family for their trust, support and encouragement to me to do the best in my life, • Prof. Dr. Gary Strobel from Montana University, US, and Dr. Hj. Idris Abu Seman, Head of Ganoderma and Diseases Research for Oil Palm Unit of Malaysian Palm Oil Board for their valuable and constructive comments, • My colleague at Sarawak Biodiversity Centre and Swinburne University of Technology Sarawak for their support and encouragements, • Norhayati Ahmed Sajali and Prof. Dr. Sepiah Muid, who assisted me in identification and encouraged me to persevere until completion of this project iii • Luming Chen and Onn May Ling , who assisted me in proof-read my thesis Thank you very much……….. iv Declaration I hereby declare that this thesis contains no material which has been accepted for the award to the candidate of any other degree or diploma, except where due reference is made in the text of this thesis. To the best of the candidate’s knowledge contains no material previously published or written by another person except where due reference is made in the text of this thesis. Where the work is based on joint research or publications, I have disclosed the relative contribution of the respective workers or authors. Noreha Mahidi 4 September 2015 v Table of Contents Content Page Abstract i Acknowledgements iii Declaration v Table of Contents vi List of Abbreviations x List of Figure xi List of Table xiii List of Appendix xv Chapter 1 Introduction and Literature Reviews 1.1 Soil Borne Fungus: Enemies in Agricultural Sector 1 1.2 Infection Court of Basal Stem Disease (BSR) 3 1.3 Biological Control Agent for BSR 6 1.4 Endophytic Fungi: New Candidates for Biological Control Agent 9 1.5 Discovery of Muscodor and Its Roles 10 1.6 Borneo: Sarawak an Ideal Location for Potential Untapped Resources 13 1.7 Aim of the Thesis 15 Chapter 2 Isolation and Characterization of Endophytic Muscodor-like Isolates Obtained from Cinnamomum javanicum in Sarawak 2.1 Introduction 16 2.2 Materials and Methods 19 2.2.1 Collection of Plant Samples 19 2.2.2 Isolation of Putative Endophytic Fungi Producing 21 Volatile Chemicals 2.2.3 Preparation of Standard Strains 22 2.2.3.1 Muscodor albus, cz-620 22 vi 2.2.3.2 Plant Pathogenic Fungi: Rhizoctonia solani, 23 Phytophthora capsici, Ganoderma boninense and Fusarium oxysporum 2.2.3.3 Preparation of Isolation Plate 23 2.2.4 In vitro Screening of Putative Endophytic Fungi Producing 23 Volatiles Chemicals 2.2.5 In vitro Screening of Endophytic Fungi Producing 24 Volatile Compounds 2.2.6 Preliminary Identification 25 2.2.6.1 Morphology of Colony 25 2.2.6.2 Molecular Identification 26 2.2.7 Storage 28 2.2.8 Viability and Productivity Test 29 2.3 Result and Discussion 30 2.3.1 Plant Collection 30 2.3.2 Isolation of Endophytic Fungi 31 2.3.3 In-vitro Screening of Endophytic Fungi Producing Volatile Chemicals 33 2.3.4 Preliminary Identification 34 2.3.5 Maintenance and Preservation 43 2.4 Conclusion 45 Chapter 3 Novel Endophytic Fungus from Borneo, Sarawak, Malaysia 3.1 Introduction 46 3.2 Materials and Methods 48 3.2.1 Morphological Identification 48 3.2.2 Analyses of the Chemical Composition in Volatile Chemicals 50 Produced by L3R3a and L5R1c 3.3 Results and Discussion 51 3.4 Conclusion 63 vii Chapter 4 Effect of Physicochemical Conditions on the Efficiency of Muscodor sarawak and Muscodor padawan as Biological Control Agent of Ganoderma boninense 4.1 Introduction 65 4.2 Materials and Methods 66 4.2.1 Effect of Inoculum Age 66 4.2.2 Effect of Culture Media 67 4.2.3 Effect of Temperature 67 4.2.4 Effect of pH 67 4.3 Results and Discussion 68 4.3.1 Anti-Ganoderma Volatile Chemicals Produced in All Stages 68 of Growth of the Test Strains 4.3.2 Media Composition Affects Efficiency of Muscodor padawan, 70 but not Muscodor sarawak, in producing Anti-Ganoderma Volatile Chemicals 4.3.3 250C and 300C are the Best Temperature for Muscodor sarawak 72 to Produce anti-Ganoderma Volatile Chemicals 4.3.4 pH affects the capability of Muscodor padawan in Producing 74 Anti-Ganoderma Volatile Chemicals 4.4 Conclusion 75 Chapter 5 Efficiency of Muscodor sarawak and Muscodor padawan in Preventing Ganoderma boninense From Infecting Oil Palm Seedlings 5.1 Introduction 77 5.2 Materials and Methods 78 5.2.1 In vitro Screening on the Capability of Barley Infected with 78 Muscodor padawan and Muscodor sarawak to Produce Volatiles Anti-Ganoderma Chemicals, Using a Double Plate Assay System 5.2.2 In vitro Screening on the Capability of Muscodor padawan and 79 Muscodor sarawak to Produce Non-volatiles Anti-Ganoderma Chemicals, Using a Dual Cultures Assay System.