Diversity and Roles of Mycorrhizal Fungi in the Bee Orchid Ophrys apifera By Wazeera Rashid Abdullah April 2018 A Thesis submitted to the University of Liverpool in fulfilment of the requirement for the degree of Doctor in Philosophy Table of Contents Page No. Acknowledgements ............................................................................................................. xiv Abbreviations ............................................................................ Error! Bookmark not defined. Abstract ................................................................................................................................... 2 1 Chapter one: Literature review: ........................................................................................ 3 1.1 Mycorrhiza: .................................................................................................................... 3 1.1.1Arbuscular mycorrhiza (AM) or Vesicular-arbuscular mycorrhiza (VAM): ........... 5 1.1.2 Ectomycorrhiza: ...................................................................................................... 5 1.1.3 Ectendomycorrhiza: ................................................................................................ 6 1.1.4 Ericoid mycorrhiza, Arbutoid mycorrhiza, and Monotropoid mycorrhiza: ............ 6 1.1.5 Orchid mycorrhiza: ................................................................................................. 7 1.1.5.1 Orchid mycorrhizal interaction: ....................................................................... 8 1.2 Rhizoctonia systematics: ................................................................................................ 8 1.2.1 Rhizoctonia as a pathogen: ...................................................................................... 9 1.2.2 Rhizoctonia as a mycorrhizal symbiont: ................................................................. 9 1.3 Isolation of orchid symbiont fungi: .............................................................................. 10 1.4 Molecular Identification of fungi: ................................................................................ 10 1.4.1 Barcoding sequences for fungi: ............................................................................. 11 1.4.2 Molecular methods in Rhizoctonia classification: ................................................ 12 1.5 Next Generation Sequencing (NGS) Technologies: .................................................... 13 1.5.1 Illumina sequencing: ............................................................................................. 14 1.5.2 NGS application: ................................................................................................... 15 1.5.3 Transcriptomes in non-model plants: .................................................................... 15 1.6 Family Orchidaceae: ................................................................................................... 16 1.6.1Terrestrial orchids: ................................................................................................. 16 1.7 Aims of this thesis: ....................................................................................................... 19 2 Chapter two: Life history of Ophrys apifera. .................................................................. 21 2.1 Introduction: - ............................................................................................................ 21 2.1.1 Life history of orchid plant: .................................................................................. 21 2.1.2 Genus Ophrys: ...................................................................................................... 25 2.1.2.1 Ophrys apifera: .............................................................................................. 25 2.2 Materials and Method: .............................................................................................. 27 2.2.1 Study area - Biosciences plants: ............................................................................ 27 2.2.2 Data recording ....................................................................................................... 27 2.2.3 Environmental data: .............................................................................................. 28 2.3 Results: ........................................................................................................................ 29 2.3.1 Bee orchid growth cycle: ...................................................................................... 29 2.3.1.1 Vegetative development: ................................................................................ 29 2.3.1.2: Flower shoots development: ......................................................................... 31 i 2.3.1.3: Relationship between vegetative and reproductive development: ................ 33 2.4 Discussion: .................................................................................................................. 36 3 Chapter three: Seed germination of Ophrys apifera in vitro. ........................................ 38 3.1 Introduction: .............................................................................................................. 38 3.1.1 Orchid seeds: ......................................................................................................... 38 3.1.1.1 Orchid seeds structure: ................................................................................... 38 3.1.1.2 Interaction between orchid seeds and fungi during germination: .................. 38 3.1.2 Laboratory culture of orchid seeds:....................................................................... 39 3.1.2.1 Decontamination of capsules and seeds: ........................................................ 40 3.1.2.2 Culture medium: ............................................................................................ 41 3.1.2.3 Growth regulators: ......................................................................................... 42 3.1.4 Culture of seedlings asymbiotically: ..................................................................... 43 3.1.5 Other factors that affect seed germination in vitro:............................................... 43 3.1.5.1 Effects of temperature on the seed germination: ............................................ 43 3.1.5.2 Effects of light on seed germination: ............................................................. 44 3.1.5.3 Effect of seed maturity on germination in vitro: ............................................ 44 3.1.6 Germinating genus Ophrys in vitro: ...................................................................... 45 3.2 Materials and Method: .............................................................................................. 46 3.2.1 Collection of orchid capsules: ............................................................................... 46 3.2.2 Equipment: ............................................................................................................ 46 3.2.3 Culture medium composition: ............................................................................... 46 3.2.4 Seed isolation: ....................................................................................................... 46 3.2.5 Culture Condition:................................................................................................. 46 3.2.6 Inoculation of fungi into seed germination cultures: ............................................ 48 3.3 Results: ........................................................................................................................ 49 3.3.1 Developmental stages of seed germination in vitro: ............................................. 49 3.3.2 Effects of media on in vitro seed germination and protocorm formation: ............ 49 3.4 Discussion: .................................................................................................................. 52 3.4.1 In vitro cultivation:................................................................................................ 52 3.4.2 Factors that impact on germination: ...................................................................... 52 4 Chapter four: Isolation and identification of orchid mycorrhizal fungi: .................... 53 4.1 Introduction: - ............................................................................................................ 53 4.1.1 Overview: .............................................................................................................. 53 4.1.2 Isolation of fungi from orchid roots: ..................................................................... 53 4.1.3 Identification fungi from orchid roots: .................................................................. 54 4.1.4 Methods for isolation and estimation of fungi in soil: .......................................... 55 4.1.5 Growth of fungi in culture: ................................................................................... 55 4.1.5.1 Effect of different culture media and temperature ......................................... 55 4.1.6 Isolation of DNA of fungi from soil: .................................................................... 56 ii 4.1.7 Internal Transcribed Spacer (ITS):........................................................................ 57 4.1.7.1 ITS primers and limitation: ...........................................................................