Effects of endophytic actinobacteria on lucerne growth and the development of its N2-fixation symbiosis with rhizobia A thesis submitted for the award Doctor of Philosophy at Flinders University of South Australia Hoang Xuyen Le Department of Medical Biotechnology School of Medicine, Faculty of Medicine, Nursing and Health Sciences Flinders University of South Australia 2015 Declarations I certify that this thesis does not contain material which has been accepted for award of any degree of diploma, and to the best of my knowledge and belief it does not contain any material previously published or written by another person except where due reference is made in the text of this thesis or in the notes. Hoang Xuyen Le i Contents Declarations .............................................................................................................................. i List of tables ............................................................................................................................ ix List of figures .......................................................................................................................... xi Acknowledgment .................................................................................................................. xiii Abbreviations .........................................................................................................................xiv Abstract .................................................................................................................................. xv Chapter 1 ................................................................................................................................ 1 Introduction and Literature review ..................................................................................... 1 1.1 Global importance of legumes and nitrogen fixation ......................................................... 2 1.1.1 General information on legumes ................................................................................. 2 1.1.2 Lucerne globally and in Australia .............................................................................. 3 1.2 Importance of soil micro-flora to legume production ........................................................ 4 1.2.1 Rhizobia and nitrogen fixation .................................................................................... 4 1.2.1.1 Rhizobium spp. ..................................................................................................... 4 1.2.1.2 The nodulation process ........................................................................................ 5 1.2.1.3 Lucerne symbiosis .............................................................................................. 12 1.2.2 Effects of other soil microorganisms on plant growth and nitrogen fixation by legumes .............................................................................................................................. 12 1.2.2.1 Mycorrhiza spp. ................................................................................................. 12 1.2.2.2 Trichoderma spp. ............................................................................................... 14 1.2.2.3 Pseudomonas spp. .............................................................................................. 14 1.2.3 Endophytic actinobacteria ........................................................................................ 15 1.2.3.1 Definition of endophyte ...................................................................................... 15 1.2.3.2 General characteristics of actinobacteria and endophytic actinobacteria ........ 16 1.2.3.3 Occurrence and diversity of endophytic actinobacteria in plants ..................... 19 1.2.3.4 Evidence of actinobacterial effects on agricultural plants ................................ 21 1.2.3.5 Potential mechanisms of action ......................................................................... 24 1.3 Summary, potential for legume improvement and critical knowledge gaps. ................... 28 1.4. Research plan (Objectives) ............................................................................................. 29 1.4.1 Aims of research ....................................................................................................... 29 1.4.2 Hypothesis ................................................................................................................. 29 1.4.3 Research plan ............................................................................................................ 29 Chapter 2 .............................................................................................................................. 31 ii General materials and methods .......................................................................................... 31 2.1 Legume seeds, rhizobia and actinobacteria ...................................................................... 32 2.2 Treatment of seeds and application of inoculants ............................................................ 33 2.2.1 Surface sterilisation of seeds..................................................................................... 33 2.2.2 Isolation, growth and application of actinobacteria ................................................. 33 2.2.3 Growth of Rhizobium ................................................................................................ 34 2.2.4 Plant growth media, nutrition, sowing and water supply ......................................... 34 2.2.5 Harvest of plants ....................................................................................................... 35 2.3 Data collection and analysis ............................................................................................. 35 2.4 Identification of endophytic actinobacteria by 16S rRNA gene amplification and sequencing.............................................................................................................................. 35 2.4.1 DNA extraction ......................................................................................................... 36 2.4.2 Quantify DNA concentration .................................................................................... 37 2.4.3 PCR 16S rRNA .......................................................................................................... 37 2.4.4 Clean up and sequencing the PCR products ............................................................. 38 Chapter 3 .............................................................................................................................. 39 Isolation and characterisation of actinobacterial endophytes from pasture legumes ... 39 3.1. Introduction ..................................................................................................................... 40 3.2 Materials and methods ..................................................................................................... 42 3.2.1 Source of legumes ..................................................................................................... 42 3.2.2 Isolation of endophytic actinobacteria ...................................................................... 42 3.2.2.1 Surface-sterilization procedure .......................................................................... 42 3.2.2.2 Media for isolation ............................................................................................. 43 3.2.2.3 Purification of actinobacterial endophytes ........................................................ 43 3.2.3 Characterisation of endophytic actinobacteria ........................................................ 43 3.2.3.1 Morphology (frequency and distribution) .......................................................... 43 3.3 Results and discussion ..................................................................................................... 44 3.3.1 Isolation of endophytic actinobacteria from legumes ............................................... 44 3.3.2 Morphological characterisation of actinobacteria ................................................... 47 3.4 Conclusion ....................................................................................................................... 49 Chapter 4 .............................................................................................................................. 50 Effects of actinobacteria on lucerne germination, seedling growth, and tolerance of soil- borne disease......................................................................................................................... 50 4.1 Introduction ...................................................................................................................... 51 4.2 Materials and methods ..................................................................................................... 54 iii 4.2.1 Effects of actinobacteria on lucerne germination ..................................................... 54 4.2.1.1 Lucerne germination on agar ............................................................................ 54 4.2.1.2 Lucerne growing in a sandy loam ...................................................................... 54 4.2.2 Inhibition of the growth of pathogenic fungi on agar ............................................... 55 4.2.3 Effect of actinobacteria on the development of root damage