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The Role of Helper Bacteria in Facilitating Mycorrhization of Biserrula Pelecinus L., a Pasture Legume New to Australia

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The Role of Helper Bacteria in Facilitating Mycorrhization of Biserrula Pelecinus L., a Pasture Legume New to Australia The role of helper bacteria in facilitating mycorrhization of Biserrula pelecinus L., a pasture legume new to Australia David James Orchard Graduate Diploma in Science (Botany), University of New England, Armidale, NSW, Australia Bachelor of Arts (Hons), Australian National University, Canberra, ACT, Australia Submitted to Charles Sturt University for the degree of Doctor of Philosophy School of Agricultural and Wine Sciences Faculty of Science March, 2020 Contents List of Tables .................................................................................................................. iv List of Figures ................................................................................................................. vi Certification of Authorship ........................................................................................... ix Acknowledgements .......................................................................................................... x Abstract ........................................................................................................................... xi Chapter 1. Mycorrhiza helper bacteria in the rhizosphere of Biserrula pelecinus L. ........................................................................................................................................... 1 1.1. Introduction .......................................................................................................... 1 1.2. Plant-microbe interactions in the rhizosphere................................................... 2 1.3. Microbial diversity in the mycorrhizosphere..................................................... 7 1.4. Mycorrhiza helper bacteria ............................................................................... 10 1.5. The root receptivity hypothesis ......................................................................... 21 1.6. Biserrula as a target species .............................................................................. 28 1.7. Conclusion ........................................................................................................... 31 1.8. Aims ..................................................................................................................... 32 1.9. Thesis structure .................................................................................................. 32 Chapter 2. Observation and quantification of arbuscular mycorrhizas in the roots of the pasture legume Biserrula pelecinus L. .............................................................. 34 2.1. Introduction ........................................................................................................ 34 2.2. Format of experiments ....................................................................................... 36 2.3. Experiment 1: Quantification of mycorrhizas in the roots of field- and trap culture-grown biserrula ............................................................................................ 36 2.4. Experiment 2: Identification of mycorrhizas in the roots of field- and trap culture-grown biserrula ............................................................................................ 41 2.5. Experiment 3: Recruitment of AM fungi to the root environment of biserrula in a pasture environment ......................................................................... 44 2.6. Results .................................................................................................................. 47 2.7. Discussion ............................................................................................................ 54 2.8. Conclusion ........................................................................................................... 57 Chapter 3. Identification of mycorrhiza helper bacteria in the rhizosphere of Biserrula pelecinus L. .................................................................................................... 59 3.1. Introduction ........................................................................................................ 59 3.2. Materials and methods ....................................................................................... 61 The role of helper bacteria in facilitating mycorrhization of the pasture legume Biserrula pelecinus L. ii 3.3. Results .................................................................................................................. 70 3.4. Discussion ............................................................................................................ 88 3.5. Conclusion ........................................................................................................... 92 Chapter 4. Ecological interactions in the rhizosphere of Biserrula pelecinus L. and their influence on MHB activity .................................................................................. 93 4.1. Introduction ........................................................................................................ 93 4.2. Materials and methods ....................................................................................... 96 4.3. Results ................................................................................................................ 103 4.4. Discussion .......................................................................................................... 108 4.5. Conclusion ......................................................................................................... 113 Chapter 5. Characterisation of the rhizosphere microbiota of Biserrula pelecinus L. across a two-season legume-cereal rotation ......................................................... 114 5.1. Introduction ...................................................................................................... 114 5.2. Materials and methods ..................................................................................... 116 5.3. Results ................................................................................................................ 123 5.4. Discussion .......................................................................................................... 140 5.5. Conclusion ......................................................................................................... 144 Chapter 6. Summary and conclusions ....................................................................... 146 6.1. Introduction ...................................................................................................... 146 6.2. Aims of the research ......................................................................................... 146 6.3. Key findings ...................................................................................................... 147 6.4. Synthesis ............................................................................................................ 149 6.5. Future research ................................................................................................. 154 6.6. Conclusion ......................................................................................................... 155 References .................................................................................................................... 158 Appendix 1. Mycorrhiza helper bacteria in ectomycorrhizas ................................ 193 Appendix 2. Structure of experiments addressing ecological interactions in the rhizosphere (Chapter 4)……………………………………………………………..202 The role of helper bacteria in facilitating mycorrhization of the pasture legume Biserrula pelecinus L. iii List of Tables Chapter 1. Table 1-1. Evidence of a helper effect on mycorrhization, growth of fungal mycelium, or plant growth parameters in arbuscular mycorrhizas ................................................... 14 Chapter 2. Table 2-1. Primers and sequencing conditions for nested PCR ..................................... 44 Table 2-2. Primers and sequencing conditions for ITS diversity profiling .................... 46 Table 2-3. Frequency and intensity of mycorrhization in field- and pot-grown biserrula ......................................................................................................................................... 48 Table 2-4. BLAST matches for spore DNA ................................................................... 50 Table 2-5. Relative abundance of common AM fungi in rhizosphere, bulk, and field soils ................................................................................................................................. 52 Table 2-6. Diversity index values for ITS diversity profiles by soil compartment ........ 55 Chapter 3. Table 3-1. Diversity profiling primers and sequencing conditions ................................ 62 Table 3-2. 16S primers and sequencing conditions ........................................................ 65 Table 3-3. Genus-specific primers used in this study..................................................... 65 Table 3-4. Bacterial cultures used in each trial .............................................................. 67 Table 3-5. Diversity index values for 16s diversity profiles by soil compartment ........ 72 Table 3-6. Differentially abundant bacterial taxa in the biserrula rhizosphere and the surrounding field soil ...................................................................................................... 76 Table 3-7. BLAST matches for putative MHB strains
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