Thomas James Thirkell Thesis
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The Influence of Nitrogen Source on the Nutrition of Arbuscular Mycorrhizal Plants Thomas James Thirkell PhD University of York Biology September 2016 Abstract Arbuscular mycorrhizal fungi (AMF) form a symbiosis with 70% of land plants and may contribute significantly to plant phosphorus (P) and nitrogen (N) acquisition in exchange for photosynthetic carbon. AMF mainly acquire N as simple, inorganic molecules such as ammonium and nitrate, but whether AMF display a preference for either source, or can increase plant N content is uncertain. Furthermore, soil nutrient availability, especially that of N, may determine the extent to which a fungus engages in nutrient-for-C trade with plants; further experimental validation of this suggestion is warranted. Experiments ranging from Petri plate microcosms to field trials were carried out to address these questions. Plant N uptake via AMF was traced using stable isotope 15N, having been added to hyphal-only compartments which allow access to AMF, while excluding plant roots. AMF species Glomus aggregatum and Rhizophagus irregularis were used individually to inoculate plants allowing comparisons between AMF isolates, while N uptake preferences were tested by providing 15N as ammonium, nitrate or ammonium nitrate. R. irregularis contribution to plant nutrient uptake was sufficient to increase host biomass in chapter 2, but limited evidence of AMF preference for nitrate or ammonium-N was seen in any experiment. N acquisition by AMF was highest in a Petri microcosm experiment when supplied as ammonium nitrate. Functional diversity among AMF species was observed in terms of plant growth and nutrient uptake, and carbon acquisition from the plant: G. aggregatum was less beneficial to plant partners than R. irregularis. Experimental field-trial data suggests that soil nutrient levels influence both the community structure of AMF and the extent to which they engage in N-for-C trade with plant partners. These findings highlight the complexities and potential significance of the AM route for N uptake both in simplified experimental systems and full-scale commercial crop field trial plants. 2 Table of Contents Abstract........................................................................................................................ 2 Table of Contents ......................................................................................................... 3 List of Tables ............................................................................................................. 10 List of Figures ............................................................................................................ 12 Acknowledgements .................................................................................................... 21 Author’s declaration ................................................................................................... 23 Chapter 1. ...................................................................................................................... 24 General Introduction ...................................................................................................... 24 1.1. Rationale ............................................................................................................. 24 1.2. Plant mineral nutrition ......................................................................................... 25 1.2.1. Nitrogen as a plant nutrient ........................................................................... 26 1.2.2. The Terrestrial N cycle ................................................................................. 26 1.2.3. Soil Nitrogen ................................................................................................ 28 1.2.4. Plant root N acquisition ................................................................................. 31 1.2.5. Phosphorus as a nutrient................................................................................ 32 3 1.2.6. Soil Phosphorus ............................................................................................ 32 1.2.7. Phosphorus acquisition ................................................................................. 33 1.3. Mycorrhizas ........................................................................................................ 34 1.3.1 The Arbuscular Mycorrhizal symbiosis .......................................................... 34 1.3.2. AMF physiology ........................................................................................... 35 1.3.3. Multiple nutrient uptake pathways ................................................................ 37 1.3.4. Arbuscular Mycorrhizal N uptake ................................................................. 38 1.4. Control of AMF nutrient trade ............................................................................. 40 1.5. Crop plants and AMF .......................................................................................... 42 1.5.1. AMF for sustainable agriculture .................................................................... 43 1.6. AMF uptake of fertiliser N ............................................................................... 45 1.7. Studying AM symbioses ...................................................................................... 46 1.8. Aims and hypotheses ........................................................................................... 47 Chapter 2. ...................................................................................................................... 49 Resolving the 'nitrogen paradox' of arbuscular mycorrhizas: fertilization with organic matter brings considerable benefits for plant nutrition and growth .................................. 49 4 2.1 Introduction .......................................................................................................... 49 2.2 Materials and Methods ......................................................................................... 52 2.2.1 Microcosm Design ......................................................................................... 52 2.2.2 AMF Inoculum .............................................................................................. 54 2.2.3 Nutrient Addition ........................................................................................... 55 2.2.4 Patch Material ................................................................................................ 55 2.2.5 Harvest and Analysis ..................................................................................... 56 2.2.6 Statistical Analysis ......................................................................................... 57 2.3 Results.................................................................................................................. 58 2.4 Discussion ............................................................................................................ 64 Chapter 3. ...................................................................................................................... 67 Nitrogen source preference of arbuscular mycorrhizal plants across different scales: Petri plates to glasshouse microcosms .................................................................................... 67 3.1. Introduction ......................................................................................................... 67 3.2. Materials and Methods ........................................................................................ 72 3.2.1. Experiment 1 (Petri plates) ............................................................................ 72 5 3.2.1.1. Experimental setup ................................................................................. 72 3.2.1.2. Arbuscular Mycorrhizal Inoculum .......................................................... 73 3.2.1.3. 15N addition treatments ........................................................................... 74 3.2.1.4. Harvest and Analysis .............................................................................. 74 3.2.1.5. Statistical Analysis ................................................................................. 75 3.2.2. Experiment 2 (Allium cepa microcosms) ....................................................... 76 3.2.2.1. Experimental setup ................................................................................. 76 3.2.2.2. Arbuscular Mycorrhizal Fungi................................................................ 77 3.2.2.3. Nutrient addition .................................................................................... 78 3.2.2.4. 14C labelling ........................................................................................... 79 3.2.2.5. Growth chamber..................................................................................... 81 3.2.2.6. Harvest and Analysis .............................................................................. 81 3.2.2.7. Statistical Analysis ................................................................................. 83 3.3. Results. ............................................................................................................... 84 3.3.1. Experiment 1 - Petri dish microcosm ............................................................. 84 3.3.2. Experiment 2 – Allium cepa microcosms ....................................................... 85 6 3.4. Discussion ..........................................................................................................