Plant Foliar Phosphorus and Silicon Content Changes in Response to Root
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PLANT FOLIAR PHOSPHORUS AND SILICON CONTENT CHANGES IN RESPONSE TO ROOT FUNGAL COMMUNITIES AND SILICON ENRICHMENT Rosemary Erin Haskell PhD University of York Biology August 2017 Abstract Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that supply limiting nutrients, largely phosphorus (P), to plants in exchange for carbon. Silicon (Si) is an important defence element for plants and several reports have observed a relationship between AMF colonisation and Si uptake. It is unknown how diverse soil microbial communities affect foliar Si and P concentrations, and whether improvements in foliar Si concentrations due to AMF colonisation are observed in field conditions. This is the first study to document the dual effects of AMF and Si application in a non-crop species (Brachypodium sylvaticum) and the effect of different microbial communities, on plant uptake and deposition of Si and P. An initial glasshouse experiment used a single species AMF inoculum in combination with a Si enrichment treatment to investigate the effect on foliar Si and P concentration. The results showed that AMF improved the uptake of Si and P compared to non-colonised plants, but that different mechanisms for uptake are likely. Introducing microbial communities isolated from agricultural and woodland environments as inocula in a controlled environment showed that microbial diversity alters the efficacy of Si and P supply, and that improvements in the supply of these were not directly related to AMF colonisation. Finally, B. sylvaticum plants from woodland were sampled across two years. The results of this sampling did not show any benefit of AMF on Si and P uptake, but did reveal significant differences in P concentration over time irrespective of fungal colonisation. Comparisons between studies using high throughput sequencing demonstrates that the methods commonly used in mycorrhizal studies may be overlooking important interactions with un- recorded organisms in the soil and roots of experimental plants. Ecologically relevant studies incorporating long-term repeated sampling are required to fully understand how microbial communities can improve Si and P nutrition in plants. 2 Table of Contents Abstract ..........................................................................................................................................2 Table of Contents ...........................................................................................................................3 List of Tables .................................................................................................................................7 List of Figures ................................................................................................................................9 Acknowledgements ......................................................................................................................13 Author’s Declaration ....................................................................................................................14 1 General Introduction ............................................................................................................15 1.1 Rationale ......................................................................................................................15 1.2 Arbuscular mycorrhizal fungi ......................................................................................16 1.2.1 Biology and structure of the arbuscular mycorrhizal symbiosis ..........................16 1.2.2 Diversity and distribution of AMF species ..........................................................18 1.2.3 AMF and plant stress tolerance ............................................................................18 1.3 Silicon ..........................................................................................................................20 1.3.1 Silicon availability, uptake and deposition in plants ............................................20 1.3.2 Si and plant defence .............................................................................................24 1.4 AMF and Si ..................................................................................................................25 1.5 Illumina MiSeq Amplicon Sequencing ........................................................................27 1.6 Thesis aims and hypotheses .........................................................................................28 2 Methods development ..........................................................................................................30 2.1 Substrate choice experiment ........................................................................................30 2.1.1 Introduction and background ...............................................................................30 3 2.1.2 Materials and methods .........................................................................................30 2.1.3 Results ..................................................................................................................33 2.1.4 Discussion ............................................................................................................37 2.2 Plant host selection for mycorrhizal and silicon manipulation experiments. ...............39 2.2.1 Winter wheat and mycorrhizal colonisation ........................................................39 2.2.2 Plant host screening experiment...........................................................................42 2.3 Verifying mycorrhizal cultures ....................................................................................47 2.3.1 Rationale ..............................................................................................................47 2.3.2 Materials and methods .........................................................................................47 2.3.3 Results ..................................................................................................................49 2.3.4 Discussion ............................................................................................................49 2.4 Overview of High-Throughput Sequencing methods ..................................................52 2.4.1 Sample preparation ..............................................................................................52 2.4.2 Illumina Miseq sequencing ..................................................................................53 2.4.3 Sequence processing ............................................................................................53 3 Colonisation by Rhizophagus irregularis increases root uptake efficiency of silicon and phosphorus in Brachypodium sylvaticum ....................................................................................60 3.1 Introduction ..................................................................................................................60 3.2 Materials and methods .................................................................................................61 3.3 Results ..........................................................................................................................62 3.3.1 Plant growth and colonisation ..............................................................................62 3.3.2 Foliar Si and P content .........................................................................................62 4 3.4 Discussion ....................................................................................................................67 4 How does mycorrhizal community affect Si and P content of Brachypodium sylvaticum plants? ..........................................................................................................................................71 4.1 Introduction ..................................................................................................................71 4.2 Materials and methods .................................................................................................74 4.2.1 Preparation of biological materials ......................................................................74 4.2.2 Experimental set-up and harvest ..........................................................................74 4.2.3 Illumina Miseq sequencing ..................................................................................75 4.2.4 Statistical analysis ................................................................................................76 4.3 Results ..........................................................................................................................77 4.3.1 Plant growth and colonisation ..............................................................................78 4.3.1 Foliar Si and P content .........................................................................................79 4.3.2 Fungal Diversity ...................................................................................................81 4.3.3 Relationships between fungal diversity and plant growth parameters .................89 4.4 Discussion ....................................................................................................................94 5 Community dynamics of fungi colonising Brachypodium sylvaticum roots and effects on foliar silicon and phosphorus concentrations ...............................................................................98 5.1 Introduction