The microbiology of lean and obese soil Frances Patricia Jones September 2016 Department of Geography and Environmental Science, University of Reading, UK Department of AgroEcology, Rothamsted Research, UK Submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy Abstract The bacterial genus Bradyrhizobium is biologically important within soils, with different representatives found to perform a range of functions including nitrogen fixation through symbioses, photosynthesis and denitrification. The Highfield experiment at Rothamsted provides an opportunity to study the impact of plants on microbial communities as it has three long-term contrasting regimes; permanent grassland, arable and bare fallow (devoid of plants). The bare fallow plots have a significant reduction in soil carbon and microbial biomass. Bradyrhizobium has been shown by metagenomic studies on soil to be one of the most abundant and active groups including in bare fallow soil indicating that some phenotypes are adapted to survive in the absence of plants. A culture collection was created with isolates obtained from contrasting soil types from Highfield in addition to woodland soil, gorse (Ulex europeaus) and broom (Cytisus scoparius) root nodules. The collection’s phylogeny has been explored by sequencing housekeeping genes to determine whether soil treatment affects the core genome. One grassland and one bare fallow isolate had their genome sequenced and differences have been assessed to establish their potential for a range of functions and to direct future experiments. The functional diversity of the collection has been investigated using carbon metabolism assays to identify key substrates and determine whether the isolates group according to soil treatment. Symbiosis capacity and role in nitrogen cycling has been examined using nodulation tests, anaerobic growth on nitrate and nitrous oxide production and reduction through denitrification. A high level of diversity can be seen throughout the collection with differences being linked to niche adaptation. Understanding more about Bradyrhizobium could give clues on how above ground management impacts a key group within the soil community. Furthermore, the first assembled genomes of two non-symbiotic Bradyrhizobium strains isolated from soil provide an important resource for microbiology and soil ecology. i Table of contents 1 Abstract ................................................................................................................................................ i Table of contents ................................................................................................................................ ii List of tables ........................................................................................................................................ x List of figures ...................................................................................................................................... xi Abbreviation list ............................................................................................................................... xiv Statement of original authorship ..................................................................................................... xvi Acknowledgements ......................................................................................................................... xvii Dedication ...................................................................................................................................... xviii 1 Introduction ............................................................................................................................... 1 1.1 Soil microbial communities ................................................................................................ 1 1.2 Abiotic and biotic effects on soil microbial communities .................................................. 3 1.3 Plant-microbe interactions................................................................................................. 5 1.4 Soil microbial ecology and agriculture ............................................................................... 8 1.5 The Fabaceae plant family ............................................................................................... 11 1.6 Rhizobiales and the Bradyrhizobiaceae ........................................................................... 12 1.7 Nodulation ....................................................................................................................... 16 1.8 The microbial nitrogen cycle ............................................................................................ 22 1.8.1 Nitrogen fixation ...................................................................................................... 22 1.8.2 Denitrification .......................................................................................................... 22 1.9 The Highfield experiment and Bradyrhizobium ............................................................... 23 1.10 Project objectives ............................................................................................................. 25 1.10.1 Aims .......................................................................................................................... 25 2 General materials and methods ............................................................................................... 27 2.1 Culture medium ............................................................................................................... 27 2.2 Growth conditions............................................................................................................ 27 2.3 DNA extraction ................................................................................................................. 27 2.4 Polymerase chain reaction (PCR) mixture ........................................................................ 28 2.5 Agarose gel electrophoresis and PCR product purification ............................................. 28 ii 2.6 DNA sequencing and sequence analysis .......................................................................... 28 2.7 Storage of isolates ............................................................................................................ 29 3 Isolation & identification of Bradyrhizobium from soil and root nodules ............................... 30 3.1 Introduction ..................................................................................................................... 30 3.1.1 Isolation of bacteria from root nodules ................................................................... 30 3.1.2 Isolation of bacteria from soil .................................................................................. 31 3.1.3 Medium design and growth conditions ................................................................... 31 3.1.4 Key characteristics of Bradyrhizobium for the isolation method............................. 32 3.1.5 Culture medium for Bradyrhizobium isolation ......................................................... 32 3.1.6 Use of 16S rRNA gene for identification .................................................................. 33 3.1.7 Aims .......................................................................................................................... 33 3.2 Materials and methods .................................................................................................... 34 3.2.1 Description of study sites ......................................................................................... 34 3.2.2 Isolation of free-living Bradyrhizobium isolates ....................................................... 37 3.2.3 Isolation of symbiotic Bradyrhizobium isolates ....................................................... 37 3.2.4 Bradyrhizobium specific 16S primer design ............................................................. 38 3.2.5 PCR optimisation ...................................................................................................... 41 3.2.6 PCR amplification ..................................................................................................... 42 3.2.7 Reference isolates included in the culture collection .............................................. 42 3.2.8 Sequence analysis .................................................................................................... 43 3.3 Results .............................................................................................................................. 45 3.3.1 Creation of a Rothamsted Bradyrhizobium culture collection ................................. 45 3.3.2 16S sequence analysis .............................................................................................. 51 3.4 Discussion ......................................................................................................................... 55 3.4.1 Summary of the Bradyrhizobium culture collection ................................................ 55 3.4.2 Isolation method of Bradyrhizobium from soil and nodules ................................... 55 3.4.3 Importance of a culture collection ........................................................................... 56 4 Multilocus sequence analysis of the Bradyrhizobium culture collection ................................. 57 iii 4.1 Introduction ....................................................................................................................