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Scottish P. sylvestris populations have been extensively researched, using monoterpene and isozyme markers, but differentiation among populations is too low to identify origins or gene flow. Protocols were developed for the analysis of chloroplast DNA (cpDNA) Restriction Fragment Length Polymorphisms (RFLP) in P. sylvestris to determine whether cpDNA markers could be used for these purposes. Nine populations from throughout the range of pine in Scotland were sampled. Two individuals from each of these populations were surveyed using seven restriction enzymes and 13 probes from P. contorta, a total of 91 probe/enzyme combinations. The cpDNA genome of P. sylvestris was found to be about 119 kilobase pairs in length. 100% of the length of the genome was sampled, and 0.52% of the sequence length. No variation was found in any individual. These results were compared with a survey of P. sylvestris from China, Sweden and Turkey. There is no evidence that the cpDNA genotype of Scottish pine differs in any way from these varieties, suggesting that the cpDNA genome of P. sylvestris is homogeneous over a large part of the species' range. A survey of 191 individuals for 1 probe/enzyme combination revealed one variant individual, which appeared to be heteroplasmic for two cpDNA haplotypes. It was not possible to determine whether this was due to biparental inheritance of somatic mutation within that individual. The implications of these results for further research on the structure and inheritance of cpDNA in gymnosperms, the use of cpDNA as a genetic market in P. sylvestris, and the conservation of Scottish populations of this species are discussed. Finally, it is suggested that an increased collaboration between molecular biologists and ecologists is the best way to approach studies using sophisticated molecular techniques to measure genetic diversity in natural populations. In this way, the potential of this approach to improve the management of genetic resources can be fully realised.

Global food demand is expected to rise by up to 62% by 2050, with an increasing population and finite land available for food production. Denitrification causes loss of fixed nitrogen from soil systems, thereby reducing crop productivity. Current nitrogen cycling models cannot fully explain the variation in nitrogen use efficiency at the field scale; this variation is thought to be associated with microbial community dynamics. The dynamics of denitrification and denitrifying communities are affected by the physicochemical properties of the soil, and therefore the land management practices which alter them. In this thesis, we look at the effects of land management history and innate environmental variation using a long-term factorial field trial, a working commercial farm, and laboratory-based experiments. By studying the denitrifier community composition, abundance and diversity in the context of environmental data, we were able to address the question of how specific land management practices and innate spatiotemporal variation affects the distribution of denitrifiers within arable soils. It was found that bacterial and denitrifier communities are largely unaffected by land management history, and cluster predominantly along environmental gradients. Additionally, there was a high degree of spatiotemporal variation in soil denitrifying communities, with implications for future sampling strategies as well as computer modelling. Finally, it was established that isolation of bacteria from the environment is a viable method for culturing non-model organisms that can be characterized and used to inform nitrogen-cycling models. In this combination of well-established agricultural field trial and working commercial farm, the major identifiable determinant of bacterial community dissimilarity was a blend of physicochemical factors. The importance of selection on bacterial communities, relative to ecological drift, may have consequences for attempts to understand and model nutrient availabilities in arable farming systems, and improve agricultural productivity to feed the growing global population.

European foulbrood (EFB), a disease of honey bee larvae (Apis mellifera), is caused by Melissococcus plutonius. This bacterium has a worldwide distribution, found wherever honey bees are kept. EFB outbreaks vary in severity, with treatment options of shook swarm, oxytetracycline antibiotic application and colony destruction available to manage this disease. M. plutonius bacterial isolates have previously been differentiated using a multi- locus sequence typing scheme (MLST), with a focus on tracing the source of new EFB outbreaks. A putative toxin gene, melissotoxin A, has been shown to correlate with virulence in previous larval infection studies. Whole genome sequencing of approximately 50 M. plutonius isolates suggests that current strain typing (ST) methods are likely insufficient to separate the most common isolates in the UK, such as ST3. Statistical analysis of the relationship between isolate possession of a gene, melissotoxin A, and type of treatment used on the EFB outbreak, showed a negative relationship between the toxin presence and incidence of colony destruction. Using laboratory reared honey bee larvae, M. plutonius virulence was tested at the strain level. Within these experiments, it was found that isolates possessing the melissotoxin A gene did not cause a significantly higher larval mortality rate, and expected virulence phenotypes, based on clonal complex type, wasn't always observed for strains tested. Anecdotally, treatment success of EFB with OTC may vary dependent on strain type present in the infection. Isolates of M. plutonius were tested for OTC resistance in vitro by using minimum inhibitory concentration (MIC) assays and several antibiotic resistant strains were identified. A complete understanding of all aspects of EFB, and the causative bacteria M. plutonius, is essential to improve treatment strategy, and therefore honey bee health, in the future.

Monitoring blanket bog condition and functions is critical in ensuring the ecosystem services they provide, especially as their environment becomes increasingly pressured. Microbial DNA-based monitoring tools allow the assessment of soil communities, but our knowledge of the microbial processes underpinning ecosystem service provision is limited. Specifically, (a) how microbial communities vary with management and climate, (b) how the microbial community is related to environmental variables, and (c) whether microbial community measurements can be used to predict water quality and carbon fluxes, including methane. Here, microbial community taxonomy and fungal community function was measured on UK blanket bog with different management, habitat, or restoration status using sequencing techniques, alongside a novel carbon partitioning mesocosm experiment. Water quality variables were measured alongside this experimental setup, along with a suite of environmental, chemical, and soil edaphic variables. Fungal, bacterial, and archaeal microbial communities changed with management and location. Microbial communities were highly variable but showed some categorisation based on climatic conditions. Communities subject to managed grouse moor burning were frequently different from every other habitat category. Concentrations of zinc, magnesium, and calcium were strongly associated with microbial community structure, alongside soil temperature, rainfall, and the abundance of Calluna vulgaris. Blanket bogs with a legacy of pollution contained specifically adapted microbial taxa. Grouse moor burning was linked to changes in fungal trophic groups, with potential consequences for water quality. Changes to the fungal and bacterial communities, alongside the abundance of sedges, were linked to changes in methane flux, and fungal community change was linked to soil respiration. The findings here indicate that ecosystem services can be linked to management and habitat status via microbial processes, but that our knowledge remains incomplete. This thesis provides the basis for further in-depth experiments and recommends focusing on a functional, rather than a taxonomy, based approach.

Intensive conventional agricultural practices, including conventional tillage and short rotations of annual crops, degrades soil organic carbon (SOC) stores, harms soil organisms and degrades macroaggregates important for soil structure and vital ecosystem services. More sustainable regenerative farming practices, for example reintroducing grass-clover leys into arable rotations, need to be adopted urgently to reduce non-renewable inputs and disturbance, increase organic matter inputs, and allow beneficial soil organisms to recover. The main aims of this thesis were to elucidate the mechanistic basis of soil changes caused by grass-clover leys in arable rotations by characterising changes in soil microbial communities, metabolomes and their impacts on soil aggregation and C storage. Introducing a three-year ley into conventionally managed arable fields increased proportions of soil macroaggregates (>2000 μm) by 5.4-fold, achieving similar proportions as the undisturbed adjacent hedgerow. Bulk SOC rose from 20.3 to 22.6 Mg ha-1 (0.77 Mg C ha-1 yr-1) but was not statistically significant. Macroaggregate-associated and protected OC increased from 2.0 Mg ha-1 to 9.6 Mg ha-1. Simultaneously, fungal communities significantly changed under ley, with increased prevalence and diversity in the Ascomycota and Glomeromycota phyla, but no significant changes identified in soil bacteria. Biosurfactants, lignans and flavonoids were upregulated in the soil metabolome, possibly associated with aggregate stability. Finally, a one-year earthworm manipulation experiment revealed that soil macroaggregates, and associated OC and N, all increase with increased earthworm numbers, being particularly driven by endogeics. This thesis affirms the benefits of grass-clover leys in arable rotations, evidencing their involvement in improving soil structure and quality associated with changes to the soil metabolome, microbiome and earthworm populations. These results provide justification for the rewarding of farmers for including leys in arable rotations for the biological, chemical and physical benefits to soil health which are in the interest of society.

Potassium (K) is essential for plant growth and health, featuring in a vast array of activities within the plant. Deficiencies in K nutrition cause serious issues regarding plant growth and crop yield. Polyhalite (K2SO4.MgSO4.2CaSO4.2H2O) is a hydrated evaporate mineral which can be used directly as a K fertiliser. However, there has been limited work on the suitability and efficacy of polyhalite compared to traditionally used K fertilisers such as potassium chloride (KCl) and potassium sulphate (K2SO4). The aims of this thesis were to explore the efficacy of polyhalite as a K fertiliser compared to KCl and K2SO4 and evaluate its effect on rice growth traits and nutrient content in both balanced and unbalanced fertiliser regimes. No differences in rice biomass were discovered between different K fertiliser regimes. However nutrient analyses showed inconsistent results to the different K fertilisers. A range of rice cultivars was then assessed for their response to different K fertiliser regimes. No significant variation between individual cultivars was observed in response to different K fertiliser sources with respect to biomass or nutrient content. Additionally, grouping cultivars by potassium use efficiency provided few changes in nutrient content responses. Finally, microbial communities in the plant rhizosphere were characterised for taxonomic diversity to determine if and how these properties are affected by different K fertilisers. In nutritionally balanced fertiliser regimes, the bacterial populations of the rice plant rhizosphere were not influenced by treatment although harvesting timepoint did affect their composition. Conversely, comparison of unbalanced K regimes revealed differences in bacterial species composition, but little change was observed in the total number or distribution of species of the bacterial populations. The findings of this work can be used to inform similar investigations using polyhalite as a K fertiliser as well as future work investigating K fertiliser effects on microbial populations.

Rhizobia are agriculturally important bacteria capable of forming symbiosis with legumes and fixing atmospheric nitrogen which sustainably improves plant productivity and soil fertility. The Rhizobium leguminosarum species complex is highly genetically diverse and contains five genetically distinct genospecies. Significant phenotypic diversity is also displayed within Rhizobium leguminosarum; however, no phenotypes are genospecies-exclusive. The importance of the broad genetic diversity of Rhizobium leguminosarum and its influence on phenotypic diversity and rhizosphere-associated interactions are unclear. In this thesis, Rhizobium leguminosarum symbiovar trifolii (Rlt) intraspecies diversity was investigated by assessing the genetic and phenotypic variation of white clover nodule Rlt from agricultural field managements across Europe. This thesis identified that the significant genetic diversity of Rlt can manifest in substantial transcriptional and phenotypic variation across strains, and this diversity can influence plant-mediated symbiont selectivity and competitive strain interactions. A novel multiplexed high-throughput amplicon sequencing approach, MAUI-seq, was developed to improve detection of chimeras and other erroneous sequences for confident determination of intraspecies diversity from environmental samples. Using this method, significant Rlt nodule population diversity was identified between clover genotypes due to the combined effects of plant-host filtering and geospatial variation in allele frequencies of individual genes. Investigation of multiple Rlt strain transcriptomes demonstrated that genospecies displayed differences in core genome expression which was associated with phenotypic growth traits and putative differences in bacterial metabolism. Genomic and transcriptomic variation was utilised to identify transcriptional units conserved across strains. Pairwise growth competition experiments between Rlt strains further showed that significant competitive variation is evident and potentially associated with genospecies differences. This research demonstrates that utilising multiple strains can aid identification of species-specific traits by considering the representative variation within a species. The work presented here has laid the groundwork for future investigation into the implications of intraspecies diversity for symbiotic effectiveness in the rhizobia-legume symbiosis.

Intensive conventional agriculture allows us to increase crop yields in line with global demand, but it puts future yields and food security at a disadvantage due to the increased vulnerability to changes in the environment that intensive systems are less able to withstand. Arbuscular mycorrhizal fungi (AMF) are a key target for increasing agricultural sustainability as they moderate aspects of ecosystem functioning including plant productivity, nutrient cycling, soil structural maintenance, water relations and pathogen regulation. Restoring AMF functioning could be of importance to restoring degraded soil properties to confer climate change resistance and resilience. The main aims of this thesis were (a) to investigate how various agricultural management practices impact the community composition and diversity of AMF, and (b) to explore the causal pathways of AMF diversity and community composition in conferring benefits to soil health and functioning through crop yields and water stress mitigation. Overall, the thesis considered AMF communities against whole-community functional phenotypes and soil health under contrasting agricultural management practices. This included AMF inoculation of soils, the inclusion of grass-clover leys in arable rotations and contrasting tillage intensities. The thesis increases our knowledge of the link between management practices, soil health and crop yields that can be used to inform management choices in real-world agricultural situations. This is particularly important for grass-clover leys which are a relatively understudied management practice. From the findings of this thesis, it is recommended that future studies employ a reductionist approach to assessing AMF function under variable situations using a combination of targeted mechanistic experiments and larger holistic experiments. These experiments would assess individual AMF functions under different environmental contexts that will provide a trait-based framework of individual function and compliment them with larger scale community experiments that can expand upon the mechanistic knowledge gathered to begin to predict community-level function.

Over recent decades salt marshes have been restored as a cost-effective response to coastal biodiversity loss and flood management. Previous research on established sites has demonstrated that restored marshes have significantly different sediment properties than natural marshes which appears to broadly impact ecosystem function. A study was conducted to examine the differences in natural and realigned salt marshes in terms of plant biodiversity, sediment characteristics (bulk density, water content, pH and nutrients) and microbial communities in Colchester Essex, UK. We studied three pairs of natural and realigned salt marshes of different ages, 13, 62, 118 years since breaching of the sea wall. Furthermore, we studied monthly changes of sediment characteristics of a newly realigned site from breach to 14 months of tidal inundation. In addition to monthly sediment changes, we placed invertebrate exclusion chambers in the newly realigned marsh to examine the effect of bioturbation in changing sediment characteristics and microbial communities. Sediment characteristics of our realigned marshes were significantly different than those of the natural marshes. In addition, natural marshes displayed higher variability and heterogeneity in nutrient and water content than our realigned marshes. Within our newly realigned salt marsh we observed that despite the different starting sediment characteristics, 14 months after inundation our realigned site was broadly similar to the natural marsh but only on the top 5cm of sediment, indicating the presence of a relic agricultural layer which can affect the hydrology and development of the system. Macro-invertebrate colonization has shown that it can influence the geochemical characteristic and microbial communities of sediment in a newly realigned salt marsh. Microbial communities' composition and abundances within a newly realigned marsh are significantly different from natural marshes 14 months post inundation.

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.

Metaldehyde is a molluscicide that is used to prevent crop damage by slugs and snails. It is frequently found in drinking water sources at concentrations above the legal maximum, and causes >90% of water quality failures in England. There is currently no economical, widely used treatment to remove metaldehyde pollution from water. Prior to the work presented here, it was known that metaldehyde is degraded biotically in soil, but the nature and identity of organisms responsible was unknown. Two novel metaldehyde degrading bacterial strains were isolated from domestic soil that can utilise metaldehyde as a sole carbon and energy source; Acinetobacter calcoaceticus E1 and Variovorax E3. Evidence that acetaldehyde is the primary metabolite of metaldehyde is presented: the metaldehyde dependent expression of an aldehyde dehydrogenase in A. calcoaceticus E1 was found, and the maximum metabolic flux of acetaldehyde was found to be greater than that of metaldehyde in cells grown using metaldehyde. The genomes of the isolated organisms were acquired. Comparative genomics of the two isolates showed that their novel catalytic ability was not dependent on genes shared between them, despite being isolated from the same soil sample. Comparison of the A. calcoaceticus E1 genome against other Acinetobacter that cannot utilise metaldehyde yielded a strong candidate for the primary metaldehyde degrading enzyme that has characteristics consistent with an enzyme that catalysed ether hydrolysis of isochorismate in its ancestral form and has evolved to hydrolyse ether bonds in metaldehyde.

Environmental change will have significant impacts on microbial ecosystems. Microorganisms dominate most biogeochemical pathways, and environmental perturbations may alter these functions. Such functions include nutrient cycling, pollution abatement and greenhouse gas emission, and it is paramount that the impact of environmental change on ecosystems is understood. High throughput DNA sequencing provides a window into complex microbial communities and their functional potential, thus allowing us to empirically study how such communities respond to predicted future environments. There are, however, caveats and challenges associated with such technologies, particularly with converting billions of sequencing base calls into species and function counts. This thesis firstly quantifies the performances of sequence annotation tools and parameters using a simulated metagenome. It is found that tools differ in performance, and that parameter selection can significantly reduce annotation accuracy e.g. One Codex correctly annotated many sequences at the genus level, whereas MG-RAST RefSeq produced many false positive annotations. The results provide a guideline to quantitatively inform researchers about the impacts of certain choices on annotation performance, and show that some published studies may be drawing incorrect conclusions. This thesis also investigates the impacts of increased flooding frequency and duration on soil microbial ecosystems, in line with predicted climate change. Increased frequency has significant impacts on biodiversity, community composition and potential function. SkyLine, a novel, continuous gas flux measuring system, was used to record CO2 and CH4 fluxes. Increased flooding duration significantly reduced CH4 oxidation and increased CO2 assimilation, with the combined global warming potential of these gasses reduced.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a damaging disease of honey bees (Apis mellifera) with a global distribution. AFB infects the honey bee larvae through spore-contaminated larval food and once infected the larvae will die within twelve days. AFB infection leads to the eventual death of the honey bee colony. In many countries the best control method is thought to be the destruction of the colony, to destroy the hardy, infectious spores. Therefore infection nearly always leads to the death of the colony. In the UK cases of the disease have decreased in recent years due to statutory control methods, however sporadic outbreaks occur each year. Many advances have been made in our knowledge of the mechanisms of infection by P. larvae in recent years however, a high resolution, standardised method of strain typing is required to track disease spread and understand outbreak sources. This thesis describes the development of the first multilocus sequence typing (MLST) scheme for P. larvae. MLST is the gold standard for pathogenic bacteria typing. It is based on the sequencing of sections of 6-10 housekeeping genes. MLST is standardised, as primer and allele sequences can be made available for other researchers. The new MLST scheme was used to describe previously undetectable patterns of distribution at a global level as well as at a national level. Humans and bees are implicated in the movement of the disease over different spatial scales. Using the MLST scheme a diverse group of isolates were selected for whole genome sequencing. For the first time multiple genomes were compared within and between genotypes. The ability of the MLST scheme to describe relationships amongst sequence types (STs) was tested by comparing phylogenies based on core genes and MLST sequences. I describe the discovery of seven plasmids in four STs previously unknown to harbour plasmids.

The detection and control of American Foulbrood (AFB) is made more challenging due to a lack of evidence regarding the virulence mechanisms employed by this honeybee pathogen. Whilst incidence of this pathogen within the UK has recently declined, ~100 colonies were identified as infected with AFB in 2011 (to end of September), so AFB should still be considered a serious threat to honeybee health. It is known that within the species many phenotypes exist, and the infection caused by the phenotypes differs greatly. This PhD thesis presents several advances towards a greater understanding of the intra-specific differences occurring within the species. Chapter 2 evaluates the use of 16S rRNA sequencing as a method of Paenibacillus larvae identification, as well as exploring the use of this ribosomal subunit for differentiation of the species. The sequencing of two housekeeping (purH and PyrE) genes assesses the potential of a Multi Locus Sequence Typing (MLST) method, as a means of subspecies differentiation. Chapter 3 assesses what can be inferred from use of Enterobacterial Repetitive Intergenic Consensus (ERIC) sequence fingerprinting, with regards to prior knowledge genetic differences. Lateral Flow Devices (LFDs), a commonly used diagnostic tool, are tested to ensure P. larvae isolates representing all 4 ERIC types are detected. In Chapter 4 an in-vitro honeybee rearing method is employed to observe the correlation between proteolytic activity of isolates and in-vitro virulence. The method is applied to a wider range of reference isolates, to observe the intra-species differences existing. The ability to produce large numbers of viable spores is explored as a potential difference existing between ERIC types I and IV. Whole genome shotgun sequencing is used in chapter 5 to perform comparative genomic studies on 4 P. larvae isolates also utilising 646 contigs from a previous sequencing project. The possible presence of plasmid DNA is explored, through GC content analysis. The genetic basis of a sporulation phenotypic difference is examined by BLAST analysis of orthologous genes. In Chapter 6 the findings of this thesis are discussed in more detail, and potential areas of further study are identified.