The primary aim of this project was to identify and characterise Fusarium species associated with the basal rot of Allium species and internal fruit rot of sweet peppers in the UK. The secondary objective was to develop quick molecular markers to identify Fusarium oxysporum f. sp. cepae (FOC) causing onion basal rot. Isolates representing diverse Fusarium species taken from onions, garlic, shallot and leeks obtained from different production and processing sites in the UK were collected. F. proliferatum was found for the first time to be a causal agent of onion basal rot in the UK, but F. oxysporum was by far the most common species and F. oxysporum isolates belonged to at least two different genotypes based on a sequence comparison of several “housekeeping” genes, and overall, appeared to be polyphyletic. None of the housekeeping genes studied correlate with pathogenicity. Secreted in xylem (SIX) genes offer more promise for the specific identification of F. oxysporum formae speciales (Lievens et al., 2009a) and a homologue of the SIX7 gene was found only in a few FOC isolates suggesting that SIX7 is not absolutely necessary for pathogenicity. Whole genome sequencing of a FOC isolate was carried out in order to understand pathogenicity and identify novel effector genes. This work revealed the presence of further homologues of published SIX genes, namely SIX3, SIX5 and SIX9. The presence of SIX3 and SIX5 has only been reported from F. oxysporum f. sp. lycopersici previously. Additionally, screening of eleven new candidate effector genes suggested that FOC isolates have different gene sets which correspond to the continuous variation of aggressiveness found within the FOC population. Fusarium lactis, F. proliferatum and F. solani were identified in association with internal fruit rot of sweet pepper obtained from three different production sites in the UK.

The use of crop protection products (CPPs) is essential to improve crop production levels and feed the rising global population. Safety testing of CPPs is also essential to ensure that their use does not adversely affect human health or the environment. Currently, laboratory-based studies typically over-estimate the environmental persistence of CPPs in the environment. The inclusion of environmental variables that are currently omitted from laboratory studies, such as non-UV light, could reduce the disparity between laboratory and field degradation studies. The inclusion of light resulted in a significant reduction in extractable parent compound for benzovindiflupyr, chlorotoluron, prometryn, imidacloprid, and fludioxonil compared to dark conditions. In contrast, a significantly slower rate of cinosulfuron transformation was observed under light compared to dark conditions. In a separate experiment, terminal restriction fragment length polymorphism showed that under light conditions, the soil surface (0-3 mm) harboured distinct phototroph, bacterial and fungal communities compared to the underlying bulk soil (3-12 mm), or dark incubated soil. 454 pyrosequencing revealed that light selected for diazotrophs at the soil surface, including Nostoc punctiforme, in addition to heterotrophic bacteria, particularly within the phylum Firmicutes. Metatranscriptomic analysis showed that light also selected for actively transcribing photosynthetic eukaryotes at the soil surface, such as Chlorophyceae and Saccharomycetes, in addition to heterotrophs, such as Nostocaceae. Finally, light selected for major soil functions such as photosynthesis, and reduced the alpha and beta diversity of predicted protein coding regions at the soil surface. This work has important implications for CPP regulatory studies and soil surface management practices.

Post harvest discolouration in lettuce is an increasingly important problem due to the shift in the market for prepacked processed salads. Variation in post harvest discolouration was recorded in a lettuce diversity set of 28 accessions representative of the lettuce genepool. The parents of the WHRI lettuce mapping population, Saladin and Iceberg were included in the diversity set. They showed significantly different responses for discolouration and the difference between them was representative of a major part of the variation seen in the diversity set. F7 RILs derived from a cross between Saladin and Iceberg were suitable for genetic analysis of post harvest discolouration. As a precursor to the genetic analysis, a good quality linkage map based on the F7 Sal x Ice population was generated. Significant genetic variation in the post harvest response was demonstrated for these RILs. Twenty-one significant QTL were identified for post harvest discolouration traits, and the markers linked to the QTL can be used for marker assisted selection. Significant but weak correlations were recorded between discolouration and important agronomic traits, however as these were not highly correlated this means that post harvest discolouration and agronomic traits can generally be independently selected for by breeders without having to compromise on other traits. Research was also initiated to understand the metabolic changes underlying the phenotype change. Significant variation in levels of metabolites related to post harvest discolouration including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and total phenolic content (TPC) was observed in RILs with extreme phenotypes. The differences in metabolite levels were significantly correlated with the discolouration phenotypes. Work was also initiated to identify candidate genes associated with the biosynthetic pathway responsible for discolouration (phenylpropanoid pathway) in an attempt to identify genes underlying QTL. Twenty-three genes have been placed on the Sal x Ice map using comparative genomic approaches. Some of these co-locate within the region of a discolouration QTL and are therefore candidate genes for the QTL effect. Mutants with altered post harvest discolouration phenotypes were also confirmed in this study; however the point of mutation could not be identified although it is thought to be downstream of PPO. This indicates that a desired phenotype with reduced levels of post harvest discolouration can be achieved by two approaches to breeding; using natural and induced variation. This study has provided the tools and knowledge to do this.

Predicted climate change and increasing global populations suggest that water will become an increasingly scarce and valuable commodity. Breeding plants which produce equivalent yields with reduced water input is therefore vital to sustain and increase crop production in the future. The phytohormone, abscisic acid (ABA), is important in controlling plant responses to water stress. It may be possible to improve water use efficiency (WUE) by genetically modifying tomato and other species to maintain elevated levels of ABA under optimal (unstressed) conditions, thereby manipulating an intrinsic signalling mechanism which is known to mediate drought-induced alterations of stomatal behaviour. ABA is synthesised via the oxidative cleavage of C40 epoxycarotenoid precursors, a reaction catalysed by the key enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). Pure breeding transgenic tomato lines constitutively over-expressing LeNCED1, known as sp5 and sp12, both have elevated ABA concentrations, which reduce stomatal conductance under optimal (unstressed) growth conditions, thus conserving soil water during periods when corresponding wild type (WT) control plants were inefficient in its use. Under well-watered conditions, whole plant transpiration efficiency (TEp) was significantly greater in both 'high ABA' lines (sp12 and sp5) than in WT plants. The over-expression of LeNCED1 was combined with over-expression of a gene (LeBCH2) encoding β-carotene hydroxylase (BCH), an enzyme acting earlier in the ABA biosynthetic pathway. These 'double transgene' lines (G28 and G29) consistently exhibited further improvements in ABA accumulation and TEp relative to corresponding 'single transgene' parental lines. Lines G28 and G29 respectively exhibited 37 and 54 % improvements in TEp relative to WT controls. When evaluated as a potential 'high ABA' rootstock, it was found that the 'double transgene' G29 line did not provide a sufficiently strong root-sourced signal to affect the stomatal behaviour of scions. To increase ABA biosynthesis in the roots further, a programme designed to combine the over-expression of three ABA biosynthetic genes (LeNCED1; LeBCH2; LePSY1) was initiated with the objective of obtaining a rootstock which produced sufficient ABA to affect stomatal behaviour when grafted onto WT scions. Unfortunately, there was insufficient time to complete this work by the end of the period reported in this thesis, although the programme is ongoing.

The time of bolting is an important factor in lettuce production because it affects the yield and quality of the harvested crop. Bolting is promoted by higher temperatures and is an increasing problem for growers with the current trend for warmer summers. Lettuce plants that are in the early stages of bolting are visibly indistinguishable from non-bolting plants, however there are changes in the biosynthesis of secondary metabolites which are produced to protect the young floral bud from insect attack. These compounds give the lettuce plant a bitter taste and render the crop unsaleable. The development of late bolting varieties, which would have a greater ‘holding ability’ in the field, would result in reduced crop losses and an extension to the growing season. In many plants, the timing of the transition from vegetative growth to flowering is controlled by environmental cues which serve to communicate growth conditions favourable for sexual reproduction and seed maturation. Studies in Arabidopsis have led to the identification of several different pathways that come together to regulate flowering time. Little research has been done on these response pathways in lettuce, however, research has shown that components of these pathways are conserved between Arabidopsis and other crop species. The aim of this project is to isolate genes regulating flowering time in lettuce in order that novel alleles of these genes can be used to manipulate bolting time. A lettuce BAC library has been screened and homologues of eight Arabidopsis flowering time genes, principally from the autonomous pathway, have been isolated. Functional orthologues of FLOWERING LOCUS T (FT) and the autonomous pathway gene, FLK have been characterised in lettuce, suggesting that there is conservation of the genes involved in flowering time in Arabidopsis and lettuce. Lettuce lines with a range of bolting times, including lines which bolt significantly later than wild-type have been identified from EMS mutagenised populations of cultivated lettuce and a diversity set of wild lettuce. Homozygous lines from a Lactuca sativa cv. Larissa EMS population with a reproducible late bolting phenotype when tested under commercial growing conditions have been identified. These lines have been made available to Rijk Zwaan® for inclusion in future breeding programs aimed at delaying bolting and improving the ‘holding’ ability’ of commercial lettuce crops. Genomic sequence of selected lettuce flowering time genes have been compared between the late bolting lines and wild-type looking for polymorphisms that may account for the late bolting phenotype. Polymorphisms within these genes were identified in some of the late bolting lines, however through analysing the polymorphism in segregating backcross populations they have been shown not to be causing the late bolting phenotype. Transcriptome sequencing has also been performed to identify polymorphisms in other, possibly novel, genes which may be causing the late bolting phenotype, as yet, no mutation segregating within the late bolting lines has been identified.

The rhizosphere has a rich fungal microbiome, including parasites, commensals and mutualists. An important group in the rhizosphere are assumed to be the arbuscular mycorrhizal fungi (AMF), which live in symbiosis with around 80% of plant species. AMF have been shown to increase plant yield, biomass, disease resistance, and shoot P. Plants exchange carbon in the form of sugars for nutrients assimilated by AMF. There is little known about AMF in association with Miscanthus giganteus, a productive bioenergy crop grown in the UK and abroad. Work was carried out to characterise the abundance, organisation, importance, function and stability over space and time of rhizosphere fungi and AMF in M. giganteus roots. Field samples from Lincolnshire were analysed using staining and molecular techniques, including small subunit rRNA gene terminal restriction fragment length polymorphism, clone libraries and amplicon pyrosequencing, and meta-transcriptomics. M. giganteus was also grown in a number of pot experiments, with various treatments including fungal inoculations and fungicide application. A number of fungal phyla were found in the roots, particularly Ascomycota, the composition of which shifted over time and exhibited diurnal patterns of activity. Fungi enhanced plant growth by a third, and were functionally active in the roots in the meta-transcriptome. AMF communities were found at much lower relative abundances in roots, and inoculation with AMF did not enhance M. giganteus growth. The work highlights the importance of the whole root mycobiome to plant growth and health, and the relatively small role Glomeromycota play in M. giganteus comparison with other fungi. The work also demonstrated the dynamic nature of fungal activity over hours, months, and years, and the complex interactions the fungal community has with environmental variables.

The oomycete Hyaloperonospora arabidopsidis (Hpa) is the causal agent of downy mildew of Arabidopsis thaliana; a system that can be used as a model for the study of plant-pathogen interactions. In order for successful colonisation, biotrophic pathogens such as Hpa suppress or evade plant defences through secretion of effector proteins into the plant to manipulate and disrupt the host immune system. Alignment of oomycete effector proteins has revealed a conserved amino acid sequence at the N-terminus with the consensus sequence RxLR (arginine, any, leucine, arginine), thus allowing the use of Bioinformatic approaches to identify putative effector proteins in the Hpa genome. Studying effector action and their targets in the host may help elucidate important components of the plant defence response, eventually leading to more durable crops. Expression of the Hpa effector HaRxL21 in planta has been shown to alter host susceptibility to Hpa, Botrytis cinerea and Pseudomonas syringae. Here the interaction targets of HaRxL21 are presented and interaction with the transcriptional co-repressor TOPLESS (TPL) has been validated in planta using BIFC and Co-IP. Using deletion and mutation analysis, the specificity of the interacting protein domains has been identified as between the CTLH domain of TPL and Leucine residues within the EAR motif of HaRxL21. Microarrays have revealed effects of HaRxL21 on host transcription, particularly up regulation of genes involved in ABA signalling and a decreased induction of SA responsive genes upon SA induction. Finally, work has been carried out to determine the biochemical function of HaRxL21, showing an increased stability of TPL in the presence of this effector.

A collection of heritage variety accessions were characterised using Amplified Fragment length Polymorphisms (AFLPs) (200 accessions ) and multivariate analysis of morphological characters (366 accessions); key features of interest for the conservation of Plant Genetic Resources were the identification of diversity within and between accessions. Motivations and practices of heritage variety growers were explored using questionnaires. Heritage varieties are herein defined as traditional crop varieties that have a historical origin of over 40 years, are non-hybrid and non-GMO and are of cultural/heritage value to their users; they are part of the suite of plant genetic resources currently utilised by growers and of potential use to plant breeders in the future. A large range of morphological and genetic diversity was present between accessions in all crops; in addition, diversity was found within accessions, particularly in Vicia faba, Daucus carota and Cucumis sativum. Comparisons between data sets were made for diversity, relationships, comparisons with commercial standards and identifying potential duplicates. The synthesis of both data sets highlighted the three potential duplicates for further investigation by HSL (all in Pisum sativum). The findings highlight the importance of heritage varieties and the Heritage Seed Library, both culturally and in terms of conservation for present and future use.

Perennial ryegrass (Lolium perenne L.) is one of the most economically and environmentally important grass species for the temperate zone. It maintains effective self-incompatibility (SI), which promotes outbreeding as well as limits the efficient production of inbred lines and hybrids. SI in L. perenne is controlled by the S and Z loci, mapping to linkage groups 1 and 2, respectively. None of the gene products has been identified so far. Comparative mapping has identified regions on rice chromosomes 5 (R5) and 4 with synteny to regions of L. perenne genome containing the S and Z loci, respectively. Markers were developed from the syntenic rice genomic region to refine the S and Z maps. The closest flanking markers had a map distance of 2 cM from S and 0.2 cM from Z. SI cDNA libraries were developed from in-vitro pollinated stigma subtracted with unpollinated stigma to identify SI components and SI response related genes. Through a BLAST search, candidates identified from the SI libraries that were orthologous to sequences on the S and Z flanking regions on rice R4 and R5 were the prime candidate SI genes. Altogether ten SI candidate genes were identified with incompatible response associated differentially expression pattern: a rapid increase in expression within two minutes after pollen-stigma contact and reaching a maximum between 2-10 minutes, implying their roles in the SI response. Attempts were carried out to determine the linkage relationships between the identified candidates and the S or Z loci. Large fine scale mapping populations were developed individually for the S and Z loci to generate high resolution maps of S and Z towards map-based cloning. Tightly linked markers were identified mapping at a distance of 1.4 cM from S and 0.9 cM from Z. The studies performed in this project have implications on both the underlying genetic control and the associated biochemical responses involved in L. perenne SI. The closely linked markers for S or Z could be applied in future marker assisted selection breeding programmes and map-based cloning.

Clubroot disease, caused by the soil borne microorganism, Plasmodiophora brassicae is a significant disease of cruciferous crops as it results in stunted plant growth and reduced yield. The aim of this thesis was to investigate the effect of environmental factors on the viability of P. brassicae resting spores. Methods of detecting and quantifying the organism were optimised, and several different methodologies were investigated as potential bases for a viability assay. A molecular based seedling assay which quantified the rate of germination and subsequent root infection was determined to be an effective assay system. Investigation of several factors within a laboratory setting demonstrated that calcium stimulated the germination of resting spores, but subsequently reduced the level of infection in Brassica root tissue. Magnesium displayed the same effects but was not as significant as calcium. Ammonium displayed the same effects as calcium and magnesium however the level of ammonium applied was also significant. The commonly reported occurrence of lower incidence of clubroot disease at high pH was supported by finding less spore germination at high pH. Temperature was significant to P. brassicae spore germination and subsequent root infection, with the optimum temperature for germination and infection combined being 18.5-19oC. Low temperature (5oC) reduced spore germination but did not have a significant effect on the zoospores infective capability following germination. Prediction of environmental conditions within field settings was achieved using a simulation model specifically designed to incorporate parameters anticipated to influence P. brassicae viability. Monitoring the DNA equivalent levels of P. brassicae spores in soils over the course of crop growth allowed determination of predictive significant factors, and the results from these investigations supported the findings of the seedling assay. Calcium, magnesium, nitrogen and pH were among the factors significant to the level of P. brassicae spores within the soil.