Downy mildew of lettuce caused by Bremia lactucae is a serious disease resulting in yield loss. The population structure of the pathogen in the UK is poorly understood. This PhD project concentrated on developing molecular markers to differentiate the genotypic variation of B. lactucae populations, with the aim of improving methods to investigate lettuce - Bremia interactions. Thirty-seven B. lactucae isolates (including single-spore and new field isolates) were collected and characterized for virulence using the conventional International Bremia Evaluation Board (IBEB) differential set. Microsatellite markers (SSR, ISSR) were investigated for Bremia race specific marker development. Three isolates of B. lactucae were characterized by ISSR (inter simple sequence repeat) primers, although the polymorphic DNA could not be cloned in this project due to the highly variable results of the ISSR process. Some microsatellite repeats were found in B. lactucae isolates sequences that amplified by Plasmopara viticola (grape downy mildew) SSR markers. The development of Simple Sequence Repeat (SSR) markers from Bremia genomic DNA was not successful, which might result from the primers used being unsuitable for Bremia microsatellite enrichment. Bremia specific ITS primers were used for quantitative PCR. RxLR primers obtained from UC Davis (USA) were tested using the collection of B. lactucae isolates. RxLR1 primers distinguished between isolates BL801 and BL806. Eight SNPs were identified in three isolates amplified by RxLR5. No polymorphism was observed on the gel for the remaining RxLR primers on single spore races. Unrefined field isolates showed more polymorphisms on the gel than single spore isolates. The phenotypic differences between these two isolates have been identified by the IBEB differential set. Microscopy and qPCR quantification were used to investigate the compatible and incompatible interactions. The results suggest that BL801 is more virulent than BL806, as more infection structures were observed in IBEB resistant cultivars. Results of qPCR and spore count/unit weight of cotyledons showed that BL801 and BL806 were significantly different. The qPCR quantification results from 4 and 5 dpi were correlated with the spore count/unit weight of cotyledons. Although further work is required to develop race specific markers, the methods used in this project demonstrate the potential use of molecular markers to investigate lettuce - Bremia interactions.

The scientific case made by the AUTHOR’S comparative Brassica oleracea genomics work is presented through 5 peer reviewed research papers. In order to achieve a comprehensive understanding of the evolution of B. oleracea the identification of unique genome characteristics, established using comparative genomics, is required. The genome characteristics established within these papers deliver significant contributions to original knowledge. These include a detailed illustration of how macro scale synteny varies markedly between the B. oleracea and A. thaliana genomes; unambiguous integration of the B. oleracea cytogenetic and genetic linkage maps; a cross species characterisation of a large collinear inverted segmental duplication on a single B. oleracea chromosome establishing that the relative physical distances have stayed approximately the same; retrotransposon copy number estimations and characterisation of their genomic organisation and isolation, characterisation and cross species analysis of a C genome specific repeat. For each paper the AUTHOR’S individual scientific contribution to each aspect of the work is described in detail. Both individually and as a body of work these publications substantially advance the fields of comparative, Brassica and genomic research.

There is a consensus that the development of successful Integrated Pest Management strategies requires a detailed understanding of pest biology. In the case of the currant lettuce aphid (Nasonovia ribisnigri), an economically important pest aphid of lettuce, sources of such information are limited. This study considers key aspects of N. ribisnigri biology which influence its control. In particular, it makes comparisons between biotypes which succumb to (wild-type) or overcome (resistance-breaking), the host plant resistance (Nr-gene) in commercial lettuce cultivars. Experiments on the effects of temperature and photoperiod on the development of N. ribisnigri showed no differences between wild-type and resistance-breaking biotypes. At low temperatures (5, 10 and 15ºC), wild-type biotypes developed to adulthood on resistant cultivars, indicating that the Nr-gene is temperature sensitive. A linear regression between development rate and temperature estimated a lower developmental threshold of around 4.7ºC. Nasonovia ribisnigri usually overwinters as a diapausing egg but overwintering nymphs/adults have been observed. In the laboratory eggs were obtained at 12ºC 13L:11D. Sequential sampling of eggs from the field suggested that diapause ended between late January and early February. Post-diapause development was estimated to take <50 day-degrees using a LDT of 4.7°C. Nasonovia ribisnigri survived the winter as nymphs/adults on Veronica arvensis in the Midlands. Other weed species were suitable hosts in the labratory: Chichorium intybus, Crepis capillaris, Lapsana communis, Hieracium aurantiacum, Hieracium pilosella, Veronica spicata and Veronica officinalis. Field trials, using sequentially planted plots of lettuce, and applying 'exclusion' and pesticidal treatments indicated that natural enemies and emigration regulate aphid populations in the summer and contribute to the mid-summer crash. A large-scale screen of 96 cultivars and wild relatives of lettuce identified new sources of resistance against wild-type and resistance-breaking biotypes. Results from this study can be used to inform further development of an Integrated Pest Management strategy for this pest.

Amaranthus tuberculatus (Common Waterhemp) is a prevalent, problematic weed in Midwestern USA, where genetically-modified crops are widely grown, resulting in multiple annual applications of glyphosate. Such practices provide the selection pressure for the evolution of glyphosate resistance. Evolutionary theory predicts that adaptation to novel stresses, such as herbicide application, will incur a cost in the original (herbicide-free) environment. This project aims to identify whether glyphosate resistance in a population of Amaranthus tuberculatus was associated with a fitness cost in the absence of glyphosate. Initial dose response experiments on the study population determined a resistance index of 3.2 compared to a standard sensitive population. To generate appropriate material for comparisons between phenotypes, individual plants were cloned and their resistance status determined by glyphosate application. Parent plants were designated as resistant or susceptible and appropriate crosses were performed to generate seed lines. The incomplete segregation of resistant and susceptible seed lines indicated that resistance was controlled by a quantitative trait. EPSPS gene amplification was elucidated as the primary mechanism of glyphosate resistance. There was a strong positive relationship between half-sibling seed family LD50 and relative EPSPS gene copy number indicating that resistance was primarily caused by target-site gene amplification. Subsequent fitness experiments used seed families to determine that no fitness trade-off was associated with resistance (or gene copy number) when plants are grown without competition, in glasshouse and polytunnel growth environments. However, a small growth penalty was associated with elevated gene copy number in plants grown in a polytunnel, without competition (10% biomass reduction at +20 EPSPS copies), this led to competition experiments. A fitness cost was associated with resistance under intra-phenotypic competition; however, the cost was mitigated under inter-specific competition with maize. A cost was associated with glyphosate resistance and this cost was influenced by interactions with biotic and abiotic factors.

The effects of temperature, irradiance, supplementary UV-B and RlFR ratios on volatile oil compounds and plant growth parameters in basil plants have been determined. The base temperature for plant growth, the variation in chemical composition between leaves, the variation in chemical composition during the day and the effect of storage at 4°C for 24 h were also determined. Basil is a warm climate plant and its base temperature for growth is 10.9°C. The optimum temperature for plant growth is 25°C and this temperature also enhances the volatile oil content in leaves. Plants grown at 25°C for two weeks were taller and possessed more dry matter and larger leaves than plants grown at other temperatures. The total volatile oil content in fresh leaves was three times the level compared with plants at lSoC. Temperature also affected the composition of volatile oils. Warm conditions resulted in the accumulation of eugenol and cis-ocimene. whereas cool temperatures resulted in more camphor and trans-p-farnesene. There was no effect, however, on the relative contents of 1,8-cineole and linalool. Treatments with alternating temperature that supplied the same accumulated day degrees (ADD), but with a different sequence of temperatures, did not affect most of the plant growth parameters, however, volatile oil content and composition were strongly affected by the temperature regime of the final two weeks. The higher the temperature before harvesting, the higher the volatile oil content and the relative content of eugenol produced. Basil plants grow well in full sun, however they can tolerate light shade. Heavy shade (75% and 50% shade) resulted in small plants with reduced dry matter, and the volatile oil content in fresh leaves was five times lower than in control plants. Heavy shade significantly increased the content of methyl eugenol, but strongly decreased the contents of eugenol and Iinalool. Two weeks treatment with supplementary UV-B (ultraviolet - B) light resulted in short plants with higher dry matter and thicker leaves. It also stimulated the synthesis of volatile oil compounds, i.e. phenyl-propanoids (eugenol) and terpenoids (notably 1,8-cineole and linalool). There was no effect, however, on volatile oil composition. Use of supplementary light to produce a high RlFR (red / far-red light) ratio resulted in shorter plants with less dry matter and smaller leaves. But the volatile oil content of the leaves was greatly increased. The content of eugenol was decreased whereas the content of ~-myrcene was increased. There were no effects on the relative contents of 1,8-cineole, linalool and other compounds. There were no differences in the volatile oil content and composition of fresh basil leaves harvested during the daytime, i.e. between morning (9.00am) and late afternoon (5.00pm). After storage for 24 h at 4°C in dark conditions, there were no differences in volatile oil content and composition in fresh leaves. There was a great difference, however, in the content and composition of volatile oils between young and mature leaves. The sensory analysis showed that trained panellists could perceive different intensities of volatile oils and consumers preferred the stronger intensity of volatile oils in fresh basil leaves.

Tomato is an economically important crop that can be devastated by many root infecting pathogens. The development of alternative and sustainable crop cultivation techniques and disease control methods is a must for the tomato industry, due to more strict government regulations and concerns over the sustainability of conventional chemical-intensive agriculture (Dixon and Margerison, 2009). In this thesis, the molecular fingerprinting method Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and next generation sequencing method (pyrosequencing) were used, targeting ITS1, ITS2 and 23S ribosomal DNA to characterize and examine microbial community assemblages in the rhizosphere of tomato. These molecular techniques were employed alongside traditional cultivation, microscopy and plant health assessment techniques to determine the effects of growth media, plant age and disease control methods on rhizosphere microbial populations and tomato root health. Plant age and media were found to significantly affect microbial community assemblages; conversely, microbial populations were not altered by soil amendments or rootstock disease control measures used. These findings suggest that the factors influencing rhizosphere community structure can be ranked by importance. Furthermore, if the most influential factors are kept consistent then rhizosphere microbial structures are robust and difficult to perturb with changes in a factor contributing less control over microbial community composition. No direct link between crop health assessments and rhizosphere microbial community diversity or presence of root pathogens could be established. Furthermore, high abundance of potential pathogens and poor crop health assessments during the growing season did not always result in poor health or disease symptoms at the end of cropping assessment in our trials. These results imply that many factors control the rhizosphere competence and ecological role of different species, ultimately affecting the outcome of disease. As no known methods are capable of efficiently assessing the fate of total microorganisms in the rhizosphere over time and space, this study could be considered as part the ‘descriptive phase’ in this field (Kent and Triplett, 2002). Pyrosequencing increased the resolution and confidence of rDNA analysis compared to T-RFLP, identifying organism within samples to a genus and often species level. Advances in next generation sequencing and analytical tools and pipelines associated with this analysis are likely to develop as these methods become common practice. With this in mind, next generation sequencing represents the future approach for resolving complex microbial communities in environmental samples.

The brown planthopper, Nilaparvata lugens, is a pest of rice in tropical regions. Its direct feeding results in loss in yield and plant death ("hopper bum"). Several compounds that stimulate insect attraction have been detected in rice plants colonised by N. lugens, including 1,2-dimethoxybenzene or veratrole. Electro-physiological studies and highresolution gas chromatography have identified veratrole as an attractant of N. lugens. Veratrole is a product of salicylic acid, a derivative of the phenyl propanoid pathway. Salicylic acid is decarboxylated to catechol, a step which is encoded by salicylate hydroxylase. Catechol is subsequently methylated to veratrole, which is released as a volatile compound from rice leaves. Mature scutellum-derived rice calli from (Oryza sativa) cv.Taipei 309 were transformed, using microprojectile bombardment, with pROB5 containing the hpt gene conferring resistance to the antibiotic hygromycin and pSLJ7307 carrying the nahG gene derived from Pseudomonas putida and coding for the enzyme salicylate hydroxylase. Following selection on hygromycin-containing medium, 17 independent transgenic rice plants were regenerated from >3600 bombarded calli, with a transformation frequency of 0.47%. Transgenic plants were confirmed by RT-PCR. Plant lines were classified as high expressors (10 lines) and low expressors (7 lines) depending on salicylate hydroxylase production. All transgenic lines exhibited higher enzyme activity than wild-type plants. Transgenic plants produced had altered metabolism for antioxidant enzymes such as catalase, ascorbate peroxidase and superoxide dismutase and reactive oxygen species such as hydrogen peroxide. Plants unable to accumulate salicylic acid exhibited delayed transcription of pathogenesis related genes and may therefore be compromised in their ability to respond to pathogen attack and mechanical wounding. Enhanced veratrole production was corroborated using gas chromatography of volatiles released from transgenic undamaged and mechanically damaged plants. Bioassays indicated that N. lugens were more attracted to high expressing plants than to wild-type plants, making more visits to areas containing transgenic rice leaves than areas containing non-transformed leaves and spending longer in these areas. Manipulating the production of veratrole by enhancing salicylate hydroxylase activity has therefore modified attraction of the N. lugens for high expressing nahG positive rice plants.

The production of new rose cultivars by sexual crossing is problematic and time consuming due to sexual incompatibility. the failure of seeds to genninate. and to a limited gene pool. Biotechnology provides an obvious alternative for the creation of genetic novelty in rose. This thesis focuses on the development of novel approaches, based on embryo rescue, pollen cryopreservation, protoplast and transformation technologies. A reproducible embryo rescue technique was developed in which embryos were excised and genninated on agar solidified medium containing a basic salt mixture and carbohydrate. The choice of carbohydrate and the growth conditions employed were demonstrated to markedly affect the percentage germination and subsequent plantlet development. This technique was used to greatly increase the production of F, hybrid progeny when compared to conventional germination methods. The failure of sexual crosses between several English rose cultivars was shown to be due to a combination of low pollen viability and to the operation of a pollen-style incompatibility mechanism (probably of the gametophytic self-incompatibility type). Degree of flower opening and method of pollen dehiscence were shown to significantly affect pollen viability. A technique was developed for the effective cryopreservation of English rose pollen. Using this technique it was possible to store pollen at ultra-low temperatures without any significant loss in viability. Such a technique compared favourably with conventional techniques (refrigeration and freezing) in which a loss in viability over time was demonstrated to occur. In vitro shoot cultures of English rose were established on MS-based media containing BAP. GA3 and NAA following the treatment of explants with an antioxidant solution to negate the effects of phenolic oxidation. The production of callus was shown to be genotype dependant and lacked regeneration potential. Rhizogenic responses were observed in leaf discs of two cultivars however shoot regeneration was not observed. Using a variety of enzyme mixtures it was possible to isolate protoplasts from both In vitro leaf material and from cell suspensions. Both mesophyll and cell suspension derived protoplasts were cultured to a microcallus stage. Plating density, growth regulator concentration and the use of antioxidants were all demonstrated to have a significant effect on the protoplast plating efficiency. Rhizogenesis was achieved from mesophyll protoplast-derived calli. Protoplasts, sometimes labelled with a fluorescent marker, were subjected to both chemical and electrofusion. Using micromanipulation, heterokaryons, formed during electrofusion, were recovered. Such heterokaryons, when cultured. underwent division and formed microcalli which subsequently developed into calli. The hybrid nature of such calli were conftrmed by isozyme analysis, determination of ploidy level and RAPD analysis. The introduction of a plasmid containing a gus marker gene into zygotic embryos of English rose was shown to be possible. This was achieved by microprojectile-mediated DNA delivery using a laboratory built electrical discharge device. The efficiency of this technique was influenced by the concentration of microprojectiles and DNA used. And by firing distance and choice of DNA construct. The relevance of this study and its applications, in the context of rose breeding are discussed.

Conidia of A. linicola germinated over a wide range of temperatures (5 - 25°C) on both agar and leaves. Germination started within 2 h after inoculation at temperatures between 10°C and 25°C, either on agar or on leaves. At 5°C, there were lag periods of 2 and 4 h before the initiation of germination on agar and on leaves, respectively. Germinating A. linicola conidia were very sensitive to drying between 2 and 6 h after inoculation. In the presence of leaf wetness, light applied before the initiation of germination delayed the germination process and decreased the length of the germ tubes. Light applied after the onset of germination decreased both the percentage of conidia which germinated and the length of the germ tubes. In the absence of leaf wetness, light applied before or after the initiation of germination stopped the germination process or decreased the percentage of conidia which germinated, respectively. Conidia of A. Linicola germinated by producing germ tubes and occasionally by producing secondary conidia. Formation of appressoria was inhibited at 5°C. Penetration of the leaf tissues started 12 h after inoculation at 15°C and occurred mainly directly through the epidermal cells and occasionally through stomata. A. linicola is a "diurnal sporulator". In vitro most isolates sporulated only after exposure to diurnal NUV-light. However, for some isolates exposure to diurnal NUV-light did not seem to induce sporulation unless the mycelium was wounded and grown on a medium rich in CaCO3 (S-medium) at high relative humidity. In vivo sporulation of A. linicola was increased after induction by light. The greatest numbers of conidia were produced under continuous leaf wetness and alternating dark/light periods (12 h each). Under these conditions the number of conidia produced increased with increasing temperature from 10°C to 20°C. Alternating 15°C/10°C or 20°C/15°C day/night temperatures decreased the number of conidia produced compared with the constant temperatures 15°C and 20°C, respectively. In controlled environment studies, infection of linseed plants by A. Linicola and development of symptoms was affected by the leaf wetness period, its interaction with temperature and by the light conditions. Eight hours of leaf wetness were sufficient to initiate the disease at 25°C but not at 15°C when a longer period of 10 h was needed. Infection of linseed plants by A. Linicola occurred under interrupted leaf wetness periods at 15°C, but the incidence and severity of the disease was lower than that under continuous leaf wetness. The disease incidence on stems and the disease severity on leaves was negatively correlated with the length of the light period applied immediately after inoculation. Disease incidence and severity increased with increasing inoculum concentration from 1 x 10³ to 1 x 105 conidia ml-¹. The cotyledons appeared to be more susceptible to A. linicola infection than the leaves when the same inoculums density was used. A. linicola was detected on 12 of the 20 seed samples tested and on six of them at a high incidence (> 50%). Seed seems to be the main source of primary inoculum as the pathogen was effectively transmitted from infected seeds to the emerging seedlings. Infected linseed stem debris, volunteer linseed plants and the weed Veronica agrestis were also sources of primary inoculum for the infection of linseed crops by A. linicola. Structures resembling chlamydospores formed in the mycelium and conidia of A. linicola seem to be involved in the survival of the pathogen in stem debris. Conidia of A. linicola were mainly dispersed by the wind (air-borne conidia) and their dispersal followed seasonal and diurnal periodicities, which were influenced by the weather conditions and the incidence of the disease in the crop. The greatest numbers of A. linicola conidia were collected by the Burkard spore sampler on the first dry day following periods of rain, between 12:00 h and 13:00 h and during the period between flowering and harvest of the crop (July - September). Bait plants were more efficient than the Burkard spore sampler in detecting A. linicola conidia present in the crop early in the growing season. The number of A. linicola conidia dispersed within a linseed crop decreased with increasing height above ground, but some conidia were collected 80 cm above the crop canopy. The number of A. linicola conidia dispersed downwind from a line inoculum source decreased with increasing distance from the source and by the end of the growing season conidia were collected by up to 40 m from the source. When the A. linicola disease gradients were studied from point or line inoculum sources, the disease incidence decreased with increasing distance from the inoculum source. By the end of the growing season, the disease was detected 20 or 60 m from the point or line inoculum sources, respectively. Multiple applications of iprodione or prochloraz sprays to control A. Linicola infection in the crop, especially the seed-borne phase of the pathogen, and to increase crop yield gave variable results depending on the weather conditions and the incidence of the disease in the crop. Multiple applications of benomyl or chlorothalonil sprays had either no effect or increased the incidence of the disease in the crop.

Lodging, the permanent displacement of plant stems from the vertical, occurs on average once every four years in UK wheat crops, when it reduces the yield and bread making quality of grain. Lodging usually occurs in the summer and control is most commonly sought by applying growth retarding chemicals in the spring. This thesis develops a method of predicting which crops are prone to lodging so that spring lodging controls can be targeted most effectively. A model was developed in association with this study which calculates stem and root lodging risk from summer-time plant, weather and soil factors. The spread of the plant's root plate, the stem diameter and the number of shoots per plant were shown to have very strong influences on lodging. Structural rooting depth, stem failure yield stress, height at centre of gravity and the rate at which stems oscillate in wind (natural frequency) were also important, but less influential. Methods of predicting the most important lodging-associated plant characters from crop observations in spring were developed from the literature. These were then tested through experiments in 1995 and 1996 with factorial combinations of crops sown in late September and late October, at 500 seeds m-2 and 250 seeds m-2 and with large and small levels of residual soil nitrogen. Early sowing, dense seed rates and fertile soils all increased stem and root lodging, with sowing date having the greatest influence. Plants sown at high densities had small root plates and were poorly anchored; they also had fewer shoots causing a smaller leverage. Early sown plants had shoots with a high centre of gravity and slow natural frequency, causing a greater leverage. Plants sown early on fertile soils had narrow, weak stems. Final shoot number per plant was predicted with good precision (R2=094) from spring plant number m-2 and maximum shoot number m-2 using a model of tiller survival. Stem diameter was predicted with moderate precision (R2=057) from spring canopy size and shoot number m-2, via a calculation of the amount of dry matter partitioned to each stem base. Root plate spread showed a linear and inverse relationship to spring plant density (R2=0.48), mainly as a result of variation in the length of the rigid roots and in the width of the plant base. It is concluded that early season crop observations have the potential to predict the values of the most influential lodging-associated plant characters, from which a model of lodging can calculate the proneness of crops to stem or root lodging in time for remedial action. The next steps would be to develop prediction schemes for other plant characters which influence lodging and test all the predictions in a wider range of crops, sites and seasons.