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Potatoes respond to mechanical stress by invoking a precisely controlled biochemical reaction leading to production of the pigment melanin. Predisposition to produce melanin is variety specific, this specificity being conveyed by a range of genetic, environmental and biochemical factors. A well characterised response of plant tissues to perturbation by biotic or abiotic stress factors is the rapid generation of oxygen based free radicals - active oxygen species (AOS). Due to their nature as high-energy, largely unstable, radicals, AOS have been implicated in a wide range of deleterious effects upon plant tissues as well as being involved in a range of signalling cascades within cells. This project attempted to demonstrate a link between AOS and other associated oxidative factors, and blackspot bruise potential. It was demonstrated that in response to mechanical impact, potato tubers respond by producing a well-defined level of superoxide free radicals. The amount of superoxide generation was found to be nearly perfectly correlated with susceptibility to blackspot bruise when investigated over eight potato varieties. By using inhibitors and scavengers of free radicals it was shovra that superoxide and not hydrogen peroxide was directly responsible for the effects upon pigment synthesis. Furthermore studies upon polyphenol oxidase (PPO), the key enzyme in the synthesis of melanin, demonstrated that a significant enhancement of activity was noted in the presence of superoxide radicals, lending support to animal studies which suggest that PPO utilizes superoxide radicals more readily and more efficiently than its usual co-substrate molecular oxygen. Associated with the production of superoxide radicals was the demonstration of modification to tuber proteins, specifically the detection of secondary carbonyl groups, a modification known to be the direct result of exposure to AOS. This study quantified the carbonyl levels and once again these were almost perfectly correlated with susceptibility to blackspot bruise formation. Once the correlation of oxidative factors and blackspot bruise susceptibility was established then studies were undertaken to demonstrate the effect of a range of metalions upon both blackspot bruise susceptibility and oxidative protein modifications. These studies suggested that potassium and zinc both had negative effects on blackspot susceptibility however only potassium had a negative effect on secondary carbonyl accumulation. Studies on tubers impacted twice indicated that the normal 'polarity' of the tuber in terms of susceptibility to blackspot bruise could be influenced and this was confirmed by studies on superoxide generation which showed that the second point of impact always had a reduced blackspot susceptibility and reduced superoxide generation. Finally a model is presented to incorporate this novel information regarding the role of AOS in blackspot bruise susceptibility together with existing knowledge of the biochemistry within this system, to propose a system in which AOS play an integral and wide-ranging role.

Until recently strawberries grown in the United Kingdom were grown in open fields, the plants and fruit were exposed to the British weather. This resulted in a short 6 week harvest period where the fruit was often damaged by rain and infected by Botrytis cinerea. Strawberry growers started to use polythene tunnels to extend the cropping season, protect the fruit from rain damage and reduce the incidence of infection by B. cinerea. However the conditions produced by the polythene tunnels were ideal for the growth and development of Podosphaera aphanis (strawberry powdery mildew). Growers are now under pressure from the retailers to reduce the amount of fungicides that they use to control P. aphanis. The symptoms related to P. aphanis infection have been identified (leaf cupping, visible mycelium and red blotches) and a progression has been established: From the symptom progression two new s~ring methods for the identification of P. aphanis infections were developed wmch have greater relevance to current cultivation methods than the previous method. The source of initial inoculum for newly planted and established sites was identified. The inoculum was planted into new sites on the plants coming from the propagators and overwintering on plants within established sites. This was contrary to what the growers believed. They were basing their early season tunnel management on keeping the perceived air borne infection out of their tunnels. A rule based prediction system has been developed that has the potential to reduce the number of fungicide applications applied by the growers. The prediction system ensures that fungicide applications are not applied too close together. Potassium Bicarbonate has been shown to provide comparable control of P. aphanis to that achieved with Systhane (Myclobutanil). Significantly better control of P. aphanis was achieved using a new (at the time) product, Fortress (Quinoxyfen). There were significant differences in the resistance °to infection by P. aphanis displayed by different cultivars of strawberry. Elsanta, the cultivar favoured by the retailers was not one of the most resistant. Control of inoculum . already present on plants as they are being planted could be achieved by dipping the plant in Systhane. Growers are under considerable pressure from the retailers to reduce the amount of fungicides used to control P. aphanis. Growers could achieve this by implementing the recommendations made in this report.

Fusarium ear blight (FEB) is a disease of wheat and small grain cereals, caused by the fungi Fusarium culmorum and Fusarium graminearum. The disease causes premature bleaching of spikelets and shrivelling of the grain can result in a direct yield loss. Mycotoxins such as deoxynivalenol produced by the fungus can reduce grain quality. Breeding for resistant wheat cultivars is considered one of the best control options. Previously identified resistance has been reported in the Chinese genotype Sumai 3. The principal aim of this project was to identify novel sources of resistance to FEB. Twenty four wheat genotypes were evaluated for resistance to FEB in this project. Molecular markers linked to previously identified QTL from Sumai 3 conferring resistance were used to confirm their absence in the genotypes under investigation, and revealed that none of the genotypes under investigation contained all of the QTL for resistance. Field trials conducted over two years screening for resistance demonstrated that, although not statistically similar to Sumai 3, levels of disease were below 10% in some of the Chinese genotypes. Follow up experiments using reporter strains of Fusarium graminearum explored the accumulation of fungal biomass and the expression of the gene Tri5, which is essential for DON biosynthesis. Fungal biomass levels were not significantly different between genotypes; however expression of the Tri5 gene was significantly lower in the genotype Alsen. A previously developed wheat leaf seedling bioassay was also explored. Scanning electron microscopy revealed the presence of fungal hyphae in advance of the visible lesion during the infection course of F. culmorum. Inoculation with a Tri5 mutant strain of F. graminearum demonstrated that a lack of mycotoxin production altered the lesion type. This project has successfully identified potential novel resistance mechanisms and the future prospects for the control of this disease are discussed.

Cucumber mosaic virus (CMV) has emerged as one of the most serious threats to tomato cultivation in Greece. In the present study the effects of Benzothiadiazoles (BTH) and pyraclostrobin against mechanically or aphid-transmitted CMV in tomato plants, of hybrid F1 Clodin, were investigated in greenhouse experiments. BTH was confirmed as capable of inducing systemic acquired resistance (SAR) in tomato seedlings against CMV, while pyraclostrobin was not. Responses to BTH application and/or CMV inoculation on Spanish tomato hybrid Delos (BTH, BTH+CMV, CMV treatments) were monitored during winter and spring season in Greece. In both seasons the SAR derived from BTH application suppressed CMV. BTH treatment presented increased plant growth, fruit size and marketable tomato yield compared to CMV and BTH+CMV treatments, whereas decreased compared to healthy control. CMV treatment caused the most severe stunting of tomato plants among the examined treatments and resulted in yield loss of marketable fruits, although the total fruit number was higher versus to other treatments. Cont/d.

Potato is one of the most important food crops in the world, and viruses are largely responsible for the degeneration of this vegetatively propagated crop. At least 35 viruses have been reported to infect potato naturally. The majority and the most economically important ones are vectored by aphids. The objective of this study was to conduct molecular and biological investigations into virus transmission mechanisms, including developing diagnostic methods to help to control the spread of aphid-borne potato viruses, and disrupting the vectoring ability of their aphid vectors by insecticide spray. One way to control the spread of aphid-borne viruses is to control their aphid vector, but this is not always feasible as the majority of aphid-borne potato viruses, including the most important ones, are transmitted non-persistently, being acquired within a very short time before agrochemicals can act. Thus an alternative approach to controlling this class of viruses is through a full understanding of the interaction between the virus, the host plant and the aphid vector, which was the first objective of this project. In this respect, some aphid cuticle proteins were identified to interact with potato virus Y helper component (HC-Pro) through screening of an aphid cDNA expression library, and their potential role in virus transmission was discussed. Moreover, the concept of short retention of non-persistent viruses inside their aphid vectors was challenged; the results show that PVY can be retained inside its aphid vector for a long time but it is not transmissible. This novel finding together with binding to aphid cuticle proteins, generated some new ideas about transmission mechanisms that were proposed and discussed. In addition, the effect on aphid vectoring ability of the plants used to rear aphid colonies, as a virus source, and as a virus recipient was investigated. From laboratory studies of aphid transmission, it was concluded that the transmission efficiency of PVY was significantly affected by the host plant species used to rear M. persicae, or that used as a virus recipient plant. The availability of sensitive and cheap virus detection methods is critical for early detection and control of potato viruses. In this project a sensitive fully recombinant ELISA was developed and validated for routine testing of potato leafroll virus. This technology can be applied to detect other potato viruses and has the potential to replace the commonly used immune reagent antisera.

In contrast to the negative impacts of landscape change on many aspects of biodiversity, scavenging bird species, like gulls (Larus spp.), have reacted positively and expanded their ranges. This in tum has brought a number of problems, including; damage to town centres, an increased risk of disease transmission to humans, domestic animals and livestock, an increased risk of collision with aircraft and a threat to vulnerable seabird populations. A great deal of money has been invested in measures to mitigate these problems. However, these have often been hampered by a failure to understand the ecology of the system concerned. This study employs a variety of statistical techniques to investigate factors related to the spatial and temporal distribution of gulls, the possible problems they may cause and the efficacy of measures to reduce the impact of these problems. Using structural equation modelling (SEM) it was possible to show that in contrast to other groups, like corvids which use landfill sites close to their roosts throughout the year, gulls rely most heavily on landfill sites as a source of food during the winter. However, analysis of the spatial distribution of winter gull roosts using negative binomial generalised linear models (GLMs) showed that only roosts of the black-headed gull (Larus 'ridibundasv were positively influenced by proximity to landfill sites. In contrast to the winter, when roosts were widely distributed, during the summer roosts had a coastal distribution. The problems posed by gulls to air safety and human health were investigated by analysing the spatial patterns of accidents and the incidence of salmonella carriage by 3 wild birds. In the first, bivariate k-means clustering revealed that strikes on Royal Air Force (RAF) aircraft by gulls were clustered within the 6 km surrounding major (>1000 individuals) gull roosts and landfill sites. These results were used to identify additional areas capable of supporting large numbers of gulls, and hence likely to represent a threat to aircraft in the future. I used survival analysis to investigate temporal and spatial patterns in wild bird salmonella. Passerines were more likely to be infected with salmonella than non-passerines and further analysis was indicative of gulls having a higher rate of salmonella infection than other non-passerines. Salmonella prevalence in wild birds was greatest during the winter and spring, and in areas with large populations of cattle. Having investigated the factors determining where gulls were and the risks they posed to aircraft safety and human health, I analyse the efficacy of a range of management techniques to control problem gull populations, using linear mixed effects models (LMEs). This revealed that techniques with occasional lethal events were the most effective. By using a range of statistical techniques, it was possible to disentangle a series of complex and often interacting relationships between gulls, the landscape and humans.

Rhizoctonia so/ani is the causative agent of wirestem on Brassica crops. R. so/ani is a species complex comprising genetically. distinct groups known as anastomosis groups (AGs). Knowledge of which AGs are responsible for disease is necessary to formulate appropriate management strategies. Important knowledge about the specific AGs responsible for disease in UK Brassica crops and where in the production chain the fungus became associated with plants was lacking. A survey of UK .f3rassica o/eracea crops was completed to. establish which anastomosis groups of Rhizoctonia so/ani were associated with module raised plants. No R. so/ani was recovered from plants collected directly from UK propagators. R. so/ani was identified from asymptomatic stem bases collected from field crops using classical mycology, suggesting field inoculum is important. Such data suggests UK propagation houses have high standards of hygiene and future research should concentrate on elucidating the field biology of R. so/ani. The pathogenicity of the recoveredi.isolates was demonstrated across a range of crops, including B. o/eracea. However, the isolates were not pathogenic to monocotyledonous species suggesting these may act as effective break crops. Sequence data were generated from ribosomal and ~-tubulin regions and the anastomosis groups of the R. so/ani isolates identified using parsimony and Bayesian based phylogenetic methods. Both methods suggested the majority of isolates recovered from the stem bases of UK B. o/eracea plants belonged to AG2-1 and all but one of the remaining isolates belonged to AG4 HGii. These data are consistent with research from the USA, however this is the first report for UK crops. AG determination using nucleotide sequence information proved more successful and less time consuming than classical mycological approaches. AG2-1 formed three distinct clusters in all analyses suggesting this' subgroup is genetically diverse, a conclusion supported by the problems encountered when investigating AGs using classical mycology. The monophyly of genera Ceratobasidium and Thanatephorus were investigated using constrained analyses, however no firm conclusions could be drawn to accept or reject this hypothesis. Protocols were devel~edto' deteet AG 'and ·spedfic sub~groups in plant and ·soil samples using real-time peR. Soil testing suggested AG2-1 was more frequently detected in samples collected from the upper 10 cm of fields used for B. oleracea production. Such information is consistent with other research and suggests the growth of R. solani may be limited by lack of air filled pores within soil matrixes. The molecular methods were used to investigate the spatial and temporal association of R. solani with field grown B. oleracea plants. The molecular protocols confirmed that R. solani AG2-1 became rapidly associated with a large number of B. oleracea plants. The principle arguments for such an association hinged on identifying the dominant behaviour expressed by the fungus. Rapid colonisation of the root system and stem base would benefit saprophytic behaviour of R. solani as the fu.ngus could capitalise when the crop lifecycle was complete. As a pathogen the benefits of early colonisation are clear and perhaps the reason for low disease levels can be explained by the absence of suitable environmental conditions for disease to progress. A third hypothesis could be that R. solani may form mycorrhizal associations with B. oleracea crops.

Rice blast disease is caused by the heterothallic ascomycete fungus Magnaporthe oryzae and is one of the most severe diseases of cultivated rice throughout the world. The PMK1 (Pathogenicity Mitogen-activated protein Kinase) gene in M. oryzae has been identified to play diverse roles during pathogenesis-related development. PMK1 regulates appressorium formation and infectious hyphal growth. PMK1 is functionally related to Saccharomyces cerevisiae FUS3 and KSS1 MAPK genes which regulate the transcription factor Ste12. The STE12 homologue in M. oryzae, MST12, has also been identified and is known to be essential for appressorium mediated penetration and infectious growth. These observations imply that Pmk1 regulates a diverse set of targets important in both the initiation of appressorium development and the subsequent stages of invasive growth. In addition, the Mst12 transcription factor may function downstream of Pmk1 to regulate genes involved in appressorial penetration and infectious hyphal growth. We have used a comparative proteomic study between Guy11 (isogenic wild type), ∆mst12 and ∆pmk1 mutants to understand which genes are induced during appressorium formation and may be regulated by Mst12 and Pmk1. We carried out two-dimensional gel electrophoresis with proteins extracted from conidia that had germinated on a hydrophobic surface after 6h, 12h, 16h and 24h of incubation. M. oryzae underwent major changes in protein abundance and expression during the first 6 hours of spore germination in Guy11 which suggested that much of protein synthesis associated with appressorium morphogenesis and virulence occurs precociously during conidium germination on rice leaf surface. More than 394 differentially expressed proteins during conidium germination and appressorium formation have been identified by mass spectrometry. We confirmed many proteins already known as determinants of pathogenicity in M. oryzae such as enzymes involved in melanin biosynthesis or fatty acid β-oxidation. However, we also identified proteins absent or lowly induced in the Δpmk1 and Δmst12 mutants involved in cell wall reorganisation, secondary metabolism, lipid metabolism, photomorphogenesis or found as proteins of unknown functions. We generated 28 targeted gene deletion mutants to test the putative function of proteins predicted by proteomics analysis to be associated with appressorium development. We obtained 7 mutants showing a significant reduction in virulence and confirmed importance of regulated proteolysis during appressorium development but also elucidated new processes involved in infection such as the phosphoinositide pathway and three proteins of unknown function.

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