Plant responses to simultaneous biotic and abiotic stresses involve the activation of multiple signalling pathways that often interact in a synergistic or an antagonistic manner. Recent studies have shown that the plant response to a combination of stresses differ to those that occur when exposed to a single stress. The aim of the present study is to investigate the response of wheat (Triticum aestivum) to both salinity and aphid Sitobion avenae infestation, applied singly and in combination, at the physiological and transcriptional levels, to provide a better understanding of the impact of abiotic and biotic interactions and cross tolerance in wheat. These two forms of stress were selected since they are known to have a major impact on crop productivity. Wheat genotype 122-1 was shown to be tolerant to salt (160 mM NaCl) on the basis of biomass; accumulating high levels of Na+ in the shoots and was partially resistant to aphids in terms of fecundity. Pretreatment of this genotype with salt significantly (p < 0.001) reduced aphid fecundity (by 37%) relative to its control, indicating enhanced resistance to aphids. This positive interaction between salt and aphid stress was investigated at 6h and 24h post aphid infestation on the wheat transcriptome. Microarray analysis indicated common and specific gene expression patterns of the 61.290 transcripts differentially regulated in response to salt stress alone, aphid infestation alone and dual stress compared to the controls. Dual stress specifically and strongly increased the transcription level of the following genes assigned to jasmonate synthesis encoding lipoxygenase (LOX), abiotic stress (heat), miscellaneous enzyme families (acid and other phosphatases) at 6h, and secondary metabolism (phenylpropanoids) at 24h. Furthermore, based on functional classification analysis, several categories which were shown to be significantly activated by dual stress such as cytokinin hormone synthesis and MAP kinases signalling were not, however, significantly changed under either salt stress or aphid infestation alone. The current study demonstrated that jasmonate hormone signalling pathways antagonized those of salicylic acid under dual stress and aphid infestation at 6 h. Results suggests that the interaction between combined salinity and aphids stresses caused distinct alteration in gene expression patterns that could not be detected under either of the two stresses when applied individually. This study proposes that the activation of specific genes involved in the acquisition of defence/tolerance, such as those encoding cytochrome P450 and MYB domain transcription factor families, due to prior exposure to salt may enhance subsequent tolerance to aphids. The present study sheds light on candidate genes with putative functions in the crosstalk and the acquisition of cross tolerance and provides new insights on wheat response to multiple stress conditions. Such information is a prerequisite for enhancing crop tolerance to a broad-spectrum of stress.

Published research showed there was a link between the abundance of septoria diseases (Zymoseptoria tritici and Parastagonospora nodorum) and sulphurous acid in acid rain. Historical records showed that P. nodorum was at a peak in 1970s while Z. tritici became more abundant at 1980s. Another, small scale, study showed that both pathogens were affected by sulphur, but Z. tritici was reduced more by sulphur than P. nodorum. These studies suggested it could be useful to investigate the effect of sulphur fertilizer under larger scale, field, conditions. Thus, the aim of this project was to study if sulphur fertilizer affects the abundance of these pathogens, and to study if sulphur fertilization alters the yield and quality of wheat under disease pressure. A further question was whether silicon fertilizer affects both pathogens and the yield and quality of wheat. A final aspect of this thesis was to develop a way to quantify pathogens by using qPCR assay on large field samples. A glasshouse experiment showed that all wheat cultivars were susceptible to available pure isolates of both pathogens, but the proportion of leaves infected by these isolates varied between cultivars. Cv. Paragon showed a high proportion of infected leaves in comparison with Gallant and Maris Huntsman (M. Huntsman). Three successive field experiments were conducted. Z. tritici infected all plots, so it was not possible to estimate changes in competitive effects due to the fertiliser regimes. In the field experiments a variety of measurements were used at different growth stages to assess the diseases. The proportion of leaves infected with, and disease severity of, Z. tritici were not significantly affectedly by sulphur fertilization in 2013. In two further experiments, proportion of leaves infected with, and spore concentration of, Z. tritici were reduced by sulphur fertilization. In the 2014-2015 experiment disease severity of Z. tritici was significantly reduced by sulphur fertilization. Although autumn spray and seed inoculation with P. nodorum did not succeed, a spring spray inoculation was successful in the 2014-2015 experiment. In this experiment, proportion of leaves infected and disease severity of both fungi were reduced by sulphur fertilization. but Z. tritici was reduced more than P. nodorum. These results agree with the historical trends. Yield and quality measurements results showed that sulphur fertilization increased yield and the wheat quality was improved. This result was obtained when the sulphur fertilization was split and applied at multiple times throughout the growing season of wheat (Table 6).

To study the auxin metabolism of etiolated oat seedlings a modification of the straight growth method of assaying hormones, using wheat coleoptile cylinders has been employed. Wheat coleoptiles detached from the endosperm are subjected to high and low temperature treatment, and exposure to dim red light during handling, prior to cutting from them the 10 mm cylinders used in the assay. Such cylinders grow less in sucrose-buffer solution than do those not so treated, but their response to externally applied IAA is not affected. In this way a very small amount of IAA ( 0.005,ug ) in 0.5 ml of sucrose-buffer can be detected with certainty. The auxin content of the wheat coleoptiles used to provide the cylinders for the assay has been investigated. Heating and chilling, employed either separately or in combination, destroys native hormone; the reduced growth of the cylinders may thus be accounted for. Exposure to dim red light for the period used in the experiments has little effect on hormone content. The growth of the mesocotyl and coleoptile of etiolated oat plants can be modified differentially by using a number of environmental conditions as follows Treatment Mesocotyl Coleoptile Carbon dioxide promotion depression Ethanol slight promotion depression Nitrates slight promotion promotion after 5th day Sucrose slight promotion strong depression Heating strong depression transient promotion Illumination strong depression transient promotion Samples of plants grown under these conditions were harvested on successive days in darkness and immediately frozen in solid carbon dioxide. They were then dissected without allowing them to thaw and auxins were extracted in cold ether; purified; chromatographed, and assayed.

The molecular interaction between Rhynchosporium commune and its host barley was studied to gain a better understanding of the pathogen during infection and provide further characterisation of resistance in barley, using a combination of bioinformatics, transcript expression analysis, proteomics and confocal microscopy. Expression analysis of potential effector sequences identified novel candidate effectors Rc_10934, Rc_2091 and Rc_2835 which showed the highest abundance during the biotrophic infection. A further two novel candidates Rc07_03591 and Rc07_02334 and a LysM containing protein (RcLysM3) were identified using a proteomic analysis of infected plant apoplast. Further apoplastic analysis revealed some of the most abundant proteins that are present in R. commune’s infection toolkit. Cell wall degrading enzymes (CWDEs), virulence factors and proteins involved in detoxification were all highlighted as some of the main key players of pathogenesis. A large family of LysM domain containing proteins was later identified in the R. commune genome. Expression profiling revealed the upregulation of some of the transcripts during infection, indicating a potential role in pathogenesis, whereas others were expressed in vitro indicating potential functions for the proteins in fungal growth and development. RcLysM3 containing 3 LysM domains and sharing similarities with the well-known C. fulvum Ecp6 effector was selected for further characterisation. Bioinformatics predictions showed a high affinity for chitin binding which was confirmed in vitro. Binding analysis revealed that it can also bind chitosan but not plant cell wall polysaccharides, indicating that it is potentially involved in the evasion of plant immune responses. The presence of the effector was also identified in the apoplast of infected barley leaves using a proteomic approach. R. commune strain expressing GFP was used to characterise differences in pathogen growth and colony morphology in response to different genetic backgrounds of barley using lines carrying the Rrs3 (Abyssinian), Rrs4 (CI11549) and Rrs13 (BC line 30) genes and barley landraces with uncharacterised resistance. Rrs1 resistance was further analysed using comparative proteomics to identify proteins differentially expressed in resistant and susceptible cultivars. Pathogenesis related proteins - chitinase, glucanase and thaumatin-like protease, were identified in the barley apoplastic fluid and were shown to be upregulated during infection. In addition, serine carboxypeptidase and purple acid phosphatase proteins were identified that were novel to the barley resistance interaction but have been identified in other incompatible interactions as defence related proteins. The final chapter of this thesis is dedicated to the analysis of asymptomatic growth of R. commune on the model dicotyledonous plant N. benthamiana and analysis of effector transcription during growth on a non-host. R. commune growth was shown to be confined to the leaf surface, with no evidence of plant cell deterioration in transgenic N. benthamiana plants expressing an mRFP-tagged plasma membrane protein. This system could be used for further research into non-host interactions and provides insights into the growth of R. commune on alternative plant species.

Genetic resistance to Wheat streak mosaic virus (WSMV) offers the most attractive and environmentally safe strategy for disease control. While effective resistance in hexaploid bread wheat (Triticum aestivum) has recently been described in only one case, the Wsm2 gene, more successful resistance has been introgressed from the related hexaploid wheatgrass, Thinopyrum intermedium, as the Wsm1 and Wsm3 genes. In the current study, fluorescent in situ hybridization (FISH) with genomic DNA from Th. intermedium, Aegilops tauschii and repetitive DNA probes was applied to four breeding populations of wheat-Th. intermedium, previously tested for WSMV-resistance. Three different wheat-Th. intermedium recombinant chromosomes, the well-known 4Ai#2S.4DL and two novel, 1B and 3D, were identified to be associated with WSMV-resistance. These novel introgressed genes from Th. intermedium were designated as Wsm4 and Wsm5 respectively. The Wsm4 gene was pinpointed to a 6% interstitial map region on the 1BS flanked by Xgwm4144 and Xgwm1100 markers. Six new PCRmarkers, five linked to Wsm1 and one to Wsm4 were identified. Molecular markers now provide a good coverage of the 4Ai#2S arm for effective marker assisted selection and the new genes increase our arsenal to combat the disease. Two highly repetitive satellite DNA families, Afa and pSc119.2, were isolated for the first time from Th. intermedium and their diversity in respect to copies from other Triticeae species were investigated. They showed contrasting evolutionary dynamics leading to time dependent or independent homogenization of Afa and pSc119.2 sequences. Both repeats are excellent cytological markers and characterized the 4Ai#2S chromosomal arm, in the alien wheat lines and the Th. intermedium genome. Southern hybridization, with methylation sensitive and insensitive restriction enzymes and immunostaining with anti-5-methyl-cytosine antibodies were employed to assess DNA methylation. Overall, no massive changes were evident in the wheat genome, however the alien arm showed reduced cytosine methylation which is characteristic for actively transcribing chromatin.

Temperate or pooid cereal crops, such as wheat, barley and oat, represent a major world food source much of which is attributable to the storage capabilities of the cereal endosperm and influences of domestication selection. Brachypodium distachyon has recently become established as a genome-sequenced model system and the first wild member of the Pooideae be studied in detail. We provide the first detailed description of grain development in Brachypodium assessing its suitability as a model for grain development in crop species. Cellular and molecular mapping of developing Brachypodium endosperm domains reveals significant differences in aleurone differentiation reflecting differences in grain filling and endosperm storage reserves. We extend this survey of grain morphology and endosperm organisation to a wider sample of the Pooideae incorporating both wild and cultivated species. Focusing on the functionally important aleurone domains, distinct patterns of grain tissue organisation are described. Results indicate that organizational features are correlated to species’ ecological and grain quality characters and that the modified aleurone region, absent in Brachypodium, may be a feature of only a subset of cereals, specifically the Triticeae tribe. A more systematic candidate gene approach focusing on transcription factors was initiated in attempts to find the genes underpinning this variation. The identification of orthologous key regulatory genes with both similar and contrasting patterns of expression provides information on the differences and conservation of grain developmental pathways amongst the Pooideae. To investigate function of candidate genes, publically available insertional mutants for major MADS-box and YABBY genes were obtained and characterized, while simultaneously attempting to establish genetic transformation protocols to enable RNAi analyses of other candidate genes.

Research was conducted at Reading University over three growing seasons into alternatives to herbicides m spnng cereals by identifying traits that enhance crop competitive ability and exammmg the role of seed quality in Integrated Weed Management (IWM~. Nine spring wheat genotypes, spring Barley, spring Triticale and a mixture of spring wheat genotypes Paragon and Belvoir were monitored for their competitive ability in three weed treatments, i.e. weed free and with model weeds Sinapis alba L. or Avena sativa L. The ranking of competitive ability (CA) was estimated from both crop tolerances to weed pressure (i.e. a low crop grain yield loss - CYL) and weed suppression. Genotypic differences in CA were repeatable, with CYL of 28%, 39% and 28-75% for barley, triticale and wheat in 2003, respectively. Barley also suppressed weed biomass by 59% more than wheat. Among wheats, Axona consistently maintained higher yields (10% CYL in 2002 and 46% in 2003). Paragon suppressed weeds by 63% in 2002 and 50% in 2003 whereas cv. Status was a poor competitor. The wheat mixture reduced CYL but had no significant effect on weed suppression. An important observation for variety trials was that the ranking for grain yields in weed free plots was inconsistent with those with weeds. Competitive genotypes tended to be taller and have rapid early growth at GS 15 giving early ground cover, a higher absolute growth rate at GSI5, a shorter mean time to emergence and higher early leave area. On seed quality and its interactions with agronomic practices such as seeding rate and sowing date, doubling seed rate improved crop competitiveness if the crop was sown at the same time as weeds rather than a week later. When a vigorous seed lot was used, higher number of seedlings, ground cover, harvest index and number of fertile tillers per m2 and lower mean time to emergence contributed to its higher crop grain yield. High vigour seeds suppressed total weed biomass up to 89,-43 and 63% in three experiments, the suppression varying between individual weed species. For example, when natural weed infestations were present, high vigour seeds suppressed Convolvulus arvensis L. by 52.5% and Solanum nigrum L. by 95.5%. One particular feature of this thesis was an attempt to understand why high vigour seeds are more competitive. The results indicated that more than 75% of the variations in grain yield and 81 % of the variation in weed dry mutter suppression can be accounted for due to achieving more rapid emergence than the weeds.

Modern annual arable crops are susceptible to outbreaks of pests due mainly to the uniform habitats that agricultural fields represent. Cereals are particularly prone to infestations of aphids, which may damage the crop directly through herbivory, or indirectly by acting as vectors of disease. These insects, however, have a large range of invertebrate natural enemies, which under certain circumstances, can maintain infestations below economically damaging levels. Greater habitat complexity at landscape and farm scale usually leads to more diverse assemblages of natural enemies at the field scale, but such diversity less often translates to a higher risk for pests. When higher natural enemy diversity is associated with lower levels of pest control, intraguild predation (IGP) is often cited as one of the primary antagonsitic mechanisms. IGP occurs where predators not only compete for the same resource, but also partake in a trophic interaction with one another. Controlled experiments suggest that the niche proximity of predators relative to each other and their shared prey may help predict the outcome of multiple- predator interactions. The primary aim of this thesis was to assess levels of IGP amongst generalist invertebrate predators and to elucidate their spatial patterns, in fields of winter wheat (<italic>Triticum aestivum</italic>), an important cereal crop in North-West Europe. The chief objective was to establish the regulatory abilities of these predators in the control of aphids. Post-mortem gut content analysis using PCR was used to establish the intensity of IGP by two polyphagous predators, the carabid beetles <italic>Pterostichus melanrius</italic> and <italic>P. madidus</italic>, on a number of insectivorous linyphiid spiders and their shared aphid prey. Each of the spiders tested was found to suffer IGP. Predation rates were adjusted using data from controlled feeding trials and resampled using Monte Carlo models to test the hypothesis that predation was density-dependent. In one experiment, the web-occupying linyphiid <italic>Tenuiphantes tenuis</italic> was consumed by up to a third of <italic>P. melanarius</italic>. Predation rates by carabids on the linyphiid <italic> Bathyphantes gracilis</italic> were consistently lower than expected. <italic> B. gracilis</italic> also relies principally on its web to capture prey, but builds these webs significantly higher in the wheat stem than <italic>T. tenuis </italic>. Preferences for intraguild prey species more likely to hunt aphids on the ground, the tetragnathid spider <italic>Pachygnatha degeeri</italic>, and the linyphiids Erigone spp. (<italic>E. atra</italic> and <italic>E. dentipalpis </italic>), were less consistent than those species more dependent on their webs to hunt aphids and other prey. While many factors may contribute to the outcomes of multi-predator interactions, these findings broadly supported the hypothesis that niche proximity of intraguild predators is positively related to levels of disruption due to intraguild interactions.

No description available.

To meet the nutritional requirements of a growing population, world agricultural output must increase by 70-100% by 2050. However, worldwide wheat yields are close to reaching a 'glass ceiling'. In order to meet the challenges of a rapidly growing population, scarcity of resources and a trend toward high-protein diets, traditional agriculture has looked towards molecular biology for insight and innovation for the future. Studying the mechanisms underlying DNA damage and repair is crucially important to facilitating generation of novel variation and developing breeding techniques. The Mismatch Repair (MMR) system is a highly-conserved series of pathways responsible for maintaining genomic integrity in prokaryotes and eukaryotes. The constituent proteins repair DNA mismatches and prevent meiotic recombination between homoeologous sequences. Work in Arabidopsis, tobacco and tomato has shown that plants with an impaired MMR system accumulate point mutations and undergo increased homoeologous recombination, which may be useful in breeding programs. Two key MMR genes are explored: MutS Homologue 2 (MSH2) acts as a heterodimer with MSH3, MSH6 or MSH7 to detect point mutations, insertions and deletions. Post Meiotic Segregation increased 2 (PMS2) is a homologue of the MutL gene, which co-ordinates repair of mismatches. The two genes were cloned and sequenced from elite cultivar Cadenza, and the sequences subsequently compared to next generation sequencing data from Chinese Spring. Three homoeologous copies of each gene were identified in the genome of wheat, and two of these were sequenced from the transcriptome. The expression of MSH2 was studied using qPCR in the transcriptome of Cadenza and four gamma-irradiated deletion lines, generated in a Paragon background. The four deletion lines showed varying responses: from down-regulation of MSH2 expression by 57% to upregulation by 82%. The expression of the individual homoeologues of MSH2 were also explored using a qPCR based method and by examining RNAseq data, but this was inconclusive.