Field strains, W x R hybrids and inter-specific hybrids of the eyespot pathogen <I>Pseudocercosporella herpotrichoides </I>were characterised using traditional morphological and more recently developed molecular identification techniques which distinguished between the W and R pathotypes. The pathogenicity of these strains and hybrids to wheat, barley and rye was determined and related to their morphological and molecular characterisation and highlighted the unreliability of some of these techniques. Characterisation of the hybrids revealed the genetic inheritance from the parental strains which generally was greater from the R-type parent with some W x R- hybrids showing an intermediate morphology and others showing novel pathogenicity. An examination of the ability of the strains and hybrids to produce infection plaques <I>in vitro</I> found that infection plaque production could be related to the strain/hybrid pathogenicity on wheat, barley and rye. This indicates a requirement of infection plaques for successful host colonisation. No evidence was found of secondary metabolites produced in fungal culture filtrates using a wheat cell suspension assay and a wheat root inhibition assay which were involved in pathogenicity or host symptom induction. In a comparative study over time on wheat and rye, the development of visual disease symptoms was related to microscopic infection structures and the amount of fungal DNA present in the stem base. This revealed different infection strategies of the W and R pathotypes, the W-types being 'slow and stead' and the R-types being 'fast and furious' in their infection. The infection structures of both W and R-types were found to be always in advance of visual disease symptoms and limited colonisation of the host could occur without visual disease symptoms being present.

Senescence of plant roots is reviewed and related to laboratory and field observations on root turnover (death and replacement) of groundnut (<I>Arachis hypogaea</I> L.). Root death was assessed by browning, which correlated with failure of cells to plasmolyse after staining with neutral red. For five cultivars of groundnut grown in pasteurised soil in transparent tubes, death of root laterals commenced 3 - 4 weeks after sowing; root laterals died in proximal regions and new laterals formed in distal regions as the tap root extended. The half-life of individual roots was 3.7 - 4.4 weeks for all cultivars. Up to the time of plant maturity (14-20 weeks for different cultivars) 72.7% - 83.2% of cumulative (total) root length had died. Similar patterns were seen in field-grown plants in Malawi. Two pathogens of groundnut (<I>Aspergillus niger, Fusarium oxysporum</I>) and two saprophytes (<I>Idriella bolleyi</I> from cereals and <I>Mucor hiemalis</I> from groundnut) were induced or selected for tolerance of benomyl or pimaricin. The mutants had reduced hyphal extension rates on agar but sporulation similar to the wild-type parents in liquid culture. Spore suspensions of these mutants and of <I>Aspergillus flavus</I> were inoculated onto groundnut root laterals of different ages in tubes of soil. After 3 weeks, <I>A. niger</I> and <I>F. oxysporum</I> were not recovered; in other studies <I>A. niger</I> was found to germinate poorly in rhizosphere soil. <I>A. flavus, I. bolleyi</I> and <I>M. hiemalis</I> were recovered in higher numbers on young roots (lower in soil profile) than on older roots. Both growth at the expense of nutrients released early during root senescence <I>(A. flavus, I. bolleyi</I>) and spread in percolating water (<I>M. hiemalis</I>) are suggested to explain these findings. Spores of <I>M. hiemalis</I> (pimaricin-tolerant) were applied to groundnut hypocotyls and to the surface on non-planted soil in an experimental field plot in Malawi. The fungus spread rapidly and progressively down the rhizosphere, reaching at least 58 cm during 51 days, but it did not move far (7 cm) in non-planted soil.

No description available.

Background: Phloem-feeding insects cause significant crop damage worldwide, but despite this little is understood about how plants protect and defend themselves from these threats. Phloem-feeding insects are very specific in their mode of feeding and present a unique stress on plant fitness. Not only do these insects feed for long periods of time on host plants, but they also act as vectors for plant viruses. The Brown planthopper (BPH)-Rice and Aphid-Arabidopsis systems provide good models for studying the induced responses in plants to phloem-feeding insects. Results: In BPH-rice interaction studies, the results showed that 29% of differentially expressed genes in response to BPH feeding were involved in stress responses in plants. Of particular interest was the differential expression of genes encoding the pathogen related proteins β-1,3-glucanase 1, 2 and 5 genes and genes encoding callose synthase 1, 3 and 5. QRT-PCR results have shown that genes encoding callose synthase 1 and 5 (GSL1 and GSL5) were highly expressed in both the moderately resistant IR64 and the resistant IR70 rice cultivars; they were however down regulated in the BPH susceptible cultivar TN1. Similarly, genes encoding the GTP binding protein were expressed to higher levels in cultivars IR64 and IR70 in response to BPH feeding, compared to TN1. In contrast, genes involved in callose degradation, namely β-1,3-glucanase genes 1, 2 and 5 (Gns1/Osg1, Gns2 and Gns5) were highly expressed in the susceptible cultivar in response to BPH feeding; Osg1 and Gns2 were not expressed in either IR64 or IR70, while β- Gns5 was down regulated in both resistant cultivars, compared to the susceptible cultivar (TN1). This differential gene expression in response to BPH feeding might suggest an important role for these genes in plant defence against phloem-feeding insects. Further studies demonstrated that the exogenous application of hydrogen peroxide to the susceptible rice cultivar TN1 improved resistance of this cultivar to BPH to moderate. GTP binding protein, Callose synthase GSL1 and GSL5 genes were up-regulated, while β-1,3-glucanase genes Gns1, 2, 3 and 5 were down-regulated in response to BPH feeding, suggesting that reactive oxygen species generated under hydrogen peroxide treatment might play a role in bringing about the responses leading to resistance. In aphid-Arabidopsis interaction studies, aphid bioassays showed that oxidative signal inducible protein kinases (Oxi1 serine-threonine MAPKs), β-1,3-glucanase Gns1, Gns2 and Gns3 mutants were resistant to aphid feeding and they could survive until the seeding stage when infested. However, Camta3-1, Camta3-2 (calmodulin binding transcription activators), and the Oxi1 null mutant (oxidative signal inducible with no-function) died in response to aphid infestation before reaching the seeding stage. Furthermore, Col-0 (Columbia) and WS2 (Wisconsin) wild type backgrounds for Oxi1 and Oxi1 null mutant respectively, died quickly under aphid feeding. Gene expression analysis using QRT-PCR on the aphid resistant Oxi1 mutant and the susceptible parental line demonstrated that transcripts for callose synthase gene GSL5 were expressed at a higher level in the Oxi1 mutant compared to Col-0. Whilst β-1,3-glucanase Gns1, 2, 3 and 5 genes were down-regulated in the Oxi1 mutant in response to aphid feeding, β-1,3-glucanase Gns2 gene was induced in Col-0 to high levels in response to aphid feeding. Application of hydrogen peroxide putatively induced the oxidative inducible signalling (Oxi1 serine-threonine) MAPKs. Induction of Oxi1 stimulated callose production probably via a Ca2+ signalling pathway. Application of hydrogen peroxide to Col-0 improved the resistance level of this susceptible line in response to aphid feeding. Transcript expression analysis demonstrated that GSL5 was expressed at high levels in response to aphid feeding, while β-1,3-glucanase Gns2 gene was down-regulated in response to hydrogen peroxide treatment. In addition Gns1, 3 and 5 genes were not expressed in response to aphid feeding. Interestingly, hydrogen peroxide increased the susceptibility of the Oxi1 mutant to aphid attack. Conclusion: β-1,3-glucanase Gns2 gene might play an important role in plant susceptibility to phloem feeding insects in both monocots and dicots. Evidence from the present study suggests that callose synthase GSL5 plays an important role in plant defence against insects and may be a key gene in insect/wound response in plants. The application of hydrogen peroxide induces Oxi1 serine-threonine MAPKS and increased callose production via a Ca2+ signalling pathway and caused a down-regulation of β-1,3-glucanase Gns 1, 2, 3 and 5 genes. Over expression as well as down-regulation of Oxi1 may increase plant susceptibility to phloem feeding (BPH-aphids) insects suggesting that specific levels of Oxi1 are required.

The extensive applications of insecticides in agriculture and public health require appropriate methods to monitor their ecological and toxicological effects on target and non-target organisms. C.elegans was used as a model organism in this project as it has been successfully used to assess the toxicity of environmental pollutants including those in contaminated soil. C.elegans was used here to test various concentrations of insecticides/nematicides on wild-type and mutant worms. Direct observation and counting of pharyngeal muscle contraction was carried out and showed that incubation with anthelmintic or insecticide produced a concentration dependent inhibition of pharyngeal pumping in control animals. Results obtained showed that the CCA-l T-type calcium chatmelmay be such a target for pyrethroids and DDT. Worms lacking functional CCA-l (strain JD21) were less sensitive to both DDT and deltamethrin compared with wild-type N2 worms as pharyngeal pumping was reduced by DDT in N2 and JD21 st rains with ICso values of 909.2ppm and 9942ppm respectively and by deltamethrin with ICso values of 877.5ppm and 50527ppm respectively after a 1 h treatment. JD21 won11S were also more motile compared with N2 worms. EAT-2, an acetylcholine receptor subunit, may be affected by levamisole which is an acetylcholine receptor agonist, as DA465 worms lacking EAT -2 were less sensitive to levamiso le compared with N2 especially at lower concentrations.

The work presented in this thesis documents studies on the impact of RNA interference (RNAi) on potential control target genes and how this alters the phenotype of the pre-parasitic stage of the plant parasitic nematodes (PPN), Globodera pallida and Meloidogyne incognita. A wide range of targets were selected across all the main tissue types within the worms (hypodermis, muscle, nervous system, subventral gland, dorsal gland) and significant knockdown was achieved (using short-interfering [si]RNAs) in all tissue types except the dorsal gland. Overall, the data strongly support the utility of PPN J2s as a model for functional genomics studies using RNAi. Further work was carried out on selected targets including the neuropeptide-encoding genes, Gp-flp-30 and Gp-flp-31 which are thought to be only expressed in PPN species. These targets showed reduced target transcript levels following RNAi, however not to the same degree as most of the previously targeted genes; their silencing did not induce a significant phenotype suggesting they are involved in functions other than normallocomotion/chemotaxis. We also targeted Gp-flp-21 and its putative receptor encoding gene Gp-flp-21 R which in Caenorhabditis elegans modulate sociality. Silencing either gene did not alter PPN motility but did disrupt positive chemotaxis, consistent with a role in chemosensation and consistent with the FLP-21 R being the FLP-21 receptor. Finally, the work reported here has shown that silencing Gp-ace-2, which encodes an acetylcholinesterase, results in complete paralysis of the parasites and prevents them from infecting their host plant and thus complete their life cycle. These data provide functional validation for a variety of PPN control targets.

Parasitic nematodes infect plants and animals to undermine global food production. They are the causative agents of a range of neglected tropical diseases, inflicting significant human morbidity and mortality, predominantly within the world's most impoverished communities. The development of anthelmintic resistance threatens the primary mode of parasite control and necessitates the development of alternatively management strategies. This thesis incorporates bioinformatic and reverse genetic analyses in an attempt to investigate the neuromuscular biology of parasitic nematodes. Firstly, a reciprocal BLAST-based gene mining methodology was employed to generate a pan-phylum view of FMRFamide-like peptide (flp) and flp-receptor gene diversity, revealing previously unrecognised variation within the FLPergic gene profiles present within the genomes of parasitic nematodes. These new data identify the most highly conserved neuropeptide ligand and receptor encoding genes within the FLPergic signalling system and facilitate future efforts to deorphanise and therapeutically exploit nematode FLP-receptors for parasite control. Secondly, this thesis attempts to optimise a gene silencing platform for the validation of novel anthelmintic targets within the neuromuscular signalling system of the model parasite Ascaris suum. The data presented characterise the spread and development of RNA interference (RNAi) within adult A. suum and demonstrate significant and potent transcript knockdown for multiple tissue-specific gene targets post-dsRNA injection. Despite this 100 % success rate, no post-RNAi phenotypes were recorded over the course of these investigations. This was inspite of efforts to: (i) target putatively essential genes; (ii) employ a highly quantitative electrophysiology-based phenotypic assay; (iii) improve the RNAi trigger delivery methodology; and (iv) develop a novel strategy to uncover a post-RNAi phenotype. The data described here highlight the potential utility of RNAi in adult A. suum and warrant further investigation

This thesis reports the results of investigations concerning the potential range and global colonisation ability of selected insect pests of oilseed rape (<I>Brassica napus</I> L.). The Scottish distribution, abundance and phenology of three pest species, the cabbage seed weevil, <I>Centorhynchus assimilis</I> Payk (Coleoptera. Curculionidae), the brassica pod midge, <I>Dasineura brassicae</I> Winn.

With the aim of finding simple and effective disease-control measures, laboratory experiments described in this thesis tested the effects of various cations (Ca²⁺, Mg²⁺, K⁺) and potentially fungitoxic compounds (saponins, gramicidin S, ethanol) on different stages of the life cycle of two pathogenic zoospore-forming fungi, <i>Pythium aphanidermatum </i>and <i>Phytophthora parasitica.</i> Also, nutrient irrigation solutions from various experimental treatments in a large tomato-cropping glasshouse trial at Horticultural Research International (HRI, Yorkshire) were tested for effects on the two fungi in laboratory conditions. Cations, fungitoxic compounds and experimental irrigation solutions were tested for effects on the following aspects of fungal behaviour: mycelial growth, production of sporangia, release of zoospores from sporangia, zoospore motility, zoospore encystment and cyst germination. When tested on individual stages of the life cycle, high concentrations of Ca²⁺ and Mg²⁺ in nutrient broth reduced mycelial growth by <i>Py. aphanidermatum </i>but not <i>Ph. parasitica.</i> Sporangial production by <i>Ph. parasitica</i> in mineral salts solution was unaffected by amendments of Ca²⁺, Mg²⁺ or K⁺, but these amendments suppress the ability of sporangia to subsequently liberate zoospores into water; the exception was 5 mM Ca²⁺ which was markedly enhanced subsequent zoospore release. Increasing concentrations of Ca²⁺, Mg²⁺ and K⁺ in the solution that bathed pre-formed sporangia of either fungus reduced the number of zoospores that were released. These three cations also suppressed the proportion of zoospores that remained motile, and increased the proportion of vortex-encysted zoospores that would germinate. When tested cumulatively on all stages of the life cycle K⁺ was more effective than Ca²⁺ in suppressing the infection related behaviour of both fungi. These experiments suggest that increasing the ratio of K⁺ to Ca²⁺ in bathing solutions should suppress the ability of both fungi to spread and cause disease. The trial undertaken by HRI failed to demonstrate natural suppression in a semi-commercial irrigation system, which was the principal trial objective. Consequently the analyses presented in this thesis were unable to determine at which points of the infection sequence the agents of natural suppression might act.

Despite the significant progress achieved in molecular biology in the last few years, our knowledge about the mechanisms of plant resistance against necrotrophic and non-host pathogens is still rudimentary. Here, we report the isolation and characterization of three Arabidopsis mutants selected for altered resistance against B. cinerea. Mutations in the Increased Botrytis Resistance (IBRJ) gene resulted in significant resistance against B. cinerea and A. brassicicola, another necrotrophic fungus. Interestingly, ibri plants also exhibited enhanced susceptibility to the host and non-host bacterial pathogens P. syringae pv. tomato and P. fluorescens, respectively. Conversely, resistance against B. gram mis f. sp. tritici and E. cichoracearum was not affected, suggesting JBRJ is required for resistance against some, but not all pathogens. By TAIL-PCR IBRJ gene was found to be allelic to asymetric leaves 1 (as1). AS1 belongs to the R2R3 subfamily of the MYB-domain and has been extensively studied because of its central function in leaf development. Interestingly, neither as2 or erecta mutants or the conditional line KNJ are affected in resistance against B. cinerea or PstDC3 000. These results suggest that as1-mediated disease resistance and susceptibility is independent from the AS1 -dependent pathway regulating leaf development. Moreover, we show that AS1 function in disease resistance & susceptibility is conserved in at least three evolutionary divergent plant species, suggesting AS1 function in disease resistance is relatively ancient. The analysis of a substantial series of as1 double mutants in Arab idopsis revealed that both the production and perception of jasmonate (JA) and ethylene (ET) are required for as1-mediated disease resistance against necrotrophic pathogens. Moreover, the expression of JA/ET-dependent defence genes was shown to be accelerated in as1 plants in response to B. cinerea challenge. While as1 resistance against necrotrophic pathogens seems to be associated with enhanced expression of JA/ET - dependent genes, susceptibility against the different strains of host and non-host bacterial pathogens occurred in the presence of normal PR-1 expression and salicylic acid accumulation, suggesting susceptibility towards bacterial pathogens is not associated with defects in the activation of SA signalling pathway. In sum, our findings indicate that AS1 is a negative regulator of resistance against necrotrophic fungi and a positive regulator of nonhost resistance and basal protection against bacterial pathogens in both Arabidopsis and other plant species. In contrast to ibr1, the ibr2 and ebsl (Enhanced Botrytis susceptible1) mutants seem to specifically affect resistance against B. cinerea. The ibr2 was mapped to a short interval on chromosome five. Despite ibr2 cloning is not accomplished, we speculated that the mutation is in an alpha-tubulin gene because ibr2 plant morphology resembles the phenotype of the previously characterized lefty1 and lefty2 mutants, which also encode alpha-tubulins. Our speculation is supported by the presence of two alpha-tubulins in the mapped region on chromosome 5, close to the predicted location of ibr2. The Botrytis susceptible line ebs1 co-segregates with basta resistance, indicating that the mutated gene is tagged. However, the T-DNA inserted contains additional sequences on its left border that prevented successful cloning. The ebs1 plants show impaired resistance against B. cinerea, but unaffected resistance against virulent and avirulent strains of PstDC3 000, suggetsing EBS1 plays a more specific role in resistance against necrotrophic pathogens. Further characterization of ibr2 and ebs1 is required in order to elucidate their role in plant disease resistance.