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To ascertain phylogenetic relationships in the genus, the ITS region (internal transcribed spacers) of 18-26S nuclear ribosomal DNA repeat was sequenced for 32 species (35 taxa) of <I>Bupleurum </I>(31 species sequenced for the first time). Sequences were obtained from all the species of the area study, with the exception of two (one a doubtful taxon). Taxa from all the currently most generally accepted sections (<I>Bupleurum, Reticulata, Diaphyllum, Isophyllum </I>and <I>Tenoria</I>) and subsections of the genus were included. As an outgroup, sequences of two species of another basal genus of <I>Apioideae,</I> <I>Anginon</I> Raf., were also obtained. In total 68 samples were sequenced which allowed confirmation of sequence by replication for several of the taxa, and provided information on the number of nucleotide changes found within taxon. A phylogeny derived from cladistic analysis of the aligned ITS sequences clearly shows the division of the genus in two main clades (100% supported in the strict consensus tree). This division is supported also by the analysis of the 5.8S sequence alone (conserved gene between ITS1 and ITS2). Based on these results, which are almost completely supported by morphological characters, two new subgenera are proposed: <I>Bupleurum </I>and <I>Penninervia comb. nov. </I>The proposed new subgenus <I>Penninervia, </I>a more basal group, includes all pinnate-reticulate veined species of the genus (<I>B. angulosum </I>L., <I>B. foliosum </I>Salzm. ex DC., <I>B. fruticosum </I>L., <I>B. gibraltarium </I>Lam. and <I>B. stellatum </I>L.), and also <I>B. rigidum </I>that has itself a unique type of venation. The association of these species has never been proposed before (not even the pinnate-reticulate species alone). Subgenus <I>Bupleurum</I>, as defined here, includes all the taxa with typical parallel-veined leaves, and represents the vast majority of the species of the genus. The groups presented within each subgenus are informally treated as neither morphological or sequence data are conclusive and therefore no formal rank is attributed. Final cladistic analysis including sequences from 4 other basal <I>Apioideae </I>genera (outgroup: <I>Anginon, Heteromorpha </I>Cham. and Schltdl., <I>Pleurospermum </I>Hoffm. and <I>Physospermum </I>Cusson ex Jussieu), confirms <I>Bupleurum </I>as a monophyletic group. In this analysis, <I>Heteromorpha </I>appears as the closest genus, with <I>Anginon </I>being the most divergent.

In laboratory experiments, Ca²⁺, other divalent cations and various Ca²⁺-modulating treatments were tested for effects on germination of zoospore cysts and of sporangia of three isolates of <i>Phytophthora infestans</i> and sometimes for an isolate of<i> P. palmivora. </i>The aim was to determine whether manipulation of Ca²⁺ or Ca²⁺-mediated events has the potential for control of potato late blight caused by <i>P. infestans. </i>This work was based on previous evidence for a central role of Ca²⁺ in the infection sequence from zoospores of phytopathogenic <i>Phytophthora, Pythium </i>and <i>Aphanomyces </i>spp. Zoospore cyst germination of both <i>P. infestans </i>and <i>P. palmivora</i> was suppressed by early post-encystment addition of the chelators EGTA or BAPTA, indicating a requirement for external Ca²⁺ or other polyvalent cations, by calcium channel blockers (La³⁺, Gd³⁺, verapamil) or by amiloride, indicating a requirement for flux of Ca²⁺ or other ions across the cyst membrane, by calmodulin antagonists (calmidazolium, dibucaine, trifluoperazine) and by intracellular calcium antagonists (caffeine, TMB-8), indicating a role for both calmodulin and for Ca²⁺ release from intracellular stores. Supplements of Ca²⁺ or other divalent cations (Ba²⁺, Mg²⁺) also suppressed cyst germination, but sometimes partly or completely overcame the suppression by other treatments if applied early as post-treatments. Germination of sporangia of <i>P. infestans</i> by hyphal outgrowth (direct germination, at <i>c. </i>20°C) or zoosporogenesis (indirect germination, at <i>c.</i> 12°C) was suppressed by the same treatments as applied to zoospore cysts. These treatments sometimes caused rapid sporangial death, assessed microscopically by irreversible changes in cytoplasmic organisation. Their suppressive or toxic effects were generally more pronounced when sporangia were incubated to induce indirect rather than direct germination. The suppression caused by Ca²⁺-modulating treatments could be rescued only partly by simultaneous or early post-application of divalent cations. Even potentially mild chemical treatments (0.1% pectin or 5 mM orthophosphate) caused rapid (20-30 min) death of sporangia (especially when incubated for indirect germination). Simultaneous or early post-treatments with divalent cations could only partly reverse the suppression.

The aims of the research were to develop and apply live cell imaging techniques using confocal microscopy to image and analyse vesicle trafficking, organelle dynamics and molecular localization at high spatial and temporal resolution in filamentous fungi. The dyes FM4-64, and to a lesser extent, FM1-43, were used to analyse vesicle trafficking, and as general probes to image hyphal tip growth, branching, septum formation, hyphal fusion, conidiophore development and the early stages of protoperithecium development. <i>Neurospora crassa </i>was the main system studied, although a taxonomically diverse range of other species were also analysed. Uptake of FM4-64 into hyphae was shown to be time- and energy-dependent, consistent with internalization being mediated by endocytosis. FM4-64 proved to be an excellent vital stain for different cell components including the apical vesicle cluster (AVC), vacuolar network and mitochondria. The dye was used to compare the morphology of the AVC in 15 different species. The green fluorescent protein (GFP) is a recombinant fluorescent probe that can be targeted to organelles and used to label specific proteins within living cells. Confocal microscopy was used to image transformants of <i>Aspergillus nidulans</i> expressing GFP targeted to the vacuolar network, mitochondria, nuclei, spindle pole bodies, endoplasmic reticulum, and Golgi cistemae. In addition, a range of vital fluorescent dyes were used to image organelles, and results from these studies were compared to those obtained with GFP transformants. Vacuolar staining revealed an extensive tubular network in actively growing regions of hyphae, whereas in sub-apical regions large spherical vacuoles were observed. Division of a single mitochondrion was imaged in <i>Aspergillus nidulans</i> expressing GFP targeted to mitochondria. Potentiometric dyes (especially rhodamine-123) indicated that the membrane potential of mitochondria in growing hyphal tips was higher than that in sub-apical regions.

This thesis describes investigations of DNA synthesis in the myxomycete, Physarum polycephalum, using an isotope dilution technique capable of measuring macromolecular synthesis directly, in contrast to methods which follow the incorporation of a radioactive precursor. Using petri dish and large surface cultures, the pattern of either nuclear or total DNA accumulation during the mitotic cycle was determined in three media with cycle lengths varying between 9 and 12 h. In common with previously published results, no G1 phase was detected. Statistical analysis suggested that, within the range examined, changes in cycle length were confined mainly, if not solely, to G2, S phase remaining constant at approximately 120 min. During G2 an amount of synthesis was detected which ranged from 10 to 27% of nuclear DNA, more than could be due to nucleolar DNA. In most cases, DNA content showed less than a doubling between divisions, and possible causes of this and of G2-synthesis are discussed. Examining DNA synthesis in the presence of cycloheximide which at 20 mug per ml could, within 15 min, inhibit by 95% the incorporation of carbon-14 labelled lysine, it was found that (i) inhibition of replication was detectable after 20 min exposure but, at shorter times, the method employed was insufficiently sensitive to detect inhibition consistently; (ii) residual synthesis of approximately 15 - 20% of total or nuclear DNA can occur during 3 h exposure to cycloheximide (20 mug per ml) and (iii) approximately 50% inhibition of protein synthesis by 0.25 mug per ml cycloheximide produced no detectable effect on the level of DNA synthesis. Other experiments described are: (i) preliminary attempts to construct an in vitro replicating system from plasmodial homogenates, assaying synthesis by isotope dilution; (ii) measurements in small plate cultures of macromolecular synthesis between inoculation and M1, and DNA synthesis within the inoculum region; (iii) determinations and statistical analyses of the growth rate of microplasmodia and surface plasmodia under various conditions.

Physarum polycephalum is a Myxomycete which grows as uninucleate amoebae or multinucleate plasmodia. The aim of this work was to isolate mitotic cycle mutants in an apogamic strain (CLd) of P. polycephalum and to exploit the natural mitotic synchrony of plasmodia for their analysis. The first stage (Chapter 3) involved isolation of temperature-sensitive growth mutants. Such mutants can be isolated by testing amoebal clones or plasmodia derived from amoebal clones. Testing amoebae is much easier, but previous work indicated that most mutations expressed in the plasmodial phase could only be isolated by testing plasmodia. The evidence, however, was equivocal, so both methods were used to isolate mutants, and their merits compared. It was concluded that over 70% of mutants isolated were expressed in both amoebae and plasmodia. Thus, mutations expressed in plasmodia can be isolated by testing amoebae. The second stage (Chapter 4) involved identification of mitotic cycle mutants amongst the temperature-sensitive mutants. Asynchronous cultures of amoebae or plasmodia of the mutants were transferred from permissive to nonpermissive temperature and observed microscopically. Of more than 100 mutants screened, three had altered mitotic indices and one was defective in cell division. The third stage (Chapter 5) involved preliminary characterization of these putative cell cycle mutants. Synthesis of macromolecules in plasmodia, and the DNA contents of plasmodial and amoebal nuclei were measured. These measurements were complemented by time-lapse cinematography of amoebal cultures (Chapter 6). It was concluded that none of the four mutants was completely blocked in cell cycle progression Despite limited success, the results indicated that future attempts to isolate cell cycle mutants of P. polycephalum should be more successful.

This is the first study to integrate and correlate the effect of ecophysiological factors on the life cycle of Aspergillus flavus by carrying out complementary work on gene expression of the aflatoxin gene cluster, with growth, sporulation and phenotypic toxin production. This information was used to understand the role of ecological factors on key biosynthetic genes and examine the use of such information for control of aflatoxin production using RNA interference. Ecological studies showed the profiles for growth, sporulation and aflatoxin B1 (AFB1) production with optimum ranges of water activity (aw) and temperature for AFB1 production being identified. A. flavus grew faster at 0.99 aw at all temperatures, but optimally at 30-35°C. The highest amount of asexual conidia was produced at 0.95 aw followed by 0.90 aw and then 0.99 aw at all temperatures examined. Interestingly, the partitioning of AFB1 into biomass, medium and spores showed that at 0.99 aw, about 50% of the mycotoxin was present in the biomass and the medium, with very little present in the spores. However, as water stress was imposed there was a switch to a significantly higher channelling of AFB1 (about 45%) into the spores, especially at 0.95 and 0.93 aw levels. A microarray analysis was used to examine the effect of aw x temperature interactions on the relative expression of the aflatoxin gene cluster for the first time using A. flavus NRRL 3357. This showed that under mild stress conditions (20°C/0.99 aw) several of the cluster genes, in particular aflS and aflJ, were highly induced concomitant with high levels of phenotypic AFB1 production. Highest amounts of AFB1 were produced in all conditions where aflS expression was elevated. When the ratio between the normalised expression data of the aflS/aflR genes was generated, high ratios were obtained at 25°C and 30°C at 0.99 and 0.95 aw and low ratios at 25°C and 30°C at 0.90 aw. This is in agreement with the AFB1 production profile. Cont/d.

Chapter 1 describes deoxynivalenol and zearalenone mycotoxins as secondary metabolites of Fusarium fungi. Metabolism of Fusarium fungi is discussed in the light of deoxynivalenol and zearalenone biosynthesis. Consequences of the presence of toxic fungal metabolites in cereal products are highlighted. Chapter 2 focuses on production of material for degradation and binding studies i.e. gram quantities of both mycotoxins. Procedures of deoxynivalenol and zearalenone production by fermentation were successfully designed and executed. The identities of the compounds were confirmed by NMR. Additionally, radio labelled deoxynivalenol was produced using developed fermentation methods. Chapter 3 investigates degradation of deoxynivalenol and zearalenone under typical cereal processing conditions. Acidic and basic degradation models were developed and executed. Degradation products of deoxynivalenol and zearalenone were isolated and characterised by LC-MS and NMR. NMR time course experiments were successfully performed resulting in mechanistic and kinetic information of deoxynivalenol and zearalenone degradation. Chapter 4 presents studies on binding of deoxynivalenol to food components such starch and flour. Previously produced 14C-labelled deoxynivalenol served as a tracer during binding studies. Influence of extraction procedures on deoxynivalenol bound to starch was investigated. The existence of binding of deoxynivalenol to flour was observed. Speculations on the nature of binding were made.

Mycosphaerella graminicola is an important pathogenic fungus of the wheat that causes Septoria tritici blotch. The pathogenesis of M graminicola differs from most plant pathogenic fungi because M graminicola mostly infects the host via stomata, not by formation of appresorium. The latent and biotrophic period can last up to three weeks until a rapid collapse of mesophylls and the nectrotropic stage begins, indicating that toxins are being released. Attempts to isolate the secondary metabolites from four strains of M graminicola: IP0323, STII, STl6 and ST93, that could be associated with the toxin production has led to the isolation of MG 1, MG2 and anthranillic acid. It was noted that the strains produced a low amount of yield and low amount of metabolites, indicating that the genes are tightly regulated. Epigenetic modifiers 5-azacytidine and suberohydroxamic acid were treated to IP0323, with no new gene expression. The genome of M graminicola has been sequenced, and indicated that M graminicola has 10 polyketide synthase and 1 polyketide-nonribosomal peptide synthetase hybrid, which could be involved in pathogenicity. Three PKS were selected: MgPKS2, MgPKS8 and MgPKS9 for heterologous expression in Aspergillus oryzae. The heterologous expression with eGFP fused to the respective genes indicated that: MgPKS2 was not expressed in A. oryzae, probably due to the inability of A. oryzae to splice the five introns of MgPKS2; MgPKS8 was expressed, due to the positive eGFP result, however there was no new metabolite detected in the LCMS; MgPKS9 was not expressed, probably the same reason as MgPKS2. MgPKS9 was used to check the intron splicing ability of A. oryzae because there are only two putative introns in MgPKS9. It was observed that removal of both introns gave positive eFGP results and that the presence of Intron 1 halted MgPKS9 expression in A. oryzae. LCMS analysis of positive transformants of MgPKS9 without both introns however showed no new metabolite production. This indicated heterologous expression of M graminicola in A. OIyzae is not favourable probably due to these reasons: problems in introns splicing, incorrect protein folding, protein being degraded, lack of special ACPS and lack of unique starter unit.

Uridine uptake by Schizosaccharomyces pombe is by a single step reaction with a Km of 3.15x10-5M. If the uridine concentration in the medium exceeds 2mM, uridine uptake appears to be chiefly by diffusion. The rate of uptake does not appear to limit the rate at which 3H-uridine is incorporated into RNA. In synchronous cultures, the rate of uridine uptake parallels the rate of its incorporation, but changes in the uptake rate occur 0.1 cell cycle after those in incorporation rate. The specific activity of the 3H-uridine pool in such cultures remains constant. Inhibitors which alter the relationship between the rates of uptake and RNA synthesis alter the size of the uridine pool. The relationship between pulse length and the distribution of 3R-uridine in various species of RNA was examined electrophoretically. After 1 min. of labelling, all radioactivity was in the ribosomal precursor, heterodisperse and 4-5 S RNA fractions. After pulses of 10 min or longer, label is distributed in essentially a steady-state labelling pattern. In synchronous cultures, the rate of RNA synthesis doubles at the time of DNA synthesis. When cultures are treated with the inhibitors of DNA synthesis, 2'-deoxyadenosine and 1-phenylethanol, they go through a period of adjustment during which the rate of RNA synthesis no longer is proportional to the DNA content of the cell. After cells have adjusted, however, the relationship between DNA and RNA synthesis is restored. Following a step-down in culture conditions the various RNA species are transcribed in approximately the same proportions as in control cultures. But the total rate of RNA synthesis is depressed and the processing of ribosomal precursor RNA is inhibited. The results are discussed and possible control mechanisms are considered.