The nutrition and physiology of the mycoparasites <I>Pythium oligandrum,</I> <I>P. mycoparasiticum </I>and <I>P. acanthophoron</I> differed from that of plant pathogenic <I>Pythium </I>spp., but with minor variations between strains and species of the mycoparasites. <I> P. oligandrum,</I> <I>P. mycoparasiticum </I>and <I>P. acanthophoron </I>did not use inorganic nitrogen. <I>P. oligandrum </I> and <I>P. mycoparasiticum</I> required exogenous thiamine, whereas <I>P. acanthophoron </I>was self-sufficient for thiamine. The mycoparasites grew on mannitol as a carbon source in the presence of cholesterol, calcium or both, but the effects of calcium and cholesterol differed between the mycoparasites, and isolates of <I>P. mycoparasiticum </I>were intolerant of ethanol, used as the solvent for cholesterol. <I>P. oligandrum. P. acanthophoron </I>and <I>P. mycoparasiticum,</I> tested as single strains, showed different growth responses to ergosterol, cholesterol and β - sitosterol. These mycoparasites were less tolerant of elevated NaC1 concentrations than were <I>P. ultimum </I>and <I>P. aphanidermatum</I>, and grew much better in complex undefined media than in defined media. Of various media tested, 3% molasses gave maximum oospore production, Oospores of <I>P. oligandrum </I> and <I>P. acanthophoron </I>showed maximum 17 - 19% germination after 18 h incubation on agar at 25<SUP>o</SUP>C, when oospores were harvested from 5-week cultures on molasses medium. The apparent viability of oospores, determined by tetrazolium bromide stain, was 75% and 90% respectively, contrasting with the low germinability. Oospores required an ageing period of 35 days, which was nutrient-independent, before they showed maximum germination. Storage of oospores in air-dried culture biomass reduced their germinability. Of many treatments used, the maximum percentage germination (<I>c.</I>30%) was obtained after treatment with 0.001% potassium permanganate. However, culture-produced oospores of <I>P. mycoparasiticum</I> consistently failed to germinate and seemed to be non-viable when stained with tetrazolium bromide. The low germinability of oospores of mycoparasitic <I>Pythium </I>spp. in general, especially after dry storage, could be a major limitation to their use as biocontrol agents of plant pathogens.

No description available.

The species <i>Eucalyptus camaldulensis </i>Dehnh. is of great importance in Mediterranean, sub-tropical and tropical countries for the production of domestic products, such as poles, posts timber and fuelwood. Some members of the genus <i>Eucalyptus</i> are reported to form both arbuscular- (AM) and ecto-mycorrhizas (EM). The main objectives of this study were to look at the host-symbiont interactions between <i>E. Camaldulensis</i> and AM and EM fungi, and interactions between the two mycorrhizal types. The initial aim of the project was to determine suitable experimental conditions for the formation of both types of mycorrhizas on <i>E. camaldulensis</i> seedlings. Two experiments, the first with AM fungi and the second with EM fungi, were set up successively using vermiculite-peat (VP) and sand-perlite (SP) as growth media, and 10 mg 1^-1 and 30 mg l^-1 phosphorus (P) Ingestad's nutrient solution in each case. <i>Glomus intraradices</i> Schenck and Smith. isolate UT 143-2 and <i>Pisolithus tinctorius</i> (Pers.) Coker and Crouch isolate PTE were used as the test AM and EM fungus respectively. Results showed that both AM and EM colonisation were very low (1-6%) but even so AM inoculation had a significantly depressive growth effect on seedlings of <i>E. camaldulensis.</i> In both experiments VP was found to be the best medium for both the growth of seedlings and the formation of mycorrhizas. A subsequent experiment using one nutrient concentration (5 mg 1^-1 P) and three AM and six EM isolates with VP as the growth medium resulted in colonisation of up to 20% by two AM fungi (<i>Glomus clarum</i> Nicolson and Schenck. isolate BR148-1 and <i>Gigaspora rosea</i> Nicholson and Schenck isolate FL105-5) but none of the EM fungi used in that experiment formed any mycorrhizas. The fourth experiment using three AM inocula (including two from the previous experiment and one from a trap culture of Bangladeshi soil) and four nutrient regimes (Ingestad's 2.5,5.0,10 and 20 mg 1^-1P) resulted in 30-50% colonisation; most colonisation was by <i>G clarum</i> BR148-1 and was greater at 10 mg 1^-1P (>50%). AM colonisation again resulted in a negative growth response of <i>E. camaldulensis </i>seedlings. In a similar experiment using five isolates of <i>P. tinctorius, </i>only isolate K55 resulted in colonisation >15% most of which occurred at 2.5 mg 1^-1 (>25%) while the other isolates resulted in <1% colonisation.

The aims of this research were to analyze the Ca²⁺ and physiological responses to mechanical perturbation and hypo-osmotic shock in <i>Neurospora crassa. </i>Cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) was measured by expression of codon optimized aequorin in wild type and deletion mutants in which genes encoding different components of the Ca²⁺ signalling machinery had been deleted. The [Ca²⁺]_c responses of germ tubes, vegetative hyphae and conidia were characterized. Ca²⁺ signatures produced in response to mechanical perturbation or hypo-osmotic shock were analysed to identify which components of the Ca²⁺ signalling machinery were responsible for generating these signatures. The involvement of multiple proteins in the [Ca²⁺]_c responses to mechanical perturbation and hypo-osmotic shock of germ tubes was identified. The Ca²⁺ signature and germ tube swelling produced in response to mechanical perturbation were both dependent on the influx of external Ca²⁺ and the MID1 mechanosensory protein. The plant antifungal proteins (defensins), MsDef1, RsAFP2, MtDef2, and MtDef4, were all found to have distinct, stimulus specific effects on the [Ca²⁺]_c responses to mechanical perturbation and hypo-osmotic shock. The mycovirus antifungal protein KP4 exhibited no inhibitory effect on the [Ca²⁺]_c response to either stimulus. This analysis provided the basis for the development of a high throughput assay for the discovery of antifungal compounds that target Ca²⁺ signalling and homeostasis.

The primary aim of this thesis was to isolate temperature sensitive genetic mutants of Schizosaccharomyces pombe which are defective in DNA replication. 30 presumptive. (DNA) mutants were isolated after nitrosoguanidine mutagenesis on the basis of their having a defect in the S phase period of the cell cycle. The mutations are all recessive and were allocated to 9 unlinked nuclear genes (cde 10, 17, 18, 19, 20, 21, 22, 23, 24). Physiological characterization revealed that (1) Mutants of cdc 10, 20, 22 are probably defective in the initiation of S phase, (2) mutants of cdc 21, 23 are defective in the process of DNA synthesis, (3) mutants of cdc 18, 19 are not defective in bulk DNA synthesis and their to functions are completed prior to or independently of S phase at the permissive temperature, and (4) mutants of cdc 17, 2.4 are not defective in bulk DNA synthesis but their is functions cannot be completed at the permissive temperature if DNA synthesis is inhibited by hydro urea. cdc 17-K42 possesses 41 major thermo-sensitive phenotypes: a is lethality, sensitivity, a defect in the joining of nascent DNA strands, and an abnormally high level of DNA ligase. The DNA ligase assays suggest that this mutation is located in the structural gene for DNA ligase. Mutants of cdc 24 are also defective in the joining of nascent tWA strands, but at a much higher molecular weight level. The secondary aim of this thesis was an analysis of the control of S phase in S. pombe. This was investigated by growth of cells under nitrogen limitation in a chemostat at different growth rates. Only the bl phase of the cell cycle is extended as the generation time is increased under these conditions. The initiation of DNA replication may be dependent on the cell attaining a critical size. Several techniques necessary for a study of DNA replication in S. pombe were specially developed during this work.

The membrane-bound enzyme, inositol phosphorylceramide synthase (IPC synthase), encoded by <i>AUR1, </i>has been identified in yeast and fungi. Since IPC synthase is unique to fungi, there is an opportunity for development of a species-specific enzyme inhibitor that might have low toxicity to the host for use as a fungicide. Michaelis-Menten kinetic parameters have been determined for <i>S.</i> <i>cerevisiae </i>IPC synthase using Triton X-100 solubilised microsomes (phosphatidylinositol K_M 357µM and fluorescent ceramide, N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-ceramide K_M 5.22µM). The fluorescent ceramide was found to exhibit substrate inhibition at high concentration (K_I 125.69µM). When the substrate inhibition parameter was considered the apparent K_M of N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-ceramide increased to 11.42µM. Both <i>B. cinerea </i>and <i>S. cerevisiae </i>Aur1p have been cloned, recombinantly overexpressed and purified using affinity tags. <i>B. cinerea </i>Aur1p was overexpressed in <i>E. coli </i>Top 10™ cells in inclusion bodies as an N-terminal Glutathione-S-transferase fusion. However, IPC synthase activity was low and expression in <i>P. pastoris </i>was investigated. A large number of constructs were prepared and successful intracellular overexpression was achieved for both <i>B. cinerea </i>and <i>S. cerevisiae </i>Aur1p in KM71H cells (Mut^S) as C-terminal c-myc His₆ fusions. Although expression was achieved with both full length and N-terminally truncated forms, the full length proteins had greater activity. <i>B. cinerea </i>Aur1p was far more active when expressed in <i>P. pastoris </i>rather than <i>E. coli. </i>Native <i>S. cerevisiae </i>IPC synthase was found to be functionally glycosylated with high mannose content and the increased activity is probably due to the correct processing and post translational modifications occurring in the eukaryotic system. Native IPC synthase activity was demonstrated in <i>P. pastoris </i>and an isolation method based on affinity chromatography has been identified for native <i>P. pastoris </i>and <i>B. cinerea </i>Aur1p.

No description available.

No description available.

This work is concerned with the molecular analysis of ascomycete mating type genes of various <I>Sordaria</I> species. Work previously published has reported the cloning and characterisation of mating type genes from several <I>Neurospora </I>species. In heterothallic species the genotype at the mating type locus (mtA or mta) determines the mating type. Homothallic species, which proceed through the sexual cycle without the need to mate, have no obvious mating types but molecular analysis has been used to demonstrate the presence of mating type genes in species with this life cycle. <I>Neurospora </I>species and <I>Sordaria </I>species both belong to the Sordariaceae and are closely related. Several λ clones containing putative <I>Sordaria</I> mating type genes from heterothallic and homothallic species had been isolated previously using <I>N. crassa </I>mtA and mta probes. In this study the mtA-1 gene of the heterothallic species <I>S. sclerogenia </I>was subcloned from a λ clone and sequenced. The equivalent gene from <I>S. equina </I>(a homothallic species containing only the mtA sequence) was also subcloned and sequenced. A λ clone for the species <I>S. fimicola</I> was found to hybridise with both the mtA and mta probes. <I>S. fimicola </I>is a homothallic species containing mtA and mta in the same nucleus. On using the lambda clone it was found that the mtA and mta genes are linked in this species. All the <I>Sordaria</I> mtA-1 genes contained putative DNA binding domains, α domains. The mta-1 gene sequenced from <I>s. fimicola </I>contained a putative HMG box. The <I>S. equina</I> mtA-1 gene was expressed in a sterile <I>N. crassa </I>mta mutant and was found to restore mating type function to the mutant. The mtA-1 gene did not however confer homothallic behaviour on the recipient mutant. <I>S. equina </I>and <I>S. sclerogenia </I>contain a 59bp common region following on from the mtA-1 gene which is conserved in both these species and in <I>Neurospora </I>species. A variable region continues on from the common region in <I>S. equina </I>and <I>S. sclerogenia </I>and in <I>Neuropora </I>species.

This member of the Muoorales was first found by Schröter (1) near Baden in 1677, and again in 1879, growing on Paxillus involutus. In 1904 it was found on Gomphidius visoidus in the Peloponnese and sent to Vuillomin (2), and in 1927 Ling-Young (3) found it on Boletus soaber in the forests of the Puy do Dame. In addition, an unnamed species of Dicranophora found by Thaater on Boleti at Kittery Point, Maine, U. S. A., was briefly described by Blakeslee (4) in his classic paper of, 1904. These are the only recorded appearances of the fungus in nature, up to the present. The accounts referred to above are short and incomplete and differ on a number of, point$. Blakeslee and Ling-Young were oonoerned only with the sexual stage. Vuillemin, whose paper is the only one published on Dioranophora alone, dealt chiefly with the sporangial stage. The ohief charaoteristios of the fungus, as described by Sohröter (5), may be summarised thus. -- Protoplasm yellowish-red. Sporangia of two types, (1) large, with conical columolla, and small elliptical spores, and (2) small, with 2- or 3-pointed forked columella and 1-2 much larger reniform spores. Zy gospore spherical, chestnut-brown, smooth or with fine warts, suspensors very unequal, the one swollen, the other thread-like.