No description available.

This thesis described the experimental analysis of several aspects of multicellular development in <I>Sordaria brevicollis</I>, a filamentous ascomycete which has previously been considered to be exclusively heterothallic. Sexual reproduction in <I>S. brevicollis</I> requires the formation of protoperithecia, which must usually be crossed by fertilisation with spermatia of the opposite mating type before perithecial development can take place. The morphology of protoperithecia and crossed perithecia has been examined using a variety of microscopical techniques. Also described is the formation of two additional types of multicellular structure, which have not been characterised previously, uncrossed perithecia, and vegetative hyphal aggregates (VHAs). Uncrossed perithecia are produced by homokaryons of both mating types, although the phenomenon is more commonly observed in strains of <I>mt</I>A. The term is used to cover a number developmental stages, which range from enlarged protoperithecia, that form ostioles but exhibit no further differentiation, to perithecia which develop (at least) rudimentary necks. Although the majority of uncrossed perithecia are empty, a proportion of the fruitbodies which develop on <I>mt</I>A homokaryons have been found to contain a few ascospores, arranged in linear, 8 -spored asci. Homokaryotic spore production has never been observed in strains of <I>mta</I>. The genetic mechanisms underlying the development of ascospores in uncrossed perithecia have been examined using heterokaryon analysis. Heterokaryons constructed using spore colour mutants were used to show that meiosis and recombination occurred during the formation of ascospores in uncrossed perithecia, but the recombination frequency (calculated from the percentage of symmetrical M II asci) was lower than that from a comparable heterothallic cross. It was also shown that the frequency of third division overlap was significantly higher in the asci from uncrossed perithecia than in those from crossed perithecia. Observations based on homothallic and heterothallic development have been used to construct and evaluate two models for nuclear behaviour during the formation of protoperithecia. The evolution of homothallism and heterothallism in <I>Sordaria</I> and <I>Neurospora </I>has also been considered in the light of the facultative homothallism seen in <I>S. brevicollis. </I>

The aim of this study was to monitor changes in cytosolic free calcium ([Ca2+]c in filamentous fungi using codon-optimised aequorin under different conditions In order to analyse Ca 2+ signalling in these organisms, and to use the recombinant aequorin method in fungicide mode-of-action studies, and as novel toxicant biosensor. Calcium signalling is little understood in filamentous fungi largely because easv and routine methods for Ca 2+ measurement in living hyphae have previously been unavailable. Recently a new method for measuring cellular Ca 2+ based on using codon-optimised recombinant aequorin, has been developed and used throughout the present study. The calibration method to convert light detected from aequorin expressing strains into [Ca 2+]c concentrations was optimised and critically evaluated. It was concluded that codon-optimised aequorin can provide excellent qualitative measurements of fungal [Ca2+]c but that precise quantification of [Ca2+]c using this method need to be treated with caution. Three external stimuli (mechanical perturbation, hypo-osmotic shock and high external CaC12, but not hyper-osmotic shock) were found to transiently increase [Ca 2+]c levels and to generate specific [Ca 2+]c signatures. Different parameters of the Ca 2+ signature (rise time, amplitude and full width half maximum) were quantified. Transient [Ca2+]c increases were also observed in response to cold and heat shock. Using Ca2+ channel blockers (LaC13, KP4, ryanodine, nifedipine, TN1B-8, verapamil), the Ca 2+ chelator BAPTA, and [Ca2+]c agonists (A23187, caffeine and cyclopiazonic acid), it was shown that the [Ca 2+]c increases resulting from hypo-osmotic shock and high external CaCl2 are predominantly due to the influx of Ca 2+ from the external media through plasma membrane Ca 2+ channels. The [Ca 2+ 1C increases resulting from mechanical perturbation seem to arise from both extracellular and intracellular sources. My results indicate that filamentous fungi possess a number of the components of the calcium signalling machinery found in other eukaryotic cells.

No description available.

No description available.

One of the major aims of the study was to characterise spore germination using light microscopy and to investigate the role and relative importance of each of the three conidial cells for successful germination. Analysis of germ tube growth rate showed that there are two modes of germination: a conidium produces either a single germ tube or two slightly slower growing germ tubes. Results from scanning electron microscopy and fluorescence microscopy studies suggest that germ tube emergence is asymmetric and directed towards the substratum. A method was developed by which individual conidial cells could be selectively killed, using localised laser irradiation, a technique which has not been previously used with fungal cells. The data suggest that isolated apical and basal cells are able to germinate and form appressoria. The middle cell never germinated, even when it was the only living cell in a conidium. Furthermore, there was evidence that the apical cell, which germinates most frequently, exerts "apical dominance" over the basal cell. Very little is known about organelle morphology and distribution within living fungal spores, and even less is known abut organellar dynamics during spore germination. Another major aim of this study was to characterise organellar organisation and dynamics in living and potentially pathogenic conidia throughout germination. The analysis used confocal microscopy and vital stains. To obtain biologically meaningful results, an <i>in vitro</i> system which mimicked <i>in vivo</i> conditions as closely as possible was developed and optimised for confocal microscopy. A range of potentially vital dyes were screened as organelle stains in living conidia during germination. Nuclei (stained with SYTO 11), vacuolar compartments (stained with cDFFDA), mitochondria (stained with Rhodamine 123), and the apical vesicle cluster (stained with FM4-64) were identified and characterised during germination. Each cell was shown to contain a single nucleus that changed position within the cell during germination but otherwise did not exhibit extensive movement.

No description available.

<i>Pythium aphanidermatum</i> (Edson) Fitz., <i>Pythium catenulatum</i> Matthews and <i>Pythium dissotocum</i> Drechs. were compared for responses to amino acids and sugars in tests involving taxis, zoospore encystment and cyst germination <i>in vitro</i>. Comparisons were made between the fungi and for each fungus at these three stages of development, which are parts of the normal sequence leading to infection of host roots or colonisation of non-living substrata from zoospores. Other substances tested for induction of encystment or germination <i>in vitro</i> were cellulose film, crab shell, uronic acids or uronate-containing compounds, partially characterised polysaccharides from plants, root mucilages and cations. Motility attributes of the fungi were compared and, for <i>P. aphanidermatum</i> only, were characterised in the presence of several ions and compounds that interfere with calcium-mediated events (EGTA, dibucaine, trifluoperazine, lanthanum, verapamil, amiloride, A3187 and TMB-8). Amino acids were tested for competitive effects in chemotaxis assays <i>in vitro</i>. Calcium and EGTA were also tested for effects on adhesion of encysted spores to glass slides. Accumulation and encystment of <i>P. aphanidermatum</i> on detached wheat roots was studied on microscope slides, with or without prior treatment of roots with calcium alginate gel, methylene blue, alcian blue, India, ink ruthenium red and lectins that bind residues of fucose, D-glucose, D-mannose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Many experiments involved video-recording zoospore responses and subsequent analysis of videotapes. Evidence is presented for an effect of calcium and other divalent cations on zoospore motility patterns, cyst adhesion and induction of germination. Several amino acids and sugars elicited zoospore taxis, encystment, or cyst germination but sometimes differently for different fungi or at these different stages of development. Competition experiments enabled some of these responses to be related to proposed receptor functions, and in some cases amino acids could overcome inhibition of germination caused by the presence of calcium-modulators.

The role of external signals (particularly the substratum surface, light and nutrients) in regulating the pre-penetration phase of <I>Magnaporthe grisea</I> (Herbert) Barr [anamorph, <I>Pyricularia grisea</I> Sacc.] was analysed on rice leaves, artificial substrata and in liquid suspension. Surface contact was found to be essential for appressorium induction but not conidium germination. Both a high surface hydrophobicity and light favoured the formation of short differentiated germ tubes and large numbers of appressoria, but neither factor was essential for their induction. Light intensity had a graded effect on the lengths of differentiated germ tubes but not on the number of appressoria formed. No single sugar or nitrogen source was found to have a marked influence on appressorium formation. However, a high concentration of a complete nutrient source prevented differentiation in the dark but not in the light. Higher numbers of appressoria differentiated on rice leaves than on artificial substrata suggesting that the host provides additional factors, and thus a more conducive environment, for promoting appressorium formation. Overall, the results indicated that the pre-penetration phase of rice blast infection involves a programme of growth and differentiation triggered at conidium germination and regulated by multiple signals from the host and environment. The role of cytosolic pH (pH<SUB>c</SUB>) in growing germ tubes during conidium germination, germ tube growth and appressorium formation was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1).

The work in this thesis was designed to study the role of Ca<SUP>2+</SUP> in zoospore biology, and extends previous findings by comparing effects of Ca<SUP>2+</SUP> and Ca<SUP>2+</SUP> -modulating treatments in zoospores and zoospore cysts of <I>Pythium </I>spp., <I>Phytophthora </I>spp. and <I>Aphanomyces euteiches. </I>Zoospore motility was studied by videomicroscopy to compare the behaviour of <I>Phytophthora infestans, Ph. parasiticia </I>and <I>Ph. plamivora aphanidermatum, Py. dissotocum, Py. catenulatum </I>and <I>A. euteiches. </I>Zoospores of all fungi swam in an extended helix, but the <I>Phytophthora </I>spp. and <I>A. euteiches </I>made frequent changes of direction when swimming in water or buffer, whereas the <I>Pythium </I>spp. did not do so. Chelation of external Ca<SUP>2+</SUP> with EGTA or BAPTA buffer caused zoospores of all fungi to swim in a straight path, reduced random turning, and prevented the spores from flanging direction when they collided with obstacles. <I>Phytophthora </I>zoospores accumulated at the surface of suspensions, but this was abolished in the presence of EGTA, whereas <I>Pythium </I>zoospores always accumulated at the base of suspensions. Isotropic backgrounds of some amino acids (e.g. L-glutamic acid) induced an irregular motility pattern; this was overridden by EGTA but restored by subsequent addition of Ca<SUP>2+</SUP>. These findings are discussed in relation to host-location behaviour by zoospores. A role of Ca<SUP>2+</SUP> in germination of cysts, induced by agitation of zoospores of <I>Py. aphanidermatum </I>and <I>Ph. parasitica, </I>was demonstrated by reduced or suppressed germination in the presence of Ca<SUP>2+</SUP> -uptake inhibitors (La<SUP>3+</SUP>, verapamil), Ca<SUP>2+</SUP> chelators (BAPTA), calmodulin-antagonists (trifluoperazine, calmidazolium) or compounds that affect intracellular Ca<SUP>2+</SUP> stores (caffeine, TMB-8). The critical concentrations and times of application of these treatments were determined, and some divalent cations and organic nutrients (sugars, amino acids) were shown partly to reverse the suppression of germination. A link was demonstrated between Ca<SUP>2+</SUP> fluxes and zoospore development into cysts.