No description available.

No description available.

No description available.

No description available.

The aims of this research were to develop and an aequorin-based approach for measuring cytosolic Ca²⁺ ([Ca²⁺]_c) in living hyphae of <i>Neurospora crassa </i>and to use this method to investigate the contribution of individual proteins to the generation of the specific Ca²⁺-signatures associated with [Ca²⁺]_c transients. Molecular and genomic methods were also used to identify Ca²⁺-signalling proteins in <i>Neurospora crass, Aspergillus fumigatus </i>and <i>Magnaporthe grisea.</i> Results confirmed that a reliable method for the quantitative measurement of [Ca2+]c in living <i>N. crassa</i> hyphae had been developed with the aequorin reporter system. This method was used to characterise Ca²⁺-signatures in <i>N. crassa </i>in response to (a) mechanical perturbation, (b) hypo-osmotic shock and (c) high external Ca²⁺ under different environmental conditions. Ca²⁺-signatures in response to these stimuli were shown to have a unique set of characteristics in response to each stimulus. These characteristics were apparent under all the conditions tested. Ca²⁺-signatures in response to the three stimuli were measured in wild-type <i>N. crassa</i> treated with Ca²⁺ antagonists and agonists and in untreated mutant strains of <i>N. crassa</i> compromised in Ca²⁺-signalling. In each case, differences in Ca²⁺-signatures could be quantitatively measured. Cloning of the <i>cot-4</i> gene in the <i>cot-4</i> morphological mutant of <i>N. crassa</i> showed it to encode the catalytic subunit of calcineurin, a Ca²⁺/calmodulin dependent protein phosphatase. An analysis of the genomes of <i>N. crassa</i>, <i>A. fumigatus </i>and <i>M. grisea</i> identified for the first time, many of the key Ca²⁺-signalling proteins present in filamentous fungi. An inventory of Ca²⁺-signalling proteins in filamentous fungi is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these proteins.

Single beam and holographic optical tweezer micromanipulation have been explored. The single beam system used was a simple, compact, easy-to-use, safe and robust optical tweezer setup mounted on a standard research grade light microscope. It was specifically designed to produce high quality images and to be used with brightfield, phase contrast, differential interference contrast and fluorescence optics. The holographic optical tweezer system used involved the creation of multiple traps and complex patterns of light, and was employed with a range of laser wavelengths. The various optical tweezer systems were used in a wide range of applications to trap and micromanipulate whole fungal cells, organelles within cells, and synthetic beads. The experimental studies demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; create ‘pseudowalls’ of light to control hyphal growth over extended distances; make piconewton force measurements; investigate the tethering of organelles within cells; mechanically stimulate hyphal tips; produce stable, fixed arrays of cells; and, deliver chemicals to localized regions of hyphae. A significant finding was that germ tubes seem to generate significantly lower growth forces than leading vegetative hyphae. An assessment of the photodamage caused to spores showed that ungerminated spores could be trapped for short periods of time without damage, but could not germinate whilst being continuously trapped. However once germinated, spores continually trapped for 25 min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.

In <i>N. crassa, </i>a specialized hypha, the trichogyne, grows out chemotropically from the ascogonium (female cell) towards a sex pheromone releasing male cell which is commonly a macroconidium of opposite mating type. Following macroconidium-trichogyne fusion, the male and female nuclei were formed to be arrested in nuclear division. The female nuclei became immobilized, rounded up and clumped together whilst all of the male nuclei from the macroconidium moved unidirectionally and sequentially towards the ascogonium with an ‘inchworm-like’, repeated elongation and condensation pattern of movement. Dynein, kinesins and myosins played a role in regulating perithecial formation and the behaviour of male and female nuclei during mating. The dynein subunits DYN2, DLC, DIC and DYN27, the kinesins NKIN2 and KAR3, and the myosin MYO2 encoded by the female influenced male nuclear behaviour whilst the dynein subunit RO-3, the kinesin KIP2 and the myosins MYO1 and MYO2 encoded by male influenced female nuclear behaviour. These results showed that motor proteins derived from the male and female cooperate to regulate the movement of male nuclei and that male-female nuclear recognition occurs immediately following macroconidium-trichogyne fusion. Disruption of microtubule and actin polymerization, and inhibition of myosin activity, inhibited perithecial development and perturbed male nuclear behaviour during mating. A new type of hypha produced by conidia, the conidial sex tube (CST), was discovered. It was found to be included by sex pheromone from the opposite mating type and to be regulated by red, green and blue light.

No description available.

Fungicide/fungus interactions were analysed on the wheat leaf surface using single 0.2 <i>μ</i>l droplet of azoxystrobin and the pathogens <i>Blumeria graminis</i> and <i>Puccinia recondita</i>. Although no observable activity was observed on established colonies of <i>P. recondita, </i>azoxystrobin was shown to have potent curative and preventative activity on <i>B. graminis</i> over distances of up to 7 mm within 24 h. Micro-phosphorimaging of wheat leaves showed that azoxystrobin translocated up to 9 cm after 24 h. Wax stripping of leaves indicated that the majority of movement occurred acropetally. These results indicated that azoxystrobin diffusion through wax and epidermal cells was responsible for preventative and curative activity over short time periods (within 7 h of treatment). Quantitative analysis of radiolabelled fungicide uptake using combustion analysis and scintillation counting showed that approximately 9% of applied azoxystrobin from 0.2 <i>μ</i>l fungicide droplets (125 g/L azoxystrobin) was taken into the leaf within 24 h. Live-cell imaging techniques were developed to study the biology and physiology of mitochondria in hyphae of <i>Neurospora crassa</i>, <i>Aspergillus nidulans </i>and <i>Botrytis cinerea. </i>The vital fluorescent probes DASPMI, Mitotracker Green, Rhodamine 123, FM4-64 and FMI-43, and GFP targeted to mitochondria were assessed for this purpose. On the basis of these data an assay for mitochondrial activity was developed using the potentiometric marker Rhodamine 123. Mitochondria were shown to be more tightly packed at the tips of growing hyphae than in sub-apical regions. Inhibitor studies (with azoxystrobin and FCCP) demonstrated that mitochondrial morphology was significantly altered during inhibition (mitochondria became spherical and retracted from the hyphal tip as growth rate decreased) and that membrane potential was significantly reduced. Fungicide uptake experiments supported the conclusion that the main uptake pathway for strobilurin fungicides is by diffusion.

In recent years, consumer perceptions are that they would like minimum levels of preservatives or even preservative free food. However, this leads to higher risks of microbial spoilage problems, especially due to growth of spoilage fungi, which are capable of growth at intermediate environmental conditions. Studies have been carried out to evaluate the effect of different preservatives at optimal and sub-optimal concentrations on growth, biosynthesis of mycotoxin production at a molecular and phenotypic level for Fusarium graminearum (Tri5) and trichothecene production and Penicillium verrusocum (otapksPv) and ochratoxin A (OTA) production. These were complimented by studies on development of RNAi approaches to inhibit key regulatory genes in the biosynthetic pathways for mycotoxins in these two species. Additional studies were carried out to develop a rapid technique for RNA extraction from fungal biomass. Initial liquid media based studies identified the growth boundaries of a range of 20 spoilage fungi including 3 mycotoxigenic species in relation to preservatives and pH. This showed that up to the legal allowable concentrations of sorbic and benzoic salts at pH 3.0 all strains were capable of growth after 24h. With the exception of F. graminearum all the other species and strains of spoilage fungi were able to grow in these conditions. The use of a mixture of preservatives, a common practise in the food industry, proved effective at inhibiting growth of most spoilage fungi for 21 days at the EU legal limits. Over the EU legal limit of 250 ppm of potassium sorbate mixed with 150 ppm of sodium benzoate only Aspergillus niger had observable growth. Mixtures of weak organic acids, fumaric and malic acid, with the preservative potassium sorbate was shown to be effective at inhibiting growth below the legal limits of use of these food additives, even though the presence of potassium sorbate appears to be fundamental to the inhibition effect, of these natural food additives. Moreover the presence of fumaric acid stimulated growth of Aspergillus flavus. The extraction of high quality total RNA from low amounts of mycelium showed that up to 3 times higher yields can be achieved while improving RNA integrity and overall quality. This development also reduced the time required to extract fungal RNA and the risk of cross-contamination showing the potential use in high throughoutput gene expression studies. In vitro and In situ studies demonstrated the risk of using single sub-optimal antifungal compounds to inhibit growth and mycotoxin production. For F. graminearum, while growth was reduced, the Tri5 gene expression and trichothecenes type B production were stimulated in the presence of thyme essential oil, Prochloraz and BHA. This was also shown with P. verrucosum where otapksPv gene expression and OTA production were stimulated at different water conditions by the presence of sub-optimum concentrations of thyme essential oil and Prochloraz. The antioxidant BHA was able to reduce both otapksPv expression and OTA production in P. verrucosum. The use of siRNA oligonucleotides to silence Tri5 and otapksPv demonstrated that both F. graminearum and P. verrucosum possess the RNAi pathway machinery. In both mycotoxigenic fungi the expression of the target key biosynthetic pathway was knocked down. The optimum levels of the designed siRNA molecules were of between 10 and 25 nM for the molecules targeting P. verrucosum otapksPv. Even though gene silencing using siRNA molecules is transient the effect on otapksPv was still observable after 15 days. This lead to a 3 to 5 times a reduction in the amount of OTA. On the other hand, the silencing of Tri5 in F. graminearum was only detectable in the first 6 days after transfection.