Many microorganisms, and in particular fungi, are able to grow over a wide pH range. Thus, these microorganisms must possess some regulatory mechanism or system that senses the environmental pH signal and ensures that gene expression of certain molecules is tailored to the pH of the environment (Penalva and Arst, 2002). In Aspergillus species and several other fungi, pH regulation is mediated by seven genes viz. palA, palB, palC, palF, palH, palI and the global pH regulatory gene, pacC (MacAbe et al, 1996; Negrete-Urtasun, 1999; Denison, 2000). The activated form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes encoding acid phosphatases (Negrete-Urtasun, 1999). PacC (and its homologues) also positively regulates genes involved in penicillin biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans (Penalva and Arst, 2002). It has also been hypothesised that pacC may negatively regulate aflatoxin biosynthesis, a carcinogenic secondary metabolite in several species of Aspergillus (Keller et al, 1997). To elucidate the role of pacC a novel method of post-transcriptional gene silencing known as RNA interference was utilized. This method involved the cloning of a partial pacC gene fragment first in the forward and then the reverse orientations in a fungal expression cassette to create an RNA interference (RNAi) vector. The unique structure of this vector would allow the cloned fragments to be expressed and the resulting RNA to immediately form a double stranded stem-loop structure or short hairpin RNA (shRNA; McDonald et al, 2005). The formation of this shRNA, in turn, would be responsible for activating the endogenous RNA degradation complexes that would lead to mRNA degradation and subsequent gene silencing (Liu et al, 2003; Kadotoni et al, 2003; McDonald et al, 2005). The results presented here have shown that confirmed pacC RNAi mutants produced aflatoxins irrespective of environmental pH (i.e. the mutants produce aflatoxins under acidic and alkaline conditions). Thus, pacC is essential for pH regulation of aflatoxin production in A. flavus. There are numerous other biological (e.g. presence of oxylipins, lipooxygenases) and non-biological factors (pH, carbon source etc.) which affect maize colonisation and aflatoxin production by A. flavus (Burrow et al, 1996; Wilson et al, 2001; Calvo et al; 2002; Tsitsigiannis et al, 2006). However, all the genetic mechanisms involved have as yet not been identified. It has been shown by Caracuel et al (2003) that pacC acts as a negative virulence regulator in plants and these workers have hypothesised that PacC prevents expression of genes that are important for infection and virulence of the pathogen. Therefore the physiological effects that pacC silencing had on the growth, conidiation and pathogenicity of A. flavus mutants were also investigated. The results of this study showed that pacC does not play a significant role in primary growth and development but does affect conidial production. SEM results showed that mutants have many “open ended” phialides and poorly developed conidiophores. This would suggest that pacC activation of conidial production genes is also required. Furthermore, pacC RNAi silencing severely impaired the ability of the A. flavus mutants to infect and cause damage on maize. The results obtained here are similar to that of pacC null mutants in A. nidulans, C. albicans and F. oxysporum which also exhibited low pathogenicity (Davis et al, 2000; Fonzi, W.A, 2002; Caracuel et al, 2003; Bignell et al, 2005 and Cornet et al, 2005). This study indicates that pathogenicity of A. flavus on maize is directly related to the structural integrity of conidia, which in turn is greatly influenced by PacC. This gene is a global transcriptional regulator and may either repress or activate one or many genes in each of the above pathways (Penalva and Arst, 2002). Studies on the genetic mechanisms of pacC regulation on these pathways are needed to elucidate the mechanisms of activation or repression of these genes.

In the first of two parts of the investigation, the stimulation of Armiil aria mellea (Fr.) Quél., a common root-rotting fungus, by Aureobastdium pullulans (DeBary) Arnaud was studied. A. pullulans is an ubiquitous imperfect fungus, inhabiting soil and other substrates. It was shown to produce in culture a volatile, heat stable, neutral substance which stimulated the mycelial development, rhizomorph initiation and rhizomorph elongation of A. mellea. A. pullulans is the only microorganism reported so far to stimulate the growth of A. mellea. The effect of ethanol on the growth of A. mellea was similar to the effect of a cell-free filtrate from a liquid culture of A. pullulans. The cell-free filtrate was shown by gas chromatography to contain ethanol. Ethanol supplied daily as 50 ppm in a glucose-asparagine medium resulted in a ten-fold increase in the number of rhizomorphs produced by A. mellea. The effects of 3-indolylacetic acid, ɣ-(indole-3)-n-butyric acid, the sodium salt of 2,4-dichlorophenoxyacetic acid and tryptophane on the growth of A. mellea were tested, but the stimulatory effect of A. pullulans was not reproduced by them. One rhizomorph tip of A. mellea could develop an extensive rhizomorph system in autoclaved soil if the stimulatory substance produced by A. pullulans was available. In the second part of the investigation the effect of soil moisture and the related effect of some soil-inhabiting Fungi imperfecti on the spread through soil of the root-rotting Basidiomycete Coniopnora puteana (Schum. ex Fr.) Karst.were studied. A soil moisture level of 20 - 25% saturation was satisfactory for the growth of C. puteana in non-sterile soil. At this moisture level C. puteana was able to grow out from a small alder disc inoculum in the centre of a petri dish and invade alder discs at the periphery of the dish. In wetter conditions (50% saturation and higher) it was unable to grow out into the soil. In autoclaved soil the cptimum moisture level for the growth of C. puteana was between 75% and 100% saturation. Small amounts of non-sterile soil were added to autoclaved soil at different moisture levels, with an effect on the growth of C. puteanas similar to that of completely non-sterile soil. Trichoderma viride Pers. ex Fr., a known antibiotic producer, was inoculated in autoclaved soil and produced a greater inhibition of C. puteana in the wetter treatments than in drier ones. The inhibitory effect of Acti-dione (cycloheximide), an antifungal antibiotic active in the pH range 3-5, was examined in non-sterile soil end in autoclaved soil. The same concentration of antibiotic resulted in a greater inhibition of the growth of C. puteana at the higher moisture levels than at the lower ones. / Science, Faculty of / Botany, Department of / Graduate

Inoculation experiments were carried out to determine the relation between bark moisture level of certain host species and their susceptibility to facultative parasites. In these experiments, cuttings of 1-to 3-year old host material and the mycelial mat of the pathogens contained in an agar cylinder were used. In the first instance, fungi that were known or found in association with bark lesions were considered: these were Cryptodiaporthe salicella (Fr.) Petrak on Salix scouleriana Barratt (Scouler willow), Dactylosporium sp. and Fusarium sp. on Acer macrophyllum Pursh. (broadleaf maple, Libertella sp. on Cornus stolonifera (Michx.) var. occidentalis (T. and G.) C. L. Hitchc. (western dogwood), Melanconis sp. on Alnus rubra Bong. (red alder). The results demonstrated that fungi normally associated with lesions of living host material proved to be pathogenic when the relative turgidity of the host bark was lowered from the field level of above 80 per cent to the range of 69 to 77 per cent. Secondly, an attempt was made to determine if correlation existed between bark moisture level and canker development by fungi not known, and not found to occur in association with lesions of some hosts. The following fungi and hosts were considered: C. salicella on red alder (Alnus rubra Bong), trembling aspen (Populus tremuloides Michx.), bitter cherry (Prunus emarginata Dougl.), black cottonwood (Populus trichocarpa Torrey and Gray), western dogwood (Cornus stolonifera var. occidentalis), and on broadleaf maple (Acer macrophyllum Pursh.); Fusarium sp. on red alder, bitter cherry, western dogwood, and on Scouler willow; Libertella sp. on red alder, bitter cherry, broadleaf maple, and on Scouler willow; Melanconis sp. on bitter cherry, western dogwood, broadleaf maple and on Scouler willow. It was shown that all of these parasites extended their host ranges, to varying extent when the bark moisture level was reduced to levels within the range of 69 to 77 per cent, or in some instances to the range of 41 to 67 per cent of saturation. Cuttings with as low bark moisture levels as 41 per cent appeared to be viable as indicated by the production of roots and (or) shoots. / Science, Faculty of / Botany, Department of / Graduate

Seven seed lots of Douglas fir seed which became heavily contaminated with mold during germination tests also had low germination values. A number of fungi were isolated from the seed coat and from within surface-sterilized seed sampled from these seed lots. Several of the fungi were found to be capable of attacking the radicles of germinating seeds under the conditions of the pathogenicity tests. Some of the fungi, both pathogenic and non-pathogenic, inhibited the germination of seed treated with the spores or mycelium of the fungi during a period of stratification. The results obtained indicate that the lower germination values of the heavily contaminated seed lots were due, in part at least, to the activity of the fungi capable of attacking the germinating seed and to fungi which inhibited the sprouting of the seed by other means. Other factors relating to the treatment of the seed prior to the germination tests which would adversely affect the germination are discussed in relation to this study. / Forestry, Faculty of / Graduate

The relatively poor immunogenicity of proteinaceous materials obtained from dermatophytes has been demonstrated. The use of incomplete Freund's adjuvant failed to enhance antibody formation to mold antigens. Many cross-reactions occurred between the antisera of rabbits immunized with various antigenic preparations from dermatophytes, as demonstrated by cross-precipitin tests and skin testing. The occurrence of common antigens among the dermatophytes has been confirmed. Pleomorphic strains did not differ markedly in their immunogenicity as compared to their respective non-pleomorphic strains. Skin testing, upon rabbits with dermatophytic antigens elicited responses which were either of the immediate or of the delayed type. Indications were obtained that toxic principles were present in some of these antigens. Skin tests provided better evidence of differentiation between various species and strains than did the precipitin tests. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A three part study was carried out to examine rheological, colloidal and floe microstructural aspects of industrial brewing yeast strains. Following a review of the literature, the rheological properties of four yeast strains (two flocculent ale and lager types and their non-flocculent variants) were examined. In related colloidal studies, orthokinetic flocculation rates of these strains as well as their cell surface charge were determined. Floc microstructure was characterized using both light and scanning electron microscopy. In a summary chapter, the cell floc model (a modification of Hunter's elastic floc model) was used to the explain the rheological and colloidal behaviour of brewing yeast suspensions. Flow behaviour studies of the commercial yeast suspensions suspended in a calcium-containing sodium acetate buffer revealed that yeast flocculent characteristics had an important influence on their suspension flow behaviour. As cell concentrations increased, suspension flow properties become increasingly non-Newtonian and could be described by the Casson model at low rates of shear and the Bingham model at shear rates above 100 s⁻¹. The cell floc model was proposed to explain the Bingham flow behaviour of these csuspensions. The Bingham yield stress in these suspensions was believed to be a function of the orthokinetic capture coefficient, cell volume and the energy to break up doublet cells. Increasing temperature tended to lower the Bingham yield stress in lager strains and increase the yield stress in ale strains. A semi-empirical explanation for the viscosity increase of deflocculated cell suspensions and the estimation of pseudo-capture coefficients was presented. Furthermore, studies of the flow behaviour of yeast strains suspended in decarbonated ale and lager beer revealed that: 1) suspensions of flocculent strains show higher yield stress values than their non-flocculent variants, 2) ale strain suspensions tended to have higher yield values than the lager strains and 3) yeast dispersed in beer had higher yield stress values than when suspended in buffered calcium suspensions. This last observation was believed to reflect the influence of ethanol on the cell binding process which has important implications for future measurements of yeast flocculation. Colloidal studies revealed for the first time, that the orthokinetic rate of flocculation of brewing yeast cells could be modelled by a first order equation, as predicted by fundamental colloid theory. While subject to considerable variation, measured rate constants led to the calculation of orthokinetic capture coefficients. Yeast cell zeta potential values generally agreed with literature data but could not be employed in the DLVO model of colloid flocculation to explain measured orthokinetic capture coefficient values. Examination of the cell zeta potential data indicated that the data had non-normal distributions. SEM examination of the four industrial yeast strains suggested that a number of distinct structures mediated cell-to-cell interaction and that intra-strain differences occurred. These findings, along with the observation of non-normal surface charge distributions, indicated that these industrially pure strains had undergone substantial variation. Treatment of the flocculent cells with pronase tended to reduce cell-to-cell contacts. In the summary chapter the cell floe model was employed to describe the rheological behaviour of the yeast suspensions. Estimation of the force needed to separate doublet yeast cells were made using critical shear rate data (i.e., the point at which Bingham flow begins). This estimate was similar to that reported for single antibody bonds and may be due to the presence of lectin-like structures on the yeast cell wall. / Land and Food Systems, Faculty of / Graduate

A study into the distribution of thermophilic and thermotolerant fungi in a spruce-pine wood chip pile in Prince George was carried out. Five treatments consisting of pine, spruce, incorporation of wood fines into spruce, sterilized spruce and sterilized spruce inoculated with a Ptychogaster sp. were examined. Samples of wood chips buried at six different locations in the chip pile were examined after 3, 6 and 12 months storage periods. From 100 randomly selected chips from each sample the fungi were isolated on 2% malt, 0.5% malic acid and 2% agar at 25° and 45°C. Data on temperature during storage and acidity of wood chips, moisture content and weight loss at the time of sampling were recorded for the six positions in the wood chip pile. Thermophilic fungi colonized the inner regions while thermotolerant fungi inhabited the outer regions of the wood chip pile. Among the thermophilic fungi, listed according to frequency of isolation were Byssochlamys emersonii Stolk-Apinis, Allescheria terrestris Apinis, Sporotrichum thermophile Apinis, Thermoascus aurantiacus Miehe and Humicola lanuginosa (Griffonand Maublanc) Bunce. The most common thermotolerant fungi were Aspergillus fumigatus. Fresenius and Chrysosporium pruinosum (Gilman and Abbot) Comb. Nov. Fungal distribution was generally related to position in the wood chip pile. Of the associated factors temperature (17°-45°C) was most strongly related to fungal distribution, whereas acidity of wood chips and moisture content did not vary greatly between positions. Incubation of wood samples on cultures demonstrated the ability of all the common thermophilic and thermotolerant fungi to cause weight loss of lodge pole pine Pinus contorta Dougl. varlatifolia, ponderosa pine Pinus ponderosa Laws. and spruce Picea glanca (Moench) Voss sap wood samples. These weight losses varied from 0.65% to 25% after six weeks incubation. Temperature, medium and type of wood affected the ability of the fungi to cause weight loss. No synergistic or antagonistic effects' existed between the thermophilic fungi. Chemical analysis of degraded wood indicated that the thermophilic fungi utilized the arabinose fraction of the hemicellulose preferentially. / Forestry, Faculty of / Graduate

Cultural studies were carried out with haploid strains of Tremella mesenterica Fr., T. encephala Pers. and T. subanomala Coker to determine the conditions and course of conjugation. Under the conditions of the experiments, the optimum pH for growth was 4.7 for all three species and so also was the optimum for conjugation in mixed isolates of T. encephala and T. mesenterica. A time lapse sequence was photographed to follow the course of conjugation in mixed isolates of T. mesenterica. Conjugation hormones such as those reported for T. mesenterica by Bandoni (1965) were demonstrated for the other two species. These hormones passed through dialysis membrane into agar. Extremely dilute suspensions of T. mesenterica haplonts were pulse exposed to semipurified hormone extracts. These suspensions were filtered and the cells were observed on the dried filter cleared with glacial acetic acid. Conjugation tube production terminated with removal of the hormone. Growth and conjugation in T. mesenterica were unaffected by the antibiotic cycloheximide. / Science, Faculty of / Botany, Department of / Graduate

The development of our knowledge of the metabolism, nutrition., and physiology of the rust fungi has been restricted because of our inability to grow these fungi in axenic culture. Most of the physiological and biochemical investigations on rust fungi have been limited to studies of the germination and development of germ tubes. It is not known, whether failure of the rusts to grow axenically is associated with a metabolic block in essential biochemical pathways. The uredial stage of an Australian isolate (ANZ 126-6, 7) of wheat stem rust was grown on a relatively simple medium. Successful in vitro growth was obtained on a medium containing Czapek's minerals, glucose and an organic nitrogen source viz. yeast extract, peptone or BSA. Vegetative growth producing a discrete colony appeared to be dependent upon high density seeding, when conventional dry seeding techniques were followed. When a spore suspension in gelatin was used for inoculation consistent, reproducible vegetative growth at low density seeding was obtained. Water spore suspensions failed to support any growth on the same series of media, under identical conditions. Sodium citrate, host extract, and host protein fractions in general inhibited growth whereas pectin increased the lag phase. Spore-like bodies were obtained inside the colony, which were devoid of any pigmentation. It is suggested that the combination of physical and chemical factors surrounding the germinating spore play an important role in the in vitro development of rust fungi. / Land and Food Systems, Faculty of / Graduate

M.F. Dutton