Dissertations / Theses on the topic 'Fungi'

Endocytosis is little understood in filamentous fungi. For some time it has been controversial as to whether endocytosis occurs in filamentous fungi. A comparative genomics analysis between Saccharomyces cerevisiae and 10 genomes of filamentous fungal species showed that filamentous fungi possess complex endocytic machineries. The use of the endocytic marker dye FM4-64, and various vesicle trafficking inhibitors revealed many similarities between endocytosis in the filamentous fungus Neurospora crassa, and endocytosis in budding yeast and mammalian cells. Actin polymerization was found to be crucial for endocytosis in N. crassa, and the microtubule cytoskeleton seemed to be necessary for long distance movement of putative early endosomes. Brefeldin A (BFA) blocked vesicular transport to the Spitzenkörper. Three putative endocytic proteins (WASP, clathrin light chain and Rab5) were labelled with fluorescent proteins in N. crassa. WASP-GFP was found to localise to motile, punctate structures in the plasma membrane just behind the hyphal apex in growing hyphae. This localisation changed to the hyphal apex when growth was temporarily arrested, indicating a possible role in endocytosis and polarized growth. Clathrin light chain-GFP was found to be concentrated in a region just behind the Spitzenkörper, which is consistent with there being a high concentration of clathrinmediated endocytosis in this region. Clathrin light chain-GFP also labelled putative Golgi and this labelling was found to be BFA sensitive, whereas BFA did not have a detectable effect on FM4-64 internalisation and organelle staining. GFP-Rab5 labelled putative early endosomes and decorated microtubules. Knock-outs of putative endocytic proteins in N. crassa, generated as part of the Neurospora genome consortium gene knock-out project, were analysed for defects in endocytosis. 14 out of 17 gene knock-outs were found to be ascospore lethal. The Rab5 knock-out was viable, but did not show a detectable effect on the endocytic internalisation of FM4-64 or its pattern of staining. However, it did exhibit a defect in sexual crossing.

Forest fires have been the major stand-replacing/modifying disturbance in boreal forests. To adapt to fire disturbance, different strategies have evolved. This thesis focuses on wood fungi, and the effect of forest fire on this organism group. In many ways it is a study on adaptation to forest fire, in concurrence with adaptation to dry open habitats. In Paper I we study increased heat resistance in  mycelia from species prevalent in fire prone environments. Fungi were cultivated on fresh wood and exposed to different temperatures. Species prevalent in fire affected habitats had a much higher survival rate over all combinations of time and temperature compared to species associated with other environments. Based on this results the competitiveness was tested after temperature stress (paper II), three fire associated species, were tested against three non fire associated species. All fire associated species had a clear advantage after heat treatment, conquering a larger volume of wood than its competitor. In paper III we studied the effect of heat shock on decomposition rate, 18 species was tested. Species were cultivated and monitored for CO2 accumulation for 8 weeks and then heat shocked. All species including non fire associated species seemed to up-regulate decomposition after heat shock, this response was more pronounced in fire associated species. To look at the possible effect of forest fire on population structure (Paper IV), we developed 29 SNP/INDELs for Phlebiopsis. gigantea. We amplified the marker containing fragments in 132 individuals of P. gigantea in 6 populations, 3 which were found in areas affected by forest fire and 3 in unaffected areas. We found no genetic structure in accordance to forest fire. However we detected geographic structure, which stands in contrast to earlier studies. This might be due to the method, using SNP´s and number of individuals in the study. Finally we collected cross-sections of decayed logs to evaluate the number of fungal species domains that are likely to be hit when drilling a saw-dust sample in a log. We used these estimates to simulate how many species that will be found by a certain number of samples. We found that in 99% of the

Endophytic fungi inhabit the living tissue of a host plant for at least a portion of their life cycle. While some researchers have shown that various endophytic fungi participate in litter decomposition, we do not know whether such fungi are actually saprotrophic, meaning that they can obtain energy from litter. Therefore, I determined if endophytic fungi are saprotrophs using leaf litter as the energy source. All 49 tested isolates were found to be saprotrophic. To compare the saprotrophic capacities of fungi from different habitats, which produce different types of litter, a universal litter proxy needs to be used. I hypothesized that pure cellulose would be an adequate proxy for litter for in vitro studies because of its abundance in litter. This was tested in the first study. Saprotrophic capacity on pure cellulose was not highly correlated with that on leaf litter. I conclude, therefore, that cellulose may not be a good proxy for leaf litter. Some endophytic fungi are biotrophs, presumably acquiring energy from photosynthate produced by the host plant. This suggests that the level of exposure to sunlight by the plant should influence the competitive ability of such fungi. If saprotrophic endophytic fungi do exist, they ought to be less competitive against biotrophic endophytic fungi in leaves receiving full sunlight than in shaded leaves. I, therefore, hypothesized that the frequency of saprotrophy will be influenced by the level of sun exposure of the leaf from which the fungi were isolated. This was tested in the second study. Moreover, because closely related organisms ought to be more similar to each other than more distantly related organisms, I also hypothesized that saprotrophic capacity has a strong phylogenetic component, which was also tested in the second study. Unexpectedly, isolate identity within genus accounted for far more variability in saprotrophic capacity than genus identity, and sun exposure did not have a significant effect on saprotrophy. These results suggest that saprotrophic capacity may not be highly consequential in the ecology of these organisms.

Molecular technology has shown the classical, morphologically defined groupings of sequestrate cortinarioid fungi to be artificial and in need of revision. However, these same molecular studies have highlighted morphological characters, such as spore shape and ornamentation, that have proved useful for distinguishing phylogenetically informative groups. This observation underpins the hypothesis of this study: that the numeric analysis of selected morphological characters can provide the same picture of the diversity of, and relationships among, sequestrate cortinarioid fungi as that recovered from phylogenetic analysis of rDNA sequence data. Sequestrate fungi are those in which the spores mature inside an enclosed fruit body, remaining there until the fruit body decomposes or is eaten. For the purposes of this thesis the following genera are considered to contain cortinarioid sequestrate fungi: Auritella, Cortinarius, Dermocybe, Descomyces, Hymenogaster, Hysterogaster, Inocybe, Protoglossum, Quadrispora, Setchelliogaster and Timgrovea. This thesis focussed on Australian representatives of these fungi to address the hypothesis outlined above. Four analysis methods were applied to each of three datasets (morphological, rDNA and combined data) in a comparative approach to test the stated hypothesis. The four analysis methods were two multivariate methods: cluster analysis and ordination (by principal coordinates analysis), and two phylogenetic methods: maximum parsimony and Bayesian analysis. Low bootstrap support and Bayesian partition probabilities for phylogenetic analyses of the morphological data indicated this dataset had little to no phylogenetic signal discernable by parsimony and Bayesian analyses. Different analyses of the morphological data differed in the way they grouped the collections. The type of clustering method used affected the pattern of relationships recovered. The coding of the data had a much more substantial effect on the patterns of relatedness suggested by the multivariate analyses. Despite the low level of phylogenetic information and agreement between analyses of the morphological data it was found that some collections were consistently grouped together. This included the separation of the Cortinarius-like collections from the Descolea-like collections and the relatively consistent grouping of some pairs of collections and some larger groups. Thus, despite the limited phylogenetic signal of the small morphological dataset and the artefacts of coding, some relatively consistent groups were recovered. Separate analyses of the Cortinarius-, Descolea- and Hebeloma-like ITS sequences recovered similar patterns to published phylogenies. The inclusion of more sequestrate taxa and a greater sample of Australian collections than previous studies, indicated that both Timgrovea subgenera nest among the Descolea-like collections and that hitherto undiscovered lineages of Descolea-like fungi are represented among the collections in Australian herbaria. The Cortinarius-like fungi fall within clades recognised by published phylogenies. Similar topologies were supported by both Parsimony bootstrap and Bayesian partition probability values for analyses of the molecular data including the separation of Cortinarius-like collections from Descolea-like collections. However neither of these methods of analysis and evaluation yielded well-resolved deeper nodes for either of these two major clades. Comparable clades/clusters of Cortinarius- like and Descolea-like collections were found in all analyses of the molecular data. Thus phylogenetically distinct groups of cortinarioid sequestrate fungi could be consistently distinguished using ITS molecular data, but not confidently related to one another. The ratio of molecular to morphological characters (741:16) meant the patterns observed for the combined analyses were more similar to those observed in analyses of the molecular data than those of the morphological data. This included the recovery of substantially similar clades/clusters to those recovered by analyses of the molecular data alone. The value of combining the morphological and molecular data as analysed is questioned despite the congruence of the datasets according to the Incongruence-Length Difference test. Differences between the molecular and combined datasets arose primarily where the molecular data grouped collections that were also grouped by the morphological data. The numeric analysis of the selected morphological characters as carried out in this study did not recover the same pattern of groups and relationships among the cortinarioid sequestrate fungi as phylogenetic analyses of ITS data. The composition of groups recovered using the morphological data alone or as part of the combined dataset, and the relationships between those groups, differed from those recovered from the molecular data alone; although there were similarities between groups recovered from different datasets. The ability of this thesis to conclusively address its fundamental hypothesis was compromised by limitations of the study such as taxon sampling, character selection, character coding and the poor resolution of the ITS phylogeny. Acknowledging these limitations, and that some similar groups were recovered, the results of this thesis do not support its stated hypothesis that the numeric analysis of selected morphological characters can provide the same picture of the diversity of, and relationships among, sequestrate cortinarioid fungi as recovered from phylogenetic analysis of rDNA sequence data.

Molecular technology has shown the classical, morphologically defined groupings of sequestrate cortinarioid fungi to be artificial and in need of revision. However, these same molecular studies have highlighted morphological characters, such as spore shape and ornamentation, that have proved useful for distinguishing phylogenetically informative groups. This observation underpins the hypothesis of this study: that the numeric analysis of selected morphological characters can provide the same picture of the diversity of, and relationships among, sequestrate cortinarioid fungi as that recovered from phylogenetic analysis of rDNA sequence data. Sequestrate fungi are those in which the spores mature inside an enclosed fruit body, remaining there until the fruit body decomposes or is eaten. For the purposes of this thesis the following genera are considered to contain cortinarioid sequestrate fungi: Auritella, Cortinarius, Dermocybe, Descomyces, Hymenogaster, Hysterogaster, Inocybe, Protoglossum, Quadrispora, Setchelliogaster and Timgrovea. This thesis focussed on Australian representatives of these fungi to address the hypothesis outlined above. Four analysis methods were applied to each of three datasets (morphological, rDNA and combined data) in a comparative approach to test the stated hypothesis. The four analysis methods were two multivariate methods: cluster analysis and ordination (by principal coordinates analysis), and two phylogenetic methods: maximum parsimony and Bayesian analysis. Low bootstrap support and Bayesian partition probabilities for phylogenetic analyses of the morphological data indicated this dataset had little to no phylogenetic signal discernable by parsimony and Bayesian analyses. Different analyses of the morphological data differed in the way they grouped the collections. The type of clustering method used affected the pattern of relationships recovered. The coding of the data had a much more substantial effect on the patterns of relatedness suggested by the multivariate analyses. Despite the low level of phylogenetic information and agreement between analyses of the morphological data it was found that some collections were consistently grouped together. This included the separation of the Cortinarius-like collections from the Descolea-like collections and the relatively consistent grouping of some pairs of collections and some larger groups. Thus, despite the limited phylogenetic signal of the small morphological dataset and the artefacts of coding, some relatively consistent groups were recovered. Separate analyses of the Cortinarius-, Descolea- and Hebeloma-like ITS sequences recovered similar patterns to published phylogenies. The inclusion of more sequestrate taxa and a greater sample of Australian collections than previous studies, indicated that both Timgrovea subgenera nest among the Descolea-like collections and that hitherto undiscovered lineages of Descolea-like fungi are represented among the collections in Australian herbaria. The Cortinarius-like fungi fall within clades recognised by published phylogenies. Similar topologies were supported by both Parsimony bootstrap and Bayesian partition probability values for analyses of the molecular data including the separation of Cortinarius-like collections from Descolea-like collections. However neither of these methods of analysis and evaluation yielded well-resolved deeper nodes for either of these two major clades. Comparable clades/clusters of Cortinarius- like and Descolea-like collections were found in all analyses of the molecular data. Thus phylogenetically distinct groups of cortinarioid sequestrate fungi could be consistently distinguished using ITS molecular data, but not confidently related to one another. The ratio of molecular to morphological characters (741:16) meant the patterns observed for the combined analyses were more similar to those observed in analyses of the molecular data than those of the morphological data. This included the recovery of substantially similar clades/clusters to those recovered by analyses of the molecular data alone. The value of combining the morphological and molecular data as analysed is questioned despite the congruence of the datasets according to the Incongruence-Length Difference test. Differences between the molecular and combined datasets arose primarily where the molecular data grouped collections that were also grouped by the morphological data. The numeric analysis of the selected morphological characters as carried out in this study did not recover the same pattern of groups and relationships among the cortinarioid sequestrate fungi as phylogenetic analyses of ITS data. The composition of groups recovered using the morphological data alone or as part of the combined dataset, and the relationships between those groups, differed from those recovered from the molecular data alone; although there were similarities between groups recovered from different datasets. The ability of this thesis to conclusively address its fundamental hypothesis was compromised by limitations of the study such as taxon sampling, character selection, character coding and the poor resolution of the ITS phylogeny. Acknowledging these limitations, and that some similar groups were recovered, the results of this thesis do not support its stated hypothesis that the numeric analysis of selected morphological characters can provide the same picture of the diversity of, and relationships among, sequestrate cortinarioid fungi as recovered from phylogenetic analysis of rDNA sequence data.

Endophytes are mutualistic fungi living in green tissue of all plants examined so far.Some of these fungi can produce compounds that are beneficial to the host plant, and it isalso known that some pathogenic fungi live parts of their lives as endophytes. Endophyticinteractions have been well characterized in various grasses, but much is unknown abouttheir interactions with trees. One reason for this is that the fungal biodiversity is muchlarger among endophytes in trees than in grasses, another is that screening for endophytestakes a lot of work. The goal of this thesis work was to develop a polymerase chainreaction (PCR) based method that is simple, fast and reliable for detection of endophytesin aspens. Eleven primer pairs were designed, each pair specific for one fungus. Afteroptimization and evaluation four of the primer pairs were found to be both specific andsensitive, and could detect fungus in DNA preparations from leaf samples.

Four aspects of horizontal genetic transfer during heterokaryon formation were examined in the asexual pathogen Fusarium oxysporum f.sp. cubense (Foe): 1) variability based on method of heterokaryon formation 2) differences in nuclear and mitochondrial inheritance 3) the occurrence of recombination without nuclear fusion 4) the occurrence of horizontal genetic transfer between distantly related isolates. The use of non- pathogenic strains of Fusarium oxysporum as biocontrol agents warrants a closer examination at the reproductive life cycle of this fungus, particularly if drag resistance or pathogenicity genes can be transmitted horizontally. Experiments were divided into three phases. Phase I looked at heterokaryon formation by hyphal anastomosis and protoplast fusion. Phase II was a time course of heterokaryon formation to look at patterns of nuclear and mitochondrial inheritance. Phase III examined the genetic relatedness of the different vegetative compatibility groups using a multilocus analysis approach. Heterokaryon formation was evident within and between vegetative compatibility groups. Observation of non-parental genotypes after heterokaryon formation confirmed that, although a rare event, horizontal genetic transfer occurred during heterokaryon formation. Uniparental mitochondria inheritance was observed in heterokaryons formed either by hyphal anastomosis or protoplast fusion. Drag resistance was expressed during heterokaryon formation, even across greater genetic distances than those distances imposed by vegetative compatibility. Phytogenies inferred from different molecular markers were incongruent at a significant level, challenging the clonal origins of Foe. Mating type genes were identified in this asexual pathogen Polymorphisms were detected within a Vegetative Compatibility Group (VCG) suggesting non-clonal inheritance and/or sexual recombination in Foe. This research was funded in part by a NIH-NIGMS (National Institutes of Health-National Institute of General Medical Sciences) Grant through the MBRS (Minority Biomedical Research Support), the Department of Biological Sciences and the Tropical Biology Program at FIU.

The interaction between several species of arbuscular mycorrhizal fungi, micropropagated strawberry plants and Phytophthora fragariae, the pathogen that causes red stele disease of strawberry plants, was investigated. The optimum temperature for germination of zoospore cysts of P. fragariae in vitro was found to be 15°C, and growth of the emerging germ tube was significantly orientated towards the strawberry root tip. Cyst germination was reduced in the presence of a mycorrhizal strawberry root. Elsanta was more susceptible to P. fragariae than the cultivar Rhapsody. A low level of colonisation of Elsanta with the arbuscular mycorrhizal fungi Glomus mosseae, Glomus intraradices or Glomus fistulosum resulted in a significantly greater amount of total phosphorus in plant shoots compared to non-mycorrhizal plants, although further increases in the percentage of root colonisation by the fungi had no effect on the plants. The presence of these mycorrhizal fungi had no effect on disease due to subsequent inoculation of the plants by P. fragariae. Increasing colonisation of Elsanta by Scutellospora nodosa was correlated with a significant increase in plant size and additional phosphorus uptake. However, these same plants exhibited greater levels of disease due to the following inoculation with P. fragariae. A low level of root colonisation of Elsanta by Acaulospora scrobiculata caused significant increases in plant size and phosphorus uptake up to a threshold level of root colonisation beyond which further increases had no affect on the plant. The results are discussed in relation to the utilisation of specific strains of arbuscular mycorrhizal fungi as inoculants of micropropagated strawberry plants of particular cultivars with the potential to increase plant growth and reduce the level of disease due to soil-borne plant pathogens.

Two field studies were conducted to assess the potential benefit of arbuscular mycorrhizal (AM) inoculation of elite strawberry plants on plant multiplication, and fruit yield, under typical nursery conditions, in particular soils classified as excessively rich in P. To study plant productivity, five commercially in vitro propagated elite strawberry cultivars ('Chambly', 'Glooscap', 'Joliette', 'Kent', and 'Sweet Charlie') were not inoculated with AM fungi or were inoculated with either a single species (Glomus intraradices), or a mixture of species (G. intraradices, Glomus mosseae, and Glomus etunicatum). AM inoculation was found to impact strawberry plant productivity in a soil with excessive P levels. The AM fungi introduced into the field by inoculated mother plants established a mycelial network in the soil through colonization of the daughter plant roots, however, persistence of colonization was determined to below (<12% in inoculated plant roots). In soils excessively rich in P, individual crop inoculation may be the only option for management of the symbiosis, as the host and non-host rotation crops, planted prior to strawberry production, had no effect on plant productivity or soil mycorrhizal potential.
To study the impact of AM inoculation on fruit production, three commercially grown strawberry cultivars (Glooscap, Joliette, and Kent) were not inoculated with AM fungi or were inoculated with either G. intraradices or G. mosseae. AM fungi impacted the fruit yield, with all inoculated cultivars producing more fruit than noninoculated cultivars during the first harvest year. The percentage of root colonization could not be used to explain the differences in total fruit yield during the first harvest year, or the increase in total fruit yield the second harvest year.
We wished to examine the effects of various P treatments on C metabolism within the intraradical mycelia (IRM) of the fungus. Specific primers were developed for the Glomus intraradices glucose-6-phosphate dehydrogenase (G6PDH) gene. Real-time quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) was used to measure the gene expression of the G. intrarardices G6PDH gene in response to external P conditions of colonized transformed carrot roots. The results showed a significant down-regulation of G6PDH in the IRM of G. intraradices when cultures were grown in a high P (350 muM P) medium compared to those grown in the low P (35 muM P) medium. The down-regulation may suggest a reduction in the C flow from the host to the fungus. There was no effect on G6PDH expression following a two-hour incubation with additional P applications (No P, low P and high P).

Interactions are all around us, and as humans we may use words and gestures to communicate our intentions. At the micro level of fungi, communications are replaced by chemical signals and structure. These interactions fall into three distinctive categories: synergistic, where organisms help each other, as is the case with ectomycorrhizal fungi and tree roots, deadlock, or combat, where organisms fight for or defend a resource. When it comes to fungi-tree interactions, the fungi group of basidiomycetes fall into the latter category. At the onset of fungal infection, a living tree defends itself by producing resinous substances such as terpenes. These compounds are frequently found in hydrodistilled turpentine, which makes turpentine a prime source of antifungal compounds. A D-optimal design of fractionated turpentine together with gas chromatography (GC) coupled to a mass spectrometer was employed to find the most biologically active constituent of turpentine. Growth rate of Coniophora puteana was used to assess the efficacy of the mixed fractions. The partial least squares projection model had an excellent predictive power (R2 = 0.988, Q2 = 0.825) and validity. A putative sesquiterpene was identified as the most active compound for inhibiting fungal growth. The model was corroborated by an external validation assay employing preparative GC. After the death of a tree, fungi are no longer hindered by secondary metabolites from the tree. Instead, other interspecies interactions and intraspecies interactions, such as fungi-fungi interactions, occur. We found that when the white-rot fungus Heterobasidion parviporum and brown-rot fungus Gloeophyllum sepiarium interact with each other, amino acids are used to a higher extent. Amino acids may be used to produce antifungal compounds to hinder the other species from growing. Lysine in particular was utilized to a greater extent during interaction. Glutamine was the only amino acid that increased in concentration. Glutamine might be exuded or converted by enzymes from already existing glutamic acid. Dry weights suggest that the fungi were in a deadlock and that nutrient limitation might be a determining factor. It seemed that H. parviporum was favoured by a decrease in pH while the opposite pattern may be true for G. sepiarium.

A study on cuticle-degrading enzymes (CDE) of three hyphomycete entomopathogens has produced information on enzyme types, levels, characteristics, mode of action, regulation, sequence of production, cellular localisation and production during host penetration. This is the first critical work on CDE of any entomopathogen. Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as sole carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular and bound enzymes corresponding to the major components of insect cuticle e.g. 3 endo-proteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase and N-acetylglucosaminidase. Considerable variations occurred in levels of production between spp. and even within a sp., but endo-proteases were exceptional in being produced in large amounts by all the isolates. CDE were produced rapidly and sequentially in culture. The first activities to appear (< 24 h) were those of the proteolytic complex, chitinases were always produced substantially later. Properties of CDE were investigated in terms of pH and tem- erature optima, substrate specificity, molecular weight, iso-electric point, mechanism of substrate degradation and the effect of specific inhibitors. Studies with culture filtrates and purified CDE revealed that substrates in intact cuticles are amenable to degradation but the prior action of protease is necessary for significant degradation of the chitin. Staining of chitin by a fluorescent lectin (FITC-WGA) and calcofluor only in cuticles from which protein has been removed (by protease or KOH) also suggests initial masking of chitin. This and determination of amino acid composition of peptides solubilised by endo-protease revealed the potential of CDE in studying the physicochemical structure of insect cuticles. The apparently localised action of CDE during host penetration may result from molecular sieving, binding to fungal walls or binding to cuticle. The first possibility is lessened by the small size of the endo-enzymes (<34 K daltons) which could allow diffusion via the various canals which traverse cuticle. However, cuticle effectively binds (ionically) CDE, and also activities of several CDE remain partly bound in various ways to hyphae and conidia (by ionic binding to walls, by disulphide bonds, and on or within membrane structures). The involvement of proteolytic enzymes in infection was suggested by their presence in conidia, penetration structures, and infected cuticle (detected histochemically and following extraction from cuticles). Also the constitutive production of endo- and exo-proteases lends weight to their possible significance in parasitism as synthesis will be subject only to catabolite repression. Chitinase is induced by N-acetylglucosamine and was not detected in infected cuticle. Possible mechanisms and significance of enzymic degradation of cuticle during infection are discussed, particularly in comparison to host penetration by phytopathogenic fungi.

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.

Dissertation (PhD)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: In the yeast Saccharomyces cerevtstee, two biochemical pathways ensure that activated cytoplasmic or peroxisomal acetyl-groups are made available for mitochondrial energy production when the cells utilise non-fermentable carbon sources. The first pathway is the glyoxylate cycle, where two activated acetyl-groups are incorporated into each cycle, which releases a C4 intermediate. This intermediate is then transported to the mitochondria where it can enter the tricarboxylic acid cycle. The second pathway is the carnitine shuttle. Activated acetyl-groups react with carnitine to form acetylcarnitine, which is then transported to the mitochondria where the acetyl group is transferred. In this study it was shown that the deletion of the glyoxylate cycle specific citrate synthase, encoded by CIT2, results in a strain that is dependent on carnitine for growth on non-fermentable carbon sources. Using a /::"cit2 strain, mutants affected in carnitine-dependent metabolic activities were generated. Complementation of the mutants with a genomic library resulted in the identification of four genes involved in the carnitine shuttle. These include: (i) the mitochondrial and peroxisomal carnitine acetyltransferase, encoded by CAT2; (ii) the outer-mitochondrial carnitine acetyltransferase, encoded by YA T1; (iii) the mitochondrial carnitine translocase, encoded by CRC1; and (iv) a newly identified carnitine acetyltransferase, encoded by YAT2. All three carnitine acetyltransferases are essential in a carnitine-dependent strain. The dependence on exogenous carnitine of the /::"cit2 strain when grown on nonfermentable carbon sources suggested that S. cerevisiae does not biosynthesise carnitine. Measurements using electrospray mass spectrometry confirmed this hypothesis. As a result an investigation was initiated into carnitine biosynthesis in order to genetically engineer a S. cerevisiae strain that could endogenously biosynthesise carnitine. The filamentous fungus, Neurospora crassa, was one of the first organisms used in the seventies to identify the precursor and intermediates of carnitine biosynthesis. However, it was only about twenty years later that the first genes encoding these enzymes where characterised. Carnitine biosynthesis is a four-step process, which starts with trimethyllysine as precursor. Trimethyllysine is converted to hydroxytrimethyllysine by the enzyme trimethyllysine hydroxylase (TMLH). Hydroxytrimethyllysine is cleaved to trimethylamino-butyraldehyde by the hydroxytrimethyllysine aldolase (HTMLA) releasing glycine. Trimethylaminobutyraldehyde is dehydrogenated to trimethylamino-butyrate (y-butyrobetaine) by trimethylamino-butyraldehyde dehydrogenase (TMABA-DH). In the last step, ybutyrobetaine is converted to t-carnltine by y-butyrobetaine hydroxylase (BBH). The N. crassa TMLH homologue was identified in the genome database based on the protein sequence homology of the human TMLH. Due to the high amount of introns predicted for this gene, the cDNA was cloned and subjected to sequencing, which then revealed that the gene indeed had seven introns. Functional expression of the gene in S. cerevisiae and subsequent enzymatic analysis revealed that the gene coded for a TMLH. It was therefore named cbs-1 for "carnitine biosynthesis gene no. 1JJ. Most of the kinetic parameters were similar to that of the human TMLH enzyme. Following this, a genomic copy of the N. crassa BBH homologue was cloned and functionally expressed in S. cerevisiae. Biochemical analysis revealed that the BBH enzyme could biosynthesise L-carnitine from y-butyrobetaine and the gene was named cbs-2. In addition, the gene could rescue the growth defect of the carnitinedependent Scii? strain on non-fermentable carbon sources when y-butyrobetaine was present. This is the first report of an endogenously carnitine biosynthesising strain of S. cerevisiae. The cloning of the remaining two biosynthesis genes presents particular challenges. To date, the HTMLA has not been characterised on the molecular level making the homology-based identification of this protein in N. crassa impossible. Although the TMABA-DH has been characterised molecularly, the protein sequence is conserved for its function as a dehydrogenase and not conserved for its function in carnitine biosynthesis, as in the case of TMLH and BBH. The reason for this is probably due to the fact that the enzyme is involved in other metabolic processes. The use of N. crassa carnitine biosynthesis mutants would probably be one way in which to overcome these obstacles. The !1cit2 mutant proved useful in studying carnitine related metabolism. We therefore searched for suppressors of !1cit2, which resulted in the cloning of RAS2. In S. cerevisiae, two genes encode Ras proteins, RAS1 and RAS2. GTP-bound Ras proteins activate adenylate cyclase, Cyr1 p, which results in elevated cAMP levels. The cAMP molecules bind to the regulatory subunit of the cAMP-dependent kinase (PKA), Bcy1 p, thereby releasing the catalytic subunits Tpk1 p, Tpk2p and Tpk3p. The catalytic subunits phosphorylate a variety of regulators and enzymes involved in metabolism. Overexpression of RAS2 could suppress the growth defect of the Sclt? mutant on glycerol. In general, overexpression of RAS2 enhanced the proliferation of wild-type cells grown on glycerol. However, the enhancement of proliferation was much better for the !1cit2 strain grown on glycerol. In this respect, the retrograde response may play a role. Overexpression of RAS2 resulted in elevated levels of intracellular citrate and citrate synthase activity. It therefore appears that the suppression of !1cit2 by RAS2 overexpression is a result of the general upregulation of the respiratory capacity and possible leakage of citrate and/or citrate synthase from the mitochondria. The phenotype of RAS2 overexpression contrasts with the hyperactive RAS2val19 allele, which causes a growth defect on glycerol. However, both RAS2 overexpression and RAS2val19activate the cAMP/PKA pathway, but the RAS2val19dependent activation is more severe. Finally, this study implicated the Ras/cAMP/PKA pathway in the proliferation effect on glycerol by showing that in a Mpk1 strain, the growth effect is blocked. However, the enhanced proliferation was still observed in the Mpk2 and Mpk3 strains when RAS2 was overexpressed. Therefore, it seems that Tpk1 p plays an important role in growth on non-fermentable carbon sources, a notion that is supported by the literature.
AFRIKAANSE OPSOMMING: In die gis Saccharomyces cerevtstee, is daar twee metaboliese weë waarmee geaktiveerde asetielgroepe na die mitochondrium vervoer kan word wanneer die sel op nie-fermenteerbare koolstofbronne groei. Die een weg is die glioksilaatsiklus, waar die geaktiveerde asetielgroepe geïnkorporeer word in die siklus en dan vrygestel word as Ca-intermediêre. Hierdie intermediêre word dan na die mitochondrium vervoer waar dit in die trikarboksielsuursiklus geïnkorporeer word. Die ander weg is die karnitiensiklus, waar geaktiveerde asetielgroepe met karnitien reageer om asetielkarnitien te vorm wat dan na die mitochondrium vervoer word waar dit die asetielgroep weer vrygestel. Hierdie studie het getoon dat die delesie van die glioksilaatsiklus spesifieke sitraatsintetase, gekodeer deur CIT2, die gisras afhanklik maak van karnitien vir groei op nie-fermenteerbare koolstofbronne. Deur gebruik te maak van 'n ócit2 gisras, kon mutante, wat geaffekteer is in karnitien-verwante metaboliese aktiwiteite, gegenereer word. Komplementering van die mutante met 'n genomiese biblioteek het gelei tot die identifisering van vier gene betrokke by die karnitiensiklus. Hierdie gene sluit in: (i) die mitochondriale en die peroksisomale karnitienasetieltransferase, gekodeer deur CAT2; (ii) die buite-mitochondriale karnitienasetieltransferase, gekodeer deur YAT1; (iii) die mitochondriale karnitientranslokase, gekodeer deur CRC1; en (iv) 'n nuutgeïdentifiseerde karnitienasetieltransferase, gekodeer deur YAT2. Daar benewens, is ook gewys dat al drie karnitienasetieltransferases noodsaaklik is in 'n karriltienafhanklike gisras. Die afhanklikheid van eksogene karnitien van die ócit2 gisras, wanneer dit gegroei word op nie-fermenteerbare koolstofbronne, was aanduidend dat S. cerevisiae nie karnitien kan biosintetiseer nie. Metings deur middel van elektronsproeimassaspektrometrie het hierdie veronderstelling bevestig. Gevolglik is 'n ondersoek deur ons geïnisieer in die veld van karnitienbiosintese om 'n S. cerevisiae gisras geneties te manipuleer om karnitien sodoende endogenies te biosintetiseer. Die filamentagtige fungus, Neurospora crassa, was een van die eerste organismes wat in die sewentiger jare gebruik is om die voorloper en intermediêre van karnitienbiosintese te identifiseer. Dit was egter eers sowat twintig jaar later dat die eerste gene wat vir hierdie ensieme kodeer, gekarakteriseer is. Karnitienbiosintese is 'n vierstap-proses wat met trirnetlellisten as voorloper begin. Trimetiellisien word omgeskakel na hidroksi-trimetiellisien deur die ensiem trimetiellisienhidroksilase (TMLH). Hidroksietrimetlelllsien word dan gesplits om trimetielaminobuteraldehied te vorm deur die werking van die hidroksitrimetiellisienaldolase (HTMLA) met die gevolglike vrystelling van glisien. Trimetielaminobuteraldehied word dan na trimetielaminobuteraat (y-butirobeteïen) deur trimetielaminobuteraldehied dehidrogenase (TMABA-DH) gedehidrogeneer. In die laaste stap word y-butirobeteïen deur middel van die y-butirobeteïen hidroksilase (BBH) na L-karnitien omgeskakel. Op grond van die proteïenvolgordehomologie in die genoomdatabasis tussen die menslike TMLH en N. crassa se TMLH is laasgenoemde geïdentifiseer. As gevolg van die groot getal introns wat vir hierdie geen voorspel is, is die cDNA-weergawe daarvan gekloneer en aan volgordebepaling onderwerp. Dit het getoon dat die geen inderdaad sewe introns bevat. Funksionele uitdrukking van die geen in S. cerevisiae en ensiematiese analise het getoon dat die geen vir 'n TMLH kodeer en is gevolglik cbs-1 genoem; dit staan vir "karnitien biosintese geen no. 1tt. Meeste van die kinetiese parameters was ook soortgelyk aan die van die menslike TMLH-ensiem. Hierna is 'n genomiese kopie van N. crassa se BBH-homoloog gekloneer en funksioneel in S. cerevisiae uitgedruk. Biochemiese analise het getoon dat die uitgedrukte BBH-ensiem L-karnitien vanaf y-butirobeteïen kan biosintetiseer en die geen is cbs-2 genoem. Daar benewens kon die geen die groeidefek van die karnitien-afhanklike tlcit2-gisras ophef wanneer dit op nie-fermenteerbare koolstofbronne in die teenwoordigheid van y-butirobeteïen aangekweek is. Hierdie is die eerste verslag oor 'n endogeniese karnitien-biosintetiserende ras van S. cerevisiae. Die klonering van die oorblywende twee karnitienbiosintetiserende gene het sekere uitdagings. Tot op datum, is die HTMLA nog nie tot op genetiese vlak gekarakteriseer nie, wat dan die homologie-gebaseerde identifikasie van hierdie proteïen in N. crassa onmoontlik maak. Alhoewel die TMABA-DH geneties gekarakteriseer is, is die proteïenvolgorde ten opsigte van sy funksie as 'n dehidrogenase gekonserveer, maar nie vir sy funksie in karnitienbiosintese soos in die geval van TMLH en BBH nie. Die rede hiervoor is moontlik omdat die ensiem ook in ander metaboliese prosesse betrokke is. Die gebruik van N. crassa karnitienmutante sal moontlik een manier wees om hierdie probleme te oorkom. Die tlcit2-mutant het handig te pas gekom vir die bestudering van karnitienverwante metabolisme. Dus is daar vir onderdrukkers van die tlcit2-mutant gesoek wat gelei het tot die klonering van die RAS2-geen. In S. cere visiae , kodeer twee gene vir Ras-proteïene, RAS1 en RAS2. GTP-gebonde Ras-proteïene aktiveer adenilaatsiklase, Cyr1 p, wat verhoogde intrasellulêre cAMP-vlakke tot gevolg het. Die cAMP bind aan die regulatoriese subeenheid van die cAMP-proteïenkinase (PKA), Bcy1 p, en daardeur word die katalitiese subeenhede, Tpk1 p, Tpk2p en Tpk3p, vrygestel. Die katalitiese subeenheid fosforileer 'n verskeidenheid van reguleerders en ensieme betrokke by metabolisme. Ooruitdrukking van RAS2 het die groeidefek van die tlcit2-mutant op gliserolonderdruk. Oor die algemeen, verbeter die ooruitdrukking van RAS2 die proliferasie van die wildetipe op gliserol bevattende media. Alhoewel, die verbetering van proliferasie was baie meer opmerklik in die tlcit2-gisras. In hierdie verband, speel die gedegenereerde response dalk 'n rol. Ooruitdrukking van RAS2 het verhoogde intrasellulêre vlakke van sitraat- en sitraatsintetase-aktiwiteit tot gevolg gehad. Dit wou dus voorkom asof die onderdrukking van die ócit2-groeidefek deur RAS2 se ooruitdrukking die gevolg was van algemene opreguiering van respiratoriese kapasiteit en die lekkasie van sitraat en/of sitraatsintetase uit die mitochondria. Die fenotipe van RAS2 ooruitdrukking kontrasteer die hiperaktiewe RAS2va / 19 alleel, wat 'n groeidefek op gliserol media veroorsaak. Alhoewel beide RAS2-00ruitdrukking en RAS2va / 19 die cAMP/PKA-weg aktiveer, is gevind dat die RAS2va/19-afhanklike aktivering strenger is. Ten slotte, die cAMP/PKA-weg is in die proliferasie effek op gliserol media geïmpliseer deur te wys dat in 'n Mpk1-gisras, die groeieffek geblokkeer is. Alhoewel, die verbeterde proliferasie is steeds waargeneem in die Mpk2-en Mpk3-gisrasse toe die RAS2-geen ooruitgedruk is. Dus, dit wil voorkom asof Tpk1 p 'n belangrike rol in die groei van gisselle op nie-fermenteerbare koolstofbronne speel; 'n veronderstelling wat deur die literatuur ondersteun word.

Stipitatic acid 12 is an aromatic seven-membered ring produced by Talaromyces stipitatus (previously known as Penicillium stipitatus). The identification of this secondary metabolite changed the concept of aromaticity and paved the way for the structural elucidation of many non-benzenoid aromatic natural products called tropolones. Stipitatic acid has undergone one of the most detailed isotopic labelling studies to understand how nature could form such an aromatic seven-membered ring under biological conditions. The incorporation of labelled acetate units into the carbon skeleton of stipitatic acid pointed to a polyketide origin for this fungal tropolonoid metabolite. Further labelling study using radioactive molecules of 3-methylocinaldehyde 29 suggested an oxidative ring expansion rearrangement for the formation of the tropolone seven-membered ring.

Sexual reproduction is an important process amongst eukaryotic organisms, with one function being to maintain genetic variation. The idea that complex eukaryotic species can persist for millions of years in the absence of sex defies fundamental evolutionary dogma, yet a group of organisms known as ancient asexuals were thought to have evolved clonally under deep evolutionary time. Prominent among these are the arbuscular mycorrhizal fungi (AMF), which are obligate plant symbionts that colonize the root cells of plants and extend their hyphae into the soil assisting the plant in acquiring key nutrients. Unlike most eukaryotes, AMF cells are multinucleate with thousands of nuclei moving through a continuous cytoplasm. Genomic analyses have identified a putative mating-type (MAT) locus within the nuclear genomes of model AMF Rhizophagus irregularis, a region that in other fungi dictates the process of sexual reproduction. Additional findings demonstrated that AMF strains carry one of two nuclear organizations. They can be either homokaryotic (AMF homokaryons), where all nuclei within the cytoplasm are virtually identical, or heterokaryotic (AMF dikaryons), where two MAT-locus variants co-exist within the cytoplasm. Despite a lack of observable traits indicative of sex, this homo/heterokaryotic dichotomy is reminiscent of the nuclear organization of sexual fungi. My research aims to build on these findings to investigate the actual role of the MAT-locus in driving AMF reproduction. To address this, I build my thesis into three main chapters. The first chapter reviews our current understanding of AMF genetics and what drives genome evolution in these organisms. The second chapter establishes a relatively easy, inexpensive, and reproducible approach to genotype known MAT variants of R. irregularis in natural and experimental conditions. The last chapter uses experimental crossings between strains to assess cytoplasmic compatibility and nuclear exchange. I demonstrate that dikaryotic spore progenies can be formed after co-culturing two distinct AMF homokaryotic strains. Further analyses of various genomic regions also reveal possible recombination in homokaryotic spore progenies from co-cultures. Overall, this research provides new experimental insights into the origin of genetic diversity in AMF. These findings open avenues to produce genetically new AMF strains in the lab using conventional crossing procedures and provide a glimpse of the mechanisms that generate AMF genetic diversity in the field.

Endophytic fungi from Cassia fistula or golden shower, a well known medicinal plant in Thailand and Asia, were isolated from trees growing in three geographical separate sites. These locations were Kanchanaburi, Nakhon Ratchasima and Bangkok and were selected to allow comparisons between their endophytic assemblages and to evaluate these data in relation to differences in plant diversity and density and local environment. Kanchanaburi which was the site closest to a natural forest situation provided the highest number of isolates with Bangkok, where the trees were isolated individuals, having the least. Members of the Xylariaceae proved to be common and frequent isolates especially species of Xylaria and Daldinia but Nemania and Hypoxylon were also obtained. Phomopsis was also well represented and clearly was dominant at the Kanchanaburi site. Species of Fusarium, Colletotrichum, Penicillium, Nigrospora, Coprinus and Psathyrella were also identified but were occasional isolates. Differences in endophytic assemblages between samples obtained early in the rainy season (July, 2001) with those sampled towards the end of the rainy season (December, 2001) were found to occur in the Nakhon Ratchasima samples with over twice as many isolates obtained from the December samples. This is likely to be a reflection on the longer exposure period to the potential inoculum of these leaves. A total of 956 endophytic isolates were obtained from the three sites with samples from Kanchanaburi (December 2000) and Bangkok and Nakhon Ratchasima in July 2001 with a further samples from Nakhon Ratchasima in December, 2001. Isolations were also made from different anatomical regions of the leaf, leaf lamina, midrib and veins. There were no appreciable differences in either the number of isolates obtained or an association between leaf area and specific fungal species. Identification of many xylariaceous endophytic isolates is well known to be problematic since Xylaria species rarely produce their anamorphic form in culture and virtually no members of the Xylariaceae develop their teleomorph in culture. Therefore molecular techniques were used to compare DNA sequences of the ITS region from a selection of endophyes with sequences obtained from teleomorphic material, or cultures derived from teleomorphs of identified and authenticated Xylariaceae. Comparisons were also made with data held in GenBank. This enabled the identity of a number of taxa to be made although more sequences from Xylaria species are required for future investigations. A number of non-xylariaceous taxa were also named as a result of DNA sequence comparisons. Secondary metabolites from the xylariaceae were also investigated and their metabolite profiles used to support identifications. The metabolite profiles proved to be a useful tool to confirm doubtful endophytic isolates when their DNA sequences could not place them with certainty in a right group. Together with extracts from other endophytic species, their inhibitory effects on bacteria and fungi were tested. Cassia endophytes were found to show low antimicrobial activity. However, they may later be shown to have other activities when when tested e. g. anti-malarial, anti-cancer and anti-HIV.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第10274号
農博第1346号
新制||農||869(附属図書館)
学位論文||H15||N3795(農学部図書室)
UT51-2003-H695
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 熊谷 英彦, 教授 井上 國世, 教授 江崎 信芳
学位規則第4条第1項該当

Thesis (Ph. D.)--Illinois State University, 1997.
Title from title page screen, viewed June 7, 2006. Dissertation Committee: Roger C. Anderson (chair), Anthony E. Liberta, Mathew J. Nadakavukaren, Derek A. McCracken, R. Michael Miller. Includes bibliographical references (leaves 68-77) and abstract. Also available in print.

The inclusion of the species of polypores with rose-colored context in the genus Fomitopsis is proposed in this study. Temperature responses demonstrated that these fungi can be placed in three groups according to their optimum temperature for growth: (1) Fungi with an optimum at 25 C, Fomitopsis rosea; (2) Fungi with an optimum at 28 C, Fomitopsis feei, Fomitopsis lilacino-gilva, Fomitopsis cajanderi, and Fomitopsis carnea; (3) Fungi with an optimum at 30-34 C, Fomitopsis dochmius, F. cajanderi, and Fomitopsis cupreo-rosea. Decay tests showed that Fomitopsis feei and Fomitopsis lilacino-gilva reported only on hardwoods are also capable of decaying conifers "in vitro", causing weight losses up to 40% after 20 weeks. It was confirmed that all these fungi were brown rotters. An heterothallic, bipolar behavior was demonstrated for F. feei and agreed with the behavior previously reported for two other members on this complex, F. rosea and F. cajanderi. Due to the failure of the other isolates to fruit in culture, their sexual behavior could not be determined. Monokaryotic x monokaryotic pairings and monokaryotic x dikaryotic pairings showed homogenic or heterogenic incompatibility between members of the same species or different species. Vegetative incompatibility was found when pairing heterokaryotic dikaryons of different isolates of the same species which demonstrated genetic diversity in the populations. Dikaryotization did not occur when heterokaryotic dikaryons of F. lilacino-gilva, F. dochmius, F. carnea, and F. cupreo-rosea were paired with monokaryons of F. feei, F. cajanderi, or F. rosea which supported the concept of genetically different species.