This work consists of an introduction, six chapters dealing with various aspects of the response of mycorrhizal fungi to copper, and a brief conclusion. The first chapter examines the enzyme tyrosinase in several ectomycorrhizal fungi and shows that its activity is altered in these fungi in response to copper. Polyamines are also examined in this chapter, and it is shown that their levels are altered in some ectomycorrhizal fungi due to copper stress but not in others. The second chapter uses transmission electron microscopy to demonstrate that copper is bound to the hyphae of ectomycorrhizal fungi grown on solid media, but the location of the binding varies between fungal species. In vitro copper tolerances of a number of ectomycorrhizal species are compared in this chapter and differences in tolerance are evident between species and between different isolates of the same species. In the third chapter, four ectomycorrhizal fungi and one nonmycorrhizal fungus are evaluated for their ability to improve the growth of Japanese Red Pine under conditions of copper stress. Improvement of pine seedling growth is not correlated with in vitro copper tolerance of the fungus, but is related to the degree of compatibility between host and fungus. Despite differences in in vitro tolerance between three isolates of the same species, there are no differences in the effect of the isolates on the tree host under conditions of copper stress. Ectomycorrhizal fungi were also inoculated in pairs on pine seedlings and the competitive abilities of the fungi are compared under stressed and nonstressed conditions. The fourth chapter discusses the results of inoculation of pine with a nonhost fungus which stimulates dichotomous branching of the root system. The compound responsible for the branching is demonstrated to be indole-3-acetic acid (IAA), a plant growth hormone. The final two chapters deal with endomycorrhizal fungi. In the first of the two, inoculation of onion with an endomycorrhizal fungus demonstrates that the fungus probably plays no direct role in the response of the plant to heavy metals, based on biomass production, nutrient uptake, and photosynthetic rate. The last chapter demonstrates that the vascular plants found on abandoned mines in Virginia and North Carolina are well colonized by endomycorrhizal fungi; thus, an absence of these fungi is not a reason for the limited natural recolonization of the mine spoils. / Ph. D.

No / Rosnecatrone 7 is a phytotoxic metabolite isolated from cultures of a virulent strain of the fungus Rosellinia necatrix. The compound is identified as 5-hydroxy-4-hydroxymethyl-1-(1-hydroxy-3-methylbut-2-enyl)-3-[(E)-propenyl]-7-oxabicyclo[4.1.0]hept-3-en-2-one by chemical and physical methods. The absolute configuration is determined

Fungi associated with Engelmann spruce and subalpine fir were studied in a high altitude area of western Wyoming. Thirty permanent plots were established and their mean stand age determined. Three age classes were delineated: young (78 years), mature (127 years), and old-age (216 years). Stand and soil parameters including density, basal area, and soil pH, P, K, Ca, organic matter, and Mg concentrations were used to define similarities in stand characteristics. Sporocarps of higher basidiomycetes and ascomycetes were collected and identified, and ectomycorrhizal root tips sampled from each plot. Pure cultures of the basidiomycetes were routinely attempted. Mycorrhizal syntheses were subsequently attempted with both tree species using successful pure cultures. Thirty-nine species of higher fungi were collected during the study. Distribution of sporocarps throughout the age classes revealed a distinct fungal flora in each age class. Greatest density of species appeared in the mature stands. Non-mycorrhizal fungi were more abundant in the mature stand while mycorrhizal species were more abundant in the old-age stand. Mycorrhizal root counts increased from young to old-age stands. Low numbers of sporocarps and mycorrhizal rootlets were collected from the young stand. It seems evident from this study that a more diverse assemblage of higher fungi is present in mature and old-age forests and a progressive increase of fungal species from young to old-age stands supports the hypothesis that fungal succession is occurring in the study area. / Master of Science

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

Rhizoctonia solani, Pythium aphanidermatum, and Macrophomina phaseolina were isolated from Euphorbia lathyris grown in fields near Tucson, Arizona. R. solani occurred as a damping-off organism in the fall. P. aphanidermatum infected seeds, seedlings, and mature plants in laboratory and greenhouse tests. Although P. aphanidermatum was infrequently isolated from field plants in Arizona, it was a major pathogen of greenhouse plants growing at high temperatures in nonsterile soil. M. phaseolina was a major pathogen. Infection of E. lathyris roots occurred within 1 mo of an October 1980 planting, but symptoms were not significant until June. Sclerotia of this fungus ranged in numbers from 1 to 246 sclerotia/g field soil. Population densities of 0.2 sclerotium/g soil were sufficient to cause more than 90% plant death in field plots. Some plants infected with M. phaseolina were growing in an area newly cleared of native desert vegetation. Subsequently, M. phaseolina was found in uncultivated soils from four vegetative communities in southern Arizona at elevations from 600 to 2,000 m; the fungus also was recovered from roots of several symptomless native plants.

O processo de transformação microbiana de metabólitos secundários é uma técnica emergente no Brasil e que apresenta enorme potencial para obtenção de substâncias biotransformadas com novas e diferentes moléculas, as quais podem apresentar melhor desempenho de suas atividades biológicas. O objetivo do presente projeto foi efetuar transformações microbianas de metabólitos secundários oriundos da espécie vegetal Viguiera robusta Gardn. (família Asteraceae), com ênfase nas lactonas sesquiterpênicas (LSTs), para posterior avaliação de sua citotoxicidade frente a linhagens celulares. As etapas de isolamento e purificação de LSTs foram realizadas através de métodos cromatográficos e a elucidação estrutural através de métodos espectroscópicos. A avaliação da atividade citotóxica dos metabólitos biotransformados foi proposta em virtude de estudos iniciais indicarem potencial citotóxico da LST budleína A frente às linhagens SK-BR-3 (adenocarcinoma de mama) e células leucêmicas JURKAT. / The microbial transformation of secondary metabolites is an emerging technique in Brazil with high potential to obtain biotransformed compounds with new and different molecules which can show enhanced biological activities. Herein, microbial transformations of secondary metabolites from Viguiera robusta Gardn. (family Asteraceae), with emphasis in sesquiterpene lactones (STLs), were proposed with the aim to evaluate their cytotoxic activity against tumor cells lines. Isolation and purification of STLs were carried out through chromatographic methods and structural elucidation by spectroscopic methods. The evaluation of cytotoxic activity of the biotransformed metabolites was proposed due to the fact that previous studies indicated potential cytotoxic activity of the STLs budlein A against SK-BR-3 and JURKAT cells lines.

Fungi are key components in forest ecosystems, being involved in decomposition of plant biomass and the cycling of nutrients in forest soils. Despite their importance little is understood about the influence forest management practices, such as long-term prescribed burning and forest conversion are having on soil fungal communities. Part of the work described in this thesis investigated the effects of long-term repeated prescribed burning on the total soil fungal community, the diversity of mycelial communities of ectomycorrhizal fungi and the influence of biennial prescribed burning on the cellulolytic soil fungal community using stable isotope probing techniques. The influence of long-term repeated prescribed burning on soil fungal communities was investigated through a series of studies conducted at Peachester State Forest, Queensland, Australia. This site has been the centre of a long-term repeated prescribed burning experiment, established since 1972, consisting of plots subjected to biennial, quadrennial or no burning. Denaturing gradient gel electrophoresis (DGGE) was used to show that long-term prescribed burning significantly altered the total fungal community structure in the top 10 cm of soil, when compared with unburned plots. Hyphal ingrowth bags, used to target ectomycorrhizal (ECM) mycelia in soil, along with DGGE analysis, indicated that profiles of the soil fungal community from 2 yr burn plots significantly differed from those of the 4 yr burn and unburned plots. Following analysis of clone assemblages from the different burn regimes, results indicated that this difference reflected an altered ECM fungal community composition. 13C stable isotope probing (SIP), following the incubation of soil with 13C labelled cellulose, and DGGE analysis was found to significantly alter the active fungal community in the upper 10cm of soil at Peachester State Forest. Fewer active fungi in the 2 yr burn plots were found to have incorporated 13C compared to the unburned plots, strongly suggesting that the activities of cellulolytic fungi were negatively affected by the 2 yr burning treatment. The thesis also incorporated work that assessed the effect of forest conversion from native eucalypt to Pinus elliottii plantation on the soil fungal community at Beerburrum State Forest, Queensland, Australia. ITS and 18S RNA and DNA were used, along with terminal restriction fragment length polymorphism (T-RFLP) and DGGE analysis, indicating that total and active fungal communities differed significantly between the native eucalypt forest and first rotation P. elliottii plantation. This suggested that the conversion from native eucalypt forest to P. elliotti plantation significantly altered the soil fungal community at the Beerburrum site. / Doctor of Philosophy (PhD)

We investigated the root endophytic fungi of lupine using four approaches: (1) occurrence of fungal colonization in field-collected roots; (2) growth response of L. latifolius to inoculation with two types of fungi; (3) structure of root colonizations of Pinus and Lupinus by Phialocephala fortinii, a septate endophytic fungus of lupine; and (4) comparison root morphology, mycorrhizal colonization and natural ¹⁵5N-abundance N₂ fixation of three legumes. In part 1, three species of Lupinus were never observed to have fungal colonization; nine species were colonized by VA mycorrhizal fungi; seven species were colonized by fungi with septate hyphae which often formed intracellular scierotia, here called septate endophytes. In part 2, shoot weight of 16 week old L. latifolius seedlings in the greenhouse was significantly reduced by Glomus spp. in one experiment; p. fortinii significantly increased nodule weight in one experiment and reduced it in the other. In part 3, P. fortinii colonized root epidermal and cortical cells in the root hair zone on ultimate lateral pine roots, as well as cortical and epidermal cells of primary roots of Pinus and Lupinus. Fungal colonization was inter- and intracellular with scierotia forming in cells of both hosts. Labyrinthine tissue, a type of fungal differentiation which occurs in the Hartig net of ectomycorrhizae, formed sporadically on pine roots. In part four, Roots of Lupinus albicaulis cv. hederma had a significantly larger proportion of coarse roots (> 1 mm diameter) and significantly less mycorrhizal colonization than two other legumes, Medicago lupinula and Trifolium hybridum. Estimated aboveground N derived from fixation ranged from 6.1 to 39.9 kg per hectare (average = 22.0 kg/ha) and did not vary significantly among species. / Graduation date: 1992

The central aim of this study was to determine if indigenous fungi of an oil-contaminated soil could be effectively used in a low-cost bioremediation of the soil. Since some of the contaminant had been present at the site for over two decades, the indigenous microbial species had been subjected to specific selection pressures for a protracted period, thus facilitating key enzymatic capabilities for hydrocarbon degradation. Analysis of the pertinent influential parameters of soil bioremediation indicated that an ex situ technique, utilising the catabolic activities of the indigenous soil fungi, was a feasible low-cost option. Fungi were isolated from the contaminated soil through a variety of techniques. The abilities of these isolates to degrade the contaminant oil and a range of representative hydrocarbon molecules was evaluated by a systematic screening programme. Sixty-two isolates were initially examined for their growth potential on hydrocarbon-supplemented agar. A bioassay, utilising hydrocarbon-impregnated filter paper discs, was then used to examine the abilities of 17 selected isolates to catabolise three representative hydrocarbon molecules (hexadecane, phenanthrene and pristane) in different concentrations. In the same bioassay, the influence of a co-metabolite (glucose) on growth potential was also examined. Eight fungal species: Trichophyton sp.; Mucor sp.; Penicillium sp.; Graphium sp.; Acremoniwn sp.; Chaetomium sp.; Chrysosporium sp.; and an unidentified basidiomycete were then selected. Liquid batch cultures with a hydrocarbon mixture of hexadecane, phenanthrene, pristane and naphthalene facilitated quantitative analysis (HPLC) of the hydrocarbon catabolic abilities of the selected isolates. Ex situ bioremediation was evaluated at laboratory-scale by both bioaugmentation and biostimulation in soil microcosm trials. During the course of the study, total petroleum hydrocarbon (TPH) concentration (U.S. EPA Method 418.1) was used as a simple and inexpensive parameter to monitor hydrocarbon disappearance in response to soil treatments. Soil microbial activities were estimated by use of a fluorescein diacetate hydrolysis bioassay. This was found to be a reliable and sensitive method to measure the activity of respiring heterotrophs as compared with the unreliable data provided by plate counts. In the bioaugmentation trial, the eight selected isolates were individually used to inoculate (30% v/v) the contaminated soil. The highest rate of biodegradation (50.5% > than the non-sterile control) was effected by an Acremonium species after 50 days incubation (25°C). The second highest rate of biodegradation (47% > than the non-sterile control) was achieved with a soil treatment of sterile barley/beer waste only. Comparable rates of hydrocarbon degradation were achieved in simple biostimulation trials. Thus, due to its lower cost, biostimulation was the preferred remediation strategy and was selected for further laboratory investigation. Common agricultural or industrial lignocellulosic wastes such as: wood chips; straw; manure; beer brewery waste; mushroom compost; and spent mushroom substrate were used as soil treatments, either alone or in combination. The effect of the addition of a standard agricultural fertiliser was also examined. The highest level of biodegradation (54.4% > the non-sterile control) was recorded in a microcosm supplemented (40% v/v) with chicken manure. Finally, an ex situ bioremediation technique was examined in a pilot-scale field trial. Wood chips and chicken manure were co-composted with the contaminated soil in a low-cost, low-maintenance bioremediation system know as passive thermal bio venting. Extensive monitoring of the thermal environment within the biopile was made as an indirect measure of microbial activity. These data were then used to optimise the composting process. Three-dimensional graphical representations of the internal temperatures, in time and space, were constructed. From these graphs, it was determined that an inner core region of approximately 500 cm3 provided a realistic simulation of conditions within a full-scale biopile. During this trial a TPH reduction of 68% was achieved in 130 days. The findings of this research suggested that the utilisation of fungal catabolism is applicable to soils contaminated with a wide range of hydrocarbon contaminants. Passive thermal bioventing offers a bioremediation strategy which is highly suitable for South African conditions in terms of its low level of technological sophistication, low maintenance design and, most importantly, its relatively low cost. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

Bibliography: leaves 172-180 / vi, 180 leaves, [29] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Pathology, 1983