A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. / Phytoremeditation of mine tailings provides the most cost-effective means of alleviating their pollutant effects. Research has shown that successful revegetation of mine tailings can be optimised by providing appropriate microbial symbionts for the plants. The aim of this study was to assess the arbuscular mycorrhizal (AM) status of trees currently being used for phytoremediation trials of mine tailings in the Welkom gold fields, and to determine the AM fungal diversity of these sites. The Acacia spp. analysed were growing on rehabilitated gold and uranium mine tailings which had undergone different rehabilitation regimes. Planted acacia trees which had been inoculated with crude AM fungal inocula were present on one mine tailing site as compared to the second mine tailing site on which the acacias were naturally colonisers and the site had been ameliorated with garden refuse. Root and slime samples were collected in early spring and half if each initial sample was used immediately for colonisation analysis and to identify AM fungi through molecular analysis of the small subunit rRNS gene sequences; the other half of each sample was used to produce trap cultures which were used later for colonisation and molecular analysis. Total AM fungal colonisation of initial samples for planted acacies was 19 % and for naturally colonising acacias was 66 %. The total AM fungal colonisation of trap culture samples for planted acacias increased to 32 % and for naturallhy colonising acacias it increased to 78 %. Spore counts of initial samples averaged 402 spores per 100 g-1 soil for planted acacias and 455 spores per 100 g-1 soil for naturally colonising acacias. For trap culture samples, spore counts decreased by approximately 50 %. The AM fungi identified fell within 8 genera, namely, Diversispora, Rhizophagus, Scutellospora, Claroideoglomus, Cetraspora, Sclerocystis, Glomus and Redecker. The study represents a first report utilising molecular biosystematics with AM fungal DNA from colonised roots as the template. The results will assist in making decisions about future AM fungal surveys and applying AM fungal inoculum in phytoremediation trials of mine waste sites. Key words: Phytoremediation, mine tailings, arbuscular mycorrhizal (AM) fungus, Acacia, molecular identification, SSU rRNA gene sequence

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No description available.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2016. / South Africa, as one of the leaders in mining industry, due to the variety and quantity of minerals produced, has been and is still producing a number of mine tailings which are contaminated by heavy metals. Heavy metals are very harmful to plants and especially to human beings and animals due to their non-biodegradable nature. The problem of environmental metal pollution could be combated by the establishment of Arbuscular Mycorrhiza (AM) vegetation on the surface of mine tailings. Besides the toxicity of the substrate, such areas usually lack essential nutrients (mainly N, P, and K) and organic matter. AM fungi contribute to soil structure by forming micro- and macro- soil aggregates within the net of external hyphae. Their presence may reduce stress caused by lack of nutrients or organic matter and increase plant resistance to pathogens, drought and heavy metals. Therefore, mycorrhizal fungi may become the key factor in successful plant revegetation of heavy-metal-polluted areas by promoting the success of plant establishment and increasing soil fertility and quality. The aim of this project was to identify AM fungi from a number of heavy metal sites in South Africa using both morphological and molecular techniques, followed by the evaluation of heavy metal distribution and localisation in mycorrhizal roots. Soil samples were collected from three different provinces, namely: Gauteng, Mpumalanga and North West provinces. The sites were selected based on their historical and current heavy metal contamination. Indigenous AM fungal isolates (which are adapted to local soil conditions) can stimulate plant growth better than non-indigenous isolates. AM fungal spores were isolated from 100g of representative soil sample by the wet sieving and decanting method, followed by assessment of spore numbers and infective propagules. The spores of a subset of the pot samples were mounted on microscope slides in polyvinyl lactic acid glycerol and identified by morphological characteristics to the level of genus or species. Most of the spores counted were observed in a 45 μm sieve. These spores were tiny and had different sizes, colours and shapes. The majority of the observed spores were small, brown and oval in shape. For morphological identification, plant roots were stained and hyphae were found to be the most abundant in roots. For molecular identification, two sets of nested PCR primers, namely NS1 & NS4 coupled with AML1 & AML2, were employed in this study due to their ability to amplify all subgroups of arbuscular mycorrhizal fungi (AM fungal, Glomeromycota), while excluding sequences from other organisms. Through both morphological characteristics and molecular identification, the following fungal genera were identified for the first time in the studied sites in South Africa. The study identified a total of 14 AM fungal genera and 55 AM fungal species, which are: Glomus (15), Acaulospora (11), Scutellospora (6), Gigaspora (6), Rhizophagus (3), Funneliformis (3), Archaeospora (2), Claroideoglomus (2), Ambispora (2), Sclerocystis (1), Fuscutata (1), Entrophospora (1), Diversispora (1), Paraglomus (1). Both Glomus and Acualospora have been observed to be the highest occurring genera in the analysed soil samples, followed by Scutellospora and Gigaspora and others mentioned. PIXE technique was successful in localising elemental concentration in both plant roots and AM fungal structures, as well as in indicating the large vesicles in root tissue. AM fungal structures in the outer cortex or outer epidermal layer of the root cross-sections were observable, as shown by the more significantly enriched Si in the vesicles and arbuscules. Distinctive elemental maps can be used to localise sites of colonisation and verification of the symbiotic nature of the tissue. This indicates that a range of metals can be sequestered in AM fungal structures above levels in surrounding host root tissue, and demonstrates the potential of Micro-PIXE to determine metal accumulation and elemental distribution in mycorrhizal plant roots and inter-and intracellular AM fungal structures. This research highlights the potential of AM fungi for inoculation of plants as a prerequisite for successful restoration of heavy metal contaminated soils. It also illustrates the importance of AM fungal diversity in selected high heavy metal (HM) sites in RSA, particularly in the North West and the Gauteng gold mining slime dams. Therefore, phytoremediation of mine tailings by mycorrhizal plants seems to be one of the most promising lines of research on mine tailings contamination by heavy metals. The strategies which evolved during this project have great potential for phytoremediation of toxic mining sites, and thus can help mitigate the environmental problems, especially in the mining waste sites. / LG2017

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No description available.

In this study, we have demonstrated that inoculation of bean seeds with non-pathogenic binucleate Rhizoctonia (np-BNR) at sowing protected bean seedlings from infection of R. solani. Using quantitative real-time RT-PCR (QRT-PCR), transcript levels of defense genes encoding 1,3-beta-glucanase (GLUC), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) in one-week old bean seedlings was monitored during np-BNR and R. solani interaction. The results revealed that protection effect of np-BNR correspond to a systemic suppression of these three defense genes' expression from significant higher level elicited by R. solani to the level of non-infected plants. This indicates that bio-protection by np-BNR isolates is not correlated to activation of these three defense genes' expression. Similar suppression was achieved for pre-colonization of bean seedlings with arbuscular mycorrhizal (AM) Glomus introradices on GLUC gene expression, although the AM fungus did not significantly reduce rot symptoms. Possible mechanisms implicated in down-regulation during plant-pathogen and np-BNR or AM interaction are discussed.

Copper hydroxide-treated surfaces are commonly used to control roots in horticultural production systems, although the particulars of Cu movement from the treated surface are largely unknown. The rate and temporal pattern of Cu mobility from in-ground Cu-treated growing baskets, and the fate of this Cu, was studied. In a field experiment involving Acer platanoides, an alternative Cu formulation consisting of Cu metal powder was determined to move slightly more slowly from the basket fabric than Cu(OH)2 over the first season while providing adequate root control. Addition of Glomus intraradices inoculum to the basket system increased the mobility of Cu metal and had no effect on Cu(OH) 2. This suggests that VAM fungi can actively solubilize Cu metal. These results also confirm that the chemistry of the Cu, possibly in addition to the chemistry of the resin, determines Cu movement from the treated surface. The fate of Cu from in-ground baskets planted with Acer jinnala was elucidated in a second field experiment. After one field season, the majority of Cu initially on the basket fabric had been deposited in the soil both in- and outside the basket. While Cu recovered in leachate represented only a tiny fraction of basket Cu, the concentration of Cu in leachate exceeded acceptable limits in the majority of samples. This indicates that the use of Cu(OH) 2-treated baskets in the field may have a negative impact on groundwater quality.

Both lettuce and pepper crops are routinely grown as transplants in Quebec. Previous research has shown that both of these plants respond well to endomycorrhizae with a resulting improvement in plant growth and yield. Although many studies have been conducted on endomycorrhizal inoculation of transplants most were looking at the end result, i.e. yields, and did not focus on changes occurring at the transplant stage. This series of experiments was conducted to look at the effect of inoculation at the transplant stage. Lettuce (Lactuca sativa cv. Parris Island Cos) and pepper (Capsicum annuum cv. Hybrid Bell Boy) were inoculated with Glomus intraradix Schenck & Smith and Glomus versiforme (Karst.) Berch. Experiments involved testing the effect of different growing media, different container volumes and different light sources on the growth of endomycorrhizal lettuce and pepper transplants. In all experiments the amount of growth enhancement due to endomycorrhizal inoculation was low or negligible. Colonization rates were generally low, probably due to low light levels. Transplants grown in larger volume containers were generally larger than those grown in smaller containers. The transplants grown in the soil and compost based media were larger than those transplants grown in the artificial media. Growth of lettuce and pepper transplants was affected differently depending on the light source used. Some interactions between the light source and the endomycorrhizal treatments was observed.

The main project was an assessment of the effect that colonization by five different Vesicular-arbuscular mycorrhiza (VAM) cultures have on the growth of the indigenous-grasses Themeda triandra and Trachypogon spicatus, when grown on coalmine topsoil. With unamended topsoil, VAM showed the ability to significantly increase the growth of the grasses compared to non-VAM control plants. The amount of effect varied with the VAM inoculum culture type, with a VAM culture originally from the Cape Flats being the most effective. In a second trial, soil fertilized with nitrogen, potassium and low concentrations of phosphate (P) was used. Again VAM displayed the ability to improve grass plant growth. The increase in P caused the Large spore inoculum to become the most effective. This indicated that different VAM cultures are inhibited to different degrees by an increase in phosphate fertilization. The low level of VAM infection, in both trials, seemed to preclude most of the VAM associated nutrient uptake control. Varying reports have been published on the effect of fertilization on VAM infection and colonization. In an attempt to further elucidate the role of fertilizer in VAM inhibition, rhizosphere soil from a long term fertility trial near Witbank, S.A. was sampled. Amcoal environmental services fertilized forty-two plots with varying concentrations of nitrogen, potassium, phosphate and lime to assess the growth of a variety of grasses. The trial had been maintained for ten years before sampling was completed for this project. Samples from each plot were taken from the rhizosphere soil of the most prominent grass (Digitaria eriantha). VAM spores were extracted from all the samples and five different types of spores were identified and counted for each sample. By comparing spore counts from each plot, the effect that the fertilizer regime had on the VAM on that plot could be assessed. Variation in the concentrations of nitrogen (N) and potassium had no significant effect on VAM colonization. Very low concentrations of N could not be assessed as all plots had been initially top dressed with nitrogen fertilizer. Phosphate (P) fertilizer concentration had a marked effect on spore concentrations. There was a significant increase in spore concentration as P levels were increased from zero P fertilization to 80kgs P/ha. Further increase in P to ≥ 60kgs P/ha resulted in a significant decrease in spore concentrations. From this it would appear that a low level of soil P is needed to give maximum VAM colonization and further increase in soil P causes VAM inhibition. Lime ameliorated the VAM inhibition caused by high concentrations of P. Increase in P caused spore concentrations of low abundance propagules (LAP) too decreased more rapidly than high abundance propagules (HAP). In high P soils VAM with LAP would eventually be eliminated from the system resulting in a decrease in VAM diversity. A project was attempted to use the recently developed Randomly Amplified Polymorphic DNA in conjunction with the Polymerase Chain Reaction (RAPD PCR) techniques to identify different VAM families. The technique causes the amplification of segments of DNA which can be visualized by gel electrophoresis and staining. Band patterns formed can be related to the VAM of origin and hence used in identification of that VAM. An attempt was made to amplify DNA from a single spore in this manner which would, in conjunction with morphological observations, make identification of VAM easier and more accurate. Problems with either releasing the DNA from the spores, or substances in the spore inhibiting the PCR reaction made obtaining band patterns difficult. After many PCR attempts, varying extraction methods and PCR conditions, no repeatable results could be obtained and work on this project was discontinued. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.