The overall objective of this thesis was to investigate how the arbuscular mycorrhizal (AM) symbiosis can affect the production of phytochemicals (antioxidants; rosmarinic and caffeic acid, RA & CA) in the shoots of basil (Ocimum basilicum L.). As a result of an increasing interest in natural/herbal medicines, more effort is now needed to produce herbal products of better quality, i.e. higher and standardised phytochemical concentrations. Thus, it was hypothesised that the naturally occurring AM fungi (AMF) could play an important role in improving the growth and phytochemical concentrations in medicinal herbs such as basil, as organic methods of cultivation are increasingly sought after to grow such plants. Despite a reasonable amount of information available in the literature on the changes of phytochemical concentrations in the roots of host plants following AM colonisation, very little is known about such processes in the aerial part of such plants. Furthermore, basil has hardly been studied as a host plant in AM research, and very little is known of its responsiveness to AM colonisation. As AMF are well known to improve phosphorus (P) uptake in their host plant, the first objective of this work was to obtain AM and non-mycorrhizal (NM) plants matched for tissue P concentrations and growth rates. Only under such conditions would it then be possible to separate benefits derived from improved plant P uptake from non-nutritional benefits. It was found that basil is highly responsive to P, and that under low or little P supply it is quite dependent on the AM symbiosis in order to grow. However, growth depressions were observed when growing basil in winter with Glomus intraradices, suggesting that the fungal symbiont can act as a strong sink of carbon (C) under such conditions. Thus, in order to obtain AM and NM plants with matched growth rates and tissue P concentrations, it was found that basil needed to be cultivated in summer in a soil/sand mixture with a ratio of 1:3 (w/w), along with 0.2 g/kg CaHPO4 and 25% of AM inoculum (AM plants). Under these conditions, AM plants grew as well as NM plants and G. caledonium and G. mosseae were shown to increase the concentrations of RA and CA in the shoots of basil, but not in roots. Such results were not an indirect effect of improved P uptake. In order to understand the mechanisms by which AMF increased RA and CA concentrations in basil, further experiments were set up to investigate the effect of 1) AM developmental stages, 2) nitrogen (N) supply and 3) phytohormone changes on the production of RA and CA in the shoots. None of these factors was found to contribute to increases in antioxidants in basil under AM symbiosis. Therefore, the mechanisms by which AMF affect RA and CA concentrations in basil still remain unknown. A final experiment was carried out to investigate the potential of an AM fungus to improve the growth of basil when challenged with a specific pathogen Fusarium oxysporum f.sp. basilici (Fob), which causes significant production losses. The results showed that inoculation of basil with G. mosseae not only improved plant growth compared to NM plants, but also conferred a protective effect against Fob. However, shoot antioxidant concentrations (RA, CA, total phenolics and essential oils) were not increased in AM plants compared to NM plants, and the mechanism of protection against Fob could not be elucidated. Due to the high variability of RA and CA concentrations obtained in AM plants in different experiments, it cannot be concluded that AMF confer an absolute advantage over uninoculated plants if the main concern is to obtain standardised concentrations of phytochemical in basil. On the other hand, the key results presented in this thesis do indicate that inoculating basil with AMF can be beneficial to improve its growth as well as antioxidant concentrations, compared to NM plants grown under similar conditions. Such results could be of potential interest to basil growers who wish to cultivate this medicinal herb organically (i.e. low P supply and no chemical fertilisers added). / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1330324 / Thesis (Ph.D) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

Proteins of the nuclear envelope play important roles in a variety of cellular processes e.g. transport of proteins between the nucleus and cytoplasm, co-ordination of nuclear and cytoplasmic events, anchoring of chromatin to the nuclear periphery and regulation of transcription. Defects in proteins of the nuclear envelope and the nuclear pore complexes have been related to a number of human diseases. To understand the cellular functions in which nuclear envelope proteins participate it is crucial to map the functions of these proteins. The present study was done in order to characterize the role of three different proteins in functions related to the nuclear envelope in the yeast Saccharomyces cerevisiae. The arginine methyltransferase Rmt2 was demonstrated to associate with proteins of the nuclear pore complexes and to influence nuclear export. In addition, Rmt2 was found to interact with the Lsm4 protein involved in RNA degradation, splicing and ribosome biosynthesis. These results provide support for a role of Rmt2 at the nuclear periphery and potentially in nuclear transport and RNA processing. The integral membrane protein Cwh43 was localized to the inner nuclear membrane and was also found at the nucleolus. A nuclear function for Cwh43 was demonstrated by its ability to bind DNA in vitro. A link to nucleolar functions was demonstrated by genetic analysis. Furthermore, Cwh43 is interacting with signalling pathways perhaps acting as a sensor for signals transmitted from the cytoplasm to the nucleus. The Myr1 protein was found to be membrane-associated and to interact with proteins involved in vesicular traffic. Overexpression of Myr1 affects nuclear morphology and nuclear pore distribution suggesting a function in membrane dynamics. In conclusion, the presented results aid in a deeper understanding of functions related to the nuclear envelope in revealing a novel link between arginine methylation and the nuclear periphery, identifying a novel inner nuclear membrane protein and a new membrane-associated protein.

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.

Cereal crops such as maize and sorghum are economically important in South Africa (SA) as a staple food diet. In order to meet the needs of South Africa’s growing population, higher yields in crop production need to be attained. However, the two major stress factors that affect yield production and require primary attention are nutrient deficiencies and pest infestations. Research is now being focused on certain endophytes that have become a valuable tool for agriculture as they protect crops against the above-mentioned stresses. The endophyte focused on in this study was Arbuscular Mycorrhizal fungi (AMF). This research was aimed at developing an in vitro culture system for SA cereal crops to enable interaction studies of endophytes. This dissertation is divided into two parts; the first part focused on the development of an in vitro culture system, the assessment of sorghum plant growth and exudate production in the presence of the Glomus intraradices strain. The results indicated that sorghum produces the required root exudates in the second stage of growth. Using high pressure liquid chromatography with mass spectrometry (HPLC/MS), it was noted that sorghum produced phytochemicals as chemoattractants for the respective endophytes. However, it was documented that when the plant underwent certain stresses they produced exudates, which acted as phytotoxic compounds that destroyed symbiotic organisms around sorghum rhizophere. The second part focused on optimization of the surface sterilization of maize seeds. The results indicated that maize contained unidentified endophytes, which negatively affected plant development. Surface sterilization of maize seeds was accomplished. The successful in vitro development can be used for future use to study plant development. Understanding plant development and interaction with symbiotic endophytes would not only be of great benefit but would also make it easier to create a biocontrol agent in vitro, which would bring about high crop yields at cost-effective prices and would be less labour intensive. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Methamphetamine interacts with vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine (DA) uptake and promotes DA release from presynaptic vesicles, increasing cytosolic DA available for methamphetamine-induced reverse transport by DA transporters. By inhibiting VMAT2, lobelane, a defunctionalized, saturated lobeline analog, decreases methamphetamine-evoked DA release and methamphetamine self-administration in rats. In this dissertation structure-activity relationships around the lobelane structure were investigated on racemic lobelane analogs with varying methylene linker lengths at central piperidine ring. Affinity for dihydrotetrabenazine (DTBZ) sites on VMAT2 and for inhibition of VMAT2 function was determined to be 0.88-63 and 0.024-4.6 µM, respectively, and positively correlated. The most potent and selective analog, (±)-cis-2-benzyl-6-(3-phenylpropyl)piperidine [(±)-GZ-730B], for VMAT2 uptake was identified as the lead. The ability of (±)-GZ-730B to inhibit methamphetamine-evoked [3H]DA release from striatal synaptic vesicles and endogenous DA release from striatal slices was determined. The lead analog-induced inhibition of methamphetamine-evoked vesicular [3H]DA release did not translate to inhibition of methamphetamine-evoked DA release in the more intact striatal slices. Moreover, poor water solubility of these lobelane analogs prohibited further in vivo work. Subsequent work focused on analogs with the C-3 and C-4 carbons in the piperidine ring eliminated to afford racemic acyclic lobelane analogs. Generally, acyclic analogs exhibited greater water solubility and less lipophilicity compared to lobelane. Acyclic analogs exhibited affinities (Ki = 0.096-17 μM) for [3H]DTBZ sites that correlated positively with affinity (Ki = 3.3-300 nM) for inhibition of [3H]DA uptake. Pure enantiomers of potent racemic analogs were synthesized, and found to potently, selectively, and competitively inhibit [3H]DA uptake at VMAT2 and to release vesicular [3H]DA in a biphasic manner. Lead enantiomer (R)-N-(1-phenylpropan-2-yl)-3-phenylpropan-1-amine [(R)-GZ-924] inhibited methamphetamine-evoked [3H]DA release from striatal synaptic vesicles, but not from the more intact striatal slices. Surprisingly, (R)-GZ-924 inhibited nicotine-evoked [3H]DA overflow from striatal slices, revealing nonspecific effects. Importantly, (R)-GZ-924 inhibited methamphetamine self-administration in rats. However, the analog also inhibited food-maintained responding, revealing a lack of specificity. The lead analog will not be pursued further as a pharmacotherapy due to the lack of specificity. Further evaluation of the pharmacophore is needed to discover analogs which specifically inhibit the neurochemical and behavioral effect of methamphetamine.

A time course study was conducted to monitor disease development and expression of the defense-related genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and hydroxyproline-rich glycoprotein (HRGP) in bean (Phaseolus vulgaris L.) plants colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices , and post-infected with the soil-borne pathogen Rhizoctonia solani. Pre-colonization of bean plants by the AM fungus did not significantly reduce the severity of rot symptoms. RNA blot analysis revealed a systemic increase in transcript levels of the four defense-related genes in response to R. solani infection. On the other hand, pre-colonization of bean plants with G. intraradices elicited no change in PAL, CHS and CHI transcripts, but an increase of HRGP transcripts in leaves was detected. A differential and systemic alteration in the expression of all four defense genes was observed in AM beans post-infected with R. solani. Depending on the time after infection with R. solani and the tissue examined, varying responses from stimulation, suppression, to no change in transcript levels were detected.