>Magister Scientiae - MSc / Nitric oxide (NO) has been shown to orchestrate multiple defense responses to both abiotic and biotic stress. Importantly, elevation of nitric oxide content in plants by using nitric oxide generating compounds has been shown to enhance plant tolerance to abiotic stresses such as salt and drought via up-regulation of genes involved in the regulation of plant responses to abiotic stress. In this study, the effect(s) of nitric oxide (generated from 10 μM of the nitric oxide donor DET/NO) on the expression of a novel soybean cystatin gene (Glyma20g08800), lipid peroxidation, caspase-like activity and cell death in salt (150 mM)-stressed soybean leaves, roots and nodules were investigated. Salt treatment resulted in elevated lipid peroxidation, caspase-like activity and increased cell death in organs studied while the observed detrimental effects of salt stress were reversed by NO treatment. Salt stress suppressed the expression of Glyma20g08800 while the levels of expression of Glyma20g08800 returned towards those of unstressed plants when the salt-stressed plants were supplemented with nitric oxide (DETA/NO). Furthermore, promoter sequences of GmCYS1p626 and three of its homologues (Glyma20g08800, Glyma14g04250 and Glyma18g12240) were analyzed for putative abiotic stress and/NO cisregulatory elements based on co-expression analyses using bioinformatics. Several abiotic stress induced transcription factors (TFs) were identified and were hypothesized to be co-acting either directly or indirectly through additional factors in the regulation of soybean cystatin expression in response to NO and abiotic stress. Taken together, these results highlight the possibility of using NO to drive high levels of expression of cystatins during salt stress and lead to accumulation of the cystatin to levels that are sufficient to inhibit salt stress-induced caspase-like activity, which will inhibit salt stress-induced cell death and thus enhance the tolerance of the plant to salt stress and possibly tolerance to drought stress as well.

>Magister Scientiae - MSc / Nitric oxide (NO) is a signaling molecule involved in mediating plant responses to various biotic and abiotic stresses. Major abiotic stresses (drought, salinity, cold) induce common cellular responses, causing osmotic stress in plants. This results in oxidative stress due to increased production of reactive oxygen species (ROS). The increased ROS levels simultaneously induce the antioxidative system (including antioxidant enzymes such as superoxide dismutase) that regulates ROS toxicity and enhance stress tolerance in plants. It is suggested that the scavenging of ROS by antioxidant enzymes can be controlled by NO. The aim of this study was to evaluate the role of exogenously applied NO on soybean (Glycine max L. Merr.) during osmotic stress, with the purpose of determining the effects of NO on the superoxide dismutase (SOD) activity in response to osmotic stress. This study also aimed at identifying and characterising SOD isoforms induced in soybean in response to osmotic stress and exogenous NO. To achieve these aims, soybean plants were treated with sorbitol (to induce osmotic stress), an NO donor [2,2'-(hydroxynitrosohydrazono)bis-ethanimine, DETA/NO] and its respective control (Diethylenetriamine, DETA). The results showed that exogenous NO alleviated osmotic stress-induced damage by reducing the superoxide radical content, lipid peroxidation levels and also maintaining cell viability in soybean leaves, nodules and roots. Only two SOD isoforms i.e. manganese SOD (MnSOD) and copper/zinc SOD (CuZnSOD) were identified and characterised in soybean leaves and roots, iron SOD (FeSOD) was not induced. The isoforms identified exhibited low SOD activity in response to osmotic stress, with the exception of a few isoforms that had increased activity. The SOD activity was regulated by exogenously applied NO. The enzymatic activity of SOD isoforms was up-regulated by exogenous NO, except for a few SOD isoforms that were not responsive to NO. The results also showed that the increased SOD activity was associated with reduced lipid peroxidation levels. The results obtained from this study suggest that exogenous NO improves osmotic stress tolerance in soybean by regulating and increasing the SOD activity of only specific isoforms. The increased SOD activity maintains the redox homeostasis balance by detoxifying and controlling the superoxide radical levels, subsequently reducing lipid peroxidation and maintaining cell viability.

>Magister Scientiae - MSc / This study investigated the effects that drought stress imposed on the growth and development of soybean plants. Soybeans were initially observed at the whole-plant level in order to identify the physical changes that had taken place in response to drought. Further investigation of the effects of drought stress on Soybean plants were quantified at the molecular level. Physical changes of soybeans in response to drought stress were typified by the change in leaf morphology and pigmentation. At the molecular level, it was observed that drought stress resulted in the accumulation of hydrogen peroxide in soybean leaves, which was met by elevated levels of lipid peroxidation. The effects of drought on the modulation of (and interplay between cystatins) cysteine protease (caspase-like) activity and programmed cell death (PCD) were also investigated. Total caspase-like activity and cell death were enhanced in response to water deficit despite the up-regulation in gene expression of the cystatin Glyma14g04250. The cystatin Glyma18g12240 was not expressed in soybean leaves, whilst the gene expression of the cystatin Glyma20g08800 remained unchanged in response to drought. This study was aimed at the characterization of two single domain soybean cystatins, namely, Glyma14g04250 and Glyma20g08800 which could potentially be overexpressed in transgenic soybean plants in an attempt to alleviate the effects of drought stress. / National Research Foundation (NRF)

It has been suggested that the Category 1 weed, Campuloclinium macrocephalum (Less) D.C has allelopathic potential, which would, at least partially, explain its apparent success as an alien plant in South Africa. Studies were done on the plant’s root, stems and leaves to determine where the strongest allelopathic potential can be found. Once it was determined that the leaves held the strongest potential, bioassay studies were conducted on lettuce (Lactuca sativa), Eragrostis tef, Eragrostis curvula and Panicum maximum with positive results found for C. macrocephalum’s allelopathic potential. Electron microscopy was performed to determine whether allelopathic substances originate and/or are stored on the surfaces of the leaf. Positive results proved that there are possible sources of allelochemicals on both adaxial and abaxial surfaces of young and mature leaves. A dipping experiment involving dichloromethane then followed to determine the solubility of the contents of the glands found on the leaf surfaces. It can be deduced from results of all of the experiments performed that C. macrocephalum is potentially allelopathic to dicotyledonous species and to grasses. Structures found on the leaves of the plant could possibly contain the allelochemicals used by the plant to ensure its successful invasive growth habits in South Africa. The allelopathic effects that this weed will have on desirable species should be considered within the broader context of its ability to interfere with those species. In this regard its competitive ability should also be studied. Campuloclinium macrocephalum is fast invading susceptible areas of South Africa; if continuous research on control and eradication of this plant is not carried out soon, the country could suffer grave economic losses. Copyright / Dissertation (MInstAgrar)--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted

Marine water contains large numbers of fish spoilage bacteria and pathogens including V. cholerae, V. parahaemolyticus, and V. vulnificus. Consumption of contaminated seafood could lead to the transmission of these pathogens to humans. Accurate identification of suspected pathogens and spoilage organisms is important to ensure consumer safety and a long shelf life. This project assessed the bacterial quality of hake during processing. Some attention was also given to pilchards and horse mackerel. The results showed a sharp increase in the mesophilic and sucrose fermenting Vibrio species counts in hake after filleting. It has been suggested that this contamination occurred during processing from biofilms present in the sea-water distribution system. During the study 257 strains isolated from Thiosulphate citrate bile salts sucrose (TCBS) agar were screened to determine the presence of the pathogenic Vibrios amongst these isolates. It was difficult to distinguish between V. alginolyticus and V. parahaemolyticus species due to high sequence similarity in their 16S rRNA genes. Final identification of the isolates required a polyphasic approach and it was found that none of the pathogenic Vibrios were present but that the Vibrio isolates mainly belonged to V. alginolyticus. As it was suspected that the main source of contamination was the treated sea water used during processing the ability of chlorine, ozone and hydrogen peroxide to prevent biolfilm formation was examined. The behaviour of two V. alginolyticus strains (V590 and V595) isolated from the processed hake was evaluated. These strains formed biofilms faster than the V. alginolyticus LMG4409 type strain, but were similar to V. parahaemolyticus LMG2850. Biofilms formed by these bacteria were resistant to 4 mg/l chlorine and to 2 mg/l ozone, but were inhibited by 0.05 % and 0.2 % hydrogen peroxide for biofilms initiation and mature biofilms respectively. The close resemblance of atypical V. alginolyticus isolates to V. parahaemolyticus may indicate the ability of pathogens to survive under similar conditions. Effective decontamination and quality assurance strategies are therefore required when processing fish to prevent disease outbreaks. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Bovine respiratory disease complex (BRD) consists of a largely single clinical entity of bronchopneumonia that is usually associated with the assembly of large numbers of especially weaner cattle into a feedlot environment. It has a multifactorial aetiology and develops as a result of complex interactions between environmental factors, host or animal factors and pathogens. It is often difficult to determine the exact role played by the various pathogens involved in an individual outbreak of disease. None of the many organisms isolated will on their own, reliably reproduce the natural disease in experimental animals. Observations from research studies and clinical experience have indicated that the presence of mycoplasmas increases the severity of respiratory disease. However, the role of Mycoplasma spp. in BRD complex as a primary or secondary pathogen remains controversial. The various stresses associated with the feedlot causes a breakdown of the defense mechanisms that normally hold the nasal infections in check, resulting in a rapid proliferation of virulent Mannheimia haemolytica serotype A1 in particular and the spread to the lower respiratory tract. The various viruses and mycoplasmas have however been shown to have the same effect as stress on the Pasteurella populations of the nasal mucosa. More than 10 species of Mycoplasma have been isolated from the bovine respiratory tract, but not all are pathogenic. They are able to act as a stress-causing agent, leading to a decreased host defense mechanism by altering the immune responsiveness or by causing tissue damage and thereby allowing bacteria to invade and colonise the lung and so causing a severe pneumonia. M. bovis and M. dispar are the more important lung isolates, with M. bovis being the most invasive and destructive and has been shown to increase the severity of calf pneumonias. M. bovis has been isolated from bovine pneumonias, arthritis, mastitis, tendosynovitis, genitalia, keratoconjunctivitis and is considered to be the primary pathogen in endemic pneumonia in younger calves. According to the literature, mycoplasmas are isolated from 25% to 80% of pneumonic lungs in feedlot cattle and the aim of the study was to identify the isolation rates in South African feedlots over a period of 2000 to 2004. To achieve this, 446 transtracheal aspirates (TTA’s) were collected from more than 25 feedlots around South Africa, except for the western Cape. Collection criteria included: pulled for respiratory disease; febrile (≥40ºC); depressed; anorexia and/or lack of rumen fill; nasal discharge or failure to clean muzzle; cough; increased respiratory rate >40 and most importantly, no prior treatment. Samples were also collected from 31 ‘healthy’ animals as controls. Samples collected were used for Mycoplasma isolations, as well as the aerobic bacteria to establish an antibiogram profile of bacteria commonly isolated in cattle feedlots. Mycoplasma spp. were isolated from 52.8% of samples taken from sick animals, with 67 out of 201 isolates (33.3%) being identified as M. bovis. According to the literature, M. bovis, M. haemolytica or P. multocida are isolated from bronchial lavage fluids from healthy calves in only a few cases, with estimates being put at 5 – 10% levels for Mycoplasma. Isolation rates of Mycoplasma spp. from healthy animals in this study was 22.7%, which was considerably higher than anticipated and could possibly be due to problems with the definition of a healthy animal. Although the number of samples from healthy animals was relatively small in this study, it was possible to show that there was a statistically significant association between Mycoplasma isolation and respiratory disease, p = 0,001 and with an odds ratio (OR) of 3,75 in cattle from those feedlots included in the study and thereby proving the hypothesis put forward. / Dissertation (MMedVet (Bovine Medicine))--University of Pretoria, 2007. / Production Animal Studies / MMedVet / Unrestricted

Along its Red Sea coast Eritrea possesses not less than 350 Islands. Dahlak Kebir, the largest of all is located approximately 50kms off the coast of Massawa, the busiest port of Eritrea. Topographically, relatively low relief limestone encircled by clean waters, abundant coral reefs and bountiful of fish species are the dominant features of the Islands. Nevertheless, their rich resources are so far not harnessed. They are inhabited by small fishing communities. One could only witness a small hotel and few houses scattered here and there, except for the presence of a military base on both Dahlak Kebir and in the near by Nakura Islands. The archaeological site of the Necropolis, a large early Islamic cemetery, and nearby ancient cisterns and pre Islamic cemetery, lies on the southwestern side of the Island. The story of Dahlak and its ruins are clear testimony that the archipelago has a mine of very rich history that needs to be explored. In the 7th century, for instance, it was through these Islands that the Islamism has infiltrated and eventually penetrated deep into various parts of Eritrea and Ethiopia. In between the beginning of the 8th and mid-9th century, the archipelago was conquered and occupied by various external powers such as the Byzantine Empire. In the 13th century, during the period of internal uprising in the Arab world, Dahlak established its own autonomous Sultanate. In latter years, Portuguese and Ottoman Kingdoms used to control the Islands and to assert their supremacy in the Red Sea. In 16th century, however, Dahlak was exclusively occupied by the Ottomans and was used as a launching ground to conquer and control the costal part and highland of Eritrea. Finally, in the 1870s Mohammed Ali of Egypt became master of the Islands. Dahlak was also known to have been the centre of trade for slaves on the Horn from the beginning of its occupation by Ottomans until its occupation of the Islands and the Red Sea coast by the Egyptians. (Eritrean travel and trade manual 2000) The prospect of the Dahlak Archipelago for the development of a successful tourist industry in Eritrea could undoubtedly be feasible. The low-lying coral chains where these volcanic Islands are located is amazingly tantalizing, fascinating and picturesque beyond description. Environmentally unpolluted, they are hygienic health-wise and could therefore be the primary attractions for tourists. Clean water and warm climate being their outstanding features, tourists could easily be lured to enjoy themselves without much ado about worrying their welfare, given their graceful natural environment is protected from deteriorations by contamination. White sandy beaches, protective bays and lagoons, provide excellent conditions for diving, snorkeling, sailing wind surfing and other sport activities. (Travel Trade Manual of Eritrea, 2000) / Dissertation (MPhil (Tourism Management))--University of Pretoria, 2008. / Tourism Management / MPhil / unrestricted

Natural and anthropogenic environmental variables pressures exert fitness pressures on our flora and vegetation and necessitate an understanding of the mechanisms of plant responses to those pressures. Overgrazing and desertification are examples of the stressors that affect plant community composition, on both large and small scales. In order to investigate the plant community response to these stressors it has been shown that it is more practical and informative to group plant species according to their functional characteristics into functional types than attempting to predict plant response solely based on previous range distribution data. The present study involved a survey of the dunefield vegetation of several privately owned farms as well as a communal farm in the southern Kalahari. The farms were subjectively chosen to represent a gradient from underutilized to overutilized. Plant community surveys were conducted on the farms and the dominant plant species were sampled and researched for selected functional traits to explore possible relationships between traits and grazing pressure. Ordination techniques were used to identify plant functional grazing response types. The identification of plant functional types through easy measurable traits will be a great aid in modelling and longterm environmental monitoring approaches. Of the three dunefield habitats, the dune street habitat was identified as the most vulnerable to overgrazing. A reduction in cumulative cover and an increase in bare surface cover indicated increased grazing pressure in the dune crest habitat. In the dune streets shrub cover and annual grass cover increased when veld was overgrazed. Functional analysis of 112 Kalahari plant species for a set of 47 plant traits showed that species clustering were strongly related to vegetative characteristics. The emerging clusters were strongly associated with major life forms, which comprised phanerophytes of different size classes, geophytes and herbaceous perennials, as well as herbaceous annuals. These clusters were identifiable even with the inclusion of reproductive traits. Selected vegetative traits are good indicators of plant response to land-use intensity since these traits lend competitive advantages to plant persistence under utilisation pressure. Plant height, spinescence, specific leaf area, leaf dry matter content, leaf sclerophylly and stem density were found to be examples of such traits. Phanerophyte and chamaephyte dominated functional groups were mostly associated with lower range condition and hemicryptophyte dominated groups with higher range condition, whereas herbaceous therophyte dominated groups did not show any specific association. / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

Nitrogen-fixing diazotrophic root-nodule bacteria are of immense economic importance because of their symbiosis with leguminous plants. Diazotrophic bacteria infect the host legume root inducing the formation of nodules in which the bacteria (also termed rhizobia) replicate and synthesize the enzyme nitrogenase. This enzyme catalyzes the reduction of atmospheric dinitrogen to ammonia for subsequent use by the plant as a major source of nitrogen. Nitrogen is an essential element required by plants for growth and synthesis of protein and is usually the most limiting element in agricultural production as well as being the most expensive component of fertilizer. The aim of my study was to determine the diversity and taxonomy of a specific group of root nodule bacteria associated with different species of Lebeckia. The genus Lebeckia Thunb. (Family Leguminosae, subfamily Papilionoideae, tribe Crotalarieae) comprises some 33 plant species. These plants are mainly indigenous to the southern and Western Cape regions of South Africa. They are divided into shrubby trifoliate-leaf species in the sections Calobota, Stiza and Viborgioides and suffrutescent unifoliate needle-leaf species in the section Lebeckia. Plants of this genus are adapted to harsh environmental conditions such as are found in the Karoo and Namaqualand. Several Lebeckia species are beneficial, such as L. spinescens and L. multiflora, which are valuable as pasture legumes and well grazed by animals especially in winter. All the species have ecological value because of their nitrogen-fixing symbiosis with rhizobia. To my knowledge, no attempts have been made in the past to investigate these microsymbionts of Lebeckia. Root nodules were collected from Lebeckia species at a wide variety of localities in the western and southern Cape regions of South Africa. Indigenous rhizobia isolated from these nodules were investigated for their nodulation abilities on their respective host plants as well as on non-host promiscuous legumes, cowpea and siratro. The isolates were then characterized using random amplified DNA fingerprinting followed by DNA sequencing of selected isolates. Results presented in this study showed that the indigenous South African genus Lebeckia is nodulated by diverse rhizobia from both α- and β-Proteobacteria. The first chapter contains a literature review of symbiotic nitrogen fixation that includes a general description of the biology, inoculant technology and the taxonomy of legumes and their rhizobia. The genera within the tribe Crotalarieae (such as Crotalaria, Lotononis, and Aspalathus) were discussed with special reference to the genus Lebeckia. Technical methods used for the classification of rhizobia were also reviewed. Non-DNA-based methods such as host specificity, substrate utilization, antibiotic resistance, morphological characters and biochemical properties as well as DNA based fingerprinting methods (ARDRA, RFLP RAPD, and AFLP), DNA sequence information, analysis of whole genomes, DNA-DNA hybridization and polyphasic approaches were outlined. The second chapter describes the isolation of 79 rhizobial isolates from the root nodules of 10 Lebeckia species. The isolates were purified and tested for nodulation and nitrogen fixation on cowpea and siratro as well as their host plants. All the isolates fixed nitrogen on their respective Lebeckia hosts, whereas 56% of the strains were effective on cowpea and 77% on siratro. The third chapter describes initial comparison and screening of the isolates by SP-PCR fingerprinting analysis. DNA profiles showed that most of the isolates grouped according to host plant species rather than geographical location. Isolates selected from different clusters were subjected to partial 16S rDNA gene sequencing to confirm their taxonomic identity. This revealed that Lebeckia is nodulated by diverse genera of root nodule bacteria from both the α-Proteobacteria (Bradyrhizobium, Mesorhizobium, and Sinorhizobium) as well as the β-Proteobacteria (Burkholderia). The final chapter (Chapter 4) provides concluding remarks on my results and possible future avenues of research on the Lebeckia rhizobia. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Concern for the severe loss of biodiversity of grassland species is often voiced. Plantation forestry is known to cause extensive and long lasting disturbance of the natural environment in particular in areas such as the mountain grassland of the Drakensberg escarpment. The survey was conducted in the Graskop area on sites within pine plantations along the Treur River bordering the Blyde River Nature Reserve. The results showed that restoration of plant species biodiversity through natural succession, on cleared plantation sites, required periods longer than seven years and that the regeneration of a great many of the indigenous forb species remained uncertain. Two survey areas were selected and within each survey area, sampling sites were selected to represent three categories of vegetation namely, undisturbed grassland and sites where pine trees had been removed three and seven years previously. Samples of plants were collected from each of these sampling sites. Analysis by means of a classification technique determined the species composition of the disturbed sites in relation to that of the intact grassland. Wheel point surveys were also carried out on these sites to provided information on the vegetation cover, as well as the degree of species regeneration on each site. The variation in vegetation composition of the various plots was ana lysed by means of Detrended Correspondence Analysis (DCA) and Two-Way Indicator Species Analysis (TWINSPAN). The results showed three distinct species assemblages which corresponded with the three categories of sampling sites. Also that the indigenous forbs species comprised 68% of all the sampled species most of which had failed to regenerate in either the three or seven year cleared plots. The most successful grass species in establishing and persisting in all sites were, Eragrostis curvula and Loudetia simplex. The fern, Pteridium aquilinum had a high prevalence on the disturbed grassland plots and the threat of its invasion of these habitats is compounded by the physical disturbance which eradication methods cause. Planning for grassland restoration involves cognizance of the complexity of grassland ecology, the influence of a multiplicity of environmental factors and the proximity of donor sites to the disturbed areas. Applying international restoration techniques used in grasslands of different origins to those of South African landscapes could result in disappointing and costly efforts. At best any attempt at managing grassland diversity should be preceded by an holistic investigation into the environmental conditions particular to the specific terrain and thereafter maintaining a conservative approach of allowing natural succession. The threat of invasive exotic species should be integral to conserving the integrity of the remaining intact natural grasslands in South Africa. It is acknowledged that in this study regeneration of grassland species on plantation sites cleared of pine trees three and seven years previously does not include a long time span such as needed for succession to take place but is useful in showing a trend in species re-colonization to resemble the vegetation of intact grassland, as well as highlighting the absence of a great number of indigenous forb species. Copyright / Dissertation (MInstAgrar)--University of Pretoria, 2012. / Geography, Geoinformatics and Meteorology / Unrestricted