Item Open AccessMolecular changes of sorghum cell suspension Cultures in response to exogenous abscisic acid(University of the Free State, 2022) Muthego, Dakalo; Ngara, R.Abiotic stresses reduce the growth and productivity of crops, thus threatening food security. It is therefore, important to develop crops that can withstand harsh environmental conditions in order to ensure availability of food. In general, plants have developed a wide range of mechanisms in response to these abiotic stresses. For example, under stress conditions, plants undergo molecular changes which include alterations in gene, protein and metabolite expression patterns that are mostly regulated by the plant hormone abscisic acid (ABA). ABAregulated stress responsive pathways are well studied in the model plant Arabidopsis (Arabidopsis thaliana), yet similar processes in sorghum (Sorghum bicolor), a drought tolerant crop, are not yet fully understood. The aim of the study was to investigate the biochemical properties and protein expression patterns of sorghum cell suspension cultures in response to exogenous ABA. White sorghum cell suspension cultures were used, and at eight days postsubculture, the cultures were treated with 100 μM ABA prepared in 70% (v/v) methanol. For control cells, an equal volume of 70% (v/v) methanol was added, and both treatment groups were incubated with shaking at 27℃ for 72 hours. Analysis of cell viability using the 3-(4,5- dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that ABA does not affect the viability of the cells at 0, 24, 48 and 72 hours. However, exogenous application of ABA for 72 hours resulted in increased accumulation of proline in the sorghum cells relative to the controls. Furthermore, proteins were extracted from the cells, as total soluble proteins (TSP), and from the culture medium, as culture filtrate proteins (CF) 72 hours after the exogenous ABA treatment. The protein profiles of the two proteomes were visually analysed on Coomassie brilliant blue-stained one-dimensional sodium dodecyl sulfate polyacrylamide gels. The gels showed that the two proteomes were of good quality even under control conditions. Furthermore, following the 72-hour ABA treatment, proteins were differentially expressed in both the TSP and CF proteomes. Moreover, isobaric tags for relative and absolute quantitation (iTRAQ) method and mass spectrometry were used to identify and quantify the differentially expressed proteins. A total of 725 and 256 proteins were identified in the TSP and CF proteomes, respectively. Of all these, 46 and 82 were ABA-responsive in the TSP and CF, respectively, and 8 proteins were common to both proteomes. Signal peptide analysis revealed that the majority of TSP found in the intracellular matrix did not have a predicted signal peptide (72%), while the majority of CF proteins found in the extracellular matrix contained signal-peptides (82%). Amongst these differentially expressed proteins in both the TSP and CF proteomes, the majority of them proteins were involved in metabolism with 37% and 35%, followed by defence with 24% and 24%, respectively. However, the metabolic processes in the CF were mainly related to carbohydrate metabolism. The signal transduction functional group was only unique to the TSP fraction, while transporters, and cell structure functional groups were unique to the CF protein fraction. The differentially expressed proteins are well-known stress proteins such as peroxidases and superoxide dismutases whose levels change under abiotic stresses. Together with causing an increase in proline content, a known osmoprotectant, exogenous ABA does indeed act as a stress phytohormone. Furthermore, these results showed that ABA influences the differential expression of both intracellular and extracellular matrix proteins, possibly suggesting the importance of both cell compartments in stress response. Furthermore, these two compartments have different roles in stress responses as suggested by the results. Therefore, the application of exogenous ABA could be the way forward to further understand plant stress response networks, and possibly to develop crops that can survive under any abiotic stress. Item Open AccessGenotype and environmental effects on maize grain yield, nutritional value and milling quality(University of the Free State, 2022) Khajoane, Tsietso Jeanett; Mbuma, N. W.; Labuschagne, M. T.; Ramburan, S.In sub-Saharan Africa and other regions in the world, many people rely on maize as their primary food. To guarantee food security, high yielding and nutritious maize hybrids must be bred. Breeding for increased maize grain yield, nutritional quality traits and milling quality allows diversification and an increase in maize production. It also helps in the alleviation of malnutrition in countries that rely on maize as their dietary source. This research was conducted in order to: 1) determine the genotype and environmental effects on maize grain yield, nutritional quality traits, and milling quality, 2) determine the interrelationship among grain yield, nutritional quality traits and milling quality in maize genotypes and 3) to evaluate genotype by environment interaction for grain yield and to determine the grain yield stability of maize hybrids. Eighteen maize genotypes (nine commercial and nine experimental hybrids) were planted using a randomized complete block design (RCBD), replicated six times at seven sites representing the diverse agro-ecologies where maize is predominantly grown in South Africa. Genotype and genotype by environment interaction effects were highly significant (P ≤ 0.001) for all traits, indicating the existence of variability in the maize breeding populations. On average, broad sense heritability (H²) of nutritional quality traits, milling quality and defective grain (DEFG) ranged from 30.86 to 82.50%, which indicated that the phenotypic differences were mostly attributed to genotypic effects. Low H² (17.63%) for grain yield was observed, which indicated that phenotypic differences observed were mostly attributed to environmental factors. High performing genotypes were identified, such as G15-Ex (grain yield, fat and milling quality), G16-Ex (protein and low moisture), G11-Ex (starch) and G14-Ex (fibre). Genotype G2-C and G4-Ex had low mean values for DEFG. The findings in this study provided variation that can be exploited in breeding programmes to improve maize. Significant and positive correlation was found for protein content with grain yield, indicating that these traits could be selected and improved simultaneously. Milling quality was positively correlated with grain yield, protein, fat and low moisture, indicating that multiple trait selection would be possible. Starch was negatively associated with protein content and grain yield, suggesting that the improvement of starch will have a negative effect on maize grain yield and protein content. The clustered heat map identified three clusters of maize hybrids, which were 1) G1-C, G7-C, G9-C, G13-Ex, G14-Ex, G16-Ex and G17-Ex, associated with high protein and fibre content, 2) G4-Ex, G5-C, G6-C, G8-C and G11-Ex, associated with high grain yield, fat, moisture and fibre content and 3) G3-C, G10-C, G12-Ex, G15-Ex and G18-Ex, associated with high milling quality and fat content. Additive main effects and multiplicative interaction analysis (AMMI) identified experimental genotypes G4-Ex, G15-Ex and G17-Ex as high yielding and the most stable genotypes, which suggested that these genotypes have broad adaptation. Genotypes G8-C and G11-Ex were high yielding but unstable. The GGE scatter plot identified high yielding genotypes that showed specific (G2-C, G7-C, G8-C, G16-Ex and G17-Ex) and broad (G1-C, G4-Ex, G13-Ex and G15-Ex) adaptation in test environments and revealed two mega environments. Therefore, testing maize genotypes in different environments is important to determine their adaptability and stability before cultivar release and recommendation for commercial production. Maize hybrids with improved grain yield and nutritional quality may be used to alleviate challenges associated with malnutrition. Item Open AccessInfluence of different selenium application methods on the physiology and morphology of drought-stressed edamame(University of the Free State, 2022) Sekhurwane, Masego; Moloi, M. J.Drought is one of the common stress factors accelerated by anthropogenic climate change contributing to crop losses across the globe. Since South Africa is classified as semi-arid, production of drought sensitive crops such as edamame (Glycine max L. Merrill) is a challenge. Over the recent years, selenium application gained attention of biologists due to its ability to reduce the adverse impacts of drought stress on crops. However, the effective method of selenium application for inducing tolerance to drought stress in edamame is not recorded. Therefore, the aim of this research was to determine the influence of different selenium application methods (seed dressing, foliar spray and soil drench) on the physiological, biochemical and morphological responses of two edamame cultivars (tolerant UVE14 and susceptible UVE17) under drought stress. The study also established relationships between the physiological, biochemical, and morphological characteristics under drought stress in edamame. The research was conducted under controlled conditions in a greenhouse. Before drought stress induction (30% water holding capacity, WHC) at the third trifoliate leaf stage (vegetative stage 4, V4), selenium was applied using different application methods [i.e., seed dressing (before sowing), foliar spray and soil drench (at first trifoliate stage)]. Optimally watered (100% WHC) plants treated with different selenium application methods served as positive controls. Physiological and biochemical data collection took place at flowering stage (reproductive stage 2, R2) and pod filling stage (R4), whereas morphological data was collected at maturity (R8) stage. The physiological responses included different chlorophyll a fluorescence parameters, stomatal conductance (gs), chloroplast pigments, electrolyte leakage (EL) and relative water content RWC). The biochemical parameters included superoxide dismutase (SOD), hydrogen peroxide (H₂O₂), guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and protein content. Results showed that the physiological and biochemical parameters were significantly different for selenium application methods at flowering and pod filling stages under drought stress. Water levels in the soil affected the physio-biochemical responses of edamame cultivars under different selenium application methods. Also, cultivars responded differently under different selenium application methods. The soil drench method was more efficient because it increased total performance index (PItotal),total chlorophyll content, stomatal conductance, RWC, H₂O₂, carotenoid, APX, GPX and SOD. Morphological data further agreed with the physio-biochemical data, showing that selenium application through soil drenching and seed dressing increased the number of seeds per plant (SPP) for drought-stressed UVE17 cultivar. The soil drenching treatment on drought-stressed edamame resulted in a high significant mean difference in the number of pods per plant (PPP), SPP and seed mass per plant (SMP), compared to the foliar and seed application methods. Correlation analysis provided evidence that increased PPP under selenium soil drench application was positively associated with high RWC, PIabs, PItotal. In addition, SOD and GPX were positively correlated to plant height (PH) and PPP for drought-stressed edamame under selenium soil drench application. Furthermore, carotenoid content positively correlated with PPP, SMPP and SPP for drought-stressed edamame under selenium seed dressing application. This study shows that under drought stress, application of selenium as a soil drench treatment is the most effective method for improving tolerance of edamame to drought stress, followed by the seed dressing method. Therefore, this work provides essential information for combating the negative effects of drought stress, which can positively contribute to food security in South Africa. Item Open AccessPhysio-morphological and biochemical traits of dibutyldithiophosphate treated drought-stressed edamame(University of the Free State, 2022) Khoza, Minah Bongiwe; Moloi, M. J.; Bowden, N.Edamame (Glycine max L. Merrill) has recently received attention in South Africa due to its high nutritional value. Production of edamame in South Africa is limited since the country consists of arid and semi-arid regions, with insufficient water available for irrigation. Dibutyldithiophosphate, a biodegradable chemical that slowly releases hydrogen sulfide, could be a solution based on recent studies that indicate H₂S acts as a signaling molecule for tolerance induction during various environmental stresses. This study investigated the impact of dibutyldithiophosphate on the physiological, biochemical and morphological characteristics of drought-stressed edamame cultivars (UVE14 and UVE17). Furthermore, the study established the relationships between the physiological, biochemical and the morphological characteristics [plant height, branches per plant, total seeds per plant, total seed mass per plant, and pods per plant] of drought-stressed edamame under different dibutyldithiophosphate concentrations. Dibutyldithiophosphate was directly applied around the seed during sowing before drought stress (30% water holding capacity) introduction at the third trifoliate leaf stage. Drought stress adversely affected the physiological, biochemical and morphological responses of the edamame cultivars. The efficacy of dibutyldithiophosphate differed according to the cultivars, growth stages (flowering and pod-filling), concentrations of dibutyldithiophosphate (0, 0.1, 1, and 2 mg/mL), as well as the water level (drought or optimal watering). The concentrations required to activate the physiological and biochemical responses were generally higher in drought-stressed UVE17 than UVE14 cultvar. To improve drought tolerance in edamame, dibutyldithiophosphate application upregulated the photosynthetic capacity (increased total chlorophyll content, chlorophyll-a, chlorophyll-b, and photochemical reflective index), which resulted in increased accumulation of sugars (total soluble sugars) and proline. The increase in total soluble sugars and proline indicate that dibutyldithiophosphate application increases the osmotic and antioxidative potential of drought-stressed edamame. Additionally, results of this study showed that dibutyldithiophosphate application decreased membrane damage by increasing the antioxidative capacity [superoxide dismutase, ascorbate peroxidase, flavonoid reflective index, and carotenoid reflective index] in drought-stressed edamame. Application of dibutyldithiophosphate also improved drought tolerance in a susceptible cultivar (UVE17) by increasing pods per plant (0.1 mg/mL), branches per plant (1 mg/mL), and total seed per plant (2 mg/mL). For drought-stressed UVE14, application of dibutyldithiophosphate increased branches per plant (all concentrations), pods per plant (0.1 and 1 mg/mL) and total seed per plant (2 mg/mL). Since there were more positive correlations between the physiological, biochemical and morphological characteristics under 0.1 and 1 mg/mL, these dibutyldithiophosphate concentrations could be recommended for drought tolerance stimulation in edamame. As a result, the hypothesis that application of dibutyldithiophosphate enhances the physiological, biochemical and morphological responses of drought-stressed edamame is accepted. In addition, it is acknowledged that application of dibutyldithiophosphate on drought-stressed edamame increases the relationships between physiological, biochemical and morphological responses. Item Open AccessEvaluating cowpea mutant genotypes for grain yield and nutritional value in South Africa(University of the Free State, 2022) Ntswane, Moshieng; Mbuma, N. W.; Labuschagne, M. T.; Shandu, S. F.Cowpeas produce a substantial amount of grain, which is a significant source of vitamins, minerals and protein to disadvantaged people with limited access to adequate nutrients. Improved cowpea genotypes have been introduced through new breeding techniques such as mutagenesis to increase the phenotypic, genetic and nutritional diversity of the crop. The objectives of this study were: 1) to determine the phenotypic diversity and characterise cowpea mutants and normal genotypes for grain yield and yield components, to identify superior cowpea mutants and normal genotypes and to determine the correlation between all measured characteristics, 2) to evaluate the variability of cowpea mutants and normal genotypes for protein content, selected mineral elements, phytic acid and the potential bioavailability of iron (Fe) and zinc (Zn), to identify superior cowpea mutants and normal genotypes, and to determine the interrelationship between all measured characteristics, and 3) to determine genotype by environment (GE) interaction, to identify superior genotypes for grain yield and to determine the adaptability and stability of cowpea mutants and normal genotypes in South Africa. Thirty-one cowpea genotypes (16 Namibian mutants, seven International Institute of Tropical Agriculture (IITA) genotypes and eight South Africa genotypes) were planted in five different environments in South Africa during the 2021/2022 cropping season. Significant (P ≤ 0.05) genotype and GE interaction effects were observed for grain yield, yield components, protein content, minerals, phytic acid and potential mineral bioavailability. Broad-sense heritability (H2) values above 50% were observed for yield components, protein, mineral elements, phytic acid and potential mineral bioavailability, while low H2 values below 50% were observed for grain yield and Boron (B), indicating the complexity in selection and genetic improvement of these traits. Superior Namibian mutants (ShR10P12, ShR3P4, ShR4P1 and BrR11P2), IITA genotype (98K-476-8) and South African genotype (Enchore) for grain yield were identified. Superior Namibian mutants (ShL3P7-2, ShR3P4, ShR4P1, BrR11P11, BrR4P11, NKL9P7, NKR8P9, NKR9P9 ShR2P11, BrR11P2, ShL2P7, ShR3P4 and NKRuP5), IITA genotypes (98K-476-8, IT82E-18, IT93K-452-1, IT99K-573-2-1 and ITOOK 1263), and South African genotypes (Oloyin, Orelu, Pan 311, Bechuana White, Enchore and Glenda) for protein content, Fe and Zn concentration were also identified. The IITA genotype (IT93K-452-1) and South African genotypes (Oloyin and Orelu) had a potential of good Fe bioavailability. All cowpea mutants and normal genotypes had a potential of poor Zn bioavailability. High yielding and stable Namibian mutants (NKL9P7, ShR10P12 and ShR2P11), IITA genotype (ITOOK 1263) and South African genotype (Agrinawa) were identified. Two mega-environments, namely, 1) Taung and Mafikeng, and 2) Mafikeng, Bloemfontein, Polokwane and Potchefstroom were identified, indicating broad adaption of the genotypes. Potchefstroom and Taung were identified as ideal environments for evaluation of cowpea genotypes. Significant positive correlations between grain yield with almost all yield components were observed. Significant positive correlations of protein content with mineral elements and phytic acid were also observed, indicating the potential to simultaneously select these traits. Namibian mutants (NKR1P3, BrR11P2, ShL2P7, ShR2P11 and ShR10P12), IITA genotype (98K-476-8) and South African genotypes (Glenda, Dr Saunders, Enchore and Oloyin) were associated with high grain yield. Namibian mutants (ShR10P12, ShR3P4, ShR9P5, BrR11P11, BrR11P2, BrR4P11, NKR1P3, NKR9P9 and NKRuP5), IITA genotypes (IT07K-292-10, IT07K-318-33, IT82E-18 and IT99K-573-2-1), and South African genotypes (Agrinawa, Bechuana White and Dr Saunders) were associated with high protein content, manganese (Mn), phytic acid, Molar ratio of phytic acid with iron (PA:Fe), Molar ratio of phytic acid with zinc (PA:Zn), and ash content. These genotypes have a potential of long-term profitability to the agricultural production industry. Item Open AccessTriazole fungicide sensitivity among South African Puccinia graminis f. sp. tritici isolates(University of the Free State, 2022) Du Toit, Isabella; Visser, B.; Boshoff, W. H. P.Wheat is an important crop and a key component in the diet of people, particularly in developing countries such as South Africa (SA; Pathak and Shrivastav, 2015). The crop is produced worldwide and is considered the most important source of food (Igrejas and Branlard, 2020). Two closely related species Triticum aestivum L. (bread wheat) and T. durum (durum wheat) are widely cultivated with global production dominated by countries including China, India, Russia, Ukraine and the United States of America (USA; OECD-FAO, 2020). During the 2019 to 2021 production seasons, SA produced an average of 1.8 million metric tons of bread wheat (CEC-Sagis, 2021), with nearly 42.2% of the total wheat yield contributed by spring wheat cultivars planted under dry land in the Western Cape province (Galal, 2021). Rust diseases are present in most areas where wheat is cultivated (Kolmer, 2005; Pretorius et al., 2020). Stem rust of wheat and barley (Hordeum vulgare L.), caused by Puccinia graminis f. sp. tritici Eriks. and E. Henn. (Pgt), is considered the most damaging wheat disease in history (Pretorius et al., 2017). Recent reports indicate this pathogen’s re-emergence in areas after decades of absence (Lewis et al., 2018; Tsushima et al., 2022). Kernels harvested from rust infected wheat are nutrient poor and low in test weight, while healthy kernels are plump and nutrient rich (Agrios, 2005). Disease control is important to maintain the production of high yielding wheat and is achieved through different control strategies, including fungicide application, a strategy widely practiced in SA (Roelfs et al., 1992; Soko et al., 2018; Anonymous, 2020b). Rust pathogens are known for their ability to overcome monogenic sources of resistance, rapid dispersal over thousands of kilometres (Visser et al., 2019) and exponential rate of development into epidemics under favourable conditions. Stem rust mainly affects the stems of wheat during grain fill, as such fungicide applications are considered less effective if not applied preventatively (Wanyera et al., 2009; Tadesse et al., 2010). The effective chemical control of stem rust depends on application timing, adequate downward translocation of the active ingredient (a. i.) or coverage of the stem area (Bayer Crop Science, n.d.). The occurrence of fungicide insensitivity in populations of fungal pathogens limits the efficacy and period during which fungicides remain effective. Loss in fungicide efficiency contributes to increased costs associated with new fungicide development (Ma and Michailides, 2005). Fungicide insensitivity is the acquired and heritable decrease in the sensitivity of isolates to the a. i. present within a fungicide formulation (Brent and Hollomon, 2007). The widespread, repeated, and incorrect fungicide application as well as overreliance on a particular a. i. increases the risk of fungicide insensitivity. However, it may also occur without prior exposure in a phenomenon known as cross-resistance where insensitivity develops for different fungicides having a. i. with similar modes of action (Kang et al., 2019; McGrath, 2001). This insensitivity is usually to the mode of action of the a. i. of the specific fungicide and is a result of selection pressure exerted on the fungal population (Brent and Hollomon, 2007). Item Open AccessMetabolic and proteomic responses of sorghum cell cultures to polyethylene glycol-induced osmotic stress(University of the Free State, 2022) Mosea, Dipuo; Ngara, Rudo; Chivasa, StephenClimate change, population growth, and the emerging water crisis are negatively affecting agricultural productivity and, thus, food security. Climate change results in drought, which is a major osmotic stress. Osmotic stress triggers the overproduction and accumulation of reactive oxygen species, which result in oxidative stress. Plants also respond to drought stress by using a wide range of biochemical and molecular mechanisms. Examples include osmotic adjustment, antioxidant defense systems, the production of phytohormones, and changes in gene and protein expression patterns. Most of the major cereal crops are drought sensitive. However, sorghum (Sorghum bicolor) is well-adapted to survive under hot and dry conditions. Sorghum is thus a potentially good model system among cereals in drought stress response studies. The study aimed to evaluate sorghum cell cultures' metabolic and proteomic responses to polyethylene glycol (PEG)-6000- induced osmotic stress. The viability of the white sorghum cell cultures was monitored using the MTT (3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay after the cells were treated with 10 and 20% PEG osmotic stress. The levels of 16 metabolites and a sugar was analysed using hydrophilic interaction chromatography (HILIC) liquid chromatography-mass spectrometry (LC-MS) for control and 10 and 20% PEG-treated white sorghum cell suspension cultures. After 72 hours of 10 and 20% PEG-induced osmotic stress treatments, total soluble proteins (TSP) were extracted, and quantified using the Bradford assay and separated using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) to determine the quality of the extracts. An isobaric tag for relative and absolute quantitation (iTRAQ) analysis was conducted in the extracts followed by bioinformatics analysis of the positively identified osmotic stress-responsive proteins. The cell viability assays showed that both 10 and 20% PEG affected the metabolic activities of white sorghum cell cultures differently and, indeed, triggered osmotic stress, and the cell viability of the stressed samples declined relative to the control. In response to the imposed osmotic stress levels, the metabolic profiles showed a dramatic decline in six amino acids namely leucine (Leu), methionine (Met), phenylalanine (Phe), serine (Ser) threonine (Thr), and valine (Val). A total of 177 and 229 white sorghum cell-cultured total soluble proteins were identified for the 10 and 20% PEG treatment experiments, respectively. Of these identified proteins, 28 and 48 were responsive to the 10 and 20% PEG treatments, respectively. Additionally, the study identified responsive proteins such as germins, peroxidases, and histones as proteins of interest because they were either uniquely responsive to severe stress or commonly responsive to the imposed osmotic stress. The results obtained added to the knowledge that can be used in breeding programmes for the improvement of cereal crops that are susceptible to drought stress. Item Open AccessPhysiological, biochemical and root proteome responses of maize seedlings to drought stress(University of the Free State, 2022) Moloi, Lerato; Ngara, R.; Gokul, A.A primary limiting factor for maize during its growing period is drought stress. With the decrease in yield and biomass triggered by drought stress in maize, drought has become a critical issue that must be considered due to the changing climate conditions. This study aimed to comparatively analyse the morpho-physiological, biochemical and root proteome changes of two maize varieties in response to drought stress. In this study, two maize varieties CN07/8-224 and CN07/8-292 with unknown drought phenotypes were used. The plant growth experiments were conducted in a growth chamber for mild stress with ~ 54% and ~48% of soil moisture content and a greenhouse for severe stress with ~51% and ~43% soil moisture content. In both growth experiments, 7-day-old maize seedlings were exposed to a 14-day drought stress treatment, while continuing to water the controls every other day. Thereafter, a range of morpho-physiological parameters such as shoot and root length/weight, leaf relative water content, chlorophyll and carotenoid content were measured. In addition, biochemical parameters such as the level of lipid peroxidation and hydrogen peroxide, as well as the activities of superoxide dismutase (SOD), and ascorbate peroxide (APX) were measured. The root proteome was analysed using the isobaric tags for absolute and relative quantitation (iTRAQ) method coupled with liquid chromatography- tandem mass spectrometry to identify differentially expressed proteins in response to drought stress. To annotate the proteins, bioinformatics studies were carried out by using the UniProt and InterPro databases. The Gene Ontology (GO) annotation and protein families were allocated to each protein that showed differential expression in response to the drought stress. Then, the proteins were functionally categorized using a modified version of the Bevan classification scheme. The results of the study showed some significant differences between the drought treated plants and control plants of CN07/8-224 and CN07/8-292, as well as between the maize varieties. Results indicated that both maize varieties experienced reductions in chlorophyll a, b, and carotenoids during drought stress. In addition, there were differences in the amounts of chlorophyll that accumulated in the various varieties. These reductions may be attributed to a reduced water supply observed in soil moisture content, which reduced the synthesis of photosynthetic pigments in maize plants. However, only the drought-stressed CN07/8-224 maize variety lengthened its roots to adapt to limited water supply. Higher lipid peroxidation and hydrogen peroxide levels correlated with increased antioxidant activities of CN07/8-292 drought-stressed roots as compared to control plants. This could be because there was more significant ROS detoxification under drought stress, which led to higher activities of SOD and APX in comparison to control plants. The root samples of CN07/8-224 and CN07/8-292 maize varieties were used to conduct iTRAQ analysis. A total of 1 232 and 1 558 proteins were positively identified in CN07/8-224 and CN07/8-292 varieties, respectively in response to mild drought stress conditions. A total of 116 and 146 of these proteins were responsive to the mild stress. The CN07/8-224 and CN07/8-292 varieties shared seven drought-responsive proteins, while the rest were unique to each variety. Collectively, the drought responsive proteins of both maize varieties were involved in primary/secondary metabolism, disease defence, and protein synthesis, all of which are involved in the response of maize varieties to drought. However, the expression of drought responsive proteins varied across the maize varieties. Overall, physiological and biochemical results have shown how the CN07/8-224 and CN07/8-292 maize varieties responded to drought stress using a range of mechanisms. In addition, strong root system architecture, as shown by higher root length and fresh root weight in CN07/8-224 variety under drought stress, could be responsible for the improved growth and greater stress tolerance. On the other hand, CN07/8-292 variety might happen to be sensitive to drought stress relative to CN07/8-224 based on the observed results. The obtained results would help researchers to find solutions on the impact of gene expression under drought. Item Open AccessEcology and conservation status of six medicinal plants commonly used to treat diabetes in the eastern Free State, South Africa(University of the Free State, 2013-12) Pitso, Teboho Raymond; Ashafa, Anofi Omotayo TomDiabetes mellitus is a global disease with an extreme effect on the quality of life of the patients and it is increasing among South Africans. South African population is predominantly black and medicinal plants represent an important asset to their livelihoods as it is to many people in developing countries. The communities from both rural and urban areas still rely on medicinal plants for their primary health care and income generation. However, the growing population and the general field collection practices of healers, herbalists and commercial gatherers are posing a serious extinction threat to the wild populations of medicinal plants. Six medicinal plants viz.: Hypoxis hemerocallidea, Dicoma anomala, Morella serrata, Gazania krebsiana, Xysmalobium undulatum and Eriocephalus punctulatus were identified to be the most commonly used to treat diabetes in the eastern Free State. The purpose of this investigation was to (1) conduct a survey on the herbarium collection of six medicinal plants used to treat diabetes (2) conduct a survey to determine the collecting practices of traditional healers and herbalist and the impact these practices have on the wild populations and (3) to conduct an ecological survey to determine the current population trends of the six selected plants in their natural habitats. A total of six herbaria (Uniqwa Herbarium, Sterkfontein Dam Nature Reserve Herbarium, Geo-Potts Herbarium, National Museum Herbarium, Free State National Botanical Garden Herbarium and Bews Herbarium) were surveyed and there were only a few specimen of the studied plants found in four surveyed herbaria. The collection labels generally lacked information on the precise location, distribution and frequency of occurrence, and most of them were very old. The present study showed that most of the traditional medicine practitioners were women with a low education level and they had little appreciation of the impact of their activities on wild populations of the medicinal plants. The indiscriminate collecting practices posed a serious extinction threat to the plants used in traditional medicine. The ecological survey identified two species Eriocephalus punctulatus and Morella serrata as potentially threatened within the study area. Herbaria should strive to become aligned with the "13-point strategy to meet conservation challenges" and this would make integral part of conservation strategies. Extensive study to determine the extent of the threat to the two identified species needs to be undertaken and their status on the Red Data List need to be revised. Traditional medicine practitioners (healers, herbalists and commercial gatherers) need to be informed about sustainable usage of natural resources and about environmental legislation regarding their business. Strong working relationships between government (Department of Environmental Affairs), Universities, South African National Parks and traditional medicine practitioners need to be established to protect the environment. Failure to use medicinal plants in sustainable way will have a negative impact on both the biodiversity and to the general health of the population. Item Open AccessInoculation techniques and evaluation methodologies for Sclerotinia sclerotiorum head and stem rot in sunflower and soybean(University of the Free State, 2018-01) Bester, Marlese Christine; McLaren, N. W.; Rothmann, L. A.Sclerotinia stem and head rot on soybean and sunflower are caused by the fungal plant pathogen Sclerotinia sclerotiorum. This pathogen can be responsible for up to 75.0% yield loss. A major constraint in the effective control of Sclerotinia in sunflowers and soybeans is the absence of stable resistance. Cultivar selection as a management strategy remains the ultimate goal in reducing Sclerotinia outbreaks among South African producers. Quantification of cultivar responses to pathogen and environmental stimuli could assist the selection and breeding of cultivars with tolerant to high disease potentials. The production of S. sclerotiorum ascospores and sclerotia were optimised in the laboratory. Maize meal and soybean stubble as substrates for sclerotia production, produced the largest sclerotia with mean masses 0.0292 mg per sclerotium, while sunflower meal produced the lightest sclerotia with a mean mass of only 0.0099 mg per sclerotium. Several literature sources have stated that a pre-conditioning step of 4 - 10°C is needed in order for sclerotia to germinate carpogenically. Results of this study indicate that no pre-conditioning is needed for South African isolates of S. sclerotiorum to form apothecial stipes and that the optimum temperature for apothecia development is 16°C. However, results indicated that extending pre-conditioning of sclerotia at 4°C by 4 - 8 weeks, will produce a higher number of apothecia. Regression analysis indicated that apothecial stipes take approximately five days longer to develop for each week of pre-conditioning. Size of sclerotia also affected the number of apothecial stipes and sclerotia 27 mm in length produced up to 118 apothecial stipes, while the smallest sclerotia, 5 mm in length only produced 14 apothecial stipes. The viability of the pathogen in infected sunflower stems whether milled into a fine powder or shredded into pieces of various sizes, was evaluated every 30 days for 12 months under laboratory conditions. All the infected stubble sizes were able to produce both S. sclerotiorum mycelium and sclerotia on Potato Dextrose Agar after 12 months. After the optimisation of Sclerotinia inoculum, efficacy of inoculum source, application technique and timing on disease severity under greenhouse and field conditions were evaluated on both soybean and sunflower. The techniques used were liquid spray mycelium, milled grain mycelium, sclerotia planted adjacent to seedlings and an ascospore suspension. A head punch method, whereby a hole is punched into a flowering head and a S. sclerotiorum colonised sorghum grain is inserted, was also applied to sunflowers. Treatments were applied to sunflower at bud, flower and head- ripening growth stage while on soybean, the liquid spray mycelium, milled grain mycelium and ascospore suspension were applied at flowering stage. A significant difference was observed between both sunflower and soybean greenhouse and field trials. Overall the technique that yielded the highest levels of Sclerotinia disease was the milled grain mycelium. A negative correlation was observed between soybean greenhouse and field trials, while a poor correlation was observed between sunflower greenhouse and field trials. This illustrated the importance of using multiple environments when screening for Sclerotinia resistance, especially under field conditions, as well as the need to consider the high genotype x environment interactions that occur. Performing resistance evaluations in the greenhouse alone, will limit the value of screening techniques. Field evaluations of resistance in soybean and sunflower cultivars were carried out in Delmas and Greytown respectively, over several planting dates. The cultivars were inoculated with milled grain mycelium and liquid spray mycelium. Regression analyses were used to determine the response type as well as the relationship between observed stem or head rot incidence within a cultivar and disease potential. The disease potential was defined as the mean disease severity within a planting date over all the cultivars within a trial. The regression model Y=aXb was used where Y is the observed incidence within a cultivar, X is the disease potential and a and b are regression parameters. Three cultivar response types were identified based on the b parameter i.e. cultivars tolerant of increasing disease potential (b>1), cultivar intolerant to increasing disease potential (b<1) and cultivars showing a linear relationship with changing disease potential (b≈1). The Sclerotinia potential required to initiate disease onset and the rate of disease increase subsequent to onset were calculated by re- arrangement of model parameters. The soybean cultivar, PAN 1454, responded less rapidly to changing stem rot potentials, having an onset potential of 19.3% and a subsequent response rate of 0.5 per potential unit increase, making it more tolerant to increasing disease potentials, while P 64 T 39 had an onset potential of 1.0% and a response rate of 3.4 per potential unit increase, making it less tolerant of increasing Sclerotinia potential. The sunflower cultivar, P 65 LC 54, responded less rapidly to changing head rot potentials, having an onset potential of 18.9% and a response rate of 0.6 per potential unit, while P 65 LL 02 had an onset potential of 2.2% and a response rate of 1.9 per potential unit increase. The level of lignin, total proteins, chitinase and β-1, 3-glucanase in the leaves of soybean and sunflower cultivars collected at different growth stages, were evaluated to determine the correlation between these resistance components and Sclerotinia disease development. No correlation was recorded. It was concluded that the reaction of soybean and sunflower cultivars to changing disease potentials is an essential component of any resistance screening study and genotype x environment interactions must be quantified. The use of multiple criteria is essential if the behaviour of the pathosystem is to be understood and if stable resistance of commercial value is to be obtained. Item Open AccessMolecular responses of sorghum cell suspension cultures to high temperature stress(University of the Free State, 2018-12) Ngcala, Mamosa Gloria; Ngara, RudoHigh temperatures and frequent drought episodes limit plant growth and development. Ultimately, low crop yields are realised and food insecurity follows. Therefore, there is a need to develop crops that can tolerate extreme temperatures as part of adapting to the global climate change. Sorghum (Sorghum bicolor (L) Moench), a naturally drought tolerant crop, which survives in hot and dry environments was used in this study. The aim of the study was to evaluate the molecular responses of ICSB 338 sorghum cell suspension cultures to high temperature stress. ICSB 338 sorghum cell suspension cultures were exposed to heat stress at 35 and 40oC for 72 hours. Analysis of the cells’ metabolic activity indicated that the cells could survive at both temperatures for 72 hours. However, when Arabidopsis cell suspension cultures were exposed to 40oC for the same period, a significant decrease in cell viability was observed. These results suggest that sorghum is more heat tolerant than Arabidopsis. The proline and glycine betaine content of ICSB 338 cell cultures was determined, following heat stress treatment at 40oC for 72 hours. A decrease in proline content was observed during the stress treatment period. On the other hand, glycine betaine was not detectable at all in the cell culture during the entire stress treatment period. Furthermore, a western blotting experiment was performed to detect the expression pattern of HSP70 in sorghum and Arabidopsis cells in response to heat stress. The results indicated that HSP70 was highly expressed at 40oC in both cell lines. In addition to the metabolic and biochemical changes that occur in plant cells in response to heat stress, protein and gene expression is also altered. Secreted proteins were extracted from the ICSB 338 culture medium, quantified and gel electrophoresed. Furthermore, the differential protein expression analysis of the extracellular matrix (ECM) proteins, following heat stress at 40oC was conducted using isobaric tags for relative and absolute quantitation (iTRAQ) technology. A total of 290 proteins were positively identified. Of these, 231 (80%) were predicted to contain a signal peptide whereas 59 (20%) did not. This indicates that most proteins were targeted for secretion via the classical secretory pathway into the ECM. Of the 290 proteins, 105 were responsive to heat stress with putative functions in metabolism (31%), disease/defence (30%), protein destination and storage (21%), signal transduction (6%) and energy (3%), while 9% had unclear classifications. However, most of the identified proteins (69%) were uncharacterised, possibly indicating their novelty in heat stress response. The expression analysis of ten target heat stress responsive genes from the proteomic dataset and other heat shock marker genes was conducted using quantitative real time-polymerase chain reaction in a time-course experiment (qRT-PCR). For all the genes analysed, differential expression patterns were observed in response to the heat stress. The observed gene and protein expressional changes indicate that sorghum is responsive to heat stress. The knowledge gained could be applied in breeding programmes for the development of heat tolerant crops to alleviate food insecurity in hot and arid regions. Item Open AccessA comparative physiological, proteomic and biochemical analysis of sorghum seedlings under salt stress(University of the Free State, 2018-11) Moloi, Sellwane Jeannette; Ngara, Rudo; Shargie, NemeraSoil salinity negatively affects plant growth and development, causing crop losses, worldwide. Understanding plant response mechanisms towards salt stress is thus an important step in ensuring food security. Sorghum [Sorghum bicolor (L.) Moench] is a drought and moderately salt tolerant crop and thus provides a potentially good model system for stress response studies. In this study, a comparative physiological, biochemical and proteomic analysis of two sorghum varieties was conducted under salt stress. SA 1441 is drought tolerant, while ICSB 338 is both drought and salt susceptible. The sorghum seedlings were grown in soil until the V3 growth stage before salt-stress treatment with 0-400 mM NaCl for seven days was applied. Physiological parameters such as relative water content, leaf water loss, stomatal conductance, chlorophyll content, shoot and root length and weight were measured following the salt-stress treatment. The results indicated a statistically significant decrease in the measured parameters with an increase in salt stress in ICSB 338, compared to SA 1441. Furthermore, the leaf relative water content of ICSB 338 was significantly lower than that of SA 1441. Proline and glycine betaine accumulation was also analysed in both sorghum varieties across all NaCl treatments. The results showed that proline played a major role in osmotic adjustment in both sorghum varieties when compared to glycine betaine. However, SA 1441 accumulated more proline levels at higher salt treatment levels than ICSB 338. Based on the physiological and biochemical results, the 100 mM NaCl treatment was selected for proteomic analysis. The isobaric tags for relative and absolute quantitation (iTRAQ) proteomic method was used to identify root and leaf salt-stress responsive proteins in both sorghum varieties. In the root proteome, 522 and 544 proteins were positively identified in SA 1441 and ICSB 338 sorghum varieties, respectively. From these root proteins, 26 (SA 1441) and 31 (ICSB 338) were responsive to 100 mM NaCl-induced salt stress. Most of the differentially expressed root proteins were involved in disease/defence functions in both sorghum varieties. In the leaf proteome, 829 (SA 1441) and 591 (ICSB 338) proteins were positively identified. From these leaf proteins, 75 (SA 1441) and 24 (ICSB 338) were responsive to salt-stress. Most of the differentially expressed leaf proteins were associated with disease/defence functions in SA 1441, followed by energy and metabolism in both sorghum varieties. Collectively, the physiological and proteomic results suggest that SA 1441, the drought tolerant variety was more protected against the salt stress better than ICSB 338, the drought and salt susceptible variety. Therefore, the results also highlight the differences in salt tolerance between the two sorghum varieties and possibly reinforce the cross-link between drought and salt-stress response mechanisms. The results of this study can be used as reference tools in studies focusing on the differences in salt stress tolerance between varieties. Item Open AccessVegetation ecology of Soetdoring Nature Reserve: pan, grassland and karroid communities(University of the Free State, 2002-09) Janecke, Beanelri Benene; Du Preez, P. J.; Venter, H. J. T.English: The main aim of this study was to identify, classify, describe and ecologically interpret the plant communities and their variations for the pans, grassland and karroid grassland of Soetdoring Nature Reserve and to compare it to other similar units where possible. A further aim of the study was to provide the Department of Environmental Affairs & Tourism of a baseline study of the grassland and pan vegetation and to provide results which could serve as an ecological basis for future management, conservation and research. Soetdoring Nature Reserve is situated in the Free State Province, about 35 kilometres north-west of Bloemfontein and covers approximately 6 000 ha. The Modder River divides the reserve in two and the Krugersdrif Dam is also included in the reserve‟s boundaries. The main aim was achieved by undertaking a phytosociological investigation by means of the Braun-Blanquet method. The total data set consists of 229 relevés and 171 species. After refinement, the Braun-Blanquet procedures yielded 17 plant communities. Phytosociological tables were compiled for each of the pan, grassland and karroid grassland, and a synoptic table for the total data set, in order to determine the communities and their variants. An ordination algorithm (DECORANA) (Hill 1979b) was also used to indicate the floristic relationships among the vegetation units. The pan unit was classified into five communities and eleven subcommunities. Two pans are present in the reserve, on the southern and northern side of the Modder River respectively. The northern pan was classified as a Grass Pan and characterised by Cynodon transvaalensis, Panicum schinzii and Echinochloa holubii. The southern pan was classified as a Diplachne Pan and is dominated by Diplachne fusca and Eragrostis bicolor. Two permanent earth dams and a hot spring are also present inside the southern pan basin. Earth Dam A is dominated by Eleusine coracana and Phyla nodiflora, while Earth Dam B is characterised by Cyperus bellus and Eragrostis biflora. The vegetation of the hot spring consists of a dominant zone of Phragmites australis, surrounded by a dense zone of Juncus rigidus. The grassland unit was divided into grassland and karroid communities, due to the state of degradation thereof. The classification resulted in five grassland communities and seven karroid communities. The grassland communities are characterised by climax grasses, with the dominant species being Themeda triandra and Digitaria eriantha. The karroid communities are dominated by dwarf karroid shrubs, like Salsola glabrescens, Rosenia humilis and Felicia muricata, as well as subclimax and pioneer grasses, like Eragrostis obtusa, Chloris virgata and Aristida adscensionis. This study provides important information on especially the pans in the reserve, since little information is available for the vegetation of pans in the Free State. The chapters on pans serve to bring all the available information together and to apply the information to the reserve‟s pans. This study is further of importance in indicating the degree of disturbance in the grassland unit. The impact of the animals and the importance of these mentioned areas for the game in the reserve, were taken into consideration for each vegetation type. Item Open AccessWater quality of the Modder River(University of the Free State, 1998-11) Koning, Nadene; Grobbelaar, J. U.; Roos, J. C.English: The Modder River is a relatively small river which drains an area of 7 960 km² , in the central region of the Free State Province, South Africa and has a mean annual runoff of 184 x 10⁶ m³ . Botshabelo is a city which was developed in the catchment area of the river and its sewage outflows are discharged into the Klein Modder River, a tributary of the Modder River. This study was conducted in order to determine seasonal and spatial patterns in the system, the influence of Botshabelo's sewage outflow on the water quality of the river, as well as the presence of any toxic compounds. It was determined that the Modder and Klein Modder Rivers do not follow distinctive seasonal patterns in terms of chemical parameters, however, N0₃-N and P0₄-P concentrations usually increased with increasing flow in the river. Physical parameters such as turbidity, flow and temperaturefollowed distinctive seasonal patterns. Turbidity and flow was high during the rainy season and temperature followed the air temperature. The Modder River is a very turbid system, influenced by physical (flow, turbidity and temperature) as well as chemical factors (high nutrient availability). The Modder River showed similarities with other South African rivers, such as the Vaal and Orange Rivers, in terms of turbidity/conductivity relationships and the range of physical and chemical parameters. Phytoplankton growth also showed distinctive seasonal patterns, with low chlorophylla concentrations in the winter and higher chlorophyll-a concentrations when temperatures became more favourable. Diatoms (especially Cyclotella sp., Stephanodiscus sp. and Nitzchia sp.) dominated the algal community in both the Klein Modder and Modder Rivers for most of the time, with the euglenophyte, Trachelomonas dominating occasionally. In the Klein Modder River, algal blooms occurred more frequently, as well as with higher concentrations than in the Modder River. This could be ascribed to the higher nutrient concentrations in the Klein Modder River, which, together with low flow conditions, provide favourable conditions for algal growth. There were periods when the nutrient concentrations in the waters of the Modder and Klein Modder Rivers were low, however, Botshabelo has an enrichment effect on the water quality, in terms of the nutrient concentrations. The inflow of the Klein Modder River into the Modder River caused on average, a 112 % increase in P0₄-P₃, a 171 % increase in N0-N nitrates and a 50 % increase in chlorophyll-a concentration. However,the Modder River showed a self-purification capacity and nutrient concentrations decreased significantly downstream to Mazelspoort, restoring the water to almost the quality of the "unpolluted" reference point. Based on toxicity tests performed with Selenasfrum capricornufum and Daphnia pulex, no high concentrations of potentially toxic compounds were found in either the Klein Modder or Modder River. However, the occasional presence of heavy metals can not be excluded. Bacteria concentrations were high in both rivers and may pose a threat to human and animal health. The use of a water quality model (PC-QUASAR) on the Modder River system, showed no results to predict the conditions in the rivers and for planning and management purposes, since the model could not be calibrated with the available data. The parameters of the model also showed great insensitivities regarding manipulation of important parameters. Because the Modder River is a very turbid system, it is also important that light availability be taken into account in any forecasting procedures m³ . Botshabelo is a city which was developed in the catchment area of the river and its sewage outflows are discharged into the Klein Modder River, a tributary of the Modder River. This study was conducted in order to determine seasonal and spatial patterns in the system, the influence of Botshabelo's sewage outflow on the water quality of the river, as well as the presence of any toxic compounds. It was determined that the Madder and Klein Modder Rivers do not follow distinctive seasonal patterns in terms of chemical parameters, however, N0₃-N and P0₄ -P concentrations usually increased with increasing flow in the river. Physical parameters such as turbidity, flow and temperaturefollowed distinctive seasonal patterns. Turbidity and flow was high during the rainy season and temperature followed the air temperature. The Modder River is a very turbid system, influenced by physical (flow, turbidity and temperature) as well as chemical factors (high nutrient availability). The Modder River showed similarities with other South African rivers, such as the Vaal and Orange Rivers, in terms of turbidity/conductivity relationships and the range of physical and chemical parameters. Phytoplankton growth also showed distinctive seasonal patterns, with low chlorophylla concentrations in the winter and higher chlorophyll-a concentrations when temperatures became more favourable. Diatoms (especially Cyclotella sp., Stephanodiscus sp. and Nitzchia sp.) dominated the algal community in both the Klein Modder and Modder Rivers for most of the time, with the euglenophyte, Trachelomonas dominating occasionally. In the Klein Modder River, algal blooms occurred more frequently, as well as with higher concentrations than in the Modder River. This could be ascribed to the higher nutrient concentrations in the Klein Modder River, which, together with low flow conditions, provide favourable conditions for algal growth. There were periods when the nutrient concentrations in the waters of the Madder and Klein Modder Rivers were low, however, Botshabelo has an enrichment effect on the water quality, in terms of the nutrient concentrations. The inflow of the Klein Modder River into the Modder River caused on average, a 112 % increase in P04-P, a 171 % increase in N0₃ -N nitrates and a 50 % increase in chlorophyll-a concentration. However,the Modder River showed a self-purification capacity and nutrient concentrations decreased significantly downstream to Mazelspoort, restoring the water to almost the quality of the "unpolluted" reference point. Based on toxicity tests performed with Selenasfrum capricornufum and Daphnia pulex, no high concentrations of potentially toxic compounds were found in either the Klein Madder or Modder River. However, the occasional presence of heavy metals can not be excluded. Bacteria concentrations were high in both rivers and may pose a threat to human and animal health. The use of a water quality model (PC-QUASAR) on the Modder River system, showed no results to predict the conditions in the rivers and for planning and management purposes, since the model could not be calibrated with the available data. The parameters of the model also showed great insensitivities regarding manipulation of important parameters. Because the Modder River is a very turbid system, it is also important that light availability be taken into account in any forecasting procedures Item Open AccessAssessing genetic diversity and identification of Microcystis aeruginosa strains through AFLP and peRRFLP analyses.(University of the Free State, 2003-12) Oberholster, Paul Johan; Grobbelaar, J. U.; Botha-Oberholster, A. M.English: There are 150 cyanobacterial genera and approximately 2 000 species known in the world. More than 40 of these have toxin producing strains. Cyanobacteria, commonly known as blue-green algae, are often present in small numbers together with a diverse assemblage of other photosynthetic algae that naturally occur in surface water worldwide. However, under conditions of warm temperatures, minimal water movement and elevated concentrations of phosphorus in a water body, cyanobacteria may frequently become dominant and form thick scums of floating algal cells. These dense aggregations of floating cells, termed 'blooms', presents a number of water quality problems; most often offensive odours and tastes, and sometimes biotoxins that can be divided into alkaloid neurotoxins and cyclic peptide hepatotoxins, commonly from the genus Microcystis and released in waterbodies. The neurotoxins act chiefly at neuromuscular junctions and cause rapid death because of respiratory paralysis. The hepatotoxins act on the hepatocyte cytoskeleton and cause intrahepatic haemorrhage and centrilobular necrosis. Clinically the hepatotoxin most often causes peracute or acute death, or subacute poisoning with signs such as icterus and hepatogenous photosensitivity. Currently cyanobacterial taxonomy does not provide an unequivocal system for the identification of toxigenic and bloom-forming genus Microcystis. The ambiguities that exist in the cyanobacterial taxonomy are due to the expressed variability, minor morphological and developmental characteristics that are used for identification. In this study geographically unrelated axenic strains of Microcystis aeruginosa were obtained from the Pasteur Institute, France (PCC); the National Institute for Environmental Studies, Japan (NIES); the Institute of Freshwater Ecology, UK (CCAP); the Pflanzen Physiologisches Institut, Universitat Gottingen, Germany (SAG) and the University of the Free State, South Africa (UV) culture collections. Nonaxenic strains were collected from Hartbeespoort, Rietvlei and Roodeplaat Dams in South Africa. After screening 20 primer combinations on a subset of strains eight IRDye700™-labeled EcoR1 primer pairs were selected for amplified fragment length polymorphism (AFLP) analysis to determine the genetic relationship of these geographically unrelated strains. A total of 909 bands were amplified from the eight primer combinations, of which 665 were informative, 207 non-informative and 37 monomorphic, with an average of 83.12 polymorphic bands per primer combination. The genetic relationship among all the Microcystis aeruginosa strains based on the combination of data obtained with the eight primer combinations was analysed employing the Unweighted Pair Group Method using Arithmetic Means (UPGMA) algorithm and presented as a dendrogram. In the dendrogram, the strains from Rietvlei (UP01) and Hartbeespoort Dams (UP04) grouped together and were thus genetically closer to each other, than to the strain from the Rhoodeplaat Dam (UP03). The Japanese strains (NIES88, NIES89, NIES90, NIES99, NIES299) also grouped separate from the other strains, with NIES90 and NIES299, genetically closest to each other. Interestingly, Microcystis aeruginosa strain PC7806 that originated from The Netherlands, also grouped within this group. Microcystis aeruginosa strains CCAP1450/1 (UK), UV027 (South Africa) and PC7813 grouped together, and are genetically closer to the UP-strains, than any of the other strains. In the present study, AFLP analysis proved useful for the identification of genetic diversity and analysis of population structure within Microcystis aeruginosa. In order to link the identification of strains with toxicity, the utility of the mcyB gene sequence for identification of strains was tested. Based on conserved motifs present in known sequences of mcyB four primer pairs were designed. Using the primer pairs Tax 3P/2M, Tax 1P/1M, Tax 7P/3M and Tax 10P/4M, the mcyB gene from PCC7813 and UV027 were sequenced, resulting in fragments of 2174 and 2170 base pairs in size, respectively. The obtained sequences were analyzed using nucleotide BLASTN annotation of the Basic Local Alignment Search Tool (BLAST). The sequence alignment indicated high homology to other published sequences in GenBank (AY034601 for pee7813 and AY034602 for UV027; e-value = 0.0). Upon further analysis of the sequences it was obvious that there are several base differences between the sequences of the two strains, which led us to investigate the potential of using differences in restriction sites, and thus insertions/deletions (indels) in nucleotide sequence to discriminate between the other M. aeruginosa strains, as well as using the mcyB gene to discern between M. aeruginosa and M. wesenbergii in raw water samples. A vast number of restriction sites were identified with differences followed by restriction digest of the specific polymerase chain reaction (PCR) mcyB gene fragment. This work demonstrates that PCR assays provide a useful indicator of toxicity as well as the identification of taxonomical characteristics between laboratory cultures and environmental isolates. A number of questions arise from the present study and future research therefore needs to address the following issues: • Are there more than one Microeystis aeruginosa strain / "population" present at a given time in a specific water reservoir? Do these populations change through the season? What role does the individual populations play in a cyanobacterial bloom? Thus, the dynamics and structure of populations need to be clarified. • Which mcy gene in the cluster is mostly responsible for toxin production? Does the expression of the genes correlate with gene structure/sequence? What role does the environment play in determining the level of expression, and thus toxin production? Item Open AccessAssessment of South African bread wheat cultivars for milling quality(University of the Free State, 2003) Aucamp, J. C.; Van Deventer, C. S.; Labuschagne, M. T.English: The objectives of this research were to assess South African winter and facultative bread wheat cultivars for milling quality, to study the effect of genotype x environment interaction on the stability of the wheat quality and to characterise the cultivars for milling performance. Thirteen hard red varieties were planted at six localities throughout the Free Sate at two planting dates, during 1997 to 1999. Betta-DN, Gariep, Molen, SST 367, SST 966 and Tugela-DN were planted at the first planting date. Betta-DN, Caledon, Elands, Gariep, Limpopo, PAN 3235, PAN 3349, SST 124, SST 972 and Tugela-DN were planted at the second date. Material was evaluated for test weight, thousand kernel weight, kernel diameter, kernel hardness, moisture content, vitreous kernels, break flour yield, flour yield, flour colour and flour protein content. Combined ANOVA's for the characteristics was performed over environments. Cultivar differences were determined by means of the least significant difference at individual localities. The analysis indicated highly significant differences among genotypes, environments and GXE interactions, for most of the measured traits. Genotypes contributed significantly to the variance in BFLY, DIAM, TKW and Hl. Environment had a large effect on TW, MOIST, VK, FLY, FCL and FPC. Significant GXE interactions were present for most of the quality traits. Canonical variate analysis was used to differentiate between cultivar groups and indicated that TKW, DIAM, HI, BFLY and TW could effectively differentiate between cultivars. The groups observed with regard to the mentioned characteristics were Gariep with Betta-DN and Molen with SST 966. Tugela-DN and SST 367 were not similar to any other cultivars. At the second planting date, Tugela-DN grouped with SST 124. Betta-DN, Caledon, Elands, Gariep, Limpopo and PAN 3235 formed a group. SST 972 and PAN 3349 were not similar to each other or to the other cultivars. The correlation matrix was performed, to calculate phenotypic relationships between quality traits. TKW had positive correlations with DIAM and these two characteristics indicated positive correlations with MOIST. HI correlated positively to TW and also correlated positively to VK. HI and VK correlated negatively to BFLY. High negative correlations were found between HI and FLY. FLY had positive correlations with BFLY, but correlated negatively with FLC. Stepwise multiple regressions indicated that only small percentages of the variation in TW, HI, BFLY, FLY and FLC could be explained by the other kernel and milling characteristics. HI explained the variation in the TW the most effectively. VK and MOIST could be regarded as the most important variance predictors for HI. Predictors for BFLY were kernel size and HI. These characteristics could define 41.6 to 63.3% of the variation in BFLY. Kernel hardness measurements, HI, BFLY and VK were the most definable for flour yield, together they interpreted up to 50.0% of the variation in FLY. TW explained less than 4% of the variation in FLY; therefore TW is not always reliable in predicting flour extraction. The kernel size explained less than 9.6 to 20.7% of the variation in FLY, illustrating the difficulty of predicting flour yield by indirect calculations. The most important variable in predicting FCL was FLY. AMMI analysis of variance was performed to investigate the influence of the GXE interaction on milling quality. Every locality by year combination was treated as a separate environment. Analysis confirmed differences in genotype performance, that there were varying growth conditions among the localities and the presence of GXE interactions. Interaction effects on TW, percentage VK, FLY, FCL and FPC, were large. IPCA 1 stability scores were compared to the AMMI stability values. Molen was the most stable cultivar at the first planting date. At the second planting date, PAN 3235 and Limpopo were the stable cultivars. Betta-DN, SST 124 and Caledon were also quite stable. Tugela-DN, PAN 3349, as well as the hybrid cultivars SST 966 and SST 972, were unstable with regard to milling quality. Milling performance, as calculated by flour extraction, ash contents and flour colour in various formulas, revealed Gariep and Betta-DN as the high performing cultivars (first planting date), while Molen and Tugela-DN reflected poorer performances. The most effective performing cultivars at the second planting dates were SST 124, PAN 3235, Betta-DN and Limpopo. PAN 3349, SST 972 and Tugela-DN indicated poorer milling performances. Item Open AccessAn investigation into the possible causes of decline in the Acacia Erioloba population of the Kathu area(University of the Free State, 2001) Van der Merwe, Karien; Du Preez, P. J.; Potgieter, G. P.English: Concerned residents have been reporting a decline in the A. erioloba population of the Kathu area to the Northern Cape Nature Conservation Service (NCNCS) since the early 1980's. This is the third study on this subject initiated by the NCNCS, and aims to elucidate the possible causes of decline in the A. erioloba population of the Kathu area. Five investigation sites were identified: the Khai-Apple Nature Reserve (KANR); the farms Lylyveld and Dernaneng, both situated down-wind with regard to the Sishen lscor Iron Ore Mine (SllOM); the Sishen Golf Course; and the farms Swarthaak and Knapdaar, both situated upwind with regard to the SllOM. The Sandveld Nature Reserve on the Free State side of the Bloemhof Dam, served as control site. In order to identify potential problem areas regarding the A. erioloba population of the Kathu area, the A. erioloba population structure of the study and control areas were compared. Irregularities in the structure of the study area indicated that some factor(s) is either a) preventing individuals from reaching middle height classes, or b) kill ing trees in these classes. To eliminate one of these options, another structure study was conducted, comprising dead individuals only. Results indicated that both factors (a+ b) play a role. By means of questionnares that were distributed th roughout the region, three major factors, potentially harmful to the A. eriofoba population, were identified. They are the mining activities of the SllOM, management strategies and natural phenomena. Two aspects of the SllOM were postulated to be potentially harmful to the A. erioloba population: the mine dust formed as a by-product of mining activities; and the lowering of ground water levels. Scanning electron microscopy confirmed that A. erioloba leaves are indeed damaged by mine dust. Through further investigations the effect of mine dust on the transpiration rate, chlorophyll a and b content and protein content of A. erioloba leaves were determined. The results of this study indicated that mine dust has no marked effect on any ofthese variables. The effect of mine dust on the germination of A. erioloba seeds and the growth potential of its embryos were also determined. No inhibitive effect was detected on either of these processes - in fact, mine dust seemed to stimulate both. No trials regarding the effect of the lowering of ground water levels by the SllOM on A. erioloba were conducted, due to its impracticality. Instead, with the aid and insight of geohydrologists it was concluded that the lowering of ground water levels does not affect A. erioloba. The aquifer underneath the mining area is seperated from the other two aquifers in the area (situated under Kathu and the KANR) by a dolorite dyke, which is impermeable to water. This was confirmed in the present study by comparing the water pumping rates of the SllOM to bore hole levels throughout the region. Three management strategies applied in the Kathu area were identified as being potentially harmful to A. erioloba: the overstocking of browsers and mixed feeders in the KANR; pod removal; and the uninformed use of chemicals. The current stocking rate of the KANR compared to its current browsing capacity, revealed that the reserve is grossly overstocked. This results in the suboptimal regeneration of A. erioloba, as seedlings cannot reach the stage where browsing doen not prove to be fatal anymore. Pod removal also inhibits optimal reg eneration, as no A. erioloba soil seed bank was found. This implies that no genetic variation in the A. erioloba pululation is built up over time, which may prove to be fatal in the event of future changes in environmental conditions. The uninformed use of non-specific chemicals in an attempt to eradicate A. mellifera on farms is furthermore also eradicating A. erioloba. One natural phenomenon was hypothesised to be inhibiting to the regeneration of A. erioloba, namely seed predation by Bruchidae. Germination trials revealed that bruchid seed predation inhibits the regeneration of A. erioloba. It is, however, compensated for by producing a relatively large seed yield per tree in an attempt to over-saturate predators. Management recommendations on the effective management of A. erioloba in the Kathu area were made. Recommendations included correct stocking rates, increasing community involvement in conservation, veld monitoring and the correct use of chemicals Item Open AccessAn assessment of endophytic fungi in needles of three pinus spp. cultivated in South Africa(University of the Free State, 1999-11) Kriel, Wilma-Marie; Swart, Wijnand J.; Crous, Pedro W.English: Endophytes, in the strict sense, are organisms that cause symptom less infections in plants. As symptomless mutualists, they can act as biocontrol agents of herbivorous insects and plant diseases. They can also be indicative of host vitality and environmental pollution. Some endophytes, however, are latent pathogens with an endophytic phase. Pine needles are colonised by various species of endophytic fungi. It is well-known that the endophytic biota vary with host species, as well as with time. Therefore the aim of this study was to qualitatively and quantitatively compare endophyte populations within the canopies of Pinas patula, P. radiata and P. elliottii, during winter and summer, and within the canopies of two separate P. radiata trees, over different seasons. Endophytic fungi were isolated from pine needles, sampled in different seasons from various positions within the canopy, by plating surface-sterilised needle sections onto cornmeal agar supplemented with antibiotics. In the first study a significant difference (P < 0.05) in endophyte numbers between samples collected during winter and summer was observed. Pinus patula and P. elliottii were more intensively colonised during winter than summer. In P. radiata, however, the number of fungi isolated in summer was significantly higher than in winter, but the fungal species isolated were consistent. Cydsneusms minus and a sterile white yeast-like fungus were most commonly isolated. Cyclsneusms minus is a latent pathogen causing autumn needle cast, and sterile yeast-like fungus is suspected to be a true endophyte. Similar endophytic fungal species were isolated in the second study, performed on two eight-year-old P. radiata trees. Samples were taken during four seasons from an isolated, solitary tree and one growing in an even-aged, plantation nearby. Five needle fascicles of four different age groups were collected from each tree. One needle per fascicle, including the fascicle sheath, was cut into 12 sections and used for the isolations. In general, fewer endophytes were isolated from the solitary tree than the plantation tree. Qualitative and quantitative differences in endophyte populations were observed within needles as well as between needle age groups and seasons. The aim of the third study was to conduct a qualitative assay of enzyme production of 2 t predominant fungal endophytes isolated from the pine needles. The enzymes assayed included cellulase, pectinase, lipase, laccase, phenol oxidase, protease, B-glucosidase, cytochrome oxidase, and peroxidase. Results were consistent with attributes associated with leaf penetration and longterm residence of fungi within pine needles. All fungi screened produced at least two of the enzymes assayed. Different substrate utilisation patterns suggest biochemical partitioning of nutritional resources by endophytes. The ubiquitous presence of lipolytic activity in all isolates tested, suggests the ability to lyse cuticular waxes in order for penetration to occur. The tolerance of most tested fungi to tannic acid (phenol oxydase production) suggests low sensitivity to phenolic compounds (tannins etc.) normally present in pine needle tissue. Management practices in plantations that reduce or increase the environmental stress on individual trees, thereby influencing endophytic populations, could have distinct beneficial or negative effects on the general vitality of trees. The most important effects of certain practices would be the influence they have on latent pathogens such as C minus. Further investigation of foliar endophytic fungi of intensively managed pine plantations is therefore justified with a view to understand the effects management practices have on their ecology. Item Open Access'n Ondersoek na die antibakteriese eienskappe van Carpobrotus edulis L.(University of the Free State, 1999-11) Van der Watt, Elmarie; Pretorius, J. C.; Kemp, K.English: In the folk medicine of South Africa, many traditional medicinal plants are used by both the rural and a small percentage of the city population. One of these plants is Carpobrotus edulis L. An investigation of the chemical composition as well as the anti-microbial properties of this plant was the main objective of this study. Overviews on traditional medicine in South Africa and the family Mesembryanthemaceae are given. According to the available literature little is known about the chemical composition, active substances in as well as pharmacological properties of C. edulis, but most of the published information deals with the ecological importance of the plant. In this study the anti-bacterial properties of the plant were confirmed and three semipurified fractions were initially isolated by applying different chromatography techniques. During purification of active substances, with prior removal of tannins, six anti-bacterial compounds were isolated of which five were identified namely rutin, neohesperidin, hyperoside, cactecin, ferulic acid while one flavonoid remained unidentified. The conclusion from this study is that the anti-bacterial properties of the plant are related to the presence of the tlavonoids rutin, neohesperidin, hyperoside, cactecin and femlic acid as well as one unknown flavonoid. The most prormsmg observation during this study was the selective inhibition of gram negative bacteria, known to be more resistant than gram positive bacteria to antibioticums. Item Open AccessWater quality of the upper Orange River(University of the Free State, 2000-05) Venter, Gertruida C.; Roos, J. C.; Van Wyk, P. W. J.English: The Upper Orange River is defined as the region between the source in the Drakensberg (Lesotho) and the Orange-Vaal confluence. Information on the water quality, phytoplankton composition and the influence of the dams on the Upper Orange River is limited. This study was conducted in order to determine seasonal and spatial patterns in the river system., as well as to determine the influence of the tributaries and the two major dams on the river system. Physical, chemical and biological factors were taken into account. Hypolimnetic water released from the Gariep and Vanderkloof Dams caused a significant decrease in the temperature downstream of the dams. The dams acted as sediment traps and caused the TSS and turbidity in the river system to decrease. A decrease in turbidity resulted in an increase in the light penetration. This created more favourable conditions for primary production and led to algal blooms in the dams. The dams are used for hydro-electric power generation and almost completely diminished seasonal flow patterns. This changed the Upper Orange River into a highly regulated system. The interference with seasonal flow patterns in the river is probably one of the main reasons for outbreaks of the pest blackfly, Simulium chutteri downstream of the dams. The oxygen-depth profile in the Gariep Dam during summer stratification indicated relatively high dissolved oxygen concentrations in the epilimnion (8.2 mg.l-1) and anoxic, near anaerobic, conditions (0.1 mg.l-1) in the hypolimnion. The oxygen decline in the hypolimnion was indirectly the cause of high algal production in the epilimnion during algal blooms. The average pH of the Upper Orange River system was mainly alkaline at 8.1, with an average total alkalinity of 95.2 mg CaC03.rl. This suggested that the Upper Orange River is a wellbuffered system. The average conductivity in the Upper Orange River was relatively low at 19.6 mS.m-1, but increased significantly since 1980. Major ions at Aliwal North occurred in the following proportions, i.e. Ca> Na > Mg > K for cations and CO3 > Cl> SO4 for anions. In the Gariep Dam and at Marksdrift the proportions were: Na > Ca > Mg > K for cations and CO3 > Cl > SO4 for anions. The average P04-P concentration was relatively low at 28.3 µg.l-1 and the average TP concentration was 107 µg.I-1. The average N03-N concentration was high at 312 µg.l-1 and increased in the dams and downstream probably due to agricultural fertilizers and nitrogen fixation by algae. The average chlorophyll a concentration ranged between 0.4 and 1084 µg.I-1, with and average concentration of 10.8 µg.l-1. The very high chlorophyll a concentration was due to a bloom of Microcystis sp. during February in the Gariep Dam. Phytoplankton dynamics manifested themselves in seasonal cycles. Cyanobacteria and Bacillariophyceae dominated during warmer months. The Chlorophyceae replaced cyanobacteria during cooler months. Light penetration in the Gariep Dam and the Vaal River had a major effect on the primary production. The maximum primary production in the Gariep Dam was 14.6 mg C. mg Chla-1 h-1 and in the Vaal River the maximum primary production was 76 mg C. mg Chla-l. h-1. According to the primary production in the Gariep Dam it can be classified as mesotrophic, while the high primary production in the Vaal River indicated eutrophic conditions. The dams exert major influences on the physical, chemical and biological parameters in the river. Low SASS4 scores were obtained and are an indication of poor water quality. This could be ascribed to disturbances by the dams. The serial discontinuity concept (SDC) is applicable in the Upper Orange River. It is likely that agricultural runoff, as well as urban and industrial pollution from the Vaal River catchment would mask the downstream recovery after the Orange- Vaal confluence.