Thesis (MSc. (Horticulture)) -- University of Limpopo, 2016. / Pelargonium sidoides has numerous medicinal applications, with economic potential to serve as a future cultigen in smallholder farming systems. However, it is highly susceptible to the root-knot (Meloidogyne species) nematodes, without any identifiable nematode resistant genotypes. Nemarioc-AL and Nemafric-BL phytonematicides, with cucurbitacin A and cucurbitacin B active ingredients, respectively, are being researched and developed as an alternative to synthetic nematicides at the University of Limpopo. However, since active ingredients in phytonematicides are allelochemicals, the two phytonematicides have the potential of inducing phytotoxicity on crops protected against nematode damage. The objectives of the study, therefore, were (1) to determine the non-phytotoxic concentration of Nemarioc-AL phytonematicide on plant growth of P. sidoides, and (2) to determine the non-phytotoxic concentration of Nemafric-BL phytonematicide in plant growth of P. sidoides. Cuttings were raised in 30-cm-diameter plastic pots containing 10 000 ml steam-pasteurised river sand and Hygromix-T at 3:1 (v/v) under microplot conditions in autumn (March-May) and repeated in spring (August October) 2015. After establishment each plant was inoculated with 5 000 eggs and second-stage juveniles (J2s) of M. javanica. Six treatments, namely, 0, 2, 4, 6, 8 and 10% concentrations of each phytonematicide on separate trials were arranged in a randomised complete block design, with seven replicates. At 56 days after inoculation, in Experiment 1, Nemarioc-AL phytonematicide, treatment significantly (P ≤ 0.05) affected plant height, dry root mass and root galls, contributing 62, 69 and 70% to total treatment variation of the three variables, respectively. Relative to untreated control Nemarioc-AL phytonematicide increased plant height and dry root mass by 34 to 61% xxi and 20 to 76%, respectively, with a slight decrease by 5% in plant height at the highest concentration. However, the material decreased root galls by 5 to 50%. Significant (P ≤ 0.05) plant variables were subjected to Curve fitting-allelochemical respond dosage model, to generate biological indices which were used to compute the mean concentration stimulation point (MCSP) using the relation: MCSP = Dm + Rh/2 and the overall sensitivity value (∑k). In Experiment 1, MCSP = 6.18% and ∑k = 3. Plant variables and increasing concentration of phytonematicide exhibited quadratic relations. Treatments reduced nematode variables, at all levels including at the lowest, but the effect were not different. In Experiment 2, Nemarioc-AL phytonematicide treatment effects were not significant on plant variables except for root galls, but were significant for root nematodes except for eggs. Data for plant variables in Experiment 2 were not subjected to Curve fitting-allelochemical respond dosage model because they were not significant (P ≤ 0.05). In Experiment 1, Nemafric-BL phytonematicide treatment significantly (P ≤ 0.05) affected plant height and root galls, contributing 63 and 67% to total treatment variation of the two variables, respectively. Relatively to untreated control, plant height was increased by 10 to 36%, while root galls was reduced by 2.43 to 60%. In Experiment 1, MCSP = 2.87% and ∑k = 3. Concentrations of Nemafric-BL phytonematicide significantly (P ≤ 0.05) reduced eggs, juveniles and Pf at all levels including at the lowest, but the effect were not significant different, with treatments contributing 78, 72 and 90% to the total treatment variation. In Experiment 2, Nemafric BL phytonematicide treatment effects were not significant on plant variables except for root galls, but were significant for root. In conclusion, Nemarioc-AL and Nemafric-BL xxii phytonematicides could be applied at the lowest concentration of 2% where it was shown to be effective in suppressing population densities of M. javanica. / Agricultural Research Council (ARC), National Research Fund (NRF) , Flemish Inter university Council of Belgium and Land Bank Chair of Agriculture ─ University of Limpopo

Anecdotal evidence of two South African Geranium species (Pelargonium reniforme and Pelargonium sidoides) from the United Kingdom with regard to plants being used against tuberculosis, which lacked scientific evidence’ prompted us to investigate these two plants for their antimicrobial properties. The German herbal remedy (‘Umckaloabo’) is prepared from these two plant species and is currently being sold for bronchitis. Acetone, chloroform and ethanol extracts were investigated against three bacteria (pathogens causing bronchitis), three fungi (fungal species associated with the upper and lower respiratory tract) and Mycobacterium tuberculosis. This is the first report on the extracts’ activity against Moraxella catarrhalis, and three fungi (Asperigilus niger, Rhizopus stolonifer and Fusarium oxysporum). Acetone and ethanol root extracts of P. sidoides and its combination with P. reniforme exhibited activity against bacteria at 5.0 mg/ml concentration. The fungi were significantly inhibited by the acetone and ethanol extracts of P. reniforme and the ethanol extract of P. sidoides at a concentration of 5.0 mg/ml. Antituberculosis activity was observed on acetone, chloroform and ethanol root extract of P. reniforme and chloroform extract of P. sidoides at 5.0 mg/ml concentration. The isolation and purification of compounds were attempted using two different approaches, of which the second approach resulted in isolation of four compounds and two flavonoids. One flavonoid (epigallocatechin) is isolated for the first time from P. sidoides. Laboratory investigations showed no activity of compounds isolated against M. tuberculosis. As Mycobacteria are intracellular pathogens, antimycobacterial activities may be due to either direct or indirect effects. Though the compounds in this study did not show antituberculosis activity, it can be speculated that the anecdotal evidence of TB-patients could be due to their immunostimulant activity. / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005. / Plant Science / unrestricted

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)

Tuberculosis (TB) is an infectious and fatal disease that ranks as the second leading killer worldwide. It is caused by Mycobacterium tuberculosis (Mtb) which is an obligate intracellular parasite that colonizes the alveolar macrophages of the immune system. The major health concern associated with TB is its co-infection with HIV and the development of strains with multi-drug resistance. The elimination of TB has been hindered due to the lack of understanding of the survival strategies used by this pathogen. Thus, research towards discovering new effective antibacterial drugs is necessary and a group of Mtb kinase enzymes were targeted in this study because these enzymes are crucial for metabolism, pathogenesis and, hence, the survival of Mtb. Kinases are a group of structurally distinct and diverse proteins that catalyze the transfer of the phosphate group from high energy donor molecules such as ATP (or GTP) to a substrate. The phosphorylation of proteins modifies the activity of specific proteins which is subsequently used to control complex cellular processes within Mtb. The starting point of this research targeted eight specific Mtb kinases namely; Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase. The aim of this project was to subclone the gene sequences for these eight recombinant Mtb kinases and express them in Escherichia coli, to purify the proteins and determine their activity. In the effort to find new lead compounds, the final stage of this study focused on the basic screening of the TB kinases against an extract prepared from Pelargonium sidoides, a medicinal plant, to identify any inhibitory effects. Although this traditional medicinal plant has been broadly researched and extensively used to treat TB, there is still a lack of understanding of this plant’s scientific curative effect. Various molecular and biochemical methods were used to achieve the aims of this project. The putative gene sequence was obtained from the annotated genome of H37Rv, deposited at NCBI as NC_000962.2. The genes encoding the kinases were successfully PCR-amplified from genomic DNA, cloned into an expression vector in-frame with a C- or N-terminal 6-histidine-tag and expressed in E. coli BL21 (DE3). The purification of the protein was complex, but various different methods and techniques were explored to obtain sufficient amounts of protein. The functional characterization of the kinases involved an HPLC enzyme assay that showed that the recombinant kinases were active. These enzymes were then screened against the potential inhibitory compounds in P. sidoides using enzyme assays to generate dose-response curves. This allowed an effective comparison not only of the Mtb kinases’ activity under normal conditions but also the kinases’ activity in the presence of a potential inhibitor. Overall, the inhibition of the enzymes required the presence of higher concentrations of the P. sidoides extract. However, the SK enzyme results presented a significantly higher inhibition and the lowest IC50 value, in comparison to the other kinases, which makes this kinase an attractive potential drug target against TB. In summation, cloning and purification of SK was successful, resulting in a concentration of 2030 μg/ml of purified enzyme and its activity analysis demonstrated enzyme functionality. This activity was reduced to zero in the presence of 1 x 102 mg/ml dilution of P. sidoides plant extract. This research conducted has extended the quality of information available in this field of study. These interesting results, proposing and identifying SK as a suitable potential target can be a starting point to significantly contribute and progress in this field of research, with the eventual goal of developing a drug to combat this fatal disease. / Life Sciences / M. Sc. (Life Sciences)