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Plant material
Plant clones of the same age were prepared for the greenhouse experiment under room temperature (25°C), light, irrigation and growth medium before trial commencement. To generate vegetative clones, plantlets were prepared through division of mother clumps of B. frutescens and T. violacea. Plantlets were established in the growth trays filled with vermiculite comprising pine-bark medium. The two mother plant species were obtained from the Vegetable and Ornamental Plant Institute (VOPI) of the Agricultural Research Council (ARC). Seeds of L. dysophylla were collected from a wild population in the Akasia municipality west of Pretoria-North and germinated in the growth trays filled with vermiculite at a depth of 1.25 cm. Almost 100% seed germination was achieved after two weeks. Seeds from one of the mature L. dysophylla plants in the greenhouse were germinated again under the same conditions to minimise genetic variability. Approximately 100% germination was again achieved within two weeks. Seedlings were grown for a period of up to six weeks in the greenhouse at the Experimental Farm of the University of Pretoria.
Seedlings were irrigated with 500 ml of distilled water every two days.
Growth of vegetative and seedling clones
After reaching a height of 10 to 15 cm with at least two leaves, seedlings were transplanted into large pots (27 cm diameter x 25 cm height, c. 14 L capacity) filled with potting-mix. Potting-mix comprised four parts loam soil, two parts sand, one part manure and two parts compost (Netshiluvhi, 1999). Seedlings were then subjected to different water stress conditions comprising irrigation with 500, 200, 100 and 50 ml of distilled water every two days under room temperature in the greenhouse. Irrigation was in the morning for the entire period of the experiment. Each water treatment consisted of four pots containing one plant per pot.
The entire experiment ran for a period of 26 weeks.
Phytochemical analysis
Chemical constituents of the extracts were analysed by thin layer chromatography (TLC) using aluminium backed TLC plates (Merck, silica gel 60 F24). The TLC plates were developed in the three mobile systems of differing polarity established in the Phytomedicine Laboratory of the University of Pretoria (Kotze & Eloff, 2002). The mobile systems used were; chloroform/ethyl acetate/formic acid (CEF: intermediate) (5:4:1), benzene:ethyl acetate:ammonia (BEA: non-polar) (9:1:0.1) and ethyl acetate:methanol:water (EMW: polar) (40:5.4:5). The chromatograms were examined under UV light (250 and 360 nm, Camac Universal lamp TL-600) to detect UV active absorbing spots. The plates were then sprayed with vanillin spraying reagent (0.1% vanillin dissolved in 28 ml methanol and 1 ml sulphuric acid) and heated at 100°C to optimal colour development. The position of the visible compounds on the TLC plate was established by calculating the retardation factor (Rf), which is the distance compound travelled divided by the distance the solvent had travelled from the origin.
Bioautography assay
The chromatograms (not sprayed with vanillin spray reagent) were left overnight to dry in a draft of cold air to remove the eluents and then sprayed with a concentrated suspension of actively growing cells of bacteria or fungi (Masoko & Eloff, 2006). This method relies on the direct growth inhibition or killing of pathogens on contact with the active band (Begue & Kline, 1972). The sprayed plates were incubated overnight at 38°C in a chamber at 100% relative humidity to allow the pathogens to grow on the plates.
After overnight incubation, bioautograms were sprayed with an aqueous solution of 2 mg/ml ρ- iodonitrotetrazolium violet (INT) (Sigma). Thereafter, bioautograms were incubated for 30 minutes to observe clear zones indicating growth inhibition of pathogens by bioactive compounds in the extracts. A set of chromatograms sprayed with vanillin-sulphuric acid was used as reference for bioautograms displaying areas of inhibition. The Rf values of active zones were correlated with those bands on the reference chromatograms.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) values (mg/ml) were determined after two-fold serial dilution (e.g. 10, 5, 2.5,1.25, 0.63, 0.32, 0.16, 0.08) of extracts with a concentration of 10 mg/ml beyond where no inhibition of growth of test bacteria was observed. This method was used to evaluate the antibacterial activity of extracts (Eloff, 1998a). Plant extracts (100 μl) in triplicate for each experiment were serially diluted two-fold with water in 96-well microlitre plates. A similar volume 100 μl of the actively growing test organism cultures was added to each well and the cultures were incubated overnight at 37°C under 100% relative humidity. As an indicator of bacterial growth, 40 μl of 0.2 mg/ml of ρ-iodonitrotetrazolium violet (INT) dissolved in water was added to each microplate well before being incubated for an hour or two (Eloff, 1998b). The MIC value was recorded as the lowest concentration that inhibited growth of bacteria. The colourless tetrazolium salt acts as an electron acceptor. It is reduced to a red-coloured formazan product by biologically active pathogens (Eloff, 1998b). Clear zones indicated inhibition of the growth of bacteria after incubation with INT. The experiment was repeated twice to confirm the results, and three replicates were included in each experiment.
Total activity
Total activity was also used as a parameter to measure the effects of temperature stress on plant activity.
Total activity value (ml/g) measures the total antibacterial activity present in different plants by dividing the quantity extracted (mg) from 1 gram of plant material with the MIC value in mg/ml (Eloff, 2000). It indicates the degree to which the active compounds in one gram of plant material can be diluted and still inhibit growth of pathogens. Total activity value is calculated by dividing the quantity in mg extracted from 1 gram of plant material (mg/g) with the MIC in (mg/ml). The higher the total activity in ml/g of a plant extract, the more effective the plant is.
Chapter 1 Introduction
1.1 Problem statement
1.2 General literature survey
1.3 Aim and objectives
Chapter 2 Antibacterial activity of acetone leaf extracts of three tree species from areas receiving different rates of annual rainfall
Abstract
2.1 Introduction
2.2 Materials and methods
2.3 Results
2.4 Discussion and conclusion
Chapter 3 Does water stress affect antibacterial activity of Tulbaghia violacea and Hypoxis hemerocallidea?
Abstract
3.1 Introduction
3.2 Materials and methods
3.3 Results and discussion
Abstract
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4. Discussion
Chapter 5 Effect of temperature stress on antimicrobial activity of three medicinal plants
Abstract
5.1 Introduction
5.2 Materials and methods
5.3 Results and discussion
Chapter 6 Antioxidant activity of acetone leaf extracts of plants growing under induced temperature and water stress conditions
Abstract
6.1 Introduction
6.2 Materials and methods
6.3 Results and discussion
Chapter 7 General discussion
References
Appendices
