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Environmental stress effects on the phytochemistry and bioactivity responses of a South African medicinal bulbous plant, Tulbaghia violacea Harvey (Alliaceae)
Deteriorating living and environmental conditions have contributed to the increasing prevalence of diseases in plants and animals. In humans, accumulation of abnormally high levels of free radicals in the tissues has been implicated in many non-communicable diseases, such as diabetes, cancer, arthritis, ischemia, gastritis, obesity and asthma. Worldwide, there is recognition of need to improve plant and animal health. Tulbaghia violacea (Alliaceae) is a medicinal plant that is extensively harvested by traditional healers in the wild for its medicinal uses and if this practice continues, it may result in an unsolicited decline of the species in situ. Therefore, there is a need for cultivation of this species. Plant cultivation in a controlled environment for conservation purposes as well as the enhancement of yield and quality is gaining favour among farmers and consumers. The main aim of this study was to investigate the effects of altering the growing conditions by applying environmental stresses on the plant growth, antifungal and antioxidant activities of T. violacea, with the view of enhancing the future cultivation of this species for pharmaceutical companies, traditional healers and the horticulture industry. This study was divided into two parts, and the first part, which was further sub-divided into two separate preliminary experiments, is presented in chapter three. Simultaneous assessments of the effects of i) varied pH levels (pH 4, pH 6, pH 8) and ii) light intensity on plant growth, antioxidant-content and -capacity of extracts of T. violacea were carried out. The second part of the thesis consisted of a more detailed assessment of the above-mentioned independent variables and interactions thereof on plant growth, and antifungal activity of extracts of T. violacea. Results obtained from the first part of the study, showed that plants exposed to pH 6 showed a marked increase in plant height (from 25-37 cm) after 2 months of treatment although, generally, the variations of the different growth parameters among the pH treatments were not significant (p > 0.05). Antioxidant-contents and -capacity were not significantly different (p > 0.05) when pH treatments were compared. However, a high polyphenol content value (of 3 mg/g) occurred in leaves of plants exposed to pH 8. Overall, comparatively, there was no significant difference (p > 0.05) in antioxidant-content and -capacity when pH treatments. In the light experiment, decreasing light intensity led to the elongation of plant height. A higher mean shoot length of 34.6 cm was obtained under low light compared to normal light (26.5 cm) two months post-treatment. The results obtained in this study indicated that light had a significant affect (p < 0.05) on the vegetative growth of this species. In contrast, normal light intensity yielded higher antioxidant-content and -capacity. The polyphenol and flavanol content were fluctuating between the averages of 5.8 mg/g to 8.5 mg/g. Overall, there was a significant difference (p < 0.05) in the antioxidant-content and -capacity when low and normal light intensity treatments compared. In conclusion, both normal light intensity and at pH 8 induced better antioxidant results. In the second part of the study, chapter four, one-month old T. violacea plantlets were grown under two light intensities (low light and normal light) in a greenhouse and concurrently exposed to varying pH levels: pH 4, pH 6 and pH 8. Plants exposed to normal light received natural sunlight through the roof of the greenhouse, while low light intensity (40% reduction) was achieved using shade nets. Plants were drip irrigated with Nutrifeed fertilizer. Plant growth parameters such as height and fresh and dry weights were determined. Leaf samples were analysed for macro-and micro-nutrients contents. Antifungal tests were carried out on the plant extracts from the various treatments in an antifungal bioassay (minimum inhibitory concentration [MIC]). The experimental data collected were analysed using one and two-way analyses of variance (ANOVA), and Tukey HSD was used to separate the means at p < 0.05 level of significance. Varied effects of different pH levels (4, 6 and 8) and light intensities (low and normal) on plant height, and fresh and dry weights were recorded in the current study. A significant interactive (df, 2; F = 0.001; p < 0.001) effect between pH and light on fresh weight was observed. The results revealed that there was a significant difference (df, 2, 57; F = 12.63; p < 0.001) in dry weights with plants under normal light intensity and pH 4 treatment (8.285 ± 0.802 g) producing the highest dry weight. There was a significant interaction (df, 2; F = 6.4; p < 0.001) between pH and light intensity on plant dry weight. Extracts from plants grown under normal light intensity showed stronger antifungal activity at pH level 4, and MIC values ranged from 0.18 ± 0 to 0.375 ± 0.04 mg/ml at 6h and 1.5 ± 0 to 0.97 ± 0.18 mg/ml at 18h. In conclusion, this study demonstrated the interactive effects of pH and light intensity on the growth of T. violacea. These findings also confirmed that it is possible to enhance the cultivation of T. violacea under greenhouse conditions. Chapter 5 focused on the interactive effects of pH and watering regime on plant growth, nutrient uptake and antifungal activity of T. violacea plant extracts, grown hydroponically. The results showed that there were significant differences (p < 0.05) on plant growth parameters amongst the different watering regimes under normal light intensity. Broadly, two trends occurred in the results: firstly, more macro-nutrients were taken up by plants in the higher frequency watering intervals as opposed to higher tissue micronutrient nutrient values for plants grown under the lower light intensity conditions. The levels of N, P, K, Mg nutrient uptake differed significantly in plants (p < 0.001) among watering interval periods. On the other hand, plants simultaneously exposed to extended watering intervals of 21-day and low light intensity showed more bioactivity of the crude extracts against F. oxysporum in the MIC bioassay. Based on the current results, a combination of shorter watering interval and normal light intensity favoured plant growth and development, while plants grown under low light intensity with longer watering interval showed good bioactivity. Broadly, these results demonstrated that varying pH, light intensity, and watering regime can influence plant growth, secondary metabolite contents and antifungal activity of crude extracts of T. violacea. These findings will contribute to the current body of knowledge around cultivation of indigenous medicinal plants. The study will further benefit the conservation of medicinal plant initiatives, increased income of small-scale farmers and potentially promote indigenous knowledge by increasing the availability of South African medicinal plants.
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