A significant increase in both leaf and plant artemisinin was observed among plants that were maintained under water deficit compared to well-watered plants . However, Selmar and Kleinw€achter argued that since drought stress also reduces growth and biomass production in most plants, drought stress-related increase in natural product concentrations does not mean that the rate of biosynthesis of natural products in the plants has increased. Another important and well-studied factor that influences the morphological and physiological processes in plants is light . When plants are exposed to low light intensity, they tend to have elongated leaves, and increased leaf surface area and plant height.High light intensity can induce plants to produce high starch and carbohydrate contents, which contribute positively to their biomass . Baligar et al. reported that in legumes, growth, nutrient uptake and use-efficiency ratios were higher at higher Photosynthetic Photon Flux Density than at lower PPFD. However, when plants are simultaneously exposed to more than one stress factor, the responses are more complex. For instance, it was observed that shading alleviated the negative impact of drought on leaf traits and biomass characteristics of Acer buergerianum Miq . On the other hand, there was no interactive effect between light and water treatments on biomass accumulation in Quercus suber L. seedlings . Holmgren argued that shading could reduce the impact of drought by limiting loss of water in soil during evaporation.
Puertolas et al. and Quero et al. described three hypotheses to predict the possible responses of plants to the interactive effects of water stress and light availability. These are trade-off hypothesis — plants that are adapted to deep shade may adapt relatively poorly to drought than other plants growing under higher light levels, hydroponic nft system facilitation hypothesis — shade enhances survival and physiological status of plants by decreasing evaporative demands and radiation loads, and orthogonal hypothesis — the combined effects of shade and water-shortage are independent, and their impacts are orthogonal. Puertolas et al. further argued that interactive responses are influenced by plant species, the intensity of water stress, the range of light intensities, the traits considered and seedling age or environmental conditions. Currently, however, reports that address the interaction of light and water stresses on bioactivity of medicinal plant extracts are scarce. Medicinal plants are an important source and inspiration for discovery of new products for drug development . Consequently, many research activities have focused on the manipulation of these secondary metabolites in plants and yields of medicinal materials in order to meet the demands of the pharmaceutical industry, traditional healers and the cosmetics industry . In South Africa, T. violacea bulbs and leaves are traditionally used for treatments of gastrointestinal ailments, asthma, fever and tuberculosis; the leaves are used to treat cancer of the oesophagus . Previously, crude extracts from T. violacea showed good antimicrobial activities against bacterial strains . T. violacea has been shown to have similar antibacterial and anti-fungal activities as Allium sativum . T. violacea is rich in sulphur-containing compounds including thiosulfinate marasmicin which exhibit significant antimicrobial activities . Kubec et al. isolated R– S- cysteine-4-oxide from the rhizomes of T. violacea. The sulphur compounds in T. violacea are unstable and J€ager and Stafford reported that grinding the rhizome material in liquid nitrogen and extraction with ethanol yielded the best results and the sulphur compounds in the rhizomes decreased rapidly upon storage, after harvest.
Methyl alpha-D-glucopyranoside, a bio-active compound that can selectively kill cancer cells was successfully isolated from T. violacea using apoptosis-guided purification.Tulbaghia violacea is regularly harvested from the wild by traditional healers, a practice that may cause decline of the species’ populations in the wild . The persistent high demand might eventually place T. violacea at risk of extinction . Hence, there is a need to develop optimum cultivation protocols that will ensure improved crop yield and quality of medicinal materials. Plant growth parameters, such as dry and fresh weights, plant height, and anti-fungal activity of plant extracts are useful indicators of yield and quality of medicinal materials. Since ambient environmental conditions during cultivation can influence plant physiology, plant health and crop yield, a greenhouse is a perfect facility for manipulating most exogenous factors like humidity, light, temperature and water. Furthermore, growing plants in greenhouses could help circumvent many challenges, such as land availability, water availability, season, climate, pests and diseases, which are major concerns with conventional cultivation of indigenous plant species . The objective of this study was to assess the individual and interactive effects of light intensity and watering regime on plant growth, nutrient uptake and anti-fungal activity of extracts of T. violacea plants, grown hydroponically.Data of the different plant growth parameters were recorded at the end of the experiment. The height of the plant was recorded at two months post treatment using a measuring tape. The number of leaves was enumerated at two months post-treatment. At the end of the experiment, plants were harvested and fresh weight was immediately measured. In order to determine the dry weight, harvested plants were placed separately in paper bags and dried in a thermo-oven at 70 C, and the dried plant samples were weighed.The anti-fungal activity was evaluated using the minimum inhibitory concentration value obtained in a microdilution assay. Fusarium oxysporum f. sp. glycines strain obtained by courtesy of the Phytomedicine Programme, University of Pretoria, South Africa was used as the pathogenic agent in the bioassay. The F. oxysporum strain was subcultured from stock agar plates and grown into nutrient broth for 4 h.
The concentration of fungal spores in the nutrient broth was determined using a haemocytometer. One hundred microlitres of solution containing crude acetone extracts of plant roots was serially diluted with sterile distilled water in 96-well microplates . The fungal suspension was added to each well of a 96- well microplate . Forty micro litre of 0.2 mg ml-1 of piodonitrotetrazolium chloride dissolved in sterile distilled water was added to each microplate well, sealed in a plastic bag and incubated at 37 C and 100% RH. Acetone was used as a negative control. The MIC values were recorded after 6, 12 and 18 h. There were three replicates per treatment and per watering interval. The MIC value and the weight of the extract obtained following acetone extraction were used to determine the Total Activity . The unit of TA is ml g-1 and it indicates the degree to which the active compounds in one g of plant materials can be diluted and still inhibit the growth of the tested microorganisms .The watering regime significantly affected the growth parameters as leaf number, fresh and dry weights, and plant height reduced with increasing watering intervals. Generally, the shorter watering interval had higher fresh and dry mean weights compared to plants exposed to the longer watering intervals. These results are consistent with the findings of Xego et al. , which showed that more abundant growth in Siphonochilus aethiopicus correlated with shorter watering intervals. In another study, water deficit due to long watering intervals had significant negative effects on plant height, leaf number, and induced a higher biomass of adventitious and tap roots of mango . Interestingly, the positive effects of a shortened watering interval on growth and biomass observed in this study correlated well with the increased tissue macro-nutrients contents. This observation provides a plausible explanation of the mechanism through which watering interval can influence growth of plants. Higher tissue nitrogen content increases plant growth rates and shifts plant biomass partitioning to above ground structures . On the other hand, a decrease in water availability can reduce nutrient uptake, transportation and availability . Nitrogen availability and the internal N status of plants correlate positively with shoot∶root ratios . Potassium and phosphorus are other essential macro-nutrients that affect physiological processes and influence plant growth and metabolism .
Variable effects of shading on growth parameters, such as plant height, leaf number and dry and fresh weights were recorded in this study. The plants grown under the 40% shade produced significantly higher mean height and lower fresh and dry weights of aerial parts than those grown in the 0% shade treatment and short watering intervals . However, under the longest watering interval and 40% shading, higher number of leaves and higher dry weights were recorded when compared to 0% shading. These results are in agreement with that of Zervoudakis et al. on Salvia officinalis L., which showed that dry mass, number of leaves and physiological parameters had a strong positive correlation with the light intensity, and plant’s height and leaf photosynthetic pigments were increased among low light treated plants. Fiorucci and Fankhauser postulated that low photosynthetically active radiation can induce pronounced phenotypic responses in some species, such as elongation of stem-like structures, elevation of leaves, as well as reduced branching and acceleration of flowering. Under a short-term low irradiance , chlorophyll b transiently increased in Brassica campestris, but extension of shading time to a 15-day period led to significant decreases in relative chlorophyll a and anthocyanin . There are few studies done on the interactive effects of low light intensity and limited water on plant growth and secondary metabolite synthesis . In the present study, plants subjected to both the 21-day watering interval and low light intensity produced more leaves than those grown under the higher light intensity and equivalent watering interval. Research done by Sack et al. reported that shading could mitigate the negative impact of water stress. Under limited light, nft channel plants may accumulate carbohydrates in leaves; these soluble sugars may reduce water loss through turgor maintenance and reduction of stomatal aperture . L€ of et al. reported an interaction between irradiance and water stress on biomass partitioning in Fagus sylvatica seedlings. Yang et al. argued that plants in shade invest more to produce shoots and leaves than biomass. Broadly, these studies corroborate our finding that watering regime and light intensity have intercative effects on plant growth parameters. Hazrati et al. reported that about 50% of total solar radiation and irrigation after depleting 40% of soil water content were the most efficient treatments for chlorophyll fluorescence and pigments of Aloe vera L. A key finding in this study is that shading alleviated the negative effects of water deficit stress on plant growth. This is in agreement with the report of Guo et al. , in which drought alleviated shading effects on Acer buergerianum Miq., and the above ground facilitation hypothesis .
Nevertheless, it is worth mentioning that the responses of plants to water and light stress also depend on species. For example, Liu and Su reported that under low light, Taxus yunnanensis produced larger leaves and a higher shoot axis length per unit dry mass under high light, whereas the leaf size and biomass yield of T. chinensis were not sensitive to light. Secondary metabolites play an important role in plant defence. They protect plants against pathogens and herbivory . Drought stress can induce plants to produce higher concentrations of secondary metabolites. In this study, although there were no significant differences in the MIC values between plants in low and high light intensities, acetone bulbous root extracts of the plants that were exposed to the longest watering interval and 40% shading yielded the highest total activity. The increase in total activity suggests that there was an interaction between watering interval and light intensity in relation to the yield of acetone extract during extraction. Hazrati et al. reported that conditions of irradiance of full sunlight and water deficit stress favoured increased anthocyanin production in Aloe vera. In conclusion, broadly, three trends occurred in the results. Firstly, the total weight of T. violacea increased with shorter watering intervals under high irradiance. Secondly, shading alleviated the negative effect of water deficit stress on plant growth. Thirdly, the longest watering interval plants had the highest total activity of bulbous root extracts. Furthermore, light intensity and watering interval had significant interactive effects on anti-fungal activity and plant growth. These results also suggested that nutrient supply and subsequent tissue nutrient levels might be modulating the responses, such as plant growth and biomass, and anti-fungal activity of plant extracts in relation to light intensity and watering regime. Future studies should investigate the interactive effects of water deficit and shading on production of bio-active compounds in T. violacea.