Johan C. Pretorius

Purification and identification of active antibacterial components in Carpobrotus edulis L.

Very little is known about the chemical composition of Carpobrotus edulis, also known as Hotnotsfig or sourfig. However, some claims have been made in the past by traditional healers, regarding its usage as a medicinal plant. In this investigation it was initially illustrated that a crude methanolic extract of the plant exhibits strong anti-bacterial activity. Subsequently, the crude extract was fractionated by means of liquid-liquid chromatography, tannins removed by means of LH20 column chromatography and bioactive fractions with antibacterial properties isolated by means of preparative thin layer chromatography. Five bioactive compounds, individually or collectively responsible for the antibacterial property of C. edulis, were purified from an active ethyl acetate fraction. These compounds were initially identified as flavanoids using standard fingerprinting methods and eventually identified as rutin, neohesperidin, hyperoside, cactichin and ferulic acid using flavanoid standards. A sixth flavanoid with antibacterial activity was also purified but could not be identified in this way. The latter is currently isolated in larger volume for identification through nuclear magnetic resonance spectroscopy.

In vitro and in vivo antifungal activity of crude extracts and powdered dry material from Ethiopian wild plants against economically important plant pathogens

In Ethiopia extracts from specific plants are used traditionally as natural fungicides in small scale farming systems where synthetic chemicals are out of reach of the average subsistence farmer while no scientific base exists for this practice. Subsequently, methanolic crude extracts from Dolichos kilimandscharicus and Maerua subcordata roots as well as Phytolacca dodecandra berries were screened in vitro for antifungal activity against Botrytis cinerea Pers.:Fr., Fusarium oxysporum Schlechtend.:Fr., Sclerotium rolfsii Sacc., Rhizoctonia solani Kühn,, Botryosphaeria dothidea (Moug.:Fr.) Ces & De Not. and Pythium ultimum Trow, using an agar diffusion method. Compared to the other plants as well as specific standard fungicides for each pathogen, the root extract of D. kilimandscharicus showed the highest broad-spectrum in vitro antifungal activity by inhibiting the mycelial growth of three of the six test organisms. Additionally, in vivo antifungal activity of dry powdered material from these plants against sorghum covered (Sporisorium sorghi) and loose kernel (Sporisorium cruentum) smuts was screened under field conditions by artificially inoculating separate sets of sorghum seed with smut spores before treating with plant material or two traditionally used fungicides, Thiram® and Apron Plus® that served as positive controls. Although the incidence of both loose and covered kernel smuts were significantly reduced by material from all plant species, the dry powdered berries of P. dodecandra were most effective. Compared to the untreated control, treatment with the plant material as well as standard fungicides resulted in significant yield increases. It was concluded that a rationale has been established for further investigation into the structured utilization of natural vegetation indigenous to Ethiopia in the agricultural industry.

Tulbaghia violacea L. I: In vitro antimicrobial properties towards plant pathogens

The antimicrobial properties of crude methanol extracts of above- and below-soil parts of Tulbaghia violacea were quantified by means of an agar diffusion method against 6 plant pathogenic bacteria and 7 fungi. The growth of 3 out of the 6 bacteria, Clavibacter michiganensis, Ralstonia solanacearum, and Xanthomonas campestris, was significantly inhibited by crude extracts of both below-soil and aerial parts of T. violacea, whereas the growth of Pseudomonas syringae, Erwinia carotovora, and Agrobacterium tumefaciens was unaffectedl. Compared with the standard fungicide, both the aerial and below-soil extracts of T. violacea significantly inhibited the mycelial growth of 6 of the 7 test fungi, Botrytis cinerea, Sclerotium rolfsii, Rhizoctonia solani, Mycosphaerella pinodes, Botryosphaeria dothidea, and P. ultimum, whereas only the below-soil extract inhibited the mycelial growth of Fusarium oxysporum significantly. The broad-spectrum antifungal activity shown by the crude T. violaceae extracts supplied a rationale for a further investigation into the in vivo activity of the extracts under glasshouse and field conditions.

Tulbaghia violacea L. II: In vivo antifungal properties towards plant pathogens

In vitro antifungal activity of crude extracts from Tulbaghia violacea against 6 economically important plant pathogenic fungi was previously reported. The in vivo control of Mycosphaerella pinodes, causative of Ascochyta blight, by different concentrations of a crude aerial part extract of T. violacea was subsequently followed qualitatively and quantitatively in terms of lesions that developed over a 6-day period at 20°C on detached pea (Pisum sativum) leaves. Infection by M. pinodes spores was prevented when the extract was applied both before and after inoculation, confirming complete inhibition of spore germination, whereas no phytotoxic effect was observed on the leaves. Additionally, the control of sorghum covered (Sporisorium sorghi) and loose (S. cruentum) kernel smuts by seed treatment with the crude extract was tested under field conditions. Before planting, different sorghum seed lots were inoculated separately with spores from the 2 pathogens at a rate of 0.5% per kg (w/w), followed by treatment with the crude extract, at a concentration of 2.0 mg/mL, 24 h later. A standard fungicide, Thiram (65 W), was applied as a positive control at a rate of 0.25% per kg (v/w). Disease incidence was quantified during harvest and expressed as percentage infected plants. Seed treatment with the extract significantly (P < 0.05) reduced the incidence of both sorghum loose and covered smut diseases, compared favourably with the standard fungicide, and resulted in significant yield increases compared to the untreated control.

Bio-stimulatory properties in seeds of plants from the families Caryophyllaceae and Fabaceae with application potential in agriculture

Abstract A comparative investigation into the bio-stimulatory activity of seed suspensions from two South African plant species belonging to each of the families Caryophyllaceae and Fabaceae were conducted using ComCat®, a commercially available bio-stimulant, as a positive control. These included Acacia erioloba and Acacia karroo from the family Fabaceae and Dianthus basuticus and Pollichia campestris from the family Caryophyllaceae. Three different bio-assays were applied to screen for possible bio-stimulatory activity in the seed suspensions. These included the effect on the respiration rate of monoculture yeast cells, germination of seeds from five vegetable crops and subsequent root growth in seedlings. The study confirmed that seed suspensions of A. eri-oloba and P . c a m p e s tr is were consistently most active in terms of all three bio-assays used to test for bio-stimulatory activity. Following liquid-solid extraction of crude extracts from these two species by using organic solvents with increasing polarity, a very small amount of active extract was recovered from P. campestris seeds. Subsequently, only the semi-purified and active ethyl acetate fraction of an A. erioloba crude extract was purified further. By means of column- and preparative thin layer chromatography the active compound was isolated, purified and identified as 2-ethyl-hexyl phthalate by means of nuclear magnetic resonance (NMR) spectroscopy. The latter was proven not to be an artefact of the extraction process. It is concluded that sufficient evidence in terms of the application potential of an A. erioloba extract in agriculture has been produced to warrant further investigation under field conditions.

The effect of soaking injury in seeds of Phaseolus vulgaris L. on germination, respiration and adenylate energy charge

Submerging Phaseolus vulgaris cv. Top Crop seed in air-saturated water for 16 h markedly depresses subsequent germination. This is termed soaking injury. The postulate by Norton (1986) that soak-injured seeds merely run out of available energy was investigated. Soaking in air-saturated water reduced the total respiration (V T ; in terms of O 2 uptake) of excised axes and cotyledons resulting in lower ATP levels. The contribution of both the mitochondrial cytochrome respiratory pathway (V CYT ) and the alternative respiratory pathway (V ALT ) to the total respiration (V T ) was significantly lower in these axes. However, the adenylate energy charge (AEC) values for both axes and cotyledons excised from soak-injured seeds did not drop below 0.6, the threshold value indicating death of plant material. Differences between the ultrastructure of mitochondria in the radicle tips of axes from unsoaked and water-soaked seeds were observed. However, the capacity to operate the Krebs cycle was similar in all axes and soak-injured seeds were still capable of germinating when the testas were removed or the seeds dried. This means that axes contained a sufficient amount of carbohydrates and potential energy to germinate after soaking. Moreover, soak-injured seeds produced ethanol at a rate which was five times higher than that of unsoaked control seeds, both during the soaking and post-soaking periods. Together with the low rate of oxygen consumption by soak-injured seeds during the same soaking and incubation time, it appears that soak-injured seeds lack the potential to switch from an anaerobic to an aerobic respiration pattern causing low ATP levels in these axes. It is speculated that the latter seems to be the result of limited pyruvate supply, probably due to accelerated and prolonged fermentation or futile combustion through other pathways or both.

The effect of soaking injury in bean seeds on protein synthesis in embryonic axes

The capacity for protein synthesis in embryonic axes of Phaseolus vulgaris L. cv. Top Crop seeds was reduced markedly by soaking in airsaturated and CO 2 -saturated water. The former treatment induces soaking injury while the latter treatment prevents injury. After soaking, the capacity for protein synthesis in axes of seeds soaked in air-saturated water declined and the pattern of synthesis remained almost similar to that at the time of termination of soaking. However, the capacity and pattern of protein synthesis in axes of seeds soaked in CO 2 -saturated water recovered subsequent to soaking and approached that of non-soaked seeds.

In vitro and in vivo bio-stimulatory properties of a Lupinus albus L. seed suspension

During an extended screening program of plant extracts for bio-stimulatory activity, a seed suspension (SS) of Lupinus albus L. cv. Betsuhana White was identified as most promising. By testing a concentration range of SS using three in vitro bio-assays [i.e. respiration rate of monoculture yeast (Saccharomyces cerevisiae) cells, germination rate of seeds from selected crops, and subsequent seedling growth], 5 mg/L was identified as the optimal concentration in terms of in vitro bio-stimulatory activity. A commercially available bio-stimulant, ComCat®, was used as a positive control, while a glucose solution for respiration tests and distilled water for germination tests served as negative controls. Compared with the controls, significant (P < 0.05) increases in the respiration rate of monoculture yeast cells and seedling root growth in the majority of test crops after treatment with SS confirmed its in vitro bio-stimulatory potential. Seedling root growth compared favourably with results obtained with the commercial bio-stimulant. Subsequently, the in vivo yield response of selected crops to foliar treatment with SS was investigated under field conditions using ComCat® as a positive control. All trials were laid out in a complete randomised block design. Foliar applications of SS at 5 mg/L under a drip irrigation system contributed to significant (P < 0.05) yield increases in lettuce (by 20.0 t/ha) and carrots (by 22.2 t/ha), which compared favourably with the positive control, but not in beetroot and cabbage. Beetroot yield was enhanced whereas cabbage yield was decreased markedly, but not significantly, by the SS treatment, compared with the negative control. The results confirmed both the in vitro and in vivo bio-stimulatory potential of a seed suspension of L. albus cv. Betsuhana White in terms of its ability to manipulate early seedling growth and enhance the yield of selected vegetable crops.

A triglyceride from Lupinus albus L. seed with bio- stimulatory properties

In vitro and in vivo bio-stimulatory activity of a Lupinus albus cv. Bethuana white Seed Suspension (SS) was previously documented using vegetable crops. This was repeated for two grain crops, maize and wheat, in an attempt to provide a rationale for the isolation and identification of the active compound(s) involved. In all cases, besides negative controls, the response to treatment with SS was compared to that of a commercial bio-stimulant, ComCat ® . At an optimum concentration of 5 mg l -1 SS significantly enhanced seedling growth and yield in both crops. Subsequently, activity directed liquid-liquid extraction of ground L. albus seed resulted in an ethyl acetate extract containing most of the biostimulatory activity. Two in vitro bio-assay procedures were employed to appraise the in vitro biostimulatory properties of semi-purified fractions as well as pure compounds contained in it. The highly active ethyl acetate extract was fractionated further by means of adsorption column chromatography resulting in eighty five fractions that were combined into twelve on the basis of similarities in thin layer chromatography profiles. Four of these fractions showed significant bio-stimulatory activity and were pooled. From this one compound was isolated, purified and identified as glyceryl trilinoleate by means of nuclear magnetic resonance spectroscopy.

A TRIGLYCERIDE FROM LUPINUS ALBUS L. SEED WITH BIO-STIMULATORY PROPERTIES

In vitro and in vivo bio-stimulatory activity of a Lupinus albus cv. Bethuana white Seed Suspension (SS) was previously documented using vegetable crops. This was repeated for two grain crops, maize and wheat, in an attempt to provide a rationale for the isolation and identification of the active compound(s) involved. In all cases, besides negative controls, the response to treatment with SS was compared to that of a commercial bio-stimulant, ComCat ® . At an optimum concentration of 5 mg l -1 SS significantly enhanced seedling growth and yield in both crops. Subsequently, activity directed liquid-liquid extraction of ground L. albus seed resulted in an ethyl acetate extract containing most of the biostimulatory activity. Two in vitro bio-assay procedures were employed to appraise the in vitro biostimulatory properties of semi-purified fractions as well as pure compounds contained in it. The highly active ethyl acetate extract was fractionated further by means of adsorption column chromatography resulting in eighty five fractions that were combined into twelve on the basis of similarities in thin layer chromatography profiles. Four of these fractions showed significant bio-stimulatory activity and were pooled. From this one compound was isolated, purified and identified as glyceryl trilinoleate by means of nuclear magnetic resonance spectroscopy.