South Africa has offered the world two indigenous aromatic plants from which commercially important natural products have been developed: Pelargonium graveolens (and its hybrids) the source of geranium oil and Agathosma betulina, from which ‘Buchu’ oil is produced. Despite the historical use of ‘Buchu’ and the commercial interest developed around this coveted indigenous resource the (limited) research has not been coherently assembled. This overview aims to unite aspects on the botany, traditional and modern day uses, chemistry and pharmacological data on ‘Buchu’ which is undeniably one of South Africa’s most renowned botanical assets.

The antimicrobial, anti-oxidant and cytotoxic activities of the extracts obtained from 17 indigenous Agathosma species (19 samples) were investigated in order to validate the historic and continued use of Agathosma species in traditional healing. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) method on four pathogens, i.e. Staphylococcus aureus (ATCC 12600), Bacillus cereus (ATCC 11778), Klebsiella pneumoniae (NCTC 9633) and Candida albicans (ATCC 10231). The anti-oxidant activity was evaluated using the 2,2- diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, while the cytotoxic properties was determined using the MTT (3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide) cellular viability assay. Agathosma ovata (round-leaf) displayed the best activity against S. aureus and B. cereus with MIC values of 0.16 mg/ml and 0.13 mg/ml, respectively. Most of the extracts had moderate to poor activity in the DPPH assay with the exception of A. capensis (Gamka) and A. pubigera which were the two most active species in the assay (IC50 values of 24.08±4.42 μg/ml and 35.61±0.86 μg/ml). The results obtained from the ABTS assay differed from that of the DPPH assay. All extracts showed greater activity in the ABTS assay with A. namaquensis and A. capensis (Besemfontein) being the most active species (IC50 values of 15.66±4.57 μg/ml and 19.84±0.09 μg/ml). Agathosma lanata (IC50 value of 26.17±9.58 μg/ml) and A. ovata (round-leaf) (IC50 value of 25.20±6.30 μg/ml) proved to be the most toxic in the MTT assay. Agathosma arida, A. collina, A. hirsuta, A. pubigera, A. roodebergensis, A. stipitata and A. zwartbergense also displayed some degree of toxicity.

Abstract Few in vitro screening assays for biological activities of plant extracts consider the potential effect of the gastrointestinal system on orally consumed plant extracts. Crude water and methanol extracts of Tarchonanthus camphoratus (wild camphor) and Agathosma betulina (‘buchu’) were prepared and exposed to simulated gastric fluid and simulated intestinal fluid during dissolution studies to address this aspect. The crude extracts and resulting simulated gastric fluid and simulated intestinal fluid products were screened for antimicrobial activity against Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Proteus vulgaris (ATCC 33420). The T. camphoratus crude extract exhibited antimicrobial activity which was reduced after exposure to simulated gastric fluid. After exposure to simulated intestinal fluid no antimicrobial activity was detected, which suggests chemical alteration or degradation of the active compounds. For A. betulina, the crude water extract and simulated gastric fluid product exhibited no antimicrobial activity, while the simulated intestinal fluid product exhibited antimicrobial activity. This suggests activation of antimicrobial constituents during exposure to simulated intestinal fluid. The chemical composition profiles of the crude extracts and products were determined by means of liquid chromatography coupled to an ultraviolet detector (LC-UV) and a mass spectrometer (LC-MS) to qualitatively assess the effect of exposure to simulated gastrointestinal conditions on the chemical composition of the extracts. In many cases, the peak area of compounds decreased after exposure to simulated gastric fluid and simulated intestinal fluid, while the peak area of other compounds increased. Thus, it can be deduced that the antimicrobial activity and chemical composition was altered after exposure to intestinal conditions during dissolution studies.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy in Microbiology at the University of Stellenbosch. / ENGLISH ABSTRACT: The interaction between a soil yeast, Cryptococcus laurentii and a medicinal plant, Agathosma betulina (Berg.) Pillans (Rutaceae), was studied. Cryptococcus laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina and liquid chromatographytandem mass spectrometry analysis revealed that the yeast was capable of producing polyamines such as cadaverine and spermine. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when A. betulina seedlings grown under axenic and low nutrient conditions were inoculated with C. laurentii CAB 578 and cultivated for five months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. Furthermore, fluoromicroscopy, in combination with the fluorescent probes Calcofluor White and Fungolight revealed the presence of metabolic active yeast colonies on the rhizoplane. The first part of the study thus provided evidence for a symbiosis between A. betulina and C. laurentii CAB 578. During the second part of the investigation, the effect of this symbiosis on quantitative elemental distribution in A. betulina roots grown under axenic, nutrient-poor conditions was assessed using micro-particle-induced x-ray emission spectrometry. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers and endophytic C. laurentii CAB 578 in root tissues were located using fluorescence microscopy and transmission electron microscopy, respectively. The results showed that the average concentrations of iron, manganese and phosphorus were significantly (P < 0.05) higher within roots of yeast-inoculated plants, compared to control plants. It was shown that the yeast was not a root endophyte and that elemental enrichment in the epi/exodermal-outer cortical tissues correlated with the presence of Casparian bands in the exodermal cells of both treatments. This was the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont. In the final part of the investigation, the effect of C. laurentii CAB 578 on the photosynthetic nitrogen, phosphorus and water-use efficiencies, as well as the carbon economy of A. betulina was studied. Agathosma betulina plants inoculated with C. laurentii CAB 578, as well as controls, were grown under axenic conditions and the following parameters measured: Apparent photon yield, foliar nitrogen and phosphorus concentrations, leaf dark respiration, maximum photosynthetic rate, photosynthetic nitrogen-use efficiency, photosynthetic phosphorus-use efficiency, photosynthetic wateruse efficiency, root construction cost, stomatal conductance, substomatal CO2 and transpiration rate. The data showed that the higher photosynthetic resource-use efficiencies in yeast-inoculated plants were a consequence of higher maximum rates of CO2 assimilation, which was not related to foliar nitrogen and phosphorus content. We hypothesize that photosynthetic stimulation in yeast-inoculated plants was a result of the increased demand for photosynthates of the yeast-root symbiosis. In summary, the study revealed that a symbiosis exists between A. betulina and the soil yeast C. laurentii CAB 578. This interaction has a significant effect on the size of the yeast population as well as on the physiology of the plant. / AFRIKAANSE OPSOMMING: Die interaksie tussen ‘n grondgis, Cryptococcus laurentii, en ‘n medisinale plant, Agathosma betulina (Berg.) Pillans, is ondersoek. Cryptococcus laurentii CAB 578 is vanuit die risosfeer van A. betulina in sy natuurlike omgewing geisoleer en vloeistof chromatografie tandem massa spektrofotometriese analise het bewys dat die gis poliamiene insluitend kadaverien en spermien produseer. Dit is bevind dat die eksogene aanwending van poli-amiene wortelgroei bevorder. Hierdie bevinding staaf die waargenome 51% verhoging in wortelgroei van mikroob-vrye A. betulina saailinge geinokuleer met C. laurentii CAB 578 en gekweek vir vyf maande onder lae nutriënt kondisies in ‘n glashuis. Met gebruik van die grond verdunningsplaat-metode, is dit verder bewys dat gisgetalle hoër was in die teenwoordigheid van wortels as in die omliggende grond. Dit is ook bewys met die gebruik van die fluoressente peilers Calcofluor White en Fungolight, in kombinasie met fluoressensie-mikroskopie, dat metabolies aktiewe giste die wortels se oppervlak gekoloniseer het. Die eerste deel van die studie het dus bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Tydens die tweede deel van die ondersoek is die effek van C. laurentii CAB 578 op die konsentrasie en verspreiding van elemente binne A. betulina wortels, gekweek onder lae-nutriënt, mikroob-vrye kondisies, bepaal met behulp van mikro-partikel geinduseerde X-straal emissie spektrofotometrie. Om die interpretasie van heterogene verspreidingspatrone van die onderskeie elemente te ondersteun, is daar met behulp van fluoressensie en transmissie-elektron-mikroskopie vir apoplastiese versperrings en endofitiese C. laurentii CAB 578 in die wortelweefsel getoets. Dit is bevind dat die gemiddelde konsentrasie van fosfaat, mangaan en yster beduidend (P < 0.05) hoër was in gis-geinokuleerde plante, as in kontrole plante. Die gis was nie ‘n wortel endofiet nie en elementale verryking in die epi/eksodermale-buitenste korteks weefsels het gekorreleer met Casparian bande in die eksodermale selle van beide behandelings. Hierdie was die eerste verslag wat die rol van ‘n grondgis as ‘n nutriënt-bekommende mikrosimbiont vir plante beskryf het. In die laaste gedeelte van hierdie ondersoek is die effek van C. laurentii CAB 578 op die fotosintetiese fosfaat, stikstof en water-verbruiksdoeltreffendheid, asook die koolstof ekonomie in mikroob-vrye Agathosma betulina plante geinokuleer met C. laurentii CAB 578 asook kontrole plante bestudeer. Die volgende parameters is getoets: Blaar donker respirasie, blaar fosfaat en stikstof konsentrasies, fotosintetiese fosfaatverbruiksdoeltreffendheid, fotosintetiese stikstof-verbruiksdoeltreffendheid, fotosintetiese water-verbruiksdoeltreffendheid, huidmond konduktansie, huidmond CO2 konsentrasie, klaarblyklike foton opbrengs, maksimum fotosintetiese spoed, wortel konstruksie-koste, en transpirasie spoed. Die resultate het getoon dat die hoër maksimum fotosintestiese spoed in gis-geinokuleerde plante gelei het tot ‘n hoër fotosintetiese verbruiksdoeltreffendheid van fosfaat, stikstof en water en dat dit nie verband gehou het met blaar fosfaat en stikstof konsentrasies nie. Dit word voorgestel dat die stimulasie van fotosintese in gisgeinokuleerde plante ‘n gevolg is van die verhoogde aanvraag na fotosintaat deur die giswortel simbiose. Om op te som, die bevindings van hierdie studie het bewys dat ‘n simbiose tussen A. betulina en C. laurentii CAB 578 bestaan. Hierdie simbiose het ‘n beduidende effek op die populasie grootte van die gis sowel as die fisiologie van die plant.

Faculty of Sciences School of Pharmacy and Pharmacology 0000073k moollaaneesa@yahoo.com / As part of an investigation of the biological activities of South African plants and due to their extensive traditional use and lack of scientific evidence, a phytochemical and pharmacological investigation was performed on 17 indigenous Agathosma species (19 samples). The chemical composition of the essential oils was determined using gas chromatography coupled to mass spectroscopy (GC-MS). Analysis resulted in the identification of 333 compounds. To evaluate the chemical similarities and differences, cluster analysis was used to assess the essential oil composition of the samples. The results showed qualitative and quantitative differences amongst the taxa. The essential oils of Agathosma hirsuta and A. zwartbergense are particularly rich in citronellal, hence they are tightly clustered in the dendrogram obtained from the cluster analysis. Linalool, myrcene and limonene are the major constituents of both A. capensis (Gamka) and A. capensis (Besemfontein). Qualitative and quantitative differences are noted in the chemical compositions of the leaf oils of Agathosma capensis (Gamka) and A. capensis (Besemfontein). Agathosma arida and A. lanata are united in a single cluster due to the compounds β-pinene, linalool and spathulenol being major components in both species. The presence of 1,8-cineole in large quantities in both Agathosma namaquensis (23.5%) and A. ovalifolia (9.7%), unites them in a single cluster. A wide chemical variability for the essential oils of indigenous Agathosma species has been demonstrated. There was considerable variation in the percentage oil yield of the essential oils. Agathosma hirsuta produced the highest yield (1.15%) whilst A. ovalifolia produced the lowest yield (0.16%). vi Previous studies have revealed that the coumarin and flavonoid components of Agathosma species are responsible for their biological activities. High performance liquid chromatography (HPLC) was used to document the non-volatile composition of Agathosma species and to establish if phenolic patterns were present amongst the species. All species were found to be rich in flavonoids (i.e. flavones and flavonols). Many of the compounds detected were common to most of the species. A pure coumarin, puberulin, was identified in the diethyl ether extract of Agathosma ovata (round-leaf) and detected in the dichloromethane and methanol (1:1) extract of A. namaquensis. Agathosma species have been used traditionally to treat a wide variety of infections. They has been used as a cough remedy, for the treatment of colds and flu, kidney and urinary tract infections, for the treatment of cholera and other stomach ailments. Based on the extensive use and lack of scientific evidence, a study was embarked upon to determine its bioactivity. Using the disc diffusion assay as a preliminary screening and thereafter the minimum inhibitory concentration (MIC) assay, the antimicrobial activity of the essential oils and non-volatile compounds was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gram-negative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. All of the extracts proved to be active against the four pathogens tested with the exception of Agathosma bathii which showed poor activity against Klebsiella pneumoniae (MIC value of 32mg/ml). The extracts exhibited stronger activity against the pathogens as compared to the essential oils. Both the essential oils and extracts exhibited higher activity towards the Gram-positive bacteria than the Gram-negative bacterium, with the extract of Agathosma ovata (round-leaf) displaying the greatest vii activity against Staphylococcus aureus (MIC value of 0.156mg/ml) and Bacillus cereus (MIC value of 0.125mg/ml). The extract of Agathosma parva displayed the greatest activity against Candida albicans and Klebsiella pneumoniae (MIC value of 1.5mg/ml). Amongst the essential oils, Agathosma pungens proved to be the most active against the Gram-positive pathogen, Bacillus cereus (MIC value of 3mg/ml). Agathosma collina was the most active against Candida albicans (MIC value of 3mg/ml) whilst A. zwartbergense proved to be the least active against most of the tested pathogens. The antimicrobial activity of the essential oils may be ascribed to oxygenated constituents, such as 1,8-cineole, linalool and carvacrol. The activity of the extracts may be ascribed to constituents such as flavonoids, coumarins and alkaloids. Due to the availability and accessibility of Agathosma ovata, a seasonal variation study was performed on the chemical composition of the essential oils and how this may impact on the antimicrobial activity. Furthermore, this species has recently been earmarked for commercial development by the flavour and fragrance industry and information on variability is required to establish the harvesting protocol. Ten samples were harvested in total. There was a substantial variation in the oil yield throughout the year, ranging from 0.23% in early Spring to 0.85% in late Autumn. A higher yield was observed during the flowering season as compared to the non-flowering season. Oil yields were low during Summer (0.44%-0.48%) which may have been due to the low oil content in stems and higher proportion of stems after flowering. The proportion of oil-rich green leaves also decreased markedly, hence affecting the yield. Overall the yields were dependant on the season harvested and proportion of plant parts distilled. viii The chemical composition of the essential oils was determined using GC-MS and resulted in the identification of 145 compounds in 10 of the samples. All samples contained a large number of common monoterpenes and had very similar compositions, with minor quantitative variation. Some components common to all samples include: sabinene, p-cymene, β-pinene, α-pinene, α-thujene, myrcene, limonene, linalool and terpinen-4-ol. Sabinene was found to be the most dominant component in all samples, ranging between 25.6% and 44.4%. Myrcene levels dropped sharply between the beginning of Spring and end of Summer, from 14.9% to 1.0%. β-pinene followed a similar trend, peaking during Spring and decreasing during the Summer months. The lowest levels of linalool (4.3%), myrcene (1.0%), β-pinene (3.9%), limonene (1.9%) and sabinene (25.6%), occurred during the Summer months when the temperatures were high. There was a Springtime increase in the levels of β- pinene, terpinen-4-ol, linalool, sabinene, limonene and p-cymene in the non-flowering Agathosma ovata. These changes may have been due to the higher proportion of young leaves during Spring, which may have oil compositions slightly different to those of mature leaves. A rare thiol derivative (tr) that could not be identified was detected in the March sample. Many of the changes were associated with flowering and the results obtained reveal that the chemical composition of the essential oil of Agathosma ovata is subject to seasonal variation. Using the MIC assay, the antimicrobial activity of the essential oils was assessed on two Gram-positive bacteria, Staphylococcus aureus and Bacillus cereus, one Gramnegative bacterium, Klebsiella pneumoniae, and one yeast, Candida albicans. The study demonstrated differences in the potency of antimicrobial activity of the essential oils distilled each month. The Winter samples were more active against Bacillus cereus, Staphylococcus aureus and Klebsiella pneumoniae. Activity in mid Spring ix was greater against Staphylococcus aureus (MIC value of 3mg/ml) and Klebsiella pneumoniae (MIC value of 3mg/ml), whilst activity decreased in Summer. There was a correlation between the concentrations of the active compounds each month and the oils antimicrobial activity. The results reveal that the antimicrobial activity of the essential oil of Agathosma ovata may not depend on the level of one component but rather the ratio of several components. ‘Buchu’ has been used traditionally as a general tonic and medicine. Tonics generally have a high anti-oxidant content in order to promote the overall well-being of the user. The anti-oxidant properties of the essential oils and non-volatile compounds was investigated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Only the non-volatile compounds exhibited activity. Their activities may be ascribed to the flavonoid components. Most of the species portrayed moderate to poor activity in the DPPH assay with the exception of Agathosma capensis (Gamka) (IC50 value of 24.08 + 4.42μg/ml) and A. pubigera (IC50 value of 35.61 + 0.86μg/ml) which were two of the most active species, although their activities were inferior when compared to vitamin C. The results from the ABTS assay differed from that of the DPPH assay. All extracts showed greater activity in this assay with Agathosma namaquensis (IC50 value of 15.66 ± 4.57μg/ml) and A. capensis (Besemfontein) (IC50 value of 19.84 ± 0.09μg/ml) being the most active species. This may be due to the ABTS assay having an additional reaction system. ‘Buchu’ has been used traditionally as an antipyretic, topically for the treatment of burns and wounds and for the relief of rheumatism, gout and bruises. The antix inflammatory activity of the essential oils and non-volatile compounds was assessed using the 5-lipoxygenase (LOX) assay. Only the essential oils exhibited activity. All proved to be active with the exception of Agathosma stipitata which was UV active and caused interference. This was due to its major compounds neral (39.9%) and geranial (10.1%) which absorbed strongly at 234 nm and hence rendered its spectrophotometric measurement impossible. The essential oil of Agathosma collina displayed the most promising activity (IC50 value of 25.98 ± 1.83μg/ml). It is well known that many herbal medicines can have adverse effects, in which case it is necessary to evaluate the benefit-risk profile. The toxic effects of Agathosma species have been poorly studied and no information is available in this regard. Hence the toxicity profile of the non-volatile compounds and essential oils was assessed on transformed human kidney epithelium (Graham) cells using the microculture tetrazolium (MTT) cellular viability assay. The extracts of Agathosma lanata (IC50 value of 26.17 ± 9.58μg/ml) and A. ovata (round-leaf) (IC50 value of 25.20 ± 6.30μg/ml) proved to be the most toxic, whilst the extracts of Agathosma bathii, A. capensis (Besemfontein), A. betulina, A. crenulata and A. namaquensis did not prove to be toxic at the concentrations tested. Serial dilutions displayed different inhibitions of cell growth and the species proved to be toxic in a dose-dependant manner. The essential oils of all 19 species proved to be much more toxic (IC50 values < 0.0001μg/ml) than a plant-derived compound that is considered relatively safe, namely quinine (IC50 value of 136.06 ± 4.06μg/ml). The toxicities of the essential oils may be due to compounds like methyl chavicol, eugenol, methyl eugenol, pulegone and methyl salicylate whilst the toxicities of the extracts may be due to the alkaloid and coumarin components.

Magister Scientiae Dentium - MSc(Dent) / Candida albicans is a clinical fungal isolate that is most frequently isolated from different host niches, and is implicated in the pathogenesis of several fungal infections, including oral candidiasis. The pathogenesis and antifungal resistance mechanisms of Candida species are complex and involve several pathways and genes. Oral candidiasis incidence rates are rapidly increasing, and the increase in resistance to conventional antifungals has led to the need to develop innocuous and more efficacious treatment modalities. The purpose of this study was to explore a single pot process for phytosynthesis of zinc oxide nanoparticles (GZnO NPs) and to assess their antifungal potential.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The Mountain Fynbos biome, a division of the Cape Floristic Region (CFR), is home to round-leafed Buchu [Agathosma betulina (Berg.) Pillans], one of South Africa’s best-known endangered herbal medicinal plants. Agathosma betulina is renowned as a traditional additive to brandy or tea, which is used for the treatment of a myriad of ailments. In its natural habitat, A. betulina thrives on mountain slopes in acid and highly leached gravelly soils, with a low base saturation and low concentrations of organic matter. To adapt to such adverse conditions, these plants have formed mutualistic symbioses with arbuscular mycorrhizal (AM) fungi. In this study, the effect of indigenous AM taxa on the physiology of A. betulina is investigated. In addition, the AM taxa responsible for these physiological responses in the plant were identified using morphological and molecular techniques. Agathosma betulina was grown under glasshouse conditions in its native rhizosphere soil containing a mixed population of AM fungi. Control plants, grown in the absence of AM fungi, were included in the experimentation. In a time-course study, relative growth rate (RGR), phosphorus (P)-uptake, P utilization cost, and carbon (C)-economy of the AM symbiosis were calculated. The data showed that the initial stages of growth were characterized by a progressive increase in AM colonization. This resulted in an enhanced P-uptake in relation to non-AM plants once the symbiosis was established. Consequently, the lower P utilization cost in AM plants indicated that these plants were more efficient in acquiring P than non-AM plants. When colonization levels peaked, AM plants had consistently higher growth respiration. This indicated that the symbiosis was resulting in a C-cost to the host plant, characterized by a lower RGR in AM plants compared to non-AM plants. Arbuscular mycorrhizal colonization decreased with increasing plant age that coincided with a decline in P-uptake and growth respiration, along with increases in RGR to a level equal to non-AM plants. Consequently, the AM benefit was only observed during the initial stages of growth. In order to identify the AM fungi in planta, morphological and molecular techniques were employed, which indicated colonization by AM fungi belonging to the genera Acaulospora and Glomus. Phylogenetic analyses of a dataset containing aligned 5.8S ribosomal RNA gene sequences from all families within the Glomeromycota, including sequences obtained during the study, supported the above mentioned identification. / AFRIKAANSE OPSOMMING: Die Fynbos bergbioom, ‘n onderafdeling van die Kaapse Floristiese Streek, huisves rondeblaar Boegoe [Agathosma betulina (Berg.) Pillans], een van Suid Afrika se bekendste bedreigde medisinale plante. Agathosma betulina is bekend vir sy gebruik as tinktuur vir die behandeling van verskeie kwale. Die plant kom voor in bergagtige streke, in suur en mineraal-arm grond, met ‘n lae organiese inhoud. Gevolglik, om aan te pas by hierdie ongunstige kondisies, vorm die plante simbiotiese assosiasies met blaasagtige, struikvormige mikorrisa (BSM). In die huidige studie is die effek van hierdie BSM op die fisiologie van A. betulina ondersoek. Die identiteit van die BSM is ook gevolglik met morfologiese en molekulêre identifikasie tegnieke bepaal. Agathosma betulina plante is onder glashuis kondisies in hul natuurlike grond gekweek, wat ‘n natuurlike populasie van BSM bevat het. Kontroles is ook in die eksperiment ingesluit en hierdie stel plante is met geen BSM geïnokuleer nie. Gevolglik is die relatiewe groeitempo, fosfor opname, fosfor verbuikerskoste asook die koolstof ekonomie van die plante bereken. Die data het getoon dat die eerste groeifase gekarakteriseer is deur toenames in BSM kolonisasie vlakke. Dit het tot ‘n hoër fosfor opname in BSM geïnokuleerde plante gelei. Die laer fosfor verbuikerskoste gedurende hierdie fase het aangedui dat die plante wat geïnokuleer is met BSM oor beter meganismes beskik het om fosfor uit die grond te bekom. Toe BSM kolonisasie vlakke gepiek het, was groei respirasie hoër in BSM geïnokuleerde plante as in die kontroles. Dit het aangedui dat die BSM kolonisasie van plante tot hoër koolstof kostes vir hierdie plante gelei het, wat weerspieël is in die laer groeitempo van die BSM geïnokuleerde plante. Die BSM kolonisasie vlakke het gedaal met toenemende ouderdom van hul gasheer plante, wat gekarakteriseer is deur ‘n laer opname van fosfor en laer groei respirasie, tesame met ‘n toename in relatiewe groeitempo tot vlakke soortgelyk aan die van die kontrole plante. Die BSM voordele vir die plant is dus net gedurende die eerste groeifase waargeneem. Die BSM wat verantwoordelik is vir hierdie fisiologiese veranderinge is gevolglik geïdentifiseer met behulp van morfologiese en molekulêre tegnieke en dit is gevind dat BSM wat behoort tot die genera Acaulospora en Glomus binne hierdie plante voorkom. Filogenetiese analise gegrond op opgelynde 5.8S ribosomale RNA geen volgordes afkomstig van al die families binne Glomeromycota asook volgordes gevind in die studie, het die bogenoemde identifikasie gestaaf.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / An increase in the demand of buchu (Agathosma betulina) oil has lead to an increase in the commercial cultivation of buchu in fields and also in hydroponic systems. A nutrient solution for hydroponically grown buchu is still required to ensure optimal growth and yield. ASNAPP (Agribusiness in Sustainable Natural African Plant Products) South Africa has done some trials to achieve optimal EC and pH in the nutrient solution. Phosphate concentrations in the nutrient solution might play a significant role due to reports by a variety of researchers on the sensitivity of Protea plants to phosphate. Buchu and Proteas are both part of the Fynbos biome and are found in regions with similar soil (sandy soils with a low pH and mineral contents) and climatic conditions. Two separate experiments were conducted to determine the effect of increasing phosphate concentrations (ranging from 0.00 to 1.40 me L-1) in the nutrient solution on buchu growth. The first experiment was done in a plastic covered structure with a pad and fan and the objective of this trial was to determine the effect of increasing phosphate concentrations in the nutrient solution on the general growth, biomass production, oil composition, mortality rate and chemical composition of the buchu plants. The second experiment was done in a glasshouse with mechanical temperature control and the aim of this trial was to determine the response of buchu to increasing concentrations of P at two different root temperatures. A chemical analysis of the plants was done and the general growth, yield and root mass were recorded to determine the response of buchu plants to the phosphate and temperature treatments. In the greenhouse experiment an optimum growth and yield response of buchu plants was found at a phosphate concentration of 0.7 me L-1 in the nutrient solution. Phosphate concentrations lower or higher than 0.7 me L-1 lead to a decrease in growth and yield. An increase in the phosphate concentration in the nutrient solution lead to a general increase in N, P, K, Ca, Mg and B content in the buchu plants and a decrease in Fe content. The mortality rate of the buchu plants increased with an increase in the phosphate concentration from 0.0 to 1.4 me L-1 in the nutrient solution. The phosphate concentration in the nutrient solution only made a significant difference on one major component of the buchu oil which was Ψ-Diosphenol, but no general trend with Ψ-Diosphenol content and P concentration could be found and the significant difference in Ψ-Diosphenol observed in this trial may only have been due to genetic variation between the plants. The effect of the different root temperatures in the glasshouse experiment was very clear. The buchu plants grown at the high root temperature (20°C) produced a higher yield and better overall growth than the plants grown at lower (10°C) temperatures. The buchu plants grown at 20°C had a significantly higher N, K, Na and B content than plants grown at 10°C. Buchu plants grown at 10°C showed no significant response in terms of growth and yield to the phosphate concentration in the nutrient solution, but plants grown at 20°C exhibited growth and yield peaks at phosphate concentrations of 0.35 and 1.4 me L-1. The peak observed in the plants growth at high phosphate concentrations is unexplainable and can possibly be ascribed to the limitation of the plants per experimental unit and/or amount of replications. The increase in P concentration in the nutrient solution caused a general increase in N, P and K content in the buchu plants. A significant interaction between the phosphate concentration and root temperature was observed for the P, Mn en Zn contents of the plants which meant that the buchu plants respond differently towards phosphate concentrations at different root temperatures.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006. / The development of a reliable and reproducible method for the genetic characterisation and identification of the commercially important Agathosma species was investigated. Previous research attempts aimed at developing a reliable and reproducible method of identifying these Agathosma species failed, mostly because these studies were based on phenotypic traits and these methods were therefore influenced by environmental factors. In this study amplified fragment length polymorphisms (AFLPs) were successfully used to quantify the genetic variation between the Agathosma species and as a result three distinct groups could be identified. The data obtained were elaborated with the Dice genetic similarity coefficient, and analysed using different clustering methods and Principle Coordinate Analysis (PCoA). Cluster analysis of the genotypes revealed an overall genetic similarity between the populations of between 0.85 and 0.99. The AFLP-based dendrogram divided the populations into three major groups: (1) the A. serratifolia and A. crenulata populations, (2) the putative hybrid, A. betulina X A crenulata populations, and (3) the A. betulina populations, confirming that this technique can be used to identify species. The question of hybridisation was also clarified by the results of the PCoA, confirming that the putative hybrid is not genetically intermediately spread between the A. crenulata and A. betulina populations, and that it is genetically very similar to A. betulina. The putative hybrid can therefore rather be viewed as a genetically distinct ecological variant of A. betulina. As the AFLP technique cannot be directly applied in large-scale, routine investigations due to its high cost and complicated technology, the development of polymerase chain reaction (PCR)-based molecular markers, able to accurately identify the species, was undertaken. Due to the superior quality of A. betulina oil, the development of such markers is especially critical for this species. Several species-specific AFLP markers were identified, converted to sequence characterised amplified regions (SCARs) and ultimately single nucleotide polymorphisms (SNPs) were characterised. The developed SCARs were unable to distinguish between the species. The conversion of AFLP fragments to SCARs is problematic due to multiple fragments being amplified with the AFLP fragment of interest. The diagnostic feature of the SNP-based markers was not sensitive enough, since this technique could not distinguish between the A. betulina and A. crenulata and/or the putative hybrid populations. The SNPs that were characterised were found not to be species-specific; they were only specific to the particular clone. Although a quick and robust marker specific for A. betulina has not yet been developed, this study sets the stage for future genetic studies on Agathosma species. Such a marker, or set of markers, would be an invaluable contribution to a blooming buchu oil industry.

Thesis (MScAgric (Agronomy)--University of Stellenbosch, 2007. / The Cape Floristic Region is a highly distinctive phytogeographical unit which is recognized as a floral Kingdom on its own. Buchu (Agathosma betulina) plants fall under this important Kingdom. Buchu is one of the traditional medicinal plants originating in the Western Cape province of South Africa and the essential oil derived from the leaves is exported in large volumes. Due to high demand, under supply, restrictions of wild harvesting and high prices for Buchu essential oil, growers have started to introduce and commercialize this species as a crop. This commercialization of Buchu necessitated agronomic research to optimize production techniques. The objective of this study was to determine the optimum pH range for the cultivation of high yielding Buchu with acceptable essential oil quality under controlled conditions and compare this with the conditions in the natural habitat. Plant, soil and climatic data were gathered from eleven sites in the natural habitat of Buchu (A. betulina) in the Cederberg Mountains. At all sites most rainfall occurred from May to September, while high temperatures were recorded in summer. Soil analyses indicated low levels of nutrients and low soil pH, ranging from 3.7 to 5.3 at all the sites studied. Low levels of nutrients were also obtained from foliar analysis collected from plants at each of the different sites. Chemical analyses of the essential oil indicated that the plants were from a high quality diosphenol chemotype. In the greenhouse experiment, five different pH levels (pH 33.99, 4-4.99, 5-5.99, 6-6.99 and 7-7.99) were evaluated to determine the effect on growth, yield and quality of A. betulina. Complete nutrient solutions were used to irrigate the plants grown in pots filled with a sand and coco peat mixture. Although the plants subjected to the pH treatment of 4-4.99 tended to have the highest growth rate and yield, this did not differ significantly (P=0.05) from plants subjected to pH values between 3 and 6.99. In contrast, the pH 7-7.99 treatment lead to reduced growth and lower vegetative yields. Levels of nutrients obtained from the leaf mineral analysis differed significantly with different pH treatments. High pH levels resulted in high nitrogen, phosphorus, sodium, manganese and boron contents, but lower contents of copper. Nitrogen, phosphorus, calcium and zinc were higher than those recorded for plants from their natural habitat, but still within the norm reported for most plants. Levels of manganese, sodium, magnesium and copper were found to be more or less similar to the values obtained in plants from the natural habitat. No significant differences were found in essential oil quality in response to the pH treatments. However, high pulegone levels (10.8 to 13.2 %) were obtained from all the treatments in the greenhouse experiment. The high levels of this essential oil constituent could have a negative effect on the marketability of the oil and this aspect may need some attention in future studies.