Alfred Botha

Publications of Alfred Botha

• Diversity and Characterization of Culturable Fungi from Marine Sediment Collected from St. Helena Bay, South Africa.

  Authors: Marnel Mouton, Ferdinand Postma, Jac Wilsenach, Alfred Botha

  Microbial ecology. 03/2012;

  Marine fungi are known to originate from a wide variety of habitats within the marine environment. Marine sediment represents one environmental niche, with most fungi occurring in these sedimentsMarine fungi are known to originate from a wide variety of habitats within the marine environment. Marine sediment represents one environmental niche, with most fungi occurring in these sediments being facultative marine fungi with terrestrial origins. It has not been proven whether these fungi merely survive the harsh environmental conditions presented by the ocean sediment, as opposed to playing an active role in this ecological niche. During this study, marine sediment was collected from St. Helena Bay, on the west coast of the Western Cape, South Africa. Using dilution, enrichment, and repetitive culturing techniques, 59 fungal isolates were obtained from marine sediments and identified to at least genus level using morphological and molecular methods. Moreover, a series of tests were performed to characterize the physical and physicochemical attributes of the isolates. Results showed that the isolates not only survived but also had the potential to grow in the natural conditions present in this environment. Extracellular cellulase was produced by the filamentous fungal isolates indicating their probable role in detrital decay processes and therefore the carbon cycle on the ocean bed. Also, denitrification patterns were observed when isolates were grown in liquid media amended with NaNO(2), NaNO(3) , and (NH(4))SO(4), implicating that these fungi have the potential to play an active role in denitrification, co-denitrification, and ammonification phases of nitrogen cycles occurring in the marine sediments.

• The lignicolous fungus Coniochaeta pulveracea and its interactions with syntrophic yeasts from the woody phylloplane.

  Authors: Andrea van Heerden, Willem H van Zyl, Christiaan W Cruywagen, Marnel Mouton, Alfred Botha

  Microbial ecology. 05/2011; 62(3):609-19.

  The yeast-like fungus Coniochaeta pulveracea was studied with regard to its novel lignocellulolytic activities and the possible effect thereof on yeasts from the woody phylloplane. An enrichmentThe yeast-like fungus Coniochaeta pulveracea was studied with regard to its novel lignocellulolytic activities and the possible effect thereof on yeasts from the woody phylloplane. An enrichment procedure was used to isolate C. pulveracea from a decaying Acacia tree, and the identity of the isolate was confirmed using morphology, as well as molecular and phylogenetic techniques. This isolate, as well as strains representing C. pulveracea from different geographical regions, were compared with regard to optimum growth temperature and enzyme activity to representatives of closely related species. These include strains of Coniochaeta boothii, Coniochaeta rhopalochaeta, and Coniochaeta subcorticalis. Plate assays for cellulase and xylanase activity indicated that all representatives of the above-mentioned species were able to produce extracellular hydrolytic enzymes and were also able to degrade birchwood toothpicks during a 50-day incubation period at 30°C. To test the ability of these fungi and their enzymes to release simple sugars from complex cellulosic substrates, filtrates obtained from liquid cultures of Coniochaeta, cultivated on carboxymethyl cellulose (CMC) as sole carbon source, were analyzed using high-performance liquid chromatography analysis. Consequently, the presence of mono- and disaccharides such as glucose and cellobiose was confirmed in these culture filtrates. Two subsequent experiments were conducted to determine whether these simple sugars released from woody material by Coniochaeta may enhance growth of phylloplane yeasts. In the first experiment, representatives of Coniochaeta were co-cultured with selected yeasts suspended in agar plates containing birchwood toothpicks, followed by examination of plates for colony formation. Results indicated that Coniochaeta growth on the toothpicks enhanced growth of nearby yeast colonies in the agar plates. In the second experiment, representatives of selected yeasts and Coniochaeta species were co-cultured on CMC and xylan-containing plates where after yeast colony formation was recorded on the plates. Saccharomyces cerevisiae strains, engineered to utilize specific wood degradation products, i.e., cellobiose or xylose, as sole carbon source were used as positive controls. While it was found that cellobiose released from CMC was assimilated by the yeasts, no evidence could be obtained that xylose released from xylan was used as carbon source by the yeasts. These ambiguous results could be ascribed to secretion of nutritious metabolic end products, other than the products of fungal xylanases.

• Micro-particle-induced X-ray emission mapping of elemental distribution in roots of a Mediterranean-type sclerophyll, Agathosma betulina (Berg.) Pillans, colonized by Cryptococcus laurentii.

  Authors: Karen J Cloete, Wojciech J Przybylowicz, Jolanta Mesjasz-Przybylowicz, Alban D Barnabas, Alexander J Valentine, Alfred Botha

  Plant, cell & environment. 06/2010; 33(6):1005-15.

  The role of rhizosphere yeasts as plant nutrient-scavenging microsymbionts in resource-limited Mediterranean-type heathlands is unknown. This study, therefore, focused on quantitative elementalThe role of rhizosphere yeasts as plant nutrient-scavenging microsymbionts in resource-limited Mediterranean-type heathlands is unknown. This study, therefore, focused on quantitative elemental distribution within the roots of a medicinal sclerophyll, Agathosma betulina (Berg.) Pillans, grown under nutrient-poor conditions, and colonized by Cryptococcus laurentii. Micro-particle-induced X-ray emission (PIXE) was used to assess quantitative elemental distribution within the roots of A. betulina inoculated with viable C. laurentii, as well as within roots of control plants that received autoclaved yeast. To aid in the interpretation of heterogeneous elemental distribution patterns, apoplastic barriers (Casparian bands) in root tissues were located using fluorescence microscopy. In addition, root cross-sections were examined for endophytic C. laurentii using light and transmission electron microscopy (TEM). The average concentrations of P, Fe and Mn were significantly (P < 0.05) higher in roots of yeast-inoculated plants, compared to control plants. Casparian bands were observed in the exodermal cells of both treatments, and the presence of these bands was correlated with elemental enrichment in the epi/exodermal-outer cortical tissues. Light and TEM micrographs revealed that the yeast was not a root endophyte. This is the first report describing the role of a soil yeast as a plant nutrient-scavenging microsymbiont.

• The osmotic stress tolerance of basidiomycetous yeasts.

  Authors: Obakeng M Tekolo, Jean McKenzie, Alfred Botha, Bernard A Prior

  FEMS yeast research. 02/2010; 10(4):482-91.

  The growth and accumulation of intracellular polyols at reduced water activity (a(w)) were studied in 40 basidiomycetous yeast strains. The growth of most strains showed greater tolerance to NaClThe growth and accumulation of intracellular polyols at reduced water activity (a(w)) were studied in 40 basidiomycetous yeast strains. The growth of most strains showed greater tolerance to NaCl than sorbitol at the same a(w). No strain was able to grow below 0.90a(w). (13)C nuclear magnetic resonance spectroscopy revealed that glycerol was the major solute accumulated intracellularly by all the yeasts when grown to 0.96a(w) (NaCl). Arabitol or mannitol was also accumulated in some yeasts, whereas a few only accumulated glycerol. Analysis of six yeasts in detail revealed that the intracellular glycerol concentrations of five yeasts increased significantly when grown at 0.96a(w) (NaCl or sorbitol) compared with growth at 0.998a(w). Arabitol and mannitol concentrations also increased, but not to the same degree. Intracellular potassium concentrations decreased when grown at 0.96a(w) (NaCl or sorbitol) and sodium increased, but only when grown at 0.96a(w) (NaCl). The survival of nine strains was evaluated in soil cultures and it was found that all grew at 100% field capacity, whereas at lower field capacity, only some strains grew or survived. The growth of basidiomycetous yeasts appears to be more sensitive to reduced a(w) than ascomycetous yeasts.

• Yeast infection in a beached southern right whale (eubalaena australis) neonate.

  Authors: Marnel Mouton, Desray Reeb, Alfred Botha, Peter Best

  Journal of wildlife diseases. 08/2009; 45(3):692-9.

  A female southern right whale (Eubalaena australis) neonate was found stranded on the Western Cape coast of southern Africa. Skin samples were taken the same day from three different locations on theA female southern right whale (Eubalaena australis) neonate was found stranded on the Western Cape coast of southern Africa. Skin samples were taken the same day from three different locations on the animal's body and stored at - 20 C. Isolation through repetitive culture of these skin sections yielded a single yeast species, Candida zeylanoides. Total genomic DNA also was isolated directly from skin samples. Polymerase chain reaction analysis of the internal transcribed spacer region of the fungal ribosomal gene cluster revealed the presence of Filobasidiella neoformans var. neoformans, the teleomorphic state of Cryptococcus neoformans. Fungal infections in cetaceans seem to be limited when compared to infections caused by bacteria, viruses and parasites. However, Candida species appear to be the most common type of fungal infection associated with cetaceans. To our knowledge this is the first report of a C. zeylanoides infection in a mysticete, as well as the first report of a dual infection involving two opportunistic pathogenic yeast species in a cetacean.

• Establishing a risk-assessment process for release of genetically modified wine yeast into the environment.

  Authors: Heidi Schoeman, Gideon M Wolfaardt, Alfred Botha, Pierre Van Rensburg, Isak S Pretorius

  Canadian journal of microbiology. 08/2009; 55(8):990-1002.

  The use and release of genetically modified organisms (GMOs) is an issue of intense public concern and, in the case of food and beverages, products containing GMOs or products thereof carry the riskThe use and release of genetically modified organisms (GMOs) is an issue of intense public concern and, in the case of food and beverages, products containing GMOs or products thereof carry the risk of consumer rejection. The recent commercialization of 2 GM wine yeasts in the United States and Canada has made research and development of risk assessments for GM microorganisms a priority. The purpose of this study was to take a first step in establishing a risk-assessment process for future use and potential release of GM wine yeasts into the environment. The behaviour and spread of a GM wine yeast was monitored in saturated sand columns, saturated sand flow cells, and conventional flow cells. A widely used commercial Saccharomyces cerevisiae wine yeast, VIN13, a VIN13 transgenic strain (LKA1, which carries the LKA1 alpha-amylase gene of Lipomyces kononenkoae), a soil bacterium (Dyadobacter fermentens), and a nonwine soil-borne yeast (Cryptococcus laurentii) were compared in laboratory-scale microcosm systems designed to monitor microbial mobility behaviour, survival, and attachment to surfaces. It was found that LKA1 cells survived in saturated sand columns, but showed little mobility in the porous matrix, suggesting that the cells attached with high efficiency to sand. There was no significant difference between the mobility patterns of LKA1 and VIN13. All 3 yeasts (VIN13, LKA1, and C. laurentii) were shown to form stable biofilms; the 2 S. cerevisiae strains either had no difference in biofilm density or the LKA1 biofilm was less dense than that of VIN13. When co-inoculated with C. laurentii, LKA1 had no negative influence on the breakthrough of the Cryptococcus yeast in a sand column or on its ability to form biofilms. In addition, LKA1 did not successfully integrate into a stable mixed-biofilm community, nor did it disrupt the biofilm community. Overall, it was concluded that the LKA1 transgenic yeast had the same reproductive success as VIN13 in these 3 microcosms and had no selective advantage over the untransformed parental strain.

• Evidence of Symbiosis Between the Soil Yeast Cryptococcus laurentii and a Sclerophyllous Medicinal Shrub, Agathosma betulina (Berg.) Pillans.

  Authors: Karen Cloete, Alexander Valentine, Marietjie Stander, Louisa Blomerus, Alfred Botha

  Microbial ecology. 11/2008;

  The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentiiThe interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 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. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.

• Enhancement of Rooibos (Aspalathus linearis) aqueous extract and antioxidant yield with fungal enzymes.

  Authors: Mia Pengilly, Elizabeth Joubert, Willem H van Zyl, Alfred Botha, Marinda Bloom

  Journal of agricultural and food chemistry. 07/2008; 56(11):4047-53.

  The leaves and stems of the Rooibos plant ( Aspalathus linearis) are used for the production of an herbal tea known for its health promoting properties, which have been linked to its flavonoidThe leaves and stems of the Rooibos plant ( Aspalathus linearis) are used for the production of an herbal tea known for its health promoting properties, which have been linked to its flavonoid content but which is substantially reduced by the traditional processing method employed. Selected food-grade fungi were screened for their potential to improve the yield of soluble matter extracted from rooibos plant material. Fungal cocktails of hydrolyzing enzymes enhanced either the yield of soluble solids ( Lentinula edodes and Rhizopus oryzae cultured in yeast peptone-wheat straw medium) or the yield in antioxidants from fermented rooibos ( R. oryzae cultured in potato dextrose or yeast peptone-wheat straw medium). When applied to green rooibos, L. edodes (cultured in yeast peptone-wheat straw medium) enhanced the release of soluble solids as well as color formation, leading to semifermented rooibos with a relatively high aspalathin content, compared to fermented rooibos.

• Soil properties that impact yeast and actinomycete numbers in sandy low nutrient soils.

  Authors: Jo-Marie Vreulink, Annerina Esterhuyse, Karin Jacobs, Alfred Botha

  Canadian journal of microbiology. 01/2008; 53(12):1369-74.

  To explore the beneficial qualities or detrimental consequences of cultureable soil yeasts, it is important to understand which physicochemical soil properties most impact populations of theseTo explore the beneficial qualities or detrimental consequences of cultureable soil yeasts, it is important to understand which physicochemical soil properties most impact populations of these unicellular fungi in their natural habitat. The goal of this study was to determine which soil properties dictate yeast numbers in pristine sandy, low nutrient soils within a semi-arid region. A correlation matrix of the data obtained for 19 different environmental variables indicated a negative correlation between soil pH and yeast numbers. Using general regression models, it was demonstrated that soil pH and copper concentration were the 2 variables that correlated best with soil yeast counts in these soils. However, soil moisture content was found to be the environmental factor with the most impact on cultureable actinomycetes and heterotrophic microbes. The study also demonstrated that divalent cation availability might impact the size of both yeast and prokaryote populations in these soils.

• 3-hydroxy fatty acids found in capsules of Cryptococcus neoformans.

  Authors: Olihile M Sebolai, Carolina H Pohl, Piet J Botes, Catharina J Strauss, Pieter W J van Wyk, Alfred Botha, Johan L F Kock

  Canadian journal of microbiology. 07/2007; 53(6):809-12.

  Using immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fattyUsing immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fatty acids in Cryptococcus neoformans. Our results suggest that these oxylipins accumulate in capsules where they are released as hydrophobic droplets through tubular protuberances into the surrounding medium.

• Phytase activity in Cryptococcus laurentii ABO 510.

  Authors: Jason van Staden, Riaan den Haan, Willem H van Zyl, Alfred Botha, Marinda Viljoen-Bloom

  FEMS yeast research. 06/2007; 7(3):442-8.

  Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayedTen Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.

• Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab'jeva & Reshetova) Golubev.

  Authors: Adriana L Botes, Jeanette Lotter, Owen H J Rhode, Alfred Botha

  Systematic and applied microbiology. 02/2005; 28(1):27-33.

  Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxideIsolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (+/-)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (+/-)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.

• Oxylipin Formation in Fungi: Biotransformation of Arachidonic Acid to 3-Hydroxy-5,8-tetradecadienoic Acid byMucor genevensis

  Authors: Carolina H. Pohl, Alfred Botha, Johan L.F. Kock, Dennis J. Coetzee, Piet J. Botes, Tankred Schewe, Santosh Nigam

  Biochemical and Biophysical Research Communications.

  The soil fungusMucor genevensiswas shown to convert exogenous arachidonic acid to the oxylipin 3-hydroxy-5Z,8Z-tetradecadienoic acid (3-HTDE) as determined by gas chromatography/mass spectrometry.The soil fungusMucor genevensiswas shown to convert exogenous arachidonic acid to the oxylipin 3-hydroxy-5Z,8Z-tetradecadienoic acid (3-HTDE) as determined by gas chromatography/mass spectrometry. This metabolite was only found in the aqueous supernatant together with free linoleic acid, but not in the final fungal biomass. In contrast, the corresponding primary arachidonic acid metabolite (3R)-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (3-HETE), which has been earlier shown to be produced by the yeastDipodascopsis uninucleata,could not be detected. These observations may be plausibly explained by a retroconversion byM. genevensisof arachidonic acid to linoleic acid before the latter is metabolised to 3-HTDE.

• Interspecies differences in the enantioselectivity of epoxide hydrolases in Cryptococcus laurentii (Kufferath) C.E. Skinner and Cryptococcus podzolicus (Bab’jeva & Reshetova) Golubev

  Authors: Adriana L. Botes, Jeanette Lotter, Owen H.J. Rhode, Alfred Botha

  Systematic and Applied Microbiology.

  Isolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxideIsolates representing Cryptococcus laurentii and Cryptococcus podzolicus, originating from soil of a heathland indigenous to South Africa, were screened for the presence of enantioselective epoxide hydrolases for 2,2-disubstituted epoxides. Epoxide hydrolase activity for the 2,2-disubstituted epoxide (±)-2-methyl-2-pentyl oxirane was found to be abundantly present in all isolates. The stereochemistry of the products formed by the epoxide hydrolase enzymes from isolates belonging to the two species (11 isolates representing C. laurentii and 23 isolates representing C. podzolicus) was investigated. The enantiopreferences of the epoxide hydrolases for 2,2-disubstituted epoxides of these two species were found to be opposite. All strains of C. laurentii preferentially hydrolysed the (S)-epoxides while all C. podzolicus isolates preferentially hydrolysed the (R)-epoxides of (±)-2,2-disubstituted epoxides. These findings indicate that the stereochemistry of the products formed from 2,2-disubstituted epoxides by the epoxide hydrolase enzymes of these yeasts should be evaluated as additional taxonomic criterion within the genus Cryptococcus. Also, the selectivity of some epoxide hydrolases originating from isolates of C. podzolicus was high enough to be considered for application in biotransformations for the synthesis of enantiopure epoxides and vicinal diols.

• Application of fatty acid profiles in the identification of yeasts

  Authors: Alfred Botha, Johan L.F. Kock

  International Journal of Food Microbiology.

  In order to develop a rapid yeast identification technique using fatty acid profiles, an extensive survey has been conducted in our laboratory on the long-chain fatty acid composition (CLFAC) ofIn order to develop a rapid yeast identification technique using fatty acid profiles, an extensive survey has been conducted in our laboratory on the long-chain fatty acid composition (CLFAC) of yeasts representing the endomycetous and basidiomycetous yeast domain. It was accomplished by cultivating the yeast strains under standardized conditions in a synthetic liquid medium. When stationary phase was reached the cells of each culture were freeze-dried and the CLFAC was examined by gas chromatography. It was found that the fatty acid profile obtained for each strain was reproducible. However, as work progressed, it became clear that variation exists within species and that the relative percentages of some strains from different species may overlap.Identification of species could therefore not be achieved in all attempts, even when the resolution of the fatty acid analyses was enhanced by using capillary columns, useful for the detection of minor fatty acids. When used in isolation, CLFAC analyses is therefore not a generally applicable identification technique for yeast species. However, the technique was found to be a valuable chemotaxanomical tool to distinguish between strains of certain species, species of certain genera and species from particular environments.The technique currently finds application in the South African food and beverage industry as a quick, cheap and easy way to distinguish between strains of Saccharomyces cerevisiae. It is also used by an industry which produces bioprotein from Geotrichum candidum, to determine fungal contaminants in a quality control process.