Microbial, Biochemical and Food Biotechnology

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Open Access
Ondersoekinge oor die afbraak van aromatiese verbindings deur mikro-organismes
(University of the Free State, 1960-01) Scott, De B.; Lütjeharms, W. J.
English: 1. In a survey of the relevant literature the methods of investigation, the systematic and biochemical aspects and the significance of microbial degradation of aromatic compounds has been discussed. 2. In a preliminary investigation it was found that nonselectivily isolated strains of streptomycetes dit not grow on a mineral salt medium with a simple aromatic compound as sole source of carbon. 3. Bacteria, fungi and one Nocardia strain able to use aromatic compounds as sole source of carbon and energy, were isolated from enrichment cultures. 4. From turbidimetric growth measurements it was found that optimum cell development of the bacteria and actinomycete investigated in a medium with a low concentration of aromatic substrate in most cases took place within two days. 5. The oxidation of a number of aromatic compounds by the bacteria and actinomycete isolated, was studied with the aid of the direct method of Warburg. 6. The method used for the calibration of manometers and Warburg flasks is described. 7. The technique of sequential induction was applied to determine the pathway of microbial degradation of the aromatic compounds investigated. With this technique the following hitherto unpublished reactions of bacteria have been found: (i) Salicylic acid - gentisic acid. (ii) Saligenin - salicylic aldehyde - salicylic acid - catechol and gentisic acid. (iii) Syringic acid - gallic acid. (iv) Guaiacol - catechol. (v) Vanillin - vanillic acid - protocatechuic acid. (vi) Veratric aldehyde - veratric acid - vanillic acid - protocatechuic acid. The degradation of phenol, benzoic acid, mandelic acid, p-cresol, m-hydroxybenzoic acid and anisic acid by bacteria, and of toluene by Nocardia sp., as studied with manometrical methods, was found to be identical with the pathway already described for bacteria and fungi. The degradation pathway of o- and m-cresol could not be established with certainty with the method used.
Open Access
Seisoenvariasie in die aminosuursamestelling van rumenprotosoë
(University of the Free State, 1978-02) Cilliers, Johannes Jacobus le Roux; Du Toit, P. J.
English: The rumen content of sheep was fractionated and analyzed. Samples were obtained over a year, at three week intervals, from a group of six Merino sheep on natural veld. The rumen contents of another group receiving specified crude protein feed and kept indoors under controlled conditions, were also analyzed. Rumen samples were fractionated into protozoal-, bacterial-, fibrous- and soluble fractions by a procedure of centrifugation. Sampling and conditions under which the sheep were kept as well as fractionating procedures are described and discussed. The rumen samples as well as the different fractions were analyzed by various means to determine the effects of seasonal variation. Dry mass, nitrogen (micro-Kjeldahl technique) and amino acid composition of these fractions were determined. In some cases the total carbohydrate- and tryptophane contents were determined. Observation of the different fractions by microscope was used for comparative purposes. For analytical determinations, including hydrolysis procedures, all sampLes were well mixed, homogenized where necessary, freeze-dried and pulverised to facilitate representative sampling. The amount of sample needed for amino acid determination was calculated from the nitrogen content. Samples were hydrolized for 22 hours at 110 °C in constant boiling HCl under vacuum. Hydrochloric acid was removed by evaporation and freeze-drying after hydrolysis and the amino acids transferred quantitavely into buffer for analysis. Amino acid analysis were conducted on a Beckman Model 120 C amino acid analyzer according to a standardized procedure. The analysis were controlled for accuracy and values compensated for losses of certain amino acids caused by acid hydrolysis. According to results, the nitrogen as well as the total amino acid content varied with seasonal variations with minimum values at the end of the winter. Each amino acid, viewed as a percentage of the total amino acids in the fraction, remained constant within certain limits over the full duration of the experiment. Certain essential amino acids, i.e. Lys, lIe and Arg occured at comparatively higher concentration levels in the protozoal fraction, whereas Thr, Ala, Tyr and Met were present at higher levels in the bacterial fraction as compared to the rumen sample. Pro and His seem to be degraded to a marked extent in the rumen, whereas Asp and Glu occured at relatively high concentration levels in all fractions, including the feed. The quality and composition of fodder have a marked effect upon the microflora of the rumen. The sheep on crude protein feed displayed much higher levels of protozoa (dry mass) than bacteria. The opposite pattern was observed with sheep on the veld. Protozoa from sheep on crude protein feed also had higher concentration levels of Lys, Tyr, Arg, Asp and Glu but lower ,levels of Ala and Gly. Bacterial fractions from both groups showed very little difference in amino acid composition. These results seem to indicate a definite role for protozoa in the rumen especially when the animal is fed a high quality fodder. In addition to preferentially supplying certain essential amino acids (protozoa used as feed) these organisms also seem to aid in the break-down of complex macromolecules present in the feed. In this respect a glycoside hydrolase, obtained from the protozoal fraction of grazing sheep, was partially purified and studied. The enzyme was purified by ethanol fractionation as well as chromatographic procedures. The optimum pH as well as the activity towards various carbohydrates was also determined. The occurence of specific hydrolases in the different micro-organisms is also affected by the feed supplied to the animal. It seems essential that further studies specifically concerning the metabolic role of protozoa in the rumen should be done.
Open Access
Long-chain fatty acid compositions and volatile metabolite patterns of yeasts associated with wine
(University of the Free State, 1987-05) Tredoux, Hendrik Gabriël; Kock, J. L. F.
A) In Chapter 1 the need for a yeast identification system in the wine industry is highlighted. The definition, as well as taxonomic development of the ascomycetous yeasts are discussed as well as the problems encountered. B) In Chapter 2 the cellular long-chain fatty acid compositions of 103 yeast strains representing 38 species related to the wine industry were determined gaschromatographically. It was possible to differentiate between most species examined as well as between some strains within species. A correlation was observed between long-chain fatty acid composition and complexity of cell differentiation, genetic recombination, carbon source- and ethylamine utilization and resistance to cycloheximide. A phylogenetic scheme for the genus Kluyveromyces was constructed on the basis of the abovementioned features. C) Chapter 3 includes the use of volatile metabolites in the identification of winery-associated yeasts. According to the results it was possible to differentiate between the Saceh. cerevisiae and S. pombe strains. D) A Discussion and Conclusions is presented in Chapter 4. This includes a discussion on the identification of wine yeasts and the relation between long-chain fatty acid composition, pseudomycelium formation, genetic recombination, carbon source- and ethylamine utilization and resistance to cycloheximide. A possible relation between the similarity in long-chain fatty acid compositions and DNA homology between yeasts strains is indicated. The use of volatile metabolites in the identification of wine yeasts is also discussed.
Open Access
Design, synthesis and expression in different hosts of a gene coding for a small multifunctional peptide
(University of the Free State, 1994-01) Van der Merwe, Walda Brenda; Pretorius, G. H. J.; Kotzé, H. F.
Platelets and coagulation both play a pivotal role in thrombosis, one of the major causes of death in Western society. It is therefore not surprising that potent inhibitors are being developed to inhibit platelet function or coagulation. In this study we developed a multifunctional peptide that would not only inhibit thrombin, but will also prevent platelet aggregation. A 29 amino acid peptide was designed comprising three inhibitory regions: 1, a part of hirudin, a potent direct antithrombin; 2, fibrinopeptide A, also an inhibitor of thrombin and 3, an amino acid sequence (arqgly- asp) essential for binding to the fibrinogen receptor on platelets to prevent fibrinogen binding. The protein sequence was reverse translated and two 60-mers with an overlap of 22 base pairs were synthesized. After filling in the ends, the gene was cloned into a yeast expression and secretion vector, pMFα8. The construct was sequenced to verify correct orientation of the gene and transformed into yeast. The expected peptide of approximately 3 kDa could not be seen on SDS-PAGE gels, nor could inhibition of thrombin or platelet aggregation be shown. However, Northern hybridization analysis revealed the presence of the mRNA. The introduction of a methionine residue at the N-terminus of the peptide and the use of protease-deficient yeast strains as expression hosts, did not improve peptide production. Another expression system, the inducible yeast expression vector pYES2, was then employed. Although no peptide could be detected, Northern blotting showed the presence of high levels of the mRNA. Optimal codons for expression in yeast were selected in the design of the gene, but the absence of a detectable level of peptide could be due to a low translation rate or due to proteolysis. Alternatively, the gene was cloned into a regulatable E. coli vector, pQE-32, and expressed intracellularly in E. coli. No band of the correct size could be detected on SDS-PAGE gels, which might be due to the extremely small molecular size of the peptide.
Open Access
The isolation of gamma-linolenic acid producing mucoralean fungi
(University of the Free State, 1997-11) Strauss, Tersia; Botha, A.; Kock, J. L. F.
English: Members of Mucorales are known to produce the high value fatty acid gamma-linolenic acid [18:3(ω6)]. Although few studies have been conducted, it is known that the type of carbon source included in the medium, influences the production of 18:3(ω6) by these fungi. The range of carbon sources on which mucoralean fungi are able to grow and produce 18:3(ω6), is still mostly unknown. Another factor that influences the quantities of 18:3(ω6) that are being produced by these fungi, is the specific fungal strain that is used in the process. Consequently, in this study it was decided first to investigate the ability of different mucoralean fungi to grow and produce 18:3(ω6) on a wide range of carbon sources. Isolation media for obtaining new strains from nature, which utilize carbon sources obtainable from industrial effluents, would subsequently be developed. The influence of 38 different carbon sources on growth and consequent 18:3(ω6) content of the lipids produced by four mucoralean fungal strains were therefore investigated. The strains represented the species Morlierella afpina, Mucor circinelloides, Mucor ffavus and Thamnosfyfum piriforme. The representatives of M. circinelloides and M. ffavus respectively utilized 25 and 23 of the 38 carbon sources in the series. The highest percentages 18:3(ω6) obtained with the representatives of M. circinelloides and M. ffavus were 27.17 % and 36.40 % respectively. In contrast, the highest percentages 18:3(ω6) obtained with the representatives of Mo. afpina and T. piriforme were only 5.61 % and 12.84 % respectively. These two strains could respectively utilize only seven and 17 of the carbon sources. This study indicated that mucoralean fungi can grow and produce 18:3(ω6) on a variety of carbon sources, including carbon sources present in industrial effluents (e.g. starch, sucrose and acetic acid). Three selective media were subsequently developed in order to isolate mucoralean fungi from soil, using the soil plate technique. The media, which were complex, respectively contained starch, sucrose and sodium acetate as carbon sources, as well as 0.02 g/l of the anti-fungal agent, benlate. The selectivity of the media for members of Mucorales was first determined by testing the media for the ability to support growth of 134 mucoralean fungal strains representing 66 species and seven genera. The three isolation media supported growth of strains representing Absidia, Actinomucor, Backusella, Mucor, Rhizopus and Thamnostylum. The ability of the isolation media to select mucoralean fungi from a natural fungal population in soil, was then determined and representatives of the genera Absidia, Cunningham'ella, GongronelIa, Mucor and Rhizopus were obtained. The results further showed that by using selective media in combination with a relatively non-selective medium, instead of the non-selective medium alone, more mucoralean taxa could be isolated from a particular soil sample. Mucoralean fungal isolates that were obtained from the soil sample, were subsequently evaluated for growth and 18:3(ω6) production in media containing starch, sucrose or glucose as sole carbon sources. Isolates representing the families Absidiaceae, Cunninghamellaceae and Mucoraceae were inoculated in complex media containing the above mentioned carbon sources. It was found that all the isolates were able to produce 18:3(ω6) on all three carbon sources. However, significant differences in volumetric 18:3(ω6) concentrations reached on different carbon sources were noted for each isolate investigated. The highest volumetric concentrations of 18:3(ω6) were obtained with an isolate representing R. stolonifer on starch (0.130 gii) and glucose (0.134 gii) as carbon sources. In order to prove that the isolates obtained using the above-mentioned isolation media, are able to grow in an industrial effluent, some of the isolates representing different families, were grown in a medium prepared from an industrial effluent containing dextrins, galactans and starch as carbon sources. The lipids of the isolates which reduced the COD value of the effluent the most, were analysed. It was found that these isolates were able to produce 18:3(ω6). This study has therefore shown that it is possible to construct isolation media to isolate 18:3(ω6) producing mucoralean fungi from a natural fungal population. It was also found that such isolates can be used to produce biomass and 18:3(ω6) from carbon sources present in industrial effluents.
Open Access
Utilisation of edible oils and GLA production by Mucor in the presence of acetate
(University of the Free State, 1998-10) Badenhorst, Jacqueline; Kock, J. L. F.
English: Surveys launched across South Africa indicate that many frying establishments abuse their frying oils and fats during the frying process, resulting in degradation and concomitant production of potentially toxic oxidation products. Some of these compounds have been shown to be toxic to animals and in human in vitro studies. Consequently, strict regulations under the Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act 54 of 1972) were published on 16 August 1996. It is now an offense to use or sell used cooking oil or fat for human consumption containing high levels of these degradation products. Since frying establishments are not allowed to discard their used oils and fats by selling to the public for consumption or dumping into municipal drainage systems, it is important that these oils and fats are collected for re-use in another form. Consequently, the aim of this study was the biotransformation of used oil wastes (containing no toxic substances) to high value lipids containing gamma-linolenic acid (GLA). This polyunsaturated fatty acid is prescribed for the treatment of eczema. In order to achieve this, Mucor circinelloides f. circinelloides CBS 108.16 was first grown on 40 gil unused sunflower oil and, as expected, produced neutral lipids (NL) similar in fatty acyl composition to the original oil. The apparent repression of the Á6 fatty acid desaturation was partially reversed when cells were grown on oil (30 g/l) and sodium acetate (10 gil) as mixed substrates resulting in an increase in GLA content. Furthermore, a three-fold increase in oil substrate utilisation and doubling of biomass production to 19.1 gil occurred when sodium acetate was added to the oil substrate. When sodium acetate (10 gil) was added to a growth medium containing used cooking oil (UCO) similar results were obtained. This experimental procedure was repeated for seven additional Mucor strains and again the stimulatory effect of sodium acetate in combination with UCO was obvious. Next, the effect of different UCO concentrations in the presence of 10 g sodium acetatell on biomass and lipid production was investigated in Mucor circinelloides CBS 108.16. According to our results, a maximum biomass concentration of 48 gIl consisting of 82 % oil yielding about 35 g NUl and up to 900 mg GLNI was achieved. The addition of 30 g UCO/I in combination with 10 g sodium acetate/l proved to be the optimum UCO concentration in order to obtain maximum GLA yield. Similar results with this strain were obtained when UCO was replaced with fresh unused cooking oil. When these experiments were repeated with linseed oil and sodium acetate as sole carbon sources, much less GLA was produced (351 mg GLNI). According to bioreactor studies, the effects of sodium acetate addition can be attributed to the change in pH of the medium during cell growth in the presence and absence of acetate. In the absence of sodium acetate the pH decreased to 2.2, whereas in its presence it increased to about pH 8.0. During metabolism of sunflower oil in the presence of sodium acetate, the percentage of saturated fatty acids in the extracellular lipids increased, suggesting a higher specificity of the fungal lipase for unsaturated fatty acids. When the sodium acetate was omitted from the medium and the pH gradually increased according to a pH profile mimicking the natural increase in pH found in the medium containing sodium acetate, similar results as in the presence of sodium acetate were obtained. This observation indicated that the pH increase alone during cultivation was responsible for the increased sunflower oil utilisation, biomass and GLA production.
Open Access
Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess
(University of the Free State, 1998-11) Morakile, Gontse; Kock, J. L. F.
English: The ability to preserve microorganisms can be considered a major . biological achievement. Of special importance is the understanding of the principles of culture preservation with minimal occurrence of contamination, genetic and viability change. At present, cryopreservation is considered the most successful preservation method for yeasts yielding high survival levels and good phenotypic stability. As a result, one of the aims of this study was the application and evaluation of a cryopreservation protocol used in the maintenance of a Saccharomyces cerevisiae strain used by a major brewing company in South Africa. In order to ensure that only pure and stable yeasts with high viability are used after revival from maintenance protocol, it is essential that appropriate, rapid and inexpensive quality control methods are implemented. Elaborate and time consuming tests are used today in the brewing industry and include estimation of mutants and bacteria using Wallerstein Laboratory Nutrient Medium (WLN), estimation of respiratory deficient (RDs) yeasts using wort agar overlaid with Triphenyl- Tetrazolium-Chloride, detection of the wild yeasts using the Swartz-Differential Medium (SDM) protocol, estimation of the non-Saccharomyces species using Lysine-Medium (LYS), detection of the lactose assimilating and lactose fermenting microorganisms using the Lactose-Peptone-Broth (LP) and detection of brewery bacteria using the Universal Liquid Medium (ULM). According to the results, a decrease in the percentage RDs as well as Variants (i.e. other mutants of Saccharomyces cerevisiae) was evident in brewing inocula when maintained through cryopreservation. When yeasts from slants (not cryopreserved) and yeasts subjected to cryopreservation were cultivated in wort contained in round bottom flasks, no significant changes in the percentage RDs, variants or maximum growth rate (J.lmax) could be detected. A 4x3x3x2 nested experimental design was performed in order . to determine the sources of variation in yeast viability, stability and contamination after preservation in liquid nitrogen for 136 days. Consequently, results on Variants and RDs suggest that the largest source of variation in the cryopreservation maintenance process was the error arising from the analytical tests. Cryopreservation also influenced the variation in the number of RDs obtained, though to a lesser extend. No contamination was found to occur during the cryopreservation protocol. From this study it is now possible to construct statistical quality control charts that can be used as an aid in the manufacture of brewing inocula maintained through cryopreservation. Another aim of this study was the evaluation of chemotaxonomie characters such as sterols and polar lipids in, as a first step, determining contamination of preserved yeasts with closely related species. According to the results, total lipid content as well as polar lipid fractions and associated fatty acid (FA) composition showed no obvious taxonomic value as the different Saccharomyces species could not be differentiated. Both linoleic acid (18:2) and linolenic acid (18:3) were detected in strains characterised by the absence of these FAs in total FA composition, a situation believed to be due to the 'dilution' of these FAs by the dominating NL fraction. Sterol content showed promise in the demarcation of the Saccharomyces sensu strictu group.
Open Access
The Amylostereum symbiont of Sirex noctilio in South Africa
(University of the Free State, 1998-12) Slippers, Bernard; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.
English: In Chapter 1 of this thesis, the literature pertaining to the symbiosis between Sirex noctilio and Amy/ostereum areo/atum in the Southern Hemisphere, is reviewed. It is evident from this review that S. noctilio and A. areo/atum have become established throughout the pine growing regions of the Southern Hemisphere, despite measures to prevent its introduction. Unlike its relative unimportance as a pathogen in the Northern Hemisphere, this fungal-insect complex has resulted in great losses to softwood industries during a number of severe outbreaks in the Southern Hemisphere. The use of biological control agents in combination with preventative silvicultural practices, has been shown to be very effective in controlling Sirex in Australasia. It is, however, also evident from this review that despite the rather large collection of knowledge concerning the wasp and its control, information regarding the population structure and phylogenetic relationships of the fungal symbiont of Sirex, is scarce. The recent introduction of S. noctilio into South Africa and its confinement to a rather small area in this country provided the opportunity to study the population of its fungal symbiont in detail. Results from Chapter 2 suggest that the fungus has a very narrow genetic base in South Africa and that the introduction of Sirex into this country was limited. The genetic base of A. areolatum in Brazil and Uruguay is similarly uniform. Of even greater interest is the fact that South Africa and Brazil share a common vegetative compatibility group and, thus, a common origin of A. areo/atum and S. noctilio. Moreover, field isolates from the Southern Hemisphere appear to be closely related, which indicates that Sirex might have spread among countries of the Southern Hemisphere and were not necessarily new introductions from the Northern Hemisphere. Isolates of the fungus associated with the biocontrol nematode, De/adenus siricidicola, are, however, distinct from isolates from other Southern Hemisphere populations of the fungus. This could negatively influence the efficacy of the nematode as biocontrol agent in countries to which the nematode has been distributed. Boidin and Lanquetin (1984) report triangular mating incompatibility between isolates from the different Amy/ostereum spp. Results of Chapter 3 support their conclusions by clearly showing that A. areolatum is more distantly related to A. chailletii, A. laevigatum and A. ferreum, than these three species are to each other. The relationship between the latter three species is, however, more clearly defined in Chapter 3 where it is shown that A. ferreum and A. laevigatum are most closely related to each other. One isolate collected from Sirex areolatus, and, therefore, expected to be A. chailletii, was most closely related to A. laevigatum and A. ferreum. Neither of the latter species has, however, been implicated in associations with woodwasps. Furthermore, the data from this study show that Amylostereum spp. group with neither Stereum nor Peniophora, as has been previously hypothesised, but rather with Echinodontium tinctorium. This grouping was included in a larger clade that included species of Russula, Heterobasidion, Lentinellus and Auriscalpium. Analysis of DNA sequence data derived from the nuc-IGS-rDNA in Chapter 4 supported the phylogenetic relationships of the Amylostereum spp. inferred in Chapter 3. Similarly, the isolate obtained from S. areolatus, did not group with any of the four species of Amylostereum and might represent a new species or a distinct group in of one of the current species. Isolates of A. areolatum associated with both S. noctilio and S. juvencus contained four heterogenic sequences in the DNA region analysed. These heterogenic sequences were contained in each isolate of the fungus in one of five combinations. Neither the heterogenic sequences included in the fungal isolates, nor the different combinations of these sequences, separated the populations of A. areolatum associated with different wasp species. Despite the heterogenic nature of this DNA region in some isolates, RFLP analysis was used effectively to distinguish between the different species of Amylostereum. The work presented in this thesis represents the first molecular. view of the phylogeny of the genus Amylostereum, as well as that of some of the Amylostereum spp. associated with woodwasp species. It is clear from Chapter 5 that these findings now provide a powerful tool to give a clearer picture of the taxonomy and evolution of these fungi, as well the ecology of their symbiosis with woodwasps. The study of the genetic structure of the fungal populations associated with woodwasps also gives new insight into the geographical origin and history of both the insects and their associated fungi.
Open Access
Diseases of Acacia mearnsii in South Africa, with particular reference to ceratocystis wilt
(University of the Free State, 1998-12) Roux, Jolanda; Wingfield, M. J.
English: The Acacia mearnsii industry is a relatively small, though very profitable industry in South Africa. Wood derived from A. mearnsii is currently in greater demand than that of either pine or eucalyptus in South Africa. Despite the importance of this industry, very little attention has been given to the genetic improvement, disease tolerance or general improvement of A. meamsii as a forestry species. The result has been that, during the last few decades, pathogens have become adapted to, and spread through plantations of this tree. Although relatively little research has been conducted on the impact of pathogens on A. mearnsii, this situation has changed during the past nine years, and particularly since the identification of Ceratocystis wilt. The planting of exotics has many advantages over native plants. In South Africa, exotic forestry species, such as Eucalyptus spp., Pinus spp. and A. mearnsii were introduced to halt the uncontrolled logging of native forests. These native forests were logged mainly for furniture and building material, but also for fuel wood, resulting in the near complete destruction of South Africa's native forests. The introduced exotics prevented the further destruction of these forests and soon became a large industry. This was particularly due to the fact that it was found that they also had a superior growth rate when compared to native species. This accelerated growth rate brought rapid results from breeding trials and, thus, a relatively rapid improvement of the material planted. Because they had been separated from their natural enemies, these trees were also initially disease free. The A. mearnsii industry has, and will continue, to face many problems and challenges from pests and diseases. After the initial phase in which the tree was removed from the pathogens affecting it in it's native range, it faced attacks by native South African pests and diseases. These can spread from native Acacia species, or from any other native plants in the same, or even different families. Exotic, mono culture industries are also constantly under threat from the introduction of pathogens from other countries, including the country of origin. This can be done by the introduction of new germ plasm or on any other plant species or plant material brought into a country. Because A. mearnsii is now planted as a monoculture, in contrast to it's native situation, diseases and pests can potentially be much more severe and will spread more rapidly and widely throughout even aged and genetically uniform stands. Propagation of A. mearnsii has, recently, advanced considerably and this is concurrent with increased demand for this wood on world markets. Lessons learned from eucalypt and pine forestry need, however, to be heeded to save unnecessary losses and time. With the advent of vegetative propagation of A. mearnsii in South Africa, it is important to include disease screening trials at the early stages of the development of clones. In order to do this, a knowledge of all possible pathogens of A. mearnsii is needed. This includes pathogens known in South Africa and those that occur beyond the borders of the country. It is also necessary to have a detailed knowledge of the biology and population structure of these pathogens in order to gain an impression of the possible success of control measures. This thesis is a compilation of work conducted on some of the known pathogens of A. mearnsii in South Africa. It also includes a large component dealing with the identification and clarification of previously unknown pathogens of A. mearnsii. It, therefore, does not focus only on diseases of A. mearnsii, but includes a chapter on a disease of Eucalyptus. The causal agent of this disease has, however, also recently been found on A. mearnsii in South Africa and this chapter aims at elucidating the possible origin of the isolates from South Africa. It also illustrates the potential threat of this pathogen to the A. mearnsii industry. South Africa is a semi-arid country that regularly suffers from severe drought. Forestry activities in the country are also mainly restricted to areas with poorer soil and where agriculture cannot be pursued on a profitable basis. Factors such as drought, hail, frost and sub-optimal soil conditions can all contribute to increased stress on trees. Under these conditions, many fungi can act as opportunistic pathogens, causing large scale losses. They often live as endophytes within their hosts, not causing any negative affect until the onset of stress. At this stage, they spread throughout trees, preventing them from recovering from the stress condition and leading to cankers and tree death. Careful management, particularly site/species matching, is required to minimise losses caused by these pathogens. This thesis provides a basis for future research on the development of management strategies to control diseases of A. mearnsii in South Africa. Information, however, also provide valuable knowledge for forestry industries outside South Africa by highlighting the threat of exotic pathogens and the importance of strict quarantine measures to prevent the spread of pathogens. This is true for the movement of not only A. mearnsii material, but as was seen here, the movement of any forestry products, since many pathogens have a wide host range. Although the thesis is comprised of a series of individual entities, these all provide information regarding the hygiene of A. mearnsii plantations. This thesis thus aims at identifying future focus points for intensive research, while at the same time focusing on those pathogens that have been known to the South African industry for a longer period of time. Chapter one provides a review of the available literature on diseases affecting not only A. mearnsii, but also other Acacia spp. important to the forestry industry, world wide. It also highlights some of the uses of these species in the countries where they are planted. The multi-purpose use of Acacia spp. is an important aspect emerging from this review. In many countries, Acacia spp. are not only planted as forestry species but are also used for soil reclamation, nitrogen fixation and fodder. The main focus of the chapter, however, is on the A. mearnsii industry in South Africa, with a brief discussion on all the diseases currently known to occur in the country. It is concluded that much research is still needed to reduce the impact of these diseases and to ensure that the Industry functions optimally. Ceratocystis albofundus must be considered as one of the most important pathogens of Acacia spp., world-wide. Currently this pathogen occurs only in South Africa, but if it is to spread to other countries, large scale losses will be incurred. It may also affect, not only A. mearnsii, but most likely many other plant species. Breeding programmes for A. mearnsii in South Africa focus strongly on this pathogen. In Chapter two, the population diversity of C. albofundus was investigated and compared with data for other Ceratocystis spp., using nuclear and mitochondrial DNA fingerprinting. It was found that the C. albofundus population has a greater genetic diversity than any of the species with which it was compared. This will thus mean that intensive breeding programmes will be necessary to ensure durability of disease tolerance. It also supports previous hypotheses that C. albofundus is native to South Africa and may be a temperate species, not found in tropical areas where its close relative, C. fimbriata, commonly occurs. The first unequivocal report of C. fimbriata and Ch. elegans from A. mearnsii is presented in Chapter three. Both these fungi were isolated from dying trees with typical symptoms of Ceratocystis wilt caused by C. albofundus. Both were shown to be capable of causing disease to seedlings under green house conditions. It was, however, found that C. albofundus is more virulent than either Ch. elegans or C. fimbriata. Both isolates were identified using molecular and morphological approaches. Unfortunately only one isolate of each exists and surveys to obtain additional samples continue to be a priority. The first report of a wilt disease of Eucalyptus, caused by Ceratocystis fimbriata in the Republic of the Congo in West Africa is recorded in Chapter four. This is not only the first report of C. fimbriata as a pathogen of Eucalyptus in Africa but is also one of the few unequivocal reports of this fungus from the continent. Pathogenicity of C. fimbriata on Eucalyptus spp. was confirmed in glass house tests. In this Chapter, C. fimbriata and C. albofundus from A. mearnsii, and C. fimbriata from Eucalyptus in Brazil were also compared to the C. fimbriata from the Congo. Comparison of the lTS region of the rRNA operon showed that isolates from all three areas grouped together in a clade of C. fimbriata, separate from European isolates. Sequence data showed that C. fimbriata from A. mearnsii in South Africa is nearly identical to the fungi from Eucalyptus in Brazil and Congo, suggesting that they may have a common origin. These findings stress the importance of sound quarantine measures to prevent the introduction of potentially devastating pathogens to South Africa. It is not yet known why C. fimbriata has not caused more diseases on A. mearnsii or Eucalyptus spp. in the country, but the situation will need to be monitored closely. Apart from C. albofundus, there are many other fungi that cause disease of A. mearnsii in South Africa. Chapter five reports on a species of Seiridium that was isolated from stem cankers on A. mearnsii. Morphological and molecular comparisons, as well as pathogenicity studies have shown that the species from A. mearnsii is similar to those species responsible for Cypress canker in many parts of the world. It also confirms previous reports that the taxonomy of the three Seiridium spp. causing cypress canker needs re-evaluation, since molecular data support the view that the three species, represent a single taxon. Pathogenicity trials on mature Cuppressus lusitanica and on A. mearnsii trees showed that both the cypress and A. mearnsii isolates are capable of causing lesions on both hosts. Many of the fungi isolated from diseased A. mearnsii during the current and previous studies of diseases resulted in the isolation of fungi, commonly found as latent pathogens on other forest trees. Chapter six encompassed a survey of the endophytic fungi of A. mearnsii, with the specific aim of identifying possible pathogens. Thirty different fungal taxa were found as endophytes of the xylem and rachi. These included F. graminearum and Botryosphaeria dothidea, which are known pathogens. During periods of environmental stress, these fungi can apparently cause disease. This is especially true because A. mearnsii is often planted on marginal sites in South Africa. Chapter seven represents the first report of Fusarium graminearum from A. mearnsii and presents evidence for the fungus being involved in disease of A. mearnsii. This pathogen was first isolated during 1994-95 disease surveys, but was not identified due to the fact that cultures on artificial media did not sporulate. In the current study, additional isolates were obtained from stem cankers and die-back symptoms and the fungus was identified based on β-tubulin gene sequences. Field inoculations using F. graminearum showed extensive lesion formation in the xylem. Previously, this Fusarium sp. was known only as a pathogen of maize and wheat in various parts of the world. Results of this study are, therefore, enigmatic and intriguing.
Open Access
Molecular taxonomy and mating type genes in Ceratocystis sensu stricto
(University of the Free State, 1999-01) Witthuhn (née Strydom), Regina Cornelia; Wingfield, B. D.; Wingfield, M. J.; Harrington, T. C.
English: Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants. In the research presented in this thesis, I have developed a rapid and reliable PCR-based RFLP identification method for species of Ceratocystis. A 1.6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small and large sub-unit rRNA genes, the 5.8S rRNA gene and the internal transcribed spaeers 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (AluI, DraI, HaeIII and RsaI) produced RFLPs that separated the species of Ceratocystis. The 1.6 kb PCR products, from the better known species of Ceratocystis, were sequenced and phylogenetically analyzed. The delimitation of taxa was consistent with results of previous studies using isozymes and rDNA sequence analysis. Ceratocystis coerulescens is a well-known cause of blue stain in spruce and pine. It was shown that C. coerulescens encompasses at least five morphological types. I compared isolates of C. coerulescens sensu lato and morphologically similar species on the basis of lTS DNA sequences. A 600 bp fragment within the ribosomal DNA operon, including the 5.8S rRNA gene and ITS 1 and 2, was amplified, sequenced and analyzed using PAUP. The five morphological types previously known as C. coerulescens, and the two other taxa from conifers, formed a strongly supported monophyletic group that includes all the Ceratocystis species occurring primarily on conifers. The species from hardwood trees, C. eucalypti, Ch. australis and Ch. neocaledoniae, also formed a monophyletic group, sister to the conifer group. The fourth species from hardwoods, C. virescens, formed a group basal to the two sister groups. The phylogeny of species in the C. coerulescens complex based on ITS DNA sequences were compared to the phylogeny based on the MAT-2 HMG box DNA sequences. A 210 bp PCR fragment of part of the MA T-2 HMG box of species in the C. coerulescens complex was amplified. C.fimbriata was used as the outgroup taxon and was distinct from the other Ceratocystis species studied. The species from conifers formed a single clade, sister to the clade formed by the species from hardwoods. Phylogenetic analysis of the MAT-2 HMG box DNA sequences differed slightly from the phylogenetic analysis based on ITS DNA sequences. Based on ITS DNA sequences C. vireseens was basal to the other species in the C. coerulescens complex, while C. laricicola and C. polonica could not be separated from each other. Differences in the MAT-2 HMG box DNA sequences for the latter two species clearly showed them to be as distinct from each other. Recent mating studies on C. coerulescens have prompted a study of the expression of mating type genes in species of Ceratocystis sensu stricto. C. eucalypti is strictly heterothallic. Most other Ceratocystis species, including C. virescens, C. coerulescens and C. pinicola are homothallic. The MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. Part of the MAT-2 idiomorph in C. eucalypti, C. vireseens and C. pinicola was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA binding motif. The expected ~300 bp PCR products were cloned and sequenced. Specific primers were designed that amplified a 210 bp fragment only in MAT-2 isolates of C. eucalypti, C. vireseens and C. pinicola. This fragment was absent from the self-sterile (MAT -1) progeny of C. vireseens and C. pinicola, confirming the deletion of MAT-2 during uni-directional mating type switching. The known DNA sequence data for the C. eucalypti MAT-2 mating type idiomorph was increased from 280 bp to 1 371 bp, using TAIL-peR and uneven PCR.
Open Access
Oomycetes associated with citrus and eucalypts root rot in South Africa
(University of the Free State, 1999-03) Maseko, Bongani; Coutinho, Teresa; Wingfield, Michael J.; Wolfaardt, Francois
English: Research conducted in this thesis explores the role that Phytophthora and Pythium species play in citrus and eucalypt root rot in South Africa. The first chapter presents an extensive literature review with special emphasis on the importance of Phytophthora and Pythium spp. to the South African Citrus and Forestry Industries. This chapter is divided .into two parts. Part one deals with biotic and abiotic factors that contribute to citrus decline. Part two focuses on recently published work on root diseases of exotic forest trees species planted 'commercially in South Africa. Special emphasis was placed on P. cinnamomi since it is thought to be the most important pathogen in the forestry industry. Results obtained in Chapter Two indicate that a wide variation in tolerance and susceptibility to P. cinnamomi exits amongst half-sib families of E. fraxinoides. Field mortality of 52 % and 30% was recorded at two trial sites following natural infection. Seven disease tolerant families were identified with the potential to be used for commercial propagation. Eucalyptus fraxinoides families tolerant to P. cinnamomi were more reliably identified using stem inoculation of young trees relative to selecting families that survived the disease after planting in the .field. In Chapter Three half-sib families of E. fraxinoides and E. smithii were evaluated for tolerance to P. cinnamomi using stem inoculations in the greenhouse. Three replicate trials for each species were conducted at different times of the year. Disease tolerance and susceptibility varied between and among the different E. fraxinoides and E. smithii families. No correlation was found between the results obtained from the replicate trials of E. fraxinoides and E. smithii. The technique used was found to be unreliable for screening E. fraxinoides and E. smithii families for tolerance to P. cinnamomi. Results of a preliminary survey in selected citrus nurseries and orchards in the Northern and Mpumalanga provinces of South Africa are presented in Chapter Five. A total of 320 Phytophthora and Pythium isolates were recovered from the rhizosphere of diseased citrus trees and diseased plant material. Phytophthora nicotianae and Pythium irregulare where the most frequently isolated species. Sixteen isolates of P. citrophthora were successfully recovered. This species has previously been presumed to be absent from the sampled regions. In the last Chapter of this thesis, pathogenicity tests were conducted on Phytophthora and Pythium spp. associated with citrus root rot. Phytophthora isolates proved to be pathogenic when inoculated into citrus fruit while Pythium spp. where either weakly or non pathogenic. Contrary results were found when the lupin assay was conducted since all Pythium spp. proved pathogenic. Phytophthora isolates were also found to be pathogenic on Rough Lemon (RL) and Troyer Citrange (RTC) rootstocks whereas Pythium spp were either weakly pathogenic or avirulent. No difference in susceptibility was observed between RL and RTC. The fruit _assay and stem inoculation techniques were found to be reliable, inexpensive and rapid results were obtained. Results obtained in this study indicated that Phytophthora spp. play an important role in citrus and eucalypt root rot. Artificial stem inoculation using a single virulent isolate of P.cinnamomi proved to be a reliable technique for screening young eucalyptus trees in the field. However, contrary results were obtained when this technique was used to screen half-sib seedlings of E. fraxinoides and E. smithii in the greenhouse. Eight Pythium and two Phytophthora spp. were isolated from the rhizosphere soil and declining citrus trees. Phytophthora species proved to be pathogenic when inoculated into citrus fruit and on Rough Lemon and Troyer Citrange rootstocks. On the other hand Pythium species screened proved to be nonpathogenic or only weakly pathogenic and thus probably play an insignificant role in citrus decline.
Open Access
Determining the canning quality of small seeded white beans (Phaseolus vulgaris L.)
(University of the Free State, 1999-05) De Lange, Anna Francina; Osthoff, G; Labuschagne, M. T.
English: It is important to the producers, processors and consumers in south Africa to have dry bean cultivars with acceptable canning quality. Therefore, dry bean breeders needs sui table screening methods to evaluate the various lines at an early stage (F4) when only small amounts of seed are available. A micro-canning method to evaluate canning beans .in tomato sauce has been developed and compared to the commercial processing procedures with comparable results. External factors that influenced the canning quality like the water quality were investigated. This micro-canning method could therefore be used to investigate the effect of genotype and environment interactions that significantly influenced the canning quality. The objectives of this study were to: o Evaluate different qeriet i.c material for use as parents in the breeding program. $ To obtain a better understanding of canning quality characteristics of beans and to ensure that the most important characteristics are evaluated and the component interrelationships. • Determine the genotypic, environmental and genotype x environment interactions that influenced the canning quality. • To ascertain the patterns of interrelationships of the canning quality parameters and chemical analysis. • To investigate the patterns and relationships between standard and choice grade cultivars. e To investigate stability of locali ties between seasons as well as the clustering of different environments and seasons. Small seeded white beans, carioca and yellow haricot beans were used to determine variability in canning quality but as a result of a lower canning quality of coloured beans, only small seeded white beans were used for further investigations. As result of the investigations of different a characteristics, only the seed size, water absorption during soaking and canning, the texture and subjective visual appearance evaluations were used to determine canning quality. These characteristics were interrelated but single no parameter could explain variation' in canning quality. Canonical correlation analysis was used to determine to what extent variation of chemical components was responsible for differences in canning quality and these results indicated that mainly potassium and calcium would influence the water absorption and texture, respectively. Canonical variate analysis was used to determine the difference between unacceptable, standard and choice grade cultivars. A model was described from these analysis that could be applied to independent data sets that results in coordinates that differentiates the lines or cultivars as unacceptable, standard and choice grade. Significant interactions between genotype, environmental and seasonal effects for canning quality traits indicated that cultivar responses to variation in localities and seasons differ. Environmental effects resulted in inconsistent quality measurements since trait expression is strongly influenced by genotype x environment interactions. Results from this study suggested that the difference between cultivars could therefore be due to a complex interaction of the chemical and structural composition, which is genetically determined and influenced by the anv i ronmen t . as well as the changes that occur during processing. The Additive Main Effects and Multiplicative Interaction (AMMI) model mainly grouped KwaZulu-Natal separately as a region with poor canning quality. The rest of South Africa's localities grouped different for each season. Resulting from this investigation, several recommendations can be made: c Breeding material should be tested fo~ more than one season in order to select superior and more stable lines. o Elimination of canning quality evaluations of KwaZulu-Natal could improve the genetic progress. • Exploitation of the interactions by breeding for specific adaptation in a region of homogeneous area. Demarcating one or two areas in South Africa for the exclusive production of small white canning beans could improve the overall canning quality of the small white bean production in South Africa.
Open Access
Utilization of wood-decay fungi for biokraft pulping of softwood
(University of the Free State, 1999-05) Wolfaardt, Jacobus Francois; Rabie, C. J.; Wingfield, M. J.
English: The forest products industry is one of the most important earners of foreign exchange for South Africa. The major focus of the industry is the production of pulp with an annual capacity of 2,4 million tons. Wood from plantations of exotic trees is the most important source of fibre, but other fibre sources are also used. Biotechnology can play a significant role in the industry to produce high value products at lower cost and could reduce the environmental impact of conventional processes. Biopulping is potentially the most important of these biotechnological processes, because it can influence all downstream operations. The aim of this study was, therefore, to develop a biopulping process for the treatment of softwood at the Sappi Ngodwana kraft mill in Mpumalanga. Initially, 278 strains of wood-decay fungi were collected from various natural habitats. This collection represents a diversity of fungal families and included species that have not previously been recorded from South Africa. The first step in selecting suitable fungal strains for biopulping was to characterize different groups on the basis of the enzymes that they produce and their oxidase reactions. The suitability of these strains for the pre-treatment of softwood chips prior to kraft pulping was subsequently assessed by evaluating their influence on kappa number, yield and strength properties of pulp. Seven of the strains tested were able to reduce the kappa number of pulp significantly, without having a significant influence on the pulp yield. These strains were more efficient than strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora that have been patented for other biopulping applications. Treatment of wood with strains of Peniophora sp., an unidentified specie as well as two strains of Stereum hirsutum resulted in pulp with improved strength properties. The envisaged biopulping process aimed at treating wood chips in outside chip storage with a biopulping fungus. The aim of one study was to investigate conditions such as temperature, moisture, CO2 and microbial populations that develop in a chip pile, and to determine the suitability of the chip pile for colonization by biopulping fungi. High temperatures and high moisture levels were observed in some areas of the chip pile, which suggested that part of the pile was unsuitable for colonization by mesophilic fungi. It will, therefore, be necessary to manage the chip pile to maintain a suitable environment for biopulping. Problems were experienced with poor colonization of freshly chipped softwood by biopulping fungi. The effect of contaminating microbes and inhibitory compounds present in wood was, therefore, studied. It was found that the inhibition of biopulping fungi by α-pinene and by contaminating microbes were both very important. The inhibition by microbes, as well as by extractives, was mitigated by a short steam treatment of wood chips. Steaming for ten minutes under atmospheric pressure could be an economical method to improve colonization by biopulping fungi. Alternatively biopulping fungi with good competitive ability and tolerance to monoterpenes could be selected. Pinus patula wood chips were pre-treated with a selected strain of Stereum hirsutum to determine the optimal conditions for the kraft pulping of pre-treated softwood and to do an economic evaluation of the process. Chips were pulped on a small scale under various pulping conditions. Lignin content, yield, and viscosity of the pulp as well as alkali consumption were evaluated. The results were used to develop models for biokraft pulping. This study showed that biopulping can reduce the kappa number of pulp or reduce the pulping time. Pulp yield losses were relatively small when pulps with low kappa number were produced. Increased alkali consumption was, however, an important factor in the economic evaluation.
Open Access
Factors associated with coniothyrium canker of Eucalyptus in South Africa
(University of the Free State, 1999-05) Van Zyl, Leonel Merwe; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.
English: English: In chapter one of this thesis, the literature pertaining to the genus Coniothyrium and its importance in plant pathology, is reviewed. Special attention is given to Coniothyrium species associated with Eucalyptus but the focus is on Eucalyptus stem canker pathogen, C. zuluense. Coniothyrium zuluense is an important pathogen in South Africa and has, since its discovery, become widespread throughout plantation areas of KwaZulu-Natal. The current means for reducing the impact of this disease is to plant disease resistant species and clones of Eucalyptus. It is evident from this review that very little information is available pertaining to the biology, reproductive system, or the population structure of C. zuluense. Such information is essential for managing the disease successfully in the future. The strategy currently used to reduce the impact of Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Results of the study represented in chapter two showed that there is considerable variation in colony colour and pathogenicity of a large collection (344) of C. zuluense isolates. Conidial morphology and growth requirements are, however, similar for all isolates tested. The considerable variation in pathogenicity indicates that C. zuluense has been present in South Africa for an extended period of time, or that virulence is changing rapidly due to strong directional selection pressure. Results of the taxonomic and pathogenicity studies in chapter two, suggest that the C. zuluense population is well established. In chapter three, the population diversity of 108 C. zuluense isolates, differing in their pathogenicity to a susceptible Eucalyptus clone, was investigated using Amplified Fragment Length Polymorphism (AFLP) technology. Results indicated that the level of genetic diversity is relatively low, but higher than expected for an asexually reproducing pathogen. Genetic similarity values also indicated a significant population differentiation between different plantation regions (subpopulations), suggesting that gene flow, together with selection, might be responsible for most of the gene diversity. New epidemics would, therefore, not be as a result of the emergence of new aggressive strains, but would rather be due to the introduction of susceptible Eucalyptus species, together with environmental conditions favouring disease development. A Coniothyrium species associated with similar symptoms to those associated with C. zuluense in South Africa was observed on E. camaldulensis in Thailand in 1996. It was previously thought that C. zuluense was restricted to South Africa. In chapter four, I show using morphological and molecular comparisons, as well as pathogenicity studies, that C. zuluense and the Coniothyrium sp. from Thailand are the same organism. This is, thus, the first record of this important Eucalyptus stem canker pathogen, C. zuluense, outside South Africa. Bacteria commonly exude from necrotic cankers on severely infected Eucalyptus clones in plantations. In chapter five, it was shown that bacteria associated with Coniothyrium canker in the field are species of the genus Pantoea. These species were identified based on 16S rDNA sequence data as P. ananatis pv. ananatis and a species closely related to P. stewartii subsp. stewartii. It was also shown that a synergistic interaction between C. zuluense and both Pantoea species exists. Inoculation studies, using both Pantoea species together with C. zuluense isolates, resulted in a significant increase in pathogenicity as opposed to inoculations where the bacterial and fungal isolates were used alone. Future studies should consider the presence or absence of both bacteria species in disease development in Thailand. During plant-pathogen interactions, pathogens are known to produce cell wall degrading enzymes, in particular pectin degrading enzymes. Polygalacturonase (PG) is the first enzyme produced during such interactions and is known to be a determining factor in pathogenicity. Chapter six showed that C. zuluense isolates and both Pantoea species, P. ananatis pv. ananatis and an unknown Pantoea sp., produce PG. Experimental assays show that levels of PG activity for both Pantoea spp. are significantly higher than those obtained for C. zuluense isolates. As PG is the first enzyme produced during disease development it is hypothesised that the two Pantoea species might play a significant role in the development of Coniothyrium canker. Production of PG could also be used as an assay to evaluate pathogenicity in different isolates of C. zuluense. Pathogen-produced cell wall-degrading enzymes play a key role in activating plant defence responses. Most inducible defence responses are the result of transcriptional activation of genes. Various plant resistance (R) genes, as well as pathogenesis-related proteins, such as polygalacturonase inhibiting proteins (PGIPs), have been linked with resistance to various fungal and bacterial pathogens. In chapter seven, a partially sequenced resistance gene from disease resistant E. grandis clone, TAG 5, was shown to be similar to a gene associated with a disease resistance gene in Arabidopsis thaliana. The most exciting aspect of this study was, however, the discovery of a shift in reading frame of this gene for the susceptible Eucalyptus clone, ZG 14. The complete sequence of this gene should provide a more complete view of its importance in disease resistance. Screening for similar interruptions in the open reading frame of various commercially available Eucalyptus clones could significantly speed up breeding programmes aimed at producing improved disease resistant clones.
Open Access
Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins
(University of the Free State, 1999-06) Botes, Adriana Leonora; Smit, M. S.; Litthauer, D.
English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).
Open Access
The hydrolysis of linalyl acetate and α-terpinyl acetate by yeasts
(University of the Free State, 1999-06) Thomas, Elias; Smit, M. S.; Litthauer, D.
English: Hydrolysis of esters by means of hydrolases such as proteases (Jones and Beck, 1976), lipases (Santiello et al., 1993) and esterases (Boland et al., 1991) has become a well established method for the resolution of racemic mixtures. The first aim of the present study was to screen the yeast culture collection of the University of the Orange Free State for yeast isolates which can be used for the enantioselective hydrolysis of rac-linalyl acetate and rac-α-terpinyl acetate, which are tertiary alcohol ester terpenes, respectively. We screened 74 yeast strains from 17 genera as well as 29 unclassified isolates. Approximately 16% of the strains screened contained tertiary alcohol hydrolase activity. Whole cell experiments, enzyme purification and characterisation were attempted on one of the hydrolases of interest obtained from Trichosporon sp. UOFS Y-0117. Whole cell experiments on reported optimal hydrolase activity in the presence of 1% maltose in a defined media (YNB). The effect of various co-solvents was also documented with a low concentration of ethanol (2.4% v/v) producing superior hydrolase activity. No toxicity, to the microbe, was observed by rac-linalyl acetate (up to 200mM) due to the cell membrane present. The use of digitonin proved that substrate transport across the cell membrane is not a reaction rate determining step. The re-usability experiment showed a significant decrease in hydrolase activity (ca 30% after 1 cycle) as the same batch of cells was exposed to substrate and product. The results also show an optimal pH of 7.5 and temperature of 30°C which coincides with physiological conditions and literature. Protein purification was attempted on a cell free extract once we determined that the hydrolase was intracellular. Small scale evaluations of different chromatographic resins ranging from ion exchange, hydrophobic interaction and affinity chromatography followed. Large scale experiments with gel filtration resins were also attempted. Purification steps were largely unsuccessful and we decided to continue with a DEAE fraction which produced a superior yield (244%). Characterisation experiments, using the DEAE active fraction, followed in which we explored the effect of rac-linalyl acetate concentrations. Enzyme inhibition and protein denaturation at low rac-linalyl acetate concentrations (detected at ca 65-100mM) is significant compared to whole cells (not detected at 200mM). This hydrolase is also an esterase. Specific amino acid modification reagents results indicate the presence of a serine and histidine amino acid present in the catalytic centre i.e. the hydrolase belongs to the serine hydrolase family. A metal chelating reagent EDTA and various metal cations had no effect on hydrolase activity. pH-stability experiments indicate a pH of 7.5 to be optimal for the retention of hydrolase activity in whole cells and crude enzyme preparation. Thermostability experiments show whole cells are four times more stable than the crude enzyme preparation at 4°C. The energy of inactivation required for activity loss is lower in whole cells (47.43kJ) compared to crude enzyme preparation (91.77kJ). The probability for an event to occur which causes inactivation is relatively low (1.8075 events/h). The converse applies to the crude- enzyme preparation (1.17514 events/h). Thus the crude enzyme is 6x108 less stable than the hydrolase present in the whole cell.
Open Access
Mycotoxin contamination of maize in relation to insect infestation, agricultural practices and agroecology in the Republic of Cameroon
(University of the Free State, 1999-09) Ngoko, Zachee; Marasas, W. F. O.; Wingfield, M. J.; Cardwell, K. F.
English: Maize (Zea mays L.), the staple food crop of the majority of the population of Cameroon, is damaged by insects and diseases from the fields to the stores. As a result, the quantity and the quality of harvested grain is reduced. This study was undertaken to identify constraints associated with the production and post-harvest losses of this commodity in two ecological zones ofCameroon from 1995 to 1997. Farmers' perceptions of diseases and pests play an important role in their acceptance of new pest management technologies. From the survey conducted to assess their perceptions, farmers reported that borers (Busseola fusca) were the main constraint to maize production in the Humid Forest and Western Highlands. Locusts (Zonecerus variegatus) and rodents were the second most important limiting factor in the Humid Forest. The storage weevil (Sitophilus zeamais) was the most damaging storage insect. Diseases were not generally known by farmers who could only recognize smuts and ear rots by the visible damage caused by them. While the period of the outbreaks of insect infestation was not reported with precision, most farmers reported that diseases occurred at the mid-season. Control practices were not well established. Disease surveys conducted from 1995 to 1997, revealed that lowland blight (Bipolaris maydis, Diplodia leaf spot (Stenocarpella macrospora) and sheath blight (Rhizoctonia solani) were the most important maize diseases in the Humid Forest, while highland blight (Exserohilum turcicum) and grey leaf spot (Cercospora zeae-maydis) prevailed in the Western Highlands. Phaeosphearia leaf spot (Phaeosphaeria maydis) was specific to the Western Highlands with a negative relationship with grey leaf spot. Busseola fusca infested maize plants at all stages of growth with high prevalence in the Humid Forest. The identification of factors -affecting maize yield demonstrated that diseases, insects and their interactions with soil infertility, soil texture, weeds, and maize varieties were responsible for the reduction of maize production. Yield reductions were 30% and 33.6% respectively, in the Humid Forest in 1995 and 1996 due to Stenocarpella macrospora, Puccinia polysora and Rhizoctonia solani. In the Western Highlands, Cercospora zeaemaydis, Busseola fusca, stem diseases, and physiological spot caused yield reductions of 51.2%, and 37.9% in 1996 and 1997, respectively. Mycological and chemical analyses of maize grain collected from 72 farmers' stores showed that several pathogens were associated with grain quality deterioration. Nigrospora spp. were the most frequently isolated fungi on kernels, followed by Fusarium moniliforme and Fusarium graminearum. Aspergillus spp. were rare in both zones. Fumonisin B1, deoxynivalenol and zearalenone were detected in maize samples at levels ranging from 300 to 26,000ng/g, 100 to 1300 ng/g, and 50 to 110 ng/g, respectively. This is the first report on the natural occurrence of these Fusarium mycotoxins in maize in Cameroon. Surveys conducted to identify the biological and physical factors that enhanced the infection of maize kernels by fungi and the contamination with fumonisin , identified several agricultural techniques related to grain quality in the Western Highlands. Harvesting in June (11.1 %) or July (23.6%), sorting right from the field (16.7%), drying over the fireplace with husk (19.4) or without husk (33.3%) and storing shelled grain in bags (19.4%) or boxes (9.7%) reduced fumonisin contamination. Continuous production of maize on the same field, harvesting in August, and the infestation by the weevil Sitophilus zeamais were factors that increased fumonisin contamination. Crop rotation, sorting maize during all the post-harvest processes and the treatment of maize grain with appropriate insecticides should decrease the risk of contamination by fumonisin. Continuing collaborative research should aim at understanding farmers' needs and priorities, investigating the epidemiology of maize diseases, screening for resistance to the most important maize diseases and improving harvesting, sorting, drying and storing methods in Cameroon.
Open Access
Mucoralean fungi present in soil from arid regions in South Africa
(University of the Free State, 1999-11) Seabi, Buti Oscar; Botha, A.; Viljoen, B. C.
English: The aim of the first part of the study was to investigate the ecological niche of mucoralean fungi in arid soil, with specific reference to the position these fungi occupy in the biogeochemical cycle of nitrogen. Consequently, selected mucoralean taxa occurring frequently in soil habitats, including strains from culture collections, as well as isolates obtained from a soil sample from arid Upper Nama Karoo, were used to evaluate in vitro growth to determine nitrogen sources and aw tolerances. Nine mucoralean fungal genera including 18 species were examined for the ability to utilise a series of nitrogen containing compounds and to grow at an aw of 0.955 on solid media. The nitrogen concentration in the media was 0.1 g.r1 and the series of nitrogen containing compounds were ammonium chloride, asparagine, sodium glutamate, sodium nitrite and potassium nitrate. The genera were Actinomucor Schostak., Backusella Hesselt. & J.J. Ellis, Cunningha'fnella Matr., GongronelIa Ribaldi, MortierelIa Coem., Mucor Fresen., Rhizomucor Lucet & Costantin., Rhizopus Ehrenb. and Thamnostylum Arx & H. P. Upadhyay. Thirty-nine fungal strains obtained from culture collections (CBS, MUFS and PPRI), as well as 12 soil isolates from the Karoo, were tested. All the species and strains tested in this study were able to utilise asparagine and glutamate. Strains belonging to CunninghamelIa, Mucor racemosus Fresen., Rhizopus microsporus Tiegh. and Rhizopus stolonifer (Ehrenb.: Fr.) VuilI. were unable to utilise ammonium chloride. Strains of CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus microsporus and Rhizopus stolonifer were unable to grow on nitrate as sole nitrogen source. Nitrite was found to be toxic to species belonging to CunninghamelIa, MortierelIa, Rhizomucor, Rhizopus and Thamnostylum. Members of GongronelIa, MortierelIa, Mucor racemosus, Rhizomucor and Thamnostylum were unable to grow at an a, of 0.955. The aim of the second part of the study was firstly to get an indication whether the mucoralean diversity of the Karoo, as observed in the first part of the study and in the records obtainable from literature, differs from data on mucoralean diversity from other arid regions. The latter included data from literature and what could be found in a soil sample taken from Kimberley Thorn Bushveld. Secondly, the aim was to test the isolates obtained from the Kimberley Thorn Bushveld soil sample in order to further explore the ability of mucoralean fungi to utilise the above mentioned series of nitrogen sources and to grow at an a, of 0.955. In addition, selected mucoralean taxa occurring frequently in soil habitats were tested for the ability to survive elevated temperatures in soil. It was found that the following species of the Mucorales may be encountered in the arid soil of the Karoo; Actinomucor elegans, CunninghamelIa echinulata, MortierelIa isabellina, Mucor circinelloides, Rhizomucor species, Rhizopus oryzae Went. Prins. Geerl. and Rhizopus stolonifer. Future surveys would reveal if genera like Absidia, GongronelIa and Zygorrhynchus, which have been isolated from arid regions, also occur in Karoo soil. Representatives of mucoralean taxa occurring in arid Karoo soil were able to utilise organic as well as inorganic oxidised nitrogen sources. However, at the concentration tested in this study, nitrite was found to be toxic to representatives of CunninghamelIa, Mortierell8, Rhizomucor and Rhizopus. Nitrate could not be utilised by CunninghamelIa, MortierelIa, Rhizomucor and Rhizopus stolonifer. Whether this inability to utilise inorganic nitrogen sources would prevail 'during oligotrophic growth in soil, remains a question to be addressed by future research. Representatives of the above mucoralean taxa occurring in arid soil were able to survive 55°C for 14 h in soil.
Open Access
An isolation procedure for arachidonic acid producing mucoralean fungi
(University of the Free State, 1999-11) Paul, Ida; Botha, A.; Kock, J. L. F.
English: Soil plates with malt extract agar and an incubation temperature of 5°C were used to selectively isolate representatives of the genus MorfierelIa from Alti Mountain Grassland soil. Fungi in the soil sample able to grow under these conditions amounted to a total of 2640 colony forming units per gram soil. Circa 94% of the total fungal isolates represented MorfierelIa subgenus MorfierelIa. The rest of the colony-forming units consisted of Mucor isolates (6.0%) and higher fungi (1.5%). Subsequently, the total lipids were extracted from the MorfierelIa isolates after cultivation on malt extract gelatine (MEG). The lipids were methylated using trimethyl sulphonium hydroxide and the methylated fatty acids in the lipids were identified using gas chromatography. The percentage arachidonic acid [20:4(ω6)], relative to other cellular long-chain fatty acids, was calculated. All the MorfierelIa strains isolated at 5°C from the Alti Mountain Grassland soil sample were found to produce 20:4(ω6). In the next part of the study, the radial growth rate on MEG was determined at 5°C and 20°C, for these MorfierelIa strains originally isolated at 5°C from Alti Mountain Grassland soil. To compare the growth of these strains with growth of other MorfierelIa strains, the radial growth rate at 5°C and 20°C was also determined for culture collection strains of MorfierelIa isolated at 25°C from Dry Sandy Highveld Grassland soil. In addition, 20:4(ω6) production in the 25°C isolates of MorfierelIa on MEG at 20°C was compared with the production of 20:4(ω6) in the MorfierelIa strains originally isolated at 5°C from Alti Mountain Grassland soil. The results indicated that all of the strains of MorfierelIa subgehus MorfierelIa originally isolated at 25°C and 5°C,were psychrotrophic and hence capable of growth at 25 °C, 20°C and 5°C. The results further indicated that the low temperature isolation procedure would be as suitable to isolate 20:4(ω6) producing MorfierelIa from soil, than isolation procedures utilizing complex media and 25°C as incubation temperature. However, this initial screening for 20:4(ω6) production was conducted on cultures grown on solid media (MEG). Consequently, four strains of M. alpine, which produced the highest percentage 20:4(ω6), were selected for further testing. Two of these strains were originally isolated at 5 °C from Alti Mountain Grassland soil and two were culture collection strains originally isolated at 25 °C from Dry Sandy Highveld Grassland soil. A reference strain of M. alpina obtained from an internationally recognized culture collection was also included in these experiments. Arachidonic acid accumulation in the neutral lipids of submerged cultures, were determined using two liquid media commonly used for this purpose. The media were Glucose Yeast Extract (GY) medium, and Hansson and Dostalek (HD) medium. Lipid analyses were conducted by harvesting fungal biomass in the stationary phase using filtration. The biomass was freeze dried and the total lipids extracted. The lipids were fractionated using column chromatography and the fatty acids of the neutral lipids were analyzed as methyl esters using gas chromatography. The volumetric concentration of 20:4(ω6) in the neutral lipids of each culture was subsequently calculated. The accumulation of 20:4(ω6) in M. alpina, originally isolated from Alti Mountain Grassland soil at 5 °C, was comparable to 20:4(ω6) accumulation in the reference strain obtained from an internationally recognized culture collection. However, the highest volumetric concentration of 20:4(ω6) in the neutral lipids obtained with the 5 °C isolates, was significantly lower than the highest volumetric concentration of 20:4(ω6) in the neutral lipids of M. alpina strains isolated at 25 °C from Dry Sandy Highveld Grassland soil. These results, therefore indicate that the low temperature isolation procedure, utilizing malt extract agar and an incubation temperature of 5 °C is not suitable for the isolation of MortierelIa strains producing high volumetric concentrations of 20:4(ω6) in the neutral lipids. However, it should be borne in mind that only five MortierelIa strains were tested for 20:4(ω6) production in this second part of the study. To confirm these results many more strains isolated at 5 °C and 25 °C from different soil habitats should be tested for 20:4(ω6) production in submerged cultures.
Open Access
The production of 3-hydroxy fatty acids by the yeast Dipodascopsis uninucleata and its implications
(University of the Free State, 1999-11) Venter, Pierre; Kock, J. L. F.; Coetzee, D. J.
English: In 1991, a novel eicosanoid namely 3-hydroxy-5, 8, 11, 14-eicosatetraenoic acid (3-HETE) was uncovered in the yeast Dipodascopsis uninucleata by van Dyk and co-workers. Strikingly, the production of this compound was found to be sensitive to low concentrations of aspirin and indomethacin. With this as background, a study was conducted that unveiled the possible biochemical pathway used by this yeast for the production of 3-HETE. Here, various fatty acids were fed to D. uninucleata, and the extracted samples analysed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography - mass spectrometry. It was found that 3-hydroxylation of fatty acids in D. uninucleata requires a 5Z, BZ - diene system either directly or following initial incomplete l3-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy - 5Z, BZ, 11Z, 14Z - eicosatetraenoic acid (3R - HETE), which rules out normal l3-oxidation as a biosynthetic route. Consequently, studies on the biological dynamics and distribution of 3-hydroxy oxylipins in D. uninucleata followed. The occurrence of oxylipins was mapped by immunofluorescence microscopy (IF) in fixed cells, with or without cell walls, using an antibody raised against 3R-HETE. This antibody turned out to cross-react with other 3-hydroxy oxylipins. These compounds were detected in situ in gametangia, asci, as well as between released aggregating ascospores. Aspirin (1mM), which is known to suppress the formation of 3-hydroxy oxylipins from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as cell aggregation, suggesting a prominent regulatory role of 3-hydroxy oxylipins for the latter. Since these oxylipins are associated with the aggregation of sexual cells in D. uninuc/eata, the next step was to screen for the presence of these compounds in aggregating cells of other yeasts such as the biotechnological important Saccharomyces cerevisiae. It was found that oxylipins such as 3-hydroxy-8:0 and 3-hydroxy-10:0 are produced over the growth cycle of the flocculating yeast Saccharomyces cerevisiae ATCC 26602. Using oxylipin specific antibodies in IF studies, it was demonstrated that these compounds are synthesized continuously from an early stage of growth and are associated with the cell wall and are present between flocculating cells. Similar results were obtained with a NewFlo phenotype flocculent brewing yeast strain. This implicated the involvement of oxylipins in cell aggregation. Further investigations using scanning- and transmission electronmicroscopy, indicated that changes in the depositing of lipid rich osmiophilic layers in the yeast followed the same pattern as the IF results. Immunogold studies verified the presence of oxylipins in these osmiophilic layers. It was uncovered that the oxylipin containing osmiophilic layers play an important role in cell aggregation. Surprisingly, further investigations implicated the presence of aspirin sensitive 3-hydroxy oxylipins in the LPS layer of the Gram-negative bacterium Escherichia co/i. In this study it was found that aspirin, at moderate concentrations, influences the biosynthesis of the endotoxic 3-hydroxylated myristic acid in the Lipid A of Gram-negative bacteria. This discovery therefore suggests an important role for aspirin as a therapeutic agent in the treatment of LPS mediated diseases