The dimorphic fungus Yarrowia lipolytica is an n-alkane assimilating yeast. During nalkane oxidation toxic fatty aldehydes are formed that are further oxidized by fatty aldehyde dehydrogenases (FALDH) to carboxylic acids that then enter the β-oxidation pathway. Very little research emphasis has been placed on FALDHs in yeasts and their precise role in n-alkane metabolism. This study aimed at contributing to the limited knowledge of yeast FALDHs and in particular the four putative Y. lipolytica FALDHs (YlFALDH1 - 4) that were recently identified in the fully sequenced genome of Y. lipolytica E150. The contribution made from this study to YlFALDHs was with reference to their promoter expression and subcellular localization. The promoter and terminator of each YlFALDH was initially used to construct reporter cassettes in conjunction with β-galactosidase (lacZ) by utilizing the Sticky-end PCR (SEP) and Enzyme-free cloning methods. These two methods proved to be unsuitable for the expression study. The promoter region of each YlFALDH was then cloned into the pINA781 expression vector containing lacZ to study the expression further. With the aid the pINA781 integrative vector and qualitative plate assays, with 5-bromo-4-chloro-3- indolyl-β-D-galactosidase (X-gal), it was observed that the promoters of YlFALDH1 and 2 were inducible by dodecane and hexadecane but not by oleic acid, glucose or glycerol. The promoter of YlFALDH2 also seemed to display the same level of transcriptional strength as the inducible POX2 promoter. Induction of the YlFALDH3 and 4 promoters was not observed. Localization of the YlFALDH proteins was studied with the aid of green fluorescent protein (GFP) from Aequorea victoria and putative localization sequences (LS) from each YlFALDH isozyme. The putative Y. lipolytica LSs comprised of the last 150 bp of the COOH-terminal of the YlFALDH proteins. These LSs were modeled from Rattus norvegicus FALDH that possesses a 35 amino acid hydrophobic protein anchor at its COOH-terminal. For localization studies, chimerical JMP5 molecules were created with an inducible ICL1 promoter, GFP and putative Y. lipolytica LS from each isozyme. Chimerical molecules were also constructed with a pKOV136 vector that contained a constitutive TEF promoter, a GFP-LS fragment (from a JMP5-chimera) and LIP2 terminator. No fluorescence was observed with epifluorescence or confocal laser microscopy when either of the JMP5- or pKOV136-chimeras were transformed into Y. lipolytica E150 and Po1g respectively. Consequently the subcellular localization could not be identified.

Not available

Acetylsalicylic acid (ASA)-sensitive 3-hydroxy (3-OH) oxylipins were uncovered in 1991 in the yeast Dipodascopsis uninucleata. Since then, various similar oxylipins were found to be widely distributed in fungi. Interestingly, 3-OH oxylipins were reported to play a role in ascospore release from enclosed asci, where they are involved in assisting nano-scale gear-like (D. uninucleata); sliding (Dipodascus); drilling (Eremothecium sinecaudum) and piercing movements (E. ashbyi and E. coryli). In Ascoidea africana, a 3- OH 10:1 oxylipin was found to be associated with hat-shaped ascospores carried inside ellipsoidal asci. However, in this study no function was proposed for this oxylipin. Since only one species representing the genus Ascoidea was studied, it became the aim to further expand this study to also include A. corymbosa and A. rubescens. Using confocal laser scanning microscopy (CLSM) on cells stained with fluorescein-coupled 3-OH oxylipin specific antibodies, this study suggests that oxylipins are specifically associated with ascospores and not vegetative cells of A. corymbosa and A. rubescens. Using gas chromatography - mass spectrometry (GC-MS) the oxylipin, 3-OH 17:0, was identified in A. corymbosa. Here, oxylipin-coated razor sharp ascospore brims may play a role in rupturing the ascus to affect forced release of hat-shaped ascospores. Literature suggests that 3-OH oxylipins are produced by β-oxidation or fatty acid synthesis in mitochondria of yeasts. Since these oxylipins accumulate in sexual cells (asci), increased mitochondrial activity is therefore expected in these structures. Strikingly, this assumption is supported in this study. Using selective fluorescence mitochondrial staining and CLSM, evidence is provided that mitochondrial function is much higher in asci containing increased amounts of 3-OH oxylipins compared to the corresponding asexual vegetative cells. Furthermore, when ASA, a mitochondrial inhibitor, was added in increased concentrations to cultures of Ascoidea, the sexual stage was found to be more sensitive. Ascospore liberation from asci was first inhibited followed by asci formation while some vegetative growth could still be observed.

Psittacine beak and feather disease (PBFD) is a dermatological condition that affects both captive and wild psittacine birds worldwide. In Southern Africa, 10 â 20% of breeding stocks are lost due to the disease each year. PBFD threatens the survival of the indigenous endangered Cape parrot (Poicephalus robustus) as well as the black-cheeked lovebird (Agapornis nigrigenis). The disease is characterized by roughly symmetrical feather loss, feather abnormalities, anorexia and immunosuppression. In advanced cases of the disease beak and claw deformities are present. The causative agent of PBFD is Beak and feather disease virus (BFDV), a circovirus belonging to a diverse group of circoviruses within the family Circoviridae. BFDV has a circular single stranded DNA genome consisting of seven open reading frames (ORFs); three of these ORFs are conserved amongst all isolates of BFDV. ORF 1 encodes the Rep protein, ORF 2 the coat protein (CP) which is also the epitopic protein of the virus and ORF 5, whose function remains unclear. BFDV cannot be cultivated in tissue/cell culture or in embryonated eggs. The inability to cultivate the virus has hampered the development of diagnostic tests and a vaccine as preventative measure against the disease. BFDV is a genetically diverse virus. Researchers have demonstrated that there are at least eight different lineages of BFDV, where Southern African isolates group into three unique genotypes. Many studies have been performed which indicated the diversity of BFDV, but so far no studies have been done which link this genetic diversity to the possibility of the existence of more than one strain of BFDV. This led to the aims of the present study which were to investigate antigenicity of BFDV isolates belonging to different genotypes and then the subsequent bacterial expression of six isolates of BFDV that were genetically different. The entire CP genes of six isolates were amplified with polymerase chain reaction (PCR) and subsequently sequenced. Phylogenetic analysis of sequence data showed that the isolates from this study grouped into lineage one as was described by Heath and co-workers (2004). Amino acid sequences from the isolates from each lineage was applied in an in silico prediction algorithm in order to establish the possibility of more than one strain of BFDV. The predictions indicated that isolates from Australia and South Africa had the same antigenic profile. However, isolate BCL1-ZAM and LK-VIC each produced their own antigenic profile. This indicated the distinct possibility that there is more than one strain of BFDV and that at least one antigenic determinant was situated at the N-terminus of the CP. However, these results have to be confirmed by conducting in vitro studies. Attempts were made to express the full length CP genes of six isolates in BL21(DE3) Escherichia coli with the pET-28b(+) vector. Neither polyacrylamide gel electrophoresis (PAGE) nor Western blotting indicated the presence of recombinantly expressed protein in any of the studies conducted. The codon adaptation index (CAI) for BFDV was calculated to be 0.250, which indicated that the CP had a 25% possibility of being expressed in E. coli due to codon incompatibility. From this study it can be suggested that before attempting expression of any gene in E. coli the CAI should be calculated. It was also concluded from this work that no further attempts to express the CP gene in E. coli should be conducted.

The history and the background of dairy products, specifically fermented milk products and foodborne diseases were reviewed. The constructed literature review also gave details of the microbial and nutritional compostion of fermented milks and examples of African traditional fermented milks. Health benefits of these dairy products were also included. Furthermore, the most common foodborne pathogens and their survival in dairy products were discussed. The survival of Escherichia coli O157:H7, non pathogenic Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. and Shigella spp in plain and fruit yoghurt during cold storage at 4 °C was investigated. The survival of these microbial pathogens ranged between 1-14 days in both types of yoghurt, with Staphylococcus aures being the most sensitive pathogen to the yoghurt enviroment. Lactic acid bacteria and yeasts counts remained fairly stable at an average of 8 log cfuml-1 and below 10cfuml-1 respectively. A reduction in pH was noticed during yoghurt storage. The high numbers of LAB and low pH played a major role in the inhibition of the food borne pathogens over time in both plain and fruit yoghurt. The survival of the same microbial pathogens in fruit yoghurt during temperature abuse at 25°C was investigated. There was a rapid die-off of the food borne pathogens at this temperature. Their survival in the yoghurt ranged between 1-3 days. There was a rapid decrease in pH and an increase in the LAB and these together with the high temperature played a major role in the higher death rate of the foodborne pathogens in the fruit yoghurt. Yeasts grew to high levels that caused the yoghurt to spoil. When comparing yoghurt stored at 4 °C and 25°C a conclusion was made: yoghurt stored at 4 °C will be of good quality but will not be regarded as safe as pathogens survival for a long period, and yoghurt stored at 25°C will be regarded as safe but will not be of good quality as high yeasts counts will result in spoilage. The microbial pathogens were also inoculated into yoghurt and their survival at 4 °C after temperature abuse at 12 °C and 37 °C for 4-6 hours was studied. The pathogenic microorganisms died-off at a higher death rate during temperature abuse at high temperatures than at a low temperature at 4 °C. At 4 °C the death rate decreased and the survival in yoghurt ranged between 1-11 days. A rapid decrease in pH and a slight increase in LAB were observed during and after temperature abuse. The yeast increased during the storage of yoghurt. This study showed that inhibition of foodborne pathogens is high at higher temperatures. The survival of the microbial pathogens in milk during the production of Sethemi at 25 °C and 37 °C was studied. The pathogens increased, growing to high counts during the first 24hrs of fermentation. After 24hrs growth of some pathogens was inhibited, however, all the pathogens could still be detected even at the end of fermentation at both temperatures. Yeasts grew to some extend and the lactic acid bacteria grew to high levels during the fermentation of milk at both temperatures. The lactose produced by LAB decreased with time and there was a slight increase in galactose. A rapid increase in lactic acid was noted after a few hours of fermentation, this caused a decline in pH which resulted in the inhibition of pathogens. The inhibition was most effective at 37 °C.

Recent contamination of the environment with Cr(VI) through various industrial activities is becoming an increasing problem to which microbial Cr(VI) reduction could prove an important alternative solution compared to currently available chemical and physical treatment options. Cr(VI) is highly toxic and has been shown to be a mutagen and carcinogen, whereas Cr(III) is considered relatively innocuous. A variety of Cr(VI)-resistant and reducing bacteria have been isolated from diverse environments both contaminated and uncontaminated with Cr(VI). In 1999, Kieft and co-workers isolated a Thermus scotoductus strain from groundwater of a South African gold mine at a depth of 3.2 km. This thermophilic bacteria was shown to be able to readily reduce a variety of metals including Fe(III), Mn(IV), Co(III)-EDTA, U(VI) and Cr(VI). This ubiquitous nature of Cr(VI) reducing bacteria and the fact that Cr(VI) rarely occurs naturally in the environment, has led several researches to proposed that these chromate reducing properties is the serendipitous activity of enzymes with other primary physiological functions. Thermus scotoductus SA-01 has the ability to tolerate up to 0.5 mM Cr(VI) during growth in a complex organic medium and reduce Cr(VI) under growth and non-growth conditions. The rate of chromate reduction is dependent on pH, temperature, initial Cr(VI) concentration and is abolished by metabolic inhibiters. Chromate reduction was catalyzed by cellular extracts using NADH as an electron donor indicating the chromate reduction mechanisms to be enzymatic. Subcellular fractionation studies indicated that Thermus scotoductus SA-01 possesses more than one chromate reduction mechanisms. A novel cytoplasmic chromate reductase was purified to homogeneity and shown to couple the oxidation of NAD(P)H to the reduction of Cr(VI). This homodimeric protein consisted of monomers of approximately 36 kDa with a non-covalently bound FMN and required the divalent metal Ca2+ for activity. The enzyme was optimally active at 65°C and a pH of 6.3, reducing 2 mol of NAD(P)H per mol Cr(VI), suggesting a mixed one- and two-electron transfer mechanism. N-terminal sequencing and screening of a genomic library of T. scotoductus SA-01 using a DIG-labelled DNA probe, revealed the cytoplasmic chromate reductase to be encoded for by an ORF of 1050 bp under the regulation of an E. coli Ï70-like promoter. Sequence analysis showed the chromate reductase to be related to the Old Yellow Enzyme (OYE) family and in particular some xenobiotic reductases. The membrane-associated chromate reductase was also purified to homogeneity and shown to be distinct from the above mentioned cytoplasmic chromate reductase. The membrane-assocated reductase appears to be peripherally-associated with the membrane of T. scotoductus and consists of two identical subunits of approximately 48 kDa. The chromate reductase contained a non-covalently bound FAD co-factor and was optimally active at 65°C and a pH of 6.5. Through N-terminal sequencing and screening of a genomic library, the membrane-associated chromate reductase was identified as homologous to the dihydrolipoamide dehydrogenase gene, encoded for by a 1386 bp ORF and located within a probable pyruvate dehydrogenase operon. Although neither of these enzymes are dedicated physiological chromate reductases, their catalytic efficiency toward Cr(VI) as substrate proved to be superior than that found for other chromate reductases described in literature, which include the nitroreductases and quinone reductases isolated from Pseudomonas putida and Escherichia coli Both the cytoplasmic and membrane-associated chromate reductases were heterologously overproduced in E. coli and T. thermophilus as active, soluble enzymes. Kinetic studies of the recombinant proteins showed that the recombinant chromate reductases expressed in T. thermophilus were more catalytic efficient than their E. coli-expressed counterparts. Although structurally no differences could be observed using UV-vis and circular dichroism spectroscopy, the T. thermophilus-expressed recombinant cytoplasmic chromate reductase proved to be more stable under extreme chemical and thermal conditions.

Cytochrome P450 monooxygenases are enzymes capable of efficiently hydroxylating hydrophobic substrates with high regio-, stereo-, and enantioselectivies; under mild reaction conditions. They are therefore attractive alternatives to traditional chemistry for the synthesis of hydroxylated products. Their use in large scale applications has however been hindered by their requirement for continuous supply of reducing cofactors. Self-sufficient P450s occur, containing fused hydroxylase and reductase domains in a single polypeptide. They exhibit higher activities than any other reported P450s. Yarrowia lipolytica is a yeast capable of efficient degradation of hydrophobic substrates and growth on alkanes as sole carbon source. The intricate pathways involved in hydrophobic substrate metabolism within this yeast involve initial hydroxylation by P450s. Y. lipolytica contains 12 alkane and fatty acid hydroxlyase encoding genes CYP52F1 to CYP52F12. This yeast has been widely tested for biotransformations of inexpensive substrates to more valuable products. Y. lipolytica has also been investigated as a host for the heterologous expression of diverse foreign proteins, including cytochrome P450s. Various genetic tools are available for this purpose, including strong, inducible promoters, (e.g. POX2 and ICL); the defective selection marker ura3d4 for integration of cloned genes in higher copy numbers; specific targets for integration; and customized host strains. Heterologous expression of two CYP genes, by Y.lipolytica has previously been investigated in our research group. These were CYP53B1, encoding a benzoatepara- hydroxylase, and CYP557A1, a putative alkane and fatty acid hydroxylase. They were cloned in single and multiple copies under the control of the POX2 promoter. Problems were encountered with the POX2 promoter when whole cells were used for biotransformations, since native P450s were also induced by the fatty acids used as inducers for the POX2 promoter, and thus interfered with activity determination of cloned P450s. A further limitation to this study was the lack of appropriate negative control strains for accurate comparisons. In the current study, the same P450s were cloned into Y. lipolytica under the control of the ICL promoter. More appropriate negative controls strains were also constructed. Both ethanol and oleic acid can induce the ICL promoter. Ethanol induction of the CYP53 expressing strains resulted in lower whole cell activities than oleic acid induction. It has been reported in the literature that activities of enzymes expressed under the ICL promoter in cell-free extracts from cells induced with either oleic acid or ethanol were comparable. It therefore seemed that ethanol was repressing other components of the metabolic pathways for the degradation of hydrophobic substrates in the cells. Additionally no convincing activity was discernable for the ICL regulated CYP557 containing strains compared to the controls under POX2 regulation, and negative control strains. Continuous ethanol addition again led to reduced activities compared to situations where ethanol was added only once, confirming the metabolic inhibition by ethanol. It can be concluded that ethanol induction of the ICL promoter is not viable for whole cell biotransformations of hydrophobic substrates. Different inducers which will repress induction of the endogenous CYP genes but will allow induction of hydrophobic substrate uptake systems must be identified if the ICL promoter is to be used for the expression of cloned CYP genes. The CYP102A1 gene encoding the self-sufficient subterminal fatty acid hydroxylase from Bacillus megaterium was also cloned into Y. lipolytica under the POX2 and ICL promoters. Strains containing this gene did not display detectable subterminal hydroxylation of fatty acids or 4-nonyloxy-benzoic acid which was used as a fatty acid analogue. Significantly increased cytochrome c reductase activities were however detected in the soluble fraction of cell free extracts from test strains compared to the negative control strains. Microsomal activities were comparable in cells with and without cloned CYP102A1. This indicated that the CYP102A1 was functionally expressed, but located in the soluble fraction, where it was unlikely to interact with the hydrophobic substrates. It was therefore concluded that Y. lipolytica is not a suitable host for whole cell biotransformations using the CYP102A1 enzyme.

Candida albicans and C. dubliniensis are two closely related pathogenic yeast species, sharing many phenotypic characteristics which make it difficult to differentiate them, especially in clinical samples. As a result of the similarities between these species, identification techniques, based on phenotypic characteristics, have been developed. In this study some of these techniques and virulence factors were used to characterise strains belonging to these species and to select phenotypically dissimilar strains for further study. This was performed to evaluate if the effect of arachidonic acid (20:4) on these strains were the same, even though they are phenotypically different. Candida albicans and C. dubliniensis can form biofilms which play an important role during infection. During C. albicans infection, 20:4, a long-chain polyunsaturated fatty acid (PUFA), derived from the phospholipids (PLs) of the infected host cell membrane, serves as carbon source and precursor for eicosanoid production. Conflicting results are presented in literature regarding the effect of 20:4 on morphogenesis in C. albicans. In addition, the effect of 20:4 on growth and morphology of C. dubliniensis is unknown. Microscopic examination and enzyme activity assay indicated that 1 mM 20:4 had little to no effect on growth and metabolic activity of planktonic cells and biofilms, as well as on the morphology and viability of the cells in the biofilms. The uptake of PUFAs by yeasts is necessary for utilisation as metabolic fuels, cellular building blocks and the production of signalling molecules. However, there are no definitive studies regarding the uptake and cellular metabolism of 20:4 by these pathogenic yeasts. The uptake and incorporation of 20:4 by planktonic cells and biofilms of selected strains of C. albicans and C. dubliniensis were evaluated by subjecting residual and cellular lipids from planktonic cells and biofilms, grown in the presence and absence of 20:4, to gas chromatography and gas chromatography-mass spectrometry. Strain specific variation in 20:4 uptake and incorporation into different lipid fractions of planktonic cells and biofilms were found. In addition, eicosanoids produced by biofilms in the presence of 20:4 were extracted and it was found that biofilms of these strains were capable of producing 3-hydroxy fatty acids from 20:4. Arachidonic acid can be incorporated into the PLs of yeasts, influencing saturation in cell membranes. It is suggested that the effectiveness of antifungals may depend upon the level of unsaturation and ergosterol content of the membrane, therefore the effect of 20:4 on the cell membrane and susceptibility of C. albicans and C. dubliniensis biofilms towards amphotericin B and clotrimazole were also determined. This was performed by confocal laser scanning microscopy, determination of mitochondrial metabolism, unsaturation index of the PL fractions and ergosterol content of the membranes of biofilms grown in the presence and absence of 20:4. The results indicated that 20:4 influences PL unsaturation and ergosterol content of both C. albicans and C. dubliniensis type strains, increasing susceptibility to the antifungals. Pre-treatment of biofilms with PUFAs may result in the reduction in antifungal dose needed to inhibit biofilms.

Boerewors is a popular dish in South Africa, and it is classified as a ground meat product. It is a fresh sausage product and the quality of the sausage is affected by the growth of microorganisms and oxidation spoilage. Boerewors is currently preserved with 450 mg/kg sulphur dioxide (SO2). The SO2 has antimicrobial, antioxidant and sensory properties in the boerewors but SO2 may also have negative effects on consumer health. Natural preservatives have become important in the food industry because consumers are moving away from consuming chemically preserved food. Rosemary extract is a natural preservative that has been shown to have antimicrobial and antioxidant properties. Another natural preservative that has been studied is chitosan which is a deacetylated form of chitin derived from the shell of crabs and shrimps and the cell wall of fungi and also has been shown to have antimicrobial and antioxidant properties. One of the aims of the study was to evaluate the microbiological quality of boerewors produced in different regions of Bloemfontein and comparing the quality of boerewors from small butcheries with supermarkets. The boerewors of 50% of the butcheries in Bloemfontein were subjected to microbial evaluation by using aerobic plate counts (APC), psychrotolerant plate counts (PPC), Enterobacteriaceae counts, coliform counts, yeasts and moulds counts and determination of the presence of Staphylococcus aureus and Escherichia coli. No Staph. aureus was found in any of the boerewors samples. The results of this study demonstrated that the microbial quality of boerewors sold in the different regions of Bloemfontein retail outlets did not differ significantly between the regions. The incidence of E. coli in the central region may indicate that the socio-economic status has an effect on the quality, though the socio-economic status was not investigated in this study. The significantly high coliform counts in boerewors purchased from the butcher shops indicate that poor hygienic conditions exist in butcher shops compared to the supermarkets. That may be related to staff being trained in hygienic practices in the corporate supermarket environment, while no training or very little training takes place in butcher shops. The second aim was to determine the effectiveness of rosemary extract, chitosan and fungi) and sulphur dioxide (SO2) as a conventional preservative, applied individually and in combination with each other, on improving the quality of boerewors. Eight boerewors treatments were formulated as follows: control (Con), 450 mg/kg SO2 (S), 260 mg/kg rosemary extract (Ros), 10 g/kg chitosan (Chi), 260 mg/kg rosemary extract + 100 mg/kg SO2 (Ros+S), 10 g/kg chitosan +100 mg/kg SO2 (Chi+S), 260 mg/kg rosemary extract + 10 g/kg chitosan (Ros+Chi) and 260 mg/kg Rosemary extract + 10 g/kg Chitosan + 100 mg/kg SO2 (Ros+Chi+S). Water activity (aw), microbial properties, colour stability, lipid stability and sensory analysis were conducted on the different sausage treatments. There were no significant differences between the aw of the different treatments. Chitosan and chitosan in combination with other preservative types had a significant effect (p<0.001) on reducing the total bacterial counts, coliforms and Enterobacteriaceae counts which were comparable to the results of the SO2 treatment. Chitosan, however, had a better effect on decreasing yeasts and moulds counts than SO2. Chitosan also showed good colour properties which were comparable to that of the 450 mg/kg SO2 preserved sausage. Rosemary extract showed a comparable lipid stability to those of the S treatment and it showed significantly (p<0.001) lower TBARS values when compared to the Chi treatment. The Ros treatment had a positive effect on the sensory taste of the sausages when compared to the Chi treatment, but the S treatment was still preferred by the sensory panel. The reduced level of 100 mg/kg SO2 showed good synergistic effects in combination with chitosan as antimicrobial, colour stabilizer and in combination with rosemary as antioxidant and improving the sensory score of the sausages. Keywords: boerewors, natural preservatives, ro

In 2007, the Kock group published the Aspirin Antifungal Hypothesis showing a clear link between oxylipin production, mitochondrial activity and acetylsalicylic acid (ASA, aspirin) sensitivity in respiring as well as non-respiring yeasts. This hypothesis suggests that mitochondrial inhibitors such as ASA selectively inhibits parts of yeast life cycles, especially the sexual stage. According to the hypothesis, mitochondrial β-oxidation products such as 3-OH oxylipins are present in elevated amounts in yeast sexual structures (asci) and lesser amounts in vegetative asexual structures (hyphae and single cells). This suggests increased mitochondrial activity in asci. Consequently, ascomycetous yeast sexual structures should be more sensitive to mitochondrial inhibitors compared to vegetative asexual structures. The purpose of the study became to assess if the Aspirin Antifungal Hypothesis could be expanded to also include other mitochondrial inhibiting drugs as well as other structures present in fungal life cycles where increased mitochondrial activities are expected. In this study, the anti-inflammatory drugs ASA, ibuprofen, indomethacin, salicylic acid and benzoic acid as well as anticancer drugs such as Lonidamine, also known for inhibiting mitochondrial activity in mammalian cells, were found to be antifungal and specifically target the sexual stage of yeast. This is shown by a unique yeast bioassay, with the mitochondrion-dependent sexual dispersal structure producing many ascospores, riboflavin production, and hyphal morphology of the notorious yeast plant pathogen Eremothecium ashbyi serving as indicators. These drugs affect this yeast in a similar way as found under oxygen limitation conditions by inhibiting sexual structure development (most sensitive), riboflavin production, and yielding characteristically wrinkled and granular hyphae, presenting a unique âanoxicâ morphological pattern. Only drugs associated with mitochondrial inhibiting activity presented such a pattern. This bio-assay may find application in the preliminary screening for novel drugs from various sources with possible mitochondrial inhibiting actions. In another part of the study, the effects of antimitochondrial compounds on asexual fungal spore dispersal structures in the pathogens Aspergillus fumigatus and Rhizopus oryzae were investigated. When anti-mitochondrial ASA and other anti-mitochondrial non-steroidal antiinflammatory drugs (NSAIDs) were added to A. fumigatus and R. oryzae, asexual fungal spore-releasing structures were targeted first at lower concentrations. Similar results were obtained when oxygen was limited. These asexual fungal spore-releasing structures contained increased levels of mitochondrial activity compared to hyphae. Increased mitochondrial activity may be necessary for the formation of asexual fungal spore dispersal structures of these fungi. Consequently, mitochondrial inhibitors may serve as effective antifungals to combat asexual fungal spore dispersal of these pathogenic fungi. In this study, the Aspirin Antifungal Hypothesis is expanded to also include various antiinflammatory compounds, anticancer drugs, plant extracts, traditional medicines and others â many showing anti-mitochondrial activity. These compounds should be further investigated to determine their minimum inhibitory concentrations (MICs) and application to combat plant and human fungal pathogens. In this study, the hypothesis is also expanded to include asexual fungal dispersal structures with increased mitochondrial activity.