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Spontaneous mutagenesis can be divided into three main steps: the introduction of DNA damage and lesions, damage recognition and DNA repair. All sources of spontaneous mutagenesis originate from within the cell itself, e.g., polymerase errors cause DNA mismatches and reactive oxygen species alter the chemical composition of DNA bases. The combined effects of all these processes influence spontaneous genomic mutation rates, which are thought to be a characteristic of individual species and/or groups of species. Although much is known about different mutagens and how they cause mutations the sequence context of these mutations are less well understood. The results of this MSc study on mutation in the filamentous fungus Fusarium circinatum showed that the 5ʹ and 3ʹ neighbouring bases of a single nucleotide polymorphism can significantly influence the type of substitution that occurred leading to the formation of mutational motifs. This was the case for both sets of genes examined (core housekeeping and non-ribosomal protein synthetase genes), whose evolution is known to differ. The fact that none of the identified motifs are shared between the two sets of genes could indicate that the cellular mutagens and/or repair machinery function differently for the two gene groups. Furthermore, none of the mutable motifs that have been identified for the well-known mutagens in model organisms could be detected in the fungus, which suggests that mutagens and/or DNA repair mechanisms of this fungus are unique. Although limited information is available for non-model eukaryotes, an estimate for the rate at which mutations arise across the genome of F. circinatum could be a good starting point for comparisons of its evolutionary rate to those of its close relatives. This was accomplished using a fluctuation analysis involving nitrate non-utilizing mutation reversion. Although mutation rate determined in this study is probably not precisely accurate, it represents a good starting point for future comparative studies on the evolutionary rate of Fusarium species. As a whole this study laid the foundation for a better understanding of spontaneous mutagenesis at specific sites in certain groups of genes as well as across the genome of the economically important plant pathogen F. circinatum. Restricted until August 2017 / Dissertation (MSc)--University of Pretoria, 2013. / gm2013 / Microbiology and Plant Pathology / Unrestricted

Cowpea, Vigna unguiculata (L.) Walp. is an important edible legume crop gaining recognition in the world generally and in Africa particular. The seed is a very good source of vegetable protein for millions of people living in sub-Saharan Africa. Cowpea seed are susceptible to global post-harvest losses caused by insect damage, microbial deterioration and other factors especially when stored at high ambient temperatures and relative humidities. The detection of Fusarium species and fumonisins associated with cowpea seed was primarily investigated in this study. Secondly, the antifungal activity of extracts from three Rubiaceae plant species was evaluated against five Fusarium species. Column chromatography was used in an attempt to isolate potential compounds from the plant extracts. Cowpea seed samples were obtained from a local market of a rural community in Mpumalanga Province, South Africa and were analysed for seed mycoflora and various fungal genera including, Aspergillus spp., Fusarium spp. (Fusarium oxysporum. F. solani, F. subglutinans, F. verticillioides and F. proliferatum), Penicillium spp., Chaetomium spp., Chrysonilia spp., Cladosporium spp., Monascus spp., Phoma spp., Mucor spp., Alternaria alternata, Epicoccum spp. and Lasiodiplodia theobromae were isolated from the seed samples. Some Fusarium species including F. oxysporum, F. verticillioides, F. subglutinans, F. solani and F. proliferatum were cultured on maize patty media and analysed for fumonisin production. Fumonisin detection was conducted using the VICAM Affinity method. Only Fusarium subglutinans and F. oxysporum produced fumonisin B analogues ranging between 1.1 - 4.3 ìg/g and 3.47 - 31.66 μg/g, respectively. Neither Fusarium verticillioides, F. solani nor F. proliferatum revealed any production of fumonisin B. Fumonisin B analogues were found to be present in all the cowpea seed samples with levels ranging between 6.2 - 59 μg/g. Purified fumonisin B1 was used to investigate the phytotoxic effects of the toxin in vitro and in vivo on cowpea seedling growth and emergence and also on the ultrastructure of the seed tissue. Surface-disinfected seeds were imbibed in sterile distilled water amended with FB1 at various concentrations (10, 25, 50 and 100 μg/ml) and dry seeds and seeds imbibed in sterile distilled water served as the controls. Percentage germination was determined in vitro and in vivo according to the International Seed Testing Association (ISTA) rules. Root and shoot length was measured after 8 days. Compared to the controls all the concentrations significantly decreased seed germination and the highest concentrations of FB1 (50 and 100 μg/ml) inhibited root and shoot elongation. Transmission electron microscopy (TEM) was used to study sections of the cotyledon and embryonic tissue of cowpea seed. Samples were treated with different concentrations of FB1. Compared to the controls, the degree of damage to the vacuoles, cell walls and lipid bodies of cowpea seed tissue was directly proportional to the concentrations. The efficacy of crude extracts from Morinda citrifolia, Gardenia brighamii, and Psychotria capensis was investigated against various Fusarium species by two methods. The microtitre plate doubling dilution method and bioautography method were used to test methanol, acetone, and dichloromethane extracts of the leaves for their antifungal activity. Psychotria capensis acetone extracts showed the best inhibition against F. proliferatum and F. verticillioides with an MIC of 1.5 mg/ml and an MFC of 1.5 mg/ml. The dichloromethane extracts exhibited no activity against the Fusarium species tested. Column chromatographic purification of methanolic extracts of the leaves of P. capensis led to the isolation and identification of two compounds namely β-sitosterol and a carotenoid derivative. / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Thesis (Ph.D. (Chemistry)) -- University of Limpopo, 2021 / Fusarium species are among pathogenic organisms responsible for massive yield and quality losses in crop production. They cause crop diseases in the field and during storage, and some species are capable of producing mycotoxins which contaminate products and threaten consumer s’ health. Conventional synthetic fungicides are available for the control of Fusarium pathogens, however, their applications have been restricted or discouraged due to their harmful effect on the environment, livestocks and human health. There are also reports about fungal-resistance to available fungicides. Moreover, the synthetic chemicals are not affordable to smallholder farmers and to some extent, they are not recommended for applications in organic farming. As an alternative to these fungicides, selected medicinal plant species were investigated as sources of natural chemicals or compounds with potential to be developed into plant-based fungicides to control Fusarium pathogens. This study aimed to identify antifungal extracts among the selected medicinal plant species which could be used to develop plant-based fungicides to control Fusarium diseases. It also focused on isolation and characterization of antifungal compounds from selected medicinal plant species. Thirteen medicinal plant species (Combretum erythrophyllum (Burch.) Sond , Melia azedarach L, Solanum mauritianum Scop, Nicotiana glauca Graham, Schotia brachypetala Sond, Lantana camara L, Combretum molle R. Br. ex G. Don, Quercus acutissima Carruth, Olea europaea L, Vangueria infausta Burch, Withania somnifera (L.) Dunal, Harpephyllum caffrum Bernh and Senna didymobotrya (Fresen.) H.S. Irwin & Barneby) were selected from literature based on their reported strong antimicrobial activity against human and/or animal pathogens. The leaves of these plant species were collected, shade-dried and extracted with water, petroleum ether, ethyl acetate and acetone. Extractant yield was recorded and each extract was evaluated for antifungal activity using a micro-dilution assay against nine Fusarium pathogens (Fusarium verticillioides, Fusarium proliferatum, Fusarium subglutinans, Fusarium graminearum, Fusarium solani, xxvii Fusarium oxysporum, Fusarium semitectum, Fusarium chlamydosporum and Fusarium equiseti). Similar solvent extracts from different plant species that demonstrated MIC value of less than 0.1 mg/ml against the same pathogen were combined and evaluated for antifungal activity. The interation effect of combined extracts was determined by calculating their fractional inhibitory concentration index (FICI) in order to determine their possible synergistic, additive, indifference or antagonistic antifungal activity against tested pathogens. Plant extracts demonstrating synergistic and or additive interaction were further evaluated in combination and individually for in vivo antifungal activity against maize seed Fusarium pathogens. At least, one of the extracts obtained from these medicinal plant species showed strong antifungal activity with minimum inhibitory concentration (MIC) of less than 0.1 mg/ml against at least one of the tested pathogens. Of the four solvent extracts evaluated, acetone and ethyl acetate extracts showed stronger antifungal activity compared to petroleum ether and water extracts. Of the nine pathogens tested, F. proliferatum was the most susceptible and was strongly inhibited (MIC < 0.1 mg/ml) by 41 plant extracts whilst F. equisite was found to be resistant with MIC < 0.1 mg/ml by only three plant extracts. In total, each pathogen was tested against 52 plant extracts. There were 17, 16 and 15 extracts from C. erythrophyllum, S. mauritianum and Q. acutissima, respectively, with MIC values less than 0.1 mg/ml. These species were the most active when tested individually. Schotia brachypetala was found to be the least active medicinal plant with only seven extracts demonstrating very strong activity (MIC < 0.1 mg/ml) against the tested pathogens. Minimum inhibitory dilution (MID) or total activity was also calculated and it was found that water and acetone extracts had the highest MID, followed by ethyl acetate extracts while petroleum ether extracts recorded the lowest. Of all plant extracts tested against the nine pathogens, 59 plant extracts demonstrated MID values of more than 1000 ml/g. Out of the 348 extract combinations evaluated, 116 and 87 extract combinations demonstrated synergistic and additive antifungal activity, respectively. The strongest activity xxviii recorded for the combined extracts resulted from synergistic interaction with MIC value of 0.001 mg/ml against F. proliferatum and F. verticilloides. Combined acetone extract of C. erythrophyllum and Q. acutissima was very active (95.75% inhibition) against F. verticilloides inoculated on maize seeds while individual preparation from M. azedarach acetone extract demonstrated 97.10% inhibition against F. proliferatum. The extracts showing good antifungal activity (≥ 50% inhibition) were further tested for phytotoxicity on maize seed germination and the lowest recorded seed germination was 86.25%, resulting from Q. acutissima ethyl acetate extract. Combined acetone extract of C. erythrophyllum and Q. acutissima did not significantly affect maize seedling growth when compared to negative control (water treatment). All plant extracts that showed strong activity (MIC < 0.1 mg/ml) when tested using micro-dilution assay were spotted on thin layer chromatography (TLC) bioautographic assay to establish and determine the number of active compounds or bands. The white spots observed on the chromatograms indicated the presence of antifungal compounds. Combretum erythrophyllum, W. somnifera and L. camara exhibited the presence of antifungal compounds against 7, 5 and 4 pathogens, respectively. Hence, these plant species were selected for isolation of antifungal compounds where open column chromatography and preparative TLC were used for compound purification. At least, three isolated fractions from the three plant species were found to be active (MIC values ranging from 0.0098 to 0.625 mg/ml) against more than five pathogens. The fractions were also found to contain different levels of phytochemicals such as glycosides, flavonoids, steroids, and terpernoids. The structures of isolated compounds or fractions were determined using nuclear magnetic resonance (NMR) and mass spectroscopic (MS) techniques. A mixture of apeginin (4′,5,7-trihydroxyflavone) and salvigenin (5-hydroxy-6,7,4'-trimethoxyflavone) isolated from the leaves of C. erythrophyllum showed strong antifungal activity (MIC values ranging from 0.01 mg/ml to 0.63 mg.ml) against 5 tested Fusarium pathogens. Also isolated from C. erythrophyllum was a derivative of maslinic acid and it has xxix shown antifungal activity with MIC values ranging from 0.08 mg/ml to 0.63 mg/ml against 6 tested pathogens. On the other hand, lantadene A (22- angeloyloxy-9-hydroxy-3-oxo-olean-12-en-28-oic acid), boswellic acid (11-keto-β-boswellic acid) and boswellic acid glycoside isolated from the leaves of Lantana camara showed good activity (MIC values ≤ 0.63 mg/ml) against one or more Fusarium pathogens. Withaferin A (4β,27-dihydroxy-1-oxo-5β,6β-epoxywitha-2-24-dienolide) glycoside isolated from the leaves of Withania somnifera showed antifungal activity with MIC value of 0.16 mg/ml against F. verticilloides. This study demonstrated potential applications of medicinal plant extracts as cheap, accessible and sustainable source of eco-friendly pesticides for fighting crop diseases in organic and smallholder farming. The extracts can be used as treatment agents to control maize seed spoilage during post-harvest storage. Additionally, characterised antifungals may serve as scaffold compounds during commercial synthesis of plant-based fungicides. / Agricultural Research Council (ARC) and National Research Foundation (NRF)

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life & Consumer Sciences / M. Sc. (Life Sciences)

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. Aflatoxins and fumonisins are among the most toxic mycotoxins. They are a significant risk factor for a cocktail of chronic health conditions including cancer of the liver, oesophagus and kidney, teratogenicity, neural tube defects, interference with lipid metabolism, a weakened immune system and a negative impact on micronutrient absorption in both man and animals. This study compared natural contamination of peanuts, peanut butter and sorghum from Gaborone, Botswana and Bulawayo, Zimbabwe with aflatoxins and fumonisins. In total 34 peanut samples, 34 sorghum samples and 11 peanut butter samples were collected randomly from retail shops and informal markets in the two cities. Fungal contamination was determined using standard mycology methods. Aflatoxin and fumonisin contamination was determined using HPLC-FLD. A. flavus/parasiticus species were detected in 66% and 100% of randomly analysed peanut samples from Bulawayo and Gaborone respectively and 27% (3/11) of peanut butter samples from Bulawayo. 67% of randomly analysed sorghum samples from Bulawayo showed A. flavus/parasiticus and Fusarium species contamination while none of the randomly analysed sorghum samples from Gaborone showed any fungal contamination. Furthermore aflatoxins were not detected in any of the sorghum samples; however 61% (11/18) of the Bulawayo sorghum samples showed fumonisin contamination (Range: 8 – 187 ng/g). Three of the peanut samples from Bulawayo were contaminated with aflatoxins (range: 6.6 – 622 ng/g) and no aflatoxins were detected in Gaborone peanuts. All 11 peanut butter samples from Bulawayo were contaminated with aflatoxins (Mean: 73.5 ng/g, Range: 6.8-250 ng/g) and AFB1 was the most prevalent. These preliminary results indicate that peanut butter and peanuts from Bulawayo are contaminated with high levels of aflatoxins. Stricter policing of regulations should be implemented to ensure compliance by manufacturers and public health interventions implemented in vulnerable communities. / Life and Consumer Sciences / M. Sc. (Life Sciences)