Made available in DSpace on 2014-06-12T15:50:48Z (GMT). No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Lectinas são proteínas que se ligam a carboidratos e glicoconjugados. Rizomas de Microgramma vaccinifolia têm ampla utilização na medicina popular no tratamento de hemoptises e hematúria. Os objetivos deste trabalho foram isolar, caracterizar, avaliar as atividades tóxica, antibacteriana e antifúngica da lectina de rizoma de M. vaccinifolia (MvRL) e identificar as raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici por biologia molecular. As proteínas do extrato de rizoma (ER) foram fracionadas com sulfato de amônio fornecendo a fração 0-60% (F0-60%). Atividade hemaglutinante (AH) e concentração de proteína foram determinadas em ER e F0-60%. MvRL foi isolada por cromatografia da F0-60% em Sephadex G-25. A AH de MvRL foi avaliada em presença de carboidratos, glicoproteínas, preparações contendo carboidrato das raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici, em diferentes temperaturas, valores de pH e na presença de íons. Eletroforese em gel de poliacrilamida de MvRL foi realizada em condições nativas (PAGE) e desnaturadas (SDS-PAGE). O efeito de MvRL sobre Artemia salina, bactérias e fungos foi também avaliado. O DNA das raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici foi isolado utilizando marcadores moleculares ISSR e RAPD. MvRL aglutinou eritrócitos humanos e sua AH foi inibida por manose, soro fetal bovino e preparações de F. oxysporum f.sp. lycopersici. A AH de MvRL foi termoestável, ativa em pH 5,0 e dependente de íons. PAGE revelou MvRL como uma proteína ácida e SDS-PAGE revelou banda polipeptídica glicosilada de massa molecular 17 kDa. Cromatografia de gel filtração definiu a massa molecular nativa de MvRL como 100 kDa indicando a lectina como um agregado molecular. MvRL foi tóxica sobre A. salina (CL50 de 154,16 μg/mL), não exibiu atividade antibacteriana e apresentou atividade antifúngica. MvRL foi mais ativa em inibir o crescimento da raça 3 de F. oxysporum f.sp. lycopersici. Os marcadores moleculares foram adequados para avaliar a variabilidade genética das raças. Em conclusão MvRL é uma lectina com atividade antifúngica e o efeito sobre F. oxysporum f.sp. lycopersici foi diferente para as três raças

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No. of bitstreams: 1 TESE_Patricia Migliorini.pdf: 2682211 bytes, checksum: 8bdf76d1693cb07dba37b177863fdbef (MD5) Previous issue date: 2018-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O presente trabalho teve por objetivo: a) avaliar o efeito de diferentes doses e fontes de Si por meio do recobrimento de sementes sobre a qualidade física, fisiológica e sanitária de sementes assim como, determinar a concentração de silício e cálcio absorvidos em plântulas de feijão; b) avaliar o desempenho de plântulas inoculadas com Fusarium oxysporum f. sp. phaseoli e recobertas com diferentes fontes de Si, durante a emergência e na expressão dos mecanismos bioquímicos, envolvidos nas respostas pós infecção em diferentes momentos e; c) avaliar o efeito do recobrimento de sementes com Si em resposta à infecção em uma cultivar suscetível e resistente ao Colletotrichum lindemuthianum, sobre o potencial fisiológico e sanitário de sementes e plântulas, durante o desenvolvimento inicial da cultura do feijão. Testou-se diferentes doses (0; 45; 90; 135 e 180g Si 100 kg-1 de sementes) e fontes de Si [Agrosilício®, silicato de cálcio e casca de arroz carbonizada (CAC)] via recobrimento de sementes, inoculados ou não pelo método de restrição hídrica. O recobrimento das sementes composto por Agrosilício®, CAC e silicato de cálcio não altera a qualidade física das sementes e proporciona plântulas vigorosas, sem comprometer a emergência final. Sementes recobertas com doses de silicato de cálcio e CAC elevam a concentração de Si nas raízes, mas reduzem a concentração de cálcio (Ca), enquanto que o Agrosilício® aumenta a concentração de Si em folhas e do Ca nas raízes. Diferentes fontes e doses de Si não controlam patógenos associados às sementes de feijão. A associação do F. oxysporum f. sp. phaseoli nas sementes prejudica a emergência e crescimento das plântulas. O Si tem pouco efeito nas respostas em plântulas não inoculadas, mas eleva a concentração de lignina. Na presença do F. oxysporum f. sp. phaseoli, o Si desencadeia melhor desempenho inicial das plântulas, favorece e potencializa os mecanismos de defesa. A fonte de Si fornecido pelo Agrosilício® proporciona uma antecipação rápida inicial da peroxidase o que pode ter amenizado e atrasado os danos do patógeno nas plântulas. A CAC como fonte de Si, embora tenha contribuído para a potencialização das enzimas de defesa nos períodos mais avançados da infecção, não se mostrou eficiente no controle da incidência da doença. O recobrimento das sementes com Si diminui a incidência e a severidade de C. lindemuthianum e proporciona maior crescimento das plântulas de feijão. O efeito de respostas potencializadas pelo Si tem maior expressividade na cultivar de maior suscetibilidade. / The a im of this study was: a) to evaluate the effect of different doses and sources of Si through the coating of seeds on the physical quality, physiological and sanitary conditions of seeds as, to determine the concentration of silicon and calcium absorbed in bean seedlings; b) to evaluate the performance of seedlings inoculated with Fusarium oxysporum f. sp. phaseoli and covered with different sources of Si, during emergence and in the expression of biochemical mechanisms, involved in post-infection responses at different times and; c) to evaluate the effect of seed coating with Si in response to infection in a susceptible and resistant Colletotrichum lindemuthianum, on the physiological and sanitary potential of seeds and seedlings, during the initial development of the bean culture. Different doses were tested (0; 45; 90; 135 and 180g Si 100 kg-1 of seeds) and sources of Si [Agrosilício®, calcium silicate and rice husk ash (AHC)] by seed coating, whether or not inoculated by the water restriction method. The covering of seeds composed of Agrosilício®, AHC and calcium silicate, does not change the physical quality of the seed, provides vigorous seedlings, without compromising the end emergency. Seeds coated with doses of calcium silicate and AHC, increase the concentration of Si in roots, but reduce the concentration of calcium (Ca), while the Agrosilício® increases the concentration of Si in leaves and Ca in the roots. Different sources and doses of Si do not control pathogens associated with bean seeds. The association of F. oxysporum f. sp. phaseoli in seeds impairs seedling emergence and growth. Si has little effect on responses in uninoculated seedlings, but raises lignin concentration. In the presence of F. oxysporum f. sp. phaseoli, Si triggered better initial seedling performance, favored and enhanced the defense mechanisms. The Si source supplied by Agrosilício® provides early rapid anticipation of peroxidase, which may have ameliorated and delayed the damage of the pathogen in the seedlings. The AHC as a source of Si, although it has contributed to the potentiation of defense enzymes in the later stages of infection was not efficient in controlling the incidence of the disease. The seed coating with Si decreases the incidence and severity of C. lindemuthianum and provides greater growth of the bean seedlings. The effect of responses potentiated by Si has greater expressiveness in the cultivar of greater susceptibility.

Doctor of Philosophy / Department of Agronomy / Guihua Bai / Growing resistant cultivars is an economically effective method to control wheat disease Fusarium head blight (FHB) caused by Fusarium graminearum. Ninety-five wheat lines mainly from China and Japan were evaluated for resistance to initial infection (type I), spread of symptoms within a spike (type II), and deoxynivalenol (DON) accumulation in infected grains (type III). Most of lines were resistant or moderately resistant, 15 lines had DON content lower than 2 ppm and six lines showed a high level of resistance for all the three types. Deoxynivalenol content was significantly correlated with type II, but not type I resistance. Fifty-nine of the ninety-five lines were evaluated for genetic diversity on the basis of amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSRs). Genetic relationships among these lines were consistent with pedigrees and their geographic distribution. Chinese lines had broader genetic diversity than Japanese lines. Sumai 3 is a widely used Chinese variety for FHB-resistant breeding in the US and elsewhere. Haplotype patterns of the SSR markers linked to FHB resistance quantitative trait loci (QTL) on chromosomes 3BS, 5AS and 6BS of Sumai 3 indicated that only a few Sumai 3 derivatives carry all of these Sumai 3 QTL. SSR data also suggested that these QTL in Sumai 3 were derived from Chinese landrace Taiwan Xiaomai. Some highly resistant lines may carry novel QTL for FHB-resistance QTL, and need further investigation. A mapping population of 139 recombinant inbred lines derived from the cross of Wangshuibai (resistant Chinese landrace)/Wheaton (susceptible cultivar) was genotyped with more than 1300 SSR and AFLP markers. Five QTL for type I resistance were detected on chromosome arms 3BS, 4BS, 5DL, 3AS, and 5AS; seven QTL for type II resistance on 3BS, 1AL, 5AS, 5DL, 7AL, and 3DL; and seven QTL for type III resistance on 3BS, 5AS, 1AS, 5DL, 1BL, and 7AL. These QTL together explained 31.7%, 64%, and 52.8% of the phenotypic variation for FHB type I, II, and III resistance, respectively. QTL on 5AS, the distal end of 3BS, and 5DL contributed to all three types of resistance. FHB resistance QTL identified in Wangshuibai can be used in developing wheat cultivars with enhanced FHB resistance by pyramiding FHB resistance QTL from other sources.

M.Tech. (Biomedical Technology) / A study was conducted with the aim to determine the incidence and contamination levels of fumonisin (FB) and FB producers in South African compound feeds. A total of 90 compound feed samples were screened for FB producing Fusarium species conventionally based on the morphological macroscopic and microscopic features. Data revealed that Fusarium spp. were most prevalent in feeds for chicken with an incidence rate of 34% recorded followed by cattle (6%) and pig feed (1%), meanwhile no Fusarium spp. was recovered from any of the horse feed analyzed. Similar samples were also analyzed for FB and in general, data indicate that 68% of samples were positive for FB1 which was the most frequent, ranging from 24 to 5515 μg/kg (mean concentration: 796.5 μg/kg). Quantification of fum 1 gene in 30 animal feed samples was performed by quantitative real time-polymerase chain reaction (qRT-PCR). Detection as well as quantification of fum 1 gene in animal feed was made possible but a positive correlation between fum 1 gene and FB levels was not established, however, an association between fungal contamination, FB and the determined fum 1 gene concentrations was evident in this study. The cytotoxic effect of FB extract from feeds was evaluated in vitro on human lymphocyte cells. Data obtained revealed that cell viability of lymphocytes was strongly influenced by both the concentration of toxin and duration of exposure. This study demonstrates that in South Africa, animals are constantly exposed to FB via consumption of feeds contaminated with the toxin even though the levels obtained are within the acceptable levels. Their presence thus highlights the need for proper quality control measures to be put in place at every step in the animal feed production chain.

The main objective of this thesis was the elucidation of the host-pathogen interaction between Pinus patula and Fusarium circinatum. This was accomplished by studying differential gene expression at the molecular level. Therefore, the first chapter reports the use of PCR-based methods in gene discovery and transcriptome analysis. The use of these techniques in the identification of novel transcripts in host-pathogen interactions addressed. These examples illustrate the differences and strong features of each technique. Chitinases are linked to defence responses in plants. In chapter tw0, the induction of chitinases in P. patula was assessed at both the protein and genetic level. Western blot analysis and enzyme activity assays indicate that chitinase enzyme is not detected a part of the defence response by P. patula after infection by F. circinatum. This was further confirmed by the lack of significant induction of two Pinus chitinase genes, LP6 and PSCHI4, as determined by RT-PCR analysis. Partial DNA sequence homologues for the LP6 and PSCH14 genes were determined and compared with a variety of plant chitinases. The low levels of detectable chitinase induction in P. patula might explain the high levels of susceptibility to the pitch canker fungus observed in seedlings of this tree. Pinus patula, the most widely planted species in South Africa, is highly susceptible to infection by F. circinatum. In chapter three, suppression subtractive hybridisation was used to elucidate the changes taking place at the molecular level early on in this interaction. Most of the identified transcripts shared homology to both biotic and abiotic stress in plants. The induction of one fragment, displaying homology to phytocyanin proteins, as followed through RT-PCR. Induction levels for this fragment differed significantly between less and more susceptible plants. Although most of the sequences isolated in this study can be Iiked to stress, most have not been linked with specific plant-pathogen interactions. This raises questions in regard to the function of these genes in host-pathogen interactions. Further research identify the function of these sequences in the defence response will be needed. These sequences can also be tested against a family of Pinus trees to ascertain if they will be useful in marker assisted selection. A molecular analysis of culture degeneration and pathogenicity of F. circinatum was attempted in chapter four. In this chapter, the differential induction of transcripts in F. circinatum was determined against several other Fusarium spp. Several of he identified fragments shared homology with stress related proteins. One transcript shared homology to a polyketide synthase, FUM5, that could be linked to fumonisin production in other Fusarium spp. ELISA detected no fumonisin production, although the FUM5 transcripts were detected. The identification of all the transcripts could provide a basis for more intensive gene discovery studies in F. circinatum and other Fusarium spp. The induction of these sequences in different isolates needs to be studied to prove their function in F. circinatum. This study also complements several other studies that studied the morphological characteristics of culture degeneration. Resistance gene analogues have been reported from a diverge set of plant species. In chapter five, degenerate PCR amplification was used to isolate TI-NBS-LRR like resistance gene analogues from a range of Pinus species. These sequences w re further characterised through comparative analysis with previously reported Pinus resistance gene analogues. Through motif analysis, several of the known conserved motifs found in NBS domains were identified and conservation with other plant NBS motifs is indicated. The P-Ioop and GLPL motifs displayed a high level of conservation on amino acid level with other plant NBS motifs. However, slight differences in several of the conserved regions were observed when the Pinus analogues were compared with Arabidopsis thaliana. The identification of differences between angiosperm and gymnosperm NBS sequences indicates that design of new degenerate probes and primers for the isolation of more ancient NBS sequences is needed. Further, phylogenetic and structural analyses of these sequences will also aid in understanding the relationship between angiosperm and gymnosperm NBS sequences. The knowledge gained from such a study will highlight the similarities between angiosperm and gymnosperm defence responses. This study represents the first detailed report on RGA in Pinus. / Thesis (PhD)--University of Pretoria, 2006. / Genetics / Unrestricted

In the interactions between fungal endophytes and their hosts, the host may benefit through protection against pathogens and pests, growth promotion and tolerance to abiotic stresses. Non-pathogenic Fusarium oxysporum endophytes of banana have been shown to reduce the damage caused by the Cosmopolitus sordidus and the burrowing nematode Radopholus similis. The mode of protection against the burrowing nematode involves induced resistance, but the molecular basis of this resistance yet to be demonstrated. It has further been reported that protection of the host by multiple endophytes can lead to better control of target pests, probably because of the multiple modes of action involved. This phenomenon, however, has not been fully demonstrated for F. oxysporum endophytes of banana. This study aimed to investigate the molecular and biochemical basis of endophyte protection of East African Highland bananas (EAHB) against C. sordidus and R. similis. Expression of banana defence-related genes following endophyte inoculation and R. similis challenge varied greatly between the nematode-susceptible cv Nabusa and the nematode-tolerant cv Kayinja. In cv Nabusa, only the peroxidase (POX) and lectin genes were responsive to endophyte colonization of roots, or R. similis challenge. POX and lectin activities were significantly down-regulated 2 and 33 days after endophyte inoculation (dai), respectively. In cv Kayinja, endophyte colonization resulted in transient up-regulation of POX and a down-regulation of endochitinase (PR-3), lectin, pectin acetylesterase (PAE), phenylalanine ammonia-lyase (PAL) and PIR7A (peroxidase). Similar to systemic acquired resistance, PR-1 and catalase activities were up-regulated in the cv Kayinja 33 dai. Genes involved in signal transduction, cell wall strengthening, jasmonic acid pathway and defence molecule transport were differentially expressed in endophyte-inoculated plants. The expression profiles of four defence-related genes following endophyte inoculation and R. similis challenge were studied using quantitative real-time PCR. ABC transporter, Β-1,3-glucan synthase, coronatine insensitive 1 (COI1) and lipoxygenase (LOX) were up-regulated following endophyte inoculation. Β-1,3-glucan synthase and COI1 were highly up-regulated following R. similis challenge of endophyte-inoculated plants of the susceptible cv Nabusa, while COI1 and LOX were highly up-regulated following nematode challenge of endophyte-inoculated plants of the tolerant cv Kayinja. However ABC transporter gene activity was not up-regulated following nematode challenge of plants of both cultivars. UP-regulation of phenylpropanoid pathway enzymes PAL, POX and PPO has been observed in roots following colonization by both pathogenic and non-pathogenic fungi. In the current study, endophyte inoculation resulted in down-regulation of PAL activity in both a susceptible (cv Nabusa) and tolerant (cv Yangambi) banana. In cv Nabusa, endophyte inoculation primed PAL activity for up-regulation 30 days post nematode challenge (dpnc). However, in cv Yangambi PAL activity was up-regulated 7 dpnc irrespective of endophyte inoculation. Endophyte inoculation transiently up-regulated POX in cv Nabusa, but activity reduced to the levels in the controls 30 dai. Similar to PAL, R. similis challenge of endophyte-inoculated plants of Nabusa caused significant up-regulation of POX 7 dpnc. Nematode challenge of control plants of cv Yangambi resulted in a non-significant up-regulation of POX compared with non-challenged controls, but a significant up-regulation compared to all endophyte-inoculated plants. PPO activity was transiently up-regulated in cv Nabusa and down-regulated in cv Yangambi 7 dai. For all treatments, PPO activity was significantly reduced between 7 dai and 120 dai (60 dpnc). Fusarium oxysporum endophyte isolates Emb2.4o and V5w2 were successfully marked with benomyl- and chlorate resistance and transformed with fluorescent protein genes, while Eny1.31i, Eny7.11o and V4w5 were marked with benomyl resistance only. Most mutants and fluorescent protein transformants maintained resistance to the selective chemical on PDA and after plant colonization. Benomyl- and chlorate-resistant mutants were successfully used to determine actual plant colonization percentages by inoculated endophytes. Similarly, GFP transformants were successfully used to ascertain the pattern of endophytic root colonization in vivo. In plants dually inoculated with isolates Emb2.4o BR 8 and V5w2 CHR 9, both isolates were recovered from roots and rhizomes 4 weeks after inoculation, but isolate V5w2 CHR 9 proved a better colonizer of the two tissue types. Root colonization by isolate V5w2 CHR 9 was boosted when inoculated dually with Emb2.4o BR 8, while that by Emb2.4o BR 8 was reduced in the presence of V5w2 CHR 9. Where growth advantages were observed for dually inoculated plants, it occurred where plants were challenged with R. similis. In the absence of pests, control plants showed better growth than endophyte-inoculated plants. On the other hand, weevil challenge of control plants resulted in significant reductions in plant height, number of live roots and root fresh weight. Dual endophyte inoculation resulted in a significant reduction in R. similis populations in nematode only challenged plants, compared with plants inoculated with Emb2.4o BR 8 singly and control plants challenged with the nematode. In one replicate banana weevil damage to the outer and inner pseudostem base, and the inner rhizome were significantly reduced for dually-inoculated plants. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

An assessment of the effect that conventional storage structures, used by smallscale farmers in northern Kwa-Zulu Natal and southern Mozambique, had on germination and vigour of maize seeds was conducted. The survey confirmed that the methods of storing the seed decreased the quality of the maize seeds. Storing maize in the field was good as a short-term solution as initial germination was 100%. Following storage at suboptimum conditions, germination dropped to 25.3%. Commercially treated maize seeds were compared to the test samples collected. After storage, the commercially treated seeds maintained a germination percentage above 75. Untreated maize seeds were treated with fungicides at the recommended dosages. Thereafter the seeds were subjected to germination and vigour tests according to methods outlined by the International Seed Testing Association. All treatments maintained percentage germination above 75. Apron® XL had the highest percentage germination of 83. This trend was also found following the cold test and greenhouse emergence. None of the treatments differed significantly from the control. In this study none of the treatments caused major imbibition damage as indicated by the percentage weight increase and the low leachate conductivity (1012-1271 ìScm-1g-1). The effect of accelerated ageing (AA, 2 and 4 days) and long-term storage (3 and 6 months) on germination and vigour of treated maize seeds was investigated. In the untreated control and treatments there was a gradual decrease in germination following ageing and storage of the seeds. Apron® XL failed to germinate after 3 months. The decrease in germination was mirrored by the leachate conductivity readings. Thiram was the only treatment to maintain germination after 6 months storage. The seeds were planted in two greenhouse trials to assess the performance of the treatments in vivo. The first trial evaluated the emergence and second the emergence and control of Fusarium graminearum. Results from the first trial showed that following 2 d AA, seeds treated with Thiram had the highest percentage emergence (70.7) followed by Celest® XL (68) and the untreated control (62.7). Following inoculation, a similar trend was seen for the treatments and the untreated control. In relation to the percentage seedlings emerged, the control had the highest percentage diseased seedlings. Celest® XL had the lowest percentage diseased seedlings (10, 2 and 1) but failed to germinate after 6 months storage. Thiram was the only treatment to emerge after 6 months storage. The ultrastructural changes in embryonic roots of the untreated control, Celest® XL and Apron® XL were investigated using transmission electron microscopy. These seeds were subjected to 48 hr rapid imbibition and 2 d AA. The most obvious difference between the untreated control, Apron® XL and Celest® XL was the number and position of the vacuoles. In contrast the lipid layer was still attached to the cell wall in the Apron® XL and Celest® XL treatments but in the untreated control they appeared more concentrated in the cytoplasm. This study proved that Thiram was the best treatment among the fungicides tested. However, these results need to be confirmed using a larger range of maize seed lots. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

A survey involving 71 farmers from rural communities in Mpumalanga, South Africa was conducted to gather information regarding the importance and utilisation of cowpea. Cowpea was rated third most important in contributing to household security, preceded by maize and vegetable production. Cowpea was mainly produced for own consumption, as a source of income and as fodder for livestock to a lesser extent. The crop was used by 8.5% of the farmers for medicinal purposes. Results indicated that 20% of the farmers encountered problems with mouldiness during storage, with insect infestation to a lesser degree. Cowpea seed samples from South Africa and Benin, West Africa were analysed for seed mycoflora and various fungal genera, particularly Aspergillus, Phoma and Lasiodiplodia were recorded. The results indicated an array of Fusarium spp. including F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. scirpi, F. semitectum and F. subglutinans. The seed samples and the F. proliferatum isolates, cultured on maize patty media, were analysed for fumonisin production. Samples were extracted with methanol/water (70:30) and cleaned-up on strong anion exchange solid phase extraction cartridges. High-performance liquid chromatography with pre-column derivatisation using o-phthaldialdehyde was used for the detection and quantification of FB1, FB2 and FB3. The cowpea cultivars from South Africa had levels of FB1 ranging between 0.12 – 0.61 µg/g. All the F. proliferatum isolates produced FB1, FB2 and FB3 with total fumonisin concentration levels between 0.80 - 25.30 µg/g. The highest level of FB1 detected was 16.86 µg/g. Surface-disinfected seeds were imbibed in sterile distilled water amended with FB1 to yield final concentrations of 10, 25, 50 and 100 ppm. Percentage germination was determined in paper towels according to the International Seed Testing Association (ISTA) rules. Root and shoot length was measured after 9 days. All the toxin concentrations significantly decreased seed germination whilst root and shoot elongation was inhibited by the 50 and 100 ppm concentrations. Embryonic seed tissue treated with FB1 indicated compaction of the protoplasm and separation of the plasma membrane from the cell wall. Lipid bodies accumulated and seemed to line the cell wall. Acetone and ethanol extracts of the leaves of two cowpea cultivars exhibited significant inhibition of the growth of fungal plant pathogens at 5.0 mg/ml, with the exception of Fusarium equiseti. The growth of some fungi, in particular Alternaria alternate, was also reduced by lower concentrations of certain extracts. Acetone extracts of the Bechwana White cultivar inhibited growth of Staphylococcus aureus and Enterococcus faecalis at 2.5 mg/ml and Bacillus cereus, B. subtilis and Enterobacter cloacae at 5.0 mg/ml. Ethanol extracts of the same cultivar showed antibacterial activity against E. faecalis and E. cloacae at 5.0 mg/ml. This study represents the first report on the natural occurrence of fumonisins on cowpea seed and the potential of F. proliferatum isolates from cowpea seed to produce fumonisins. The phytotoxic effects of FB1 on cowpea seeds as well as the antimicrobial potential of cowpea leaf extracts were demonstrated for the first time. / Thesis (PhD)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Taxol (generic name paclitaxel), a plant‐derived antineoplastic agent, was originally isolated from the bark of the Pacific yew, Taxus brevifolia. Obtaining taxol from this source requires destruction of trees. It has been used alone or in combination with other chemotherapeutic agents for the treatment of breast, ovarian as well as many other types of cancer, including non‐small cell lung carcinoma, prostate, head and neck cancer, and lymphoma, as well as AIDSrelated Kaposi’s sarcoma. The mode of action of taxol against a number of human cancer cells is by preventing the depolymerization of tubulin during cell division. This molecule increases microtubule stability in the cell and induces apoptosis. From yew trees, the yield of taxol is usually between 0.004 to 0.1% of the dry weight. The commercial isolation of 1 Kg of taxol requires about 6 to 7 tons of T. brevifolia bark obtained from 2000‐3000 well‐grown trees. The limited supply of the drug has prompted efforts to find alternative sources of taxol. Alternative methods for taxol production, such as chemical synthesis, tissue and cell cultures of the Taxus species are expensive and give low yields. A fermentation process involving any microorganism would be the most desirable means to lower the cost and increase availability. The first report on the isolation of taxol‐producing fungi from Taxus brevifolia appeared in 1993 (Stierle, et al., 1993). Several taxol‐producing fungi have been identified since, such as Taxomyces andreanae, Taxodium disticum, Tubercularia sp., Pestalotiopsis microspora, Alternaria sp., Fusarium maire and Periconia sp (Li, et al., 1996, Strobel, et al., 1996a, Strobel, et al., 1996b, Li, et al., 1998b, Ji, et al., 2006, Xu, et al., 2006). This thesis investigates the isolation of an endophytic fungus, isolated from the stem cuttings of Taxus celebica, which produces taxol and related taxanes. We observed morphological and cultural characteristics and analyzed the sequences of rDNA ITS from the strain. The isolated fungus grew on potato carrot agar (PCA) medium at 25 °C and the colonies were white to off‐white, floccose, with irregular margins. The reverse side of the culture was cream in color. The morphology was examined microscopically following staining with cotton blue in lactophenol. Cultures produced macroconidia on slender, 85 μm long phialides. The macroconidia were 25‐40 X 3.75 μm. Cultures also produced round or oval microconidia. Analysis of the ITS and D1/D2 26S rDNA sequence revealed 99 % identity with Fusarium solani voucher NJM 0271. Based on its morphological, cultural characteristics and 26S rDNA sequence, the fungus was identified as F. solani. This fungus is different from the previously reported endophytic taxol‐producing species of Fusarium. Taxol and baccatin III, produced by this fungus, were identified by chromatographic and spectroscopic comparison with standard compounds. The amount of taxol produced by F. solani in potato dextrose liquid medium is low (1.6 μg l‐1) (Chakravarthi, et al., 2008). We further investigated different growth media and various factors of cultivation to select the medium and conditions that maximize production of taxol and other taxanes by this fungus. F. solani was grown in five well‐defined culture media under stationary and shake conditions separately for various time intervals and the amounts of taxol, baccatin III and other taxanes produced were estimated by competitive immunoassay. The modified flask basal medium (MFBM) was shown to yield the highest production of taxol (128 μg l‐1) which is 80 times more than when grown in potato dextrose liquid medium, baccatin III (136 μg l‐1) and total taxanes (350 μg l‐1) under shake conditions. From our results the highest taxol production of F. solani was achieved when cultured in MFBM. The production in MFBM was 80 times higher than that cultured in the potato dextrose liquid medium. In conclusion, it was shown that the culture medium plays a major role in taxol and other taxanes production and fungal growth. MFBM is the best medium, among the media studied, to produce taxol and other taxanes. The higher concentrations of NH4NO3, MgSO4, KH2PO4 and FeCl3 in the FBM medium seem important for production of taxol and other taxanes. These results can be considered as starting‐point for the research directed to improve taxol and baccatin III production by F. solani via different approaches including fermentations, strain improvement and genetic engineering techniques. Finally, in order to get more insights into the mode of action of this fungal taxol and baccatin III (for the first time), their apoptotic activity on different cancer cell lines was determined. We elucidated the biochemical pathways leading to apoptotic cell death after fungal taxol‐ and baccatin III‐ treatment in different cancer cell lines. Experiments are done on various cancer cell lines namely JR4 Jurkat (T‐cell leukemia), J16 Bcl‐2 Jurkat T cells, HepG2 (hepatoma), caspase‐8‐deficient Jurkat T cells, HeLa (human cervical carcinoma), Ovcar3 (human ovarian carcinoma) and T47D (human breast carcinoma) cells. We were able to demonstrate that both fungal taxol and baccatin III can induce apoptosis in all the cell lines tested, by flow cytometric analysis. Hallmarks of apoptosis following the signaling pathway to far more upstream‐located events were investigated using biochemical and cell biological methods. It has shown that during fungal taxol‐ and baccatin III‐induced apoptosis, DNA is degraded resulting in a increased number of hypodiploid cells reaching up to 65‐70% after 48 h. Disruption of mitochondrial membrane potential was examined by flow cytometric analysis using mitochondrial membrane potential sensitive dye JC‐1 and JR4‐Jurkat cells were shown to undergo significant loss of mitochondrial membrane potential loss of mitochondrial membrane potential reaching up to 70% in 6 nM fungal taxol and 65 % in 3.5 μM baccatin III after 36 h. These results were similar to those observed with standard taxol and baccatin III. We further investigated the role of caspases in fungal taxol‐ and baccatin III‐induced apoptosis, caspase‐8‐deficient Jurkat cells, Bcl‐2‐over‐expressed J16‐Jurkat cells and caspase inhibitors were used. Results derived from caspase‐8‐deficient Jurkat cells show that caspase‐8 is not involved in fungal taxol‐ and baccatin IIIinduced apoptosis of Jurkat cells. Using the pan‐caspase inhibitor (Z‐VAD‐FMK), caspase‐9 inhibitor (Z‐LEHD‐FMK), caspase‐3‐inhibitor (Z‐DEVD‐FMK), caspase‐2‐ inhibitor (Z‐VDVAD‐FMK) and caspase 10‐inhibitor (Z‐AEVD‐FMK), it was shown that caspase‐10 is involved in fungal taxol‐ and baccatin III‐ induced apoptosis in JR4‐Jurkat cells. It was also shown that inhibitors of caspases‐9, ‐2 or ‐3 partially inhibited fungal taxol‐ and baccatin III‐ induced apoptosis, whereas the caspase‐ 10 inhibitor totally abrogated this process. With the use of a fluorescence microscope, several morphological features characteristic of apoptosis such as condensed chromatin and apoptotic bodies were identified in fungal taxol‐ and baccatin III‐treated JR4‐Jurkat and HeLa cells. DNA fragmentations were shown by agarose gel electrophoresis method. Our work showed that treatment of JR4‐ Jurkat and HepG2 cells with fungal taxol and baccatin III induces apoptosis as shown by DNA ladder formation. Herein it was demonstrated that fungal taxol and baccatin III have a similar mechanism of action, but the efficacy of fungal taxol to induce apoptosis is higher. In summary, fungal baccatin III is found to be effective in inducing apoptosis similar to taxol but at higher concentration and both fungal taxol and baccatin III induce apoptosis via caspase‐10 and mitochondrial pathway in Jurkat cells. In conclusion, the present study describes isolation of a taxol‐producing endophyte F. solani IISc.CJB‐1. The growth requirements of this fungus for production of taxol, baccatin III and other taxanes were studied. The apoptotic activity of taxol and baccatin III (for the first time) was observed. In addition, our results show that the culture medium plays a major role in taxol and other taxanes production and fungal growth. Among the media studied, modified flask basal medium (MFBM) is the best to produce taxol and other taxanes. It is evident from this data that this fungal strain can be promising candidate for large‐scale production of taxol and related taxanes.

Management of Fusarium wilt of banana, one of the most important diseases of agricultural crops, is complicated and involves the consideration of factors such as the biology, epidemiology and population structure of the pathogen, and genetic resources and production practices of the crop. The development of an integrated disease management programme, therefore, is of great importance in countries where the Fusarium wilt pathogen, Fusarium oxysporum f.sp. cubense Foc, has been introduced into banaria fields, and where resistant cultivars are not acceptable to local markets. To achieve this, it is important to investigate new management strategies and to review methods that have been less successful in the past. These management practices need to be practical and affordable. Since certain cultural practices have proven to be effective, management practices that could compliment them should be considered. This thesis has attempted to investigate such practices in order to develop an integrated disease management programme for Fusarium wilt of banana. One of the most important findings of this study, was that the surface sterilant previously used to prevent the introduction of the Fusarium wilt into uninfected areas in South Africa, are not effective. The sterilants Sporekill and Prazin proved to be highly effective, and are now recommended to replace the sterilants previously used. Several fungicides reduced mycelial growth of Foc in vitro, with the OMI fungicides and Benomyl found to be the most effective. The same fungicides reduced the disease severity of Fusarium wilt in the greenhouse significantly, especially when they were applied as root dip treatments. None of the fungicides found effective against Foc have been evaluated in the field against Foc before. The next step, therefore, would be to evaluate root dip treatments combined with drench treatment in the field. Although it is expected that these fungicides might have a negative effect on the microbial populations in the soil, this has yet to be investigated. Fungicides may even weaken or stress the pathogen, making it more vulnerable for the action of an effective biocontrol agent or agents. Chemical activators are probably one of the most attractive strategies to combat Fusarium wilt of banana, since it stimulate the plants' own defence system. Banana plantlets were found to be quite sensitive to the amount and method whereby chemical activators were applied. The activator benzo-(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester induced resistance against Foc on the susceptible Williams cultivar in the greenhouse, but not in the field. In field studies, environmental conditions were much more variable than in the greenhouse, which made it difficult to evaluate the effectiveness of chemical activators. Sodium nitroprusside and a product containing the harpin protein showed promising results on the Williams and DRSI cultivars, respectively. These activators need to be considered as part of an integrated disease management programme. Since they are not directly applied to the soil, they will not have a negative effect on the microbial populations in the soil. Several Fusarium isolates had been collected from banana fields with disease suppressive soils in Kiepersol, South Africa. Most of these isolates were F. oxysporum, and with the exception of one isolate, proved to be non-pathogenic to banana plants. A PCR-based restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer region of the ribosomal RNA operon grouped the non-pathogenic F. oxysporum isolates into 12 distinct genotypes. A great diversity could be seen among the non-pathogenic isolates compared to the pathogenic Foc isolates. The known-biological control agent F047 grouped with three of the South African isolates, while the one pathogenic isolate grouped with the pathogenic Foc from diseased Cavendish bananas in South Africa By using PCR-RFLPs, we were able to rapidly characterize the structure of non-pathogenic isolates of F. oxysporum in disease suppressive soils in Kiepersol. This could assist us in our search for potential biological control agents for Fusarium wilt of banana. Representative isolates from the 12 genotype groups were selected for evaluation of Fusarium wilt suppressive properties in banana. These non-pathogenic F. oxysporum isolates appeared to be good biological control candidates and was compared to known biological control agents and commercial biological control products. Fourteen of the non-pathogenic isolates, the combination of two Trichoderma strains form suppressive soils in South Africa, and two Pseudomonas fluorescens isolates were found to significantly reduce Fusarium wilt development in the greenhouse. The commercial products Patostop, B-rus and a mixture of arbuscular mycorrhizae were also found to suppress the disease severity of Foc significantly. The well-know biological control agent F047 proved to be not effective. Results concluded that two of the non-pathogenic F. oxysporum isolates and the two P. fluorescens isolates, one of which was the well-known WCS 417, were the most effective of all the agents evaluated. Since combinations of biocontrol agents may provide even more consistent and effective control than a single agent, future research will include the combination of biocontrol agents found effective in this study. It would also be of great value to determine the mode of action of these isolates, so that isolates with different modes of action could be combined to enhance the suppression effect. Biological control can be a very useful component of an integrated disease management programme, since the effective agent or agents can easily be established on tissue culture banana plantlets before they are planted in the field. AFRIKAANS : Een van seker die mees belangrikste grondgedraagte siektes in lanbou, is Fusarium verwelksiekt van piesangs. In Suid-Afrika, is die siekte verantwoordelik vir emstige verliese in die piesang produksie. Aangesien daar geen weerstandbiedende kultivars beskikbaar is wat deur die mark aanvaar word nie, is dit van kardinale belang dat 'n geintegreerde siekte beheer program vir Suid-Afrika ontwikkel word. Voordat so 'n program saamgestel kan word, is dit belangrik dat verskeie faktore aangaande die patogeen en piesang poduksiepraktyke in ag geneem moet word. Beheermaatreëls moet prakties en bekostigbaar wees, en moet die reeds bestaande praktyke kan bevoordeel. Studies wat in hierdie tesis aangebied word, oorweeg beheermaatreëls wat gekombineer kan word met die huidige praktyke, nadat vorige praktyke ook in ag geneem is. Daar word gesoek na nuwe meer doeltreffende en ekonomiese metodes om siektes te beheer. Metodes wat doeltreffend aangewend kan word om die voorkoms van die siekte te vermirider. In vitro en in vivo studies het getoon dat die DMI swamdoders en Benomil die groei van die patogeen en die ontwikkeling van Fusarium verwelksiekte die meeste onderdruk. Die beste resultate is in die glashuis gevind nadat die wortels van plante in die middels geweek is. Positiewe resultate is ook verkry met die grondtoediening van Benomil 'n week nadat plante geplant is in Foc geïnfekteerde grond. Die chemiese beheer van Fusarium verwelsiekte kan verder ondersoek word deur die effek van die grondtoedienings en wortelbehandelings in die veld te ondersoek. Daar word egter verwag dat die swamdoders moontlik 'n negatiewe uitwerking op die mikrobiese aktiwiteit in die grond kan veroorsaak. Die gebruik van effektiewe ontsmettingmiddels is uiters belangrik vir die voorkomende beheer van Fusarium verwelkdiekte op piesangs. Die ontsmettingmiddel, koper oxichloried, wat tot onlangs in Suid Afrika gebruik was, is ondoeltreffend gevind vir ontsmettingsdoeleindes. Prazin en Sporekill, twee omgewingsvriendelike middels, is baie effektief gevind en word dus aanbeveel vir die ontsetting van voertuie, skoene en veld toerusing. Chemiese plant aktiveerders stimuleer plante om hulleself te beskerm deur middel van weerstandsmeganisms. Piesang plante het sensitiwiteit getoon toonoor die konsentrasie en die toedieningsmetode van hierdie chemiese aktiveerders. In die glashuisproewe het die aktiveerder benzo-(1,2,3)thiadiazole-7-carbothioic suur S-metiel ester weerstand gestimuleer in die Williams kultivar. As gevolg van veranderende toestande in die veld was dit moeiliker om die chemiese aktiveerders se werking te evalueer. Nogtans het die middels natrium nitroprussied en 'n produk wat die protein harpin bevat die voorkoms van siekte op die Williams en DRS 1 plante verlaag. Chemiese aktiveerders behoort sterk oorweeg te word as deel van 'n geintegreerde beheer program, aangesien chemiese aktiveerders nie direk tot die grond aangewend word nie, en geen negatiewe uitwerking op die natuurlike mikrobiese populasies in die grond uitoefen nie. Verskeie Fusarium isolate is geisoleer vanuit siekte onderdukkende gronde in die Kiepersol area van Suid-Afrika. Die meeste van die isolate is geidentifiseer as F. oxsysporum. 'n PKR-gebaseerde restriksie fragment lengte polimorfisme (RFLP) ontleding van die "intergenic spacer region" van die ribosomale DNS operon het die niepatogeniese F. oxysporum isolate in 12 verskillende genotypes opgedeel. 'n Groot diversiteit was sigbaar onder die nie-patogeniese isolate in vergelyking met die patogeniese foc isolate. Die bekende beheer agent, Fo47 het gegroepeer saam met drie van die Suid Afrikaanse nie-patogene. Hierdie tegniek het ons in staat gestel om die nie-patogeniese populasie van onderdrukkende gronde in Kiepersol vinnig te karakteriseer en potentiele biologiese agente te identifiseer. Verteenwoordigende isolate van die 12 genotipiese groepe wat geidnetifiseer is, is geselekteer vir verdere evaluasie. Dit is gevind dat die isolate goeie kandidate vir moontlike bio-beheer agente maak. Die onderdrukkingsvermoe van die nie-patogene is vergelyk met die van bekende bio-beheer agente en komersiele produkte wat beskikbaar is. Veertien van die nie-patogene, die kombinasie van twee Trichoderma spp., en twee Pseudomonas fluorescens isolate het die siekte ontwikkeling van Fusarium verwelking merkwaardig onderdruk in die glashuis. Die komersiele produkte Patostop®, B-rus en die kombinasie van twee mycorrhizae isolate is ook gevind om die voorkoms van siekte te verlaag. Die wel-bekende biobeheer agent Fo47 is oneffektief gevind teen Fusarium verwelksiekte van piesangs. Resultate van die studie het bewys dat twee van die nie-patogeniese F.Oxysporum isolate en twee P. Fluorescens isolate, waarvan een die welbekende WCS 417 is, uiters effektiewe beheer agente teen Foc is. Toekomstige studies sal fokus op die kombinasie van die bio-beheer agente wat die meeste potensiaal getoon het in die studie, asook hulle meganismes van werking. Biologiese beheer is van groot waarde vir 'n geïntegreerde beheer program. Dit kan maklik met bestaande beheer maatreëls gekombineer word en potensiële biologiese beheer agente kan vooraf op weefselkultuur plante in die kwekery gevestig word. / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted