Genus Trichoderma - anamorph of the genus Hypocrea (Hypocreaceae, Hypocreales, Sordariomycetes, Ascomycota) belongs to saprotrophic filamentous micromycetes. The representatives of this genus are known primarily as cosmopolitan inhabitants of soil ecosystems, including leaf litter and woody debris. This work summarizes the results of the study of isolates belonging to the genus Trichoderma originating from the territory of the Czech Republic, mainly from soil and leaf litter. During the study, altogether 267 isolates were analysed. For all of the isolates the following methods were used: phenotypic characterisation, UP-PCR fingerprinting, gene sequencing in tef 1α and ITS regions, and in the strain AK 115/00 also in rpb2 region. The main result of the study was the identification of 21 species of genus Trichoderma belonging into 4 groups and discovery of one isolate of species yet unknown to science. Species T. crassum, T. gamsii, T. ghanense, T. rossicum and T. longipile were found in the Czech Republic for the first time. According to the literature review and results of this study, 34 different Trichoderma species is currently known from the Czech Republic. In the work there is also discussed low reliability of identification of certain species based only on phenotypic characters. Key words:...

Although pharmaceutical industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by infectious microorganisms has increased. For a long period of time, plants have been a valuable source of natural products for maintaining human and animal health. The use of plant compounds for pharmaceutical purposes has gradually increased worldwide. This is because there are many bioactive constituents in plants which hinder the growth or kill microbes. Plants could be considered a potential gold mine for therapeutic compounds for the development of new drugs. In this study, sixteen South African plant species were selected based on their antibacterial activity after a wide screening of leaf extracts of tree species undertaken in the Phytomedicine Programme, University of Pretoria. Literature search excluded eleven plants because of the work already performed on their antibacterial activities, while Pavetta schumaniana was found toxic and thus not included in the screening. The remaining four plants namely; Buxis natalensis, Macaranga capensis, Dracaena mannii and Garcinia livingstonei were screened for antibacterial activity by determining the minimum inhibitory concentrations (MIC) against 4 nosocomial bacterial pathogens Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa, and also by using bioautography. The extracts of Macaranga capensis, Garcinia livingstonei, Diospyros rotundifolia and Dichrostachys cinerea had good antibacterial activity with MIC values of 0.03, 0.04, 0.06 and 0.08 mg/ml against different pathogens. The average MIC values of the plant extracts against all the tested pathogens ranged from 0.23-1.77 mg/ml. S. aureus was the most susceptible bacterial pathogen with average MIC of 0.36 . The extract of Diospyros rotundifolia was the most active with an average MIC against all the organisms of 0.23 mg/ml. The extracts of Buxus natalensis, Dracaena mannii, and Pittosporum viridiflorum, Acacia sieberiana, Erythrina lattissima, Cassine papillosa and Pavetta schumanniana had lower antibacterial activity. G. livingstonei was selected for further work on the basis of its good activity. The bulk acetone extract of Garcinia livingstonei (20g) was subjected to solvent-solvent fractionation which yielded seven fractions. Only the chloroform and ethyl acetate fractions showed good bioactivity in the microdilution assay and bioautography. Column chromatography was used to isolate two bioactive biflavonoids from the ethyl acetate fraction. The structures of the two compounds were elucidated using nuclear magnetic resonance (NMR) spectroscopy, and were identified as amentoflavone (1) and 4′ monomethoxyamentoflavone (2). These two compounds have been previously isolated from plants that belong to the Clusiaceae. The two compounds were isolated in sufficient quantity with a percentage yield of 0.45% for amentoflavone and 0.55% for 4′ monomethoxyamentoflavone from 20 g crude acetone extract. The antibacterial activity was determined against four nosocomial bacterial pathogens (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa). The MIC values ranged from 8-100 μg/ml. Except for Staphylococcus aureus which showed resistance to amentoflavone at >100 μg/ml. All the other tested organisms were sensitive to both compounds. It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. Based on this, the antioxidant activities of the two isolated compounds were tested using the Trolox assay. The two flavones had good antioxidant activity. Amentoflavone had a Trolox equivalent antioxidant capacity (TEAC) of 0.9. The second compound 4′ monomethoxyamentoflavone had a TEAC value of 2.2 which is more than double the antioxidant activity of Trolox, a vitamin E analogue. To assess the safety of the two compounds on cell systems, cytotoxicity was determined using a tetrazolium based colorimetric assay (MTT assay) using Vero monkey kidney cells. The compounds indicated little to low toxicity against the cell line with cytotoxic concentration (CC50) of 386 μg/ml and >600 μg/ml for compound 1 and 2 respectively. Berberine (used as the control toxic substance) had a CC50 of 170 μg/ml. The Ames genotoxicity assay is used to assess the mutagenic potential of drugs, extracts and phytocompounds. The compounds isolated in this study were assayed for genotoxicity using the Salmonella typhimurium TA98 strain. Amentoflavone was genotoxic at the concentration of 100 μg/plate, but 4′ monomethoxyamentoflavone was inactive at the highest concentration of 400 μg/plate tested. The results of the antibacterial, antioxidant and cytotoxicity testing were encouraging and indicated the potential usefulness of Garcinia livingstonei in traditional medicine and drug discovery. However, the genotoxicity assay revealed potential mutagenic effects of amentoflavone, a compound isolated from the plant. Therefore, it is suggested that application of Garcinia livingstonei extracts in the treatment of human and animal ailments be done with caution to avoid mutagenic effects on the treated subjects. A relatively small change in the structure of the two compounds by replacing an hydroxyl group with a methoxy group had a major effect in increasing antibacterial and antioxidant activity and in decreasing cellular and genotoxicity. This illustrates the potential value of modifying a molecule before its possible therapeutic use. Copyright / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

The HIV/AIDS pandemic has dramatically altered patterns of morbidity and mortality in sub-Saharan Africa during the last two decades. In the absence of HIV vaccine, microbicides may offer viable option for protection against HIV infection. Microbicides are products that are applied topically inside the vagina or rectum that act to impede transmission of HIV and other sexually transmitted diseases. Small human chemokines such as RANTES (regulated upon activation, normal T cell expressed and secreted) are currently been investigated as microbicides candidates. A number of N-terminally modified RANTES analogues such as 5P12 and 6P4 with a much higher antiviral potency have been developed and they have strong potential for use as microbicides. Since plants offer an alternative option for cost effective production of protein therapeutics, we evaluated the feasibility of expressing 5P12 and 6P4 in Nicotiana benthamiana species. 5P12 is considered the most promising candidate for use in the microbicide pipeline because it inhibits HIV infection through cellular receptor antagonism. Hence its feasibility of expression was also evaluated in Lycopersicon esculentum (tomato). The two analogues were transiently expressed in the selected plant species via agrobacterium-mediated transfection. For expression in N. benthamiana, two different vectors (pTRA and MagnICON) were used to deliver the two analogues for transient expression. About 6-8 weeks-old N. benthamiana plants were agroinfiltrated via needle injection and vacuum infiltration methods and targeted to four subcellular compartments viz: apoplast, chloroplast, cytosol and endoplasmic reticulum (ER). The agroinfiltrated leaves were replanted, grown in a tissue culture laboratory and harvested after different periods. For expression in L. esculentum, the MagnICON constructs were used to deliver the 5P12 gene into four different developmental stages of tomato fruits viz: mature green (MG), breaker (B), pink (P) and ripe (R) via needle injection. The agroinjected tomato fruits were incubated in a dark cupboard and harvested after different periods. xiii Proteins were extracted from the harvested material and evaluated for 5P12 and 6P4 expression. ELISA results showed expression of 5P12 and 6P4 in N. benthamiana leaves which was detectable at 3-9 days post infiltration (dpi). Similar results were obtained for 5P12 and 6P4, consequently only results for 5P12 are reported. The vacuum infiltrated leaves of both pTRA and MagnICON constructs led to higher yields than the needle injected leaves. The highest yields were obtained with the MagnICON constructs. The highest 5P12 expression level of 603 μg/kg fresh weight leaf tissues (~0.024% TSP) was obtained in the apoplast at 9 dpi. The pTRA constructs had the highest expression levels of 0.63μg/kg FW in the cytosol at 3 dpi. 5P12 was also detectable at 3-9 dpi in L. esculentum, based on ELISA results. The highest 5P12 expression of 23.56 μg/kg FW and pH 4.75 tissues was obtained at the MG stage in the apoplast at 9 dpi. Western blot analysis confirmed the size of plantmade 5P12. Moreover, the plant extracts had anti-viral activity and were not toxic to TZM-bl cells. Our results show that the RANTES can be made in both N. benthamiana and L. esculentum and that the levels are not different from other systems reported previously. Furthermore, this is the first report that a chemokine has been expressed in plants. The quantities expressed were low making the commercial development of a microbicide from these species impractical. However, production of bulky leaf material may enhance the quantities. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Paraclinical Sciences / unrestricted

Gas phase kinetics and thermochemistry of several halogenated species relevant to atmospheric, combustion and plasma chemistry were studied using experimental and ab initio theoretical techniques.

Recent use of molecular methods to revisit taxonomically problematic diatom species revealed severe limitations of morphological species concept. Characterization of diatom species which was based solely on the morphology of their frustules often generated too broad species boundaries which inevitably lead to wrong conclusions about their ecology and distribution. Widespread opinion that many diatom species are cosmopolitan generalists resulted in a theory that dispersal of diatoms is not limited by geographical distance. However, a number of recent studies showed that dispersal of diatoms is governed by the same rules which matter for macroorganisms. Proposed master thesis addresses the topic of diversity and dispersal in diatom species complex Frustulia rhomboides sensu lato in Europe and New Zealand. Results suggest that: (1) although revealed molecular variability in this complex shows clear ecological and biogeographical signal, it is not correlated in morphology. This is another support to general need for adoption of different species concept in diatoms. (2) A considerably uneven ratio in species diversity of genus Frustulia found in ecological similar habitats in Europe and New Zealand supports the idea that diatom dispersal is limited and stressed the need for studies dealing with biogeography.

The Nile crocodile (Crocodylus niloticus) is one of 23 extant crocodilian species, and has been farmed in southern Africa since the 1960s. For the crocodile industry, chronic stress and its often negative consequences are a concern, since stressors can negatively affect animal production as well as the health of the crocodiles. When confronted with a stressor, an individual displays a stress response consisting of a suite of physiological and behavioral alterations to cope with the challenge. So far, however, no method for determining stress-related responses in Nile crocodiles has been established. In other crocodilians, the assessment of physiological responses to stress, like the related alterations in glucocorticoid concentrations, has already been done, but only by using an invasive approach, with the disadvantage of a possible handling-induced stress response. By establishing a non-invasive technique to monitor glucocorticoid levels in captive Nile crocodiles based on faecal hormone analysis, this study not only made an important contribution to a better understanding of stress and related hormonal changes in Nile crocodiles, but also provided a solid basis for developing similar non-invasive tools to collect information on the level of stress experienced by other crocodilians. Specifically the study aimed 1) to assess adrenocortical activity in Nile crocodiles by measuring faecal glucocorticoid metabolite (FGM) concentrations, and 2) to characterise changes in FGM levels in captive Nile crocodiles in relation to different housing conditions. An adrenocorticotropic hormone (ACTH) challenge was performed on 10 sub-adult crocodiles at Le Croc crocodile farm, South Africa, resulting in serum corticosterone levels of up to ~1200 %, 1 - 5 hours post-injection, above the pre-injection levels. An additional 8 individuals were exposed to electric immobilisation and handling only (control group), which resulted in a 20 – 2700 % elevation in serum corticosterone concentrations, indicating that handling was already a sufficient stressor. FGM levels in 3 singly housed animals (2 ACTH challenge; 1 handling only) reached peaks of 136 – 380 % above pre-injection levels at about 7 to 15 days following treatment, demonstrating that non-invasive hormone monitoring can be used for assessing adrenocortical function in captive Nile crocodiles based on FGM analysis. By assessing the impact of group size (n = 1, 2, or 4 individuals) on FGM levels, highest mean hormone values were found in the paired animals. A possible explanation for this finding could be that the necessary re-grouping for the study resulted in an unstable group composition, especially for the paired animals of similar size, which is reflected in comparable higher FGM concentrations. However, future research would be necessary to investigate this potential relationship in more detail. My study created opportunities to improve the management and welfare of farmed crocodiles in terms of more appropriate housing conditions and husbandry for these animals. Finally, the now established non-invasive method for monitoring adrenocortical function in Nile crocodiles provides a solid basis for further studies focusing on monitoring factors influencing adrenocortical function in populations of Nile crocodiles in the wild. / Dissertation (MSc)--University of Pretoria, 2012. / Paraclinical Sciences / unrestricted

Odonata is an order of charismatic insects, commonly called dragonflies and damselflies, that provide a rewarding study system to answer questions such as those related to diversity and biogeography. Despite being relatively well-studied compared to other insect orders, odonates have suffered from inconsistencies and disagreements about proper morphological terminology that provide barriers to incoming workers. These disagreements have subsequently led to confusion and the incorrect application of terms. Here, we clarify the terminology and propose standard terminology for terminal appendages. This terminology is employed in a taxonomic revision of Vanuatubasis Ober & Staniczek, 2009. This endemic genus of damselfly found on the island archipelago of Vanuatu and was previously only known from three species. Here, we formally describe and treat all known species of Vanuatubasis including the association of females for known species. We also describe new species Vantuatubasis evelynae sp. nov., V. insularivorum sp. nov., V. kapularum sp. nov., V. nunggoli sp. nov., V. punicea sp. nov., and V. xanthochroa sp. nov. from material collected in Vanuatu. An illustrated key to both males and females of all species within Vanuatubasis is provided as well as distributions for all known species. Finally, we explore the biogeographical patterns in Vanuatu using this genus. We reconstruct a phylogeny of Vanuatubasis, in the context of its two sister genera, to begin unraveling the complex biogeography of this lineage. We test hypothesis of dispersal from Fiji, routes of colonization across the archipelago, and how relationships reflect geographic proximity in the genus. The results provide a vital first step in understanding the faunistic patterns within Vanuatu, as well as broader patterns across the Pacific.

Kikuyu (Pennisetum clandestinum) is highly productive during summer and autumn and capable of supporting high cattle stocking rates., The winter and spring production of kikuyu, however, is low, while forage quality, and consequently milk production per cow, is also low compared to temperate grass species. The aim of this study was to determine the dry matter yield, botanical composition, nutritional value, grazing capacity and milk production potential of irrigated kikuyu over-sown with Italian ryegrass (Lolium multiflorum var. italicum), Westerwolds ryegrass (Lolium multiflorum var. westerwoldicum) or perennial ryegrass (Lolium perenne) under an intensive grazing system with Jersey cows. Calibrations for the rising plate meter (RPM) were developed for the kikuyu-ryegrass systems. These calibrations were evaluated for seasonal variation, linearity and were also combined over seasons, treatments and years to develop a calibration equation that could be used by dairy farmers in the region. The three kikuyu based pasture systems reached their peak growth rates during different months and seasons. All treatments experienced lower growth rates during winter, while peak growth rates occurred during spring for the Italian ryegrass treatment; summer for the Westerwolds ryegrass treatment and late spring/early summer for the perennial ryegrass treatment. All three treatments had similar total annual dry matter yields (kg DM ha-1) during the first year of the study. However, during year 2 the PR treatment had a higher annual DM production than IR and WR treatments. The ability of the PR treatment to maintain DM production during periods when the other treatments underwent a dip in production (WR during spring and IR during summer) enabled the PR treatment to maintain a higher annual DM production during year 2 than the systems based on annual ryegrass species. As the kikuyu component increased in kikuyu-ryegrass pastures from winter to summer, the DM and NDF content increased, while the ME content decreased. All three treatments were deficient in Ca throughout the study and deficient in P during summer and autumn for high producing dairy cows. The Ca:P ratio was below the recommended ratio of 1.6:1 for high producing dairy cows throughout the study. The grazing capacity of all three kikuyu-ryegrass systems was lower during winter and autumn than during spring and summer. The seasonal grazing capacity of the perennial ryegrass treatment, however, was more evenly distributed than that of the Italian and Westerwolds ryegrass treatments. The perennial ryegrass treatment had a lower butterfat and milk production per lactation than the Italian and Westerwolds ryegrass treatments during both years, but had the highest milk production per ha. The latter was a result of the higher annual grazing capacity achieved by the perennial ryegrass treatment. It was thus concluded that, because kikuyu over-sown with perennial ryegrass supported a higher number of animals and had a more evenly distributed fodder-flow, it allowed for higher animal production per ha than kikuyu over-sown with annual ryegrass varieties such as Italian and Westerwolds ryegrass. The pre-grazing and post-grazing regressions of all three the kikuyu based pastures developed for the RPM differed over seasons and years, primarily due to the change in botanical composition from ryegrass based pastures during winter to kikuyu-based pastures in the summer and the associated change in pasture structure. The post-grazing regressions developed during the study did not have a lower degree of accuracy (R2 values) than the pre-grazing regressions. The generalised RPM regression equations developed for kikuyu-ryegrass pastures (consisting of large data sets pooled over treatments and years) could be of use to farmers in the surrounding area, but are not recommended for research purposes due to the large errors and variation associated with such regressions. In the event that farmers employ these calibrations it is important that pasture type and pasture management practices be similar to those utilised during the study. The decision on which kikuyu-ryegrass system to utilize should be based on the specific conditions prevalent on a particular farm, an economic analysis on and a comparison between the three systems, as well as the particular fodder-flow program requirements within the pasture system. / Dissertation (MSc(Agric))--University of Pretoria, 2010. / Plant Production and Soil Science / unrestricted

Medicinal plants have played an important role in drug discovery, with many pharmaceutical products originating from plants. Isolation and characterization of antibacterial compounds is still relevant today because of continuing development of resistance of bacteria to antibiotics. The aim of the study was to evaluate the antibacterial activity of leaf extracts of nine tree species (Acalypha sonderiana, Androstachys johnsonii, Dracaena mannii, Drypetes natalensis, Funtumia africana, Necepsia casteneifolia, Oncinotus tenuiloba, Turraea floribunda, and Xylia torreana) selected from the Phytomedicine Programme Database based on good antimicrobial activities. The next step was to select the most active plant species and to isolate and characterize the antibacterial compounds. A serial microplate dilution method was used to determine the minimal inhibitory concentration and bioautography was used to determine the number of antibacterial compounds in the extract and their Rf values. Four nosocomial infection pathogens (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus) were used as test organisms. Extracts of all the plant species were active with average MIC values ranging from 0.13 to 2.0 mg/ml against the four bacteria. MIC values as low as 0.08 mg/ml was obtained with F. africana and O. tenuiloba extracts against S. aureus. In bioautography seven of the nine leaf extracts had activity with clear zones of inhibition on bioautograms against the red background. F. africana was active against all four bacteria while O. tenuiloba had selective activity against P. aeruginosa with clear bands on the bioautogram. F. africana was chosen for further investigation because (a) it had good antibacterial activity against the four tested bacteria with MIC value as low as 0.08 mg/ml, (b) there were several active compounds against all the tested bacteria based on bioautography, (c) it is common in nature, and (d) as far as our literature survey could ascertain there was no published information on the antimicrobial activity of this plant species. The bulk powdered leaves of F. africana were extracted with acetone. The acetone extract was fractionated into five fractions (hexane, chloroform, butanol, H2O and 30% H2O in methanol) using solvent-solvent fractionation, to group the phytochemicals based on their polarity. Hexane and chloroform fractions were the most active with MIC values as low as 0.02 mg/ml for the chloroform fraction. One of the traditional uses of F. africana is to treat burns. As a result, the crude extract and its five fractions were also tested for anti-inflammatory activity using both the COX-1 and COX-2 assays. The crude extract and the hexane and chloroform fraction had moderate activity against both cyclooxygenase 1 and 2. The chloroform fraction was more active than the crude extract (59.7 ± 1.4%)with an inhibition of 68.2 ± 6.6%. Because there was no activity in the aqueous extracts and traditional healers usually use water as extractant, the pain relief experiences traditionally must be due to another anti-inflammatory mechanism. One antibacterial compound was isolated from the hexane fraction using column chromatography with silica gel as the stationary phase and a hexane ethyl acetate gradient as the mobile phase from low to high polarity. The isolated compound was identified as methyl ursolate using nuclear magnetic resonance (NMR) and mass spectrometry. Methyl ursolate has been isolated from a number of plant species. However, this is the first report on the isolation from Funtumia genus and the first report of its antimicrobial activity. Previous phytochemical investigation from the stem bark of F. africana led to the isolation of steroidal alkaloids of the conanine group. Methyl ursolate had a low activity with MIC values of >250 μg/ml against the four tested bacteria, but had better activity against five fungal (Candida albicans, Cryptococcus neomeforms, Fusarium oxysporum, Penicillium janthinellium, and Rhizoctonia solani) species with an MIC value of 63 μg/ml against F. oxysporum. The chloroform fraction had excellent activity with an MIC of 20 μg/ml and may be developed to become a useful complex drug. The more than one hundred fold lower activity of the isolated methyl ursolate compared to the activity of the chloroform fraction from which it was isolated, provides strong evidence of synergism. This may be good model system for studying synergism in antimicrobial preparations. / Dissertation (MSc)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Les écosystèmes des eaux souterraines sont de plus en plus reconnus pour leur faune endémique, phylogénétiquement ancienne et écologiquement spécialisée. Avec plus de 425 espèces décrites, les amphipodes niphargidés constituent la famille des eaux souterraines la plus riche en espèces au monde et un système modèle intéressant pour la biologie de l'évolution. Cependant, les scientifiques doivent faire face à des données incomplètes et biaisées en raison de trois déficits majeurs: le déficit Linnéen pour la taxonomie, le déficit Darwinien pour la phylogénie, et le déficit Wallacien pour la biogéographie. La présente thèse vise à évaluer l'importance de ces déficits chez les niphargidés, ouvrant ainsi la voie pour y remédier. Le premier chapitre est une évaluation des effets de la découverte d'espèces cryptiques (une des causes du déficit Linnéen) sur notre compréhension des modèles de distribution à grande échelle de la diversité des niphargidés. Contrairement à ce que l'on attendait, les espèces cryptiques putatives sont réparties de manière homogène le long des gradients environnementaux, et leur découverte ne modifie donc pas notre compréhension des modèles de distribution. Le deuxième chapitre analyse l'importance de l'application des techniques moléculaires à la taxonomie des niphargidés. En étudiant le genre Microniphargus, la morphologie seule s'est avérée peu informative en raison de la pédomorphose et de l'homoplasie. L'utilisation de marqueurs ADN a permis d'attribuer le genre à une famille différente (Pseudoniphargidae), venant éclaircir les relations phylogénétiques au sein des Niphargidae (et contribuant ainsi à remédier au déficit Darwinien). Le troisième chapitre traite du rôle des régressions et transgressions marines sur la distribution des niphargidés en utilisant la biogéographie moléculaire et une modélisation biogéographique innovante (afin de remédier au déficit Wallacien). Les résultats soutiennent l'idée que la dispersion a joué un rôle essentiel dans la biogéographie historique des niphargidés, en montrant que leurs voies de dispersion sont corrélées à des événements paléogéographiques anciens. Enfin, le quatrième chapitre traite de la taxonomie, de la phylogénie et de la biogéographie d'un clade de niphargidés distribué dans la région des Alpes et des Carpates, et illustre un cas de discordance mitonucléaire dans la délimitation d'espèces vivant dans des zones affectées par les glaciations quaternaires. Une histoire complexe de divergence de lignées évolutives et de contacts secondaires pendant les fluctuations climatiques du Pléistocène explique la plus grande variabilité de l'ADN mitochondrial par rapport aux marqueurs nucléaires. Dans une telle situation, la description formelle d'espèces cryptiques basée sur le seul barcodage de l'ADN mitochondrial, comme dans certains articles récents sur les niphargidés, n'est pas recommandée. Cette thèse ouvre plusieurs perspectives pour des recherches futures basées sur la taxonomie intégrative et la modélisation biogéographique, permettant aux niphargidés très diversifiés de jouer un rôle majeur dans la surveillance des écosystèmes des eaux souterraines. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished