The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
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- Date Issued: 2015
Evaluation and identification of microbial contaminants in polyherbal medicines used for the treatment of tuberculosis in Amathole District Municipality, Eastern Cape Province, South Africa
- Authors: Famewo, Elizabeth Bosede
- Date: 2018
- Subjects: Tuberculosis -- Treatment -- South Africa Mycobacterial diseases -- South Africa -- Eastern Cape Medicinal plants -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/9680 , vital:34818
- Description: Tuberculosis is caused by Mycobacterium tuberculosis. The emergence of drug-resistant strains of this organism has become a global public health problem. In the continuing search for effective treatment, polyherbal medicines offer a great hope in the development of alternative drugs for the treatment of tuberculosis. The use of herbal formulations for therapeutic purposes has significantly increased in the developed and developing countries because of their curative property, less toxicity and minimal side effects. However, there is little information on their safety and effectiveness in the literature. To address this, polyherbal medicines used for the treatment of tuberculosis in the Eastern Cape Province of South Africa were evaluated. Ethno-medicinal survey was conducted through semi-structured questionnaires coupled with informal conversations with the herbal sellers in five communities in the study area. Bacterial and fungal DNA was extracted from the polyherbal medicines purchased. A fragment of the bacterial 16S rRNA gene and internal transcribed spacer region of the fungal rRNA operon were amplified with universal primers 27F and 518R, and ITS1 and ITS4 respectively. Following standard procedures, the amplicons were finally run on Illumina’s MiSeq platform. Furthermore, the remedies were screened against Mycobacterium tuberculosis H37Rv using Middlebrook 7H9 media and MGIT BACTEC 960 system. Agar dilution method was used to determine the minimum inhibitory concentration (MIC) of the remedies against eight bacteria and three fungi isolates. The herbal preparations were assayed for their toxicity using hatchability success and larval mortality of Artemia salina Leach. Finally, their nutritive properties were analysed using an inductively coupled plasma optical emission spectrometer for mineral analysis while the vitamins were determined using standardized methods A total of nine polyherbal preparations were collected. The herbs used for the preparation of these remedies belong to 20 families. Apiaceae [5(25 percent)] was the most prominent plant family used, followed by Liliaceae [4(20 percent)], Strychnaceae [4(20 percent)], Rutaceae [4(20 percent)] and Hypoxidaceae [3(15 percent)]. The two most frequently used plants were Allium sativum L. (Liliaceae) and Strychnos decussata (Pappe) Gilg. (Strychnaceae). Rhizomes was the most common parts used, followed by the roots and barks. The herbal medicines were prepared mainly by infusion and decoction. The presences of pathogenic and non-pathogenic bacteria were identified in the polyherbal medicines. Generally, the most common bacteria identified from the samples were Bacillus sp., Enterobacter sp., Klebsiella sp., Rahnella sp., Paenibacillus sp., Clostridium sp. and Pantoea sp. The predominant mycoflora obtained belongs to different genera or species of fungi; these include Alternaria, Candida, Ramularia, Cladosporium, Penicillium, Aspergillus and Malassezia. The susceptibility testing revealed that all the remedies contain anti-tubercular activity against M. tuberculosis H37Rv at concentrations below 50 ug/ml. Seven of the polyherbal preparations showed activity at concentrations below 25 ug/ml. The MIC values exhibited inhibitory activity at 1.562 μg/ml. However, isoniazid showed more inhibitory activity against M. tuberculosis at 0.05 μg/ml when compared to the polyherbal remedies. The inhibitory activity of the polyherbal medicines based on the overall MIC revealed that Hogsback first site (HBfs) and Fort Beaufort (FB) remedies were the most active remedies against the bacterial isolates at the concentration of 2.5 mg/mL. Among the nine herbal formulations, only King Williams Town site A (KWTa) remedy showed activity against Aspergillus niger and Aspergillus fumigatus with the MIC valve of 2.5 mg/mL. While King Williams Town site C (KWTc) and Hogsback third site (HBts) had the highest activity at 1.25 mg/mL against Candida albicans, the remaining remedies were active at 2.5 mg/mL. The percentage hatchability of 44.42 percent, 42.96 percent and 39.70 percent were observed in A. salina cysts incubated with herbal preparations from KWTa, HBfs and HBts respectively. The hatching success of the cysts in these remedies was significantly higher than the positive control (nystatin) and the negative control (sea water) at p < 0.05. The mortality of A. salina nauplii incubated in Alice (AL), King Williams Town site B (KWTb) and KWTc remedies were significantly higher than when larvae were incubated in both controls. Based on Meyer’s index, the LD50 of each polyherbal medicine was between 2.9 and 4.0 mg/ml, the LD50 values greater than 1 mg/ml, an indication that they are not toxic. The polyherbal preparations were found to be rich in vitamins and mineral nutrients. Calcium was the highest macronutrient detected while the lowest nutrient was phosphorus. Iron was the highest micronutrient in the majority of the polyherbal preparations while the lowest value was recorded for copper. Vitamin C was absent in the herbal preparations while vitamin A and E were detected. This study provides significant ethno-medicinal information on polyherbal medicines used for the treatment of TB in the study area. The presence of the identified bacteria and fungi in the herbal formulations is a cause for concern. However, the ability of the remedies to possess activity against Mycobacterium tuberculosis and other pathogenic microorganisms associated with tuberculosis infection makes them potential sources of new antimycobacterial agents. Also, they are rich sources of mineral nutrients, and are as well non-toxic, thus, they are safe for consumption. In view of their anti-tubercular properties, this study has provided a better understanding of the reasons why TB-patients make use of these polyherbal formulations. Also, the study supports the folkloric use of polyherbal medicines in the treatment of tuberculosis in the Eastern Cape Province, South Africa.
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- Date Issued: 2018
Joint Modelling Inference for Longitudinal and Time To Event Data with Application to Biomarkers in Medical and Clinical Studies
- Authors: Azeez, Adeboye Nurudeen
- Date: 2020
- Subjects: Biochemical markers Bayesian statistical decision theory
- Language: English
- Type: Thesis , Doctoral , PhD (Biostatistics)
- Identifier: http://hdl.handle.net/10353/18476 , vital:42543
- Description: In the past couple of decades, longitudinal and survival data analysis have emerged as important and popular concepts of biostatistics and statistics for disease modelling. In recent years, these two statistics concepts have been combined to develop a joint model for longitudinal and survival data analysis. The Joint model is a simultaneous modelling application of longitudinal and survival data while taking into account a possible association between them. In this thesis, three sub-topics (Conditional score approach, estimating equation approach, and modified Cholesky decomposition approach) are utilised to model the association if the independence assumption is violated. Using the conditional score approach, the study investigated the association between longitudinal covariates and the time-to-event process to examine the within-subject measurement error that could influence estimation when the assumption of normality and mutual independence is violated. Given the assumption violation, I proposed an estimating equation approach based on the conditional score to relax parametric distributional assumptions for repeated measures of random effects. I jointly modelled the time-dependent biomarkers and event times using the Cox model with intermittent time-dependent covariates measure, in which the longitudinal model was used to characterize the biomarker underlying (unobservable) trajectory and incorporated as a latent time-dependent covariate in the survival model to predict failure times. Estimates of the parameters were obtained by a restricted maximum likelihood estimate (REML). A modified Cholesky decomposition method was used to capture the within-subject covariance for a positive definite and symmetric matrix, with the assumption that the observed data from different subjects are independent. I illustrated the proposed method by a real data set from a lung study and simulation. An extension to the joint model of longitudinal-survival data was also proposed, in which the longitudinal data has a cumulative and weighted effect on the hazard event function. Using a Bayesian parametric method, I proposed a skewed weighted probability density function to estimate the parameters. The weighted cumulative effect used enabled different longitudinal profiles to be incorporated over time in calculating the hazard ratio between the subjects. The proposed functions provide greater flexibility for modelling the association structure of different longitudinal and survival sub-model. The focus was on the association between the biomarker (serum creatinine, sCr) and the development of end-stage renal disease (ESRD). Since the effect of sCr biomarker is anticipated to be a cumulative effect, with the development of sCr biomarker over time leading to progressively higher damage of the kidney. The approach was applied a simulation for validation of the proposed method
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- Date Issued: 2020