Theses and Dissertations (Biochemistry, Genetics and Microbiology (BGM))

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Mitochondrial genomes from the Ceratocystidaceae and species boundaries in Ceratocystis
(University of Pretoria, 2024-11) Duong, Tuan A.; Wingfield, Brenda D.; Kanzi, Aquillah M.; anien95@gmail.com; Viljoen, Anien
The fungal family Ceratocystidaeceae contains several genera that are considered highly pathogenic and able to cause disease on a wide range of hosts. The taxonomic history of Ceratocystidaceae species has been under debate for many years due to the lack of morphologically distinguishing characteristics between the species and genera. Chapter one of this thesis provides a broad overview of the speciation process from defining species to studying the barriers that prevents gene flow between populations or prevents hybrid zygotes from developing before and/or after fertilization. Different speciation genomic approaches, their advantages and limitations are also discussed. The chapter also provides a brief overview of the taxonomy, species delineation and the controversies associated with this process in the Ceratocystidaceae, focusing specifically on the Ceratocystis fimbriata sensu lato complex. The second chapter presents the work on characterisation of mitochondrial (mt) genomes for 18 species in 10 genera in the Ceratocystidaceae. The aim of this chapter was thus to assemble, characterise and comparatively analyse mt genomes from multiple species and genera in the Ceratocystidaceae. The work of chapter 3 focuses on the taxonomic confusion that has historically plagued the C. fimbriata s.l complex. The aim of this chapter was to use whole genome SNP data from a large collection of isolates of C. fimbriata sensu lato complex to investigate species boundaries, phylogenetic relationships, and the genetic differentiation between these closely related species. Taken together, the body of the work presented in this dissertation contributes to the current understanding of mitochondrial genome evolution, species boundaries, and the evolutionary relationship among species and genera within the Ceratocystidaceae. These findings pave the way for future research aimed at exploring the biology, pathogenicity, speciation, and host adaptation of the important group of fungi.
The effect of mpc1/mpc2 overexpression in intraerythrocytic Plasmodium falciparum parasites
(University of Pretoria, 2024-11) Niemand, Jandeli; Birkholtz, Lyn-Marie; u18001964@tuks.co.za; Voges, Suzelle
Pyruvate functions as a metabolic switch between aerobic and anaerobic metabolism, allowing a shift to an alternate metabolic pathway when required. The mitochondrial pyruvate carrier heterocomplex (MPC), composed of MPC1 and MPC2, has been identified as the transport complex responsible for pyruvate transport into the mitochondria. In P. falciparum, the putatively annotated mpc1 (pf3d7_1340800) and mpc2 (pf3d7_1470400) genes have yet to be characterised. In this study, the mpc1 and mpc2 genes in P. falciparum parasites were investigated using an overexpression approach. A transgenic P. falciparum parasite line constitutively expressing mpc1/mpc2 above basal level was established, and increased MPC abundance was confirmed. The transgenic parasites were then compared to the wild-type to confirm that the genetic modification allowing the mpc1/mpc2 overexpression did not negatively affect intraerythrocytic parasite proliferation, survival, or morphology. Likewise, mitochondrial viability, abundance of other mitochondrial metabolism proteins, and parasite sensitivity to compounds inhibiting mitochondrial function was also unaffected. To gain insight into the biology surrounding the activity of the mpc1/mpc2 genes, the downstream effects of mpc1/mpc2 overexpression in the transgenic and wild-type P. falciparum parasites lines were compared. Chemical interrogation with an MPC inhibitor indicated a reduction in parasite sensitivity to the inhibitor upon mpc1/mpc2 overexpression, whereas no difference in the parasite sensitivity was observed when treated with other types of inhibitors. The overexpression of mpc1/mpc2 resulted in reduced lactate production, as expected, since less pyruvate would remain in the cytosol to be converted into lactate. Additionally, mpc1/mpc2 overexpression promoted parasite survival during glutamine starvation as expected, since more pyruvate can enter the mitochondria to drive the TCA cycle to compensate for the lack of glutamine. Therefore, increased mpc1/mpc2 expression resulted in biological changes consistent with the expected biological responses of increased MPC activity, confirming the annotation of these genes as a MPC in intraerythrocytic P. falciparum parasites.
Genomics insights into the global evolution and antibiotic resistance of the Mycobacterium tuberculosis complex
(University of Pretoria, 2025-02) Reva, Oleg; u17288632@tuks.co.za; Muzondiwa, Dillon
Mycobacterium tuberculosis (Mtb) recently reclaimed its status as the leading global cause of death from a single infectious agent after three years of COVID-19 holding the top position. We used Mtb whole genome sequencing data (WGS) to explore the diversity of human-adapted lineages of Mtb. Using publicly available datasets, we curated and characterised a large global WGS dataset of more than 9000 Mtb strains sampled across the globe. Based on the distribution of single nucleotide polymorphisms, we performed lineage classification, drug resistance predictions and molecular clock estimations to characterise the global evolution of Mtb and create a non-redundant global reference dataset. Our data suggested that public Mtb WGS datasets are highly redundant, and redundancy minimisation is required before analysing large datasets. We next sought to explore the evolutionary dynamics that shaped the genetic landscape of the African continent which has been suggested as the origins of Mtb. We demonstrate that Lineage 2 and Lineage 4 are the most dominant on the continent. Using Maximum Likelihood and Bayesian phylogenetic techniques, we mapped identified drug resistance-associated mutations to time-resolved phylogenies. We estimated that drug resistance on the continent emerged at multiple events, with the earliest emergence of drug resistance occurring in the mid-20th century. We also identified the presence of resistance mutations associated with recently introduced drugs in isolates that were sampled prior to the use of these drugs. Using Bayesian skyline coalescent inference, we observed an expansion in the Mtb population in Africa in timelines that coincided with increased migration from Europe and Asia into Africa. We also inferred a population expansion of Mtb at the time when HIV prevalence was at its peak on the continent. We next sought to understand the evolutionary dynamics of Lineage 2 and Lineage 4 Mtb in the Southern Africa Development Community (SADC) region, a part of the continent which carries the highest burden of HIV/TB coinfection. We demonstrate that the heterogeneity of Mtb Lineage 2 diversity in the SADC region is under-characterised. We identify 13 sublineages of Lineage 2 in the region from our analysis. To explore the origins of SADC Lineage 2 and Lineage 4, we employed two phylogeographic approaches and both of them place East Asia as the origin of Lineage 2 and Europe as the origin of Lineage 4. We also infer that the two lineages were introduced through multiple introduction events with South Africa as a central hub for the dispersion of the lineages northwards. Taken together, our phylogeographic analysis and our Bayesian skyline results suggest that migration and colonialism played a role in shaping the diversity of the two Lineages in SADC. Lastly, using mathematical models, drug susceptibility testing data and genomic data, we sought to model the epistatic dynamics that govern drug resistance in Mtb. We obtained co-dependency estimates that represent the probability of one mutation emerging after another mutation. We then created networks and traced the trajectories from drug susceptibility status to pre-XDR-TB status.
Detection, dispersal and global distribution of Ceratocystis species on Eucalyptus
(University of Pretoria, 2024-10) Barnes, Irene; Wingfield, Michael J.; u14049423@tuks.co.za; Lynn, Kira Mary Theresa
In this thesis, Detection, dispersal, and global distribution of Ceratocystis species on Eucalyptus, the candidate investigated the species boundaries and transmission mechanisms of two economically important fungal pathogens. Utilizing a dataset of 1174 isolates collected from 11 countries and 8 plant hosts, population genetics, phylogenetic analysis, and morphological comparisons were employed to confirm that Ceratocystis manginecans and C. eucalypticola are distinct species, resolving long-standing taxonomic confusion. Hybridization events between these species were also identified clarifying ambiguity in the literature. The candidate developed a cutting-edge qPCR-based HRMA diagnostic tool enabling rapid species identification of these fungal pathogens directly from infected plant material and fungal cultures. This tool was successfully applied to reveal key dispersal mechanisms, including insect-mediated and airborne transmission. These findings offer valuable insights into pathogen detection, dispersal, and species boundaries, providing crucial tools for effectively managing the spread of these economically significant pathogens within the global forestry industry.
The efficacy of mycolic acid-enhanced PLGA nanoparticles for rifampicin delivery in tuberculosis treatment
(University of Pretoria, 2024-08) Lemmer, Yolandy; Verschoor, Jan Adrianus; kruger.goosen@gmail.com; Goosen, Kruger
This study explored the efficacy of nanoparticle-encapsulated rifampicin formulations for treating high Mycobacterium tuberculosis (M. tuberculosis) bacillary loads using a guinea pig model. Two formulations were compared: rifampicin encapsulated in poly(lactic-co-glycolic acid) (PLGA/RIF) and PLGA/RIF coated with mycolic acid (PLGA/RIF/MA), against traditional rifampicin treatment and control groups. Data obtained from this study will guide optimisation for future drug efficacy testing, particularly for targeted TB treatment. The guinea pig model was chosen due to its physiological and immunological similarities to human TB infection. Detailed investigations included clinical monitoring, macropathological and histopathological assessments, and bacterial load quantification to evaluate treatment efficacy, side effects, and overall animal health. Key findings indicated differences in survival rates, clinical signs of TB progression, and bacterial load reduction among the treatment groups. The PLGA/RIF group showed promising results in terms of survival rates and bacterial load reduction, suggesting potential benefits of nanoparticle-encapsulated drug formulations. However, the addition of mycolic acid in the PLGA/RIF/MA formulation did not significantly enhance treatment outcomes compared to PLGA/RIF alone, highlighting the complexity of optimizing nanoparticle formulations for TB treatment and the need for further research. The study also addressed the challenges of achieving statistical significance in animal model research, particularly in pilot studies with limited sample sizes and high variability. It emphasized the need for method refinement to lower variability and increase method repeatability and reproducibility, leading to more statistically powered studies to confirm preliminary findings and fully assess the efficacy of nanoparticle-encapsulated TB treatments. Additionally, the study recommended serological testing for TB biomarkers as a method for early TB detection in animal models, potentially enabling earlier treatment initiation and providing insights into infection dynamics and treatment response. Overall, this study laid the groundwork for further exploration of nanoparticle-based drug delivery systems in TB treatment. It underscores the considerations in designing targeted drug delivery, the challenges and potential of PLGA as a drug carrier, and the scope for innovative approaches to improve TB treatment efficacy and patient outcomes. Future studies are encouraged to build on these findings, refine animal models, and explore novel early diagnostic methods to advance TB research and treatment.
Analysis of rabies surveillance in selected African localities and prospects for continent-wide improvements
(University of Pretoria, 2024-10) Nel, Louis Hendrik; Wright, Nicolette; aylamalan@gmail.com; Malan, Ayla Janina-Bertha
Despite being a vaccine-preventable disease, rabies remains a significant public health concern throughout the developing world in particular, including all the countries of mainland Africa, where the disease results in the deaths of more than 25,000 people annually. Through a systematic review of rabies in African countries, a situational analysis highlighted the multifaceted challenges that contribute to the persistence of rabies and the current strategies employed by countries towards rabies elimination. Based on this, six critical barriers to rabies elimination could be identified: high domestic dog populations, inadequate surveillance and reporting, limited access to post-exposure prophylaxis (PEP) and vaccines, economic constraints, cultural factors, and low public awareness. This further highlighted the persistent nature of rabies across most African nations, emphasising the need for effective dog population management strategies and mass dog vaccination campaigns to break the cycle of rabies transmission. Furthermore, inadequate surveillance systems and reporting mechanisms result in a severe underreporting of rabies cases in both humans and animals, leading to an underestimation of the true burden of rabies. As such, national surveillance frameworks need to be implemented and should incorporate multidisciplinary partners and collaborative efforts between neighbouring countries. In an effort to supplement limited surveillance data by predicting gaps in the data, spatio-temporal analyses and data analyses were done using 21 years of laboratory-derived surveillance data from South Africa. The detailed spatio-temporal analysis presented here showed significant disease outbreaks in the eastern half of the country, where the highest human and dog populations could be found. Notably, the study identified underreported districts with limited surveillance, suggesting that enhanced monitoring could reveal larger disease clusters and enable targeted interventions. The analysis also captured the interplay between domestic and wildlife rabies, emphasising the need for integrated control measures. This case study based on South Africa provided evidence of the value of this approach to demonstrate the underestimation of the burden of rabies in the face of poor surveillance data – which is reality for all of Africa. Therefore, replication of this approach in other African countries and regions could add much value to the demonstration of disease burden and the direction of intervention strategies. Finally, considering the de facto challenges of rabies surveillance, a novel rapid diagnostic protocol was designed and evaluated for its potential to provide substantial improvements to the routine diagnosis and surveillance of the disease. In this protocol, the techniques for brain sampling were much simplified and lateral flow devices (LFDs) were implemented in a study that spanned over 28 months on the Unguja island of Zanzibar. Since the LFDs were implemented, a significant increase in the samples collected and sent for diagnostic testing could be seen. This work not only highlighted that LFD devices were highly specific and sensitive in diagnostically screening rabies cases but also showed that their implementation boosted the surveillance network on the island, leading to a more active surveillance approach. This, coupled with real-time reporting, enabled a meaningful increase in both active and passive surveillance across the island of Zanzibar, facilitating rapid outbreak responses, such as targeted and strategic vaccinations to rapidly break transmission cycles before rabies can spread and affect more animals and people. This further highlights the importance of novel techniques to improve overall surveillance.
Functional characterization of candidate Persea americana nucleotide-binding leucine rich repeat (PaNLR) genes during Phytophthora cinnamomi infection
(University of Pretoria, 2024-11) Van den Berg, Noelani; Swart, Velushka; susanna.anbu@up.ac.za; Anbu, Susanna Pearl
Avocado (Persea americana) production is threatened by Phytophthora cinnamomi, the causative agent of Phytophthora root rot (PRR). Current control measures rely on phosphite-based fungicides and partially-resistant rootstocks like Dusa®. However, the immune mechanisms underlying Dusa®'s resistance remain unclear. This study investigated nucleotide-binding leucine-rich repeat (NLR) proteins in Dusa®, which play a critical role in effector-triggered immunity (ETI) and hypersensitive response (HR). Using a Nicotiana benthamiana-P. cinnamomi model system, we identified the biotrophic and necrotrophic phases of infection, applying this knowledge to study avocado NLRs. Six PaNLR candidates were selected based on expression patterns, defense motifs, and effector recognition potential. Transient expression assays revealed that five PaNLRs caused HR only upon pathogen introduction, while one induced cell death independently. Cellular localization studies showed most PaNLRs reside in the nucleus during the resting stage, with one in the cytoplasm. Upon P. cinnamomi infection, all candidates triggered a strong HR, effectively restricting pathogen spread. These findings suggest that PaNLRs adopt diverse strategies to induce immunogenic cell death, balancing resource conservation and rapid defense activation. This study provides the first molecular characterization of PaNLRs, offering insights into avocado immunity and pathways for enhancing resistance in this economically important crop.
Characterisation of the WAK / WAKL gene family, with a focus on members in Persea americana implicated in defense against Phytophthora cinnamomi
(University of Pretoria, 2024-10-31) Swart, Velushka; Van den Berg, Noelani; aaronharvey0627@gmail.com; Harvey, Aaron Thomas
Phytophthora cinnamomi is a devastating oomycete pathogen, which has a significant negative impact on global avocado (Persea americana) production. The wall associated kinases (WAKs) and wall associated kinase-likes (WAKLs) are pattern recognition receptors which detect fragmented pectin (oligogalacturonides formed during pathogen infection) and activate downstream plant defence responses. The identification and charaterisation of this gene family shows many inconsistencies which needed to be addressed before delving into the avocado WAK/WAKL gene family and describing which member are implicated in the defence response of P. cinnamomi. Thus, this dissertation aimed to critically evaluate the in silico characterization of the WAK and WAKL gene family as a whole and then in avocado, with a particular focus on the genes involved in defence against P. cinnamomi. The study begins with a critical evaluation of current methodologies for identifying and classifying WAK/WAKL genes across various plant species, revealing inconsistencies in identification, classification and characterisation protocols that hinder broader gene family conclusions. The review provides a streamlined approach to help increase the standardisation in this gene family so that all genes defined as WAKs or WAKLs contain the same properties, such as a standardised set of predicted protein domains in the downstream proteins. The review also provides recommendations for characterising the family to allow a broader description to be concatenated for large-scale analyses in the future. The core of the research is the comprehensive in silico characterization of the avocado WAK/WAKL (PaWAK/WAKL) gene family. The study describes the PaWAK/WAKL family composition, gene placement and structure, the protein properties and cellular localisation, phylogenetic relationships, their expression patterns during pathogen infection, and the potential impact of cis-acting elements in targeted regulation during defence. Protein 3D structures and the binding affinities of WAK/WAKLs and the damage-associated molecular pattern, oligogalacturonide, were predicted for defence-implicated proteins. This dissertation examines the PaWAK/WAKL on a genomic, transcriptomic, and proteomic level to identify differences between the partially-resistant Dusa® and susceptible R0.12 rootstocks (while also assessing the partially-resistant LeolaTM) to predict how these factors, in combination, contribute to the increased defence efficiency against P. cinnamomi. These findings highlight the significance of specific PaWAK/WAKL genes in avocado's defence against P. cinnamomi on a multi-omic level. This research not only enhances our understanding of this disease interaction but also serves as a foundation for developing molecular tools to screen for resistant rootstocks for commercial use.
Assessment of groundwater quality for irrigation and drinking purposes in the Limpopo Granulite-Gneiss region, Limpopo Province, South Africa
(University of Pretoria, 2024-09) Claassen, Marius; mirrander.n@gmail.com; Ndhlovu, Nothando Mirrander
The global freshwater availability studies have classified South Africa as water stressed in 1999 and approaching water scarcity by the year 2025. While groundwater is available everywhere, it is not always in suitable quality meaning some groundwater dependent rural communities lack adequate information about the water quality status of their groundwater supplies. While there may be significant groundwater research studies done, the outputs never reach the impacted communities. Groundwater accounts for nearly 70% of rural domestic water supply in Limpopo Province. Furthermore, rapid population growth and more frequent drought events have led to reduced surface water supply and increased in groundwater abstraction, putting groundwater resources under enormous pressure. As communities grow, more groundwater is abstracted, and land use changes to more paved roads, houses, shopping centres, and parking lots, decreasing groundwater recharge. A total of 319 groundwater samples, from 17 monitoring sites located within Limpopo granulite-gneiss region, collected between 2000 and 2017, were analysed in order to evaluate its suitability for drinking and irrigation purposes. The abundance of cations and anions are showing Na+>Ca2+>Mg2+>K and HCO3->Cl->SO42->NO3-+NO2->F-, respectively. The weighted average values show major anions are dominant over the major cations. Na+ accounts for 48% of the cations and HCO3- accounts for 41% of the anions. The pH of groundwater in the study area ranges from 6,9 to 9,2; with over 83,7% of the samples falling within the pH 7,5 to 8,5 range; while 5,0% of the samples are within the pH 6,5 to <7,5 range; and 11,3% samples are within the pH >8,5 to 9,2 range. TDS values range from 107 to 2426 mg/L with a weighted average of 1122 mg/L. At least 57% of the samples are categorically brackish and 43% are fresh. On the Piper diagramme, the water samples are mainly plotting on Ca-Mg-HCO3 type (zone 5) and mixed (Ca-Mg-Cl-SO4 and Ca-Mg-HCO3) type (zone 9), Ca-Mg-HCO3 type indicating carbonate (temporary) hardness. The Gibbs diagramme indicates that groundwater chemistry is controlled mainly by evaporation dominance mechanisms, while high Ca2++Mg2+ concentrations relative to HCO3- concentrations, indicate silicate weathering involving reverse ion exchange. Pearson correlation analysis shows a very strong positive correlation of Na+ with Cl- (0,80), SO42- (0,81), and F- (0,78). Suitability for irrigation assessment results show that all the groundwater samples were suitable for irrigation purposes based on sodium adsorption ratio, residual sodium carbonate, and permeability index. However, for the Kelly ratio index, seven monitoring sites are unsuitable for irrigation purposes. Over 71% of the water samples have concentrations of nitrate higher than the WHO and SANS241 recommended guideline value of ≤11 mg/L, making 15 out of 17 monitoring sites unsuitable for drinking. However, the remaining two sites failed drinking water suitability in TDS, Na, Cl, SO4, and F based on SANS241and WHO, guidelines. To mitigate methemoglobinemia, a simpler, cheaper, and more immediate approach that is within the capabilities of communities is recommended.
In vitro activity and mode of action of the antimicrobial peptide CPF-P2 against Escherichia coli ATCC 700928
(University of Pretoria, 2024-09-16) Gaspar, A.R.M. (Anabella Regina Marques); Bester, Megan J.; Ibrahim, Mohammed Auwal; molebogengonkaetse@gmail.com; Onkaetse, Molebogeng Refemetswe
A major challenge associated with antimicrobial resistance (AMR) is the lack of new drugs especially for the treatment of infections caused by Gram-negative bacteria. Antimicrobial peptides (AMPs) present an alternate solution to the crisis of AMR, as AMPs are novel antimicrobials with unique modes of action and potentially reduced risk of developing antibiotic resistance. Previous studies have identified the antimicrobial potential of the frog skin-derived AMP, the caerulein-precursor fragment-P2 (CPF-P2). To advance the development of this AMP for therapeutic applications, the aim of this study was to further evaluate the antimicrobial activity, mode of action, cytotoxicity and stability against Escherichia coli ATCC 700928. Against E. coli ATCC 700928, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of CPF-P2 was 16 ± 0.7 μg/mL (6.08 ± 0.27 μM), with an MIC/MBC ratio of 1 indicating that this AMP is bactericidal. Against E. coli biofilms, the minimum biofilm prevention concentration (MBPC) of CPF-P2 was 640 μg/mL (243.2 μM), whereas both the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were >2560 μg/mL (> 972.8 μM). This implies that although some biofilm prevention occurred, against established biofilms CPF-P2 was ineffective. Only at 128 μg/mL (48.64 ± 2.64 μM) and 256 μg/mL (97.28 ± 2.25 μM) CPF-P2, did human erythrocyte haemolysis occur while some cytotoxicity was observed against HaCaT cells at 256 μg/mL (97.28 ± 4.81 μM) after 24 hours exposure. The mode of action studies included the determination of the killing kinetics, kinetics of membrane permeabilisation and the subsequent effects on bacteria ultrastructure. At the MBC of CPF-P2 (i.e. 16 μg/mL) the killing time was 120 minutes, mediated by membrane permeabilisation which was time- and concentration-dependent. Ultrastructural studies with scanning electron microscopy, confirmed membrane targeting with structural changes to cell shape, ruptured cell membranes and leakage of cellular content with evidence of blebbing. The stability of CPF-P2 in physiological environments was evaluated. CPF-P2 retained activity in 5% foetal bovine serum (FBS) but lost activity in 25% FBS. Pre-incubation with trypsin also resulted in complete loss of CPF-P2 activity. In conclusion, CPF-P2 was identified as bactericidal, selective for planktonic E. coli with a killing time of 120 minutes at its MBC. The bactericidal activity is mediated by membrane permeabilisation leading to associated changes in cell morphology. Its activity against biofilms was limited and was compromised in physiological environments. Nonetheless, this study identified CPF-P2, as a promising template for the further development of analogues for topical infections.
Effect of large herbivore decomposition on the succession of Mesic-grassland soil microbiomes
(University of Pretoria, 2024-11-19) Cowan, Don A.; Lebre, Pedro Humberto; jacques.fch@gmail.com; Fouché, Jacques
Plant detritus is abundant in grassland but decomposes slowly and is relatively nutrient-poor, whereas animal carcasses are labile and nutrient-rich. Although nutrients from carcasses are highly sought-after, historically, they have been considered insignificant due to their brief decomposition period and minor contribution to the overall landscape nutrition. Recent studies have demonstrated that carcasses significantly alter long-term soil properties at an ecosystem scale. There is a paucity of empirical evidence of the temporal scale of functional and structural succession of the soil microbiome during and after carcass decomposition. Over a period of eighteen months, this study evaluated the functional and structural succession of the soil microbiomes beneath ten Connochaetes taurinus (wildebeest) carcasses. Functional succession was measured by the utilisation of 31 ecologically relevant carbon substrates using BiologTM EcoPlatesTM. Metagenomic analysis of 16S rRNA genes evaluated the bacterial community structural succession. Functional analysis results indicated that most soil microbial processes beneath the carcasses were accelerated for a limited period but resulted in an enduring increase in functional diversity. Substrate utilisation shifted successively and remained evident throughout the study period. Conversely, bacterial diversity was significantly reduced and dissimilar to control soil, although it recovered incrementally to the control soil levels within eighteen months. Biomarkers at different taxonomic levels were identified at various postmortem intervals up to eighteen months
Application of RNA sequencing for the identification of microorganisms at the tick-host interface
(University of Pretoria, 2024-08-06) Maritz-Olivier, Christine; Olivier, Nicholas Abraham; u18057102@tuks.co.za; Archary, Merishka
The cattle tick Rhipicephalus microplus, is an invasive hematophagous ecto-parasite in South Africa and is associated with high economic loss in the cattle industry. This tick species transmits pathogens such as Anaplasma marginale, Babesia bigemina and the virulent B. bovis, resulting in high morbidity and mortality in cattle populations. Recently, an RNA virus potentially associated with human disease has been detected in this tick species highlighting the need for proactive pathogen surveillance. The aim of this study was to use Next-generation sequencing to actively detect known and novel pathogens circulating in R. microplus and cattle populations in two South African provinces, Mpumalanga and KwaZulu Natal. RNA-sequencing analyses revealed that R. microplus has a highly diverse virome. Two viruses were detected in the majority of R. microplus samples, Wuhan tick virus 2 (WTV2) and Jingmen tick virus (JMTV), the latter is potentially associated with human disease. To monitor the presence of JMTV and the ubiquitous WTV2, PCR-RFLPs were developed as rapid diagnostic tests. Furthermore, 16S sequencing and microbiome analysis revealed the presence of several genera containing pathogenic bacteria that are a concern for human and animal health such as Coxiella, Anaplasma and Ehrlichia. Using R. microplus ticks reared on pathogen free cattle enabled the prediction of a core microbiome. The analyses suggested the presence of the Coxiella endosymbiont. NGS is an essential tool detecting and monitoring known, emerging and novel pathogens enabling the implementation of prophylactic measures to limit the spread of disease.
Genomic insights into the pathogenicity and host adaptation in species of the Leptographium wageneri complex
(University of Pretoria, 2024) Duong, Tuan A.; Wingfield, Brenda D.; Wingfield, Michael J.; deanne.duplessis@up.ac.za; Du Plessis, Deanné
Fungal vascular wilt pathogens cause destructive diseases in many agriculturally important crop and tree species. Many of these pathogens are soil-borne and enter their hosts via pre-existing structures, or with the aid of insect vectors. However, there is remarkably little knowledge available regarding the molecular mechanisms of infection and pathogenicity in these fungi. Species in the Leptographium wageneri complex cause black stain root disease of conifers. This disease is characterised by black staining of the lower stems, roots, and tracheids, leading to wilt and tree death. There are three host specific species in the complex: L. wageneri, L. pseudotsugae and L. ponderosum. The genetic mechanisms underlying their pathogenicity and host adaptation have not been identified. To better understand potential pathogenicity associated factors, comparative genomic analysis was performed by comparing the genomes of species in the L. wageneri complex with those from related non-pathogenic species that reside in the same genus as well as genomes of pathogenic and non-pathogenic species in the Ophiostomataceae. The in vitro and in planta expression of an identified laccase gene was investigated to reveal its role in pathogenicity. Furthermore, laccasedeletion mutants were generated using the CRISPR-Cas9 gene editing system, followed by a pathogenicity trial on Pinus patula seedlings. Finally, a genome-wide approach was used to identify genomic regions under selection that might explain the host specialization in species of the L. wageneri complex. Genome sequences were generated for the three species in the L. wageneri complex and the nonpathogenic L. douglasii. The results revealed that species in the L. wageneri complex have larger genomes, higher gene numbers and a higher genome-wide content of transposable elements. Overall, both pathogenic and non-pathogenic species in the Ophiostomataceae have similar numbers of pathogenicity associated factors, such as secondary metabolite clusters, CAZymes, and effectors. Phylogenetic analyses indicated that the laccase gene has been horizontally acquired by species of the L. wageneri complex and L. douglasii. Expression analysis revealed that the laccase gene is up regulated in planta. The pathogenicity trial conducted with laccase-deletion mutants confirmed the essential role of the laccase gene in pathogenicity. Whole-genome SNP data were used to investigate the evolutionary relationships and genetic patterns of diversification between species of the L. wageneri complex. Structure analysis revealed three distinct clusters, each representing a species in the complex. Evolutionary analyses indicated that L. wageneri is more closely related to L. pseudotsugae, than to L. ponderosum. Selection analysis revealed several genomic regions underwent positive selection, that could explain their differentiation and host associations. Finally, I have discussed how genes within these genomic regions that encode for a heterochromatin incompatibility protein, a protein kinase and a Multi-drug transporter protein could underly the diversification and host specification in the species of the L. wageneri complex.
Importance of an arginine-glycine-aspartic acid (RGD) motif in the VP7 protein of African horse sickness virus to mediate insect cell binding
(University of Pretoria, 2024-07) Theron, Jacques; Van Staden, Vida; buyensariel@gmail.com; Buyens, Ariel Renée Monique
African horse sickness, a rapidly fatal disease of equines, is caused by African horse sickness virus (AHSV) which is transmitted to its vertebrate hosts through the bites of midges belonging to the Culicoides genus. The outer capsid of the virus surrounds an inner core composed of the structural proteins VP3 and VP7. The outermost core protein VP7 has an arginine-glycine-aspartate (RGD) tripeptide motif, which is one of the ligand sites recognized by the integrin family of cell surface receptors. To evaluate the ability of the AHSV VP7 protein to bind to insect cells and the functional significance of the VP7 RGD motif in this interaction, core-like particles (CLPs) were produced with the baculovirus expression system by co-expression of VP3 and sVP7, which encodes a soluble version of the AHSV-4 VP7 protein. Insect cell binding assays indicated that the CLPs were capable of binding to Culicoides sonorensis-derived cells, indicating a role for VP7 in this process. Next, recombinant baculoviruses expressing mutant sVP7 proteins in which the RGD motif was either deleted or mutated were synthesized. Except for deletion of the RGD motif, the RGD-mutated sVP7 proteins were able to form trimers and, when co-expressed with VP3, formed CLPs that retained the ability to bind to insect cells. These results therefore indicate that VP7 facilitates binding of CLPs to insect cells and that this interaction occurs in an RGD-independent manner.
Identification and molecular characterisation of the necrosis and ethylene-inducing peptide 1-like proteins (NLPs) of Phytophthora cinnamomi
(University of Pretoria, 2024-07) Van den Berg, Noelani; katelyn.baird@fabi.up.ac.za; Baird, Katelyn
This research began with identifying the total number of NLP-encoding genes from the P. cinnamomi transcriptome. Phylogenetic analysis was performed in conjunction with expression analysis from previously generated RNA-seq data of P. cinnamomi infection of two avocado rootstocks. Seven candidate genes were selected, based on the presence of the NLP motif, expression patterns of the genes and their phylogenetic similarities to known NLPs. The coding sequence for these candidates were confirmed with Sanger sequencing after cloning into E. coli. The resulting protein structures were visualised and analysed for similarity and an attempt was made to ascertain functionality, however, more research is needed to determine this. This study also investigated the genes responsible for ethylene production in plants to find a relationship between P. cinnamomi infection and ethylene induction. The same RNA-seq data was used to identify and assess the genes encoding the three enzymes for ethylene biosynthesis and their expression during infection. The findings from this study provide a solid framework for future research into the molecular mechanisms of action of the NLPs during infection. The individual functions of P. cinnamomi NLPs have yet to be discovered and the understanding of which NLPs act under which mechanisms is still a mystery.
The identification of Oomycetes associated with plum orchards in the Western Cape Province, South Africa
(University of Pretoria, 2019-12) Coutinho, Teresa A.; Bose, Tanay; mateka.modiba@up.ac.za; Modiba, Mateka Patience
The aim of this thesis was to determine the role played by oomycetes in plum tree decline observed in the Western Cape Province of South Africa from 2016-2018. At this time, extreme drought conditions were experienced in the province. Thus, the focus of this study was to identify and characterize oomycetes isolated from both diseased plum tree tissue and rhizosphere soil, and to test their pathogenicity on two plum cultivars. Chapter 1 reviewed previous literature on plant diseases caused by oomycetes and P. syringae pv. syringae, and how the disease triangle and climate change influenced disease development. Temperature and moisture were reported as factors that influence disease development by weakening plant hosts when conditions are unfavorable, and also they influence pathogen occurrence and establishment. The literature also highlighted the importance of the interrelations of stress factors involved in decline, including the effect of co-infection in a single host plant. Chapter 2 of this thesis focused on conducting field surveys and sampling five plum orchards in the Western Cape Province. During the survey, a few trees displayed symptoms of bleeding cankers, which suggested that an oomycete might be a possible causal agent. Isolations from the diseased plant material and soil samples were conducted followed by molecular identifications. Six oomycetes species were identified, which are: Phytophthora multivora, Phytopythium vexans, Pythium coloratum, P. diclinum, P. irregulare and P. ultimum. Phytopythium vexans was the only oomycetes that was isolated from infected plant material. The pathogenicity of P. multivora and P. vexans (isolated in this study) was determined in Chapter 3 on Sun kiss plum cultivars in a greenhouse environment. Pseudomonas syringae pv. syringae was included because bacterial canker symptoms were concurrently observed in the field. The pathogenicity trials showed that neither P. vexans nor P. multivora were able to cause symptoms and were not re-isolated from the inoculated seedlings. Seedlings infected with P. syringae showed symptoms typical of bacterial canker and the pathogen was re-isolated from the infected seedlings. Co-infection trials revealed that seedlings inoculated with P. multivora and P. syringae had larger lesion size compared to other combinations.
Symbiotic nitrogen fixation efficiency of native rhizobia in selected South African legume crops
(University of Pretoria, 2019-06) Steenkamp, Emma Theodora; Hassen, Ahmed; khumbudzomashau@gmail.com; Ndhlovu, Khumbudzo
This Master’s dissertation reports about the screening and characterization of selected alpha and beta rhizobial isolates from wild legumes in South Africa for their nodulation and nitrogen fixation properties on the cultivated legumes lucern (Medicago sativa L), cowpea (Vigna unguiculata L) and siratro (Microptilium atropurpeum D.C) under glasshouse and field conditions. The rhizobia were initially in-vitro characterized for their tolerance to various abiotic stresses and most of the strains were found to be tolerant to extremes of environmental factors such as acidity, aluminium toxicity, salinity and temperature. They were then screened for nodulation and nitrogen fixation efficacy under glasshouse and field conditions. Additional in-vitro screening for essential plant growth promoting traits including the production of siderophores, indole acetic acid, ACC-deaminase and phosphate solubilization was conducted. Most of the isolates from the wild legumes, i.e., 7 strains (6 Bradyrhizobium and 1 Paraburkholderia nodulated cowpea, 1 Bradyrhizobium strain nodulated lucerne and 13 strains (3 Paraburkholderia and 10 Bradyrhizobium strains) nodulated siratro, in the glasshouse experiment with a statistically significant number of nodules (p > 0.05). Plant biomass, including fresh weight and dry weight, were significantly improved by Bradyrhizobium strains 10BB and Arg68 in cowpea and siratro compared to un-inoculated controls. Five strains for cowpea, six strains for siratro and one strain for lucerne were selected as the best strains for field trial. After harvest, cowpea plant biomass were significantly increased when inoculated with Bradyrhizobium sp. Arg68 followed by Paraburkholderia sp. KB15 with significant increase in the amount of fixed nitrogen. There was significant difference in the amount of nitrogen fixed when inoculated with different strains of rhizobia. In siratro, plant biomass was increased after inoculation with Bradyrhizobium sp. Fp1c strain followed by Bradyrhizobium sp. 10BB although the amount of nitrogen fixed had significant different and same applies to lucerne with no nodules formed on control plant. All of the Bradyrhizobium strains tested positive for the presence of nifH gene while Bradyrhizobium strains Arg68 and Arg62 strains contained the nodC genes. The study has generated important baseline data, which can be used for further development of the rhizobial strains as legume inoculants for cowpea, siratro and lucerne, but warrants further nodulation screening study in these and other legumes of similar cross inoculation groups with cowpea, lucerne and siratro.
Characterisation of secondary metabolite pathways in the Ceratocystidaceae
(University of Pretoria, 2019-12) Wingfield, Brenda D.; Steenkamp, Emma Theodora; Van der Nest, Magrieta Aletta; Sayari, Mohammad
Members of Ceratocystistidaceae (phylum Ascomycota, class Sordariomycetes) include fungal pathogens that cause diseases on a broad spectrum of hosts, leading to substantial economic losses globally. The objective of this thesis was to provide insights into the secondary metabolite pathways of this family. For this purpose, we used whole genome sequences of 23 different members of Ceratocystistidaceae. Our results showed that all of the genomes contained putative clusters containing a reducing and non-reducing type I PKS as well as a type III PKS. Phylogenetic analyses of non-reducing-PKS-I and also PKS-III suggested that these genes were already present in the ancestor of the Ceratocystidaceae. By contrast, the various reducing type I PKS-containing clusters identified in these genomes, appeared to have distinct origins during the evolution of this family. Although one of the identified clusters potentially allows for the production of melanin, their functional characterization will undoubtedly reveal many novel and important compounds implicated in the biology of the Ceratocystidaceae. We have also found two highly conserved nonribosomal peptide synthetase genes in all genomes of Ceratocystidaceae and their potential products were predicted. These findings help to better understanding of the diversity and evolution of NRPS biosynthesis pathways in this family. We further, optimized an Agrobacterium mediated transformation system for Ceratocystis. This will allow for the functional characterization of the genes and genetic elements underlying the biological properties of this important fungus and its relatives. The average ergosterol content of different genera of Ceratocystidaceae was different from each other. We also identified all possible terpenoid related genes and biosynthetic clusters in all genomes used in this study. We found a highly conserved terpenoid gene cluster containing some of the ergosterol biosynthetic genes in all genomes. An additional terpenoid gene cluster was also identified in all the Ceratocystidaceae with geranylgeranyl pyrophosphate as a core gene, which could be involve in diterpenoid production. The outcomes of this thesis shed light on our knowledge of secondary metabolite biosynthesis pathways in Ceratocystidaceae.
Diversity, specificity and admixture in the Sirex - Amylostereum - Deladenus symbiosis
(University of Pretoria, 2019-09) Slippers, Bernard; Garnas, Jeff; Fitza, Katrin Nathalie Elsbeth
Biological control is an important management tool to deal with the rapidly increasing number of invasive pests of plantation forests globally. It is important to consider the genetic diversity of both the pest and biological control populations to understand geographic population structure, patterns of invasion, genotype-genotype interactions and potential adaptability. This thesis examined patterns of genetic diversity and specificity of the Sirex–Deladenus–Amylostereum complex. Mitochondrial sequence data and nuclear microsatellite markers were used to characterize the diversity in a global collection of D. siricidicola from both native and non–native regions. The data revealed the presence of three distinct lineages, from North America (Lineage A; non-native), the Southern Hemisphere (Lineage B; non-native) and Spain (Lineage C; native). Interestingly, samples from Chile represented an admixed population of lineages A and B. The global study showed evidence of substantially genetic diversity present globally which could be used to augment the reduced genetic diversity in the Southern Hemisphere biological control populations. The three D. siricidicola lineages were shown to be able to interbreed in culture. The admixed offspring of one of the crosses showed a significant increase in its reproductive rate on the slowest growing fungal isolate, when compared to the parental strains. Experimental admixture suggests the possibility and advantage of introducing more genetic diversity in biological control programs. As the symbiotic fungus A. areolatum of the pest wasp S. noctilio plays a crucial role in the mass production and influences the performance of the biological control agent, the fidelity of the Sirex –Amylostereum association was studied in native Siricids in Japan and their fungal associates. It was shown that the association was not species specific. Sirex nitobei was associated not only with Amylostereum areolatum, but also Amylostereum chailletii. Urocerus sp., previously associated with A. laevigatum, carried A. chailletii. Vegetative compatibility test revealed high clonality both among A. areolatum and A. chailletii in association with these wasps. Together with previous studies it seems that the host tree plays a more critical role in selection of Amylostereum species than the wasp. The thesis illustrates the importance of studying the genetic diversity of biological control agents and the potential of augmenting the genetic diversity in these populations as a mean to improve adaptability.
Diversity of rhizobial Methylobacterium species associated with indigenous legumes in South Africa
(University of Pretoria, 2019-12) Venter, S.N. (Stephanus Nicolaas); Steenkamp, Emma Theodora; Muema, Esther K.; moyanasanele@gmail.com; Moyana, Sanele B.
The genus Methylobacterium includes a variety of pink pigmented and cream white facultatively methylotrophic bacteria that are characterized by their ability to mainly utilize methanol as a carbon source. Methylobacterium includes only one known nitrogen fixing species (Methylobacterium nodulans), which was initially isolated from root nodules of the legume Crotalaria podocarpa, in Senegal. Additional Methylobacterium strains able to fix atmospheric nitrogen with members of Crotalaria and Listia legumes native to Southern Africa have since been isolated. The aim of this study thus was to investigate the taxonomic position and delineate the diversity of Methylobacterium isolates associated with Crotalaria and Listia species native to South Africa. This was achieved by employing housekeeping gene phylogenies and various phenotypic tests. Of the original 92 isolates investigated, 29 belonged to the genus Methylobacterium. Aligned sequences from the isolates, together with reference and outgroup sequences obtained from the National Center for Biotechnology Information (NCBI) database, were used for constructing phylogenetic trees. To confirm the phylogenetic results, phenotypic characterization tests were conducted. The phylogenetic analyses of the housekeeping genes of the Methylobacterium isolates grouped them into two clusters (A and B). Group A isolates were closely related to M. nodulans, while Group B formed a different cluster, grouping with a well-known Methylobacterium strain 4-46. From the results, it was clear that only isolates obtained from Crotalaria clustered in Group A with M. nodulans, whereas all Listia isolates and two Crotalaria isolates clustered in Group B. Results from this study showed that single phylogenies of 16S rRNA, recA and rpoB best delineated Methylobacterium isolates. Carbon utilization tests did not provide results that could be used for the separation of the Methylobacterium isolates according to the two assigned groups.

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