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

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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.
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.
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.
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.
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.
Discovery of Phytophthora cinnamomi RxLR effector genes expressed in avocado during infection
(University of Pretoria, 2019-12) Van den Berg, Noelani; Prabhu, Sreedhara Ashok; Joubert, Melissa
Phytophthora cinnamomi is an oomycete that targets a broad range of plants, including several economically important forestry and agricultural crops. It is the causal agent of Phytophthora Root Rot, and causes significant economic losses within the agricultural and forestry industries. Recently, the use of effector molecules by pathogenic oomycetes during plant infection has become a subject of great interest to researchers. One class of these molecules, the RxLR effectors, has become a focus of Phytophthora research, and hundreds of putative RxLR effector genes have been predicted by bioinformatic analysis of Phytophthora genomic sequences. The characterization and validation of these effectors remains an ongoing process. This study identified several P. cinnamomi RxLR genes upregulated during infection of a susceptible avocado rootstock. The genes were subjected to in silico analysis of expected RxLR characteristics and prediction of coding regions from the genomic sequence. Predictions were then validated by analysis of DNA sequences and the use of RNA-seq data, which were used to manually annotate these effector genes. The final prediction of RxLR proteins was then compared to the sequences of validated RxLRs in other Phytophthora species to enable inference of possible functions of the annotated genes. In this study, a total of 25 P. cinnamomi candidate RxLRs were identified, which were proposed to play a role during avocado infection. While expression of this number of candidate RxLRs is relatively small, these candidates may represent effectors which are expressed specifically in this host-pathogen interaction, or may be a set of “elite” effectors which contribute to virulence in all hosts. The candidate genes were analysed for the presence of the desired motifs, and a subset of 16 RxLRs were chosen for further analysis. The expression profiles of these genes were investigated further, and it was found that four of the candidates were expressed most highly at 24 hpi, which correlates with expression profiles of RxLRs in other species. Twelve of the candidate RxLRs had expression profiles which were not similar to those which have been demonstrated for other RxLRs, while four were not significantly upregulated during specific timepoints of infection. These results warrant further investigation to determine the relevance of these unique expression profiles, which may present new insights into expression patterns of RxLR effectors. Several of the 16 candidate effector genes were present in multiple copies in the P. cinnamomi genome, providing evidence for their roles in plant infection. Transcriptome data was used to manually annotate the genes, and the resulting protein predictions for most of the candidates were different from those originally predicted by gene prediction software. Not one of the prediction software used in this experiment accurately predicted the coding regions for all the genes – providing a substantial argument for the need for manual annotation of candidate effectors. Phylogenetic analysis allowed functional inferences to be made for five of the candidate effectors, based on their shared evolutionary history with RxLRs characterized in other Phytophthora species. While no functional assays have been carried out for these candidate effectors yet, their identification as putative RxLRs presents a starting point for further investigation into their functions in planta. This study presents the first report of P. cinnamomi RxLRs with confirmed sequences and expression profiles, and as such offers the first insights into infection of avocado by this pathogen at the molecular level.
Taxonomic characterization of Paraburkholderia tuberum-like rhizobia from diverse legumes
(University of Pretoria, 2020-01) Steenkamp, Emma Theodora; Venter, S.N. (Stephanus Nicolaas); Beukes, Chrizelle W.; mavimalazarus@gmail.com; Mavima, Lazarus
Paraburkholderia tuberum is an indigenous South African bacterial species and one of the first rhizobia to be described in the class Beta-proteobacteria. In the past two decades, numerous strains identified as P. tuberum or P. tuberum-like were isolated from several papilionoid legumes in South Africa, as well as from South and Central American Mimosa species. These two groups of isolates were found to contain different nodulation and nitrogen-fixation genes, suggesting that they might belong to different species. The main aim of this study was, therefore, to investigate the taxonomic status of these presumed P. tuberum isolates previously recovered from the root nodules of diverse legumes from the Core Cape Region (CCR) of South Africa, from the Caatinga, the Cerrado and the Pantanal of Brazil, and from western Mexico. The South African isolates associate with several genera in the Papilionoideae (e.g., Cyclopia, Virgilia, Hypocalyptus, Podalyria and Aspalathus) and with Vachellia karroo from the mimosoid clade. The Brazilian and Mexican isolates associate with several Mimosa species from the mimosoid clade in the Caesalpinioideae. A set of 31 South African, 28 South American and 2 Central American isolates were assigned to potential species using genealogical concordance analysis. This was accomplished by identifying unique and consistent clades across the genealogies inferred from the nucleotide sequences of independent housekeeping genes (i.e., atpD, gyrB, gltB, rpoB, acnA, pab and 16S rRNA). Subsequently, overall genome relatedness data and phenotypic characteristics were determined to seek support for the putative species. In this way, three species of rhizobial Paraburkholderia were discovered and described. The first is indigenous to South Africa, for which we propose the name P. podalyriae sp. nov. with the type strain WC7.3bT (= LMG 31413T; SARCC 750T). The other two species have Brazilian origins, and we propose the names P. simonii sp. nov. with the type strain JPY169T (= LMG 31411T; SARCC751T) and P. youngii sp. nov. with the type strain JPY251T (= LMG 31412T; SARCC752T).
Unisexual reproduction in filamentous Ascomycete Fungi, with particular reference to Huntiella moniliformis
(University of Pretoria, 2019) Wingfield, Brenda D.; Wingfield, Michael J.; Van der Nest, Magrieta Aletta; Wilken, Markus; Wilson, Andrea Melissa
English: Many filamentous ascomycete fungi are capable of sexual reproduction, though the exact mechanisms they use can differ from species to species. Those that require a compatible partner are termed heterothallic, while those that can sexually reproduce in isolation are termed homothallic. The major aim of this thesis was to further our understanding of a unique type of sexual reproduction known as unisexuality. While unisexual species harbour genes typically associated with heterothallic species, they are capable of independent sexual reproduction. Utilizing a variety of bioinformatic and molecular tools, I was able to show that unisexual reproduction is likely derived from heterothallism. I also propose that this transition is possible with a few small changes to the MAT genes and pheromone response pathway. In all three of the unisexual filamentous ascomycete fungi I investigated, mutations in the secondary MAT genes resulted in significant gene truncations or the deletion of functional domains- leading to non-functional proteins. Furthermore, these species also exhibited atypical pheromone response pathways. Given that similar changes are seen in three unisexual species from unrelated genera, I suggest that the mechanism that enables unisexual reproduction is highly conserved. Using a CRISPR-Cas9-based genome editing system, I was able to take the first steps towards experimentally mimicking unisexual behaviour in a heterothallic species, by the truncation of a secondary MAT gene in H. omanensis. Future research will thus focus on disruption of the pheromone response pathway in this species.
Interaction of African horse sickness virus inclusion bodies with cellular translation machinery to compartmentalise virus protein synthesis
(University of Pretoria, 2019-11) Van Staden, Vida; Venter, Eudri; litiayssel@gmail.com; Yssel, Litia
African horse sickness virus (AHSV) is an orbivirus in the Reoviridae family that causes severe disease in horses, with major economic implications. The viral genome consists of ten double-stranded RNA segments, encoding seven structural viral proteins (VP) plus four non-structural (NS) proteins. The non-structural proteins serve to enhance the viral life cycle by influencing viral morphogenesis, replication or egress. In orbiviruses, NS2 multimers form dense cytoplasmic matrices termed viral inclusion bodies (VIBs). It has until recently been assumed that the VIBs of AHSV merely serve as replication factories and sites wherein virus particle assembly occurs. However, various different viruses have been shown to compartmentalise virus protein synthesis within cytoplasmic replication factories, which is believed to contribute to enhancing the viral life cycle. Little is known about how AHSV utilises the host translational machinery for its replicative advantage. In this study, the distribution and morphology of AHSV VIBs were characterised at different stages of the replication cycle. The VIBs were shown to increase both in size and abundance up until a certain time point after infection. The formation of the VIBs from NS2 precursors was characterised by making use of plasmid-expressed fluorescent NS2-eGFP. Preliminary results from live-cell imaging revealed the formation of the VIBs to be a dynamic process, involving the progressive coalescence of small NS2 foci in a random fashion. Using a biochemical assay followed by confocal microscopy analyses, it was investigated whether the VIBs are sites of protein synthesis. Our results showed that active translation was substantially enhanced within the VIBs of AHSV-4, AHSV-5 and BTV-10, surpassing the intensities of cytoplasmic protein synthesis. This indicates virus-mediated compartmentalisation of translation within the VIBs as a broad functionality amongst the orbiviruses. We next determined whether different eukaryotic ribosomal components and translation initiation factors localise to the VIBs of AHSV. Interestingly, we observed differential distribution patterns of these components, indicating distinct sub-structural domains of the VIBs. The ribosomal component L11 and initiation factor eIF3θ localised throughout the VIBs, with elevated intensities towards the central regions, whereas ribosomal component S3 and initiation factor eIF4E localised to the VIB perimeters. These localisation patterns were shown to be solely mediated by NS2. These results indicate a novel functionality of the VIBs, by serving as hubs for virus protein synthesis. It still remains to be elucidated how translation is orchestrated within the VIBs, however our preliminary results could indicate to the potential for sub-domains of the VIBs being involved in specific functions regarding translation. Overall, this indication of translation compartmentalisation within the VIBs could elucidate a strategy whereby AHSV enhances the viral infection cycle.
Genomic insights into the biology and evolution of Botryosphaeriaceae
(University of Pretoria, 2019-09) Slippers, Bernard; Wingfield, Michael J.; Nagel, Jan Hendrik
The Botryosphaeriaceae is a large family of fungi including many plant pathogenic species that cause diseases of important plants such as fruit trees, grapevine, eucalypts, pines and wheat. Members of this family are considered to be latent pathogens that infect their hosts without causing symptoms and often become pathogenic only after the plant host experiences environmental stress such as extreme temperatures, drought or physical damage. The aim of this study was to investigate the genetic factors influencing the infection and reproductive biology of the Botryosphaericeae and to develop molecular markers for future application in population genetic studies on these fungi. This was achieved by acquiring genome sequence data for species of prominent genera in the Botryosphaeriaceae and through the subsequent analyses of these data. Results showed that intact mating type genes exist in all Botryosphaeriaceae considered and that frequent, independent transitions from heterothallism to homothallism have occurred in this family. It was further shown that the Botryosphaeriaceae genomes contain high numbers and diversities of secreted hydrolytic enzymes and secondary metabolite biosynthetic gene clusters, which were correlated to genome size and the total number of predicted genes. Most importantly, they were also correlated with the host ranges of the species. A large number of highly transferable microsatellite markers were developed for the genera Lasiodiplodia and Neofusicoccum. These markers were capable of amplification in many species residing in these two genera and were shown to be able to distinguish a large number of multilocus genotypes from sample populations. Overall, this study has provided valuable insights into the biology and evolution of the Botryosphaeriaceae. It has also raised important questions that future research should consider.
Genetic dissection of growth and wood properties in a nested, half-sib Eucalyptus hybrid pedigree
(University of Pretoria, 2019-12) Myburg, Alexander Andrew; Mizrachi, Eshchar; julia.candotti@gmail.com; Candotti, Julia
Eucalyptus is important for the forestry industry due to its excellent growth and wood properties. In crop species, nested multi-parent populations have been used to increase the power and resolution of quantitative trait loci (QTL) detection. These populations have predominantly been used in species in which recombinant inbred lines can be generated and have not been fully exploited in outcrossing species such as Eucalyptus. To determine if multi-parent mapping approach can be used effectively for genetic dissection in Eucalyptus, we made use of an existing F1 hybrid trial series, consisting nine E. grandis pollen parents and eight E. urophylla seed parents. The population has many full-sib (FS) families nested within half-sib (HS) families and was planted across four different sites. The objectives of this MSc study were to i) construct genetic linkage maps of one E. grandis pollen parent and one E. urophylla seed parent of the multi-parent population, ii) analyse transmission ratio distortion of mapped markers in the F1 hybrid progeny to identify hybrid compatibility barriers, iii) map QTLs underlying growth and wood properties in the two pure species parental maps. We constructed framework genetic linkage maps for the E. grandis pollen parent and the E. urophylla seed parent. A total of 388 (E. grandis HS family, n = 349) and 422 (E. urophylla HS family, n = 367) single nucleotide polymorphisms (SNP) markers were included in the linkage maps resulting in an average marker density of 2.4 cM. Using the genetic linkage maps, we identified 15 and 23 QTLs underlying growth and wood properties for the E. grandis and E. urophylla HS family, respectively. We identified large to medium effect QTLs, with the percentage of variance explained ranging from 3.06% to 36.58%. We identified different QTLs across the sites which suggests that the traits are affected by genotype-by-environment interaction. We analysed segregation distortion of the markers included in the framework genetic linkage maps within HS families, FS families and sites. We found that there is a large amount of segregation distortion (between 0 – 29.38% distortion) and that the patterns of distortion varied for individual FS families planted across multiple sites and single sites with multiple FS families. We were also able to identify potential pre- and postzygotic barriers to hybrid compatibility through the analysis of segregation distortion of dead and living trees. Taken together, these results show that there are both parent specific interactions, that are dependent on the environment, which underlie hybrid compatibility. In this study, we applied an approach whereby genetic linkage maps can be constructed and QTL identified in an outcrossing multi-parent mapping population. We show that multi-parent populations hold promise for studying hybrid compatibility, as diverse founders are crossed resulting in a number of F1 hybrid progeny. The results of this study show that this approach can be applied in existing F1 hybrid breeding trials for more fine scale genetic dissection of complex trait variation as well as hybrid compatibility of E. grandis and E. urophylla.
Effects of ecology on Ficus population genetic structure in southern Africa
(University of Pretoria, 2020-02) Greeff, Jacobus Maree; Van Noort, Simon; Compton, Stephen; sophia15_djy@outlook.com; Deng, Jun-Yin
South African forests have become fragmented due to both historical events and current human activities. Fragmentation may have increased population differentiation by reducing the gene flow between plants isolated in fragments. The genetic effects of fragmentation on species, especially plants, have received little attention in South Africa. Fig trees are keystone species in South African forests that provide food for many frugivores. Their population genetic structure has never been studied in South Africa. In my study, the population genetic structures of three forest fig trees (Ficus bizanae, Ficus craterostoma and Ficus sur) with different habitat specificity, geographical distribution, and level of endemicity were studied to explore the ecological factors (both non-biotic and biotic) that may contribute to their population differentiation. First, I did a phylogeographic and population genetic study on the forest specialist Ficus species, F. craterostoma, from forests covering the species' range in South Africa. Chapter two reports this work. Three putative refugia were inferred, which played an important role in maintaining genetic diversity and facilitating recolonization by F. craterostoma after the LGM. In addition, comparing the genetic structures of nuclear and cytoplasmic DNA I found that nuclear DNA in F. craterostoma may be resistant to fragmentation while cytoplasmic elements are more vulnerable to fragmentation. This difference stems from the reduced effective population size of cytoplasmic DNA, but seed dispersal must also be limited whereas pollen dispersal, facilitated by a mutualistic pollinator fig wasp, is very extensive. In fact, a novel analysis that compared genetic variability with a combined forest size, suggested that forests within a 61 km radius of one another are genetically connected via the tree’s fig wasps. Differences in the biology of species can lead to differences in their responses to habitat fragmentation. Therefore, a comparison of genetic structure among species with different biologies may provide an understanding of the effect of fragmentation. I compared the population genetic structures of three Ficus species (F. bizanae, F. craterostoma and F. sur) on both large and fine scales and report on these in my third and fourth chapters respectively. At a large scale, the two forest specialists have a stronger genetic structure compared to the habitat generalist (F. sur). Within the forest specialists, the species with a limited distribution (F. bizanae) showed stronger genetic structure, indicating local pollen and seed dispersal. At the fine scale, I found significant spatial genetic structure (SGS) in all three Ficus species, which may be due to limited seed dispersal between fragmented forests. Among them, F. bizanae had the highest positive kinship coefficient within a short distance as well as the highest sp statistic. This indicates that in addition to limited seed dispersal, there may also be limited pollen dispersal in F. bizanae. An increasing SGS of F. craterostoma was detected from saplings to adults at Ingeli forest (ING), which suggests that recent population fragmentation has stopped such long-distance immigration. Non-random mortality caused by microenvironmental selection may also contribute to the contrasting age-related fine scale SGS.
Molecular and bioinformatic analysis of the Persea americana (Mill.) NPR1-dependent defence signalling pathway in response to Phytophthora cinnamomi infection
(University of Pretoria, 2019-12) Van den Berg, Noelani; Naidoo, Sanushka; backer.robert@gmail.com; Backer, Robert
The global avocado industry has experienced significant growth throughout the past two decades, with annual production doubling over that time. However, increased production is accompanied by an ever-increasing threat from a variety of pests and diseases. Phytophthora root rot (PRR) is currently considered the most devastating disease of avocado, causing significant economic losses annually. The causal agent, Phytophthora cinnamomi, is a hemibiotrophic oomycete; as such, it utilises both biotrophic and necrotrophic infection strategies to overwhelm its host. Plants use numerous phytohormone regulated defence response pathways, depending on the infection strategy employed by their pathogen. Typically, defence against biotrophic pathogens applies the salicylic acid (SA)-dependent defence response pathway; whereas defence against necrotrophic pathogens is associated with the jasmonic acid/ethylene (JA/ET)-dependent pathway. Notably, the nonexpressor of pathogenesis-related genes 1 (NPR1) co-transcription factor is crucial to most SA-dependent defence gene expression. Furthermore, it is essential to the establishment of systemic acquired resistance, a plant-wide state of heightened defence readiness. However, the mechanisms required to achieve SAR and effectively defend against different pathogens is exceedingly complex. Therefore, this dissertation aimed to identify and characterise NPR1-like proteins in Persea americana (avocado). Furthermore, this study attempted to understand the response of several NPR1 pathway-associated genes, both over-time and comparatively between PRR susceptible and partially resistant avocado rootstocks. A total of five NPR1-like coding genes were described in P. americana. Initial in silico analyses suggested that three PaNPR1-like proteins could be involved in defence; two of these, PaNPR1 and PaNPR2, were likely associated with positive SAR regulation while PaNPR4 probably served an opposing role. Meanwhile, the remaining two, PaNPR3 and PaNPR5, were most likely involved in tissue development. These suspicions were later confirmed by expression analysis following phytohormone application, P. cinnamomi inoculation and tissue-specific sampling. Interestingly, significant differences were observed when comparing the expression of the several PaNPR1-like genes in the PRR susceptible (R0.12) and partially resistant (Dusa®) rootstocks. Therefore, we identified and annotated 116 orthologs of Arabidopsis thaliana NPR1 pathway genes in the P. americana genome. Using dual RNA-sequencing data, we characterised the expression of all 116 genes over time in the PRR susceptible rootstock R0.12. Additionally, we compared the expression of the NPR1 pathway-associated genes between R0.12 and the partially PRR resistant rootstock, Dusa®. Our observations suggest that SAR was established in both avocado rootstocks; additionally, expression of the majority of NPR1 pathway-associated genes is regulated to some extent following P. cinnamomi challenge. However, significant differences were evident when comparing expression in R0.12 and Dusa®. Primarily, our observations suggest that the SA-defence response pathway is suppressed more effectively in Dusa® following the establishment of SAR. Thus, Dusa® likely responds more appropriately to the pathogen’s necrotrophic phase of infection. The work presented here represents the first step in fully characterising and understanding the NPR1 pathway in P. americana and its role in resistance against PRR. Furthermore, we believe that this study will form part of the foundation for further functional characterisation of disease resistance pathways in avocado.
Adaptive and maladaptive sex ratios in a pollinating fig wasp
(University of Pretoria, 2020-01) Greeff, Jacobus Maree; karinapentz@gmail.com; Pentz, Karina
When a trait enhances fitness and arose as a result of natural selection, it is termed an adaptation. The optimization strategy employs selection thinking which makes the explicit assumption that the globally best trait will be fixed. Organisms, do not always respond optimally across all environments encountered as the strength of selection is finite. An excellent example of optimality is skewed sex ratios predicted by Hamilton's local mate competition (LMC) theory. It occurs when brothers compete in isolated groups for matings. Hamilton showed mathematically that in such groups, the unbeatable sex ratio is female-biased and this bias increase as the number of mothers that contribute to isolated patches decrease. One taxon that has been used to test LMC is the fig wasps. Fig wasps are haplodiploid, with unfertilized haploid eggs developing into males and fertilized diploid eggs into females. One or a few mothers crawl into a fig and lay all their eggs within, resulting in extreme local mate competition and sib mating. Sibmating increases relatedness of the daughter to the mother as genes from the father are identical by descent to the mother. Relatedness to sons is unaffected. This relatedness asymmetry selects for even more female-biased ratios. Thus, an extra daughter instead of a son will decrease local mate competition, and an extra daughter provides a higher fitness to the mother due to relatedness. There are two mechanisms that can explain how fig wasps sex ratios are adjusted to the number of mothers in a patch. First, a byproduct of a constraint on clutch size, where mothers lay all or most of their sons first, followed by daughters. Mothers do not change their behaviour, but limited oviposition space prevents them from laying all their eggs destined to be daughters. Second, a facultative strategy where mothers sense other ovipositing females and adjust the number of sons they lay. We studied sex ratio adjustments in Ceratosolen galili, the cuckoo wasp that oviposits in Ficus sycomorus, which is pollinated by C. arabicus. Different numbers of C. galili foundresses and a mix of these species were entered into figs to study the mechanism of sex ratio adjustment. We found that mothers adjust their sex ratios facultatively when another foundress is present, and there is no constraint on clutch size. However, if the fig becomes saturated with eggs from three foundresses, the mothers’ clutches are constrained, and sex ratios are also adjusted as a result of a byproduct. Since these foundress numbers are frequently observed in nature, selection effectively solved this problem. However, C. galili mothers often share figs with C. arabicus and C. galili should not adjust their sex ratios in their presence of C. arabicus. However, C. galili erroneously adjust their sex ratio facultatively in the presence of C. arabicus. Erroneous adjustment can be considered a maladaptation. Interestingly, the lack of pollination behaviour of C. galili is not sanctioned by reduced offspring survival.
Systems biology of organellar carbon metabolism in Eucalyptus xylogenesis
(University of Pretoria, 2020-01) Mizrachi, Eshchar; Myburg, Alexander Andrew; desri.pinard@fabi.up.ac.za; Pinard, Desré
Trees play a pivotal role in the global carbon cycle through the fixation and storage of carbon as polysaccharide and phenolic biopolymers in the secondary cell walls of xylem fibre cells (wood). Driven by the need to mitigate climate change, the utilization of wood as a source of renewable lignocellulosic biomass is undergoing a technological renaissance. Fundamental research in understanding and modelling xylogenesis as a developmental program remains crucial, as most carbon in living biomass is present in the secondary growth tissues of terrestrial plants. Although systems biology approaches in poplar, Eucalyptus and Arabidopsis have made massive strides in unravelling the complex genetic regulation underpinning xylogenesis, we know little of how the genome-bearing organelles of the cell, the plastids, and mitochondria, are integrated into the system. As the location of many carbon metabolic pathways in the cell, plastids and mitochondria play a crucial role in carbon allocation during wood formation. Despite this, plastid and mitochondrial biology have largely been ignored in the study of xylogenesis, and the vast majority of plant organellar research is focused on photosynthetic tissues. The work presented in this thesis aimed to provide a basis for understanding the role of plastid and mitochondrial biology during xylogenesis, using Eucalyptus as a model. This was achieved through three research objectives: (i.) The analysis of coregulated expression modules of nuclear-encoded plastid and mitochondrial-targeted genes generated from 156 xylem transcriptomes from a Eucalyptus grandis × E. urophylla F2 interspecific backcross population; (ii.) The assembly and annotation of the plastid and mitochondrial genomes of E. grandis, along with analysis of intergenomic DNA transfers and their expression; and (iii.) Comprehensive analysis of the transcriptomes of E. grandis plastids and mitochondria using total RNA, polyA-selected RNA, and small RNA sequencing data to understand the regulation of plastids and mitochondria in three tissues representing carbon source (mature leaf), sink (immature xylem) and transport (secondary phloem) tissues. This research has improved our understanding of carbon allocation during xylogenesis, provided a resource for future studies in Eucalyptus organellar biology and is the first to look at the transcriptomes of secondary growth plastids and mitochondria. Important findings from this work include that the regulation of plastid and mitochondrial metabolism is highly integrated with xylem development and the circadian clock. Plastid specific associations with the central circadian clock and epigenetic regulation show that the central functions of plastid retrograde signalling in leaf and green vascular tissues are conserved in wood formation. Finally, analysis of organellar transcriptomes in multiple tissues has shown for the first time that nuclearencoded polymerases uniquely drive plastidial gene expression during wood formation and that (as yet unknown) nuclear-encoded RNA-binding proteins may be involved in the active upregulation of selected plastid-encoded genes to facilitate nonphotosynthetic metabolism in the plastids of tree sink tissues. In conclusion, the thesis makes an evidence-based argument for a specific plastid type – the xyloplast – to advance research in the field of wood formation and organellar biology.
Comparative genomics and pathogenicity of Xanthomonas vasicola
(University of Pretoria, 2020-01) Coutinho, Teresa A.; Wicker, Emmanuel; McFarlane, Sharon; none; Zim, Nomakula Y.
In the mid-2000s an outbreak of bacterial blight and dieback caused by Xanthomonas vasicola pv. vasculorum was observed on a single Eucalyptus grandis clone in KwaZulu-Natal. It was suggested that this outbreak was as a result of a host jump from sugarcane. Therefore, the purpose of this dissertation is to determine the host range of X. vasicola strains and to identify the genetic determinants which may have played a role in this pathogen’s ability to jump to a new host. This dissertation will be presented in three independent chapters. Chapter 1 will review previous literature on the changes that take place in a bacterial genome leading to its adaptation to a changing environment. The chapter will include sections on the modifications of existing genes by mutations, gene duplications and gene rearrangements, acquisition of new genes by horizontal gene transfer, and the loss of genes as pathogens become specialised to their hosts. In chapter 2, the host range of X. vasicola strains isolated from different hosts will be tested. Host’s include the monocotyledonous plant species banana, maize, sorghum, sugarcane and the dicotyledonous plant species Eucalyptus grandis. The significance of the differences observed between the number of infected plants and the severity of the disease symptoms will be determined. In chapter 3, the differences between the genomes of five Xvv isolated from E. grandis and X. vasicola isolated from other hosts will be determined. The genomes of five Xvv strains from Eucalyptus were sequenced on the Illumina HiSeq 2500 platform. The chromosomes and plasmids were assembled and annotated, and the genomes were compared to those of other X. vasicola strains isolated from different hosts. These genome differences may have played a role in their adaptation to a new host.

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