Theses and Dissertations (Microbiology and Plant Pathology)

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Diversity of endophytic fungi associated with branches of Berchemia discolor in the Limpopo Province of South Africa
(University of Pretoria, 2022) Coetzee, Martin Petrus Albertus; Steenkamp, Emma Theodora; cindy.emofairy@gmail.com; Ramokgano, Cindy
Previously, studies on Berchemia discolor trees focused on their pharmacological and nutritional properties. Despite the ecological, economical and pharmaceutical importance, little is known regarding the diseases affecting the species. One recently published paper dealing with fungi associated with B. discolor in Kenya reported 12 ascomycete species that cause dieback and cankers. Thus, the studies presented in this dissertation are the first that attempted to identify fungi associated with B. discolor in South Africa. A total of 29 species were tentatively identified from samples of B. discolor collected at different collection sites in the Limpopo Province of South Africa. These species were classified based on multi-gene DNA sequencing and the species belonged to the 17 genera within 10 families. out of the 29 species, eight species are potentially new to science. Botryosphaeriaceae species were the most notable and predominant in the natural ecosystems. Of the 29 species, 12 species overlapped on branches with dieback and asymptomatic branches, nine were isolated from branches with dieback, while 8 species were obtained from asymptomatic branches. No conclusive evidence could be found that the species obtained from the branches of B. discolor are the causal agents of the dieback disease, as Koch’s postulate was not applied in this study through pathogenicity trials. This study, however, should be seen as a foundational study as limited samples were collected from one province. The results from the chapters presented in this dissertation warrant further research in which the sampling areas and the number of sampled trees should be expanded in order to realise the full extent of the fungal species diversity on B. discolor and their potential impact on the health of these trees in South Africa.
Diseases of coffee with particular reference to those affecting stems and roots in Colombia
(University of Pretoria, 2014) Roux, Jolanda; Wingfield, Michael J.; Alvaro, L.; Gaitan, B.; mike.wingfield@fabi.up.ac.za; Castro Caicedo, Bertha Lucía
Coffee trees belong to the botanical genus Coffea (family Rubiaceae). Arabica coffee (Coffea arabica) originated in Ethiopia and was first cultivated in Yemen in the 15th century. Arabica coffee was introduced to the Asian and American continents during the 17th and 18th century respectively. Today, this species accounts for 70% of the world coffee production, with C.canephora (robusta) constituting ~30% of the production. Coffee has contributed significantly to the economic and cultural development of the countries where it is grown. In 2013, the exporting countries produced approximately 8.5 million metric tonnes (MMT) of green coffee beans, with Brazil, Vietnam, Indonesia and Colombia as the main producers. Significant research programs have focused on providing high-yielding C. arabica cultivars with resistance to pests and diseases, drought, low temperatures and acidic soils. In Colombia, coffee is grown on 915 793 hectares, sustained by approximately 563 000 Colombian coffee growing families. Colombia exported 0.7 MMT of coffee in 2013, worth approximately USD 3 500 000 000. Coffee production has been significantly threatened by leaf rust caused by the fungus Hemileia vastatrix, which is present in all coffee growing countries globally, and results in substantial economic losses. In Colombia two groups of soil borne pathogens, Rosellinia species that cause root rot diseases, and Ceratocystis species which causes canker (“llaga macana”) disease, also reduce productivity. This review presents a summary of knowledge regarding coffee species, their economic importance, breeding of coffee to improve production and details of the latter two diseases, primarily in Colombia.
Seed-borne fungi of herbs cultivated in South Africa and evaluation of non-chemical seed treatments to control Alternaria sp. on coriander
(University of Pretoria, 2015-01) Aveling, Terry A.S.; Kritzinger, Quenton; terry.aveling@up.ac.za; Mangwende, Edgar
Seed-borne mycoflora associated with eight herb seed species, viz. basil (Ocimum basilicum L.), chives (Allium schoenoprasum L.), coriander (Coriandrum sativum L.), dill (Anethum graveolens L.), parsley [Petroselium crispum (Mill.) Fuss], sage (Salvia officinalis L.), thyme (Thymus vulgaris L.) and wild rocket [Diplotaxis tenuifolia (L.) DC.], and their effects on seed germination were studied. Studies on the pathogenicity of isolated seed-borne Alternaria tenuissima (Kunze) Wiltshire were also performed; whereof evaluations of non-chemical methods were conducted to control the aforementioned pathogen. Seed health tests detected ten genera of fungi associated with herb seed lots, which included Alternaria, Aspergillus, Cladosporium, Curvularia, Epicoccum, Fusarium, Penicillium, Rhizoctonia, Rhizopus and Trichoderma. It was observed that Alternaria, Aspergillus, Fusarium and Penicillium were the predominant fungi. This study represents the first record of seed-borne fungi associated with herbs of the Apiaceae and Lamiaceae plant families in South Africa. Findings of seed germination tests showed that all herb seed lots were above their minimum acceptable levels, except for seed lots of dill and wild rocket. In addition, correlation analysis showed that incidence of seed-borne fungi was positively correlated with the number of diseased seedlings raised from the aforementioned seed lots (r= 0.239, p<0.01). Pathogenicity tests showed that the seed-borne fungus, A. tenuissima, was both seed-transmitted and pathogenic on coriander. In -vitro screening tests to investigate the antifungal effects of plant extracts of Allium sativum L, Carica papaya L, Datura stramonium L, Lantana camara, Tagetes minuta and Zingiber officinale Roscoe were conducted against pathogenic A. tenuissima. Based on the agar infusion method, it was observed that most acetone and ethyl acetate extracts effectively inhibited growth of A. tenuissima when applied at low concentrations, which had minimum inhibitory concentration (MIC) values of ≤5 mg/ml. However, most water extracts demonstrated poor antifungal activities; thus, recorded high MIC values (>10 mg/ml). From the antifungal tests using the disc diffusion method, the ethyl acetate extract of Allium, acetone extracts of Datura and Zingiber, and the water extract of Lantana were selected for further evaluations in the greenhouse as they demonstrated good antifungal activities against A. tenuissima. Furthermore, a preliminary in vitro study was conducted to examine the optimum hot water treatment temperature-time combination that effectively controls A. tenuissima associated with coriander seeds. Findings of this study showed that seeds soaked in hot water at 54ºC for 15 mins resulted in a considerable reduction of the incidence of A. tenuissima with minimal effect on seed germination. However, soaking coriander seeds at temperatures above 54ºC significantly lowered seed germination. Naturally infected coriander (cultivar American long) seeds treated with plant extracts of Allium, Datura, Lantana and Zingiber; hot water at 54ºC for 15 mins and biological control agents of Bacillus subtilis (Ehrenberg) Cohn and Trichoderma harzianum Rifai were evaluated for their effects on seed germination, seedling emergence and incidence of Alternaria leaf spot disease incited by A. tenuissima. This study showed that all seed treatments effectively lowered the incidence of A. tenuissima on coriander, which resulted in improvement of percentage seed germination. However, Datura extracts negatively affected seed germination as it resulted in higher numbers of abnormal seedlings. Greenhouse experiments showed that sowing treated seeds significantly increased seedling emergence and seedling growth. Thus, seedlings raised from treated seeds were significantly longer and had broader leaf surface area, which contributed to higher seedling fresh and dry mass compared to untreated seeds. The incidence and severity of Alternaria leaf spot disease was less pronounced on coriander seedlings raised from coriander seeds treated with extracts of Allium, Zingiber and the biological control agent, Bacillus sp.
Antigenic site determination on a SAT2 foot-and-mouth disease virus using a chicken antibody phage display library
(University of Pretoria, 2013-11) Maree, Francois Frederick; Vosloo, Wilna; jacques.theron@up.ac.za; Opperman, Pamela Anne
Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. An outbreak of FMD not only severely decreases livestock productivity, but also impacts on both the local and export trade of susceptible animals and their products. This, in turn, negatively impacts the economy of affected countries. Of the seven serotypes that exist for FMD virus (FMDV), the three South African Territories (SAT) types display greater intratypic genomic and antigenic variation than the traditional “Euro-Asian” types. Although antigenic variation represents an important adaptive strategy of FMDV, especially in its maintenance host, it contributes to the decrease of vaccine cross-protection in the field, thus rendering available vaccines less effective. Knowledge of the amino acid residues that comprise the antigenic determinants will allow for the structural design of vaccine seed viruses that may provide improved protection against specific outbreak strains. The SAT2 type viruses, which are responsible for most of the FMD outbreaks in domestic animals in southern Africa, are the most variable of the SAT serotypes. In order to identify antigenic regions present on a SAT2 FMDV, two approaches were followed. In the first approach, a SAT2 vaccine strain, ZIM/7/83, was panned with a naïve chicken phagedisplayed library. Three unique SAT2/ZIM/7/83-specific phage-scFvs were obtained. Of these, phage-scFv2 was able to neutralize the SAT2/ZIM/7/83 virus and following sequencing of neutralization-resistant virus variants, an antigenic site was mapped to include residue 159 of the VP1 capsid protein. In the second approach, genetically modified viruses were generated in which known and predicted epitopes of SAT2/ZIM/7/83 were replaced with those of a disparate virus, SAT2/KNP/19/89, to determine the role of known SAT2 epitopes and to identify new potential antigenic regions. Following characterization of the epitopereplaced mutant viruses and studies with SAT2-specific monoclonal antibodies, two additional antigenic sites were mapped to include residues 71-72 of the VP2 capsid protein. The information gained from this study will not only increase the knowledge of the antigenic sites of SAT2 viruses and aid in identifying more suitable vaccine strains for SAT2 viruses, but is also the first step towards the production of a SAT2-specific epitope-based vaccine.
Characterization of sex-pheromone receptor genes of Fusarium species and other Sordariomycetes
(University of Pretoria, 2014-06-30) Steenkamp, Emma Theodora; Fourie, Gerda; Van der Merwe, Nicolaas Albertus (Albie); Wingfield, Brenda D.; tondai.kone@gmail.com; Kone, Tondani
Fungi are capable of both sexual and asexual reproduction. Sexual reproduction involves karyogamy followed by meiosis (Burnett 1968; Alexopoulos, Mims, Blackwell 1996), while asexual reproduction involves neither meiosis nor karyogamy but mitosis. Asexual reproduction in fungi, like with many other eukaryotes and prokaryotes, occurs by fragmentation, budding, or fission (Burnett 1968; Alexopoulos, Mims, Blackwell 1996). The mitotic spores that are formed through fungal asexual reproduction are known as conidiospores or conidia (singular = conidium). These are formed on hyphae or on conidiophores that are modified hyphal branches (Burnett 1968; Alexopoulos, Mims, Blackwell 1996; Leslie, Summerell, Bullock 2006).
Analysis of temporal and spatial changes in major dissolved salts in the Vaal River system over a 40 year period
(University of Pretoria, 2022) Van Niekerk, Harold; samsonakpotu@yahoo.com; Akpotu, Samson Oghenemauro
In this study, the fluctuation and variation in salinity composition of the Vaal River and its catchment area was investigated between 1975-2015. Long-term data sets gathered from a 40-year span collected across nine monitoring station in the study catchment area was analysed. The statistical tool applied for the data analysis is the Maucha diagram, as well as the Origin software used in plotting graphs. These graphs revealed several trends and patterns. The graphs and the Maucha diagrams reflected long-time changes in salts composition and concentration in the water of the Vaal River and its catchment area over a 40-year period. From the data analysed, the graphs plotted, and the Maucha diagrams, the salinity of the Vaal River was majorly total alkalinity (TAL) and sulfate (SO4) dominated, with a substantial amount of Calcium (Ca2+). However, the concentration of other salts remained relatively constant during the duration of study. Analysis of 5-year averages of TAL, SO4 and total dissolved solids (TDS) concentration showed that zones and sub-catchment uniquely impacted variation in salinity levels and composition. Furthermore, salinity levels and composition in zones 1 and 4 were greatly impacted by water from the Lesotho Highlands and the Orange River, respectively. The salinity level changes of the Vaal River were mainly attributed to anthropogenic sources such as pollution from (mining) industries, increased urbanisation, economic expansion, paving of roads, increasing population, water transfer in and out of the river’s catchment area and irrigation outflow from agricultural activities. Based on the salinity concentration of the Vaal River and the effect on humans and the environment, routine continuous monitoring and sampling programmes are recommended to be carried out by the National government. This will help to make informed decision on managing the salinity levels of the Vaal River and mitigating any negative attendant consequence of increased salinity.
Molecular genetic analysis of some enzootic rabies viruses of southern Africa
(University of Pretoria, 1997) Nel, Louis Hendrik; Jaftha, Julian Bernard
Rabies viruses are known to be able to infect a broad range of warm-blooded animals. In South Africa the disease is maintained in different animal species including dogs, jackals, bateared foxes and a variety of members of the viverridae family. These include the different mongoose species (principally the yellow mongoose), genets, suricates and a variety of small carmvores. The antigenic variation within the nucleoprotein gene has previously been investigated in efforts to characterise various isolates of rabies viruses in southern Africa. It was noted that two antigenically distinct groups are cocirculating in the country. In this study, the investigation into the epidemiology of rabies in South Africa was extended by molecular genetic analysis of a large number of isolates from wildlife and domestic animal hosts. Geographically and temporally distinct rabies viruses were previously studied by comparative nucleotide sequence analysis of DNA fragments encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region. Based on this analysis two main rabies virus groups were identified. Members of the first group infect canid host species and are closely related to the European rabies strains, while the viruses belonging to the second group circulate within different viverrid species. Although isolates of mainly mongoose origin were initially analysed, considerable heterogeneity within this group was evident. The current study was consequently undertaken to include rabies virus isolates from other viverrid host species. Following the same approach as the previous investigators (Nel et al., 1993 & von Teichman et al., 1995) four genetically distinct clusters were indicated within the viverrid lineage. These clusters corresponded closely to the geographic origin of the virus isolates independent of specific viverrid host. The results suggest genetic divergence and independent evolution of the viverrid viruses within geographically isolated regions. Spillover or cross-infection, where a viverrid virus is recovered from a canid host and vice versa, could not be attributed to a new rabies virus and were most likely initiated by interspecies transmission events. These results suggest little modification of the virus following infection of an atypical host. A phylogeny of the rabies virus variants in southern Africa was established based on sequence variation within the abovementioned genome regions. Although such an approach is most informative when compared to other methods, it is time-consuming and laborious. The use of strain-specific oligonucleotides was therefore investigated for rapid strain differentiation. Two oligonucleotides were designed based on the nucleotide sequences of the cytoplasmic domain of the glycoprotein and the G-L intergenic region. These oligonucleotides together with a common downstream primer were used to amplify DNA fragments of characteristic size, allowing for discrimination between the two rabies biotypes.
Development of a recombinant adenoviral immunocontraceptive vaccine (Ad-GKT) for use in domestic dogs
(University of Pretoria, 2021) Nel, Louis Hendrik; Wright, Nicolette; u15062555@tuks.co.za; Arnold, Danielle Patricia
Rabies is a viral disease caused by the rabies lyssavirus (RABV). Despite effective rabies vaccines for humans and animals, this disease continues to pose a major public health challenge, causing an estimated 59 000 human deaths each year, over 99% of which are caused by the domestic dog (Canis familiaris). Current methods of dog population management used in rabies control programs are ineffective. Surgical sterilisation does not reach enough of the dog population to curb population densities and contraceptives need to be administered at a specific phase in the oestrous cycle or cause a range of side effects. Immunocontraception in dogs would allow rabies vaccination coverage to be maintained, in turn reducing the burden of rabies on public health. The aim of this study was to develop an immunocontraceptive vaccine for dogs capable of eliciting a stronger immune response than that of previously constructed vaccines allowing for effective dog population management and allowing rabies vaccination coverage to be maintain, in turn reducing the burden of rabies on public health. By stabilising the dog population size, the 70% vaccination coverage required to interrupt rabies transmission within a population can be maintained. The immunocontraceptive vaccine constructed in this study contained two reproductive hormones, namely GnRH and kisspeptin, in the hope of eliciting a stronger contraceptive effect than either of these could produce alone, as well as the partial tetanus toxoid gene as an immune stimulant. The nucleic acid GnRH, kisspeptin and partial tetanus toxoid gene (GKT) insert fragment was PCR amplified from a DNA construct (pVAC-GKT) and was cloned into the adenoviral vector using In-fusion cloning technology. Transfection of pAdeno-X 293 cells was confirmed using green fluorescent microscopy and expression of the Ad-GKT mRNA in cell culture was confirmed using real-time RT-PCR. The antigenicity of the Ad-GKT construct was evaluated using female Swiss Webster mice. An indirect ELISA was used to detect seroconversion of the GnRH and Kisspeptin insert fragments. The Ad-GKT construct was successful in eliciting an immune response against GnRH and kisspeptin. Future research should include a comparative study to determine the antigenicity of the Ad-GnRH1 and Ad-GKT constructs in a canine trial for potential use in rabies control programs.
Ophiostomatoid fungi associated with fungus farming insects in South Africa
(University of Pretoria, 2021) Duong, Tuan A.; Wingfield, Michael J.; De Beer, Z. Wilhelm; janine.nel@fabi.up.ac.za; Nel, Wilma Janine
Symbiosis is the term used to describe the different forms of communal life that can exist between two unlike organisms. Primarily this involves interaction in one of three forms: mutualism where both partners benefit from their association; antagonism where one or both partners are harmed by their association; and commensalism where one partner derives benefit but the other is neither harmed nor profited from their association. Many different forms of symbioses have been described between insects and microbes and these include short, simple interactions as well as obligate associations. Aside from humans, sophisticated agricultural farming practices have only been found for three insect groups, colloquially known as fungus-farmers. These three groups – the attine ants, the macrotermitinae termites, and the ambrosia beetles - each independently evolved an obligate mutualism with fungal partners that they actively cultivate and maintain within their nests and utilize as a primary source of nutrition. The primary focus of this PhD thesis was that of the association that exists between bark and ambrosia beetles and their respective fungal partners and the genomic signatures of these associations. Additionally, the unexpected and interesting discovery of Ophiostomatoid fungi from fungus-growing macrotermitinae termites was also investigated. Very little research on ambrosia beetles and their fungal partners has been conducted in South Africa. However, the accidental introduction of the devastating Polyphagous Shot Hole Borer into the country has sparked renewed interest into this topic. The primary focus of the research included in this thesis has consequently been on ambrosia beetles in South Africa. This is also the topic of the literature review (Chapter 1) at the start of the thesis. However, given the many commonalities regarding their associations with fungi, we have also included a study that led to the discovery of Ophiostomatoid fungi associated with fungus-farming termites. The background literature relating to this association is provided in the introduction to that study and it is also a topic that has been thoroughly treated in a number of recent reviews. In Chapters 2-4 I explore the diversity of Ophiostomatoid fungi associated with fungusfarming insects in South Africa. In Chapter 2, I report four species of Ophiostomatalean species associated with commonly found ambrosia beetles in the country. One of these species is reported from South Africa for the first time and two are described as novel species. In Chapter 3, I report for the first time the presence of the granulate ambrosia beetle and its Microascalean associate, Ambrosiella roeperi, in South Africa and highlight the potential threat that this beetle and its fungal symbiont poses to South Africa’s agricultural industry. In Chapter 4, I investigate three species of Ophiostomatalean fungi discovered on the abandoned Termitomyces fungus combs of fungus-farming termites. Using both culture- based and genomics techniques, I describe these newly discovered Ophiostomatalean fungi and investigate their distinct lifestyle. In Chapter 5, I delve more deeply into the relationship shared between Sordariomycete fungi known to be associated with arthropods. In this chapter, I attempt to elucidate how the relationship with their arthropod partners has influenced their genomic evolution. Using whole genome sequences and comparative genomics methods I investigate the traits shared amongst the Ascomycete ambrosia fungi, as well as investigate how these fungi differ genetically from their close relatives. Finally, as a supplementary chapter to this thesis, I provide a comprehensive record of bark and ambrosia beetle species present in South Africa. This is justified by the fact that other studies included in this thesis involved extensive trapping of bark and ambrosia beetles, many of which had not previously been recorded in the country. This record increases the currently recorded number of these insects from South Africa from 163 to 260. Additionally, 22 species are reported from the county for the first time. It was decided to place this chapter as supplementary material and not within the main body of the thesis because it did directly capture the common theme of Ophiostomatoid fungi associated with fungus-farming insects.
Functional characterization of pathogenicity genes in Fusarium circinatum
(University of Pretoria, 2021) Steenkamp, Emma Theodora; Wingfield, Brenda D.; Wingfield, Michael J.; Coetzee, Martin Petrus Albertus; mmatshepho.phasha@up.ac.za; Phasha, Mmatshepho Malekgale
The genus Fusarium includes some of the most destructive pathogens of agricultural and forestry crops. Within this genus, Fusarium circinatum is an important pathogen that causes severe disease on pine plants of all ages and is thus responsible for huge economic losses to forestry industries worldwide. In this thesis, the roles of putative pathogenicity genes (RAS2 and FUB1) were investigated in F. circinatum. This was done by generating ras2 and fub1 knockout mutants, and studying their phenotypes alongside F. circinatum wild type strain FSP34. The phenotypes included hyphal growth, conidiation, fertility, secondary metabolite production and virulence. Transcriptomes of these mutants and the wild type strain were also sequenced, and gene expression patterns were compared. Results from the phenotypic studies showed that deletion of RAS2 caused reduced growth of F. circinatum colonies as well as reduced virulence of the fungus on Pinus patula seedlings. The results also showed that the lack of the RAS2 gene in the knockout mutant strain slowed conidia germination, and resulted in fewer perithecia being produced in a sexual cross where this strain was a female. Phenotypic studies with the fub1 knockout mutant showed that disruption of the FUB1 gene caused an abolishment in the production of the fusaric acid secondary metabolite by F. circinatum. This disruption also caused the fub1 knockout mutant to produce smaller lesions on P. patula seedlings than the wild type strain. Analyses of gene expression patterns in the ras2 knockout mutant and wild type strain revealed that the deletion of RAS2 caused downregulation in numerous genes putatively involved in F. circinatum development and pathogenesis including transcriptional regulators and components of the mitogen-activated protein kinase (MAPK) signalling pathway. Results from analyses of gene expression patterns in the fub1 knockout mutant and the wild type strains also revealed a number of downregulated genes possibly involved in pathogenesis, and an upregulation genes of the bikaverin biosynthetic cluster. Collectively, the findings from this thesis show that RAS2 and FUB1 are virulence factor genes in F. circinatum. The findings also suggest that the Ras2 protein is a master regulator that controls growth, development and virulence in this fungus, and that it does so in a MAPK-dependent manner. This work will contribute to our knowledge of the molecular basis and mechanisms used by fungi in general to cause disease on their hosts.
Detection and characterization of genetically diverse paramyxoviruses from African bats
(University of Pretoria, 2013-09) Markotter, Wanda; Nel, Louis Hendrik; Weyer, Jacqueline; wanda.markotter@up.ac.za; Mortlock, Marinda
In past years, the potential of bats as reservoir for paramyxoviruses was clearly underestimated. Research of the 21st century now provides evidence that bats play an important role as reservoir and host to these viruses. The aim of this study was to detect the presence of any novel paramyxoviruses that may be circulating in bats across Africa. The specific objectives included the screening of specimen panels of insectivorous as well as frugivorous bat species collected from a number of African countries. Two broadly-reactive universal primer sets targeting the Paramyxovirinae subfamily and the Respiro-, Morbilli- and Henipavirus genera were used in two semi-nested PCR reactions. Bat kidney was selected as target organ and bats were sampled from several countries across Africa (Cameroon, Democratic Republic of the Congo, Kenya, Nigeria, South Africa and Swaziland). Based on amino acid analysis it was determined that approximately 31 putative viral species were detected. Viruses detected, clustered phylogenetically with known genera namely Henipavirus, Morbillivirus and the newly proposed Jeilongvirus. Several viral sequences clustered outside the known genera and might belong to yet unclassified genera in the Paramyxovirinae subfamily. Viral exchange between different bat species was also observed in several occasions where sampling from geographically distant locations was done. The ability of some bat species, e.g. Eidolon helvum, to migrate over large distances, likely contributes to the spread of specific virus lineages over significant geographical space. The propensity for many bat species to roost communally is another likely contributor to enhanced virus transmission events. Due to the vast genetic variability among paramyxoviruses in nature, insight into these viruses will be vital in understanding their pathogenic nature and the possible threat they may pose to public and veterinary health sectors. Propagation and isolation in cell-cultures as well as full-genome sequence analysis will be a foremost requirement in future research of these viruses. Clearly, there are geographical limitations in this study which emphasizes the need for a One Health approach from all African countries that will greatly contribute to future research on paramyxoviruses.
Epidemiological modeling of rabies transmission pathways in dog rabies endemic KwaZulu-Natal, South Africa
(University of Pretoria, 2013-09-27) Nel, Louis Hendrik; Markotter, Wanda; loui.nel@up.ac.za; Mollentze, Theodorus Bernardus
In the fight against endemic infectious diseases – which disproportionately affect the developing world – the effective use of scarce resources is of paramount importance. For vaccine preventable diseases, vaccination campaigns should be of optimal efficiency, a goal which is dependent on effective disease surveillance as well as a thorough understanding of the disease’s spatial epidemiology. Several recent approaches show great promise in allowing us to understand the high resolution spatial aspects of epidemic disease spread following a single introduction, but do not account for the complexities inherent to endemic diseases. This thesis describes the development and use of novel techniques that can be applied to better understand endemic diseases and epidemics originating from multiple introductions, towards improved control and eventual elimination.
Barcoding of South African bat species and evaluation of their natural exposure to lyssaviruses
(University of Pretoria, 2013-07) Markotter, Wanda; Nel, Louis Hendrik; wanda.markotter@up.ac.za; McCulloch, Stewart D.
The genus Lyssavirus, currently consisting of 12 confirmed and two putative species, all of which are capable of producing the lethal encephalitic disease known as rabies. Due to the long history and common knowledge of the prototype virus, rabies virus the other members of this genus, which have only been discovered since the 1960’s, are commonly referred to as the rabies-related viruses. Rabies virus is largely maintained throughout the world by on-going viral cycles within non-volant carnivores, whilst the rabies-related members are typically associated with chiropteran populations.
The diversity and structure of Escherichia coli populations in fresh water environments
(University of Pretoria, 2013) Venter, S.N. (Stephanus Nicolaas); Brözel, V.S. (Volker Siegfried), 1963-; fanus.venter@up.ac.za; MacRae, Sarah Catherine
Escherichia coli is a well known commensal inhabitant of the gastrointestinal tract of both humans and animals and a highly diverse species. The physiology, biochemistry and genetics of E. coli have been studied extensively over many decades. However, these studies have focussed predominately on the pathogenic and commensal isolates. It has been described that E. coli typically exists in two environments, the primary environment being the gastrointestinal tract of the host and the secondary environment being that environment outside of the host (water, soil and sediments). Upon introduction into the environment outside of the host, the numbers of E. coli steadily decline. Generally, where E. coli is present in the external environment and where its numbers are maintained it is due to a constant direct faecal input from the host. This short lifespan in the environment outside of the host forms the basis for the use of E. coli as an indicator organism for faecal contamination in water systems. In contrast, multiple studies have shown that some E. coli strains have the ability to survive and persist in the external environment in the absence of faecal input from the host. With a large pan-genome and the possibility of horizontal gene transfer (HGT) of desirable traits, E. coli have the potential to adapt to a variety of different niches overcoming drastic changes in conditions in its new environment. In addition, adaptation to the secondary environment is facilitated by the presence of soils and sediments, where in an aquatic environment they provide a source of nutrients and protection from the drastic change in conditions. Here, E. coli has the ability to occupy a new niche and become naturalised within an aquatic environment. The aim of this masters project was to examine and characterise the diversity of E. coli isolates collected from two South African freshwater environments namely, the Roodeplaat and Rietvlei Dams, Pretoria. Specific research questions addressed in this study include: (1) are their unique and genetically differentiated sub-populations within the aquatic environments sample? (2) Is there a link between the unique sub-populations and their sample site? (3) Finally, what is the relationship between sub-populations in terms of gene flow and population structure? Understanding E. coli’s population structure and ecology may shed some light on its evolution and potential to adapt to new environments. Following phylogrouping, AFLP and phylogenetic analysis of the rpoS and uidA genes, the results indicated that the population was highly diverse with the majority of strains grouping together with the sewage isolates. Furthermore, population structure analyses concentrating on gene flow and genetic differentiation revealed that possible environmental groups exist within the population. In particular, two groups of E. coli isolates associated with aquatic plants showed restricted gene flow and definite genetic differentiation. These two groups can also be observed in the rpoS and uidA phylogenetic analyses where they consistently group together in the absence of sewage isolates. These findings demonstrate that some E. coli are not only able to survive outside of their host but have undergone some level of niche separation within the secondary environment. These results raise important questions into the accuracy of using E. coli as an indicator organism. In the long term, this study may aid in understanding the population dynamics of E. coli and the implications of environmental strains on using E. coli in assessing water quality.
Comparative study of Epicoccum sorghinum in Southern Africa
(University of Pretoria, 2014-04) Marais, Gert J.; Steenkamp, Emma Theodora; emma.steenkamp@up.ac.za; Van der Nest, Ariska
The Coelomycetous genus, Phoma, is defined as filamentous fungi that produce pycnidial conidiomata with monophialidic, doliiform to flask-shaped conidiogenous cells. Host specificity was regarded as an important characteristic in identifying Phoma and this Saccardoan system, together with only minor differences in morphological characteristics between species, led to the description of a high number of species with no true taxonomic relevance. Species were extensively revised by Boerema and co-authors in 2004 and reduced to 223 taxa divided into nine sections, although not all species were considered. Experience was still required to accurately differentiate between species. Phoma section Peyronellaea was characterised by alternarioid dictyochlamydospores, epicoccoid shaped chlamydospores and/or unicellular chlamydospores that looked like pseudosclerotia. This section was later dissolved and the genus Peyronellaea re-instated. Phoma sorghina belonged to this section, and has a worldwide distribution. It is considered as a weak secondary parasite of plants that produce metabolites such as mycotoxins, phytotoxins and anthraquinones. Since its first description in 1878 by Saccardo as Phyllosticta sorghina until 1973, when it was named Phoma sorghina, it has been renamed numerous times based on morphological characteristics. It was moved to Epicoccum based on phylogenetic and morphological characteristics in 2010. The aim of this review is to discuss the complexity of the taxonomic challenges in the genus, Phoma, with special reference to Epicoccum sorghinum. In addition, an attempt is also made to demonstrate the importance of E. sorghinum as a plant pathogen and the threat it poses to human health.
Development and evaluation of a recombinant vaccine against Clostridium perfringens type D epsilon toxin
(University of Pretoria, 2013) Theron, Jacques; Crampton, Michael; Louw, Maureen E.; Tovhowanir@gmail.com; Ralikhwatha, Tovhowani Daphney
Enterotoxemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens Type D. To combat the disease, a vaccine consisting of formalin-inactivated Type D toxin is available, but several concerns regarding its production have been raised. The development of efficacious recombinant subunit vaccines can provide a means whereby many of the production problems may be eliminated or minimized. However, the production of purified antigens is often associated with weakened antigen immunogenicity. New vaccine strategies therefore rely on the incorporation of effective adjuvants, which stimulate the innate immune response and, in turn, activate the adaptive immune response. An increasing number of studies have demonstrated that flagellin is a potent activator of a broad range of cell types involved in innate and adaptive immunity. Consequently, the aim of this investigation was to produce different recombinant vaccine candidates, inclusive of a flagellin-epsilon toxin fusion protein, for preventing Type D enterotoxemia. Attempts at expressing the native gene sequence for the Type D epsilon toxin in Escherichia coli have been characterized by low levels of expression. This may be due to differences in the codon bias between the heterologous gene and E. coli. Therefore, in this study, a completely synthetic codon-optimized gene encoding the epsilon toxin was obtained. The full-length etx gene (etx0), an etx gene lacking the first nine amino acids of the signal peptide sequence (etx1), as well as a flagellin-toxin fusion gene composed of the etx1 gene and a truncated hag gene of Bacillus halodurans Alk36 (Δhag::etx1) were expressed in E. coli BL21-Gold(DE3) cells. Expression of the respective recombinant proteins, designated ETX0, ETX1 and NC-ETX1, respectively, were confirmed by Western blot analysis using anti-epsilon toxin antibodies. The ETX0 protein was expressed in a soluble form, whereas the ETX1 and NC-ETX1 proteins were insoluble and accumulated in inclusion bodies. In an attempt to optimize expression of the recombinant proteins, different cultivation media and concentrations of the promoter inducer were evaluated. Under optimized expression conditions, the yields of ETX0, ETX1 and NC-ETX1 were determined to be 828 mg/l, 395 mg/l and 525 mg/l, respectively. Despite having expressed the recombinant proteins with an N-terminal hexahistidine tag, the recombinant ETX0 protein could not be purified with nickel (Ni2+) chelate affinity chromatography. This was due to removal of the affinity tag as a consequence of intracellular processing of the N-terminal signal peptide sequence. The recombinant ETX0 protein was consequently purified to near homogeneity by ion exchange chromatography. The recombinant ETX1 and NC-ETX1 proteins were purified from inclusion bodies by affinity chromatography under denaturing conditions and then refolded in TBS buffer. The yield of purified ETX0, ETX1 and NC-ETX1 were determined to be 430 mg/l, 400 mg/l and 340 mg/l, respectively. The cytotoxicity of these purified recombinant proteins was assayed in vitro against Madin-Darby canine kidney (MDCK) cells, the results of which indicated that NC-ETX1 was not toxic towards the cells and both ETX0 and ETX1 displayed moderate toxicity towards the cells. Cumulatively, the results indicated that the respective proteins were expressed to high levels in E. coli and large amounts of purified proteins could be obtained. These recombinant proteins can in future be evaluated as vaccine candidates against C. perfringens Type D enterotoxemia.
Pathogenicity and immunogenicity of recombinant rabies viruses expressing the Lagos bat virus matrix and glycoprotein genes
(University of Pretoria, 2015) Markotter, Wanda; Nel, Louis Hendrik; joe.kgaladi@gmail.com; Kgaladi, Joe
Lagos bat virus (LBV) is a phylogroup II lyssavirus exclusively found in Africa. Previous studies have shown that this virus is lethal to mice after intracranial (i.c.) and intramuscular (i.m.) inoculation. Pathogenicity determinants of LBV are yet to be determined. The antigenic composition of LBV differs substantially from that of the rabies virus (RABV) and current rabies vaccines do not provide cross protection against LBV and other phylogroup II lyssaviruses. LBV is associated with Pteropodidae bat species and although no human infections have been reported to date, fatal spill-over into dogs, cats and a mongoose have been reported. To investigate the potential role of the LBV matrix (M) protein and glycoprotein (G) in the pathogenesis, reverse genetics technology was used to construct recombinant viruses. The genes encoding the G protein or the M and G protein of the attenuated RABV strain SPBN were replaced with those of LBV (LBVAFR1999) resulting in SPBN-LBVG and SPBN-LBVM-LBVG, respectively. In addition, to evaluate the immunogenicity of the LBV G, the recombinant RABV SPBNGAS-LBVG-GAS was constructed that contained the LBV G inserted between two mutated RABV G genes (termed GAS). Multi-step growth curves showed that SPBN, SPBNGAS-GAS-GAS (a recombinant RABV containing three G protein genes) had the highest growth rate followed by SPBN-LBVG and SPBNGAS-LBVG-GAS. While there was no statistically significant difference between the growth rate of these viruses (p>0.05), the growth rate of SPBN-LBVM-LBVG was lower than that of the other viruses, including LBVAFR1999. The single-step growth curves yielded similar results, with SPBNGAS-GAS-GAS, SPBNGAS-LBVG-GAS and SPBN producing the highest titres and SPBN-LBVM-LBVG and LBVAFR1999 again producing the lowest titres. The results from both growth curves indicated that both the M and G protein of LBV control the growth rate of the virus and thereby playing a role in pathogenicity. All the viruses − with a single exception, viz. SPBNGAS-GAS-GAS − were lethal to mice after i.c. inoculation, although the pathogenicity of SPBNGAS-LBVG-GAS was lower compared to the other recombinant viruses. Mice inoculated with LBV and SPBN-LBVM-LBVG had the highest percentage mortality (100%) and the shortest mean incubation period, while those inoculated with SPBNGAS-LBVG-GAS had the lowest percentage mortality (20%) and the highest incubation period. Following i.m. inoculation, only LBVAFR1999 and SPBN-LBVM-LBVG were lethal to mice, indicating that both the M and G protein of LBV play a role in the pathogenesis of LBV. Serum from mice inoculated with SPBNGAS-GAS-GAS and RABISIN (a commercial rabies vaccine used for dogs) cross-neutralised RABV and DUVV, while no detectable VNA were observed for LBV and MOKV. These findings emphasise the already known concept that vaccines derived from RABV cross-neutralise against DUVV, but not against LBV and MOKV. Most interestingly, serum collected from mice inoculated i.m. with SPBNGAS-LBVG-GAS cross-neutralised phylogroup I and II [RABV, LBV, Duvenhage virus (DUVV) and Mokola virus (MOKV)] lyssaviruses, indicating that this recombinant virus has a potential to be used for the development of a pan-lyssavirus vaccine.
Quantification and characterization of the Streptomyces complex causing common scab of potatoes in South Africa
(University of Pretoria, 2014) Van der Waals, Jacquie E. (Jacqueline Elise); Estiene@gmail.com; Jordaan, Estiene
Common scab is a disease that results in corky lesions on the surface of potatoes. These lesions can be variable in size, shape and as a result of secondary infection may appear extremely cryptic. The disease is caused by Streptomyces, a group of Actinomycetes that are largely saprophytic. For this reason lesions may also contain saprophytic Streptomyces which makes isolation and detection of pathogenic species on potatoes difficult. Symptom expression is thought to be attributed to environmental factors and prevalence of pathogenic species. Common scab causing Streptomyces isolates were collected from most of the potato producing regions in South Africa. These isolates were characterized based on morphology, physiology and sequenced to confirm identity and determine their closest related species. A diverse range of other Streptomycetes, similar in morphology and physiology to Streptomyces scabiei (Lambert & Loria), were frequently isolated during this study from common scab lesions. Most Streptomycetes isolated from common scab lesions were S. scabiei or Streptomyces europaeiscabiei (Bouchek-Mechiche). No Streptomyces acidiscabies (Lambert & Loria) or Streptomyces turgidiscabies (Miyajima) were found. Streptomyces stelliscabiei (Bouchek-Mechiche) appears to be more geographically restricted in South Africa. The presence of txtAB, tomA and nec1 genes were investigated within pathogenic as well as non-pathogenic isolates associated with common scab lesions. Some isolates were pathogenic but lacked any of the pathogenicity / virulence genes; while others were non-pathogenic but showed the presence of at least one of the three genes. However it seems that the combined presence of all three these genes is a good indicator of the pathogenicity of an isolate when compared with the tuber slice assay for pathogenicity. Although common scab does not decrease yield, it results in smaller progeny tubers. A complex interaction exists between common scab causing Streptomycetes and their soil environment – increasing soil pH from 6.5 to 8.5 decreases disease incidence and severity. Symptoms were not found to be linked to species, soil pH or soil moisture. An increase in initial inoculum contributes to an increase in disease incidence and severity. In future uncertainty regarding the pathogenicity gene makeup of the South African Streptomyces complex must be resolved. This will include sequencing the most important genes related to pathogenicity and comparing South African isolates with the isolates from other countries. The detection of these genes with Thaxtomin A expression (HPLC) should also be examined. Should these two factors co-exist it will then be possible to design primers specific to South African pathogenic Streptomyces spp. that could possibly be used for quantitative detection pre-plant.
Characterization of Fusarium species from Pinus and Eucalyptus nurseries in Colombia and South Africa
(University of Pretoria, 2013) Steenkamp, Emma Theodora; Wingfield, Michael J.; Wingfield, Brenda D.; darryl.herron@fabi.up ac.za; Herron, Darryl Arthur
The success of the forestry industry in the world and particularly the Southern hemisphere can be attributed to the choice of fast and easy-growing exotic pine and eucalypt tree species, which have been planted separated from their natural enemies. In South Africa, species of Pinus, Eucalyptus and an Acacia species have been planted to sustain commercial forestry. This industry in South Africa is at risk, however, because of pest and pathogen movement around the world. Various species of Fusarium represent some of the most serious threats to the forestry industry. The genus includes a large number of species, many of which are important plant pathogens with host ranges that include species in Pinus, Eucalyptus and Acacia. An important and well known example in South African commercial forestry is Fusarium circinatum, which causes the disease known as pitch canker. Research on this pathogen has advanced our understanding and ability to identify the pathogen rapidly and to establish measures that will contain its spread. Identification of pathogens such as species of Fusarium represents a first step towards developing control measures. Diseases caused by emerging pathogens are becoming more complex due to exacerbating factors such as the effect that climate change might have on host-pathogen interactions. Understanding of all these factors should contribute to an enhanced capacity to protect forest plantations in the future.
Phylo- and comparative genomics of the Pantoea core genome
(University of Pretoria, 2014) Venter, S.N. (Stephanus Nicolaas); Coetzee, Martin Petrus Albertus; Coutinho, Teresa A.; Steenkamp, Emma Theodora; marike.duplessis@fabi.up.ac.za; Du Plessis, Marike
The delineation of bacterial species and genera has always been problematic as a clear definition of these concepts are lacking. In an attempt to classify bacteria into workable groups, operational criteria have been applied to delimitate boundaries for these taxonomic ranks. This approach has unfortunately led to artificial groupings that are often not comparable in terms of diversity in different groups of bacteria. A classification system needs to reflect natural groupings to depict the evolution of bacteria and predict the phenotypic and genetic diversity for these groups. In order to understand the forces that play a role in the evolution of a bacterial genus a review of the current literature was presented in Chapter 1. The major focus was on vertical inheritance and how this process can be used to depict the evolutionary path of members belonging to the same genus. The largest amount of genetic material in any one cell is thought to have been transferred from parent to progeny, supporting the idea that the vertical signal is recoverable and can in fact be the dominant signal present in the genome when looking at conserved genes. The effect of horizontal gene transfer (HGT) on the evolutionary picture obtained by vertical descent was also discussed. The core genome of a genus is defined as the genes conserved between all species of a genus and are thought to mostly include genes that are essential for the survival of members of that particular genus. In Chapter 2, the hypothesis was tested that the boundaries used to delineate genera could be based on an analysis of the shared core genome. For this purpose coherence within the core genome of the genus Pantoea was investigated. The core was characterised in terms of its functional diversity through Clusters of Orthologous Genes (COGs) and compared to the core genomes of other bacterial genera. It was seen that the core genome does give an indication of the coherence of a genus and that shared genome content can be used as a tool to delimitate genera. Previous taxonomic studies have shown that species in the genus Pantoea are well defined but that the phylogenetic relationships between these species are not well elucidated. Generally accepted approaches for phylogenetic inference, like 16S rRNA gene trees and multi-locus sequence analysis (MLSA), does not give sufficient resolution to determine the deeper evolutionary relationships between these species. In Chapter 3, phylogenomic analyses were performed to determine if a robust phylogeny, reflecting the evolutionary history of the genus, can be obtained using the core genome of the genus. The core genome as well as subsets thereof (based on COGs), was used for phylogenetic inference, to obtain a robust phylogeny for the genus.

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