Doctoral Degrees (Biochemistry)

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An assessment of the diversity and pathogenicity of Potato leafroll virus in South Africa
(Stellenbosch : Stellenbosch University, 2018-03) Roos, Wiets Gideon; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Potato production in South Africa has steadily intensified through improved pivot irrigation. Since potatoes are vegetatively propagated the potato industry is continuously threatened by Potato leafroll virus (PLRV) which is responsible for increasing yield losses in South Africa. Effective management of PLRV is dependent on its accurate detection in seed potato stocks. PLRV is a small spherical plant virus consisting of a protein capsid and an RNA genome of approximately 5900 bases. This virus is phloem restricted and is vectored by, most notably, by the green peach aphid. Plant RNA viruses pose a threat due to their high mutation rate and the ability to adapt rapidly to a changing environment. To effectively manage PLRV infection, detection of virus infection in seed potatoes is paramount. In this study, five field trials were carried out in potato production fields, to compare the commonly used DAS-ELISA with RT-PCR for PLRV detection. From the results obtained it was concluded that DAS-ELISA detection greatly underestimates the number of infected samples when compared to RT-PCR. The hotter climate of the Sandveld region appears to inhibit PLRV accumulation in infected plants and these infections then remain undetected by DAS-ELISA. PLRV isolates were sequenced and phylogenetic and bioinformatic analyses were performed to identify and characterise local variants of PLRV. PLRV isolates found in the Sandveld region were closely related to PLRV isolates from Australia. Some of these isolates had recombined with variants commonly found in Europe, Asia and the Americas as well as with those similar to PLRV isolates from Peru and Germany. Three locally produced cultivars were graft-inoculated with two PLRV isolates that represent the two main variant groups found to assess symptom development and yield reduction. Symptom development in locally produced cultivars was typical for PLRV. A yield loss resulted from this infection with no difference between the Australian type and the European/Australian recombinant type. The proteins produced by the newly sequenced isolates were further analysed in comparison to other isolates found worldwide. The variation in the proteins produced by the newly sequenced isolates was mainly due to recombination between distinct groups of PLRV in the 5’ half of the genome and through mutation in the 3’region of the genome. A differential RT-PCR was designed to distinguish, in a single reaction, between the Australian type and the European/Australian recombinant type of PLRV. This revealed that simultaneous infections with both types occurred commonly, and could explain why recombination has occurred.
Cloning and identification of genes involved in the interaction between the bacterial stone fruit pathogen Pseudomonas syringae pv. syringae strain NV and plum trees
(Stellenbosch : Stellenbosch University, 2001-03) Appel, Maryke; Bellstedt, D. U.; Mansvelt, E. L.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Bacterial canker of stone fruit, caused by Pseudomonas syringae pv. syringae, is one of the most destructive crop diseases in South Africa. Chemical control has failed completely and effective long-term management strategies will have to rely on the breeding of resistant host trees. To assist in such breeding programmes, investigations into the molecular basis of the interaction between P. s. pv. syringae and stone fruit trees have been undertaken in collaboration with the ARC-Fruit, Wine and Vine Research Institute in Stellenbosch. The aim of this dissertation was to clone and identify genes that are involved in interaction between the bacterial canker pathogen and stone fruit trees. In the first part of the study, the harpin encoding gene of a local strain of the pathogen, P. s. pv. syringae NV, was amplified in a polymerase chain reaction (PCR) strategy with primers based on the hrpAZB sequences of the bean pathogen, P. s. pv. syringae 61. Sequencing of this hrpZpssNvgene revealed a high degree of homology (96%) between the harpin encoding genes and harpin proteins of the two strains. The hrpZpssNvgene was subsequently cloned into the pMAL-c2 expression vector and expressed in Escherichia co/i. This system was used for the production of purified, biologically active, recombinant HrpZpSSNV protein. In the second part of the study, differential display (DD) technology was used to identify genes that are induced in stone fruit trees in response to P. s. pv. syringae and/or its harpin elicitor. For this purpose, actively growing shoots of two Prunus sa/icina cultivars, the moderately resistant cv. 'Laetitia' and the highly susceptible cv. 'Songold' were treated with recombinant harpinpssNvprotein or live P. s. pv. syringae NV bacteria. An untreated control and wounding control was included in the experiment. Total RNA was isolated for comparative mRNA analysis 24 hours after treatment. DD profiles were generated with fifteen primer combinations. Eight candidate bands were re-amplified, cloned and sequenced. Reverse transcription PCR was employed to verify the expression patterns of the cloned bands in the original RNA sample set. Two bands, DDc and DD4 were shown to be differentially expressed between treatments and/or cultivars, while no differences in the expression levels of the remaining six bands (DDa, DDe, DD3, DD5, DD6 and DD7) were observed. BLAST similarity searches yielded significant matches for DDe, DD4 and DD7 with plant defense-related genes.
The development of a DNA vaccine against Mycoplasma nasistruthionis sp. nov. for use in ostriches
(Stellenbosch : Stellenbosch University, 2015-12) Wium, Martha; Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Mycoplasma nasistruthionis sp. nov. str. Ms03 (Ms03) is one of three Mycoplasma species that were identified from ostriches. Mycoplasmas infections have been implicated in ostrich chick mortalities, growth retardation and downgrading of ostrich carcasses. Currently there is no vaccine available for the treatment of mycoplasmosis in ostriches. This study investigated the development of DNA vaccines against Ms03 infections in ostriches. To this end, the Ms03 genome was sequenced and annotated. The vaccine candidate gene, oppA, was identified within the genome sequence and characterized before DNA vaccines containing the oppA were developed and tested. The genome of Ms03 was sequenced and the resulting 172 contigs were annotated. This dissertation presents the first Ms03 draft genome and annotation which contributed to the understanding of Ms03 as a miniature genetically independent organism. In Ms03, genome replication, cell division, RNA transcription, protein translation and glycolysis resemble that of the closely related Mycoplasma synoviae 53. Purine and pyrimidine metabolism was incomplete and de novo synthesis thereof was not possible. Amino acid synthesis in Ms03 was mostly absent and only the genes that convert aspartate to asparagine and glycine to serine were found. More importers than exporters were annotated owing to the lack of synthesis pathways in Ms03, which is typical for mycoplasmas that have parasitic life styles. Two oligopeptide permease (opp) operons were annotated within the Ms03 genome. The potential of the oppA as a vaccine candidate gene was evaluated by investigating the need for a substrate-binding domain (OppA) as part of the OppBCDF transporter within Mycoplasma species. An oppA homologue could be identified for each oppBCDF operon in all species and therefore must play an essential role in oligopeptide transport. All mycoplasmas (except for hemoplasma) had one, two or three opp operons that could be divided into three types (Type A, B and C). Each type had unique InterPro and MEME domains and motifs which together with the phylogenetic analysis suggest unique roles in their survival under different conditions. Ms03 had a Type A and a Type B opp operon, the Type A oppA was used as vaccine candidate gene. The Type A oppA was cloned and site-directed mutagenesis was used for codon correction before the mutated gene was sub-cloned into three DNA vaccine vectors. The three DNA vaccines (pCI-neo_oppA, VR1012_oppA and VR1020_oppA) were used to vaccinate ostriches and the OppA-antibody response was analysed by ELISA. The VR1020_oppA and pCI-neo_oppA constructs elicited a primary immune response in ostriches, indicating that the OppA protein was expressed in vivo and was immunogenic. This can therefore be viewed as the first step in the development of a DNA vaccine for the control of mycoplasma infections in ostriches.
Immunological and epidemiological investigations in South African ostriches and penguins
(Stellenbosch : Stellenbosch University, 2004-04) Botes, Annelise; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry
ENGLISH ABSTRACT: Newcastle disease (NO) and mycoplasma infections in ostriches have considerable economic implications for the South African ostrich industry in that NO is a limiting factor in the export of ostrich products to the European Union and mycoplasma infections cause stock losses, reduced production, reduced hatchability and downgrading of carcasses. In the first section of this dissertation, the role of passively acquired and mucosal immunity in protection of ostrich chicks against Newcastle disease virus (NOV) was investigated. Ostrich hen serum IgG and yolk IgY were isolated and characterized, and the transfer of maternal anti-NOV antibodies to the egg yolk was determined using an enzyme-linked immunosorbent assay (ELISA). Results indicated that anti-NOV antibodies were successfully transferred from the ostrich hen to the egg yolk. In addition, ostrich IgA was isolated, characterized and rabbit anti-ostrich IgA antibodies produced and used for measuring mucosal anti- NOV IgA antibodies produced in response to mucosal vaccination. Results indicated that the live La Sota vaccine stimulates IgA production and thus mucosal immunity in ostrich chicks. In the second section of this dissertation, ostrich mycoplasmas were isolated and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches carry three unique mycoplasmas, which are phylogenetically quite divergent. The 16S rRNA gene sequences of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches by PCR. The last section of this dissertation focuses on avian malaria in African penguins and the management of this disease during rehabilitation. The Foundation for the Conservation of Coastal Birds (SANCCOB) is a seabird rescue and rehabilitation centre, which is largely dedicated to the rehabilitation of diseased, injured and oiled penguins. Significant mortalities due to avian malaria occur at this facility. The aim of this study was the development of an ELISA for the purpose of assessing the natural levels of anti-Plasmodium antibodies in African penguins on entry into the SANCCOB facility and during rehabilitation. Results indicated significant increases in anti- Plasmodium antibody levels after entry, which was not influenced by oiling. Infection with malaria and not parasite recrudescence was viewed to be the cause of this increase, indicating a possible role of the SANCCOB facility in exposing penguins to avian malaria.
Isolation and characterisation of a polygalacturonase-inhibiting protein (PGIP) and its encoding gene from Vitis vinifera L.
(Stellenbosch : Stellenbosch University, 2001-12) De Ascensao, Ana; Pretorius, I. S.; Vivier, Melane A.; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Polygalacturonase-inhibiting proteins (PGIPs) are present in the cell walls of a variety of plant species. These proteins have been shown to specifically inhibit endopolygalacturonases (endo-PGs) secreted by invading fungal pathogens as part of the induced disease resistance mechanism of plants. This is the first report on the isolation and characterisation of a pgip gene from Vitis vinifera L., designated grapevine pgip1. A single open reading frame encoding a deduced polypeptide of 333 amino acids with a predicted molecular mass of 37.1 kOa and a calculated isoelectric point of 8.61 was identified from a 5.6 kb subgenomic fragment of V. vinifera cv Pinotage. Nucleotide and derived amino acid sequence analysis of grapevine pgip1 showed significant homology with other characterised PGIP encoding genes and revealed features characteristic of PGIPs found in several other plant families. Genomic DNA analysis showed that grapevine pgip1 belongs to a small multigene family in Vitis cultivars. From Northern blot analysis it was evident that expression of the PGIP family is both tissue- and developmental stage specific. The grapevine pgip1 was transiently expressed in Nicotiana benthamiana L. with potato virus X (PVX) as a vector. Grapevine PGIP1 isolated from crude protein extracts of PVX-infected N. benthamiana were tested and showed inhibitory activity against polygalacturonases (PGs) from Botrytis cinerea. Grapevine PGIPs have not previously been purified and characterised. Molecular analyses have confirmed that PGIPs are typically encoded by multigene families and that the inhibitor specificities and kinetics of the isolated proteins differ within and among species. In this study, two PGIP isomers from V. vinifera berries were isolated. The one isomer, designated PGIP-A, was partially purified and had a molecular mass of 39 kOa, whereas the other PGIP, designated PGIP-B, was purified and had a molecular mass of 42 kOa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) and Western blot analysis. Both proteins were cell wall-bound. Enzymatic deglycosylation confirmed that PGIP-B is a glycosylated protein. Grapevine PGIP-A showed strong inhibitory activity against a homogeneous PG from Aspergillus niger and to a lesser extent against PG from Fusarium moniliforme, but was unable to interact with a crude PG preparation from B. cinerea. Grapevine PGIP-B was able to strongly inhibit PGs from B. cinerea as well as from Colletotrichum gleosporoides, yet showed no inhibition towards PG from A. niger. The grapevine pgip1 gene was expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in tobacco plants via Agrobacterium tumefaciensmediated transformation. Transgenic tobacco plants expressing the grapevine PGIP (gPGIP1) were used to demonstrate the effectiveness of this inhibitor against fungal PGs and to investigate whether gPGIP1 influences disease development. Northern blot analysis identified 19 transgenic plants expressing pgip1 transcript levels. Crude PGIP extracts from the transgenic tobacco plants inhibited PGs from B. cinerea and C. gleosporoides, but not PG from A. niger. Leaves from untransformed tobacco plants, from transgenic tobacco lines showing high and low PG inhibition, and from transgenic plants that did not express pgip1, were inoculated with B. cinerea. Transgenic leaves showed a reduction in the size of necrotic lesions of macerated tissues of approximately 45% relative to control and non-expressing transgenic leaves. The results from the heterologous expression of gPGIP1, together with the results from the protein purifications and inhibition studies, indicate that the isolated grapevine pgip1 gene encodes the isolated PGIP-B isomer. This work has ; established a good model system to study certain aspects of plant-pathogen interactions in grapevine. Heterologous expression of gPGIP1 has demonstrated that PGIP inhibition of fungal PGs slows disease development of B. cinerea in planta.
Molecular phylogenetic relationships within the subtribe Disinae (Orchidaceae) and their taxonomic, phytogeographic and evolutionary implications
(Stellenbosch : Stellenbosch University, 2007-03) Bytebier, Benny (Benny Leopold Germaine); Bellstedt, D. U.; Linder, H. Peter; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Twenty five years after the last major morphological revision, phylogenetic relationships were inferred on the basis of a new DNA dataset for the African orchid subtribe Disinae, which includes the large genus Disa and the small genus Schizodium. One nuclear gene region (ITS) and two plastid gene regions (trnLF and matK) were sequenced for 136 ingroup, representing 70% of all known Disinae species, as well as for 7 outgroup taxa. The combined data matrix contained 4094 characters and was analysed using parsimony and Bayesian inference. The generic status of Schizodium can no longer be supported, as it is deeply embedded within the genus Disa. Furthermore, the currently recognised subgenera do not reflect the phylogenetic relationships. Several of the currently recognised sections are monophyletic, others contain misplaced elements, while some are polyphyletic. These results necessitate a re-classification of the Disinae. A monotypic subtribe Disinae and a subdvision of Disa into eighteen sections is formally proposed. These sections are monophyletic, well-supported, morphologically distinguishable and are delimited to maximize the congruence with the previous classification. All currently known species are enumerated and assigned to sections. Likelihood optimisation onto a dated molecular phylogeny is subsequently used to explore the historical biogeography of Disa, as well as of three other Cape lineages (Irideae p.p., the Pentaschistis clade and Restionaceae), to find out where these lineages originated and how they spread through the Afrotemperate region. Three hypotheses have been proposed: (i) a tropical origin with a southward migration towards the Cape; (ii) a Cape origin with a northward migration into tropical Africa and (iii) vicariance. None of these hypotheses, however, has been thoroughly tested. In all cases, tropical taxa are nested within a predominantly Cape clade and there is unidirectional migration from the Cape into the Drakensberg and from there northwards into tropical Africa. Dating estimates show that the migration into tropical East Africa has occurred in the last 17 million years, consistent with the Mio-Pliocene formation of the mountains in this area. The same technique is then utilised to reconstruct the temporal occurrence of ancestral ecological attributes of the genus Disa. The first appearance of species in the grassland and savanna biomes, as well as in the subalpine habitat, are in agreement with the existing, reliable geological and paleontological information. This suggests that phylogenies can be used to date events for which other information is lacking or inconclusive, such as the age of the fynbos biome and the start of the winter rainfall regime in southern Africa. The results indicate that these are much older than what is currently accepted and date back to at least the Oligocene.
Phylogenetics of the genus Erica and anthocyanin synthesis gene expression in Erica plukenetii
(Stellenbosch : Stellenbosch University, 2017-03) Le Maitre, Nicholas; Bellstedt, D. U.; Pirie, M. D.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: The drivers of species radiations are central to questions about the evolution of diversity. The flora of the Cape Floristic Region (CFR) is particularly diverse, has exceptionally high level of endemism and may have radiated at an exceptionally high rate. Various drivers of this radiation have been proposed, including climate change, fire, niche adaptation, persistence of lineages and shifts in pollination syndrome. Erica is the largest genus in the CFR but its radiation has not been well studied phylogenetically. A multiple marker phylogeny would be significant in establishing its radiation rate and further elucidating the role and importance that factors such as biogeography and pollinator shifts, have played in driving its radiation specifically and in the CFR flora in general. Floral colour shifts between red and white flowers have been shown to be important in switches between pollination syndromes. The anthocyanin pathway produces coloured anthocyanins that colour the flowers of plants, also in Erica. A multiple chloroplast and ITS marker region phylogeny was constructed for 597 accessions. Automated and manual alignment strategies were used to generate phylogenies and found to not be significantly different. Overall the phylogeny showed African species are descended from European species and that Mascarean and Drakensberg species may share a common ancestor with Cape species. A single Cape clade is present, sister to one anomalous species, and the sub clades reveal structure primarily related to biogeography and not morphology. Both flower colour and pollination syndrome are highly labile and multiple switches have occurred between anemophily, entomophily and ornithophily. Red flowers and ornithophily have evolved independently on at least 14 occasions. In red flowered Erica plukenetii whole genome sequencing approaches using Illumina NGS sequencing were used to obtain sequences of the anthocyanin pathway genes and their trans-acting regulatory genes. RT-PCR and RT-qPCR were used to measure the expression of these genes in two populations of red-, pink- and white-flowered E. plukenetii. Expression of the CHS and the ANS genes were found to be reduced in white flowers in these populations respectively. Sequencing of the promoter regions of these genes in red-, pink- and white-flowered plants revealed mutations in the promoter binding sites of the white flowered plants that likely are the cause of anthocyanin synthesis enzyme gene down regulation and consequent loss of flower colour. Biogeographical factors and shifts between pollination syndromes that potentially result from changes in red anthocyanin synthesis contributed to the loss of anthocyanin production are therefore likely important drivers of the radiation of Erica in the CFR.
A study of the strain evolution and recombination of South African isolates of Potato virus Y
(Stellenbosch : Stellenbosch University, 2012-12) Visser, Johan Christiaan; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry. The incidence of this virus has greatly increased over the past 20 years. In previous studies nonrecombinant strains of PVY, PVY N and PVY O, were detected in South African potatoes. In a recent study the occurrence of non-recombinant strains of PVY in South African potatoes was shown to have decreased while infection by more virulent recombinant strains, PVY NTN and PVY N-W, had increased dramatically. Infection of potato plants with PVY may cause stunted growth and mosaic or necrotic leaf symptoms which in turn can lead to a significant reduction in yield. Highly virulent recombinant PVY isolates as well as some of the non-recombinant strains may cause potato tuber necrotic ringspot disease (PTNRD) which may result in losses of 10% to total crop failure. For this reason investigation of infection by local recombinant isolates on local cultivars was important. To this end a representative number of isolates were selected for whole genome sequencing based on the relative occurrence of the various isolates in South Africa. A number of these sequenced isolates were subsequently used to infect local cultivars of potato in order to investigate the influence of genetic variation within the viral genome on symptom expression. In this study 27 South African isolates of PVY were sequenced through overlapping RT-PCR fragments. Seven of these isolates, six PVY NTN and one PVY N-W, were used to mechanically infect four local cultivars of potatoes under greenhouse conditions. The infected plants were monitored to establish the rate of systemic spread using a highly sensitive qRT-PCR and resulting tubers were visually screened for PTNRD. Highly variable recombinant isolates appear to be less virulent than the more conserved recombinant isolates possibly indicating molecular determinants for pathogenicity. For this reason the amino acid sequences of the South African isolates were compared to those of international isolates and scrutinized for variation and substitutions. Some South African isolates displayed amino acid substitutions unique to the specific isolate, making them unlike those found internationally. Substitution rates throughout the amino acid sequences differed greatly, with some isolates displaying hardly any changes whilst others varied a great deal from overseas isolates. Certain regions, many of which had specific functions, were more conserved than others. This study further investigated the recombination events within the PVY genome using reticulate phylogenetic analysis, molecular dating and network construction techniques. Unlike existing approaches, the one described in this study neither assumes an underlying strictly bifurcating species tree nor assumes prior knowledge of processes underlying deviations between individual gene trees. Through the use of the resulting robust time calibrated phylogeny, the patterns of diversification and recombination in PVY may be placed in the historical context of human cultivation of potatoes. Through the use of these techniques the study aimed to test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. From these analyses it can be deduced that recombinant strains of PVY were imported into South Africa.

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Browsing Doctoral Degrees (Biochemistry) by browse.metadata.advisor "Bellstedt, D. U."

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