Masters Degrees (Genetics)
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Browsing Masters Degrees (Genetics) by browse.metadata.advisor "Burger, Johan T."
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- ItemThe characterisation and partial sequencing of the grapevine chloroplast genome(Stellenbosch : Stellenbosch University, 2004-03) Rose, B. A. (Beverley Ann); Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome.
- ItemCharacterisation of microRNA expression profiles of Vitis vinifera in response to grapevine leafroll-associated virus 3 infection(Stellenbosch : Stellenbosch University, 2017., 2017-03) Aldrich, Dirk Jacobus; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.ENGLISH ABSTRACT: Grapevine leafroll disease (GLD) is endemic to all grape-growing regions of the world and is considered the most significant grapevine viral disease. Grapevine leafroll-associated virus 3 (GLRaV-3) is considered the primary cause of GLD and in South African vineyards five genetic variant groups (I, II, III, VI and VII) have been confirmed. Small RNAs (sRNAs) have been shown to play a significant role in a plant’s response to biotic and abiotic stress. This has led to a growing interest in evaluating sRNAs, such as microRNAs (miRNAs), for their role in mediating gene regulation in response to virus infections. In this study, stem-loop RT-qPCR probe-based assays were utilised for miRNA quantitation in GLRaV-3 positive and negative grapevines. A set of own-rooted Cabernet Sauvignon plants representing GLRaV-3 variant groups I, II, III and VI has been established from cuttings of highly symptomatic GLRaV-3 infections found in commercial vineyards. These plants were sampled and screened to yield the first data set. Additionally, young Cabernet Sauvignon plants were established and graft-inoculated with single infections of the five known variants of GLRaV-3 found in South African vineyards. All these plants were maintained in a climate-controlled greenhouse and sampled twice, six months apart, to yield two data sets. A fourth data set comprised of GLRaV-3 positive and negative Cabernet Sauvignon plants sampled from various vineyards in Stellenbosch. Eleven miRNAs were quantified in both infected and healthy grapevine samples. Putative miRNA targets were predicted and annotated using in silico analyses. These targets were subsequently quantified in both greenhouse and field samples using a SYBR Green RT-qPCR assay. This study validated statistically significant differences in virus concentrations, expressed as virus concentration ratios (VCRs), in plants singly infected with different GLRaV-3 variants. Interestingly, no difference in mean VCRs were observed between data sets, despite notable differences in plant age, duration of GLRaV-3 infection, scion/rootstock combination and growing conditions. Several miRNAs showed statistically significant expression modulation between infected and healthy samples. miRNA expression between data sets varied substantially and a greater miRNA/target response was observed in plants with more established GLRaV-3 infections. The lack of significant differences in mean VCRs between data sets, coupled with the consistent modulation of certain miRNAs in plants that have likely been infected for longer is a promising result. This finding could indicate that successful inhibition of further virus replication by plant defence mechanisms occurred and that these miRNAs and their targets are implicated in this response. The predicted targets for these miRNAs are genes involved in disease resistance, apoplastic processes, oxidation-reduction processes and growth and developmental processes. Additionally, possible variant-specific miRNA responses to infection were observed across all data sets, which could aid in elucidating possible biological differences between variants of GLRaV-3.
- ItemThe characterisation of selected grapevine cultivars using microsatellites(Stellenbosch : Stellenbosch University, 2002) Ross-Adams, Helen Esther; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics and IPB.ENGLISH ABSTRACT: Grapevine supports one of the oldest industries in South Africa today, and is also of significant international importance. With increasing international trade and the transport of fruit and other grapevine-derived products between borders, it has become increasingly important for South African farmers and viticulturalists to ensure their products conform to strict international market requirements if they are to remain competitive. Such requirements include the correct and accurate identification of berries and wines according to cultivar. In light of this, 26 different wine, table grape and rootstock cultivars, as well as a number of clones from KWV's core germplasm collection were characterised at 16 microsatellite marker loci. Microsatellite markers are known for their high level of informativeness, reliability and reproducibility, and are widely used in the identification and characterisation of plant varieties, population analyses and forensic applications. Unique allelic profiles were obtained for all but two plants, which proved to be identical at all loci considered, and thus 'clones'. These profiles were collated to form a database, containing the DNA fingerprints of each sample at each locus. The relative levels of informativeness of each marker used were also determined, and compared with those found in the literature. Six markers proved to be highly informative, and are promising in the potential application of this technology to other cultivars. The applicability of microsatellite markers to such studies is confirmed; this approach could easily be extended to include any number of cultivars of national and international interest. The results of such an investigation would have important implications for both the farming and commercial industries alike.
- ItemCharacterising the viral and microbial diversity in old and young grapevines(Stellenbosch : Stellenbosch University, 2017-12) Oosthuizen, Kristin; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: There is anecdotal evidence suggesting that old vines produce wines of higher quality than young vines. In South Africa, vines are generally regarded ‘old’ when they reach 35 years of age, while ‘young’ vines are less than ten years old. Grapevines are susceptible to a large spectrum of pathogens that have negative impacts on grape quality and yield. This crop is also colonised by diverse endophytic microorganisms that play an important role in plant growth, health and productivity. To date, limited molecular research has been performed to determine the complexity of the pathogenic and endophytic communities in old vines. This study aimed to characterise the viral and fungal profiles of old and young Pinotage grapevines, using next-generation sequencing in a metagenomics approach. To determine the viral diversity, double-stranded RNA was extracted from phloem to enrich for virus-specific nucleic acids, and sequenced on an Illumina platform. High-quality reads were assembled into contigs and classified through BLAST analysis against the NCBI database. Additionally, the reads were mapped to a database consisting of known grapevine virus and viroid genome sequences. Reverse-transcription PCR detection assays were performed to validate the presence of the identified viruses. The fungal communities were characterised by extracting total DNA from the vascular tissues of the cane, followed by amplification of the ITS2 region, and deep amplicon sequencing. The ITS2 sequences were clustered into operational taxonomic units at a 97% identity threshold and taxonomically classified through BLAST analysis against the UNITE database. Viruses of the families Closteroviridae, Betaflexiviridae and Tymoviridae, and four pospiviroids were detected. The virus community was more diverse in the old vines, with 31 and 16 virus variants detected in the old and young vines, respectively. This was expected, since old vines have been exposed to viral pathogens for a longer period. The economically important grapevine leafroll-associated virus 3 was the most abundant species present in the samples, consistent with previous surveys of vineyards in the Western Cape. Grapevine Syrah virus 1, and possibly grapevine rupestris vein feathering virus, was identified for the first time in South African grapevines, expanding the global distribution of the virus(es). The amplicon data revealed the presence of different filamentous and yeast-like fungal taxa commonly associated with grapevines, including species of Alternaria, Aureobasidium, Cladosporium and Epicoccum. Several pathogens of grapevine trunk diseases and postharvest rot, and endophytic species with biocontrol properties were detected. The young-vine sample group showed greater fungal diversity, as determined by three alpha diversity metrics, although not statistically significant. It may be speculated that the fungal community of old vines is more accustomed to the environment, and therefore less diverse. No differences were observed between the old and young vines, with regards to the community composition. The data generated in this study has contributed to research on the complex viral and fungal communities inhabiting old vines.
- ItemConstruction of a cDNA library for the vine mealybug, Planococcus ficus (Signoret)(Stellenbosch : Stellenbosch University, 2008-12) Holm, Kora; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.The vine mealybug, Planococcus ficus (Signoret), is a severe pest of grapevine in many grape and wine producing countries around the world. It is renowned not only for the considerable damage it infers to grapevine of its own accord, but in particular for its role in transmitting deleterious viral diseases such as grapevine leafroll disease, Kober stem grooving, Shiraz disease and corky bark. Incidentally, it is an exceptionally tenacious antagonist of grapevine, being resistant to both chemical and biological control mechanisms. As a result, finding an effective strategy for P. ficus control has become a main priority of viticultural industries worldwide. Possible implementation of biotechnological approaches to pest management has resulted in a need for P. ficus genetic data - of which there are currently very little available. The transcribed genes of an organism can be captured in a cDNA library, and the sequences of the various transcripts can then be characterized. In this study altogether five cDNA libraries were constructed from the transcribed sequences of Planococcus ficus (Signoret). Instrumental to their construction was the identification of an RNA extraction protocol that provided large quantities of high quality RNA from mealybugs. The five cDNA libraries were the result of a set of modifications to the Creator™ SMART™ cDNA Library Construction Kit (used for Primary Library construction), and differed mainly with regards to range of insert sizes they contain. Whereas an abundance of short fragments were found in the Primary Library (42% of screened inserts 60.5 kb, and 20% >1 kb), the Fractionated Libraries contained inserts of specific size ranges that were more-or-less equally represented. The broadest size range was found in Fractionated Library 4, for which a uniform distribution over the range 0.25 kb - 4 kb was observed. Average insert sizes of Fractionated Libraries 1 to 4 were estimated at 0.25 kb, 0.5 kb, 1 kb and 2 kb respectively. These results demonstrated the importance of using a protocol designed to circumvent the bias towards incorporation of shorter transcripts in cDNA libraries. Although the libraries were not exhaustively analyzed, the outcome of a pilot investigation indicated that 41% of the submitted sequences had matches in the non-redundant database of the National Center for Biotechnology Information (NCBI, E-value 6 10-5), and that approximately 82% of these were of insect origin. Moreover, two potential targets for an RNAi-mediated approach to P. ficus pest control were identified. With one exception, these sequences seemed to be unique to arthropods. Future research needs to investigate the efficiency by which these sequences are able to constrain P. ficus proliferation, and their suitability for grapevine transformation.
- ItemThe construction of an expression vector for the transformation of the grape chloroplast genome(Stellenbosch : Stellenbosch University, 2003-12) Robson, Julia; Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species.
- ItemThe construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines(Stellenbosch : Stellenbosch University, 2004-12) Van Eeden, C. (Christiaan); Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
- ItemThe determination of the spatial and temporal distribution of Aster Yellows phytoplasma in grapevine(Stellenbosch : Stellenbosch University, 2015-04) Smyth, Natalie; Burger, Johan T.; Souza Richards, Rosineide; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: South Africa is ranked amongst the top ten for wine production internationally. Viticulture contributes immensely to the economy, which justifies research into the pathogens that may negatively affect wine production. Aster Yellows phytoplasma was reported in South African vineyards in 2010 and has since been an ongoing problem for grape farmers in affected areas. Throughout the world, phytoplasma diseases such as Grapevine Yellows have caused detrimental effects on the vines, often resulting in death. The limited knowledge on prevention and control of the pathogen can be attributed to the lack of full understanding of the epidemiology and accurate diagnosis. The aim of this study was to determine the spatial distribution of Aster Yellows phytoplasma in individual grapevines and to record a possible temporal or seasonal distribution. The recovery phenotype phenomenon was encountered during the study and surveys were conducted in order to determine whether recovery was permanent. In order to perform the studies, a reliable assay to accurately detect the pathogen in grapevines was required. A comparison between three assays was completed in furtherance of deciding which to use for the further experimentation. The three assays included a nested PCR utilizing universal primers, a Real-Time PCR using Syto9 as a double stranded DNA specific dye and a Real- Time PCR with a TaqMan® probe using an identical dilution series. Of the three assays tested, the nested PCR proved to be the most sensitive diagnostic procedure, detecting Aster Yellows phytoplasma in very low titers and was thus used for diagnostics in further experiments. In order to determine the spatial patterns of Aster yellows phytoplasma infection, leaf, petiole, trunk, root and cane samples were taken from three whole grapevine plants. Phloem scrapings obtained from the cane samples yielded more positive results in comparison to the other parts of the plant tested. Not only do phytoplasmas display an erratic spatial distribution, but also have a tendency to change over time. Thirty symptomatic grapevines were sampled over one and a half growing seasons, with results concluding that February yielded the most positive diagnoses. Fifty plants that had been previously pruned back and no longer displayed symptoms were also sampled in 2013 and 2014, and all yielded negative results over both years. This study contributes to comprehension of Aster Yellows phytoplasma epidemiology and ultimately the advancement of accurate diagnosis.
- ItemDetermination of the virus diversity associated with Grapevine leafroll disease(Stellenbosch : Stellenbosch University, 2015-04) Molenaar, Nicholas; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Vitis vinifera is the woody crop most susceptible to intracellular pathogens. Currently 70 pathogens infect grapevine, of which 63 are of viral origin. Grapevine leafroll-associated virus 3 (GLRaV-3) is the type species of the genus Ampelovirus, family Closteroviridae. It is considered to be the primary causative agent of Grapevine leafroll disease (GLD) globally; however, the etiology of GLD is not completely understood. Here we report on the viral populations present in GLD symptomatic grapevines across the Western Cape province, South Africa. A widespread survey was performed to screen 315 grapevines for 11 grapevine-infecting viruses using RT-PCR. Additionally, GLRaV-3 variant groups were distinguished with high-resolution melt (HRM) curve analysis used in conjunction with real-time RT-PCR. Members of the family Closteroviridae were detected with the highest frequency, particularly GLRaV-3 that was detected in 87% of tested plants. Nextgeneration sequencing (NGS) is capable of detecting known and novel viruses without prior knowledge of viral sequences and when used in a metagenomic approach is able to detected viral populations within diseased vines. A total of 17 grapevine samples were subjected to NGS using either an Illumina MiSeq or HiSeq 2500 instrument to determine the virome within GLD vines. Collectively, more than 190 million reads were generated through NGS. Read datasets were trimmed and filtered for quality and subjected to both read-mapping and de novo assembly. Contigs assembled de novo were analyzed with BLAST (Basic Local Alignment Search Tool) against the NCBI (National Centre for Biotechnology Information) database and it was determined that GLRaV-3 was the best-represented virus, comprising 97.5% of the assembled contigs. Grapevine virus F (GVF) was detected for the first time in South African vineyards through de novo assemblies and the complete genome sequence validated through direct Sanger sequencing. The complete genome of GVF isolate V5 spans 7 539 nucleotides and shares 89.11% nucleotide identity to existing GVF genomes. The data generated through this study will assist in further understanding the etiology of GLD, support the current hypothesis of GLRaV-3 as the primary contributor to GLD, aid in understanding virus associations in diseased vines and potentially develop systems in which to control disease spread and symptom severity.
- ItemThe development of a diagnostic assay for nepoviruses in grapevine(Stellenbosch : Stellenbosch University, 2015-12) Frazenburg, Lolita; Burger, Johan T.; Souza Richards, Rosineide; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV.
- ItemDevelopment of alternative diagnostic protocols for Candidatus Phytoplasma asteris and Coniella granati Sacc. (Syn. Pilidiella granati)(Stellenbosch : Stellenbosch University, 2019-04) Page, Lucan Dylan; Burger, Johan T.; Linus Opara, Umezuruike Linus; Perold, Willem; Maree, H. J.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: The Republic of South Africa (RSA) is an integral part of the global fruit exporting chain. Currently, South Africa is ranked eighth in the world in wine production, exporting 40% of all locally produced wine. Another emerging fruit industry is pomegranates, of which RSA currently ranks fourth in the Southern hemisphere, exporting 88% of its pomegranate produce. However, pathogens affect the yield of these industries, warranting further research. Two of these pathogens are Candidatus Phytoplasma asteris (AY phytoplasma), a phytoplasma that infects grapevine, and Coniella granati, a fungus that infects pomegranates. AY phytoplasma was first reported in RSA in 2010 and C. granati was first reported in RSA in 2017. Currently, methods used for the detection of both pathogens rely on time-consuming nested-PCR assays that require trained technicians and equipment such as thermocyclers. The aim of this project was to develop a functional diagnostic method for the rapid detection of AY phytoplasma and C. granati. This project also aimed to compare current diagnostic assays to a new isothermal diagnostic assay, namely recombinase polymerase amplification (RPA). Additionally, this project in collaboration with the department of Electronic and Electrical Engineering at Stellenbosch University is developing a microfluidic device for detection of these fruit pathogens, based on the RPA assay. Isothermal RPA diagnostic assays for both AY phytoplasma and C. granati worked rapidly, effectively decreasing the time required to determine results. Comparisons between PCR and RPA diagnostic assays determined that PCRs were slightly more sensitive; however, the RPA was much faster. In situ tests of disease symptomology of pomegranate fruits replicated results found in literature. The biological groundwork for the microfluidic device was also laid; this was done by means of specificity tests, using biotinylated capture probes and streptavidin-coated magnetic beads. This study advances the understanding of modern diagnostic assays compared to traditional diagnostic assays, reporting effective detection of plant pathogens in a short time. Overall, the RPA diagnostic was faster at detecting C. granati than the PCR, saving an estimated hour from start to finish, while the AY RPA successfully detected AY phytoplasma in an hour and twenty minutes compared with ten hours using the AY nested-PCR assay.
- ItemThe efficacy of the antimicrobial peptides D4E1, VvAMP-1 and Snakin1 against the grapevine pathogen aster yellows phytoplasma(Stellenbosch : Stellenbosch University, 2013-03) Spinas, Nicole Lotte; Burger, Johan T.; Stephan, Dirk; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression. The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv "Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system. In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a "recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups. This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine.
- ItemEngineering Brome mosaic virus as a potential drug delivery nanoparticle for prostate cancer(Stellenbosch : Stellenbosch University, 2017-03) Lee, Nadine; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.ENGLISH ABSTRACT: Cancer is a leading cause of annual mortality worldwide. Prostate cancer is the second most prevalent type of cancer after breast cancer. The conventional treatment options that are currently available are not optimal due to their non-specificity as well as treatment often failing. Recent advances have turned to nanotechnology as the future of cancer therapy, and viral nanoparticles (VNPs) in particular are promising delivery vehicles. VNPs provide a protein scaffold that is relatively easy to modify while being biodegradable. Plant viruses specifically can be purified with ease at high concentrations and are safe for use in humans. Brome mosaic virus (BMV) is an icosahedral virus selected for this study due to its stability under a range of experimental conditions, such as pH and temperature, and its robustness in chemical conjugation experiments. BMV was also selected for the availability of modifiable amino acids on its exterior surface. The goal of this project was to engineer BMV as a potential delivery nanoparticle for prostate cancer treatments. Wild type BMV was purified from Nicotiana benthamiana and particles quantified using transmission electron microscopy as well as dot blots. The virus particles were modified by conjugation of two fluorescent molecules, Alexa Fluor-647 and Cy5, to the glutamic acid residues on the exterior surface of the BMV capsid. Two peptides, PKRGFQD-C and SNTRVAP-C, were conjugated to the solvent-exposed lysine residues using three SM(PEG)n crosslinkers of different lengths. These peptides respectively target the receptors α-2-macroglobulin and GRP78, which are found on the cell surface of androgen-independent prostate cells. The SM(PEG)24 crosslinker could successfully conjugate the peptides. When the fluorescent labeling was performed first, the peptide conjugation was unsuccessful. As an alternative, the fluorescent molecule and peptides were both conjugated to the lysine residues. The VNPs were assessed in normal and cancerous prostate cell lines for non-specific and targeted uptake. This was assessed using fluorescence microscopy and flow cytometry. The uptake of the VNPs was 75% for PKRGFQD-C and 95% for SNTRVAP-C in the PC3 cell line, which is indicative of late-stage androgen-independent cancer. The uptake in VCaP (early stage androgen-dependent cancer) was lower than for PNT2 (normal prostate cells). We consider these results positive as the VNPs will most likely target the androgen-independent cells. This study demonstrated that BMV, as a candidate VNP, can successfully be modified with a fluorescent molecule and targeting peptide in order to specifically target prostate cancer cells.
- ItemAn evaluation of the efficacy of antimicrobial peptides against grapevine pathogens(Stellenbosch : University of Stellenbosch, 2011-03) Visser, Marike; Burger, Johan T.; Stephan, D.; University of Stellenbosch. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: This study investigated the use of antimicrobial peptides (AMPs) as possible source of resistance against a range of pathogens in grapevine. Whilst the ultimate aim would be to express AMPs in grapevine, the development of transgenic grapevine is time consuming and therefore pre-screening of potential AMPs is necessary. These small molecules, of less than 50 amino acids in length, are expressed by almost all organisms as part of their non-specific defence system. In vitro pre-screening of AMP activity is valuable but is limited since the activity on artificial media may differ from the AMP activity in planta. These tests are also restricted to pathogens which can be cultured in vitro. These limitations can be overcome by using transient expression systems to determine the in planta activity of AMPs against pathogens of interest. In this study transient systems were used to express AMPs in developed plant tissue to test their efficacy against grapevine pathogens such as Agrobacterium vitis, Xylophilus ampelinus and aster yellows phytoplasma. Aster yellows phytoplasma, which was recently discovered in local vineyards, is known to cause extensive damage and therefore pose a great threat to the South African grapevine industry. To study the in planta effect of AMPs against the abovementioned pathogens, transient expression vectors were constructed expressing either of the AMPs D4E1 or Vv-AMP1. D4E1 is a synthetically designed AMP known to be active against bacteria and fungi, while Vv-AMP1, isolated from grapevine berries, has already shown activity against fungi. In a transient approach in grapevine, the expression of foreign genes from viral and non-viral vectors was confirmed by expression of the marker genes β-glucuronidase and Green Fluorescent Protein, while tissue-printing immunoassays confirmed viral replication and systemic spread in Nicotiana benthamiana. The viral vectors were based on the phloem-limited virus grapevine virus A. Only Agrobacterium-mediated 35S transient expression vectors were used for AMP in planta activity screening since the viral-mediated expression in grapevine was insufficient for screening against A. vitis and X. ampelinus as it was restricted to phloem tissues after whole-leaf infiltration. No phytoplasma-infected material could be established and as a result AMP activity screening was only performed against the A. vitis and X. ampelinus. Quantification of the bacteria was performed by qPCR. Vv-AMP1 did not show activity against either of the two bacteria in planta while D4E1 was found to be active against both. The observed in planta activity of D4E1 correlated with the in vitro activity as measured in an AMP plate bioassay. In contrast to in vitro screenings, the in planta AMP activity screening might give a more accurate representation of the potential antimicrobial activity of the peptide in a transgenic plant environment. This study proved that transient expression systems can be used as a pre-screening method of AMP activity in planta against grapevine pathogens, allowing the screening of various AMPs in a relatively short period of time before committing to transgenic grapevine development.
- ItemEvaluation of two pathogen-derived resistance strategies for Grapevine leafroll-associated virus 3(Stellenbosch : Stellenbosch University, 2015-04) Suidgeest, Faira; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.ENGLISH ABSTRACT: Grapevine leafroll disease (GLD), caused by the members of the family Closteroviridae, is one of the most economic important viral diseases affecting grapevine. Grapevine leafroll associated virus 3 (GLRaV-3), of the genus Ampelovirus, is the most widespread member of the leafroll associated virus family. To prevent the spread of GLD, management strategies such as rogueing and insect vector control are required to limit crop losses. Alternative control strategies based on genetic modification of the grapevine genome, such as pathogen-derived resistance (PDR), is proven to be effective in conferring resistance to several viruses. Therefore, the focus of this study was to evaluate pathogen-derived resistance strategies for GLRaV-3 using the following two approaches; 1) evaluation of transgenic plants expressing a dysfunctional GLRaV-3 heat shock protein 70 homolog (HSP70h) in order to confer resistance against GLRaV-3, and 2) the construction of artificial microRNAs (amiRNAs) to use as a tool for silencing specific sequences of GLRaV-3 in the grapevine host and the development of an amiRNA-mediated silencing validation system. In the first part of this study, six transgenic plant lines (plant lines #1, #3, #9, #14, #15 and #17) as well as a non-modified plant line, were inoculated with GLRaV-3 by grafting buds of each onto GLRaV-3 infected plant material. After approximately five months, GLRaV-3 virus titres of all grafted plants were quantified relative to two reference genes using RT-qPCR. Results were evaluated by comparing the relative virus titre of each transgenic plant line to that of the non-modified control plant line. Results showed that resistance levels of plant line #3 was significantly enhanced (>99%) and remarkably, plant line #14, showed to be more susceptible to the virus. The second part of the study was the construction and validation of amiRNAs targeting GLRaV-3 sequences. Two 21 nt regions of GLRaV-3 were successfully incorporated into miRNA backbone vvi167b of grapevine. Moreover, target constructs were developed by incorporating corresponding GLRaV-3 target sequences into the 3’ UTR of a green fluorescence protein (GFP) gene. Subsequently, the target constructs were co-infiltrated with the constructed amiRNA in Nicotiana benthamiana and GFP expression levels were quantified to determine the silencing efficiency of the amiRNAs. Results showed that the amiRNAs were successful in silencing the GFP target construct, however, they were not specific in silencing exclusively their corresponding target. These amiRNA constructs are ideal for further viral studies to determine the efficiency of silencing GLRaV-3 in GLD infected grapevines.
- ItemThe expression of Dianthin 30, a ribosome inactivating protein(Stellenbosch : Stellenbosch University, 2003-03) Maree, H. J. (Hans Jacob); Burger, Johan T.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. Institute for Plant Biotechnology (IPB)ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are currently classified as rRNA N-glycosidases, but also have polynucleotide: adenosine glycosidase activity. RIPs are believed to have anti-viral and anti-fungal properties, but the exact mechanism of these proteins still need to be elucidated.The mechanism of resistance however, appears to be independent of the pathogen. For resistance the RIP terminates virus infected plant cells and stops the reproduction and spread of the virus. Transgenic plants containing RIPs should thus be resistant to a wide range of viruses. The ultimate goal of the larger project of which this forms part is the development of virus resistant plants. To monitor the expression of a RIP in a transgenic plant a detection method had to be developed. Antibody detection of the RIP was decided upon as the most cost effective method. The RIP, Dianthin 30 from Dianthus caryophyllus (carnation), was used and expressed in bacterial and insect expression systems. The bacterial expression experiments were done using the pET expression system in BL21(DE3)pLysS cells. The expression in this system yielded recombinant protein at a very low concentration. Expression experiments were also performed in insect tissue culture with the baculovirus vector BAC-TO-BAC™.With this system the expression was also too low to be used for the production of antibodies. A Dianthin 30 specific peptide was then designed and then produced by Bio-Synthesis. This peptide was then used to raise antibodies to detect Dianthin 30. These antibodies were tested on Dianthus caryophyllus proteins. To establish if this detection method was effective to monitor the expression in plants, tobacco plants were transformed with Agrobacterium tumefaciens containing Dianthin 30 in the pART27 plant expression vector. The putative transformed plants were analysed with peR and Southern blots.
- ItemThe incidence and distribution of grapevine yellows disease in South African vineyards(Stellenbosch : Stellenbosch University, 2014-04) Carstens, Roleen; Burger, Johan T.; Stephan, Dirk; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: South Africa is ranked eighth in the world as far as international wine production is concerned and in terms of area under bearing vines South Africa is ranked 12th. In 2011 the wine industry contributed R4 204.4 million to the South African economy in state revenue from wine products. The importance of viticulture to the economy of South Africa forces the industry to limit the effect of all disease causing pathogens in order to keep their competitive edge. Aster yellows (AY) phytoplasma 16SrI-B subgroup was reported for the first time in grapevine (Vitis vinifera L. (Vitaceae)) in South Africa in 2006. Worldwide phytoplasma diseases of grapevine cause serious damage ranging from lower yields to the death of vines. The lack of knowledge about the epidemiology of AY disease makes it difficult to determine the impact of the disease on the South African wine industry. The aim of this study was to conduct surveys in disease-affected vineyards in the Vredendal region to determine the incidence and spatial distribution of the disease in a variety of cultivars. The field surveys based on visual symptoms of AY disease were confirmed by polymerase chain reaction (PCR). A survey was also conducted in and around AY-infected vineyards in search of possible alternative host plants of the phytoplasma. Spatial distribution of AY-affected vines were analysed using the PATCHY spatial analysis package. A rapid decline of AY-affected Chardonnay eventually leading to the death of vines was observed, confirming the sensitivity of Chardonnay towards grapevine yellows infections. Symptomless AY infections occurred and AY could not be detected in all symptomatic vines, which indicate uneven distribution of AY in individual vines. Molecular analyses using PCR-RFLP showed that all vines sampled in the Vredendal vicinity contained AY phytoplasma only. No phytoplasmas were present in any weeds or other possible host plants tested. Although the mean yearly disease incidences of Chardonnay (29.95%) and Chenin blanc (16.64%) were higher than Pinotage (5.80%) over the four-year survey period, there was no statistically significant difference between the disease incidences of these three cultivars. The mean yearly disease incidence showed a trend over time and the disease incidence of the first year was significantly lower than that of the other years. Chardonnay showed a cumulative disease incidence of 37.77% at the end of the 4-year study which means that Chardonnay vineyards can be 100% AY infected in ten years’ time. Spatial distribution patterns of AY-infected vines were mostly non-random with clustering of disease affected vines along and across vine rows. With the exception of one vineyard, aggregation of AY-affected vines mostly occurred on the edge of vineyards adjacent to infected vineyards. This epidemiological study gives an indication of the sensitivity of the different cultivars towards AY, the tempo of spreading and the future impact of the disease on the South African wine industry. It also contributes valuable information towards the development of a management strategy for grapevine yellows disease in South African vineyards.
- ItemInvestigating the recovery phenotype phenomenon in Aster Yellows-infected Grapevine(Stellenbosch : Stellenbosch University, 2017-12) Van der Vyver, Ane; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: South Africa is ranked as the seventh largest wine producing country in the world. Grapevine is one of the most important crops, which warrants extensive research on pathogens and diseases that impact vine health. Aster Yellows (AY) phytoplasma was first identified in South African vineyards in 2010, and poses a major threat to local vineyards. The pathogen symptomatology results in substantial grape yield loss and in many cases death. Though no treatment for AY-infections have been commercialised, a common practice among farmers have been to inflict physiological and chemical stresses on infected plants resulting in the induction of a recovery phenotype. It is unknown whether this recovery is permanent. The aim of this study was to identify an AY-infected vineyard and induce a recovery in half of the sample group, after which the AY-infection status of the plants was monitored over two years. Furthermore, the AY genetic diversity of isolates in the vineyard were investigated to ensure that any observed recovery is not due to false negative diagnostics. The effect of possible viral pathogens on recovery phenotype induction in AY-infected vines was also investigated. A triple-nested PCR assay allowed for the identification of 40 AY-infected and 40 healthy plants in February 2016, after which half of each experimental group was coppiced to induce a recovery phenotype. A large-scale remission in AY-infection was observed throughout the vineyard, both in coppiced and uncoppiced plants. Through RFLP assays and Sanger sequencing, a single genetic variant was observed in the studied vineyard, thereby suggesting that the observed recovery was a true one. Grapevine viruses were found in almost all of the AY-positive plants before coppicing, with all healthy plants being virus free. This changed after coppicing however, where a large remission in virus infections was seen post coppicing in AY-positive plants. Additionally, viruses were identified in a small number of AY-negative plants after coppicing. The presence of viruses seemed to have no effect on recovery phenotype induction. This study contributes to our understanding of recovery phenotype induction, reporting a large-scale remission of the pathogen even in the absence of coppicing.