Browsing by Author "Page, Lucan Dylan"

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    Development 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.