Development of alternative diagnostic protocols for Candidatus Phytoplasma asteris and Coniella granati Sacc. (Syn. Pilidiella granati)

Page, Lucan Dylan (2019-04)

Thesis (MScAgric)--Stellenbosch University, 2019.

Thesis

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.

AFRIKAANSE OPSOMMING: Die Republiek van Suid-Afrika (RSA) is 'n integrale deel van die globale vrugte uitvoerketting. Veertig persent van alle plaaslik geproduseerde wyne word uitgevoer. en Suid-Afrika is die agste grootste wynprodusent. 'n Opkomende vrugtebedryf in Suid-Afrika is granate. Suid-Afrika is tans die vierde grootste granaatprodusent in die suidelike halfrond en 88% van alle granaatprodukte word uitgevoer. Patogene beïnvloed egter die opbrengs en oeste van hierdie industrië, daarom word verdere navorsing geregverdig. Twee van hierdie patogene is Candidatus Phytoplasma asteris (AY fitoplasma), 'n fitoplasma wat wingerde infekteer, en Coniella granati, 'n swam wat granate infekteer. AY fitoplasma is in 2010 en C. granati is in 2017 vir die eerste keer in SA gerapporteer. Tans is diagnotiese metodes wat gebruik word vir die diagnosering van beide patogene, afhanklik van tydrowende polimerase ketting reaksies (PKRs), wat opgeleide tegnici en toerusting, soos termosikleerders, benodig om uitgevoer te word. Die doel van hierdie projek was om 'n funksionele deteksie metode te ontwikkel vir die vinnige identifisering van AY fitoplasma en C. granati. Hierdie projek het ook daarop gefokus om huidige diagnostiese toetse te vergelyk met ‘n nuwe isotermiese diagnostiese toets, naamlik rekombinase polimerase amplifikasie (RPA). Daarbenewens ontwikkel hierdie projek in samewerking met die department van Ingenieurwese by die Universiteit Stellenbosch 'n mikrofluidiese toestel vir die indentifisering van hierdie vrugtepatogene. Isotermiese RPA diagnostiese toetse vir beide AY fitoplasma en C. granati het vinnige resultate gelewer. Dus was effektief minder tyd van begin tot einde benodig vir suksesvolle identifikasie van bogenoemde patogene. Vergelykings tussen PKR diagnostiese toetse en RPA diagnostiese toetse het bepaal dat PKR’s effens meer sensitief was, maar die RPA was heelwat vinniger. In situ toetse van die simptome van siektes van granaatvrugte het die resultate in literatuur bevestig. Die biologiese fondasie vir die mikrofluidiese toestel is ook gelê met behulp van spesifisiteits-toetse gebiotinileerde DNS-fragmente en streptavidien-bedekte magnetiese sfere te gebruik.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/106017
This item appears in the following collections: