Browsing by Author "Van Wyk, Lezan"

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    An investigation into the potato leafroll virus problem in the Sandveld region, South Africa
    (Stellenbosch : Stellenbosch University, 2017-12) Van Wyk, Lezan; Bellstedt, D. U.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Potato leafroll virus (PLRV) is responsible for significant yield losses in the South African (SA) potato industry. PLRV incidence in the Sandveld region has increased dramatically over the past 15 years. Enzyme-linked immunosorbent assay (ELISA) is used for routine testing by the SA Seed Potato Certification Scheme to diagnose PLRV infection, but many countries have changed to reverse transcription polymerase chain reaction (RT-PCR) for detection of PLRV because of its greater sensitivity. This project aimed to develop and validate a probe-based quantitative real-time reverse transcription PCR (RT-qPCR) to detect PLRV in potatoes and obtain an assessment of PLRV incidence in the Sandveld region, SA. This project also aimed to confirm infection in aphids and characterise aphid transmitted PLRV isolates by sequencing. Finally, this project aimed to apply a next-generation sequencing (NGS) technology to identify and characterise isolates, to compare non-coding 5’ and 3’ regions of the genome and lastly, to identify unknown viruses and other pathogens that possibly occur in potatoes in the Sandveld region. Suitable primers and a TaqMan probe were designed to develop a highly sensitive RT-qPCR detection method for PLRV. An amplified complementary DNA (cDNA) was cloned into a plasmid and used for assay quantification and validation. Thereafter, potato leaves were tested over a full calendar year and results were compared to vector pressure. Overall high infection levels were found, but in certain times of the year low infection levels were found due to low vector pressure. SA tubers were also tested with this method. This study indicates that the SA Potato Certification Scheme should reconsider the use of ELISA as the method for PLRV detection and replace it with the described RT-qPCR method. Secondly, the presence of PLRV in aphids was confirmed with RT-qPCR. A whole PLRV genome was amplified and sequenced after extraction from an infectious aphid. This generated whole PLRV genome was aligned in a data matrix with other whole genome sequences. Phylogenetic analysis of the whole genomes revealed that the aphid extracted PLRV isolate grouped with eight other SA isolates from the Sandveld region. Lastly, Ion Torrent was used to obtain information about further PLRV isolates present in the Sandveld region. Samples with low Cq values corresponded to a high number mapping, coverage and sequencing depth of small interfering RNAs (siRNAs). Three complete genomes were obtained by mapping siRNAs to the reference sequence, as de novo assembly could not obtain contigs longer than 700 nucleotides. Phylogenetic analysis of the whole genomes revealed that three of the samples grouped with an Australian isolate and seven SA isolates. The remaining isolate grouped with nine other SA isolates. Minor variation between upstream and downstream non-coding regions was seen. No other potato or unknown viruses were identified, but an unknown fungus was identified in all samples which needs further investigation.