Browsing by Author "Aldrich, Dirk Jacobus"

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    Characterisation of citrus tristeza virus-induced stem pitting in citrus
    (Stellenbosch : Stellenbosch University, 2023-12) Aldrich, Dirk Jacobus; Maree, Hans Jacob; Bester, Rachelle; Burger, Johan Theodorus; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV) is the most important viral pathogen of citrus and causes several disease syndromes in different citrus hosts. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The exact mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study aimed to utilise CTV infectious clone mutants in a reverse genetics approach to study stem pitting induction. A panel of recombinant CTV clones was generated focussing on the open reading frames (ORFs) implicated in stem pitting induction and pathogenicity, namely p33, p18, p13 and p23. ORF replacements from severe- and mild-pitting South African CTV isolates were introduced into the mild-pitting infectious clone (CTV-fl6 - genotype T36) to determine if severe stem pitting could be induced. Stem pitting assessments were complemented with the determination of virus concentration ratios in ‘Mexican’ lime and ‘Duncan’ grapefruit to relate stem pitting outcomes to virus concentration. The various infectious clone and wild type CTV infections yielded a broad range of stem pitting outcomes and specific ORF replacements associated with enhanced stem pitting were identified. Plant responses to different stem pitting pressures were further assessed by untargeted metabolite profiling and the quantitation of the stress-responsive phytohormones, abscisic acid, jasmonic acid and salicylic acid. In both citrus hosts, the metabolite profiling yielded fourteen statistically significant compounds that differed between stem pitting groups. These compounds were mainly phenolic acids and phenolic glycosides and are known to function as antioxidant and stress signalling molecules. Significant differences in phytohormone content were also found between test groups, particularly in plants that were severely stunted. Stem pits were also characterised at the molecular anatomical level using a combination of known and novel techniques to better understand the nature of the xylem and phloem tissues impacted by severe pitting. Established methods such as biological staining and fluorescence microscopy were used to visualise CTV-induced stem pitting and virus localisation. The utility of two novel technologies that have not previously been used to study CTV-induced stem pitting, namely high-resolution CT scanning and serial block face scanning electron microscopy was also evaluated. This study contributed to our understanding of CTV-induced stem pitting in citrus. Importantly, the use of CTV infectious clones for South African CTV research was established, which can allow for targeted follow-up experiments to further characterise the local citrus-CTV pathosystem. This can also provide novel opportunities for potentially employing CTV infectious clones as a crop protection tool against other important citrus diseases on the African continent.
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    Characterisation 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.