Browsing by Author "Cook, Glynnis"

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    Characterization of citrus tristeza virus variants and their influence on symptom expression in grapefruit
    (Stellenbosch : Stellenbosch University, 2019-04) Cook, Glynnis; Maree, H. J.; Burger, Johan T.; Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics.
    ENGLISH ABSTRACT: Citrus tristeza virus (CTV), a member of the family Closteroviridae, was responsible for large scale destruction of citrus, especially in the Americas, due to tristeza disease and necessitated a production switch to less sensitive rootstocks. CTV however continues to affect citrus through the stem-pitting disease phenotype which is especially problematic in sweet orange, grapefruit and lime cultivars. In South Africa, the productive lifespan of grapefruit trees was severely affected by stem-pitting, requiring early tree replacement with an associated lag in production. This affect was later mitigated by applying cross-protection, a management strategy using non-stem-pitting sources of CTV, but without prior knowledge of which CTV strains were responsible for stempitting or which strains were present in the cross-protection sources. To understand the disease and unravel mechanisms underlying cross-protection, it is necessary to characterise CTV strains to investigate both virus-host- and strain-interactions. The aim of this study was firstly to identify single-strain isolates belonging to different strains, to characterise them biologically and to determine full-genome sequences. These characterised CTV isolates were further used in a complementation study to investigate possible synergistic interactions affecting stem-pitting. Complete viral genomes of eight single-strain isolates were determined during the study. Two commercial grapefruit cultivars, ‘Star Ruby’ and ‘Marsh’, were used in a glasshouse trial to evaluate the ability of specific strains to induce stem-pitting in single or mixed infections. Evaluation over four years showed that symptom expression of mild strains did not result in altered symptom expression when in combination with each other. Importantly demonstrating that there was no additive effect on stem-pitting expression with multiple isolates. Relative quantitation of the strains in ‘Marsh’ and ‘Star Ruby’ plants indicated that the individual strain concentrations were not significantly altered when in combination with the other strains. A valuable discovery made within this project was the characterisation of two variants of the T68 strain, derived from the same GFMS12 source, but displaying differences in stem-pitting severity in grapefruit. This finding demonstrates the co-existence of severe and mild variants of the same strain in one source and provides an explanation for the presumed strain segregation event observed for the GFMS12 cross-protection source that resulted in the discontinuation of the source for use in cross-protection of grapefruit. The characterisation of these variants will further assist in the identification of the sequence determinants for stem-pitting in grapefruit.
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    Citrus tristeza virus genotype detection using high-throughput sequencing
    (MDPI, 2021-01-23) Bester, Rachelle; Cook, Glynnis; Maree, Hans J.
    The application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.
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    In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection
    (BioMed Central, 2017) Visser, Marike; Cook, Glynnis; Burger, Johan T.; Maree, Hans J.
    Background: Small RNA (sRNA) associated gene regulation has been shown to play a significant role during plant-pathogen interaction. In commercial citrus orchards co-infection of Citrus tristeza virus (CTV) and viroids occur naturally. Methods: A next-generation sequencing-based approach was used to study the sRNA and transcriptional response in grapefruit to the co-infection of CTV and Citrus dwarfing viroid. Results: The co-infection resulted in a difference in the expression of a number of sRNA species when comparing healthy and infected plants; the majority of these were derived from transcripts processed in a phased manner. Several RNA transcripts were also differentially expressed, including transcripts derived from two genes, predicted to be under the regulation of sRNAs. These genes are involved in plant hormone systems; one in the abscisic acid, and the other in the cytokinin regulatory pathway. Additional analysis of virus- and viroid-derived small-interfering RNAs (siRNAs) showed areas on the pathogen genomes associated with increased siRNA synthesis. Most interestingly, the starting position of the p23 silencing suppressor’s sub-genomic RNA generated a siRNA hotspot on the CTV genome. Conclusions: This study showed the involvement of various genes, as well as endogenous and exogenous RNA-derived sRNA species in the plant-defence response. The results highlighted the role of sRNA-directed plant hormone regulation during biotic stress, as well as a counter-response of plants to virus suppressors of RNA-silencing.
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    Towards the validation of high-throughput sequencing (HTS) for routine plant virus diagnostics: measurement of variation linked to HTS detection of citrus viruses and viroids
    (BioMed Central, 2021-03-22) Bester, Rachelle; Cook, Glynnis; Breytenbach, Johannes H. J.; Steyn, Chanel; De Bruyn, Rochelle; Maree, Hans J.
    Background: High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods: Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results: The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions: This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.