Browsing by Author "Steyn, Chanel"

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    Sensitivity of citrus rootstocks to two citrus viroids and the influence on tree growth
    (Stellenbosch : Stellenbosch University, 2022-03) Steyn, Chanel; Fourie, Paul; Cook, Glynnis; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Despite the supply of disease-free propagation material to the industry by the South African Citrus Improvement Scheme, citrus viroids, particularly citrus dwarfing viroid (CDVd) and hop stunt viroid (HSVd), are occasionally detected in commercial orchards. Locally, these viroids were associated with stunting of trees on sensitive trifoliate hybrid rootstocks. New rootstock cultivars are developed and, in addition to their horticultural performance, it is important to evaluate the effects of viroid infections. A field trial was established consisting of ‘Midknight’ Valencia on ten different rootstocks, including a trifoliate (‘Rich16-6’), two non-trifoliates (Rough Lemon ‘Cairn’ and ‘Rangpur’ lime) and seven trifoliate hybrids (‘US-812’, ‘MxT’, ‘C-22 Bitters’, ‘C-54 Carpenter’ and ‘C-57 Furr’ citrandarins, as well as ‘Carrizo’ and ‘C-35’ citranges). The scions were graft-inoculated with CDVd as a single infection, and in combination with HSVd to investigate potential viroid interactions. Transmission detection was notably erratic in scions on some rootstocks, particularly MxT and C-22, indicating that rootstocks may influence viroid distribution within scions. To study the effect of rootstocks on spatial distribution of CDVd and HSVd within scions on C-35, Carrizo, MxT and Rough Lemon, a relative RT-qPCR assay was developed, using GAPC2 (Glyceraldehyde-3- phosphate dehydrogenase C2) and UPL7 (Ubiquitin-protein ligase 7) reference genes for qPCR data normalization, to determine viroid concentration ratios from inner and outer scion canopy positions. Large variation in concentration ratios were seen for both viroids, demonstrating uneven spatial distribution, especially for CDVd in scions on C-35 and Rough Lemon, but also HSVd in scions on C-35. Despite this variation, the inner canopy proved to be a more reliable sampling position for detection as higher viroid titres were mostly determined from this position. Absolute CDVd and HSVd quantification was done from inner canopies of scions on the ten rootstocks. CDVd copies ranged from 1 to 831, and HSVd copies were determined in a broader range from 8 to 3963. Rootstock cultivar had a significant influence on viroid titres in the scion. Scions on C-22 and Rich16-6 had significantly lower CDVd titres than C-57, Carrizo, ‘Rangpur’ lime, Rough Lemon and US-812. However, scions on MxT and Rich16-6 accumulated significantly fewer HSVd copies than C-35, C-57, Carrizo and ‘Rangpur’ lime, indicating that viroid species accumulation differed for each rootstock. CDVd titres were not significantly influenced by co-infection with HSVd, but HSVd reached significantly higher copy numbers than CDVd in scions on all ten rootstocks, with as much as a 24-fold increase of HSVd compared to CDVd copies in scions on C-35. CDVd and HSVd genome sequences generated from a scion of each rootstock, 19 months post inoculation, were determined and mutations were detected within the pathogenic regions of both viroids. The significance of these mutations requires further investigation. After 2 years of field growth, no significant influence of viroid infections on either trunk circumference or canopy volume, were observed. However, impact on growth is expected only after 4 to 5 years in the field. This study indicated that rootstock cultivar influenced the spatial distribution, accumulation and genetic variability of two citrus viroids within ‘Midknight’ Valencia scions. The quantitative real-time RT-PCR assays developed in this study will be used in further analysis of this field trial to monitor viroid titres in both scions and rootstocks to unravel the complex interactions of the viroids with both scion and rootstocks.
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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.