Characterization of citrus tristeza virus variants and their influence on symptom expression in grapefruit

Thesis (PhDAgric)--Stellenbosch University, 2019.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.AFRIKAANSE OPSOMMING: Sitrus tristeza virus (CTV), 'n lid van die familie Closteroviridae, was verantwoordelik vir grootskaalse vernietiging van sitrus, weens tristeza siekte en het 'n produksie veradering na minder sensitiewe onderstamme genoodsaak. CTV het egter steeds 'n nadelige invloed op sitrus weens stamgleuf wat veral problematies is in soetlemoen, pomelos en lemmetjie kultivars. In Suid-Afrika is die produktiewe lewensduur van pomelo-bome verlaag weens stamgleuf, wat vroeë boomvervanging met 'n gepaardgaande produksieverlaging vereis het. Hierdie invloed is later verminder deur die toepassing van kruisbeskerming, 'n bestuurstrategie waar ligte bronne van CTV toegepas word, maar sonder voorafgaande kennis van watter CTV-rasse verantwoordelik is vir stamgleuf of watter rasse teenwoordig is in die bronne. Om die siekte te verstaan en meganismes onderliggend aan kruisbeskerming te ontleed, is dit nodig om CTV-rasse te karakteriseer om beide virus-gasheer- en rasinteraksies te ondersoek. Die doel van hierdie studie was om eerstens enkelras isolate wat aan verskillende rasse behoort, te identifiseer, om hulle biologies te karakteriseer en om volledige-genoom-nukleotiedvolgordes te bepaal. Hierdie gekarakteriseerde CTV-isolate is verder gebruik in 'n komplementeringsstudie om moontlike sinergistiese interaksies te ondersoek wat stamgleuf beïnvloed. Vollengte virale genome van agt enkelras-isolate is tydens die studie bepaal. Twee kommersiële pomelo kultivars, 'Star Ruby' en 'Marsh', is in 'n glashuisproef gebruik om die vermoë van spesifieke rasse te evalueer om stamgleuf in enkel- of gemengde infeksies te veroorsaak. Evaluering oor vier jaar het getoon dat simptoom uitdrukking van ligte rasse nie gelei het tot 'n veranderde simptoom uitdrukking wanneer hul in kombinasie met mekaar voorgekom het nie. Dit is belangrik om aan te toon dat daar geen toevoegende effek op stamgleuf uitdrukking met veelvoudige isolate was nie. Relatiewe hoeveelheids bepaling van die rasse in 'Marsh' en 'Star Ruby' plante het aangedui dat die individuele raskonsentrasies nie beduidend verander in kombinasie met die ander rasse nie. 'n Waardevolle ontdekking wat in hierdie projek gemaak is, was die karakterisering van twee variante van die T68-ras, afkomstig van dieselfde GFMS12-bron, maar met verskille in stamgleuf uitdrukking in pomelo's. Hierdie bevinding demonstreer die gelyktydige bestaan van strawwe en ligte variante van dieselfde ras in een bron en verskaf 'n verduideliking vir die vermoedelike ras segregasie gebeurtenis waargeneem in die GFMS12 kruisbeskerming bron, wat gelei het tot die staking van die bron vir gebruik as kruisbeskerming van pomelo's. Die karakterisering van hierdie variante sal verder help met die identifisering van die volgorde-bepaling van stamgleuf in pomelo's

Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing

CITATION: Bester, R., Cook, G. & Maree, H.H. 2021. Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing. Viruses, 13(2):1-17. https://doi.org/10.3390/v13020168The original publication is available at https://www.mdpi.com/journal/viruses/aboutThe 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.Publishers versio

Citrus Bent Leaf Viroid Present in Citrus in South Africa

Currently six viroid species are recognised which infect the genera Citrus and Poncirus, with an additional tentative new species reported. Citrus bent leaf viroid (CBLVd) has been reported from various citrus growing regions world-wide, but has not been formally documented from South Africa. CBLVd was detected in field samples in various citrus growing regions in South Africa during routine diagnostic analyses conducted since 2011. The detection and sequence verification of CBLVd from field samples is reported in this study. Biological confirmation of CLBVd could be done for one sample that was shown to contain a single viroid infection. Bent-leaf symptom expression was observed after slash inoculation of sample RNA to the ‘Etrog’ citron indicator host. This study was a retrospective analysis, of previously identified CLBVd-positive samples, to document the long-standing presence of CBLVd in South Africa

First report of citrus virus A in citrus in South Africa

High-throughput sequencing (HTS) of citrus indicator hosts, originally inoculated from field samples and showing transient chlorotic flecking or oak leaf patterns, revealed the presence of the first South African variant of citrus virus A (CiVA). This virus was first identified in citrus in Italy and was classified as a member of the second species (Coguvirus eburi) of the genus Coguvirus within the order Bunyavirales. The South African sequence variants of CiVA share 95.26-95.55% (RNA1) and 94.82-97.85% (RNA2) nucleotide sequence identity with the CiVA sequences from the Italian isolate. The discovery of CiVA in South African citrus orchards indicates a wide distribution of CiVA and further investigations are required to determine the association of CiVA with citrus disease symptoms. &nbsp

Additional file 2: Figure S1. of In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection

Biological process based network of genes differentially expressed across both grapefruit varieties. Figure S2. Molecular function based network of genes differentially expressed across both grapefruit varieties. Figure S3. Cellular component based network of genes differentially expressed across both grapefruit varieties. (PDF 2159 kb

Additional file 1: Table S1. of In silico analysis of the grapefruit sRNAome, transcriptome and gene regulation in response to CTV-CDVd co-infection

Results for the transcript differential expression analysis of ‘Marsh’ plants. Transcripts were considered to be differentially regulated as a result of infection if a |log2 fold change| > =1 and padj < =0.05 were observed. Table S2. Results for the transcript differential expression analysis of ‘Star Ruby’ plants. Transcripts were considered to be differentially regulated as a result of infection if a |log2 fold change| > =1 and padj < =0.05 were observed. Table S3. Results for the gene differential expression analysis across both ‘Marsh’ and ‘Star Ruby’ plants. Table S4. Biological process based characterisation of genes differentially expressed across both grapefruit varieties. Table S5. Molecular function based characterisation of genes differentially expressed across both grapefruit varieties. Table S6. Cellular component based characterisation of genes differentially expressed across both grapefruit varieties. (XLSX 392 kb

Evaluating high-resolution computed tomography to study citrus tristeza virus-induced stem pitting

Citrus tristeza virus (CTV) is the most important viral pathogen of citrus. CTV-induced stem pitting negatively impacts grapefruit and sweet orange production. The mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study evaluated the utility of high-resolution CT scanning as a tool to study stem pitting in live citrus material. CT scans were used to easily identify pits based on differences in tissue density. Stem pits were also mapped and modelled three-dimensionally along the length of the stem. Nano-CT scanning proved to be a potentially valuable, non-destructive method for stem pitting characterization in citrus

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

CITATION: Bester, R., et al. 2021. 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. Virology Journal, 18:61, doi:10.1186/s12985-021-01523-1.The original publication is available at https://virologyj.biomedcentral.comPublication of this article was funded by the Stellenbosch University Open Access FundBackground: 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.https://virologyj.biomedcentral.com/articles/10.1186/s12985-021-01523-1Publisher's versio

Evaluating high-resolution computed tomography to study citrus tristeza virus-induced stem pitting

Citrus tristeza virus (CTV) is the most important viral pathogen of citrus. CTV-induced stem pitting negatively impacts grapefruit and sweet orange production. The mechanisms of stem pitting development in CTV-infected citrus remain unclear. This study evaluated the utility of high-resolution CT scanning as a tool to study stem pitting in live citrus material. CT scans were used to easily identify pits based on differences in tissue density. Stem pits were also mapped and modelled three-dimensionally along the length of the stem. Nano-CT scanning proved to be a potentially valuable, non-destructive method for stem pitting characterization in citrus

Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids

The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study