Validation of High-Throughput Sequencing (HTS) for Routine Detection of Citrus Viruses and Viroids.

The management of plant diseases relies on the accurate identification of pathogens that requires a robust and validated tool in terms of specificity, sensitivity, repeatability, and reproducibility. High-throughput sequencing (HTS) has become the method of choice for virus detection when either a complete viral status of a plant is required in a single assay or if an unknown viral agent is expected. To ensure that the most accurate diagnosis is made from an HTS data analysis, a standardized protocol per pathosystem is required. This chapter presents a detailed protocol for the detection of viruses and viroids infecting citrus using HTS. The protocol describes all the steps from sample processing, nucleic acid extraction, and bioinformatic analyses validated to be an efficient method for detection in this pathosystem. The protocol also includes a section on citrus tristeza virus (CTV) genotype differentiation using HTS data.

Investigating Protein-Protein Interactions Between Grapevine Leafroll-Associated Virus 3 and Vitis vinifera.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

Identification of Interactions between Proteins Encoded by Grapevine Leafroll-Associated Virus 3.

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

Utilisation of a mitochondrial intergenic region for species differentiation of fruit flies (Diptera: Tephritidae) in South Africa.

BACKGROUND: Fruit flies (Diptera: Tephritidae) comprise species of agricultural and economic importance. Five such fruit fly species are known to affect commercial fruit production and export in South Africa: Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis. Management practices for these pests include monitoring, application of pest control products, post-harvest disinfestation measures and inspection of consignments both prior to shipment and at ports of entry. In activities relating to monitoring and inspection, accurate identification of these pests to species level is required. While morphological keys for adult stages of these fruit fly species have been well developed, morphological keys for earlier life stages remain problematic. In instances where closely related species cannot be reliably distinguished morphologically, there is a need for molecular tools to assist in identifying these five fruit fly species during surveillance practices, where sequencing-based approaches would be beneficial. RESULTS: Two complete mitochondrial genomes were assembled for each fruit fly species investigated using high throughput sequencing data generated in this study. A single primer set was designed to amplify a region between tRNAile and tRNAmet. The amplicon consists of a partial segment of tRNAile, intergenic region I (tRNAile - tRNAgln), the complete sequence of tRNAgln, intergenic region II (tRNAgln - tRNAmet), and a partial segment of tRNAmet. PCR amplicons were generated for 20 specimens of each species, five of which were colony adult males, five colony larvae, and 10 wild, trap-collected specimens. Upon analysis of the amplicon, intergenic region I was identified as the most informative region, allowing for unambiguous identification of the five fruit fly species. The similarity in intergenic region II was too high between C. rosa and C. quilicii for accurate differentiation of these species. CONCLUSION: The identity of all five fruit flies investigated in this study can be determined through sequence analysis of the mitochondrial intergenic regions. Within the target amplicon, intergenic region I (tRNAile - tRNAgln) shows interspecific variation sufficient for species differentiation based on multiple sequence alignment. The variation in the length of intergenic region I is proposed as a potential tool for accurately identifying these five fruit flies in South Africa.

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.

A multiplex PCR assay for the identification of fruit flies (Diptera: Tephritidae) of economic importance in South Africa.

The fruit fly (Diptera: Tephritidae) species, Ceratitis capitata, Ceratitis cosyra, Ceratitis rosa, Ceratitis quilicii, and Bactrocera dorsalis are of economic importance in South Africa. These agricultural pests cause extensive damage to a range of commercially produced fruit, primarily for export. These pests are of phytosanitary significance, and their presence in fruit-producing regions in South Africa has led to restrictions in export trade of fresh produce. Accurate identification of these flies, particularly at immature stages intercepted in fruit consignments originating from South Africa, is essential but remains an ongoing challenge. A rapid and accurate identification assay to differentiate these five species is needed for inspection and pest surveillance. High throughput sequencing data were generated for each of the five fruit fly species, and five sets of species-specific primers were designed for use in a multiplex PCR. Each primer set amplifies an amplicon of a different size for each species allowing for accurate identification. PCR sensitivity tests demonstrate that the limit of detection for this assay is 10 ng and 4 ng of DNA when extracted from larvae and adult specimens, respectively. The assay developed can be applied in fruit inspection and survey activities within the country and at ports of entry.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of plum viroid I (PlVd-I).

Plum viroid I (PlVd-I) is found in marbling and corky flesh diseased plum trees in South Africa. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the high-throughput detection of PlVd-I was developed. This assay can be performed on crude extracts and detection can either be a pH dependent colorimetric reaction or a real-time fluorescent signal reaction. The false discovery rate was shown to be low and no decrease in sensitivity was detected compared to RT-PCR. The RT-LAMP assay allows for the fast and cost-effective detection of PlVd-I that will curtail the distribution of infected plant material.

Distribution and Genetic Diversity of Coguvirus eburi in South African Citrus and the Development of a Real-Time RT-PCR Assay for Citrus-Infecting Coguviruses.

Citrus virus A (CiVA), a novel negative-sense single-stranded RNA virus assigned to the species Coguvirus eburi in the genus Coguvirus, was detected in South Africa with the use of high-throughput sequencing after its initial discovery in Italy. CiVA is closely related to citrus concave gum-associated virus (CCGaV), recently assigned to the species Citrus coguvirus. Disease association with CiVA is, however, incomplete. CiVA was detected in grapefruit (C. paradisi Macf.), sweet orange [C. sinensis (L.) Osb.], and clementine (C. reticulata Blanco) in South Africa, and a survey to determine the distribution, symptom association, and genetic diversity was conducted in three provinces and seven citrus production regions. The virus was detected in 'Delta' Valencia trees in six citrus production regions, and a fruit rind symptom was often observed on CiVA-positive trees. Additionally, grapefruit showing symptoms of citrus impietratura disease were positive for CiVA. This virus was primarily detected in older orchards that were established prior to the application of shoot tip grafting for virus elimination in the South African Citrus Improvement Scheme. The three viral-encoded genes of CiVA isolates from each cultivar and region were sequenced to investigate sequence diversity. Genetic differences were detected between the Delta Valencia, grapefruit, and clementine samples, with greater sequence variation observed with the nucleocapsid protein (NP) compared with the RNA-dependent RNA polymerase (RdRp) and the movement protein (MP). A real-time detection assay, targeting the RdRp, was developed to simultaneously detect citrus-infecting coguviruses, CiVA and CCGaV, using a dual priming reverse primer to improve PCR specificity.

A Review of the 'Candidatus Liberibacter africanus' Citrus Pathosystem in Africa.

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as 'Candidatus Liberibacter africanus'. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five 'Ca. L. africanus' subspecies have been identified in various rutaceous species, with 'Ca. L. africanus subsp. clausenae' the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.

Concerning the Etiology of Syrah Decline: A Fresh Perspective on an Old and Complex Issue Facing the Global Grape and Wine Industry.

Syrah decline, first identified in Southern France in the 1990s, has become a major concern in the global grape and wine industry. This disease mainly affects Syrah (Shiraz) grapevines. Characteristic symptoms include the bright and uniform reddening of leaves throughout the canopy in late summer or early fall; the appearance of abnormalities on the trunk, mainly at the graft union (swelling, pits, grooves, and necrosis); and a reduction in vine vigor, yield and berry quality. Diseased vines may die a few years after disease onset. Damages to the vine are even more pronounced in cool climate regions such as Ontario (Canada), where the affected vines are subjected to very cold and prolonged winters, leading to large numbers of vine deaths. Despite the extensive efforts of the global grape research community over the past few decades, the etiology of this disease remains unclear. In this study, we conducted extensive analyses of viruses in declining Syrah vines identified in commercial vineyards in the Niagara region (Ontario, Canada) through high-throughput sequencing, PCR, RT-PCR and the profiling of genetic variants of select viruses. Multiple viruses and viral strains, as well as three viroids, were identified. However, an unequivocal causal relationship cannot be established between Syrah decline and any of these viruses, although the possibility that certain virus or genetic variants, or both in combination, may contribute to the disease cannot be excluded. Gleaning all information that is available to date, we feel that the traditional approach and an insistence on finding a single cause for such a complex disorder in a woody perennial fruit crop involving grafting will prove to be futile. We hope that this study offers new conceptual perspectives on the etiology of this economically important but enigmatic disease complex that affects the global grape and wine industry.

Complete genome sequence of a grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant from South Africa.

High-throughput sequencing (HTS) was used to construct the virome profile of an old grapevine-leafroll-diseased grapevine (Vitis vinifera). De novo assembly of HTS data showed a complex infection, including a virus sequence with similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 7090 nucleotides and has four open reading frames (ORFs). Genome organisation and phylogenetic analysis identify this virus as a divergent variant of grapevine Roditis leaf discoloration-associated virus (GRLDaV) with 90% nucleotide sequence identity to isolate w4 (NC_027131). This is the first genome sequence of a South African variant of GRLDaV.

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.

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.

Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing.

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.

Grapefruit Field Trial Evaluation of Citrus Tristeza Virus T68-Strain Sources.

Determination of virus genomes and differentiation of strains and strain variants facilitate the linkage of biological expression to specific genetic units. For effective management of stem pitting disease of citrus tristeza virus (CTV) by cross-protection, an understanding of these links is necessary. The deliberate field application of a biological agent such as a virus first requires a thorough assessment of the long-term impact before it can be applied commercially. Three CTV sources were genetically characterized as different variants of the T68 strain, and their long-term effects on stem pitting and production were investigated. The different CTV sources were inoculated to 'Star Ruby' grapefruit trees and evaluated for a number of biological parameters in a field trial in the Limpopo Province of South Africa over a 10-year period. Significant differences were observed in stem pitting severity, impact on tree growth, yield, and the percentage of small fruit produced. These T68 variants were also associated with different stem pitting phenotypes. The variants differed in only 44 nucleotide positions across their genomes, and these minor genetic differences can therefore be used to identify possible genome regions affecting stem pitting.

Citrus Tristeza Virus Isolates of the Same Genotype Differ in Stem Pitting Severity in Grapefruit.

Two isolates of the T68 genotype of citrus tristeza virus (CTV) were derived from a common source, GFMS12, by single aphid transmission. These isolates, named GFMS12-8 and GFMS12-1.3, induced stem pitting with differing severity in 'Duncan' grapefruit (Citrus × paradisi [Macfad.]). Full-genome sequencing of these isolates showed only minor nucleotide sequence differences totaling 45 polymorphisms. Numerous nucleotide changes, in relatively close proximity, were detected in the p33 open reading frame (ORF) and the leader protease domains of ORF1a. This is the first report of full-genome characterization of CTV isolates of a single genotype, derived from the same source, but showing differences in pathogenicity. The results demonstrate the development of intragenotype heterogeneity known to occur with single-stranded RNA viruses. Identification of genetic variability between isolates showing different pathogenicity will enable interrogation of specific genome regions for potential stem pitting determinants.

Genomic characterisation of a newly identified badnavirus infecting ivy (Hedera helix).

High-throughput sequencing (HTS) was used to investigate ringspots on ivy (Hedera helix) leaves. De novo assembly of HTS data generated from a total RNA extract from these leaves yielded a contig with sequence similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 8,885 nucleotides and has three open reading frames (ORFs). Genome organisation and phylogenetic analysis identifies this newly identified virus as a new member of the genus Badnavirus for which we propose the name "ivy ringspot-associated virus" (IRSaV).

A Plum Marbling Conundrum: Identification of a New Viroid Associated with Marbling and Corky Flesh in Japanese Plums.

Over the past 2 decades, fruit symptoms resembling a marbling pattern on the fruit skin or corking of the fruit flesh were observed on Japanese plums in South Africa, resulting in unmarketable fruit. The ability of high-throughput sequencing (HTS) to detect known and unknown pathogens was exploited by assaying affected and unaffected fruit tree accessions to identify the potential aetiological agent of marbling and/or corky flesh disease. In this study, it is shown that the disease is associated with a previously undescribed small RNA with typical viroid structural features. The potential viroid was the only pathological agent consistently detected in all symptomatic trees by HTS, and the association with the symptoms was confirmed in field surveys over two seasons. To date, this RNA was not detectable by RT-PCR in seedlings raised from seeds collected from infected trees. Although the autonomous replication of this viroid-like RNA was not proven, it was shown to be transmissible by grafting and associated with a range of symptoms that include marbling on the fruit skin, corky flesh, reduced fruit size, irregular shape, and uneven fruit surface depending on the cultivar. Moreover, the circular RNA genome, consisting of 317 nucleotides, strongly supports that this viroid-like RNA is most likely a viroid for which the name plum viroid I (PVd-I) is proposed. The primary structure of this viroid showed a less than 90% nucleotide sequence identity to viroids of the genus Apscaviroid, with which it has close phylogenetic relationships and shares conserved structural motifs.

ICTV Virus Taxonomy Profile: Closteroviridae.

Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.

Draft Genome Sequence of a "Candidatus Phytoplasma asteris"-Related Strain (Aster Yellows, Subgroup 16SrI-B) from South Africa.

Here, we report the draft genome sequence of a phytoplasma discovered in grapevine. The genome size is 600,116 nucleotides (nt), with 597 predicted open reading frames. It is most similar to a maize bushy stunt phytoplasma of group 16SrI-B (aster yellows). The possible presence of a 3,833-nt plasmid was also noted.

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies.

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.