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Comparisons with RT-qPCR have shown that HTS has a similar level of sensitivity for the detection of several potato viruses and demonstrated the contribution of the bioinformatics approach, since targeted analysis by mapping reads improved the sensitivity 10-fold (Santala and Valkonen,2018). Specificity Specificity is an important criterion in the adoption of any diagnostic technique. Because the identification of an agent is based on sequence data, specificity of HTS-based diagnostics is expected to be more predictable and less prone to unexplained cross reactions or false negative results caused by unexpected interactions of reagents with target or host nucleic acids or proteins. Reproducibility and repeatability As obtaining a diagnostic result by HTS is a multi-phasic process, the approach, platform, bioinformatic strategy, interpretation all need to be considered for reproducibility. [...]far there have been only limited investigations into the reproducibility of these various phases. Appropriate use of negative controls during the process and introduction of cut-offs based on signal-to-noise is a solution used in many routine testing laboratories deploying other, similarly sensitive techniques.

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.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

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 tRNA ile and tRNA met . The amplicon consists of a partial segment of tRNA ile , intergenic region I (tRNA ile - tRNA gln ), the complete sequence of tRNA gln , intergenic region II (tRNA gln - tRNA met ), and a partial segment of tRNA met . 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 (tRNA ile - tRNA gln ) 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.

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.

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.

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.

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.

The evolutionary history of the exclusively grapevine (Vitis spp.) infecting, grapevine leafroll-associated virus 3 (GLRaV-3) has not been studied extensively, partly due to limited available sequence data. In this study we trace the evolutionary history of GLRaV-3, focussing on isolate GH24, a newly discovered variant. GH24 was discovered through the use of next-generation sequencing (NGS) and the whole genome sequence determined and validated with Sanger sequencing. We assembled an alignment of all 13 available whole genomes of GLRaV-3 isolates and all other publicly available GLRaV-3 sequence data. Using multiple recombination detection methods we identified a clear signal for recombination in one whole genome sequence and further evidence for recombination in two more, including GH24. We inferred phylogenetic trees and networks and estimated the ages of common ancestors of GLRaV-3 clades by means of relaxed clock models calibrated with asynchronous sampling dates. Our results generally confirm previously identified variant groups as well as two new groups (VII and VIII). Higher order groups were defined as supergroups designated A to D. Supergroup A includes variant groups I-V and supergroup B group VI and its related unclassified isolates. Supergroups C and D are less well known, including the newly identified groups VII (including isolate GH24) and VIII respectively. The inferred node ages suggest that the origins of the major groups of GLRaV-3, including isolate GH24, may have occurred prior to worldwide cultivation of grapevines, whilst the current diversity represents closely related isolates that diverged from common ancestors within the last century.

The global importance of apple as a fruit crop necessitates investigations into molecular aspects of the processes that influence fruit quality and yield, including plant development, fruit ripening and disease resistance. In order to study and understand biological processes it is essential to recognise the range of molecules, which influence these processes. Small non-coding RNAs are regulatory agents involved in diverse plant activities, ranging from development to stress response. The occurrence of these molecules in apple leaves was studied by means of next-generation sequencing. 85 novel microRNA (miRNA) gene loci were predicted and characterized along with known miRNA loci. Both cis- and trans-natural antisense transcript pairs were identified. Although the trans-overlapping regions were enriched in small RNA (sRNA) production, cis-overlaps did not seem to agree. More than 150 phased regions were also identified, and for a small subset of these, potential miRNAs that could initiate phasing, were revealed. Repeat-associated siRNAs, which are generated from repetitive genomic regions such as transposons, were also analysed. For this group almost all available repeat sequences, associated with the apple genome and present in Repbase, were found to produce siRNAs. Results from this study extend our current knowledge on apple sRNAs and their precursors significantly. A rich molecular resource has been created and is available to the research community to serve as a baseline for future studies.