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.
Over the past two decades fruit symptoms resembling a marbling pattern on the fruit skin or corking of the fruit flesh was 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 and irregular shaped 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
, with which it has close phylognetic relationships and shares conserved structural motifs.
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.
•A one-step RT-LAMP assay was developed for the detection of plum viroid I (PlVd-I).•The RT-LAMP assays can be performed on crude extracts from leaf material.•Low false discovery rate and no decrease in sensitivity compared to RT-PCR.•Low-cost detection assay to limit the distribution of infected planting material.
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Viruses in the family
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
, which is available at ictv.global/report/closteroviridae.
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 of GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from
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
. 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 to interact with p20A in yeast but could not be demonstrated
. 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
could lead to GLD.
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. 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.sup.ile and tRNA.sup.met. The amplicon consists of a partial segment of tRNA.sup.ile, intergenic region I (tRNA.sup.ile - tRNA.sup.gln), the complete sequence of tRNA.sup.gln, intergenic region II (tRNA.sup.gln - tRNA.sup.met), and a partial segment of tRNA.sup.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. 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.sup.ile - tRNA.sup.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.
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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.
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Abstract
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.
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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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