Explore the vast range of titles available.

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21–22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

Rapid and safe access to new plant genetic stocks is crucial for primary plant industries to remain profitable, sustainable, and internationally competitive. Imported plant species may spend several years in Post Entry Quarantine (PEQ) facilities, undergoing pathogen testing which can impact the ability of plant industries to quickly adapt to new global market opportunities by accessing new varieties. Advances in high throughput sequencing (HTS) technologies provide new opportunities for a broad range of fields, including phytosanitary diagnostics. In this study, we compare the performance of two HTS methods (RNA-Seq and sRNA-Seq) with that of existing PEQ molecular assays in detecting and identifying viruses and viroids from various plant commodities. To analyze the data, we tested several bioinformatics tools which rely on different approaches, including direct-read, de novo, and reference-guided assembly. We implemented VirusReport, a new portable, scalable, and reproducible nextflow pipeline that analyses sRNA datasets to detect and identify viruses and viroids. We raise awareness of the need to evaluate cross-sample contamination when analyzing HTS data routinely and of using methods to mitigate index cross-talk. Overall, our results suggest that sRNA analyzed using VirReport provides opportunities to improve quarantine testing at PEQ by detecting all regulated exotic viruses from imported plants in a single assay.

Summary Co‐infection of apple trees with several viruses/viroids is common and decreases fruit yield and quality. Accurate and rapid detection of these viral pathogens helps to reduce losses and prevent virus spread. Current molecular detection assays used for apple viruses require specialized and expensive equipment. Here, we optimized a CRISPR/Cas12a‐based nucleic acid detection platform for the diagnosis of the most prevalent RNA viruses/viroid in apple, namely Apple necrotic mosaic virus (ApNMV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). We detected each RNA virus/viroid directly from crude leaf extracts after simultaneous multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) with high specificity. Positive results can be distinguished by the naked eye via oligonucleotide‐conjugated gold nanoparticles. The CRISPR/Cas12a‐RT‐RPA platform exhibited comparable sensitivity to RT‐qPCR, with limits of detection reaching 250 viral copies per reaction for ASPV and ASGV and 2500 copies for the others. However, this protocol was faster and simpler, requiring an hour or less from leaf harvest. Field tests showed 100% agreement with RT‐PCR detection for 52 samples. This novel Cas12a‐based method is ideal for rapid and reliable detection of apple viruses in the orchard without the need to send samples to a specialized laboratory.

Apples (Malus domestica), one of Iran’s oldest cultivated fruit crops, hold considerable economic importance. In this study, 170 apple leaf samples representing various commercial cultivars were collected across the country. RT-PCR screening targeted five common apple-infecting viruses and two viroids: apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple green crinkle-associated virus (AGCaV), apple mosaic virus (ApMV), apple scar skin viroid (ASSVd), and hop stunt viroid (HSVd). To identify additional or novel agents, 40 RT-PCR-negative samples were pooled into two composite groups and analyzed using next-generation sequencing (NGS). NGS was also performed on individual samples with mixed infections to retrieve full genomes. RT-PCR confirmed the presence of ACLSV, ASPV, ASGV, AGCaV, ApMV, and HSVd. NGS further revealed three additional pathogens: citrus concave gum-associated virus (CCGaV), apple hammerhead viroid (AHVd), and apricot vein clearing-associated virus (AVCaV), which were subsequently detected across the collection by RT-PCR. AGCaV was most prevalent (47.6%), followed by ACLSV (45.8%), HSVd (27.6%), AVCaV (20.5%), ASGV (17%), AHVd (15.2%), ASPV (14.1%), CCGaV (4.7%), and ApMV (3.5%). Mixed infections occurred in 67% of samples. Phylogenetic analysis based on CP genes (ACLSV, ASGV, AGCaV) and full genomes (AVCaV, AHVd) clustered Iranian isolates together, suggesting a common origin. This is the first report in Iran of AGCaV, CCGaV, ApMV, and AVCaV in apple, and notably, the first global report of AVCaV in a non-Prunus host. The findings provide the first comprehensive assessment of the sanitary status of apple trees in Iran.

Virome analysis was performed on 174 grape genetic resources from the National Agriculture and Food Research Organization, Japan. A total of 20 bulk samples was prepared by grouping the vines into batches of 6–10 plants. Each of the bulk samples was analyzed using high-throughput sequencing, which detected 27 viruses and 5 viroids, including six viruses and one viroid reported in Japan for the first time (grapevine viruses F, L, and T, grapevine Kizil Sapak virus, grapevine Syrah virus 1, grapevine satellite virus, and grapevine yellow speckle viroid 2). In addition, a novel vitivirus was detected with a maximum nucleotide sequence identity of only 58% to its closest relative, grapevine virus A (GVA). The genome of this novel virus was 7,461 nucleotides in length and encoded five open reading frames showing the typical genomic structure of vitiviruses. Phylogenetic trees of vitiviruses placed it in a distinct position nearest to GVA or grapevine virus F (GVF) in genomes and amino acids of deduced replication-associated protein (RAP) and coat protein (CP). The amino acid sequence identities of RAP and CP with GVA, GVF, and other vitiviruses were a maximum of 53% and 73%, respectively, which were significantly below the species demarcation threshold of 80% in the genus. The low identity and phylogenetic analyses indicate the discovery of a novel vitivirus species provisionally named grapevine virus P.

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.

Background The use of next-generation sequencing has become an established method for virus detection. Efficient study design for accurate detection relies on the optimal amount of data representing a significant portion of a virus genome. Findings In this study, genome coverage at different sequencing depths was determined for a number of viruses, viroids, hosts and sequencing library types, using both read-mapping and de novo assembly-based approaches. The results highlighted the strength of ribo-depleted RNA and sRNA in obtaining saturated genome coverage with the least amount of data, while even though the poly(A)-selected RNA yielded virus-derived reads, it was insufficient to cover the complete genome of a non-polyadenylated virus. The ribo-depleted RNA data also outperformed the sRNA data in terms of the percentage of coverage that could be obtained particularly with the de novo assembled contigs. Conclusion Our results suggest the use of ribo-depleted RNA in a de novo assembly-based approach for the detection of single-stranded RNA viruses. Furthermore, we suggest that sequencing one million reads will provide sufficient genome coverage specifically for closterovirus detection.

Viroids represent a threat to the citrus industry and also display an intricate matter for citrus tristeza virus (CTV) control as most of the commercial citrus rootstocks that are resistant/tolerant to CTV appear to be highly susceptible to viroid infection. Therefore, a detailed knowledge of the viroid’s incidence and distribution, along with the assessment of unexplored epidemiological factors leading to their occurrence, are necessary to further improve control measures. Herein, a large-scale epidemiological study of citrus viroids in five districts, 38 locations and 145 fields in Greece is presented, based on the analysis of 3005 samples collected from 29 cultivars of six citrus species. We monitored the occurrence of citrus exocortis (CEVd), hop stunt (HSVd), citrus dwarfing (CDVd), citrus bark cracking (CBCVd), and citrus bent leaf (CBLVd) viroids, and addressed their epidemiological patterns and factors shaping their population structure. Our results show a high frequency and wide distribution of four viroids in all areas and in almost all hosts, whereas CBLVd occurrence was restricted to Crete. Mixed infections were found in all districts in which a wide spread of viroids was observed. We identified a potential pathogens’ different preferences that could be partially explained by the host and cultivar, including the type of infection (single or mixed) and the number of viroids in the mixed infections. Overall, this work provides the first detailed epidemiological study on citrus viroids, enriching our knowledge for the implementation, production, and distribution of certified citrus propagative material, and the development of sustainable control strategies.

A comprehensive diagnostic method of known plant viruses and viroids is necessary to provide an accurate phytosanitary status of fruit trees. However, most widely used detection methods have a small limit on either the number of targeted viruses/viroids or the number of samples to be evaluated at a time, hampering the ability to rapidly scale up the test capacity. Here we report that by combining the power of high multiplexing PCR (499 primer pairs) of small amplicons (120-135bp), targeting 27 viruses and 7 viroids of fruit trees, followed by a single high-throughput sequencing (HTS) run, we accurately diagnosed the viruses and viroids on as many as 123 pome and stone fruit tree samples. We compared the accuracy, sensitivity, and reproducibility of this approach and contrast it with other detection methods including HTS of total RNA (RNA-Seq) and individual RT-qPCR for every fruit tree virus or viroid under the study. We argue that this robust and high-throughput cost-effective diagnostic tool will enhance the viral/viroid knowledge of fruit trees while increasing the capacity for large scale diagnostics. This approach can also be adopted for the detection of multiple viruses and viroids in other crops.

Pospiviroid species are transmitted through capsicum and tomato seeds. Trade in these seeds represents a route for the viroids to invade new regions, but the magnitude of this hazard has not been adequately investigated. Since 2012, tomato seed lots sent to Australia have been tested for pospiviroids before they are released from border quarantine, and capsicum seed lots have been similarly tested in quarantine since 2013. Altogether, more than 2000 seed lots have been tested. Pospiviroids were detected in more than 10% of the seed lots in the first years of mandatory testing, but the proportion of lots that were infected declined in subsequent years to less than 5%. Six pospiviroid species were detected: Citrus exocortis viroid, Columnea latent viroid, Pepper chat fruit viroid, Potato spindle tuber viroid, Tomato chlorotic dwarf viroid and Tomato apical stunt viroid. They were detected in seed lots exported from 18 countries from every production region. In many seed lots, the detectable fraction (prevalence) of infected seeds was estimated to be very small, as low as 6 × 10−5 (~1 in 16,000; CI 5 × 10−6 to 2.5 × 10−4) for some lots. These findings raise questions about seed production practices, and the study indicates the geographic distributions of these pathogens are uncertain, and there is a continuing threat of invasion.