Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants

Abstract

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.

Nanovirseq: dsRNA sequencing for plant virus and viroid detection by Nanopore sequencing

“…First, plant-derived reads were incorrectly assigned to Citrus exocortis viroid (CEVd) in the CO-9-86J sample. This issue was also reported in a previous study, where a plant sequence was misannotated as CEVd (72). In contrast, we did not observe this misannotation in our dsRNAcD and dsRNA-MiSeq datasets of the same sample.…”

Enhancing biosecurity against virus disease threats to Australian grain crops: current situation and future prospects

“…Recent advances in high-throughput sequencing (HTS) and bioinformatic analysis have provided considerable opportunities to upgrade virus surveillance for plant health services and virus discovery, so this subject has recently received considerable worldwide attention (Massart et al, 2014;Adams and Fox, 2016;Massart et al, 2017;Pecman et al, 2017;Adams et al, 2018;Villamor et al, 2019;EPPO, 2022;Lebas et al, 2022;AlcalaB riseño et al, 2023;Fontdevila Pareta et al, 2023), including within Australia (Barrero et al, 2017;Piper et al, 2019;Maina et al, 2021;Whattam et al, 2021;Gauthier et al, 2022;Lelwala et al, 2022;Mackie et al, 2022;Maina et al, 2022). Moreover, the cost of HTS is declining at a significant rate due to advances in sequencing technology, so its application as a routine diagnostic tool is becoming increasing attractive (Villamor et al, 2019;Lebas et al, 2022).…”

“…Notably, whole genome sequencing can be costly when used for routine massive parallel sequencing. Therefore, over the last five years, considerable efforts have been underway in Australia to explore the possible integration of rapid diagnostics innovation involving HTS genome based smart tools into plant health and biosecurity programs (Barrero et al, 2017;Piper et al, 2019;Maina et al, 2021;Whattam et al, 2021;Gautier et al, 2022;Lelwala et al, 2022;Mackie et al, 2022). The following Sections describe examples of the most promising, cost-effective approaches suitable for use by plant biosecurity and other plant health authorities.…”

“…Under these circumstances, preventing incursions of damaging viruses and their virus vectors, and eradicating or containing any that establish successfully, is becoming an increasingly difficult challenge for biosecurity authorities located within different world regions and countries. Fortunately, rapid technological advances that improve the effectiveness of virus diagnosis in preborder, border and postborder situations are providing new opportunities to detect viruses earlier and more reliably (Jones, 2014;Martin et al, 2016;Kreuze, et al, 2023;Waite et al, 2022;Adams and Fox, 2016;Barrero et al, 2017;Pecman et al, 2017;Bronzato-Badial et al, 2018;FAO, 2019;Piper et al, 2019;Liefting et al, 2021;Maina et al, 2021;Whattam et al, 2021;Gauthier et al, 2022;Lelwala et al, 2022;Mackie et al, 2022;AlcaláBriseño et al, 2023).…”

“…The most significant pathway for long-distance plant virus dissemination is via virus-infected vegetative planting material, fruits and seeds distributed via trade supply chains involving ship, plane, railway or road transport. However, international germplasm exchange for plant breeding purposes also contributes (Thresh, 1980;Thresh, 1982, Bos, 1992Lapierre and Signoret, 2004;Jones, 2009;Sastry, 2013;Jones, 2020;Jones, 2021;Whattam et al, 2021;Lenzen et al, 2023). Moreover, the movement via trade supply chains of viruliferous arthropod vectors adhering to vegetative planting material and cut flowers, or viruliferous nematode vectors and resting spores of fungal vectors in infested soils, constitutes another major pathway for long-distance plant virus dissemination (Lecoq et al, 2003;Jones, 2009;Jones and Barbetti, 2012;Fereres, 2015;Wamaitha et al, 2018;Jones and Naidu, 2019).…”