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

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.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Side-by-Side Comparison of Post-Entry Quarantine and High Throughput Sequencing Methods for Virus and Viroid Diagnosis.

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.

Evolution of Plant Virus Diagnostics Used in Australian Post Entry Quarantine.

As part of a special edition for MDPI on plant virology in Australia, this review provides a brief high-level overview on the evolution of diagnostic techniques used in Australian government Post-Entry Quarantine (PEQ) facilities for testing imported plants for viruses. A comprehensive range of traditional and modern diagnostic approaches have historically been employed in PEQ facilities using bioassays, serological, and molecular techniques. Whilst these techniques have been effective, they are time consuming, resource intensive and expensive. The review highlights the importance of ensuring the best available science and diagnostic developments are constantly tested, evaluated, and implemented by regulators to ensure primary producers have rapid and safe access to new genetics to remain productive, sustainable and competitive.

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes.

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

BACKGROUND: Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. RESULTS: We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. CONCLUSIONS: We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.