High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of

Wheat streak mosaic tritimovirus (WSMV) seriously damages wheat worldwide. We report analyses of new complete ORF (CO) sequences from seven Australian isolates with 56 COs and 128 coat protein (CP) genes sequenced previously. Eleven CO and three CP sequences were recombinants so were removed from our analyses. Patristic distances of maximum‐likelihood phylogenies of nonrecombinant (n‐rec) CO sequences and their CP sequences were closely correlated (R = 0.994, p ≤ 0.00001). The phylogeny of all 188 n‐rec CP genes had four well‐supported phylogroups (I–IV): phylogroup I (one Mexican sequence), phylogroup II (six Iranian sequences), phylogroup III (48 sequences) and phylogroup IV (133 sequences), each with basal Iranian sequences and either mostly European (phylogroup III) or mostly American (phylogroup IV) terminal sequences. Australian and South American sequences formed a phylogroup IV subcluster within a Pacific Northwest USA cluster. Unlike the Iranian, South American and European populations, the North American and Australian populations demonstrated recent population imbalance. Sample collection dates of 40 CO sequences are known, allowing WSMV phylogeny dating by RTDT methodology. The most recent WSMV ancestor was dated at 1456 CE, and the Australian cluster at 1998.7 CE, only 2–3 years before WSMV was first reported. Tritimoviruses originated in central Eurasia, WSMV first entering wheat in its Middle East domestication centre and one basal lineage being taken to Mexico after the Spanish conquest, whereas the other most basal lineage spread throughout Iran, before spreading to other world regions. Probable future spread to other world regions of additional WSMV phylogroups, and of interphylogroup recombinants, constitutes a biosecurity concern.

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Rose cryptic virus-1 (RoCV1) also known as Rosa multiflora cryptic virus is a partitivirus affecting roses, one of the most important ornamental crops worldwide. RoCV1 has previously been reported in the US, Canada and New Zealand, and has now been identified in the United Kingdom for the first time. Using High Throughput Sequencing (HTS) RoCV1 sequences were found in two samples collected in 2007 and 2012. This discovery led to the development of a RT-qPCR (TaqMan) assay for the detection of this virus. As part of a rose virus survey in the UK, 251 samples were analysed using the newly developed RoCV1 RT-qPCR test, following ELISA analysis for other common rose viruses. The results of the RT-qPCR test were confirmed using published conventional PCR primers and Sanger sequencing of amplified products. Results suggest that RoCV1 could have been infecting roses in the UK since at least 2007, with a large number of recently collected samples (43%) found to be infected. Cryptoviruses are not thought to cause direct economic losses in their plant host, although it is not clear what impact they might have in mixed infections.Open Access This article is distributed under the terms of the Creative Comm ons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

High Plains wheat mosaic virus (HPWMoV) causes a serious disease in major wheat-growing regions worldwide. We report here the complete or partial genomic sequences of five HPWMoV isolates from Australian wheat samples. Phylogenetic analysis of the nucleotide sequences of the eight genomic segments of these five isolates together with others from Genbank found all eight genes formed two lineages, L1 and L2. L1 contained a single isolate from Colorado in the North American Great Plains Region (GPR), and L2 had two unresolved clusters, A and B, of isolates from Australia and the GPR. A quarter of the L2B isolate sequences of the nucleocapsid gene (RNA3) were recombinant, which is unexpected as little evidence of recombination exists in viruses with negative single-stranded RNA genomes. Phylogenies calculated from the amino acid sequences of HPWMoV’s RNA-dependent RNA-polymerase (RNA1), glycoprotein (RNA2), and nucleocapsid protein (RNA3) showed they were closest to those of Palo Verde broom virus. However, its movement protein (RNA4) was closer to those of Ti ringspot-associated and common oak ringspot-associated viruses, indicating the RNA4 segments of their ancestors reassorted to produce the current emaraviruses. To avoid increased yield losses from co-infection, biosecurity measures are advised to avoid HPWMoV introduction to countries where wheat streak mosaic virus already occurs.

The seeds were imported from Asia and samples were screened in compliance with import testing requirements. A total of 3,000 seeds were tested for Tomato mottle mosaic virus (ToMMV; genus Tobamovirus) using the RT-PCR test (F-5476 and R-6287) from Levitzky et al. (2019). A product of the expected size (811 bp) was obtained in all subsamples (12 subsamples each of 250 seeds) and subsequently sent for Sanger sequencing. Sequences from four of these samples were compared against those in GenBank, and confirmed to be ToMMV (Accession Nos. OK334226, OK334230-32), with ≥99% nucleotide and 99-100% amino acid identity compared with the exemplar strain (KF477193; YP_008492931.1). To obtain the whole genome of the isolate, the 12 subsamples were bulked and sequenced by high throughput sequencing (HTS) as described by Fox et al. (2019) and bioinformatic analysis using the Angua pipeline (Fowkes et al., 2021). The whole genome of ToMMV was obtained (6375 bp; OK334224), and confirmed after a BLAST search (99.6% identity KF477193.1; Figure 1).The genome of Tomato mosaic virus (ToMV) was also found (OK334225).

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