High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

Biological characteristics of 11 Potato virus S (PVS) isolates from three cultivated potato species (Solanum spp.) growing in five Andean countries and 1 from Scotland differed in virulence depending on isolate and host species. Nine isolates infected Chenopodium quinoa systemically but two others and the Scottish isolate remained restricted to inoculated leaves; therefore, they belonged to biologically defined strains PVS^A and PVS^O, respectively. When nine wild potato species were inoculated, most developed symptomless systemic infection but Solanum megistacrolobum developed systemic hypersensitive resistance (SHR) with one PVS^O and two PVS^A isolates. Andean potato cultivars developed mostly asymptomatic primary infection but predominantly symptomatic secondary infection. In both wild and cultivated potato plants, PVS^A and PVS^O elicited similar foliage symptoms. Following graft inoculation, all except two PVS^O isolates were detected in partially PVS-resistant cultivar Saco, while clone Snec 66/139-19 developed SHR with two isolates each of PVS^A and PVS^O. Myzus persicae transmitted all nine PVS^A isolates but none of the three PVS^O isolates. All 12 isolates were transmitted by plant-to-plant contact. In infective sap, all isolates had thermal inactivation points of 55 to 60°C. Longevities in vitro were 25 to 40 days with six PVS^A isolates but less than 21 days for the three PVS^O isolates. Dilution end points were 10⁻³ for two PVS^O isolates but 10⁻⁴ to 10⁻⁶ with the other isolates. Complete new genome sequences were obtained from seven Andean PVS isolates; seven isolates from Africa, Australia, or Europe; and single isolates from S. muricatum and Arracacia xanthorhiza. These 17 new genomes and 23 from GenBank provided 40 unique sequences; however, 5 from Eurasia were recombinants. Phylogenetic analysis of the 35 nonrecombinants revealed three major lineages, two predominantly South American (SA) and evenly branched and one non-SA with a single long basal branch and many distal subdivisions. Using least squares dating and nucleotide sequences, the two nodes of the basal PVS trifurcation were dated at 1079 and 1055 Common Era (CE), the three midphylogeny nodes of the SA lineages at 1352, 1487, and 1537 CE, and the basal node to the non-SA lineage at 1837 CE. The Potato rough dwarf virus/Potato virus P (PVS/PRDV/PVP) cluster was sister to PVS and diverged 5,000 to 7,000 years ago. The non-SA PVS lineage contained 18 of 19 isolates from S. tuberosum subsp. tuberosum but the two SA lineages contained 6 from S. tuberosum subsp. andigena, 4 from S. phureja, 3 from S. tuberosum subsp. tuberosum, and 1 each from S. muricatum, S. curtilobum, and A. xanthorrhiza. This suggests that a potato-infecting proto-PVS/PRDV/PVP emerged in South America at least 5,000 years ago, became endemic, and diverged into a range of local Solanum spp. and other species, and one early lineage spread worldwide in potato. Preventing establishment of the SA lineages is advised for all countries still without them.

There is only limited knowledge of the presence and incidence of viruses in peas within the United Kingdom, therefore high-throughput sequencing (HTS) in combination with a bulk sampling strategy and targeted testing was used to determine the virome in cultivated pea crops. Bulks of 120 leaves collected from twenty fields from around the UK were initially tested by HTS, and presence and incidence of virus was then determined using specific real-time reverse-transcription PCR assays by testing smaller mixed-bulk size samples. This study presents the first finding of turnip yellows virus (TuYV) in peas in the UK and the first finding of soybean dwarf virus (SbDV) in the UK. While TuYV was not previously known to be present in UK peas, it was found in 13 of the 20 sites tested and was present at incidences up to 100%. Pea enation mosaic virus-1, pea enation mosaic virus-2, pea seed-borne mosaic virus, bean yellow mosaic virus, pea enation mosaic virus satellite RNA and turnip yellows virus associated RNA were also identified by HTS. Additionally, a subset of bulked samples were re-sequenced at greater depth to ascertain whether the relatively low depth of sequencing had missed any infections. In each case the same viruses were identified as had been identified using the lower sequencing depth. Sequencing of an isolate of pea seed-borne mosaic virus from 2007 also revealed the presence of TuYV and SbDV, showing that both viruses have been present in the UK for at least a decade, and represents the earliest whole genome of SbDV from Europe. This study demonstrates the potential of HTS to be used as a surveillance tool, or for crop-specific field survey, using a bulk sampling strategy combined with HTS and targeted diagnostics to indicate both presence and incidence of viruses in a crop.

Forty-seven potato virus A (PVA) isolates from Europe, Australia, and South America’s Andean region were subjected to high-throughput sequencing, and 46 complete genomes from Europe (n = 9), Australia (n = 2), and the Andes (n = 35) obtained. These and 17 other genomes gave alignments of 63 open reading frames 9,180 nucleotides long; 9 were recombinants. The nonrecombinants formed three tightly clustered, almost equidistant phylogroups; A comprised 14 Peruvian potato isolates; W comprised 37 from potato in Peru, Argentina, and elsewhere in the world; and T contained three from tamarillo in New Zealand. When five isolates were inoculated to a potato cultivar differential, three strain groups (= pathotypes) unrelated to phylogenetic groupings were recognized. No temporal signal was detected among the dated nonrecombinant sequences, but PVA and potato virus Y (PVY) are from related lineages and ecologically similar; therefore, “relative dating” was obtained using a single maximum-likelihood phylogeny of PVA and PVY sequences and PVY’s well-supported 157 CE “time to most common recent ancestor”. The PVA datings obtained were supported by several independent historical coincidences. The PVA and PVY populations apparently arose in the Andes approximately 18 centuries ago, and were taken to Europe during the Columbian Exchange, radiating there after the mid-19th century potato late blight pandemic. PVA’s phylogroup A population diverged more recently in the Andean region, probably after new cultivars were bred locally using newly introduced Solanum tuberosum subsp. tuberosum as a parent. Such cultivars became widely grown, and apparently generated the A × W phylogroup recombinants. Phylogroup A, and its interphylogroup recombinants, might pose a biosecurity risk.[ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .