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International audience; Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Besides, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries, while the most prevalent virus was NSPaV, followed by PaLV. An analysis of the genetic diversity of these viruses was also conducted. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed either in graft-inoculated GF305 indicators, or in different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution and prevalence of peach-infecting luteoviruses, and suggest these viruses are likely asymptomatic in peach under most circumstances.

50 Pág., In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratifcation vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV, This work was supported in part through Laulima Govern ment Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Gov ernment Solutions, LLC under Contract No. HHSN272201800013C. This work was also funded in part by Contract No. HSHQDC 15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges support from the Mississippi Agri cultural and Forestry Experiment Station (MAFES), USDA-ARS pro ject 58-6066-9-033 and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project, under Accession Num ber 1021494.

International audience; Modern agriculture has influenced plant virus emergence through ecosystem simplification, introduction of new host species, and reduction in crop genetic diversity. Therefore, it is crucial to better understand virus distributions across cultivated and uncultivated communities in agro-ecological interfaces, as well as virus exchange among them. Here, we advance fundamental understanding in this area by characterizing the virome of three co-occurring replicated Poaceae community types that represent a gradient of grass species richness and management intensity, from highly managed crop monocultures to little-managed, species-rich grasslands. We performed a large-scale study on 950 wild and cultivated Poaceae over 2 years, combining untargeted virome analysis down to the virus species level with targeted detection of three plant viruses. Deep sequencing revealed (i) a diversified and largely unknown Poaceae virome (at least 51 virus species or taxa), with an abundance of so-called persistent viruses; (ii) an increase of virome richness with grass species richness within the community; (iii) stability of virome richness over time but a large viral intraspecific variability; and (iv) contrasting patterns of virus prevalence, coinfections, and spatial distribution among plant communities and species. Our findings highlight the complex structure of plant virus communities in nature and suggest the influence of anthropogenic management on viral distribution and prevalence.

We report the discovery of a new flavi-like virus identified in wild carrots (Daucus carota subsp. carota), using a double-stranded (ds)RNA high-throughput sequencing (HTS) approach. The new virus, tentatively named "carrot flavi-like virus 1" (CtFLV-1), has a large genome of 21.8 kb that harbours a single open reading frame encoding a 7,078-aa polyprotein with conserved RNA helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains. The new virus is phylogenetically related to recently described flavi-like viruses from arthropods, but its closest relative is a plant-associated virus, gentian Kobu-sho-associated virus (GKSaV). A pairwise comparison showed that these two viruses share 38.4% amino acid (aa) sequence identity in their polyproteins and 73% and 47.8% aa sequence identity in their conserved RdRp and Hel domains, respectively. Based on their similar genome organization and phylogenetic relationship, GKSaV and CtFLV-1 could form the basis for a new genus of plant-associated viruses, possibly within the family Flaviviridae, for which the name "Koshovirus" is proposed.

Modern agriculture has influenced plant virus emergence through ecosystem simplification, introduction of new host species, and reduction in crop genetic diversity. Therefore, it is crucial to better understand virus distributions across cultivated and uncultivated communities in agro-ecological interfaces, as well as virus exchange among them. Here we advance fundamental understanding in this area by characterizing the virome of three co-occurring replicatedPoaceaecommunity types that represent a gradient of grass species richness and management intensity, from highly managed crop monocultures to little-managed, species-rich grasslands. We performed a large-scale study on 950 wild and cultivatedPoaceaeover two years combining untargeted virome analysis down to virus species level with targeted detection of three plant viruses. Deep sequencing revealed i) a diversified and largely unknownPoaceaevirome (at least 51 virus species/taxa), with an abundance of so-called persistent viruses; ii) an increase of virome richness with grass species richness within the community; iii) a stable virome richness over time but a large viral intraspecific variability; and iv) contrasted patterns of virus prevalence, co-infections and geographical distribution among plant communities and species. Our findings highlight the complex structure of plant virus communities in nature and suggest the influence of anthropic management on viral distribution and prevalence.

The complete genome sequence of a previously uncharacterized sugarcane-infecting virus of the genus Ampelovirus was obtained by high-throughput sequencing (HTS) and rapid amplification of cDNA ends. This genome consists of 13,114 nucleotides (nt) and harbours seven open reading frames. Phylogenetic and pairwise identity analyses with the complete or near-complete genome sequences of 16 isolates revealed that they all belong to a new Ampelovirus species, family Closteroviridae. These 16 isolates were distributed in three phylogenetic groups and potential recombinant events were identified for only one of these isolates. An immunocapture-reverse transcription-PCR (IC-RT-PCR) assay was developed with antibodies previously produced against a clostero-like virus that had not yet been recognized by the International Committee on Taxonomy of Viruses but for which the name sugarcane mild mosaic virus (SCMMV) had been proposed. The new ampelovirus was detected by IC-RT-PCR, and Sanger-sequenced amplicons confirmed the identity of the virus based on HTS data. SCMMV was detected in 65 of 350 samples of quarantined sugarcane germplasm using RT-PCR targeting the hsp70 gene. These samples originated from Africa, the Caribbean, North America, South America and South-east Asia, thus revealing that SCMMV is present in many sugarcane-growing regions of the world. The complete or near-complete sequences reported here represent the first genomic sequences for SCMMV. Furthermore, the newly developed RT-PCR diagnostic assay will facilitate the understanding of SCMMV epidemiology and will help improve management practices.

The analysis by high throughput sequencing (HTS) and RT-PCR of Spanish pomegranate fruits showing yellow rings revealed the presence of viroid isolates closely related to fig isolates of apple dimple fruit viroid (ADFVd). The analysis of pomegranate public RNASeq data (Sequence Reads Archives, SRAs) from Israel provided evidence for the presence of similar ADFVd isolates in pomegranate trees in this country. In addition, reads or contigs of plum viroid I (PVd-I) isolates were also identified in two of the analyzed SRA datasets from Israel, suggesting the presence of this second viroid in pomegranate. Full length ADFVd genomic sequences have been recovered, increasing knowledge on the diversity of this viroid and on the pomegranate virome in which only four viruses and one viroid had previously been reported.

High-throughput sequencing (HTS) and sequence mining tools revolutionized virus detection and discovery in recent years and implementing them with classical plant virology techniques results to a powerful approach to characterize viruses. An example of a virus discovered through HTS is Solanum nigrum ilarvirus 1 (SnIV1) (familyBromoviridae), which was recently reported in various solanaceous plants from France, Slovenia, Greece, and South Africa. It was likewise detected in grapevines (Vitaceae) and severalFabaceaeandRosaceaeplant species. Such a very diverse host association is atypical for ilarviruses, thus warranted further investigation. In this study, modern and classical virological tools were combined to accelerate the characterization of SnIV1. Through HTS-based virome surveys, mining of sequence read archive datasets, and literature search, SnIV1 was further identified from diverse plant and non-plant sources globally. SnIV1 isolates showed relatively low variability compared to other phylogenetically related ilarviruses. Phylogenetic analyses showed a distinct basal clade of isolates from Europe, while the rest formed clades of mixed geographic origin. Furthermore, systemic infection of SnIV1 inSolanum villosumand its mechanical and graft transmissibility to solanaceous species were demonstrated. Near identical SnIV1 genomes from the inoculum (S. villosum) and inoculatedNicotiana benthamianawere sequenced, thus partially fulfilling Koch’s postulates. SnIV1 was shown to be seed-transmitted and potentially pollen-borne, has spherical virions, and possibly induces histopathological changes in infectedN. benthamianaleaf tissues. Overall, this study provided information to better understand the diversity, distribution, and pathobiology of SnIV1, but whether it could emerge as a destructive pathogen remains uncertain.FundingEU Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-ITN) project no. GA 813542Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection and Slovenian Research Agency (ARRS) funding no. P4-0165, P4-0407, J4-4553Balik Scientist Program (Republic Act 11035) of the Department of Science and Technology– Philippine Council for Agriculture, Aquatic, and Natural Resources Research and Development (DOST–PCAARRD), Republic of the PhilippinesThe Belgian FPS Health Food Chain Safety and Environment under Project RT18/3 SEVIPLANT

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the familyBetaflexiviridaein the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.

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, mycetes, 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., Scientific publication adapted from the deliverable 2.2 of the Valitest project - www.valitest.eu

Emaravirus (Order Bunyavirales; Family Fimoviridae) is a genus comprising over 20 emerging plant viruses with a worldwide distribution and economic impact. Emaraviruses infect a variety of host plants and have especially become prevalent in important long-living woody plants. These viruses are enveloped, with a segmented, single-stranded, negative-sense RNA genome and are transmitted by eriophyid mites or mechanical transmission. Emaraviruses have four core genome segments encoding an RNA-dependent RNA polymerase, a glycoprotein precursor, a nucleocapsid protein, and a movement protein. They also have additional genome segments, whose number varies widely. We report here that the proteins encoded by these segments form three main homology groups: a homolog of the sadwavirus Glu2 Pro glutamic protease; a protein involved in pathogenicity, which we named “ABC”; and a protein of unknown function, which we named “P55”. The distribution of these proteins parallels the emaravirus phylogeny and suggests, with other analyses, that emaraviruses should be split into at least two genera. Reliable diagnosis systems are urgently needed to detect emaraviruses, assess their economic and ecological importance, and take appropriate measures to prevent their spread (such as routine testing, hygiene measures, and control of mite vectors). Additional research needs include understanding the function of emaravirus proteins, breeding resistant plants and clarifying transmission modes.

International audience; A novel potyvirus was identified in symptomatic hedge mustard (Sisymbrium officinale (L.) Scop.) and wild radish (Raphanus raphanistrum L.) in France. The nearly complete genome sequence of hedge mustard mosaic virus (HMMV) was determined, demonstrating that it belongs to a sister species to turnip mosaic virus (TuMV). HMMV readily infected several other members of the family Brassicaceae, including turnip, shepherd's purse (Capsella bursa-pastoris), and arabidopsis. The identification of HMMV as a Brassicaceae-infecting virus closely related to TuMV leads us to question the current scenario of TuMV evolution and suggests a possible alternative one in which transition from a monocot-adapted ancestral lifestyle to a Brassicaceae-adapted one could have occurred earlier than previously recognized.Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.all OK

We report the complete genome sequencing of a novel member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae) infecting pineapple. The full genome of this virus was obtained from total RNAs extracted from pineapple leaf samples collected in Reunion Island, using a combination of high-throughput sequencing technologies. It is 6,757 nt long, excluding the poly(A) tail, and shares all the hallmarks of vitiviruses. Phylogenetic analyses performed on the replication-associated protein and capsid protein gene sequences unambiguously place this new virus, for which we propose the name “pineapple vitivirus A”, in the Vitivirus genus.

Following a request of the European Commission, EFSA and ANSES, beneficiary of the EFSA tasking grant on horizon scanning for plant pests (GP/EFSA/ALPHA/2017/02), developed a methodology to order by risk non‐regulated pests recently identified through the monitoring of media and scientific literature. The ranking methodology proposed at the end of the pilot phase was based on the scoring of pests under evaluation following 16 criteria related to the steps of the pest risk assessment scheme. The multicriteria matrix of scores obtained was then submitted to the multicriteria analysis method PROMETHEE. The pilot methodology was tested on a limited number of pests (14 pests identified during the monitoring activity, and 4 ‘control’ pests whose well‐known risk should be reflected either in a positive or negative score), then applied on all non‐regulated pests identified through the media and scientific literature monitoring in the first 2 years of the project. After having collected feedback from the targeted final users (EU risk managers), the methodology underwent a few refinements: (i) implementation of the methodology to a set of already assessed reference pests from EFSA opinions, (ii) exclusions of three criteria from the scoring phase, (iii) identification of pests proposed for further action (‘positive’ pests), using a threshold defined after scoring the reference pests.

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 preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material., This article is based upon work from the work package 2 of the project VALITEST (https://www.valitest.eu/), supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 773139.

9 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP), The self-replicating polerovirus-associated RNAs have recently been classified as tombusvirus-like associated RNAs (tlaRNAs). In a metagenomic comparison of the virome of wild (Daucus carota subsp. carota) and cultivated (D. carota subsp. sativus) carrots, four tlaRNAs were identified: carrot red leaf virus-associated RNA (CtRLVaRNA), beet western yellows virus-associated RNA (BWYVaRNA), and two other tlaRNAs detected for the first time in carrots, arracacha latent virus E-associated RNA (ALVEaRNA) and a new tlaRNA tentatively named carrot red leaf virus-associated RNA 2 (CtRLVaRNA-2). Their genomic sequences show a typical tlaRNA genome organization with a very short 5′ untranslated region (UTR), a long 3′ UTR and consecutive open reading frames (ORFs), ORF1a and ORF1b, separated by an amber stop codon, leading to the production of an ORF1a/ORF1b RdRp fusion protein. The genetic diversity and prevalence of these various agents were analysed in carrot populations in France and Spain. CtRLVaRNA was the most frequently detected in both wild and cultivated carrot populations and was genetically more heterogeneous than ALVEaRNA and CtRLVaRNA-2. ALVEaRNA was detected both in France and Spain and showed preferential association with cultivated carrots. In contrast, CtRLVaRNA-2 was only detected from one region of France and was preferentially associated with wild carrot populations. Analysis of the virome of individual plants showed the presence of CtRLV but not that of the expected helper virus for ALVEaRNA and BWYVaRNA, suggesting a degree of flexibility in their associations with helper viruses, which in turn may have biological and epidemiological consequences., This study was funded by the European Union through the Horizon 2020 Marie Skłodowska‐Curie Actions Innovative Training Network (H2020 MSCA‐ 60 ITN) project ‘INEXTVIR’ (GA 813542).

Cordyline virus 1 (CoV1) is a velarivirus that has so far only been reported in ornamental Ti plants (Cordyline fruticosa). Using high-throughput sequencing, we identified CoV1 infection in yam accessions from Vanuatu. Using a specific RT-PCR assay, we found that CoV1 is also present and highly prevalent in Dioscorea alata, D. cayenensis, and D. trifida in Guadeloupe. Phylogenetic analysis showed that CoV1 isolates infecting yam in Guadeloupe display a low level of molecular diversity. These data provide insights into the transmission of CoV1 in yam in Guadeloupe.

Mite-borne viruses belonging to the genus Allexivirus (family Alphaflexiviridae) commonly occur on garlic in many parts of the world. There are usually asymptomatic and cause small damage to the plants, but often occur in mixed infection with potyviruses and carlaviruses, with synergistic effects reducing crop quality and leading to higher losses (Taglienti et al. 2017). Their occurrence on Tunisian garlic crops was studied here in the same garlic germplasm collection mentioned in a previous first report (Ayed et al. 2019). Leaf samples from a total of 66 garlic accessions were tested by DAS-ELISA using specific antibodies (DSMZ, Germany) against garlic virus A (GarV-A), garlic virus B (GarV-B) and garlic virus C (GarV-C). These serological tests showed individual virus incidence of respectively 56.4%, 67.7% and 10%. Our findings corroborate with the results of Chodorska et al (2012). In order to confirm the presence of these viruses, RT-PCR assays were performed using total RNAs extracted using two silica-capture extraction procedures according to Foissac et al (2005) and specific primers targeting the coat protein genes of the various viruses. These primers, designed for the present study are (GarV-A-F: 5' YCTYTTCTCHYTDGCHTGGACYTG 3' and GarV-A-R: 5' RCCYTTCCTAGACCARTTRGCRGG 3' for GarV-A; GarV-B-F: 5' TGGGCYTGYTACCACAAYGGATC 3' and GarV-B-R 5' TCTGCGCGVGTGGADACCATRTT 3' for GarV-B; GarV-C-F: 5' ARGAYCTYTTYTCMCTYGCRTGGGC 3' and GarV-C-R: 5' GGAGGYTCRTGAATYTGTTGTTG 3' for GarV-C). The viruses were detected by a two-step RT-PCR as described by Marais et al (2015). PCRs consisted of one cycle at 95 °C for 5 min; followed by 40 cycles of denaturation at 95 °C for 45 s, annealing at 45 °C for 45 s, and elongation at 72 °C for 45s; and a final extension step at 72 °C for 10 min. Products of the expected size (214 bp for GarV-A, 363 bp for GarV-B and 439 bp for GarV-C) were amplified from 58 (88%), 47 (71%) and 56 (85%) accessions, respectively. Forty three samples (65%) were co-infected by the three viruses. Higher numbers of positives revealed by RT-PCR especially in the case of GarV-C may reflect the higher sensitivity and efficiency of this technique compared to ELISA. Direct sequencing of selected amplicons of the expected size obtained for GarV-A, -B, and -C Tunisian isolates was performed and the sequences submitted to GenBank, validating the specificity of the three RT-PCR assays. The two sequenced GarV-A isolates (MK599147 and MN995836) shared 98% nucleotide (nt) sequence identity with each other, and 93-94% identity with the closest isolate in GenBank, the "G118" isolate from China (MN059320). The three sequenced GarV-B isolates (MN995829 to MN995831) shared 88-98% nt identity with each other. For "GarV-B 18.1" (MN995830) and "GarV-B 36.2" (MN995831) the closest isolate was "1109.1" (JX682828) from Spain (92-93% nt identity). For "GarVB 17.2" (MN995829), the closest isolate was "B-Sp-3" (LC97167) from Spain (90% nt identity). The sequenced GarV-C isolate (MN995834) showed the highest sequence nt identity (93%) with the "GarV-9" isolate (HQ724848) from Spain. To our knowledge this is the first report of the presence of GarV-A, -B and -C in Tunisia. The presence of these allexiviruses may pose a threat to the preservation of the Tunisian garlic germplasm and, more broadly, to garlic production in Tunisia. For this reason, the scrupulous identification of viruses occurring in garlic plants will help to use the appropriate strategy to decrease viral incidence in garlic growing area.