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Potato virus V (PVV) causes a disease of potato (Solanum tubersosum) in South and Central America, Europe, and the Middle East. We report here the complete genomic sequences of 42 new PVV isolates from the potato’s Andean domestication center in Peru and of eight historical or recent isolates from Europe. When the principal open reading frames of these genomic sequences together with those of nine previously published genomic sequences were analyzed, only two from Peru and one from Iran were found to be recombinant. The phylogeny of the 56 nonrecombinant open reading frame sequences showed that the PVV population had two major phylogroups, one of which formed three minor phylogroups (A1 to A3) of isolates, all of which are found only in the Andean region of South America (Peru and Colombia), and the other formed two minor phylogroups, a basal one of Andean isolates (A4) that is paraphyletic to a crown cluster containing all the isolates found outside South America (World). This suggests that PVV originated in the Andean region, with only one minor phylogroup spreading elsewhere in the world. In minor phylogroups A1 and A3, there were two subclades on long branches containing isolates from S. phureja evolving more rapidly than the others, and these interfered with dating calculations. Although no temporal signal was directly detected among the dated nonrecombinant sequences, PVV and potato virus Y (PVY) are from the same potyvirus lineage and are ecologically similar, so “subtree dating” was done via a single maximum likelihood phylogeny of PVV and PVY sequences, and PVY’s well-supported 157 ce “time to most common recent ancestor” was extrapolated to date that of PVV as 29 bce. Thus the independent historical coincidences supporting the datings of the PVV and PVY phylogenies are the same; PVV arose ≥2,000 years ago in the Andes and was taken to Europe during the Columbian Exchange, where it diversified around 1853 ce, soon after the European potato late blight pandemic. PVV is likely to be more widespread than currently realized and is of biosecurity relevance for world regions that have not yet recorded its presence. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Lettuce ring necrosis virus (LRNV), genus Ophiovirus, was detected by the Netherlands Institute for Vectors, Invasive plants and Plant health (NIVIP) in June and November of 2021 in two samples of chili pepper fruits (Capsicum spp.), both in mixed infection with other viruses. The first sample originated from a production site in Belgium (Sample ID: 40009704) and the second from a production site in the Netherlands (Sample ID: 41115269). One of the fruits of 40009704 showed a light purple circular pattern, while fruits from 41115269 showed colored (ring)spots. The samples were analyzed using Illumina sequencing on a NovaSeq 6000 platform (PE 150) as described previously (Hammond et al., 2021), obtaining 39.9M and 22.8M total reads for 40009704 and 41115269. The corresponding sequence read archives (SRA) were deposited in the NCBI SRA database under BioProject accession number PRJNA917231. From both samples, the nearly complete genome of LRNV (RNA1-4) was obtained and deposited in GenBank (40009704, OQ160823- OQ160826 (7616, 1799, 1502, 1382 nt, mapped reads: 40K, 12K, 114K, 12K , average read coverage (ARC): 0.8K, 0.9K, 11.3K and 1.1K); 41115269, OQ160827- OQ160830 (7616, 1801, 1518, 1389 nt, mapped reads: 112K, 7K, 357K, 55K reads, ARC: 2.2K, 0.6K, 34K and 5.8K)). The shared sequence identities with the Genbank reference sequence of LRNV (NC_006051-NC_006051) were 99.2 and 99.2% (RNA1), 99.1 and 99.1% (RNA2), 98.3 and 98.8% (RNA3), 99.0 and 98.9% (RNA4) for 40009704 and 41115269 respectively. The shared sequence identities between 40009704 and 41115269 were 99.9 (RNA1), 99.0 (RNA2), 99.1 (RNA3) and 99.5% (RNA4). In addition to LRNV, the ophiovirus ranunculus white mottle virus (RWMV) was detected in both samples (OQ160831-OQ160834; OQ160835-OQ160838), while the tobamovirus pepper mild mottle virus (PMMoV) was present in the fruits of 41115269 (OQ160839). Since RWMV has been associated with leaf symptoms in pepper (Gambley et al., 2019; Rivarez et al., 2022) and the colored (ring)spots of 41115269 were very similar to reported symptoms of PMMoV-infected pepper fruits (Martínez-Ochoa et al., 2003), it remains unclear whether LRNV contributed to the observed symptoms. Additionally, LRNV was detected in tomato (Solanum lycopersicum) in Belgium in 2020. In the frame of a metagenomic survey using Virion-Associated Nucleic Acids (VANA)-based protocol (Maclot et al., 2021) on a Nextseq 500 platform (PE 150), partial genome sequences of LRNV were detected in two pools of tomato plants. One pool was made of 44 asymptomatic cultivars from a non-commercial grower (one sample per cultivar) yielding 118K total reads of which 84, 59, 335, and 18 reads mapped on RNA1, 2, 3, and 4, covering 35%, 69%, 100% and 55% of the genome, respectively. The other pool consisted of 15 plants from one cultivar from a production site yielding 3.1M total reads of which 6 and 5 reads mapped on RNA3 and 4, respectively. The detection of LRNV was confirmed for both pooled samples using the real-time RT-PCR method, targeting the CP gene, as described by Maachi et al. (2021). To our knowledge this is the first report of LRNV in pepper anywhere in the world. Additionally, although the disease lettuce ring necrosis in lettuce (Lactuca sativa) has been described in Belgium and the Netherlands before the causal agent was identified (Bos & Huijberts, 1996), this is the first official report of this virus in Belgium and the Netherlands. This publication resulted from pre-publication data sharing of sequences and biological data among plant virologists to provide more context to two independent findings (Hammond et al., 2021). Bos, L., & Huijberts, N. (1996). Lettuce ring necrosis, caused by a chytrid-borne agent distinct from lettuce big-vein ‘virus’. European Journal of Plant Pathology, 102(9), 867-873. https://doi.org/10.1007/bf01877057 Gambley, C., Persley, D., & Thomas, J. E. (2019). First record of Ranunculus white mottle virus from Australia. New Disease Reports, 40(1), 13-13. https://doi.org/10.5197/j.2044-0588.2019.040.013 Hammond, J., Adams, I. P., Fowkes, A. R., McGreig, S., Botermans, M., Oorspronk, J. J. A., Westenberg, M., Verbeek, M., Dullemans, A. M., Stijger, C. C. M. M., Blouin, A. G., Massart, S., De Jonghe, K., Heyneman, M., Walsh, J. A., & Fox, A. (2021). Sequence analysis of 43‐year old samples of Plantago lanceolata show that Plantain virus X is synonymous with Actinidia virus X and is widely distributed. Plant Pathology, 70, 249-258. https://doi.org/10.1111/ppa.13310 Maachi, A., Torre, C., Sempere, R. N., Hernando, Y., Aranda, M. A., & Donaire, L. (2021). Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops. Microorganisms, 9(5). https://doi.org/10.3390/microorganisms9051043 Maclot, F. J., Debue, V., Blouin, A. G., Fontdevila Pareta, N., Tamisier, L., Filloux, D., & Massart, S. (2021). Identification, molecular and biological characterization of two novel secovirids in wild grass species in Belgium. Virus Res, 298, 198397. https://doi.org/10.1016/j.virusres.2021.198397 Martínez-Ochoa, N., Langston, D. B., Mullis, S. W., & Flanders, J. T. (2003). First Report of Pepper mild mottle virus in Jalapeno Pepper in Georgia. Plant Health Progress, 4(1). https://doi.org/10.1094/php-2003-1223-01-hn Rivarez, M. P. S., Kogej, Z., Jakos, N., Pecman, A., Seljak, G., Vucurovic, A., Ravnikar, M., Mehle, N., & Kutnjak, D. (2022). First Report of Ranunculus White Mottle Ophiovirus in Slovenia in Pepper with Yellow Leaf Curling Symptom and in Tomato. Plant Dis, PDIS08211624PDN. https://doi.org/10.1094/PDIS-08-21-1624-PDN

With the emergence of high throughput sequencing (HTS) technologies, the discovery of new plant viruses has outpaced their biological characterization. However, it is crucial to understand the biology of these viruses to evaluate the risks they pose for the production of crops and natural ecosystems and to manage them properly. In 2018, Physostegia chlorotic mottle virus (PhCMoV) was detected in Austria in aPhysostegiaplant (Lamiaceae) using HTS, and subsequent prepublication data sharing associated the presence of the virus with severe fruit symptoms on important crops like tomato, eggplant, and cucumber across nine European countries. This discovery led to a collaborative effort to understand better the virus’s genetic diversity, host range, symptomatology, and distribution. Still, specific knowledge gaps remained. In this study, the authors address these gaps by examining the transmission mode, prevalence, and disease severity of PhCMoV. Bioassay and field survey confirmed the causal association between the presence of the virus and symptoms on tomato and eggplant. The investigation also mapped out the historical and geographic footprint of the virus, spanning back 30 years and including a new location, Switzerland. Based on field survey, PhCMoV was found to naturally infect 11 new host plant species across seven families, extending the host range of PhCMoV to 20 plant species across 14 plant families. Greenhouse assays with mechanical inoculation showed that yield losses could reach 100% depending on the phenological stage of the plant at the time of infection. The study also identified a polyphagous leafhopper species (Anaceratagalliasp.) as the natural vector of PhCMoV. PhCMoV was widespread in diversified vegetable farms in Belgium where tomato is grown in soil, occurring in approximately one-third of such farms. However, outbreaks were sporadic and it can be suggested that they were associated with specific cultural practices, such as the cultivation of perennial plants in tomato tunnels that can serve as a host for both the virus and its vector. To further explore this phenomenon and better manage the virus, studying the ecology of theAnaceratagalliaevector would be beneficial.

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.

The number of small-scale diversified vegetable growers in industrialized countries has risen sharply over the last ten years. The risks associated with plant viruses in these systems have been barely studied in Europe, yet dramatic virus emergence events, such as tomato brown fruit rugose virus, sometimes occur. We developed a methodology that aimed to understand better the implications related to viruses for tomato production in Belgian’s vegetable farms by comparing growers’ perception of the presence of viral symptoms (visual inspection) with non targeting detection of nearly all viruses present in the plants by high throughput sequencing technologies (HTS). Virus presence and impact were interpreted considering the farm’s typology and cultural practices, the grower’s professional profiles, and visual inspection of plant-viral-like symptoms. Overall, The data indicated that most growers have limited understanding of tomato viruses and are not concerned about them. Field observations were correlated to this perception as the prevalence of symptomatic plants was usually lower than 1%. However, important and potentially emergent viruses, mainly transmitted by insects, were detected in several farms. Noteworthy, the presence of these viruses was correlated with the number of plant species grown per site (diversity) but not with a higher awareness of the growers regarding plant viral diseases or a higher number of symptomatic plants. In addition, both HTS and perception analysis underlined the rising incidence and importance of an emergent virus: Physostegia chlorotic mottle virus. Overall, the original methodology developed here, combining social science with HTS technologies, could be applied to other crops in other systems to identify emergent risks associated with plant viruses and can highlight the communication needed toward growers to mitigate epidemics.

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.

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

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.

Cassava is one of the most important staple crops in Africa and its production is seriously damaged by viral diseases. In this study, we identify for the first time and characterize the genome organization of novel ampeloviruses infecting cassava plants in diverse geographical locations using three high-throughput sequencing protocols [Virion-Associated Nucleotide Acid (VANA), dsRNA and total RNA], and we provide a first analysis of the diversity of these agents and of the evolutionary forces acting on them. Thirteen new Closteroviridae isolates were characterized in field-grown cassava plants from the Democratic Republic of Congo (DR Congo), Madagascar, Mayotte, and Reunion islands. The analysis of the sequences of the corresponding contigs (ranging between 10,417 and 13,752 nucleotides in length) revealed seven open reading frames. The replication-associated polyproteins have three expected functional domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). Additional open reading frames code for a small transmembrane protein, a heat-shock protein 70 homolog (HSP70h), a heat shock protein 90 homolog (HSP90h), and a major and a minor coat protein (CP and CPd respectively). Defective genomic variants were also identified in some cassava accessions originating from Madagascar and Reunion. The isolates were found to belong to two species tentatively named Manihot esculenta-associated virus 1 and 2 (MEaV-1 and MEaV-2). Phylogenetic analyses showed that MEaV-1 and MEaV-2 belong to the genus Ampelovirus, in particular to its subgroup II. MEaV-1 was found in all of the countries of study, while MEaV-2 was only detected in Madagascar and Mayotte. Recombination analysis provided evidence of intraspecies recombination occurring between the isolates from Madagascar and Mayotte. No clear association with visual symptoms in the cassava host could be identified. ispartof: VIRUSES-BASEL vol:13 issue:6 ispartof: location:Switzerland status: published

Plantain virus X was first recognized by the ICTV as a species in the genus Potexvirus in 1982. However, because no sequence was available for plantain virus X (PlVX), abolishing the species was proposed to the Flexiviridae working group of the ICTV in 2015. This initiated efforts to sequence the original isolates from Plantago lanceolata samples. Here we report the full‐genome sequencing of two original isolates of PlVX, which demonstrate that the virus is synonymous to Actinidia virus X, a species previously reported from kiwifruit (Actinidia sp.) and blackcurrant (Ribes nigrum). PlVX was previously noted to be widespread in the UK in P. lanceolata. This report additionally presents novel data on the distribution and diversity of PlVX, collected at the same site as the original UK isolates, and from three independent surveys, two in the Netherlands and one in Belgium. This study also includes two new host records for PlVX, Browallia americana and Capsicum annuum (sweet pepper), indicating the virus is more widespread and infects a broader range of hosts than previously reported. This stresses the importance of surveys of noncultivated species to gain insight into viral distribution and host range. This study also demonstrates the value of generating sequence data for isolates retained in virus collections. Additionally, it demonstrates the potential value in prepublication data sharing for giving context to virus detections such as the four independent studies here which, when combined, give greater clarity to the identity, diversity, distribution, and host range of plantain virus X.\ud \ud

International audience; High throughput sequencing was performed on virion-associated nucleic acids (VANA) from a pool of fifty asymptomatic rough bluegrasses (Poa trivialis L.) collected in a Belgian grazed pasture. Bioinformatics analyses produced some contigs presenting similarities with secovirid genomes, in particular nepoviruses and waikaviruses. Three distinct positive-sense single-stranded RNAs including 5? and 3? UTR were reconstructed and they represented two novel viruses infecting rough bluegrass, for which the provisional names poaceae Liege nepovirus A (PoLNVA, 7298 nt for RNA1 and 4263 nt for RNA2) and poaceae Liege virus 1 (PoLV1, 11,623 nt) were proposed. Compared to other Secoviridae members, the highest amino acid identity reached 90.7 % and 66.7 % between PoLNVA and nepoviruses for the Pro-Pol and CP regions respectively, while PoLV1 presented the highest amino acid identity with waikaviruses but with lower identities, i.e. 41.2 % for Pro-Pol and 25.8 % for CP regions, far below the ICTV demarcation criteria for novel secovirid. Based on sequence identity and phylogenetic analyses, PoLNVA was proposed to belong to the genus Nepovirus and PoLV1 as an unclassified secovirids. Detection of the two novel viruses was confirmed in high prevalence in rough bluegrass and ten other wild Poaceae species (Agropyron repens, Agrostis capillaris, Apera spica-venti, Anthoxanthum odoratum, Cynosorus cristatus, Festuca rubra, Holcus lanatus, Lolium perenne, Phleum bertolini and Phleum pratense) by RT-PCR and Sanger sequencing, revealing a diverse host range within Poaceae for these novel secovirids. Seed transmission was evaluated and confirmed for PoLNVA.

A new strain of Ageratum latent virus (AGLV) was found in symptomless leaves of Veronica bollonsii, V. breviracemosa and V. dieffenbachii - the native shrubs in New Zealand. The partial genome sequence of the AGLV from V. bollonsii has 85.9–89.1% amino acid similarity to the Australian isolate AGLV-1998. This is the first record of the AGLV in Veronica species and in New Zealand.

Peas are an important legume for human and animal consumption and are also being used as green manure or intermediate crops to sustain and improve soil condition. Pea production faces constraints from fungal, bacterial, and viral diseases. We investigated the virome of German pea crops over the course of three successive seasons in different regions of pea production to gain an overview of the existing viruses. Pools from 540 plants, randomly selected from symptomatic and asymptomatic peas, and non-crop plants surrounding the pea fields were used for ribosomal RNA-depleted total RNA extraction followed by high-throughput sequencing (HTS) and RT-PCR confirmation. Thirty-five different viruses were detected in addition to nine associated nucleic acids. From these viruses, 25 are classified as either new viruses, novel strains or viruses that have not been reported previously from Germany. Pea enation mosaic virus 1 and 2 were the most prevalent viruses detected in the pea crops, followed by pea necrotic yellow dwarf virus (PNYDV) and turnip yellows virus which was also found also in the surrounding non-legume weeds. Moreover, a new emaravirus was detected in symptomatic peas in one region for two successive seasons. Most of the identified viruses are known to be aphid transmissible. The results revealed a high virodiversity in the German pea fields that poses new challenges to diagnosticians, researchers, risk assessors and policy makers, as the impact of the new findings are currently unknown., Graphical Abstract German pea virome.

UMR BFP - Equipe Virologie; International audience; Since the establishment of the genus Vitivirus, several additional viruses have been sequenced and proposed to represent new species of this genus. Currently, the International Committee on Taxonomy of Viruses recognizes 15 vitivirus species. The report of new vitiviruses that fail to completely adhere to the species demarcation criteria, the incorporation of non-vitivirus grapevine viruses in the unofficial "naming system", and the existence of non-grapevine vitiviruses lead to inconsistencies in classification. In this report, we give a brief overview of vitiviruses and use currently available information to clarify the present status of the vitivirus taxonomy.

Vitiviruses are ssRNA(+) viruses in the family Betaflexiviridae (subfamily Trivirinae). There are currently 10 ICTV recognized virus species in the genus; nevertheless, the extended use of NGS technologies is rapidly expanding their diversity and official recognition of six more have been proposed recently. Here, we present the characterization of a novel virus from grapevine, which fits the genomic architecture and evolutionary constraints to be classified within the Vitivirus genus. The detected virus sequence is 7607 nt long, including a typical genome organization of ORFs encoding a replicase (RP), a 22 kDa protein, a movement protein, a coat protein (CP) and a nucleic acid binding protein. Phylogenetic analyses based on the predicted RP and CP proteins unequivocally place the new virus within the Vitivirus genus. Multiple independent RNAseq data confirmed the presence of the detected virus in berries at diverse developmental stages. Additionally, we detected, confirmed, and assembled virus sequences from grapevine samples of distinct cultivars from America, Europe, Asia and Oceania, sharing 74.4%–97.8% nt identity, suggesting that the identified virus is widely distributed and diverse. We propose the name grapevine virus L (GVL) to the detected Vitivirus. Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Soltero Brisbane, Reid. Foundation Plant Services; Estados Unidos Fil: Voncina, Darko. University of Zagreb; Croacia Fil: Almeida, Rodrigo P.. University of California at Berkeley; Estados Unidos Fil: Blouin, Arnaud G.. No especifíca; Fil: Al Rwahnih, Maher. University of California at Berkeley; Estados Unidos Fil: Gómez Talquenca, Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Mendoza-San Juan. Estación Experimental Agropecuaria Mendoza; Argentina Fil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; Argentina

Red clover vein mosaic virus (RCVMV) is an important virus of leguminous crops that can cause devastating losses. During a routine survey of legumes conducted on the South Island of New Zealand, RCVMV was found in mixed infections in clover plants with Alfalfa mosaic virus and White clover mosaic virus. The full-length sequence of the New Zealand isolate RCVMV-NZ from clover shared 96% nucleotide sequence identity with a chickpea isolate previously described from Washington (United States). Targeted surveys of pea, faba bean, and pasture crops showed that RCVMV-NZ is widespread on the South Island in New Zealand. This isolate is causing mild if any symptoms on experimental hosts and naturally infected plants.

Grapevine geminivirus A (GGVA), a member of the family Geminiviridae, was originally described from the United States on Vitis vinifera, cultivars Black Beet and Nagano Purple, imported from Korea (Al Rwahnih et al. 2017). Since its initial characterization, GGVA has also been reported from the United States, China, and Korea in diverse Vitis spp. (Al Rwahnih et al. 2017; Fan et al. 2017; Jo et al. 2016). The transmission of GGVA by means of a vector and the economic impact of this virus are still unknown. In 2016, a small-scale, untargeted virus survey was done to assess the efficacy of high-throughput sequencing of small RNA (sRNA). Leaf samples from 20 vines were collected from the New Zealand Winegrowers’ germplasm collection, Lincoln, New Zealand. During the survey, GGVA was identified in an asymptomatic sample of Vitis vinifera, an interspecific cross Seibel 7052 (VID 280). sRNA was extracted using a mirVana miRNA Isolation Kit (Thermo Fisher Scientific). A library was prepared using the TruSeq Small RNA Sample Prep Kit (Illumina) and sequenced with 50-bp single-end reads (small RNA-Seq) on an Illumina HiSeq2000 at the Beijing Genomics Institute (Hong Kong), and a total of 32.6 million reads was obtained. When the reads were mapped to the reference sequence of GGVA (GenBank accession no. KX618694), 163,611 reads were assembled with a mean coverage level of 1,183× across the genome, yielding a near complete 2,902-nt GGVA genome sequence. Mapping was done using Bowtie2 (version 2.2.4) (Langmead and Salzberg 2012). Three viruses (grapevine leafroll-associated virus 2, grapevine rupestris stem pitting associated virus, and grapevine asteroid mosaic associated virus) and one viroid (hop stunt viroid) were also detected in this sample using the YABI Virus Surveillance and Diagnosis (VSD) toolkit (Barrero et al. 2017). The grapevine was resampled in July 2018, and the full genome was amplified by PCR using two abutting primers, 23-1F/2R (Fan et al. 2017). BLAST analysis of the sequence obtained (2,905 nt; GenBank accession no. MK690474) showed 99% nt similarity to the full genome of GGVA isolate Tamar (GenBank accession no. KX618694). The virus was detected in only one vine out of 20 (from 12 different cultivars) sequenced by sRNA-Seq from the same germplasm collection. Twenty additional grapevine samples from the South Island tested negative for GGVA by a species-specific PCR using the primers V1-F1/1R (Fan et al. 2017). To the best of our knowledge, this is the first report of GGVA in New Zealand.

A novel virus, with characteristics of viruses classified within the genus Vitivirus, was identified from a sample of Vitis vinifera cv. Chardonnay in New Zealand. The virus was detected with high throughput sequencing (small RNA and total RNA) and its sequence was confirmed by Sanger sequencing. Its genome is 7507 nt long (excluding the polyA tail) with an organisation similar to that described for other classifiable members of the genus Vitivirus. The closest relative of the virus is grapevine virus E (GVE) with 65% aa identity in ORF1 (65% nt identity) and 63% aa identity in the coat protein (66% nt identity). The relationship with GVE was confirmed with phylogenetic analysis, showing the new virus branching with GVE, Agave tequilina leaf virus and grapevine virus G (GVG). A limited survey revealed the presence of this virus in multiple plants from the same location where the newly described GVG was discovered, and in most cases both viruses were detected as co-infections. The genetic characteristics of this virus suggest it represents an isolate of a new species within the genus Vitivirus and following the current nomenclature, we propose the name “Grapevine virus I”.

We report a sequence of a novel vitivirus from Vitis vinifera obtained using two high-throughput sequencing (HTS) strategies on RNA. The initial discovery from small-RNA sequencing was confirmed by HTS of the total RNA and Sanger sequencing. The new virus has a genome structure similar to the one reported for other vitiviruses, with five open reading frames (ORFs) coding for the conserved domains described for members of that genus. Phylogenetic analysis of the complete genome sequence confirmed its affiliation to the genus Vitivirus, with the closest described viruses being grapevine virus E (GVE) and Agave tequilana leaf virus (ATLV). However, the virus we report is distinct and shares only 51% amino acid sequence identity with GVE in the replicase polyprotein and 66.8% amino acid sequence identity with ATLV in the coat protein. This is well below the threshold determined by the ICTV for species demarcation, and we propose that this virus represents a new species. It is provisionally named “grapevine virus G”.

Beetroot (Beta vulgaris subsp. vulgaris) is becoming increasingly popular in Germany with an increase in field production from 1,205 ha in 2013 to 1,826 ha in 2018 (Behr, 2019). It is estimated that EU-wide 24,000 ha of beetroot were…

Serological methods are routinely used for the detection of many economically important grapevine viruses. The most commonly used format is the enzyme-linked immunosorbent assay (ELISA) which provides a robust, sensitive and rapid method to screen large numbers of samples from the field. A number of companies provide high-quality ELISA kits against most of the main viral pathogens infecting grapevines. Although virus titre shows seasonal fluctuations and the viruses may be unevenly distributed in vines, particularly for recent infection, ELISA provides reliable diagnosis if samples are collected at the optimal time in the specified vine tissue. Relative quantification can be achieved and the dynamic range of the assay can be extended by calculation of reaction rates from a kinetic assay and interpolation of these rates onto a dose-response curve. Additionally, comparing reaction rates obtained using monoclonal or polyclonal antibodies for detection can assist the identification of novel serotypes of a virus. Results from ELISA should be supplemented by molecular tests in critical situations, and vice versa, since some viral strains may not be detected by one or other type of tests. Antibodies can also be used to trap viruses or double-stranded RNA (immunocapture), thus providing an inexpensive and simple way to concentrate and purify viral RNA for subsequent molecular analyses. Further techniques that may assist serological detection of viruses are described.

Grapevine leafroll–associated virus-3 (GLRaV-3) is transmitted from infected grapevines to healthy vines by mealybugs. The stylet penetration behaviour of Pseudococcus longispinus (Hemiptera: Pseudococcidae) was electronically monitored on leaves of grape plants to quantify the time taken to access the phloem and the duration of phloem ingestion. More of the first instar nymphs than of the adults showed sustained (>10 min) phloem ingestion; however, nymphs were affected adversely by longer monitoring periods. The probing and phloem ingestion periods of adults on GLRaV-3-infected Red Globe and uninfected Pinot noir were not significantly different from each other. Most adults showed the first phloem ingestion and the first sustained phloem ingestion after 12 h and after 24 h of their first probe, respectively. Free (unwired) and electrical penetration graph (EPG)-monitored (wired) P. longispinus adults were tested for acquisition of virus after different acquisition access periods (AAP). Acquired GLRaV-3 RNA in extracts of whole insects was detected by reverse transcription and polymerase chain reaction amplification. For the unwired adult mealybugs, 30 % that had an AAP of 48 h and 90 % that had an AAP of 72 or 96 h were positive for GLRaV-3. Five out of 24 (21 %) of the wired mealybugs allowed at least 24 h APP (1.8–44.4 h of phloem ingestion) were positive for GLRaV-3. None of the adult mealybugs acquired virus during the first 24 h of AAP. We conclude that GLRaV-3 acquisition by P. longispinus generally occurs within 24–72 h AAP with as little as 1.8 h of phloem ingestion.

The complete genomes of tobamovirus isolates from Plantago major L. from New Zealand (NZ-439), Plantago sp. from Germany (Kons 1105), Actinidia chinensis (Actinidia-AC) and A. deliciosa (Actinidia-AD) from China were sequenced and compared to previously published tobamovirus genomes. Their genome organization and phylogenetic analysis of the putative replicase component, replicase readthrough component, movement protein, coat protein and complete genome placed all four isolates in subgroup 3 of the tobamoviruses. The complete genomes differed from each other by

Although a number of protocols have been developed for detection of viruses at the genus or family level, universal approaches to detect and identify unknown viruses are still required. High-resolution tandem mass spectrometry was used to identify accurately peptide masses and their constituent sequences from partially purified plant virus preparations. Analysis of the peptide fragment masses against a virus database using pattern-matching algorithms identified sequences with homology to known virus peptides and also predicted peptides using de novo sequence analysis. This method provided sufficient information to confirm the identity of two known viruses that were included as controls (Cucumber mosaic virus and Tomato spotted wilt virus) and to identify unknown viruses in six viral isolates. The unknown viruses have been identified as four common viruses (Alfalfa mosaic virus, Tobacco streak virus, Citrus leaf blotch virus and Ribgrass mosaic virus), and two novel viruses (a potexvirus and a vitivirus). The identification of viruses from five distinct families by the tandem mass spectrometric determination of their coat protein demonstrates that this is a useful method for initial virus identification. This method, complemented with molecular or immunological procedures, provides a rapid and convenient way to identify both known and novel plant viruses.

Next-generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to justify their use in large-scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double-stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time-consuming and labour-intensive. We present an alternative method to enrich dsRNA from plant extracts using anti-dsRNA monoclonal antibodies in a pull-down assay. The extracted dsRNA can be amplified by reverse transcriptase-polymerase chain reaction and sequenced by next-generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand-native ornamental plant and a weed, respectively. Of the sequence data obtained, 31-74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.

Leaves of Plantago spp. growing in two regions of New Zealand were sampled and tested for the presence of tobamoviruses. Transmission, immunological and molecular studies revealed the presence of both Turnip vein clearing virus (TVCV) and Ribgrass mosaic virus (RMV). The phylogenetic analysis of complete coat protein amino acid sequences grouped these viruses in the RMV and TVCV clusters of sub group 3 tobamoviruses. This is the first confirmed report of these viruses from New Zealand.

A ssRNA virus from kiwifruit (Actinidia spp.) was identified as a member of the family Betaflexiviridae. It was mechanically transmitted to the herbaceous indicators Nicotiana benthamiana, N. clevelandii, N. glutinosa and N. occidentalis. The complete genome was comprised of three ORFs and a 3’poly (A) tail. Phylogenetic analysis of the entire genome indicated it was a novel member of the genus Citrivirus (family Betaflexiviridae). The complete nucleotide sequence differed from that of citrus leaf blotch virus (CLBV) by ~ 26 %. The movement protein (ORF2) and coat protein (ORF3) shared 95-96 % and 90-92 % amino acid sequence identity, respectively, with CLBV. The replicase polyprotein (ORF1) was distinctly different from published CLBV sequences, with 78-79 % amino acid sequence identity, while the 5’ UTR and 3’ UTR differed from CLBV by 28 % and 29 %, respectively. The sequence differences indicate that the citrivirus from Actinidia is either a divergent strain of CLBV or a member of a new citrivirus species.

Two co-infecting novel vitiviruses from Actinidia chinensis were identified from mechanically inoculated Nicotiana occidentalis. Both virus genomes were sequenced and share 64% nucleotide identity. Their overall structure is typical of vitiviruses, with five open reading frames (ORFs) and a polyadenylated 3′ end. Open reading frame 4 (ORF4) encodes the coat protein, the most conserved gene of the vitiviruses, in which they share 75% amino acid identity, 61-68% with grapevine virus B, 55-59% with grapevine virus A, and 37-42% with grapevine virus E. Based on the molecular criteria for species demarcation in the family Betaflexiviridae, these are two novel viruses, tentatively named Actinidia virus A and Actinidia virus B.

Ornamental flower bulbs are an increasingly important crop in New Zealand. In general, production is dependent on overseas breeding programmes and regular imports of propagation material, in particular from The Netherlands. Toenable the implementation of appropriate phytosanitary controls, a targeted survey was done in 2005–2006 to determine the presence of viruses in the most important bulb genera. Using serological and molecular methods, 10 viruses (Alstroemeria mosaic virus, Freesia sneak virus [syn. Freesia leaf necrosis-associated ophiovirus], Hippeastrum mosaic virus, Hyacinth mosaic virus, Nerine latent virus, Nerine yellow stripe virus, Ornithogalum virus 3, Tulip mosaic virus, Veltheimia mosaic virus and Vallota mosaic virus) were identified for the first time in New Zealand. In addition eleven new hosts of viruses reported previously in New Zealand were detected; these new associations include one not reported previously overseas (Tomato spotted wilt virus in Ornithogalum). None of the newly reported viruses cause significant diseases and it is unlikely that they will affect bulb production significantly.

Soil-borne wheat mosaic virus (SBWMV) was identified using transmission electron microscopy, enzymelinked immunosorbent assay (ELISA) with SBWMV-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) tests with SBWMV-specific primers and sequence comparison in two out of five samples collected from a wheat (Triticum aestivum) plant showing severe leaf mosaic. The plant was also infected with Barley stripe mosaic virus and Barley yellow dwarf virus-MAV and -PAV. A further 28 out of 200 wheat samples tested positive for SBWMV using ELISA. A comparison of 11 New Zealand SBWMV isolates indicated that they were all identical and had 98% nucleotide sequence identity with SBWMV isolates from the United States, subgroup New York-Illinois. At the location of the SBWMV outbreak the vector, Polymyxa graminis, was detected by PCR and the identity was confirmed by sequencing. This is the first report of SBWMV in New Zealand.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .