New Disease ReportsVolume 44, Issue 2 e12043 NEW DISEASE REPORTOpen Access Potato spindle tuber viroid detected from Solanum sisymbriifolium seed in trade A. Fowkes, A. Fowkes orcid.org/0000-0001-7918-441X Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Skelton, A. Skelton Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorL. Frew, L. Frew Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Buxton-Kirk, A. Buxton-Kirk Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorR. Ward, R. Ward Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorV. Harju, V. Harju orcid.org/0000-0003-3247-6698 Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorR. Weekes, R. Weekes Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Fox, Corresponding Author A. Fox Adrian.Fox@fera.co.uk orcid.org/0000-0003-1280-1836 Fera Science Ltd., York Biotech campus, Sand Hutton, York, United Kingdom Correspondence A. Fox, Fera Science Ltd., York Biotech campus, Sand Hutton, York, United Kingdom. Email: Adrian.Fox@fera.co.ukSearch for more papers by this author A. Fowkes, A. Fowkes orcid.org/0000-0001-7918-441X Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Skelton, A. Skelton Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorL. Frew, L. Frew Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Buxton-Kirk, A. Buxton-Kirk Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorR. Ward, R. Ward Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorV. Harju, V. Harju orcid.org/0000-0003-3247-6698 Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorR. Weekes, R. Weekes Fera Science Ltd., York Biotech campus, Sand Hutton, York, United KingdomSearch for more papers by this authorA. Fox, Corresponding Author A. Fox Adrian.Fox@fera.co.uk orcid.org/0000-0003-1280-1836 Fera Science Ltd., York Biotech campus, Sand Hutton, York, United Kingdom Correspondence A. Fox, Fera Science Ltd., York Biotech campus, Sand Hutton, York, United Kingdom. Email: Adrian.Fox@fera.co.ukSearch for more papers by this author First published: 27 October 2021 https://doi.org/10.1002/ndr2.12043AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Solanum sisymbriifolium (sticky nightshade) originates from South America and was initially introduced to Europe as an ornamental plant. However, the species has recently been added to the European and Mediterranean Plant Protection Organisation alert list due to the high fecundity of the species, leading to the plant being considered a noxious weed in Sicily and coastal areas in the Mediterranean region (European and Mediterranean Plant Protection Organisation, 2021). With the reduction in availability of chemical controls for potato cyst nematode (PCN; Globodera pallida and G. rostochiensis) the species has recently received increased interest for use as a trap crop. This is because the root exudates promote hatching of PCN juveniles but do not allow establishment of nematode populations. Throughout 2018, 19 samples consisting of 1000 seeds each, and one sample of 3000 seeds, were submitted to Fera Science Ltd. from seed lots of S. sisymbriifolium, to be tested for Potato spindle tuber viroid (PSTVd). These were submitted as part of commercial "due diligence" testing prior to importing seed into the UK for efficacy trials. Each of the seed lots was subdivided into sub-samples of 250 seeds, i.e. four sub-samples for each of the 1000 seed samples, and 12 sub-samples for the 3000 seed sample. RNA was extracted from the seeds using a magnetic bead extraction method (Kingfisher Flex, Thermo Scientific, UK) and tested by real-time RT-PCR using an assay that detects PSTVd, Tomato chlorotic dwarf viroid and Tomato planta macho viroid (Boonham et al., 2004). Five samples tested positive using this assay. To identify the species of pospiviroid detected, RNA extracts were tested by RT-PCR with a Verso 1-step RT-PCR Reddymix kit (Thermo Scientific, USA) using pospi 1 FW and pospi 1 RE primers (Verhoeven et al., 2004). Bands were detected of the correct size (c. 196 bp) for sub-sample extracts from five samples. The resulting PCR products were sent for sequencing using the above primer sets (Sequiserve, Germany or Eurofins, Germany). PSTVd was confirmed in all five samples by sequence comparison using BLAST, with >99.4% identity to an isolate from The Netherlands (KY936876), and the sequences were added to GenBank (MZ226444-MZ226448). Due to the samples being submitted during commercial testing, the origin of the seed samples is unknown. Whilst S. sisymbriifolium has only previously been listed as an experimental host of PSTVd (Diener, 1979), it has previously been intercepted in this host by the Netherlands Plant Protection Service. These interceptions indicate that the species is a natural host of PSTVd, and contaminated seeds are present in commercial seed lots of this host which could pose a plant health risk through the introduction of the viroid (Netherlands Food and Consumer Product Safety Authority, 2017). Given the increased interest in the use of S. sisymbriifolium as a trap crop for PCN management in potato rotations, the role of this crop as a host of PSTVd and the risk of seed transmission and transmission via roots should be further investigated. ACKNOWLEDGEMENTS This work was partially funded under the Defra-Fera Long Term Service Agreement. REFERENCES Boonham N, González Pérez L, Mendez MS, Lilia Peralta E, Blockley A, Walsh K, Barker I, Mumford RA, 2004. Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. Journal of Virological Methods 116, 139– 146. https://doi.org/10.1016/j.jviromet.2003.11.005CrossrefCASPubMedWeb of Science®Google Scholar Diener TO, 1979. Viroids and viroid diseases. New York, NY: John Wiley & Sons. Web of Science®Google Scholar European and Mediterranean Plant Protection Organization, 2021. EPPO Alert List – Solanum sisymbriifolium (Solanaceae). https://www.eppo.int/ACTIVITIES/plant_quarantine/alert_list_plants/solanum_sisymbriifolium. Accessed 12/03/2021. Google Scholar Netherlands Food and Consumer Product Safety Authority, 2017. First finding of Potato spindle tuber viroid (PSTVd) in seeds of Solanum sisymbriifolium, originating in Asia. https://english.nvwa.nl/documents/plant/plant-health/pest-reporting/documents/first-finding-of-potato-spindle-tuber-viroid-pstvd-in-seeds-of-solanum-sisymbriifolium-originating-in-asia. Accessed 14/10/2021. Google Scholar Verhoeven JThJ, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW, 2004. Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology 110, 823– 31. https://doi.org/10.1016/j.jviromet.2003.11.005CrossrefWeb of Science®Google Scholar Volume44, Issue2October–December 2021e12043 ReferencesRelatedInformation

InMay 2018, tomato leaves (Solanum lycopersicum cv. Delisher) exhibiting symptoms of necrotic patches were collected from a glasshouse in Southern England. It was suspected that the samples were infected with Tomato chlorosis virus (ToCV), genus Crinivirus, as ToCV had been detected in a different variety on the same holding. Analysis was done by high throughput sequencing (Fox et al., 2019). The presence of ToCV could not be confirmed. A total of 3,453,344 reads were produced from the sample and 652 were found to represent the complete genome sequence of Southern tomato virus (STV), genus Amalgavirus (GenBank Accession No.MW266062) with an average coverage of 34 and with 100% nucleotide sequence identity to the original isolate of STVdiscovered in2009 (EF442780.1). To confirm the presence of this virus, a specific real-timeRT-PCR for STV (Table 1)was designed using PRIMER EXPRESS v.2 (Life Technologies Co., USA). The sample tested positive using this assay.

In March 2017, 52 dried-leaf samples of brassicaceous plants from the Philippine Highlands, one of which was a sample of cabbage (Brassica oleracea var. capitata; Fig. 1), were submitted to Fera Science Ltd., via the Centre for Agriculture and Bioscience International (CABI). The plants were all displaying virus-like, yellowing symptoms (Reeder et al., 2017). Initially, samples were bulked together and tested by ELISA for the presence of viruses known to occur in the Brassicaceae including Cucumber mosaic virus (CMV) (Agdia, USA), Turnip mosaic virus (TuMV) and Turnip yellows virus (TuYV) (Loewe, Germany). All the bulked samples gave negative results for CMV, TuMV and TuYV, and the samples were also tested individually for the presence of Turnip yellow mosaic virus (TYMV) (DSMZ, Germany). All Chinese cabbage (Brassica rapa subsp. pekinensis) samples were positive for TYMV as described by Reeder et al. (2017). However, the Brassica oleracea var. capitata sample tested negative for TYMV and was therefore screened for virus infection using an Illumina MiSeq as described by Adams et al. (2014). A total of 857,018 reads were obtained from the sample, 924 were assembled to yield the complete coding sequence of a polerovirus (GenBank Accession No. MW537050). The average sequencing depth of this contig was 31. Comparison at the whole genome level initially suggested that the isolate was either Brassica yellows virus or TuYV with significant nucleotide identity (>94%) to examples of both viruses. Recombination analysis using RDP 4 (Martin et al., 2015) for both TuYV (NC003743) and BrYV (NC016038) type isolates suggested that this isolate has a small recombination event in the P3-P5 region. Filardo et al. (2021) have proposed that TuYV and BrYV are the same species and describe isolates with a similar P3-P5 recombination as within a clade (group 2) of their unified TuYV species. Therefore, this isolate has been identified as containing a group 2 TuYV. A specific real-time RT-PCR primer/probe set was designed based on both TuYV and BrYV sequences. The primer/probe sequences are as follows TuYV-F2: 5′-GCCGCYTGTTTCTCAGTTCTG-3′; TuYV-R2: 5′-RACTAACCACGAGTAAAGAAGCTCAA-3′; TuYV-P2 (probe): [FAM] 5′-ACGAGTTGCGGCAYGATCCAGC[BHQ1] -3′. Testing the Brassica oleracea var. capitata sample by the real-time RT-PCR assay yielded a positive result, whereas the sample had tested negative by ELISA, suggesting the ELISA does not detect all strains of TuYV. TuYV is widespread in cruciferous plants across mainland China, South Korea and Japan (Zhang et al., 2016) but this is the first report of TuYV in the Philippines. Further work is needed to determine the prevalence of TuYV in cruciferous plants in the Philippines and identify the key aphid vectors. This information could then be used to inform future management strategies. This testing was partially funded through the Defra-Fera Long Term Service Agreement. The authors would like to thank CABI for their support.

In July 2019 a sample of tomato leaves (Solanum lycopersicon cv. Piccolo) was submitted to Fera Science Ltd, from a grower in the South of England. The sample was sent in following the appearance of mosaic symptoms, which had spread throughout the crop in the affected glasshouse. Symptoms were consistent with a viral infection and had emerged following inoculation with a mild strain of Pepino mosaic virus (PepMV) as a cross-protection treatment, however symptoms had persisted post-inoculation. The sample was tested using ELISA kits for the presence of PepMV (Bioreba, Switzerland) plus Tobacco mosaic virus, Tomato brown rugose fruit virus (ToBRFV) and Tomato mosaic virus (DSMZ, Germany), according to the manufacturers' instructions. The sample tested positive for the presence of ToBRFV (Tobamovirus) and PepMV but negative for the other viruses. The ToBRFV result was confirmed by conventional RT-PCR using ToBRFV-specific primers ToBRFV-F/ToBRFV-R (Alkowni et al., 2) and with generic tobamovirus primers 514 Tombamo-s1/515 Tobamo-as1 (Menzel et al., 3). PCR products of the expected sizes (560 bp for both primer sets) were obtained, sequenced and confirmed to be ToBRFV by nucleotide sequence comparison (GenBank Accession No. MN210325 and MN210326 respectively). Genome sequences for ToBRFV and PepMV in the sample were also obtained by sequencing a TruSeq ribosome depleted plant leaf library (Illumina, UK) on a MiSeq sequencer (Illumina, UK). Sequencing and analysis were done as described previously (Adams et al., 1). The genome of ToBRFV (MN182533) had between 99.7-99.9% identity to genomes of ToBRFV isolates from Israel (KX619418.1), Jordan (KT383474.1), Germany (MK133095.1, MK133093.1), Mexico (MK319944.1) and Palestine (MK165457.1). The genome of PepMV (MN182534) had high identity (99.6%) to that of an isolate of PepMV commonly used for cross protection (Menzel et al., 3). ToBRFV was first reported from symptomatic crops in Israel in 2014, with the presence of the virus being confirmed following a similar outbreak in Jordan in 2015, with recent reports from Germany (2018) and Turkey (2019) (EPPO, 2019) and China (Yan et al., 4). The virus overcomes the tomato tobamovirus-resistance gene TM-22. Fruit from ToBRFV-infected plants are known to mature irregularly and can be mottled with yellow or brown spots making fruit unmarketable. The virus may also pose a risk to pepper crops (EPPO, 2019). Following reporting of the presence of ToBRFV the grower voluntarily removed all plants from the affected glasshouse and destroyed these in accordance with guidance from the UK National Plant Protection Organization. Work is being conducted to investigate the possible source of the outbreak. This work was funded under the Defra-Fera Long Term Service Agreement

New Disease ReportsVolume 39, Issue 1 p. 23-23 ArticleOpen Access Potato spindle tuber viroid detected in seed of uncultivated Solanum anguivi, S. coagulans and S. dasyphyllum collected from Ghana, Kenya and Uganda A. Skelton, Corresponding Author A. Skelton anna.skelton@fera.co.uk Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Buxton-Kirk, A. Buxton-Kirk Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fowkes, A. Fowkes Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorV. Harju, V. Harju Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorR. Ward, R. Ward Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorL. Frew, L. Frew Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorO. Wagstaff, O. Wagstaff The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorT.R. Pearce, T.R. Pearce The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorJ. Terry, J. Terry The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorJ. Dickie, J. Dickie The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorC. Cockel, C. Cockel The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorD.O. Nyamongo, D.O. Nyamongo Kenya Genetic Resources Research Institute, Nairobi, KenyaSearch for more papers by this authorL.M. Aboagye, L.M. Aboagye Plant Genetic Resources Research Institute, Bunso, GhanaSearch for more papers by this authorJ.M. Wasswa, J.M. Wasswa Plant Genetic Resources Centre, Entebbe, UgandaSearch for more papers by this authorA. Fox, A. Fox Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this author A. Skelton, Corresponding Author A. Skelton anna.skelton@fera.co.uk Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Buxton-Kirk, A. Buxton-Kirk Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fowkes, A. Fowkes Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorV. Harju, V. Harju Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorR. Ward, R. Ward Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorL. Frew, L. Frew Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorO. Wagstaff, O. Wagstaff The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorT.R. Pearce, T.R. Pearce The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorJ. Terry, J. Terry The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorJ. Dickie, J. Dickie The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorC. Cockel, C. Cockel The Royal Botanic Gardens, Kew, Millennium Seed Bank, Wakehurst Place, Ardingly, West Sussex, RH17 6TNSearch for more papers by this authorD.O. Nyamongo, D.O. Nyamongo Kenya Genetic Resources Research Institute, Nairobi, KenyaSearch for more papers by this authorL.M. Aboagye, L.M. Aboagye Plant Genetic Resources Research Institute, Bunso, GhanaSearch for more papers by this authorJ.M. Wasswa, J.M. Wasswa Plant Genetic Resources Centre, Entebbe, UgandaSearch for more papers by this authorA. Fox, A. Fox Fera Science Ltd., Sand Hutton, York, YO41 1LZ UKSearch for more papers by this author First published: 20 June 2019 https://doi.org/10.5197/j.2044-0588.2019.039.023Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Adapting Agriculture to Climate Change (Crop Wild Relatives) is a project, jointly managed by the Global Crop Diversity Trust and the Millennium Seed Bank, Royal Botanic Gardens, Kew (RBG Kew). As part of this project, seed of 30 wild taxa related to Solanum melongena (aubergine) were collected from seven countries. These Solanum species grow wild in Africa and are used for food and/or medicinal purposes. No virus or viroid symptoms were seen on the plants at collection. Initial cleaning and drying of collected seed was carried out in-country before being shipped to RBG Kew, where debris was removed, and the samples x-rayed, counted and sealed in universal glass bottles and stored at -20°C. In June 2018, 98 seed samples (100 seeds per sample) of the solanaceous species were sent from RBG Kew to Fera Science Ltd. to be tested for Potato spindle tuber viroid (PSTVd) to facilitate export to the World Vegetable Center, Taiwan for pre-breeding research. This pre-breeding research is the initial step aimed at identifying important genetic diversity within crop wild relatives to enable subsequent breeding of domesticated crops that are better adapted to future climatic conditions. Seed samples were of eight species, namely Solanum anguivi, S. anomalum, S. cerasiferum, S. coagulans, S. dasyphyllum, S. incanum, S. macrocarpon and S. virginianum. Solanum anguivi and S. dasyphyllum were considered priority species in the current project, due to the lack of existing collections. Due to the small consignment size, an initial screen was done on a subsample of 10 seeds per sample. RNA was extracted from the seeds using a magnetic bead extraction method and tested by real-time RT-PCR using an assay that detects PSTVd, Tomato chlorotic dwarf viroid and Tomato planta macho viroid (Boonham et al., 2). Eight of the samples tested positive using this assay: five samples of S. anguivi, two of S. dasyphyllum and one of S. coagulans. To identify the species of pospiviroid detected, RNA extracts were tested by RT-PCR with a Verso 1-step RT-PCR Reddymix kit (Thermo Scientific, USA) using 3H1-F and 2H1-R primers (Shamloul et al., 4) and pospi 1 FW and pospi 1 RE primers (Verhoeven et al., 2). Bands were detected of the correct size (c. 196 bp) for all eight samples using the Verhoeven et al. (5) primers and for three samples using the Shamloul et al. (4) primers (c. 360 bp). The resulting PCR products were sent for sequencing using the above primer sets (Sequiserve, Germany). PSTVd was confirmed in all eight samples and the sequences added to GenBank (Table 1). Herbarium specimens collected alongside the seed collections were also tested by real-time RT-PCR using the Boonham et al. (2) assay. Two of the herbarium samples tested positive and were tested by RT-PCR using the Verhoeven et al. (5) assay. The resulting PCR products were sent for sequencing, PSTVd was identified and sequences added to GenBank (Table 1). The available sequence for the related herbarium and seed samples shared 98-99% homology. Table 1. Seed species, authorin’ for binomial name, geographic origin and GenBank accession numbers for associated sequence Species (Authoriny) Country of collection Region Date of collection GenBank Accession No. of viro id sequence GenBank Accession No. of related herbarium specimen Solanum anguín (Lam.) Ghana Brong-Ahafo, Sunyani November 2016 MK330993 Solanum anguín (Lam.) Uganda Central Region, Mityana April 2016 MK330986 Solanum anguín (Lam.) Uganda Central Region, Mityana April 2016 MK3 30990 Solanum anguín (Lam.) Uganda Western region. Kisoro August 2016 MK330989 Solanum anguín (Lam.) Uganda Western region, Kibaale August 2016 MK330995 MK330992 Solanum coagulons (Forssk.) Kenya Rift Valley, Samburu August 2016 MK330994 MK330991 Solanum dasyphyllum (Schumach & Thonn.) Uganda Central region, Gomba July 2016 MK33098S Solanum dasyphyllum (Schumach & Thonn.) Uganda Centred region, Gomba April 2016 MK330987 This is the first report of a pospiviroid being detected in S. anguivi, S. coagulans and S. dasyphyllum, confirming the potential for pospiviroids to be distributed through non-commercial seed. There are very few reports of PSTVd in Africa; the viroid was detected in Egypt in potato and in Nigeria (CABI, 3) and in Ghana in tomato (Batuman et al., 1). These data indicate that PSTVd may be more widely distributed than suggested by current reports. PSTVd-infected seed collections and related herbarium specimens were destroyed or transferred under quarantine licence to Fera Science Ltd. for research purposes. Figure 1Open in figure viewerPowerPoint Acknowledgments This testing was funded through the Defra-Fera Long Term Service Agreement. The Adapting Agriculture to Climate Change (Crop Wild Relatives) project is funded by the Government of Norway. References 1Batuman O, Çiftçi OC, Osei MK, Miller SA, Rojas MR, Gilbertson RL, 2019. Rasta disease of tomato in Ghana is caused by the pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid. Plant Disease (in press). 10.1094/PDIS-10-18-1751-RE 2Boonham N, Pérez LG, Mendez MS, Peralta EL, Blockley A, Walsh K, Barker I, Mumford RA, 2004. Development of a real-time RT-PCR assay for the detection of Potato spindle tuber viroid. Journal of Virological Methods 116, 139– 146. 10.1016/j.jviromet.2003.11.005 3 CABI (2018) Potato spindle tuber viroid (spindle tuber of potato) datasheet. CABI Invasive Species Compendium. https://www.cabi.org/cpc/datasheet/43659. (Accessed 1 April 2019). 4Shamloul AM, Hadidi A, Zhu SF, Singh RP, Sagredo B, 1997. Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants. Canadian Journal of Plant Pathology 19, 89– 96. 10.1080/07060669709500580 5Verhoeven JTJ, Jansen CCC, Willemen TM, Kox LFF, Owens RA, Roenhorst JW, 2004. Natural Infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology 110, 823– 831. 10.1007/s10658-004-2493-5 Citing Literature Volume39, Issue1January 2019-June 2019Pages 23-23 FiguresReferencesRelatedInformation

In January 2016, 35 leaf dried samples of chilli pepper (Capsicum sp.) were submitted to Fera Science Ltd, from six districts in Rwanda: Ruhango, Nyanza, Rulindo, Kayonza, Nyagatare and Kirehe. The samples were sent in following the appearance of a suspected virus in the chilli pepper crops. Symptoms before drying included distorted leaves, stunting and mosaic (Fig. 1). Chilli pepper is an important horticultural crop in Rwanda; in 2017, 27 tones chilli pepper were exported, mainly to the UK, The Netherlands and Belgium, worth approximately US $54,000 (National Agricultural Export Development Board, Rwanda). Following a previous finding of an unknown potyvirus in a sample of Capsicum with the same symptoms from Rwanda in June 2015, the samples were tested by ELISA with generic potyvirus antisera from the Leibniz-Institut DSMZ (Braunschweig, Germany). A potyvirus was detected by ELISA in all the samples. To try to identify the potyvirus by sequencing, the samples were tested by PCR using the P9502 and CPUP potyvirus primers (van der Vlugt et al., 3). However, the presence of a potyvirus could not be confirmed in this way. The samples were also tested by ELISA for known potyviruses found in Capsicum, including Chilli veinal mottle virus (DSMZ) and Potato virus Y (Bioreba, Reinach, Switzerland). These ELISA tests were negative. Therefore, one of the samples from the Ruhango region was screened using an Illumina MiSeq as described by Adams et al. (1). Sequences for the following viruses were derived from the MiSeq run and added to GenBank: Pepper veinal mottle virus (PVMV, genus Potyvirus; GenBank Accession No. MG470801, Pepper yellows virus (PeYV, genus Polerovirus; MG470802) which is a newly described virus detected in pepper (Lotos et al., 2), Cucumber mosaic virus (CMV, genus Cucumovirus; MG470798, MG470799 and MG470800) and a novel virus which is tentatively a member of the genus Enamovirus (MG470803). Real time PCR assays were designed (Table 1) to PVMV and PeYV and all the samples were tested using these assays. Both PVMV and PeYV were detected in samples from Ruhango, Nyanza and Nyagatare. However, PeYV but not PVMV, was detected in samples from Kayonza, Rulindo and Kirehe. Following the sequencing results the samples were also tested by ELISA to confirm CMV infection (Agdia, Elkhart, Indiana, USA). CMV was detected in the sample tested by next generation sequencing (Ruhango region) and in samples from Nyanza and Rulindo, but not in the samples from Kayonza, Nyagatare and Kirehe. This is the first confirmed report of PVMV and PeYV in Rwanda and the first detection of a novel enamovirus. Work is ongoing to try to characterise the enamovirus. This testing was partially funded through the Defra-Fera Long Term Service Agreement. The authors would like to thank CABI for supporting this work through the Plantwise Programme.

New Disease ReportsVolume 38, Issue 1 p. 25-25 ArticleOpen Access Plantago asiatica mosaic virus detected in Lilium in the UK V. Harju, Corresponding Author V. Harju val.harju@fera.co.uk Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorH. Tozer, H. Tozer Plant Health and Seed Inspectorate, Animal and Plant Health Agency, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZSearch for more papers by this authorA. Dible, A. Dible Plant Health and Seed Inspectorate, Animal and Plant Health Agency, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZSearch for more papers by this authorA. Buxton Kirk, A. Buxton Kirk Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fowkes, A. Fowkes Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Skelton, A. Skelton Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fox, A. Fox Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this author V. Harju, Corresponding Author V. Harju val.harju@fera.co.uk Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorS. Forde, S. Forde Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorH. Tozer, H. Tozer Plant Health and Seed Inspectorate, Animal and Plant Health Agency, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZSearch for more papers by this authorA. Dible, A. Dible Plant Health and Seed Inspectorate, Animal and Plant Health Agency, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZSearch for more papers by this authorA. Buxton Kirk, A. Buxton Kirk Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fowkes, A. Fowkes Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Skelton, A. Skelton Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this authorA. Fox, A. Fox Fera Science Ltd, National Agri-food Innovation Campus, Sand Hutton, York, YO41 1LZ UKSearch for more papers by this author First published: 05 December 2018 https://doi.org/10.5197/j.2044-0588.2018.038.025Citations: 2AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat In April 2018, a sample of Oriental hybrid lily (Lilium spp.) with distinctive symptoms was submitted to Fera Science Ltd. for diagnosis from a nursery in southern England by the Plant Health and Seed Inspectorate (PHSI). From the rusty-coloured, necrotic streaking on the leaves it was initially suspected that Plantago asiatica mosaic virus (PlAMV, genus Potexvirus) was the causal agent (Fig.1). Figure 1Open in figure viewerPowerPoint Total RNA was extracted from the sample using magnetic beads and tested with a Verso 1-step RT- PCR Reddymix kit (Thermo Scientific, USA) using generic potexvirus primers, reverse Potex 4 and forward Potex 5 primers (Miglino et al., 5). A 280 bp amplicon was purified and sent for sequencing (Genbank Accession No. MK005152). This sequence had 99% identity to PlAMV (KY807785). The result was confirmed using another RT-PCR assay with primers corresponding to the PlAMV coat protein gene sequence (Parrella et al., 6), and the same PCR master mix as used previously, to generate a 1 kb amplicon (MK005151). Sequence comparisons confirmed that the virus was PlAMV with 99% identity to Accession No. KX245539. The infected plants identified in 2018 were grown in the UK from bulbs originating in The Netherlands, where PlAMV has previously been reported (Anonymous, 2). A lily sample was diagnosed by Fera as being infected with PlAMV in 2012, (Anderson et al., 1). These bulbs were grown in Spain and intercepted in the UK by PHSI, and the virus was not thought to have established. The lily samples in 2012 and 2018 were both from plants or propagation material originating in another country however the 2018 outbreak is the first report of PlAMV being detected from lilies actually grown on-site in the UK. PlAMV has been reported in several countries in Europe, the Americas, Korea, China and Japan and can infect natural hosts including Plantago asiatica (Russian Far East, Korea), Nandina domestica (USA), Rehmannia glutinosa (Korea) and Lilium and Primula spp (Japan), as well as a number of wild plant hosts (CABI, 3). The extent of the 2018 outbreak was limited to a few glasshouse plants. Virus symptoms were not seen during site inspections in previous years. PlAMV is not regulated in the UK however necrotic streaking on the leaves makes infected lilies unsaleable. Losses of up to 80% have been reported in commercial cut-flower lily production in The Netherlands (Anonymous, 2). Virus control is managed by the growers as this potexvirus can be transmitted both in contaminated soil and during the bulb washing processes via contaminated water (de Kock, 4). Appropriate phytosanitary recommendations were put in place in the affected glasshouses to prevent further spread. Acknowledgments This work was supported by Defra under its Plant Health Long-Term Service Agreement (LTSA) with Fera Science Ltd. References 1Anderson H, Fox A, Matthews-Berry S, Reed P, Skelton A, 2012. Rapid pest risk analysis for Plantago asiatica mosaic virus. The Food and Environment Research Agency, York, UK. https://secure.fera.defra.gov.uk/phiw/riskRegister/downloadExternalPra.cfm?id=4074. Accessed 30 August 2018. 2 Anonymous (2010) Pest report -The Netherlands: Plantago asiatica mosaic virus on Lilium spp. Ministry of Agriculture, Nature and Food Quality, The Netherlands. https://145.12.37.103/txmpub/files/?p_file_id=2001424. Accessed 30 August 2018. 3 CABI, 2018. Plantago asiatica mosaic virus. https://www.cabi.org/isc/datasheet/121894. Accessed 30 August 2018. 4deKock MJD, Slootweg G, Aanholt JTM, Lemmers MEC, Pham KTK, Dees RHL, Boer FA, Hollinger TC, 2013. Understand and combat groundbreaking spread of PIAMV and TVX [in Dutch]. Lisse, The Netherlands: Business Unit Bloembollen, Boomkwekerij & Fruit. 5Miglino R, Jodlowska A, vanSchadewijk AR. 2006. Detection and identification of a novel potexvirus infecting Allium by paramagnetic beads, ssRNA isolation and one tube RT-PCR assay with a new potexvirus genus primer set. Acta Horticulturae 722, 285– 292. 10.17660/ActaHortic.2006.722.35 6Parrella G, Greco B, Pasqualini A, Nappo. AG, 2015. Plantago asiatica mosaic virus found in protected crops of lily hybrids in southern Italy. Plant Disease 99, 1289. 10.1094/PDIS-03-15-0281-PDN Citing Literature Volume38, Issue1July 2018-December 2018Pages 25-25 FiguresReferencesRelatedInformation

In September 2017, a sample of mulberry leaf (Morus alba cv. Capsrum) was submitted from a nursery in Worcestershire, to Fera Science Ltd. Over 100 trees were affected with a chlorotic oak leaf line pattern on the fully developed leaves which was consistent with viral infection (Fig. 1). The sample was tested by ELISA for Arabis mosaic virus, Raspberry ringspot virus, Tomato black ring virus (TBRV), Tomato spotted wilt virus (all antisera provided by DSMZ, Germany), Impatiens necrotic spot virus, Strawberry latent ringspot virus, TBRV (Bioreba, Switzerland) and Cucumber mosaic virus (CMV) (Agdia, USA). Healthy control mulberry was not available, so a composite of several plants was used, including tomato and Nicotiana tabacum. ELISA testing was negative except for a positive reaction for CMV. To confirm the CMV positive result, the mulberry was inoculated onto Chenopodium quinoa, N. glutinosa, N. hesperis, N. occidentalis P1 and N. tabacum. Twenty-one days post inoculation no virus symptoms were seen. The sample was also tested by real-time PCR for CMV (Table 1), CMV was not detected. After the initial testing asymptomatic mulberry leaves were also tested by ELISA for CMV and again a positive ELISA reaction was obtained. To further investigate the virus infection status, the sample was screened using an Illumina MiSeq as described by Adams et al. (1). From these data the presence of Prunus necrotic ringspot virus (PNRSV, genus Ilarvirus, family Bromoviridae) and Mulberry cryptic virus 1 (suggested acronym MuCV1, tentative member of the family Partitiviridae) was inferred. The sequences were submitted to GenBank, Accession Nos. MH282499 and MH282498, respectively. No CMV sequences were detected. Considered with the real-time PCR and sap inoculation results, the CMV ELISA reaction was an erroneous result. Given the likely cross reaction of mulberry leaf homogenate with the CMV antisera, mulberry may be a problematic host for ELISA testing. To confirm the PNRSV finding the sample was tested by RT-PCR using primers C and D (Sanchez-Navarro et al, 3) and the resulting PCR product was sent for sequencing. PNRSV was confirmed (MH282500). Cryptic viruses generally induce no or only very mild symptoms (Boccardo et al, 2), therefore the oak leaf-like pattern seen may be caused by PNRSV, or by a synergistic effect with Mulberry cryptic virus 1. This is the first report of PNRSV in mulberry. As the only other report of Mulberry cryptic virus 1 is an incidental detection from mulberry in Fayetteville, Arkansas, USA (GU145316.1) (I. Tzanetakis, pers. comm.), this is the first report of MuCV1 in Europe. This testing was funded through the Defra-Fera Long Term Service Agreement.

In 2012, vegetable farmers in the Philippine highlands (Buguias and Benguet provinces) first observed unusual yellowing symptoms in Chinese cabbage (Brassica rapa subsp. pekinensis) planted in their fields. The symptoms were pronounced vein clearing, vein yellowing and a bright yellow mosaic. Similar symptoms were again seen in 2013 with more farmers reporting serious crop losses. The Benguet State University Plant Pest Clinic initially identified the problem as a viral disease but were unable to identify the cause further. The disease continued to affect farmers in subsequent years and in a varietal field trial conducted from October 2015 to February 2016 the incidence ranged from 10-32%. In January of 2017 during a field visit to the provinces of Buguias and Benguet, virus-like symptoms were observed on Chinese cabbage (Figs. 1-3), rocket (Eruca sativa; Fig. 4), cabbage (Brassica oleracea), pak choi (Brassica rapa subsp. chinensis) and radish (Raphanus raphanistrum subsp. sativus). In addition to the virus symptoms the plants were observed to be heavily infested with adult striped flea beetle (Phyllotreta striolata). The viral disease was widespread on Chinese cabbage infecting approximately 25% of the crop (Fig. 5). Symptomatic leaves were taken from the brassicaceous plants at 17 sampling locations. In total leaves from 48 Chinese cabbage plants were selected as well as leaves from individual plants of pak choi, rocket, radish and cabbage. The leaf samples were packaged up and sent to the Diagnostic and Advisory Service, CABI, UK for further analysis. On examination the samples were subsequently forwarded onto Fera Science Ltd for virus testing. The dried samples were bulked together for virus testing using ELISA. All samples tested negative for known viruses of Brassicaceae, such as Cucumber mosaic virus, Turnip mosaic virus and Turnip yellows virus. However, the bulked sample was positive for Turnip yellow mosaic virus (TYMV, genus Tymovirus). In order to ascertain how widespread the virus was, representative leaf samples from each host at each location were tested for TYMV individually using ELISA. All of the leaf samples of Chinese cabbage tested positive as did one sample of rocket. However, there were no positives for TYMV for leaf samples of cabbage, pak choi or radish. To confirm the ELISA results several of the samples were tested by RT-PCR using a TYMV primer set (Lee & Rho, 2015). Amplicons of the expected size were detected (491 bp) in all the Chinese cabbage samples tested and the rocket sample. The PCR products for one of the Chinese cabbage samples and the rocket sample were sent for sequencing. Consensus sequence data was obtained using Mega 4.1 software. Nucleotide sequences were compared by searching the BLASTn database, confirming TYMV in both samples (95% nucleotide sequence identity to GenBank Accession No. X07441). The sequences were added to GenBank (MF576298, Chinese cabbage; and MF576299, rocket). TYMV is confined almost entirely to the Brassicaceae family, and has been reported in the former Czechoslovakia, Italy, Spain, UK, Japan, Canada, Australia and New Zealand. However, to the best of the authors' knowledge this is the first report of this virus in the Philippines. The virus is seed transmitted with relatively low rates of transmission (2.5-2.9%) (Alfaro-Fernández et al., 1) and this may have been how it arrived in the Philippines. Transmission in the field is by flea-beetles, species of Phyllotreta and Psylliodes, and the mustard beetle (Phaedon cochleariae) including its larvae. Future studies to investigate the transmission and management options for the virus in the Philippines are being planned.

In August 2016 a sample of Veronica gentianoides cv. Tissington White was submitted to Fera Science Ltd. from a nursery in West Sussex. The sample was submitted with symptoms of severe distortion and mottling and these symptoms were widespread throughout the nursery which could affect their marketability. The sample was tested by ELISA for Cucumber mosaic virus (Agdia, USA), Arabis mosaic virus, potyviruses, Tobacco ringspot virus, Tomato ringspot virus and Tomato spotted wilt virus (DSMZ, Germany), Impatiens necrotic spot virus and Strawberry latent ringspot virus (Bioreba, Switzerland). The sample was negative for all viruses tested. The sample was subsequently tested for Tobacco rattle virus using RT-PCR with specific primers (Mumford et al., 3) but also tested negative. To check for the presence of virus the sample was then mechanically inoculated onto Chenopodium quinoa, Nicotiana benthamiana, N. hesperis and N. occidentalis P1. Thirteen days post inoculation all plants showed symptoms such as chlorotic spots, distortion and necrosis (Figs. 1-2). RNA was extracted from symptom-bearing leaves of N. benthamiana using an RNeasy Plant Mini Kit (Qiagen, UK), and a sequencing library prepared using the Scriptseq complete plant root kit (Illumina, UK) and sequenced along with other indexed samples on an Illumina MiSeq using a 2times300 bp V3 kit. 61,857 paired reads were produced and analysed as described by Adams et al. (1). Ten contigs totalling 8,236 bp were identified having between 94-96% identity at the nucleotide level to substantial parts of the bipartite genome of Broad bean wilt virus 2 (BBWV-2). The protein sequence of the combined coat proteins had between 88-98% identity to that of coat proteins of different isolates of BBWV-2 found on GenBank. Figure 3 shows a neighbour joining tree (1000 replicates) produced using MEGA6 (Tamura et al., 5) of the coat protein genes of this isolate (GenBank Accession No. MF429951) compared to other members of the family Secoviridae. This confirms it as a member of the species BBWV-2. To confirm the identification, material from N. benthamiana was tested by ELISA (DSMZ, Germany; reference number AS-0862, and LOEWE, Germany; reference number 07017S) using a positive control (PC-0862) and was found to be positive with both antisera. BBWV has previously been found on V. scutellata in New York (Rist & Lorberr, 4) though it was not specified whether BBWV-1 or BBWV-2 was present. BBWV-2 has been previously detected in Digitalis and Salvia officinalis in the UK (Mumford et al., 2). This is thought to be the first confirmed finding of BBWV-2 in Veronica. This testing was funded through the Defra-Fera Long Term Service Agreement.

Symptoms consistent with European mountain ash ringspot-associated virus (EMARaV), genus Emaravirus, have been historically observed across the UK on Sorbus aucuparia (rowan or European mountain ash) (Cooper, 2). The virus was recently confirmed across Scotland (Robel et al., 6). EMARaV has also been detected by molecular methods in Scandinavia, Russia and the Czech Republic (Grimová et al., 4). In England, there was an initial finding of EMARaV from rowan leaves exhibiting chlorotic ring and line-pattern symptoms (Fig 1.) at the Yorkshire Arboretum, North Yorkshire, in July 2013. A site-specific survey of Sorbus spp. was conducted by Fera in July 2014. The virus was also included as a 'priority pest' in the Observatree citizen science project, with further symptomatic samples of Sorbus spp. submitted from sites across England. To confirm the presence of the virus in these samples, total RNA was extracted and tested by RT- PCR (Mielke et al., 5). Amplicons of the expected size for RNA 2 (300 bp) and RNA 3 (204 bp) were sent for sequencing (Table 1). These were used to construct maximum likelihood (1000 replicates) phylogenetic trees including a representative selection of other EMARaV sequences available on GenBank. EMARaV was confirmed at the Yorkshire Arboretum in S. aucuparia subsp. sibirica, and S. glomerulata, S. rehderiana, S. sp. aff. scalaris and S. pteridophylla, representing first host records for the latter four species. The virus was additionally detected from S. aucuparia from Crawley, West Sussex, Amotherby and Coneythorpe Bank (both North Yorkshire), and in Sorbus spp. from southwest London. A neighbour joining phylogenetic tree generated from the partial RNA2 sequences obtained from the UK findings using MEGA6 (Fig. 2) confirmed the viruses as EMARaV. It also showed that the main outbreak in the Yorkshire Arboretum and Amotherby, c. 5 miles away, may have originated from the same source but that the original 2013 outbreak at the Yorkshire Arboretum, Coneythorpe Bank and London may be distinct from this outbreak and each other (approximately 2% sequence divergence). No RNA2 sequence was obtained from the Crawley sample. Similar results were obtained for RNA3 (data not presented). Following initial findings in Scotland, a UK pest risk analysis was conducted (Defra, 3). These findings support the analysis's hypothesis that the virus is present in a broad range of Sorbus species and confirms that EMARaV is present in several different regions of England with the infections likely to be from multiple previous incursions. We would like to thank J. Grimshaw (The Yorkshire Arboretum), S. Redstone (RBG-Kew), H. Carter (Tree Health England) and Observatree volunteers for their help in collecting samples. Observatree is a citizen science project funded by EU-Life and Defra.

In March 2016, samples of eight Solanum jasminoides plants were submitted to Fera Science Ltd., from a nursery in southeast England. The samples were sent to the laboratory as part of plant health monitoring for latent viral diseases,…

In February 2014 a sample of Viola tricolor ssp. tricolor was submitted to Fera from outdoor bedding plants grown in Yorkshire, UK. The sample was sent in following the appearance of symptoms including bleaching and distortion (Fig. 1). The sample was tested by ELISA for Cucumber mosaic virus (CMV) and with generic antisera for the presence of potyviruses. The sample was negative in each case. In October 2014 a further Viola sample was submitted from a grower in Wiltshire, UK, with up to 10% of the stock affected by similar symptoms. Again, the results of ELISA testing for CMV and potyviruses were negative. Samples were screened using an Illumina MiSeq as described by Adams et al. (2). The tripartite genome (GenBank Accession Nos. RNA1-KX196166, RNA2-KX196165, RNA3-KX196164) of a putative sub-group 4 ilarvirus was recovered from the samples having 96% similarity to Viola white distortion associated virus (VWDaV) (GU168941; Fig. 2). To confirm the presence of the virus in the samples a real-time PCR assay was designed (F: 5'-AAGGGAAGAAAGCGAAGACTGTT-3', R: 5'-TCACAGAGTGATCAACCAATCGT-3', probe: 5' Fam-TAGAGATGCCAAACCCGTAACTAAAACG-BHQ1 3') from consensus sequences from this study and the sequences described by Cuiffo et al. (2). In each case the samples were positive for the presence of VWDaV. Cuiffo et al. (2) associated the presence of VWDaV with symptoms consistent with pansy mottle syndrome, a disorder recognised since the 1960's. To investigate this link, during the summer of 2015, samples were collected from nurseries across England (West Sussex, Hertfordshire and East Yorkshire) which had pansy plants exhibiting bleaching and/or distortion. Samples of asymptomatic pansies from the same cultivars were also submitted as a ‘healthy control’. Between 63.5-73.3% of the 300 pansies tested from affected cultivars were positive for VWDaV with no clear correlation to observable symptom or geographic origin. This report supports the conclusions from Cuiffo et al. (2) that the virus is probably disseminated widely in germplasm of Viola spp. Initial sample testing was supported by the Defra-Fera Long Term Service Agreement. Sample collection, testing and data analysis of the follow up surveillance work was supported through AHDB-Horticulture project PO 016a ‘The role of environmental factors in the incidence of pansy mottle syndrome’.

In October 2014 a stand of coppiced Corylus avellana (hazel) growing in Surrey (UK) was found exhibiting symptoms of yellowing of leaves, dieback around the leaf margin, a lack of density of the tree canopy and proliferation of small thin …

Nasturtium officinale (watercress) is a semi-aquatic, high value, leaf and stem crop. As a member of the family Brassicaceae (Cruciferae), it has a variety of culinary, medicinal and cosmetic uses. In 2009 watercress samples from Spain…