Dietary analysis of herbivorous insects relies on successfully eliminating surface contamination. If this cannot be performed reliably, then it will not be possible to differentiate between plants that the insect is feeding on and plants the insect has been in contact with, either directly or via pollen. Methods in the literature often use bleach and alcohol washes to remove contamination. We perform a controlled metabarcoding baseline study on a herbivorous, xylem-feeding insect, the Meadow Spittlebug (Philaenus spumarius), using Oxford Nanopore Technologies (ONT) sequencing, and identify possible contamination that persists after washes. Despite the reported success of methods in the literature, we find that contamination is still present, leading to possible false-positive results. We hypothesise that pollen is the main source of contamination, its robust nature making it difficult to remove, and conduct a further three experiments with the goal of removing pollen from the surface of Philaenus spumarius. This study investigates the effectiveness of robust bleach/Tween/alcohol washes, sterile gut excision (including combined with Distel application), and ultraviolet light as alternative sterilisation approaches. Overall, our findings indicate that we are unable to remove surface contamination and still detect signals that may originate in the gut. In no experiment did we unequivocally detect plant DNA that originated in the P. spumarius gut.

In March 2018 a sample of daffodil (Narcissus sp.) was submitted to the plant clinic at Fera Science Ltd from a farm in South West England. The sample was exhibiting symptoms consistent with a viral disease of an unknown cause and the disease was distributed sporadically throughout the crop (the incidence was not recorded). Symptoms included chlorotic streaking and leaf distortion (Figure 1). A single plant sample was submitted for testing. Initial tests for the presence of viruses were done using ELISA. The assays used included generic potyvirus, and specific Arabis mosaic virus (DSMZ, Germany) and Cucumber mosaic virus tests (Agdia, USA). A positive reaction was obtained with the potyvirus ELISA. RNA was extracted by magnetic bead extraction using an InvimagVirus DNA/RNA mini kit (Invitek GmbH, Germany) (Fox et al., 2019). A potyvirus genus-level RT-PCR (van der Vlugt et al., 1999) was used to confirm the identity of the potyvirus detected by ELISA. A PCR product of the expected size [c. 650 bp] was obtained and sent for Sanger sequencing to Sequiserve Gmbh (Germany). The amplicon sequencing result confirmed the presence of a potyvirus and identified the virus as Snowdrop virus Y (SVY) with 99% nucleic acid identity to GenBank Accession No. MH886519.1. reported from Australia. The amplicon was deposited in GenBank (OP871789). The sample was also subjected to high throughput sequencing (HTS) using a TruSeq kit on the Illumina MiSeq (Fowkes et al., 2021). The resulting HTS data provided complete coding sequences (deposited in Genbank) of the following potyviruses and a macluravirus, respectively; Narcissus late season yellows virus (OP871786, 99% identity to LC664185.1), SVY (OP871788, 99% identity to LC757029.1) and Narcissus latent virus (NLV, OP871787, 99% identity to KX979913.1). This report constitutes the first known record of SVY in daffodil from the UK. The metagenomic sequence data for this sample were deposited under bio project code PRJNA892468. In April 2020, another daffodil sample was submitted with foliar symptoms of browning and streaking from South East England. Approximately 50% of the plants in the field were affected. This sample was tested by ELISA as described above, with the following additional tests: Broad bean wilt virus I and II, and Tobacco necrosis virus (Loewe, Germany). A potyvirus was detected and identified by conventional PCR to be SVY (OP871785, 99% identity to MH886519.1) as described above. The Sanger sequencing having been performed by Eurofins (Germany). Further testing with Illumina MiSeq HTS yielded partial genomes for NLV (OP871781, 99% identity to KX979913.1); Nerine latent virus (genus Carlavirus; OP871782, 99% identity to ON228217.1), SVY (OP871783, 99% identity to LC757029.1) and Turnip yellows virus (TuYV, genus Polerovirus; OP871784, 91% identity to KU521235.1). The metagenomic sequence data for this sample were deposited under bio project code PRJNA 892468. TuYV was confirmed by real-time RT-PCR (Buxton-Kirk et al., 2021). This is the first host record of TuYV in Narcissus. SYV was first detected in snowdrop in 2008 in the UK (EU927399). The presence of SVY and TuYV, along with the other viruses identified, in diseased daffodils may suggest that virus infection provides a constraint on production in the daffodil industry. Further work needs to be conducted to ascertain the distribution and prevalence of these viruses in UK narcissus production.

New Disease ReportsVolume 46, Issue 1 e12118 NEW DISEASE REPORTOpen Access First report of Grapevine red globe virus in grapevine in the United Kingdom Madeline Dixon, Madeline Dixon orcid.org/0000-0002-4281-1245 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAimee Fowkes, Aimee Fowkes orcid.org/0000-0001-7918-441X Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorClare Hogan, Clare Hogan Animal and Plant Health Agency, Sand Hutton, York, UKSearch for more papers by this authorIan Adams, Ian Adams Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorSam McGreig, Sam McGreig Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorHollie Pufal, Hollie Pufal School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, UKSearch for more papers by this authorRichard Ward, Richard Ward orcid.org/0000-0002-7085-025X Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorValerie Harju, Valerie Harju orcid.org/0000-0003-3247-6698 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAnna Skelton, Anna Skelton orcid.org/0000-0002-5418-1930 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAdrian Fox, Corresponding Author Adrian Fox [email protected] orcid.org/0000-0003-1280-1836 Fera Science Ltd, Sand Hutton, York, UK Correspondence Adrian Fox, Fera Science Ltd, Sand Hutton, York, YO41 1LZ, UK. E-mail: [email protected]Search for more papers by this author Madeline Dixon, Madeline Dixon orcid.org/0000-0002-4281-1245 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAimee Fowkes, Aimee Fowkes orcid.org/0000-0001-7918-441X Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorClare Hogan, Clare Hogan Animal and Plant Health Agency, Sand Hutton, York, UKSearch for more papers by this authorIan Adams, Ian Adams Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorSam McGreig, Sam McGreig Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorHollie Pufal, Hollie Pufal School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, UKSearch for more papers by this authorRichard Ward, Richard Ward orcid.org/0000-0002-7085-025X Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorValerie Harju, Valerie Harju orcid.org/0000-0003-3247-6698 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAnna Skelton, Anna Skelton orcid.org/0000-0002-5418-1930 Fera Science Ltd, Sand Hutton, York, UKSearch for more papers by this authorAdrian Fox, Corresponding Author Adrian Fox [email protected] orcid.org/0000-0003-1280-1836 Fera Science Ltd, Sand Hutton, York, UK Correspondence Adrian Fox, Fera Science Ltd, Sand Hutton, York, YO41 1LZ, UK. E-mail: [email protected]Search for more papers by this author First published: 18 September 2022 https://doi.org/10.1002/ndr2.12118AboutSectionsPDF 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 August 2019 a sample of grapevine (Vitis sp.) leaves was submitted to Fera Science Ltd. from a vineyard in the south of England. The sample was sent following the appearance of an unknown disease. Symptoms included red colouration and flecking (Figures 1, 2). The sample was tested by DAS-ELISA for the presence of Arabis mosaic virus (DSMZ, Germany), Raspberry ringspot virus (DSMZ, Germany), Strawberry latent ringspot virus, Tobacco ringspot virus (Agdia, USA), Tomato black ring virus (DSMZ, Germany and Bioreba, Switzerland), Tomato ringspot virus (Loewe, Germany), and plate trap ELISA for potyviruses using a generic potyvirus antisera (DSMZ, Germany). The sample gave negative results for all tests. FIGURE 1Open in figure viewerPowerPoint Grapevine leaf showing red colouration FIGURE 2Open in figure viewerPowerPoint Grapevine leaf showing red flecking The sample was then tested by high throughput sequencing (HTS) on a MiSeq Sequencer (Illumina, UK) (Fowkes et al., 2021). The total number of reads for the sample was 342,422 with 5 (0.001%) being mapped to the genome of Grapevine red globe virus (GRGV, genus Maculavirus). The sequence contig of 217 nt obtained by HTS was checked by nucleotide comparison through BLAST against publicly available sequences. The partial sequence of the replicase gene had 94% identity to a GRGV sequence (GenBank Accession No. MZ451066.1), a historical isolate sequenced from the Sidney live plant collection, country of origin unknown (Mike Rott, CFIA, Pers. Comm.). The genome fragment of GRGV was uploaded to GenBank (ON187032). Sequencing data has been submitted to the NCBI short read archive, BioProject (PRJNA817416). Other grapevine viruses were also inferred from the sequence data, namely Grapevine rupestris stem pitting-associated virus (genus Foveavirus, ON187034) and Grapevine fleck virus (genus Maculavirus, ON187033). In each case these have been previously reported from the UK (Silva et al., 2017). To further confirm infection by GRGV, the sample was tested by RT-PCR using primers specific for GRGV (Ruiz-Garcia et al., 2018). A product of 418 bp was obtained (ON187031). The product was analysed by Sanger sequencing (MWG GmbH, Germany) and checked by nucleotide sequence comparison against sequences publicly available through BLAST. The sequence had 95% identity to the sequence of a GRGV isolate from Washington State, USA (MT749359.1). GRGV was first reported in Italy (Sabanadzovic et al., 2000) and has since been found in China, Croatia, France, Germany, Greece, Iran, Slovenia, Spain and the USA (Nourinejhad Zarghani et al., 2021; EPPO Global database, 2022). No specific symptoms have been associated with GRGV (Ruiz-Garcia et al., 2018), and as the detection of GRGV was in co-infection with other viruses, no conclusion can be drawn on the cause of the observed symptoms. To our knowledge this is the first report of GRGV in the UK. This suggests a broader distribution than previously reported and indicates the need for further surveillance of grapevine to establish current distribution. ACKNOWLEDGEMENTS This work was funded under the Defra-Fera Long Term Service Agreement. REFERENCES EPPO (2022) EPPO Global Database. Available at: https://gd.eppo.int/taxon/GRGV00. [Accessed 21 June 2022] Fowkes, A.R., McGreig, S., Pufal, H., Duffy, S., Howard, B., Adams, I.P. et al. (2021) Integrating high throughput sequencing into survey design reveals turnip yellows virus and soybean dwarf virus in pea (Pisum sativum) in the United Kingdom. Viruses, 13, 2530. https://doi.org/10.3390/v13122530 Nourinejhad Zarghani, S., Khalili, M., Dizadji, A. & Wetzel, T. (2021) First report of grapevine red globe virus in grapevine in Iran. Journal of Plant Pathology, 103, 661. https://doi.org/10.1007/s42161-021-00749-w Ruiz-García, A.B., Nourinejhad Zarghani, S., Okic, A., Olmos, A. & Wetzel, T. (2018) First report of grapevine red globe virus in grapevine in Germany. Plant Disease, 102, 1675. https://doi.org/10.1094/PDIS-01-18-0105-PDN Sabanadzovic, S., Abou-Ghanem, N., Castellano, M.A., Digiaro, M. & Martelli, G.P. (2000) Grapevine fleck virus-like viruses in Vitis. Archives of Virology, 145, 553– 565. https://doi.org/10.1007/s007050050046 Silva, G., Lecourt, J., Clover, G.R.G. & Seal, S.E. (2017) First report of Grapevine fanleaf virus infecting grapevine in the United Kingdom. New Disease Reports, 36, 2044- 0588. https://doi.org/10.5197/j.2044-0588.2017.036.009 Volume46, Issue1July 2022e12118 FiguresReferencesRelatedInformation

Zymoseptoria tritici is the causal agent of Septoria tritici blotch, the most important disease affecting wheat crops and responsible for up to 50% loss in yield. Azoles and SDHIs are the main groups of fungicides used to control the disease. The efficacy of these fungicides has decreased in recent years due to resistance development. Fungicide resistance can be a result of mutations in the target genes, mutations in upstream regulatory elements that result in over-expression of target proteins and the overexpression of transporters that remove toxic compounds from cells. In the current study nanopore sequencing was used to analyse isolates, mock communities and field samples to determine the pathogen population composition related to azole resistance. Despite the presence of sequencing errors, the method was able to effectively differentiate the sequences of different haplotypes present in both mock and field populations for haplotypes that were relatively common in the overall population. However, haplotypes present at a frequency lower than 5% of the total population, could not be accurately distinguished from haplotypes generated through background sequencing errors. The nanopore protocols are rapid and inexpensive, enabling the routine profiling of haplotypes within pathogen populations at the start of the season or between sprays, facilitating the selection of the most appropriate fungicide to control them, yet limit further selection for azole resistance.

The family Apiaceae comprises approximately 3700 species of herbaceous plants, including important crops, aromatic herbs and field weeds. Here we report a study of 10 preserved historical or recent virus samples of apiaceous plants collected in the United Kingdom (UK) import interceptions from the Mediterranean region (Egypt, Israel and Cyprus) or during surveys of Australian apiaceous crops. Seven complete new genomic sequences and one partial sequence, of the apiaceous potyviruses apium virus Y (ApVY), carrot thin leaf virus (CaTLV), carrot virus Y (CarVY) and celery mosaic virus (CeMV) were obtained. When these 7 and 16 earlier complete non-recombinant apiaceous potyvirus sequences were subjected to phylogenetic analyses, they split into 2 separate lineages: 1 containing ApVY, CeMV, CarVY and panax virus Y and the other CaTLV, ashitabi mosaic virus and konjac virus Y. Preliminary dating analysis suggested the CarVY population first diverged from CeMV and ApVY in the 17th century and CeMV from ApVY in the 18th century. They also showed the “time to most recent common ancestor” of the sampled populations to be more recent: 1997 CE, 1983 CE and 1958 CE for CarVY, CeMV and ApVY, respectively. In addition, we found a new family record for beet western yellows virus in coriander from Cyprus; a new country record for carrot torradovirus-1 and a tentative novel member of genus Ophiovirus as a co-infection in a carrot sample from Australia; and a novel member of the genus Umbravirus recovered from a sample of herb parsley from Israel.

Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.

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

In July 2019, a sample of Rosa spp.was submitted to Fera Science Ltd. via the Royal Horticultural Society (RHS) gardening advice service. The sample (ID220) was sent in following the appearance of unknown symptoms including mottling, yellow/white patching, thin texture and a pink colour in the leaves. RNA was extracted using a CTAB method adapted from Adams et al. (1), with the 4M LiCl incubation performed overnight at 4°C. The sample was tested for common rose viruses using RT-qPCR (Table 1). A positive result was achieved for Rose cryptic virus 1. Subsequently, the sample was analysed by high throughput sequencing (HTS) using a TruSeq Stranded Total RNA Library Prep Plant kit (Illumina Inc., USA) for library preparation. A MiSeq instrument and a MiSeq Reagent Kit v3 (600-cycle) (Illumina Inc.) were used to run the library. The run generated 569,452 reads for the sample, and data was analysed as described by Fox et al. (2). Three fragments of rose spring dwarf-associated virus (RSDaV) were identified (234, 251 and 229 bp; GenBank Accession Nos. MT993839-MT993841). A BLAST+ search found sequences with high sequence identity in both nucleotide (92.11-93.59% identity, EU024678.1) and amino acid comparisons (94.34-100%, YP_001949737.1; YP_001949736.1; YP_001949738.1). RT-PCR amplification using specific primers (Salem et al., 5) was performed to confirm the result, and a product of the expected size (418 bp) was obtained. ApMV-F-TGG TGG AGG ATT ACG ATG AAA GTA ApMY-R-TTT GAA ACC CTT TCG GTC CAT ApMY-Pe-[FAM]-CGA AAG GTC CGA ATC-[MGB-NFQ] Malandraki et al. (2017). PLoS One 12, e0180S77. doi: 10.1371 journal.pone.01S0S77 ArMV-CP-F-TAG CCC TTG GAG ACA ATC CT ArMV-CP-R-CCT CCA AAT CCC ACA TTA AC ArMV-CP-Pe-[FAM]-TGC CCA TAT GAT AGC TTG TCA TGG AC-[BHQ1] Wei et al. (2011). Australasian Plant Pathology 41. 93-98. doi: 10.1007. s13313-011-0095-1 CMV-F-GCT TGT TTC GCG CAT TCA A CMV-RI-GAG GCA GRA ACT TTA CGR ACT GT CMV-RII-TGA AGG TAC TTT CCG AAC TGT AAC C CMV-Pe-[FAM]-TTA ATC CTT TGC CGA AAT TTG ATT CTA CCG T GTG-[TAMRA] Skelton et al. (2018). Yen Disease Reports 37, 23. doi:10.5197/j.2044-058S.2018.037.023 INSV-120F-CTT CTT TAC C.AA CAA CCG TGA AAA INSY-19SR-AGA TTG CCT ATT CTT GAG GAA GGA INS V-14 5Pe-[FAM] - ATT CAG AAC ATG ACT ACT GC-[MGB] Department for Environment. Food & Rural Affairs (2005) Integrated management of viruses and thrip vectors in protected ornamentals. UK: SID 5 (2 05). PNRSV10F-TTC TTG AAG GAC CAACCG AGA GG PNRSV1OR-GCT .AAC GCA GGT .AAG ATT TCC AAG C PNRSYPe-[FAM] ATG TCT TGC TGG TCG ATG 3[MGB-NFQ] Marbot et al. (2003). Plant Disease 87. 1344-1348. doi: 10.1094 pdis.2003.S7.11.1344 RpRSV-1699F-GTT GTG TTG CTT CCC AGG GTA T RpRS V-17SOR-YAA .AAC CAR SGG TGC ATA TTC TTT RpRSY-1723Pe-[FAM]-TGC AGA CCT GGG AAA AGG AGG TTA ATC CT-[BHQ1] Monger & Mumford (2010). https://horticulture.ahdb.org.uk/sites/default/files/research_papers/SF_84 RoCVl-2-Fw-TGA TCG ACC AAA GTT GCA ACC RoCV 1-2-Rv-GAA GAT AAG ACA ATG CAG TCA CTT TCT T RoCY1-2-Pe-[FAM]-ATT CGG ACT GAA TTT GCT A-[MGB-NFO] RRV-F-GAT TAC CTT GTA GCC AAT TAC TTC TAA CTG RRV-R-CAT CTT C.AA. TGA TAT GCT CAA TTT AGT TAA. RRY-Pe-rFAMl TGT GTT TGC ACT GTT GAC - [MC-B -NFQ] |SLRSY-194F - CAT CTC CAA ART GCT CMT TTC A SLRSY-192F- ACC TCC TTC AAA. GTG TTC CTT TCA SLRSY-271R-GYC CRC TAG CTT CTG CCT CRC SLRSV-275R-TGT AGT CCA CTC GAT TCT GTC TCA C SLRSY-224Pe-[AM]-TTG GGT GYC CRT GCA ARC AGC ATA CT-[BHQ1] Monger & Mumford (2010). https://horticulture.ahdb.org.uk/sites/default/files/research_papers/SF_84 TRV-1466F-CAT GCT AAC AAA TTG CGA AAG C TRV-1553R-TAC AGA CAA ACC ATC CAC AAT TAT TTT TRY-1489Pe-[FAM]-ACG TGT GAC ACC AAC CAT GTC AGC AAC T-[TAMRA] Mumford et al. (2000). Phytopathology 90. 448-453. doi: 10.1094/phyto.2000.90.5.44S TRSY-F-GGG GTG CTT ACT GGC AAG G TRSV-R-GC.A CCA GCG TAA GAA CCC AA TRS Y-Pe- IE AMI -T G A TTT GCG GCG TAC TG-[MGB] EPPO (2017). Bulletin OEPP/EPPO Bulletin. 47, 135-145. doi:10.1111/epp.12376 ToRSV-F-GAA TGG TTC CCA GCC ACT T ToRSV-R-AGT CTC AAC TTA ACA TAC CAC ToRSV-Pe-[FAM]-AGG ATC GCT ACT CCT CCG TCA AC-[BHQ] Tang et al. (2014). Journal ofVirological Methods, 201, 38-43. doi:10.1016/j.jviromet.2014.02.011 TSWV-F- CTC TTG ATG ATG CAA AGT CTG TGA TSWV- R -TCT CAA AGC TAT CAA CTG AAG CAA TAA TSWY Pe-IFAMl-AGG TAA GCT ACC TCC CAG CAT TAT GGC AAG-[TAM] EPPO (2004). Bulletin OEPP EPPO Bulletin 34,271-279. doi: 10.111l/j.l365-233S.2004.0072S.x To assess the spread of RSDaV in the UK, 171 roses were analysed using the RT-PCR assay. Samples were collected as part of a survey of rose viruses in the UK and both asymptomatic and symptomatic leaf samples, consistent with virus infections symptoms (mottling, yellow veining, distortion, and ringspots) were included. Only one sample (ID140) resulted positive for RSDaV, and no symptoms were identified. Previous analysis showed this sample was positive for Arabis mosaic virus by ELISA and RT-qPCR. The RT-PCR product (418 bp) from both RSDaV-positive samples (ID220, 140) were sequenced, and nucleotide comparisons showed a 98.51-99.02% identity with sequences in GenBank (HM236366.1; HM2363641; HM236362.1; HM236364.1). Amino acid comparison showed a 98.51-100% identity with previously published sequences (ADK78852.1; ADK78851.1). RSDaV has previously been found in the USA (Salem et al., 5), Chile (Rivera & Engel, 4), and New Zealand (Milleza et al., 3). This is the first report of RSDaV in Europe. Further samples (4) were submitted to the RHS and Fera Science Ltd. Plant Clinic, showing the previously described unknown symptoms. They were tested by RT-PCR and also sequenced by HTS and tested negative for RSDaV. The cause of these symptoms is not believed to be of viral origin. Authors would like to thank Dr Maher Al Rwahnih for providing a positive control. The work was funded through the Defra-Fera Long Term Service Agreement, the RHS and Newcastle University.

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

Ullucus tuberosus (ulluco) is a tuber‐forming species that has become a novel crop in highland and temperate maritime climates. Eight viruses have been previously reported infecting Ullucus, including Andean potato latent virus (APLV), a quarantine virus within the European Union. No reference sequences have been published for the viruses previously described from U. tuberosus. Plants grown in the UK for the internet trade were tested for the presence of quarantine viruses using ELISA and real‐time RT‐PCR. ELISA positive results were obtained for APLV and multiple other viruses. A similar suite of viruses was detected at a second outbreak site linked to horticultural trade. Virus identification was by high‐throughput sequencing (HTS) using a ribosomal RNA (rRNA)‐depleted total RNA approach. Analysis of viral contigs indicated the presence of several novel viruses closely related to, but not consistent with, the viruses indicated by ELISA. Further confirmatory testing by real‐time RT‐PCR indicated that two tymoviruses, tentatively named Ullucus tymovirus 1 and Ullucus tymovirus 2, were more closely related to each other (85% identity), than to APLV or Andean potato mild mosaic virus (63–66% identity). APLV could not be confirmed from either site by either HTS or PCR. A novel tobamovirus (Ullucus tobamovirus 1) was only detected at the initial outbreak site. A novel polerovirus (Ullucus polerovirus 1) and a distinct genotype of Papaya mosaic virus were detected from both outbreak sites. Deploying HTS during a plant health outbreak demonstrates the potential of this approach to give rapid, accurate diagnosis.