First Report of Rose Spring Dwarf‐associated Virus in Rosa Spp. in United Kingdom

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.