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.

Species of Rudbeckia, often known as black-eyed Susan or coneflower, are used as bedding plants in UK gardens. The large, daisy-like flowerheads typically have bright yellow petals and a conspicuous black centre which may be raised into a cone shape. During the summer of 2016, Rudbeckia fulgida var. sullivantii cv. Goldsturm plants, purchased in the preceding May from a nursery in south-east England, were observed with black leaf spots in outdoor beds at the Royal Horticultural Society (RHS) Garden Wisley. Lower leaves exhibited small black lesions in July, which coalesced as the season progressed leading to complete necrosis of the bottom leaves and spotting on higher leaves. Spots showed no halos and became necrotic only later in the season. Flowering did not appear to be reduced. Pycnidia within the leaf spots were epiphyllous, 50-75 μm diameter, with a neck protruding slightly above the leaf surface. Conidia were filiform, 30-60 times 1.5-2 μm, with three septa. A single-spore isolate was obtained on water agar and cultured on potato dextrose agar. Living cultures were deposited in the RHS culture collection held at RHS Garden Wisley (Accession No. RHS454672) and at Westerdijk Fungal Biodiversity Institute, Netherlands (Accession No. CBS145765). The internal transcribed spacer (ITS) region of rDNA, the β-tubulin (Btub) gene and the translation elongation factor 1-alpha (EF1) gene were amplified using the primers ITS4:ITS5, T1:B-Sandy-R, and EF1-728F:EF2, respectively, according to the method by Verkley et al. (5). The DNA amplicons were sequenced (GenBank Accession Nos. MN093336 (ITS), MN105980 (Btub) and MN166626 (EF1)). The ITS sequence differed by one base pair from the only ITS sequence available for Septoria rudbeckiae (JQ677043). No previous sequences were available for comparison for Btub and EF1 and none of the available sequences had more than 90% identity. Pathogenicity was confirmed by spraying Rudbeckia fulgida sullivantii ‘Goldsturm’ plants with a conidial suspension (1 × 106 conidia/ml) prepared from spores from 21-day-old cultures on potato dextrose agar, incubated at 20°C with a 12 hr light/ 12 hr dark cycle. Plants were kept in high humidity for 72 hr after inoculation. After four weeks, black lesions were observed on leaves. Pycnidia and conidia consistent with S. rudbeckiae were found within each lesion. Control plants sprayed with sterilised water showed no symptoms. Septoria rudbeckiae was described from the USA (Ellis & Halsted, 1) where it is now widespread causing disfigurement of Rudbeckia in gardens. Although the fungus has been recorded from a number of different Rudbeckia species, Rudbeckia fulgida var. sullivanti cv. Goldstrum has been recognised in the USA as one of the most susceptible cultivars. Septoria rudbeckiae has also been reported from Canada, Bulgaria and Romania (Farr & Rossman, 2), Turkey (as Septoria sp.; Gumrukcu, 3) and Korea (Park, 4). To our knowledge, this is the first report of S. rudbeckiae in the United Kingdom. The authors would like to thank Jane Renshaw for aid in preparation of fungal cultures.

Species of Rudbeckia, often known as black-eyed Susan or coneflower, are used as bedding plants in UK gardens. The large, daisy-like flowerheads typically have bright yellow petals and a conspicuous black centre which may be raised into a…

Thunbergia alata, often known as black-eyed Susan vine, is used in UK gardens as an annual climber in bedding schemes and hanging baskets. Flowers are typically shades of yellow, orange and red with a black centre. Plants germinated onsite from commercial seed were observed with dark leaf spots in outdoor beds at RHS Wisley in July 2017. Initial symptoms were dark circular spots surrounded by a yellow halo. These developed into circular necrotic lesions with a small white, central spot (Figs. 1-2). Lesions coalesced to produce early leaf necrosis but plants continued to flower profusely through to the end of the season. Microscopic inspection of leaf lesions revealed brown conidia with extremely long beaks on unbranched conidiophores, developing on the necrotic tissues (Figs. 3-4). Mature conidia had ellipsoid or obclavate bodies, (79-) 85-108 (-117) × (15-) 17-29 (-31) μm with 8-9 (-11) primary transverse septa and (2-) 5-6 (-8) euseptate cells. Beaks were (150-) 185-310 (-350) μm in length, tapering from a base (3.6-) 4.1-7.0 (-7.3) μm in diameter. These features identify the casual fungus as Alternaria thunbergiae described by Simmons & Alcorn (3) from leaf spots on Thunbergia alata collected in Queensland, Australia. Although 90% of the conidia were narrower than the original description of A. thunbergiae (27-32 μm), 98% were broader than A. iranica (17-20 μm, Simmons & Ghosta, 4) which was placed in synonymy with A. thunbergiae by Woudenberg et al. (5) using a five-gene phylogenetic analysis. A single-spore isolate was obtained on potato dextrose agar and deposited in the RHS culture collection held at RHS Wisley (RHS400616) and at the Westerdijk Fungal Biodiversity Institute, Netherlands (CBS145627). The internal transcribed spacer (ITS) region of rDNA and the glyceraldehyde-3-phosphate (GAPDH) gene were amplified and sequenced (GenBank Accession Nos. MK295816 and MK307897, respectively). The ITS sequence was identical to two ITS sequences for A. thunbergiae already held in GenBank (KJ718257 and KJ718258) and differed from a third available sequence (KJ718259) by one base pair. The GAPDH sequence was identical to all three GAPDH sequences for A. thunbergiae available in GenBank (KJ718084, KJ718085 and KJ718086). To confirm pathogenicity, damp filter paper discs on which the fungus was sporulating, were placed on the leaves of young Thunbergia alata plants kept at ambient temperature in natural light conditions. Discs were removed after a 48-hour period in which the plants were kept at 100% humidity. Pale, necrotic lesions developed after two weeks. Spores typical of A. thunbergiae were found on the lower leaf surface of lesions, isolated and confirmed as A. thunbergiae using ITS and GAPDH sequences. Control plants, on which damp sterile filter paper discs were placed, showed no symptoms. In addition to Australia, A. thunbergiae has been reported from Florida, USA (Leahy, 1), from Rio de Janeiro, Brazil (Melo, 2) and from Miandoab, Iran on Allium cepa as A. iranica (Simmons & Ghosta, 4). To our knowledge, this is the first record of A. thunbergiae in the United Kingdom and Europe. Whilst the infected mature plants continued to flower well at RHS Wisley, the unsightly foliage caused a reduction in plant quality. These symptoms could have significant negative implications for growers producing planting material. A pressed specimen of Thunbergia alata showing typical symptoms has been deposited at the Royal Botanic Gardens, Kew as K(M)257596. The authors would like to thank Jane Renshaw for aid in preparation of fungal cultures and Saskia Harris for preparation and labelling of herbarium specimens.

Santolina chamaecyparissus, also known as cotton lavender, is a dwarf Mediterranean shrub often grown for its aromatic foliage and as a dwarf hedging plant. In 2013, S. chamaecyparissus plants were sent to the Royal Horticultural Society from a garden in Derbyshire with decayed roots and lesions at the base of the stems. To isolate the pathogen, 1-2 mm pieces of necrotic tissue from the lesion leading edge were placed on Phytophthora selective media (SMA; amended as per Brasier et al., 1) and incubated at 20°C for three days. In addition, pieces of necrotic stem tissue were placed into apple (cv. Granny Smith) baits and incubated in the dark at 20°C for up to one week. As soon as lesions formed on the apples the lesions were placed onto SMA plates for isolation. Hyphal tips were transferred onto carrot agar (CA) in order to obtain pure cultures. Sporangia formed readily when CA plugs of an actively growing culture were floated in filtered pond water. The sporangia (Fig. 1) were ovoid to obpyriform in shape, ranging between 22-79 (55.6) x 15-49 (37.2) μm in size (average l/b ratio 1.50; n = 46), papillate and primarily non-caducous, but occasionally caducous with a short pedicel (4-7 μm). Oogonia were readily produced on CA, globose and measured between 24-39 (32.8) μm (n = 70). The paragynous (occasionally amphigynous) antheridia were spherical or club-shaped and measured 10-16 (12.9) x 12-23 (15.6) μm (n = 33). Oospores were aplerotic and had a diameter of 21-35 (28.3) μm (n = 64). Chlamydospores were observed on CA after at least two weeks growth. They were intercalary or terminal and had a diameter of between 25- 42 (33.6) μm (n = 56). Based upon morphological characteristics, which agreed with the original description by Kröber & Marwtiz (4), the pathogen was identified as Phytophthora tentaculata. The molecular identification of an isolate (RHS252983) as P. tentaculata was confirmed by sequencing the ITS region using a semi-nested PCR reaction as described by Henricot & Waghorn (3). The ITS of the RHS252983 isolate (GenBank Assession No. MG761692) was identical to other P. tentaculata isolates (KF501392, KF667505, AF266775 and FJ802009). Six plants of S. chamaecyparissus cv. Pretty Carol were inoculated at the stem base with 3 mm plugs from a seven-day-old P. tentaculata culture (RHS252983) grown on CA as described by Henricot & Waghorn (3). The plants were kept in a grow dome at ambient room temperature and natural light conditions. After 21 days, the plants showed signs of wilting and decline when compared to control plants inoculated with plugs of CA only (Fig. 2). Necrotic lesions extended up and down the stems with lengths of 50-190 (114) mm. In comparison, agar controls yielded lesions of 15-30 (20) mm. Phytophthora tentaculata was successfully re-isolated from the lesion margins of the inoculated plants and confirmed by DNA sequencing. Phytophthora tentaculata has been recorded in North America (Rooney-Latham & Blomquist 5), Asia (China, Japan) and several European countries (Italy, Germany, Spain and The Netherlands) (Farr & Rossman, 2). In California, it has been found in restoration nurseries providing planting stock for forest and other environmental settings with the potential to cause lasting environmental damage (Rooney-Latham et al., 6). To our knowledge, this is the first report of P. tentaculata in the UK.

Santolina chamaecyparissus , also known as cotton lavender, is a dwarf Mediterranean shrub often grown for its aromatic foliage and as a dwarf hedging plant. In 2013, S. chamaecyparissus plants were sent to the Royal Horticultural Society…

In September 2014, a sample of Japanese rose, Kerria japonica, exhibiting severe defoliation and stem lesions was submitted to RHS Gardening Advice from a garden in West Yorkshire, England (Fig. 1). Further samples were subsequently received from 12 locations across England (Fig. 2). Leaves of infected samples exhibited numerous small red-brown spots (1-5 mm diameter) with dark purple borders (Fig. 1). Spots were visible on both leaf surfaces and sometimes numbered in the hundreds on a single leaf. In humid conditions clusters of white spores were visible in the centre of the spots. As the infection progressed the spots coalesced and the leaves turned yellow through to brown and fell from the stems. Stem lesions appeared as purple-brown, slightly-sunken elliptical cankers which remained visible on the stems throughout the year. Cankers which girdled the stem resulted in extensive stem die-back. Upon microscopic examination it was determined that the symptoms were caused by the fungus Blumeriella kerriae (Stewart, 5). Blumeriella kerriae is widespread on K. japonica in America causing twig and leaf blight, but has not previously been recorded on any host plant in the UK. Acervuli were scattered across each spot/lesion. Conidia were filiform, curved, hyaline, 45-94 times 1.4-5.6 μm (mean 69 times 3.5 μm) (n = 60) (Fig. 3). Observations differed to Stewart's original description which described 1-septate conidia; observed conidia were mainly 2-3 septate, with occasional 1-septate conidia. A single spore isolate on potato dextrose agar was obtained and deposited in the RHS Wisley culture collection (JS20160615). The internal transcribed spacer (ITS) region of rDNA was amplified (White et al., 6) and sequenced (GenBank Accession No. KY929501). There were no previous DNA sequences for B. kerriae available in GenBank. Fungal ITS sequences for species within the family Dermateaceae, including Blumeriella jappii (Pederson et al., 2010), were obtained to place KY929501 within a phylogenetic tree. Sequences were aligned with the MUSCLE v3.8.31 algorithm (Edgar, 1). The beginning and end of the alignment, where base callings were ambiguous, were excluded from the analysis. Phylogenetic trees were constructed through Bayesian inference analysis performed in MrBayes v3.2.6 (Ronquist et al., 4) with the GTR + I + G model identified by MrModeltest v2.3 (Nylander, 2). The analyses were run for 1,000,000 generations, sampling every 1000 generations. Trees from the first 25% of the sampled generations were discarded. Phylogenetic analysis placed B. kerriae (KY929501) in a cluster with the closely related cherry leaf pathogen B. jappii (Fig. 4). To confirm pathogenicity, a conidial suspension (~105 conidia/ml, sterile distilled water, 0.2% Tween-20) was prepared by harvesting fresh conidia from stem lesions. All observed spores were consistent with B. kerriae. The spore suspension was sprayed onto newly emerged leaves of K. japonica plants. Inoculated plants were held in a humidity chamber for 48 h and thereafter placed in a glasshouse. After 13 days, typical leaf spot symptoms developed on the leaves of inoculated plants. Blumeriella kerria conidia were produced from acervuli in the leaf spots, fulfilling Koch's postulates. A control plant treated with sterilised water and Tween-20 remained symptomless. Kerria japonica is a valuable garden and hedging plant with high tolerance for poor environmental conditions. Until now it has had no serious pest or disease problems in the UK. The spread of Blumeriella kerriae in the UK will change how K. japonica is used in UK horticulture and may result in gradual loss of the plant from retail. Thanks to Mrs Jenny Denton and Dr Geoff Denton for initial sample handling, and Ms. Jane Renshaw and Dr Fay Newbery for aid in preparation of fungal cultures.

In September 2014, wilting plants were observed in a trial of Impatiens hawkeri New Guinea Group hybrids at RHS Garden Wisley. The stems of affected plants were defoliated (Fig. 1) and black staining was noted in the vascular tissues when cut stems were examined in cross section. Stems with staining were incubated in a humid chamber and after three days abundant verticils and conidia of a Verticillium species were produced, corresponding to the dark, stained areas of the stems (Fig. 2). The fungus usually went on to produce microsclerotia suggestive of Verticillium dahliae when the specimens had been incubated for seven days. Natural infection was thus confirmed on 26 cultivars and pure cultures of V. dahliae from Impatiens SunPatiens Vigorous Magenta 'Misato Fg3' and Impatiens 'Impacwhi' were isolated onto potato dextrose agar and used for infection assays and molecular analysis. The ITS region was sequenced with ITS1F (Gardes & Bruns, 2) and ITS4 (White et al., 5). The sequences (GenBank Accession Nos. KT970068-KT970070) were identical to V. dahliae isolates from, for example, tomato (FJ900167, GU461607 and GU461610), cotton (GU461609) and grapevine (FJ475122). Three isolates were deposited into the RHS Plant Pathology culture collection (Accession Nos. M10A6, M10D5 and M10E5). Plug plants of Impatiens cvs. SunPatiens Blush Pink ‘Sakimp013‘ and SunPatiens Vigorous Magenta were potted into 9 cm pots in a 3:1 (v/v) mix of John Innes No. 2 potting mix:vermiculite. After four weeks' growth, the roots were washed and placed for 30 minutes in conidial suspensions (c. 107 conidia/ml) of V. dahliae (M10E5) in sterile distilled water, or water alone, before transferring into fresh potting mix as above. Two plants per treatment were placed in a growth room maintained at a temperature of 20°C day/16°C night and a humidity of 55%. Plants showed signs of wilting, defoliation and vascular tissue staining four weeks post inoculation (Fig. 3). Uninoculated plants showed no symptoms of wilt. Verticillium dahliae was recovered from the vascular tissue in stained stems of inoculated plants. This is the first record of Verticillium wilt on Impatiens New Guinea Group hybrids. The disease has been recorded on other species of Impatiens such as I. balsamina (French, 1) and I. walleriana (Taylor, 4). However, this report is of particular interest as New Guinea Group hybrids have been grown as a substitute for I. walleriana (busy Lizzie), which is very susceptible to the downy mildew pathogen Plasmopara obducens (Jennings, 3). New Guinea Group hybrids are not thought to be susceptible to P. obducens.

In autumn 2013, anda range of bulbs of ornamental Allium species was planted in the Royal Horticultural Society's garden at Wisley to assess their vigour and aesthetic qualities. The trial included 179 accessions of plant material, representing 32 species or hybrids, from six nurseries in the United Kingdom or The Netherlands. Yellow flecking and striping with occasional reddening on the leaf surfaces and leaf curling was observed on many of the plants during April 2015 (Figs. 1-4). Leaf samples were collected from symptom-bearing plants of 20 accessions. RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, Manchester, UK) and tested by RT-PCR using broad-spectrum primers for carlaviruses and tospoviruses (Agdia, Elkhart, USA), potyviruses (Marie-Jeanne et al., 5) and tobraviruses (Jones et al., 4). No samples tested positive for tospoviruses but all tested positive using at least one of the three other primer sets. A. jesdianum ‘Pendjikent’, A. nigrum and A. siculum (syn. Nectaroscordum siculum) tested positive using the carlavirus primers but it was not possible to sequence the product. The remaining amplicons of the expected size were directly sequenced in both directions and were identified by BLAST analysis; representative sequences were deposited in GenBank (Table 1). GenBank Accession Nos. KT223098, KT223099, KT223100, KT223101, KT223102 and KT223103 had 98, 98, 99, 92, 99 and 85 % nucleotide sequence identity with Leek yellow stripe virus (AB194628), Onion yellow dwarf virus (KR025485), Ornamental onion stripe mosaic virus (OrOSMV; EU042750) Pea early browning virus (PEBV; X14006) Tobacco rattle virus (JX144383), and Turnip mosaic virus (TuMV; AB701697), respectively. The limited sequence identity of the Allium TuMV isolate with other isolates of the species may suggest that the isolate belongs to a distinct strain. This is the first report of PEBV infecting Allium and does not accord with Brunt et al. (3) who reported that A. cepa was not experimentally susceptible to the virus. The remaining viruses have been reported previously from Allium spp. However, with few exceptions such as Noda et al. (6), the specific identity of ornamental hosts has not been reported and therefore this is the first report of many of these virus-host associations. This is also the first report of OrOSMV in the UK. Since the early 1800's ornamental Allium species have been grown widely in UK gardens as herbaceous perennials. They are prized for their architectural qualities and large, colourful umbels that attract pollinating insects. They are generally hardy and well suited to most UK soil types (Block, 1). The increasing importance of ornamental Allium (including Nectaroscordum) is illustrated by the 8% rise in production area over the last 4 years (to 228 ha in 2014/15) in The Netherlands (Bloembollenkeuringsdienst, 2). The authors thank G. Denton (RHS Gardening Advice), M. Heath and K. Robbirt (RHS Trials) for diagnostic assistance and horticultural information.

California poppy (Eschscholzia californica) is grown in UK gardens as an annual for its colourful display. In October 2012, diseased samples of California poppies (cv. Summer Sun) collected in a garden in Shropshire (West Midlands) were received at the advisory service at RHS Wisley. The plants were grown from seeds and about 80% of the plants were affected. Symptoms included white oval-shaped leaf spots surrounded by a dark border (Fig. 1). The spots were 1 mm in length or less on the lower and upper leaf surfaces and on the stems. The leaves became wilted and blackened. Microscopic examination revealed the presence of globose or polygonal hyaline to brown ustilospores measuring 8.3-14.8 times 7.2-12.8 μm (average 10.4 times 8.4 μm), wall 2-layered and 0.4-1.5 μm thick (Fig. 2). Long, filiform, slightly bent basidiospores measuring 24-62 times 2 μm (average 43.2 times 2 μm) were observed. According to Vánky (pers. com.), these have been observed in the genus Entyloma and, after or without conjugation, can produce secondary or tertiary sporidia. Sporidia were also observed and these were hyaline, cylindrical, mostly curved, unicellular, rounded at one end and tapered at the other end. They measured 9.4-16.5 times 2-3.6 μm (average 13.9 times 2.8 μm). The symptoms and the hyaline, densely aggregated spores embedded within the leaf tissue were characteristic of a smut fungus belonging to the genus Entyloma. Morphological examination and host association of the fungus suggested it should be referred to Entyloma eschscholziae Harkness, the only species known from this host (Vánky, 6). To obtain additional evidence, DNA was extracted from infected plant material using the Plant DNAeasyTM mini kit (Qiagen, Germany) according to the manufacturer's instructions. The ITS region was amplified using ITS 1F and ITS 4B primers (Gardes & Bruns, 3) and sequenced (GenBank Accession No. KC456226). The DNA sequence was 100% identical to an uncultured Basidiomycota detected in grassland soils in California (HM240159). The sequence was also closely related to Entyloma species. The closest match to DNA sequences determined to the species level within GenBank was with E. bidentis (coverage 84%, 98% identity, AY854963) and E. eryngii-plani (coverage 84%, 98% identity, AY081034). Sequences of E. eschscholziae were not available in GenBank. In general, the molecular work carried out by Begerow et al. (1) supported a species concept for the genus Entyloma based on host specificity. Altogether, based on the morphological characteristics, sequencing of the ITS and host specificity, the smut was confirmed as Entyloma eschscholziae (Harkness, 4; Vánky 6; Farr & Rossman, 2). The original description of this species does not include an anamorph (Harkness, 4; Vánky 6) and although conidia were observed by Savile, (5), these were not described. A voucher specimen was deposited at the Royal Botanic Gardens, Kew, UK (K(M)179304). A subsequent collection on this host from the Royal Botanic Gardens, Kew was obtained by A.M. Ainsworth on 22 Oct. 2012, accessed as K(M)179357. This is the first report of Entyloma eschscholziae causing smut in the UK and also in Europe. Until now, this disease has only been reported in California and New Zealand (Farr & Rossman, 2).

The Food andEnvironment Research Agency, Sand Hutton, York, YO41 1LZ, UKAgastache is a genus of 30 species of aromatic perennials in the familyLamiaceae. In June and July 2009, cultivars of Agastache sp. ‘TangerineDreams’, A. mexicana ‘Red Fortune’, A. aurantiaca ‘Apricot Sprite’ andA. cana ‘Purple Pygmy’ at Wisley gardens were infected by downy mil-dew.Thesymptomswerechloroticleafspotseventuallyturningbrown.Adowny mildew-like growth was observable underneath the leaf surfaces.Microscopic examination revealed conidiophores typical of Peronos-pora.Conidiophoreswerestraight,monopodially branched (typically 4–6 times), 137–325 lm in length. Ultimate branchlets were sinuous andobtuse, the longer measuring 12–20 lm, the shorter 5–8 lm. Conidiawere olive brown, broadly ellipsoidal to subglobose and measured 22–30 · 15–25 lm.Nooosporeswerefound.TheITSofisolatesfoundoncultivarsofA.mexicana‘RedFortune’andAgastachesp.‘TangerineDreams’(GenBankAccessionNos.GQ390794and GQ390795) showed 99% homology to a new Peronospora speciesfound on coleus and basil recently named as Peronospora belbahrii(Thines et al., 2009). Specimens were deposited at Kew (K(M)163651andK(M)163652).Tosupportthemolecularidentification,conidiawererubbed from infected Agastache leaves onto leaves of four differenthealthy coleus cultivars (‘Winsome’, ‘Mrs Pilkington’, ‘Roy Pedley and‘DurhamGala’).Afterinoculation, aplastic bagwasplaced onplantsfor48 hrs. The plants were misted with water twice a day and left outdoorswhere the temperature ranged from 14 Cto27 C. Controls were notinoculated. After 14 days, the same downy mildew was found on cvs.‘Winsome’and‘DurhamGala’whilsttheotherplantsremainedhealthy.This is the first record of P. belbahrii on Agastache and in the UK. Theonly other downy mildew species recorded on Agastache is P. lophantibut this species has been found to be conspecific with P. lamii (Shin &Choi, 2006). Peronospora belbahrii is distinct from P. lamii (Belbahriet al.,2005). It was firstreported from Africain1933,then much later in1993 in the USA and in 1999 in Europe where it is causing epidemics(Thines et al., 2009).The fungus poses serious threats to production ofcoleus and other Lamiacae grown commercially. Statutory action hasnowbeentakenagainstthispathogen.References

Aizoaceae is the largest family of succulent plants containing 127 genera and about 2500 species of which most are endemic to arid or semi-arid parts of southern Africa, with a few occurring in Australasia and the Americas. South African Aizoaceae have long been cultivated in British gardens since they were introduced in the second half of the 17th Century. Some, notably Carpobrotus edulis, have become naturalised by the coast in south west England and compete with native species. In summer 2007 and 2008, diseased samples of Delosperma cooperi, Lampranthus‘Tresco Orange’, L. roseus and Lampranthus sp., both genera belonging to the Aizoaceae, were received at the Royal Horticultural Society, Wisley and at the Royal Botanic Gardens at Kew. These samples originated from private gardens in East Sussex, West Sussex and Devon. Infected plants were covered with chalky blister-like pustules typical of white blister (Albugo spp.). For samples of both genera, microscopic examination revealed oospores in the leaf tissue that were spherical, 50–61 × 53–61 µm (average 56 × 57 µm), with a two-layered wall, 4–8 µm thick. The outer layer was reticulated, the alveoli angular and irregular, 3–6 µm wide. Conidiophores were cylindrical (50 × 21 µm). Conidia were cubical, globose or elliptical, 16–23 × 13–23 µm (average 19 × 16 µm). The morphological characteristics fit the description of Albugo trianthemae (Wilson 1908; Waterhouse 1975). Material from East Sussex on L. roseus was deposited in Kew (Accession No. K(M) 159 430). The ITS region of the isolate infecting Delosperma was amplified following Bonants et al. (1997), cloned and sequenced (GenBank Accession No. FJ234425) but with no ITS sequences of Albugo trianthemae available in GenBank, results of the sequence search came back as unique. Albugo trianthemae causes yellowing and poor development, and may lead to shrivelling of shoots and death of the host. It has been recorded on various genera belonging to the Aizoaceae in New Zealand, Australia, Ghana, Sudan and southwestern United States (Waterhouse, 1975; Farr et al., 2008). However, it has not previously been recorded on Delosperma before but has been found on Lampranthus in Australia and New Zealand (Farr et al., 2008). This is the first report of Albugo trianthemae in the UK and indeed Europe. A second species on this host family, A. austroafricana occurs on Aizoon in South Africa. It is distinguished from A. trianthemae in having warted rather than reticulate oospores (Waterhouse, 1975).

The influence of in vitro conditioning and rooting of microcuttings with different concentrations of sucrose (0–6%) and types of carbohydrate (sucrose, glucose, maltose, fructose and sorbitol) on their in vivo establishment was investigated using Potentilla fruticosa cultivar ‘Tangerine’ and Ficus lyrata. Maximum values for shoot height, fresh weight and dry weight were obtained for both species 4 weeks after transferring to in vivo conditions when previously conditioned with 2 or 4% sucrose. Plantlet establishment was high (85–97.5%) and only declined (15–62.5%) when 0% sucrose was used. Shoot height, fresh weight and dry weight of Potentilla were greatest when conditioned with sucrose, glucose or maltose. Similar results were obtained for Ficus except that maltose gave significantly less shoot growth than glucose or sucrose. For both species sorbitol gave the least growth followed by fructose. Plant establishment was high (85–100%) for all carbohydrate types tested. The ways in which the carbohydrate in the culture medium may influence the quality of acclimatized plants and interact with the morphology of the cultured species are discussed.