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First Report of Clover yellow mosaic virus in Postentry Quarantine Tulipa gesneriana Plants Imported from the United States

Veerakone, S., Tang, J., Zheng, A., Ward, L. I., Mason, C.
Plant disease 2018 v.102 no.2 pp. 464
Chenopodium giganteum, Clover yellow mosaic virus, DNA-directed RNA polymerase, Stellaria media, Tulipa gesneriana, Verbena, Vicia, alfalfa, apples, bulbs, chlorosis, color, crop production, cultivars, cut flowers, economic valuation, electron microscopy, faba beans, flowers, genes, hosts, indicator species, leaves, nucleic acids, oligodeoxyribonucleotides, peas, quarantine, reverse transcriptase polymerase chain reaction, reverse transcription, risk factors, sap, virion, viruses, Germany, New Zealand, United States
Tulipa, a genus of bulbous plants in the lily family, is cultivated as an ornamental in many countries because of its large showy flowers. In October 2016, a plant of Tulipa gesneriana cultivar Ile de France, showing chlorotic streaks on leaves and color breaking on flowers, was received at the Plant Health and Environment Laboratory, Auckland, New Zealand (NZ). This plant originated from bulbs imported from the United States and was being held in a postentry quarantine facility in Southland, NZ. Electron microscopy of a crude sap preparation from the symptomatic leaf tissue revealed the presence of potex-like filamentous virus particles. Extract from infected leaf tissue was mechanically inoculated onto a range of herbaceous species. Chlorotic local lesions and mild systemic chlorosis were observed on Chenopodium amaranticolor and C. quinoa 4 weeks postinoculation. Total nucleic acid (NA) was extracted from both original and symptomatic indicator plants using a Kingfisher ML extraction machine (Thermofisher Scientific, Waltham, MA) with an InviMag plant kit (Stratec, Berkenfeld, Germany; cat. no. 7437300250), following the manufacturer’s instructions. Total NA extracted from Tulipa was tested by reverse-transcription (RT) PCR using a broad-spectrum primer set for potexviruses (van der Vlugt and Berendsen 2002). A product of the expected size (730 bp) was obtained, and the amplicon was sequenced directly. The BLAST search in GenBank for the obtained sequence showed a maximum identity of 83% nucleotide (nt) with the RNA polymerase gene of Clover yellow mosaic virus (ClYMV) (accession no. D29630). To confirm the identification of ClYMV, RT-PCR was performed using specific forward (5′-GGACACTCAGCCTTCACCAACT) and reverse (3′-CCTGTCYGCTTCYTTGTAGTTTTG) primers designed to amplify a 508-bp fragment in the 3′ terminal region of this virus. A one-step RT-PCR kit from Invitrogen (Life Technologies) was used with the following cycling conditions: 50°C for 30 min, 94°C for 3 min, and then 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30s, followed by a final extension at 72°C for 5 min. The NA from the original host was tested by RT-PCR with the specific primers. An amplicon of the expected size was obtained, directly sequenced, and deposited in GenBank (MF666671). BLASTn analysis revealed the highest identity (95%) with a ClYMV isolate from Verbena (AY936202) and a much lower identity (85%) with a ClYMV isolate from Vicia (D29630). ClYMV has been reported to occur naturally in clover, alfalfa, common chickweed, peas, broad beans, apple, and verbena (Adams and Antoniw 2005; Mumford et al. 2005), although only a few sequences of broad bean and verbena isolates are available in GenBank to date. Cut flowers are an increasingly important commodity in NZ, and tulips are a significant crop. The presence of ClYMV could become an important threat to crop production, and economic value could be potentially affected. Considering that potexviruses can spread easily by mechanical means, the risk of mechanical spread to other ornamental hosts via propagation tools is possible. To our knowledge this is the first finding of ClYMV in tulip in the world.