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First Report of Milk Vetch Dwarf Virus Infecting Lily in Korea

Choi, H., Jo, Y., Zhou, Y., Cho, W. K.
Plant disease 2019 v.103 no.8 pp. 2144
Aphis craccivora, Astragalus sinicus, Carica papaya, Cucumber mosaic virus, DNA, Glycine max, Lilium, Lily mottle virus, Lily symptomless virus, Milk vetch dwarf virus, Nicotiana tabacum, Pisum sativum, Plantago asiatica, Plantago asiatica mosaic virus, RNA, RNA libraries, Vicia faba, alternative hosts, bulbs, certification, cultivars, faba beans, flowers, leaves, nucleotide sequences, peas, plant diseases and disorders, plant viruses, plantations, sequence analysis, soybeans, surveys, tobacco, vegetative propagation, viruses, Japan, Massachusetts, South Korea
Milk vetch dwarf virus (MDV) is a member of the genus Nanovirus in the family Nanoviridae, composed of eight genomic DNA segments, and is transmitted by aphids (Shirasawa-Seo et al. 2005). MDV infects legume plants including broad beans (Vicia faba L.), peas (Pisum sativum L.), soybeans (Glycine max [L.] Merr.), and Chinese milk vetch (Astragalus sinicus L.), causing yellowing and dwarf symptoms. In addition, MDV infects tobacco (Nicotiana tabacum L.) (Yang et al. 2016) and papaya (Carica papaya L.) (Lal et al. 2018). In June 2018, five out of seven oriental lilies (Lilium × hybridum), cultivar ‘Medusa’ cultivated in Iksan, South Korea, showed strong yellowing in the leaves and dwarf symptoms. To confirm the viral nature of this lily disease, we pooled five flower samples and extracted total RNAs using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Using the NEBNext Ultra RNA Library Prep Kit for Illumina, following the manufacturer’s instructions (NEB, Ipswich, MA), we generated a single RNA-sequencing library, which was further paired-end sequenced (2 × 100 bp) using Illumina’s HiSeq 2000. Raw sequence reads were assembled by a Trinity program with default parameters, and the assembled contigs were subjected to a BLASTn search against NCBI’s viral reference database (Jo et al. 2018). We identified four contigs (39 reads) ranging from 457 to 736 bp and two contigs (218 reads) with 994 bp specific to MDV segments DNA-M and DNA-S, respectively. We only identified MDV-associated contigs through RNA sequencing. An assembled contig of 994 bp in length, referred to as the MDV segment DNA-S isolate Medusa, was blasted against NCBI’s nucleotide database. The BLASTn result showed that the MDV segment DNA-S isolate Medusa shared 91% nucleotide identity with broad bean No.9 in Japan (GenBank AB044387.1). To confirm the presence of MDV in the sample, polymerase chain reaction (PCR) was conducted with MDV DNA-S-specific primers, including 5′-ACAGCTGTCTTTGCTTCGTCTCCA-3′ (position 232 to 255) and 5′-CCTTCATTAATGAAGGGCATTATTGG-3′ (position 950 to 925), based on the MDV reference genome sequence (GenBank NC_003646.1). We amplified a 719-bp PCR product that was cloned, followed by Sanger sequencing. The sequence result confirmed that the MDV isolate Medusa (GenBank MK433284) shared 93% nucleotide identity with MDV isolate VU (GenBank KY070245.1). Moreover, we also obtained the complete sequence of the MDV DNA-M segment (GenBank MK370719) by PCR and Sanger sequencing. We examined 16 other asymptomatic lily cultivars naturally grown in five different plantations for MDV infection through PCR using the same primers and protocol described above. We examined three plants per cultivar. Out of 16 lily cultivars, only Medusa plants (all three) were infected by MDV. We confirmed the absence of the cucumber mosaic virus, lily mottle virus, lily symptomless virus, and Plantago asiatica mosaic virus in the Medusa plants by reverse transcription PCR, indicating a single infection of MDV in the Medusa cultivar. Our study revealed the alternative host of MDV in lily for the first time; however, nothing is known for aphids, which could transmit MDV in Korea. Because lilies are vegetatively propagated, a bulb certification program should include MDV. Moreover, a survey for the presence of Aphis craccivora, the known vector for MDV (Inouye et al. 1968) in lily fields should be carried out, to consider the possibility of the natural spread of this virus. To our knowledge, this is the first report of MDV infecting lily in the world.