Orchid is one of the most popular and commercially important cultivated flowers in the world. Among many orchid species, Dendrobium species are popular cut flowers and potted plants in South Korea. In March 2019, 10 Dendrobium orchid plants in a greenhouse in Daegu, South Korea showed large chlorotic blotches, mosaic and mottle symptoms. One leaf each from the 10 symptomatic orchid plants by leaf dip-preparations and transmission electron microscopy (JEM-1400; JEOL Inc., Tokyo, Japan) after leaf dip-preparations (Brenner and Horne 1959; Richert-Pöggeler et al. 2019). Typical potyvirus-like particles of flexuous and filamentous shape and ∼ 760 × 15 nm length/width were observed in all tested samples. The presence of potyvirus was confirmed by serological detection with a commercially available ImmunoStrip® for potyvirus group (Agdia, Elkhart, USA). In contrast, a negative result was obtained for a virus-free Dendrobium plant by the serological test. The two most common viruses in orchids, namely odontoglossum ringspot virus (ORSV) and cymbidium mosaic virus (CymMV) in all Dendrobium samples were not detected in any samples by an ImmunoStrip® for ORSV and CymMV (Agdia, Elkhart, USA). To determine the species of the virus, total RNA was extracted from all 10 ImmunoStrip®-positive samples using the RNeasy plant mini kit (Qiagen, Hilden, Germany). Subsequently, reverse transcription-PCR (RT-PCR) products (~1,625 bp) were amplified using potyvirus- specific primer pair (Gibbs and Mackenzie, 1997) and sequenced by the Sanger method at Macrogen (Seoul, South Korea). Sequencing results showed 100% nucleotide identity among 10 samples. Thus, one sequence was chosen for identification of virus species using sequence comparison. BLASTn analysis showed that the nucleotide sequence and its deduced amino acid sequence of the amplicon shared 95.4-98.7% and 96.2-99.6% identity to multiple clover yellow vein virus (ClYVV) sequences (e.g., accession no. AB011819) in GenBank. To further confirm the presence of ClYVV and determine if other viral agents were present in the samples, total RNA from three of the 10 symptomatic plants was depleted of ribosomal RNAs and subjected to high-throughput sequencing (HTS) analysis on a HiSeq 4000 platform (Macrogen Inc., Seoul, South Korea). A total of 3,764,432, 4,203,881, and 4,139,775 of 150-bp paired-end clean reads were obtained for the three samples. After de novo assembly of the reads with Trinity (Haas et al. 2013), 5, 6 and 7 contigs were obtained and searched with BLASTn against NCBI viral refseq database. Eighteen contigs from all three samples sized at 2,176-9,432 nt exhibited 94.0-97.9% nucleotide identity with the complete genome sequences of other ClYVV isolates (e.g., accession no. AB011819) deposited in Genbank; no other viruses were identified by HTS. The complete genome sequence (9,585 nucleotides in length) of ClYVV Dendrobium isolate (ClYVV-Den) was determined using ClYVV-specific primers (Takahashi et al., 1997) and the sequence of CIYVV-Den was deposited to GenBank (Accession no. LC506604). Together, these results support that symptomatic Dendrobium orchids were infected with ClYVV-Den in this study. ClYVV has been previously reported affecting Calanthe orchids in Japan (Inouye et al., 1988; Ikegami et al., 1995). Our results suggest that ClYVV may be detrimental to the production of Dendrobium orchids or commercial ornamental crops in South Korea. To our knowledge, this is the first report of ClYVV in Dendrobium sp. in South Korea.
HTS-Based Diagnostics of Sugarcane Viruses: Seasonal Variation and Its Implications for Accurate Detection
