First report of Plum pox virus strain D isolate in peach (Prunus persica) in Korea

Plum pox virus(PPV; genus Potyvirus), the causal agent of Sharka disease of stone fruit trees (genus Prunus), is designated as a quarantine virus in Korea. To investigate the presence of viruses infecting peach trees (Prunus persica), 36 peach leaf samples showing viral symptoms such as mosaic, vein clearing, and malformation were collected at Cheongdo Peach Experimental Station in Cheongdo County, Korea, in May 2015. RNA sequencing was performed to identify the causal agent of the symptoms. All collected samples were combined into one sample, and total RNA was extracted from the pooled sample using an Easy-Spin Total RNA Extraction Kit (Intron, Daejeon, Korea). RNA sequencing was carried out on an Illumina HiSeq2500 sequencer by Theragen Etex Bio Institute (Suwon, Korea) and sequence analysis was performed by SeqGenesis (Daejeon, Korea), as described previously (Lim et al. 2015). One large contig (9,786 bp) of the analyzed viral 430 contigs corresponded to the genome of PPV-D isolate Ou1 (GenBank accession no. AB545926, 99% nucleotide identity with 100% coverage). To validate the RNA sequencing results, total RNA was extracted from all collected samples and carried out one-step reverse transcription (RT)-PCR with a PPV-specific primer set (5′-AATTTGCGTGTTTTCGTTCC-3′ and 5′-ATGGCGAAGTCTCAGTTGCT-3′). Nine of the 36 samples gave an amplicon of the expected size (437 bp) and the three selected positive amplicons were purified and sequenced. A BLAST search showed that the nucleotide sequences of the amplicons were most closely related to PPV-D isolate K27 (KR028387). Presence of PPV was also confirmed using amplification with the PPV-D strain specific primer set (P1 5′-ACCGAGACCACTACACTC CC-3′ and PD 5′-CTTCAACGACACCCGTACGG-3′), producing a 168 bp amplicon, according to the diagnostic protocols (Chirkov et al. 2016;Olmos et al. 1997). Electron microscopy of negatively stained preparations of crude sap extracted from three positive leaf samples showed that the sap contained filamentous rod-shaped particles approximately 700 to 800 nm long. For determination of the nucleotide sequence of the coat protein of the PPV isolate Cheongdo, the 1,137 bp amplicon obtained by RT-PCR with the primers PPV-CP-F (5′-GCATCTGAGACAGAAATTGAGC-3′) and PPV-CP-R (5′-CAACTGGATGATTAGACTCTCAC-3′) was cloned and sequenced. The nucleotide sequence (990 bp) of the coat protein was deposited into GenBank (LC159485). Sequence analysis showed that the PPV isolate Cheongdo has highest nucleotide sequence identity (99%) to PPV-D isolate K27 (KR028387) from Ukraine and was very close to typical PPV-D isolates from Russia, Poland, and Japan. To our knowledge, this is the first report of PPV in peach in Korea and the Korean isolate belongs to the PPV-D strain. Sharka, caused by PPV, is the most devastating and widespread disease of Prunus worldwide (Chirkov et al. 2016) and could be a serious threat to peach production in Korea. Therefore, a large-scale survey is required to determine the incidence of PPV with official management for the rapid eradication of the virus.