First Report of Malus domestica Virus A Infecting an Apple Tree in South Korea

Abstract

Apple (Malus domestica) is economically important and widely grown in South Korea. Many viruses and viroids infect apple trees, reducing the productivity of orchards (Baek et al. 2019; James et al. 2013). Apple stem grooving virus (ASGV), apple mosaic virus (ApMV), apple latent spherical virus (ALSV), apple chlorotic leaf spot virus (ACLSV), apple green crinkle associated virus (AGCaV), apple stem pitting virus (ASPV), and apple scar skin viroid (ASSVd) are the major viral pathogens that infect apple (Cho et al. 2016). Malus domestica virus A (MdoVA), a putative member of Velarivirus (Closteroviridae), has been identified from an apple tree in the Czech Republic (Koloniuk et al. 2019; Rubio et al. 2013). From August to September 2018, 11 asymptomatic and 11 symptomatic apple leaf samples showing mild mosaic symptoms, necrotic leaf spots, mottling, leaf distortion, or yellowing were collected from Uiseong (n = 4; cv. Fuji), Yeongcheon (n = 4; cv. Hongro), and Jangsu (n = 14; Fuji = 8 and Hongro = 6). Total RNA was extracted from two symptomatic and two asymptomatic samples using a HiYield Total RNA Mini Kit (Plant; RBC Bioscience). Four cDNA libraries were constructed and sequenced by the Illumina HiSeq4000 system at Macrogen. A total of 219,160,492 paired-end raw reads were generated from the four libraries with the Illumina HiSeq4000 sequencer. The raw reads were quality-trimmed and assembled into 387,422 contigs of 201 to 16,053 nt using Trinity v2.1.1 software. BLASTn analysis of the contigs indicated that the selected samples were infected with several apple viruses, including ASPV, ASGV, ACLSV, AGCaV, and apple hammerhead viroid (AHVd). A BLASTn search of the obtained contigs also detected seven contigs (741 to 5,138 nt) related to MdoVA (GenBank MN548734). The nucleotide (nt) similarities between these contigs and the MdoVA reference sequence were 88.1 to 93.9%. Specific primer sets (i.e., MdoVA-SK_F3 [nt position 2,496 to 2,515]: 5′-GTTGGCTCATGACAGTTTGC-3′; MdoVA-SK_R3 [3,722 to 3,741]: 5′-GGATGTGGTAAGGTAGTACA-3′; MdoVA-SK_F9 [9,269 to 9,288]: 5′-GTACATGAGGACTATCCTCG-3′; and MdoVA-SK_R9 [10,464 to 10,483]: TCTGACTTTCCTGAGGAACC-3′) were designed to confirm the presence of MdoVA in some of the apple leaf samples. Total RNA was extracted from the 22 samples individually and subjected to RT-PCR using SuPrimeScript RT-PCR Premix (GeNet Bio) with MdoVA-specific primers. The expected fragments of 1,246 and 1,215 bp were only detected in the sole asymptomatic Fuji sample that was included for high-throughput sequencing, suggesting that the virus was not associated with any of the symptoms observed. This sample was used to determine the complete genome sequence of this South Korea MdoVA isolate (MdoVA-SK). Specific primer sets were designed based on the seven contig sequences. All designed primer pairs successfully produced overlapping RT-PCR fragments of the expected size from the sample. PCR products were purified with an AccuPrep PCR Purification Kit (BIONEER) and cloned into an RBC TA cloning vector (RBC Bioscience). At least four clones of each fragment were Sanger sequenced. The obtained overlapping sequences were consistent with the original assembled contigs from paired-end RNA sequencing. The 5′- and 3′-terminal sequences were obtained using a 5′- and 3′-Rapid Amplification of cDNA Ends kit (RACE version 2.0; Invitrogen) based on the MdoVA-SK specific 5′ and 3′ RACE primers. The complete genome sequence of MdoVA-SK (17,004 nt) was assembled using DNAMAN v5.2.10 (Lynnon Biosoft) and deposited in GenBank (MN901923). BLASTn analysis showed that MdoVA-SK shared 91.3% sequence identity at the complete genome level with the sole MdoVA isolate (J1, MN548734) in GenBank. At the individual gene level, the identities between the isolates were 88.6 to 99.0%. This is the first report of MdoVA naturally infecting apple trees in South Korea and the second report in the world. Although the virus did not appear to be associated with any disease symptoms in apple trees, more research is needed to understand its biological and pathological properties.