First Report of Apple Luteovirus 1 and Apple Rubbery Wood Virus 1 on Apple Tree Rootstocks in Korea

Abstract

Recently, several tentative new viruses have been discovered from apple trees (Malus pumila) using high-throughput sequencing (Liu et al. 2018; Rott et al. 2018); therefore, we have also investigated the existence of viruses infecting apple tree rootstocks, including new or emerging viral species, in Korea. Leaf samples, exhibiting yellowing and mottled symptoms, from 10 rootstocks (P1, Ottawa 11, Ottawa 13, Ottawa 5M, MM102, MM115, M5, M7, M10, and JM7) at the Apple Research Institute located in Gunwi and 20 rootstocks (M9) at a farmer’s apple orchard located in Cheongju were collected in September 2017. All samples were mixed into one pool, and an Illumina HiSeq2500 system was used to produce 100 bp paired-end RNA sequencing reads (a total of approximately 30 Gbp) at Theragen Etex Bio Institute (Suwon, Korea). Raw data treatment was performed using SG-VIPdb at SeqGenesis (Daejeon, Korea), as described by Lim et al. (2015). The high-throughput RNA sequencing generated contigs derived from two recently discovered viruses (apple luteovirus 1 [ALV-1] and apple rubbery wood virus 1 [ARWV-1]) not reported in Korea, as well as five viruses (apple chlorotic leaf spot virus, apple green crinkle associated virus, apple stem grooving virus, apple stem pitting virus, and apricot latent virus [Cho et al. 2016]) already identified in apple trees cultivated in Korea. NCBI BLAST searches showed that the 6,361-nt ALV-1 contig (3,507 mapped reads) shared 94% nucleotide sequence identity (93% query coverage) with a PA8 isolate (GenBank accession no. MF120198), the only ALV-1 isolate publicly available, and the 1,062-nt ARWV-1 contig (576 mapped reads) shared 99% nucleotide sequence identity (99% query coverage) with a 4342-5 isolate (MF062138). To confirm the presence of ALV-1 and ARWV-1, ALV-1-specific primers, 5′-CAAATCGAGCAAGAGGAGGC-3′ and 5′-TTTGCCAGTCCAACAAGTCG-3′ (expected size of 443 bp), and ARWV-1-specific primers, 5′-CCACTTGCTTACTGTTTCTGC-3′ and 5′-TGGACCATGCAGCATATGAA-3′ (expected size of 617 bp), were designed using sequences of each contig. All individual leaf samples were used to extract total RNA with a WizPrep Plant RNA Mini Kit (wizbiosolutions, Seongnam, Korea), and reverse transcription polymerase chain reaction (RT-PCR) was conducted using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea) with the specific primers. Of the 30 rootstock samples, six (M9) were found positive for ALV-1, and 11 (nine M9, one P1, and one JM7) were positive for ARWV-1. Among the virus-infected samples, four M9 samples were found to be coinfected by both ALV-1 and ARWV-1. One RT-PCR amplicon from a diagnosis of each viral infection was purified using an AccuPrep PCR Purification Kit (BIONEER, Daejeon, Korea) and sequenced at Macrogen (Daejeon, Korea). The NCBI BLAST searches of the amplicon sequences showed that the ALV-1 CJ15 isolate (from M9 rootstock; MH708164) had 97% nucleotide sequence identity to the PA8 isolate, and the ARWV-1 CJ11 isolate (from M9 rootstock; MH714536) had 98% nucleotide sequence identity to a 982-11 isolate segment S (MF062127). Because all ALV-1- and/or ARWV-1-infected samples were confirmed to be coinfected with more than two other viruses already identified in Korea mentioned above, a further survey is needed to clearly understand the symptoms caused by the viral infections. To the best of our knowledge, this is the first report on the existence of ALV-1 and ARWV-1 infecting apple tree rootstocks in Korea.