First report of apple hammerhead viroid infecting apple trees in South Korea

Abstract

In August and September 2018, apple trees of ‘Fuji’ and ‘Hongro’ cultivars looked abnormal with poor growth. Suspecting infection by viruses, leaf samples were collected from a total of 22 Fuji and Hongro apple trees in Uiseong, Yeongcheon, and Jangsu in Korea. To perform comparative analysis of virus infections, leaves from a healthy and a diseased tree of Fuji and Hongro apple cultivars were chosen. Total RNA was extracted from each of the four selected samples with a HiYield Total RNA Mini Kit (Plant; RBC Bioscience, Taipei, Taiwan), and paired-end RNA sequencing was conducted using an Illumina HiSeq4000 system at Macrogen (Daejeon, Korea). Analysis of the de novo assembled contigs showed that the apple trees were infected with several viruses including apple green crinkle associated virus, apple stem grooving virus, apple stem pitting virus, and apricot latent virus. In addition, the samples were also infected with apple hammerhead viroid (AHVd), recently identified in apple trees (Szostek et al. 2018; Zhang et al. 2014). AHVd was found in three samples but not in the abnormal-looking Hongro sample. We tried to determine if there is a correlation between AHVd infection and poor growth of apple trees. To detect AHVd in 22 apple leaf samples, AHVd-specific primers (5′-TTAGCCTTCCTGATGAGTCC-3′ and 5′-TGTGTCTACTTAAAGACTCAC-3′) were designed using the contig sequences and 18 AHVd sequences available in GenBank. RNA extracted from the leaf samples was subjected to reverse transcription (RT) polymerase chain reaction (PCR) using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea) with the specific primer set. RT-PCR tests confirmed that 17 out of the 22 samples were infected with AHVd: five out of six healthy-looking Fuji samples, five out of six abnormal-looking Fuji samples, four out of five healthy-looking Hongro samples, and three out of five abnormal-looking Hongro samples. Thus, there was no correlation between presence of AHVd and poor growth. To obtain AHVd sequences from healthy-looking and abnormal-looking samples of the two cultivars, Sanger sequencing of RT-PCR amplicons was conducted at Macrogen. Each RT-PCR amplicon was purified using an AccuPrep PCR Purification Kit (BIONEER, Daejeon, Korea) and cloned into the RBC T&A Cloning Vector (RBC Bioscience, Taipei, Taiwan). Three clones per RT-PCR were analyzed. Interestingly, the RT-PCR amplicons from Fuji samples were 2 nucleotides (nt) longer than those from Hongro samples. NCBI BLASTn searches showed that 374-nt AHVd sequences from the healthy-looking or abnormal-looking Fuji samples (GenBank accession nos. MK690629 and MK690630, respectively) had the highest nucleotide sequence identities of 95.72 and 94.65% (100% query coverages) to isolate AHVd-17-Japan (GenBank: MG662376). The two partial AHVd sequences were 9 nt longer than the corresponding sequence of the isolate AHVd-17-Japan. Additional BLASTn searches showed that both 372-nt AHVd sequences (GenBank: MK690627 and MK690628) from the healthy-looking or abnormal-looking Hongro samples had the highest nucleotide sequence identity (98.12%; 100% query coverage) to AHVd isolate Italy2 (GenBank: MH049330). To the best of our knowledge, this is the first report on the presence of AHVd infecting apple trees in Korea. It is still not known if AHVd can cause any disease on apple or other orchard crop plants, and future studies need to address this.