First Report of Apple Hammerhead Viroid in the United States, Japan, Italy, Spain, and New Zealand

In June of 2017, a ‘Honeycrisp’ apple (Malus pumila Mill.) cultivar from central Washington exhibiting shoot decline and dieback symptoms was submitted for diagnosis to the Washington State University Clean Plant Center Northwest (WSU-CPCNW). Total RNA was extracted (RNeasy Plant Mini Kit) from blade, midrib, and petiole tissue of leaves removed from the symptomatic plant. High-throughput sequencing (HTS) library preparation (Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Plant) and sequencing (Illumina HiSeq 50 Cycle Single Read Sequencing version 4 platform) was performed by Huntsman Cancer Institute (University of Utah, Salt Lake City). Sequence data analysis (CLC Genomics Workbench version 10.1.1) showed that the plant was infected with apple stem grooving virus, apple stem pitting virus, apple chlorotic leaf spot virus, apple green crinkle-associated virus, and apple hammerhead viroid (AHVd), a circular RNA containing hammerhead ribozymes first identified in China (Zhang et al. 2014) and recently shown to be a bona fide viroid (Serra et al. 2018). Approximately 23.5 million 50 nt raw reads were trimmed, filtered, and mapped against the Malus domestica genome to remove host sequences. Contigs were constructed by de novo assembly of the remaining 775,909 reads then subjected to BLAST analysis against the NCBI nucleotide, viroid, and virus reference databases. A single 451-nt contig covering the entire AHVd genome was assembled from 42,925 reads with a mean read coverage of 4,729. This isolate shared 90.2% identity with the AHVd reference sequence KR605506 from China (Zhang et al. 2014) and 88.4% with the Pacific Gala isolate MF402929 from Canada (Messmer et al. 2017). AHVd presence in this plant was confirmed by one-step reverse transcription polymerase chain reaction (RT-PCR) using the primers AHVd-13F_PG and AHVd-12R_PG (Messmer et al. 2017). The sequence of the Washington isolate was deposited in NCBI GenBank under accession number MG662372. On the basis of routine HTS and RT-PCR results, we screened 24 additional apple plants held in isolation and under quarantine that had been submitted by breeding programs and nurseries during 2016 to 2017 to the WSU-CPCNW from the United States and other countries. Screening by RT-PCR, as described above, indicated AHVd presence in 10 additional samples. HTS data were available for 8 of the 10 positive samples and confirmed AHVd presence in an additional sample from Washington, one from Ohio (U.S.A.), one sample each from Japan and New Zealand, and two samples each from Italy and Spain. Full-length or partial sequences of these eight isolates were deposited in NCBI GenBank under accession numbers MG662373 to MG662376, MH049330 to MH049332, and MH049334. Taken with the earlier reports (Messmer et al. 2017; Zhang et al. 2014), these results indicate that AHVd has a worldwide distribution. The aligned region spanning 30 to 427 nt of these additional isolates was compared with the reference sequences. These eight isolates shared 82 to 90.5 and 86.2 to 93.9% identities, respectively, with the AHVd reference sequences KR605506 from China and isolate Pacific Gala (MF402929) from Canada. AHVd has thus far been detected only in apple, indicating a single species, despite some sequence identities below the arbitrary cutoff of 90% (Serra et al. 2018). Although AHVd has been detected in apple trees with trunk splitting (Messmer et al. 2017) and shoot decline and dieback (this study), none of the other apples surveyed in this study or others (Serra et al. 2018) exhibited disease symptoms. Further work is needed before this recently described and apparently widespread viroid can be definitively associated with a disease.