Occurrence and sequence diversity of apple hammerhead viroid in major apple-producing regions of northern China

Apple hammerhead viroid (AHVd) is an emerging pathogen with a global distribution in apple-growing regions. To clarify its occurrence and distribution in northern China, long non-coding RNA (lncRNA) sequencing combined with RT-PCR was employed to screen 502 apple samples from commercial orchards in Hebei, Liaoning, Inner Mongolia, Shandong, and Shaanxi. AHVd was detected in 34.7% of samples, with infection rates ranging from 25.6% to 37.5% across the five provinces. Notably, 93.7% of AHVd-positive samples were co-infected with multiple viruses, with one extreme case harboring ten distinct pathogens. Quantitative RT-PCR revealed that AHVd accumulation was significantly higher in branch phloem than in leaves or fruit peels. Phylogenetic analysis demonstrated that Chinese AHVd isolates formed a distinct monophyletic group (Group II), although varieties clustered with Italian, Canadian, and German lineages. Sequence analysis identified nucleotide variations exclusively localized within stem-loop I, including a 5-nt deletion and a 10-nt insertion, without disrupting the conserved rod-shaped and highly branched secondary structure. These findings elucidate the epidemiology, genetic diversity, and tissue-specific distribution of AHVd in northern China, providing a foundation for improved diagnostic protocols and management strategies.