First report of hop stunt viroid ( <i>Hostuviroid impedihumuli</i> ) infecting mulberry trees ( <i>Morus alba</i> L.) in Slovakia

Hostuviroid impedihumuli (hop stunt viroid, HSVd) is a widespread viroid species, a small, circular, single-stranded RNA phytopathogen with a genome of approximately 294–303 nucleotides (Flores et al., 2020). HSVd has a broad host range (Ortolá and Daròs, 2023), infecting multiple plant families, with grapevine (Vitis vinifera L.) considered its most prevalent host and putative evolutionary origin (Sano et al., 2001; Kawaguchi-Ito et al., 2009). Despite this wide host range, HSVd has only rarely been reported in mulberry in Europe (Elbeaino et al., 2012), with most documented isolates originating from Asia (Ma et al., 2023; Shilpa et al., 2024). Sampling of Morus alba L. (white mulberry, n = 41) and M. nigra L. (black mulberry, n = 13) was conducted in western Slovakia throughout 2024–2025 across multiple locations during the main fruit ripening period (May–July). Although HSVd-associated symptoms have been reported in mulberry (Shilpa et al., 2024), all sampled Slovak plants were asymptomatic, consistent with previous European reports (Elbeaino et al., 2012). All leaf samples were screened for HSVd by real-time quantitative PCR using in-house designed primers HSVd_101F (5`-CCAGCGAGAGGCGTG-3`, sense)/HSVd_239bR (5`-GAACCGAGAGGTGATGCC-3`, antisense) and probe (HSVd_133_P1: 5`-CTCTGGAGTAGAGGCTCTGCCTTCGAAACA-3`, FAM-labeled). Reactions were performed using TaqMan™ Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Samples were further analyzed by reverse transcription PCR using proofreading TaKaRa Ex Taq polymerase (Takara Bio Inc., Shiga, Japan) with primers HSV-78P and HSV-83M (Sano et al., 2001), enabling amplification of the full-length viroid genome. Amplicons were directly sequenced by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). Overall, 22 of 54 samples tested positive for HSVd by both methods. Interestingly, all positives originated from M. alba (~ 54% host-specific prevalence), whereas M. nigra samples tested negative. RT-qPCR indicated low HSVd accumulation (typically several thousand copies/µL), confirming that infections in mulberry are generally low-titer and may pose challenges for detection using conventional RT PCR (Elbeaino et al., 2012). Sequence analysis of 12 single-variant isolates (Genbank accession numbers PZ281875-PZ281886) showed that all detected HSVd variants fall within the known grapevine-derived sequence background, with no mulberry-specific mutations, supporting previous report (Shilpa et al., 2024). All these sequence variations corresponded to two clusters (“6A” and “7A”) defined by seven position-specific mutations previously identified within a grapevine population in Slovakia (Alaxin et al., 2023). Most sampled trees were also located in open agro-ecological environments, thus further indicating a common grapevine origin. Moreover, mixed “6A/7A” nucleotide signals were observed in 10 isolates by direct Sanger sequencing, likely reflecting population heterogeneity and mutual coinfections of mulberry by both HSVd clusters. The ability of mulberry isolates to retain their full infectivity across herbaceous hosts was previously demonstrated (Ma et al., 2023; Shilpa et al., 2024), suggesting that the common mulberry could pose as an active ecological hub or secondary host for HSVd infection dissemination with potential epidemiological relevance. Our results extend the information on HSVd incidence in mulberries in European region.