Cucumber mosaic virus (CMV; Cucumovirus, Bromoviridae) is an omnipresent virus characterized by a large host range and high genetic variability. Using high-throughput sequencing, we have characterized near complete genomes of 14 Slovak CMV variants from different plant hosts. Of these, three variants originated from the Papaveraceae species (oilseed poppy, common poppy and great celandine), previously poorly described as CMV natural hosts. Based on a BLAST search and phylogenetic analysis, the Slovak CMV isolates can be divided into two genetically different Groups, Ia and II, respectively. The SL50V variant, characterized by a divergent RNA2 sequence, potentially represents a reassortant variant. In four samples (T101, SL50V, CP2, MVU2-21), the presence of satellite CMV RNA was identified along with CMV. Although mechanically transmitted to experimental cucumber plants, the role of satellite RNA in the symptomatology observed could not be established due to a complex infection of original hosts with different viruses.

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the “6A” and “7A” groups based on the particular stretch of adenines at nucleotide positions 39–44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation.

Analysis of the viromes of three symptomatic Fabaceae plants, i.e., red clover (Trifolium pratense L.), pea (Pisum sativum L.), and common bean (Phaseolus vulgaris L.), using high-throughput sequencing revealed complex infections and enabled the acquisition of complete genomes of a potyvirus, bean yellow mosaic virus (BYMV). Based on phylogenetic analysis, the Slovak BYMV isolates belong to two distinct molecular groups, i.e., VI (isolate FA40) and XI (isolates DAT, PS2). Five commercial pea genotypes (Alderman, Ambrosia, Gloriosa, Herkules, Senator) were successfully infected with the BYMV-PS2 inoculum and displayed similar systemic chlorotic mottling symptoms. Relative comparison of optical density values using semi-quantitative DAS-ELISA revealed significant differences among virus titers in one of the infected pea genotypes (Ambrosia) when upper fully developed leaves were tested. Immunoblot analysis of systemically infected Alderman plants showed rather uneven virus accumulation in different plant parts. The lowest virus accumulation was repeatedly detected in the roots, while the highest was in the upper part of the plant stem.

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.

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.

Peanut stunt virus (PSV; genus Cucumovirus , family Bromoviridae ) was identified infecting black locust ( Robinia pseudoacacia L.) in Slovakia, representing an unusual host for this legume-infecting virus. Using high-throughput sequencing, three nearly complete tripartite genomes of PSV (designated AG6, AG19, and AG25) were obtained from naturally infected black locust trees. These isolates showed high mutual nucleotide identity (96.8–99.0%) but approximately 20% divergence from typical PSV isolates originating from legume hosts. Phylogenetic analyses placed the Slovak isolates within a distinct clade together with previously reported black locust PSV isolates from Hungary, supporting host-associated divergence. Additional RT-PCR screening revealed that 7 of 30 tested black locust trees were PSV-positive. All infected trees exhibited symptoms, while no asymptomatic PSV infections were detected. Notably, the black locust isolates were not detected using commercially available polyclonal anti-PSV antibodies, likely due to substantial divergence in capsid protein amino acid sequences. Biological assays demonstrated that black locust isolate AG6 remained infectious to several typical Fabaceae hosts following mechanical inoculation, including peanut, common bean, pea, and soybean, as well as Nicotiana benthamiana , with cultivar-dependent symptom expression observed in common bean and pea. Furthermore, PSV-associated satellite RNA was detected in two of seven PSV-positive samples, and its complete genomic sequence was determined, representing the first report of this satellite RNA in black locust. Altogether, these findings demonstrate the presence of genetically and serologically distinct PSV isolates in R. pseudoacacia and provide new insights into PSV diversification and host adaptation.