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

This study investigated the biochemical responses of resistant and susceptible varieties of common bean (Phaseolus vulgaris) to bean common mosaic virus (BCMV) infection. In the study, colour parameters (L, a, b, chroma and hue angle), total phenolic content, total flavonoid content, proline, ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, total soluble protein, hydrogen peroxide, malondialdehyde, chlorophyll content and antioxidant enzyme activities (catalase, ascorbate peroxidase and superoxide dismutase) were assessed. Results indicate that BCMV-infected susceptible plants exhibited significant reductions in chlorophyll a, chlorophyll b, and chlorophyll a/b ratio, impairing their photosynthetic capacity. In contrast, resistant varieties maintained higher chlorophyll levels and exhibited less oxidative stress. The infection also induced increases in total phenolic and flavonoid contents across both plant types, with the resistant variety showing a more robust accumulation, indicating a stronger defence response. Antioxidant enzyme activities, including catalase, ascorbate peroxidase, and superoxide dismutase, were elevated in both varieties; however, the resistant plants demonstrated significantly higher activity levels. These findings underscore the differential biochemical responses between resistant and susceptible bean plants, highlighting the complex interactions between BCMV and host defence mechanisms. Understanding these distinctions is crucial for developing effective management strategies against BCMV.

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.

This study investigated the effects of two plant viruses as biotic elicitors on hyoscyamine production in Datura stramonium L. callus cultures. Their effects were compared with those of acetylsalicylic acid, Norit buffer, and mechanical callus injury. The results demonstrated varying effects of the elicitors and differences in hyoscyamine production over time, with some acting more as inhibitors than elicitors. Eighteen days after the start of elicitation, the highest hyoscyamine production was observed in calli treated with tomato mosaic virus, reaching 4.06 +/- 0.50 mg per gram dry weight of callus. Elicitation with cucumber mosaic virus increased hyoscyamine levels to 2.55 +/- 0.68 mg/g of dry weight, while the application of 0.1 mM ASA resulted in 2.35 +/- 0.18 mg/g of dry weight. In untreated control callus, hyoscyamine production was 1.02 +/- 0.01 mg/g of dry weight. Mechanical damage to callus tissue led to hyoscyamine production of 2.76 +/- 0.7 mg/g of dry weight after 23 days of elicitation, compared to 1.71 +/- 0.17 mg/g of dry weight in untreated callus. In contrast, treatment with Norit buffer and 0.5 mM ASA reduced hyoscyamine production. The use of extracts from virus-infected plants as elicitors in in vitro plant cultures has not been reported to date. Both viruses in extracts demonstrated a positive effect on hyoscyamine production, particularly tomato mosaic virus. These findings clearly indicate that plant viruses are effective elicitors of secondary metabolite production in in vitro cultures.

Grapevine yellow speckle viroid 1 (GYSVd-1) is a widespread subviral pathogen affecting grapevine (Vitis vinifera L.), with potential implications for viticulture. This study investigates the prevalence, genetic variability, intrahost population polymorphism and secondary RNA structure of GYSVd-1 in Slovakia, where its molecular characteristics have been largely unexplored. Leaf samples from 49 grapevine plants across western and southern Slovakia were analyzed using RT-PCR, followed by sequencing and phylogenetic analyses. GYSVd-1 was detected in 73% of the samples, demonstrating high prevalence rates across vineyards in Slovakia. Sequence analysis revealed a high degree of genetic similarity among Slovak isolates, with nucleotide identity ranging from 96.2% to 100%, consistent with low variability reported globally. Phylogenetic analyses showed no specific grouping of Slovak isolates. Intrahost variability, assessed via subcloning of RT-PCR products, demonstrated the presence of random mutations among viroid clones, consistent with quasispecies nature of viroids in their hosts. These findings suggest that both interhost and intrahost viroid populations evolve under similar pressures to maintain structural stability essential for viroid function. Secondary RNA structure analysis of Slovak GYSVd-1 isolates revealed that all unambiguous sequences exhibited the characteristic rod-like conformation. In contrast, a significant proportion of sequences from mixed populations failed to form proper rod-like structures, suggesting that these variants may lack biological viability. Overall, this study unveils the prevalence of GYSVd-1 in Slovak vineyards and provides valuable insights to guide future phylogenetic studies and structural analyses of viroids.

The disease “sharka”, caused by Potyvirus plumpoxi (plum pox virus), is the most harmful viral disease affecting stone fruits. The virus spreads over long distances through illegal and insufficiently controlled exchange of infected propagative plant material. Once established in an area, the virus spreads locally through vegetative propagation of infected plant material, and naturally through aphid-vectors. Previously considered a European problem, sharka has now been reported in 54 Prunus-growing countries in all continents except Oceania, although the disease has been eradicated from the United States of America. The economic cost of the disease in the 28 years from 1995 to 2023 is estimated to be €2.4 × 109, equivalent to approx. 0.17% of the stone fruit industry’s value. This includes more than over €2 × 109 in direct fruit losses, €1.4 million from international rejection of symptomatic fruit, and over €100 million in eradication and disease limitation costs. Indirect costs include €137 million, mainly associated with ELISA analyses, and approx. €130 million in costs related to research and science networks. Cumulative global losses from the sharka pandemic since the decade 1910/20 probably surpass €13 × 109. These outlays exclude indirect trade costs, economic losses, genetic erosion of traditional cultivars, and the costs of developing new cultivars tolerant or resistant to plum pox virus. The decline in these costs compared to the previously evaluated €10 billion from the 1970s to 2006 is analyzed. Four case studies (for Spain, Turkey, Chile, and Greece) illustrate different sharka scenarios and management strategies.

This collaborative work by over 180 researchers from 40+ countries addresses the challenges posed by “phantom agents”—putative pathogenic agents named in literature without supporting data on their existence. Those agents remain on regulatory lists, creating barriers in trade and plant certification. Historically identified based solely on symptoms, these agents lack isolates or sequence data, making reliable detection or risk assessment impossible. After reviewing over 120 such agents across 10 key plant genera, we recommend their removal from regulatory lists and call for revised standards aligned with modern diagnostics. This effort seeks to streamline germplasm exchange, benefiting global agriculture by removing the constraints imposed by phantoms.

Over the past two decades, plant viral vectors have emerged as a powerful tool for the production of recombinant proteins in plants. Among the different plant viruses engineered to carry foreign genes of interest in their genomes, potyviruses have gained attention due to their polyprotein expression strategy and broad host range. To date, at least eleven different species belonging to the genus Potyvirus have been used for heterologous gene expression in both their natural and experimental hosts. This review article provides an overview of the current state of potyvirus-based plant viral vectors, discussing the advantages and limitations of these systems. We also discuss the future challenges and potential applications of potyvirus-based expression vectors, including the production of vaccines, nanoparticles, therapeutics, and metabolic engineering. Overall, we highlight the potential of potyvirus-based vectors as a versatile tool for recombinant protein production in plants.

Cucumber mosaic virus (Bromoviridae) is a worldwide widespread pathogen affecting more than 1200 plant species. Although more attention is paid to CMV research on agricultural crops, vegetables or ornamental plants, this work was focused on the analysis of several weeds and wild plants as potential natural hosts of CMV. For this purpose, samples from two different agroecological locations were tested by DAS-ELISA using CMV-specific antibodies, revealing nine CMV-positive plant species. CMV isolates from greater burdock (Arctium lappa L.), rough hawksbeard (Crepis biennis L.), wood avens (Geum urbanum L.), prickly lettuce (Lactuca serriola L.), black medick (Medicago lupulina L.), bitter dock (Rumex obtusifolius L.), field sowthistle (Sonchus arvensis L.), white clover (Trifolium repens) and white violet (Viola alba Besser.) were further characterized by sequencing the complete CP gene, disclosing their affiliation to the CMV Group II. We have shown that weeds and wild plant species can be effective hosts for CMV and can serve as virus reservoirs from which the virus can be spread to cultivated crops.

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.

During the screening of lettuce (Lactuca sativa L.) samples from the United Kingdom in 2015 using high throughput sequencing (HTS) technology, the genome of a novel virus was identified in a plant showing symptoms of stunting and yellowing. The virus genome consisted of two RNA molecules; an RNA1 of 6001 nt encoding a polyprotein with protein cofactor, helicase, protease and RNA-dependent RNA polymerase motifs and an RNA2 of 6553 nt encoding a polyprotein with viral movement and coat protein motifs. Independently, in 2018, an analysis of ribodepleted total RNA from a dandelion (Taraxacum officinale L.) displaying leaf malformation and puckering revealed a complex infection involving a secovirus related to the UK lettuce virus. Moreover, a small survey confirmed the presence of the secovirus in environmental dandelion samples in Slovakia. Detailed analysis of these sequences suggests they all belonged to the same putative species, a member of the subgenus Stramovirus, genus Sadwavirus, family Secoviridae named lettuce secovirus 1 with the tentative Latin binomial Sadwavirus lactucae.

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer.

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.

Prune dwarf virus (PDV) infection affects the production of stone fruits ( Prunus spp.) around the world, including in Turkey. In this study, we analyzed the available nucleotide (nt) and amino acid (aa) sequences of P1, P2, movement protein (MP), and coat protein (CP) genes to assess the molecular variability and structure of non-Turkish and Turkish PDV populations. Global isolates were clustered into three major groups in nt-based phylogenetic trees of MP and CP. Comparison of geographically different isolates showed that the MP aa sequences were more conserved than the CP aa sequences. The major aa changes in MP and CP showed the most effect on group 3 isolates. MP and CP-based analyses revealed the lowest number of variable sites ( S ), total number of mutations (η), average number of nt differences between sequences ( k ), and nt diversity (per site) (π) values in group 3 isolates, indicating high genomic similarities. The Turkish isolates manifested the highest S and η, and the second highest k and π values were observed in the comparison with CP, confirmed the high intra-group divergence of the Turkish isolates belonging to the three phylogroups. A stronger purifying selection pressure was observed in the MP region than in the CP region. Neutrality tests deduced negative values for all the MP and CP phylogroups and positive values for the P1, P2, Bulgarian, and Polish populations. MP and CP comparisons showed the divergence of the three phylogroups, as revealed by the results of the K S *, K ST *, Z *, and S nn permutation statistical tests. The fixation index ( F ST ) values suggested that non-Turkish PDV isolates had a higher genetic distance compared with Turkish isolates from group 3 and the Turkish isolates from the other two groups.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

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.

High-throughput sequencing of two lettuces showing virus-like symptoms in France provided evidence of infection by members of the family Secoviridae. One plant (JG1) had a complex mixed infection that involved, among others, a novel waikavirus (lettuce waikavirus 1) and two isolates of a sequivirus related to lettuce mottle virus (LeMoV). The second lettuce plant (JG2) was singly infected by LeMoV. Complete genomic sequences were obtained for all four isolates and, in addition, near complete genome sequences were obtained for other LeMoV or LeMoV-related isolates (from French cultivated and wild lettuces and from a Brazilian cultivated lettuce) and for two isolates of another family Asteraceae-infecting sequivirus, dandelion yellow mosaic virus (DaYMV). Analysis of these genomic sequences allows the proposal of tentative genome organization for the various viruses and clarification of their phylogenetic relationships. Sequence and host range comparisons point to significant differences between the two sequivirus isolates identified in the JG1 plant and LeMoV isolates from France and Brazil, suggesting they belong to a novel species for which the name lettuce star mosaic virus is proposed.

Phaseolus vulgaris alphaendornavirus-1 (PvEV-1, family Endornaviridae) was identified by ribodepleted total RNA high-throughput sequencing in the virome of two bean plants (Phaseouls vulgaris L.) grown in a garden in western Slovakia. Two nearly complete PvEV-1 genomes (ca. 14.06 kb, named PV1 and PV2) were assembled, showing 99.9% nucleotide identity, while their nucleotide identity with the reference PvEV-1 genome (NC_039217) reached 98.4%. Two primer pairs spanning the viral helicase encoding region and sequence upstream of the RNA-dependent RNA polymerase were designed and used to confirm the presence of the virus in the original bean samples by RT-PCR. A subsequent search for PvEV-1 presence in Slovakia was focused on two groups of samples: 1) bean plants grown under open field conditions and sampled during the vegetation period and 2) bean accessions grown from seeds obtained from a Slovak and French bean germplasm collection. Based on RT-PCR results, 4 out of 15 bean samples from open fields and 12 out of 21 bean accessions from the curated germplasm collection tested PvEV-1-positive. Interestingly, sequencing of RT-PCR products revealed that all amplified isolates are identical in the two amplified genomic portion which is also identical to those of the PV1 and PV2 isolates. These results suggest a relatively high incidence of PvEV-1 in bean in Slovakia. This is the first evidence and characterization of PvEV-1 from bean plants in Europe.

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.