Evaluation of Hop Varieties (Humulus lupulus L.) for Their Response to HLVd, HSVd, and CBCVd Viroids in tissue cultures

Evaluation of Hop Varieties (Humulus lupulus L.) for Their Response to HLVd, HSVd, and CBCVd Viroids in tissue cultures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Evaluation of Hop Varieties (Humulus lupulus L.) for Their Response to HLVd, HSVd, and CBCVd Viroids in tissue cultures Helena Volk, Patricija LAP, Andreja ČERENAK, Jernej JAKŠE This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5282171/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Feb, 2025 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract This study evaluates the susceptibility of various hop varieties to three viroids: Hop Latent Viroid (HLVd), Hop Stunt Viroid (HSVd), and Citrus Bark Cracking Viroid (CBCVd), all of which pose significant threats to Slovenian hop production. The experiment was conducted under in vitro conditions, involving the cultivation of 13 different hop varieties in tissue cultures, alongside the in vitro synthesis of viroid transcripts and subsequent inoculation of the plants. The plants were then monitored for growth, development, and their responses to viroid infection. The findings reveal the complex interactions between hops and viroids, with successful inoculation observed in all varieties. However, infection rates varied, with HSVd showing the highest rates of infection, followed by CBCVd and HLVd. This research enhances the understanding of viroid dynamics in hop plants, which is crucial for developing effective disease management strategies. Despite the insights gained, no resistance or tolerance to the viroids was identified in any of the tested hop varieties. hops tissue culture viroid CBCVd HLVd HSVd Figures Figure 1 Figure 2 Figure 3 Figure 4 Main Text The common hop ( Humulus lupulus L.) is classified as an herbaceous perennial plant and belongs in the hemp family (Cannabacae). Hop cultivation plays a crucial role for the brewing industry, providing the essential oils and bitter acids necessary for flavouring and preserving beer. Viroids are small, single-stranded, circular RNA molecules with a size of 246 to 401 nucleotides. These molecules do not encode proteins, but can replicate, move, and evade the host's defence system (Darós, 2016). To date, four different hop viroids have been discovered: Hop Latent Viroid (HLVd) (Puchta et al., 1988), Hop Stunt Viroid (HSVd) (Sasaki and Shikata, 1977), Apple Fruit Crinkle Viroid (AFCVd) (Sano et al., 2004) and Citrus Bark Cracking Viroid (CBCVd) (Jakše et al., 2015). All these viroids were found in European hop fields (Radišek in sod., 2012; Jakše et al., 2015), except for AFCVd, which only occurs in Japan. HLVd spreads among hop plants primarily by vegetative propagation and often shows no clear symptoms of infection (Barbosa et al., 2005). This has made HLVd the most widespread viroid in hop fields. In contrast, HSVd is not as widespread, however it has numerous host plants, and the disease has a significant economic impact in hops. CBCVd was identified in 2007 in Slovenian hop fields as the cause of a previously unknown disease characterized by visible and distinct symptoms that lead to plant death within 3 to 5 years (Jakše et al., 2015). The mechanisms by which viroids cause disease are still unclear, though their symptoms resemble viral infections, suggesting transcriptional or post-transcriptional gene silencing as a mode of action. Since viroids are present in small quantities, they are not thought to deplete RNA nucleotides in host cells. Many hosts show no symptoms and are considered "latent," but viroids can multiply rapidly in susceptible plants. A single nucleotide change can alter the severity of the disease. Mechanical transmission through contaminated tools is common, while seed or pollen transmission is rare. Viroids infect plants by entering and replicating in the chloroplast or nucleus, then spreading between cells via plasmodesmata and through the phloem (Navarro et al., 2021). Infected plants activate RNA silencing mechanisms, including both transcriptional and post-transcriptional silencing, to defend against viroids (Mishra et al., 2018; Navarro et al., 2021). The aim of this study was to test 13 hop varieties for their susceptibility to the viroids HLVd, HSVd and CBCVd, three viroids which pose a threat to European hop fields, in tissue culture conditions. Hop cultivation ends in late August and the plant remains dormant until late spring. As a tall plant cultivated on special trellises, evaluating symptoms throughout the year in a smaller space using tissue culture could expedite the assessment process. The expectation was that symptoms would develop earlier in tissue cultures and that we would discover new resistant or tolerant hop varieties and to confirm the resistance and tolerance of two hop varieties (Styrian Wolf and Styrian Cardinal, respectively) that had shown these characteristics in initial field studies (Radišek et al., 2022). A Murashige and Skoog medium was used for micropropagation of hop (Frant in sod., 2001) varieties: Atlas, Blisk, Buket, Cekin, Cicero, Styrian Eureka, Styrian Gold, Styrian Kolibri, Wye Target, Bobek, Styrian Wolf, Styrian Cardinal, and Celeia. The dimeric RNA transcripts used for viroid inoculation were prepared from plasmids containing the dimeric sequence of the corresponding viroid. Plasmids were isolated from overnight cultures of E. coli DH5α using the High Pure Plasmid Isolation Kit (Roche), linearized with a restriction enzyme, and reverse transcribed using a reverse transcription kit following the manufacturer's protocols. Specifically, 1 µg of linearized plasmid was used in a 20 µL transcription reaction, incubated at 37 °C for 4 hours in a thermocycler. Table 1 lists the combinations of restriction enzymes and reverse transcription kits used for reverse transcription to produce the positive strand of the viroid transcript. Table 1: Enzymes and reverse transcription kits used for viroid transcript synthesis Viroid Plasmid carrier Enzyme used Reverse transcription kit CBCVd pBSII-CBCVd HincII (Thermo Scientific) MEGAscript™ T7 Transcription Kit (Thermo Scientific) HLVd pBSII-HLVd Eco53kI (NEB) MEGAscript™ T3 Transcription Kit (Thermo Scientific) HSVd pGEM-T-Easy-HSVd BmrI (NEB) MEGAscript™ SP6 Transcription Kit (Thermo Scientific) Inoculation of hop plants with HLVd, HSVd and CBCVd transcripts was carefully performed on selected uniform plants to allow accurate monitoring of disease symptoms. Inoculum was prepared in a volume of 50 µL by mixing 150 µg of each viroid transcript with 0.5 mM K 2 HPO 4 , with an additional viroid-free control inoculum prepared only with 0.5 mM K 2 HPO 4 . The largest and healthiest leaf sitting lowest on the plant was selected for inoculation. Each plant was inoculated with 1 µl of the desired inoculum, which was smeared over the leaf with an L-shaped plastic rod to create small lesions through which the viroid could enter and spread in the plant (Figure 1). Four plants per variety were inoculated, while two plants served as controls. Figure 1: Inoculation of hop tissue cultures with viorid RNA transripts Eight weeks after inoculation, total nucleic acids were isolated (Kump and Javornik, 1996) from newly developed shoots and viroid presence was evaluated by RT-PCR (Jakše in Radišek, 2005) using the appropriate primer sequences (Bernad and Duran-Vila, 2006 for CBCVd; Hataya et al., 1992 for HLVd and Matousek et al., 2003 for HSVd). We confirmed that all hop cultivars were successfully infected with all transcripts (Figures 2, 3 and 4). We also noted that the varieties 'Bobek' and 'Wye Target' were not viroid free plants since HLVd infection was confirmed in starting material. HSVd showed highest mean infection rate since 94 % of inoculated plants were successfully infected with it. The mean infection rate of CBCVd was also high, 90 % and the mean infection rate of HLVd, naturally the most widespread viroid in hop was surprisingly low at 55 %. A more detailed overview of the infection rates is presented in Table 2. Figure 2: Results of agarose gel electrophoresis of RT-PCR of HLVd inoculation of hop plants Figure 3: Results of agarose gel electrophoresis of RT-PCR of HSVd inoculation of hop plants Figure 4: Results of agarose gel electrophoresis of RT-PCR of CBCVd inoculation of hop plants Table 2: Number and % of infected plants inoculated with HLVd, HSVd or CBCVd viroid HLVd HSVd CBCVd All 3 viorids Hop cultivar no. % no. % no. % no. % Atlas 2 50 4 100 4 100 10 83 Blisk 3 75 4 100 2 50 9 75 Buket 3 75 4 100 3 75 10 83 Cekin 1 25 3 75 4 100 8 67 Cicero 2 50 3 75 3 75 8 67 Styrian Eureka 3 75 4 100 4 100 11 92 Styrian gold 3 75 3 75 4 100 10 83 Styrian Kolibri 2 50 4 100 3 75 9 75 Wye Target n.a. 4 100 4 100 8 100 Bobek n.a. 4 100 4 100 8 100 Styrian Wolf 2 50 4 100 4 100 10 83 Styrian Cardinal 1 25 4 100 4 100 9 75 Celeia 2 50 4 100 4 100 10 83 All cultivars 24 55 49 94 47 90 120 81 Legend: “no.” – number of viroid positive plants out of 4 inoculated per viroid; “n.a.” – not applicable, the plants have been HLVd-infected prior the experiment. The symptoms of plants infected with HLVd, HSVd, and CBCVd viroids can be challenging to recognize, especially under certain conditions like tissue culture, where plants do not fully develop visible symptoms such as cones or tall growth. In the field, HLVd infections can affect metabolic pathways, leading to chlorosis, slower growth, and smaller cones, but these symptoms were not observed in our tissue-cultured plants, which did not significantly differ from the controls. Similarly, HSVd symptoms, typically appearing years after infection and including curling leaves, smaller cones, and reduced alpha acid content, were absent in our study. Both viroids infected all cultivars with no resistance or tolerance, with infection rates for HLVd and HSVd ranging from 25 % to 100 %, as shown in Table 2. CBCVd infections are known to cause early mild yellowing and curling of leaves in the field, but our CBCVd-infected plants also did not show significant symptoms in tissue culture. While field observations suggest that Styrian Wolf is the only hop that cannot be infected with CBCVd (Radišek et al., 2022), our study successfully infected it with 100 % efficiency using in vitro viroid transcripts. We hypothesize that the differences between our results and field conditions stem from varying viroid transmission methods and plant growth environments, with field infections primarily occurring mechanically through low levels of infected sap, whereas our study used high concentrations of synthetic viroid transcripts. Future research should explore lower concentrations and RNA extracts from infected plants to better replicate field conditions. The tissue cultures were maintained for an additional 3 months until the plants depleted the cultivation media and required subculturing. A visual evaluation conducted at that time again yielded no significant information on viroid symptoms due to uneven growth. This aligns with previous studies, such as Marton et al. (1982), who found no significant effect of Citrus Exocortis Viroid (CEVd) on the growth of tomato callus and suspension cultures. Recent research on viroids and in vitro plant cultivation has primarily focused on viroid elimination (Bettoni et al., 2022; Yang et al., 2024) or detection (Wan Chow Wah and Symons, 1997; Lee et al., 2022), rather than symptom development. Our findings further highlight that visual symptoms may not reliably indicate viroid presence in tissue culture, emphasizing the need for molecular detection techniques like RT-PCR for accurate identification. Hop growers face significant challenges from various pathogens, including viroids, which can be mitigated by using viroid-free planting material. Breeding efforts focus on developing virus- and viroid-free genotypes that are resistant to diseases and pests (Oset Luskar et al., 2021). This research aimed to assess the susceptibility of various hop cultivars to HLVd, HSVd, and CBCVd infections, which threaten European hop fields. While we expected to find resistance or tolerance in some cultivars, all tested varieties were infected by the three viroids. Surprisingly, HLVd, the most widespread viroid, had the lowest infection potential (55 % average), while CBCVd and HSVd exhibited higher infection rates, with averages of 90 % and 94 %, respectively. RT-PCR confirmed successful infection in nearly all samples inoculated with HSVd. Declarations Acknowledgments: This research was financially supported by the Slovenian Research and Innovation Agency project Z4-3214 and by the Slovenian Research and Innovation Agency program P4-0077. Statements and Declarations: Competing Interests : The authors declare that they have no financial or non-financial competing interests to disclose. References Barbosa, C. J., Pina, J. A., Bernad, L., Serra, P., Navarro, L., & Duran-Vila, N. (2005). Mechanical transmission of citrus viroids. Plant Disease , 89 (7), 749–754. https://doi.org/10.1094/PD-89-0749 Bernad, L., & Duran-Vila, N. (2006). A novel RT-PCR approach for detection and characterization of citrus viroids. Molecular and Cellular Probes , 20 (2), 105–113. https://doi.org/10.1016/j.mcp.2005.11.001 Bettoni, J. C., Fazio, G., Costa, L. C., Hurtado-Gonzales, O. P., Rwahnih, M. A., Nedrow, A., & Volk, G. M. (2022). Thermotherapy followed by shoot tip cryotherapy eradicates latent viruses and apple hammerhead viroid from in vitro apple rootstocks. Plants , 11 (5), 582. https://doi.org/10.3390/plants11050582 Darós, J. A. (2016). Viroids: Small noncoding infectious RNAs with the remarkable ability of autonomous replication. In A. Wang & X. Zhou (Eds.), Current research topics in plant virology (pp. 295–322). Springer. https://doi.org/10.1007/978-3-319-32919-2_13 Ferant, N., Javornik, B., & Luthar, Z. (2001). Regeneracija hmelja ( Humulus lupulus L.) pri cv. Aurora. Hmeljarski bilten , 8 , 19–25. Hataya, T., Katsuyuki, H., Suda, N., Nagata, T., Shifang, L., Itoga, Y., Tanikoshi, T., & Shikata, E. (1992). Detection of hop latent viroid (HLVd) using reverse transcription and polymerase chain reaction (RT-PCR). Annals of the Phytopathological Society of Japan , 58 , 677–684. https://doi.org/10.3186/jjphytopath.58.677 Jakše, J., & Radišek, S. (2005). Vzpostavitev identifikacijskega sistema za določanje hmeljevega latentnega viroida. Hmeljarski bilten , 12 , 49–57. Jakše, J., Radišek, S., Pokorn, T., Matousek, J., & Javornik, B. (2015). Deep-sequencing revealed Citrus bark cracking viroid (CBCVd) as a highly aggressive pathogen on hop. Plant Pathology , 64 (1), 831–842. https://doi.org/10.1111/ppa.12325 Kump, B., & Javornik, B. (1996). Evaluation of genetic variability among common buckwheat (Fagopyrum esculentum Moench) populations by RAPD markers. Plant Science , 114 , 149–158. https://doi.org/10.1016/j.plantsci.1996.05.010 Lee, H. J., Han, Y. S., Cho, I. S., & Jeong, R. D. (2022). Development and application of reverse transcription droplet digital PCR assay for sensitive detection of apple scar skin viroid during in vitro propagation of apple plantlets. Molecular and Cellular Probes , 61 , 101789. https://doi.org/10.1016/j.mcp.2021.101789 Marton, L., Duran-Vila, N., Lin, J. J., & Semancik, J. S. (1982). Properties of cell cultures containing the citrus exocortis viroid. Virology , 122 (2), 229–238. https://doi.org/10.1016/0042-6822(82)90223-9 Matousek, J., Orctová, L., Patzak, J., Svoboda, P., & Ludvikova, I. (2003). Molecular sampling of hop stunt viroid (HSVd) from grapevines in hop production areas in the Czech Republic and hop protection. Plant Soil and Environment , 49 (4), 168–175. https://pse.agriculturejournals.cz/pdfs/pse/2003/04/06.pdf Mishra, A. K., Kumar, A., Mishra, D., Nath, V. S., Jakše, J., Kocábek, T., Killi, U. K., Morina, F., & Matoušek, J. (2018). Genome-wide transcriptomic analysis reveals insights into the response to citrus bark cracking viroid (CBCVd) in hop (Humulus lupulus L). Viruses , 10 (10), 570. https://doi.org/10.3390/v10100570 Navarro, B., Flores, R., & Di Serio, F. (2021). Advances in viroid-host interactions. Annual Review of Virology , 8 , 305–325. https://doi.org/10.1146/annurev-virology-091919-092331 Oset Luskar, M., Čerenak, A., Radišek, S., Košir, I. J., & Čeh, B. (2021). Ministrstvo za kmetijstvo, gozdarstvo in prehrano in Inštitut za hmeljarstvo in pivovarstvo. Opisna sortna lista za hmelj 2021 . Republika Slovenija. Puchta, H., Ramm, K., & Sanger, H. L. (1988). The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops. Nucleic Acids Research , 16 (10), 4197–4216. https://doi.org/10.1093/nar/16.10.4197 Radišek, S., Majer, A., Jakše, J., & Javornik, B. (2012). First report of hop stunt viroid infecting hop in Slovenia. Plant Disease , 96 (4), 592. https://doi.org/10.1094/PDIS-08-11-0640-PDN Radišek, S., Guček, T., Dobrajc, M., Štajner, N., Matoušek, J., & Jakše, J. (2022). Symptomology of citrus bark cracking viroid on hop and impact to Slovenian hop production. In VIROID 2022: Abstracts of papers presented at the VIROID 2022 conference: Viroids, Viroid-like RNAs and RNA viruses: 14–16 September 2022 Crete, Greece (p. 16). [COBISS.SI-ID 130006275]. Sano, T., Yoshida, H., Goshono, M., Monma, T., Kawasa, H., & Ishizaki, K. (2004). Characterization of a new viroid strain from hops: Evidence for viroid speciation by isolation in different host species. Journal of General Plant Pathology , 70 (3), 181–187. https://doi.org/10.1007/s10327-004-0105-z Sasaki, M., & Shikata, E. (1977). Studies on the host range of hop stunt disease in Japan. Proceedings of the Japan Academy, Series B. Physical and Biological Sciences, 53 (3), 103–108. https://doi.org/10.2183/pjab.53.103 Wan Chow Wah, Y. F., & Symons, R. H. (1997). A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples. Journal of Virological Methods , 63 (1–2), 57–69. https://doi.org/10.1016/s0166-0934(96)02115-5 Yang, C., Nguyen, V. A., Nulu, N. P. C., Kalaipandian, S., Beveridge, F. C., Biddle, J., Young, A., & Adkins, S. W. (2024). Towards pathogen-free coconut germplasm exchange. Plants , 13 (13), 1809. https://doi.org/10.3390/plants13131809 Cite Share Download PDF Status: Published Journal Publication published 07 Feb, 2025 Read the published version in European Journal of Plant Pathology → Version 1 posted Editorial decision: Minor revisions 27 Nov, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers invited by journal 21 Oct, 2024 Editor invited by journal 19 Oct, 2024 Editor assigned by journal 18 Oct, 2024 First submitted to journal 17 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5282171","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":368414942,"identity":"91cdcd2c-749b-457d-8ac2-e2a4474d9210","order_by":0,"name":"Helena 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15:22:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":441402,"visible":true,"origin":"","legend":"\u003cp\u003eResults of agarose gel electrophoresis of RT-PCR of HLVd inoculation of hop plants\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5282171/v1/8e11256aa0e8edb62a34afc4.png"},{"id":68200235,"identity":"ab4841ba-59ed-4a08-8964-649216b11a9d","added_by":"auto","created_at":"2024-11-04 15:22:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":445032,"visible":true,"origin":"","legend":"\u003cp\u003eResults of agarose gel electrophoresis of RT-PCR of HSVd inoculation of hop plants\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5282171/v1/de771d0cafc9ecb298703e3c.png"},{"id":68200237,"identity":"272096a9-1c2f-449e-a346-7e34a814276c","added_by":"auto","created_at":"2024-11-04 15:22:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":425638,"visible":true,"origin":"","legend":"\u003cp\u003eResults of agarose gel electrophoresis of RT-PCR of CBCVd inoculation of hop plants\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5282171/v1/22068670ab49c27b7744325d.png"},{"id":75930549,"identity":"881b32da-047b-4c9b-a7cb-b7a8781bf083","added_by":"auto","created_at":"2025-02-10 16:13:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1813872,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5282171/v1/3682c906-95c2-4fa5-ae28-37116b0b2dff.pdf"}],"financialInterests":"","formattedTitle":"Evaluation of Hop Varieties (Humulus lupulus L.) for Their Response to HLVd, HSVd, and CBCVd Viroids in tissue cultures","fulltext":[{"header":"Main Text","content":"\u003cp\u003eThe common hop (\u003cem\u003eHumulus lupulus\u003c/em\u003e L.) is classified as an herbaceous perennial plant and belongs in the hemp family (Cannabacae). Hop cultivation plays a crucial role for the brewing industry, providing the essential oils and bitter acids necessary for flavouring and preserving beer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eViroids are small, single-stranded, circular RNA molecules with a size of 246 to 401 nucleotides. These molecules do not encode proteins, but can replicate, move, and evade the host\u0026apos;s defence system (Dar\u0026oacute;s, 2016). To date, four different hop viroids have been discovered: Hop Latent Viroid (HLVd) (Puchta et al., 1988), Hop Stunt Viroid (HSVd) (Sasaki and Shikata, 1977), Apple Fruit Crinkle Viroid (AFCVd) (Sano et al., 2004) and Citrus Bark Cracking Viroid (CBCVd) (Jak\u0026scaron;e et al., 2015). All these viroids were found in European hop fields (Radi\u0026scaron;ek in sod., 2012; Jak\u0026scaron;e et al., 2015), except for AFCVd, which only occurs in Japan. HLVd spreads among hop plants primarily by vegetative propagation and often shows no clear symptoms of infection (Barbosa et al., 2005). This has made HLVd the most widespread viroid in hop fields. In contrast, HSVd is not as widespread, however it has numerous host plants, and the disease has a significant economic impact in hops. CBCVd was identified in 2007 in Slovenian hop fields as the cause of a previously unknown disease characterized by visible and distinct symptoms that lead to plant death within 3 to 5 years (Jak\u0026scaron;e et al., 2015).\u003c/p\u003e\n\u003cp\u003eThe mechanisms by which viroids cause disease are still unclear, though their symptoms resemble viral infections, suggesting transcriptional or post-transcriptional gene silencing as a mode of action. Since viroids are present in small quantities, they are not thought to deplete RNA nucleotides in host cells. Many hosts show no symptoms and are considered \u0026quot;latent,\u0026quot; but viroids can multiply rapidly in susceptible plants. A single nucleotide change can alter the severity of the disease. Mechanical transmission through contaminated tools is common, while seed or pollen transmission is rare. Viroids infect plants by entering and replicating in the chloroplast or nucleus, then spreading between cells via plasmodesmata and through the phloem (Navarro et al., 2021). Infected plants activate RNA silencing mechanisms, including both transcriptional and post-transcriptional silencing, to defend against viroids (Mishra et al., 2018; Navarro et al., 2021).\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to test 13 hop varieties for their susceptibility to the viroids HLVd, HSVd and CBCVd, three viroids which pose a threat to European hop fields, in tissue culture conditions. Hop cultivation ends in late August and the plant remains dormant until late spring. As a tall plant cultivated on special trellises, evaluating symptoms throughout the year in a smaller space using tissue culture could expedite the assessment process. The expectation was that symptoms would develop earlier in tissue cultures and that we would discover new resistant or tolerant hop varieties and to confirm the resistance and tolerance of two hop varieties (Styrian Wolf and Styrian Cardinal, respectively) that had shown these characteristics in initial field studies (Radi\u0026scaron;ek et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA Murashige and Skoog medium was used for micropropagation of hop (Frant in sod., 2001) varieties: Atlas, Blisk, Buket, Cekin, Cicero, Styrian Eureka, Styrian Gold, Styrian Kolibri, Wye Target, Bobek, Styrian Wolf, Styrian Cardinal, and Celeia. The dimeric RNA transcripts used for viroid inoculation were prepared from plasmids containing the dimeric sequence of the corresponding viroid. Plasmids were isolated from overnight cultures of \u003cem\u003eE. coli\u003c/em\u003e DH5\u0026alpha; using the High Pure Plasmid Isolation Kit (Roche), linearized with a restriction enzyme, and reverse transcribed using a reverse transcription kit following the manufacturer\u0026apos;s protocols. Specifically, 1 \u0026micro;g of linearized plasmid was used in a 20 \u0026micro;L transcription reaction, incubated at 37 \u0026deg;C for 4 hours in a thermocycler. Table 1 lists the combinations of restriction enzymes and reverse transcription kits used for reverse transcription to produce the positive strand of the viroid transcript.\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;1: Enzymes and reverse transcription kits used for viroid transcript synthesis\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 12.4792%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eViroid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.802%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlasmid carrier\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.6273%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEnzyme used\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.0915%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReverse transcription kit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4792%;\"\u003e\n \u003cp\u003eCBCVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.802%;\"\u003e\n \u003cp\u003epBSII-CBCVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.6273%;\"\u003e\n \u003cp\u003eHincII\u003cbr\u003e\u0026nbsp;(Thermo Scientific)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.0915%;\"\u003e\n \u003cp\u003eMEGAscript\u0026trade; T7 Transcription Kit\u003cbr\u003e\u0026nbsp;(Thermo Scientific)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4792%;\"\u003e\n \u003cp\u003eHLVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.802%;\"\u003e\n \u003cp\u003epBSII-HLVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.6273%;\"\u003e\n \u003cp\u003eEco53kI\u003cbr\u003e\u0026nbsp;(NEB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.0915%;\"\u003e\n \u003cp\u003eMEGAscript\u0026trade; T3 Transcription Kit\u0026nbsp;\u003cbr\u003e\u0026nbsp;(Thermo Scientific)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 12.4792%;\"\u003e\n \u003cp\u003eHSVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.802%;\"\u003e\n \u003cp\u003epGEM-T-Easy-HSVd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 23.6273%;\"\u003e\n \u003cp\u003eBmrI\u003cbr\u003e\u0026nbsp;(NEB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45.0915%;\"\u003e\n \u003cp\u003eMEGAscript\u0026trade; SP6 Transcription Kit (Thermo Scientific)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eInoculation of hop plants with HLVd, HSVd and CBCVd transcripts was carefully performed on selected uniform plants to allow accurate monitoring of disease symptoms. Inoculum was prepared in a volume of 50 \u0026micro;L by mixing 150 \u0026micro;g of each viroid transcript with 0.5 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, with an additional viroid-free control inoculum prepared only with 0.5 mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. The largest and healthiest leaf sitting lowest on the plant was selected for inoculation. Each plant was inoculated with 1 \u0026micro;l of the desired inoculum, which was smeared over the leaf with an L-shaped plastic rod to create small lesions through which the viroid could enter and spread in the plant (Figure 1). Four plants per variety were inoculated, while two plants served as controls.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;1: Inoculation of hop tissue cultures with viorid RNA transripts\u003c/p\u003e\n\u003cp\u003eEight weeks after inoculation, total nucleic acids were isolated (Kump and Javornik, 1996) from newly developed shoots and viroid presence was evaluated by RT-PCR (Jak\u0026scaron;e in Radi\u0026scaron;ek, 2005) using the appropriate primer sequences (Bernad and Duran-Vila, 2006 for CBCVd; Hataya et al., 1992 for HLVd and Matousek et al., 2003 for HSVd).\u003c/p\u003e\n\u003cp\u003eWe confirmed that all hop cultivars were successfully infected with all transcripts (Figures 2, 3 and 4). We also noted that the varieties \u0026apos;Bobek\u0026apos; and \u0026apos;Wye Target\u0026apos; were not viroid free plants since HLVd infection was confirmed in starting material. HSVd showed highest mean infection rate since 94 % of inoculated plants were successfully infected with it. The mean infection rate of CBCVd was also high, 90 % and the mean infection rate of HLVd, naturally the most widespread viroid in hop was surprisingly low at 55 %. A more detailed overview of the infection rates is presented in Table 2.\u003c/p\u003e\n\u003cp\u003eFigure 2: Results of agarose gel electrophoresis of RT-PCR of HLVd inoculation of hop plants\u003c/p\u003e\n\u003cp\u003eFigure 3: Results of agarose gel electrophoresis of RT-PCR of HSVd inoculation of hop plants\u003c/p\u003e\n\u003cp\u003eFigure 4: Results of agarose gel electrophoresis of RT-PCR of CBCVd inoculation of hop plants\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;2: Number and % of infected plants inoculated with HLVd, HSVd or CBCVd viroid\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 20.915%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHLVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 20.915%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHSVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 20.915%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCBCVd\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 20.915%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAll 3 viorids\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHop cultivar\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eno.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eno.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eno.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eno.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e%\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eAtlas\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e83\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eBlisk\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e75\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eBuket\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e83\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eCekin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e67\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eCicero\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e67\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eStyrian Eureka\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e92\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eStyrian gold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e83\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eStyrian Kolibri\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e75\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eWye Target\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003en.a.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e100\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eBobek\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003en.a.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e100\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eStyrian Wolf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e83\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eStyrian Cardinal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e75\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003eCeleia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e83\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 16.3399%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAll cultivars\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e24\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e\u003cem\u003e49\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e\u003cem\u003e94\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 9.80392%;\"\u003e\n \u003cp\u003e\u003cem\u003e47\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 11.1111%;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 10.4575%;\"\u003e\n \u003cp\u003e\u003cem\u003e81\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eLegend: \u0026ldquo;no.\u0026rdquo; \u0026ndash; number of viroid positive plants out of 4 inoculated per viroid; \u0026ldquo;n.a.\u0026rdquo; \u0026ndash; not applicable, the plants have been HLVd-infected prior the experiment.\u003c/p\u003e\n\u003cp\u003eThe symptoms of plants infected with HLVd, HSVd, and CBCVd viroids can be challenging to recognize, especially under certain conditions like tissue culture, where plants do not fully develop visible symptoms such as cones or tall growth. In the field, HLVd infections can affect metabolic pathways, leading to chlorosis, slower growth, and smaller cones, but these symptoms were not observed in our tissue-cultured plants, which did not significantly differ from the controls. Similarly, HSVd symptoms, typically appearing years after infection and including curling leaves, smaller cones, and reduced alpha acid content, were absent in our study. Both viroids infected all cultivars with no resistance or tolerance, with infection rates for HLVd and HSVd ranging from 25 % to 100 %, as shown in Table 2. CBCVd infections are known to cause early mild yellowing and curling of leaves in the field, but our CBCVd-infected plants also did not show significant symptoms in tissue culture. While field observations suggest that Styrian Wolf is the only hop that cannot be infected with CBCVd (Radi\u0026scaron;ek et al., 2022), our study successfully infected it with 100 % efficiency using \u003cem\u003ein vitro\u003c/em\u003e viroid transcripts. We hypothesize that the differences between our results and field conditions stem from varying viroid transmission methods and plant growth environments, with field infections primarily occurring mechanically through low levels of infected sap, whereas our study used high concentrations of synthetic viroid transcripts. Future research should explore lower concentrations and RNA extracts from infected plants to better replicate field conditions.\u003c/p\u003e\n\u003cp\u003eThe tissue cultures were maintained for an additional 3 months until the plants depleted the cultivation media and required subculturing. A visual evaluation conducted at that time again yielded no significant information on viroid symptoms due to uneven growth. This aligns with previous studies, such as Marton et al. (1982), who found no significant effect of Citrus Exocortis Viroid (CEVd) on the growth of tomato callus and suspension cultures. Recent research on viroids and \u003cem\u003ein vitro\u003c/em\u003e plant cultivation has primarily focused on viroid elimination (Bettoni et al., 2022; Yang et al., 2024) or detection (Wan Chow Wah and Symons, 1997; Lee et al., 2022), rather than symptom development.\u003c/p\u003e\n\u003cp\u003eOur findings further highlight that visual symptoms may not reliably indicate viroid presence in tissue culture, emphasizing the need for molecular detection techniques like RT-PCR for accurate identification. Hop growers face significant challenges from various pathogens, including viroids, which can be mitigated by using viroid-free planting material. Breeding efforts focus on developing virus- and viroid-free genotypes that are resistant to diseases and pests (Oset Luskar et al., 2021). This research aimed to assess the susceptibility of various hop cultivars to HLVd, HSVd, and CBCVd infections, which threaten European hop fields. While we expected to find resistance or tolerance in some cultivars, all tested varieties were infected by the three viroids. Surprisingly, HLVd, the most widespread viroid, had the lowest infection potential (55 % average), while CBCVd and HSVd exhibited higher infection rates, with averages of 90 % and 94 %, respectively. RT-PCR confirmed successful infection in nearly all samples inoculated with HSVd.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThis research was financially supported by the Slovenian Research and Innovation Agency project Z4-3214 and by the Slovenian Research and Innovation Agency program P4-0077.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and Declarations: Competing Interests\u003c/strong\u003e: The authors declare that they have no financial or non-financial competing interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarbosa, C. J., Pina, J. A., Bernad, L., Serra, P., Navarro, L., \u0026amp; Duran-Vila, N. (2005). Mechanical transmission of citrus viroids. \u003cem\u003ePlant Disease\u003c/em\u003e, \u003cem\u003e89\u003c/em\u003e(7), 749\u0026ndash;754. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PD-89-0749\u003c/span\u003e\u003cspan address=\"10.1094/PD-89-0749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernad, L., \u0026amp; Duran-Vila, N. (2006). A novel RT-PCR approach for detection and characterization of citrus viroids. \u003cem\u003eMolecular and Cellular Probes\u003c/em\u003e, \u003cem\u003e20\u003c/em\u003e(2), 105\u0026ndash;113. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mcp.2005.11.001\u003c/span\u003e\u003cspan address=\"10.1016/j.mcp.2005.11.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBettoni, J. C., Fazio, G., Costa, L. C., Hurtado-Gonzales, O. P., Rwahnih, M. A., Nedrow, A., \u0026amp; Volk, G. M. (2022). Thermotherapy followed by shoot tip cryotherapy eradicates latent viruses and apple hammerhead viroid from in vitro apple rootstocks. \u003cem\u003ePlants\u003c/em\u003e, \u003cem\u003e11\u003c/em\u003e(5), 582. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants11050582\u003c/span\u003e\u003cspan address=\"10.3390/plants11050582\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDar\u0026oacute;s, J. A. (2016). Viroids: Small noncoding infectious RNAs with the remarkable ability of autonomous replication. In A. Wang \u0026amp; X. Zhou (Eds.), \u003cem\u003eCurrent research topics in plant virology\u003c/em\u003e (pp. 295\u0026ndash;322). 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Towards pathogen-free coconut germplasm exchange. \u003cem\u003ePlants\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e(13), 1809. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants13131809\u003c/span\u003e\u003cspan address=\"10.3390/plants13131809\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hops, tissue culture, viroid, CBCVd, HLVd, HSVd","lastPublishedDoi":"10.21203/rs.3.rs-5282171/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5282171/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluates the susceptibility of various hop varieties to three viroids: Hop Latent Viroid (HLVd), Hop Stunt Viroid (HSVd), and Citrus Bark Cracking Viroid (CBCVd), all of which pose significant threats to Slovenian hop production. The experiment was conducted under \u003cem\u003ein vitro\u003c/em\u003e conditions, involving the cultivation of 13 different hop varieties in tissue cultures, alongside the \u003cem\u003ein vitro\u003c/em\u003e synthesis of viroid transcripts and subsequent inoculation of the plants. The plants were then monitored for growth, development, and their responses to viroid infection. The findings reveal the complex interactions between hops and viroids, with successful inoculation observed in all varieties. However, infection rates varied, with HSVd showing the highest rates of infection, followed by CBCVd and HLVd. This research enhances the understanding of viroid dynamics in hop plants, which is crucial for developing effective disease management strategies. Despite the insights gained, no resistance or tolerance to the viroids was identified in any of the tested hop varieties.\u003c/p\u003e","manuscriptTitle":"Evaluation of Hop Varieties (Humulus lupulus L.) for Their Response to HLVd, HSVd, and CBCVd Viroids in tissue cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-04 15:22:07","doi":"10.21203/rs.3.rs-5282171/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2024-11-27T08:21:49+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-10-21T14:19:52+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-21T08:07:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2024-10-19T06:58:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-18T15:21:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2024-10-17T06:54:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ce70c959-0143-4ea8-8cc5-f0eef9707063","owner":[],"postedDate":"November 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-10T16:06:05+00:00","versionOfRecord":{"articleIdentity":"rs-5282171","link":"https://doi.org/10.1007/s10658-025-03009-0","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2025-02-07 15:58:21","publishedOnDateReadable":"February 7th, 2025"},"versionCreatedAt":"2024-11-04 15:22:07","video":"","vorDoi":"10.1007/s10658-025-03009-0","vorDoiUrl":"https://doi.org/10.1007/s10658-025-03009-0","workflowStages":[]},"version":"v1","identity":"rs-5282171","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5282171","identity":"rs-5282171","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}