First evidence of allexiviruses in Allium plants in Ukraine and molecular characterization of their isolates

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We surveyed cultivated Allium plants collected in eight Ukrainian regions (2022–2025) and screened their samples for garlic virus B (GarV-B), garlic virus C (GarV-C) and shallot virus X (ShVX) using enzyme-linked immunosorbent assay (ELISA). GarV-B, GarV-C and ShVX were detected in 39/108 (36.1%), 23/108 (21.3%) and 21/108 (19.4%) plants, respectively, with infections which were strongly host-associated: garlic (n = 63) had high frequencies of indicated viruses (GarV-B − 61.9%; GarV-C − 36.5%; ShVX − 28.6%), whereas onion samples (n = 33) were largely negative (ShVX − 3.0%; GarV-B and GarV-C - not detected). Co-occurrence analysis within garlic revealed a nested allexivirus module in which GarV-C and ShVX occurred only in GarV-B-positive plants. RT-PCR and Sanger sequencing generated 11 partial genomes representing GarV-B, GarV-C, ShVX, GarV-A and GarV-D. Maximum-likelihood phylogenies placed Ukrainian allexivirus isolates within established global diversity and indicated both European- and Asian-affiliated lineages. These findings provide the first evidence of allexiviruses in Ukrainian Allium crops, and support their inclusion in plant health surveillance and planting-material certification. Biological sciences/Biotechnology Biological sciences/Microbiology Biological sciences/Molecular biology Biological sciences/Plant sciences Allium allexivirus garlic virus B garlic virus C shallot virus X Ukraine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Allium crops such as garlic, onion, and shallot are of high economic importance worldwide, but their production is often constrained by viral diseases. These vegetatively propagated crops are susceptible to a range of viruses, especially those in the genera Potyvirus , Carlavirus , and Allexivirus [ 1 ] Among these, allexiviruses (family Alphaflexiviridae ) are particularly notable pathogens of Allium species. The genus Allexivirus currently comprises eight recognized species infecting alliaceous plants: garlic viruses A, B, C, D, E, X, garlic mite-borne filamentous virus, and shallot virus X [ 2 ]. In addition to efficient dissemination through vegetative propagation of infected bulbs/cloves, several allexiviruses can also be transmitted by eriophyid mites, providing a mechanism for secondary spread within Allium plantings [ 4 ]. Allexivirus infections in Allium plants are typically latent or mild in symptomatology, often causing only subtle mosaic, mottling, faint chlorotic striping of leaves, or mild stunting [ 3 , 4 ]. Despite the limited visible symptoms, these viruses can have a significant economic impact on Allium crops. Infected plants suffer yield losses and quality deterioration of bulbs [ 5 ]. For example, single infections by certain garlic allexiviruses can reduce bulb weight by roughly 10–15% [ 5 ], while mixed infections (especially with potyviruses or carlaviruses) can lead to even greater yield reductions [ 6 , 7 ]. Consequently, allexiviruses are regarded as important contributors to the garlic and onion viral disease complex that undermines crop productivity and bulb quality [ 4 , 7 ]. In Allium crops, allexiviruses are ubiquitous. First detected infecting Allium in the early 1990s in Russia [ 8 ], these viruses have since been reported across Asia, Europe, Africa, Oceania, and the Americas. For instance, Allexivirus species have been identified in garlic or shallot in such countries as Japan, China, and India in Asia, Greece, Italy, Spain, and Poland in Europe, as well as in the Middle East and Africa (e.g., Iran, Sudan) and the Americas (e.g., Argentina, USA) [ 9 – 12 ]. Notably, Ukraine-neighboring countries in Eastern Europe have reported the presence of allexiviruses – for example, Russia and Poland have documented multiple garlic-infecting Allexivirus species [ 8 – 13 ]. In Ukraine, however, there have been no prior records of allexivirus infections, underscoring a significant gap in the regional knowledge of Allium virus distribution. Here we report the first detection and characterization of allexiviruses in garlic, onion, and shallot in Ukraine. This study provides the first evidence of Allexivirus presence in Allium crops in Ukraine, expanding the known range of these viruses and marking an initial step toward understanding their prevalence and impact on Ukraine’s Allium production and export. The findings contribute important new data on Allium viral pathogens in the region, with implications for crop health monitoring and management in Ukrainian agriculture. Results Prevalence of allexiviruses in Allium plants in Ukraine ELISA screening detected allexiviruses in the surveyed Allium material (Table 1 ). Across all tested samples, garlic virus B (GVB), garlic virus C (GVC) and shallot virus X (ShVX) were detected in 38 (35.3%), 23 (21.3%) and 21 (19.4%), respectively. Table 1 Serological screening of different Allium species and Ukraine’s regions for virus occurrence Species GVB GVC ShVX Garlic ( A. sativum ) Cherkasy region 1/13 0/13 0/13 Kyiv region 14/18 10/18 8/18 Odesa region 7/8 6/8 3/8 Poltava region 2/4 2/4 1/4 Ternopil region 4/4 0/4 0/4 Vinnytsia region 6/11 3/11 4/11 Zakarpattia region 3/3 2/3 1/3 Zaporizhzhia region 2/2 0/2 1/2 Subtotal ( Garlic ) 39/63 23/63 18/63 Onion ( A. cepa ) Cherkasy region 0/3 0/3 0/3 Kyiv region 0/10 0/10 1/10 Odesa region 0/8 0/8 0/8 Poltava region 0/1 0/1 0/1 Ternopil region 0/3 0/3 0/3 Vinnytsia region 0/8 0/8 0/8 Subtotal ( Onion ) 0/33 0/33 1/33 Leek ( A. ampeloprasum ) 0/4 0/4 0/4 Shallot ( A. cepa var. aggregatum ) 0/3 0/3 2/3 A. fistulosum 0/1 0/1 0/1 A. schoenoprasum 0/1 0/1 0/1 A. scorodoprasum 0/1 0/1 0/1 A. tuberosum 0/1 0/1 0/1 A. hollandicum 0/1 0/1 0/1 Total 39/108 23/108 21/108 Allexivirus detections were strongly host-associated. In A. sativum (garlic), GVB was detected in 39/63 (61.9%), while GVC and ShVX were detected in 23/63 (36.5%) and 18/63 (28.6%), respectively. In contrast, GVB and GVC were not detected in A. cepa (0/36 and 0/36, respectively), while ShVX was detected only sporadically (3/36; 8.3%). Virus-virus associations To place allexivirus detections in a mixed-infection context, we analyzed co-occurrence with potyvirus and carlavirus detections obtained in the same plants; prevalence results for those viruses in this sample set have been reported previously [ 14 , 15 ]. Using the complete-case dataset for the eight-virus panel (LYSV, OYDV, SYSV, GCLV, SLV, GVB, GVC, ShVX; n = 108), allexiviruses were commonly observed within multi-virus profiles, including combinations where two or more allexiviruses occurred together and/or co-occurred with potyviruses/carlaviruses. Because allexivirus detections were host-associated, pairwise virus-virus associations were evaluated primarily within garlic ( A. sativum ) using the complete-case dataset for the eight-virus panel (LYSV, OYDV, SYSV, GCLV, SLV, GVB, GVC, ShVX; n = 63). Two-sided Fisher’s exact tests and Pearson chi-square tests applied to 2×2 presence/absence tables identified a strongly structured allexivirus module in which GVB formed the core. GVC was never detected in the absence of GVB (23/23 GVC-positive samples were also GVB-positive; Fisher p = 4.39×10⁻⁷; χ² p = 2.0×10⁻⁶). Likewise, ShVX was found only in GVB-positive plants (Fisher p = 2.89×10⁻⁵; χ² p = 8.2×10⁻⁵), and GVC and ShVX co-occurred significantly (Fisher p = 1.95×10⁻⁶; χ² p = 1.0×10⁻⁶). Using potyvirus and carlavirus detections specifically to evaluate the co-infection context of allexiviruses, GVC showed positive associations with LYSV (Fisher p = 0.00847; χ² p = 0.00538) and with SLV (Fisher p = 0.00835; χ² p = 0.00596) in garlic. In addition, LYSV and SLV co-occurred more frequently than expected by chance (Fisher p = 0.00231; χ² p = 0.00160). Molecular confirmation and phylogenetic placement of allexivirus isolates To complement ELISA screening, selected samples were also characterized by RT-PCR and Sanger sequencing of partial allexivirus genomes. In total, 11 sequences were obtained. Amplicons generated with primers targeting garlic virus B yielded four sequences that, upon sequence identification, corresponded to two GarV-B isolates (GVB-36-23, PX761687; GVB-43-23, PX761688), one GarV-A isolate (GVA-112-22, PX766271), and one GarV-D isolate (GVD-6-22, PX766272), indicating that the targeted region can be conserved across related allexiviruses. Additional sequencing covered five GarV-C isolates (GVC-112-22, PX757648; GVC-12-25, PX757649; GVC-16-24, PX757650; GVC-43-23, PX757651; GVC-44-23, PX757652) and two ShVX isolates (ShVX-115-22, PX766273; ShVX-32-23, PX766274). Maximum-likelihood-based phylogeny reconstructed with representative sequences placed all Ukrainian isolates within their expected species clades and clarified their closest relationships. The two Ukrainian GarV-B sequences (GVB-36-23, PX761687; GVB-43-23, PX761688) were closely identical (99.04% nt identity), and were placed adjacent to the Czech isolate JX682832, sharing with it 98.08% and 98.28% nt identity, respectively (Fig. 1 ). In contrast, their nucleotide identity to the nearby Czech isolate JX682833 was significantly lower (91.38% and 91.19%). The Ukrainian GarV-A isolate (GVA-112-22, PX766271) clustered closest to Chinese sequence MN059305 (96.49% nt identity), with similar identity to another Chinese isolate MN059252 (96.67%) (Fig. 2 ). The Ukrainian GarV-D isolate (GVD-6-22, PX766272) grouped with Chinese isolates MN059369 and MN059370, showing 95.90% nucleotide identity to both (Fig. 3 ). Five Ukrainian GarV-C sequences fell within a lineage anchored by Czech isolates. GVC-43-23 (PX757651) and GVC-44-23 (PX757652) formed a strongly supported pair (99.82% nt identity) placed closest to the JX682840 Czech sequence, sharing with it 96.20% and 96.01% nt identity, respectively (and lower identity to JX682843, 93.12% and 92.93%) (Fig. 4 ). GVC-12-25 (PX757649) and GVC-16-24 (PX757650) formed a second Ukrainian pair (97.64% nt identity) that also showed its highest identity to JX682840 (96.56% and 95.47%), with lower identity to Chinese sequence MN059142 (93.84% and 92.57%). The remaining Ukrainian GVC isolate PX757648 (GVC-112-22) was closest to Czech sequence JX682846 (99.09% nt identity) and also highly similar to the Japanese one, LC097169 (98.73% nt identity). Finally, two Ukrainian ShVX sequences clustered within established ShVX diversity represented by European and Asian isolates. ShVX-115-22 was closest to French isolate MH389251 (89.53% nt identity), with similar identity to French MH389252 (89.30%). However, ShVX-32-23 showed its highest identity to French isolates MH389250 and MH389254 (83.26% nt identity), while was more distant from the Indian isolates ON986786 and OK104171 (69.30% nt identity) (Fig. 5 ). Two Ukrainian ShVX sequences shared only 82.79% nucleotide identity with each other, suggesting their different origin. Discussion This study provides the first evidence that allexiviruses infect cultivated Allium crops in Ukraine, extending the documented range of this group of garlic- and shallot-associated viruses in Europe [ 2 , 10 , 13 ]. ELISA screening revealed that GarV-B, GarV-C and ShVX were already present in surveyed material, with detections strongly structured by host: infections were frequent in garlic but rare or absent in onion. Such host skew is consistent with the epidemiology of Allium viruses in vegetatively propagated garlic, where viruses can persist and accumulate across planting cycles even when symptoms are mild or unapparent [ 1 , 4 ]. The sporadic detection of ShVX in onion nevertheless indicates that allexiviruses (or at least ShVX) can occur outside garlic-dominated contexts, supporting broader host-inclusive surveillance rather than focusing solely on garlic. Allexiviruses were commonly embedded within multi-virus profiles, and the within-garlic association structure suggests non-random assembly of infections. The nested pattern in which GarV-C and ShVX were detected only in GarV-B-positive plants, together with the significant GarV-C/ShVX co-occurrence, is compatible with repeated circulation of multi-infected planting material and shared transmission opportunities, including eriophyid mite-mediated spread reported for allexiviruses [ 4 ]. The positive associations of GarV-C with LYSV and SLV further emphasize that allexiviruses circulate within mixed infections typical of Allium pathosystems, where co-infection can exacerbate losses relative to single infections [ 5 – 7 ]. Given that yield and bulb-quality penalties have been demonstrated for allexiviruses alone and in combination with additional viruses [ 5 – 7 ], the structured co-occurrence observed here suggests that allexiviruses should be considered integral contributors to the virus complex affecting Ukrainian garlic, alongside the other Allium viruses recently reported from the country [ 14 , 15 ]. Sequencing and phylogenetic placement confirmed multiple allexivirus species and situated Ukrainian isolates within established global diversity. The clustering of Ukrainian GarV-B and several GarV-C isolates near Czech reference lineages is consistent with regional connectivity of garlic planting material within Europe and mirrors patterns reported from other European surveys [ 13 , 17 ]. In parallel, the affinities of Ukrainian GarV-A and GarV-D to Asian isolates align with the broader global dissemination of garlic viruses documented across continents [ 11 , 12 ], underscoring that introductions from multiple sources are plausible for clonally propagated crops. The substantial divergence between the two Ukrainian ShVX sequences supports multiple introductions and/or longer-term circulation with diversification, rather than a single recent origin, consistent with the heterogeneous ShVX diversity reported in different regions [ 8 , 12 ]. Finally, the recovery of GarV-A and GarV-D from amplicons generated with a GarV-B-targeted primer set highlights that conserved genomic regions can yield cross-species amplification; therefore, sequencing (or species-resolving assays) is essential when translating screening results into species-level inferences. Overall, the presence of multiple allexiviruses in Ukrainian Allium crops – often in structured mixed infections with other economically relevant viruses – supports incorporating allexiviruses into routine monitoring and planting-material health programs. Priority next steps include expanded surveys across regions and cultivars, deeper sequence coverage to better resolve introductions and local spread, and field-based quantification of yield and quality impacts under Ukrainian production conditions [ 5 – 7 ]. In practice, the findings reinforce the value of virus-tested planting stocks and strengthened phytosanitary oversight to limit accumulation and dissemination of allexiviruses and associated virus complexes in Ukraine’s Allium sector [ 1 , 5 – 7 ]. Methods Plant sampling A total of 108 Allium plants were analysed in Ukraine during 2022–2025, representing commercial plantings and private small-scale cultivation in eight regions (Cherkasy, Kyiv, Odesa, Poltava, Ternopil, Vinnytsia, Zakarpattia, and Zaporizhzhia). The dataset included primarily garlic ( Allium sativum ; n = 69) and onion (A. cepa var. cepa; n = 37), with smaller numbers of shallot, leek and other Allium spp. Plants were sampled irrespective of symptom status; both plants showing virus-like symptoms (e.g., striping, mosaic, deformation) and plants without obvious symptoms were included, consistent with our earlier Allium virus surveys. Serological screening (ELISA) All samples were screened serologically for Allexiviruses (GVB, GVC, ShVX) using commercial kits for enzyme-linked immunosorbent assay (DSMZ, Germany) following the microplate ELISA approach [ 16 ]. The results were recorded using an automatic Thermo Labsystems Opsis MR microplate reader (USA) with Dynex Revelation Quicklink software at the wavelength of 405 nm. RNA extraction, RT-PCR and Sanger sequencing For molecular confirmation and phylogenetic characterization, total RNA was extracted from approximately 100 mg of leaf tissue using the Quick-RNA Plant Miniprep Kit (Zymo Research, USA) following the manufacturer’s protocol. Reverse transcription was performed with M-MuLV reverse transcriptase (NEB, USA) and a random primer mix (NEB, USA), using incubation steps of 25°C for 5 min, 42°C for 1 h, and 65°C for 20 min. PCR amplification was carried out using GreenTaq PCR Master Mix (Thermo Fisher Scientific, USA). The cycling profile followed our established workflow (initial denaturation at 95°C for 3 min, then 30 cycles of 95°C for 30 s, annealing for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min), with annealing temperatures adjusted by target sequence as follows: 53°C for GVB and GVC, and 56°C for ShVX. Amplicons were visualized by electrophoresis in 1.5% agarose gels. Primer sets used in this study were: GVB-F 5′-TGACGGGCAAACAGCAGAATAA-3′ GVB-R 5′-ATATAGCTTAGCGGGTCCTTC-3′ and GVC-F 5′-TTGCTACCACAATGGTTCCTC-3′, GVC-R 5′-TACTGGCACGAGTTGGGAAT-3′ targeting CP + NABP region of garlic virus B and garlic virus C; ShVX-F 5′-ACCGAAATCACAGTTAACTCCTTTGG-3′ and ShVX-R 5′-TCTACGGTTGTCGATTTTGTGCGT-3′ targeting replicase of shallot virus X [ 17 ]. PCR products selected for sequencing were purified and subjected to Sanger sequencing. Chromatograms were inspected and assembled in BioEdit v7.2.5 [ 18 ]. In total, partial allexivirus sequences were generated for 11 Ukrainian isolates (two GarV-B, five GarV-C, two ShVX, plus two sequences amplified with the GarV-B primer set that were later identified as GarV-A and GarV-D). GenBank accession numbers are provided in the Results and in the figures. Sequence alignment and phylogenetic analysis Representative reference sequences were retrieved from GenBank for each virus and combined with Ukrainian sequences. Multiple sequence alignments were generated with MAFFT [ 19 ] using the L-INS-i strategy. Maximum-likelihood phylogenetic trees were inferred in IQ-TREE [ 20 , 21 ], with the substitution model selected using the program’s model-selection procedure (ModelFinder) [ 22 ], and branch support assessed using 1,000 bootstrap replicates. For presentation, bootstrap values > 50% are shown on branches. To improve readability, densely sampled clades were collapsed where appropriate; numbers in brackets indicate the count of sequences collapsed into that node. Statistical analysis of the serological dataset Virus prevalence was summarized as proportions of virus-positive plants. Mixed-infection structure and virus-virus associations were evaluated using 2×2 contingency tables on presence/absence calls, following standard categorical-data approaches [ 23 ]. Pairwise associations were tested using two-sided Fisher’s exact tests and Pearson chi-square tests, and effect size was summarized as odds ratios where applicable. Analyses were performed on complete-case subsets for the specific virus panels being compared, and results were interpreted with caution for low-frequency detections. Declarations Funding declaration The authors declare that no grants or other financial support were received for this research. Author Contribution All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Kyrylo Taher, Halyna Snihur, Tetiana Shevchenko, Iryna Budzanivska, and Oleksiy Shevchenko. The first draft of the manuscript was written by Kyrylo Taher, and all authors commented on previous versions of the manuscript. All authors read and approved the final revision of the manuscript. Data Availability The partial genome sequences generated in this study have been deposited in GenBank under accession numbers PX761687, PX761688, PX766271, PX766272, PX757648-PX757652, PX766273 and PX766274. The serological prevalence dataset and analysis outputs are available from the corresponding author upon reasonable request. References Conci, V. C., Canavelli, A. E. & Balzarini, M. G. The distribution of garlic viruses in leaves and bulbs during the first year of infection. J. Phytopathol. 158 , 186–193 (2010). King, A. M. Q. et al. 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Taher, K., Snihur, H. & Shevchenko, O. Global distribution of shallot yellow stripe virus in Allium crops with new detections in Ukraine. Eur. J. Plant. Pathol. 173 , 697–702 (2025). Clark, M. F. & Adams, A. N. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol. 34 , 475–483 (1977). Chodorska, M., Paduch-Cichal, E., Kalinowska, E. & Szyndel, M. S. Assessment of allexivirus infection in garlic plants in Poland. Acta Sci. Pol. Hortorum Cultus . 13 , 179–186 (2014). Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98 (1999). Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30 , 772–780 (2013). Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32 , 268–274 (2015). Minh, B. Q. et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol. Biol. Evol. 37 , 1530–1534 (2020). Kalyaanamoorthy, S. et al. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods . 14 , 587–589 (2017). Agresti, A. Categorical Data Analysis (Wiley, 2002). Additional Declarations No competing interests reported. Supplementary Files SupplementaryData1Sequences.fasta Cite Share Download PDF Status: Published Journal Publication published 02 May, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Mar, 2026 Reviews received at journal 12 Mar, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviews received at journal 02 Feb, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 16 Jan, 2026 Editor invited by journal 13 Jan, 2026 Submission checks completed at journal 08 Jan, 2026 First submitted to journal 08 Jan, 2026 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. 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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-8501892","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581783395,"identity":"4e576d00-6813-4973-8322-0a08ab64137e","order_by":0,"name":"Kyrylo Taher","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBACCWYGhgMPCsBsxocNRGtJMDAAsZkNidMCIqBa2CSJ0iLZzvsQaMsfeXn33meVM3ccZjA4fgC/FmlmdgOQwww3njludnPjGaCWMwn4tcgxs4H9wrhxRhrbzYdtQC0HiNRiv3H+M7ZCsJbzDwg5DKIlcb4EGxvjRpCWGwRskWwGazFO3sCTxiw5sy2dR/IGAVskzh9j/vChQs52fvsxxo+9bdZyfOcJ2AIHBgcgNA+R6oFAvoF4taNgFIyCUTDCAAAYrUOEPCRaUAAAAABJRU5ErkJggg==","orcid":"","institution":"Taras Shevchenko National University of Kyiv","correspondingAuthor":true,"prefix":"","firstName":"Kyrylo","middleName":"","lastName":"Taher","suffix":""},{"id":581783399,"identity":"d7649137-df15-4cf0-ba6d-3930ed9af46a","order_by":1,"name":"Tetiana Shevchenko","email":"","orcid":"","institution":"Taras Shevchenko National University of Kyiv","correspondingAuthor":false,"prefix":"","firstName":"Tetiana","middleName":"","lastName":"Shevchenko","suffix":""},{"id":581783400,"identity":"7b4e7bdb-a2dd-46ad-adec-e6bc92434f00","order_by":2,"name":"Halyna Snihur","email":"","orcid":"","institution":"Taras Shevchenko National University of Kyiv","correspondingAuthor":false,"prefix":"","firstName":"Halyna","middleName":"","lastName":"Snihur","suffix":""},{"id":581783401,"identity":"5c350aa2-64fb-4eef-8170-b1880bbdc9f3","order_by":3,"name":"Oleksiy Shevchenko","email":"","orcid":"","institution":"Taras Shevchenko National University of Kyiv","correspondingAuthor":false,"prefix":"","firstName":"Oleksiy","middleName":"","lastName":"Shevchenko","suffix":""},{"id":581783402,"identity":"2b9e4ee5-f3e6-4a01-860e-89ec86cc034e","order_by":4,"name":"Iryna Budzanivska","email":"","orcid":"","institution":"Taras Shevchenko National University of Kyiv","correspondingAuthor":false,"prefix":"","firstName":"Iryna","middleName":"","lastName":"Budzanivska","suffix":""}],"badges":[],"createdAt":"2026-01-02 15:23:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8501892/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8501892/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-47632-1","type":"published","date":"2026-05-02T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101424428,"identity":"a5333a64-98af-4b95-b645-392c91eccb08","added_by":"auto","created_at":"2026-01-29 14:12:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4731642,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of Ukrainian isolates of garlic virus B (GarV-B) using a maximum likelihood approach based on partial \u003cem\u003eCP\u003c/em\u003e and \u003cem\u003eNABP\u003c/em\u003egenes’ sequences. The analysis was conducted usinga partitioned model with TPM2u+F+G4 for the \u003cem\u003eCP\u003c/em\u003e partition and TN+F+G4 for the \u003cem\u003eNABP\u003c/em\u003epartition, with 1,000 bootstrap replicates. Bootstrap values \u0026gt;50 are shown. Garlic virus X (GarV-X) sequence (NC_001800) served as an outgroup.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/c6cc5bdb77927945efaa5789.png"},{"id":101424304,"identity":"5c02696c-cd6e-4ac3-8ac2-03249708205c","added_by":"auto","created_at":"2026-01-29 14:11:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5621389,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of Ukrainian isolates of garlic virus A (GarV-A) using a maximum likelihood approachbased on partial \u003cem\u003eCP\u003c/em\u003e and \u003cem\u003eNABP\u003c/em\u003egenes’ sequences. The analysis was conducted usinga partitioned model with HKY+F+G4 for the \u003cem\u003eCP\u003c/em\u003e partition and K3Pu+F+G4 for the \u003cem\u003eNABP\u003c/em\u003epartition, with 1,000 bootstrap replicates. Bootstrap values \u0026gt;50 are shown. Garlic virus D (GarV-D) sequence (NC_022961) served as an outgroup.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/a9ca9402baf66fdc367724f8.png"},{"id":101424285,"identity":"1e04d445-9a7e-4fb3-b990-445d3bb0f4c9","added_by":"auto","created_at":"2026-01-29 14:11:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7084988,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of Ukrainian isolates of garlic virus C (GarV-C) using a maximum likelihood approachbased on partial \u003cem\u003eCP\u003c/em\u003e and \u003cem\u003eNABP\u003c/em\u003egenes’ sequences. The analysis was conducted usinga partitioned model with HKY+F+I+G4 for the \u003cem\u003eCP\u003c/em\u003e partition and TIM+F+I+G4 for the \u003cem\u003eNABP\u003c/em\u003e partition, with 1,000 bootstrap replicates. Bootstrap values \u0026gt;50 are shown. Garlic virus B (GarV-B) sequence (NC_025890) served as an outgroup.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/2a27c009772d3b5345ca7995.png"},{"id":101424334,"identity":"2481b720-5e6b-45c3-8a6c-853b0339af22","added_by":"auto","created_at":"2026-01-29 14:12:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3996756,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of Ukrainian isolates of garlic virus D (GarV-D) using a maximum likelihood approachbased on partial \u003cem\u003eCP\u003c/em\u003eand \u003cem\u003eNABP\u003c/em\u003e genes’ sequences. The analysis was conducted using a partitioned model with HKY+F+I+G4 for the \u003cem\u003eCP\u003c/em\u003e partition and K3Pu+F+G4 for the \u003cem\u003eNABP\u003c/em\u003e partition, with 1,000 bootstrap replicates. Bootstrap values \u0026gt;50 are shown. Garlic virus A (GarV-A) sequence (NC_003375) served as an outgroup.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/20a5ae181231f0575b031f30.png"},{"id":101424318,"identity":"eb4234a8-0308-4693-a2d6-03ab84a4ee66","added_by":"auto","created_at":"2026-01-29 14:11:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4964240,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of Ukrainian isolates of shallot virus X (ShVX) using a maximum likelihood approachbased on partial \u003cem\u003eCP\u003c/em\u003e and \u003cem\u003eNABP\u003c/em\u003e genes’ sequences. The tree was constructed using TN+F+G4 model, with 1,000 bootstrap replicates. Bootstrap values \u0026gt;50 are shown. Garlic virus D (GarV-D) sequence (NC_0022961) served as an outgroup.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/5ccbefdacb722aafa3026a79.png"},{"id":108809226,"identity":"257fc049-2673-4f18-98da-34fbed7b29d3","added_by":"auto","created_at":"2026-05-08 15:51:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26700250,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/8343667b-e8a3-4d53-a4bf-6bc98a1a202f.pdf"},{"id":101424303,"identity":"e88219b1-209a-4935-97ef-219bd55f8f09","added_by":"auto","created_at":"2026-01-29 14:11:52","extension":"fasta","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":6287,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData1Sequences.fasta","url":"https://assets-eu.researchsquare.com/files/rs-8501892/v1/c9f2d7db25fedb96fe4963d9.fasta"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFirst evidence of allexiviruses in Allium plants in Ukraine and molecular characterization of their isolates\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eAllium\u003c/em\u003e crops such as garlic, onion, and shallot are of high economic importance worldwide, but their production is often constrained by viral diseases. These vegetatively propagated crops are susceptible to a range of viruses, especially those in the genera \u003cem\u003ePotyvirus\u003c/em\u003e, \u003cem\u003eCarlavirus\u003c/em\u003e, and \u003cem\u003eAllexivirus\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] Among these, allexiviruses (family \u003cem\u003eAlphaflexiviridae\u003c/em\u003e) are particularly notable pathogens of \u003cem\u003eAllium\u003c/em\u003e species. The genus \u003cem\u003eAllexivirus\u003c/em\u003e currently comprises eight recognized species infecting alliaceous plants: garlic viruses A, B, C, D, E, X, garlic mite-borne filamentous virus, and shallot virus X [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition to efficient dissemination through vegetative propagation of infected bulbs/cloves, several allexiviruses can also be transmitted by eriophyid mites, providing a mechanism for secondary spread within Allium plantings [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAllexivirus infections in \u003cem\u003eAllium\u003c/em\u003e plants are typically latent or mild in symptomatology, often causing only subtle mosaic, mottling, faint chlorotic striping of leaves, or mild stunting [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite the limited visible symptoms, these viruses can have a significant economic impact on \u003cem\u003eAllium\u003c/em\u003e crops. Infected plants suffer yield losses and quality deterioration of bulbs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For example, single infections by certain garlic allexiviruses can reduce bulb weight by roughly 10\u0026ndash;15% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], while mixed infections (especially with potyviruses or carlaviruses) can lead to even greater yield reductions [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, allexiviruses are regarded as important contributors to the garlic and onion viral disease complex that undermines crop productivity and bulb quality [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eAllium\u003c/em\u003e crops, allexiviruses are ubiquitous. First detected infecting \u003cem\u003eAllium\u003c/em\u003e in the early 1990s in Russia [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], these viruses have since been reported across Asia, Europe, Africa, Oceania, and the Americas. For instance, \u003cem\u003eAllexivirus\u003c/em\u003e species have been identified in garlic or shallot in such countries as Japan, China, and India in Asia, Greece, Italy, Spain, and Poland in Europe, as well as in the Middle East and Africa (e.g., Iran, Sudan) and the Americas (e.g., Argentina, USA) [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, Ukraine-neighboring countries in Eastern Europe have reported the presence of allexiviruses \u0026ndash; for example, Russia and Poland have documented multiple garlic-infecting \u003cem\u003eAllexivirus\u003c/em\u003e species [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In Ukraine, however, there have been no prior records of allexivirus infections, underscoring a significant gap in the regional knowledge of Allium virus distribution.\u003c/p\u003e \u003cp\u003eHere we report the first detection and characterization of allexiviruses in garlic, onion, and shallot in Ukraine. This study provides the first evidence of \u003cem\u003eAllexivirus\u003c/em\u003e presence in \u003cem\u003eAllium\u003c/em\u003e crops in Ukraine, expanding the known range of these viruses and marking an initial step toward understanding their prevalence and impact on Ukraine\u0026rsquo;s \u003cem\u003eAllium\u003c/em\u003e production and export. The findings contribute important new data on \u003cem\u003eAllium\u003c/em\u003e viral pathogens in the region, with implications for crop health monitoring and management in Ukrainian agriculture.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePrevalence of allexiviruses in \u003cem\u003eAllium\u003c/em\u003e plants in Ukraine\u003c/p\u003e \u003cp\u003eELISA screening detected allexiviruses in the surveyed \u003cem\u003eAllium\u003c/em\u003e material (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Across all tested samples, garlic virus B (GVB), garlic virus C (GVC) and shallot virus X (ShVX) were detected in 38 (35.3%), 23 (21.3%) and 21 (19.4%), respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSerological screening of different \u003cem\u003eAllium\u003c/em\u003e species and Ukraine\u0026rsquo;s regions for virus occurrence\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGVB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGVC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShVX\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGarlic (\u003c/b\u003e\u003cb\u003eA. sativum\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCherkasy region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKyiv region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14/18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10/18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8/18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdesa region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoltava region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTernopil region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVinnytsia region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6/11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4/11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZakarpattia region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZaporizhzhia region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSubtotal\u003c/b\u003e \u003cb\u003e(\u003c/b\u003e\u003cb\u003eGarlic\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39/63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23/63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18/63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eOnion (\u003c/b\u003e\u003cb\u003eA. cepa\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCherkasy region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKyiv region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdesa region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePoltava region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTernopil region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVinnytsia region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSubtotal\u003c/b\u003e \u003cb\u003e(\u003c/b\u003e\u003cb\u003eOnion\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eLeek (\u003c/b\u003e\u003cb\u003eA. ampeloprasum\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eShallot (\u003c/b\u003e\u003cb\u003eA. cepa\u003c/b\u003e \u003cb\u003evar.\u003c/b\u003e \u003cb\u003eaggregatum\u003c/b\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. fistulosum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. schoenoprasum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. scorodoprasum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. tuberosum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA. hollandicum\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0/1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39/108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23/108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21/108\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAllexivirus detections were strongly host-associated. In \u003cem\u003eA. sativum\u003c/em\u003e (garlic), GVB was detected in 39/63 (61.9%), while GVC and ShVX were detected in 23/63 (36.5%) and 18/63 (28.6%), respectively. In contrast, GVB and GVC were not detected in \u003cem\u003eA. cepa\u003c/em\u003e (0/36 and 0/36, respectively), while ShVX was detected only sporadically (3/36; 8.3%).\u003c/p\u003e \u003cp\u003eVirus-virus associations\u003c/p\u003e \u003cp\u003eTo place allexivirus detections in a mixed-infection context, we analyzed co-occurrence with potyvirus and carlavirus detections obtained in the same plants; prevalence results for those viruses in this sample set have been reported previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Using the complete-case dataset for the eight-virus panel (LYSV, OYDV, SYSV, GCLV, SLV, GVB, GVC, ShVX; n\u0026thinsp;=\u0026thinsp;108), allexiviruses were commonly observed within multi-virus profiles, including combinations where two or more allexiviruses occurred together and/or co-occurred with potyviruses/carlaviruses.\u003c/p\u003e \u003cp\u003eBecause allexivirus detections were host-associated, pairwise virus-virus associations were evaluated primarily within garlic (\u003cem\u003eA. sativum\u003c/em\u003e) using the complete-case dataset for the eight-virus panel (LYSV, OYDV, SYSV, GCLV, SLV, GVB, GVC, ShVX; n\u0026thinsp;=\u0026thinsp;63). Two-sided Fisher\u0026rsquo;s exact tests and Pearson chi-square tests applied to 2\u0026times;2 presence/absence tables identified a strongly structured allexivirus module in which GVB formed the core. GVC was never detected in the absence of GVB (23/23 GVC-positive samples were also GVB-positive; Fisher p\u0026thinsp;=\u0026thinsp;4.39\u0026times;10⁻⁷; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;2.0\u0026times;10⁻⁶). Likewise, ShVX was found only in GVB-positive plants (Fisher p\u0026thinsp;=\u0026thinsp;2.89\u0026times;10⁻⁵; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;8.2\u0026times;10⁻⁵), and GVC and ShVX co-occurred significantly (Fisher p\u0026thinsp;=\u0026thinsp;1.95\u0026times;10⁻⁶; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;1.0\u0026times;10⁻⁶).\u003c/p\u003e \u003cp\u003eUsing potyvirus and carlavirus detections specifically to evaluate the co-infection context of allexiviruses, GVC showed positive associations with LYSV (Fisher p\u0026thinsp;=\u0026thinsp;0.00847; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;0.00538) and with SLV (Fisher p\u0026thinsp;=\u0026thinsp;0.00835; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;0.00596) in garlic. In addition, LYSV and SLV co-occurred more frequently than expected by chance (Fisher p\u0026thinsp;=\u0026thinsp;0.00231; χ\u0026sup2; p\u0026thinsp;=\u0026thinsp;0.00160).\u003c/p\u003e \u003cp\u003eMolecular confirmation and phylogenetic placement of allexivirus isolates\u003c/p\u003e \u003cp\u003eTo complement ELISA screening, selected samples were also characterized by RT-PCR and Sanger sequencing of partial allexivirus genomes. In total, 11 sequences were obtained. Amplicons generated with primers targeting garlic virus B yielded four sequences that, upon sequence identification, corresponded to two GarV-B isolates (GVB-36-23, PX761687; GVB-43-23, PX761688), one GarV-A isolate (GVA-112-22, PX766271), and one GarV-D isolate (GVD-6-22, PX766272), indicating that the targeted region can be conserved across related allexiviruses. Additional sequencing covered five GarV-C isolates (GVC-112-22, PX757648; GVC-12-25, PX757649; GVC-16-24, PX757650; GVC-43-23, PX757651; GVC-44-23, PX757652) and two ShVX isolates (ShVX-115-22, PX766273; ShVX-32-23, PX766274).\u003c/p\u003e \u003cp\u003eMaximum-likelihood-based phylogeny reconstructed with representative sequences placed all Ukrainian isolates within their expected species clades and clarified their closest relationships. The two Ukrainian GarV-B sequences (GVB-36-23, PX761687; GVB-43-23, PX761688) were closely identical (99.04% nt identity), and were placed adjacent to the Czech isolate JX682832, sharing with it 98.08% and 98.28% nt identity, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, their nucleotide identity to the nearby Czech isolate JX682833 was significantly lower (91.38% and 91.19%). The Ukrainian GarV-A isolate (GVA-112-22, PX766271) clustered closest to Chinese sequence MN059305 (96.49% nt identity), with similar identity to another Chinese isolate MN059252 (96.67%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Ukrainian GarV-D isolate (GVD-6-22, PX766272) grouped with Chinese isolates MN059369 and MN059370, showing 95.90% nucleotide identity to both (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFive Ukrainian GarV-C sequences fell within a lineage anchored by Czech isolates. GVC-43-23 (PX757651) and GVC-44-23 (PX757652) formed a strongly supported pair (99.82% nt identity) placed closest to the JX682840 Czech sequence, sharing with it 96.20% and 96.01% nt identity, respectively (and lower identity to JX682843, 93.12% and 92.93%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). GVC-12-25 (PX757649) and GVC-16-24 (PX757650) formed a second Ukrainian pair (97.64% nt identity) that also showed its highest identity to JX682840 (96.56% and 95.47%), with lower identity to Chinese sequence MN059142 (93.84% and 92.57%). The remaining Ukrainian GVC isolate PX757648 (GVC-112-22) was closest to Czech sequence JX682846 (99.09% nt identity) and also highly similar to the Japanese one, LC097169 (98.73% nt identity).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, two Ukrainian ShVX sequences clustered within established ShVX diversity represented by European and Asian isolates. ShVX-115-22 was closest to French isolate MH389251 (89.53% nt identity), with similar identity to French MH389252 (89.30%). However, ShVX-32-23 showed its highest identity to French isolates MH389250 and MH389254 (83.26% nt identity), while was more distant from the Indian isolates ON986786 and OK104171 (69.30% nt identity) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Two Ukrainian ShVX sequences shared only 82.79% nucleotide identity with each other, suggesting their different origin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first evidence that allexiviruses infect cultivated \u003cem\u003eAllium\u003c/em\u003e crops in Ukraine, extending the documented range of this group of garlic- and shallot-associated viruses in Europe [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. ELISA screening revealed that GarV-B, GarV-C and ShVX were already present in surveyed material, with detections strongly structured by host: infections were frequent in garlic but rare or absent in onion. Such host skew is consistent with the epidemiology of Allium viruses in vegetatively propagated garlic, where viruses can persist and accumulate across planting cycles even when symptoms are mild or unapparent [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The sporadic detection of ShVX in onion nevertheless indicates that allexiviruses (or at least ShVX) can occur outside garlic-dominated contexts, supporting broader host-inclusive surveillance rather than focusing solely on garlic.\u003c/p\u003e \u003cp\u003eAllexiviruses were commonly embedded within multi-virus profiles, and the within-garlic association structure suggests non-random assembly of infections. The nested pattern in which GarV-C and ShVX were detected only in GarV-B-positive plants, together with the significant GarV-C/ShVX co-occurrence, is compatible with repeated circulation of multi-infected planting material and shared transmission opportunities, including eriophyid mite-mediated spread reported for allexiviruses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The positive associations of GarV-C with LYSV and SLV further emphasize that allexiviruses circulate within mixed infections typical of Allium pathosystems, where co-infection can exacerbate losses relative to single infections [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Given that yield and bulb-quality penalties have been demonstrated for allexiviruses alone and in combination with additional viruses [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], the structured co-occurrence observed here suggests that allexiviruses should be considered integral contributors to the virus complex affecting Ukrainian garlic, alongside the other Allium viruses recently reported from the country [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSequencing and phylogenetic placement confirmed multiple allexivirus species and situated Ukrainian isolates within established global diversity. The clustering of Ukrainian GarV-B and several GarV-C isolates near Czech reference lineages is consistent with regional connectivity of garlic planting material within Europe and mirrors patterns reported from other European surveys [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In parallel, the affinities of Ukrainian GarV-A and GarV-D to Asian isolates align with the broader global dissemination of garlic viruses documented across continents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], underscoring that introductions from multiple sources are plausible for clonally propagated crops. The substantial divergence between the two Ukrainian ShVX sequences supports multiple introductions and/or longer-term circulation with diversification, rather than a single recent origin, consistent with the heterogeneous ShVX diversity reported in different regions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Finally, the recovery of GarV-A and GarV-D from amplicons generated with a GarV-B-targeted primer set highlights that conserved genomic regions can yield cross-species amplification; therefore, sequencing (or species-resolving assays) is essential when translating screening results into species-level inferences.\u003c/p\u003e \u003cp\u003eOverall, the presence of multiple allexiviruses in Ukrainian \u003cem\u003eAllium\u003c/em\u003e crops \u0026ndash; often in structured mixed infections with other economically relevant viruses \u0026ndash; supports incorporating allexiviruses into routine monitoring and planting-material health programs. Priority next steps include expanded surveys across regions and cultivars, deeper sequence coverage to better resolve introductions and local spread, and field-based quantification of yield and quality impacts under Ukrainian production conditions [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In practice, the findings reinforce the value of virus-tested planting stocks and strengthened phytosanitary oversight to limit accumulation and dissemination of allexiviruses and associated virus complexes in Ukraine\u0026rsquo;s \u003cem\u003eAllium\u003c/em\u003e sector [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003ePlant sampling\u003c/p\u003e \u003cp\u003eA total of 108 \u003cem\u003eAllium\u003c/em\u003e plants were analysed in Ukraine during 2022\u0026ndash;2025, representing commercial plantings and private small-scale cultivation in eight regions (Cherkasy, Kyiv, Odesa, Poltava, Ternopil, Vinnytsia, Zakarpattia, and Zaporizhzhia). The dataset included primarily garlic (\u003cem\u003eAllium sativum\u003c/em\u003e; n\u0026thinsp;=\u0026thinsp;69) and onion \u003cem\u003e(A. cepa\u003c/em\u003e var. cepa; n\u0026thinsp;=\u0026thinsp;37), with smaller numbers of shallot, leek and other \u003cem\u003eAllium\u003c/em\u003e spp. Plants were sampled irrespective of symptom status; both plants showing virus-like symptoms (e.g., striping, mosaic, deformation) and plants without obvious symptoms were included, consistent with our earlier \u003cem\u003eAllium\u003c/em\u003e virus surveys.\u003c/p\u003e \u003cp\u003eSerological screening (ELISA)\u003c/p\u003e \u003cp\u003eAll samples were screened serologically for Allexiviruses (GVB, GVC, ShVX) using commercial kits for enzyme-linked immunosorbent assay (DSMZ, Germany) following the microplate ELISA approach [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The results were recorded using an automatic Thermo Labsystems Opsis MR microplate reader (USA) with Dynex Revelation Quicklink software at the wavelength of 405 nm.\u003c/p\u003e \u003cp\u003eRNA extraction, RT-PCR and Sanger sequencing\u003c/p\u003e \u003cp\u003eFor molecular confirmation and phylogenetic characterization, total RNA was extracted from approximately 100 mg of leaf tissue using the Quick-RNA Plant Miniprep Kit (Zymo Research, USA) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003cp\u003eReverse transcription was performed with M-MuLV reverse transcriptase (NEB, USA) and a random primer mix (NEB, USA), using incubation steps of 25\u0026deg;C for 5 min, 42\u0026deg;C for 1 h, and 65\u0026deg;C for 20 min. PCR amplification was carried out using GreenTaq PCR Master Mix (Thermo Fisher Scientific, USA). The cycling profile followed our established workflow (initial denaturation at 95\u0026deg;C for 3 min, then 30 cycles of 95\u0026deg;C for 30 s, annealing for 30 s, 72\u0026deg;C for 45 s, and a final extension at 72\u0026deg;C for 10 min), with annealing temperatures adjusted by target sequence as follows: 53\u0026deg;C for GVB and GVC, and 56\u0026deg;C for ShVX. Amplicons were visualized by electrophoresis in 1.5% agarose gels.\u003c/p\u003e \u003cp\u003ePrimer sets used in this study were: GVB-F 5\u0026prime;-TGACGGGCAAACAGCAGAATAA-3\u0026prime;\u003c/p\u003e \u003cp\u003eGVB-R 5\u0026prime;-ATATAGCTTAGCGGGTCCTTC-3\u0026prime; and GVC-F 5\u0026prime;-TTGCTACCACAATGGTTCCTC-3\u0026prime;, GVC-R 5\u0026prime;-TACTGGCACGAGTTGGGAAT-3\u0026prime; targeting CP\u0026thinsp;+\u0026thinsp;NABP region of garlic virus B and garlic virus C; ShVX-F 5\u0026prime;-ACCGAAATCACAGTTAACTCCTTTGG-3\u0026prime; and ShVX-R 5\u0026prime;-TCTACGGTTGTCGATTTTGTGCGT-3\u0026prime; targeting replicase of shallot virus X [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePCR products selected for sequencing were purified and subjected to Sanger sequencing. Chromatograms were inspected and assembled in BioEdit v7.2.5 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In total, partial allexivirus sequences were generated for 11 Ukrainian isolates (two GarV-B, five GarV-C, two ShVX, plus two sequences amplified with the GarV-B primer set that were later identified as GarV-A and GarV-D). GenBank accession numbers are provided in the Results and in the figures.\u003c/p\u003e \u003cp\u003eSequence alignment and phylogenetic analysis\u003c/p\u003e \u003cp\u003eRepresentative reference sequences were retrieved from GenBank for each virus and combined with Ukrainian sequences. Multiple sequence alignments were generated with MAFFT [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] using the L-INS-i strategy. Maximum-likelihood phylogenetic trees were inferred in IQ-TREE [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], with the substitution model selected using the program\u0026rsquo;s model-selection procedure (ModelFinder) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and branch support assessed using 1,000 bootstrap replicates. For presentation, bootstrap values\u0026thinsp;\u0026gt;\u0026thinsp;50% are shown on branches. To improve readability, densely sampled clades were collapsed where appropriate; numbers in brackets indicate the count of sequences collapsed into that node.\u003c/p\u003e \u003cp\u003eStatistical analysis of the serological dataset\u003c/p\u003e \u003cp\u003eVirus prevalence was summarized as proportions of virus-positive plants. Mixed-infection structure and virus-virus associations were evaluated using 2\u0026times;2 contingency tables on presence/absence calls, following standard categorical-data approaches [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Pairwise associations were tested using two-sided Fisher\u0026rsquo;s exact tests and Pearson chi-square tests, and effect size was summarized as odds ratios where applicable. Analyses were performed on complete-case subsets for the specific virus panels being compared, and results were interpreted with caution for low-frequency detections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no grants or other financial support were received for this research.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Kyrylo Taher, Halyna Snihur, Tetiana Shevchenko, Iryna Budzanivska, and Oleksiy Shevchenko. The first draft of the manuscript was written by Kyrylo Taher, and all authors commented on previous versions of the manuscript. All authors read and approved the final revision of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe partial genome sequences generated in this study have been deposited in GenBank under accession numbers PX761687, PX761688, PX766271, PX766272, PX757648-PX757652, PX766273 and PX766274. The serological prevalence dataset and analysis outputs are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eConci, V. C., Canavelli, A. E. \u0026amp; Balzarini, M. G. 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Methods\u003c/em\u003e. \u003cb\u003e14\u003c/b\u003e, 587\u0026ndash;589 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgresti, A. \u003cem\u003eCategorical Data Analysis\u003c/em\u003e (Wiley, 2002).\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Allium, allexivirus, garlic virus B, garlic virus C, shallot virus X, Ukraine","lastPublishedDoi":"10.21203/rs.3.rs-8501892/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8501892/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAllexiviruses (family \u003cem\u003eAlphaflexiviridae\u003c/em\u003e) are widespread pathogens of vegetatively propagated \u003cem\u003eAllium\u003c/em\u003e crops, but their occurrence has not previously been documented in Ukraine. We surveyed cultivated \u003cem\u003eAllium\u003c/em\u003e plants collected in eight Ukrainian regions (2022\u0026ndash;2025) and screened their samples for garlic virus B (GarV-B), garlic virus C (GarV-C) and shallot virus X (ShVX) using enzyme-linked immunosorbent assay (ELISA). GarV-B, GarV-C and ShVX were detected in 39/108 (36.1%), 23/108 (21.3%) and 21/108 (19.4%) plants, respectively, with infections which were strongly host-associated: garlic (n\u0026thinsp;=\u0026thinsp;63) had high frequencies of indicated viruses (GarV-B \u0026minus;\u0026thinsp;61.9%; GarV-C \u0026minus;\u0026thinsp;36.5%; ShVX \u0026minus;\u0026thinsp;28.6%), whereas onion samples (n\u0026thinsp;=\u0026thinsp;33) were largely negative (ShVX \u0026minus;\u0026thinsp;3.0%; GarV-B and GarV-C - not detected). Co-occurrence analysis within garlic revealed a nested allexivirus module in which GarV-C and ShVX occurred only in GarV-B-positive plants. RT-PCR and Sanger sequencing generated 11 partial genomes representing GarV-B, GarV-C, ShVX, GarV-A and GarV-D. Maximum-likelihood phylogenies placed Ukrainian allexivirus isolates within established global diversity and indicated both European- and Asian-affiliated lineages. These findings provide the first evidence of allexiviruses in Ukrainian \u003cem\u003eAllium\u003c/em\u003e crops, and support their inclusion in plant health surveillance and planting-material certification.\u003c/p\u003e","manuscriptTitle":"First evidence of allexiviruses in Allium plants in Ukraine and molecular characterization of their isolates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 14:09:56","doi":"10.21203/rs.3.rs-8501892/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-13T14:13:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T12:10:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119972586880614703201669711652520188778","date":"2026-02-19T14:20:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T14:13:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316425426266753357165298128631781038847","date":"2026-01-28T12:06:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-27T13:14:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-16T09:06:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-13T18:38:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-08T22:17:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-08T22:13:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0037920-5975-43d5-9af0-db5d126d1687","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61974652,"name":"Biological sciences/Biotechnology"},{"id":61974653,"name":"Biological sciences/Microbiology"},{"id":61974654,"name":"Biological sciences/Molecular biology"},{"id":61974655,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-08T15:12:59+00:00","versionOfRecord":{"articleIdentity":"rs-8501892","link":"https://doi.org/10.1038/s41598-026-47632-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-05-02 15:57:46","publishedOnDateReadable":"May 2nd, 2026"},"versionCreatedAt":"2026-01-29 14:09:56","video":"","vorDoi":"10.1038/s41598-026-47632-1","vorDoiUrl":"https://doi.org/10.1038/s41598-026-47632-1","workflowStages":[]},"version":"v1","identity":"rs-8501892","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8501892","identity":"rs-8501892","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}