Vector-enabled metagenomics reveals the first detection of the geminivirus beet curly top Iran virus in Europe

SHORT COMMUNICATION Vector-enabled metagenomics reveals the first 1 detection of the geminivirus b eet curly top Iran 2 virus in Europe 3 1.1 Author names 4 Laura Miozzi1†, Silvia Rotunno1†, Fulco Frascati1,2, Monica Marra1,3, Francesco Nugnes4, 5 Umberto Bernardo4, Daniele Marian1, Sofia Bertacca1, Massimiliano Ballardini5, Gian Paolo 6 Accotto1, Anna Maria Vaira1* and Emanuela Noris1 7 †These authors contributed equally to this work 8 9 L. Miozzi https://orcid.org/0000-0003-0410-8230 10 S. Rotunno https://orcid.org/0000-0002-9227-2454 11 F. Frascati https://orcid.org/0009-0007-9444-7186 12 M. Marra https://orcid.org/0000-0002-3582-1242 13 F. Nugnes https://orcid.org/0000-0001-9309-4007 14 U. Bernardo https://orcid.org/0000-0001-8190-2177 15 D. Marian - 16 S. Bertacca https://orcid.org/0000-0002-0353-7642 17 M. Ballardini - 18 G.P. Accotto https://orcid.org/0000-0002-3825-1097 19 A.M. Vaira https://orcid.org/0000-0001-6702-1162 20 E. Noris https://orcid.org/0000-0001-8656-8841 21 22 1.2 Affiliation(s) 23 1 Institute for Sustainable Plant Protection. National Research Council (CNR), 10135 Torino, 24 Italy 25 2 Department of Ecology and Biology, University of Tuscia, 01100 Viterbo, Italy; 26 3 Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy 27 4 Institute for Sustainable Plant Protection. National Research Council (CNR), 80055 Portici 28 (NA), Italy 29 5 ESASEM S.p.A. Casaleone (VR) Italy 30 31 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 1.3 Corresponding author and email address 32 Anna Maria Vaira – [email protected] 33 34 1.4 Keywords 35 BCTIV; Becurtovirus; cicadellids; agroinoculation; cucurbits; high-throughput sequencing; 36 rolling circle amplification 37 38 1.5 Repositories: 39 The beet curly top Iran virus (BCTIV, Becurtovirus betae) genome sequences have been 40 deposited in NCBI GenBank under accession no. PV972859. The HTS raw sequence reads 41 have been deposited in the NCBI Sequence Read Archive under BioProject accession no. 42 PRJNA1040478. BioSample accession numbers SAMN38260155, SAMN38260160, 43 SAMN38260161, SAMN38260162, SAMN38260167, SAMN38260169 correspond to pools 44 INS20_IT1, INS21_IT6, INS21_IT7, INS21_IT8, IS21_IT13 and INS22_IT15 respectively. 45 BCTIV infectious clone (isolate Plavit-Bt36, Ref-SKU: 029V-05683) is in Eva-Global portal 46 repository: European Virus Archive – AISBL, https://www.european-virus-archive.com/ 47 48 2. Abstract 49 Geminiviruses are among the most threatening emerging insect-borne viruses and are 50 responsible for serious outbreaks. Climate change could further exacerbate their impact on 51 crops, highlighting the need for new diagnostic approaches to manage potentially dangerous 52 situations. Vector-Enabled Metagenomics (VEM) exploits the natural ability of highly mobile 53 insects to accumulate viruses acquired from plants over time and space within an 54 ecosystem; this approach is effectual in monitoring the presence of new invasive and 55 indigenous viruses in large areas. Geminiviruses have circular single-stranded DNA 56 genomes that can be readily targeted by Rolling Circle Amplification (RCA). The combination 57 of RCA and VEM largely increases the chances of detecting geminiviruses. This approach 58 enabled us to identify the becurtovirus beet curly top Iran virus (BCTIV, Becurtovirus betae) 59 in insects collected in Europe. BCTIV is a major pathogen of sugar beet but can also infect 60 plants of other families; it is transmitted by cicadellids and has been so far detected only in 61 Iran and Anatolia (Turkey). We also show that two cucurbit species, watermelon (Citrullus 62 lanatus) and zucchini (Cucurbita pepo) are both natural and experimental hosts for BCTIV. 63 64 3. Impact statement 65 Virus infections account for almost 50% of emerging plant diseases globally and may 66 produce high crop losses, resulting in huge economic and social impact worldwide. 67 Geminiviruses, threatening both monocot and dicot plants, represent high risk for both staple 68 food and industrial crops. A peculiar diagnostic approach combining a specific geminivirus 69 enrichment reaction, the monitoring of the virome of highly mobile insects within agricultural 70 areas and the sensitivity of high throughput sequencing (HTS) was effective in producing a 71 first alert for a new polyphagous virus. The reduced cost of HTS methods further raises 72 interest in this approach, making it suitable as a first step for monitoring large areas. 73 74 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 4. Data summary 75 The authors confirm all supporting data, code and protocols have been provided within the 76 article or through supplementary data files. 77 78 5. INTRODUCTION 79 The world’s staple and nutritionally important food crops are increasingly affected by virus 80 disease pandemics and epidemics which significantly reduce their yields and quality. This 81 situation is progressively more alarming due to the growing food requirements for the human 82 population and global warming issues [1]. Emerging infectious diseases are generally 83 caused by pathogens which invade new geographical areas mainly due to the spread of their 84 natural vectors, or attack new species with increased incidence, gaining winning ecological 85 fitness [2]. The Geminiviridae family, listing fourteen Genera [3], includes viruses with single-86 stranded circular DNA (ssDNA) genomes, encapsidated into twinned icosahedral particles. 87 Geminiviruses affect both monocot and dicot plants, including important cereals, vegetables, 88 ornamentals and fiber crops. They are among the most threatening emerging plant viruses 89 and have caused serious outbreaks during the last decades across different parts of the 90 world [4]. The geminivirus beet curly top Iran virus (BCTIV, Becurtovirus betae) has a 91 monopartite genome, infects mainly sugar beet (Beta vulgaris), but also tomato, pepper, and 92 common bean [5, 6], and is transmitted by Neoaliturus (Circulifer) haematoceps (Mulsant & 93 Rey), family Cicadellidae [7]. Up to date, BCTIV has been reported only in the Asian 94 continent, i.e. in Iran and Anatolia (Turkey). 95 Metagenomics-based approaches are suitable for a wide epidemiological surveillance of 96 viral pathogens. Specifically, Vector-Enabled Metagenomics (VEM) exploits the natural 97 ability of highly mobile insects to accumulate viruses acquired from plants over time and 98 space within an ecosystem [8]. VEM has already been applied to insect vectors or their 99 predators to identify known and novel plant viruses [9-13]. 100 In this work, we carried out a VEM survey in an intensive vegetable-producing area in 101 Southern Italy, with the aim to analyze the circulating geminivirome. To increase the 102 detection of the geminivirus genomes, a Rolling Circle Amplification (RCA) step was 103 included before VEM analysis [9, 11]. The RCA-VEM procedure was applied to leafhoppers 104 (cicadellids), known to include members that can transmit geminiviruses, but can also be just 105 carriers of viruses after feeding. This strategy enabled us to detect BCTIV for the first time in 106 Italy in insects and in cucurbit plants, providing evidence of the virus presence in the 107 European continent. The ability of BCTIV to infect watermelon and zucchini was also 108 confirmed by inoculation experiments with an infectious agroclone. 109 110 6. METHODS 111 Sample collection and processing 112 Cicadellids were collected during spring and summer of 2020-2022 in cucurbit and tomato 113 fields and nearby non-cultivated areas located in the Campania region (Southern Italy). 114 Areas of 100 m x 2 m along the fields were surveyed using sweeping nets or entomological 115 aspirators. Cicadellids were transferred into collection tubes and stored in ethanol at -20°C. 116 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint A total of 74 cicadellid samples (each made of 1 to 8 individuals) were collected from 30 117 different fields, as described in Table 1. 118 In the same fields, plant samples consisting of zucchini (Cucurbita pepo), watermelon 119 (Citrullus lanatus), and tomato (Solanum lycopersicum), showing virus-like symptoms were 120 also collected; for each field, 20 representative plants were collected forming a unique plant 121 pool. The plant samples were dried by calcium chloride treatment immediately after 122 collection and stored at -20°C. 123 Total nucleic acids (TNA) extraction and RCA amplification 124 The 74 cicadellid samples were extracted using a CTAB-based method according to [14]. 125 TNA concentration was adjusted to 500 ng/µL, following Nanodrop spectrophotometer 126 (Thermo Scientific) measurement. TNAs were subsequently grouped into 6 pools according 127 to collection date and location (Table 1). 128 Each insect TNA pool (1.5 µg) was subjected to RCA, using the Sigma TempliPhi 129 Amplification kit (Sigma Aldrich), following manufacturers’ instructions. The reaction was 130 conducted at 30 °C for 30 hours [15]. RCA products were purified using the DNA Clean & 131 Concentrator Kit (Zymo Research) and 1 µg of each purified RCA product was delivered to 132 Novogene service for DNA sequencing with Illumina Novaseq 6000 (2x150bp). 133 134 Cicadellid examination 135 Cicadellids were morphologically examined based on the available descriptions and 136 diagnostic keys for the genus Neoaliturus (Circulifer) [16, 17]. Molecular identification was 137 performed by barcoding on selected BCTIV-positive individual insect DNA, amplifying a 138 fragment of about 680bp of the COI gene (Cytochrome C Oxidase subunit I) using the 139 universal primers LCO/HCO [18], followed by Sanger sequencing. Obtained sequences were 140 compared with those present in the GenBank NCBI (https://www.ncbi.nlm.nih.gov/ accessed 141 on 26-Jan-2026) database with BLASTN [19]. 142 143 Illumina High throughput Sequencing (HTS) and data analysis 144 Raw data were checked for quality and adapter contamination with FASTp (v. 0.21.0) [20]. 145 Ribosomal sequences were removed with BBMap tool (v. 38.7, 146 https://sourceforge.net/projects/bbmap/). Reads were assembled into scaffolds using 147 metaSPAdes (v. 3.15.1) [21] with k-mer sizes of 97, 107, 117, and 127; statistics on the 148 assembly were obtained with QUAST (v. 5.2.0) [22]. Redundancy reduction was performed 149 with CAP3 (Version Date 02/10/15) [23]. Only contig sequences longer than 500 bp were 150 retained and used as input for diamond BLASTx (v. 2.0.15; diamond database updated at 151 13/05/2023) [24]. Results of BLASTx search were visualized with MEGAN (v. 6.25.9) [25]. In 152 order to retrieve full-length geminiviral genomes, clean reads were mapped against the 153 BCTIV reference genome (RefSeq Acc. No. NC_010417.1) using Bowtie2 (v. 2.2.9) [26]. For 154 cicadellid identification, contigs were mapped with Bowtie2 on the COI gene sequences of 155 the Cicadellidae family available in the BOLD database [27] with default parameters. 156 For phylogenetic analysis, complete genomes and aminoacid sequences of the coat protein 157 (CP) of selected Becurtovirus isolates and reference sequences of the genus Curtovirus 158 were downloaded from NCBI Virus (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/, accessed 159 20/01/2026). These sequences were multialigned using MUSCLE embedded in MEGA11 160 software (v. 11.0.11) [28]. The IQTREE online tool (v. 3.0.1, http://iqtree.cibiv.univie.ac.at/) 161 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint [29] was used for model selection and tree inference. The obtained phylogenetic trees were 162 modified using FigTree (v.1.4.3, https://tree.bio.ed.ac.uk/software/figtree/). 163 164 Validation by end-point PCR and Sanger sequencing 165 The presence of BCTIV was validated by end-point PCR using the virus-specific primers 166 BCTIV-272-F (5’-CGAAGCTATCCAGCCTTGCT-3’) and BCTIV-831-R (5’-167 CGATCCACAATAACCCAATG-3’), amplifying a 559 bp fragment of the BCTIV-SIV isolate 168 (GenBank Acc. No. JX082259), spanning the V2 and V1 coding sequences. PCR was 169 performed using Platinum™ II Taq Hot-Start DNA Polymerase kit (Invitrogen), following 170 manufacturer’s instruction, with an annealing temperature of 55 °C. TNA extracted from the 171 field sample 150621_w_3 or from experimentally infected N. benthamiana plants was used 172 as positive control. Selected representative amplicons were Sanger-sequenced to verify 173 amplification specificity. 174 175 BCTIV agroinoculation 176 For infectivity assays, agrobacteria carrying the infectious clone of the BCTIV-SIV isolate [7] 177 were grown for 48 h at 28°C in 50 ml liquid YEB medium containing 100 µg/l kanamycin and 178 50 µg/l rifampicin, with shaking. Bacteria were pelleted and resuspended in 1.5 ml sterile 179 water. About 50 µL of the suspension were inoculated in the stems of cucurbit seedlings at 180 the first-true leaf stage. Several cultivars of watermelon (4 seedlings of cv. Sugar Baby, 2 of 181 cv. Crimson, 8 of cv. Bontà, and 4 of cv. Sentinel), 14 zucchini seedlings (cv. Genovese), 182 and 4 melon seedlings (Cucumis melo, cv. Vendram) were tested in two separate 183 experiments. Seven N. benthamiana plants at the 4-leaf stage were inoculated as positive 184 controls. All plants, including non-inoculated controls, were maintained in greenhouse at 25 185 ± 5 °C, under 16 h light/8 h dark cycle. Plants were evaluated for symptoms and tested by 186 PCR for BCTIV infection 40 days post inoculation (dpi). 187 188 7. RESULTS AND DISCUSSION 189 NGS analysis of insect pools and PCR detection 190 The high-throughput sequencing of DNAs extracted from the six cicadellid samples 191 generated a total of 837,226,900 reads. Following pre-processing and quality filtering, 192 approximately 99,328,568 to 150,180,006 reads per sample were retained for downstream 193 analysis (Supplementary Table 1). BCTIV-related sequences were identified in the three 194 pools INS21_IT_6, INS21_IT_7 and INS21_IT_8 (Table 1). Specifically, a full-length genome 195 sequence of BCTIV was recovered in pools INS21_IT_7 and INS21_IT_8, showing 99% 196 identity to the BCTIV-SIV isolate genome (Acc. No. JX082259). The INS21_IT_7-derived 197 sequence was submitted to the GenBank database with the accession number PV972859. 198 In the INS21_IT_6 pool, a partial BCTIV genome sequence of 533 nt was recovered, 199 spanning nucleotides 2435-120 of the BCTIV-SIV isolate, including the Large Intergenic 200 Region, and showing 99% identity with the BCTIV-SIV isolate. The detection of a partial 201 BCTIV sequence in this pool could result from a very low concentration of viral target DNA 202 (Supplementary Table 1) or by a technical bias in library construction [30, 31]. 203 Both genome- and CP-based phylogenetic analyses revealed a well-defined BCTIV cluster, 204 clearly distinct from other becurtoviruses (Fig. 1, Supplementary Fig. 1). Within this cluster, 205 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint two main subgroups are evident: one comprising isolates from central and Southern Iran and 206 another including isolates from Northern and Western Iran and from Turkey. The latter forms 207 a cohesive subclade with isolates from Iran’s Azerbaijan province, near the Turkish border. 208 Notably, the Italian isolate clusters within the central/Southern Iran subgroup, indicating a 209 close genetic relationship with isolates from this region. 210 211 212 213 214 215 216 Figure 1. Maximum likelihood phylogenetic tree inferred using IQ-TREE based on the complete genome 217 sequences of viral species belonging to the Becurtovirus genus, using Curtovirus reference genomes as outliers. 218 Node values represent ultrafast bootstrap supports (UFBoot) calculated from 1,000 replicates, with values ≥70% 219 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint considered well supported. The best-fit substitution model (TIM2+F+G4) was automatically selected by 220 ModelFinder. The BCTIV complete genome sequenced in this work is shown in purple. NC_015051.1 SpCTAV 221 indicates the Becurtovirus spinach curly top Arizona virus. The outliers, represented by the Curtovirus references 222 genomes (NC_0014612.1 BCTV, beet curly top virus; NC_002543.1 HrCTV, horseradish curly top virus; 223 NC_014631.1 SSCTV, spinach severe curly top virus; NC_034629.1 PepYDV, pepper yellow dwarf virus) are 224 shown in blue. Becurtovirus clades corresponding to different geographical areas are highlighted with different 225 colors: yellow indicates sequences from South-central Iran, green denotes sequences from Turkey, pink from 226 Northwest Iran, and blue from USA. 227 228 The presence of BCTIV in cicadellids was confirmed by PCR using the individual cicadellid 229 samples present in the three positive pools. Overall, 19 out of 31 samples (61%), all 230 collected in June 2021, were PCR-positive (Table 1); in detail, BCTIV was detected in 4 out 231 of 10, 8 out of 10, and 7 out of 11 samples present in the pools INS21_IT_6, 7 and 8, 232 respectively (Fig. 2a). Two representative amplified fragments were Sanger-sequenced, 233 confirming BCTIV-specific amplification. 234 235 236 Figure 2. PCR validations using BCTIV-specific primers. (a) Analysis of 31 cicadellid samples forming the insect 237 pools INS21_IT6, 7 and 8. (b) Analysis of 11 plant samples collected in/nearby fields where BCTIV-positive 238 cicadellids were found. Lanes 1, 150621_w_1; 2, 150621_z_1; 3, 150621_z_2; 4, 150621_z_4; 5, 150621_z_5; 239 6, 150621_z_6; 7, 150621_w_3; 8, 160621_z_1; 9, 160621_z_2; 10, 160621_w_1; 11, 160621_w_2, listed in 240 Table 1. (c) Analysis of seedlings artificially inoculated with the BCTIV agroclone: 14 zucchini, 18 watermelon, 241 and 4 melon seedlings, tested 40 days post inoculation. The arrow indicates the BCTIV 559bp amplified 242 fragment; M, 100bp marker; +, BCTIV positive control; nt, no template; z/m/w, zucchini, melon and watermelon 243 negative control samples. Red asterisk indicates Sanger-sequenced amplicons. 244 245 The BCTIV-positive leafhopper samples were collected mainly in the Salerno area and, to a 246 lesser extent, in Naples and Caserta areas. Although the survey was conducted across three 247 years in different growing seasons, only cicadellids collected in early summer (June 2021) 248 were BCTIV-positive, suggesting that the collection period might be relevant, possibly linked 249 to the life cycle of the insects (Table 1; Fig. 3). 250 251 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint Table 1 – Insect and plant samples analyzed in this study for the presence of beet curly top Iran virus (BCTIV). 252 Collection sites, cicadellid and plant samples where BCTIV-related sequences were identified are indicated in 253 bold. 254 Collection date Collection site Crop (n. of surveyed fields) Cicadellid pools (n. of insect samples in pool) Plant pools (20 plants/pool) July 2020 Eboli (SA) Zucchini (2) INS20_IT_1 (19) nt Watermelon (2) Tomato (3) Villa Literno (CE) Zucchini (2) June 2021 Eboli (SA) Watermelon (1) INS21_IT_6 (10) 150621_w_1 Zucchini (3) 150621_z_1 150621_z_2 150621_z_4 Montecorvino (SA) Zucchini (1) INS21_IT_7 (10) 150621_z_5 Pontecagnano (SA) Zucchini (1) 150621_z_6 Villa Literno (CE) Zucchini (1) 160621_z_1 Caivano (NA) Zucchini (1) 160621_z_2 Zucchini (1) INS21_IT_8 (11) C. Principe (CE) Watermelon (2) 160621_w_1 160621_w_2 Battipaglia (SA) Watermelon (1) nc 150621_w_3 November 2021 Villa Literno (CE) Zucchini (3) INS21_IT_13 (19) nt Giugliano (NA) Zucchini (1) Eboli (SA) Zucchini (2) Bellizzi (SA) Tomato (1) April 2022 Giugliano (NA) Zucchini (1) INS22_IT_15 (6) nt Eboli (SA) Zucchini (1) Pontecagnano (SA) Zucchini (1) Giugliano (NA) Watermelon (1) 255 SA: Salerno; CE: Caserta; NA: Napoli; nc: not collected; nt: not tested 256 Plant pool codes indicate the collection date and the crop (w, watermelon; z, zucchini), followed by a progressive number (ddmmyy_x_n) 257 258 Following the detection of BCTIV in insects collected in some cucurbit fields, we investigated 259 the plants growing in and close to the same fields, where symptoms presumably linked to 260 BCTIV infection were observed (Fig. 4 a-d). The presence of BCTIV was confirmed by PCR 261 in 2 zucchini and 2 watermelon fields out of eleven tested (Table 1; Fig. 2b) and BCTIV-262 specific amplification was confirmed on selected amplified fragments by Sanger sequencing. 263 Despite the relatively low infection rate found in the fields tested, our results highlight the 264 urgent need for further searches in this region to assess the real distribution of this virus, 265 concentrating on the major known BCTIV hosts, i.e. Beta vulgaris and Spinacia oleracea. 266 267 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 268 Figure 3. Geographical distribution of BCTIV-positive samples in the Campania region, Italy. Red and green 269 markers indicate the sites where BCTIV-positive insect samples and plant pools were identified, respectively. 270 Image modified from Wikimedia Commons. Original authors: Vonvikken and Marcomob, Public Domain. 271 272 Cicadellid analysis 273 Morphological examination of the cicadellids present in BCTIV-positive pools did not allow us 274 to identify specimens of the genus Neoaliturus (Circulifer), which includes the species N. 275 (Circulifer) haematoceps, the only known natural vector of BCTIV, previously recorded in the 276 Mediterranean areas, including Italy [32]. Consistently, no Illumina-derived contigs from the 277 tested cicadellids could be clearly assigned to this genus, based on the COI sequences 278 available in the BOLD reference database. To further investigate the occurrence of 279 Neoaliturus spp. individuals, a COI barcoding approach was adopted. Similarities ranging 280 between 90.4-99.7% were detected with the genera Exitianus and Psammotettix belonging 281 to the Cicadellidae family and similarity of 98,9% was found with the genus Laodelphax 282 within the Delphacidae family. In two cases, sequence alignment was consistent only at the 283 Cixiidae family level. Importantly, none of the amplified sequences reached at least 97% 284 identity with the Neoaliturus COI gene accession present in the GenBank reference 285 databases (Table 2). 286 Since the presence of Neoaliturus spp. could not be demonstrated in our samples by 287 morphological or sequence analyses [33, 34], it is possible that this vector is rare in the 288 environment and that the tested cicadellids only occasionally ingested BCTIV virions during 289 feeding. Alternatively, we cannot exclude the presence of a previously unrecognized BCTIV 290 vector active in the Mediterranean environment among the collected positive cicadellids, 291 potentially representing an additional risk factor for virus establishment and spread. 292 Moreover, the lack of an updated integrative taxonomic framework for Neoaliturus 293 emphasizes the need for further studies. 294 295 296 Table 2. Results of barcoding analysis of individual insects present in BCTIV-positive pools. The best hit 297 obtained by BLASTn analysis of the amplified COI sequence are shown, together with the coverage and identity 298 percentages. The levels of identity with the reference COI gene of the Neoaliturus haematoceps are also 299 indicated, the coverage percentage of the twelve COI sequences with the reference ranges from 92 to 97%. We 300 considered the identity > 97% sufficient for genus assignment [33, 34] 301 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 302 303 Expanding the BCTIV host range 304 As cucurbits have not yet been clearly established as natural hosts of BCTIV [5, 6], to 305 substantiate our findings, artificial inoculation of watermelon, melon and zucchini seedlings 306 with the BCTIV infectious clone was performed. At 40 dpi, 11 out of 18 watermelon, 1 out of 307 4 melon, and 7 out of 11 zucchini seedlings tested positive in PCR (Fig. 2c). At this time 308 point, watermelons showed reduced growth, chlorosis, greyish veins and light curling of 309 leaves, accompanied by small necrotic lesions adjacent to veins (Fig. 4e), while chlorotic 310 symptoms occurred on zucchini and melon plants. All N. benthamiana plants used as control 311 showed typical BCTIV symptoms already at 7 dpi (Fig. 4f). 312 313 pool insect ID BLASTn best hit Family coverage (%) identity (%) identity with N. haematoceps COI gene MK188548 (%) INS21_IT_6 1881-1 PP600601.1 Reptalus quinquecostatus Cixiidae 95 86,4 71,7 INS21_IT_7 1882-1 LC775130.1 Exitianus capicola Cicadellidae 90 90,4 73,8 1883-1 KR567922.1 Psammotettix sp. Cicadellidae 97 98,8 80,9 1884-1 MK292933.1 Laodelphax striatellus Delphacidae 97 98,9 70,6 1885 KR573169.1 Psammotettix confinis Cicadellidae 96 99,1 81,2 1886-1 KR573169.1 Psammotettix confinis Cicadellidae 97 99,1 80,6 1887-1 PP600593.1 Psammotettix alienus Cicadellidae 97 99,7 81 1892-1 OQ569810.1 Psammotettix confinis Cicadellidae 98 99,1 80,5 INS21_IT_8 1888 OR624912.1 Cixiidae sp. Cixiidae 97 88,2 72 1889-1 KR567922.1 Psammotettix sp. Cicadellidae 97 99,1 81 1890-1 MZ519872.1 Psammotettix sp. Cicadellidae 98 99,2 80,8 1891 KR573169.1 Psammotettix confinis Cicadellidae 97 99,1 80,4 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 314 Fig. 4. Symptoms attributable to BCTIV infection in field cultivated plants (panels A-D) and in experimentally 315 inoculated plants (panels E and F). Yellow mosaic and curling symptoms on (A and C) young zucchini plants 316 collected in fields 150621_z_1 and 150621_z_5 respectively; yellow mosaic and curling symptoms on (B and D) 317 watermelon plants collected in field 150621_w_3 and 160621_w_1 (see also Table 1). (E) Symptoms on an adult 318 leaf of a BCTIV-agroinoculated watermelon seedling (var. Sentinel, 40 dpi). (F) Typical crumpling symptoms on 319 N. benthamiana young leaves at 7 dpi, used as positive control. 320 321 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint Altogether, the agroinoculation experiments and the molecular analysis of field samples 322 support the conclusion that watermelon and zucchini represent new hosts for BCTIV and 323 confirm that melon can host BCTIV, as recently reported [35]. Indeed, although previous 324 studies reported that zucchini plants collected in Iran tested positive in ELISA using a curly 325 top virus-specific antibody, no clear identification of the infecting virus was available at that 326 time [36]. 327 328 8. CONCLUDING REMARKS 329 The emergence of plant viruses in new areas involves ecological adaptation processes 330 resulting from changes in virus−host and virus-vector associations over time and space. 331 During a surveillance project in the Mediterranean area focused on tomato and cucurbit 332 crops, we adopted an RCA-VEM approach on associated insects to investigate the 333 circulating geminivirome. This strategy led us to identify for the first time in Europe and 334 specifically in Italy the presence of BCTIV, a becurtovirus to date described only in the Asian 335 continent (Iran and Anatolia) [7, 37, 38]. 336 Overall, this study confirms that RCA-VEM is a powerful approach to conduct 337 epidemiological surveillance of circulating virome, providing prompt alerts over a 338 polyphagous and potentially invasive geminivirus. This diagnostic strategy might be adopted 339 to monitor the introduction into new areas of other dangerous geminiviruses [39, 40] and 340 associated satellites [41, 42], as well as their vectors. Such surveillance is especially crucial 341 for Southern European countries, naturally exposed to phytopathological threats from 342 warmer tropical and subtropical countries. 343 344 9. Author statements 345 9.1 Author contributions 346 L.M: data analysis, sample collection, review and editing. S.R: data analysis, laboratory 347 analyses, review and editing. F.F: laboratory analyses. M.M: laboratory analyses. F.N: 348 conceptualization, sample collection, laboratory analyses. UB: conceptualization, sample 349 collection. D.M: sample collection, laboratory analyses. S.B: laboratory analyses, review and 350 editing. M.B: sampling organization. G.P.A: sample collection, review and editing. A.M.V: 351 funding acquisition, conceptualization, writing the original draft, laboratory analyses, review 352 and editing. E.N: conceptualization, review and editing. 353 9.2 Conflicts of interest 354 The authors declare that there are no conflicts of interest 355 356 9.3 Funding information 357 This work was financially supported by Italian Ministry for University and Research (MUR) 358 through the PRIMA project GeMed – Prevention and control of new and invasive 359 geminiviruses infecting vegetables in the Mediterranean (PRIMA2018_00090 – Section 2) 360 9.4 Ethical approval 361 Not applicable 362 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted February 19, 2026. ; https://doi.org/10.64898/2026.02.18.706615doi: bioRxiv preprint 9.5 Consent for publication 363 Not applicable 364 365 9.6 Acknowledgements 366 We wish to thank Elena Zocca and Luca Bordone for greenhouse work management; 367 Simona Gargiulo, Flavia de Benedetta, and Roberta Ascolese for field samplings and 368 Fortuna Miele for the field and laboratory activities. 369 370 10. Legends of Supplementary Material 371 Supplementary Table 1. Summary statistics of bioinformatics analysis of the insect pools. 372 Supplementary Figure 1. Maximum likelihood phylogenetic tree inferred using IQ-TREE 373 based on the coat protein (CP) aminoacid sequences of viral species belonging to the 374 Becurtovirus genus, using CP Curtovirus as outliers. Node values represent ultrafast 375 bootstrap supports (UFBoot) calculated from 1,000 replicates, with values ≥70% considered 376 well supported. The best-fit substitution model (JTT+G4) was automatically selected by 377 ModelFinder. The BCTIV CP from genome sequenced in this work is shown in purple. 378 YP_0042079231.1 SpCTAV indicates the Becurtovirus spinach curly top Arizona virus. The 379 outliers, represented by the Curtovirus references genomes (NP_040559.1 BCTV, beet curly 380 top virus; NP_066183.1 HrCTV, horseradish curly top virus; YP_003966135.1 SSCTV, 381 spinach severe curly top virus; YP_009362975.1 PepYDV, pepper yellow dwarf virus) are 382 shown in blue. Becurtovirus clades corresponding to different geographical areas are 383 highlighted with different colors: the yellow rectangle indicates sequences from South-central 384 Iran, the green one denotes sequences from Turkey, pink from Northwest Iran, and blue 385 from USA. 386 387 388 11. References 389 1. Jones RAC. Global Plant Virus Disease Pandemics and Epidemics. Plants (Basel) 2021;10(2). 390 2. 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