Isolation and Characterization of Bacteriophage vB_RsoP_BMB116 Infecting Ralstonia solanacearum

Isolation and Characterization of Bacteriophage vB_RsoP_BMB116 Infecting Ralstonia solanacearum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation and Characterization of Bacteriophage vB_RsoP_BMB116 Infecting Ralstonia solanacearum Shoude Liu, Rong Wen, Qi feng Li, Ming Sun, Dong hai Peng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7097101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ralstonia solanacearum is an increasingly prominent multidrug-resistant phytopathogen. Phages are an effective alternative for treating Ralstonia solanacearum infections. In this study, the phage vB_RsoP_BMB116, which is specific to R . solanacearum , was isolated from Fujian, China. Electron microscopy revealed that vB_RsoP_BMB116 exhibited a podoviridae morphotype. The double-stranded DNA genome of vB_RsoP_BMB116 spans 83,020 bp and encodes 126 predicted unidirectionally oriented genes, of which 42 have putative functions assigned, while the remainder are hypothetical proteins. Genome analysis indicated that vB_RsoP_BMB116 is a new genus. Ralstonia solanacearum bacteriophage Podoviridae genome Plant pathogenic bacteria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key points The new Ralstonia solanacearum phage vB_RsoP_BMB116 ( novel genus) is a key resource for microbiology and biotechnology, aiding new phage therapies and biocontrol agents. Observed via transmission electron microscopy, the novel phage vB_RsoP_BMB116 belongs to the Podoviridae family. In vitro assays demonstrated that it exhibits significant application potential for the prevention and control of plant bacterial diseases. Introduction Ralstonia solanacearum , a Gram-negative pathogen, causes bacterial wilt (Hayward 1991 ). It is classified within Ralstonia solanacearum species complex (RSSC), which comprises three distinct species: R. solanacearum, R pseudosolanacearum , and R. syzygii (Prior et al. 2016 ). All three species are recognized as major bacterial wilt pathogens that contribute to substantial crop losses and are classified as significant phytopathogens (Safni et al. 2014 ; Mansfield et al. 2012 ). Bacterial wilt, triggered by the soilborne pathogen R. solanacearum , severely impacts Solanaceae crops like ginger potato, tomato, eggplant, pepper and flue-cured tobacco (Fujiwara et al. 2011 ; da Silva Xavier et al. 2018 ). The bacterium spreads via soil and water, infects roots, and invades the vascular system, causing potato brown rot and wilting (Hayward 1991 , Vasse et al. 1995 ; Alvarez et al. 2010). Bacterial wilt results in estimated global agricultural losses exceeding $ 950 million annually, posing a substantial economic burden on Solanaceae cultivation across subtropical, tropical, and temperate regions (Mansfield et al. 2012 ; Sae-Ueng et al. 2020 ). Accordingly, R. solanacearum is regarded the second most critical bacterial phytopathogen, following Pseudomonas syringae (Mansfield et al. 2012 ). The development of effective therapeutics for bacterial wilt is imperative, given the limitations of conventional agrochemicals, including resistance emergence, human health, and environmental damage (Jiang et al. 2017 ; Nischal 2015 ). Bacteriophages, the most prevalent viruses in nature, have been employed against bacterial infections since their discovery in the 1910s due to their unique antibacterial properties (Bruynoghe and Maisin 1921 ; Sharma et al. 2017 ). Their ability to infect and lyse Ralstonia spp. has led to growing interest in lytic phages as biocontrol tools for bacterial wilt (Wang et al. 2019 ), Numerous Ralstonia -infecting phages identified. In this investigation, we isolated a novel phage, vB_RsoP_BMB116, infecting R. solanacearum from tomato fields in Fujian, China. Transmission electron microscopy (TEM) revealed that vB_RsoP_BMB116 exhibited a Podoviridae morphology with an isometric head and a short, noncontractile tail. High-throughput sequencing revealed that vB_RsoP_BMB116 comprised a double-stranded (dsDNA) with a full length of 83,020 bp, a mean G + C content of 63%, and 126 open reading frames (ORFs). Due to its low genomic similarity with existing phages in the GenBank database, vB_RsoP_BMB116 is classified as a novel Ralstonia phage genus. Therefore, it has potential for application in bacterial wilt suppression. Materials and Methods Bacterial Strains and Culture protocol All 19 RSCC strains used in this investigation, including strain RS3221-2, were provided by Professor Bo Liu (Academy of Agriculture Science, Fujian; Ningde City Academy of Agriculture Science, Fujian Province, China). (Table 1 ). Seventeen of these strains, excluding the previously characterized GMI1000 and FJAT-1458, were isolated from wilt-infected plants in Fujian Province, China, and verified utilizing the 759/760 species-specific primers (Fegan and Prior 2005 ; Kai et al. 2022). All isolates were confirmed as Ralstonia solanacearum (phylotype I) via a multiplex PCR protocol as previously outlined (Fegan and Prior 2005 ; Kai et al. 2022). Aerobic cultivation of Ralstonia solanacearum was conducted at 37°C in sucrose-peptone-agar (SPA) broth containing 20 g/L sucrose, 5 g/L peptone, 0.5 g/L K₂HPO₄, and 0.25 g/L anhydrous MgSO₄, as well as on casamino acid-peptone-glucose agar comprising 5 g/L glucose, 10 g/L peptone, 1 g/L casamino acid, and 15 g/L agar (pH 7.2 ). Table 1 Host range of phage vB_RsoP_BMB116. Ralstonia solanacearum species complex Strain Phylotypes, Races Origin or reference Strains lysed by phage vB_RsoP_BMB116 R . solanacearum Rs3206 Ⅰ, 1 Wilted tomato plant (Fuzhou, China) + R . solanacearum Rs3207 Ⅰ, 1 Wilted tomato plant (Fuzhou, China) + R . solanacearum Rs3208-1 Ⅰ, 1 Wilted tomato plant (Fuzhou, China) + R . solanacearum Rs3208-2 Ⅰ, 1 Wilted tomato plant (Fuzhou, China) + R . solanacearum Rs3211 Ⅰ, 1 Wilted tomato plant (Fuzhou, China) + R . solanacearum Rs3217 Ⅰ, 1 Wilted ginger plant (Fuzhou, China) + R . solanacearum Rs3220-1 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3220-2 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3221 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3221-1 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3221-2 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3222 Ⅰ, 1 Wilted tomato plant (Ningde, China) + R . solanacearum Rs3224 Ⅰ, 1 Wilted eggplant plant (Ningde, China) + R . solanacearum Rs3225 Ⅰ, 1 Wilted eggplant plant (Ningde, China) + R . solanacearum Rs3226 Ⅰ, 1 Wilted eggplant plant (Ningde, China) + R . solanacearum Rs3228 Ⅰ, 1 Wilted pepper plant (Ningde, China) + R . solanacearum Rs3239 Ⅰ, 1 Wilted peanut plant (Quanzhou, China) - R . solanacearum GMI1000 Ⅰ, 1 (Drigues et al., 1985 ) + R . solanacearum FJAT-1458 Ⅰ, 1 (Chen et al., 2017 ) + Phage Isolation and Purification The vB_RsoP_BMB116 (novel phage) was obtained from tomato field samples in Fujian, China, employing R. solanacearum Rs3221-2 as the indicator host via the double-layer agar assay.It was isolated utilizing a modified version of a previously established enrichment protocol (Van et al. 2009). Approximately 2 g of soil samples were added 30 mL of sterile SPA broth in a 250 mL Erlenmeyer flask and incubated at 37°C with agitation at 200 rpm overnight. After centrifugation (12,000 rpm, 10 min, 4°C), the supernatant was filtered (0.22 µm PES syringe filters) to eliminate bacterial cells, yielding a sterile soil extract (Kai et al. 2022). This filtrate was mixed with exponentially growing R. solanacearum strain Rs3221-2 with 15 min incubation. Phage detection was then verified utilizing spot assay based on the double-layer agar technique. An isolated plaque was collected with a sterile pipette and subjected to five successive rounds to generate a clonal phage preparation. The phage vB_RsoP_BMB116 (novel phage) nucleotide sequences which have been newly determined submitted to a database, and the accession numbers provided is PX020966. Host Range Assay To evaluate host specificity, the spot assay was conducted using 24 Ralstonia solanacearum strains. Tenfold serial dilutions of the phage stock (starting from 10⁸ PFU/mL) were applied (5 µL per spot) onto double-layer agar plates containing target bacteria. Following overnight incubation at 37°C, plaque formation was observed. Morphological Characterization by Transmission Electron Microscopy The vB_RsoP_BMB116 morphology was examined by TEM negative staining (Kai et al. 2022). The resulting phage pellet was rinsed twice in 0.1× phosphate-buffered saline (PBS) to eliminate residual medium and resuspended in 1 mL of buffer. The suspension was applied to a carbon-coated grid, followed by 2% phosphotungstic acid (pH 6.8) negative staining for 20 s, then imaged with an H-7650 TEM at 100 kV at Huazhong Agricultural University, Wuhan. The morphological characteristics of VB_RsoP_BMB116 were determined using ten phage particles. One-Step Growth Curve The phage VB_RsoP_BMB116 one-step growth determined using standard methodology (Hyman and Abedon 2009 ; Wang et al. 2022 ). it was mixed with mid-log-phase R. solanacearum Rs3221-2 at an multiplicity of infection (MOI) of 0.1 and allowed to adsorb for 30 min. Following adsorption, cells were centrifuged (13,000 g, 1 min) to remove free phage, retaining only phage-associated bacteria. The pellet was re-inoculated into 10 mL SPA broth and incubated at 28°C under agitation (200 rpm). Phage titers were measured from samples collected every 15 min utilizing the double-layer agar plaque assay. Burst size was calculated as the ratio of released phage particles to infected bacterial cell numbers during the latency period (Wang et al. 2022 ). Phage Genome Sequencing and Annotation vB_RsoP_BMB116 genomic DNA was purified utilizing a ZnCl₂-based method, as previously described (Santos 1991 ), and analyzed through paired-end sequencing with Illumina HiSeq 2500. Genome assembly of vB_RsoP_BMB116 was performed using ABySS alignment v2.0 with varying k-mer lengths (40–75) (Jackman et al. 2017 ). The vB_RsoP_BMB116 genome was annotated via RAST v2.0, ( https://rast.nmpdr.org/ ) (Aziz et al. 2008 ), and ORF functional predictions were manually refined using BLASTp against NCBI protein databases. The tRNAs were predicted utilizing tRNAScan-SE v2.0 (Lowe and Chan 2016 ). Phage Analysis Classification Status The vB_RsoP_BMB116 genome was aligned against public databases via NCBI's BLASTn tool to identify the vB_RsoP_BMB116 genome sequence similarities ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). To screen for phages with the highest VIRIDIC and PASC values, the online tools VIRIDIC ( http://rhea.icbm.uni-oldenburg.de/VIRIDIC/ ) (Moraru et al 2020 ) and pairwise sequence comparison (PASC; http://www.ncbi.nlm.nih.gov/sutils/pasc ) (Bao et al 2014 ) were utilized to assess nucleotide-based intergenomic similarity (VIRIDIC value) and PASC values between vB_RsoP_BMB116 and other phages, respectively. A genome-wide proteomic tree of phage vB_RsoP_BMB116 with 128 phages was constructed using the online software ViPTree 3.3 ( http://www.genome.jp/viptree ). The selection criteria of the 128 reference phages were as follows: Firstly, ViPTree 3.3 was applied to construct a total evolutionary tree containing vB_RsoP_BMB116 with all the viruses in the database, from which the 38 phages with the closest affinity to vB_RsoP_BMB116 were selected. These phages also exhibited the highest homology with vB_RsoP_BMB116 in the BLASTn and PASC search phages. Secondly, representative species were randomly selected from each of the four orders ( Crassvirales , Kirjokansivirales , Methanobavirales , and Thumleimavirales ) under the Araliaceae in the latest virus classification system released by the International Committee on Taxonomy of Viruses (ICTV). Thirdly, 1–2 representative species were selected from each of the 33 independent families under the Araliaceae that did not belong to any order. Average nucleotide identity (ANI) value and in silico DNA-DNA hybridization value between phage vB_RsoP_BMB116 and proximate phages were estimated utilizing EzGenome online tools ( http://www.ezbiocloud.net/ezgenome/ani ) (Figueras et al. 2014 ) and GGDC (Meier-Kolthoff et al. 2013 ), respectively. Core genes shared between proximate phages were predicted using online VirClust tool ( http://rhea.icbm.uni-oldnburg.de/VIRCLUST ) (Moraru et al. 2023). Taxonomic analysis was conducted utilizing the ICTV classification criteria for species, genera, subfamilies, and families. Plant Experiment Application efficacy of phage vB_RsoP_BMB116 (1.05 × 10⁸ PFU/mL) in controlling bacterial wilt in tomato caused by R. solanacearum . The experiment included two groups: (1) Negative control group inoculated only with R. solanacearum strain BMB Rs3221-2, and (2) BMB Rs3221-2-inoculated group treated with phage vB_RsoP_BMB116 for biocontrol. Uniform tomato seedlings at the three-leaf-one-heart stage were selected for pot experiments, with five plants per group and two replicates. Pathogen inoculation was performed via root irrigation using a bacterial suspension (OD600 = 0.8–1.2) at 40 mL per pot. The treatment group received phage suspension (1.0 × 10⁸ PFU/mL) through root irrigation (40 mL per pot) 7 d after transplantation. The plants were cultivated at 30°C under a 12-h light/12-h dark cycles, and disease incidence and progression were monitored. Statistical Analyses Statistical differences between the two groups were analyzed utilizing an independent-samples t-test, with significance levels denoted as * P < 0.05, ** P < 0.01, and *** P < 0.001. Results Phage vB_RsoP_BMB116: Isolation and Structural morphology The vB_RsoP_BMB116 (novel phage) was obtained from tomato field samples in Fujian, China, employing R. solanacearum Rs3221-2 as the indicator host via the double-layer agar assay. Moreover, strain Rs3221-2 classified as Ralstonia solanacearum , was recovered from tomato fields and shown to cause bacterial wilt. Our results revealed that vB_RsoP_BMB116 produced clear plaques with diameters of 0.5–1 mm on the plate containing Rs3221-2 (Fig. 1 b 1 c). Phage vB_RsoP_BMB116 was further amplified and purified according to aforementioned protocols for TEM imaging and genomic DNA isolation. (Dong et al 2018 ). TEM identified vB_RsoP_BMB116 as a Podoviridae -type phage, featuring an isometric head (51 ± 2 nm) and a short, noncontractile tail (18 ± 1 nm) (Fig. 1 a). Phage vB_RsoP_BMB116 Biophysical Stability Thermal and pH Stability vB_RsoP_BMB116 remained biologically active after 1 h of exposure to buffer conditions ranging from pH 6 to 9 (Fig. 2 a). The phage maintained high activity at 20–60°C (Fig. 2 b). However, phage efficacy decreased significantly after exposure to 70°C, and total inactivation was occured at 80°C for 20 min. One-Step Growth Curve and MOI of Phage vB_RsoP_BMB116 Maximum phage activity was achieved at an MOI of 0.1 (Fig. 2 d). The growth curve of vB_RsoP_BMB116 (Fig. 2 c), showed a latent period of ~ 20 min and a ~ 60 min burst phase before reaching a stable phase. The burst size was estimated at 120 ± 15 PFU/cell. Host Range of Phage vB_RsoP_BMB116 The biocontrol capacity of vB_RsoP_BMB116 was assessed by testing its lytic activity against 19 R. solanacearum strains , comprising 17 field isolates from wilted hosts (peanut, ginger, eggplant, tomato, and pepper) and two sequenced strains, GMI1000 and FJAT-1458 (Chen et al. 2017 ; Drigues et al. 1985 ). All tested strains were classified as Ralstonia solanacearum (Table 1 ). Our findings displayed that phage exhibited a broad lytic spectrum, infecting 18 of 19 Ralstonia solanacearum strains. Genomic Analysis of Phage vB_RsoP_BMB116 The phage vB_RsoP_BMB116 genome sequence was completed using high-throughput sequencing, followed by splicing and assembly. Comprehensive genomic features of phage vB_RsoP_BMB116 is provided in Table 2 . The vB_RsoP_BMB116 has an 83,020 bp genome with 63% G + C content, cyclizability, and one fixed end (R1 = 360 > 100). Furthermore, a whole genome-comparison of vB_RsoP_BMB116 was conducted utilizing tblastn in NCBI’s sequence database. The results revealed that there was no sequence with high similarity to vB_RsoP_BMB116 in the public database, and the phage exhibited low similarity to the reported Achromobacter phage JWF. The genome-wide coverage and identity were 2% and 73.12%, respectively, indicating that vB_RsoP_BMB116 could be a novel phage. To better understand the phage-encoded proteins, the online annotation software RAST ( http://rast.nmpdr.org/ ) was utilized to annotate vB_RsoP_BMB116 whole genome, while the Proksee online software ( https://proksee.ca/ ) was utilized to generate the phage genome map. The vB_RsoP_BMB116 genome contains 126 ORFs, 33.33% of which encode known functional proteins, while the remaining ORFs can only be annotated as hypothetical proteins of unknown function. No known virulence factors and antibiotic resistance genes were identified in vB_RsoP_BMB116 genome utilizing CARD and VFDB analyses. vB_RsoP_BMB116 encodes 126 ORFs, with 10 proteins related to phage morphology, 12 proteins related to DNA replication and metabolism, and two proteins related to phage packaging. The portal protein is encoded by ORF119, and the phage terminase large subunit family protein, whose cleavage module is the lysis system i-spanin subunit Rz, is encoded by ORF84. The phage holin family protein is encoded by ORF85. We discovered that the hypothetical proteins occupied nearly 67% of all ORFs and were not predicted by the conserved structural domain analysis, which also failed to predict the presence of conserved structural domains. Given that most phages exhibit very compact gene composition, the emergence of phage genomes containing many non-functional proteins remains to be explored further. Moreover, it was initially hypothesized that these hypothetical proteins might be novel proteins, which we have not been able to define appropriately at this time. Twelve predicted proteins were associated with phage structure or assembly (Fig. 3 ; Table 2 ), including the major head protein (ORF116), portal protein (ORF119), tape measure protein (ORF107 and ORF109), tail proteins (ORF95, ORF97, ORF98, ORF101, ORF102, ORF103, and ORF105), and phage terminase large subunit family protein (ORF120). Consistent with other temperate phages, the longest phage ORF encodes the tape measure protein responsible for tail length determination (Katsura, 1987 ). The site-specific integrase (ORF 82) was identified as a putative protein reflecting the temperate nature of vB_RsoP_BMB116. Phage integrase facilitates site-specific recombination between host genomes and phage, a key step in lysogeny initiation (Rajamanickam and Hayes 2018 ). Efficient lysis of the bacterial host generally requires endolysin and holin in dsDNA phages (Young 1992 ). A holin-like protein of phage was predicted to be encoded by ORF85. Table 2 The annotation of vB_RsoP_BMB116 genome ORFs with possible functions ORF no. Location (bp) Strand Protein size (aa) Possible function start stop vB_RsoP_BMB116_11 4078 4371 + 97 response regulator transcription factor vB_RsoP_BMB116_21 7745 8176 + 143 metal-dependent phosphohydrolase vB_RsoP_BMB116_26 9184 9711 + 175 antirestriction protein vB_RsoP_BMB116_38 13292 13537 + 81 superinfection immunity protein vB_RsoP_BMB116_52 18516 19220 + 234 cell wall hydrolase vB_RsoP_BMB116_53 19334 19585 + 83 KTSC domain-containing protein vB_RsoP_BMB116_57 21056 22513 + 485 DEAD/DEAH box helicase vB_RsoP_BMB116_60 23782 24564 + 260 DNA polymerase I, thermostable vB_RsoP_BMB116_62 24737 27427 + 896 DNA polymerase vB_RsoP_BMB116_63 27522 28052 + 176 metal-dependent phohydrolase vB_RsoP_BMB116_64 28037 29383 + 448 DNA ligase vB_RsoP_BMB116_65 29446 29877 + 143 Holliday junction resolvase RecU vB_RsoP_BMB116_66 29874 30440 + 188 3'-5' exonuclease vB_RsoP_BMB116_67 30485 31465 + 326 metallophosphoesterase vB_RsoP_BMB116_69 31784 33856 + 690 SMC family ATPase vB_RsoP_BMB116_70 34159 34644 + 161 nucleoside triphosphate pyrophosphohydrolase family protein vB_RsoP_BMB116_72 34922 36205 + 427 FAD-dependent thymidylate synthase vB_RsoP_BMB116_73 36207 36788 + 193 deoxynucleoside monophosphate kinase protein vB_RsoP_BMB116_74 36873 38696 + 607 ribonucleoside-diphosphate reductase subunit alpha vB_RsoP_BMB116_75 38735 39781 + 348 ribonucleotide-diphosphate reductase subunit beta vB_RsoP_BMB116_79 41237 40824 − 137 type II toxin-antitoxin system, death-on-curing family toxin vB_RsoP_BMB116_82 42202 43329 + 375 site-specific integrase vB_RsoP_BMB116_84 44478 43936 − 177 lysis system i-spanin subunit Rz vB_RsoP_BMB116_85 44864 44478 − 128 phage holin family protein vB_RsoP_BMB116_87 45765 45214 − 183 glycoside hydrolase family 19 protein vB_RsoP_BMB116_90 47079 46384 − 231 PhoH family protein vB_RsoP_BMB116_92 48651 47389 − 420 radical SAM domain-containing protein vB_RsoP_BMB116_95 51216 50299 − 305 tail fiber domain-containing protein vB_RsoP_BMB116_96 51641 51225 − 138 enoyl-CoA hydratase/carnithine racemase-like vB_RsoP_BMB116_97 52465 51641 − 274 tail fiber assembly protein vB_RsoP_BMB116_98 54921 52465 − 818 tail fiber protein vB_RsoP_BMB116_101 60263 56667 − 1198 phage tail protein vB_RsoP_BMB116_102 60844 60263 − 193 tail assembly protein vB_RsoP_BMB116_103 61208 60849 − 119 tail protein vB_RsoP_BMB116_104 61677 61198 − 159 NlpC/P60 family protein vB_RsoP_BMB116_105 62161 61664 − 165 minor tail protein vB_RsoP_BMB116_107 67808 62553 − 1751 phage tail tape measure protein vB_RsoP_BMB116_109 68467 68081 − 128 tail length, tape measure protein chaperone vB_RsoP_BMB116_116 73679 72489 − 396 major head protein vB_RsoP_BMB116_118 75187 74111 − 358 DNA-binding protein vB_RsoP_BMB116_119 76801 75317 − 494 portal protein vB_RsoP_BMB116_120 78542 76791 − 583 phage terminase large subunit family protein vB_RsoP_BMB116_122 79188 81647 + 819 bifunctional DNA primase/polymerase Furthermore, A viral proteomic tree was constructed utilizing ViPTree (Fig. 4 ), based on whole-genome similarity score (GS) (Nishimura et al. 2017 ). Pairwise SG values comparing SNW-1 with known prokaryotic dsDNA viruses were determined, and a genomic distance-matrix was utilized to build tree via the BIONJ. The findings revealed that vB_RsoP_BMB116 clustered with several viruses ( Achromobacter phage JWF and Achromobacter phage 2 − 1). The low SG values (0.2199–0.2269) between vB_RsoP_BMB116 and related phages suggest its classification as a distinct novel taxon. In the proteomic tree, the viruses with the smallest evolutionary distance from vB_RsoP_BMB116 were Achromobacter phage JWF and Achromobacter phage 2 − 1. The ANI and isDDH values shared by vB_RsoP_BMB116 with them are 64.26%, 12.5%, and 64.85%, 12.5%, both of which are less than the boundary values of a species (ANI value > 95% and isDDH value > 70%). The results indicate that vB_RsoP_BMB116 is a new species. Phage taxonomy is undergoing a significant transformation as numerous, genome-based families have been introduced. Accordingly, the taxonomic status of vB_RsoP_BMB116 is difficult to ascertain at this moment. Genome-related phage similarity was evaluated utilizing the VIRIDIC tool. The similarity level between vB_RsoP_BMB116 and the putative phages Achromobacter phage 2 − 1 and JWF was ~ 18%, significantly below 70% threshold for phages-genus combinitation (Turner et al. 2021). Therefore, vB_RsoP_BMB116 likely constitutes a unique genus. Additional phages exhibited minimal intergenomic similarity to both vB_RsoP_BMB116 and Achromobacter phages 2 − 1 and JWF (Fig. 5 ). Biocontrol Efficacy of vB_RsoP_BMB116 in Plant disease In the negative control group inoculated only with R. solanacearum BMB Rs3221-2, tomato seedlings rapidly developed wilting symptoms (Fig. 6 a). Contrarily, the prevention group that received both R. solanacearum BMB Rs3221-2 and bacteriophage vB_RsoP_BMB116 treatment demonstrated gradual recovery of tomato seedlings, with restored healthy growth and favorable development (Fig. 6 b). The bacteriophage vB_RsoP_BMB116 demonstrated a cure rate of 91.53% against bacterial wilt disease . Discussion The agricultural use of bacteriophages offers a viable strategy for managing bacterial diseases, particularly in light of rising antibiotic resistance. In this investigation, a novel Ralstonia phage, vB_RsoP_BMB116, belonging to a new genus, was isolated and characterized. However, before being applied to production, its genome was analyzed to understand its genetic behavior. A proteomic tree was derived from high genome-similarity with other phages. VIRIDIC analysis revealed that the Ralstonia phage vB-RsoP_BMB116 may belong to a new genus. The current species demarcation standard is based on ≥ 95% genome sequence identity, indicating < 5% nucleotide divergence between viruses of the same species. Phage vB_RsoP_BMB116 showed only 73.12% similarity (2% query coverage) with Achromobacter phage JWF and 79.23% (1% coverage) with phage 2 − 1. Consequently, vB_RsoP_BMB116 is a new species. ANI, the standard for prokaryotic species demarcation, (Konstantinidis and Tiedje, 2005 ; Goris et al. 2007 ; Jain et al. 2018 ), defines members of the same species as sharing > 95% identity. The ANI between vB_RsoP_BMB116 and similar genomes was determined utilizing ( https://www.ezbiocloud.net/tools/ani ) online tool (Yoon et al. 2017 ), and it was discovered that vB_RsoP_BMB116 exhibited the highest ANI value of 62.85% with Achromobacter phage JWF. suggesting that it represents a novel species. Moreover, VIRDIC revealed that phage vB_RsoP_BMB116 belongs to a new genus that is different from other phages (Fig. 4 ). In conclusion, genomic sequence analysis revealed that Ralstonia phage vB_RsoP_BMB116 exhibited no significant genomic similarity to existing tailed viruses or known Ralstonia phages. Future exploration of its biological properties may shed light on host-phage interactions within this important bacterial genus. Although Bacteriophages provide a viable agricultural bacterial disease control strategy, yet phage therapy translation from lab to clinic faces challenges and limitations. Given its complex interactions and potential side effects, further research is needed to ensure safe, effective use in managing plant and other bacterial illnesses. Declarations Ethics approval and consent to participate The study received approval from the Huazhong Agricultural University Institutional Review Board. Given that the research solely focused on bacterial strains and their phages without involving human materials or patient information, the Review Board of the Huazhong Agricultural University granted an exemption from formal review and waived the requirement for informed consent. All procedures were conducted in line with applicable guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Author details 1 Department of State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University. Funding This research did not receive any fund. Author Contribution Liu conceptualized and designed the study; Wen performed the experiments, including sample collection, data acquisition, and laboratory analyses; Li conducted the statistical analysis and interpreted the results; Liu and Wen drafted the initial manuscript; Sun and Peng critically revised the manuscript for intellectual content; All authors reviewed and approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Acknowledgements Not applicable. Availability of data and materials No new data were generated or analyzed in this study. The findings are based entirely on existing literature. References Fegan M, Prior P (2005) How complex is the Ralstonia solanacearum species complex. Bacterial wilt disease and the Ralstonia solanacearum species complex 1:449–461 Drigues P, Demery-Laforgue D, Trigalet A, Dupin P, Samain D, Asselineau J (1985) Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants. J Bacteriol 162:504–509 Chen D, Liu B, Zhu Y, Wang J, Chen Z, Che J, Zheng X, Chen X (2017) Complete genome sequence of Ralstonia solanacearum FJAT-1458, a potential biocontrol agent for Tomato Wilt. Genome Announc 5:1 Wang K, Chen D, Liu Q, Zhu P, Sun M, Peng D (2022) Isolation and Characterization of Novel Lytic Bacteriophage vB_RsoP_BMB50 infecting Ralstonia solanacearum . Curr Microbiol 79(9):245 Van Twest R, Kropinski AM (2009) Bacteriophage enrichment from water and soil. Methods Mol Biol 501:15–21 Hyman P, Abedon ST (2009) Practical methods for determining phage growth parameters. Methods Mol Biol 501:175–202 Wang M, Du J, Zhang D, Li X, Zhao J (2017) Modification of different pulps by homologous overexpression alkali-tolerant endoglucanase in Bacillus subtilis Y106. Sci Rep 7:3321 Santos MA (1991) An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Res 19:5442 Jackman SD, Vandervalk BP, Mohamadi H, Chu J, Yeo S, Hammond SA, Jahesh G, Khan H, Coombe L, Warren RL, Birol I (2017) ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter. Genome Res 27:768–777 Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75 Lowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res 44:W54–57 Sullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualizer. Bioinformatics 27:1009–1010 Moraru C, Varsani A, Kropinski AM (2020) VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12:1 Ma Y, Pacan JC, Wang Q, Xu Y, Huang X, Korenevsky A, Sabour PM (2008) Microencapsulation of bacteriophage felix O1 into chitosan-alginate microspheres for oral delivery. Appl Environ Microbiol 74:4799–4805. 10.1128/AEM.00246-08 Li J, Li Y, Ding Y, Huang C, Zhang Y, Wang J, Wang X (2021) Characterization of a novel Siphoviridae Salmonella bacteriophage T156 and its microencapsulation application in food matrix. Food Res Int. 2021;140:110004. 10.1016/j.foodres.2020.110004 Moraru C, Varsani A, Kropinski AM (2020) VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12(11):1268 Bao YM, Chetvernin V, Tatusova T (2014) Improvements to pairwise sequence comparison (PASC): a genome-based web tool for virus classification. Arch Virol 159(12):3293–3304 Figueras MJ, Beaz-Hidalgo R, Hossain MJ, Liles MR (2014) Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis. Genome Announcements 2(6):e00927–e00914 Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14(1):1–14 Moraru C (2023) VirClust-a tool for hierarchical clustering, core protein detection and annotation of ( prokaryotic ) viruses. Viruses 15(4):1007 Dong Z, Xing S, Liu J, Tang X, Ruan L, Sun M, Tong Y, Peng D (2018) Isolation and characterization of a novel phage Xoosp2 that infects Xanthomonas oryzae pv . oryzae . J Gen Virol 99:1453–1462 Drigues P, Demery-Lafforgue D, Trigalet A, Dupin P, Samain D, Asselineau J (1985) Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants. J Bacteriol 162:504–509 Chen D, Liu B, Zhu Y, Wang J, Chen Z, Che J, Zheng X, Chen X (2017) Complete genome sequence of Ralstonia solanacearum FJAT-1458, a potential biocontrol agent for Tomato Wilt. Genome Announc 5:1 Safni I, Cleenwerck I, De Vos P, Fegan M, Sly L, Kappler U (2014) Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp . syzygii subsp . nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp . indonesiensis subsp . nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp . nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp . nov. Int J Syst Evol Microbiol 64:3087–3103 Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629 Wang X, Wei Z, Yang K, Wang J, Jousset A, Xu Y, Shen Q, Friman VP (2019) Phage combination therapies for bacterial wilt disease in tomato. Nat Biotechnol 37:1513–1520 Fujiwara A, Fujisawa M, Hamasaki R, Kawasaki T, Fujie M, Yamada T (2011) Biocontrol of ralstonia solanacearum by treatment with lytic bacteriophages. Appl Environ Microb 77:4155–4162 Da Silva Xavier, da Silva FP, Vidigal PMP, Lima TTM, de Souza FO, Alfenas-Zerbini P (2018) Genomic and biological characterization of a new member of the genus Phikmvvirus infecting phytopathogenic Ralstonia bacteria. Arch Virol 163:3275–3290 Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614–629 Sae-Ueng U, Bhunchoth A, Phironrit N, Treetong A, Sapcharoenkun C, Chatchawankanphanich O, Leartsakulpanich U, Chitnumsub P (2020) C22 podovirus infectivity is associated with intermediate stiffness. Sci Rep 10:12604 Bruynoghe R, Maisin J (1921) Essais de thérapeutique au moyen du bacteriophage. CR Soc Biol 85:1120–1121 Sharma S, Chatterjee S, Datta S, Prasad R, Dubey D, Prasad RK, Vairale MG (2017) Bacteriophages and its applications: an overview. Folia Microbiol 62:17–55 Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by pseudomonas solanacearum . Annu Rev Phytopathol 29:65–87 Jiang G, Wei Z, Xu J, Chen H, Zhang Y, She X, Macho AP, Ding W, Liao B (2017) Bacterial Wilt in China: History, Current Status, and Future Perspectives. Front Plant Sci 8:1549 Nischal PM (2015) First global report on antimicrobial resistance released by the WHO. Natl Med J India 27:241 Hayward AC (1991) Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum . Annu Rev Phytopathol 29:65–87 Vasse J, Frey P, Trigalet A (1995) Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by Pseudomonas solanacearum . Mol Plant Microbe Interact 8:241–251 Álvarez B, Vasse J, Le-Courtois V, Trigalet-Démery D, López MM, Trigalet A (2008) Comparative behavior of Ralstonia solanacearum biovar 2 in diverse plant species. Phytopathology 98:59–68 Prior P, Ailloud F, Dalsing BL, Remenant B, Sanchez B, Allen C (2016) Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species. BMC Genomics 17:90 Turner D, Kropinski AM (2021) Adriaenssens, E.M. A Roadmap for genome-based phage taxonomy. Viruses 13:506 Adriaenssens EM, Brister JR (2017) How to name and classify your phage: an informal guide. Viruses 9:70. 10.3390/v9040070 Konstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. PNAS 102:2567–2572. 10.1073/pnas.0409727102 Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. 10.1099/ijs.0.64483-0 Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. 10.1038/s41467-018-07641-9 Yoon SH, Ha SM, Lim JM, Kwon SJ, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281–1286. 10.1007/s10482-017-0844-4 Rajamanickam K, Hayes S (2018) The bacteriophage lambda CII phenotypes for complementation, cellular toxicity and replication inhibition are suppressed in cII-oop constructs expressing the small RNA oop. Viruses 10:115 Young R (1992) Bacteriophage lysis: Mechanism and regulation. Microbiol Rev 56:430–481 Katsura I (1987) Determination of bacteriophage λ tail length by a protein ruler. Nature 327:73–75 Nishimura Y, Yoshida T, Kuronishi M, Uehara H, Ogata H, Goto S (2017) ViPTree: The viral proteomic tree server. Bioinformatics 33:2379–2380 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7097101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":499581894,"identity":"23855313-d24c-4c89-8b98-a730813bee3d","order_by":0,"name":"Shoude Liu","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shoude","middleName":"","lastName":"Liu","suffix":""},{"id":499581896,"identity":"e25bfc7b-b391-4b04-bc21-fae139275a3f","order_by":1,"name":"Rong Wen","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Wen","suffix":""},{"id":499581898,"identity":"c23626e2-9ac3-44d1-af1c-7cb6b389fc9b","order_by":2,"name":"Qi feng Li","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"feng","lastName":"Li","suffix":""},{"id":499581901,"identity":"8e4c2486-c96e-46ed-ae96-d590fee81f96","order_by":3,"name":"Ming Sun","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Sun","suffix":""},{"id":499581902,"identity":"693974cd-2385-4824-aa47-8ad9e9d8318e","order_by":4,"name":"Dong hai Peng","email":"data:image/png;base64,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","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Dong","middleName":"hai","lastName":"Peng","suffix":""}],"badges":[],"createdAt":"2025-07-11 02:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7097101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7097101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88969622,"identity":"d59e941b-30bf-41d6-8bf3-ba603252be17","added_by":"auto","created_at":"2025-08-13 09:29:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4355898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhage morphology and biological characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) TEM image of vB_RsoP_BMB116, depicting phage capsids with an isomethic head (approximately 51 nm) and a short noncontractile tail (approximately 18 nm); the bar is 100 nm. (b-c) Phage plaques of vB_RsoP_BMB116 infecting Rs3221-2 in double-layer agar plates.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/046e4ce46bd49bf12f96bd1a.png"},{"id":88968362,"identity":"6637ba6b-626e-4281-99a0-1e3afae052a5","added_by":"auto","created_at":"2025-08-13 09:21:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiophysical Stability of Phage vB_RsoP_BMB116\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The pH stability of vB_RsoP_BMB116. (b) Temperature stability of vB_RsoP_BMB116. (c) The One-step growth curve of Phage vB_RsoP_BMB116. (d) Optimal MOI for phage infection.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/1f96b79f91514578260a9225.png"},{"id":88968365,"identity":"16f6010a-b794-47b2-99f6-3dc3d30de523","added_by":"auto","created_at":"2025-08-13 09:21:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2215343,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome map of vB_RsoP_BMB116. \u003c/strong\u003eAll\u003cstrong\u003e \u003c/strong\u003epredicted ORFs were classified into five categories according to gene function annotation: green, phage structural proteins; blue, DNA-packaging-related proteins; purple, DNA replication- and regulation-related proteins; red, lysis module; and orange, other proteins and hypothetical proteins.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/eaf0809083804dd23149d7b8.png"},{"id":88969623,"identity":"0b647a23-7d36-48e5-b0c1-9244c3c69a88","added_by":"auto","created_at":"2025-08-13 09:29:12","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":276494,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViPTree-generated proteomic tree illustrating vB_RsoP_BMB116 and its related phages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRight and left branches indicate host group and family-level classification, respectively. The phage vB_RsoP_BMB116 is represented by a red star.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/135b6946a08bf7c3e80feab4.jpeg"},{"id":88968368,"identity":"170aadca-265d-4b40-a0e0-00e9feb07796","added_by":"auto","created_at":"2025-08-13 09:21:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5527487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVIRIDIC heatmap indicating intergenomic similarity between vB_RsoP_BMB116 and related phages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe horizontally aligned genome fraction ranges from 0 to 1 and represents the degree of alignment between the genomes of different bacteriophages. The higher the value, the greater aligned genome proportion. The vertical genome length ratio ranges from 0 to 1 and represents the proportional relationship between the lengths of bacteriophage genomes.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/418bfce9fd9da401c002bf2d.png"},{"id":88968370,"identity":"30225b07-39ec-41cd-bd2c-28f72f1c96c7","added_by":"auto","created_at":"2025-08-13 09:21:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8517184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial Effects of Phages on Tomato Bacterial Wilt Management\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Tomato seedlings infected with BMB_Rs3221-2. (b) Tomato plants inoculated with \u003cem\u003eR.\u003c/em\u003e \u003cem\u003esolanacearum\u003c/em\u003e pathogen BMB Rs3221-2 and treated with phage vB_RsoP_BMB116 for biocontrol.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/7c6569b16353eee2f44ed69c.png"},{"id":92359819,"identity":"536c54b9-3066-42f3-9b00-4b04f091efe4","added_by":"auto","created_at":"2025-09-28 16:16:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22259506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7097101/v1/c98034ca-7155-4327-9b9a-a23ed1760af0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation and Characterization of Bacteriophage vB_RsoP_BMB116 Infecting Ralstonia solanacearum","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003e\u003cem\u003eThe new Ralstonia solanacearum phage vB_RsoP_BMB116 ( novel genus) is a key resource for microbiology and biotechnology, aiding new phage therapies and biocontrol agents.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eObserved via transmission electron microscopy, the novel phage vB_RsoP_BMB116 belongs to the Podoviridae family.\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003e\u003c/em\u003e\u003cem\u003eIn vitro assays demonstrated that it exhibits significant application potential for the prevention and control of plant bacterial diseases.\u003c/em\u003e\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eRalstonia solanacearum\u003c/em\u003e, a Gram-negative pathogen, causes bacterial wilt (Hayward \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). It is classified within \u003cem\u003eRalstonia solanacearum\u003c/em\u003e species complex (RSSC), which comprises three distinct species: \u003cem\u003eR. solanacearum, R pseudosolanacearum\u003c/em\u003e, and \u003cem\u003eR. syzygii\u003c/em\u003e (Prior et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). All three species are recognized as major bacterial wilt pathogens that contribute to substantial crop losses and are classified as significant phytopathogens (Safni et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mansfield et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Bacterial wilt, triggered by the soilborne pathogen \u003cem\u003eR. solanacearum\u003c/em\u003e, severely impacts Solanaceae crops like ginger potato, tomato, eggplant, pepper and flue-cured tobacco (Fujiwara et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; da Silva Xavier et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The bacterium spreads via soil and water, infects roots, and invades the vascular system, causing potato brown rot and wilting (Hayward \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1991\u003c/span\u003e, Vasse et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Alvarez et al. 2010). Bacterial wilt results in estimated global agricultural losses exceeding \u003cspan\u003e$\u003c/span\u003e950\u0026nbsp;million annually, posing a substantial economic burden on Solanaceae cultivation across subtropical, tropical, and temperate regions (Mansfield et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sae-Ueng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Accordingly, \u003cem\u003eR. solanacearum\u003c/em\u003e is regarded the second most critical bacterial phytopathogen, following \u003cem\u003ePseudomonas syringae\u003c/em\u003e (Mansfield et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The development of effective therapeutics for bacterial wilt is imperative, given the limitations of conventional agrochemicals, including resistance emergence, human health, and environmental damage (Jiang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nischal \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBacteriophages, the most prevalent viruses in nature, have been employed against bacterial infections since their discovery in the 1910s due to their unique antibacterial properties (Bruynoghe and Maisin \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1921\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Their ability to infect and lyse \u003cem\u003eRalstonia\u003c/em\u003e spp. has led to growing interest in lytic phages as biocontrol tools for bacterial wilt (Wang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), Numerous \u003cem\u003eRalstonia\u003c/em\u003e-infecting phages identified.\u003c/p\u003e\u003cp\u003eIn this investigation, we isolated a novel phage, vB_RsoP_BMB116, infecting \u003cem\u003eR. solanacearum\u003c/em\u003e from tomato fields in Fujian, China. Transmission electron microscopy (TEM) revealed that vB_RsoP_BMB116 exhibited a \u003cem\u003ePodoviridae\u003c/em\u003e morphology with an isometric head and a short, noncontractile tail. High-throughput sequencing revealed that vB_RsoP_BMB116 comprised a double-stranded (dsDNA) with a full length of 83,020 bp, a mean G\u0026thinsp;+\u0026thinsp;C content of 63%, and 126 open reading frames (ORFs). Due to its low genomic similarity with existing phages in the GenBank database, vB_RsoP_BMB116 is classified as a novel \u003cem\u003eRalstonia\u003c/em\u003e phage genus. Therefore, it has potential for application in bacterial wilt suppression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eBacterial Strains and Culture protocol\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll 19 RSCC strains used in this investigation, including strain RS3221-2, were provided by Professor Bo Liu (Academy of Agriculture Science, Fujian; Ningde City Academy of Agriculture Science, Fujian Province, China). (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Seventeen of these strains, excluding the previously characterized GMI1000 and FJAT-1458, were isolated from wilt-infected plants in Fujian Province, China, and verified utilizing the 759/760 species-specific primers (Fegan and Prior \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kai et al. 2022). All isolates were confirmed as \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (phylotype I) via a multiplex PCR protocol as previously outlined (Fegan and Prior \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kai et al. 2022). Aerobic cultivation of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e was conducted at 37\u0026deg;C in sucrose-peptone-agar (SPA) broth containing 20 g/L sucrose, 5 g/L peptone, 0.5 g/L K₂HPO₄, and 0.25 g/L anhydrous MgSO₄, as well as on casamino acid-peptone-glucose agar comprising 5 g/L glucose, 10 g/L peptone, 1 g/L casamino acid, and 15 g/L agar (pH 7.2\u003cem\u003e).\u003c/em\u003e\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\u003eHost range of phage vB_RsoP_BMB116.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eRalstonia solanacearum\u003c/em\u003e species complex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhylotypes, Races\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOrigin or reference\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eStrains lysed by phage vB_RsoP_BMB116\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3206\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3207\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3208-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3208-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted ginger plant (Fuzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3220-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3220-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3221\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3221-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3221-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3222\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted tomato plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3224\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted eggplant plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted eggplant plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted eggplant plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3228\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted pepper plant (Ningde, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRs3239\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWilted peanut plant (Quanzhou, China)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGMI1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Drigues et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1985\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFJAT-1458\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eⅠ, 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003ePhage Isolation and Purification\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe vB_RsoP_BMB116 (novel phage) was obtained from tomato field samples in Fujian, China, employing \u003cem\u003eR. solanacearum\u003c/em\u003e Rs3221-2 as the indicator host via the double-layer agar assay.It was isolated utilizing a modified version of a previously established enrichment protocol (Van et al. 2009). Approximately 2 g of soil samples were added 30 mL of sterile SPA broth in a 250 mL Erlenmeyer flask and incubated at 37\u0026deg;C with agitation at 200 rpm overnight. After centrifugation (12,000 rpm, 10 min, 4\u0026deg;C), the supernatant was filtered (0.22 \u0026micro;m PES syringe filters) to eliminate bacterial cells, yielding a sterile soil extract (Kai et al. 2022). This filtrate was mixed with exponentially growing \u003cem\u003eR. solanacearum\u003c/em\u003e strain Rs3221-2 with 15 min incubation. Phage detection was then verified utilizing spot assay based on the double-layer agar technique. An isolated plaque was collected with a sterile pipette and subjected to five successive rounds to generate a clonal phage preparation.\u003c/p\u003e\u003cp\u003eThe phage vB_RsoP_BMB116 (novel phage) nucleotide sequences which have been newly determined submitted to a database, and the accession numbers provided is PX020966.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHost Range Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate host specificity, the spot assay was conducted using 24 \u003cem\u003eRalstonia solanacearum\u003c/em\u003e strains. Tenfold serial dilutions of the phage stock (starting from 10⁸ PFU/mL) were applied (5 \u0026micro;L per spot) onto double-layer agar plates containing target bacteria. Following overnight incubation at 37\u0026deg;C, plaque formation was observed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological Characterization by Transmission Electron Microscopy\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe vB_RsoP_BMB116 morphology was examined by TEM negative staining (Kai et al. 2022). The resulting phage pellet was rinsed twice in 0.1\u0026times; phosphate-buffered saline (PBS) to eliminate residual medium and resuspended in 1 mL of buffer. The suspension was applied to a carbon-coated grid, followed by 2% phosphotungstic acid (pH 6.8) negative staining for 20 s, then imaged with an H-7650 TEM at 100 kV at Huazhong Agricultural University, Wuhan. The morphological characteristics of VB_RsoP_BMB116 were determined using ten phage particles.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOne-Step Growth Curve\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe phage VB_RsoP_BMB116 one-step growth determined using standard methodology (Hyman and Abedon \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). it was mixed with mid-log-phase \u003cem\u003eR. solanacearum\u003c/em\u003e Rs3221-2 at an multiplicity of infection (MOI) of 0.1 and allowed to adsorb for 30 min. Following adsorption, cells were centrifuged (13,000 g, 1 min) to remove free phage, retaining only phage-associated bacteria. The pellet was re-inoculated into 10 mL SPA broth and incubated at 28\u0026deg;C under agitation (200 rpm). Phage titers were measured from samples collected every 15 min utilizing the double-layer agar plaque assay. Burst size was calculated as the ratio of released phage particles to infected bacterial cell numbers during the latency period (Wang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhage Genome Sequencing and Annotation\u003c/b\u003e\u003c/p\u003e\u003cp\u003evB_RsoP_BMB116 genomic DNA was purified utilizing a ZnCl₂-based method, as previously described (Santos \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), and analyzed through paired-end sequencing with Illumina HiSeq 2500. Genome assembly of vB_RsoP_BMB116 was performed using ABySS alignment v2.0 with varying k-mer lengths (40\u0026ndash;75) (Jackman et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The vB_RsoP_BMB116 genome was annotated via RAST v2.0, (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rast.nmpdr.org/\u003c/span\u003e\u003cspan address=\"https://rast.nmpdr.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Aziz et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and ORF functional predictions were manually refined using BLASTp against NCBI protein databases. The tRNAs were predicted utilizing tRNAScan-SE v2.0 (Lowe and Chan \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhage Analysis Classification Status\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe vB_RsoP_BMB116 genome was aligned against public databases via NCBI's BLASTn tool to identify the vB_RsoP_BMB116 genome sequence similarities (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To screen for phages with the highest VIRIDIC and PASC values, the online tools VIRIDIC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rhea.icbm.uni-oldenburg.de/VIRIDIC/\u003c/span\u003e\u003cspan address=\"http://rhea.icbm.uni-oldenburg.de/VIRIDIC/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Moraru et al \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and pairwise sequence comparison (PASC; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/sutils/pasc\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/sutils/pasc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Bao et al \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) were utilized to assess nucleotide-based intergenomic similarity (VIRIDIC value) and PASC values between vB_RsoP_BMB116 and other phages, respectively.\u003c/p\u003e\u003cp\u003eA genome-wide proteomic tree of phage vB_RsoP_BMB116 with 128 phages was constructed using the online software ViPTree 3.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/viptree\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/viptree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The selection criteria of the 128 reference phages were as follows: Firstly, ViPTree 3.3 was applied to construct a total evolutionary tree containing vB_RsoP_BMB116 with all the viruses in the database, from which the 38 phages with the closest affinity to vB_RsoP_BMB116 were selected. These phages also exhibited the highest homology with vB_RsoP_BMB116 in the BLASTn and PASC search phages. Secondly, representative species were randomly selected from each of the four orders (\u003cem\u003eCrassvirales\u003c/em\u003e, \u003cem\u003eKirjokansivirales\u003c/em\u003e, \u003cem\u003eMethanobavirales\u003c/em\u003e, and \u003cem\u003eThumleimavirales\u003c/em\u003e) under the Araliaceae in the latest virus classification system released by the International Committee on Taxonomy of Viruses (ICTV). Thirdly, 1\u0026ndash;2 representative species were selected from each of the 33 independent families under the Araliaceae that did not belong to any order.\u003c/p\u003e\u003cp\u003eAverage nucleotide identity (ANI) value and in silico DNA-DNA hybridization value between phage vB_RsoP_BMB116 and proximate phages were estimated utilizing EzGenome online tools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ezbiocloud.net/ezgenome/ani\u003c/span\u003e\u003cspan address=\"http://www.ezbiocloud.net/ezgenome/ani\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Figueras et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and GGDC (Meier-Kolthoff et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), respectively. Core genes shared between proximate phages were predicted using online VirClust tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rhea.icbm.uni-oldnburg.de/VIRCLUST\u003c/span\u003e\u003cspan address=\"http://rhea.icbm.uni-oldnburg.de/VIRCLUST\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Moraru et al. 2023).\u003c/p\u003e\u003cp\u003eTaxonomic analysis was conducted utilizing the ICTV classification criteria for species, genera, subfamilies, and families.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlant Experiment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eApplication efficacy of phage vB_RsoP_BMB116 (1.05 \u0026times; 10⁸ PFU/mL) in controlling bacterial wilt in tomato caused by \u003cem\u003eR. solanacearum\u003c/em\u003e. The experiment included two groups: (1) Negative control group inoculated only with \u003cem\u003eR. solanacearum\u003c/em\u003e strain BMB Rs3221-2, and (2) BMB Rs3221-2-inoculated group treated with phage vB_RsoP_BMB116 for biocontrol. Uniform tomato seedlings at the three-leaf-one-heart stage were selected for pot experiments, with five plants per group and two replicates. Pathogen inoculation was performed via root irrigation using a bacterial suspension (OD600\u0026thinsp;=\u0026thinsp;0.8\u0026ndash;1.2) at 40 mL per pot. The treatment group received phage suspension (1.0 \u0026times; 10⁸ PFU/mL) through root irrigation (40 mL per pot) 7 d after transplantation. The plants were cultivated at 30\u0026deg;C under a 12-h light/12-h dark cycles, and disease incidence and progression were monitored.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStatistical differences between the two groups were analyzed utilizing an independent-samples t-test, with significance levels denoted as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePhage vB_RsoP_BMB116: Isolation and Structural morphology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vB_RsoP_BMB116 (novel phage) was obtained from tomato field samples in Fujian, China, employing \u003cem\u003eR. solanacearum\u003c/em\u003e Rs3221-2 as the indicator host via the double-layer agar assay. Moreover, strain Rs3221-2 classified as \u003cem\u003eRalstonia solanacearum\u003c/em\u003e, was recovered from tomato fields and shown to cause bacterial wilt. Our results revealed that vB_RsoP_BMB116 produced clear plaques with diameters of 0.5\u0026ndash;1 mm on the plate containing Rs3221-2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec). Phage vB_RsoP_BMB116 was further amplified and purified according to aforementioned protocols for TEM imaging and genomic DNA isolation. (Dong et al \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). TEM identified vB_RsoP_BMB116 as a \u003cem\u003ePodoviridae\u003c/em\u003e-type phage, featuring an isometric head (51\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm) and a short, noncontractile tail (18\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage vB_RsoP_BMB116 Biophysical Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThermal and pH Stability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003evB_RsoP_BMB116 remained biologically active after 1 h of exposure to buffer conditions ranging from pH 6 to 9 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). The phage maintained high activity at 20\u0026ndash;60\u0026deg;C (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). However, phage efficacy decreased significantly after exposure to 70\u0026deg;C, and total inactivation was occured at 80\u0026deg;C for 20 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOne-Step Growth Curve and MOI of Phage vB_RsoP_BMB116\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaximum phage activity was achieved at an MOI of 0.1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The growth curve of vB_RsoP_BMB116 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), showed a latent period of ~\u0026thinsp;20 min and a\u0026thinsp;~\u0026thinsp;60 min burst phase before reaching a stable phase. The burst size was estimated at 120\u0026thinsp;\u0026plusmn;\u0026thinsp;15 PFU/cell.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHost Range of Phage vB_RsoP_BMB116\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biocontrol capacity of vB_RsoP_BMB116 was assessed by testing its lytic activity against 19 \u003cem\u003eR. solanacearum strains\u003c/em\u003e, comprising 17 field isolates from wilted hosts (peanut, ginger, eggplant, tomato, and pepper) and two sequenced strains, GMI1000 and FJAT-1458 (Chen et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e; Drigues et al. \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e). All tested strains were classified as \u003cem\u003eRalstonia solanacearum\u003c/em\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Our findings displayed that phage exhibited a broad lytic spectrum, infecting 18 of 19 \u003cem\u003eRalstonia solanacearum\u003c/em\u003e strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic Analysis of Phage vB_RsoP_BMB116\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phage vB_RsoP_BMB116 genome sequence was completed using high-throughput sequencing, followed by splicing and assembly. Comprehensive genomic features of phage vB_RsoP_BMB116 is provided in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The vB_RsoP_BMB116 has an 83,020 bp genome with 63% G\u0026thinsp;+\u0026thinsp;C content, cyclizability, and one fixed end (R1\u0026thinsp;=\u0026thinsp;360\u0026thinsp;\u0026gt;\u0026thinsp;100).\u003c/p\u003e\n\u003cp\u003eFurthermore, a whole genome-comparison of vB_RsoP_BMB116 was conducted utilizing tblastn in NCBI\u0026rsquo;s sequence database. The results revealed that there was no sequence with high similarity to vB_RsoP_BMB116 in the public database, and the phage exhibited low similarity to the reported \u003cem\u003eAchromobacter\u003c/em\u003e phage JWF. The genome-wide coverage and identity were 2% and 73.12%, respectively, indicating that vB_RsoP_BMB116 could be a novel phage.\u003c/p\u003e\n\u003cp\u003eTo better understand the phage-encoded proteins, the online annotation software RAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rast.nmpdr.org/\u003c/span\u003e\u003c/span\u003e) was utilized to annotate vB_RsoP_BMB116 whole genome, while the Proksee online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca/\u003c/span\u003e\u003c/span\u003e) was utilized to generate the phage genome map. The vB_RsoP_BMB116 genome contains 126 ORFs, 33.33% of which encode known functional proteins, while the remaining ORFs can only be annotated as hypothetical proteins of unknown function. No known virulence factors and antibiotic resistance genes were identified in vB_RsoP_BMB116 genome utilizing CARD and VFDB analyses.\u003c/p\u003e\n\u003cp\u003evB_RsoP_BMB116 encodes 126 ORFs, with 10 proteins related to phage morphology, 12 proteins related to DNA replication and metabolism, and two proteins related to phage packaging. The portal protein is encoded by ORF119, and the phage terminase large subunit family protein, whose cleavage module is the lysis system i-spanin subunit Rz, is encoded by ORF84. The phage holin family protein is encoded by ORF85. We discovered that the hypothetical proteins occupied nearly 67% of all ORFs and were not predicted by the conserved structural domain analysis, which also failed to predict the presence of conserved structural domains. Given that most phages exhibit very compact gene composition, the emergence of phage genomes containing many non-functional proteins remains to be explored further. Moreover, it was initially hypothesized that these hypothetical proteins might be novel proteins, which we have not been able to define appropriately at this time.\u003c/p\u003e\n\u003cp\u003eTwelve predicted proteins were associated with phage structure or assembly (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), including the major head protein (ORF116), portal protein (ORF119), tape measure protein (ORF107 and ORF109), tail proteins (ORF95, ORF97, ORF98, ORF101, ORF102, ORF103, and ORF105), and phage terminase large subunit family protein (ORF120). Consistent with other temperate phages, the longest phage ORF encodes the tape measure protein responsible for tail length determination (Katsura, \u003cspan class=\"CitationRef\"\u003e1987\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe site-specific integrase (ORF 82) was identified as a putative protein reflecting the temperate nature of vB_RsoP_BMB116. Phage integrase facilitates site-specific recombination between host genomes and phage, a key step in lysogeny initiation (Rajamanickam and Hayes \u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e). Efficient lysis of the bacterial host generally requires endolysin and holin in dsDNA phages (Young \u003cspan class=\"CitationRef\"\u003e1992\u003c/span\u003e). A holin-like protein of phage was predicted to be encoded by ORF85.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eThe annotation of vB_RsoP_BMB116 genome ORFs with possible functions\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eORF no.\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eLocation (bp)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eStrand\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eProtein size (aa)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePossible function\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003estart\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003estop\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4078\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4371\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eresponse regulator transcription factor\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7745\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8176\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e143\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emetal-dependent phosphohydrolase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_26\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9184\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9711\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e175\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eantirestriction protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13292\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13537\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e81\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esuperinfection immunity protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18516\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19220\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e234\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ecell wall hydrolase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_53\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19334\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19585\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e83\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eKTSC domain-containing protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_57\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e21056\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e22513\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e485\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDEAD/DEAH box helicase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_60\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e23782\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24564\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e260\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA polymerase I, thermostable\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e24737\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27427\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e896\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA polymerase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_63\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27522\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28052\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e176\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emetal-dependent phohydrolase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e28037\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29383\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e448\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA ligase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29446\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29877\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e143\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHolliday junction resolvase RecU\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_66\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e29874\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30440\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3'-5' exonuclease\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e30485\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e31465\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e326\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emetallophosphoesterase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e31784\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33856\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e690\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSMC family ATPase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_70\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34159\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34644\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enucleoside triphosphate pyrophosphohydrolase family protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_72\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e34922\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36205\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e427\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFAD-dependent thymidylate synthase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_73\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36207\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36788\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e193\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003edeoxynucleoside monophosphate kinase protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_74\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36873\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e38696\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e607\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eribonucleoside-diphosphate reductase subunit alpha\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_75\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e38735\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39781\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e348\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eribonucleotide-diphosphate reductase subunit beta\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_79\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e41237\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40824\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e137\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etype II toxin-antitoxin system, death-on-curing family toxin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_82\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e42202\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43329\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e375\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003esite-specific integrase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_84\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e44478\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e43936\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e177\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003elysis system i-spanin subunit Rz\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_85\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e44864\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e44478\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e128\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ephage holin family protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_87\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45765\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e45214\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e183\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eglycoside hydrolase family 19 protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_90\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e47079\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46384\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e231\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePhoH family protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_92\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48651\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e47389\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e420\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eradical SAM domain-containing protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_95\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51216\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e50299\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e305\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail fiber domain-containing protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_96\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51641\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51225\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e138\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eenoyl-CoA hydratase/carnithine racemase-like\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_97\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52465\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e51641\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e274\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail fiber assembly protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_98\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e54921\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e52465\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e818\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail fiber protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_101\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60263\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e56667\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1198\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ephage tail protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_102\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60844\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60263\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e193\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail assembly protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_103\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61208\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e60849\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e119\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_104\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61677\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61198\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e159\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNlpC/P60 family protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_105\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e62161\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61664\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e165\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eminor tail protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_107\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67808\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e62553\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1751\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ephage tail tape measure protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_109\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e68467\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e68081\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e128\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003etail length, tape measure protein chaperone\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_116\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e73679\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e72489\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e396\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emajor head protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_118\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75187\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e74111\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e358\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDNA-binding protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_119\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76801\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75317\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e494\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eportal protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_120\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e78542\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76791\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026minus;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e583\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ephage terminase large subunit family protein\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003evB_RsoP_BMB116_122\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e79188\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e81647\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e+\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e819\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ebifunctional DNA primase/polymerase\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, A viral proteomic tree was constructed utilizing ViPTree (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), based on whole-genome similarity score (GS) (Nishimura et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e). Pairwise SG values comparing SNW-1 with known prokaryotic dsDNA viruses were determined, and a genomic distance-matrix was utilized to build tree via the BIONJ. The findings revealed that vB_RsoP_BMB116 clustered with several viruses (\u003cem\u003eAchromobacter\u003c/em\u003e phage JWF and \u003cem\u003eAchromobacter\u003c/em\u003e phage 2\u0026thinsp;\u0026minus;\u0026thinsp;1). The low SG values (0.2199\u0026ndash;0.2269) between vB_RsoP_BMB116 and related phages suggest its classification as a distinct novel taxon. In the proteomic tree, the viruses with the smallest evolutionary distance from vB_RsoP_BMB116 were \u003cem\u003eAchromobacter\u003c/em\u003e phage JWF and \u003cem\u003eAchromobacter\u003c/em\u003e phage 2\u0026thinsp;\u0026minus;\u0026thinsp;1. The ANI and isDDH values shared by vB_RsoP_BMB116 with them are 64.26%, 12.5%, and 64.85%, 12.5%, both of which are less than the boundary values of a species (ANI value\u0026thinsp;\u0026gt;\u0026thinsp;95% and isDDH value\u0026thinsp;\u0026gt;\u0026thinsp;70%). The results indicate that vB_RsoP_BMB116 is a new species. Phage taxonomy is undergoing a significant transformation as numerous, genome-based families have been introduced. Accordingly, the taxonomic status of vB_RsoP_BMB116 is difficult to ascertain at this moment.\u003c/p\u003e\n\u003cp\u003eGenome-related phage similarity was evaluated utilizing the VIRIDIC tool. The similarity level between vB_RsoP_BMB116 and the putative phages \u003cem\u003eAchromobacter\u003c/em\u003e phage 2\u0026thinsp;\u0026minus;\u0026thinsp;1 and JWF was ~\u0026thinsp;18%, significantly below 70% threshold for phages-genus combinitation (Turner et al. 2021). Therefore, vB_RsoP_BMB116 likely constitutes a unique genus. Additional phages exhibited minimal intergenomic similarity to both vB_RsoP_BMB116 and \u003cem\u003eAchromobacter\u003c/em\u003e phages 2\u0026thinsp;\u0026minus;\u0026thinsp;1 and \u003cem\u003eJWF\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiocontrol Efficacy of vB_RsoP_BMB116 in Plant disease\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the negative control group inoculated only with \u003cem\u003eR. solanacearum\u003c/em\u003e BMB Rs3221-2, tomato seedlings rapidly developed wilting symptoms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). Contrarily, the prevention group that received both \u003cem\u003eR. solanacearum\u003c/em\u003e BMB Rs3221-2 and bacteriophage vB_RsoP_BMB116 treatment demonstrated gradual recovery of tomato seedlings, with restored healthy growth and favorable development (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). The bacteriophage vB_RsoP_BMB116 demonstrated a cure rate of 91.53% against bacterial wilt disease .\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe agricultural use of bacteriophages offers a viable strategy for managing bacterial diseases, particularly in light of rising antibiotic resistance. In this investigation, a novel \u003cem\u003eRalstonia\u003c/em\u003e phage, vB_RsoP_BMB116, belonging to a new genus, was isolated and characterized.\u003c/p\u003e\u003cp\u003eHowever, before being applied to production, its genome was analyzed to understand its genetic behavior. A proteomic tree was derived from high genome-similarity with other phages. VIRIDIC analysis revealed that the \u003cem\u003eRalstonia\u003c/em\u003e phage vB-RsoP_BMB116 may belong to a new genus. The current species demarcation standard is based on \u0026ge;\u0026thinsp;95% genome sequence identity, indicating\u0026thinsp;\u0026lt;\u0026thinsp;5% nucleotide divergence between viruses of the same species. Phage vB_RsoP_BMB116 showed only 73.12% similarity (2% query coverage) with \u003cem\u003eAchromobacter\u003c/em\u003e phage JWF and 79.23% (1% coverage) with phage 2\u0026thinsp;\u0026minus;\u0026thinsp;1. Consequently, vB_RsoP_BMB116 is a new species. ANI, the standard for prokaryotic species demarcation, (Konstantinidis and Tiedje, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Goris et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jain et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), defines members of the same species as sharing\u0026thinsp;\u0026gt;\u0026thinsp;95% identity. The ANI between vB_RsoP_BMB116 and similar genomes was determined utilizing (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ezbiocloud.net/tools/ani\u003c/span\u003e\u003cspan address=\"https://www.ezbiocloud.net/tools/ani\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) online tool (Yoon et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and it was discovered that vB_RsoP_BMB116 exhibited the highest ANI value of 62.85% with \u003cem\u003eAchromobacter\u003c/em\u003e phage JWF. suggesting that it represents a novel species. Moreover, VIRDIC revealed that phage vB_RsoP_BMB116 belongs to a new genus that is different from other phages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn conclusion, genomic sequence analysis revealed that \u003cem\u003eRalstonia\u003c/em\u003e phage vB_RsoP_BMB116 exhibited no significant genomic similarity to existing tailed viruses or known \u003cem\u003eRalstonia\u003c/em\u003e phages. Future exploration of its biological properties may shed light on host-phage interactions within this important bacterial genus. Although Bacteriophages provide a viable agricultural bacterial disease control strategy, yet phage therapy translation from lab to clinic faces challenges and limitations. Given its complex interactions and potential side effects, further research is needed to ensure safe, effective use in managing plant and other bacterial illnesses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eThe study received approval from the Huazhong Agricultural University Institutional Review Board. Given that the research solely focused on bacterial strains and their phages without involving human materials or patient information, the Review Board of the Huazhong Agricultural University granted an exemption from formal review and waived the requirement for informed consent. All procedures were conducted in line with applicable guidelines and regulations.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAuthor details\u003c/h2\u003e\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research did not receive any fund.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLiu conceptualized and designed the study; Wen performed the experiments, including sample collection, data acquisition, and laboratory analyses; Li conducted the statistical analysis and interpreted the results; Liu and Wen drafted the initial manuscript; Sun and Peng critically revised the manuscript for intellectual content; All authors reviewed and approved the final version of the manuscript, and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eNo new data were generated or analyzed in this study. The findings are based entirely on existing literature.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFegan M, Prior P (2005) How complex is the \u003cem\u003eRalstonia solanacearum\u003c/em\u003e species complex. Bacterial wilt disease and the \u003cem\u003eRalstonia solanacearum\u003c/em\u003e species complex 1:449\u0026ndash;461\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDrigues P, Demery-Laforgue D, Trigalet A, Dupin P, Samain D, Asselineau J (1985) Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of \u003cem\u003ePseudomonas solanacearum\u003c/em\u003e and from three avirulent mutants. J Bacteriol 162:504\u0026ndash;509\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen D, Liu B, Zhu Y, Wang J, Chen Z, Che J, Zheng X, Chen X (2017) Complete genome sequence of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e FJAT-1458, a potential biocontrol agent for Tomato Wilt. Genome Announc 5:1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang K, Chen D, Liu Q, Zhu P, Sun M, Peng D (2022) Isolation and Characterization of Novel Lytic Bacteriophage vB_RsoP_BMB50 infecting \u003cem\u003eRalstonia solanacearum\u003c/em\u003e. Curr Microbiol 79(9):245\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Twest R, Kropinski AM (2009) Bacteriophage enrichment from water and soil. Methods Mol Biol 501:15\u0026ndash;21\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHyman P, Abedon ST (2009) Practical methods for determining phage growth parameters. Methods Mol Biol 501:175\u0026ndash;202\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang M, Du J, Zhang D, Li X, Zhao J (2017) Modification of different pulps by homologous overexpression alkali-tolerant endoglucanase in \u003cem\u003eBacillus subtilis\u003c/em\u003e Y106. Sci Rep 7:3321\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSantos MA (1991) An improved method for the small scale preparation of bacteriophage DNA based on phage precipitation by zinc chloride. Nucleic Acids Res 19:5442\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJackman SD, Vandervalk BP, Mohamadi H, Chu J, Yeo S, Hammond SA, Jahesh G, Khan H, Coombe L, Warren RL, Birol I (2017) ABySS 2.0: resource-efficient assembly of large genomes using a Bloom filter. Genome Res 27:768\u0026ndash;777\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST Server: rapid annotations using subsystems technology. BMC Genomics 9:75\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLowe TM, Chan PP (2016) tRNAscan-SE On-line: integrating search and context for analysis of transfer RNA genes. Nucleic Acids Res 44:W54\u0026ndash;57\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSullivan MJ, Petty NK, Beatson SA (2011) Easyfig: a genome comparison visualizer. Bioinformatics 27:1009\u0026ndash;1010\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraru C, Varsani A, Kropinski AM (2020) VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12:1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMa Y, Pacan JC, Wang Q, Xu Y, Huang X, Korenevsky A, Sabour PM (2008) Microencapsulation of bacteriophage felix O1 into chitosan-alginate microspheres for oral delivery. Appl Environ Microbiol 74:4799\u0026ndash;4805. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.00246-08\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00246-08\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Li Y, Ding Y, Huang C, Zhang Y, Wang J, Wang X (2021) Characterization of a novel \u003cem\u003eSiphoviridae Salmonella\u003c/em\u003e bacteriophage T156 and its microencapsulation application in food matrix. Food Res Int. 2021;140:110004. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.foodres.2020.110004\u003c/span\u003e\u003cspan address=\"10.1016/j.foodres.2020.110004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraru C, Varsani A, Kropinski AM (2020) VIRIDIC-A novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12(11):1268\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBao YM, Chetvernin V, Tatusova T (2014) Improvements to pairwise sequence comparison (PASC): a genome-based web tool for virus classification. Arch Virol 159(12):3293\u0026ndash;3304\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFigueras MJ, Beaz-Hidalgo R, Hossain MJ, Liles MR (2014) Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis. Genome Announcements 2(6):e00927\u0026ndash;e00914\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeier-Kolthoff JP, Auch AF, Klenk HP, G\u0026ouml;ker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14(1):1\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoraru C (2023) VirClust-a tool for hierarchical clustering, core protein detection and annotation of (\u003cem\u003eprokaryotic\u003c/em\u003e) viruses. Viruses 15(4):1007\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDong Z, Xing S, Liu J, Tang X, Ruan L, Sun M, Tong Y, Peng D (2018) Isolation and characterization of a novel phage Xoosp2 that infects \u003cem\u003eXanthomonas oryzae pv\u003c/em\u003e. \u003cem\u003eoryzae\u003c/em\u003e. J Gen Virol 99:1453\u0026ndash;1462\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDrigues P, Demery-Lafforgue D, Trigalet A, Dupin P, Samain D, Asselineau J (1985) Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of \u003cem\u003ePseudomonas solanacearum\u003c/em\u003e and from three avirulent mutants. J Bacteriol 162:504\u0026ndash;509\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen D, Liu B, Zhu Y, Wang J, Chen Z, Che J, Zheng X, Chen X (2017) Complete genome sequence of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e FJAT-1458, a potential biocontrol agent for Tomato Wilt. Genome Announc 5:1\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSafni I, Cleenwerck I, De Vos P, Fegan M, Sly L, Kappler U (2014) Polyphasic taxonomic revision of the \u003cem\u003eRalstonia solanacearum\u003c/em\u003e species complex: proposal to emend the descriptions of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e and \u003cem\u003eRalstonia syzygii\u003c/em\u003e and reclassify current \u003cem\u003eR. syzygii\u003c/em\u003e strains as \u003cem\u003eRalstonia syzygii subsp\u003c/em\u003e. \u003cem\u003esyzygii subsp\u003c/em\u003e. nov., \u003cem\u003eR. solanacearum\u003c/em\u003e phylotype IV strains as \u003cem\u003eRalstonia syzygii subsp\u003c/em\u003e. \u003cem\u003eindonesiensis subsp\u003c/em\u003e. nov., banana blood disease bacterium strains as \u003cem\u003eRalstonia syzygii subsp. celebesensis subsp\u003c/em\u003e. nov. and \u003cem\u003eR. solanacearum\u003c/em\u003e phylotype I and III strains as \u003cem\u003eRalstonia pseudosolanacearum sp\u003c/em\u003e. nov. Int J Syst Evol Microbiol 64:3087\u0026ndash;3103\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614\u0026ndash;629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWang X, Wei Z, Yang K, Wang J, Jousset A, Xu Y, Shen Q, Friman VP (2019) Phage combination therapies for bacterial wilt disease in tomato. Nat Biotechnol 37:1513\u0026ndash;1520\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFujiwara A, Fujisawa M, Hamasaki R, Kawasaki T, Fujie M, Yamada T (2011) Biocontrol of \u003cem\u003eralstonia solanacearum\u003c/em\u003e by treatment with lytic bacteriophages. Appl Environ Microb 77:4155\u0026ndash;4162\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDa Silva Xavier, da Silva FP, Vidigal PMP, Lima TTM, de Souza FO, Alfenas-Zerbini P (2018) Genomic and biological characterization of a new member of the genus Phikmvvirus infecting \u003cem\u003ephytopathogenic Ralstonia\u003c/em\u003e bacteria. Arch Virol 163:3275\u0026ndash;3290\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M, Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I, Salmond G, Foster GD (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13:614\u0026ndash;629\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSae-Ueng U, Bhunchoth A, Phironrit N, Treetong A, Sapcharoenkun C, Chatchawankanphanich O, Leartsakulpanich U, Chitnumsub P (2020) C22 \u003cem\u003epodovirus\u003c/em\u003e infectivity is associated with intermediate stiffness. Sci Rep 10:12604\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBruynoghe R, Maisin J (1921) Essais de th\u0026eacute;rapeutique au moyen du bacteriophage. CR Soc Biol 85:1120\u0026ndash;1121\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSharma S, Chatterjee S, Datta S, Prasad R, Dubey D, Prasad RK, Vairale MG (2017) Bacteriophages and its applications: an overview. Folia Microbiol 62:17\u0026ndash;55\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayward AC (1991) Biology and epidemiology of bacterial wilt caused by \u003cem\u003epseudomonas solanacearum\u003c/em\u003e. Annu Rev Phytopathol 29:65\u0026ndash;87\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJiang G, Wei Z, Xu J, Chen H, Zhang Y, She X, Macho AP, Ding W, Liao B (2017) \u003cem\u003eBacterial Wilt\u003c/em\u003e in China: History, Current Status, and Future Perspectives. Front Plant Sci 8:1549\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNischal PM (2015) First global report on \u003cem\u003eantimicrobial resistance\u003c/em\u003e released by the WHO. Natl Med J India 27:241\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHayward AC (1991) Biology and epidemiology of bacterial wilt caused by \u003cem\u003ePseudomonas solanacearum\u003c/em\u003e. Annu Rev Phytopathol 29:65\u0026ndash;87\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasse J, Frey P, Trigalet A (1995) Microscopic studies of intercellular infection and protoxylem invasion of tomato roots by \u003cem\u003ePseudomonas solanacearum\u003c/em\u003e. Mol Plant Microbe Interact 8:241\u0026ndash;251\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e\u0026Aacute;lvarez B, Vasse J, Le-Courtois V, Trigalet-D\u0026eacute;mery D, L\u0026oacute;pez MM, Trigalet A (2008) Comparative behavior of \u003cem\u003eRalstonia solanacearum\u003c/em\u003e biovar 2 in diverse plant species. Phytopathology 98:59\u0026ndash;68\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrior P, Ailloud F, Dalsing BL, Remenant B, Sanchez B, Allen C (2016) Genomic and proteomic evidence supporting the division of the plant pathogen \u003cem\u003eRalstonia solanacearum\u003c/em\u003e into three species. BMC Genomics 17:90\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTurner D, Kropinski AM (2021) Adriaenssens, E.M. A Roadmap for genome-based phage taxonomy. Viruses 13:506\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdriaenssens EM, Brister JR (2017) How to name and classify your phage: an informal guide. Viruses 9:70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v9040070\u003c/span\u003e\u003cspan address=\"10.3390/v9040070\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKonstantinidis KT, Tiedje JM (2005) Genomic insights that advance the species definition for prokaryotes. PNAS 102:2567\u0026ndash;2572. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.0409727102\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0409727102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM (2007) DNA\u0026ndash;DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/ijs.0.64483-0\u003c/span\u003e\u003cspan address=\"10.1099/ijs.0.64483-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:5114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41467-018-07641-9\u003c/span\u003e\u003cspan address=\"10.1038/s41467-018-07641-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoon SH, Ha SM, Lim JM, Kwon SJ, Chun J (2017) A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek 110:1281\u0026ndash;1286. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s10482-017-0844-4\u003c/span\u003e\u003cspan address=\"10.1007/s10482-017-0844-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRajamanickam K, Hayes S (2018) The bacteriophage lambda CII phenotypes for complementation, cellular toxicity and replication inhibition are suppressed in cII-oop constructs expressing the small RNA oop. Viruses 10:115\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYoung R (1992) Bacteriophage lysis: Mechanism and regulation. Microbiol Rev 56:430\u0026ndash;481\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatsura I (1987) Determination of bacteriophage λ tail length by a protein ruler. Nature 327:73\u0026ndash;75\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNishimura Y, Yoshida T, Kuronishi M, Uehara H, Ogata H, Goto S (2017) ViPTree: The viral proteomic tree server. Bioinformatics 33:2379\u0026ndash;2380\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ralstonia solanacearum, bacteriophage, Podoviridae, genome, Plant pathogenic bacteria","lastPublishedDoi":"10.21203/rs.3.rs-7097101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7097101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eRalstonia solanacearum\u003c/em\u003e is an increasingly prominent multidrug-resistant phytopathogen. Phages are an effective alternative for treating \u003cem\u003eRalstonia solanacearum\u003c/em\u003e infections. In this study, the phage vB_RsoP_BMB116, which is specific to \u003cem\u003eR\u003c/em\u003e. \u003cem\u003esolanacearum\u003c/em\u003e, was isolated from Fujian, China. Electron microscopy revealed that vB_RsoP_BMB116 exhibited a \u003cem\u003epodoviridae\u003c/em\u003e morphotype. The double-stranded DNA genome of vB_RsoP_BMB116 spans 83,020 bp and encodes 126 predicted unidirectionally oriented genes, of which 42 have putative functions assigned, while the remainder are hypothetical proteins. Genome analysis indicated that vB_RsoP_BMB116 is a new genus.\u003c/p\u003e","manuscriptTitle":"Isolation and Characterization of Bacteriophage vB_RsoP_BMB116 Infecting Ralstonia solanacearum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 09:21:08","doi":"10.21203/rs.3.rs-7097101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"49fd8e9a-bac2-47fb-bb38-a8d83aef644f","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-28T16:08:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-13 09:21:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7097101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7097101","identity":"rs-7097101","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}