Whole-genome and Comparative Genomic Analysis Reveal the Biocontrol and Plant Growth-Promoting Potential of Bacillus velezensis JN.Y2 Against Oat Anthracnose

Whole-genome and Comparative Genomic Analysis Reveal the Biocontrol and Plant Growth-Promoting Potential of Bacillus velezensis JN.Y2 Against Oat Anthracnose | 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 Whole-genome and Comparative Genomic Analysis Reveal the Biocontrol and Plant Growth-Promoting Potential of Bacillus velezensis JN.Y2 Against Oat Anthracnose Wei Quan, Ting Zhang, Chen-Lu Liu, Shao-Xuan Shi, Xi-Xi Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9114671/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Oat anthracnose, primarily caused by Colletotrichum cereale , represents a significant threat to oat production, necessitating the development of sustainable biocontrol alternatives. In this study, we characterized an oat endophytic bacterium, Bacillus velezensis JN.Y2, isolated from healthy oat leaves in Inner Mongolia. In vitro assays demonstrated that B. velezensis JN.Y2 possesses an inhibitory activity against C. cereale (74.49%) and exhibits a broad antifungal spectrum. Greenhouse and three-year multi-location field trials confirmed its beneficial biocontrol efficacy, which remained stable even under fluctuating climatic conditions and high disease pressure, consistently outperforming conventional chemical treatments. Beyond disease suppression, B. velezensis JN.Y2 significantly enhanced oat growth and grain yield, supported by its ability to produce IAA, solubilize nutrients, and secrete diverse hydrolytic enzymes. Complete genome sequencing revealed a 3.87 Mb circular chromosome containing 13 secondary metabolite BGCs and 4 AOIs for RiPPs, including Amylocyclicin and LCI. Comparative genomic analysis highlighted an "open" pangenome and identified 411 unique genes associated with specialized metabolism and environmental sensing. While B. velezensis JN.Y2 shares high sequence synteny with B. velezensis CBMB205, distinct variations in the sporulation kinase kinA and secondary metabolite pathways suggest a fine-tuned adaptation to the oat endosphere. Furthermore, biosafety evaluations confirmed a relatively high level of genetic stability and a lack of active antibiotic resistance. Collectively, these findings provide a beneficial molecular foundation for the application of B. velezensis JN.Y2 as a reliable and secure biocontrol agent in sustainable agriculture. Bacillus velezensis Oat anthracnose Biocontrol Plant growth promotion Whole-Genome Sequencing Comparative genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Oats ( Avena sativa ) are globally significant cereal crops renowned for their multifunctional properties and rich nutritional profile (Butt et al. 2008 ). In China, naked oats ( A. nuda ) are predominantly cultivated in regions such as Inner Mongolia, Hebei, and Ningxia, serving as both functional food and essential forage. However, oat cultivation is severely threatened by anthracnose, a destructive disease primarily caused by Colletotrichum cereale (Crouch and Beirn, 2009 ; Shi et al. 2023 ). Other species, including C. americae-borealis and C. graminicola , also pose significant risks to oat production (Politis, 1976 ; Wang et al. 2022 ). While chemical fungicides remain the conventional management strategy, their application is increasingly hampered by high costs, environmental impacts, and the emergence of pathogen resistance (Janisiewicz and Conway, 2010 ). Biological control using beneficial plant endophytes offers a sustainable and eco-friendly alternative to chemical treatments (Khairullina et al. 2023 ; Wang et al. 2023 ). Endophytes are recruited by plants from the rhizosphere, often through chemotactic responses to root exudates, and play a critical role in enhancing host health and resilience (Ghimire et al. 2023 ; Malfanova et al. 2013 ). Among these, the genus Bacillus is distinguished by its robust and well-documented efficacy in promoting plant growth and suppressing pathogens (Miljaković et al. 2020 ). Bacillus strains facilitate plant health through nutrient solubilization, phytohormone synthesis, and the production of a diverse arsenal of secondary metabolites, such as lipopeptides and cell wall-degrading enzymes (Alamri, 2015 ; Choub et al. 2021 ; Grahovac et al. 2023 ; Zhen et al. 2022 ). Despite the recognized potential of Bacillus species as biocontrol agents, research on endophytic strains specifically adapted to the oat microenvironment remains limited. While certain strains like B. subtilis and B. velezensis have shown promise in controlling oat pathogens, there is still a lack of specific strains widely applied for targeted disease management. Understanding the regulatory mechanisms and colonization strategies of these bacteria is essential for their effective application (Nicholson et al. 2000 ; Ye et al. 2021; Zhang et al. 2023 ). In this study, an endophytic strain, Bv. JN.Y2, was isolated from healthy oat leaves in Inner Mongolia. Its broad-spectrum antifungal potential and biocontrol efficacy against oat anthracnose were assessed through in vitro assays, greenhouse experiments, and systematic three-year multi-location field trials. Furthermore, integrated whole-genome and comparative genomic analyses were performed to characterize the genetic landscape of Bv. JN.Y2, specifically investigating evolutionary structural variations, secondary metabolite biosynthesis, and environmental sensing mechanisms. This research aims to elucidate the molecular determinants of the biocontrol and plant growth-promoting capacities of Bv. JN.Y2, providing a beneficial theoretical foundation for its stable colonization and sustainable application within the oat microenvironment. Materials and methods Strains, pathogens and culture condition The strains used in this study are listed in Table 1 . Specifically, the endophytic bacterial Bv. JN.Y2 was isolated from healthy oat leaves collected in Jining District, Ulanqab City, Inner Mongolia, China. A kanamycin-resistant mutant, Bv. JN.Y2-kan R , which was previously constructed and maintained in our laboratory, was used for colonization assays. Stock cultures were maintained at -80 ℃ in Luria-Bertani (LB) broth (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) supplemented with 30% (v/v) glycerol. For routine use, Bv. JN.Y2 was cultured on LB agar at 37 ℃ for 24 h. When culturing Bv. JN.Y2-kan R , the medium was supplemented with 30 µg/mL kanamycin sulfate. The fungal pathogens used in this study included Colletotrichum cereale , Colletotrichum fructicola , Phyllosticta sorghina , Fusarium oxysporum , Fusarium graminearum , Alternaria alternata , Rhizoctonia solani , and Didymella glomerata . All fungi were obtained from the Key Laboratory of Biopesticide Creation and Resource Utilization of Inner Mongolia Autonomous Region. Fungal stocks were stored at -80 ℃ in 25% (v/v) glycerol. For experiments, mycelial plugs from frozen stocks were inoculated onto the center of Potato Dextrose Agar (PDA, 200 g/L potato, 20 g/L glucose, 18 g/L agar) plates and incubated in the dark at 25 ℃ for 10–12 days. Table 1 Bacterial, plasmids and fungal strains used in this study. Strains/Plasmids Description Source Strains Bv. JN.Y2 Wild-type biocontrol strain isolated from oat. This laboratory Bv. JN.Y2-kan R Spontaneous kanamycin-resistant mutant ( kan R ); used for colonization assays. This laboratory B. amyloliquefaciens YN.J3 Biocontrol strain; used as a positive control for biofilm formation assays. This laboratory Fungal Pathogens Colletotrichum cereale Anthracnose pathogen This laboratory Colletotrichum fructicola Anthracnose pathogen This laboratory Phyllosticta sorghina Leaf spot pathogen This laboratory Fusarium oxysporum Root rot pathogen This laboratory Fusarium graminearum Head blight pathogen This laboratory Alternaria alternata Leaf blotch pathogen This laboratory Rhizoctonia solani Root rot pathogen This laboratory Didymella glomerata Leaf spot pathogen This laboratory In Vitro antifungal activity assays The antagonistic activity of Bv. JN.Y2 was evaluated using a dual-culture assay (Xu, 2020). A 5-mm mycelial plug of each pathogen was placed 2 cm from the edge of a PDA plate. Bv. JN.Y2 was streaked on the opposite side, 3 cm from the fungal plug. Plates inoculated only with the pathogen alone served as controls. After incubation at 25 ℃ for 5–10 days, the radial growth of the fungus in control (Rc) and treatment (Rt) plates was measured. Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. The inhibition rate was calculated as: $$\text{I}\text{n}\text{h}\text{i}\text{b}\text{i}\text{t}\text{i}\text{o}\text{n}\left(\text{%}\right)=\frac{\text{R}\text{c}-\text{R}\text{t}}{\text{R}\text{c}}\times100$$ Additionally, to assess the morphological impact, hyphae from the edge of the inhibition zone were observed under a microscope to detect potential abnormalities (Kim, 2022). To evaluate the broad-spectrum antagonistic activity of Bv. JN.Y2, the strain was tested against seven additional phytopathogenic fungi: C. fructicola , P. sorghina , F. oxysporum , F. graminearum , A. alternata , R. solani , and D. glomerata . Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. All plates were incubated under the same conditions, and the inhibition rates were calculated. The biocontrol efficacy of Bv. JN.Y2 was evaluated in a greenhouse pot experiment (Meng et al. 2016 ). A spore suspension of C. cereale was prepared from 14-day-old PDA cultures by rinsing with 0.1 M PBS, with the concentration adjusted to 1 × 10 6 spores/mL using a hemocytometer. The bacterial inoculum was prepared by harvesting late-exponential phase Bv. JN.Y2 cells through centrifugation at 4°C and 6,000 rpm for 10 min, followed by washing and resuspension in 0.1 M PBS to a final concentration of 1 × 10 8 CFU/mL. Three treatments were established: (1) Bv. JN.Y2 treatment followed by C. cereale inoculation; (2) sterile 0.1 M PBS treatment followed by C. cereale inoculation as a negative control; (3) thiophanate-methyl (TM, 1,500-fold dilution) treatment followed by C. cereale inoculation as a positive control. Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. The disease incidence, disease index and control efficacy were calculated as previously described: Grade 1, 0%; Grade 2, 50% or necrotic (McKinney, 1923 ). The disease index and control efficacy were calculated as below: $$\text{D}\text{I}=\frac{\left(\sum\left(\text{G}\text{r}\text{a}\text{d}\text{e}\times\text{n}\right)\right)}{\left(\text{N}\times5\right)}\times100$$ $$\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\text{e}\text{f}\text{f}\text{i}\text{c}\text{a}\text{c}\text{y}\left(\text{%}\right)=\frac{\left(\text{D}{\text{I}}_{\left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}-\text{D}{\text{I}}_{\left(\text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}\right)}\right)}{\text{D}{\text{I}}_{\left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}}\times100$$ Field trials Field trials were conducted during the 2023, 2024, and 2025 growing seasons at two distinct locations in Inner Mongolia: Jining District (40.92°N, 113.15°E) and Wuchuan County (41.20°N, 111.47°E). Both sites feature chestnut soil and are endemic regions for oat anthracnose, with severe annual outbreaks of C. cereale . During the trial period, annual precipitation increased significantly from approx. 340–360 mm in 2023 to over 500–600 mm in 2024 and 2025, providing a rigorous environment for evaluating biocontrol stability. The experiment followed a randomized complete block design with three treatments (five replicates each): (1) Untreated Control (CK); (2) TM, 1,500-fold dilution; and (3) Bv. JN.Y2 cell suspension (1 × 10 8 CFU/mL). Each plot was 30 m 2 (5 m × 6 m) with a 1 m buffer zone maintained between adjacent plots. The oat cultivar "Baiyan No. 2" was sown at a rate of 180 kg/ha. For the Bv. JN.Y2 treatment, seeds were soaked in the bacterial suspension for 2 h prior to sowing, followed by two foliar sprays in booting stage and heading stage. The CK group seeds were soaked in sterile water for 2 h as a control for the soaking process. For the TM treatment, only foliar sprays were applied at the same time points, with no seed soaking performed. Standard local agronomic practices were followed uniformly across all plots. In October, the disease index and grain yield (kg/ ha) were recorded. Identification and characterization of Bv. JN.Y2 The morphological characteristics of Bv. JN.Y2 were observed after incubation on LB agar at 37°C for 24 h. Colony morphology, including shape, color, margin, and surface texture, was recorded. Cell morphology, Gram reaction, and endospore formation were examined using light microscopy (Olympus, Tokyo, Japan), respectively (Zhou et al. 2022 ). Motility was assessed using semi-solid agar tubes. Physiological and biochemical characteristics were evaluated according to Bergey’s Manual of Systematic Bacteriology (Sneath et al. 1986). Catalase activity was determined by bubble formation in 3% H 2 O 2 , and oxidase activity was tested using tetramethyl-p-phenylenediamine. Hydrolytic activities were assessed on agar plates supplemented with specific substrates: starch (amylase), gelatin (gelatinase), skim milk (protease), and carboxymethyl cellulose (cellulase). The metabolic profile was further characterized through standard assays, including nitrate reduction, Voges-Proskauer (V-P), methyl red, indole production, H 2 S production, urease activity, and citrate utilization. Carbon source utilization was tested in minimal medium supplemented with 1% (w/v) of glucose, sucrose, maltose, D-mannitol, or lactose as the sole carbon source. Genomic DNA was extracted from overnight cultures of Bv. JN.Y2 using a bacterial genomic DNA extraction kit (Tiangen, Beijing, China). For taxonomic identification, the 16S rRNA gene was amplified using specific primers as detailed in Online Resource 1 (Table S1 ). The PCR amplification was performed under the following conditions: initial denaturation at 95°C for 3 min; followed by 33 cycles of denaturation at 94°C for 25 s, annealing at 57°C for 25 s, and extension at 72°C for 60 s; and a final extension at 72°C for 5 min. PCR products were purified and sequenced by Tsingke Biotechnology Co., Ltd. (Beijing, China). The resulting sequence was aligned with reference sequences of closely related Bacillus species retrieved from the NCBI database using ClustalW. A phylogenetic tree was constructed based on the sequences of 16S rRNA using the Neighbor-Joining method in MEGA 7.0 software. The reliability of the phylogenetic clusters was evaluated by bootstrap analysis with 1,000 replicates. Characterization of enzymatic activity, biofilm formation, and plant colonization Hydrolytic enzyme assays: Extracellular enzyme production was screened on agar plates supplemented with specific substrates: 1% (w/v) skim milk for protease, 1% (w/v) soluble starch for amylase (Guleria et al. 2016 ), 1% (w/v) sodium carboxymethyl cellulose for cellulase (Teather et al. 1982), 1% (w/v) apple pectin for pectinase (Kuvvet et al. 2019 ), and 0.5% (w/v) colloidal chitin for chitinase (Agrawal et al. 2012). After incubation at 30 ℃ for 3–5 days, the presence of clear halos around colonies indicated positive enzymatic activity. To visualize halos for cellulase and pectinase, plates were stained with 0.1% Congo red for 15 min and destained with 1 M NaCl to 10 min. Biofilm formation was quantified in MSgg medium using a 24-well plate assay as described previously (Liu et al. 2022 ). The hyper-biofilm-forming strain B. amyloliquefaciens YN.J3 served as a positive control. Briefly, 10 µL of an overnight culture (adjusted to OD 600 = 1.0) was inoculated into 2 mL MSgg medium and incubated statically at 37 ℃ for 48 h. Biofilms were stained with 0.1% (w/v) crystal violet, gently washed three times with 0.1 M PBS to remove planktonic cells, and subsequently solubilized with 30% (v/v) acetic acid. Biofilm biomass was quantified by measuring the absorbance at 570 nm (A 570 ). The colonization ability of Bv. JN.Y2 was evaluated using the kanamycin-resistant mutant Bv. JN.Y2-kan R (Fan et al. 2017 ). Thirty-day-old oat seedlings were inoculated via root drenched with 50 mL of a bacterial suspension (1.5 ×10 8 CFU/mL). At 1, 3, 7, 10, 20, and 30 days post-inoculation (dpi), the root, stem, and leaf tissues were harvested. Samples were surface-sterilized with 75% ethanol and 3% NaClO, rinsed five times with sterile water, and homogenized. The homogenates were serially diluted and plated on LB agar containing 30 µg/mL kanamycin. After incubation at 37°C for 24 h, the colonizing population was enumerated and expressed as CFU/g fresh weight. Evaluation of plant growth-promoting traits and growth enhancement The plant growth-promoting (PGP) potential of Bv. JN.Y2 was evaluated through multiple assays. Nitrogen metabolism and assimilation was assessed by observing bacterial growth on nitrogen-free Ashby's medium. Phosphate and potassium solubilization were determined by the formation of clear halos on Pikovskaya's agar and Aleksandrov agar, respectively. Siderophore production was detected using Chrome Azurol S (CAS) agar, where a change in color to an orange halo indicated a positive result (Ariyani, 2021; Qiu et al. 2025 ). A greenhouse experiment was conducted to evaluate the effect of Bv. JN.Y2 on oat growth, following the protocol of Wang (Wang et al. 2018 ) with minor modifications. Oat seeds (cv. Baiyan No. 2) were surface-sterilized with 75% ethanol for 30 s and 3% NaClO for 5 min, followed by five rinses with sterile water. Germinated seedlings were transplanted into pots at the two-leaf stage. Ten-day-old seedlings were inoculated via root drenching with 50 mL of the Bv. JN.Y2 suspension (1.5×10 8 CFU/mL). Control plants were treated with an equal volume of sterile water. All plants were maintained in a greenhouse at 28°C, 60% relative humidity, with a16 h/8 h (light/dark cycle). After 30 days, plant height, root length and root fresh weight were measured. To determine the root dry weight (biomass), samples were dried at 65°C for 72 h until a constant weight was achieved. Genome sequencing, assembly, and annotation High-quality genomic DNA of Bv. JN.Y2 was isolated using the SDS-CTAB protocol. A hybrid sequencing strategy was employed by Novogene Co., Ltd., utilizing both PacBio Sequel and Illumina NovaSeq PE150 platforms. The raw PacBio subreads were processed via SMRT Link (v8.0) and assembled de novo using Canu (v2.0) (Koren et al. 2017 ). To enhance sequence accuracy, the resulting assembly was polished with Illumina short reads using Pilon (v1.22), yielding a single circular chromosome. Genome annotation was performed through an integrated pipeline. GeneMarkS (v4.17) was used for coding sequence prediction (Besemer et al. 2001 ), while repetitive elements were identified via RepeatMasker and Tandem Repeats Finder (TRF) (Benson, 1999). The non-coding RNA (ncRNA) fractions, including tRNAs and rRNAs, were detected using tRNAscan-SE and rRNAmmer, respectively (Lowe and Eddy, 1997 ; Lagesen et al. 2007 ; Kalvari et al. 2018 ). Furthermore, mobile genetic elements (MGEs), such as prophages and genomic islands, were localized using phiSpy and IslandPath-DIOMB(Hsiao et al. 2003 ; Akhter et al. 2012 ; Ge et al. 2016 ). Functional characterization of the proteome was achieved by searching against multiple databases, including KEGG, GO, COG, and Swiss-Prot(Ashburner et al. 2000 ; Galperin et al. 2015 ; Kanehisa et al. 2004 ; Kanehisa et al. 2006 ; Cantarel et al. 2009 ). Carbohydrate-active enzymes were categorized using dbCAN. The final genomic landscape was visualized using a Circos map (Krzywinski et al. 2009 ). Genome mining and comparative analysis of biosynthetic gene clusters BGCs responsible for secondary metabolite production in Bv. JN.Y2 genome were predicted using antiSMASH (v8.0) (Blin et al. 2025 ). The analysis was performed using the "relaxed" detection strictness to maximize the identification of putative clusters. To characterize the genetic organization and evolutionary conservation of these clusters, a comparative analysis was performed between Bv. JN.Y2 and four closely related strains B. velezensis FZB42 (Bv. FZB42), B. velezensis SQR9 (Bv. SQR9), B. velezensis CBMB205 (Bv. CBMB205) and B. amyloliquefaciens GKT04 (Ba. GKT04). The ClusterBlast module integrated into antiSMASH was employed to identify homologous gene clusters and visualize synteny. The schematic diagrams of gene arrangement and orientation were generated directly based on the antiSMASH visualization outputs. Genomic characterization and comparative analysis of bacteriocin biosynthetic gene clusters in Bv. JN.Y2 The whole-genome sequence of Bv. JN.Y2 was subjected to comprehensive genome mining using the BAGEL4 web server to identify potential bacteriocins and Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) (Liu et al. 2020 ). The identification process integrated core peptide searches and context-based gene cluster detection. Based on genomic proximity and functional relevance, the identified loci were categorized into specific Areas of Interest (AOIs) for subsequent detailed characterization. To evaluate the evolutionary conservation and structural features of the identified core peptides, representative homologous sequences were retrieved from the NCBI GenBank and BAGEL4 databases. Multiple sequence alignment was executed using the Clustal Omega algorithm with default parameters (Lugani and Sooch, 2017 ). The resulting alignment profile was professionally rendered using ESPript 3.2 to visualize conserved residues and physicochemical properties (Peng et al. 2024 ). Absolute conservation was defined by a 100% identity threshold across all compared strains, while similarity was assessed based on the equivalence of amino acid residues. The functional annotation of the core peptides was further validated through BLASTp searches against the non-redundant protein database at NCBI. Genomic screening for acquired resistance genes and biosafety evaluation To evaluate the genomic safety of Bv. JN.Y2 for potential agricultural applications, the presence of acquired antimicrobial resistance (AMR) genes was screened using the ResFinder (Version 4.7.2) database (Kim et al., 2025 ). The whole-genome sequence was analyzed with a minimum identity threshold of 80% and a minimum length coverage of 60%. To further assess the potential for horizontal gene transfer (HGT), the chromosomal coordinates of identified AMR homologs were cross-referenced against the boundaries of genomic islands and prophage regions, which were predicted using IslandPath-DIOMB program and PHAST, respectively. Phenotypic resistance predictions were derived from the ResFinder system to ensure a comprehensive evaluation of Bv. JN.Y2 safety. Comparative genomic analysis between Bv. JN.Y2 and reference Bacillus For the comparative genomic analysis, eight representative Bacillus strains were selected to serve as taxonomic and functional references (Table 2 ). These include species type strains to establish a precise phylogenetic framework, and established model biocontrol agents to provide a comparative context for evaluating the specific PGP and antagonistic mechanisms of Bv. JN.Y2. Furthermore, distantly related lineages were included to better delineate the core and strain-specific genomic features of the genus. Whole-genome alignments and synteny analyses were conducted using MUMmer (v3.23) and LASTZ (v1.03.54) (Kurtz, et al. 2004 ; Harris, 2007 ; Chiaromonte et al. 2001 ). These tools were employed to identify Single Nucleotide Polymorphisms (SNPs), small insertions/deletions (Indels), and structural variations (SVs) across the genomes. To evaluate the evolutionary relationships, a phylogenetic tree based on genome-wide SNPs was constructed using Treebest (v1.9.2) and PhyML (v3.0). Pan-genome analysis was performed via CD-HIT (v4.6.1) to classify the core, dispensable, and strain-specific gene sets. Table 2 Strain information required for comparative genome analysis. Strain name Strain name in report Genebank accession number B. velezensis JN.Y2 Bv. JN.Y2 CP178220.1 B. licheniformis ATCC 14580 Bl.14580 CP140161.1 B. mycoides BPN36/3 Bm.BPN36.3 CP035997.1 B. subtilis subsp. subtilis str. 168 Bs.168 AL009126.3 B.velezensis SQR9 Bv.SQR9 CP006890.1 B.velezensis CBMB205 Bv.CBMB205 CP011937.1 B.velezensis FZB42 Bv.FZB42 CP000560.2 B. thuringiensis serovar berliner ATCC 10792 Bt.10792 CM000753.1 B. amyloliquefaciens GKT04 Ba.GKT04 CP072120.1 Statistical analysis All experiments were conducted with three independent biological replicates to ensure the reproducibility and reliability of the data. Within each biological replicate, five technical replicates were performed. Data are presented as mean ± standard deviation (SD). Prior to analysis, the normality of the distribution and homogeneity of variance were confirmed using Shapiro-Wilk and Levene’s tests, respectively. For the PGP experiment, an independent samples t-test was employed to evaluate the statistical significance of the differences. Statistical significance was determined using one-way analysis of variance (ANOVA). Significant differences between treatment means were evaluated using Duncan’s multiple range test and the Least Significant Difference test at a significance level of P < 0.05. All statistical computations were performed using SPSS Statistics v26.0 (IBM Corp., Armonk, NY, USA). Graphical representations were generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The final multi-panel figures and illustrations were compiled and processed using Adobe Photoshop 2021 software (Adobe Systems Inc., San Jose, CA, USA). Results Bv. JN.Y2 exhibits broad-spectrum antifungal activity and effective control to oat anthracnose An endophytic bacterium, designated as Bv. JN.Y2, was isolated from healthy oat plants. In dual-culture assays, Bv. JN.Y2 significantly suppressed the mycelial growth of C. cereale , achieving an inhibition rate of 74.49% (Table 3 ; Fig. 1A, 1B). Microscopic examination revealed that Bv. JN.Y2 induced swelling and deformation of the hyphal tips, indicating a disruption of apical hyphal extension (Fig. 1C, 1D). In greenhouse pot experiments, Bv. JN.Y2 demonstrated a control efficacy of 62.40% (Fig. 1E-G), which is better than TM. Table 3 Evaluation of the inhibitory effect of Bv. JN.Y2 on C. cereale under dual-culture and greenhouse conditions. CK Dual-culture inhibitory rate(%) Greenhouse disease incidence (%) Greenhouse disease index Greenhouse control efficacy (%) - 100.00 ± 0.00 a 47.00 ± 1.73 a - Bv. JN.Y2 74.49 ± 2.73 56.67 ± 12.58 b 17.67 ± 3.51 b 62.40 Thiophanate-methyl - 86.67 ± 7.64 c 30.67 ± 5.03 c 34.74 Note : Values are expressed as mean ± SD. Different lowercase letters (a, b, c) in the same column indicate significant differences according to Duncan's multiple range test ( P < 0.05). During the 2023–2025 trial period, annual precipitation in Wuchuan and Jining showed a significant increasing trend, shifting from near-normal levels in 2023 (approximately 340–360 mm) to historical extreme rainfall in 2024 and 2025 (over 500–600 mm). Despite the increased disease pressure from surging humidity, Bv. JN.Y2 demonstrated stable biocontrol efficacy (Table 4 ). During the initial field trials in 2023, the control efficacy of Bv. JN.Y2 was recorded at 56.01% in Jining and 64.96% in Wuchuan. These values were comparatively higher than those observed for the chemical fungicide TM, which yielded control efficacy values of 42.69% and 53.94% at the respective sites. In 2024, as annual precipitation increased, Bv. JN.Y2 continued to demonstrate stable biocontrol performance, achieving control efficiencies of 63.41% in Jining and 60.55% in Wuchuan. During the same period, the CE of the chemical treatment was recorded at 54.75% and 50.58%, respectively. By 2025, a year characterized by historically high rainfall levels, the biocontrol stability of Bv. JN.Y2 remained evident. The strain maintained control efficiencies of 56.15% in Jining and 65.64% in Wuchuan, consistently exceeding the suppression levels provided by TM (46.53% and 50.08%, respectively). Collectively, the three-year monitoring data suggest that Bv. JN.Y2 possesses a balanced and reliable capacity to suppress oat anthracnose across fluctuating environmental conditions and varying levels of disease pressure. Table 4 Control efficiency of Bv. JN.Y2 on oat anthracnose and yield in two experimental sites from year 2023 to 2025. Year Test site Treatment Disease index Control efficiency Yield (kg/ha) 2023 Ulanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region(40.92506N, 113.15690E) Control 70.00 ± 2.80 a - 2940.00 ± 164.17 a Bv. JN.Y2 30.27 ± 0.61 c 56.01 ± 0.89 3391.35 ± 80.82 b Tm 39.45 ± 5.00 b 42.69 ± 7.27 3066.15 ± 104.19 a X030 private land in Keligeng Town, Wuchuan County, Hohhot City(41.20277N, 111.473127E) Control 70.64 ± 2.01 a - 2583.45 ± 93.07 a Bv. JN.Y2 24.11 ± 2.06 c 64.96 ± 2.30 3069.85 ± 79.43 b Tm 31.69 ± 1.15 b 53.94 ± 1.67 2729.35 ± 51.08 a 2024 Ulanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region Control 68.27 ± 2.20 a - 3127.65 ± 54.98 a Bv. JN.Y2 25.20 ± 3.86 c 63.41 ± 5.63 3532.30 ± 70.99 b Tm 31.13 ± 0.90 b 54.75 ± 1.31 3234.30 ± 42.27 a X030 private land in Keligeng Town, Wuchuan County, Hohhot City Control 60.17 ± 7.32 a - 2732.10 ± 64.49 a Bv. JN.Y2 27.00 ± 2.51 c 60.55 ± 3.69 3016.60 ± 84.08 b Tm 34.00 ± 2.50 b 50.58 ± 3.63 2851.20 ± 62.50 a 2025 Ulanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region Control 71.87 ± 6.89 a - 2980.65 ± 81.68 a Bv. JN.Y2 31.33 ± 1.29 b 56.15 ± 4.27 3535.95 ± 41.93 b Tm 38.4 ± 3.82 b 46.53 ± 2.84 3065.65 ± 25.02 a X030 private land in Keligeng Town, Wuchuan County, Hohhot City Control 70.27 ± 1.22 a - 2768.75 ± 76.55 a Bv. JN.Y2 24.13 ± 1.80 c 65.64 ± 2.88 3151.50 ± 25.28 b Tm 35.07 ± 0.23 b 50.08 ± 1.18 2908.85 ± 51.72 c Note : Data are presented as mean ± SD. Different lowercase letters (a, b, c) within the same column for each location and year indicate significant differences according to Duncan's multiple range test ( P < 0.05). Control: blank control; Bv. JN.Y2: Bacillus velezensis JN.Y2; Tm: Thiophanate-methyl. To assess its antifungal spectrum, Bv. JN.Y2 was co-cultured with seven other common fungal phytopathogens. The strain exhibited significant inhibitory activity against all tested pathogens ( P < 0.05) (Fig. 2). Inhibition rates ranged from 48.39% to 81.16%, with the highest activity observed against P. sorghina (81.16%). Inhibition rates against C. fructicola , R. solani , D. glomerata , and A. alternata exceeded 50%. While inhibition rates against F. graminearum and F. oxysporum were 48.39% and 48.53%, respectively. These findings confirm that Bv. JN.Y2 possesses broad-spectrum antifungal capacity, could be potentially applied for controlling multiple plant disease, caused by fungal pathogens. Morphological, physiological, and molecular identification of Bv. JN.Y2 After incubation on LB agar at 37°C for 24 h, colonies of Bv. JN.Y2 exhibited a creamy-white, opaque appearance, characterized by irregular margins and a rugose (wrinkled) surface (Fig. 3A). Microscopic examination and physiological assays indicated that Bv. JN.Y2 is a Gram-positive, motile, rod-shaped bacterium (Fig. 3B). The formation of central ellipsoidal endospores was observed, which is a typical characteristic of the genus Bacillus (Fig. 3C). As detailed in Table 5 , Bv. JN.Y2 demonstrated relatively diverse hydrolytic capabilities, testing positive for catalase and cellulase activities, as well as the hydrolysis of starch, gelatin, and casein. The strain also showed positive results for nitrate reduction, the Voges-Proskauer (V-P) reaction, and citrate utilization. In contrast, negative results were recorded for oxidase activity, methyl red test, indole production, H 2 S production, and urease activity. Regarding carbon source utilization, the strain was capable of metabolizing glucose, sucrose, maltose, and D-mannitol, while it appeared unable to utilize lactose. These morphological and biochemical traits are consistent with the established descriptions of Bacillus velezensis . Table 5 Physiological and biochemical characteristics of Bv. JN.Y2. Characteristics Results Morphology Gram staining + Cell shape Rod Endospore + Motility + Enzyme Activity Catalase + Oxidase - Starch hydrolysis + Gelatin liquefaction + Casein hydrolysis + Cellulase + Biochemical Tests Nitrate reduction + Voges-Proskauer + Methyl Red - Indole production - Citrate utilization + H₂S production - Urease - Carbon Source Glucose + Sucrose + Maltose + D-Mannitol + Lactose - Note: + , positive response or growth; − , negative response or no growth. Data represents the mean of three independent replicates. For the preliminary taxonomic classification of Bv. JN.Y2, 16S rRNA gene sequence analysis was initially performed. PCR amplification of the 16S rRNA gene yielded a single, distinct band of the expected size upon agarose gel electrophoresis (Fig. 3D). Subsequent sequence analysis and the resulting phylogenetic tree indicated that Bv. JN.Y2 clustered within the same clade as B. velezensis GT3033 and CSQXZD26, with a bootstrap support of 94% (Fig. 3E). Although the discriminatory power of 16S rRNA sequences among closely related species within the genus Bacillus is relatively limited, this analysis provided foundational evidence for the genus-level assignment of Bv. JN.Y2. Given the high degree of genetic similarity among closely related species within this genus, a phylogenetic tree based on whole-genome SNPs was subsequently constructed to achieve a more robust identification. The SNP-based phylogenetic analysis revealed a close genetic affinity between Bv. JN.Y2 and Bv. CBMB205, supported by a bootstrap value of 99% (Fig. 3F). By integrating the morphological, physiological, and biochemical characteristics with this high-resolution genomic evidence, Bv. JN.Y2 was confirmed as B. velezensis . Hydrolase secretion, biofilm formation and colonization To further explore the potential biocontrol mechanisms of Bv. JN.Y2, its capacity for extracellular enzyme secretion and biofilm formation was evaluated. The secretion of hydrolytic enzymes is generally considered a relevant factor in both pathogen antagonism and successful endophytic colonization. In this study, distinct hydrolysis halos were observed on agar plates supplemented with protease, amylase, cellulase, and pectinase substrates (Fig. 4A–D). Specifically, proteases can participate in the degradation of structural proteins within fungal cell walls, while cellulases and pectinases may facilitate the entry and systemic spread of the endophytic bacterium within host plant tissues. Although no obvious hydrolysis halo was detected on colloidal chitin agar (Fig. 4E), Bv. JN.Y2 maintained the ability to grow using chitin as the sole carbon source, suggesting a basal chitin-utilizing capacity that might assist in fungal antagonism. Collectively, these results indicate that Bv. JN.Y2 can secrete a relatively diverse array of hydrolases, which may coordinately contribute to its biocontrol efficacy. Furthermore, biofilm formation assays revealed that Bv. JN.Y2 was capable of developing structural biofilms in MSgg medium. Quantitative analysis using crystal violet staining showed an OD 570 value of 1.62 for Bv. JN.Y2. While this production was comparatively lower than that of the hyper-producing reference strain B. amyloliquefaciens YN.J3 (OD 570 = 2.17), it still demonstrated a favorable biofilm-forming capability relative to the control (Fig. 4F, Table 6 ). To dynamically assess the spatial and temporal colonization patterns of Bv. JN.Y2 in oat plants, a kanamycin-resistant transformant (Bv. JN.Y2-kan R ), which was obtained from our laboratory collection, was utilized and tracked via plate counting. The results indicated that the bacterial population in the roots reached its peak at 1 dpi with a density of 1.21 × 10⁵ CFU·g⁻¹, and subsequently showed a gradual decrease, maintaining a level of 2.85 × 10⁴ CFU·g⁻¹ at 30 dpi. In the aerial parts, colonization in the stems and leaves peaked slightly later, reaching 8.20 × 10⁴ CFU·g⁻¹ at 3 dpi and 5.73 × 10⁴ CFU·g⁻¹ at 7 dpi, respectively, before experiencing a gradual decline (Fig. 4G). These findings suggest that Bv. JN.Y2 possesses the ability to systemically spread and establish a relatively stable population within oat tissues over an extended period. PGP traits of Bv. JN.Y2 and its effect on oat yield enhancement To comprehensively assess the PGP potential of Bv. JN.Y2, both laboratory assays and field trials were evaluated. In vitro biochemical assays revealed that Bv. JN.Y2 was capable of growing on nitrogen-free Ashby medium, and it produced clear halos on Pikovskaya and Aleksandrov agar plates, indicating its capacities for nitrogen metabolism or assimilation (Fig. 5A), and solubilization of phosphate and potassium (Fig. 5B, 5C). Furthermore, the distinct halo observed on the CAS agar medium suggested the production of siderophores (Fig. 5D). In seedling evaluations, treatment with Bv. JN.Y2 resulted in an increase in seedling height and shoot fresh weight by 19.73% and 36.73%, respectively, although the difference in dry weight was not statistically significant. Regarding root development, root length, root fresh weight, and root dry weight were enhanced by 43.03%, 40.21%, and 11.96%, respectively. Additionally, the chlorophyll SPAD value exhibited a 25.27% increase compared to the control group (Fig. 5E, Table 7 ). Table 6 Quantitative analysis of biofilm formation measured by crystal violet staining (OD 570 ) Strain OD 570 B. amyloliquefaciens YN.J3 2.17 ± 0.07 c Bv. JN.Y2 1.62 ± 0.04 b CK (LB+Msgg media) 0.37 ± 0.07 a Table 7 Effect of Bv. JN.Y2 on the growth parameters of oat seedlings. Plant height (cm) Sterile water Bv. JN.Y2 24.12 ± 0.17 a 28.88 ± 0.58 b Root length (cm) 5.88 ± 0.24 a 8.41 ± 0.49 b Shoot fresh weight (g) 0.49 ± 0.03 a 0.67 ± 0.04 b Root fresh weight (g) 0.0475 ± 0.0029 a 0.0666 ± 0.0012 b Shoot dry weight (g) 0.10 ± 0.01 a 0.11 ± 0.02 a Root dry weight (g) 0.0184 ± 0.0015 a 0.0206 ± 0.0012 b Chlorophyll content (SPAD) 27.70 ± 0.79 a 34.70 ± 0.56 b Note : Data are presented as mean ± SD. Statistical analysis was performed using an independent samples t -test. Different lowercase letters within the same row indicate significant differences between treatments ( P < 0.05). Across the three consecutive years of field trials (2023–2025) in Jining and Wuchuan, oat plants treated with Bv. JN.Y2 consistently exhibited higher grain yields compared to CK and TM groups (Table 4 ). In 2023, the application of Bv. JN.Y2 led to favorable yield increases, reaching 3391.35 kg/ha in Jining and 3069.85 kg/ha in Wuchuan. During 2024, as environmental precipitation increased, the yield-promoting effect remained relatively stable, with the Bv. JN.Y2 treatment achieving 3532.30 kg/ha and 3016.60 kg/ha at the two respective sites. In the multi-rain year of 2025, the yield in the Bv. JN.Y2-treated plots reached 3535.95 kg/ha in Jining and 3151.50 kg/ha in Wuchuan, maintaining a consistent advantage over the corresponding controls. Collectively, these findings suggest that Bv. JN.Y2 likely promotes oat growth and stably enhances crop yield across varying climatic conditions by facilitating nutrient absorption and improving overall physiological vigor. Genomic features and functional gene mining of Bv. JN.Y2 To uncover the molecular basis underlying its beneficial traits, the complete genome of Bv. JN.Y2 was sequenced and annotated. The genome consists of a single circular chromosome of 3, 871, 731 bp with a GC content of 47.26% (Fig. 6, Table 8 ). No plasmid was detected. The genome contains 3, 987 predicted protein-coding genes with an average length of 872 bp, along with 25 rRNA genes, 84 tRNA genes, and 8 sRNA genes. The complete genome sequence has been deposited in the NCBI database under the GenBank accession number CP178220.1. Functional annotation was performed across multiple databases, yielding the following assignments: COG (2, 939 genes), NR (3, 912), GO (2, 667), KEGG (3, 836), Pfam (2, 667), Swiss-Prot (3, 255), CAZy (155), and TCDB (516). Subsequent genomic screening revealed an array of genes associated with PGP activities. Specifically, gene clusters related to nitrogen metabolism and assimilation ( nifH , nifU , yutI ), Fe-S cluster assembly ( sufB , sufC , sufU , sufD ), phosphate metabolism ( pstA/B1/B2/C/S , phoA , bglA ), potassium uptake ( ktrA/C/D , kdpD ), siderophore production ( fhu , feu , dhb , ent ), and indole-3-acetic acid (IAA) synthesis (the trp operon) were identified (Online Resource 1 Table S2 ). These findings provide a genetic explanation for the physiological PGP traits observed in vitro. Table 8 The general genome feature of Bv. JN.Y2 Feature Vaule Genome size (bp) 3,871,731 G + C content 47.26% Topology Circular Plasmid 0 Total size of protein-coding genes 3,478,611 Protein-coding genes 3987 Average CDs size (bp) 872 rRNA number (total) 25 tRNA number 84 sRNA number 8 Repetitive sequence(bp) 18131 (0.4683%) CRISPR 7 Prophage 13 Gls 9 Gene cluster 12 Genes assigned to COG 2939 Genes assigned to NR 3912 Genes assigned to GO 2667 Genes assigned to KEGG 3836 Genes assigned to Pfam 2667 Genes assigned to Swiss-Prot 3255 Genes assigned to CAZy 155 Genes assigned to TCDB 516 Furthermore, a variety of genes encoding hydrolases were annotated, including amylases ( amyA ), proteases ( epr , aprE , vpr ), cellulases ( celA/B/C/F , xynB ), pectinases ( pel , pgdA , pgdB ), and chitin-related enzymes ( yxkH , yheN ). Additionally, a comprehensive set of genes essential for plant-microbe interactions and colonization was identified, encompassing those governing chemotaxis ( cheA/W/Y , mcpA/B/C ), flagellar motility ( fliG/M/N ), quorum sensing ( comA , comP , luxS , phrA , sinI/R ), and biofilm formation ( tasA , epsC , bslA ) (Online Resource 1 Table S3). Together, these genomic features establish a relatively solid genetic foundation for the biocontrol and growth-promoting capacities of Bv. JN.Y2. Discovery and comparative analysis of secondary metabolite biosynthetic gene clusters To further elucidate the molecular mechanisms underlying the biocontrol efficacy of Bv. JN.Y2, the antiSMASH tool was employed to mine its genome for BGCs. A total of 13 BGCs were identified, encompassing non-ribosomal peptide synthetases (NRPS), trans-AT polyketide synthases (transAT-PKS), hybrid PKS/NRPS systems, and terpenes (Table 9 ). Among these, seven clusters exhibited high similarity to known pathways and were predicted to direct the synthesis of surfactin, macrolactin H, bacillaene, fengycin, difficidin, bacillibactin, and bacilysin. Furthermore, Region 2 (PKS-like), Region 4 (Lanthipeptide-class-II), Region 9 (T3PKS), and multiple terpene gene clusters (Regions 3, 8, and 11) distributed at various genomic positions exhibited notably low similarity to entries in known databases. These findings suggest that Bv. JN.Y2 possesses a favorable potential for the synthesis of novel or strain-specific antimicrobial metabolites, which may relatively significantly assist in its environmental adaptation and biocontrol performance. To characterize the genetic organization of these clusters, a comparative synteny analysis was performed between Bv. JN.Y2 and four biocontrol reference strains. Visualization revealed a remarkably high degree of collinearity and synteny between Bv. JN.Y2 and the model strain Bv. FZB42 (Fig. 7). The arrangement and orientation of core biosynthetic operons (e.g., srfAA-AC , fenA-E , mlnA-G , baeJ-R , and difA-L ) were highly conserved across the analyzed B. velezensis strains. This conservation not only confirms the genetic integrity of these antimicrobial pathways in Bv. JN.Y2, but supports its taxonomic identification at the genomic level, indicating its potential as a biocontrol agent. Table 9 Predicted genes clusters involved in synthesis of secondary metabolites in Bv. JN.Y2 genome. Region Most similar known cluster Type Size Similarity Confidence Position 1 Surfactin NRPs 65, 407 bp High 264, 083–329, 490 2 Butirosin PKS-like 41, 244 bp - 865, 948 − 907, 192 3 - Terpene 20, 740 bp - 989, 236-1, 009, 976 4 - Lanthipeptide-class-ii 28, 888 bp - 1, 130, 469-1, 159, 357 5 Macrolactin H TranAT-PKS 88, 233 bp High 1, 325, 970-1, 414, 203 6 Bacillaene transAT-PKS,T3PKS,NRPS 110, 093 bp High 1, 632, 894-1, 742, 987 7 Fengycin NRPS, transAT-PKS,betalactone 137, 801 bp High 1, 807, 618-1, 945, 419 8 - Terpene 21, 883 bp - 1, 970, 646-1, 992, 529 9 - T3PKS 41, 100 bp - 2, 055, 847-2, 096, 947 10 Difficidin TransAT-PKS 106, 182 bp High 2, 211, 933-2, 318, 115 11 - Terpene-precursor 20, 890 bp - 2, 341, 409-2,362, 299 12 Bacillibactin NRP-metallophore,NRPS,Ripp-like 51, 791 bp High 2, 942, 817-2, 994, 608 13 Bacilysin Other 41, 418 bp High 3, 530, 918-3, 572, 336 In silico mining of bacteriocins and RiPPs The genomic architecture of Bv. JN.Y2 was explored using the BAGEL4 platform, which identified four distinct Areas of Interest (AOIs) dedicated to the synthesis of RiPPs (Fig. 8). Within the genomic architecture of Bv. JN.Y2, AOI_01 is situated between coordinates 2, 979, 272 and 2, 999, 461, encompassing 26 predicted open reading frames (ORFs). The primary biosynthetic product of this cluster is identified as Amylocyclicin, a circular bacteriocin recognized for its significant role in improving the competitive fitness of Bacillus species within their ecological niches. Distinct from the other synthesis-oriented clusters, AOI_02 (2, 929, 112–2, 949, 271) comprises 26 ORFs and is characterized as a regulatory locus centered on the signaling peptide ComX3. As a pivotal component of the Quorum Sensing system, ComX3 potentially modulates the secondary metabolic profile of Bv. JN.Y2, thereby orchestrating its adaptive responses to environmental stimuli without the requirement of a traditional core peptide synthesis template. Complementing these findings, AOI_03 is located at positions 243, 431 to 263, 566 and contains 19 ORFs, encoding the Linear Competition Inducer (LCI) as its core protein. LCI has been demonstrated to facilitate environmental dominance by exerting potent antimicrobial and antifungal activities against competing microorganisms. Lastly, AOI_04 (1, 132, 550–1, 158, 286) exhibits a sophisticated organizational structure with 28 ORFs and is classified as a class II lanthipeptide biosynthetic cluster. Notably, this cluster is characterized by the presence of two distinct core peptides exhibiting high homology to Haloduracin_beta, which are further associated with the Type A lanthibiotic (L_biotic_typeA) and Mersacidin-like families. This multi-component architecture is a hallmark of certain RiPPs that often exhibit enhanced bioactivity through synergistic mechanisms, potentially conferring a consistent defensive advantage to the host strain. To validate the functional robustness of these systems, multiple sequence alignments were performed using Clustal Omega and visualized via ESPript 3.2 (Fig. 9). The resulting alignment profile confirmed a high degree of evolutionary conservation across the core peptides. Specifically, the Amylocyclicin sequence in Bv. JN.Y2 exhibits 100% identity with reference circular bacteriocins from B. velezensis FZB42 and DSYZ. This is further supported by BLASTp validation, showing 99.10% identity (99% coverage) to a circular bacteriocin from B. inaquiensis with a significant E-value of 2e-74. For the LCI system, the core peptide shares 96.81% identity with unnamed B. velezensis , B. velezensis DSM 23117 and 98.94% with B. siamensis SCSIO 05746. Notably, Bv. JN.Y2 shared a significantly lower identity of only 53.33% with the model strain B. velezensis FZB42. This pronounced divergence in the LCI sequence, contrasted with the 100% identity observed for Amylocyclicin, highlights the unique evolutionary trajectory and the distinct antimicrobial profile of Bv. JN.Y2 compared to the classical model strain. Regarding the multi-component AOI_04, while AOI_04/1 shows varied homology (0-78.05%), AOI_04/2 exhibits absolute conservation with 100% identity to the homolog from B. amyloliquefaciens PM415. These molecular signatures underscore the sophisticated antimicrobial potential of the Bv. JN.Y2 genome. Genomic screening for acquired resistance genes and biosafety evaluation The genomic landscape of Bv. JN.Y2 is characterized by a relatively rich composition of MGEs, including 9 predicted genomic islands (GIs) and 13 prophage regions (Online Resource 1 Table S4, S5). This substantial dataset underscores an active evolutionary history driven by HGT, which likely contributes to the strain's genomic plasticity and its specialized adaptation to the oat endosphere. Notably, these GIs are hypothesized to harbor potential genes associated with environmental fitness, nutrient metabolism, and plant-growth promotion, which are essential for the endophytic lifestyle of Bv. JN.Y2. Consistent with this dynamic genomic architecture, ResFinder screening identified two acquired AMR homologs (Table 10 ): cfr(B) (identity: 88.61%) and tet(L) (identity: 86.87%). Spatial distribution analysis revealed that cfr(B) (52, 804–53, 839 bp) is localized within the Prophage 1 region (29, 352–103, 065 bp), providing a molecular record of an ancestral transduction event. In contrast, tet(L) (373, 529–374, 905 bp) resides within the stable chromosomal backbone, situated outside all predicted GIs and prophage sequences. Despite the historical acquisition of these elements, the ResFinder system predicted a "No resistance" phenotype across all tested antibiotic categories, including aminoglycosides and beta-lactams, for which no significant hits were found. Given the plasmid-free architecture of Bv. JN.Y2, the chromosomal localization and the "cryptic" nature of the prophage-associated cfr(B) suggest that these sequences likely represent non-functional evolutionary remnants rather than active resistance determinants. This sophisticated genomic compartmentalization appears to effectively control potential resistance risks while maintaining high genomic plasticity. Consequently, these findings underscore a relatively high level of biosafety and genetic stability for Bv. JN.Y2, supporting its potential as a sustainable and secure biocontrol agent in oat production. Table 10 Summary of AMR genes and their spatial association with MGEs in Bv. JN.Y2 AMR Gene Identity (%) Chromosomal Position (bp) MGE Association Predicted Phenotype cfr(B) 88.61 52, 804 − 53, 839 Prophage 1 (29, 352 − 103, 065 bp) No resistance tet(L) 86.87 373, 529 − 374, 905 None (Stable chromosomal backbone) No resistance Comparative genome analysis between Bv. JN.Y2 and eight reference Bacillus strains To elucidate the genetic divergence, functional variations, and evolutionary adaptation of the tested strains relative to the reference strain Bv. JN.Y2, a systematic profiling of SNPs, InDels, and SVs was conducted across all genomes (Table 11 , Fig. 10, Online Resource 1 Fig. S1 ). Among all investigated strains, Bv. CBMB205 exhibited a relatively high degree of genetic conservation compared to the reference genome. A genome-wide analysis revealed only 3 SNPs (including only 1 non-synonymous mutation) and 8 InDels within CDS regions (comprising 5 insertions and 3 deletions) (Table 11 , Fig. 10A). The SNP-based phylogenetic tree (Fig. 3F) further illustrates these evolutionary relationships: closely related B. velezensis , such as Bv. CBMB205, Bv. SQR9 and Bv. FZB42, tend to accumulate higher densities of SNPs while maintaining robust sequence synteny. Conversely, distantly related lineages like Bm. BPN36.3 and Bt. ATCC 10792 occupy basal positions in the tree, where their evolutionary divergence is primarily driven by complex structural rearrangements and poorer synteny, despite exhibiting relatively fewer detectable point mutations. This high level of sequence stability was further corroborated by the SV profiling. Similarly, other strains belonging to B. velezensis (Online Resource 1 Fig. S1 D, E, F) demonstrated relatively low structural variation and maintained robust sequence synteny, reflecting their close intra-specific phylogenetic relationships. Notably, a divergent variation pattern was observed in Bm. BPN36.3 (Online Resource 1 Fig. S1 B) and Bt. ATCC 10792 (Online Resource 1 Fig. S1 G). While these strains showed comparatively low point mutation counts—with 2, 777 and 2, 152 SNPs, 9 and 11 InDels, respectively—their macro-scale structural profiles were characterized by dense, large-scale genomic clusters. This suggests that these lineages may have undergone relatively complex sequence rearrangements or recombinations during their evolutionary trajectories. In contrast, Bs. 168 exhibited significant genetic differentiation from the reference, with the total number of SNPs reaching 141, 090. Even within the B. velezensis group, Bv. FZB42 and Bv. SQR9 accumulated 55, 591 and 37, 721 SNPs, respectively, indicating a notable level of intra-specific genetic diversity. Collectively, the frequency of SVs across the strains fluctuated between 4 and 512 events, encompassing deletions, insertions, and complex translocations. The distribution of these structural features showed a relatively strong correlation with the taxonomic distance of each strain relative to Bv. JN.Y2. These multi-dimensional variation data not only elucidate the distinct genomic signatures formed during long-term evolution, but provide a relatively solid molecular foundation for subsequent investigations into functional gene expression and environmental adaptation across these diverse strains. Furthermore, KEGG functional annotation was performed to investigate the impact of key genetic heterogeneities on the biocontrol properties and environmental fitness of Bv. JN.Y2, particularly from the dual perspectives of protein functional evolution and gene expression regulation. Bv. JN.Y2 exhibited relatively high structural stability relative to its close relative Bv. CBMB205, with key genetic divergence focused on non-synonymous SNPs in the sporulation kinase kinA . This variation suggests a potentially more sensitive regulatory strategy for sensing environmental stress. Meanwhile, the conserved core developmental framework and high sequence synteny provide a relatively solid genetic foundation for stable colonization. In comparisons with biocontrol strains such as Bv. FZB42, Bv. SQR9 and Ba. GKT04, non-synonymous SNPs and InDels were highly enriched within secondary metabolite clusters (e.g., Iturin, Fengycin, and Pks), driving the diversification of substrate specificity. In addition, localized SV-mediated rearrangements promoted the modular evolution of biocontrol genes, providing molecular evidence for its broad-spectrum antagonistic activity. Regarding environmental sensing, variations in coding regions of ComP and chemotaxis proteins, coupled with structural fine-tuning of upstream regulatory elements, imply a "precision-tuning" mechanism for responding to root exudates, thereby enhancing sensing sensitivity in soil. Compared to distant lineages like Bl. 14580 and Bm. BPN36.3, Bv, JN.Y2 underwent comprehensive differentiation ranging from large-scale rearrangements to core metabolic functions (e.g., tuf , sufB ) and stress-resistance pathways (e.g., spoIIP , uvrA/B ). Collectively, these multi-level variation patterns reinforce the survival resilience and adaptive fitness of Bv. JN.Y2 under extreme environmental conditions. Table 11 Statistics and annotation of SNPs in the genomes of eight Bacillus strains Reference Strain Sample Strain Synonymous SNP Nonsynonymous SNP Total CDS SNP Intergenic Total SNP Bv. JN.Y2 Bl.14580 21, 293 6, 428 27, 846 2, 100 29, 946 Bm.BPN36.3 1, 546 803 2, 364 413 2, 777 Bs.168 101, 661 28, 332 28, 332 130, 600 141, 090 Bv.SQR9 24, 882 9, 148 34, 142 3, 579 37, 721 Bv.CBMB205 0 1 3 0 3 Bv.FZB42 37, 095 13, 014 50, 326 5, 265 55, 591 Bt.10792 1, 164 611 1, 794 358 2, 152 Ba.GKT04 37, 898 13, 216 51, 304 5, 273 56, 577 Core-Pan analysis between Bv. JN.Y2 and eight reference Bacillus strains To characterize the evolutionary trajectories and functional divergence among nine Bacillus strains, including Bv. JN.Y2, a comparative pangenome analysis was conducted. From a total of 43, 274 identified protein-coding sequences, 12, 473 orthologous genes (pan-genes) were clustered to define the collective gene repertoire of the cohort. Within this framework, a core genome of 1, 086 genes shared across all strains was identified, likely representing the conserved genetic foundation of the group. Conversely, the dispensable genome, comprising 11, 387 genes distributed among subsets of strains, highlighted the considerable genomic plasticity of the lineage. Notably, Bv. JN.Y2 harbored 411 unique genes—a figure within a moderate range (13–1, 508) that reflects its distinct genetic identity. Dilution curve analysis further corroborated these dynamics, showing the core genome stabilizing at approximately 1,086 genes, while the pangenome expanded without reaching a saturation plateau. These findings indicate that this strain assembly possesses a typical "open" pangenome, suggesting a collective genetic repertoire with substantial potential for further expansion upon the inclusion of additional genomic data. Among the 1, 086 core genes, 524 (48.2%) were successfully annotated via the KEGG database. At the primary functional categories, Metabolism was the most prominent category (cumulative frequency: 1, 485), followed by Genetic Information Processing (143) and Environmental Information Processing (78). These results suggest that the core genome primarily sustains basal physiological activities and signal transduction, providing a stable functional framework for Bv. JN.Y2 and its relatives. Further analysis at the secondary functional categories highlighted a high prevalence of genes involved in Global and overview maps (817), Carbohydrate metabolism (158), and Amino acid metabolism (134). Specifically, specific metabolic pathways revealed 304 genes in Metabolic pathways, 60 in Carbon metabolism, and critical energy-related components like Glycolysis/Gluconeogenesis (25) and Oxidative phosphorylation (24). Regarding biocontrol and adaptation, genes for the biosynthesis of secondary metabolites (160) and antibiotics (120) exhibited high conservation, underscoring a relatively stable genetic basis for the antagonistic potential of the B. velezensis group. Additionally, the enrichment of Ribosome (45) genes supports efficient protein synthesis, while conserved Two-component systems (33) and ABC transporters (28) likely enhance environmental sensing and nutrient acquisition, collectively maintaining bacterial fitness across diverse ecological niches. In contrast to the conserved core genome, the 411 strain-specific genes of Bv. JN.Y2 are characterized by specialized functions that likely facilitate its distinct ecological adaptation. KEGG annotation reveals a notable enrichment in secondary metabolism, particularly monobactam biosynthesis, suggesting a relatively good potential for the production of unique bioactive compounds. The identification of pathways such as lysine biosynthesis and butanoate metabolism further indicates enhanced metabolic flexibility. Furthermore, unique components of quorum sensing and two-component systems reflect a specialized capacity for environmental sensing and population-level coordination. Combined with specific ABC transporters and genes involved in sporulation and stress response, these genetic features collectively reinforce the resilience and competitive fitness of Bv. JN.Y2 within its ecological niche. Discussion Members of the genus Bacillus are widely regarded as promising biological control agents, a reputation primarily attributed to their capacity to establish mutualistic endophytic relationships with host plants and suppress pathogens through multifaceted mechanisms (Reva et al. 2004 ; Hashem et al. 2019 ). In this study, the endophytic strain Bv. JN.Y2, isolated from oat stems and taxonomically identified via both 16S rRNA sequences and whole-genome SNP-based phylogenetic trees, demonstrated relatively good dual functionality in anthracnose suppression and PGP. Comprehensive genome mining of Bv. JN.Y2 revealed a substantial repertoire of genes associated with secondary metabolite biosynthesis, nutrient utilization and cycling, environmental adaptation, as well as chemotaxis and colonization. Furthermore, a systematic comparative genomic analysis was conducted to elucidate the genetic architecture of Bv. JN.Y2. Our findings indicated that, in addition to the core genome, the strain has evolved 411 unique genes and exhibited a certain degree of specificity in terms of genomic variants, including SNPs, InDels, and SVs. These unique genes and sequence variations are notably enriched in key pathways such as secondary metabolism, quorum sensing, and stress response. In particular, the genetic divergence accumulated in the sporulation kinase kinA and secondary metabolite clusters (e.g., Iturin and Fengycin) provided a relatively good basis for the diversification of substrate specificity. Collectively, the abundant genetic variation within this "open" pan-genome defines the unique genetic identity of Bv. JN.Y2, establishing a relatively good foundation for its survival resilience and competitive adaptability within complex ecological niches. The biocontrol potential of Bacillus species is generally attributed to the synergy of multiple traits, including direct antagonism facilitated by secondary metabolites from the NRPS, PKS, and RiPPs pathways (Jangir et al. 2018 ; Zhou et al. 2021 ), competition for finite resources (Kramer et al. 2020 ), and effective colonization of the rhizosphere and endophytic tissues, a process often facilitated by CWDEs and biofilm (Wang et al. 2024 ). Combined results from greenhouse assays and whole-genome annotation revealed that Bv. JN.Y2 possesses a diverse genetic repertoire encoding proteases, cellulases, and various BGCs including NRPS, PKS, and RiPPs. The co-existence of these multi-functional antimicrobial BGCs likely underpins the broad-spectrum inhibitory capacity of Bv. JN.Y2. As reported by Shen, the synergistic action of multiple secondary metabolites and CWDEs secreted by B. velezensis SH1471 effectively suppresses a wide range of pathogens, including F. oxysporum (Shen et al. 2023 ). This characteristic—simultaneous secretion of CWDEs and antimicrobial metabolites—underscores the potential of Bv. JN.Y2 as a versatile biocontrol agent for managing various plant diseases beyond oat anthracnose (Chowdhury et al. 2015 ). Furthermore, genomic mining and CAS assay results confirmed the potential of Bv. JN.Y2 to synthesize catecholate-type siderophores (e.g., bacillibactin) and its possession of sophisticated transport systems ( feu and fhu ) for mediating catecholate and hydroxamate iron uptake (Chen et al. 2007 ). This robust iron-sequestration capability likely facilitates the inhibition of pathogen proliferation under iron-limited conditions, thereby enhancing the strain’s ecological fitness within the complex rhizosphere environment (Yu et al. 2011 ). In addition, longitudinal tracking of Bv. JN.Y2-kan R demonstrated that the strain can stably colonize the roots, stems, and leaves of oat plants for at least 30 days. This persistent endophytic colonization is likely facilitated by a synergistic mechanism involving: (i) CWDEs (cellulases and pectinases) encoded by celA/B/C/F , pel , and pgdA genes, which assist in tissue penetration (Ku et al. 2018 ); (ii) mcp , che , motility-related and QS genes that orchestrate the recruitment process in response to root exudates (Zhou et al. 2016 ); and (iii) a relatively strong capacity for biofilm formation; (iv) the presence of a surfactin-encoding gene cluster, which is relatively effective in enhancing bacterial motility and promoting colonization (Gao et al. 2016 ). Our findings are consistent with those of Weng (Weng et al. 2013 ), who utilized GFP-labeling to show that B. velezensis SQR9 effectively responds to root exudates and establishes stable colonization in cucumber, subsequently protecting the host from F. oxysporum infection. Microbes promoted plant growth by secreting IAA and facilitating nutrients absorption (Idris et al. 2007 ). The genome of Bv. JN.Y2 contains the complete trp operon ( trpABCDEF ), contributing to IAA synthesis. Bv. JN.Y2 grow normally on nitrogen-free medium and core genes ( nifH , nifU ), regulatory gene ( yutI ) are presented in genome of Bv. JN.Y2. Fan proved that genetic configurations of core genes and regulatory genes giving B. velezensis FZB42 nitrogen metabolism and assimilation trait (Fan et al. 2018 ). Movever, genes related to phosphate metabolism and transport ( pstABCS , phoA ), potassium mobilization ( ktrACD , kdpD ) are also present in genome of Bv. JN.Y2, which collectively exert a relatively beneficial influence on plant growth promotion (Meena et al. 2016 ). Additionally, the genome harbors the dhb operon ( dhbACEBF ), which is responsible for the biosynthesis and transport of siderophores. This mechanism plays a relatively conducive role in chelating iron from the environment, and further supporting nutrient acquisition under iron-limited conditions (Zhang et al. 2024 ). Overall, this multi-pronged genetic toolkit explains the enhanced biomass and yield observed in oats treated with Bv. JN.Y2. Genome-wide analysis using antiSMASH and BAGEL4 identified a total of 17 BGCs. Notably, several clusters, such as those for Fengycin and Bacillaene, exhibit NRPS-PKS hybrid features, highlighting the robust potential of Bv. JN.Y2 to synthesize structurally sophisticated and functionally varied secondary metabolites (Zhao and Kuipers, 2016 ). Comparative genomic analysis with four closely related Bacillus strains further reveals that the arrangement and composition of core biosynthetic operons in Bv. JN.Y2 are highly syntenic with the model strain Bv. FZB42. This stands is in contrast to Bv. CBMB205, which displays significant rearrangements within core clusters such as fen , dhb , and dif . Such high genetic conservation and structural stability in key metabolic pathways suggest that the biocontrol efficacy of Bv. JN.Y2 may be relatively predictable across diverse ecological environments (Rabbee et al. 2019 ; Wang et al. 2015 ; Xie et al. 2016 ;). Beyond conventional PKS/NRPS pathways, Bv. JN.Y2 exhibits unique characteristics in its RiPPs. The identification of four AOIs via BAGEL4 underscores the strain's competitive advantage in complex ecological niches. These AOIs are reported to encode products that regulate bacterial quorum sensing, induce membrane damage, or inhibit cell wall synthesis in pathogenic microorganisms (Scholz et al. 2014 ; Špacapan et al. 2020 ; Wang et al. 2024 ). Sequence alignment and characterization of core peptides reveal varying levels of conservation: while Amylocyclicin and LCI show high sequence identity with reference strains—indicating substantial evolutionary stability—the Class II lanthipeptide displays lower consistency. This divergence suggests that Bv. JN.Y2 may have undergone adaptive evolution under environmental pressures, potentially leading to the emergence of novel antimicrobial compounds with specialized diversity. Given the dissemination risks associated with acquired resistance genes among environmental microbiota, systematic biosafety evaluation of functional strains is an indispensable prerequisite for safeguarding agro-ecological equilibrium and public health (Von et al. 2016). Three-year field trials across two distinct locations demonstrated that Bv. JN.Y2 consistently enhances oat growth. To assess its environmental release risks, comprehensive genomic mining of acquired resistance genes and MGEs were performed. Despite the identification of MGEs and homologs of cfr(B) and tet(L) , Bv. JN.Y2 exhibited a robust biosafety profile. Notably, its plasmid-free architecture—with all ARGs anchored exclusively within the chromosomal scaffold or prophage regions—significantly mitigates the potential for HGT (Panel, 2018). Furthermore, the "no resistance" phenotype predicted by ResFinder suggests that these sequences represent non-functional evolutionary remnants rather than active resistance determinants, further reinforcing the suitability and safety of Bv. JN.Y2 for sustainable oat production. Comparative genomics serves as a sophisticated approach for elucidating evolutionary trajectories and functional variations (Alcaraz et al. 2010 ), providing a beneficial framework for identifying the superior traits of target strains. In this study, systematic comparative genomic analysis of Bv. JN.Y2 against both closely and distantly related strains revealed its distinct genetic differentiation and functional potential. Bv. JN.Y2 exhibits high sequence conservation and synteny with the rhizosphere-derived strain Bv. CBMB205. This observation aligns with the ecotype theory proposed by Cohan, which suggests that strains occupying similar ecological niches may utilize "periodic selection" to maintain genomic consistency by purging genetic diversity (Cohan, 2002 ). Such conservation implies that Bv. JN.Y2 possesses a relatively high degree of environmental plasticity, allowing it to adapt to both rhizosphere and endophytic habitats—a trait that likely supports its broad-spectrum plant growth-promoting potential. Conversely, comparisons with distantly related strains indicate that large-scale SVs are more prevalent than point mutations. This supports the hypothesis that genomic rearrangement, rather than single-nucleotide polymorphism, serves as a primary driver of functional divergence in the long-term evolution of the Bacillus genus (Feulner and De-Kayne, 2017 ). Regarding its biocontrol mechanisms, Bv. JN.Y2 displays a metabolic profile distinct from the model strain Bv. FZB42. While Iturin is a well-known lipopeptide with antagonistic activity against various fungal pathogens (Yan et al. 2020 ), our analysis revealed a "genes-present, cluster-incomplete" phenomenon in Bv. JN.Y2. Specifically, although ituABC genes were identified, antiSMASH failed to detect a complete BGC, suggesting localized gene loss or functional recombination during evolution. This metabolic differentiation often reflects specialized niche adaptation (Steinke et al. 2021 ), indicating that Bv. JN.Y2 may rely on variations within other conserved clusters, such as Fengycin or Pks, to maintain its antagonistic efficacy and stabilize its biocontrol performance. The identification of non-synonymous mutations in the sporulation kinase kinA reflects the role of the natural variation of the phosphorelay system in environmental adaptation (Stephenson and Hoch, 2002 ). Similar to other stress-resistant Bacillus strains, Bv. JN.Y2 appears to employ fine-tuned regulation of core developmental genes to navigate fluctuating environments, establishing a molecular basis for effective colonization. Furthermore, sequence polymorphisms in ComP and chemotaxis proteins, coupled with structural variations in upstream regulatory elements, suggest a more precise root exudate sensing system. This likely drives the evolution of Bv. JN.Y2 from a "generalist" toward an "environment-specific" specialist, consistent with findings that signal transduction refinement provides a competitive advantage in soil colonization (Blake et al. 2021 ). Pangenome analysis characterized Bv. JN.Y2 as having an "open" genome. The presence of 411 unique genes—involved in monobactam synthesis, environmental signaling, transmembrane transport, and cellular stress response—suggests that HGT has facilitated the acquisition of novel metabolic features. This genetic uniqueness likely supports the strain's functional specificity and provides a solid molecular foundation for the targeted development of its beneficial traits (Olanrewaju et al. 2021 ). While these candidate variations offer valuable insights for future functional genomics, their specific biological effects require further experimental validation. Conclusion This study provides a comprehensive evaluation of Bv. JN.Y2 as a beneficial biocontrol and PGP agent tailored for oat cultivation. Through a combination of longitudinal field trials and high-resolution genomic mining, we demonstrated that Bv. JN.Y2 offers a relatively stable performance in suppressing C. cereale while simultaneously enhancing crop yields across diverse environmental conditions. The genomic architecture of Bv. JN.Y2, characterized by its "open" pangenome and diverse array of antimicrobial BGCs, underscores its favorable evolutionary adaptation to the plant endosphere. Our comparative analysis revealed that Bv. JN.Y2 maintains a unique genetic identity through 411 strain-specific genes and key regulatory variations, such as those in the kinA and comP loci, which likely contribute to its efficient environmental sensing and colonization. Moreover, the plasmid-free nature and the absence of active resistance phenotypes establish a solid basis for its biosafety in agricultural applications. In summary, Bv. JN.Y2 represents a promising candidate for the development of targeted biopesticides and biofertilizers. While the identified candidate variations and unique metabolic pathways offer valuable insights, further functional studies will be beneficial to fully elucidate the specific biological effects of these genetic determinants in complex soil-plant ecosystems. Declarations Funding This work was supported by the Science and Technology Program of Inner Mongolia Autonomous Region [Grant No. 2025YFHH0165]; the Basic Research Fund for Universities Directly Affiliated with Inner Mongolia Autonomous Region [Grant No. BR251033]; the Central Government-Guided Local Science and Technology Development Fund [Grant Nos. 2022ZY0060 and 2022ZY0065]; the National Key Research and Development Program of China [Grant No. 2023YFD1600701-5]; and the China Agriculture Research System for Oat and Buckwheat [Grant No. CARS-07-C-3]. The support from these programs and organizations has been relatively helpful in the successful completion of this research. Ethical approval Not applicable. Informed Consent Not applicable. Competing interests The authors declare that they have no competing interests or conflict of interest. Author contribution W.Q. was responsible for conceptualization, methodology, investigation, formal analysis, writing – original draft, data curation, validation, and visualization. T.Z. contributed to investigation, methodology, validation, and resources. C.L.L. performed investigation, data curation, and formal analysis. S.X.S. contributed to investigation, resources, and validation. X.X.Z. was involved in investigation, methodology, and data curation. K.H.L. contributed to investigation, software, and visualization. C.Y.W. performed investigation, resources, and formal analysis. M.M.Z. contributed to formal analysis, validation, software, and writing – review & editing. B.Z.D. and H.Y.Z. were responsible for supervision, project administration, funding acquisition, conceptualization, and writing – review & editing. All authors reviewed and approved the final manuscript. Data Availability The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. The complete genome sequence has been deposited in the GenBank database under the accession numbers CP178220.1. Acknowledgments We gratefully acknowledge the financial support provided by the following programs and organizations: the Science and Technology Program of Inner Mongolia Autonomous Region (Grant No. 2025YFHH0165); the Basic Research Fund for Universities Directly Affiliated with Inner Mongolia Autonomous Region (Grant No. BR251033); the Central Government-Guided Local Science and Technology Development Fund (Grant Nos. 2022ZY0060 and 2022ZY0065); the National Key Research and Development Program of China (Grant No. 2023YFD1600701-5); and the China Agriculture Research System for Oat and Buckwheat (Grant No. CARS-07-C-3). 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Annual Reviews Microbiol 56(1):457–487. https://doi.org/10.1146/annurev.micro.56.012302.160634 Feulner PGD, De-Kayne R (2017) Genome evolution, structural rearrangements and speciation[J]. J Evol Biol 30(8):1488–1490. https://doi.org/10.1111/jeb.13101 Yan F, Li C, Ye X et al (2020) Antifungal activity of lipopeptides from Bacillus amyloliquefaciens MG3 against Colletotrichum gloeosporioides in loquat fruits[J]. Biol Control 146:104281. https://doi.org/10.1016/j.biocontrol.2020.104281 Steinke K, Mohite OS, Weber T et al (2021) Phylogenetic distribution of secondary metabolites in the Bacillus subtilis species complex[J]. Msystems, 6(2): 10.1128/msystems. 00057 – 21. https://doi.org/10.1128/msystems.00057-21 Stephenson K, Hoch JA (2002) Evolution of signalling in the sporulation phosphorelay[J]. Mol Microbiol 46(2):297–304. https://doi.org/10.1046/j.1365-2958.2002.03186.x Blake C, Nordgaard M, Maróti G et al (2021) Diversification of Bacillus subtilis during experimental evolution on A rabidopsis thaliana and the complementarity in root colonization of evolved subpopulations[J]. Environ Microbiol 23(10):6122–6136. https://doi.org/10.1111/1462-2920.15680 Olanrewaju OS, Ayilara MS, Ayangbenro AS et al (2021) Genome mining of three plant growth-promoting Bacillus species from maize rhizosphere[J]. Appl Biochem Biotechnol 193(12):3949–3969. https://link.springer.com/article/ 10.1007/s12010-021-03660-3 Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx FullUncroppedGelImages.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Apr, 2026 Reviews received at journal 18 Apr, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers invited by journal 29 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Submission checks completed at journal 18 Mar, 2026 First submitted to journal 13 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9114671","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614893852,"identity":"c1b6d680-9a5f-48be-9f73-c3f3c3536f96","order_by":0,"name":"Wei Quan","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Quan","suffix":""},{"id":614893855,"identity":"7d909511-e6bf-4f3f-90c7-504fc2abfe4f","order_by":1,"name":"Ting Zhang","email":"","orcid":"","institution":"Inner Mongolia Autonomous Region General Station of Forestry and Grassland","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zhang","suffix":""},{"id":614893859,"identity":"e6edd4a6-ee8d-44d3-bdd4-6a38471f08f4","order_by":2,"name":"Chen-Lu Liu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chen-Lu","middleName":"","lastName":"Liu","suffix":""},{"id":614893860,"identity":"b78f1968-c223-423c-bb0a-3dd8c3c37c90","order_by":3,"name":"Shao-Xuan Shi","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shao-Xuan","middleName":"","lastName":"Shi","suffix":""},{"id":614893863,"identity":"6852b661-807f-4027-9188-2a3240e6e6ed","order_by":4,"name":"Xi-Xi Zhang","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xi-Xi","middleName":"","lastName":"Zhang","suffix":""},{"id":614893867,"identity":"45820185-5afb-47c0-aa2e-eec10b66a0cf","order_by":5,"name":"Ke-Han Liu","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ke-Han","middleName":"","lastName":"Liu","suffix":""},{"id":614893871,"identity":"ca8fcac8-1b62-46d6-8943-f0330270acc6","order_by":6,"name":"Chun-Yang Wang","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Yang","middleName":"","lastName":"Wang","suffix":""},{"id":614893873,"identity":"e4477e63-a629-4ad3-9733-948c1f372310","order_by":7,"name":"Ming-Min Zhao","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ming-Min","middleName":"","lastName":"Zhao","suffix":""},{"id":614893878,"identity":"6ba6da60-6a23-4bb7-b858-a6682107465d","order_by":8,"name":"Bao-Zhu Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie3NsUrEMBjA8a8EOuXu1hTENxA+KUTkin2VlkJdCnY6brjhprqczhWfwjeIBK5LaNeADh7CLToUhMNB5XI4WqtuDvlDCHzJLwGw2f5jAoDsdgQiRIsB/Ry7vyJuvCqn6Z8I9X2qJPxIhtWFfMpnQXjEIs4GRbN3cK4Q2omE0fW8k3iqTsflMiXHZZQyr7ijXGXolLUEdi86CeqM+9SVLupoyQ53RGRIBoUEZFEP+ZAUdVywuKgpb56RvPcT/9G8yVAnBIUSlGvzi9NDPKU4ubpMEdXaWc2niSHr/HZRn1Kmu8mwWvgv+SYIsTpr5RuehLxJbh5eJ+P9UdlNTC77MhJm0e/um0jbc2iz2Ww2gC3kiWSd3bvt/wAAAABJRU5ErkJggg==","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Bao-Zhu","middleName":"","lastName":"Dong","suffix":""},{"id":614893880,"identity":"dda3c5ca-0bac-4bc6-bd00-dfecbce06911","order_by":9,"name":"Hong-You Zhou","email":"","orcid":"","institution":"Inner Mongolia Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Hong-You","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2026-03-13 12:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9114671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9114671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106056185,"identity":"6144e5e6-31ed-4577-a2d5-787f5a772407","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2957971,"visible":true,"origin":"","legend":"\u003cp\u003eThe inhibitory effect of Bv. JN.Y2 on \u003cem\u003eC. cereale \u003c/em\u003eunder dual-culture and greenhouse conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: (A)\u003c/strong\u003e Colony morphology of the pathogen \u003cem\u003eC. cereal\u003c/em\u003e on PDA medium (Control). \u003cstrong\u003e(B)\u003c/strong\u003e Dual-culture assay showing the inhibition zone produced by strain JN.Y2 against the pathogen. \u003cstrong\u003e(C–D)\u003c/strong\u003eMicroscopic observations of hyphal abnormalities induced by Bv. JN.Y2. Images show cellular swelling, deformation, and vacuolization of the fungal mycelia and spores. Scale bar = 10 μm. \u003cstrong\u003e(E–G) \u003c/strong\u003eBiocontrol efficacy assay on detached leaves. Leaves were inoculated with \u003cem\u003eC. cereal\u003c/em\u003e mycelial spore suspension. \u003cstrong\u003e(E)\u003c/strong\u003e Negative control (CK), treated with sterile water. \u003cstrong\u003e(F)\u003c/strong\u003ePositive control, treated with the chemical fungicide thiophanate-methyl. \u003cstrong\u003e(G)\u003c/strong\u003eTreatment with the cell suspension of Bv. JN.Y2.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/285502f1ceb080679e2aa0b4.jpeg"},{"id":106056187,"identity":"abed1247-3c0a-401b-b809-c0e2d2ab9029","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":941870,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the inhibitory ability of Bv. JN.Y2 against different pathogens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e The upper panel presents the dual-culture confrontation between Bv. JN.Y2 and various fungal pathogens. Data in the lower panel represent the mean ± SD. Different lowercase letters (a, b) above the bars for each pathogen denote significant differences between CK and the Dual-culture treatment, according to an independent samples \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). CK: blank control; Dual-culture: treatment with Bv. JN.Y2.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/ba112e87d945decd3db6d9c1.jpg"},{"id":106056189,"identity":"69ab5518-29dd-46a2-9435-035a2ca019aa","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4843221,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and molecular identification of Bv. JN.Y2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: (A)\u003c/strong\u003e Colony morphology of strain Bv. JN.Y2 grown on LB agar for 24 h. \u003cstrong\u003e(B)\u003c/strong\u003e Gram staining showing Gram-positive rod-shaped cells. Scale bar = 10 μm. \u003cstrong\u003e(C)\u003c/strong\u003e Endospore staining showing central or subterminal ellipsoidal spores. Scale bar = 10 μm. \u003cstrong\u003e(D)\u003c/strong\u003eAgarose gel electrophoresis of PCR products for 16S rRNA. \u003cstrong\u003e(E)\u003c/strong\u003ePhylogenetic tree constructed based on the sequences of 16S rRNA\u003cstrong\u003e \u003c/strong\u003egene using the Neighbor-Joining method. Bootstrap values (\u0026gt;50%) are indicated at the nodes. Bv. JN.Y2 is highlighted in red. \u003cstrong\u003e(F) \u003c/strong\u003ePhylogenomic tree constructed based on whole-genome SNPs. The scale bar represents the number of substitutions per site.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/27242388d38f348aeea3d452.png"},{"id":106095008,"identity":"9f40c225-0362-40df-8ad9-c29ed7d5fcbf","added_by":"auto","created_at":"2026-04-03 11:43:55","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4144943,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of potential biocontrol characteristics of Bv. JN.Y2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e \u003cstrong\u003e(A–E)\u003c/strong\u003e Qualitative detection of extracellular hydrolytic enzymes on agar plates. Clear halos around the colonies indicate positive activities for protease, amylase, cellulase, pectinase and chitinase (A–E). \u003cstrong\u003e(F)\u003c/strong\u003e Biofilm phenotype of strains \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e YN.J3 and Bv. JN.Y2 at the air-liquid interface in MSgg medium compared to the control (MSgg+LB) after 48 h. \u003cstrong\u003e(G)\u003c/strong\u003e Colonization dynamics of Bv. JN.Y2 in the root, stem, and leaf tissues of oats. Samples were collected at 1, 3, 7, 10, 20, and 30 dpi. Data are presented as mean ± SD. Different lowercase letters (a, b, c) in the same column indicate significant differences according to Duncan's multiple range test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/b41f82af5d2ed88aca3b3134.jpeg"},{"id":106056190,"identity":"3a697d7b-56b8-463a-b38b-adf417814094","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2698719,"visible":true,"origin":"","legend":"\u003cp\u003ePotential PGP properties of Bv. JN.Y2 and its effects on oat growth.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e \u003cstrong\u003e(A–D)\u003c/strong\u003e Qualitative assays of PGP traits \u003cem\u003ein vitro\u003c/em\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Nitrogen metabolism and assimilation capacity; \u003cstrong\u003e(B)\u003c/strong\u003e Phosphate solubilization; \u003cstrong\u003e(C)\u003c/strong\u003e Potassium solubilization; \u003cstrong\u003e(D)\u003c/strong\u003e Siderophore production. \u003cstrong\u003e(E)\u003c/strong\u003e Phenotypic comparison of seedlings treated with Bv. JN.Y2 suspension compared to the sterile water treatment.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/7c343c359f0f89cd47d2b568.jpeg"},{"id":106094094,"identity":"1d4887bc-8548-452a-bd1e-51061b6534e1","added_by":"auto","created_at":"2026-04-03 11:40:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3500927,"visible":true,"origin":"","legend":"\u003cp\u003eCircular genome map of Bv. JN.Y2 and its COG function classification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eThe outermost circle represents the genomic coordinates. Moving from the outside inward, the tracks display: gene functional annotation results (COG categories), ncRNA locations, GC content, and GC skew. GC content was calculated using a window and step size of (chromosome length/1000) bp. Blue regions pointing inward indicate that the GC content is lower than the genome-wide average, while red regions pointing outward indicate the opposite; the peak height represents the magnitude of the difference from the average. GC skew was calculated using the formula (G-C)/(G+C) with a window and step size of (chromosome length/1000) bp. Green regions pointing inward indicate that the G content is lower than C, while orange regions pointing outward indicate the opposite.\u003cstrong\u003e \u003c/strong\u003eThe x-axis represents the COG functional categories, and the y-axis represents the number of annotated genes.\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/6bf45f90389ecbb487dc48d3.jpg"},{"id":106414814,"identity":"dbd8e575-a9ba-4849-9907-eb33cc829941","added_by":"auto","created_at":"2026-04-08 10:25:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3531405,"visible":true,"origin":"","legend":"\u003cp\u003eComparisons of secondary metabolites synthesis clusters in Bv. JN.Y2, Bv. FZB42, Bv. SQR9, Bv, CBMB205 and Ba. GKT04.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e The genetic organization of eight predicted gene clusters is shown. Genes are represented by arrows, with the arrowhead indicating the direction of transcription. Color coding is based on predicted function: Dark red indicates core biosynthetic genes; light red/pink indicates additional biosynthetic genes; blue indicates transport-related genes; green indicates regulatory genes; and grey indicates other genes. Key biosynthetic genes are labeled with their standard gene names, including \u003cem\u003esrf\u003c/em\u003e(surfactin), \u003cem\u003efen\u003c/em\u003e (fengycin), \u003cem\u003ebtr\u003c/em\u003e/\u003cem\u003epks\u003c/em\u003e (butirosin), \u003cem\u003emln\u003c/em\u003e(macrolactin), \u003cem\u003ebae\u003c/em\u003e (bacillaene), \u003cem\u003edhb\u003c/em\u003e (bacillibactin), \u003cem\u003ebac\u003c/em\u003e(bacilysin), and \u003cem\u003edif\u003c/em\u003e (difficidin) operons. Abbreviations: \u003cstrong\u003eBv\u003c/strong\u003e, \u003cem\u003eBacillus velezensis\u003c/em\u003e; \u003cstrong\u003eBa\u003c/strong\u003e, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/c06c6c63daa46d0ce8056ada.jpg"},{"id":106056193,"identity":"484ee46d-f9c0-47b5-abbc-65542ead5b58","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1947046,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic organization and putative functional assignment in Bv. JN.Y2 genome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e The diagram illustrates four AOIs identified via the BAGEL4 platform within the Bv. JN.Y2 genome. Horizontal tracks represent specific genomic regions, with numerical coordinates in parentheses indicating their precise locations on the chromosome.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/c97086f7bb0a282ce1d5f756.png"},{"id":106095365,"identity":"4a26e017-b353-47f7-b58f-c93a140e356e","added_by":"auto","created_at":"2026-04-03 11:47:42","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":4986884,"visible":true,"origin":"","legend":"\u003cp\u003eComparative sequence analysis and conservation profiling of core antimicrobial peptides from Bv. JN.Y2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e The figure presents the multiple sequence alignment of four core antimicrobial peptides identified within the Bv. JN.Y2 genome (corresponding to AOI_01, AOI_03, and the two subunits of AOI_04) compared with their representative homologs. The sequences were initially aligned using Clustal Omega, and the resulting genomic architecture was professionally visualized through ESPript 3.2. In this alignment profile, white characters on a red background signify absolute conservation (100% identity) across all analyzed strains, while yellow-boxed characters represent residues with similar physicochemical properties.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/4af3122726a763d6ea2c54de.png"},{"id":106056195,"identity":"2e7c1fcd-bee7-4488-996e-6b56baaecd41","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2132184,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomic analysis of Bv. JN.Y2 and eight reference strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Statistics of InDels detected in the genomes of reference strains compared to Bv. JN.Y2. Blue bars represent insertions, and red bars represent deletions. \u003cstrong\u003e(B)\u003c/strong\u003e Flower plot illustrating the core and pan-genome analysis. The central circle represents the number of core genes shared by all nine analyzed strains (1,086 genes), while the petals indicate the number of strain-specific (unique) genes for each strain. Abbreviations:\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eBv,\u003c/strong\u003e\u003cem\u003e B. velezensis\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eBa, \u003c/strong\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e;\u003cstrong\u003eBs,\u003c/strong\u003e\u003cem\u003e B. subtilis\u003c/em\u003e;\u003cstrong\u003e Bl,\u003c/strong\u003e\u003cem\u003e B. licheniformis\u003c/em\u003e;\u003cem\u003e \u003c/em\u003e\u003cstrong\u003eBm,\u003c/strong\u003e\u003cem\u003eB. megaterium\u003c/em\u003e; \u003cstrong\u003eBt,\u003c/strong\u003e\u003cem\u003e B. thuringiensis\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/35153361dc39b0a33bcc3935.png"},{"id":107479820,"identity":"99f61e84-e636-435c-8e58-02de6e99395b","added_by":"auto","created_at":"2026-04-22 01:53:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33328542,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/36b9528d-0139-4b96-8231-0d1582cf49cd.pdf"},{"id":106094202,"identity":"c6359ae1-dd3c-4d43-9bab-d0d66b5005f7","added_by":"auto","created_at":"2026-04-03 11:41:42","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6322479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/1a9c11d570c98943272dc9bf.docx"},{"id":106056188,"identity":"7e334881-da37-458b-a41b-7eeae72eac49","added_by":"auto","created_at":"2026-04-03 01:32:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":657229,"visible":true,"origin":"","legend":"","description":"","filename":"FullUncroppedGelImages.docx","url":"https://assets-eu.researchsquare.com/files/rs-9114671/v1/f4bb6bca81f4b38bcabfd736.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-genome and Comparative Genomic Analysis Reveal the Biocontrol and Plant Growth-Promoting Potential of Bacillus velezensis JN.Y2 Against Oat Anthracnose","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOats (\u003cem\u003eAvena sativa\u003c/em\u003e) are globally significant cereal crops renowned for their multifunctional properties and rich nutritional profile (Butt et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In China, naked oats (\u003cem\u003eA. nuda\u003c/em\u003e) are predominantly cultivated in regions such as Inner Mongolia, Hebei, and Ningxia, serving as both functional food and essential forage. However, oat cultivation is severely threatened by anthracnose, a destructive disease primarily caused by \u003cem\u003eColletotrichum cereale\u003c/em\u003e (Crouch and Beirn, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other species, including \u003cem\u003eC. americae-borealis\u003c/em\u003e and \u003cem\u003eC. graminicola\u003c/em\u003e, also pose significant risks to oat production (Politis, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While chemical fungicides remain the conventional management strategy, their application is increasingly hampered by high costs, environmental impacts, and the emergence of pathogen resistance (Janisiewicz and Conway, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiological control using beneficial plant endophytes offers a sustainable and eco-friendly alternative to chemical treatments (Khairullina et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Endophytes are recruited by plants from the rhizosphere, often through chemotactic responses to root exudates, and play a critical role in enhancing host health and resilience (Ghimire et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Malfanova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Among these, the genus \u003cem\u003eBacillus\u003c/em\u003e is distinguished by its robust and well-documented efficacy in promoting plant growth and suppressing pathogens (Miljaković et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e strains facilitate plant health through nutrient solubilization, phytohormone synthesis, and the production of a diverse arsenal of secondary metabolites, such as lipopeptides and cell wall-degrading enzymes (Alamri, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Choub et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Grahovac et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite the recognized potential of \u003cem\u003eBacillus\u003c/em\u003e species as biocontrol agents, research on endophytic strains specifically adapted to the oat microenvironment remains limited. While certain strains like \u003cem\u003eB. subtilis\u003c/em\u003e and \u003cem\u003eB. velezensis\u003c/em\u003e have shown promise in controlling oat pathogens, there is still a lack of specific strains widely applied for targeted disease management. Understanding the regulatory mechanisms and colonization strategies of these bacteria is essential for their effective application (Nicholson et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Ye et al. 2021; Zhang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, an endophytic strain, Bv. JN.Y2, was isolated from healthy oat leaves in Inner Mongolia. Its broad-spectrum antifungal potential and biocontrol efficacy against oat anthracnose were assessed through in vitro assays, greenhouse experiments, and systematic three-year multi-location field trials. Furthermore, integrated whole-genome and comparative genomic analyses were performed to characterize the genetic landscape of Bv. JN.Y2, specifically investigating evolutionary structural variations, secondary metabolite biosynthesis, and environmental sensing mechanisms. This research aims to elucidate the molecular determinants of the biocontrol and plant growth-promoting capacities of Bv. JN.Y2, providing a beneficial theoretical foundation for its stable colonization and sustainable application within the oat microenvironment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, pathogens and culture condition\u003c/h2\u003e \u003cp\u003eThe strains used in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Specifically, the endophytic bacterial Bv. JN.Y2 was isolated from healthy oat leaves collected in Jining District, Ulanqab City, Inner Mongolia, China. A kanamycin-resistant mutant, Bv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e, which was previously constructed and maintained in our laboratory, was used for colonization assays. Stock cultures were maintained at -80 ℃ in Luria-Bertani (LB) broth (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract) supplemented with 30% (v/v) glycerol. For routine use, Bv. JN.Y2 was cultured on LB agar at 37 ℃ for 24 h. When culturing Bv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e, the medium was supplemented with 30 \u0026micro;g/mL kanamycin sulfate.\u003c/p\u003e \u003cp\u003eThe fungal pathogens used in this study included \u003cem\u003eColletotrichum cereale\u003c/em\u003e, \u003cem\u003eColletotrichum fructicola\u003c/em\u003e, \u003cem\u003ePhyllosticta sorghina\u003c/em\u003e, \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eFusarium graminearum\u003c/em\u003e, \u003cem\u003eAlternaria alternata\u003c/em\u003e, \u003cem\u003eRhizoctonia solani\u003c/em\u003e, and \u003cem\u003eDidymella glomerata\u003c/em\u003e. All fungi were obtained from the Key Laboratory of Biopesticide Creation and Resource Utilization of Inner Mongolia Autonomous Region. Fungal stocks were stored at -80 ℃ in 25% (v/v) glycerol. For experiments, mycelial plugs from frozen stocks were inoculated onto the center of Potato Dextrose Agar (PDA, 200 g/L potato, 20 g/L glucose, 18 g/L agar) plates and incubated in the dark at 25 ℃ for 10\u0026ndash;12 days.\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\u003eBacterial, plasmids and fungal strains used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrains/Plasmids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStrains\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type biocontrol strain isolated from oat.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpontaneous kanamycin-resistant mutant (\u003cem\u003ekan\u003c/em\u003e\u003csup\u003eR\u003c/sup\u003e); used for colonization assays.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e YN.J3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBiocontrol strain; used as a positive control for biofilm formation assays.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFungal Pathogens\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum cereale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthracnose pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eColletotrichum fructicola\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthracnose pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePhyllosticta sorghina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf spot pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusarium oxysporum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoot rot pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFusarium graminearum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHead blight pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAlternaria alternata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf blotch pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRhizoctonia solani\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoot rot pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDidymella glomerata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeaf spot pathogen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThis laboratory\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eantifungal activity assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antagonistic activity of Bv. JN.Y2 was evaluated using a dual-culture assay (Xu, 2020). A 5-mm mycelial plug of each pathogen was placed 2 cm from the edge of a PDA plate. Bv. JN.Y2 was streaked on the opposite side, 3 cm from the fungal plug. Plates inoculated only with the pathogen alone served as controls. After incubation at 25 ℃ for 5\u0026ndash;10 days, the radial growth of the fungus in control (Rc) and treatment (Rt) plates was measured. Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. The inhibition rate was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{I}\\text{n}\\text{h}\\text{i}\\text{b}\\text{i}\\text{t}\\text{i}\\text{o}\\text{n}\\left(\\text{%}\\right)=\\frac{\\text{R}\\text{c}-\\text{R}\\text{t}}{\\text{R}\\text{c}}\\times100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAdditionally, to assess the morphological impact, hyphae from the edge of the inhibition zone were observed under a microscope to detect potential abnormalities (Kim, 2022).\u003c/p\u003e \u003cp\u003eTo evaluate the broad-spectrum antagonistic activity of Bv. JN.Y2, the strain was tested against seven additional phytopathogenic fungi: \u003cem\u003eC. fructicola\u003c/em\u003e, \u003cem\u003eP. sorghina\u003c/em\u003e, \u003cem\u003eF. oxysporum\u003c/em\u003e, \u003cem\u003eF. graminearum\u003c/em\u003e, \u003cem\u003eA. alternata\u003c/em\u003e, \u003cem\u003eR. solani\u003c/em\u003e, and \u003cem\u003eD. glomerata\u003c/em\u003e. Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. All plates were incubated under the same conditions, and the inhibition rates were calculated.\u003c/p\u003e \u003cp\u003eThe biocontrol efficacy of Bv. JN.Y2 was evaluated in a greenhouse pot experiment (Meng et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). A spore suspension of \u003cem\u003eC. cereale\u003c/em\u003e was prepared from 14-day-old PDA cultures by rinsing with 0.1 M PBS, with the concentration adjusted to 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e spores/mL using a hemocytometer. The bacterial inoculum was prepared by harvesting late-exponential phase Bv. JN.Y2 cells through centrifugation at 4\u0026deg;C and 6,000 rpm for 10 min, followed by washing and resuspension in 0.1 M PBS to a final concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Three treatments were established: (1) Bv. JN.Y2 treatment followed by \u003cem\u003eC. cereale\u003c/em\u003e inoculation; (2) sterile 0.1 M PBS treatment followed by \u003cem\u003eC. cereale\u003c/em\u003e inoculation as a negative control; (3) thiophanate-methyl (TM, 1,500-fold dilution) treatment followed by \u003cem\u003eC. cereale\u003c/em\u003e inoculation as a positive control. Each treatment consisted of three independent biological replicates, with five technical replicates per biological replicate. The disease incidence, disease index and control efficacy were calculated as previously described: Grade 1, 0%; Grade 2, \u0026lt; 10%; Grade 3, 11%\u0026ndash;25%; Grade 4, 26%\u0026ndash;50%; and Grade 5, \u0026gt; 50% or necrotic (McKinney, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1923\u003c/span\u003e). The disease index and control efficacy were calculated as below:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{D}\\text{I}=\\frac{\\left(\\sum\\left(\\text{G}\\text{r}\\text{a}\\text{d}\\text{e}\\times\\text{n}\\right)\\right)}{\\left(\\text{N}\\times5\\right)}\\times100$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\text{e}\\text{f}\\text{f}\\text{i}\\text{c}\\text{a}\\text{c}\\text{y}\\left(\\text{%}\\right)=\\frac{\\left(\\text{D}{\\text{I}}_{\\left(\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}-\\text{D}{\\text{I}}_{\\left(\\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}\\right)}\\right)}{\\text{D}{\\text{I}}_{\\left(\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\right)}}\\times100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eField trials\u003c/h3\u003e\n\u003cp\u003eField trials were conducted during the 2023, 2024, and 2025 growing seasons at two distinct locations in Inner Mongolia: Jining District (40.92\u0026deg;N, 113.15\u0026deg;E) and Wuchuan County (41.20\u0026deg;N, 111.47\u0026deg;E). Both sites feature chestnut soil and are endemic regions for oat anthracnose, with severe annual outbreaks of \u003cem\u003eC. cereale\u003c/em\u003e. During the trial period, annual precipitation increased significantly from approx. 340\u0026ndash;360 mm in 2023 to over 500\u0026ndash;600 mm in 2024 and 2025, providing a rigorous environment for evaluating biocontrol stability. The experiment followed a randomized complete block design with three treatments (five replicates each): (1) Untreated Control (CK); (2) TM, 1,500-fold dilution; and (3) Bv. JN.Y2 cell suspension (1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). Each plot was 30 m\u003csup\u003e2\u003c/sup\u003e (5 m \u0026times; 6 m) with a 1 m buffer zone maintained between adjacent plots. The oat cultivar \"Baiyan No. 2\" was sown at a rate of 180 kg/ha. For the Bv. JN.Y2 treatment, seeds were soaked in the bacterial suspension for 2 h prior to sowing, followed by two foliar sprays in booting stage and heading stage. The CK group seeds were soaked in sterile water for 2 h as a control for the soaking process. For the TM treatment, only foliar sprays were applied at the same time points, with no seed soaking performed. Standard local agronomic practices were followed uniformly across all plots. In October, the disease index and grain yield (kg/ ha) were recorded.\u003c/p\u003e\n\u003ch3\u003eIdentification and characterization of Bv. JN.Y2\u003c/h3\u003e\n\u003cp\u003eThe morphological characteristics of Bv. JN.Y2 were observed after incubation on LB agar at 37\u0026deg;C for 24 h. Colony morphology, including shape, color, margin, and surface texture, was recorded. Cell morphology, Gram reaction, and endospore formation were examined using light microscopy (Olympus, Tokyo, Japan), respectively (Zhou et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Motility was assessed using semi-solid agar tubes.\u003c/p\u003e \u003cp\u003ePhysiological and biochemical characteristics were evaluated according to Bergey\u0026rsquo;s Manual of Systematic Bacteriology (Sneath et al. 1986). Catalase activity was determined by bubble formation in 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and oxidase activity was tested using tetramethyl-p-phenylenediamine. Hydrolytic activities were assessed on agar plates supplemented with specific substrates: starch (amylase), gelatin (gelatinase), skim milk (protease), and carboxymethyl cellulose (cellulase). The metabolic profile was further characterized through standard assays, including nitrate reduction, Voges-Proskauer (V-P), methyl red, indole production, H\u003csub\u003e2\u003c/sub\u003eS production, urease activity, and citrate utilization. Carbon source utilization was tested in minimal medium supplemented with 1% (w/v) of glucose, sucrose, maltose, D-mannitol, or lactose as the sole carbon source.\u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from overnight cultures of Bv. JN.Y2 using a bacterial genomic DNA extraction kit (Tiangen, Beijing, China). For taxonomic identification, the 16S rRNA gene was amplified using specific primers as detailed in Online Resource 1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The PCR amplification was performed under the following conditions: initial denaturation at 95\u0026deg;C for 3 min; followed by 33 cycles of denaturation at 94\u0026deg;C for 25 s, annealing at 57\u0026deg;C for 25 s, and extension at 72\u0026deg;C for 60 s; and a final extension at 72\u0026deg;C for 5 min. PCR products were purified and sequenced by Tsingke Biotechnology Co., Ltd. (Beijing, China). The resulting sequence was aligned with reference sequences of closely related \u003cem\u003eBacillus\u003c/em\u003e species retrieved from the NCBI database using ClustalW. A phylogenetic tree was constructed based on the sequences of 16S rRNA using the Neighbor-Joining method in MEGA 7.0 software. The reliability of the phylogenetic clusters was evaluated by bootstrap analysis with 1,000 replicates.\u003c/p\u003e\n\u003ch3\u003eCharacterization of enzymatic activity, biofilm formation, and plant colonization\u003c/h3\u003e\n\u003cp\u003eHydrolytic enzyme assays: Extracellular enzyme production was screened on agar plates supplemented with specific substrates: 1% (w/v) skim milk for protease, 1% (w/v) soluble starch for amylase (Guleria et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), 1% (w/v) sodium carboxymethyl cellulose for cellulase (Teather et al. 1982), 1% (w/v) apple pectin for pectinase (Kuvvet et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and 0.5% (w/v) colloidal chitin for chitinase (Agrawal et al. 2012). After incubation at 30 ℃ for 3\u0026ndash;5 days, the presence of clear halos around colonies indicated positive enzymatic activity. To visualize halos for cellulase and pectinase, plates were stained with 0.1% Congo red for 15 min and destained with 1 M NaCl to 10 min.\u003c/p\u003e \u003cp\u003eBiofilm formation was quantified in MSgg medium using a 24-well plate assay as described previously (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The hyper-biofilm-forming strain \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e YN.J3 served as a positive control. Briefly, 10 \u0026micro;L of an overnight culture (adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.0) was inoculated into 2 mL MSgg medium and incubated statically at 37 ℃ for 48 h. Biofilms were stained with 0.1% (w/v) crystal violet, gently washed three times with 0.1 M PBS to remove planktonic cells, and subsequently solubilized with 30% (v/v) acetic acid. Biofilm biomass was quantified by measuring the absorbance at 570 nm (A\u003csub\u003e570\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eThe colonization ability of Bv. JN.Y2 was evaluated using the kanamycin-resistant mutant Bv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e (Fan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thirty-day-old oat seedlings were inoculated via root drenched with 50 mL of a bacterial suspension (1.5 \u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL). At 1, 3, 7, 10, 20, and 30 days post-inoculation (dpi), the root, stem, and leaf tissues were harvested. Samples were surface-sterilized with 75% ethanol and 3% NaClO, rinsed five times with sterile water, and homogenized. The homogenates were serially diluted and plated on LB agar containing 30 \u0026micro;g/mL kanamycin. After incubation at 37\u0026deg;C for 24 h, the colonizing population was enumerated and expressed as CFU/g fresh weight.\u003c/p\u003e\n\u003ch3\u003eEvaluation of plant growth-promoting traits and growth enhancement\u003c/h3\u003e\n\u003cp\u003eThe plant growth-promoting (PGP) potential of Bv. JN.Y2 was evaluated through multiple assays. Nitrogen metabolism and assimilation was assessed by observing bacterial growth on nitrogen-free Ashby's medium. Phosphate and potassium solubilization were determined by the formation of clear halos on Pikovskaya's agar and Aleksandrov agar, respectively. Siderophore production was detected using Chrome Azurol S (CAS) agar, where a change in color to an orange halo indicated a positive result (Ariyani, 2021; Qiu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA greenhouse experiment was conducted to evaluate the effect of Bv. JN.Y2 on oat growth, following the protocol of Wang (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with minor modifications. Oat seeds (cv. Baiyan No. 2) were surface-sterilized with 75% ethanol for 30 s and 3% NaClO for 5 min, followed by five rinses with sterile water. Germinated seedlings were transplanted into pots at the two-leaf stage. Ten-day-old seedlings were inoculated via root drenching with 50 mL of the Bv. JN.Y2 suspension (1.5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/mL). Control plants were treated with an equal volume of sterile water. All plants were maintained in a greenhouse at 28\u0026deg;C, 60% relative humidity, with a16 h/8 h (light/dark cycle). After 30 days, plant height, root length and root fresh weight were measured. To determine the root dry weight (biomass), samples were dried at 65\u0026deg;C for 72 h until a constant weight was achieved.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing, assembly, and annotation\u003c/h2\u003e \u003cp\u003eHigh-quality genomic DNA of Bv. JN.Y2 was isolated using the SDS-CTAB protocol. A hybrid sequencing strategy was employed by Novogene Co., Ltd., utilizing both PacBio Sequel and Illumina NovaSeq PE150 platforms. The raw PacBio subreads were processed via SMRT Link (v8.0) and assembled \u003cem\u003ede novo\u003c/em\u003e using Canu (v2.0) (Koren et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To enhance sequence accuracy, the resulting assembly was polished with Illumina short reads using Pilon (v1.22), yielding a single circular chromosome.\u003c/p\u003e \u003cp\u003eGenome annotation was performed through an integrated pipeline. GeneMarkS (v4.17) was used for coding sequence prediction (Besemer et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), while repetitive elements were identified via RepeatMasker and Tandem Repeats Finder (TRF) (Benson, 1999). The non-coding RNA (ncRNA) fractions, including tRNAs and rRNAs, were detected using tRNAscan-SE and rRNAmmer, respectively (Lowe and Eddy, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Lagesen et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kalvari et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Furthermore, mobile genetic elements (MGEs), such as prophages and genomic islands, were localized using phiSpy and IslandPath-DIOMB(Hsiao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Akhter et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ge et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFunctional characterization of the proteome was achieved by searching against multiple databases, including KEGG, GO, COG, and Swiss-Prot(Ashburner et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Galperin et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kanehisa et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kanehisa et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Cantarel et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Carbohydrate-active enzymes were categorized using dbCAN. The final genomic landscape was visualized using a Circos map (Krzywinski et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenome mining and comparative analysis of biosynthetic gene clusters\u003c/h3\u003e\n\u003cp\u003eBGCs responsible for secondary metabolite production in Bv. JN.Y2 genome were predicted using antiSMASH (v8.0) (Blin et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The analysis was performed using the \"relaxed\" detection strictness to maximize the identification of putative clusters. To characterize the genetic organization and evolutionary conservation of these clusters, a comparative analysis was performed between Bv. JN.Y2 and four closely related strains \u003cem\u003eB. velezensis\u003c/em\u003e FZB42 (Bv. FZB42), \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 (Bv. SQR9), \u003cem\u003eB. velezensis\u003c/em\u003e CBMB205 (Bv. CBMB205) and \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e GKT04 (Ba. GKT04). The ClusterBlast module integrated into antiSMASH was employed to identify homologous gene clusters and visualize synteny. The schematic diagrams of gene arrangement and orientation were generated directly based on the antiSMASH visualization outputs.\u003c/p\u003e\n\u003ch3\u003eGenomic characterization and comparative analysis of bacteriocin biosynthetic gene clusters in Bv. JN.Y2\u003c/h3\u003e\n\u003cp\u003eThe whole-genome sequence of Bv. JN.Y2 was subjected to comprehensive genome mining using the BAGEL4 web server to identify potential bacteriocins and Ribosomally synthesized and Post-translationally modified Peptides (RiPPs) (Liu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The identification process integrated core peptide searches and context-based gene cluster detection. Based on genomic proximity and functional relevance, the identified loci were categorized into specific Areas of Interest (AOIs) for subsequent detailed characterization. To evaluate the evolutionary conservation and structural features of the identified core peptides, representative homologous sequences were retrieved from the NCBI GenBank and BAGEL4 databases. Multiple sequence alignment was executed using the Clustal Omega algorithm with default parameters (Lugani and Sooch, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The resulting alignment profile was professionally rendered using ESPript 3.2 to visualize conserved residues and physicochemical properties (Peng et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Absolute conservation was defined by a 100% identity threshold across all compared strains, while similarity was assessed based on the equivalence of amino acid residues. The functional annotation of the core peptides was further validated through BLASTp searches against the non-redundant protein database at NCBI.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenomic screening for acquired resistance genes and biosafety evaluation\u003c/h2\u003e \u003cp\u003eTo evaluate the genomic safety of Bv. JN.Y2 for potential agricultural applications, the presence of acquired antimicrobial resistance (AMR) genes was screened using the ResFinder (Version 4.7.2) database (Kim et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The whole-genome sequence was analyzed with a minimum identity threshold of 80% and a minimum length coverage of 60%. To further assess the potential for horizontal gene transfer (HGT), the chromosomal coordinates of identified AMR homologs were cross-referenced against the boundaries of genomic islands and prophage regions, which were predicted using IslandPath-DIOMB program and PHAST, respectively. Phenotypic resistance predictions were derived from the ResFinder system to ensure a comprehensive evaluation of Bv. JN.Y2 safety.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative genomic analysis between Bv. JN.Y2 and reference\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the comparative genomic analysis, eight representative \u003cem\u003eBacillus\u003c/em\u003e strains were selected to serve as taxonomic and functional references (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These include species type strains to establish a precise phylogenetic framework, and established model biocontrol agents to provide a comparative context for evaluating the specific PGP and antagonistic mechanisms of Bv. JN.Y2. Furthermore, distantly related lineages were included to better delineate the core and strain-specific genomic features of the genus. Whole-genome alignments and synteny analyses were conducted using MUMmer (v3.23) and LASTZ (v1.03.54) (Kurtz, et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Harris, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Chiaromonte et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). These tools were employed to identify Single Nucleotide Polymorphisms (SNPs), small insertions/deletions (Indels), and structural variations (SVs) across the genomes. To evaluate the evolutionary relationships, a phylogenetic tree based on genome-wide SNPs was constructed using Treebest (v1.9.2) and PhyML (v3.0). Pan-genome analysis was performed via CD-HIT (v4.6.1) to classify the core, dispensable, and strain-specific gene sets.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStrain information required for comparative genome analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrain name in report\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenebank accession number\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\u003eB. velezensis\u003c/em\u003e JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP178220.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. licheniformis\u003c/em\u003e ATCC 14580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBl.14580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP140161.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. mycoides\u003c/em\u003e BPN36/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBm.BPN36.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP035997.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. subtilis subsp. subtilis str.\u003c/em\u003e 168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBs.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAL009126.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB.velezensis\u003c/em\u003e SQR9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.SQR9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP006890.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB.velezensis\u003c/em\u003e CBMB205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.CBMB205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP011937.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB.velezensis\u003c/em\u003e FZB42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.FZB42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP000560.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. thuringiensis serovar berliner\u003c/em\u003e ATCC 10792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBt.10792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCM000753.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eB. amyloliquefaciens\u003c/em\u003e GKT04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBa.GKT04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCP072120.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with three independent biological replicates to ensure the reproducibility and reliability of the data. Within each biological replicate, five technical replicates were performed. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Prior to analysis, the normality of the distribution and homogeneity of variance were confirmed using Shapiro-Wilk and Levene\u0026rsquo;s tests, respectively. For the PGP experiment, an independent samples t-test was employed to evaluate the statistical significance of the differences. Statistical significance was determined using one-way analysis of variance (ANOVA). Significant differences between treatment means were evaluated using Duncan\u0026rsquo;s multiple range test and the Least Significant Difference test at a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical computations were performed using SPSS Statistics v26.0 (IBM Corp., Armonk, NY, USA). Graphical representations were generated using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The final multi-panel figures and illustrations were compiled and processed using Adobe Photoshop 2021 software (Adobe Systems Inc., San Jose, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBv. JN.Y2 exhibits broad-spectrum antifungal activity and effective control to oat anthracnose\u003c/h2\u003e \u003cp\u003eAn endophytic bacterium, designated as Bv. JN.Y2, was isolated from healthy oat plants. In dual-culture assays, Bv. JN.Y2 significantly suppressed the mycelial growth of \u003cem\u003eC. cereale\u003c/em\u003e, achieving an inhibition rate of 74.49% (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;1A, 1B). Microscopic examination revealed that Bv. JN.Y2 induced swelling and deformation of the hyphal tips, indicating a disruption of apical hyphal extension (Fig.\u0026nbsp;1C, 1D). In greenhouse pot experiments, Bv. JN.Y2 demonstrated a control efficacy of 62.40% (Fig.\u0026nbsp;1E-G), which is better than TM.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of the inhibitory effect of Bv. JN.Y2 on \u003cem\u003eC. cereale\u003c/em\u003e under dual-culture and greenhouse conditions.\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCK\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDual-culture inhibitory rate(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGreenhouse disease incidence (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGreenhouse disease index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGreenhouse control efficacy (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.67\u0026thinsp;\u0026plusmn;\u0026thinsp;12.58 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.67\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e62.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThiophanate-methyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e86.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.64 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.67\u0026thinsp;\u0026plusmn;\u0026thinsp;5.03 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003eNote\u003c/b\u003e: Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different lowercase letters (a, b, c) in the same column indicate significant differences according to Duncan's multiple range test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv description=\"\" class=\"Drawing\" id=\"285172169\" name=\"图片 1\"\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDuring the 2023\u0026ndash;2025 trial period, annual precipitation in Wuchuan and Jining showed a significant increasing trend, shifting from near-normal levels in 2023 (approximately 340\u0026ndash;360 mm) to historical extreme rainfall in 2024 and 2025 (over 500\u0026ndash;600 mm). Despite the increased disease pressure from surging humidity, Bv. JN.Y2 demonstrated stable biocontrol efficacy (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). During the initial field trials in 2023, the control efficacy of Bv. JN.Y2 was recorded at 56.01% in Jining and 64.96% in Wuchuan. These values were comparatively higher than those observed for the chemical fungicide TM, which yielded control efficacy values of 42.69% and 53.94% at the respective sites. In 2024, as annual precipitation increased, Bv. JN.Y2 continued to demonstrate stable biocontrol performance, achieving control efficiencies of 63.41% in Jining and 60.55% in Wuchuan. During the same period, the CE of the chemical treatment was recorded at 54.75% and 50.58%, respectively. By 2025, a year characterized by historically high rainfall levels, the biocontrol stability of Bv. JN.Y2 remained evident. The strain maintained control efficiencies of 56.15% in Jining and 65.64% in Wuchuan, consistently exceeding the suppression levels provided by TM (46.53% and 50.08%, respectively). Collectively, the three-year monitoring data suggest that Bv. JN.Y2 possesses a balanced and reliable capacity to suppress oat anthracnose across fluctuating environmental conditions and varying levels of disease pressure.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eControl efficiency of Bv. JN.Y2 on oat anthracnose and yield in two experimental sites from year 2023 to 2025.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYear\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTest site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDisease index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eControl efficiency\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eYield (kg/ha)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUlanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region(40.92506N, 113.15690E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2940.00\u0026thinsp;\u0026plusmn;\u0026thinsp;164.17 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3391.35\u0026thinsp;\u0026plusmn;\u0026thinsp;80.82 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e39.45\u0026thinsp;\u0026plusmn;\u0026thinsp;5.00 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.69\u0026thinsp;\u0026plusmn;\u0026thinsp;7.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3066.15\u0026thinsp;\u0026plusmn;\u0026thinsp;104.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX030 private land in Keligeng Town, Wuchuan County, Hohhot City(41.20277N, 111.473127E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2583.45\u0026thinsp;\u0026plusmn;\u0026thinsp;93.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.06 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3069.85\u0026thinsp;\u0026plusmn;\u0026thinsp;79.43 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2729.35\u0026thinsp;\u0026plusmn;\u0026thinsp;51.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUlanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e68.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3127.65\u0026thinsp;\u0026plusmn;\u0026thinsp;54.98 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.41\u0026thinsp;\u0026plusmn;\u0026thinsp;5.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3532.30\u0026thinsp;\u0026plusmn;\u0026thinsp;70.99 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3234.30\u0026thinsp;\u0026plusmn;\u0026thinsp;42.27 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX030 private land in Keligeng Town, Wuchuan County, Hohhot City\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.17\u0026thinsp;\u0026plusmn;\u0026thinsp;7.32 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2732.10\u0026thinsp;\u0026plusmn;\u0026thinsp;64.49 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3016.60\u0026thinsp;\u0026plusmn;\u0026thinsp;84.08 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.58\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2851.20\u0026thinsp;\u0026plusmn;\u0026thinsp;62.50 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUlanqab Agricultural Science Institute, Jining District, Ulanqab City, Inner Mongolia Autonomous Region\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e71.87\u0026thinsp;\u0026plusmn;\u0026thinsp;6.89 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2980.65\u0026thinsp;\u0026plusmn;\u0026thinsp;81.68 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56.15\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3535.95\u0026thinsp;\u0026plusmn;\u0026thinsp;41.93 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3065.65\u0026thinsp;\u0026plusmn;\u0026thinsp;25.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eX030 private land in Keligeng Town, Wuchuan County, Hohhot City\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e70.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2768.75\u0026thinsp;\u0026plusmn;\u0026thinsp;76.55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3151.50\u0026thinsp;\u0026plusmn;\u0026thinsp;25.28 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2908.85\u0026thinsp;\u0026plusmn;\u0026thinsp;51.72 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different lowercase letters (a, b, c) within the same column for each location and year indicate significant differences according to Duncan's multiple range test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Control: blank control; Bv. JN.Y2: \u003cem\u003eBacillus velezensis\u003c/em\u003e JN.Y2; Tm: Thiophanate-methyl.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo assess its antifungal spectrum, Bv. JN.Y2 was co-cultured with seven other common fungal phytopathogens. The strain exhibited significant inhibitory activity against all tested pathogens (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;2). Inhibition rates ranged from 48.39% to 81.16%, with the highest activity observed against \u003cem\u003eP. sorghina\u003c/em\u003e (81.16%). Inhibition rates against \u003cem\u003eC. fructicola\u003c/em\u003e, \u003cem\u003eR. solani\u003c/em\u003e, \u003cem\u003eD. glomerata\u003c/em\u003e, and \u003cem\u003eA. alternata\u003c/em\u003e exceeded 50%. While inhibition rates against \u003cem\u003eF. graminearum\u003c/em\u003e and \u003cem\u003eF. oxysporum\u003c/em\u003e were 48.39% and 48.53%, respectively. These findings confirm that Bv. JN.Y2 possesses broad-spectrum antifungal capacity, could be potentially applied for controlling multiple plant disease, caused by fungal pathogens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMorphological, physiological, and molecular identification of Bv. JN.Y2\u003c/h2\u003e \u003cp\u003eAfter incubation on LB agar at 37\u0026deg;C for 24 h, colonies of Bv. JN.Y2 exhibited a creamy-white, opaque appearance, characterized by irregular margins and a rugose (wrinkled) surface (Fig.\u0026nbsp;3A). Microscopic examination and physiological assays indicated that Bv. JN.Y2 is a Gram-positive, motile, rod-shaped bacterium (Fig.\u0026nbsp;3B). The formation of central ellipsoidal endospores was observed, which is a typical characteristic of the genus \u003cem\u003eBacillus\u003c/em\u003e (Fig.\u0026nbsp;3C). As detailed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Bv. JN.Y2 demonstrated relatively diverse hydrolytic capabilities, testing positive for catalase and cellulase activities, as well as the hydrolysis of starch, gelatin, and casein. The strain also showed positive results for nitrate reduction, the Voges-Proskauer (V-P) reaction, and citrate utilization. In contrast, negative results were recorded for oxidase activity, methyl red test, indole production, H\u003csub\u003e2\u003c/sub\u003eS production, and urease activity. Regarding carbon source utilization, the strain was capable of metabolizing glucose, sucrose, maltose, and D-mannitol, while it appeared unable to utilize lactose. These morphological and biochemical traits are consistent with the established descriptions of \u003cem\u003eBacillus velezensis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysiological and biochemical characteristics of Bv. JN.Y2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eResults\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphology\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCell shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRod\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEndospore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEnzyme Activity\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatin liquefaction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCasein hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCellulase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBiochemical Tests\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVoges-Proskauer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole production\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrate utilization\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH₂S production\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCarbon Source\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD-Mannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003cb\u003eNote: +\u003c/b\u003e, positive response or growth; \u003cb\u003e\u0026minus;\u003c/b\u003e, negative response or no growth. Data represents the mean of three independent replicates.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor the preliminary taxonomic classification of Bv. JN.Y2, 16S rRNA gene sequence analysis was initially performed. PCR amplification of the 16S rRNA gene yielded a single, distinct band of the expected size upon agarose gel electrophoresis (Fig.\u0026nbsp;3D). Subsequent sequence analysis and the resulting phylogenetic tree indicated that Bv. JN.Y2 clustered within the same clade as \u003cem\u003eB. velezensis\u003c/em\u003e GT3033 and CSQXZD26, with a bootstrap support of 94% (Fig.\u0026nbsp;3E). Although the discriminatory power of 16S rRNA sequences among closely related species within the genus \u003cem\u003eBacillus\u003c/em\u003e is relatively limited, this analysis provided foundational evidence for the genus-level assignment of Bv. JN.Y2. Given the high degree of genetic similarity among closely related species within this genus, a phylogenetic tree based on whole-genome SNPs was subsequently constructed to achieve a more robust identification. The SNP-based phylogenetic analysis revealed a close genetic affinity between Bv. JN.Y2 and Bv. CBMB205, supported by a bootstrap value of 99% (Fig.\u0026nbsp;3F). By integrating the morphological, physiological, and biochemical characteristics with this high-resolution genomic evidence, Bv. JN.Y2 was confirmed as \u003cem\u003eB. velezensis\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHydrolase secretion, biofilm formation and colonization\u003c/h2\u003e \u003cp\u003eTo further explore the potential biocontrol mechanisms of Bv. JN.Y2, its capacity for extracellular enzyme secretion and biofilm formation was evaluated. The secretion of hydrolytic enzymes is generally considered a relevant factor in both pathogen antagonism and successful endophytic colonization. In this study, distinct hydrolysis halos were observed on agar plates supplemented with protease, amylase, cellulase, and pectinase substrates (Fig.\u0026nbsp;4A\u0026ndash;D). Specifically, proteases can participate in the degradation of structural proteins within fungal cell walls, while cellulases and pectinases may facilitate the entry and systemic spread of the endophytic bacterium within host plant tissues. Although no obvious hydrolysis halo was detected on colloidal chitin agar (Fig.\u0026nbsp;4E), Bv. JN.Y2 maintained the ability to grow using chitin as the sole carbon source, suggesting a basal chitin-utilizing capacity that might assist in fungal antagonism. Collectively, these results indicate that Bv. JN.Y2 can secrete a relatively diverse array of hydrolases, which may coordinately contribute to its biocontrol efficacy.\u003c/p\u003e \u003cp\u003eFurthermore, biofilm formation assays revealed that Bv. JN.Y2 was capable of developing structural biofilms in MSgg medium. Quantitative analysis using crystal violet staining showed an OD\u003csub\u003e570\u003c/sub\u003e value of 1.62 for Bv. JN.Y2. While this production was comparatively lower than that of the hyper-producing reference strain \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e YN.J3 (OD\u003csub\u003e570\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.17), it still demonstrated a favorable biofilm-forming capability relative to the control (Fig.\u0026nbsp;4F, Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo dynamically assess the spatial and temporal colonization patterns of Bv. JN.Y2 in oat plants, a kanamycin-resistant transformant (Bv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e), which was obtained from our laboratory collection, was utilized and tracked via plate counting. The results indicated that the bacterial population in the roots reached its peak at 1 dpi with a density of 1.21 \u0026times; 10⁵ CFU\u0026middot;g⁻\u0026sup1;, and subsequently showed a gradual decrease, maintaining a level of 2.85 \u0026times; 10⁴ CFU\u0026middot;g⁻\u0026sup1; at 30 dpi. In the aerial parts, colonization in the stems and leaves peaked slightly later, reaching 8.20 \u0026times; 10⁴ CFU\u0026middot;g⁻\u0026sup1; at 3 dpi and 5.73 \u0026times; 10⁴ CFU\u0026middot;g⁻\u0026sup1; at 7 dpi, respectively, before experiencing a gradual decline (Fig.\u0026nbsp;4G). These findings suggest that Bv. JN.Y2 possesses the ability to systemically spread and establish a relatively stable population within oat tissues over an extended period.\u003c/p\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePGP traits of Bv. JN.Y2 and its effect on oat yield enhancement\u003c/h2\u003e \u003cp\u003eTo comprehensively assess the PGP potential of Bv. JN.Y2, both laboratory assays and field trials were evaluated. In vitro biochemical assays revealed that Bv. JN.Y2 was capable of growing on nitrogen-free Ashby medium, and it produced clear halos on Pikovskaya and Aleksandrov agar plates, indicating its capacities for nitrogen metabolism or assimilation (Fig.\u0026nbsp;5A), and solubilization of phosphate and potassium (Fig.\u0026nbsp;5B, 5C). Furthermore, the distinct halo observed on the CAS agar medium suggested the production of siderophores (Fig.\u0026nbsp;5D). In seedling evaluations, treatment with Bv. JN.Y2 resulted in an increase in seedling height and shoot fresh weight by 19.73% and 36.73%, respectively, although the difference in dry weight was not statistically significant. Regarding root development, root length, root fresh weight, and root dry weight were enhanced by 43.03%, 40.21%, and 11.96%, respectively. Additionally, the chlorophyll SPAD value exhibited a 25.27% increase compared to the control group (Fig.\u0026nbsp;5E, Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantitative analysis of biofilm formation measured by crystal violet staining (OD\u003csub\u003e570\u003c/sub\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOD\u003csub\u003e570\u003c/sub\u003e\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\u003eB. amyloliquefaciens\u003c/em\u003e YN.J3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCK (LB+Msgg media)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of Bv. JN.Y2 on the growth parameters of oat seedlings.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePlant height (cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSterile water\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoot length (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot fresh weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoot fresh weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0475\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0029 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0666\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot dry weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRoot dry weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.0184\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0015 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0206\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0012 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChlorophyll content (SPAD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003cb\u003eNote\u003c/b\u003e: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis was performed using an independent samples \u003cem\u003et\u003c/em\u003e-test. Different lowercase letters within the same row indicate significant differences between treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAcross the three consecutive years of field trials (2023\u0026ndash;2025) in Jining and Wuchuan, oat plants treated with Bv. JN.Y2 consistently exhibited higher grain yields compared to CK and TM groups (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In 2023, the application of Bv. JN.Y2 led to favorable yield increases, reaching 3391.35 kg/ha in Jining and 3069.85 kg/ha in Wuchuan. During 2024, as environmental precipitation increased, the yield-promoting effect remained relatively stable, with the Bv. JN.Y2 treatment achieving 3532.30 kg/ha and 3016.60 kg/ha at the two respective sites. In the multi-rain year of 2025, the yield in the Bv. JN.Y2-treated plots reached 3535.95 kg/ha in Jining and 3151.50 kg/ha in Wuchuan, maintaining a consistent advantage over the corresponding controls. Collectively, these findings suggest that Bv. JN.Y2 likely promotes oat growth and stably enhances crop yield across varying climatic conditions by facilitating nutrient absorption and improving overall physiological vigor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGenomic features and functional gene mining of Bv. JN.Y2\u003c/h2\u003e \u003cp\u003eTo uncover the molecular basis underlying its beneficial traits, the complete genome of Bv. JN.Y2 was sequenced and annotated. The genome consists of a single circular chromosome of 3, 871, 731 bp with a GC content of 47.26% (Fig.\u0026nbsp;6, Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). No plasmid was detected. The genome contains 3, 987 predicted protein-coding genes with an average length of 872 bp, along with 25 rRNA genes, 84 tRNA genes, and 8 sRNA genes. The complete genome sequence has been deposited in the NCBI database under the GenBank accession number CP178220.1. Functional annotation was performed across multiple databases, yielding the following assignments: COG (2, 939 genes), NR (3, 912), GO (2, 667), KEGG (3, 836), Pfam (2, 667), Swiss-Prot (3, 255), CAZy (155), and TCDB (516). Subsequent genomic screening revealed an array of genes associated with PGP activities. Specifically, gene clusters related to nitrogen metabolism and assimilation (\u003cem\u003enifH\u003c/em\u003e, \u003cem\u003enifU\u003c/em\u003e, \u003cem\u003eyutI\u003c/em\u003e), Fe-S cluster assembly (\u003cem\u003esufB\u003c/em\u003e, \u003cem\u003esufC\u003c/em\u003e, \u003cem\u003esufU\u003c/em\u003e, \u003cem\u003esufD\u003c/em\u003e), phosphate metabolism (\u003cem\u003epstA/B1/B2/C/S\u003c/em\u003e, \u003cem\u003ephoA\u003c/em\u003e, \u003cem\u003ebglA\u003c/em\u003e), potassium uptake (\u003cem\u003ektrA/C/D\u003c/em\u003e, \u003cem\u003ekdpD\u003c/em\u003e), siderophore production (\u003cem\u003efhu\u003c/em\u003e, \u003cem\u003efeu\u003c/em\u003e, \u003cem\u003edhb\u003c/em\u003e, \u003cem\u003eent\u003c/em\u003e), and indole-3-acetic acid (IAA) synthesis (the \u003cem\u003etrp\u003c/em\u003e operon) were identified (Online Resource 1 Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These findings provide a genetic explanation for the physiological PGP traits observed in vitro.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe general genome feature of Bv. JN.Y2\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVaule\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenome size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,871,731\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG\u0026thinsp;+\u0026thinsp;C content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.26%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTopology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCircular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal size of protein-coding genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3,478,611\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein-coding genes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3987\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage CDs size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e872\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003erRNA number (total)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003etRNA number\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003esRNA number\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRepetitive sequence(bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18131 (0.4683%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCRISPR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProphage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene cluster\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to COG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2939\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to NR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3912\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to GO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to KEGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3836\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to Pfam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to Swiss-Prot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3255\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to CAZy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenes assigned to TCDB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e516\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurthermore, a variety of genes encoding hydrolases were annotated, including amylases (\u003cem\u003eamyA\u003c/em\u003e), proteases (\u003cem\u003eepr\u003c/em\u003e, \u003cem\u003eaprE\u003c/em\u003e, \u003cem\u003evpr\u003c/em\u003e), cellulases (\u003cem\u003ecelA/B/C/F\u003c/em\u003e, \u003cem\u003exynB\u003c/em\u003e), pectinases (\u003cem\u003epel\u003c/em\u003e, \u003cem\u003epgdA\u003c/em\u003e, \u003cem\u003epgdB\u003c/em\u003e), and chitin-related enzymes (\u003cem\u003eyxkH\u003c/em\u003e, \u003cem\u003eyheN\u003c/em\u003e). Additionally, a comprehensive set of genes essential for plant-microbe interactions and colonization was identified, encompassing those governing chemotaxis (\u003cem\u003echeA/W/Y\u003c/em\u003e, \u003cem\u003emcpA/B/C\u003c/em\u003e), flagellar motility (\u003cem\u003efliG/M/N\u003c/em\u003e), quorum sensing (\u003cem\u003ecomA\u003c/em\u003e, \u003cem\u003ecomP\u003c/em\u003e, \u003cem\u003eluxS\u003c/em\u003e, \u003cem\u003ephrA\u003c/em\u003e, \u003cem\u003esinI/R\u003c/em\u003e), and biofilm formation (\u003cem\u003etasA\u003c/em\u003e, \u003cem\u003eepsC\u003c/em\u003e, \u003cem\u003ebslA\u003c/em\u003e) (Online Resource 1 Table S3). Together, these genomic features establish a relatively solid genetic foundation for the biocontrol and growth-promoting capacities of Bv. JN.Y2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDiscovery and comparative analysis of secondary metabolite biosynthetic gene clusters\u003c/h2\u003e \u003cp\u003eTo further elucidate the molecular mechanisms underlying the biocontrol efficacy of Bv. JN.Y2, the antiSMASH tool was employed to mine its genome for BGCs. A total of 13 BGCs were identified, encompassing non-ribosomal peptide synthetases (NRPS), trans-AT polyketide synthases (transAT-PKS), hybrid PKS/NRPS systems, and terpenes (Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Among these, seven clusters exhibited high similarity to known pathways and were predicted to direct the synthesis of surfactin, macrolactin H, bacillaene, fengycin, difficidin, bacillibactin, and bacilysin. Furthermore, Region 2 (PKS-like), Region 4 (Lanthipeptide-class-II), Region 9 (T3PKS), and multiple terpene gene clusters (Regions 3, 8, and 11) distributed at various genomic positions exhibited notably low similarity to entries in known databases. These findings suggest that Bv. JN.Y2 possesses a favorable potential for the synthesis of novel or strain-specific antimicrobial metabolites, which may relatively significantly assist in its environmental adaptation and biocontrol performance.\u003c/p\u003e \u003cp\u003eTo characterize the genetic organization of these clusters, a comparative synteny analysis was performed between Bv. JN.Y2 and four biocontrol reference strains. Visualization revealed a remarkably high degree of collinearity and synteny between Bv. JN.Y2 and the model strain Bv. FZB42 (Fig.\u0026nbsp;7). The arrangement and orientation of core biosynthetic operons (e.g., \u003cem\u003esrfAA-AC\u003c/em\u003e, \u003cem\u003efenA-E\u003c/em\u003e, \u003cem\u003emlnA-G\u003c/em\u003e, \u003cem\u003ebaeJ-R\u003c/em\u003e, and \u003cem\u003edifA-L\u003c/em\u003e) were highly conserved across the analyzed \u003cem\u003eB. velezensis\u003c/em\u003e strains. This conservation not only confirms the genetic integrity of these antimicrobial pathways in Bv. JN.Y2, but supports its taxonomic identification at the genomic level, indicating its potential as a biocontrol agent.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePredicted genes clusters involved in synthesis of secondary metabolites in Bv. JN.Y2 genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMost similar known cluster\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSize\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSimilarity Confidence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePosition\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurfactin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNRPs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65, 407 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e264, 083\u0026ndash;329, 490\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eButirosin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePKS-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41, 244 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e865, 948\u0026thinsp;\u0026minus;\u0026thinsp;907, 192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTerpene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20, 740 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e989, 236-1, 009, 976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLanthipeptide-class-ii\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28, 888 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1, 130, 469-1, 159, 357\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMacrolactin H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTranAT-PKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88, 233 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1, 325, 970-1, 414, 203\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacillaene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003etransAT-PKS,T3PKS,NRPS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e110, 093 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1, 632, 894-1, 742, 987\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFengycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNRPS, transAT-PKS,betalactone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e137, 801 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1, 807, 618-1, 945, 419\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTerpene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21, 883 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1, 970, 646-1, 992, 529\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT3PKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41, 100 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2, 055, 847-2, 096, 947\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDifficidin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTransAT-PKS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e106, 182 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2, 211, 933-2, 318, 115\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTerpene-precursor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20, 890 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2, 341, 409-2,362, 299\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacillibactin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNRP-metallophore,NRPS,Ripp-like\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51, 791 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2, 942, 817-2, 994, 608\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacilysin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOther\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41, 418 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3, 530, 918-3, 572, 336\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eIn silico mining of bacteriocins and RiPPs\u003c/h2\u003e \u003cp\u003eThe genomic architecture of Bv. JN.Y2 was explored using the BAGEL4 platform, which identified four distinct Areas of Interest (AOIs) dedicated to the synthesis of RiPPs (Fig.\u0026nbsp;8). Within the genomic architecture of Bv. JN.Y2, AOI_01 is situated between coordinates 2, 979, 272 and 2, 999, 461, encompassing 26 predicted open reading frames (ORFs). The primary biosynthetic product of this cluster is identified as Amylocyclicin, a circular bacteriocin recognized for its significant role in improving the competitive fitness of \u003cem\u003eBacillus\u003c/em\u003e species within their ecological niches. Distinct from the other synthesis-oriented clusters, AOI_02 (2, 929, 112\u0026ndash;2, 949, 271) comprises 26 ORFs and is characterized as a regulatory locus centered on the signaling peptide ComX3. As a pivotal component of the Quorum Sensing system, ComX3 potentially modulates the secondary metabolic profile of Bv. JN.Y2, thereby orchestrating its adaptive responses to environmental stimuli without the requirement of a traditional core peptide synthesis template. Complementing these findings, AOI_03 is located at positions 243, 431 to 263, 566 and contains 19 ORFs, encoding the Linear Competition Inducer (LCI) as its core protein. LCI has been demonstrated to facilitate environmental dominance by exerting potent antimicrobial and antifungal activities against competing microorganisms. Lastly, AOI_04 (1, 132, 550\u0026ndash;1, 158, 286) exhibits a sophisticated organizational structure with 28 ORFs and is classified as a class II lanthipeptide biosynthetic cluster. Notably, this cluster is characterized by the presence of two distinct core peptides exhibiting high homology to Haloduracin_beta, which are further associated with the Type A lanthibiotic (L_biotic_typeA) and Mersacidin-like families. This multi-component architecture is a hallmark of certain RiPPs that often exhibit enhanced bioactivity through synergistic mechanisms, potentially conferring a consistent defensive advantage to the host strain.\u003c/p\u003e \u003cp\u003eTo validate the functional robustness of these systems, multiple sequence alignments were performed using Clustal Omega and visualized via ESPript 3.2 (Fig.\u0026nbsp;9). The resulting alignment profile confirmed a high degree of evolutionary conservation across the core peptides. Specifically, the Amylocyclicin sequence in Bv. JN.Y2 exhibits 100% identity with reference circular bacteriocins from \u003cem\u003eB. velezensis\u003c/em\u003e FZB42 and DSYZ. This is further supported by BLASTp validation, showing 99.10% identity (99% coverage) to a circular bacteriocin from \u003cem\u003eB. inaquiensis\u003c/em\u003e with a significant E-value of 2e-74. For the LCI system, the core peptide shares 96.81% identity with unnamed \u003cem\u003eB. velezensis\u003c/em\u003e, \u003cem\u003eB. velezensis\u003c/em\u003e DSM 23117 and 98.94% with \u003cem\u003eB. siamensis\u003c/em\u003e SCSIO 05746. Notably, Bv. JN.Y2 shared a significantly lower identity of only 53.33% with the model strain \u003cem\u003eB. velezensis\u003c/em\u003e FZB42. This pronounced divergence in the LCI sequence, contrasted with the 100% identity observed for Amylocyclicin, highlights the unique evolutionary trajectory and the distinct antimicrobial profile of Bv. JN.Y2 compared to the classical model strain. Regarding the multi-component AOI_04, while AOI_04/1 shows varied homology (0-78.05%), AOI_04/2 exhibits absolute conservation with 100% identity to the homolog from \u003cem\u003eB. amyloliquefaciens\u003c/em\u003e PM415. These molecular signatures underscore the sophisticated antimicrobial potential of the Bv. JN.Y2 genome.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGenomic screening for acquired resistance genes and biosafety evaluation\u003c/h2\u003e \u003cp\u003eThe genomic landscape of Bv. JN.Y2 is characterized by a relatively rich composition of MGEs, including 9 predicted genomic islands (GIs) and 13 prophage regions (Online Resource 1 Table S4, S5). This substantial dataset underscores an active evolutionary history driven by HGT, which likely contributes to the strain's genomic plasticity and its specialized adaptation to the oat endosphere. Notably, these GIs are hypothesized to harbor potential genes associated with environmental fitness, nutrient metabolism, and plant-growth promotion, which are essential for the endophytic lifestyle of Bv. JN.Y2. Consistent with this dynamic genomic architecture, ResFinder screening identified two acquired AMR homologs (Table\u0026nbsp;\u003cspan refid=\"Tab10\" class=\"InternalRef\"\u003e10\u003c/span\u003e): \u003cem\u003ecfr(B)\u003c/em\u003e (identity: 88.61%) and \u003cem\u003etet(L)\u003c/em\u003e (identity: 86.87%). Spatial distribution analysis revealed that \u003cem\u003ecfr(B)\u003c/em\u003e (52, 804\u0026ndash;53, 839 bp) is localized within the Prophage 1 region (29, 352\u0026ndash;103, 065 bp), providing a molecular record of an ancestral transduction event. In contrast, \u003cem\u003etet(L)\u003c/em\u003e (373, 529\u0026ndash;374, 905 bp) resides within the stable chromosomal backbone, situated outside all predicted GIs and prophage sequences. Despite the historical acquisition of these elements, the ResFinder system predicted a \"No resistance\" phenotype across all tested antibiotic categories, including aminoglycosides and beta-lactams, for which no significant hits were found. Given the plasmid-free architecture of Bv. JN.Y2, the chromosomal localization and the \"cryptic\" nature of the prophage-associated \u003cem\u003ecfr(B)\u003c/em\u003e suggest that these sequences likely represent non-functional evolutionary remnants rather than active resistance determinants. This sophisticated genomic compartmentalization appears to effectively control potential resistance risks while maintaining high genomic plasticity. Consequently, these findings underscore a relatively high level of biosafety and genetic stability for Bv. JN.Y2, supporting its potential as a sustainable and secure biocontrol agent in oat production.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab10\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 10\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of AMR genes and their spatial association with MGEs in Bv. JN.Y2\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=\"char\" char=\".\" 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\u003eAMR Gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIdentity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosomal Position (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMGE Association\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePredicted Phenotype\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\u003ecfr(B)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e88.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e52, 804\u0026thinsp;\u0026minus;\u0026thinsp;53, 839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProphage 1 (29, 352\u0026thinsp;\u0026minus;\u0026thinsp;103, 065 bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo resistance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003etet(L)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e86.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026minus;\" colname=\"c3\"\u003e \u003cp\u003e373, 529\u0026thinsp;\u0026minus;\u0026thinsp;374, 905\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone (Stable chromosomal backbone)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo resistance\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eComparative genome analysis between Bv. JN.Y2 and eight reference\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the genetic divergence, functional variations, and evolutionary adaptation of the tested strains relative to the reference strain Bv. JN.Y2, a systematic profiling of SNPs, InDels, and SVs was conducted across all genomes (Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Fig.\u0026nbsp;10, Online Resource 1 Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among all investigated strains, Bv. CBMB205 exhibited a relatively high degree of genetic conservation compared to the reference genome. A genome-wide analysis revealed only 3 SNPs (including only 1 non-synonymous mutation) and 8 InDels within CDS regions (comprising 5 insertions and 3 deletions) (Table\u0026nbsp;\u003cspan refid=\"Tab11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, Fig.\u0026nbsp;10A). The SNP-based phylogenetic tree (Fig.\u0026nbsp;3F) further illustrates these evolutionary relationships: closely related \u003cem\u003eB. velezensis\u003c/em\u003e, such as Bv. CBMB205, Bv. SQR9 and Bv. FZB42, tend to accumulate higher densities of SNPs while maintaining robust sequence synteny. Conversely, distantly related lineages like Bm. BPN36.3 and Bt. ATCC 10792 occupy basal positions in the tree, where their evolutionary divergence is primarily driven by complex structural rearrangements and poorer synteny, despite exhibiting relatively fewer detectable point mutations. This high level of sequence stability was further corroborated by the SV profiling. Similarly, other strains belonging to \u003cem\u003eB. velezensis\u003c/em\u003e (Online Resource 1 Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, E, F) demonstrated relatively low structural variation and maintained robust sequence synteny, reflecting their close intra-specific phylogenetic relationships. Notably, a divergent variation pattern was observed in Bm. BPN36.3 (Online Resource 1 Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) and Bt. ATCC 10792 (Online Resource 1 Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eG). While these strains showed comparatively low point mutation counts\u0026mdash;with 2, 777 and 2, 152 SNPs, 9 and 11 InDels, respectively\u0026mdash;their macro-scale structural profiles were characterized by dense, large-scale genomic clusters. This suggests that these lineages may have undergone relatively complex sequence rearrangements or recombinations during their evolutionary trajectories. In contrast, Bs. 168 exhibited significant genetic differentiation from the reference, with the total number of SNPs reaching 141, 090. Even within the \u003cem\u003eB. velezensis\u003c/em\u003e group, Bv. FZB42 and Bv. SQR9 accumulated 55, 591 and 37, 721 SNPs, respectively, indicating a notable level of intra-specific genetic diversity. Collectively, the frequency of SVs across the strains fluctuated between 4 and 512 events, encompassing deletions, insertions, and complex translocations. The distribution of these structural features showed a relatively strong correlation with the taxonomic distance of each strain relative to Bv. JN.Y2. These multi-dimensional variation data not only elucidate the distinct genomic signatures formed during long-term evolution, but provide a relatively solid molecular foundation for subsequent investigations into functional gene expression and environmental adaptation across these diverse strains.\u003c/p\u003e \u003cp\u003eFurthermore, KEGG functional annotation was performed to investigate the impact of key genetic heterogeneities on the biocontrol properties and environmental fitness of Bv. JN.Y2, particularly from the dual perspectives of protein functional evolution and gene expression regulation. Bv. JN.Y2 exhibited relatively high structural stability relative to its close relative Bv. CBMB205, with key genetic divergence focused on non-synonymous SNPs in the sporulation kinase \u003cem\u003ekinA\u003c/em\u003e. This variation suggests a potentially more sensitive regulatory strategy for sensing environmental stress. Meanwhile, the conserved core developmental framework and high sequence synteny provide a relatively solid genetic foundation for stable colonization. In comparisons with biocontrol strains such as Bv. FZB42, Bv. SQR9 and Ba. GKT04, non-synonymous SNPs and InDels were highly enriched within secondary metabolite clusters (e.g., Iturin, Fengycin, and Pks), driving the diversification of substrate specificity. In addition, localized SV-mediated rearrangements promoted the modular evolution of biocontrol genes, providing molecular evidence for its broad-spectrum antagonistic activity. Regarding environmental sensing, variations in coding regions of ComP and chemotaxis proteins, coupled with structural fine-tuning of upstream regulatory elements, imply a \"precision-tuning\" mechanism for responding to root exudates, thereby enhancing sensing sensitivity in soil. Compared to distant lineages like Bl. 14580 and Bm. BPN36.3, Bv, JN.Y2 underwent comprehensive differentiation ranging from large-scale rearrangements to core metabolic functions (e.g., \u003cem\u003etuf\u003c/em\u003e, \u003cem\u003esufB\u003c/em\u003e) and stress-resistance pathways (e.g., \u003cem\u003espoIIP\u003c/em\u003e, \u003cem\u003euvrA/B\u003c/em\u003e). Collectively, these multi-level variation patterns reinforce the survival resilience and adaptive fitness of Bv. JN.Y2 under extreme environmental conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab11\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 11\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistics and annotation of SNPs in the genomes of eight \u003cem\u003eBacillus\u003c/em\u003e strains\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReference Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSynonymous SNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNonsynonymous SNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal CDS SNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIntergenic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal SNP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eBv. JN.Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBl.14580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21, 293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6, 428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27, 846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2, 100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29, 946\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBm.BPN36.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1, 546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e803\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2, 364\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e413\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2, 777\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBs.168\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e101, 661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28, 332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28, 332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e130, 600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e141, 090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.SQR9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24, 882\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9, 148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e34, 142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3, 579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37, 721\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.CBMB205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBv.FZB42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37, 095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13, 014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50, 326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5, 265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55, 591\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBt.10792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1, 164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1, 794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2, 152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBa.GKT04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37, 898\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13, 216\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51, 304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5, 273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e56, 577\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCore-Pan analysis between Bv. JN.Y2 and eight reference\u003c/b\u003e \u003cb\u003eBacillus\u003c/b\u003e \u003cb\u003estrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize the evolutionary trajectories and functional divergence among nine \u003cem\u003eBacillus\u003c/em\u003e strains, including Bv. JN.Y2, a comparative pangenome analysis was conducted. From a total of 43, 274 identified protein-coding sequences, 12, 473 orthologous genes (pan-genes) were clustered to define the collective gene repertoire of the cohort. Within this framework, a core genome of 1, 086 genes shared across all strains was identified, likely representing the conserved genetic foundation of the group. Conversely, the dispensable genome, comprising 11, 387 genes distributed among subsets of strains, highlighted the considerable genomic plasticity of the lineage. Notably, Bv. JN.Y2 harbored 411 unique genes\u0026mdash;a figure within a moderate range (13\u0026ndash;1, 508) that reflects its distinct genetic identity. Dilution curve analysis further corroborated these dynamics, showing the core genome stabilizing at approximately 1,086 genes, while the pangenome expanded without reaching a saturation plateau. These findings indicate that this strain assembly possesses a typical \"open\" pangenome, suggesting a collective genetic repertoire with substantial potential for further expansion upon the inclusion of additional genomic data.\u003c/p\u003e \u003cp\u003eAmong the 1, 086 core genes, 524 (48.2%) were successfully annotated via the KEGG database. At the primary functional categories, Metabolism was the most prominent category (cumulative frequency: 1, 485), followed by Genetic Information Processing (143) and Environmental Information Processing (78). These results suggest that the core genome primarily sustains basal physiological activities and signal transduction, providing a stable functional framework for Bv. JN.Y2 and its relatives. Further analysis at the secondary functional categories highlighted a high prevalence of genes involved in Global and overview maps (817), Carbohydrate metabolism (158), and Amino acid metabolism (134). Specifically, specific metabolic pathways revealed 304 genes in Metabolic pathways, 60 in Carbon metabolism, and critical energy-related components like Glycolysis/Gluconeogenesis (25) and Oxidative phosphorylation (24). Regarding biocontrol and adaptation, genes for the biosynthesis of secondary metabolites (160) and antibiotics (120) exhibited high conservation, underscoring a relatively stable genetic basis for the antagonistic potential of the \u003cem\u003eB. velezensis\u003c/em\u003e group. Additionally, the enrichment of Ribosome (45) genes supports efficient protein synthesis, while conserved Two-component systems (33) and ABC transporters (28) likely enhance environmental sensing and nutrient acquisition, collectively maintaining bacterial fitness across diverse ecological niches.\u003c/p\u003e \u003cp\u003eIn contrast to the conserved core genome, the 411 strain-specific genes of Bv. JN.Y2 are characterized by specialized functions that likely facilitate its distinct ecological adaptation. KEGG annotation reveals a notable enrichment in secondary metabolism, particularly monobactam biosynthesis, suggesting a relatively good potential for the production of unique bioactive compounds. The identification of pathways such as lysine biosynthesis and butanoate metabolism further indicates enhanced metabolic flexibility. Furthermore, unique components of quorum sensing and two-component systems reflect a specialized capacity for environmental sensing and population-level coordination. Combined with specific ABC transporters and genes involved in sporulation and stress response, these genetic features collectively reinforce the resilience and competitive fitness of Bv. JN.Y2 within its ecological niche.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMembers of the genus \u003cem\u003eBacillus\u003c/em\u003e are widely regarded as promising biological control agents, a reputation primarily attributed to their capacity to establish mutualistic endophytic relationships with host plants and suppress pathogens through multifaceted mechanisms (Reva et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Hashem et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, the endophytic strain Bv. JN.Y2, isolated from oat stems and taxonomically identified via both 16S rRNA sequences and whole-genome SNP-based phylogenetic trees, demonstrated relatively good dual functionality in anthracnose suppression and PGP. Comprehensive genome mining of Bv. JN.Y2 revealed a substantial repertoire of genes associated with secondary metabolite biosynthesis, nutrient utilization and cycling, environmental adaptation, as well as chemotaxis and colonization. Furthermore, a systematic comparative genomic analysis was conducted to elucidate the genetic architecture of Bv. JN.Y2. Our findings indicated that, in addition to the core genome, the strain has evolved 411 unique genes and exhibited a certain degree of specificity in terms of genomic variants, including SNPs, InDels, and SVs. These unique genes and sequence variations are notably enriched in key pathways such as secondary metabolism, quorum sensing, and stress response. In particular, the genetic divergence accumulated in the sporulation kinase kinA and secondary metabolite clusters (e.g., Iturin and Fengycin) provided a relatively good basis for the diversification of substrate specificity. Collectively, the abundant genetic variation within this \"open\" pan-genome defines the unique genetic identity of Bv. JN.Y2, establishing a relatively good foundation for its survival resilience and competitive adaptability within complex ecological niches.\u003c/p\u003e \u003cp\u003eThe biocontrol potential of \u003cem\u003eBacillus\u003c/em\u003e species is generally attributed to the synergy of multiple traits, including direct antagonism facilitated by secondary metabolites from the NRPS, PKS, and RiPPs pathways (Jangir et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), competition for finite resources (Kramer et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and effective colonization of the rhizosphere and endophytic tissues, a process often facilitated by CWDEs and biofilm (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Combined results from greenhouse assays and whole-genome annotation revealed that Bv. JN.Y2 possesses a diverse genetic repertoire encoding proteases, cellulases, and various BGCs including NRPS, PKS, and RiPPs. The co-existence of these multi-functional antimicrobial BGCs likely underpins the broad-spectrum inhibitory capacity of Bv. JN.Y2. As reported by Shen, the synergistic action of multiple secondary metabolites and CWDEs secreted by \u003cem\u003eB. velezensis\u003c/em\u003e SH1471 effectively suppresses a wide range of pathogens, including \u003cem\u003eF. oxysporum\u003c/em\u003e (Shen et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This characteristic\u0026mdash;simultaneous secretion of CWDEs and antimicrobial metabolites\u0026mdash;underscores the potential of Bv. JN.Y2 as a versatile biocontrol agent for managing various plant diseases beyond oat anthracnose (Chowdhury et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, genomic mining and CAS assay results confirmed the potential of Bv. JN.Y2 to synthesize catecholate-type siderophores (e.g., bacillibactin) and its possession of sophisticated transport systems (\u003cem\u003efeu\u003c/em\u003e and \u003cem\u003efhu\u003c/em\u003e) for mediating catecholate and hydroxamate iron uptake (Chen et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). This robust iron-sequestration capability likely facilitates the inhibition of pathogen proliferation under iron-limited conditions, thereby enhancing the strain\u0026rsquo;s ecological fitness within the complex rhizosphere environment (Yu et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In addition, longitudinal tracking of Bv. JN.Y2-kan\u003csup\u003eR\u003c/sup\u003e demonstrated that the strain can stably colonize the roots, stems, and leaves of oat plants for at least 30 days. This persistent endophytic colonization is likely facilitated by a synergistic mechanism involving: (i) CWDEs (cellulases and pectinases) encoded by \u003cem\u003ecelA/B/C/F\u003c/em\u003e, \u003cem\u003epel\u003c/em\u003e, and \u003cem\u003epgdA\u003c/em\u003e genes, which assist in tissue penetration (Ku et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); (ii) \u003cem\u003emcp\u003c/em\u003e, \u003cem\u003eche\u003c/em\u003e, motility-related and QS genes that orchestrate the recruitment process in response to root exudates (Zhou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); and (iii) a relatively strong capacity for biofilm formation; (iv) the presence of a surfactin-encoding gene cluster, which is relatively effective in enhancing bacterial motility and promoting colonization (Gao et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our findings are consistent with those of Weng (Weng et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), who utilized GFP-labeling to show that \u003cem\u003eB. velezensis\u003c/em\u003e SQR9 effectively responds to root exudates and establishes stable colonization in cucumber, subsequently protecting the host from \u003cem\u003eF. oxysporum\u003c/em\u003e infection.\u003c/p\u003e \u003cp\u003eMicrobes promoted plant growth by secreting IAA and facilitating nutrients absorption (Idris et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The genome of Bv. JN.Y2 contains the complete \u003cem\u003etrp\u003c/em\u003e operon (\u003cem\u003etrpABCDEF\u003c/em\u003e), contributing to IAA synthesis. Bv. JN.Y2 grow normally on nitrogen-free medium and core genes (\u003cem\u003enifH\u003c/em\u003e, \u003cem\u003enifU\u003c/em\u003e), regulatory gene (\u003cem\u003eyutI\u003c/em\u003e) are presented in genome of Bv. JN.Y2. Fan proved that genetic configurations of core genes and regulatory genes giving \u003cem\u003eB. velezensis\u003c/em\u003e FZB42 nitrogen metabolism and assimilation trait (Fan et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Movever, genes related to phosphate metabolism and transport (\u003cem\u003epstABCS\u003c/em\u003e, \u003cem\u003ephoA\u003c/em\u003e), potassium mobilization (\u003cem\u003ektrACD\u003c/em\u003e, \u003cem\u003ekdpD\u003c/em\u003e) are also present in genome of Bv. JN.Y2, which collectively exert a relatively beneficial influence on plant growth promotion (Meena et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, the genome harbors the \u003cem\u003edhb\u003c/em\u003e operon (\u003cem\u003edhbACEBF\u003c/em\u003e), which is responsible for the biosynthesis and transport of siderophores. This mechanism plays a relatively conducive role in chelating iron from the environment, and further supporting nutrient acquisition under iron-limited conditions (Zhang et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Overall, this multi-pronged genetic toolkit explains the enhanced biomass and yield observed in oats treated with Bv. JN.Y2.\u003c/p\u003e \u003cp\u003eGenome-wide analysis using antiSMASH and BAGEL4 identified a total of 17 BGCs. Notably, several clusters, such as those for Fengycin and Bacillaene, exhibit NRPS-PKS hybrid features, highlighting the robust potential of Bv. JN.Y2 to synthesize structurally sophisticated and functionally varied secondary metabolites (Zhao and Kuipers, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Comparative genomic analysis with four closely related \u003cem\u003eBacillus\u003c/em\u003e strains further reveals that the arrangement and composition of core biosynthetic operons in Bv. JN.Y2 are highly syntenic with the model strain Bv. FZB42. This stands is in contrast to Bv. CBMB205, which displays significant rearrangements within core clusters such as \u003cem\u003efen\u003c/em\u003e, \u003cem\u003edhb\u003c/em\u003e, and \u003cem\u003edif\u003c/em\u003e. Such high genetic conservation and structural stability in key metabolic pathways suggest that the biocontrol efficacy of Bv. JN.Y2 may be relatively predictable across diverse ecological environments (Rabbee et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e;). Beyond conventional PKS/NRPS pathways, Bv. JN.Y2 exhibits unique characteristics in its RiPPs. The identification of four AOIs via BAGEL4 underscores the strain's competitive advantage in complex ecological niches. These AOIs are reported to encode products that regulate bacterial quorum sensing, induce membrane damage, or inhibit cell wall synthesis in pathogenic microorganisms (Scholz et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Špacapan et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Sequence alignment and characterization of core peptides reveal varying levels of conservation: while Amylocyclicin and LCI show high sequence identity with reference strains\u0026mdash;indicating substantial evolutionary stability\u0026mdash;the Class II lanthipeptide displays lower consistency. This divergence suggests that Bv. JN.Y2 may have undergone adaptive evolution under environmental pressures, potentially leading to the emergence of novel antimicrobial compounds with specialized diversity.\u003c/p\u003e \u003cp\u003eGiven the dissemination risks associated with acquired resistance genes among environmental microbiota, systematic biosafety evaluation of functional strains is an indispensable prerequisite for safeguarding agro-ecological equilibrium and public health (Von et al. 2016). Three-year field trials across two distinct locations demonstrated that Bv. JN.Y2 consistently enhances oat growth. To assess its environmental release risks, comprehensive genomic mining of acquired resistance genes and MGEs were performed. Despite the identification of MGEs and homologs of \u003cem\u003ecfr(B)\u003c/em\u003e and \u003cem\u003etet(L)\u003c/em\u003e, Bv. JN.Y2 exhibited a robust biosafety profile. Notably, its plasmid-free architecture\u0026mdash;with all ARGs anchored exclusively within the chromosomal scaffold or prophage regions\u0026mdash;significantly mitigates the potential for HGT (Panel, 2018). Furthermore, the \"no resistance\" phenotype predicted by ResFinder suggests that these sequences represent non-functional evolutionary remnants rather than active resistance determinants, further reinforcing the suitability and safety of Bv. JN.Y2 for sustainable oat production.\u003c/p\u003e \u003cp\u003eComparative genomics serves as a sophisticated approach for elucidating evolutionary trajectories and functional variations (Alcaraz et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), providing a beneficial framework for identifying the superior traits of target strains. In this study, systematic comparative genomic analysis of Bv. JN.Y2 against both closely and distantly related strains revealed its distinct genetic differentiation and functional potential. Bv. JN.Y2 exhibits high sequence conservation and synteny with the rhizosphere-derived strain Bv. CBMB205. This observation aligns with the ecotype theory proposed by Cohan, which suggests that strains occupying similar ecological niches may utilize \"periodic selection\" to maintain genomic consistency by purging genetic diversity (Cohan, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Such conservation implies that Bv. JN.Y2 possesses a relatively high degree of environmental plasticity, allowing it to adapt to both rhizosphere and endophytic habitats\u0026mdash;a trait that likely supports its broad-spectrum plant growth-promoting potential. Conversely, comparisons with distantly related strains indicate that large-scale SVs are more prevalent than point mutations. This supports the hypothesis that genomic rearrangement, rather than single-nucleotide polymorphism, serves as a primary driver of functional divergence in the long-term evolution of the \u003cem\u003eBacillus\u003c/em\u003e genus (Feulner and De-Kayne, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Regarding its biocontrol mechanisms, Bv. JN.Y2 displays a metabolic profile distinct from the model strain Bv. FZB42. While Iturin is a well-known lipopeptide with antagonistic activity against various fungal pathogens (Yan et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), our analysis revealed a \"genes-present, cluster-incomplete\" phenomenon in Bv. JN.Y2. Specifically, although \u003cem\u003eituABC\u003c/em\u003e genes were identified, antiSMASH failed to detect a complete BGC, suggesting localized gene loss or functional recombination during evolution. This metabolic differentiation often reflects specialized niche adaptation (Steinke et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), indicating that Bv. JN.Y2 may rely on variations within other conserved clusters, such as Fengycin or Pks, to maintain its antagonistic efficacy and stabilize its biocontrol performance. The identification of non-synonymous mutations in the sporulation kinase \u003cem\u003ekinA\u003c/em\u003e reflects the role of the natural variation of the phosphorelay system in environmental adaptation (Stephenson and Hoch, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Similar to other stress-resistant \u003cem\u003eBacillus\u003c/em\u003e strains, Bv. JN.Y2 appears to employ fine-tuned regulation of core developmental genes to navigate fluctuating environments, establishing a molecular basis for effective colonization. Furthermore, sequence polymorphisms in ComP and chemotaxis proteins, coupled with structural variations in upstream regulatory elements, suggest a more precise root exudate sensing system. This likely drives the evolution of Bv. JN.Y2 from a \"generalist\" toward an \"environment-specific\" specialist, consistent with findings that signal transduction refinement provides a competitive advantage in soil colonization (Blake et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pangenome analysis characterized Bv. JN.Y2 as having an \"open\" genome. The presence of 411 unique genes\u0026mdash;involved in monobactam synthesis, environmental signaling, transmembrane transport, and cellular stress response\u0026mdash;suggests that HGT has facilitated the acquisition of novel metabolic features. This genetic uniqueness likely supports the strain's functional specificity and provides a solid molecular foundation for the targeted development of its beneficial traits (Olanrewaju et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While these candidate variations offer valuable insights for future functional genomics, their specific biological effects require further experimental validation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides a comprehensive evaluation of Bv. JN.Y2 as a beneficial biocontrol and PGP agent tailored for oat cultivation. Through a combination of longitudinal field trials and high-resolution genomic mining, we demonstrated that Bv. JN.Y2 offers a relatively stable performance in suppressing \u003cem\u003eC. cereale\u003c/em\u003e while simultaneously enhancing crop yields across diverse environmental conditions. The genomic architecture of Bv. JN.Y2, characterized by its \"open\" pangenome and diverse array of antimicrobial BGCs, underscores its favorable evolutionary adaptation to the plant endosphere. Our comparative analysis revealed that Bv. JN.Y2 maintains a unique genetic identity through 411 strain-specific genes and key regulatory variations, such as those in the \u003cem\u003ekinA\u003c/em\u003e and \u003cem\u003ecomP\u003c/em\u003e loci, which likely contribute to its efficient environmental sensing and colonization. Moreover, the plasmid-free nature and the absence of active resistance phenotypes establish a solid basis for its biosafety in agricultural applications. In summary, Bv. JN.Y2 represents a promising candidate for the development of targeted biopesticides and biofertilizers. While the identified candidate variations and unique metabolic pathways offer valuable insights, further functional studies will be beneficial to fully elucidate the specific biological effects of these genetic determinants in complex soil-plant ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the Science and Technology Program of Inner Mongolia Autonomous Region [Grant No. 2025YFHH0165]; the Basic Research Fund for Universities Directly Affiliated with Inner Mongolia Autonomous Region [Grant No. BR251033]; the Central Government-Guided Local Science and Technology Development Fund [Grant Nos. 2022ZY0060 and 2022ZY0065]; the National Key Research and Development Program of China [Grant No. 2023YFD1600701-5]; and the China Agriculture Research System for Oat and Buckwheat [Grant No. CARS-07-C-3]. The support from these programs and organizations has been relatively helpful in the successful completion of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003eThe authors declare that they have no competing interests or conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eW.Q. was responsible for conceptualization, methodology, investigation, formal analysis, writing \u0026ndash; original draft, data curation, validation, and visualization. T.Z. contributed to investigation, methodology, validation, and resources. C.L.L. performed investigation, data curation, and formal analysis. S.X.S. contributed to investigation, resources, and validation. X.X.Z. was involved in investigation, methodology, and data curation. K.H.L. contributed to investigation, software, and visualization. C.Y.W. performed investigation, resources, and formal analysis. M.M.Z. contributed to formal analysis, validation, software, and writing \u0026ndash; review \u0026amp; editing. B.Z.D. and H.Y.Z. were responsible for supervision, project administration, funding acquisition, conceptualization, and writing \u0026ndash; review \u0026amp; editing. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request. The complete genome sequence has been deposited in the GenBank database under the accession numbers CP178220.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eWe gratefully acknowledge the financial support provided by the following programs and organizations: the Science and Technology Program of Inner Mongolia Autonomous Region (Grant No. 2025YFHH0165); the Basic Research Fund for Universities Directly Affiliated with Inner Mongolia Autonomous Region (Grant No. BR251033); the Central Government-Guided Local Science and Technology Development Fund (Grant Nos. 2022ZY0060 and 2022ZY0065); the National Key Research and Development Program of China (Grant No. 2023YFD1600701-5); and the China Agriculture Research System for Oat and Buckwheat (Grant No. CARS-07-C-3). The support from these funding sources has been relatively helpful in the successful completion of this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e The online version contains supplementary material available as Online Resource 1, which includes additional tables and figures supporting the findings of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eButt MS, Tahir-Nadeem M, Khan M, K I et al (2008) Oat: unique among the cereals[J]. 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Appl Biochem Biotechnol 193(12):3949\u0026ndash;3969. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://link.springer.com/article/\u003c/span\u003e\u003cspan address=\"https://link.springer.com/article/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12010-021-03660-3\u003c/span\u003e\u003cspan address=\"10.1007/s12010-021-03660-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus velezensis, Oat anthracnose, Biocontrol, Plant growth promotion, Whole-Genome Sequencing, Comparative genomics","lastPublishedDoi":"10.21203/rs.3.rs-9114671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9114671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOat anthracnose, primarily caused by \u003cem\u003eColletotrichum cereale\u003c/em\u003e, represents a significant threat to oat production, necessitating the development of sustainable biocontrol alternatives. In this study, we characterized an oat endophytic bacterium, \u003cem\u003eBacillus velezensis\u003c/em\u003e JN.Y2, isolated from healthy oat leaves in Inner Mongolia. In vitro assays demonstrated that \u003cem\u003eB. velezensis\u003c/em\u003e JN.Y2 possesses an inhibitory activity against \u003cem\u003eC. cereale\u003c/em\u003e (74.49%) and exhibits a broad antifungal spectrum. Greenhouse and three-year multi-location field trials confirmed its beneficial biocontrol efficacy, which remained stable even under fluctuating climatic conditions and high disease pressure, consistently outperforming conventional chemical treatments. Beyond disease suppression, \u003cem\u003eB. velezensis\u003c/em\u003e JN.Y2 significantly enhanced oat growth and grain yield, supported by its ability to produce IAA, solubilize nutrients, and secrete diverse hydrolytic enzymes. Complete genome sequencing revealed a 3.87 Mb circular chromosome containing 13 secondary metabolite BGCs and 4 AOIs for RiPPs, including Amylocyclicin and LCI. Comparative genomic analysis highlighted an \"open\" pangenome and identified 411 unique genes associated with specialized metabolism and environmental sensing. While \u003cem\u003eB. velezensis\u003c/em\u003e JN.Y2 shares high sequence synteny with \u003cem\u003eB. velezensis\u003c/em\u003e CBMB205, distinct variations in the sporulation kinase \u003cem\u003ekinA\u003c/em\u003e and secondary metabolite pathways suggest a fine-tuned adaptation to the oat endosphere. Furthermore, biosafety evaluations confirmed a relatively high level of genetic stability and a lack of active antibiotic resistance. Collectively, these findings provide a beneficial molecular foundation for the application of \u003cem\u003eB. velezensis\u003c/em\u003e JN.Y2 as a reliable and secure biocontrol agent in sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Whole-genome and Comparative Genomic Analysis Reveal the Biocontrol and Plant Growth-Promoting Potential of Bacillus velezensis JN.Y2 Against Oat Anthracnose","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-03 01:32:31","doi":"10.21203/rs.3.rs-9114671/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-29T14:31:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-18T06:30:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31394866338602286445144702735975277891","date":"2026-03-30T02:09:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-29T18:48:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T09:53:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-18T09:53:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2026-03-13T12:07:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"235357ba-e852-4d68-9cd5-f7b28441b636","owner":[],"postedDate":"April 3rd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-04-29T14:31:10+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T03:09:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-03 01:32:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9114671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9114671","identity":"rs-9114671","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}