Integrated Genomic and Metabolomic Analysis Reveals the Biocontrol Potential of Endophytic Bacillus velezensis NS13 Against Fusarium spp. in Lonicera macranthoides

Integrated Genomic and Metabolomic Analysis Reveals the Biocontrol Potential of Endophytic Bacillus velezensis NS13 Against Fusarium spp. in Lonicera macranthoides | 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 Integrated Genomic and Metabolomic Analysis Reveals the Biocontrol Potential of Endophytic Bacillus velezensis NS13 Against Fusarium spp. in Lonicera macranthoides Junpeng Qi, Zhong Chen, Sheng’e Lu, Li Liu, Han Wang, Wei Zhuo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7464262/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jan, 2026 Read the published version in BMC Microbiology → Version 1 posted 19 You are reading this latest preprint version Abstract Fusarium spp. are major fungal pathogens causing root rot. They exhibit a broad host range and high pathogenicity, leading to yield losses, reduced quality, and plant mortality. Current control measures rely primarily on chemical pesticides, with few sustainable biological options available. This study compared rhizosphere microbial diversity between healthy and diseased Lonicera macranthoides , revealing increased pathogenic fungi ( Fusarium , Plectosphaerella , p < 0.01) and reduced beneficial fungi ( Trichoderma , Chao1/Shannon, p < 0.05) in diseased plants. An endophytic Bacillus velezensis strain, NS13, was isolated from healthy roots. Plate confrontation assays showed strong inhibition of Fusarium oxysporum from L. macranthoides and other Fusarium species ( F. solani , F. graminearum , F. fujikuroi ). The 3.95 Mb genome encoded 4,060 proteins, including 96 biocontrol-related genes. AntiSMASH identified 15 secondary metabolite biosynthetic gene clusters, with five linked to antifungal, three to antibacterial activity, and seven potentially novel compounds. LC–MS/MS metabolomics detected multiple antifungal metabolites, including cyclic dipeptides, fatty acid amides (e.g., erucamide), and oleanolic acid. These results demonstrate soil microbial dysbiosis in L. macranthoides affected by root rot and confirm the broad-spectrum anti- Fusarium potential of NS13, highlighting its promise as a biocontrol resource against Fusarium pathogens in medicinal plants. Fusarium spp. Bacillus velezensis NS13 genomics Lonicera macranthoides root rot metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Fusarium spp. are ubiquitous and highly destructive fungal pathogens with broad host specificity, systemic infection capabilities, and strong environmental adaptability.[ 1 ] They rapidly infect the roots, stem bases, and vascular systems of various crops, causing devastating diseases such as root rot, stem rot, and wilting.[ 2 ] The thick-walled chlamydospores of Fusarium can persist in soil for extended periods and produce mycotoxins, such as fusaric acid, posing severe threats to agricultural safety and human health.[ 3 , 4 ] Their robust sporulation and dissemination capacities enable regional or even trans-regional spread through infected plants, irrigation water, wind, agricultural practices, or seedling trade, leading to rapid disease outbreaks.[ 5 ] The covert and systemic nature of Fusarium infections makes early detection challenging and eradication difficult, often resulting in continuous cropping obstacles.[ 6 ] The high virulence, pathogenicity, and transmissibility of Fusarium spp. render them among the most challenging soil-borne fungal pathogens to control in agricultural ecosystems.[ 7 ] Lonicera macranthoides , an important medicinal plant in China [ 8 ], faces increasing challenges from root rot during cultivation, which severely impacts yield and quality, posing a critical bottleneck to the industry’s sustainable development. Lonicera spp., closely related to L. macranthoides [ 9 , 10 ], are similarly affected by root rot during cultivation. Research indicates that the genus Fusarium induces a range of symptoms in host plants, including wilting, root rot, and bulb rot, resulting in diseases with significant economic impacts [ 11 ]. Additionally, Fusarium spp. broadly affect other plants [ 12 ], such as Polygonatum odoratum and Polygonatum cyrtonema , where Fusarium oxysporum causes substantial tuber yield losses [ 13 , 14 ]. Root rot in medicinal crops like Angelica sinensis [ 15 ], Panax ginseng [ 16 ], Panax notoginseng [ 17 ], and Codonopsis pilosula [ 18 ] is often linked to Fusarium oxysporum and Fusarium solani , with disease mechanisms closely tied to mycotoxin production, such as fusaric acid, underscoring the widespread threat of Fusarium to medicinal plants [ 19 ]. Currently, Fusarium disease control primarily relies on chemical fungicides, but their prolonged use leads to environmental pollution, pesticide residues, pathogen resistance, and soil microbial dysbiosis, necessitating green, safe, and effective alternatives.[ 20 ] Endophytic bacteria, which naturally colonize host tissues and form stable symbiotic relationships with their environment, offer unique advantages in biocontrol [ 21 ]. Their mechanisms include: 1) resource competition, where siderophores and alkaline phosphatases compete with pathogens for carbon, nitrogen, and iron [ 22 ]; 2) direct antibiosis through non-ribosomal peptides, polyketides, and chitinases [ 23 ]; 3) induced resistance via activation of phytoalexin synthesis and cell wall reinforcement; [ 24 ] and 4) growth promotion through nitrogen fixation and phosphate solubilization, enhancing plant nutrition and disease resistance. [ 25 ] These synergistic mechanisms provide a robust foundation for the application of endophytic bacteria in disease control. Bacillus velezensis , a member of the Bacillus amyloliquefaciens subgroup, is recognized for its broad-spectrum antimicrobial properties and stable biocontrol efficacy. [ 26 ] Its genome is rich in non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters [ 27 ], enabling the synthesis of antifungal and antibacterial metabolites such as iturin, fengycin, bacillomycin, difficidin, and macrolactin [ 28 ]. Studies show that B. velezensis FZB42 and its derivatives effectively suppress Fusarium -related diseases, including wheat head blight [ 29 ], tomato wilt [ 30 ], and pepper root rot [ 31 ], while stably colonizing the rhizosphere or root tissues for sustained biocontrol [ 29 ]. This species also regulates plant hormone balance, enhances stress tolerance, and improves nutrient uptake, making it an ideal biocontrol and growth-promoting agent [ 32 ]. However, its application in medicinal plants, particularly for L. macranthoides root rot, remains underexplored. Addressing this research gap, this study isolated B. velezensis NS13 from L. macranthoides roots and systematically evaluated its inhibitory effects against Fusarium oxysporum , Fusarium fujikuroi , Fusarium solani , and Fusarium graminearum , the causal agents of root rot. The antibacterial mechanisms were elucidated at the genetic and metabolic levels, providing a valuable microbial resource and theoretical basis for the green control of L. macranthoides root rot and offering new insights for sustainable disease management in medicinal plants. Methods Plant and Soil Materials Healthy and root rot-affected L. macranthoides plants and their rhizosphere soil were collected from a cultivation base in Yun’ai Village, Zhongling Town, Xiushan County, Chongqing, China (28°47′N, 108°59′E). Diseased plants exhibited typical root rot symptoms (root decay, plant wilting). Soil samples were collected using a five-point sampling method within a 10–50 cm radius from the plant’s main stem base, removing surface litter and stones, and excavating to a depth of ~ 20 cm to collect 0–2 mm soil tightly adhering to roots. Samples from each treatment (healthy/diseased) were thoroughly mixed, stored in sterile bags, kept in an icebox, and rapidly transported to the laboratory. Each treatment included three biological replicates. Soil Microbial DNA Extraction and High-Throughput Sequencing Total soil DNA was extracted using a DNA extraction kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. Fungal community analysis targeted the ITS1 region using primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) for PCR amplification. The 25 µL reaction system included 12.5 µL 2× Taq PCR MasterMix (Vazyme, China), 1 µL of each primer (10 µmol/L), 2 µL DNA template (50 ng/µL), and 8.5 µL nuclease-free water. The PCR program was: 95°C for 5 min; 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s; and 72°C for 10 min. PCR products were verified by 2% agarose gel electrophoresis, purified using a Qiagen Gel Extraction Kit (Qiagen, Germany), and sequenced on the Illumina MiSeq platform (2 × 250 bp) by Shanghai Sangon Biotech Co., Ltd. A 5–10% PhiX control was added to ensure sequencing quality. Bioinformatics and Statistical Analysis Raw sequencing data were quality-controlled using QIIME2, removing low-quality sequences (quality score < 20, length < 200 bp) and chimeras to obtain clean tags. Operational taxonomic units (OTUs) were clustered at 97% similarity using VSEARCH [ 33 ], with taxonomic annotation performed against the UNITE database (v8.3) [ 14 ]. Alpha diversity indices (Chao1 richness, Shannon diversity) were calculated using VSEARCH. Differences in alpha diversity between healthy and diseased rhizosphere soils were assessed using t-tests (p < 0.05). The relative abundance of the top 10 dominant genera was visualized using bar plots generated with the R package ggplot2( https://ggplot2.tidyverse.org/ ). Endophytic Bacteria Isolation Healthy L. macranthoides roots were collected from the Yun’ai Village cultivation base. Surface soil was removed using a sterile scalpel, followed by surface sterilization: 75% ethanol for 2 min, 3% sodium hypochlorite for 5 min, and three rinses with sterile water. Sterilized root segments were cut into 0.5 cm pieces, ground in a sterile mortar, and suspended in 10 mL 0.85% sterile saline. The suspension was serially diluted (10⁻³–10⁻⁵), and 100 µL of each dilution was spread onto LB agar plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, pH 7.0) and incubated at 28°C for 48 h. Single colonies were selected based on morphology, purified by streaking three times, and stored at 4°C. Physiological, Biochemical, and Molecular Identification of Strain NS13 Strain NS13 was cultured on LB agar at 30°C for 24 h, and colony morphology (color, shape, margin) was observed. Gram staining was performed using a kit (Solarbio, China) and observed under a microscope. For scanning electron microscopy (SEM), NS13 was cultured in LB liquid medium (37°C, 150 rpm) for 48 h, centrifuged (4,500 × g, 5 min, 4°C), washed with PBS, resuspended in 2.5% glutaraldehyde fixative, fixed at room temperature for 2 h, and stored at 4°C. For molecular identification, genomic DNA was extracted using a bacterial DNA extraction kit (Omega Bio-tek, USA) and quantified with a TBS-380 fluorometer (Turner BioSystems, USA). The 16S rRNA gene sequences were compared against the GenBank database using BLAST to confirm taxonomic status. Plate Confrontation Assay of NS13 Against Fusarium spp. Four representative plant-pathogenic Fusarium spp., covering diverse hosts and ecological adaptations, were used. All strains grew stably on PDA medium (potato 200 g/L, glucose 20 g/L, agar 15 g/L, pH 6.0). Three reference strains (Table S1 ) were obtained from the BeNa Culture Collection (BNCC), and one was previously isolated from L. macranthoides root rot rhizosphere soil by our group. Strains were stored short-term on LB slants at 4°C and long-term in 20% glycerol at -80°C. The antagonistic ability of NS13 was evaluated using a plate confrontation assay. NS13 was cultured in LB liquid medium (28°C, 150 rpm) to an OD₆₀₀ of 1.0. Fusarium strains were grown on PDA plates until the colony diameter reached ~ 8 cm, and a 6 mm diameter fungal disc was taken from the colony edge and placed at the center of a new PDA plate. Four 1.0 µL drops of NS13 culture were symmetrically inoculated 2.0 cm from the fungal disc. The control group (CK) received equal volumes of LB medium. Each treatment had three replicates. Plates were incubated at 28°C until the control colony diameter reached 8 cm. Colony diameters (average of the longest and shortest perpendicular axes, in mm) were measured for both treatment and control groups (Wang et al., 2025). Inhibition rate was calculated as: Inhibition rate (%) = [(Dc – Dt) / Dc] × 100, where Dc is the control colony diameter and Dt is the treatment colony diameter. Data were processed in Microsoft Excel, and significant differences were analyzed using Student’s t-test in SPSS (v26.0, IBM Corp., USA) (p < 0.05). Whole-Genome Sequencing and Assembly of NS13 B. velezensis NS13 was cultured in LB liquid medium (37°C, 150 rpm). Genomic DNA (gDNA) was extracted using the Invitrogen PureLink® Genomic DNA Extraction Kit (Thermo Fisher Scientific, USA), with concentration and purity assessed using a NanoDrop ND-1000 spectrophotometer, followed by purification with the Zymo Quick-DNA Kit (Zymo Research, USA). For next-generation sequencing (NGS), a ~ 400 bp insert paired-end library was constructed: gDNA was fragmented using a Covaris sonicator, end-repaired with T4 DNA polymerase, A-tailed at the 3’ end, ligated with adapters, size-selected by gel electrophoresis, and amplified with indexing PCR. The library was quality-checked using an Agilent Bioanalyzer 2100 and sequenced on the Illumina platform (150 bp paired-end) by Shanghai Biozeron Biotech Co., Ltd. For PacBio sequencing, an SMRTbell library was prepared using the Express Template Prep Kit 2.0, with fragments > 8 kbp selected using BluePippin, and sequenced on the Sequel II platform. Genome assembly was performed as follows: NGS data were quality-controlled using Trimmomatic (v0.39) [ 34 ] with parameters SLIDINGWINDOW:4:15 MINLEN:75, PacBio long reads were error-corrected, and assembly was conducted using Unicycler (v0.5.0)[ 35 ] with default parameters, followed by genome circularization using Circlator (v1.5.5)[ 36 ]. Phylogenomic Analysis of NS13 The reference genome of B. velezensis FZB42 was downloaded from NCBI ( https://www.ncbi.nlm.nih.gov ). Phylogenomic analysis was performed using the Type Genome Server (TYGS, http://tygs.dsmz.de ), constructing a maximum likelihood phylogenetic tree based on genome BLAST distance with 1,000 bootstrap replicates, visualized using PhyD3. Digital DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator 3.0 (GGDC, https://ggdc.dsmz.de/ggdc.php ) with formula 2 (100 replicates), with a species threshold of ≥ 70%. Average nucleotide identity (ANI) was computed using JspeciesWS ( https://jspecies.ribohost.com/jspeciesws/ ) with the BLAST algorithm (ANIb), with a species threshold of ≥ 95%. Annotation of Antibacterial Genes and Secondary Metabolite Biosynthetic Gene Clusters NS13 protein-coding genes were annotated using public databases (NR, Swiss-Prot, COG, GO, KEGG). Biocontrol-related genes were categorized into four groups: resource competition genes (RCG), mediating competition for space and nutrients (e.g., siderophore synthesis/transport, alkaline phosphatase); antibacterial activity genes (AAG), encoding antibiotics (NRPS/PKS), antimicrobial peptides, and cell wall-degrading enzymes (e.g., chitinase, lysozyme); induced resistance genes (IRG), activating host defense mechanisms (e.g., phytoalexin synthesis, cell wall reinforcement); and growth-promoting genes (PGPG), enhancing plant resilience through nitrogen fixation (e.g., nifH/nifD) or phosphate solubilization. Secondary metabolite biosynthetic gene clusters (BGCs) were predicted using antiSMASH (v4.1.0)[ 37 ] and compared with B. velezensis FZB42. LC-MS/MS Analysis of NS13 Metabolites NS13 fermentation broth was centrifuged (10,000 × g, 10 min, 4°C) to remove solids, and the supernatant was collected. The pH was adjusted to 2.0 with HCl to optimize metabolite extraction or precipitation. Samples were incubated at 4°C overnight to promote precipitation, re-centrifuged (10,000 × g, 10 min, 4°C), and the precipitate was extracted with acetonitrile. The extract was concentrated under reduced pressure, lyophilized, reconstituted in methanol:water (80:20, v/v), and filtered through a 0.22 µm membrane to remove particles. LC-MS/MS analysis was performed using a reverse-phase C18 column (ACQUITY UPLC BEH C18, 2.1×100 mm, 1.7 µm). The mobile phase consisted of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B), with a gradient of 0–2 min, 5% B; 2–20 min, 5%–95% B; 20–25 min, 95% B; 25–27 min, return to 5% B. The flow rate was 0.3 mL/min, column temperature was 40°C, and injection volume was 3 µL. Mass spectrometry was conducted on a high-resolution Thermo Q Exactive HF-X in positive ion mode, with a full scan range of m/z 150–2000 (resolution 70,000), using data-dependent acquisition (DDA) or targeted parallel reaction monitoring (PRM), HCD fragmentation (NCE 25–35), MS/MS resolution of 17,500, and dynamic exclusion of 15 s. Raw data were processed using Xcalibur and Compound Discoverer 3.3, with metabolite identification performed against GNPS, MassBank, and PubChem databases. Results Soil Fungal Community Diversity Analysis in Root Rot-Affected L. macranthoides Phenotypic observations of L. macranthoides root rot (Fig. 1 A) showed yellow-brown decay of fine roots progressing to main roots, with later stages exhibiting black-brown softening, leaving only the xylem, accompanied by leaf yellowing, stem base browning, growth cessation, and eventual plant death. Alpha diversity analysis (Fig. 1 B) revealed significantly lower Chao1 (richness) and Shannon (diversity) indices in the rhizosphere fungal communities of diseased plants compared to healthy ones (p < 0.05), indicating a simplified microbial community structure and disrupted homeostasis. This suggests that root rot significantly alters rhizosphere microbial community structure and diversity. Genus-level composition analysis (Fig. 1 C) revealed a marked shift in community structure. Healthy plant rhizospheres were dominated by beneficial genera such as Trichoderma (biocontrol function [ 38 ]), Saitozyma , Arxiella , Penicillium , and Aspergillus (top 5), while diseased plants showed a significant increase in pathogenic genera, including Fusarium (pathogen), Plectosphaerella , Auricularia , and Staphylotrichum . Quantitative comparisons (Figure S1 A) showed significantly reduced abundances of Trichoderma , Saitozyma , Arxiella , Penicillium , Aspergillus , and Scytalidium in diseased plants, while Fusarium , Plectosphaerella , Auricularia , and Staphylotrichum (Figure S1 B) were significantly enriched (p < 0.01), indicating a strong association between root rot and the enrichment of pathogenic fungi and depletion of beneficial microbes. Isolation and Identification of Bacillus velezensis NS13 Endophytic bacteria were isolated from L. macranthoides root tissues, and a strain with distinct morphological characteristics, resembling Bacillus , was selected after three rounds of purification and named NS13 (Fig. 2 A). It exhibited typical Bacillus morphology and was Gram-positive (Fig. 2 B, C). A phylogenetic tree based on 16S rRNA gene sequences (Fig. 2 D) showed a close relationship with the type strain B. velezensis FZB42. This taxonomic classification was supported by physiological and biochemical characteristics (Table S1 ): NS13 was negative for the Voges-Proskauer test, positive for nitrate reduction, capable of utilizing citrate and propionate as carbon sources, but unable to metabolize D-mannitol, D-xylose, or L-arabinose. It exhibited protease activity (gelatin liquefaction), starch hydrolysis, weak salt tolerance (up to 7% NaCl), and growth at pH 5.7. Plate Confrontation Assay of NS13 Against Fusarium spp. Plate confrontation assays (Fig. 3 C) demonstrated significant inhibition of F. oxysporum (from L. macranthoides root rot) by NS13, with an inhibition rate of 56.60% ± 3.88%. In pairwise confrontation assays with three other Fusarium strains, NS13 formed clear inhibition zones, with morphological changes observed at the fungal colony edges, suggesting the production of potent antifungal metabolites (Fig. 3 A, B). Quantitative analysis (Fig. 3 D) showed varying inhibition rates: 76.04% ± 1.68% against F. fujikuroi (highest), 70.14% ± 1.46% against F. solani , 56.60% ± 3.88% against F. oxysporum , and 51.59% ± 2.24% against F. graminearum . Genomic Features and Taxonomic Analysis of NS13 Hybrid assembly using PacBio RSIII and Illumina NovaSeq 6000 data yielded a complete, closed circular chromosome for B. velezensis NS13 (Fig. 4 ), with a genome size of 3.95 Mb and a GC content of 46.55%. A total of 4,060 protein-coding genes were predicted (Table S2 ), with 91.26% annotated in the NR database (Table S3 ), indicating robust metabolic capacity and environmental adaptability. Functional annotation revealed potential antifungal mechanisms: the CAZy database (Figure S2 A) identified 152 carbohydrate-active enzyme genes, including GH1, GT2, and CBM50 families, suggesting capabilities for degrading complex carbon sources and recognizing fungal cell wall components, enhancing direct fungal inhibition. KEGG annotation identified 3,463 metabolism-related genes (Figure S2 B-C), with significant enrichment in secondary metabolite biosynthesis (24.09%) and antibiotic biosynthesis (18.16%), indicating the ability to produce diverse antifungal molecules. Genes related to amino acid synthesis (10.72%) and ABC transport systems (10.1%) supported metabolite synthesis, regulation, and transport. COG annotation (Figure S3 A) showed enrichment in amino acid transport/metabolism, carbohydrate metabolism, and cell wall biogenesis, aiding activity under fungal stress. GO annotation (Figure S3 B) highlighted widespread “metabolic process” and “catalytic activity” genes, reflecting diverse metabolic and environmental response potential. Collectively, NS13’s genetic repertoire supports carbon utilization, secondary metabolite production, transmembrane transport, and fungal recognition, forming the basis of its antifungal activity. Phylogenomic analysis confirmed NS13’s taxonomic status. A 16S rRNA-based phylogenetic tree (Fig. 2 D) showed a close relationship with B. velezensis . A whole-genome-based phylogenetic tree (Figure S3 A) with multiple reference strains confirmed NS13’s closest relation to FZB42. isDDH and ANI analyses (Figure S3 B) showed similarity values exceeding 70% and 95%, respectively, meeting species delineation criteria. Based on morphological, physiological, biochemical, and phylogenomic evidence, NS13 was classified as Bacillus velezensis NS13 within the Firmicutes, Bacilli, Bacillales, Bacillaceae, and Bacillus genera. Mining of Antifungal Genes in NS13 Functional annotation revealed a systematic antifungal genetic foundation in B. velezensis NS13, with 96 biocontrol-related genes identified (Fig. 5 A): 65.63% induced resistance genes, 22.92% resource competition genes, 8.33% antibacterial activity genes, and 3.13% growth-promoting genes. Specifically (Table S5), polyketide synthase genes (e.g., K15327) synthesize antifungal polyketides [ 39 ], endoglucanase genes (K01179) degrade fungal cell walls [ 40 ], and siderophore transport genes (K23188) limit fungal iron acquisition [ 41 ]. Two-component system histidine kinases (K07777) and Fur iron regulators (K03711) may induce host resistance via JA/SA signaling and oxidative stress responses [ 42 ], while sporulation genes (K06378) support rhizosphere colonization and sustained biocontrol [ 43 ]. KEGG enrichment analysis (Fig. 5 B) identified 15 significant pathways, including antibiotic synthesis, two-component systems, and ABC transport, supporting NS13’s antifungal mechanisms via direct antibiosis, nutrient competition, and induced resistance. AntiSMASH analysis identified 15 secondary metabolite BGCs (Fig. 5 C), spanning 888.062 kb (22.49% of the genome). Eight BGCs had defined functions (Table S6), encoding antifungal metabolites like fengycin, surfactin (lipopeptides), bacillaene, difficidin (polyketides), bacillibactin (siderophore), and bacilysin (membrane-disrupting). Fengycin and surfactin disrupt fungal membranes, bacillaene inhibits chitin synthesis, and bacillibactin limits fungal iron acquisition [ 44 , 45 ]. Seven unannotated BGCs (22.04%) contained PKS-NRPS hybrid modules and unique modification enzyme domains, suggesting potential for novel antifungal compounds. Comparison with FZB42 revealed a unique locillomycin BGC in NS13, indicating broader or differential antifungal capabilities. In summary, NS13’s biocontrol genes, diverse BGCs, and detectable broad-spectrum antifungal metabolites synergistically contribute to direct antibiosis, nutrient competition, and induced resistance, forming the genetic and metabolic basis for its effective antagonism against Fusarium spp. LC-MS Analysis Reveals Antifungal Metabolites in NS13 High-resolution mass spectrometry confirmed NS13’s rich antifungal metabolite profile. Multiple compounds with potential biocontrol activity were detected (Table S7), including cyclic dipeptides with a diketopiperazine (DKP) scaffold, such as Cyclo(phenylalanyl-prolyl), 3-(1-hydroxyethyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, 3-(propan-2-yl)-octahydropyrrolo[1,2-a]pyrazine-1,4-dione, and 3-[(4-hydroxyphenyl)methyl]-octahydropyrrolo[1,2-a]pyrazine-1,4-dione. These cyclic dipeptides, known for membrane permeability and structural stability, are widely recognized for antibacterial and antifungal activity [ 46 , 47 ], and their repeated detection suggests they are key factors in NS13’s inhibition of Fusarium . Additionally, fatty acid amides with membrane-disrupting potential, such as oleamide, erucamide, and stearamide, were detected [ 48 – 50 ]. Active dipeptides like carnosine and valylproline, capable of scavenging free radicals and modulating microbial interactions, were also identified, along with phenolic antioxidants like rutin and quercetin-3β-D-glucoside, reported to inhibit fungal virulence factors [ 51 , 52 ], potentially enhancing NS13’s resilience to fungal stress. Notably, polyamines like spermine were detected, which stabilize DNA, regulate membrane permeability [ 53 ], and activate plant immune signaling, suggesting both direct antibacterial activity and enhanced host defense. In addition, triterpenoids with potential antifungal activity, such as oleanolic acid [ 54 ], and some metabolites that have not been fully annotated, such as PPK and NP-011220, were also detected, which may represent secondary metabolites unique to NS13. Further confirmation of their biological functions through structural analysis and activity validation is needed. Discussion Genomic Analysis Reveals the Genetic Basis of NS13 Biocontrol Genomic analysis serves as a powerful methodological tool, providing rich insights into the biocontrol mechanisms of Bacillus velezensis [ 55 ]. These mechanisms, including the synthesis of antibacterial compounds, competitive nutrient suppression, and induction of host resistance, are all reflected at the genomic level [ 56 ]. Furthermore, genomic analysis offers critical support for the genetic improvement of Bacillus velezensis , enabling optimization of its antifungal activity or environmental adaptability through gene editing or synthetic biology approaches, thereby enhancing its biocontrol application potential [ 57 ]. Specifically, the 3.95 Mb NS13 genome encodes 4,060 proteins, including 96 antifungal-related genes. Notably, NRPS and PKS genes, such as those responsible for fengycin and bacillaene synthesis, align with known antifungal mechanisms in B. velezensis [ 28 , 58 ]. Siderophore genes, including those for bacillibactin, support nutrient competition by limiting Fusarium iron acquisition [ 41 ]. Genes linked to induced systemic resistance, such as those regulating JA/SA signaling, suggest enhanced host defense, consistent with other plant [ 42 ]. The discovery of seven uncharacterized BGCs encoding novel PKS-NRPS hybrid enzymes highlights NS13’s potential to produce unique antifungal compounds, distinguishing it from FZB42. This genetic diversity positions NS13 as a valuable resource for biocontrol and novel metabolite discovery. Metabolomics Provides the Material Basis for NS13’s Antifungal Activity Metabolomics, as a core technical tool for analyzing microbial functional metabolites, can systematically identify various antifungal active components produced by Bacillus velezensis during its growth and metabolism, thereby providing direct evidence for clarifying the material basis of its biocontrol effects [ 59 ]. LC-MS/MS analysis identified a range of NS13 antifungal metabolites, including cyclic dipeptides [ 60 ], fatty acid amides such as erucamide, and triterpenoids such as oleanolic acid. Cyclic dipeptides disrupt fungal signaling and membrane integrity, while lipopeptides like fengycin and surfactin destabilize fungal membranes [ 61 ]. The detection of oleanolic acid [ 62 ], which is known for its antifungal properties, suggests synergy with lipopeptides, enhancing NS13’s biocontrol efficacy. Uncharacterized BGCs encoding novel metabolites further underscore NS13’s potential for developing new antifungal agents. Compared to other Bacillus strains, NS13’s unique production of locillomycin and diverse dipeptides suggests broader antifungal capabilities, potentially overcoming strain-specific efficacy limitations. These findings align with prior studies demonstrating B. velezensis efficacy against Fusarium diseases in crops like wheat and maize [ 63 , 64 ]. For instance, FZB42 produces similar lipopeptides, including fengycin and surfactin, to suppress F. graminearum [ 65 ]. However, NS13’s unique locillomycin [ 66 ] BGC and higher BGC diversity suggest enhanced antifungal potential, particularly against F. fujikuroi and F. solani. Unlike earlier studies focusing on cereals, this work extends B. velezensis application to medicinal plants, addressing a critical gap in L. macranthoides root rot control. Differences in inhibition rates, such as lower efficacy against F. graminearum compared to F. fujikuroi, may reflect species-specific susceptibility or experimental conditions, including medium and incubation time. The integrated genomic and metabolomic approach distinguishes this study from earlier phenotype-based work, providing mechanistic insights into NS13’s biocontrol efficacy. Potential and Challenges of B. velezensis NS13 in Controlling L. macranthoides Root Rot In root rot-affected L. macranthoides rhizospheres, Fusarium enrichment aligns with its established role as a major soil-borne pathogen. The observed decline in beneficial fungi such as Trichoderma and in microbial diversity, as indicated by Chao1 and Shannon indices with p < 0.05, points to microbial dysbiosis, likely driven by F. oxysporum mycotoxins such as fumonisins that suppress competing microbes [ 67 ]. The high Fusarium abundance in diseased plants confirms its role as the primary driver of root rot, consistent with its pathogenicity in related species like L. japonica . These findings highlight the need for targeted biocontrol to restore microbial balance and suppress Fusarium dominance. B. velezensis NS13 exhibited potent antagonism against F. oxysporum with 56.60% inhibition and against other Fusarium spp, with the highest efficacy against F. fujikuroi at 76.04%. This variability may reflect differences in cell wall composition or metabolic vulnerabilities among Fusarium species [ 68 ].. Clear inhibition zones in confrontation assays suggest diffusible antibacterial compounds, supported by the detection of lipopeptides such as fengycin and surfactin, as well as polyketides such as bacillaene, which are known to disrupt fungal membranes and spore germination [ 65 ]. Unlike chemical fungicides, which face resistance issues, NS13’s multi-mode action minimizes resistance development. Differential inhibition rates suggest NS13’s efficacy may be optimized for specific Fusarium strains, warranting field validation. This study elucidates the genetic and metabolic mechanisms of NS13’s antagonism against Fusarium spp., positioning it as a viable alternative to chemical fungicides and addressing environmental concerns like pesticide residues and resistance [ 65 ]. Practically, NS13 could be developed as a biofungicide for sustainable L. macranthoides cultivation, reducing yield losses and ensuring medicinal quality. The discovery of novel BGCs opens avenues for identifying new antifungal compounds with potential in medicine and agriculture. Despite its strengths, this study has limitations. Plate-based assays, while reliable, may not fully reflect NS13’s performance in complex soil environments, where abiotic factors such as pH and humidity, along with microbial interactions, may modulate efficacy. Field trials are needed to validate NS13’s biocontrol potential under natural conditions. The functions of seven uncharacterized BGCs remain speculative, requiring targeted gene knockout or heterologous expression studies for confirmation. The focus on L. macranthoides limits direct extrapolation to other crops, though NS13’s broad-spectrum activity suggests wider applicability. Future research should explore NS13’s interactions with plant immune systems and its long-term stability in rhizosphere microbiomes. Overall, this study establishes B. velezensis NS13 as a potent biocontrol agent against Fusarium-induced root rot in L. macranthoides, leveraging synergistic biocontrol genes and antifungal metabolites. Its application could transform disease management in medicinal plant cultivation, promoting sustainability. Future studies should focus on field validation, functional characterization of novel BGCs, and NS13’s role in modulating plant immunity to enhance biocontrol efficacy. Conclusions This study provides significant insights into the microbial dynamics associated with Fusarium -induced root rot in Lonicera macranthoides and highlights the potential of Bacillus velezensis strain NS13 as a sustainable biocontrol agent. The observed microbial dysbiosis in the rhizosphere of diseased plants, characterized by an increase in pathogenic fungi such as Fusarium and Plectosphaerella and a reduction in beneficial fungi like Trichoderma , underscores the ecological imbalance contributing to disease progression. The isolation of the endophytic B. velezensis NS13 from healthy roots and its demonstrated strong antagonistic activity against multiple Fusarium species, including F. oxysporum , F. solani , F. graminearum , and F. fujikuroi , positions it as a promising biocontrol candidate. Genomic analysis revealed a robust arsenal of 96 biocontrol-related genes and 15 secondary metabolite biosynthetic gene clusters, five of which are linked to antifungal activity, three to antibacterial activity, and seven potentially encoding novel compounds. The detection of antifungal metabolites, such as cyclic dipeptides, fatty acid amides (e.g., erucamide), and oleanolic acid, via LC–MS/MS further supports the strain’s efficacy. These findings not only enhance our understanding of soil microbial interactions in the context of root rot but also advocate for the development of B. velezensis NS13 as an environmentally friendly alternative to chemical pesticides for managing Fusarium pathogens in medicinal plants. Future research should focus on field trials and formulation strategies to optimize the practical application of NS13 in sustainable agriculture. Abbreviations ITS: Internal Transcribed Spacer ; LB: Luria-Bertani ; OD: Optical Density PCR: Polymerase Chain Reaction ;SEM: Scanning Electron Microscopy Declarations Clinical Trial: Not applicable Ethics approval and consent to participate : Not applicable Consent for publication : Not applicable Availability of data and materials : The datasets generated and/or analysed during the current study are available in the National Center for Biotechnology Information (NCBI) repository, primary accession code PRJNA1306347. Competing interests : The authors declare that they have no competing interests. Funding: This work was supported by the Basic Scientific Research Projects of Chongqing (Nos. 2024jbky-021, 2025jbky-008, 2024jbky-017), and the Performance Incentive Guidance Special Project of Chongqing (No. 2022jx011), as well as the Basic Research Expenses of Chongqing Science and Technology Bureau (No. 2024jbky-032). Authors' contributions J. Qi, S. Lu, and Z. Chen conceptualized the study and designed experiments to investigate microbial dysbiosis in Lonicera macranthoides root rot, including microbial diversity comparisons using Chao1/Shannon indices and p-value analyses. J. Qi and Z. Chen collected rhizosphere soil samples, isolated the NS13 strain, and conducted plate confrontation assays. J. Qi, L. Liu, and H. Zhou analyzed metabolites via LC-MS/MS. J. Qi, Z. Chen, and W. Zhuo performed genomic sequencing and AntiSMASH analysis to identify biosynthetic gene clusters. J. Qi and Z. Chen drafted the manuscript, with Y. Wang, M. Yang, Y. Yang, and F. Ren contributing to revisions and polishing. S. Lu and H. Wang provided essential strains, plant materials, and LC-MS/MS equipment. F. Ren coordinated team efforts and ensured smooth project execution. F. Ren secured funding for the study. All authors approved the final manuscript. Acknowledgements None. References Coleman JJ. 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17:19:41","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":193984,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/abc5cbcd70803b63d8f21ac9.html"},{"id":92614290,"identity":"82e99119-ed47-4fbb-92b6-c75ce964f185","added_by":"auto","created_at":"2025-10-01 17:19:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6667741,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and microbial community characteristics of \u003cem\u003eL. macranthoides\u003c/em\u003e root rot. (A) Phenotype of root rot. (B) Alpha diversity indices (Chao1 and Shannon) of rhizosphere microbial communities in healthy and diseased plants. (C) Genus-level composition of rhizosphere microbial communities in healthy and diseased plants.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/d04c1684c4900c637f54f14d.png"},{"id":92614973,"identity":"1fdf5588-79df-4b10-97e5-232017954a70","added_by":"auto","created_at":"2025-10-01 17:27:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2554468,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological characteristics and phylogenetic tree of strain NS13 based on 16S rRNA gene sequences. (A) Colony morphology. (B) Gram staining. (C) SEM image. (D) Phylogenetic tree based on 16S rRNA.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/ec8764f59c13f27ca897e286.png"},{"id":92614976,"identity":"fc5af8c4-1999-47a9-bebc-90dbe32bf337","added_by":"auto","created_at":"2025-10-01 17:27:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6553132,"visible":true,"origin":"","legend":"\u003cp\u003eAntagonistic activity of NS13 against four \u003cem\u003eFusarium\u003c/em\u003e strains. (A) Individual cultures of four \u003cem\u003eFusarium\u003c/em\u003e strains. (B) Plate confrontation of NS13 with four \u003cem\u003eFusarium\u003c/em\u003estrains. (C) Schematic of the confrontation assay. (D) Inhibition rates of NS13 against four \u003cem\u003eFusarium\u003c/em\u003e strains.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/fd47d58acdd4ce35b11633d1.png"},{"id":92614977,"identity":"d21011ea-ef80-460f-80e4-8fc57a1fb0d7","added_by":"auto","created_at":"2025-10-01 17:27:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1885926,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic circular map of NS13. Note: From outer to inner rings, the outermost ring indicates genome size; the second and third rings represent CDS on the positive and negative strands, with colors indicating different COG functional categories; the fourth ring shows rRNA and tRNA; the fifth ring displays GC content, with outward red peaks indicating regions above the average GC content and inward blue peaks indicating regions below; the innermost ring shows GC skew (G-C/G+C), with positive values favoring positive-strand transcription and negative values favoring negative-strand transcription.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/48b26c8086587f8d2b3b15f9.png"},{"id":92615565,"identity":"69a0f1d1-4f9a-4c38-bc91-c4863f40648f","added_by":"auto","created_at":"2025-10-01 17:35:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1117772,"visible":true,"origin":"","legend":"\u003cp\u003eBiocontrol characteristics of NS13 genome. (A) Statistics of four biocontrol gene categories. (B) Heatmap of gene-biocontrol mechanism associations. (C) Secondary metabolite BGC analysis.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/8f8ee80abec009374270bb67.png"},{"id":100070089,"identity":"9296ddae-63b8-4dd7-9b71-39bd5d70e409","added_by":"auto","created_at":"2026-01-12 16:16:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18991608,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/22dad130-3edb-4617-9aad-59baac6e1b95.pdf"},{"id":92614286,"identity":"c538e5df-f6ba-48d9-8269-5d0c9856e0a5","added_by":"auto","created_at":"2025-10-01 17:19:40","extension":"csv","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":13489,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5Genomicbiocontrolannotation.csv","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/c9b988c018a0242ba0524b3c.csv"},{"id":92614285,"identity":"eb457f6d-919d-46e9-b8d0-81afb09f3641","added_by":"auto","created_at":"2025-10-01 17:19:40","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26829,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7Compounds.csv","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/a2d58c8325017de4ac4762f1.csv"},{"id":92614296,"identity":"d7bd8e9f-6581-4988-9779-5736d6a6b9a0","added_by":"auto","created_at":"2025-10-01 17:19:41","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2255641,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7464262/v1/834313b18067d8e8a61686a4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated Genomic and Metabolomic Analysis Reveals the Biocontrol Potential of Endophytic Bacillus velezensis NS13 Against Fusarium spp. in Lonicera macranthoides","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eFusarium\u003c/em\u003e spp. are ubiquitous and highly destructive fungal pathogens with broad host specificity, systemic infection capabilities, and strong environmental adaptability.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] They rapidly infect the roots, stem bases, and vascular systems of various crops, causing devastating diseases such as root rot, stem rot, and wilting.[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] The thick-walled chlamydospores of \u003cem\u003eFusarium\u003c/em\u003e can persist in soil for extended periods and produce mycotoxins, such as fusaric acid, posing severe threats to agricultural safety and human health.[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] Their robust sporulation and dissemination capacities enable regional or even trans-regional spread through infected plants, irrigation water, wind, agricultural practices, or seedling trade, leading to rapid disease outbreaks.[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] The covert and systemic nature of \u003cem\u003eFusarium\u003c/em\u003e infections makes early detection challenging and eradication difficult, often resulting in continuous cropping obstacles.[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] The high virulence, pathogenicity, and transmissibility of \u003cem\u003eFusarium\u003c/em\u003e spp. render them among the most challenging soil-borne fungal pathogens to control in agricultural ecosystems.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e\u003cp\u003e\u003cem\u003eLonicera macranthoides\u003c/em\u003e, an important medicinal plant in China [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], faces increasing challenges from root rot during cultivation, which severely impacts yield and quality, posing a critical bottleneck to the industry\u0026rsquo;s sustainable development. \u003cem\u003eLonicera\u003c/em\u003e spp., closely related to \u003cem\u003eL. macranthoides\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], are similarly affected by root rot during cultivation. Research indicates that the genus \u003cem\u003eFusarium\u003c/em\u003e induces a range of symptoms in host plants, including wilting, root rot, and bulb rot, resulting in diseases with significant economic impacts [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, \u003cem\u003eFusarium\u003c/em\u003e spp. broadly affect other plants [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], such as \u003cem\u003ePolygonatum odoratum\u003c/em\u003e and \u003cem\u003ePolygonatum cyrtonema\u003c/em\u003e, where \u003cem\u003eFusarium oxysporum\u003c/em\u003e causes substantial tuber yield losses [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Root rot in medicinal crops like \u003cem\u003eAngelica sinensis\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], \u003cem\u003ePanax ginseng\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003ePanax notoginseng\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and \u003cem\u003eCodonopsis pilosula\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] is often linked to \u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eFusarium solani\u003c/em\u003e, with disease mechanisms closely tied to mycotoxin production, such as fusaric acid, underscoring the widespread threat of \u003cem\u003eFusarium\u003c/em\u003e to medicinal plants [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrently, \u003cem\u003eFusarium\u003c/em\u003e disease control primarily relies on chemical fungicides, but their prolonged use leads to environmental pollution, pesticide residues, pathogen resistance, and soil microbial dysbiosis, necessitating green, safe, and effective alternatives.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Endophytic bacteria, which naturally colonize host tissues and form stable symbiotic relationships with their environment, offer unique advantages in biocontrol [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Their mechanisms include: 1) resource competition, where siderophores and alkaline phosphatases compete with pathogens for carbon, nitrogen, and iron [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; 2) direct antibiosis through non-ribosomal peptides, polyketides, and chitinases [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; 3) induced resistance via activation of phytoalexin synthesis and cell wall reinforcement; [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and 4) growth promotion through nitrogen fixation and phosphate solubilization, enhancing plant nutrition and disease resistance. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] These synergistic mechanisms provide a robust foundation for the application of endophytic bacteria in disease control.\u003c/p\u003e\u003cp\u003e\u003cem\u003eBacillus velezensis\u003c/em\u003e, a member of the \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e subgroup, is recognized for its broad-spectrum antimicrobial properties and stable biocontrol efficacy. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Its genome is rich in non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], enabling the synthesis of antifungal and antibacterial metabolites such as iturin, fengycin, bacillomycin, difficidin, and macrolactin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Studies show that \u003cem\u003eB. velezensis\u003c/em\u003e FZB42 and its derivatives effectively suppress \u003cem\u003eFusarium\u003c/em\u003e-related diseases, including wheat head blight [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], tomato wilt [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and pepper root rot [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while stably colonizing the rhizosphere or root tissues for sustained biocontrol [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This species also regulates plant hormone balance, enhances stress tolerance, and improves nutrient uptake, making it an ideal biocontrol and growth-promoting agent [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, its application in medicinal plants, particularly for \u003cem\u003eL. macranthoides\u003c/em\u003e root rot, remains underexplored.\u003c/p\u003e\u003cp\u003eAddressing this research gap, this study isolated \u003cem\u003eB. velezensis\u003c/em\u003e NS13 from \u003cem\u003eL. macranthoides\u003c/em\u003e roots and systematically evaluated its inhibitory effects against \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eFusarium fujikuroi\u003c/em\u003e, \u003cem\u003eFusarium solani\u003c/em\u003e, and \u003cem\u003eFusarium graminearum\u003c/em\u003e, the causal agents of root rot. The antibacterial mechanisms were elucidated at the genetic and metabolic levels, providing a valuable microbial resource and theoretical basis for the green control of \u003cem\u003eL. macranthoides\u003c/em\u003e root rot and offering new insights for sustainable disease management in medicinal plants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant and Soil Materials\u003c/h2\u003e\u003cp\u003eHealthy and root rot-affected \u003cem\u003eL. macranthoides\u003c/em\u003e plants and their rhizosphere soil were collected from a cultivation base in Yun\u0026rsquo;ai Village, Zhongling Town, Xiushan County, Chongqing, China (28\u0026deg;47\u0026prime;N, 108\u0026deg;59\u0026prime;E). Diseased plants exhibited typical root rot symptoms (root decay, plant wilting). Soil samples were collected using a five-point sampling method within a 10\u0026ndash;50 cm radius from the plant\u0026rsquo;s main stem base, removing surface litter and stones, and excavating to a depth of ~\u0026thinsp;20 cm to collect 0\u0026ndash;2 mm soil tightly adhering to roots. Samples from each treatment (healthy/diseased) were thoroughly mixed, stored in sterile bags, kept in an icebox, and rapidly transported to the laboratory. Each treatment included three biological replicates.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSoil Microbial DNA Extraction and High-Throughput Sequencing\u003c/h3\u003e\n\u003cp\u003eTotal soil DNA was extracted using a DNA extraction kit (Tiangen Biotech, Beijing, China) following the manufacturer\u0026rsquo;s instructions. Fungal community analysis targeted the ITS1 region using primers ITS1F (5\u0026rsquo;-CTTGGTCATTTAGAGGAAGTAA-3\u0026rsquo;) and ITS2R (5\u0026rsquo;-GCTGCGTTCTTCATCGATGC-3\u0026rsquo;) for PCR amplification. The 25 \u0026micro;L reaction system included 12.5 \u0026micro;L 2\u0026times; Taq PCR MasterMix (Vazyme, China), 1 \u0026micro;L of each primer (10 \u0026micro;mol/L), 2 \u0026micro;L DNA template (50 ng/\u0026micro;L), and 8.5 \u0026micro;L nuclease-free water. The PCR program was: 95\u0026deg;C for 5 min; 35 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, 72\u0026deg;C for 30 s; and 72\u0026deg;C for 10 min. PCR products were verified by 2% agarose gel electrophoresis, purified using a Qiagen Gel Extraction Kit (Qiagen, Germany), and sequenced on the Illumina MiSeq platform (2 \u0026times; 250 bp) by Shanghai Sangon Biotech Co., Ltd. A 5\u0026ndash;10% PhiX control was added to ensure sequencing quality.\u003c/p\u003e\n\u003ch3\u003eBioinformatics and Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eRaw sequencing data were quality-controlled using QIIME2, removing low-quality sequences (quality score\u0026thinsp;\u0026lt;\u0026thinsp;20, length\u0026thinsp;\u0026lt;\u0026thinsp;200 bp) and chimeras to obtain clean tags. Operational taxonomic units (OTUs) were clustered at 97% similarity using VSEARCH [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], with taxonomic annotation performed against the UNITE database (v8.3) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Alpha diversity indices (Chao1 richness, Shannon diversity) were calculated using VSEARCH. Differences in alpha diversity between healthy and diseased rhizosphere soils were assessed using t-tests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The relative abundance of the top 10 dominant genera was visualized using bar plots generated with the R package ggplot2(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggplot2.tidyverse.org/\u003c/span\u003e\u003cspan address=\"https://ggplot2.tidyverse.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eEndophytic Bacteria Isolation\u003c/h3\u003e\n\u003cp\u003eHealthy \u003cem\u003eL. macranthoides\u003c/em\u003e roots were collected from the Yun\u0026rsquo;ai Village cultivation base. Surface soil was removed using a sterile scalpel, followed by surface sterilization: 75% ethanol for 2 min, 3% sodium hypochlorite for 5 min, and three rinses with sterile water. Sterilized root segments were cut into 0.5 cm pieces, ground in a sterile mortar, and suspended in 10 mL 0.85% sterile saline. The suspension was serially diluted (10⁻\u0026sup3;\u0026ndash;10⁻⁵), and 100 \u0026micro;L of each dilution was spread onto LB agar plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L, pH 7.0) and incubated at 28\u0026deg;C for 48 h. Single colonies were selected based on morphology, purified by streaking three times, and stored at 4\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003ePhysiological, Biochemical, and Molecular Identification of Strain NS13\u003c/h3\u003e\n\u003cp\u003eStrain NS13 was cultured on LB agar at 30\u0026deg;C for 24 h, and colony morphology (color, shape, margin) was observed. Gram staining was performed using a kit (Solarbio, China) and observed under a microscope. For scanning electron microscopy (SEM), NS13 was cultured in LB liquid medium (37\u0026deg;C, 150 rpm) for 48 h, centrifuged (4,500 \u0026times; g, 5 min, 4\u0026deg;C), washed with PBS, resuspended in 2.5% glutaraldehyde fixative, fixed at room temperature for 2 h, and stored at 4\u0026deg;C.\u003c/p\u003e\u003cp\u003eFor molecular identification, genomic DNA was extracted using a bacterial DNA extraction kit (Omega Bio-tek, USA) and quantified with a TBS-380 fluorometer (Turner BioSystems, USA). The 16S rRNA gene sequences were compared against the GenBank database using BLAST to confirm taxonomic status.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlate Confrontation Assay of NS13 Against\u003c/b\u003e \u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFour representative plant-pathogenic \u003cem\u003eFusarium\u003c/em\u003e spp., covering diverse hosts and ecological adaptations, were used. All strains grew stably on PDA medium (potato 200 g/L, glucose 20 g/L, agar 15 g/L, pH 6.0). Three reference strains (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were obtained from the BeNa Culture Collection (BNCC), and one was previously isolated from \u003cem\u003eL. macranthoides\u003c/em\u003e root rot rhizosphere soil by our group. Strains were stored short-term on LB slants at 4\u0026deg;C and long-term in 20% glycerol at -80\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe antagonistic ability of NS13 was evaluated using a plate confrontation assay. NS13 was cultured in LB liquid medium (28\u0026deg;C, 150 rpm) to an OD₆₀₀ of 1.0. \u003cem\u003eFusarium\u003c/em\u003e strains were grown on PDA plates until the colony diameter reached\u0026thinsp;~\u0026thinsp;8 cm, and a 6 mm diameter fungal disc was taken from the colony edge and placed at the center of a new PDA plate. Four 1.0 \u0026micro;L drops of NS13 culture were symmetrically inoculated 2.0 cm from the fungal disc. The control group (CK) received equal volumes of LB medium. Each treatment had three replicates. Plates were incubated at 28\u0026deg;C until the control colony diameter reached 8 cm. Colony diameters (average of the longest and shortest perpendicular axes, in mm) were measured for both treatment and control groups (Wang et al., 2025). Inhibition rate was calculated as: Inhibition rate (%) = [(Dc \u0026ndash; Dt) / Dc] \u0026times; 100, where Dc is the control colony diameter and Dt is the treatment colony diameter. Data were processed in Microsoft Excel, and significant differences were analyzed using Student\u0026rsquo;s t-test in SPSS (v26.0, IBM Corp., USA) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eWhole-Genome Sequencing and Assembly of NS13\u003c/h2\u003e\u003cp\u003e\u003cem\u003eB. velezensis\u003c/em\u003e NS13 was cultured in LB liquid medium (37\u0026deg;C, 150 rpm). Genomic DNA (gDNA) was extracted using the Invitrogen PureLink\u0026reg; Genomic DNA Extraction Kit (Thermo Fisher Scientific, USA), with concentration and purity assessed using a NanoDrop ND-1000 spectrophotometer, followed by purification with the Zymo Quick-DNA Kit (Zymo Research, USA). For next-generation sequencing (NGS), a\u0026thinsp;~\u0026thinsp;400 bp insert paired-end library was constructed: gDNA was fragmented using a Covaris sonicator, end-repaired with T4 DNA polymerase, A-tailed at the 3\u0026rsquo; end, ligated with adapters, size-selected by gel electrophoresis, and amplified with indexing PCR. The library was quality-checked using an Agilent Bioanalyzer 2100 and sequenced on the Illumina platform (150 bp paired-end) by Shanghai Biozeron Biotech Co., Ltd. For PacBio sequencing, an SMRTbell library was prepared using the Express Template Prep Kit 2.0, with fragments\u0026thinsp;\u0026gt;\u0026thinsp;8 kbp selected using BluePippin, and sequenced on the Sequel II platform.\u003c/p\u003e\u003cp\u003eGenome assembly was performed as follows: NGS data were quality-controlled using Trimmomatic (v0.39) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] with parameters SLIDINGWINDOW:4:15 MINLEN:75, PacBio long reads were error-corrected, and assembly was conducted using Unicycler (v0.5.0)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] with default parameters, followed by genome circularization using Circlator (v1.5.5)[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhylogenomic Analysis of NS13\u003c/h3\u003e\n\u003cp\u003eThe reference genome of \u003cem\u003eB. velezensis\u003c/em\u003e FZB42 was downloaded from NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Phylogenomic analysis was performed using the Type Genome Server (TYGS, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"http://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), constructing a maximum likelihood phylogenetic tree based on genome BLAST distance with 1,000 bootstrap replicates, visualized using PhyD3. Digital DNA-DNA hybridization (dDDH) was calculated using the Genome-to-Genome Distance Calculator 3.0 (GGDC, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggdc.dsmz.de/ggdc.php\u003c/span\u003e\u003cspan address=\"https://ggdc.dsmz.de/ggdc.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with formula 2 (100 replicates), with a species threshold of \u0026ge;\u0026thinsp;70%. Average nucleotide identity (ANI) was computed using JspeciesWS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jspecies.ribohost.com/jspeciesws/\u003c/span\u003e\u003cspan address=\"https://jspecies.ribohost.com/jspeciesws/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the BLAST algorithm (ANIb), with a species threshold of \u0026ge;\u0026thinsp;95%.\u003c/p\u003e\n\u003ch3\u003eAnnotation of Antibacterial Genes and Secondary Metabolite Biosynthetic Gene Clusters\u003c/h3\u003e\n\u003cp\u003eNS13 protein-coding genes were annotated using public databases (NR, Swiss-Prot, COG, GO, KEGG). Biocontrol-related genes were categorized into four groups: resource competition genes (RCG), mediating competition for space and nutrients (e.g., siderophore synthesis/transport, alkaline phosphatase); antibacterial activity genes (AAG), encoding antibiotics (NRPS/PKS), antimicrobial peptides, and cell wall-degrading enzymes (e.g., chitinase, lysozyme); induced resistance genes (IRG), activating host defense mechanisms (e.g., phytoalexin synthesis, cell wall reinforcement); and growth-promoting genes (PGPG), enhancing plant resilience through nitrogen fixation (e.g., nifH/nifD) or phosphate solubilization. Secondary metabolite biosynthetic gene clusters (BGCs) were predicted using antiSMASH (v4.1.0)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and compared with \u003cem\u003eB. velezensis\u003c/em\u003e FZB42.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLC-MS/MS Analysis of NS13 Metabolites\u003c/h2\u003e\u003cp\u003eNS13 fermentation broth was centrifuged (10,000 \u0026times; g, 10 min, 4\u0026deg;C) to remove solids, and the supernatant was collected. The pH was adjusted to 2.0 with HCl to optimize metabolite extraction or precipitation. Samples were incubated at 4\u0026deg;C overnight to promote precipitation, re-centrifuged (10,000 \u0026times; g, 10 min, 4\u0026deg;C), and the precipitate was extracted with acetonitrile. The extract was concentrated under reduced pressure, lyophilized, reconstituted in methanol:water (80:20, v/v), and filtered through a 0.22 \u0026micro;m membrane to remove particles.\u003c/p\u003e\u003cp\u003eLC-MS/MS analysis was performed using a reverse-phase C18 column (ACQUITY UPLC BEH C18, 2.1\u0026times;100 mm, 1.7 \u0026micro;m). The mobile phase consisted of water\u0026thinsp;+\u0026thinsp;0.1% formic acid (A) and acetonitrile\u0026thinsp;+\u0026thinsp;0.1% formic acid (B), with a gradient of 0\u0026ndash;2 min, 5% B; 2\u0026ndash;20 min, 5%\u0026ndash;95% B; 20\u0026ndash;25 min, 95% B; 25\u0026ndash;27 min, return to 5% B. The flow rate was 0.3 mL/min, column temperature was 40\u0026deg;C, and injection volume was 3 \u0026micro;L. Mass spectrometry was conducted on a high-resolution Thermo Q Exactive HF-X in positive ion mode, with a full scan range of m/z 150\u0026ndash;2000 (resolution 70,000), using data-dependent acquisition (DDA) or targeted parallel reaction monitoring (PRM), HCD fragmentation (NCE 25\u0026ndash;35), MS/MS resolution of 17,500, and dynamic exclusion of 15 s. Raw data were processed using Xcalibur and Compound Discoverer 3.3, with metabolite identification performed against GNPS, MassBank, and PubChem databases.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSoil Fungal Community Diversity Analysis in Root Rot-Affected\u003c/b\u003e \u003cb\u003eL. macranthoides\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhenotypic observations of \u003cem\u003eL. macranthoides\u003c/em\u003e root rot (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) showed yellow-brown decay of fine roots progressing to main roots, with later stages exhibiting black-brown softening, leaving only the xylem, accompanied by leaf yellowing, stem base browning, growth cessation, and eventual plant death. Alpha diversity analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) revealed significantly lower Chao1 (richness) and Shannon (diversity) indices in the rhizosphere fungal communities of diseased plants compared to healthy ones (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a simplified microbial community structure and disrupted homeostasis. This suggests that root rot significantly alters rhizosphere microbial community structure and diversity.\u003c/p\u003e\u003cp\u003eGenus-level composition analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) revealed a marked shift in community structure. Healthy plant rhizospheres were dominated by beneficial genera such as \u003cem\u003eTrichoderma\u003c/em\u003e (biocontrol function [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]), \u003cem\u003eSaitozyma\u003c/em\u003e, \u003cem\u003eArxiella\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e (top 5), while diseased plants showed a significant increase in pathogenic genera, including \u003cem\u003eFusarium\u003c/em\u003e (pathogen), \u003cem\u003ePlectosphaerella\u003c/em\u003e, \u003cem\u003eAuricularia\u003c/em\u003e, and \u003cem\u003eStaphylotrichum\u003c/em\u003e. Quantitative comparisons (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) showed significantly reduced abundances of \u003cem\u003eTrichoderma\u003c/em\u003e, \u003cem\u003eSaitozyma\u003c/em\u003e, \u003cem\u003eArxiella\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, and \u003cem\u003eScytalidium\u003c/em\u003e in diseased plants, while \u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003ePlectosphaerella\u003c/em\u003e, \u003cem\u003eAuricularia\u003c/em\u003e, and \u003cem\u003eStaphylotrichum\u003c/em\u003e (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB) were significantly enriched (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating a strong association between root rot and the enrichment of pathogenic fungi and depletion of beneficial microbes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation and Identification of\u003c/b\u003e \u003cb\u003eBacillus velezensis\u003c/b\u003e \u003cb\u003eNS13\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEndophytic bacteria were isolated from \u003cem\u003eL. macranthoides\u003c/em\u003e root tissues, and a strain with distinct morphological characteristics, resembling \u003cem\u003eBacillus\u003c/em\u003e, was selected after three rounds of purification and named NS13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). It exhibited typical \u003cem\u003eBacillus\u003c/em\u003e morphology and was Gram-positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). A phylogenetic tree based on 16S rRNA gene sequences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) showed a close relationship with the type strain \u003cem\u003eB. velezensis\u003c/em\u003e FZB42. This taxonomic classification was supported by physiological and biochemical characteristics (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e): NS13 was negative for the Voges-Proskauer test, positive for nitrate reduction, capable of utilizing citrate and propionate as carbon sources, but unable to metabolize D-mannitol, D-xylose, or L-arabinose. It exhibited protease activity (gelatin liquefaction), starch hydrolysis, weak salt tolerance (up to 7% NaCl), and growth at pH 5.7.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlate Confrontation Assay of NS13 Against\u003c/b\u003e \u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlate confrontation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) demonstrated significant inhibition of \u003cem\u003eF. oxysporum\u003c/em\u003e (from \u003cem\u003eL. macranthoides\u003c/em\u003e root rot) by NS13, with an inhibition rate of 56.60% \u0026plusmn; 3.88%. In pairwise confrontation assays with three other \u003cem\u003eFusarium\u003c/em\u003e strains, NS13 formed clear inhibition zones, with morphological changes observed at the fungal colony edges, suggesting the production of potent antifungal metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) showed varying inhibition rates: 76.04% \u0026plusmn; 1.68% against \u003cem\u003eF. fujikuroi\u003c/em\u003e (highest), 70.14% \u0026plusmn; 1.46% against \u003cem\u003eF. solani\u003c/em\u003e, 56.60% \u0026plusmn; 3.88% against \u003cem\u003eF. oxysporum\u003c/em\u003e, and 51.59% \u0026plusmn; 2.24% against \u003cem\u003eF. graminearum\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eGenomic Features and Taxonomic Analysis of NS13\u003c/h2\u003e\u003cp\u003eHybrid assembly using PacBio RSIII and Illumina NovaSeq 6000 data yielded a complete, closed circular chromosome for \u003cem\u003eB. velezensis\u003c/em\u003e NS13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), with a genome size of 3.95 Mb and a GC content of 46.55%. A total of 4,060 protein-coding genes were predicted (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), with 91.26% annotated in the NR database (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), indicating robust metabolic capacity and environmental adaptability. Functional annotation revealed potential antifungal mechanisms: the CAZy database (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA) identified 152 carbohydrate-active enzyme genes, including GH1, GT2, and CBM50 families, suggesting capabilities for degrading complex carbon sources and recognizing fungal cell wall components, enhancing direct fungal inhibition. KEGG annotation identified 3,463 metabolism-related genes (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-C), with significant enrichment in secondary metabolite biosynthesis (24.09%) and antibiotic biosynthesis (18.16%), indicating the ability to produce diverse antifungal molecules. Genes related to amino acid synthesis (10.72%) and ABC transport systems (10.1%) supported metabolite synthesis, regulation, and transport. COG annotation (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) showed enrichment in amino acid transport/metabolism, carbohydrate metabolism, and cell wall biogenesis, aiding activity under fungal stress. GO annotation (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB) highlighted widespread \u0026ldquo;metabolic process\u0026rdquo; and \u0026ldquo;catalytic activity\u0026rdquo; genes, reflecting diverse metabolic and environmental response potential. Collectively, NS13\u0026rsquo;s genetic repertoire supports carbon utilization, secondary metabolite production, transmembrane transport, and fungal recognition, forming the basis of its antifungal activity.\u003c/p\u003e\u003cp\u003ePhylogenomic analysis confirmed NS13\u0026rsquo;s taxonomic status. A 16S rRNA-based phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) showed a close relationship with \u003cem\u003eB. velezensis\u003c/em\u003e. A whole-genome-based phylogenetic tree (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA) with multiple reference strains confirmed NS13\u0026rsquo;s closest relation to FZB42. isDDH and ANI analyses (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB) showed similarity values exceeding 70% and 95%, respectively, meeting species delineation criteria. Based on morphological, physiological, biochemical, and phylogenomic evidence, NS13 was classified as \u003cem\u003eBacillus velezensis\u003c/em\u003e NS13 within the Firmicutes, Bacilli, Bacillales, Bacillaceae, and Bacillus genera.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMining of Antifungal Genes in NS13\u003c/h2\u003e\u003cp\u003eFunctional annotation revealed a systematic antifungal genetic foundation in \u003cem\u003eB. velezensis\u003c/em\u003e NS13, with 96 biocontrol-related genes identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA): 65.63% induced resistance genes, 22.92% resource competition genes, 8.33% antibacterial activity genes, and 3.13% growth-promoting genes. Specifically (Table S5), polyketide synthase genes (e.g., K15327) synthesize antifungal polyketides [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], endoglucanase genes (K01179) degrade fungal cell walls [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and siderophore transport genes (K23188) limit fungal iron acquisition [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Two-component system histidine kinases (K07777) and Fur iron regulators (K03711) may induce host resistance via JA/SA signaling and oxidative stress responses [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while sporulation genes (K06378) support rhizosphere colonization and sustained biocontrol [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. KEGG enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) identified 15 significant pathways, including antibiotic synthesis, two-component systems, and ABC transport, supporting NS13\u0026rsquo;s antifungal mechanisms via direct antibiosis, nutrient competition, and induced resistance.\u003c/p\u003e\u003cp\u003eAntiSMASH analysis identified 15 secondary metabolite BGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), spanning 888.062 kb (22.49% of the genome). Eight BGCs had defined functions (Table S6), encoding antifungal metabolites like fengycin, surfactin (lipopeptides), bacillaene, difficidin (polyketides), bacillibactin (siderophore), and bacilysin (membrane-disrupting). Fengycin and surfactin disrupt fungal membranes, bacillaene inhibits chitin synthesis, and bacillibactin limits fungal iron acquisition [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Seven unannotated BGCs (22.04%) contained PKS-NRPS hybrid modules and unique modification enzyme domains, suggesting potential for novel antifungal compounds. Comparison with FZB42 revealed a unique locillomycin BGC in NS13, indicating broader or differential antifungal capabilities.\u003c/p\u003e\u003cp\u003eIn summary, NS13\u0026rsquo;s biocontrol genes, diverse BGCs, and detectable broad-spectrum antifungal metabolites synergistically contribute to direct antibiosis, nutrient competition, and induced resistance, forming the genetic and metabolic basis for its effective antagonism against \u003cem\u003eFusarium\u003c/em\u003e spp.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLC-MS Analysis Reveals Antifungal Metabolites in NS13\u003c/h2\u003e\u003cp\u003eHigh-resolution mass spectrometry confirmed NS13\u0026rsquo;s rich antifungal metabolite profile. Multiple compounds with potential biocontrol activity were detected (Table S7), including cyclic dipeptides with a diketopiperazine (DKP) scaffold, such as Cyclo(phenylalanyl-prolyl), 3-(1-hydroxyethyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, 3-(propan-2-yl)-octahydropyrrolo[1,2-a]pyrazine-1,4-dione, and 3-[(4-hydroxyphenyl)methyl]-octahydropyrrolo[1,2-a]pyrazine-1,4-dione. These cyclic dipeptides, known for membrane permeability and structural stability, are widely recognized for antibacterial and antifungal activity [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], and their repeated detection suggests they are key factors in NS13\u0026rsquo;s inhibition of \u003cem\u003eFusarium\u003c/em\u003e. Additionally, fatty acid amides with membrane-disrupting potential, such as oleamide, erucamide, and stearamide, were detected [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Active dipeptides like carnosine and valylproline, capable of scavenging free radicals and modulating microbial interactions, were also identified, along with phenolic antioxidants like rutin and quercetin-3β-D-glucoside, reported to inhibit fungal virulence factors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], potentially enhancing NS13\u0026rsquo;s resilience to fungal stress. Notably, polyamines like spermine were detected, which stabilize DNA, regulate membrane permeability [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and activate plant immune signaling, suggesting both direct antibacterial activity and enhanced host defense. In addition, triterpenoids with potential antifungal activity, such as oleanolic acid [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and some metabolites that have not been fully annotated, such as PPK and NP-011220, were also detected, which may represent secondary metabolites unique to NS13. Further confirmation of their biological functions through structural analysis and activity validation is needed.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eGenomic Analysis Reveals the Genetic Basis of NS13 Biocontrol\u003c/h2\u003e\u003cp\u003eGenomic analysis serves as a powerful methodological tool, providing rich insights into the biocontrol mechanisms of \u003cem\u003eBacillus velezensis\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. These mechanisms, including the synthesis of antibacterial compounds, competitive nutrient suppression, and induction of host resistance, are all reflected at the genomic level [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Furthermore, genomic analysis offers critical support for the genetic improvement of \u003cem\u003eBacillus velezensis\u003c/em\u003e, enabling optimization of its antifungal activity or environmental adaptability through gene editing or synthetic biology approaches, thereby enhancing its biocontrol application potential [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpecifically, the 3.95 Mb NS13 genome encodes 4,060 proteins, including 96 antifungal-related genes. Notably, NRPS and PKS genes, such as those responsible for fengycin and bacillaene synthesis, align with known antifungal mechanisms in B. velezensis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Siderophore genes, including those for bacillibactin, support nutrient competition by limiting Fusarium iron acquisition [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Genes linked to induced systemic resistance, such as those regulating JA/SA signaling, suggest enhanced host defense, consistent with other plant [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The discovery of seven uncharacterized BGCs encoding novel PKS-NRPS hybrid enzymes highlights NS13\u0026rsquo;s potential to produce unique antifungal compounds, distinguishing it from FZB42. This genetic diversity positions NS13 as a valuable resource for biocontrol and novel metabolite discovery.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eMetabolomics Provides the Material Basis for NS13\u0026rsquo;s Antifungal Activity\u003c/h2\u003e\u003cp\u003eMetabolomics, as a core technical tool for analyzing microbial functional metabolites, can systematically identify various antifungal active components produced by \u003cem\u003eBacillus velezensis\u003c/em\u003e during its growth and metabolism, thereby providing direct evidence for clarifying the material basis of its biocontrol effects [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. LC-MS/MS analysis identified a range of NS13 antifungal metabolites, including cyclic dipeptides [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], fatty acid amides such as erucamide, and triterpenoids such as oleanolic acid. Cyclic dipeptides disrupt fungal signaling and membrane integrity, while lipopeptides like fengycin and surfactin destabilize fungal membranes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The detection of oleanolic acid [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], which is known for its antifungal properties, suggests synergy with lipopeptides, enhancing NS13\u0026rsquo;s biocontrol efficacy. Uncharacterized BGCs encoding novel metabolites further underscore NS13\u0026rsquo;s potential for developing new antifungal agents. Compared to other Bacillus strains, NS13\u0026rsquo;s unique production of locillomycin and diverse dipeptides suggests broader antifungal capabilities, potentially overcoming strain-specific efficacy limitations.\u003c/p\u003e\u003cp\u003eThese findings align with prior studies demonstrating B. velezensis efficacy against Fusarium diseases in crops like wheat and maize [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. For instance, FZB42 produces similar lipopeptides, including fengycin and surfactin, to suppress F. graminearum [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, NS13\u0026rsquo;s unique locillomycin [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] BGC and higher BGC diversity suggest enhanced antifungal potential, particularly against F. fujikuroi and F. solani. Unlike earlier studies focusing on cereals, this work extends B. velezensis application to medicinal plants, addressing a critical gap in L. macranthoides root rot control.\u003c/p\u003e\u003cp\u003eDifferences in inhibition rates, such as lower efficacy against F. graminearum compared to F. fujikuroi, may reflect species-specific susceptibility or experimental conditions, including medium and incubation time. The integrated genomic and metabolomic approach distinguishes this study from earlier phenotype-based work, providing mechanistic insights into NS13\u0026rsquo;s biocontrol efficacy.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePotential and Challenges of\u003c/b\u003e \u003cb\u003eB. velezensis\u003c/b\u003e \u003cb\u003eNS13 in Controlling L. macranthoides Root Rot\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn root rot-affected \u003cem\u003eL. macranthoides\u003c/em\u003e rhizospheres, \u003cem\u003eFusarium\u003c/em\u003e enrichment aligns with its established role as a major soil-borne pathogen. The observed decline in beneficial fungi such as \u003cem\u003eTrichoderma\u003c/em\u003e and in microbial diversity, as indicated by Chao1 and Shannon indices with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, points to microbial dysbiosis, likely driven by \u003cem\u003eF. oxysporum\u003c/em\u003e mycotoxins such as fumonisins that suppress competing microbes [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The high \u003cem\u003eFusarium\u003c/em\u003e abundance in diseased plants confirms its role as the primary driver of root rot, consistent with its pathogenicity in related species like \u003cem\u003eL. japonica\u003c/em\u003e. These findings highlight the need for targeted biocontrol to restore microbial balance and suppress \u003cem\u003eFusarium\u003c/em\u003e dominance.\u003c/p\u003e\u003cp\u003eB. velezensis NS13 exhibited potent antagonism against F. oxysporum with 56.60% inhibition and against other \u003cem\u003eFusarium\u003c/em\u003e spp, with the highest efficacy against F. fujikuroi at 76.04%. This variability may reflect differences in cell wall composition or metabolic vulnerabilities among \u003cem\u003eFusarium\u003c/em\u003e species [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e].. Clear inhibition zones in confrontation assays suggest diffusible antibacterial compounds, supported by the detection of lipopeptides such as fengycin and surfactin, as well as polyketides such as bacillaene, which are known to disrupt fungal membranes and spore germination [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Unlike chemical fungicides, which face resistance issues, NS13\u0026rsquo;s multi-mode action minimizes resistance development. Differential inhibition rates suggest NS13\u0026rsquo;s efficacy may be optimized for specific Fusarium strains, warranting field validation.\u003c/p\u003e\u003cp\u003eThis study elucidates the genetic and metabolic mechanisms of NS13\u0026rsquo;s antagonism against Fusarium spp., positioning it as a viable alternative to chemical fungicides and addressing environmental concerns like pesticide residues and resistance [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Practically, NS13 could be developed as a biofungicide for sustainable L. macranthoides cultivation, reducing yield losses and ensuring medicinal quality. The discovery of novel BGCs opens avenues for identifying new antifungal compounds with potential in medicine and agriculture.\u003c/p\u003e\u003cp\u003eDespite its strengths, this study has limitations. Plate-based assays, while reliable, may not fully reflect NS13\u0026rsquo;s performance in complex soil environments, where abiotic factors such as pH and humidity, along with microbial interactions, may modulate efficacy. Field trials are needed to validate NS13\u0026rsquo;s biocontrol potential under natural conditions. The functions of seven uncharacterized BGCs remain speculative, requiring targeted gene knockout or heterologous expression studies for confirmation. The focus on L. macranthoides limits direct extrapolation to other crops, though NS13\u0026rsquo;s broad-spectrum activity suggests wider applicability. Future research should explore NS13\u0026rsquo;s interactions with plant immune systems and its long-term stability in rhizosphere microbiomes. Overall, this study establishes B. velezensis NS13 as a potent biocontrol agent against Fusarium-induced root rot in L. macranthoides, leveraging synergistic biocontrol genes and antifungal metabolites. Its application could transform disease management in medicinal plant cultivation, promoting sustainability. Future studies should focus on field validation, functional characterization of novel BGCs, and NS13\u0026rsquo;s role in modulating plant immunity to enhance biocontrol efficacy.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides significant insights into the microbial dynamics associated with \u003cem\u003eFusarium\u003c/em\u003e-induced root rot in \u003cem\u003eLonicera macranthoides\u003c/em\u003e and highlights the potential of \u003cem\u003eBacillus velezensis\u003c/em\u003e strain NS13 as a sustainable biocontrol agent. The observed microbial dysbiosis in the rhizosphere of diseased plants, characterized by an increase in pathogenic fungi such as \u003cem\u003eFusarium\u003c/em\u003e and \u003cem\u003ePlectosphaerella\u003c/em\u003e and a reduction in beneficial fungi like \u003cem\u003eTrichoderma\u003c/em\u003e, underscores the ecological imbalance contributing to disease progression. The isolation of the endophytic \u003cem\u003eB. velezensis\u003c/em\u003e NS13 from healthy roots and its demonstrated strong antagonistic activity against multiple \u003cem\u003eFusarium\u003c/em\u003e species, including \u003cem\u003eF. oxysporum\u003c/em\u003e, \u003cem\u003eF. solani\u003c/em\u003e, \u003cem\u003eF. graminearum\u003c/em\u003e, and \u003cem\u003eF. fujikuroi\u003c/em\u003e, positions it as a promising biocontrol candidate. Genomic analysis revealed a robust arsenal of 96 biocontrol-related genes and 15 secondary metabolite biosynthetic gene clusters, five of which are linked to antifungal activity, three to antibacterial activity, and seven potentially encoding novel compounds. The detection of antifungal metabolites, such as cyclic dipeptides, fatty acid amides (e.g., erucamide), and oleanolic acid, via LC\u0026ndash;MS/MS further supports the strain\u0026rsquo;s efficacy. These findings not only enhance our understanding of soil microbial interactions in the context of root rot but also advocate for the development of \u003cem\u003eB. velezensis\u003c/em\u003e NS13 as an environmentally friendly alternative to chemical pesticides for managing Fusarium pathogens in medicinal plants. Future research should focus on field trials and formulation strategies to optimize the practical application of NS13 in sustainable agriculture.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eITS: Internal Transcribed Spacer ; LB: Luria-Bertani ; OD: Optical Density\u003c/p\u003e\n\u003cp\u003ePCR: Polymerase Chain Reaction ;SEM: Scanning Electron Microscopy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eClinical Trial:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate :\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication :\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials :\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available in the National Center for Biotechnology Information (NCBI) repository, primary accession code PRJNA1306347.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests :\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Basic Scientific Research Projects of Chongqing (Nos. 2024jbky-021, 2025jbky-008, 2024jbky-017), and the Performance Incentive Guidance Special Project of Chongqing (No. 2022jx011), as well as the Basic Research Expenses of Chongqing Science and Technology Bureau (No. 2024jbky-032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. Qi, S. Lu, and Z. Chen conceptualized the study and designed experiments to investigate microbial dysbiosis in \u003cem\u003eLonicera macranthoides\u003c/em\u003e root rot, including microbial diversity comparisons using Chao1/Shannon indices and p-value analyses. J. Qi and Z. Chen collected rhizosphere soil samples, isolated the NS13 strain, and conducted plate confrontation assays. J. Qi, L. Liu, and H. Zhou analyzed metabolites via LC-MS/MS. J. Qi, Z. Chen, and W. Zhuo performed genomic sequencing and AntiSMASH analysis to identify biosynthetic gene clusters. J. Qi and Z. Chen drafted the manuscript, with Y. Wang, M. Yang, Y. Yang, and F. Ren contributing to revisions and polishing. S. Lu and H. Wang provided essential strains, plant materials, and LC-MS/MS equipment. F. Ren coordinated team efforts and ensured smooth project execution. F. Ren secured funding for the study. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eColeman JJ. The \u003cem\u003efusarium solani\u003c/em\u003e species complex: ubiquitous pathogens of agricultural importance. Mol Plant Pathol. 2016;17:146\u0026ndash;58. https://doi.org/10.1111/mpp.12289.\u003c/li\u003e\n \u003cli\u003eSummerell BA. Resolving \u003cem\u003efusarium\u003c/em\u003e : current status of the genus. Annu Rev Phytopathol. 2019;57:323\u0026ndash;39. https://doi.org/10.1146/annurev-phyto-082718-100204.\u003c/li\u003e\n \u003cli\u003eKim H, Hwang J-Y, Shin J, Oh K-B. Inhibitory effects of diketopiperazines from marine-derived streptomyces puniceus on the isocitrate lyase of candida albicans. 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Annu Rev Microbiol. 2013;67:399\u0026ndash;416. https://doi.org/10.1146/annurev-micro-092412-155650.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fusarium spp., Bacillus velezensis NS13, genomics, Lonicera macranthoides root rot, metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-7464262/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7464262/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eFusarium spp.\u003c/em\u003e are major fungal pathogens causing root rot. They exhibit a broad host range and high pathogenicity, leading to yield losses, reduced quality, and plant mortality. Current control measures rely primarily on chemical pesticides, with few sustainable biological options available. This study compared rhizosphere microbial diversity between healthy and diseased \u003cem\u003eLonicera macranthoides\u003c/em\u003e, revealing increased pathogenic fungi (\u003cem\u003eFusarium\u003c/em\u003e, \u003cem\u003ePlectosphaerella\u003c/em\u003e, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and reduced beneficial fungi (\u003cem\u003eTrichoderma\u003c/em\u003e, Chao1/Shannon, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in diseased plants. An endophytic \u003cem\u003eBacillus velezensis\u003c/em\u003e strain, NS13, was isolated from healthy roots. Plate confrontation assays showed strong inhibition of \u003cem\u003eFusarium oxysporum\u003c/em\u003e from \u003cem\u003eL. macranthoides\u003c/em\u003e and other \u003cem\u003eFusarium\u003c/em\u003e species (\u003cem\u003eF. solani\u003c/em\u003e, \u003cem\u003eF. graminearum\u003c/em\u003e, \u003cem\u003eF. fujikuroi\u003c/em\u003e). The 3.95 Mb genome encoded 4,060 proteins, including 96 biocontrol-related genes. AntiSMASH identified 15 secondary metabolite biosynthetic gene clusters, with five linked to antifungal, three to antibacterial activity, and seven potentially novel compounds. LC\u0026ndash;MS/MS metabolomics detected multiple antifungal metabolites, including cyclic dipeptides, fatty acid amides (e.g., erucamide), and oleanolic acid. These results demonstrate soil microbial dysbiosis in \u003cem\u003eL. macranthoides\u003c/em\u003e affected by root rot and confirm the broad-spectrum anti-\u003cem\u003eFusarium\u003c/em\u003e potential of NS13, highlighting its promise as a biocontrol resource against \u003cem\u003eFusarium\u003c/em\u003e pathogens in medicinal plants.\u003c/p\u003e","manuscriptTitle":"Integrated Genomic and Metabolomic Analysis Reveals the Biocontrol Potential of Endophytic Bacillus velezensis NS13 Against Fusarium spp. in Lonicera macranthoides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-01 17:19:36","doi":"10.21203/rs.3.rs-7464262/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-15T14:39:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T04:39:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T19:40:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-10T14:35:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T09:47:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T16:24:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157743492813434687622348948614135221351","date":"2025-09-25T20:11:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156958834746546062090324492674373822672","date":"2025-09-24T09:52:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141459753676326583931322295220396412489","date":"2025-09-22T16:41:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16208187328539598589516120812034202641","date":"2025-09-22T15:30:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"122043272826105009109433312952225253272","date":"2025-09-22T12:06:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118036385010896519852260252480918221890","date":"2025-09-22T06:16:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136781069415352722377327346895707259333","date":"2025-09-20T15:13:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119454031200230275003385732105313343063","date":"2025-09-20T15:12:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-20T15:10:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-20T08:02:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-18T17:56:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-18T10:28:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-09-18T10:03:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dfb263e0-dec7-450d-9be3-40db396f4323","owner":[],"postedDate":"October 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:09:53+00:00","versionOfRecord":{"articleIdentity":"rs-7464262","link":"https://doi.org/10.1186/s12866-025-04551-x","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2026-01-05 15:59:06","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-10-01 17:19:36","video":"","vorDoi":"10.1186/s12866-025-04551-x","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04551-x","workflowStages":[]},"version":"v1","identity":"rs-7464262","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7464262","identity":"rs-7464262","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}