Bacillus amyloliquefaciens strain D-1 was isolated via the dilution plating method from the rhizosphere soil of Pseudostellaria heterophylla, which was sampled in Shibing County, Guizhou Province, China (27°4′21”N, 108°8′0″E) (Deng et al., 2023; Yuan et al., 2024; Zhou et al., 2023) Alternaria alternata strain JK3-4, a causal pathogen of leaf spot disease, was isolated from symptomatic P. heterophylla leaves (Wang et al., 2023). For the evaluation of in vivo disease resistance, the P. heterophylla cultivar ‘Guishen 1#’ was used. Luria-Bertani (LB) agar medium and potato dextrose agar (PDA) medium was used to culture strain D-1 and strain JK3-4, respectively (Yuan et al., 2024; Zhou et al., 2023).

Dual-culture antagonism assays were performed to assess the antifungal activity of strain D-1 and its metabolite extract against the toxigenic pathogen A. alternata strain JK3-4, following previously established protocols (Gong et al., 2019; Zhou et al., 2023). Briefly, a 10 μL aliquot of A. alternata strain JK3-4 conidial suspension (1.0 × 10⁵ CFU/mL) was inoculated at the center of potato dextrose agar (PDA) plates. Subsequently, to evaluate the antifungal efficacy of B. amyloliquefaciens strain D-1, its inhibitory activity against A. alternata was investigated by two independent methods. In the direct confrontation assay, a plug of the fungus was placed on a PDA plate, and 10 μL of bacterial suspension (OD₆₀₀ = 1.0) was spotted 3 cm away, with ddH₂O serving as a negative control. In the agar well diffusion assay, three wells were punched equidistantly (3 cm from the center) around a central fungal colony for the application of the test compound. Two wells were filled with 100 μL of metabolite extract, and a third well received 100 μL of methanol as a solvent control. The plates were then incubated, and the inhibition zones were measured. Each experiment was replicated three times. All plates were incubated in the dark at 28 °C for 5 days. After incubation, the radial growth of A. alternata strain JK3-4 was measured from the center of the conidial inoculation site to the edge of the mycelial colony. The antifungal inhibition rate was calculated using the following formula: Inhibition rate (%) = [(Control mycelial radius − Treated mycelial radius)/Control mycelial radius] × 100, where “Control mycelial radius” refers to the radial growth of A. alternata on ddH₂O-treated plates, and “Treated mycelial radius” refers to its radial growth on plates challenged with either B. amyloliquefaciens strain D-1 or its metabolite extract.

The genome of B. amyloliquefaciens strain D-1 (GenBank ID in NCBI: PRJNA1333658) was sequenced using a hybrid approach combining PacBio RS II single-molecule real-time (SMRT) and Illumina sequencing, which was performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Genomic DNA libraries were prepared using the NEXTFLEX Rapid DNA-Seq Kit and sequenced on an Illumina NovaSeq 6,000 platform with paired-end reads (2 × 150 bp). Raw Illumina reads were processed using fastp (v0.23.0) to remove low-quality reads, reads containing excessive ambiguous bases (N), and short reads. Whole genome of strain D-1 were assembled using Unicycler (v0.4.8), followed by error correction employing the polished clean Illumina reads using Pilon (v1.22) (Wick et al., 2017).

Genome annotation was conducted using multiple gene prediction tools: Glimmer¹ (Delcher et al., 2007), GeneMarkS (Besemer and Borodovsky, 2005), and Prodigal (Benson, 1999). Transfer RNA (tRNA) genes were identified with tRNAscan-SE (v2.0) (Chan and Lowe, 2019), and, ribosomal RNA (rRNA) were detected using Barrnap (ver. 0.9²) (Liu et al., 2019). Functional annotation of predicted genes was performed via homology-based searches using BLASTP, Diamond, and HMMER against the NR, Swiss-Prot, Pfam, GO, COG, KEGG, and CAZY databases. Secondary metabolite biosynthetic gene clusters were identified using antiSMASH (v5.1.2) with the MiBIG database.

Crude lipopeptides (LPs) were extracted using the acid precipitation method, as described in our previous study (Gong et al., 2015; Yuan et al., 2025). A single colony of B. amyloliquefaciens strain D-1 was inoculated into 20 mL of LB medium in a 100-mL Erlenmeyer flask, followed by incubation at 28 °C for 18 h. A 12-mL aliquot of the pre-culture was transferred to 200 mL of fresh LB medium in a 500-mL Erlenmeyer flask and incubated for 48 h under the same temperature conditions. The bacterial culture was centrifuged at 12,000 × g for 20 min at 4 °C to harvest the culture supernatant (cell-free fraction). The pH of the cell-free supernatant was adjusted to 2.0 using 6 M hydrochloric acid (HCl), and the mixture was stirred overnight at 4 °C to induce LP precipitation. The resulting precipitate was collected by centrifugation at 12,000 rpm for 10 min. The precipitate was washed three times with sterile ultrapure water, lyophilized (freeze-dried), and finally resuspended in methanol to obtain a crude LP solution. The identity of the extracted LPs was confirmed using reversed-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC–MS), following the protocols established in our previous research (Gong et al., 2015).

Briefly, mass spectrometry analysis was conducted using an LC–MS-20 AD system (Shimadzu Corporation, Tokyo, Japan) equipped with a triple quadrupole mass analyzer and an ESI source operated in full positive scan mode. Chromatographic separation was performed using a C18 reversed-phase column (EXT-C18, 2.7 μm × 3.0 mm × 30 mm, packed with 5 μm particles) maintained at 40 °C. The flow rate was set to 0.2 mL/min. Mass spectrometry parameters included an ESI ion source operated in positive ion detection mode, a mass range of 100–1,500 Da, a detection voltage of 1.1 kV, a ion source voltage of 4.5 kV, and an ion source current of 6.5 μA.

The inoculants, comprising fresh cells of strain D-1 (Bc) with OD600 set at 1.0, its fermentation supernatant (Bf), and crude lipopeptides (LPs), were prepared as following our previously described method (Gong et al., 2015).

P. heterophylla seedlings at the eight-leaf stage were transplanted into pots (10 cm in diameter) and acclimated in a growth chamber under a photoperiod of 16 h light/8 h dark, with day/night temperatures maintained at 25/18 °C. 10 μL of an A. alternata spore suspension (1.0 × 10⁵ CFU·mL⁻¹) was inoculated to the leaf center. At 2 days post-inoculation (dpi) with A. alternata, the seedlings were sprayed with Bc, Bf, or LPs; sterile double-distilled water (ddH₂O) was used as the control. All treatments were conducted under 90% relative humidity, with all seedlings maintained in the same growth chamber (under the aforementioned acclimation conditions). Each treatment included 8 independent seedlings. Disease severity was evaluated by lesion area method according to our previous study at 10 days post-treatment (dpt) with Bc, Bf, or LPs (Wang et al., 2023). The leaves lesion area in pathogen inoculation site was measured by ImageJ (version 1.53 t; National Institutes of Health, USA); each treatment calculated 12 leaves.

The assay was performed following the method described by Li et al. (2021). Briefly, 50 μL of a lipopeptide (LPs) solution (5 mg·mL⁻¹) was added to 5 mL of potato dextrose broth (PDB) containing 1.0 × 10⁵ CFU·mL⁻¹ spores of A. alternata. At 0, 4, 8, 12, 16, and 24 h post-inoculation (hpi), the optical density at 600 nm (OD₆₀₀) was measured using a Synergy 2 multimode microplate reader (BioTek Instruments, Winooski, VT, USA). Concurrently, the spore germination rate was determined by microscopic examination using an Olympus BX41 microscope (Olympus Corporation, Tokyo, Japan). After 5 days of incubation at 28 °C with shaking at 180 rpm, hyphal samples were collected, stained with calcofluor white (CFW), and observed under an Olympus IX73 fluorescence microscope (Olympus Corporation, Tokyo, Japan) to evaluate the treatment-induced morphological changes in A. alternata mycelia and spores.

Twenty grams of P. heterophylla tuberous roots were placed in a sterilized sterile petri dish. The tuberous roots were then infected with 1 mL of A. alternata spore suspension (1.0 × 10⁵ CFU·mL⁻¹) and treated with 1 mL of crude lipopeptide (5 mg·mL⁻¹). Control samples were incubated with 1 mL of sterile methanol. All samples were incubated at 25 °C for 20 days, with three independent biological replicates per group. Photographs were taken for documentation, and all tuberous root samples were immediately stored at −80 °C for subsequent mycotoxin analysis.

Five major mycotoxins produced from A. alternata were analyzed using the PriboFastx® MFC311 Solid-Phase Extraction Column Test Kit (Pribolab, Qingdao, China), following the kit’s recommended protocols. The analytical workflow was as follows: Pre-dried P. heterophylla tuberous roots were ground into a fine powder. Exactly 2 g of the powdered sample was accurately weighed and transferred to a centrifuge tube. Ten mL of an acetonitrile/water solution (80:16, v/v) containing 0.1% (v/v) formic acid was added to the tube. The mixture was vigorously shaken for 30 min to facilitate mycotoxin extraction, then centrifuged at 6000 rpm for 5 min to separate the supernatant from the solid residue. Four mL of the resulting supernatants were carefully transferred to a new sterile tube and purified using a PriboFastx® MFC311 SPE column to remove interfering substances. The purified filtrate was dried via liquid nitrogen evaporation to concentrate on the mycotoxins. The dried residue was re-dissolved in 8 mL of methanol to form a mycotoxin-enriched solution. Two mL of the re-dissolved solution was passed through a 0.45-μm hydrophilic polyethersulfone (PES) filter to remove particulate matter, ensuring compatibility with subsequent chromatographic analysis. The filtered sample solution was subjected to mycotoxin quantification using a high-performance liquid chromatography (HPLC) system equipped with a photodiode array (PDA) detector (Shimadzu, Shanghai, China). For accurate quantitative analysis, analytical standards of the five target mycotoxins were purchased from Sigma-Aldrich (St. Louis, MO, USA): Alternariol (AOH), Alternariol monomethyl ether (AME), Altenuene (ALT), Tenuazonic acid (TeA), Tentoxin (TEN). These standards were used to generate calibration curves, which were applied to calculate the concentration of each mycotoxin in the P. heterophylla tuberous root samples.

To detect hydrogen peroxide (H₂O₂) and callose, leaves of P. heterophylla treated with Bc, Bf, or LPs were harvested at 10 days post-treatment (dpt). H₂O₂ accumulation and callose deposition were visualized using previously described methods (Kumar et al., 2009; Millet et al., 2010; Yuan et al., 2025). Briefly, leaves were submerged in a 1 mg/mL solution of 3,3′-diaminobenzidine (DAB, pH 5.5) for 4 h, then boiled in 96% ethanol for 10 min to remove chlorophyll and stored in 96% ethanol. H₂O₂ accumulation was identified by the presence of reddish-brown staining in the treated leaves.

For callose detection, P. heterophylla leaves were fixed in a methanol: acetic acid solution (3:1, v/v) for 4 h. Following fixation, the leaves were sequentially dehydrated in 70% methanol for 2 h and in 50% methanol for an additional 2 h. After overnight hydration in deionized water, the leaves were washed three times with water and cleared by treatment with 10% (w/v) sodium hydroxide (NaOH) for 1–2 h. Following three further water washes, the leaves were stained in a solution containing 0.01% (w/v) aniline blue (Sigma-Aldrich, Shanghai, China) dissolved in 150 mM K₂HPO₄ (pH 9.5) for 3–4 h. Callose deposition was visualized using a Nikon H600L microscope under UV light illumination.

To investigate the expression of toxin biosynthesis genes in A. alternata, mycelia of A. alternata treated with LPs were collected at 20 days post-treatment (dpt). Total RNA was isolated using the TransZol Up Plus RNA Kit (TransGen Biotech Co., Ltd., Beijing, China). RNA concentration and quality were assessed using a NanoDrop 2000 microspectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and by 1% agarose gel electrophoresis. First-strand cDNA synthesis was performed via reverse transcription using total RNA as the template and Anchored Oligo(dT)₁₈ primer (0.5 μg/μL), following the protocol provided with the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech Co., Ltd., Beijing, China). The synthesized cDNA was stored at −80 °C for downstream applications.

Six mycotoxin biosynthesis genes in A. alternata—including TES1, TES, PksJ, PksA, PksI, and PksJF—were selected for quantitative real-time PCR (RT-qPCR) analysis (Table 1). The primers used in this study were intron-spanning PCR primers. The β-tubulin gene was used as the endogenous reference gene for A. alternata. Relative gene expression levels were calculated using the 2^-ΔCT method.

The results are presented as mean ± standard deviation. Differences were analyzed using Student’s t-tests and one-way analysis of variance (ANOVA) using Origin software (Version 2018, OriginLab Inc., Northampton, MA, USA).

The antifungal activity of B. amyloliquefaciens strain D-1 was evaluated using dual-culture antagonism assays, as described in our previous studies (Gong et al., 2019; Zhou et al., 2023). Results showed that both live bacterial cells (Bc) and cell-free fermentation broth (Bf) significantly inhibited the growth of A. alternata (p < 0.0001) (Figure 1A). A. alternata colonies radius was significantly reduced in the presence of strain D-1 (1.6 cm) compared with the control (2.5 cm). Similarly, treatment with the cell-free fermentation supernatant of D-1 resulted in a smaller colony radius (1.1 cm) relative to the control (2.6 cm) (Figures 1B,C). Notably, the inhibition rate of the cell-free fermentation supernatant against A. alternata reached 54.7%, which was significantly higher than that of the live D-1 strain (39.4%) (Figure 1D). Collectively, these findings demonstrate that B. amyloliquefaciens D-1 effectively suppresses A. alternata, the causal pathogen of leaf spot disease in P. heterophylla, suggesting its potential as a promising biocontrol resource for managing this phytopathogen.

Genome sequencing analysis of B. amyloliquefaciens strain D-1 revealed a genome size of 3,980,777 bp with a GC content of 46.46%. The genome encodes 3,829 protein-coding genes (CDS), 86 tRNA genes, and 27 rRNA genes (Figure 2A and Table 2).

Prediction of secondary metabolite biosynthetic gene clusters (BGCs) in strain D-1 using antiSMASH (v5.1.2) identified at least nine putative biosynthetic pathways, including those for lipopeptides, polyketides, and terpenoids. Notably, the BGCs for fengycin, difficidin, bacillaene, bacillibactin, bacilysin, and macrolactin H showed 100% similarity to their respective reference clusters in the MiBIG database, while the surfactin biosynthetic cluster exhibited 82% similarity to its reference. These findings suggest that strain D-1 has high potential to synthesize these secondary metabolites, with detailed information on each BGC provided in Table 3. Collectively, genomic analysis highlights B. amyloliquefaciens D-1’s capacity to produce lipopeptides and polyketides—key classes of antifungal metabolites—thereby reinforcing its antifungal potential.

Further Clusters of Orthologous Groups (COG) functional annotation uncovered gene clusters encoding the lipopeptide biosynthetic machinery, including those for surfactins, iturin A, and plipastatin (fengycin B). The surfactin A biosynthetic cluster contains four core genes: srfAA, srfAB, srfAC, and srfATE. The iturin A biosynthetic cluster harbors three key genes: ituA, ituB, and ituC. Additionally, a distinct BGC associated with plipastatin biosynthesis was identified, including five key biosynthetic genes: ppsA, ppsB, ppsC, ppsD, and ppsF (Figure 2B). Collectively, these genomic insights confirm that B. amyloliquefaciens strain D-1 possesses the complete genetic blueprint for synthesizing lipopeptide antimicrobial compounds, further underscoring its potential as a robust biocontrol agent against phytopathogens.

HPLC analysis identified three major compound classes, with retention times for fengycins, iturins, and surfactins observed at 38.126–59.746 min, 60.327–64.860 min, and 86.829–96.593 min, respectively (Figure 3A). Based on subsequent characterization by HPLC–ESI-MS and comparison with references (Gong et al., 2015), the molecular weight spectra of the secondary metabolites were consistent with those of cyclic lipopeptides produced by Bacillus species, including fengycins, iturins, and surfactins. Molecular ion peaks [M + H]⁺ were detected at m/z 1,436, 1,449,1,461, 1,478, 1,491 and 1,506 for fengycins (Figure 3B); at m/z 1,044, 1,058, 1,073, and 1,086 for iturins (Figure 3C); and m/z 1,008, 1,022, 1,036, and 1,050 for surfactins (Figure 3D). Each compound class exhibited a 14 Da mass differences between consecutive homologs, indicating variation in fatty acid chain lengths within each group (CH₂ = 14 Da) (Table 4). Collectively, these findings confirm that strain D-1 synthesizes three major classes of cyclic lipopeptides: fengycin, iturin, and surfactin.

A pot experiment was conducted to evaluate the biocontrol efficacy of Bc, Bf, and LPs. Foliar application of Bc, Bf, and LPs significantly suppressed the leaf spot development in P. heterophylla (Figure 4A). In contrast, water-treated control showed unrestricted mycelial colonization by A. alternata on the undersurfaces of leaves, with extensive hyphal growth observed (Figure 4B). Treatment with Bc or Bf restricted mycelial invasion, though slight dehydration was observed at pathogen inoculation sites. Notably, LP-treated leaves exhibited complete inhibition of surface mycelia, with no hyphal penetration detected on leaf surfaces (Figure 4B). The mean disease lesion area in the control group (0.66 cm²) was significantly larger than that in treatments with Bf (0.40 cm²), Bc (0.23 cm²), and LPs (0.05 cm²) groups (Figure 4C). Notably, LPs exhibited highest biocontrol activity against A. alternata, outperforming both Bc and Bf. These findings indicate that B. amyloliquefaciens strain D-1 and its metabolites, particularly lipopeptides, possess robust biocontrol potential against A. alternata-induced leaf spot disease in P. heterophylla.

Microscopic analysis revealed that treatment of A. alternata with crude lipopeptide (LPs) extract significantly inhibited mycelial development and branching (Figure 5A). Compared with the untreated control, LP-treated samples exhibited a 59.9% reduction in hyphal branch density, from 172 to 69 branches/mm² (Figure 5B). Additionally, the antifungal effect on spore germination was assessed following treatment with crude LP extract, which resulted in significant suppression of germination (Figures 5C,D). Relative to the untreated control, nearly complete inhibition of spore germination was observed after 20 h of co-incubation in LPs-treated samples (Figures 5C,D). Collectively, these findings indicate that the lipopeptides inhibit A. alternata growth and development by disrupting hyphal branching and sporulation, thereby impeding fungal proliferation.

Following 20 days of treatment with LPs on A. alternata-infected P. heterophylla tuberous roots, infection assessments demonstrated significantly reduced A. alternata colonization compared to control samples, which exhibited dense mycelial growth (Figure 6A). HPLC-FAD analysis quantified mycotoxin levels in LPs-treated and control group, revealing significant reductions in the concentrations of alternariol (ALT, 13.70 μg/g vs. 59.5 μg/g), alternarol (AOH, 8.7 μg/g vs. 467.9 μg/g), alternaria monomethyl ether (AME, 41.0 μg/g vs. 229.4 μg/g), tenuazonic acid (TeA, 110.1 μg/g vs. 737.2 μg/g), and tentoxin (TEN, 20.3 μg/g vs. 148.8 μg/g) in LP-treated samples (Figures 6B,C). Previous studies have identified polyketide synthase (PKS) genes (PKSJ, PKSA, PKSI, and PKSJF) as key biosynthetic genes for alternariol (ALT), alternariol methyl ether (AME), and tenuazonic acid (TeA), while non-ribosomal peptide synthase (NRPS) genes (TES and TES1) were found to regulate TEN biosynthesis (Yun et al., 2015). Quantitative real-time PCR (qRT-PCR) analysis revealed that relative expression levels of PKSJ, PKSA, PKSI, PKSJF, TES, and TES1 were significantly downregulated in treated samples compared to controls, showing reductions of 5.0-, 5.8-, 1.6-, 9.0-, 3.0-, and 2.5-fold, respectively (Figure 7). Collectively, these findings indicate that LPs from strain D-1 effectively suppress Alternaria mycotoxin biosynthesis—including alternariol (ALT), tenuazonic acid (TeA), tentoxin (TEN), alternariol monomethyl ether (AME), and altertoxin (AOH)—by regulating the expression of key biosynthesis genes.

H₂O₂ accumulation and callose deposition serve as a critical indicator of plants disease resistance (Wu et al., 2019). In this study, H₂O₂ accumulation (brown-yellow spots) were evaluated in P. heterophylla leaves treated with strain Bc, Bf, or LPs. Compared with control plants, reduced H₂O₂ accumulation was observed at the A. alternata inoculation sites in Bf-, Bc-, and LP-treated leaves (Figure 8A). Conversely, non-inoculated regions of treated leaves exhibited elevated H₂O₂ levels. Notably, Bc- and LP-treated leaves showed greater H₂O₂ accumulation than Bf-treated samples in non-inoculated regions.

Additionally, callose deposition (blue fluorescent deposits) was quantified in P. heterophylla leaves treated with Bc, Bf, or LPs. Relative to control plants, non-inoculated regions of treated leaves displayed increased callose accumulation. Similarly, Bc- and LP-treated leaves exhibited greater callose deposition than Bf-treated samples in non-inoculated regions (Figure 8B). These results suggest that strain D-1 and its lipopeptides can elicit a stronger oxidative stress response in planta against the pathogen, contributing to enhanced systemic resistance.

Bacillus species have emerged as pivotal biocontrol agents in agricultural systems, owing to their capacity to inhibit fungal pathogens and suppress disease development in crops, fruits, and vegetables (Bi et al., 2023; Salazar et al., 2023). These bacteria exhibit broad-spectrum antagonism against phytopathogenic fungi, including A. brassicicola (Wu et al., 2021), Magnaporthe oryzae (Li et al., 2018), Botrytis cenerea (Samaras et al., 2021; Yu et al., 2022), Ralstonia solanacearum (Yang et al., 2023), Fusarium graminearium (Gong et al., 2015), and F. oxysporum (Li et al., 2018). The present study demonstrates that B. amyloliquefaciens strain D-1 possesses significant potential for the biocontrol of A. alternata-induced diseases. The observed antifungal efficacy, particularly of the cell-free fermentation supernatant (Bf), is consistent with findings from other studies on Bacillus spp. (Zhao et al., 2022). These results highlight the potential of B. amyloliquefaciens strain D-1 as a novel biocontrol candidate for managing A. alternata-induced diseases in P. heterophylla and other economically important crops.

Genome mining of B. amyloliquefaciens D-1 revealed a rich potential for secondary metabolite synthesis, identifying nine putative biosynthetic gene clusters, including those for the key antifungal lipopeptides such as fengycin, iturin, and surfactin. The high sequence similarity (82–100%) of these clusters to reference genomes is consistent with findings in other biocontrol Bacillus strains, such as the model organism B. velezensis FZB42, which also possesses a similar repertoire of lipopeptide clusters crucial for its broad-spectrum antagonism (Chen et al., 2007). Notably, the surfactin cluster in strain D-1 exhibited a relatively lower identity (82%), suggesting potential structural variations. This result aligns with previous reports where the variations in lipopeptide synthetase gene cluster can lead to novel structural homologs with potentially enhancement bioactivity (Zeng et al., 2025). HPLC-MS analysis confirmed the production of these lipopeptide families, with mass spectra showing characteristic 14 Da intervals between homologs, corresponding to fatty acid chain length variations. This pattern of producing multiple lipopeptide homologs is a well-documented strategy in Bacillus spp., as demonstrated in B. amyloliquefaciens S76-3 (Gong et al., 2015), where the synergistic interaction between different lipopeptides resulted in superior antifungal efficacy. The ability of strain D-1 to co-produce these potent lipopeptides, particularly the high antifungal activity associated with iturin and fengycin families (Ongena and Jacques, 2008), provides a compelling molecular basis for its strong biocontrol performance observed in vitro and in planta against A. alternata.

Previous studies have demonstrated that beneficial bacteria directly antagonize soil-borne phytopathogenic fungi and oomycetes by secreting antifungal compounds, including cyclic lipopeptides and volatile organic compounds (VOCs) (Wang et al., 2024). In this study, strain D-1 was found to directly inhibit A. alternata hyphal growth, branching, and spore germination through the production of cyclic lipopeptides. Cyclic lipopeptides from Bacillus, Pseudomonas, Burkholderia, and cyanobacteria exert their antimicrobial effects via strong amphiphilic properties that enable rapid insertion into fungal cell membranes, disrupting lipid bilayers and causing cytosolic leakage (Deleu et al., 2008; Gong et al., 2015; Gu et al., 2017; Melo et al., 2016). Specifically, iturin A and plipastatin A from B. amyloliquefaciens strain S76-3 induce severe morphological changes in Fusarium graminearum conidia and hyphae, including vacuolation, cytoplasmic leakage, and cellular inactivation (Gong et al., 2015). These findings suggest that strain D-1 disrupts A. alternata cell membranes through cyclic lipopeptides, thereby inhibiting fungal proliferation.

Additionally, previous studies have shown that Bacillus species can prime systemic resistance in plants, reducing disease incidence through activation of defense mechanisms (Xia et al., 2024). These responses include H₂O₂ accumulation and callose deposition, which are hallmarks of plants immune activation (Wu et al., 2019). In this study, strain D-1 induced significant systemic resistance in P. heterophylla, as evidenced by elevated H₂O₂ accumulation and callose deposition in non-inoculated leaf regions. Specifically, Bc- and LP-treated plants exhibited significantly higher H₂O₂ levels and callose deposition compared to Bf-treated and control plants. These findings suggest that both Bc and LPs contribute to systemic resistance induction, potentially acting synergistically to enhance plants immunity.

Mycotoxin contamination, caused by phytopathogenic fungi such as Fusarium and Alternaria, represents a critical challenge to the safety of medicinal herbs and crops (Li et al., 2025). Beyond direct pathogen inhibition, an ideal biocontrol agent should also mitigate the impact of these toxic secondary metabolites. Our results indicate that lipopeptides from B. amyloliquefaciens D-1 effectively suppress the production of key Alternaria mycotoxins, including alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN). This suppression is likely achieved through the downregulation of core biosynthetic genes, a phenomenon consistent with recent findings that microbial antagonistic compounds can disrupt the transcriptional regulation of fungal secondary metabolism (Zhang et al., 2024). The ability of Bacillus-derived metabolites to interfere with mycotoxin biosynthesis pathways adds a crucial layer to their biocontrol profile, as preventing toxin accumulation is often more desirable than post-hoc removal (Yun et al., 2015).

Furthermore, certain Bacillus strains exhibit the capacity to degrade mycotoxins. This aligns with our observation of mycotoxin reduction and is supported by independent studies on strain D-1, which has been shown to biodegrade zearalenone into non-toxic metabolites (Deng et al., 2023). This dual capability, inhibiting biosynthesis and degrading existing toxins, is also reported in other biocontrol agents. For example, B. velezensis LS01 could simultaneously inhibit aflatoxin production and degrade aflatoxin B1 (Liu et al., 2023). The combination of these two mechanisms in the repertoire of strain D-1 highlights its significant potential as a multifunctional agent for comprehensive mycotoxin management in the production chain of P. heterophylla.