Cyclic lipopeptides from Bacillus amyloliquefaciens D-1 control leaf spot in Pseudostellaria heterophylla via enhancing host immunity and inhibiting Alternaria alternata

Introduction Pseudostellaria heterophylla , a valuable medicinal herb in traditional Chinese medicine, is severely threatened by leaf spot disease caused by Alternaria alternata , leading to significant yield losses and mycotoxin contamination that compromises root quality and safety. This study aimed to characterize Bacillus amyloliquefaciens strain D-1 as a potential multifunctional biocontrol agent to mitigate this pathogen. Methods Strain D-1 was isolated from the rhizosphere soil of P. heterophylla . Its antifungal efficacy was assessed through in vitro dual-culture assays and in planta pot experiments using both viable cells (Bc) and cell-free fermentation supernatant (Bf). Whole-genome sequencing was employed to identify biosynthetic gene clusters (BGCs), and high-performance liquid chromatography-mass spectrometry (HPLC-MS) was used to detect lipopeptides (LPs). Quantitative PCR and mycotoxin quantification were performed to evaluate the effects on fungal gene expression and toxin accumulation. Results Strain D-1 demonstrated strong antagonistic activity, with Bf achieving a 54.7% inhibition rate against A. alternata . Genomic analysis revealed nine putative BGCs, including those for fengycin, iturin, and surfactin. HPLC-MS confirmed the production of these LPs, which effectively disrupted hyphal branching and spore germination. In pot trials, crude LP extracts reduced disease lesion area by 92.0% and suppressed key mycotoxin biosynthesis genes (PKSJ, PKSA, etc.), leading to a 77.0–98.1% reduction in toxin levels (ALT, AME, TeA, TEN). Additionally, strain D-1 and its LPs triggered systemic resistance in P. heterophylla , evidenced by elevated H₂O₂ accumulation and callose deposition. Discussion The findings establish B. amyloliquefaciens D-1 as a novel biocontrol agent with triple mechanisms: direct antifungal action, suppression of mycotoxin biosynthesis, and induction of host systemic resistance. Its application offers a sustainable strategy to manage A. alternata in P. heterophylla cultivation, addressing both disease control and medicinal safety concerns. heterophylla cultivation, addressing both disease control and medicinal safety concerns.