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

Haixia Shi¹Shihua Zhou¹Yanping Gao¹Jiang Xue²Lang Qin¹Lingling Wang¹XIAOHONG OU¹Yanhong Wang¹Lanping Guo³Li Gu⁴Qing-Song Yuan^1*Tao Zhou¹

• ¹Guizhou University of Traditional Chinese Medicine, Guiyang, China
• ²Jiangsu University, Zhenjiang, China
• ³State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, Beijing, China
• ⁴Fujian Agriculture and Forestry University, Fuzhou, China

Pseudostellaria heterophylla (family Caryophyllaceae) is a medicinally valuable herb cultivated for its tuberous roots, which have been used in traditional Chinese medicine (TCM) for over a century. However, the leaf spot disease caused by Alternaria alternata—and the subsequent mycotoxin contamination it induces—have emerged as major threat to this medicine herbs, leading to substantial yield losses and severe degradation of root quality. This study employs an integrated phenotypic, genomic, and metabolomic approach to characterize Bacillus amyloliquefaciens strain D-1 as a multifunctional biocontrol agent against A. alternata in P. heterophylla. Strain D-1, isolated from rhizosphere soil of P. heterophylla, exhibited robust antifungal activity both in vitro and in planta. Dual-culture antagonism assays showed that both viable cells (Bc) and cell-free fermentation supernatant (Bf) of D-1 significantly inhibited A. alternata growth, with Bf achieving a 54.7% inhibition rate against the pathogen. Genome sequencing of D-1 identified nine putative secondary metabolite biosynthetic gene clusters (BGCs), including those encoding the cyclic lipopeptides (LPs) fengycin, iturin, and surfactin. High-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis confirmed the production of these LPs, which were shown to inhibit A. alternata hyphal branching and suppress spore germination. Pot experiments validated biocontrol efficacy of strain D-1, with crude LPs extracts reducing the leaf spot disease lesion area by 92.0% compared to the water-treated control. Moreover, LPs significantly downregulated the expression of A. alternata mycotoxin biosynthesis genes (PKSJ, PKSA, PKSI, PKSJF, TES, and TES1), which corresponded to a 77.0–98.1% reduction in the accumulation of key mycotoxins (ALT, AME, TeA, TEN) in infected P. heterophylla roots. Additionally, strain D-1 and its LPs induced systemic resistance in P. heterophylla, characterized by elevated hydrogen peroxide (H₂O₂) accumulation and callose deposition in non-inoculated tissues. This work establishes B. amyloliquefaciens D-1 as a novel biocontrol agent with direct antifungal activity, mycotoxin biosynthesis inhibition, and systemic resistance induction capabilities. Its application provides a sustainable solution for managing A. alternata in traditional Chinese medicine cultivation, addressing both disease incidence and medicinal safety concerns.