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

## TL;DR

A new strain of Bacillus amyloliquefaciens helps control a fungal disease in a medicinal plant by inhibiting the fungus, reducing toxins, and boosting plant immunity.

## Contribution

Discovery of a novel biocontrol agent with triple mechanisms for managing leaf spot disease in Pseudostellaria heterophylla.

## Key findings

- Strain D-1 reduced disease lesion area by 92.0% and suppressed mycotoxin levels by 77.0–98.1%.
- Lipopeptides from D-1 inhibited fungal spore germination and hyphal branching.
- D-1 triggered systemic resistance in the plant, indicated by H₂O₂ accumulation and callose deposition.

## Abstract

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.

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.

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.

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.