# Cell-Free Supernatant of Bacillus subtilis Suppresses the Mycelial Growth of Penicillium expansum by Destroying the Cell Membrane Integrity and Provoking Cell Death

## TL;DR

A natural solution using Bacillus subtilis supernatant can stop Penicillium expansum from growing on fruits, reducing spoilage and mycotoxins without harmful chemicals.

## Contribution

The study reveals that Bacillus subtilis supernatant induces cell death in Penicillium expansum by damaging membranes and triggering apoptosis-like processes.

## Key findings

- Bacillus subtilis supernatant inhibits spore germination and hyphal growth of Penicillium expansum.
- The supernatant causes membrane damage, ROS accumulation, and apoptosis-like cell death in the fungus.
- Application of the supernatant reduces disease incidence and lesion expansion in grapes and citrus.

## Abstract

Penicillium expansum causes rot in fruits such as grapes and citrus, accompanied by the production of harmful mycotoxins, leading to substantial economic losses. At present, synthetic chemical agents are widely applied to control this pathogen, yet they exert adverse impacts on the environment and human health. Accordingly, this study investigated a natural and eco-friendly control strategy, namely, the utilization of cell-free supernatant derived from Bacillus subtilis to combat P. expansum. Assays demonstrated that the cell-free supernatant of B. subtilis effectively inhibited the germination and spread of spores on postharvest fruits. The underlying antifungal mechanism involves disrupting the protective outer membrane of the fungus and triggering severe intracellular stress responses, which ultimately result in the mortality of P. expansum.

Penicillium expansum inflicts significant economic damage in the fruit and vegetable industry due to its wide distribution and ability to infect a diverse range of hosts. Therefore, developing safe and environmentally sustainable strategies to suppress the growth of this pathogen is of critical importance. Bacillus subtilis, recognized for its broad antimicrobial activity and widespread occurrence, has been widely utilized in the biological management of plant diseases. This research seeks to assess the inhibitory potential of B. subtilis against P. expansum. The cell-free supernatant (CFS) derived from B. subtilis significantly suppresses the germination of spores, germ tube extension, and hyphal development of P. expansum. It also reduces disease incidence in grapes and citrus and suppresses the expansion of lesions. Further investigation had shown that it induced mycelium reactive oxygen species (ROS) accumulation, destroyed the cell membrane integrity, led to leakage of cytoplasmic contents and induced membrane lipid peroxidation. Moreover, exposure to high concentrations of CFS results in mycelial contraction and morphological abnormalities, triggering the disintegration of intracellular organelles and markedly upregulating the expression of apoptosis-like programmed cell death genes. The self-protective response elicited by 5% CFS is insufficient to counteract the extent of cellular damage, ultimately driving cells toward a dynamic, multistage, and disintegrative form of cell death. The findings of this study offer a theoretical foundation for managing P. expansum after harvest.