Approach for quick exploration of highly effective broad-spectrum biocontrol strains based on PO8 protein inhibition

Aflatoxins, produced by *Aspergillus flavus* and *A. parasiticus*, are highly toxic and carcinogenic mycotoxins that contaminate food and feed globally, causing significant health risks and economic losses. Current control methods, including Good Agricultural Practices (GAPs) and Good Manufacturing Practices (GMPs), along with physical and chemical strategies, often prove insufficient due to nutritional loss, residue issues, or the development of resistant pathogens. Biological control, using microorganisms to inhibit aflatoxigenic *A. flavus*, presents a promising, environmentally friendly alternative. Previous research identified PO8 protein as an early warning molecule for aflatoxin production in peanuts, indicating its potential as a target for biocontrol agent development. This study investigates the feasibility of screening and developing biocontrol agents by inhibiting PO8, focusing on aflatoxin contamination in peanuts.

Extensive research explores mycotoxin reduction using *Bacillus* species and other microorganisms. Studies have demonstrated the effectiveness of various biocontrol agents in reducing aflatoxin contamination in different crops. However, a consistent and efficient screening method for broad-spectrum biocontrol agents remains a challenge. The discovery of the PO8 protein as an early warning molecule offers a novel approach to address this limitation, providing a potential target for rapid and effective biocontrol strain selection.

This study involved several key steps: 1. **PO8 Secretion Monitoring:** Ten aflatoxigenic *A. flavus* strains were cultured, and intracellular and extracellular PO8 content were measured daily using ELISA for eight days to determine optimal incubation time for subsequent experiments. 2. **Biocontrol Strain Screening:** Twelve biocontrol bacterial strains were tested for their ability to inhibit PO8 expression in *A. flavus* JXZS-118-8. The inhibition ratio of PO8 was measured at 24, 30, and 36 hours using ELISA. 3. **Broad-Spectrum Determination:** The efficacy of four selected biocontrol strains (AAC, CB, JDF, JZ) and their mixture against PO8 and aflatoxin (AFB1) production was evaluated using three aflatoxigenic *A. flavus* strains from different regions of China. PO8 and AFB1 levels were measured at 24, 30, and 36 hours using ELISA and HPLC, respectively. 4. **Peanut Inoculation Experiment:** The effectiveness of the four biocontrol strains and their mixture was tested in a peanut inoculation experiment. Peanuts were inoculated with *A. flavus*, treated with the biocontrol agents, and then PO8 and AFB1 levels were analyzed. 5. **Field Trial:** A mixture of the four biocontrol strains (BBBE) was tested in field trials at five sites across China. The abundance of *A. flavus* in peanut rhizosphere soil was determined using plate counting and colony identification methods. 6. **Analytical Methods:** PO8 levels were quantified using a Sandwich-ELISA, and AFB1 levels were determined by HPLC. Statistical analysis was performed using one-way ANOVA.

The intracellular PO8 content of *A. flavus* peaked at 48 hours, remaining stable thereafter. Five bacterial strains exhibited strong PO8 inhibition; four (*Enterobacter ludwigii*, *Brevibacillus laterosporus*, *Bacillus amyloliquefaciens*, *Bacillus mucilaginosus*) were selected for further investigation. The combination of these four biocontrol bacteria significantly increased the inhibition rate of both PO8 and AFB1 compared to individual strains, achieving almost complete inhibition in all three tested *A. flavus* strains (over 94% at 36h). In the peanut inoculation experiment, the mixed treatment group exhibited the highest inhibition rate of PO8 (99.9%) and AFB1 (99.9%). Field trials demonstrated a 50-83.3% inhibition rate of *A. flavus* in peanut rhizosphere soil across five different locations.

The findings demonstrate that inhibiting the early warning molecule PO8 is a feasible strategy for developing effective broad-spectrum biocontrol agents against aflatoxin production. The superior performance of the mixed biocontrol agents compared to single strains highlights the synergistic effects of combining multiple strains with different mechanisms of action. The successful field trials validate the effectiveness of the approach under real-world conditions. The study reveals the potential of *Enterobacter ludwigii*, *Brevibacillus laterosporus*, *Bacillus amyloliquefaciens*, and *Bacillus mucilaginosus* as promising biocontrol agents for aflatoxin prevention in peanuts. The results highlight the importance of considering regional variations in *A. flavus* strains when developing biocontrol strategies.

This study presents a novel and efficient approach for screening highly effective broad-spectrum biocontrol strains against aflatoxin contamination by targeting the PO8 protein. The successful development and validation of a mixed biocontrol agent (BBBE) demonstrates the potential of this strategy for improving food safety and promoting sustainable agriculture. Future research could focus on optimizing the formulation and application methods of BBBE, investigating the specific mechanisms of action of each bacterial strain, and testing its effectiveness against a wider range of mycotoxin-producing fungi.

The study primarily focused on aflatoxin contamination in peanuts, and the generalizability of the findings to other crops or mycotoxins needs further investigation. The field trials were conducted in specific regions of China, and additional studies are needed to confirm the effectiveness of BBBE in different geographical locations and environmental conditions. The specific mechanisms underlying the observed inhibition of PO8 and AFB1 by the biocontrol bacteria require further elucidation.