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

Aflatoxins, especially aflatoxin B1, are highly toxic, mutagenic, and carcinogenic mycotoxins produced predominantly by Aspergillus flavus and A. parasiticus, contaminating susceptible crops such as peanuts and maize in the field and during storage. Conventional control measures (GAPs, GMPs, physical and chemical strategies) have limitations including inefficiency, nutrient loss, chemical residues, and resistance development. Biological control has emerged as a safer, environmentally friendly alternative. The authors previously identified PO8, a protein produced by aflatoxigenic A. flavus, as an early warning biomarker that can predict aflatoxin contamination risk before toxin production. The research question is whether inhibiting the early warning molecule PO8 can be used as a rapid and effective strategy to screen and develop broad-spectrum biocontrol strains against aflatoxigenic A. flavus to prevent aflatoxin contamination in peanuts. The study aims to validate this PO8-inhibition-based screening approach and develop a combined biocontrol agent with broad-spectrum efficacy.

The paper situates the study within growing concerns about global aflatoxin contamination in crops and food products, citing widespread occurrence in corn and peanuts and documented outbreaks of acute aflatoxicosis. Prior strategies include physical and chemical methods with notable drawbacks, and an expanding body of work on biological control using Bacillus spp. and other microbes to suppress toxigenic Aspergillus growth and aflatoxin production. Notable studies include the use of Bacillus licheniformis volatiles, B. subtilis oxidoreductases involved in bacilysin biosynthesis for detoxification, and antagonism by B. amyloliquefaciens reducing AFB1 in maize and suppressing A. flavus on maize ears. Atehnkeng et al. reported field efficacy of mixtures of A. flavus vegetative compatibility groups to prevent aflatoxin in maize. The authors’ prior work introduced PO8 as an early warning marker and established a nanobody-based sandwich ELISA for Aspergillus detection, forming the basis for the present PO8-inhibition screening approach.

Study design encompassed: (1) characterizing PO8 secretion dynamics in A. flavus; (2) screening biocontrol bacteria by their ability to inhibit PO8 expression; (3) evaluating selected antagonists and their mixture against multiple A. flavus strains from different Chinese regions; (4) validating on peanut kernels; and (5) conducting field trials with a mixed biocontrol agent. - Strains: Ten aflatoxigenic A. flavus isolates (LNZW-1, SX-1-1, SDJY-95-1, ANHBB-14, AnhHSZ-53, Hubzhx-33, JXZS-118-8, XZCY-21-5, HNDX-8, GDZJ-15) from peanut-producing provinces. Twelve candidate biocontrol bacteria from a lab library: 2BQN19 (Stenotrophomonas sp.), 54 (Bacillus amyloliquefaciens), HS10 (Bacillus licheniformis), 1JN2 (Bacillus subtilis), 5BS2 (Bacillus subtilis), B6 (Bacillus sp.), Y2 (Bacillus cereus), DY (Bacillus licheniformis), AAC (Enterobacter ludwigii), CB (Brevibacillus laterosporus), JDF (Bacillus amyloliquefaciens), JZ (Bacillus mucilaginosus). - Culture conditions: A. flavus on DG-18 agar; conidia harvested in 0.01% Tween-80 after 5–6 days at 28 °C (dark). For liquid culture, Sabouraud’s Dextrose Broth (SDB) with 5×10^5 conidia/mL at 28±1 °C, 180 rpm. - PO8 secretion dynamics: Ten A. flavus strains grown in SDB (50 mL) for 8 days; intracellular and extracellular PO8 measured daily via ELISA to determine localization and peak times. - Screening via PO8 inhibition: A. flavus JXZS-118-8 pre-cultured 12 h in SDB (5×10^5 conidia/mL). Each of 12 bacteria added to 10^7 CFU/mL; co-cultured up to 36 h. Mycelia collected at 24, 30, and 36 h for PO8 quantification. Three biological replicates per condition; one-way ANOVA (p<0.05) for significance. - Broad-spectrum testing: Four selected bacteria (AAC, CB, JDF, JZ) and their mixture (AAC+CB+JDF+JZ) added at 10^7 CFU/mL to three A. flavus strains (LNZW-1, JXZS-118-8, GDZJ-15) after 12 h pre-culture; sampled at 24, 30, 36 h. Mycelia separated from bacterial cells by filtration and washing for PO8 ELISA; culture supernatants used for AFB1 quantification by HPLC-FLD. - Peanut inoculation: Surface-sterilized mature peanut seeds (10 g per plate) inoculated with 200 µL A. flavus conidia (5×10^5 CFU/mL). Experimental groups received 3 mL bacterial suspension (AAC, CB, JDF, JZ, or mixture); controls received water. Incubation 7 days at 28 °C (dark). Visual assessment of spore growth; PO8 (ELISA) and AFB1 (HPLC) measured. - Field trials (2021): Mixed biocontrol agent (BBBE) prepared from the four strains applied as biological bacterial fertilizer at 30 kg·hm^-2 in peanut fields at five sites: Junan (JN, Shandong), Zhengyang (ZY, Henan), Siyang (SY, Jiangsu), Xiangyang (XY, Hubei), Fuzhou (FZ, Fujian). Conventional agronomic practices followed. Rhizosphere soil sampling by five-point method; A. flavus abundance determined by plating on DG-18, colony counts (CFU/g) calculated, and identification by morphology and molecular methods. - Analytics: PO8 quantified via sandwich ELISA using PO8-specific VHH capture and rabbit polyclonal detection, HRP-conjugate and TMB read at 450 nm. Inhibition ratio (%) = [(C_PO8 − E_PO8)/C_PO8]×100%. AFB1 extracted with 80% methanol, analyzed by HPLC-FLD (C18 column; mobile phase MeOH:water 45:55; 1.0 mL/min; 30 °C; Ex 360 nm/Em 440 nm). Statistics by one-way ANOVA (SPSS 26.0, p<0.05).

- PO8 localization and timing: Intracellular PO8 constituted >99% of total PO8 over 8 days across ten A. flavus strains; intracellular PO8 rose rapidly by day 2 (≈48 h) and then plateaued. Extracellular PO8 peaked between days 2–5 depending on strain. - Screening by PO8 inhibition (A. flavus JXZS-118-8): All 12 bacterial treatments significantly reduced PO8 versus control at 24, 30, and 36 h (p<0.05). Five strains showed strongest inhibition, generally >90%: DY-E (99%, 95%, 98%), JDF-E (99%, 98%, 95%), AAC-E (97%, 98%, 95%), CB-E (99%, 94%, 85%), JZ-E (96%, 89%, 98%). Others were less consistent (e.g., 2BQN19-E: 96%, 77%, 98%; 54-E: 94%, 64%, 91%; HS10-E: 90%, 70%, 76%; 1JN2-E: 56%, 68%, 54%; 5BS2-E: 83%, 80%, 79%; B6-E: 94%, 86%, 85%; Y2-E: 89%, 87%, 83%). Four strains (AAC, CB, JDF, JZ) were selected for further study. - Broad-spectrum efficacy: Against three A. flavus strains (LNZW-1, JXZS-118-8, GDZJ-15), the four individual bacteria variably inhibited PO8 and AFB1, whereas their mixture nearly completely inhibited PO8 and AFB1 by 36 h across strains. For JXZS-118-8, both PO8 and AFB1 were almost completely inhibited in all treatments; for LNZW-1 and GDZJ-15, the mixture achieved near-complete inhibition at 36 h even when some single strains were less effective. Statistical analysis confirmed significant differences across treatments and times (p<0.05), with some non-significant pairwise time comparisons within treatments (e.g., AAC-E 24 h vs 36 h; JDF-E/JZ-E/MIX-E 24 h vs 30 h). - Peanut kernel assay: All four bacteria reduced visible spore growth versus control after 7 days at 28 °C; the mixture showed the strongest effect (minimal spores). PO8 and AFB1 levels were significantly reduced in all treatments (p<0.05), with the mixture yielding the lowest concentrations and highest inhibition rates. The mixture achieved up to 99.9% inhibition of AFB1. - Field trials: The mixed biocontrol agent BBBE reduced A. flavus abundance in peanut rhizosphere soils, with inhibition rates ranging from 50% to 83.3% across five sites: SY 83.3%, XY 76.9%, ZY 60.9%, JN 50%, FZ 50%. - Overall, mixing the four strains broadened the biocontrol spectrum and yielded superior, more consistent suppression of PO8 expression and AFB1 production than any single strain.

The study demonstrates that using the early warning molecule PO8 as a functional screening target enables rapid identification of effective biocontrol strains against aflatoxigenic A. flavus. PO8 is predominantly intracellular and peaks by 48 h, informing a practical window for inhibition assays. Screening via PO8 inhibition identified four antagonists (Enterobacter ludwigii AAC; Brevibacillus laterosporus CB; Bacillus amyloliquefaciens JDF; Bacillus mucilaginosus JZ) with strong anti-PO8 and anti-aflatoxin activity. Although individual strains exhibited strain-specific and time-dependent variability, their mixture consistently achieved near-complete inhibition of both PO8 and AFB1 in multiple A. flavus isolates from diverse regions, indicating complementary mechanisms and a broadened control spectrum. Validation on peanut kernels confirmed substantial suppression of spore growth and >95% inhibition of PO8 and AFB1, with the mixture reaching 99.9% AFB1 inhibition. Field trials across five provinces showed practical efficacy, reducing A. flavus abundance in rhizosphere soils by 50–83.3%. Collectively, these findings support the feasibility and significance of a PO8-inhibition-guided strategy to develop environmentally friendly, broad-spectrum biocontrol agents for aflatoxin source control and improved food safety.

This work establishes a new, rapid screening and development approach for aflatoxin biocontrol based on inhibiting the early warning molecule PO8. Four biocontrol strains (Enterobacter ludwigii AAC, Brevibacillus laterosporus CB, Bacillus amyloliquefaciens JDF, Bacillus mucilaginosus JZ) were identified, and their mixture (BBBE) provided broad-spectrum, high-efficacy suppression of PO8 expression and AFB1 production in vitro, on peanut kernels, and in field trials. The strategy effectively expands the control spectrum via strain combination and offers a green, practical solution for aflatoxin source control. Future work could elucidate the molecular mechanisms underlying PO8 and aflatoxin inhibition, optimize strain ratios and formulations for varied environments and crops, assess long-term field performance and microbiome impacts, and explore application to other mycotoxin-producing fungi.

- Individual biocontrol strains exhibited limited broad-spectrum efficacy; robust control required the mixture. - Field performance varied by site, with inhibition ranging from 50% to 83.3%, indicating environmental dependence. - Broad-spectrum in vitro testing used three A. flavus isolates; wider strain diversity and longer-term assessments would strengthen generalizability. - The study focused on peanuts; cross-crop applicability requires additional validation.