Introduction

Aflatoxins, derivatives of polyketide, secondary metabolites primarily produced by fungal species from the Aspergillus genus, especially, Aspergillus flavus and Aspergillus parasiticus, are a group of structurally related toxic, mutagenic, and carcinogenic mycotoxins1,2,

Methods

Strains and culture conditions

The aflatoxigenic A. flavus strains LNZW-1, SX-1-1, SDJY-95-1, ANHBB-14, AnhHSZ-53, Hubzhx-33, JXZS-118-8, XZCY-21-5, HNDX-8, and GDZJ-15 were isolated from the peanut soils in Liaoning, Shanxi, Shandong, Anhui, Hubei, Jiangxi, **zang, Hunan and Guangdong peanut-planting provinces of China, which is depicted in Fig. 6. The A. flavus strains were grown on DG-18 agar medium (protein 5 g, glucose 10 g, potassium dihydrogen phosphate 1.0 g, magnesium sulfate 0.5 g, zinc sulfate 0.01 g, copper sulfate 0.005 g, ammonium chlortronitol 0.002 g, gildamycin hydrochloride 0.05 g, agar 15 g, and 1 L water, pH adjusted to 5.6 ± 0. 2, 121 °C sterilized for 20 min). The conidia were harvested with a 0.01% Tween-80 solution after 5–6 days of culture at 28 °C in darkness. The biocontrol bacteria including 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), selected from the biocontrol strain library in our laboratory, were isolated from peanut or their rhizosphere soil, which were previously reported by Yang35, Li et al. (2019)29, and Zhang et al. (2021)36. The biocontrol bacteria strains were cultured at 28 °C for 18–24 h in an LB liquid medium, which contained 5 g of yeast extract, 10 g of peptone, and 10 g of NaCl per liter. A. flavus and the bacterial antagonists, respectively, were prepared by quantifying the conidia and bacterial cells using a hemocytometer.

Fig. 6: The source locations of A. flavus strains used and the distribution map of the field test in this research.
figure 6

In the map, the picture “plate” represents the source locations of A. flavus strains used in this work, and the picture “peanut field” represents the demonstration sites of the biocontrol agent BBBE used in field trials. In this figure, 1 cm represents a distance of 230 km in the field.

Full size image

The observation of the secretion regularity of PO8 at different times in high virulence-producing A. flavus strains

Briefly, ten A. flavus strains were grown in conical flasks containing 50 ml Sabouraud’s Dextrose Broth (peptone 10 g, glucose 40 g, 121 °C sterilized for 20 min) with 5 × 105 conidia/mL and shaken (180 rpm) at 28 ± 1 °C for 8 days. The mycelium was collected by filtration at different incubation times every 24 h under sterile conditions with sterile water and gauze, and then quick-frozen in liquid nitrogen, and freeze-dried for further use. The culture medium was also collected into the sterile tubes and then stored at −20 °C for use. Three biological replicates were set up for all control and experimental groups.

Screening of biocontrol bacterial strain based on PO8 inhibition

Briefly, the A. flavus strain JXZS-118-8 was grown in conical flasks containing 50 ml Sabouraud’s Dextrose Broth with 5 × 105 conidia/mL and shaken (180 rpm) at 28 °C for 12 h. And then, 12 biocontrol bacteria strains were added to the above conical flasks to reach a final concentration of 107CFU/mL and continued to culture for 36 h. At different incubation times (24 h, 30 h, and 36 h), the mycelia were collected into the sterile tubes and then stored at −20 °C. Three biological replicates were set up for all experimental and control groups.

Briefly, the A. flavus strains LNZW-1, JXZS-118-8, and GDZJ-15 were grown in conical flasks containing 50 ml Sabouraud’s Dextrose Broth with 5 × 105 conidia/mL and shaken (180 rpm) at 28 ± 1 °C for 12 h. And then, the suspension of four biocontrol bacteria strains (AAC, CB, JDF, JZ,) and their mixture (AAC + CB + JDF + JZ) was added to the above conical flasks to reach a final concentration of 107 CFU/mL and continued to culture for 36 h. At different incubation times (24 h, 30 h, and 36 h), the mycelia that were collected for the detection of PO8 by being filtered out of the liquid medium with sterile gauze and washed three times with sterilization water to separate mycelia from bacterial cells, and Sabouraud’s Dextrose Broth were collected into the sterile tubes and then stored at −20 °C for the detection of AFB1. Three biological replicates were set up for all experimental and control groups.

Peanut Inoculation experiment

Healthy postharvest mature peanut seeds were selected for the experiments to investigate the phenotypic data of the peanut matrices. All seeds were surface-sterilized by immersion in 70% ethanol for 1 min and rinsed with sterile distilled water three times for 1 min each, and then the moisture on the peanut surface was absorbed by sterilization filter paper. Two hundred microliters of the conidial suspension (5.0 × 105 CFU/mL) were added to the experimental and control plates, respectively. In the experimental group, 3 mL of bacterial suspension of the AAC, CB, JDF, JZ, and their mixture were added to the 10.0 g peanut seeds in a sterile Petri plate (ensure that the peanut surface is completely covered). In the control group, 3 mL of ultrapure water was added to the peanut seeds in sterile Petri plates. Then, the experimental and control group samples were placed in an incubator and cultured at 28 °C in darkness for 7 days. Three biological replicates were set up for all experimental and control groups.

Field trials

The four biocontrol bacteria strains screened out were mixed to produce biocontrol agents BBBE for use in field trials in 2021. Field trials were conducted at five sites, including Junan County (JN, Shandong Province), Zhengyang County (ZY, Henan Province), Siyang County (SY, Jiangsu Province), **angyang County (XY, Hubei Province), Fuzhou City (FZ, Fujian Province) in 2021. In experimental plots, biological bacteria fertilizer of 30 kg hm−2 was added with fertilizer material, the peanut variety was the main local variety, and the local conventional sowing method, as well as management measures, were adopted for weeding pest, and boom control. At the same time, the control plots were set.

Determination of A. flavus abundance in peanut rhizosphere soil

The method to isolate and identify A. flavus was conducted according to the method proposed by Zhang et al.37. In detail, using the five-point sampling method, 5 peanut plants were randomly selected within the range of 2 m2, and the peanut rhizosphere soil samples were collected, and they were mixed into one sample. A total of 10 samples including five control samples and five treatment samples were collected from each demonstration base. To prepare soil suspensions, 10.0 g soil samples were added to conical flasks containing 90 mL of sterile water on the ultra-clean workbench, and mixed them thoroughly by shaking at 28 °C (200 rpm/min, 2 h). After that, 50 μL of soil suspension was transferred to the sterile plates with DG-18 solid medium and spread evenly using a glass spreader. And these plates were placed in an incubator at 28 °C in darkness. The colonies were observed and counted regularly, and the hyphae were identified by morphological and molecular biology methods. The plates that were contaminated were cleaned up in time to avoid cross-contamination.

The number of aflatoxin-producing A. flavus colonies per gram of soil was calculated by the following formula:

$${\rm{The\; colony\; count}}\left({\rm{CFU}}/{\rm{g}}\right)=\left[{\rm{N}}/\left({{\rm{V}}}_{1}\times {\rm{m}}\right)\right]\times {\rm{V}}$$

Where N is the colony count of the plates, V1 is the volume of injection, m is the mass of the sample, and V is the volume of the soil suspension.

Detection of PO8 by Sandwich-ELISA

The method of PO8 extraction differs in different samples. The extraction of PO8 in mycelia was as follows: 10 mg mycelium was added into 2 ml tubes, 500 μL of 1 × PBS (0.01 mol/L) buffer, and four steel balls (3 mm in diameter) were added, grounded at 60 Hz for 30 s in beveller, 12,000 rpm, centrifuged for 10 min, and then the supernatant was filtered with a membrane of 0.22 μm (pore size), and stored at −20 °C for further use.

In the peanut inoculation experiment, the PO8 was extracted according to the following protocol: 2.5 g peanut was added into 50 ml tubes, 10 mL of 1 × PBS buffer, and four steel balls (65 mm in diameter) were added, grounded at 2500 rpm for 10 min, 4500 rpm, centrifuged for 10 min, and then the supernatant was filtered with a membrane of 0.22 μm (pore size), and stored at −20 °C for further use.

PO8 quantification was conducted by the method proposed by Wang et al. (2017) with modifications20. Sandwich ELISA was developed to quantify PO8 according to the following protocol: 96-well microtiter plates (Corning, NY, USA) were coated with 200 μL/well of capture antibody (PO8-VHH Nano-antibody) in 1 × PBS buffer at a concentration of 3 μg/mL and incubated at 4 °C overnight. Plates were rinsed six times with 350 μL/well of 1 × PBST (1 × PBS containing 0.05% Tween 20), subsequently blocked with 300 μL/well of 3% skimmed milk in 1 × PBST buffer at 37 °C for 2 h. After nine items of washings with 1 × PBST, 200 μL of serially diluted mycelia were added at 37 °C for 1 h; washing cycles were repeated and 200 μL of rabbit polyclonal antibody at a concentration of 2 μg/mL in 1 × PBS was added and incubated at 37 °C for 1 h; after washing, 200 μL HRP-labeled goat anti-rabbit IgG antibody (Solarbio, Bei**g, China, 1:5000 dilution) was incubated at 37 °C for 1 h; after nine times washing, 100 μL of TMB solution were incubated at 37 °C for 10 min; the reaction was then terminated by adding 50 μL of 2 M H2SO4 and the absorbance values were detected at 450 nm using the CMax Plus microplate reader (Molecular Devices, CA, USA). The relative reduction of PO8 was calculated by the following formula:

$${\rm{Inhibition}}\; {\rm{ratio}}\,( \% )=\left({{\rm{C}}}_{{\rm{PO}}8}-{{\rm{E}}}_{{\rm{PO}}8}\right)/{{\rm{C}}}_{{\rm{PO}}8}\times 100 \%$$

Where CPO8 is the concentration of PO8 in control groups, EPO8 is the concentration of PO8 in experimental groups.

Detection of AFB1 by High-Performance Liquid Chromatography

AFB1 extraction was conducted as follows: Briefly, aflatoxins B1 in mycelium or peanuts were extracted using 80% Methanol: water = 80:20, the supernatant was purified using a vacuum filtration system with a 25 mm membrane filter (0.22 μm pore size). Quantitative analysis of aflatoxins was performed by Agilent 1100 HPLC, equipped with a fluorescence detector (FLD), and Romer Derivatisation Unit was used in the system.

The HPLC conditions were as follows: chromatographic column: Waters Symmetry C18 5 μm, 4.6 mm × 250 mm, (C18-A analytical column (15 cm × 4.6 mm × 5 μm); injection volume: 10 μl; column temperature: 30 °C; flow rate: 1.0 ml/min; mobile phase: methanol: water = 45:55 (volume ratio); and fluorescence detection wavelength: excitation wavelength 360 nm and emission wavelength 440 nm.

Statistical analysis

One-way ANOVA was carried out to evaluate any significant difference in the control effect of biocontrol bacteria, using SPSS 26.0. The different letter means different significance levels (p < 0.05).