Bacillus velezensis strain atc-al and applications thereof

Abstract

The application discloses a bacillus velezensis ATC-AL and application thereof, the strain preservation number of which is CCTCC: M20251280. The genome of the bacillus velezensis ATC-AL has a synthetic gene cluster for synthesizing various antifungal active substances, the active substances can destroy the cell surface structure of pathogenic bacteria, induce cell apoptosis, inhibit spore development of pathogenic fungi, have a broad-spectrum inhibiting effect on various pathogenic fungi such as diaporthe phaseolorum, fusarium graminearum and aspergillus flavus, the inhibiting rate of the bacillus velezensis ATC-AL on the production of vomitoxin reaches 95%, and the inhibiting rate of the bacillus velezensis ATC-AL on the production of aflatoxin B1 reaches 100%; fungus infection experiments on sweet potatoes, corns and wheat prove that the active substances in the cell fermentation liquor of the bacillus velezensis ATC-AL can completely inhibit the corruption of diaporthe phaseolorum, fusarium graminearum and aspergillus flavus on the above food crops; and the application enriches the microbial control resource library for biologically controlling pathogenic fungi and toxin pollution.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microbial application technology and biological control, and particularly relates to a bacillus velezensis ATC-AL and application thereof. BACKGROUND

[0002] During planting, harvesting, transportation and storage, agricultural products such as grains and sweet potatoes are extremely susceptible to infection by pathogenic fungi. Among them, Fusarium graminearum, Aspergillus flavus and Ceratocystis fimbriata are the main pathogenic fungi, which not only cause grain rot and yield reduction, but also seriously threaten human and animal health and cause food safety hazards and huge economic losses due to the production of mycotoxins (such as vomitoxin, aflatoxin B1, etc.).

[0003] Aflatoxin produced by Aspergillus flavus has strong carcinogenicity, hepatotoxicity and mutagenicity, and AFB1 type is the most toxic, which is listed as a class of carcinogens by the International Agency for Research on Cancer of the World Health Organization. This toxin is extremely easy to contaminate corn, peanut, soybean and other grain and oil crops and their processed products, seriously threatening food safety and public health. On the other hand, vomitoxin (DON) produced by Fusarium graminearum also widely exists in corn, wheat and other cereals, and has obvious gastrointestinal toxicity and immunosuppressive effect. These mycotoxins have high thermal stability and are difficult to remove by processing, resulting in frequent occurrence of mycotoxin residues in grain and oil processing products, which has become one of the major barriers for the export of agricultural products and their processed products in China. At present, the main means for controlling fungal contamination and toxins during storage of agricultural products at home and abroad is still chemical preservatives and fungicides, and common agents include carbendazim, thiophanate-methyl, etc. However, chemical control has many problems such as residue risk, environmental pollution, development of drug resistance, etc., and it is difficult to meet the development needs of modern green and safe agriculture.

[0004] In recent years, biological control as a new green prevention and control technology has attracted widespread attention due to its safety, environmental protection, sustainability and other advantages. Using antagonistic microorganisms or their metabolites to inhibit the growth of pathogenic fungi has become an important direction for the prevention and control of fungal contamination during the storage of agricultural products. Among them, Bacillus velezensis has been widely studied and applied in the biological control of crop diseases due to its advantages such as production of active metabolites (such as lipopeptides, enzymes, volatile organic compounds, etc.), broad-spectrum antibacterial activity, strong stress resistance, and wide adaptability. However, most of the reported Bacillus velezensis biocontrol strains are applied in the field soil and rhizosphere environment, and the resources of Bacillus velezensis that can specifically inhibit pathogenic fungi and their toxin synthesis during storage are still limited. Therefore, it is necessary to further explore and develop high-efficiency and safe microbial resources that can be applied to the storage and preservation of sweet potatoes, corn, wheat and other food crops.

[0005] Therefore, it is urgent to screen and develop new Bacillus velezensis resources with high-efficiency and broad-spectrum antifungal activity, toxin inhibition ability, and application in the storage and prevention of agricultural products, to enrich the microbial control resource library and provide technical support for realizing green agriculture and ensuring the safety of food storage and transportation. SUMMARY

[0006] The present application aims to develop a strain of Bacillus velezensis with clear source and high safety and efficiency, and its application in preventing and controlling common fungal diseases and toxin contamination of food crops such as sweet potatoes during field and storage processes. The specific application purposes are as follows:

[0007] The first application purpose of the present application is to provide the above-mentioned Bacillus velezensis as a broad-spectrum bacteriostatic agent for inhibiting plant / animal pathogenic fungi such as C. falcatum, A. flavus, F. graminearum and A. fumigatus.

[0008] The second application purpose of the present application is to provide a strain of Bacillus velezensis to solve the problem of lack of effective biological control means for black spot disease of sweet potato caused by C. falcatum.

[0009] The third application purpose of the present application is to provide the above-mentioned Bacillus velezensis for application in controlling common toxin contamination (A. flavus toxin B1, vomitoxin, etc.) during the storage of food crops.

[0010] The fourth application purpose of the present application is to provide the above-mentioned Bacillus velezensis for application in preventing and controlling fungi and their toxin contamination of food crops such as sweet potatoes, corn and wheat during postharvest storage.

[0011] To achieve the above-mentioned application purposes, the technical solutions adopted by the present application are as follows:

[0012] The present application provides a bacillus velezensis with strong antagonistic ability, named bacillus velezensis ATC-AL, which has been preserved in China Center for Type Culture Collection on June 6, 2025, with the preservation number CCTCC: M20251280 and the preservation address being Wuhan University, Wuhan, China.

[0013] Preferably, the genome of the bacillus velezensis ATC-AL contains a synthetic gene cluster capable of synthesizing a variety of antifungal active substances. Through whole genome sequencing and secondary metabolite mining, it is confirmed that the ATC-AL strain can produce a variety of antibacterial secondary metabolites, including lipopeptides (such as iturin, fengycin) and volatile organic compounds (VOCs).

[0014] The present application provides a microbial preparation, which comprises one or more of the above-mentioned bacillus velezensis ATC-AL, bacillus velezensis ATC-AL fermentation broth, and active substances of bacillus velezensis ATC-AL. The microbial preparation prepared by using the above-mentioned bacillus velezensis ATC-AL is used for inhibiting the growth of pathogenic fungi such as aspergillus flavus and fusarium graminearum and / or the production of mycotoxins such as aflatoxin and vomitoxin.

[0015] Further, the preparation method of the bacillus velezensis ATC-AL fermentation broth is as follows: inoculating the bacillus velezensis ATC-AL strain into LB liquid medium and culturing at 37℃ for 7 days with shaking to obtain the ATC-AL fermentation broth; and the preparation method of the active substances of the bacillus velezensis ATC-AL is as follows: extracting the culture solution with an equal volume of ethyl acetate, and obtaining the active substances of the bacillus velezensis ATC-AL after rotary evaporation.

[0016] Preferably, the preparation form of the preparation is one of liquid culture, spray-dried powder, wettable granules, or suspended granules.

[0017] To achieve the first application purpose, the technical solution adopted by the present application is as follows:

[0018] The above-mentioned bacillus velezensis ATC-AL or the above-mentioned microbial preparation is applied in the broad-spectrum inhibition of plant / animal pathogenic fungi, and the pathogenic fungi are one or more of fusarium graminearum, ceratocystis fimbriata, aspergillus flavus, fusarium solani, phytophthora cinnamomi, aspergillus terreus, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, and candida albicans.

[0019] Analysis of the antagonistic activity of the above-mentioned ATC-AL strain or its cell-free supernatant fermentation broth against various major pathogenic fungi revealed that it had an inhibition rate of 100% against *Aspergillus nidus*, *Fusarium solani*, *Aspergillus flavus*, and *Aspergillus fumigatus*; and inhibition rates of 75.76%, 70.98%, 74.70%, 70.05%, 62.94%, and 50.00% against *Aspergillus nidus*, *Fusarium graminearum*, *Phytophthora infestans*, *Aspergillus niger*, *Aspergillus terreus*, and *Candida albicans*, respectively.

[0020] To achieve the second application objective mentioned above, the technical solution adopted by this invention is as follows:

[0021] The application of the aforementioned *Bacillus vesalis* ATC-AL or the aforementioned microbial preparation in the control of postharvest black spot disease in sweet potato. The *Bacillus vesalis* ATC-AL fermentation broth screened in this invention significantly inhibits the mycelial growth and spore germination of *Bacillus vesalis*. Scanning electron microscopy revealed that the pathogenic mycelia were noticeably shrunken, swollen, and deformed, exhibiting typical stress-induced damage characteristics. This fermentation product disrupts the integrity of the pathogenic cell membrane, inducing intracellular reactive oxygen species (ROS) accumulation, a decrease in mitochondrial membrane potential, and apoptosis, ultimately leading to cell death.

[0022] To achieve the third application objective mentioned above, the technical solution adopted by this invention is as follows:

[0023] The application of the aforementioned Bacillus belyssus ATC-AL or the aforementioned microbial preparation in the control of Aspergillus flavus and / or aflatoxin contamination in corn. This invention further verifies that the ATC-AL strain has a significant inhibitory effect on the toxin-producing ability of Aspergillus flavus: the specific verification process is as follows: the ATC-AL strain inhibits the toxin production of Aspergillus flavus by adding Aspergillus flavus and Bacillus flavus co-culture or gradient Bacillus flavus active substances to PDB liquid medium and YES liquid medium, culturing at 28℃ for 4 days, extracting the residual toxin in 700 μL of fermentation broth with 700 μL of dichloromethane, and detecting the aflatoxin content by thin-layer chromatography, with an inhibition rate of 100%.

[0024] To achieve the fourth application objective mentioned above, the technical solution adopted by this invention is as follows:

[0025] The application of the aforementioned Bacillus belyssus ATC-AL or the aforementioned microbial preparation in the control of Fusarium graminearum and / or vomitoxin contamination in wheat. This invention further verifies that the ATC-AL strain has a significant inhibitory effect on the toxin-producing ability of Fusarium graminearum: the specific verification process is as follows: the ATC-AL strain inhibits Fusarium graminearum toxin production by adding different gradient concentrations of Bacillus graminearum active substances to TBI liquid culture medium and culturing at 28℃ for 7 days. The vomitoxin content was detected using a vomitoxin kit from Shenzhen Fende Biotechnology Co., Ltd., and the inhibition rate was 85.76%.

[0026] Furthermore, in order to effectively control pathogen and toxin contamination of sweet potatoes, corn, wheat and other grains during storage, this invention further develops a green treatment method based on ATC-AL fermentation broth: Bacillus vesiculosus ATC-AL fermentation broth or cell-free supernatant of Bacillus vesiculosus ATC-AL fermentation broth is used to protect sweet potatoes, corn and wheat through immersion treatment, spray treatment or coating treatment.

[0027] Experiments have shown that the ATC-AL strain and its fermentation products can significantly reduce the occurrence of sweet potato black spot disease during storage and effectively control the production of corn aflatoxin and wheat scab. Its inhibition rates against aflatoxin B1 and Fusarium graminearum DON toxin can reach 100% and 95.8% or more, respectively.

[0028] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0029] The present invention provides a strain of Bacillus belyssus ATC-AL, which achieves effective biocontrol of black spot disease caused by *Aspergillus longifolius* in sweet potatoes. This strain not only significantly inhibits the synthesis of fungal toxins such as aflatoxin and vomitoxin, but also exhibits broad-spectrum inhibitory activity against a variety of important plant pathogenic fungi such as *Aspergillus flavus*, *Fusarium graminearum*, and *Aspergillus fumigatus*, demonstrating excellent application potential. Its fermentation products can be used as post-harvest treatment agents for agricultural products such as sweet potatoes, corn, and wheat to delay mold growth and toxin contamination, ensuring the safety of stored grains. The present invention is green, environmentally friendly, safe, and efficient, providing a sustainable microbial solution for the prevention and control of agricultural product diseases and the treatment of toxin contamination, and has extremely high research and application value. Attached Figure Description

[0030] Figure 1 The images show the isolation and purification effects of Bacillus belyssus ATC-AL in this invention: (A) Microscopic image of bacterial morphology after Gram staining; (B) Bacillus morphology under a scanning electron microscope; (C) Phylogenetic tree of Bacillus belyssus ATC-AL strain based on 16S rDNA gene sequence; (D) Bacillus belyssus ATC-AL produces volatile antibacterial components on plates to inhibit the growth of *Saccharomyces cerevisiae*; (E) Effects of cellulase activity, protease activity, phosphate solubilization activity, and lysis activity of ATC-AL strain.

[0031] Figure 2 This invention presents a gene cluster for synthesizing antifungal active ingredients in the genome of Bacillus belyssus ATC-AL and analyzes the antifungal activity of its fermentation broth; (A) A diagram showing the completion of the whole genome sequencing of Bacillus belyssus ATC-AL; (B) A diagram showing the inhibitory effect of supernatant fermentation broth of Bacillus belyssus ATC-AL with different proportions on the growth of *Saccharomyces cerevisiae*, where Control is the control group without fermentation broth;

[0032] Figure 3The diagram shows the antifungal activity of Bacillus belyssus ATC-AL in this invention. (A) Minimum inhibitory concentration of the active substance of ATC-AL strain against *Bacillus sacchariformis*; (B) Plate effect of the active substance inhibiting the growth of *Bacillus sacchariformis*; (C) Statistical graph of inhibition rate of *Bacillus sacchariformis* growth; (D) Inhibitory effect of the active substance on conidial development of *Bacillus sacchariformis*; (E) Statistical graph of inhibition rate of conidial germination; (F) Statistical graph of the active substance inhibiting the expression of conidial development regulatory genes abaA and flbA.

[0033] Figure 4 Schematic diagrams illustrating the disruption of cell surface structural integrity by the active substance of Bacillus belysii ATC-AL in *Saccharomyces cerevisiae*; (A) Scanning electron micrographs of *Saccharomyces cerevisiae* hyphae and conidia after treatment with the active substance of ATC-AL strain, where Control represents the control group without the active substance; (B) Heatmap of ergosterol synthesis gene expression in *Saccharomyces cerevisiae* after treatment with the active substance of ATC-AL strain; (C) Schematic diagram of the inhibition of ergosterol synthesis gene expression after treatment with the active substance of ATC-AL strain by quantitative real-time PCR; (D) Effect of cell contents release in *Saccharomyces cerevisiae* after treatment with the active substance of ATC-AL strain; (E) Fluorescence microscopy image of *Saccharomyces cerevisiae* hyphae after staining with propidium iodide (PI).

[0034] Figure 5 The diagram shows the oxidative damage induced by the active substance of Bacillus belye ATC-AL in *Saccharomyces cerevisiae*. (A) A heatmap showing the inhibition of high expression of related antioxidant genes in *Saccharomyces cerevisiae* after treatment with the active substance of ATC-AL strain. (B) A schematic diagram showing the inhibition of the expression of related antioxidant genes by the active substance of ATC-AL strain by quantitative real-time PCR. (C)-(E) Schematic diagrams showing the inhibition of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in *Saccharomyces cerevisiae* after treatment with the active substance of ATC-AL strain. (F) Fluorescence microscopy image of *Saccharomyces cerevisiae* hyphae after staining with 2,7-dichlorofluorescein diacetate (DCFH-DA, reactive oxygen species indicator). (G) Graph showing the malondialdehyde (MDA) content in *Saccharomyces cerevisiae* after treatment with the active substance of ATC-AL strain.

[0035] Figure 6A schematic diagram illustrating the induction of apoptosis in *Bacillus belyssae* ATC-AL active substance by *Bacillus belyssae*; (A) Fluorescence microscopy image of *Bacillus belyssae* hyphae treated with the active substance of ATC-AL strain and stained with JC-1; (B) Fluorescence image of *Bacillus belyssae* hyphae stained with TUNEL and DAPI, where BF is the bright field field of the microscope.

[0036] Figure 7 The diagram shows the broad-spectrum antibacterial effect of Bacillus belyssus ATC-AL and its active substances in this invention; (A) Diagram showing the broad-spectrum antibacterial effect of the ATC-AL strain; (B) Diagram showing the broad-spectrum antibacterial effect of the active substances of the ATC-AL strain.

[0037] Figure 8 The following diagrams illustrate the effects of Bacillus belyssae ATC-AL and its active substances in inhibiting toxin production by Aspergillus flavus and Fusarium graminearum in this invention: (A) Thin-layer chromatography analysis of AFB1 after co-culturing ATC-AL strains with different cell ratios in the same volume with Aspergillus flavus in PDB; (B) Statistical analysis of the relative yield of AFB1 in diagram A; (C) Thin-layer chromatography analysis of AFB1 after adding different concentrations of active substances from ATC-AL strains to the YES toxin-producing medium; (D) Statistical analysis of the relative yield of AFB1 in diagram C; (E) Statistical analysis of the yield of vomitoxin (DON) after adding different concentrations of active substances from ATC-AL strains to the Fusarium graminearum toxin-producing medium (TBI medium).

[0038] Figure 9 The following diagrams illustrate the effects of Bacillus belye ATC-AL on the inhibition of infection by *Aspergillus niger*, *Aspergillus flavus*, and *Fusarium graminearum*, as well as the toxin production after infection: (A) Inhibition effect of different concentrations of ATC-AL active substance treatment on *Aspergillus niger* infection in sweet potatoes; (B) Statistical analysis of the diameter of rotten spots in diagram A; (C) Inhibition effect of different concentrations of ATC-AL active substance treatment on *Aspergillus flavus* infection in corn kernels; (D) Statistical analysis of the number of *Aspergillus flavus* spores on corn seeds in diagram C; (E) Statistical analysis of the residual AFB1 yield on infected corn seeds; (F) Inhibition effect of different concentrations of ATC-AL active substance treatment on *Fusarium graminearum* infection in wheat seeds; (G) Statistical analysis of the DON vomitoxin yield on wheat seeds in diagram F. Detailed Implementation

[0039] The technical solution provided by the present invention will be described in detail below with reference to specific embodiments; unless otherwise specified, the reagents and materials used in the operations in the following embodiments were purchased from commercial sources.

[0040] Example 1

[0041] Isolation, screening, and identification of a strain of Bacillus belye ATC-AL.

[0042] I. Isolation and Screening of Bacillus belye ATC-AL

[0043] The *Bacillus belye* of this invention was isolated and screened from the rhizosphere of sweet potato. The specific implementation process is as follows:

[0044] (1) Isolation of sweet potato rhizosphere microorganisms

[0045] Soil samples were collected from the rhizosphere of sweet potato tubers at a depth of 3-8 cm. 10 mL of sterile water was added per gram of soil sample, and the mixture was ground for 2 minutes. The supernatant was then transferred to a 10 mL centrifuge tube. The supernatant was diluted with sterile water to three concentration gradients: 10⁻¹, 10⁻², and 10⁻³. 100 μL of each diluted solution and undiluted stock solution were spread onto LB agar plates. Each sample was repeated three times, and the samples were incubated at 37°C for 3 days.

[0046] (2) Purification and preservation of strains

[0047] Microorganisms with similar morphology were grouped together. One strain from each group was inoculated onto LB solid medium. Single colonies were picked and streaked onto fresh LB solid medium for subculturing and purification. After three subculturings, the culture was transferred to 1 mL of LB medium, incubated overnight at 37°C with shaking at 200 rpm, mixed with 60% glycerol at a 1:1 volume ratio, and stored in a cryovial at -80°C for long-term preservation.

[0048] (3) Screening of bacteria that antagonize the growth of pathogenic fungi

[0049] The isolated and purified strains were initially screened using the plate confrontation method. A vertical cross was drawn on a PDA plate with the origin as the center, and 2 μL of 10 μL of the plate was added. 7 Fresh pathogenic conidia per mL were aspirated into the center of a PDA plate, and bacterial suspension was streaked around the center using an inoculation loop. Plates inoculated only with the pathogenic fungus served as a blank control, and three replicates were prepared. The plates were incubated in the dark at 28°C. Bacteria exhibiting antibacterial activity were selected for further screening, and a strain named ATC-AL was identified.

[0050] II. Identification of the strain

[0051] The obtained ATC-AL strain was identified by morphological, molecular biological, and physiological and biochemical characteristics. The specific identification steps are as follows:

[0052] (1) Morphological identification of fungal strains

[0053] Bacillus was inoculated into LB liquid medium and cultured overnight at 37°C. The bacterial pellet was collected by centrifugation, washed with sterile PBS, and then stained with Gram stain. The bacterial color was observed under both an optical microscope and a fluorescence microscope. The bacterial cells were resuspended in 2.5% glutaraldehyde solution and incubated overnight at 4°C to fix the cells. After centrifugation, residual glutaraldehyde was washed twice with sterile PBS. The cells were then dehydrated in a gradient manner with different concentrations of alcohol (30%, 50%, 70%, 90%, 100%). After each gradient dehydration for 15 min, the supernatant was discarded by centrifugation before proceeding to the next gradient. The alcohol containing the bacterial cells was then dropped onto the clean, conductive silicon wafer and dried at no higher than 40°C. Gold sputtering was then performed to enhance the conductivity of the cells for 90 s. The cell morphology of the Bacillus was observed using scanning electron microscopy (SEM), and cell length and width were recorded. The results are as follows: Figure 1 As shown, the cells of this bacterium appear blue under a microscope. Figure 1 A), presumed to be a Gram-positive bacterium, characterized by its rod-shaped form ( Figure 1 B) The surface is not smooth and has wrinkles; the cell length is 2-4 μm and the cell width is 0.6-0.8 μm. Morphologically, this bacterium exhibits characteristics of Bacillus.

[0054] (2) Identification of physiological and biochemical characteristics

[0055] The physiological and biochemical characteristics of the ATC-AL strain were detected according to Bergey's bacterial identification method. For example... Figure 1 As shown in E, the ATC-AL strain exhibits good cellulase activity, protease activity, phosphate-solubilizing activity, and lysis activity, and can produce volatile gases with antibacterial activity. Figure 1 D), these characteristics are basically consistent with those of Bacillus.

[0056] (3) Molecular identification of bacterial strains

[0057] The bacterial culture preserved in step (2) of Part 1 of Example 1 was taken out of the -80℃ freezer and streaked onto LB solid medium using an inoculation loop. After incubation at 37℃ for 18 hours in an inverted incubator, it was inoculated into 4 ml of LB liquid medium using an inoculation loop and incubated overnight at 37℃ and 180 rpm on a shaker. The bacterial culture and primers 27F and 1492R were sent to Suzhou Genewiz Biotechnology Co., Ltd. for 16S rRNA amplicon sequencing. Primer sequences: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'. After removing impurities and homologous splicing, BLAST similarity comparison was performed in GenBank, and the phylogenetic tree of the strain is shown below. Figure 1As shown in Figure C, the ATC-AL strain provided by this invention has a 100% similarity to Bacillus velezensis. Combined with other identification methods, this indicates that the bacterium is Bacillus velezensis.

[0058] Example 2

[0059] The genome of Bacillus belyssus ATC-AL contains a cluster of synthetic genes capable of synthesizing a variety of antifungal active substances.

[0060] (1) Whole genome sequencing of the bacterial strain

[0061] 10 μL of *Bacillus belyssima* ATC-AL was inoculated into a glycerol storage tube and cultured overnight in 4 ml LB broth at 37°C with shaking at 200 rpm until the logarithmic growth phase was reached (OD600 approximately 0.6-0.8). 3-5 ml of bacterial cells were collected and centrifuged in a 1.5 ml centrifuge tube at 4°C. The supernatant was discarded, and the bacterial cells at the bottom of the tube were retained. The centrifuge tube was sealed and immediately flash-frozen in liquid nitrogen, stored at -80°C, and sent to the laboratory of Shanghai Ling'en Biotechnology Co., Ltd. on dry ice for third-generation bacterial genome sequencing. The bacterial genome contains 3,910,637 bp, with a GC content of 46.61%, and contains 4036 coding genes, 27 ribosomal RNAs, and 86 transfer RNAs (tRNAs). Figure 2 A).

[0062] (2) Discovery of antifungal active substance gene clusters in the genome of ATC-AL strain and analysis of their antifungal activity

[0063] The anti-fungal secondary metabolite gene clusters in the ATC-AL strain genome were identified using antiSMASH 8.0 (https: / / antismash.secondarymetabolites.org / ). A total of 16 potential bioactive ingredient synthesis gene clusters were identified. These included five encoding non-ribosomal peptide synthases (NRPS), one encoding a cluster containing RRE elements, three encoding transacyl-based polyketide synthases (transAT-PKS), one encoding a RiPP analog (other unspecified ribosomally synthesized and post-translational modified peptide products RiPP), three encoding terpene precursors, one encoding an NRPS analog, two encoding azole-containing RiPPs, one encoding a β-lactam, two encoding terpenes, one encoding an NRP-metal carrier, two encoding type III polyketide synthases (T3PKS), and one encoding another type of polyketide synthase (PKS analog). Sequence similarity analysis showed that gene clusters 1, 3, 6, 7, 8, 12, 14, 15, and 16 had high sequence similarity to gene clusters encoding known surfactants, phytoalexins, macrolide H, bacillus thiouracil, fumonisin, bacillus thiouracil AN, bacillus siderophores, and bacillusin, indicating that the ATC-AL strain genome contains abundant gene clusters for synthesizing antifungal active substances (Table 1 below).

[0064] Table 1 Summary of gene clusters synthesizing antifungal active components in the genome of Bacillus belyssus ATC-AL

[0065]

[0066]

[0067] (3) Determination of antifungal activity of ATC-AL strain fermentation broth

[0068] Fermentation broth of the ATC-AL strain (cultured at 37℃ and 200 rpm for 2 days) was filtered through a 0.22 μM filter membrane and added to preheated and melted PDA medium to achieve final concentrations of 0%, 1%, 5%, 10%, 20%, 30%, and 40% (total volume 10 mL). The control group consisted of an equal volume of sterile LB medium. Fresh spores of *Saccharomyces cerevisiae* were collected and diluted with 0.1% Tween 80 solution to a final spore concentration of 5 × 10⁻⁶. 6 spores / mL, 2 μL of spore suspension was spotted into the center of PDA medium, and cultured at 28℃ for 5 days. Phenotypic observation revealed that the fermentation broth of the ATC-AL strain inhibited the growth of the pathogen. When the fermentation broth concentration reached 40%, the growth of the pathogen was completely inhibited. Figure 2 B).

[0069] Example 3

[0070] Analysis of the antibacterial activity of Bacillus vesicularis ATC-AL against *Saccharomyces cerevisiae*.

[0071] (1) Preparation of fermentation broth of ATC-AL strain

[0072] The activated Bacillus from Example 1 was added to a fermenter containing 40L of LB liquid medium and cultured at 37°C with aeration for 7 days.

[0073] (2) Extraction of active substances

[0074] Add ethyl acetate to the fermentation broth at a volume ratio of 1:1, and extract by aeration and tumbling for 6 hours. Stop the aeration, and after separation, release the fermentation broth waste liquid from the lower end of the separatory funnel. Release the upper layer of ethyl acetate and add it to a rotary evaporator for rotary evaporation. After completion, dissolve the active substance extracted from each 1L of fermentation broth in 1mL of methanol.

[0075] (3) Determination of the minimum inhibitory concentration (MIC) of active substances of ATC-AL strain against *Saccharomyces cerevisiae*

[0076] Fresh spores of *Saccharomyces cerevisiae* were collected and diluted with 0.1% Tween 80 solution to achieve a final spore concentration of 5 × 10⁻⁶. 6 spores / mL. Weigh 0.015g of resporane powder and dissolve it in 10mL of sterile water to achieve a final concentration of 75μg / mL after adding it to the system, ensuring aseptic operation throughout the process. Next, perform a gradient dilution of the active substance. Wells 2-11 are the experimental groups (active substance concentrations of 0, 2, 4, 6, 8, 12, 14, 16, 18, and 20μL / mL, respectively), and well 1 is the blank control (no spore suspension added). Add 170μL LDB medium (190μL LDB medium to well 1), 20μL spore suspension (no spore suspension added to well 1), and 10μL resporane to each well from wells 2-11, with three replicates per group. After completion, seal with sealing film and wrap with aluminum foil, incubate at 28℃ for 3 days, and then photograph and observe. Resporane is an indicator of redox reactions and normally appears blue-purple. If bacterial growth in a sample can promote the reduction of resveratrol and cause a color change, the presence of viable bacteria in the sample can be determined by observing the color change from cyan or reddish-purple to white or colorless. Figure 3 As shown in Figure A, when the concentration of the active substance reaches 8 μL / mL, the color of the resplenium is blue-purple, while the color of the treatment at 0–6 μL / mL is nearly colorless, indicating that 8 μL / mL of the active substance is the minimum inhibitory concentration of *Saccharomyces cerevisiae*.

[0077] (4) Inhibition of mycelial growth of ATC-AL strain active substances on sweet potato long-beaked scab mycelium

[0078] The active substance was added to the melted PDA medium at final concentrations of 0 μL / mL, 2 μL / mL, 4 μL / mL, 6 μL / mL, 8 μL / mL, and 10 μL / mL, and then poured into 12-well plates. After the PDA medium solidified, the spore concentration was obtained by following step (3) in Example 3. 6 A solution of sweet potato long-beaked spores per mL was prepared, and 2 μL of the spore solution was spotted into the center of a PDA medium and incubated in the dark at 28°C for 3 days. Results are as follows: Figure 3 As shown in B and 3C, the growth of *Saccharomyces cerevisiae* was significantly inhibited at an active substance concentration of 8 μL / mL, which is the minimum inhibitory concentration (MIC) of the active substance against *Saccharomyces cerevisiae*. At an active substance concentration of 10 μL / mL, the growth of *Saccharomyces cerevisiae* mycelia was completely inhibited, with an inhibition rate of 100%, which is the minimum bactericidal concentration (MBC) of the active substance against *Saccharomyces cerevisiae*.

[0079] (5) Inhibition of spore germination by active substances of ATC-AL strains of *Saccharomyces cerevisiae*

[0080] The active substance was added to heated and melted water agar plates at final concentrations of 0 μL / mL, 2 μL / mL, 4 μL / mL, and 8 μL / mL. After the water agar plates solidified, the spore concentration was obtained as 5 × 10⁻⁶ spores, following step (3) in Example 3. 6 A solution of Aspergillus flavus spores per mL was prepared, and 2 μL of the spore solution was spotted onto water agar plates with different concentrations of active ingredients. The plates were incubated in the dark at 28°C for 6 h, 12 h, and 24 h to observe the spore germination rate of *Aspergillus flavus* spores at different concentrations of active ingredients.

[0081] The formula for calculating spore germination rate is as follows:

[0082] Spore germination rate (%) = Number of germinated spores / Total number of spores × 100;

[0083] The results are as follows Figure 3 As shown in D and 3E, when the concentration of the active substance was 8 μL / mL, it completely inhibited the spore germination of *Saccharomyces cerevisiae*, with an inhibition rate exceeding 95%. Quantitative real-time PCR analysis of the effect of the active substance on the expression of genes regulating sporulation development revealed that treatment with 8 μL / mL of the active substance significantly reduced the expression levels of two sporulation regulatory genes, abaA and flbA. Figure 3 F) indicates that the active substances in the ATC-AL strain can inhibit spore development by suppressing the expression of sporulation-regulating genes.

[0084] Example 4

[0085] Bacillus belysinia ATC-AL disrupts the cell surface structure of *Pseudomonas stolonifera* and induces apoptosis.

[0086] I. Effects of Bacillus belye ATC-AL on the cell surface structural integrity of *Pseudomonas aeruginosa*

[0087] (1) Effects of active substances from ATC-AL strain on the integrity of cell surface structure of *Pseudomonas aeruginosa*

[0088] Take 100 μL of freshly collected spore solution (containing 5 × 10⁻⁶ spores). 6 (Spores) were inoculated in 900 μL of PDB medium and cultured at 28 °C and 200 rpm for 12 hours. The cultured spores and mycelia were collected by centrifugation and fixed by soaking in 2.5% glutaraldehyde solution for two hours; excess glutaraldehyde was removed by centrifugation for five minutes, and then the samples were dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol, each time for 20 minutes; then centrifuged at 4 °C and 12000 rpm for 5 minutes, and the supernatant was discarded. After freeze-drying the samples for 12 hours, they were sputter-coated with gold, and the fungal structure was imaged using a scanning electron microscope. The results are as follows: Figure 4 As shown in Figure A, the untreated control group showed that the mycelia and conidia had uniform thickness, smoothness, and fullness. However, the treatment with active substances by the ATC-AL strain caused the mycelia and spores of *Saccharomyces cerevisiae* to dehydrate, become sunken, and suffer from increased surface damage.

[0089] (2) Effects of bioactive substances of ATC-AL strain on ergosterol synthesis in cell membrane component of *Polygonum longiformis*

[0090] Take freshly collected spore solution and place it in 20 mL of PDB (containing 2×10⁶ spores). 6 In a culture medium containing 1 spore, the culture was carried out at 28℃ and 200 rpm for 24 hours. The active substance was added and not added, with a final concentration of 8 μL / mL, and the culture was continued at 28℃ and 200 rpm for another 12 hours. Mycelia were collected, and total RNA was extracted and sent to Shanghai Ling'en Biotechnology Co., Ltd. on dry ice for transcriptome sequencing. Transcriptome analysis revealed that after treatment with the active substance, the expression levels of genes related to ergosterol synthesis in *A. sweet potato*, including erg2, erg4, erg25, erg26, and erg28, were significantly downregulated in *A. sweet potato*. Figure 4 B); Further verification by quantitative real-time PCR revealed that the expression levels of the above five ergosterol synthesis-related genes were significantly reduced after treatment with the active substance in the ATC-AL strain. Figure 4 C) indicates that the active substances of the ATC-AL strain inhibited the synthesis of ergosterol, a cell membrane component of *Phytophthora stolonifera*.

[0091] (3) Effects of bioactive substances of ATC-AL strain on the release of cell contents of *Saccharomyces cerevisiae*

[0092] Take 2 mL of freshly collected spore solution (containing 10... 6 Cells (1 spore) were placed in 20 mL of PDB and cultured at 28 °C and 120 rpm for 48 hours. Hyphae were collected by centrifugation, and cells were washed twice with PBS. The cells were then resuspended in PBS containing different concentrations (0, 4 μL / mL, and 8 μL / mL) of the active ingredient. Methanol was used as a negative control. The treated and control cultures were again cultured with shaking at 28 °C, and samples were taken at 0, 1, 2, 4, and 12 hours. After incubation, the cells were centrifuged at 8000 rpm for 5 minutes at 28 °C, and the absorbance of the supernatant at 280 nm was measured using a microplate reader and compared with a blank sample containing no fungi. Results are as follows: Figure 4 After treatment with active substances, the absorbance of the D,ATC-AL strain increased significantly, indicating leakage of cell contents from *Saccharomyces cerevisiae*.

[0093] (4) Effect of fluorescence microscopy on the effect of active substances of ATC-AL strain on cell membrane structure of *Phytophthora stolonifera*

[0094] Freshly collected spore solution was placed in 20 mL of PDB medium and cultured at 28°C and 200 rpm for 24 hours. Different final concentrations of active ingredient (0, 2, 4, and 8 μL / mL) were added, and the culture was continued at 28°C and 200 rpm for another 12 hours. Hyphae were collected by centrifugation, washed three times with PBS buffer, and stained with 5 mM propidium iodide (PI) at 37°C for 20 min in the dark. The stained hyphae were then washed at least three times with PBS buffer. Finally, the cells were suspended in PBS and examined under a fluorescence microscope. Results are as follows: Figure 4 As shown in Figure E, the mycelia treated with the active substance at all concentrations of ATC-AL strain exhibited strong red fluorescence signals, which increased with increasing fermentation broth concentration. The mycelia treated with 8 μL / mL of active substance showed the strongest red fluorescence intensity, while the mycelia in the control group showed weaker red fluorescence intensity. These results indicate that the treatment of the ATC-AL strain with the active substance severely disrupted the cell membrane structure of *Saccharomyces cerevisiae*, leading to the entry of PI dye into the cell.

[0095] II. Effects of bioactive substances from ATC-AL strains on the oxidative response of *Pseudomonas aeruginosa*

[0096] (1) Effects of ATC-AL active substances on the expression of related antioxidant genes in *Polygonum longiformis*

[0097] Analysis of the transcriptome results in step (2) of Part 1 of Example 4 revealed that treatment with the active substance in the ATC-AL strain significantly downregulated the expression levels of genes related to ROS scavenging in *Polygonum longiflorum*, including prx1, aox1, pox9, glrX, cat2, ccp1, sodB, sod1, sod2, and ccs1. Figure 5 A). Further validation by quantitative real-time PCR revealed that the expression levels of the four ROS scavenging-related genes aox1, pox9, ccp1, and sod2 were significantly reduced after treatment with the active substance in the ATC-AL strain. Figure 5 B).

[0098] (2) Effects of ATC-AL active substances on the activity of related antioxidant enzymes in *Polygonum longiformis*

[0099] After treatment with ATC-AL active substance, the enzyme activities of antioxidant-related enzymes in *Polygonum longifolium*, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were detected. The initial spore preparation, mycelial culture, and treatment were performed according to step (2) in Part 1 of Example 4. The results showed that treatment with 8 μL / mL of ATC-AL active substance significantly reduced the enzyme activities of SOD, POD, and CAT. Figure 5 The results (C~5E) indicate that the active substance ATC-AL can inhibit the enzyme activity of related antioxidant enzymes in *Polygonum longifolium*.

[0100] (3) Effect of active substances of ATC-AL strain on reactive oxygen species (ROS) content in the mycelium of *Saccharomyces cerevisiae*

[0101] The initial spore preparation, mycelial culture, and treatment were performed according to step (4) in Part 1 of Example 4. The cleaned mycelia were stained with 10 mM 2,7-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 30 min in the dark. After thorough washing with PBS, fluorescence microscopy revealed that mycelia treated with the active substance at various concentrations of ATC-AL strain all exhibited strong green fluorescence signals, which increased with increasing concentration of the active substance. The mycelia treated with 8 μL / mL of the active substance showed the strongest red fluorescence intensity. Figure 5 F) indicates that treatment with the active substance of the ATC-AL strain led to the accumulation of excessive ROS in fungal cells.

[0102] (4) Effect of active substances of ATC-AL strain on malondialdehyde (MDA) content in the mycelium of *Polygonum longifolium*

[0103] The initial spore preparation and mycelial culture were performed according to step (2) in Part 1 of Example 4. Mycelia were collected and ground with liquid nitrogen. 0.2g of the ground mycelia were used to detect the MDA content in the cells using the thiobarbituric acid method. The results showed that after treatment with the active substance from the ATC-AL strain, the MDA content in *Saccharomyces cerevisiae* significantly increased, with the highest MDA content observed in the 8μL / mL active substance treatment group. Figure 5 G). MDA can disrupt membrane lipid structure, and the higher its content, the more severe the damage to the cell membrane. Therefore, the active substances of ATC-AL strain can damage the cell membrane structure of *Phytophthora stolonifera*.

[0104] III. Application of ATC-AL strain active substances in inducing apoptosis in *Phytophthora stolonifera* cells

[0105] (1) Effects of active substances from ATC-AL strain on mitochondrial membrane potential of *Pycnodon dactylon* cells

[0106] The initial spore preparation, mycelial culture, and treatment were performed according to step (4) in Part 1 of Example 4. The cleaned mycelia were incubated at 37°C for 30 minutes with a JC-1 probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbonyl cyanine iodide). After thorough washing with PBS, fluorescence microscopy revealed that the intensity of red fluorescence gradually decreased with increasing concentration of the active substance, while the intensity of green fluorescence increased in the opposite direction. Figure 6 A) In normal cells, the membrane potential is normal. JC-1 crosses the mitochondrial membrane polarity to enter the mitochondria and aggregates into a red fluorescent polymer. During apoptosis, the mitochondrial transmembrane potential depolarizes, and JC-1 is released from the mitochondria as a green fluorescent monomer. These results indicate that the active substance of the ATC-AL strain leads to a decrease in the mitochondrial membrane potential and mitochondrial damage in *Phytophthora spp.* cells.

[0107] (2) Effects of active substances from ATC-AL strains on apoptosis of *Pseudomonas sacchariformis* cells

[0108] Mitochondrial transmembrane potential depolarization is one of the important indicators of apoptosis. TUNEL and DAPI fluorescent probes were further used to assess cell apoptosis after treatment with the active substance. The initial spore preparation, hyphal culture, and treatment followed the steps in Part 1 (4) of Example 4. The hyphae were stained with TdT enzyme (terminal deoxynucleotidyl transferase) and TUNEL (transferase-mediated dUTP nick-end labeling assay) reaction buffer at 37°C in the dark for 1.5 hours. After thorough washing with PBS, they were then incubated with DAPI (4',6-diamidindole-2-phenylindole) staining solution at room temperature in the dark for 30 minutes. Finally, fluorescence microscopy revealed that, compared with the control group, the intensity of red fluorescence gradually increased with increasing concentration of the active substance after treatment, while the nuclear fluorescence of DAPI-stained cells gradually decreased, indicating DNA fragmentation. Figure 6 B) This is a marker of apoptosis.

[0109] Example 5

[0110] Broad-spectrum antibacterial activity of Bacillus belysin ATC-AL

[0111] I. Broad-spectrum antifungal effect of ATC-AL strain

[0112] (1) Preparation of ATC-AL bacterial suspension and fungal spore suspension

[0113] Following the experimental steps (1) and (3) in Example 3, Bacillus belysin ATC-AL bacterial suspension and spore suspension with a final concentration of 5×10⁻⁶ were obtained respectively. 6 Spores / mL of Fusarium graminearum, Aspergillus flavus, Fusarium solani, Phytophthora spores, Aspergillus terreus, Aspergillus fumigatus, Aspergillus niger, Aspergillus niger, and Candida albicans.

[0114] (2) Confrontation experiment between ATC-AL strain and different pathogenic fungi

[0115] Using a pipette tip, draw a line on each of the following PDA plates: top, bottom, left, and right. Inoculate the center of the solidified PDA plate with 2 μL of spores from each of the following fungi: *Fusarium graminearum*, *Aspergillus flavus*, *Fusarium solani*, *Phytophthora spores*, *Aspergillus terreus*, *Aspergillus fumigatus*, *Aspergillus nidus*, *Aspergillus niger*, and *Candida albicans*. Use an unstreaked plate as a blank control. Incubate at 37°C for 3 days, measure the colony diameter, and calculate the inhibition rate. Figure 7As shown in Figure A, the ATC-AL strain exhibits broad-spectrum inhibitory activity against a variety of pathogenic fungi. The inhibition rates against *Fusarium graminearum* were 70.98%, *Echinochloa chinensis* 84.35%, *Aspergillus flavus* 61.43%, *Fusarium solani* 68.71%, *Phytophthora indicum* 74.70%, *Aspergillus terreus* 62.94%, *Aspergillus fumigatus* 55.00%, *Aspergillus nidus* 75.76%, *Aspergillus niger* 70.05%, and *Candida albicans* 50.00%. Therefore, the ATC-AL strain demonstrates excellent broad-spectrum antifungal effects.

[0116] II. Broad-spectrum antifungal effects of active substances in ATC-AL strains

[0117] The extraction of active substances from ATC-AL strains and the collection of pathogenic fungal spores were performed according to experimental steps (2) and (3) in Case Study 3, respectively. Active substances were added to melted PDA medium at final concentrations of 0 μL / mL, 8 μL / mL, 16 μL / mL, and 32 μL / mL, and poured into square grid plates. After the PDA medium solidified, 2 μL of spores from *Fusarium graminearum*, *Fusarium solani*, *Aspergillus flavus*, *Phytophthora infestans*, *Aspergillus fumigatus*, and *Candida albicans* were spotted into the center of the PDA medium and incubated in the dark at 28°C for 2 days, with three replicates per group. Results are as follows: Figure 7 As shown in Figure B, at an active substance concentration of 32 μL / mL, the inhibition rate against *Fusarium solani*, *Aspergillus flavus*, and *Aspergillus fumigatus* was 100%, and the inhibition rate against *Fusarium graminearum* was 72.85%. The growth of *Phytophthora infestans* and *Candida albicans* was also significantly inhibited, with relative inhibition rates of 29.41% and 20.34% for bacteria with diameters less than 0.5 cm. Therefore, the active substance of the ATC-AL strain also exhibits good broad-spectrum antibacterial effects.

[0118] Example 6

[0119] Application of Bacillus belye ATC-AL in inhibiting the synthesis of aflatoxin and vomitoxin

[0120] I. The inhibitory effect of co-culturing Bacillus belycetamol ATC-AL with Aspergillus flavus on Aspergillus flavus toxin production.

[0121] (1) Co-culture of ATC-AL strain with Aspergillus flavus

[0122] Following experimental steps (1) and (3) in Example 3, the final concentrations of Bacillus belysin ATC-AL bacterial suspension and Aspergillus flavus spore suspension were obtained to be 1×10⁻⁶. 7spores / mL. Aspergillus flavus and ATC-AL strain cells were mixed at ratios of 1:0.2, 1:1, and 1:5, respectively. The volume of both Aspergillus flavus and Bacillus was 500 μL, and the mixture was added to 9 mL of PDB medium. The control group was prepared by adding 500 μL of Aspergillus flavus spore solution, 500 μL of LB medium, and 9 mL of PDA medium. The mixture was incubated at 28 °C and 200 rpm for 5 days.

[0123] (2) Extraction and analysis of aflatoxin B1

[0124] After 5 days of cultivation, 700 μL of the co-culture fermentation broth was collected and aflatoxin was extracted with 700 μL of dichloromethane, then evaporated to dryness overnight. The toxin sample was reconstituted with 15 μL of dichloromethane, and the sample was transferred to a silica gel plate. The toxin was separated and detected using a developing solvent (dichloromethane:acetone = 9:1). The results are as follows: Figure 8 As shown in A and B, the aflatoxin production gradually decreased with the increase of ATC-AL strain. The inhibition efficiencies of ATC-AL strain against aflatoxin B1 were 69.89%, 79.36%, and 100%, respectively.

[0125] II. Bacillus belysin ATC-AL active substance inhibits aflatoxin production by Aspergillus flavus.

[0126] (1) Inhibitory effect of ATC-AL active substances on aflatoxin synthesis

[0127] The active substance was added to YES liquid culture medium at final concentrations of 0 μL / mL, 4 μL / mL, and 8 μL / mL, respectively. 100 μL of freshly collected 1×10⁻⁶ ppm solution was then added to each medium. 7 Aspergillus spores / mL were incubated in a 28℃ incubator in the dark for 5 days.

[0128] (2) Extraction and analysis of aflatoxin B1

[0129] The extraction and analysis of aflatoxin B1 were performed according to step (2) in Part 1 of Example 6. The results are as follows: Figure 8 As shown in C and 8D, when the concentration of the active substance is 4 μL / mL, the inhibition rate of the active substance against aflatoxin B1 can reach 100%.

[0130] III. The active substance of Bacillus belysin ATC-AL inhibits toxin production by Fusarium graminearum.

[0131] (1) Collection of fresh spores of Fusarium graminearum

[0132] For the collection of fresh Fusarium graminearum spores, refer to the experimental steps in Case Study 3 (3) and dilute the spore solution to 10. 7 per mL.

[0133] (2) Inhibitory effect of ATC-AL active substances on toxin production by Fusarium graminearum

[0134] The active substance was added to 30 mL of TBI toxin-producing liquid culture medium at final concentrations of 0, 4, and 8 μL / mL, respectively. Then, 100 μL of freshly collected 1×10⁻⁶ tungsten-containing medium was added. 7 Fusarium graminearum spores / mL were cultured at 28°C and 200 rpm in the dark for 7 days.

[0135] (3) Extraction and analysis of vomitoxin

[0136] The vomitoxin production of Fusarium graminearum at different concentrations of active substances was measured using an enzyme-linked immunosorbent assay (ELISA) kit from Shenzhen Fende Biotechnology Co., Ltd. The results are as follows: Figure 8 As shown in E, the inhibitory efficiencies of ATC-AL active substances against DON toxin were 38.33%, 83.87%, and 85.76%, respectively.

[0137] Example 7

[0138] Control effects of Bacillus vesiculosus ATC-AL on sweet potato black spot, maize aflatoxin and wheat scab.

[0139] I. Control efficacy of ATC-AL strain against sweet potato black rot

[0140] Select healthy sweet potatoes of similar size, wash them with tap water, soak them in a 1% sodium hypochlorite solution for 10 minutes, rinse them twice with sterile water, wash them with 70% ethanol for 2 minutes, and then rinse them twice with sterile water. Use a sterile dissecting needle to make four wounds approximately 5 mm deep on the waist of the sweet potato. After drying, add 20 μL of a 1×10⁻⁶ sodium hypochlorite solution to the wounds. 6 Sweet potato spore suspension at concentrations of spores / mL was prepared, and active ingredients were added to final concentrations of 0, 2, 4, and 8 μL / mL, respectively. After drying, the suspensions were placed in sterile containers. After incubation at 28°C for 20 days, the disease incidence in sweet potatoes was assessed. Figure 9 As shown in Figures A and 9B, the control group sweet potatoes exhibited large areas of black spots and depressions on their surface, with obvious black spots visible in cross-sections. The active substance treatment groups significantly reduced the occurrence and damage of black spot disease, with the active substances showing inhibition efficiencies of 44.65%, 62.89%, and 68.87% on the lesions, respectively.

[0141] II. Inhibitory effect of ATC-AL strain on Aspergillus flavus infection in maize

[0142] (1) Experiment on Aspergillus flavus infection of corn kernels

[0143] Peel the corn kernels, keeping them intact, and make a small incision at the base of the seed. The sterilization process for the corn kernels follows the method described in Part 1 of Example 7. Remove the corn seeds, blot dry with sterile filter paper, and place them in a 50ml sterile Erlenmeyer flask. Add 20ml of an active substance solution with final concentrations of 0, 4, and 8 μL / mL. The amount of methanol should be kept constant in all three groups to ensure the experiment only involves the concentration of the active substance. Place the three Erlenmeyer flasks in a shaker at 28°C, 200 rpm, for 60 minutes to allow the active substance to fully coat the corn seeds. Place two layers of sterile filter paper in each 60mm sterile petri dish and add 2ml of sterile water or the corresponding concentration of active substance to maintain a humid environment. Place the corn seeds coated with the active substance flat on the filter paper in the petri dish, and inoculate each seed with 2μL of a solution containing 10... 7 Fresh Aspergillus flavus spores (spores / mL) were incubated at 28°C in the dark for 5 days. Results are as follows: Figure 9 As shown in Figure C, when the concentration of the active substance was 4 μL / mL, the active substance significantly inhibited the infection of maize seeds by Aspergillus flavus.

[0144] (2) Collection of Aspergillus flavus spores from corn kernels

[0145] Six corn seeds from each petri dish were transferred to 50 ml centrifuge tubes, and 10 ml of 0.1% Tween 80 was added. The tubes were vortexed to elute the spores. 15 μL of the spore suspension was pipetted onto a hemocytometer and the spore count was performed using an optical microscope to calculate the spore concentration. Results are as follows: Figure 9 As shown in D, when the concentration of the active substance was 4 μL / mL, the active substance significantly inhibited the sporulation ability of Aspergillus flavus after infecting maize seeds, with an inhibition rate of 83.72%; when the concentration of the active substance was 8 μL / mL, its inhibition rate on sporulation of Aspergillus flavus was 90.70%.

[0146] (3) Extraction of aflatoxin B1 from corn kernels

[0147] Add 10 mL of dichloromethane to the mixture obtained in step (2), shake to mix, and after complete separation, transfer the lower organic phase to a new centrifuge tube. The extraction and detection of aflatoxin B1 are then performed in the same manner as step (2) in Part 1 of Example 6. The results are as follows: Figure 9 As shown in E, treatment with active substances at concentrations of 4 μL / mL and 8 μL / mL both resulted in a 100% inhibition rate against aflatoxin production.

[0148] III. Control effect of ATC-AL strain on wheat seeds infected with Fusarium graminearum

[0149] (1) Experiment on Fusarium graminearum infecting wheat seeds

[0150] Healthy wheat seeds of similar size were selected. The disinfection process for the wheat seeds followed the method described in Part 1 of Example 7. The disinfected wheat seeds were removed and blotted dry with sterile filter paper. The seeds were then placed into three 50ml sterile Erlenmeyer flasks, and 20ml of active substance solutions with final concentrations of 0, 4, and 8 μL / mL were added. The amount of methanol in all three groups was kept constant to ensure that the only variable in the experiment was the concentration of the active substance. The three Erlenmeyer flasks were placed on a shaker at 28°C and 200 rpm for 60 minutes to allow the active substance to fully coat the wheat seeds. Two layers of sterile filter paper were placed in each 60mm sterile petri dish, and 2ml of the corresponding concentration of active substance was added to maintain a humid environment. 10g of wheat seeds coated with the active substance were placed flat on the filter paper in the petri dish, and 200μL of a 10% concentration solution was prepared. 7 Spray a solution of *Fusarium graminearum* spores at a concentration of 1 / mL onto the surface of wheat, and mix thoroughly by shaking. Incubate at 28°C in the dark for 20 days. Results are as follows: Figure 9 As shown in F, when the concentration of the active substance was 16 μL / mL, the active substance significantly inhibited the infection of wheat seeds by Fusarium graminearum.

[0151] (2) Extraction of vomitoxin from wheat seeds

[0152] Weigh 2g of wheat seed sample from step (1) into a 50ml centrifuge tube, add 20ml of deionized water, shake for 5min, centrifuge at 4000r / min for 10min at room temperature, take the supernatant, dilute it 20 times, add 0.5ml of the diluted supernatant and 0.5ml of reconstitution solution to a new centrifuge tube, mix well, take 50μL of the liquid and use the vomitoxin kit from Shenzhen Find Biotechnology Co., Ltd. to measure the toxin production of Fusarium graminearum at different active substance concentrations. The results are as follows: Figure 9 As shown in G, the inhibition rates of the active substances on vomitoxin produced by Fusarium graminearum infection of wheat seeds were 44.41%, 95.23%, and 95.80%, respectively.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis ATC-AL, characterized in that, The Bacillus belyes ATC-AL was deposited at the China Center for Type Culture Collection on June 6, 2025, with accession number CCTCC: M20251280, at Wuhan University, Wuhan, China.

2. A microbial preparation, characterized in that, The active substance of Bacillus vesiculus ATC-AL as described in claim 1, Bacillus vesiculus ATC-AL, and Bacillus vesiculus ATC-AL fermentation broth are included in one or more of these. The preparation method of the active substance of Bacillus vesiculus ATC-AL is as follows: the fermentation broth of Bacillus vesiculus ATC-AL is extracted with an equal volume of ethyl acetate, and the active substance of Bacillus vesiculus ATC-AL is obtained by rotary evaporation.

3. The microbial preparation according to claim 2, characterized in that, The preparation method of the Bacillus belyssus ATC-AL fermentation broth is as follows: Bacillus belyssus ATC-AL strain is inoculated into LB liquid medium and cultured at 37°C with shaking for 7 days to obtain ATC-AL fermentation broth.

4. The microbial preparation according to claim 2, characterized in that, The formulation is in one of the following forms: liquid, spray powder, dry wettable powder, or dry wettable granules.

5. The application of the Bacillus belyssus ATC-AL of claim 1 or the microbial preparation of claim 2 in the broad-spectrum inhibition of plant / animal pathogenic fungi, wherein the pathogenic fungus is Fusarium graminearum (…). Fusarium graminearum ), sweet potato long-beaked shell fungus ( Ceratocystis fimbriata Aspergillus flavus Aspergillus flavus Fusarium solani () Fusarium solani ), Phytophthora ( Phytophthora cactorum Aspergillus terreus ( ) Aspergillus terreus Aspergillus fumigatus ( ) Aspergillus fumigatus Aspergillus nidus ( ) Aspergillus nidulans Aspergillus niger (), Aspergillus niger Candida albicans ( Candida albicans One or more of the following.

6. The application of Bacillus belyssus ATC-AL as described in claim 1 or the microbial preparation as described in claim 2 in the prevention and control of postharvest black spot disease in sweet potatoes.

7. The use of Bacillus belyssus ATC-AL as described in claim 1 or the microbial preparation as described in claim 2 in the control of aflatoxin and / or aflatoxin contamination in corn.

8. The use of Bacillus belye ATC-AL as described in claim 1 or the microbial preparation as described in claim 2 in the control of Fusarium graminearum and / or vomitoxin contamination in wheat.

9. The application according to any one of claims 6-8, wherein the specific application process is as follows: the Bacillus vesiculosus ATC-AL fermentation broth or the cell-free supernatant of the Bacillus vesiculosus ATC-AL fermentation broth is used to protect sweet potatoes, corn, and wheat through immersion treatment, spray treatment, or coating treatment.

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