Bacillus velezensis ATC-AL and application thereof

Abstract

The invention discloses a bacillus velezensis ATC-AL and application thereof. The preservation number of the strain is CCTCC (China Center For Type Culture Collection): M20251280. A genome of the bacillus velezensis ATC-AL has a synthetic gene cluster for synthesizing various antifungal active substances, the active substances of the bacillus velezensis ATC-AL can destroy cell surface structures of pathogenic bacteria, induce cell apoptosis and inhibit spore development of pathogenic fungi, and the bacillus velezensis ATC-AL has a broad-spectrum inhibition effect on various pathogenic fungi such as sweet potato coracocystis canaliculata, fusarium graminearum and aspergillus flavus; the generation inhibition rate of vomitoxin reaches 95%, and the generation inhibition rate of aflatoxin B1 can reach 100%; fungus infection experiments on sweet potatoes, corn and wheat prove that active substances in a strain cell fermentation broth can completely inhibit the decay effect of sweet potato coracocystis longirostris, fusarium graminearum and aspergillus flavus on the food crops; according to the invention, a microbial control resource library for biological control of pathogenic fungi and toxin pollution thereof is enriched.

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] The antagonistic activity analysis of the ATC-AL strain or the cell-free supernatant fermentation liquor thereof on various major pathogenic fungi shows that the inhibition rates of the ATC-AL strain on G. oryzae, F. solani, A. flavus and A. fumigatus are 100%; the inhibition rates of the ATC-AL strain on A. niger, F. graminearum, P. infestans, A. niger, A. terreus and C. albicans are 75.76%, 70.98%, 74.70%, 70.05%, 62.94% and 50.00%, respectively.

[0020] To achieve the second application purpose, the technical solution adopted by the present application is:

[0021] The application of the above-mentioned Bacillus velezensis ATC-AL or the above-mentioned microbial preparation in the prevention and treatment of black spot disease of sweet potato. The fermentation liquor of the Bacillus velezensis ATC-AL screened by the present application has a significant inhibitory effect on the mycelial growth and spore germination of G. oryzae. It is found through scanning electron microscopy observation that the pathogenic fungal mycelium is obviously shrunk, swollen and deformed, showing typical stress injury characteristics. The fermentation product destroys the integrity of the cell membrane of the pathogenic fungus, induces the accumulation of intracellular reactive oxygen species ROS, the decrease of mitochondrial membrane potential and cell apoptosis, and finally leads to the death of the fungus.

[0022] To achieve the third application purpose, the technical solution adopted by the present application is:

[0023] The application of the above-mentioned Bacillus velezensis ATC-AL or the above-mentioned microbial preparation in the prevention and control of A. flavus and / or aflatoxin pollution in corn. The present application further verifies that the ATC-AL strain has a significant inhibitory effect on the toxin production capacity of A. flavus: the specific verification process is that the ATC-AL strain inhibits the toxin production of A. flavus by adding A. flavus and Bacillus for co-culture or gradient Bacillus active substances in PDB liquid medium and YES liquid medium and culturing at 28℃ for 4d, extracting 700μL dichloromethane from 700μL fermentation liquor, and detecting the content of aflatoxin by thin layer chromatography, and the inhibition rate is 100%.

[0024] To achieve the fourth application purpose, the technical solution adopted by the present application is:

[0025] The application of the above-mentioned Bacillus velezensis ATC-AL or the above-mentioned microbial preparation in the prevention and control of F. graminearum and / or vomitoxin pollution in wheat. The present application further verifies that the ATC-AL strain has a significant inhibitory effect on the toxin production capacity of F. graminearum: the specific verification process is that the ATC-AL strain inhibits the toxin production of F. graminearum by adding different gradient concentrations of Bacillus active substances in TBI liquid medium and culturing at 28℃ for 7d, and detecting the content of vomitoxin by the vomitoxin kit of Shenzhen Fender Biotechnology Co., Ltd., and the inhibition rate is 85.76%.

[0026] Further, in order to effectively control the pathogenic bacteria and toxin pollution of food such as sweet potato, corn and wheat during the storage period, the application further develops a green treatment method based on the ATC-AL fermentation liquor: the ATC-AL fermentation liquor or the cell-free supernatant of the ATC-AL fermentation liquor is used for the protection of sweet potato, corn and wheat through immersion treatment, spraying treatment or coating treatment.

[0027] Experiments prove that the ATC-AL strain and its fermentation product can significantly reduce the occurrence of black spot disease of sweet potato during storage, effectively prevent and control the occurrence of corn aspergillus disease and wheat scab, and the inhibition rates of the ATC-AL strain and its fermentation product on aflatoxin B1 and DON toxin of fusarium graminearum are more than 100% and 95.8%, respectively.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] The ATC-AL strain of the application realizes the effective biological control of black spot disease caused by diaporthe phaseolorum, and the strain can not only significantly inhibit the synthesis of mycotoxins such as aflatoxin and vomitoxin, but also has broad-spectrum inhibitory activity on many important plant pathogenic fungi such as aspergillus flavus, fusarium graminearum and aspergillus fumigatus, and shows excellent application potential; the fermentation product of the strain can be used as a postharvest treatment agent for agricultural products such as sweet potato, corn and wheat, so as to delay mold and toxin pollution and ensure the safety of stored grain; the application is green, safe and efficient, and provides a sustainable microbial solution for the prevention and control of agricultural product diseases and the treatment of toxin pollution, and has high research and application value. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Fig. 1 is a purification effect diagram of the ATC-AL strain of the application, (A) is a microscope photograph of the bacterial morphology after gram staining; (B) is a scanning electron microscope photograph of the bacterial morphology; (C) is a phylogenetic tree of the ATC-AL strain of the bacillus based on the 16S rDNA gene sequence; (D) is a diagram showing that the ATC-AL strain of the bacillus produces volatile antibacterial components to inhibit the growth of diaporthe phaseolorum; (E) is a diagram showing the cellulase activity, protease activity, phosphorus solubility and solubility of the ATC-AL strain;

[0031] Figure 2 Fig. 2 is a diagram showing the synthesis of the antifungal active ingredient gene cluster in the genome of the ATC-AL strain of the bacillus and the antifungal activity analysis of the fermentation liquor, (A) is a diagram showing the completion of the whole genome sequencing of the ATC-AL strain of the bacillus; (B) is a diagram showing the growth inhibition effect of the supernatant fermentation liquor of the ATC-AL strain of the bacillus with different proportions on diaporthe phaseolorum, wherein Control is a control group without fermentation liquor;

[0032] Figure 3Figure (A) is the minimum inhibitory concentration of the active substance of the Bacillus velezensis ATC-AL strain on the C. batatium; (B) is the plate effect diagram of the active substance inhibiting the growth of C. batatium; (C) is the statistical diagram of the inhibition rate of inhibiting the growth of C. batatium; (D) is the inhibition effect diagram of the active substance on the conidial development of C. batatium; (E) is the statistical diagram of the inhibition rate of conidial germination; (F) is the statistical diagram of the active substance inhibiting the expression of abaA and flbA genes regulating conidial development;

[0033] Figure 4 Figure (A) is the scanning electron microscope structure diagram of the hyphae and conidia of C. batatium treated by the active substance of the Bacillus velezensis ATC-AL strain, wherein Control is the control group without active substance; (B) is the expression heat map of the ergosterol synthesis gene in the cell membrane component of C. batatium treated by the active substance of the Bacillus velezensis ATC-AL strain; (C) is the schematic diagram of the expression amount of the ergosterol synthesis gene inhibited by the active substance of the Bacillus velezensis ATC-AL strain detected by fluorescence quantitative PCR; (D) is the effect diagram of the release of cell contents of C. batatium treated by the active substance of the Bacillus velezensis ATC-AL strain; (E) is the fluorescence diagram of the hyphae of C. batatium dyed by propidium iodide (PI) detected by fluorescence microscope;

[0034] Figure 5 Figure (A) is the heat map of the high expression of the related antioxidant genes in C. batatium inhibited by the active substance of the Bacillus velezensis ATC-AL strain; (B) is the schematic diagram of the expression amount of the related antioxidant genes inhibited by the active substance of the Bacillus velezensis ATC-AL strain detected by fluorescence quantitative PCR; (C)-(E) are respectively the schematic diagrams of the superoxide dismutase (SOD) enzyme activity, peroxidase (POD) enzyme activity and catalase (CAT) enzyme activity in C. batatium inhibited by the active substance of the Bacillus velezensis ATC-AL strain; (F) is the fluorescence diagram of the hyphae of C. batatium dyed by 2,7-dichlorofluorescein diacetate (DCFH-DA, active oxygen indicator) detected by fluorescence microscope; (G) is the result diagram of the malondialdehyde (MDA) content in C. batatium treated by the active substance of the Bacillus velezensis ATC-AL strain;

[0035] Figure 6Figure for the related schematic diagram of inducing cell apoptosis of C. falcatum by Bacillus velezensis ATC-AL active substance; (A) fluorescence microscope detection of C. falcatum mycelium treated by ATC-AL strain active substance, and fluorescence diagram generated after JC-1 staining; (B) fluorescence diagram of C. falcatum mycelium after TUNEL and DAPI staining, wherein BF is the field of view of the microscope bright field;

[0036] Figure 7 Figure for the broad-spectrum bacteriostatic effect of Bacillus velezensis ATC-AL and its active substance in the application; (A) figure for the broad-spectrum bacteriostatic effect of ATC-AL strain; (B) figure for the broad-spectrum bacteriostatic effect of ATC-AL strain active substance;

[0037] Figure 8 Figure for the effect of Bacillus velezensis ATC-AL and its active substance on inhibiting toxin production of A. flavus and F. graminearum in the application; (A) thin layer chromatography analysis diagram of AFB1 after co-culturing ATC-AL strain and A. flavus in PDB with the same volume and different cell number ratio; (B) statistical analysis diagram of relative yield of AFB1 in A; (C) thin layer chromatography analysis diagram of AFB1 after adding different concentrations of ATC-AL strain active substance in YES toxin production medium; (D) statistical analysis diagram of relative yield of AFB1 in C; (E) statistical analysis diagram of DON toxin yield of F. graminearum after adding different concentrations of ATC-AL strain active substance in F. graminearum toxin production medium (TBI medium);

[0038] Figure 9 Figure for the effect of Bacillus velezensis ATC-AL on inhibiting infection and toxin production after infection of C. falcatum, A. flavus and F. graminearum in the application; (A) figure for the inhibitory effect of different concentrations of ATC-AL strain active substance on C. falcatum infection of sweet potato; (B) statistical analysis diagram of the diameter of rotten spots in A; (C) figure for the inhibitory effect of different concentrations of ATC-AL strain active substance on A. flavus infection of corn kernels; (D) statistical analysis diagram of the number of A. flavus spores on corn kernels in C; (E) statistical analysis diagram of AFB1 yield residue on corn kernels after infection; (F) figure for the inhibitory effect of different concentrations of ATC-AL strain active substance on F. graminearum infection of wheat kernels; (G) statistical analysis diagram of DON vomitoxin yield on wheat kernels in F. DETAILED DESCRIPTION

[0039] The technical solutions provided by the application are described in detail below in combination with specific examples; the reagents and materials used in the operations in the following examples are purchased from commercial channels if not specifically stated.

[0040] Example 1

[0041] Isolation, screening and identification of a bacillus velezensis ATC-AL.

[0042] I. Isolation and screening of bacillus velezensis ATC-AL

[0043] The bacillus velezensis of the present application is isolated and screened from the rhizosphere of sweet potato. The specific implementation process is as follows:

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

[0045] Soil samples are collected in the rhizosphere of sweet potato, the collection depth is 3-8 cm, 10 mL of sterile water is added per gram of soil sample, grinding for 2 min, and then the supernatant is sucked into a 10 mL centrifuge tube. The supernatant is diluted to 10-1, 10-2 and 10-3 concentration gradients with sterile water; take 100 μL of the diluent and the undiluted mother liquor, respectively, and spread on LB solid medium, repeat 3 times for each sample, and cultivate in a 37℃ incubator for 3 days.

[0046] (2) Purification and preservation of strains

[0047] The microorganisms with the same morphology are classified into a category, and a strain is inoculated in LB solid in the same category of microorganisms, and a single bacterial colony is picked and streaked on a new LB solid medium for subculture purification. After three times of subculture, inoculate into 1 mL of LB medium, shake at 200 rpm at 37℃ overnight, mix with 60% glycerol at a volume ratio of 1:1, and store in a freezer box at -80℃ refrigerator for long-term preservation.

[0048] (3) Screening of bacteria with antagonistic effect on growth of pathogenic fungi

[0049] The plate confrontation method is used to preliminarily screen the strains after isolation and purification, a vertical cross is drawn on the PDA plate with the origin as the center, 2 μL of 10 7 Fresh pathogenic fungal conidia of 10 individual / mL are added to the center of the PDA plate, and the bacterial liquid is taken with an inoculation ring and streaked around the center. The plate inoculated with pathogenic fungi only is used as a blank control, and 3 parallel plates are set. After 28℃ dark culture, the bacteria with inhibitory effect are selected for further rescreening, and a strain named ATC-AL is screened.

[0050] II. Identification of strains

[0051] The obtained ATC-AL strain is identified by morphology, molecular biology and physiological and biochemical characteristics, and the specific steps are as follows:

[0052] (1) Morphological identification of 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 C, it is found that the ATC-AL strain provided by the application has a similarity of 100% with Bacillus velezensis, and in combination with other identification methods, it is indicated that the strain is Bacillus velezensis.

[0058] Example Two

[0059] The genome of Bacillus velezensis ATC-AL contains a synthetic gene cluster capable of synthesizing a plurality of antifungal active substances

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

[0061] 10 μL of Bacillus velezensis ATC-AL was inoculated from a glycerol preservation tube into 4 ml of LB liquid and cultured at 37°C on a shaker at 200 r / min overnight to the logarithmic growth phase, OD600 was about 0.6-0.8, 3-5 ml of bacterial cells were collected, 1.5 ml centrifuge tubes were centrifuged at low temperature 4°C, the supernatant was discarded, and the bacterial cells at the bottom of the tube were reserved. The centrifuge tube was sealed, immediately frozen in liquid nitrogen, and stored at -80°C. Dry ice was sent to the laboratory of Shanghai Ling'en Biological Technology Co., Ltd. to complete the three-generation bacterial genome sequencing. The genome of the strain has 3,910,637 bp, the GC content is 46.61%, contains 4036 coding genes, 27 ribosomal RNAs and 86 transfer RNAs (tRNA) Figure 2 A).

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

[0063] The antiSMASH 8.0 (https: / / antismash.secondarymetabolites.org / ) was used to identify potential antifungal active secondary metabolite gene clusters in the genome of ATC-AL strain. A total of 16 potential active ingredient synthesis gene cluster regions were identified. Among them, five encode non-ribosomal peptide synthesis enzymes (NRPS), one encodes a gene cluster containing RRE elements, three encode transacylase polyketide synthases (transAT-PKS), one encodes a RiPP analogue (other ribosomal synthesis and post-translational modification peptide products RiPP), three encode terpene precursors, one encodes an NRPS analogue, two encode azole-containing RiPP, one encodes a beta-lactam, two encode terpenes, one encodes an NRP-metal carrier, two encode type III polyketide synthases (T3PKS), and one encodes other types of polyketide synthases (PKS analogues). Sequence similarity analysis showed that gene clusters 1, 3, 6, 7, 8, 12, 14, 15 and 16 have high sequence similarity with known gene clusters encoding surfactin, phytazolidin, macrolide H, bacillene, formosin, bacillithiazoles A-N, bacillibactin siderophore and bacillibactin, indicating that the ATC-AL strain genome has a rich gene cluster for synthesizing antifungal active substances (Table 1 below).

[0064] Table 1 Summary of antifungal active ingredient synthesis gene clusters in the genome of Bacillus velezensis ATC-AL

[0065]

[0066]

[0067] (3) Antifungal activity determination of ATC-AL strain fermentation broth

[0068] After the fermentation broth of ATC-AL strain (cultured at 37°C, 200 rpm for 2d) was filtered with a 0.22μM filter, it was added to preheated melted PDA medium to make its final concentration 0%, 1%, 5%, 10%, 20%, 30% and 40% (total volume 10mL), and the control group was added with the same volume of sterile LB medium. Fresh spore solution of C. batatium was collected and diluted with 0.1% Tween 80 solution to make the final concentration of spores 5×10 6 spores / mL, 2μL spore solution was spotted in the center of PDA medium, and phenotypic observation was made after 5d culture at 28°C. The fermentation broth of ATC-AL strain inhibited the growth of the pathogenic fungus, and when the concentration of the fermentation broth reached 40%, the growth of the pathogenic fungus was completely inhibited Figure 2 B).

[0069] Example Three

[0070] Analysis of the inhibitory activity of Bacillus velezensis ATC-AL on Diaporthe phaseolorum

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

[0072] The activated Bacillus velezensis of Example One was added to a fermenter containing 40 L of LB liquid medium, and aerated and cultured at 37°C for 7 d.

[0073] (2) Extraction of active substances

[0074] Ethyl acetate was poured into the fermentation broth at a volume ratio of 1:1, and aerated and rolled for 6 h. After stopping aeration, the fermentation broth waste liquid was discharged from the lower end of the separatory funnel after layering, and the upper ethyl acetate was discharged and added to a rotary evaporator for rotary evaporation. After completion, the active substances extracted from each 1 L of fermentation broth were dissolved in 1 mL of methanol.

[0075] (3) Detection of the minimum inhibitory concentration (MIC) of ATC-AL strain active substances on Diaporthe phaseolorum

[0076] Fresh spore liquid of Diaporthe phaseolorum was collected, diluted with 0.1% Tween 80 solution, and the final concentration of spores was 5 x 10 6 spores / mL. 0.015 g of resazurin powder was dissolved in 10 mL of sterile water, and the final concentration after adding it to the system was 75 μg / mL, and the process was ensured to be sterile. Next, the gradient of active substances was diluted in gradient, and holes 2-11 were the experimental group (the content of active substances was 0, 2, 4, 6, 8, 12, 14, 16, 18, and 20 μL / mL, respectively), and hole 1 was the blank control (without spore liquid). 170 μL of PDB medium was added to each of holes 2-11 (190 μL of PDB medium was added to hole 1), 20 μL of spore liquid was added to each of holes 2-11 (no spore liquid was added to hole 1), and 10 μL of resazurin was added to each of holes 2-11. Each group had three replicates. After completion, the holes were sealed with a sealing film and wrapped with tin foil paper, and after incubation at 28°C for 3 d, the holes were photographed and observed. Resazurin is an indicator of redox reactions and normally appears blue-purple. If the bacteria in the sample grow, they can reduce resazurin and cause a color change. According to the color change from blue-green, purple to white or colorless, it can be determined whether there are living bacteria in the sample. As shown in FIG. 1A, when the concentration of active substances reaches 8 μL / mL, the color of resazurin is blue-purple, and the color of 0-6 μL / mL treatment is close to colorless, indicating that 8 μL / mL of active substances is the minimum inhibitory concentration of Diaporthe phaseolorum. Figure 3 A, when the concentration of active substances reaches 8 μL / mL, the color of resazurin is blue-purple, and the color of 0-6 μL / mL treatment is close to colorless, indicating that 8 μL / mL of active substances is the minimum inhibitory concentration of Diaporthe phaseolorum.

[0077] (4) Inhibition of ATC-AL strain active substances on the growth of Diaporthe phaseolorum mycelium

[0078] The active substance was added to the melted PDA medium at a final concentration of 0 μL / mL, 2 μL / mL, 4 μL / mL, 6 μL / mL, 8 μL / mL, 10 μL / mL, and poured into a 12-well plate. After the PDA medium solidified, the spore solution with a concentration of 5 x 10 6 spores / mL was obtained according to the procedure of step (3) in Example 3, 2 μL of the spore solution was spotted onto the center of the PDA medium, and incubated at 28°C in the dark for 3 days. The results are shown in Figs. Figure 3 B, 3C, the growth of C. batatium was significantly inhibited when the concentration of the active substance was 8 μL / mL, which was the minimum inhibitory concentration (MIC) of the active substance against C. batatium; when the concentration of the active substance was 10 μL / mL, the growth of C. batatium was completely inhibited, with an inhibition rate of 100%, which was the minimum bactericidal concentration (MBC) of the active substance against C. batatium.

[0079] (5) Inhibition of spore germination of C. batatium by the active substance of ATC-AL strain

[0080] The active substance was added to the melted PDA medium at a final concentration of 0 μL / mL, 2 μL / mL, 4 μL / mL, 8 μL / mL, and poured into a 12-well plate. After the PDA medium solidified, the spore solution with a concentration of 5 x 10 6 spores / mL was obtained according to the procedure of step (3) in Example 3, 2 μL of the spore solution was spotted onto the center of the PDA medium, and incubated at 28°C in the dark for 3 days. The results are shown in Figs.

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

[0082] Spore germination rate (%) = number of germinated spores / total number of spores x 100;

[0083] The results are shown in Figs. Figure 3 D, 3E, when the concentration of the active substance was 8 μL / mL, the active substance completely inhibited the spore germination of C. batatium, with an inhibition rate of more than 95%. The detection of the effect of the active substance on the expression of sporulation regulatory genes by fluorescent quantitative PCR found that the expression of the two sporulation regulatory genes abaA and flbA was also significantly reduced after treatment with 8 μL / mL of the active substance Figure 3 F), indicating that the active substance of the ATC-AL strain can inhibit spore development by inhibiting the expression of sporulation regulatory genes.

[0084] Example Four

[0085] Disruption of the cell surface structure of C. batatium by Bacillus velezensis ATC-AL and induction of cell apoptosis

[0086] I. Effects of Bacillus velezensis ATC-AL on the cell surface structure integrity of C. batatium

[0087] (1) Effects of ATC-AL strain active substances on the cell surface structure integrity of C. batatium

[0088] Take 100 μL of freshly collected spore solution (containing 5 x 10 6 spores) in 900 μL of PDB medium, then incubate at 28°C, 200 rpm for 12 hours. Centrifugal collection of cultured spores and mycelium, and soak in 2.5% glutaraldehyde solution for two hours for fixation; centrifugal for five minutes to remove excess glutaraldehyde, then dehydrate the sample with 30%, 50%, 70%, 90%, 100% ethanol, each for 20 minutes; then 4°C, 12000 rpm centrifugal for 5 minutes, discard the supernatant. After freeze-drying the sample for 12 hours, take it out for gold spraying treatment, and use scanning electron microscope to take pictures of the fungal structure. The results are shown in Figure 4 A, the mycelium and conidium surface of the untreated control group is consistent in thickness, smooth and full, while the ATC-AL strain active substance treatment leads to dehydration, depression and intensifies surface damage of the mycelium and spores of C. batatium.

[0089] (2) Effects of ATC-AL strain active substances on the synthesis of ergosterol components of C. batatium cell membrane

[0090] Take the freshly collected spore solution and place it in 20 mL of PDB (containing 2 x 10 6 spores) medium, incubate at 28°C, 200 rpm for 24 hours, add and do not add active substances with a final concentration of 8 μL / mL, and continue to incubate at 28°C, 200 rpm for 12 hours. Collect the mycelium and extract total RNA, send to Shanghai Ling'en Biological Technology Co., Ltd. for transcriptome sequencing. Analysis of transcriptome results found that after treatment with ATC-AL strain active substances, the expression of genes related to ergosterol synthesis in C. batatium, including erg2, erg4, erg25, erg26, erg28, was significantly down-regulated Figure 4 B); further validation by fluorescent quantitative PCR found that the expression of the above five ergosterol synthesis related genes was significantly reduced Figure 4 C) after treatment with ATC-AL strain active substances, indicating that ATC-AL strain active substances inhibited the synthesis of ergosterol components of C. batatium cell membrane.

[0091] (3) Effects of ATC-AL strain active substances on the release of cell contents of C. batatium

[0092] Take 2 mL of freshly collected spore solution (containing 10 6 spores) in 20 mL PDB and incubate at 28°C, 120 rpm for 48 hours. Centrifuge to collect mycelium, wash the cells twice with PBS, and suspend them in PBS containing different concentrations (0, 4 μL / mL and 8 μL / mL) of active substance. Use methanol as a negative control. The treated culture and the control culture are again incubated at 28°C with shaking, and samples are taken at 0, 1, 2, 4 and 12 hours. After incubation, the cells are centrifuged at 8000 rpm for 5 minutes at 28°C, and the absorbance of the supernatant at 280 nm is measured using a microplate reader and compared with a blank sample without fungi. The results are shown in Figure 4 D, after treatment of ATC-AL strain active substance, the absorbance of the treatment group increased significantly, indicating that the cell contents of C. batatium leaked.

[0093] (4) Fluorescence microscopy detection of the effect of ATC-AL strain active substance on the cell membrane structure of C. batatium

[0094] Take a freshly collected spore solution and place it in 20 mL of PDB medium, incubate at 28°C, 200 rpm for 24 hours, add different final concentrations of active substance (0, 2, 4, 8 μL / mL) respectively, and continue to incubate at 28°C, 200 rpm for 12 hours. Centrifuge to collect mycelium, wash mycelium with PBS buffer for 3 times, and stain mycelium with propidium iodide (PI) at a concentration of 5 mM at 37°C in the dark for 20 min. Then wash the stained mycelium with PBS buffer for at least 3 times. Finally, suspend the cells in PBS and examine under a fluorescence microscope. The results are shown in Figure 4 E, the mycelium treated with ATC-AL strain active substance at each concentration has a strong red fluorescence signal, and the fluorescence intensity increases with the increase of the concentration of the fermentation broth, and the red fluorescence intensity of the mycelium treated with 8 μL / mL active substance is the strongest, while the red fluorescence intensity of the control mycelium is weak. The above results show that the treatment of ATC-AL strain active substance severely damages the cell membrane structure of C. batatium, leading to the entry of PI dye into the cell interior.

[0095] II. Effect of ATC-AL strain active substance on the oxidative response process of C. batatium

[0096] (1) Effect of ATC-AL active substance on the expression of related antioxidant genes in C. batatium

[0097] The transcriptome results in step (2) of the first part of Example Four were analyzed, and it was found that the ATC-AL strain active substance treatment led to a significant down-regulation of the expression of genes related to reactive oxygen species ROS scavenging in Diaporthe phaseolorum, including prx1, aox1, pox9, glrX, cat2, ccp1, sodB, sod1, sod2, and ccs1 Figure 5 A). Further verification by fluorescence quantitative PCR found that the expression of the above four ROS scavenging related genes aox1, pox9, ccp1, and sod2 was significantly reduced after ATC-AL strain active substance treatment Figure 5 B).

[0098] (2) Effect of ATC-AL active substance on the activity of antioxidant enzymes in Diaporthe phaseolorum

[0099] The enzyme activities of antioxidant-related enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), in Diaporthe phaseolorum after ATC-AL active substance treatment were detected. The spore preparation, mycelial culture, and treatment in the early stage were in accordance with step (2) of the first part of Example Four. The results showed that 8 μL / mL of ATC-AL active substance treatment could significantly reduce the enzyme activities of SOD, POD, and CAT Figure 5 C-5E), indicating that ATC-AL active substance could inhibit the enzyme activities of related antioxidant enzymes in Diaporthe phaseolorum.

[0100] (3) Effect of ATC-AL strain active substance on the content of reactive oxygen species (ROS) in Diaporthe phaseolorum mycelium

[0101] The spore preparation, mycelial culture, and treatment in the early stage were in accordance with step (4) of the first part of Example Four. The washed mycelium was stained with 2,7-dichlorofluorescein diacetate (DCFH-DA) at a concentration of 10 mM for 30 min in the dark at 37°C. After washing with PBS, it was found by fluorescence microscopy that the mycelium treated with ATC-AL strain active substance at various concentrations had strong green fluorescence signals, and the fluorescence intensity increased with increasing active substance concentration, with the strongest red fluorescence intensity at 8 μL / mL of active substance treatment Figure 5 F), indicating that ATC-AL strain active substance treatment led to the accumulation of excessive ROS in fungal cells.

[0102] (4) Effect of ATC-AL strain active substance on the content of malondialdehyde (MDA) in Diaporthe phaseolorum mycelium

[0103] The pre-spore preparation, mycelium culture and treatment refer to the steps (2) of the first part of Example 4. The mycelium was collected and ground with liquid nitrogen, and 0.2 g of the ground mycelium was used to detect the content of MDA in the cell by the method of thiobarbituric acid. It was found that the content of MDA in the C. batatiae increased significantly after treatment with the active substance of the ATC-AL strain, and the content of MDA in the 8 μL / mL active substance treatment group was the highest Figure 5 G). MDA can destroy the lipid structure of the membrane, and the higher the content, the more serious the damage to the cell membrane. Therefore, the active substance of the ATC-AL strain can damage the cell membrane structure of C. batatiae.

[0104] III. Application of the active substance of the ATC-AL strain in inducing apoptosis of C. batatiae

[0105] (1) Effect of the active substance of the ATC-AL strain on the mitochondrial membrane potential of C. batatiae cells

[0106] The pre-spore preparation, mycelium culture and treatment refer to the steps (4) of the first part of Example 4. The washed mycelium was incubated with JC-1 probe (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide) at 37°C for 30 min. Then it was washed with PBS and detected by fluorescence microscopy. It was found that the intensity of red fluorescence gradually weakened with the increase of the concentration of the active substance, while the green fluorescence was opposite Figure 6 A). In normal cells, the membrane potential is normal, JC-1 enters the mitochondria through the membrane polarity of mitochondria, and forms red fluorescent multimers; during apoptosis, the mitochondrial transmembrane potential depolarizes, and JC-1 is released from the mitochondria as green fluorescent monomers. The above results show that the active substance of the ATC-AL strain can cause the mitochondrial membrane potential of C. batatiae cells to decrease, and the mitochondria to be damaged.

[0107] (2) Effect of the active substance of the ATC-AL strain on the apoptosis of C. batatiae cells

[0108] Depolarization of mitochondrial transmembrane potential is one of the important indicators of apoptosis. Further use of TUNEL and DAPI fluorescent probes to assess the apoptosis of cells after treatment with active substances. The previous spore preparation, mycelium culture and treatment refer to the steps of the first part of example four (4). With TdT enzyme (terminal deoxynucleotidyl transferase) and TUNEL (transferase-mediated dUTP nick end labeling assay) reaction buffer and mycelium staining at 37°C in the dark for 1.5 hours. After washing with PBS, then incubated with DAPI (4', 6-diamidino-2-phenylindole) staining solution at room temperature for 30 minutes. Finally, using a fluorescence microscope, it was found that after treatment with active substances, compared with the control group, the intensity of red fluorescence gradually increased with the increase of active substance concentration, while the DAPI stained nucleus fluorescence gradually weakened, indicating that DNA appeared fragmentation Figure 6 B), which is a marker of apoptosis.

[0109] Example Five

[0110] Broad-spectrum antibacterial effect of bacillus velezensis ATC-AL

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

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

[0113] According to the experimental steps (1), (3) in example three, bacillus velezensis ATC-AL bacterial solution and spore solution with a final concentration of 5 x 10 6 spores / mL of fusarium graminearum spore solution, fusarium solani spore solution, aspergillus flavus spore solution, fusarium solani spore solution, phytophthora cactorum spore solution, aspergillus terreus spore solution, aspergillus fumigatus spore solution, aspergillus nidulans spore solution, aspergillus niger spore solution, candida albicans spore solution.

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

[0115] Use the gun head to dip the bacterial solution to draw a line on the upper, lower, left and right of the PDA plate, inoculate 2 μL of fusarium graminearum spore solution, fusarium solani spore solution, aspergillus flavus spore solution, fusarium solani spore solution, phytophthora cactorum spore solution, aspergillus terreus spore solution, aspergillus fumigatus spore solution, aspergillus nidulans spore solution, aspergillus niger spore solution, candida albicans spore solution in the center of the solidified PDA plate, and the line without drawing as a blank control, 37°C culture for 3d, measure the colony diameter, calculate the inhibition rate. For example Figure 7As shown in Table B, when the active substance is 32 μL / mL, the inhibition rate of ATC-AL strain on Fusarium graminearum, Aspergillus flavus and Aspergillus fumigatus is 100%, the inhibition rate on Fusarium solani is 72.85%. The growth of Pythium aphanidermatum and Candida albicans is also significantly inhibited, the diameter of the fungus is less than 0.5 cm, and the relative inhibition is 29.41% and 20.34% respectively. Therefore, the active substance of ATC-AL strain also has good broad-spectrum bacteriostatic effect.

[0116] II. Broad-spectrum antifungal effect of active substance of ATC-AL strain

[0117] The extraction of the active substance of ATC-AL strain and the collection of pathogenic fungal spore solution are respectively referred to the experimental steps (2) and (3) in Example 3. The active substance is added to the melted PDA medium at a final concentration of 0 μL / mL, 8 μL / mL, 16 μL / mL and 32 μL / mL, and poured into a square lattice hole plate. After the PDA medium is solidified, 2 μL of spore solution of Fusarium graminearum, Fusarium solani, Aspergillus flavus, Pythium aphanidermatum, Aspergillus fumigatus and Candida albicans is respectively spotted in the center of the PDA medium, and cultured at 28°C in the dark for 2d, with 3 parallel groups. The results are shown in Table B. Figure 7 As shown in Table B, when the active substance is 32 μL / mL, the inhibition rate of ATC-AL strain on Fusarium graminearum, Aspergillus flavus and Aspergillus fumigatus is 100%, the inhibition rate on Fusarium solani is 72.85%. The growth of Pythium aphanidermatum and Candida albicans is also significantly inhibited, the diameter of the fungus is less than 0.5 cm, and the relative inhibition is 29.41% and 20.34% respectively. Therefore, the active substance of ATC-AL strain also has good broad-spectrum bacteriostatic effect.

[0118] Example Six

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

[0120] I. Inhibition effect of co-culture of Bacillus velezensis ATC-AL and Aspergillus flavus on aflatoxin production

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

[0122] According to the experimental steps (1) and (3) in Example 3, the Bacillus velezensis ATC-AL bacterial solution and the spore solution of Aspergillus flavus are obtained respectively, and the final concentration of the spore solution is 1×10 7spores / mL. The cell number of Aspergillus flavus and ATC-AL strain were mixed in the ratio of 1:0.2, 1:1, 1:5, respectively, and the volume of Aspergillus flavus and Bacillus velezensis was 500 μL, which was added to 9 mL of PDB medium. The control group added 500 μL of Aspergillus flavus spore solution, 500 μL of LB medium and 9 mL of PDA, and was cultured at 28°C, 200 rpm for 5 days.

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

[0124] After 5 days of culture, 700 μL of co-culture fermentation broth was aspirated, and 700 μL of dichloromethane was used to extract aflatoxin, which was evaporated overnight. The toxin sample was remelted with 15 μL of dichloromethane, and the sample was added to a silica gel plate, and the toxin was separated and detected by developing agent (dichloromethane: acetone = 9:1). The results are shown in Figure 8 A, 8B, the yield of aflatoxin decreases gradually with the increase of the amount of ATC-AL strain, and the inhibition efficiency of ATC-AL strain on aflatoxin B1 is 69.89%, 79.36% and 100%, respectively.

[0125] II. Inhibition of aflatoxin production by Bacillus velezensis ATC-AL active substance

[0126] (1) Inhibition of aflatoxin synthesis by ATC-AL active substance

[0127] The active substance was added to the YES liquid medium at a final concentration of 0 μL / mL, 4 μL / mL and 8 μL / mL, respectively, and 100 μL of Aspergillus flavus spores with a concentration of 1×10 7 spores / mL were added to each medium, and cultured in a 28°C incubator in the dark for 5d.

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

[0129] The extraction and analysis of aflatoxin B1 refer to the steps (2) of the first part of Example Six. The results are shown in Figure 8 C, 8D, when the concentration of the active substance is 4 μL / mL, the inhibition rate of the active substance on aflatoxin B1 can reach 100%.

[0130] III. Inhibition of Fusarium graminearum toxin production by Bacillus velezensis ATC-AL active substance

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

[0132] The collection of fresh spores of Fusarium graminearum refers to the experimental steps (3) in Example Three, and the spore solution is diluted to 10 7 spores / mL.

[0133] (2) Inhibition effect of ATC-AL active substance on Fusarium graminearum toxin production

[0134] The active substance was added to 30 ml of TBI toxin-producing liquid medium at a final concentration of 0, 4, 8 μL / mL, respectively, and 100 μL of freshly collected Fusarium graminearum spores at a concentration of 1 x 10 7 spores / mL was added, and the culture was incubated at 28°C, 200 rpm, in the dark for 7 days.

[0135] (3) Extraction and analysis of vomitoxin

[0136] The toxin production of Fusarium graminearum under different concentrations of active substance was measured using a vomitoxin kit from Shenzhen Finde Biotechnology Co., Ltd. by enzyme-linked immunosorbent assay. The results are shown in Figure E, and the inhibition efficiency of ATC-AL active substance on DON toxin was 38.33%, 83.87%, and 85.76%, respectively. Figure 8

[0137] Example Seven

[0138] Preventive effect of Bacillus velezensis ATC-AL on sweet potato black spot, corn aspergillosis, and wheat scab

[0139] I. Preventive effect of ATC-AL strain on sweet potato black spot

[0140] Healthy and similarly sized sweet potatoes were selected, washed with tap water, soaked in 1% sodium hypochlorite solution for 10 minutes, washed with sterile water twice, washed with 70% ethanol for 2 minutes, and then washed with sterile water twice for standby. Four 5 mm deep wounds were made on the waist of the sweet potato using a sterile dissecting needle. After drying, 20 uL of sweet potato long-beaked conk spore solution at a concentration of 1 x 10 6 spores / mL was added to the wounds, and active substance at a final concentration of 0, 2, 4, 8 μL / mL was added, respectively. After drying, it was added to a sterile lunch box. After 20 days of incubation at 28°C, the incidence of sweet potato was detected. As shown in Figures Figure 9 A, 9B, the control group of sweet potato showed large areas of black spots and depressions on the surface, and the cross-sectional tissue showed obvious black spots. The active substance treatment group significantly reduced the occurrence and damage of black spot, and the inhibition efficiency of the active substance on the lesion was 44.65%, 62.89%, and 68.87%, respectively.

[0141] II. Inhibition effect of ATC-AL strain on Aspergillus flavus infection of corn

[0142] (1) Aspergillus flavus infection of corn kernel experiment

[0143] ​The corn kernels were peeled, kept intact, and a small hole was made at the root of the seeds. The corn seeds were sterilized according to the method described in Example 7, Part 1. The corn seeds were taken out, dried with sterile filter paper, and placed in 50 ml sterilized conical flasks, 20 ml of active substance solution with a final concentration of 0, 4, 8 μL / mL was added, and the amount of methanol was controlled to be the same in the three groups of samples, so that the experiment only had the concentration of active substance as the variable. The three conical flasks were placed in a 28°C shaker at 200 rpm for 60 min to allow the active substance to fully coat the corn seeds. Two layers of sterile filter paper were placed in each 60 mm sterile petri dish, and 2 ml of sterile water or the corresponding concentration of active substance was added to maintain a humid environment. The corn seeds coated with the active substance were placed flat on the filter paper in the petri dish, and 2 μL of fresh Aspergillus flavus spore solution containing 10 7 The results are shown in Table C. When the concentration of the active substance was 4 μL / mL, the active substance had significantly inhibited the infection of Aspergillus flavus on corn seeds. Figure 9 The results are shown in Table C. When the concentration of the active substance was 4 μL / mL, the active substance had significantly inhibited the infection of Aspergillus flavus on corn seeds.

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

[0145] The 6 corn seeds in each petri dish were transferred to a 50 ml centrifuge tube, and 10 ml of 0.1% Tween 80 was added, and vortexed to elute the spores. 15 μL of the spore solution was taken and counted under a light microscope using a hemocytometer to calculate the spore concentration. The results are shown in Table D. When the concentration of the active substance was 4 μL / mL, the active substance had significantly inhibited the sporulation ability of Aspergillus flavus after infecting the corn seeds, with an inhibition rate of 83.72%; when the concentration of the active substance was 8 μL / mL, the inhibition rate of the active substance on the sporulation of Aspergillus flavus was 90.70%. Figure 9 The results are shown in Table D. When the concentration of the active substance was 4 μL / mL, the active substance had significantly inhibited the sporulation ability of Aspergillus flavus after infecting the corn seeds, with an inhibition rate of 83.72%; when the concentration of the active substance was 8 μL / mL, the inhibition rate of the active substance on the sporulation of Aspergillus flavus was 90.70%.

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

[0147] To the mixture obtained in step (2), 10 mL of dichloromethane was added, shaken to mix well, and after complete separation, the lower organic phase was taken into a new centrifuge tube. The following steps for the extraction and detection of aflatoxin B1 were the same as steps (2) in Part 1 of Example 6. The results are shown in Table E. The inhibition rates of the active substance with concentrations of 4 μL / mL and 8 μL / mL on the toxin production of Aspergillus flavus were both 100%. Figure 9 The results are shown in Table E. The inhibition rates of the active substance with concentrations of 4 μL / mL and 8 μL / mL on the toxin production of Aspergillus flavus were both 100%.

[0148] III. Prevention of Fusarium graminearum infection of wheat seeds by ATC-AL strain

[0149] (1) Experiment of Fusarium graminearum infection of wheat seeds

[0150] Select healthy, similar size wheat seeds, the disinfection process of wheat seeds refer to the method of the first part of Example Seven. Take out the disinfection wheat seeds, dry the moisture with sterile filter paper, put the wheat seeds into 3 50ml sterilization conical flask respectively, add 20ml of final concentration 0, 4, 8μL / mL active substance solution, the amount of methanol in three groups of samples needs to be controlled to be the same, so that the experiment has only one variable of active substance concentration. Put the three conical flasks into 28℃ shaking table, 200rpm, 60min, let the active substance wrap the wheat seeds fully. Put two layers of sterile filter paper in each 60mm sterile petri dish, and add 2ml of corresponding concentration active substance to keep the wet environment. Take 10g of wheat seeds wrapped by active substance and put them flat on the filter paper in the petri dish, configure 200μL of concentration 10 7 The results are shown in Figure 9 F, when the concentration of active substance is 16μL / mL, the active substance has significantly inhibited the infection of fusarium graminearum on wheat seeds.

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

[0152] Take 2g of wheat seed sample in step (1) in a 50ml centrifuge tube, add 20ml of deionized water, shake for 5min, centrifuge at room temperature for 10min at 4000r / min, take the supernatant and dilute 20 times, then add 0.5ml of diluted supernatant and 0.5ml of reconstituted solution in a new centrifuge tube, mix well, take 50μL of liquid to measure the toxin production of fusarium graminearum under different active substance concentrations using vomitoxin kit of Shenzhen Fender Biotechnology Co., Ltd. The results are shown in Figure 9 G, the inhibition rate of active substance on the production of vomitoxin by fusarium graminearum infected wheat seeds is 44.41%, 95.23% and 95.80% respectively.

Claims

1. A strain of 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. The Bacillus belyssus ATC-AL according to claim 1, characterized in that, The genome of the Bacillus belyssus ATC-AL contains a cluster of synthetic genes capable of synthesizing a variety of antifungal active substances.

3. A microbial preparation, characterized in that, It includes one or more of the following: Bacillus vesiculosus ATC-AL, Bacillus vesiculosus ATC-AL fermentation broth, and active substances of Bacillus vesiculosus ATC-AL as described in claim 1.

4. The microbial preparation according to claim 3, 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; The preparation method of the active substance of Bacillus belyssus ATC-AL is as follows: the fermentation broth of Bacillus belyssus ATC-AL is extracted with an equal volume of ethyl acetate, and the active substance of Bacillus belyssus ATC-AL is obtained by rotary evaporation.

5. The microbial preparation according to claim 3, characterized in that, The formulation is in one of the following forms: liquid, spray powder, dry wettable powder, or dry wettable granules.

6. The application of Bacillus belyssus ATC-AL as described in claim 1 or the microbial preparation as described in claim 3 in the broad-spectrum inhibition of plant / animal pathogenic fungi, wherein the pathogenic fungi are one or more of Fusarium graminearum, Aspergillus niger, Aspergillus flavus, Fusarium solani, Phytophthora spp., Aspergillus terreus, Aspergillus fumigatus, Aspergillus nidus, Aspergillus niger, and Candida albicans.

7. The application of Bacillus berberis ATC-AL as described in claim 1 or the microbial preparation as described in claim 3 in the prevention and control of postharvest black spot disease in sweet potatoes.

8. The use of Bacillus belyssus ATC-AL as described in claim 1 or the microbial preparation as described in claim 3 in the prevention and control of aflatoxin and / or aflatoxin contamination in corn.

9. The use of Bacillus belye ATC-AL as described in claim 1 or the microbial preparation as described in claim 3 in the control of Fusarium graminearum and / or vomitoxin contamination in wheat.

10. The application according to any one of claims 7-9, wherein the specific application process is as follows: the Bacillus vesicae ATC-AL fermentation broth or the cell-free supernatant of the Bacillus vesicae 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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