Bacillus subtilis BS-9 and application thereof in prevention and treatment of corn leaf spot disease

Abstract

The invention discloses bacillus subtilis BS-9 and application thereof in prevention and treatment of corn leaf spot disease, and relates to the technical field of screening of biocontrol strains for prevention and treatment of corn leaf spot disease. The strain BS-9 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 27, 2024, the preservation number is CGMCC No.30158, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the strain is classified and named as Bacillus subtilis. The strain can effectively antagonize Epicoccum nigrum, Epicoccum broomcorn, Helminthosporium maydis, Alternaria alternata and Helminthosporium bombycis, pot experiments show that fermentation broth inoculated with the strain can improve the prevention and treatment effect on the corn leaf spot disease, and a theoretical basis is provided for subsequent development of a biocontrol microbial inoculum for the corn leaf spot disease.

Description

Technical Field

[0001] The invention relates to the technical field of screening biocontrol strains for preventing and controlling corn leaf spot disease, and more particularly to a Bacillus subtilis BS-9 and application thereof in preventing and controlling corn leaf spot disease. Background Art

[0002] Corn is an important food and feed crop, second only to rice and wheat in terms of cultivated area. However, corn cultivation is often plagued by major diseases such as leaf spot. Corn leaf spot is primarily caused by fungi, including large leaf spot, small leaf spot, Curvularia leaf spot, gray leaf spot, Alternaria leaf spot, and southern rust. These diseases occur year-round or intermittently, causing losses to corn production. Leaf spot is a significant threat to corn production. In typical years, field yields decrease by approximately 10% in diseased fields. In years when susceptible varieties are prevalent, yield reductions can exceed 50%, and in severe cases, even total crop failure. Currently, leaf spot control relies primarily on chemical control and the use of disease-resistant varieties. However, due to the complexity of the pathogen, the wide variation and differentiation of the causative bacteria, the diversity and variability of corn varieties, and improper cultivation practices driven by climate change and growing demand, effective and targeted chemical agents and disease-resistant varieties remain elusive. While disease-resistant varieties cannot meet production needs, chemical control can also lead to pesticide residues, environmental pollution, and the development of drug resistance in pathogens.

[0003] Biological control, a field with great development potential, has attracted considerable attention in recent years. Studies have shown that antagonistic bacteria are effective against leaf spot disease, and considerable progress has been made in using active organic compounds produced by biocontrol bacteria to inhibit the maize leaf spot pathogen. However, the availability of available strains remains relatively limited, and research on effective antibacterial substances is lacking. Therefore, it is necessary to identify biocontrol strains that are highly effective against maize leaf spot pathogens and to investigate the antibacterial organic compounds in their metabolites.

[0004] Therefore, how to screen a strain that can prevent and treat corn leaf spot disease is an urgent problem that those skilled in the art need to solve. Summary of the Invention

[0005] In view of this, the present invention provides a Bacillus subtilis BS-9 and application thereof in preventing and controlling corn leaf spot disease.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect of an embodiment of the present invention, a Bacillus subtilis BS-9 is provided. The BS-9 strain was deposited in the General Microbiology Center of the China Culture Collection Administration on March 27, 2024, with a deposit number of CGMCC No. 30158 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as Bacillus subtilis.

[0008] A second aspect of the present invention provides a use of Bacillus subtilis BS-9, which includes:

[0009] 1) preventing and controlling corn leaf spot disease, and preparing a microbial agent for preventing and controlling corn leaf spot disease;

[0010] 2) Antagonize the pathogenic bacteria of corn leaf spot.

[0011] Preferably, the pathogenic bacteria are Echinococcus nigricans, Echinococcus sorghum, Helicoverpa maydis, Alternaria alternata, and Helicoverpa macrospora.

[0012] A third aspect of an embodiment of the present invention provides a sterile fermentation broth of Bacillus subtilis BS-9: the primary screened strain BS-9 is inoculated into LB liquid culture medium, cultured at 28°C and 220 r / min for 3 days, and then refrigerated centrifuged (10,000 r / min, 4°C) for 20 minutes, and filtered through a 0.22 μm filter membrane to obtain a sterile fermentation broth.

[0013] A fourth aspect of the embodiments of the present invention provides a preparation for preventing and treating corn leaf spot, comprising a Bacillus subtilis BS-9 agent or a sterile fermentation broth thereof.

[0014] Through the above technical solution, it can be seen that compared with the existing technology, the present invention has screened a new biocontrol strain BS-9 that can antagonize corn leaf spot-related pathogens. After amplification and comparison of its 16S rDNA, it was determined to be Bacillus subtilis. This strain can effectively antagonize black coccus, sorghum coccus, mayd umbilical cordis, alternariae and large-spotted umbilical cordis, and pot experiments show that the fermentation liquid inoculated with this bacteria can improve the prevention and control effect of corn leaf spot, providing a theoretical basis for the subsequent development of biocontrol agents for corn leaf spot. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0016] Figure 1 The attached figures are the colony characteristics and antibacterial effect diagrams of each pathogen; among them, A shows the pathogen colony characteristics of Echinococcus nigricans, Echinococcus sorghum, Helminthosporium maydis, Alternaria alternata, and Helminthosporium macrospotted from left to right; B shows the antibacterial effects of strain BS-9 on five pathogens, Echinococcus nigricans, Echinococcus sorghum, Helminthosporium maydis, Alternaria alternata, and Helminthosporium macrospotted from left to right.

[0017] Figure 2 The attached figure shows the morphological characteristics of strain BS-9.

[0018] Figure 3 The attached figure is a phylogenetic tree of strain BS-9 constructed based on the neighbor-joining method of 16S rDNA gene sequences.

[0019] Figure 4 The attached figure shows the inhibitory effect of BS-9 metabolites on four pathogens (5d); AD: pure culture of Ephesus nigricans, Helminthosporium zeae, Alternaria alternata, and Helminthosporium macrosporum (CK); ad: plate culture containing Bacillus subtilis BS-9 fermentation broth.

[0020] Figure 5 The accompanying drawings show the potted plant protection efficacy of strain BS-9 against Alternaria leaf spot in maize, wherein CK1: 'Xinzhongyu 801' corn inoculated only with the pathogen, A1: 'Xinzhongyu 801' corn treated with prochloraz and the pathogen, B1: 'Xinzhongyu 801' corn treated with BS-9 and the pathogen; CK2: 'Mintian 986' corn inoculated only with the pathogen, A2: 'Mintian 986' corn treated with prochloraz and the pathogen, B2: 'Mintian 986' corn treated with BS-9 and the pathogen. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Soil samples were collected from Wuli Village, Chong'an Subdistrict, Wuyishan City, Fujian Province (latitude: 27°75'116", longitude: 118°3'86"). Five maize pathogens were tested: Epicoccus nigrum (the pathogen of corn leaf spot), Micrococcus sorg hum (the pathogen of corn leaf spot), Alternaria alternata (the pathogen of alternaria leaf spot), Bipolarismaydis (the pathogen of corn leaf spot), and Exs erohilum turcicum (the pathogen of corn leaf spot). These pathogens were provided by the Institute of Plant Protection, Fujian Academy of Agricultural Sciences.

[0023] Reagents and culture media

[0024] PDA medium (200 g peeled potato, 20 g glucose, 20 g agar, 1 L distilled water), PDB medium (200 g peeled potato, 20 g glucose, 1 L distilled water), NA medium (18 g agar, 3 g beef extract, 5 g peptone, 2.5 g glucose), LB liquid medium (10 g tryptone, 5 g yeast extract, 10 g NaCl), sterile water, methanol (analytical grade), ethyl acetate (analytical grade), etc.

[0025] Example 1 Isolation, purification, screening and identification of antagonistic strains

[0026] The dilution coating method was used to isolate bacteria from the soil. After the soil sample was dried, 1 g of crushed soil was weighed and diluted in 10-fold gradients to 10 -2 , 10 -3 and 10 -4 Mix the dilutions by pipetting, then pipette 200 μL of each gradient onto PDA culture medium. Apply evenly using a flame-sterilized applicator. Repeat three times for each concentration. Place the culture medium in a 28°C incubator for three days before transferring to purification.

[0027] The strains were purified by the plate streak method. The antagonistic bacteria to be purified were picked up from the culture medium with antagonistic bacteria initially screened with a sterile pipette tip, and parallel streaks were made on the surface of a sterile NA culture medium plate. One bacteria was streaked on 3 plates.

[0028] Initial screening: Using the plate standoff method, inoculate the target pathogen at the center of the culture medium and the biocontrol bacteria 2.5 cm away from the center pathogen. Inoculate at four points in total, with three replicates. Screen for biocontrol bacteria strains with good antibacterial effects.

[0029] Second screening: Take the primary screening strain and inoculate it into LB liquid medium, shake and culture at 28℃ and 220r / min for 2 days, centrifuge at 10000r / min, 4℃ for 20min, take the supernatant, and filter it through a 0.22μm filter to obtain sterile fermentation liquid. 7 Place Oxford cups at appropriate intervals on a 5 mL suspension of leaf spot pathogen spores (CFU / mL). Pour 100 μL of the biocontrol filtrate into the cups. After 30 minutes of stagnation, incubate in a 28°C incubator for 4 days. Measure the diameter of the inhibition zone and record the experimental results. Repeat each treatment three times to select strains with strong antibacterial activity and good growth for subsequent testing.

[0030] The antibacterial effects of antagonistic strains against five maize leaf spot pathogens were determined using a stand-off culture method. Three replicates were used for each treatment, and all plates were incubated in the dark at 28°C. After colonies in the control group filled the plate, the inhibition band of the experimental group was measured. Colony diameters were also measured, and the percentage of colony growth inhibition was calculated. The inhibition band was the distance between the colony edges of the antagonist and pathogen colonies.

[0031] A total of 15 single colonies were isolated from soil samples from three gradients. Three strains with an inhibition zone greater than 5 mm were initially screened and used for rescreening. After culturing the initial screened strains against the pathogens for 7 days, the BS-9 strain had an inhibition zone diameter greater than 5 mm. An inhibition spectrum experiment was conducted using the BS-9 strain against five strains of corn leaf spot pathogens. The results showed that BS-9 had excellent antibacterial effects ( Figure 1 Finally, strain BS-9 was selected for further classification and identification. The inhibition width is shown in Table 1;

[0032] Table 1 Inhibitory bandwidth (5 days) of strain BS-9 against five corn leaf spot pathogens

[0033]

[0034] Identification of antagonistic strains

[0035] Morphological and physiological identification: First, inoculate the strain onto PDA medium and incubate at 28°C for 2 days. Observe the colony morphology and preliminarily identify the strain. Then, inoculate the strain onto NA medium and observe and record the colony color, transparency, and edge characteristics. Based on the preliminary morphological identification results, perform a Gram stain reaction on the strain. Measure the VP reaction, glucose fermentation, lactose fermentation, and maltose fermentation. Refer to the "Common Bacterial Systematic Identification Manual" and the "Berger's Manual of Bacterial Identification."

[0036] The colonies are grayish white, turbid, round or irregular in shape ( Figure 2The strain was Gram-positive, with rod-shaped cells measuring 0.7-0.8 μm × 2.0-3.0 μm.

[0037] The physiological and biochemical characteristics of strain BS-9 are shown in Table 2. The results of glucose fermentation test and VP test were positive, indicating that strain BS-9 could utilize glucose, maltose and sucrose.

[0038] Table 2 Physiological and biochemical characteristics of strain BS-9

[0039]

[0040]

[0041] Note: “+” indicates a positive result; “-” indicates a negative result.

[0042] Molecular biological identification: The genomic DNA of the biocontrol bacteria was extracted using a bacterial genomic DNA extraction kit (OMEGA), and the BS-9 strain was molecularly identified by 16S rDNA sequence analysis. The 16S rDNA PCR amplification primers were bacterial 16S universal primers 27-F (5′-AGAGTTTGATCCTGGC TCAG-3′) / 1541-R (5′-AAGGAGGTGATCCAGCCGCA-3′) to amplify its sequence.

[0043] PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 90 seconds, and extension at 72°C for 2 minutes, with a final extension at 72°C for 5 minutes. The resulting PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The resulting sequences were assembled and subjected to BLAST analysis in the NCBI database. The sequences were then submitted to NCBI to obtain the corresponding GenBank accession numbers. Based on the alignment results, the 16S rDNA sequences of related strains were downloaded from GenBank. After BLAST homology comparison, a phylogenetic tree was constructed using the MEGA7.0 neighbor-joining method.

[0044] The results showed that the 16S rDNA of strain BS-9 was amplified to obtain a sequence fragment of 1477bp in length. The sequencing results were compared with the sequence fragment of Bacillus subtilis model strain (accession number: MN945436) in GenBank, and the similarity between the sequence fragment and the model strain of Bacillus subtilis (accession number: MN945436) was 99%. The 16S rDNA gene sequences of closely related Bacillus strains were selected from the GenBank database to construct a phylogenetic tree ( Figure 3The results showed that strain BS-9 clustered with Bacillus subtilis in the phylogenetic tree based on 16S rDNA. Combined with morphological characteristics and physiological and biochemical characteristics, strain BS-9 was finally identified as Bacillus subtilis. It was deposited in the General Microbiology Center of the China Culture Collection of Microorganisms with the deposit number CGMCC No. 30158 on March 27, 2024 at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and was named Bacillus subtilis.

[0045] Example 2 Determination of the efficacy of metabolites of antagonistic strains

[0046] The primary screening strain BS-9 was inoculated into LB liquid medium and cultured at 28°C and 220 rpm for 3 days. The culture was then shaken at 10,000 rpm, 4°C, and filtered through a 0.22 μm filter to obtain a sterile fermentation broth. Three gradients of 1 mL, 3 mL, and 5 mL were added to 95 mL of polydimethylsiloxane (PDA), inverted onto plates, and allowed to solidify. Four common pathogens, including Ephemerococcus nigricans, Helminthosporium zeae, Alternaria alternata, and Helminthosporium macrosporum, were selected and inoculated in the center of the plates containing BS-9 metabolites. The plates were incubated at 28°C, with three replicates per treatment. Pathogens cultured without sterile fermentation broth served as controls. Data were recorded at 3, 5, and 7 days of culture. After 7 days of culture, colony diameters were measured using the cross-hatch method, and the inhibition rate of the antagonistic strain metabolites was calculated. Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100.

[0047] Determination of the antibacterial effect of the metabolites of strain BS-9 on four common corn leaf spot pathogens Figure 4 After 5 days of constant temperature culture, it was found that the metabolites of strain BS-9 had a significant inhibitory effect on the four common corn leaf spot pathogens.

[0048] The inhibition rates are shown in Table 3;

[0049] Table 3 Inhibitory rate of metabolites of strain BS-9

[0050]

[0051] Based on the antibacterial test results of strain BS-9 and its metabolites, strain BS-9 has a high inhibition rate and obvious inhibitory effect on common corn leaf spot pathogens. Therefore, its secondary metabolites were further analyzed and identified, and its effective antibacterial components were preliminarily explored.

[0052] Example 3 Extraction of active substances from metabolites of antagonistic strains and GC-MS and LC-MS analysis

[0053] GC-MS analysis: The test strain BS-9 was inoculated into a screw-top glass vial (15 mL) containing an appropriate amount of NA, tightly sealed, and incubated at 28°C for 5 days. The vial was then preheated in a 40°C water bath for 20 minutes. A 100 μm polydimethylsiloxane (PDMS) extraction tip was inserted, and headspace extraction was performed in a 50°C water bath for 30 minutes to collect volatile organic compounds (VOCs).

[0054] GC-MS conditions: DB-5MS flexible quartz capillary column (30 mm × 0.25 mm × 0.25 μm). Carrier gas: nitrogen, flow rate: 1.0 mL min -1 , no split, inlet temperature 250℃. Heating process: initial temperature 35℃, heating 1min, 5℃·min -1 The temperature was raised to 250°C at a rate of 100°C and heated for 5 min. Mass analysis conditions: interface temperature 250°C, ion source temperature 250°C, electron energy 70 eV, scan rate 0.5 s·scan -1 Relative quantitative analysis of volatile organic compounds was performed using peak profile visualization (quadrupole mass spectrometry, NIST05 standard library). GC-MS data were first compared with the National Institute of Standards and Technology (NIST) database, and components with a similarity greater than 80% were selected for compositional analysis.

[0055] LC-MS analysis: The test strain BS-9 was inoculated into 1 L of sterile LB liquid medium, and cultured with shaking at 28°C and 150 rpm for 5 days. Then, 50 mL of fermentation broth was taken and centrifuged at 12,000 r / min for 5 min. The supernatant was retained and filtered using a 0.22 μL filter membrane. An equal volume of ethyl acetate was added to the separatory funnel and shaken to mix thoroughly, then allowed to stand. After stratification, the shaking was continued and the operation was repeated three times. The upper organic phase was collected, and the lower aqueous phase was extracted with ethyl acetate for a total of three extractions. All the extracted organic phases were concentrated and evaporated to dryness using a rotary evaporator, and the evaporated material was dissolved and evaporated three times with 3 mL of chromatography-grade methanol.

[0056] LC-MS conditions: Column: F5 (100 mm × 2.1 mm, 2.6 μm); mobile phase A was ultrapure water, mobile phase B was chromatographic-grade acetonitrile, gradient elution conditions (mobile phase B, 0-2 min, 5%; 2-8 min, 5%-60%; 8-20 min, 60%-95%; 20-25 min, 95%; 25-25.01 min, 95%-5%; 25.01-27 min, 5%); injection volume was 10 μL, flow rate was 300 μL / min, and detection wavelength was 254 nm.

[0057] After HPLC separation, the samples were analyzed by electrospray ionization (ESI) in both positive and negative ion modes using a Q-Exactive quadrupole-orbitrap high-resolution mass spectrometer. ESI source conditions were as follows: source gas 1 and 2 at 60 V, curtain gas at 30 V, source temperature at 320°C, and ion source spray voltage at ±3.5 kV. The MS scan range was 80 to 1200 Da. Secondary mass spectra were acquired using data-dependent scanning in high-sensitivity mode, with a declustering potential of ±60 V and a collision energy of 35 ± 15 eV. The initial LC-MS data were interpreted and analyzed in detail using Qualitative Analysis B.05.00 software. The data were then compared with a database to determine the major components of the crude ethyl acetate extract of the fermentation broth of strain BS-9.

[0058] The primary extract of volatile organic compounds produced by the metabolism of strain BS-9 was obtained by headspace solid-phase microextraction. The volatile compounds produced during the fermentation of BS-9 were detected and analyzed by HS-SPME-GC-MS. Among the detected volatile compounds, there were 9 components with a similarity greater than 80%, as shown in Table 4.

[0059] Table 4 GC-MS analysis of volatile organic compounds in strain BS-9

[0060]

[0061] A total of nine volatile components produced by BS-9 metabolism were identified, with a similarity greater than 80%. These components primarily included carbazoles, oxadiazine, phenol, and alkanes. Among the volatile components of BS-9 metabolites identified in this study, 2,4-di-tert-butylphenol has been shown to have strong antibacterial properties against some plant pathogens, so it was initially selected for the next step in the efficacy evaluation experiment.

[0062] Metabolite components analyzed by LC-MS

[0063] The non-volatile primary extract produced by the metabolism of strain BS-9 was obtained by ethyl acetate extraction and rotary evaporation. LC-MS results showed that the primary extract of BS-9 fermentation broth matched nine known compounds, all of which appeared as single peaks. The compound names, mass-to-charge ratios, and substance types are detailed in Table 5.

[0064] Table 5 LC-MS analysis of organic active substances of strain BS-9

[0065]

[0066]

[0067] A total of 9 components with a similarity greater than 90% were identified among the non-volatile substances produced by BS-9 metabolism, mainly including esters, acids and lipopeptides.

[0068] Example 4 Potted plant control effect of antagonistic strains

[0069] The potted control efficacy test was conducted on the disease-resistant corn variety 'Xinzhongyu 801' and the susceptible corn variety 'Mintian 986'. Healthy corn plants with a height of approximately 15 cm were selected. Three treatments were set up in the experiment (three treatments for each variety). Purified colonies of the maize Alternaria leaf spot pathogen were picked and incubated in PDB at 28°C and 220 rpm for 7 days. After filtering to remove the mycelium, the concentration was adjusted to 1×10 7 CFU / mL of pathogen spore suspension was sprayed with 20 mL of sterile water (CK), 1×10 8 CFU / mL of BS-9 fermentation broth and a 1000-fold 45% prochloraz solution were used, with 30 plants treated each time and three replicates. Routine management was performed during the experiment. Disease indexes were calculated on days 7 and 14 after inoculation to calculate control efficacy. Disease grading standards were based on the methods in "GB / T 17980.107-2004 Guidelines for Field Efficacy Tests of Pesticides (II) Part 107: Control of Corn Large and Small Leaf Spots with Fungicides."

[0070] Experimental data were entered into Excel 2007 and analyzed using DPS 7.05 for univariate analysis. Duncan's method was used to analyze the inhibitory bandwidth and seedling efficacy of the antagonistic strain BS-9 against different maize leaf spot pathogens. Disease index (%) was calculated based on disease severity: disease index (%) = [∑(number of diseased spots × disease severity value)] / (number of inoculated spots × highest disease severity value) × 100%; control rate (%) = [(disease severity index of control group - disease severity index of treated group)] / control severity index × 100%. Lesion grading was based on (Subramani and Aalbersberg 2012) with appropriate adjustments: Grade 0: susceptible at the point of a puncture wound; Grade 1: lesion diameter less than 5 mm; Grade 2: lesion diameter 5-7 mm; Grade 3: lesion diameter 7-9 mm; Grade 4: lesion diameter 9-14 mm; Grade 5: lesion diameter greater than 14 mm.

[0071] The results of the pot test on the efficacy of the fermentation liquid of strain BS-9 against corn Alternaria leaf spot (14 days) are shown in Figure 5 , indicating that the corn plants in the control group inoculated with pathogens only grew weakly, with more yellow-brown or brown spots visible on the leaves, a more serious disease severity, and wilted leaves; the corn plants treated with prochloraz after inoculation with the pathogen had fewer leaf spots; compared with the control group and the experimental group treated with prochloraz, the corn plants inoculated with the fermentation liquid of strain BS-9 and the pathogen were sturdier, with good leaf growth, fewer leaf spots, and a lighter disease severity.

[0072] As shown in Table 6, 7 days after inoculation, the BS-9 fermentation liquid achieved 79.76% and 71.51% control against Alternaria leaf spot on the resistant cultivar 'Xinzhongyu 801' and the susceptible cultivar 'Mintian 986,' respectively. Fourteen days after inoculation, the BS-9 fermentation liquid achieved 70.61% and 57.86% control against Alternaria leaf spot on 'Xinzhongyu 801' and the susceptible cultivar 'Mintian 986,' respectively. Compared with the control effect of 1000-fold 45% prochloraz, the fermentation liquid of strain BS-9 demonstrated superior control against Alternaria leaf spot. These results demonstrate that this antagonistic bacterium has an inhibitory effect on Alternaria leaf spot caused by Alternaria alternifolia and exhibits promising potential for biocontrol applications.

[0073] Table 6 Potted control effect of strain BS-9 on corn Alternaria leaf spot

[0074]

[0075] Note: - indicates empty.

[0076] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0077] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Bacillus subtilis BS-9, characterized in that The BS-9 strain was deposited in the General Microbiology Center of the China Culture Collection Administration on March 27, 2024, with the deposit number CGMCC No. 30158, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is Bacillus subtilis.

2. The use of Bacillus subtilis BS-9 according to claim 1, characterized in that The uses include: 1) preventing and controlling corn leaf spot disease, and preparing a microbial agent for preventing and controlling corn leaf spot disease; 2) Antagonize the pathogenic bacteria of corn leaf spot.

3. The use according to claim 2, characterized in that The pathogenic bacteria are nigrococcus, sorghum epoecilomyces, maydis umbilical cordis, alternariae, and macrospotted umbilical cordis.

4. A sterile fermentation liquid of Bacillus subtilis BS-9 according to claim 1, characterized in that: The preparation process is as follows: the primary screened strain BS-9 is inoculated into LB liquid culture medium, cultured at 28°C and 220 rpm for 3 days, and then refrigerated centrifuged at 4°C and 10,000 rpm for 20 minutes. The sterile fermentation broth is filtered through a 0.22 μm filter membrane.

5. A preparation for preventing and treating corn leaf spot, characterized in that: The invention comprises Bacillus subtilis BS-9 bacterial agent or its sterile fermentation liquid.

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