Bacillus subtilis GL57, rain wash resistant microbial inoculum and application of bacillus subtilis GL57 and rain wash resistant microbial inoculum

Abstract

The invention discloses bacillus subtilis GL57, a rain wash resistant microbial agent and application thereof, and belongs to the technical field of agricultural microorganisms. The preservation number of the bacillus subtilis GL57 is CGMCC (China General Microbiological Culture Collection Center) No.35080. The verification shows that the strain GL57 has an excellent inhibition effect on the Zymophila maydis, which indicates that the strain GL57 has antibacterial activity. The microbial inoculum is obtained by combining a two-stage temperature control method with a carrier compounded rain wash resistant agent process. Under the field condition, the GL57 rain-wash-resistant microbial inoculum is applied to corn leaves and has a remarkable biological control effect on corn southern leaf blight, and it is indicated that the GL57 and the microbial inoculum thereof have good application potential and prospects in the aspect of gramineous plant disease control in corn southern leaf blight epidemic areas caused by frequent rainfall.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and in particular to a strain of Bacillus subtilis GL57, a rain-resistant bacterial agent, and its application. Background Technology

[0002] Small leaf spot disease in maize, caused by *Bipolaris maydis*, a fungus belonging to the Deuteromycotina subphylum, has become one of the major diseases threatening maize production. This pathogen infects leaves and stems, hindering photosynthesis, resulting in insufficient grain filling, and in severe cases, causing lodging. Currently, control methods still primarily rely on chemical agents, such as triazole fungicides like difenoconazole and tebuconazole. However, their long-term use has led to several problems: firstly, the mutation rate of resistance genes to difenoconazole in the pathogen has increased to 37%; secondly, chemical agents significantly damage the soil microbial community, reducing soil organic matter content by 0.3-0.5 percentage points per year; and thirdly, pesticide residues have resulted in a detection rate of azoxystrobin in maize kernels as high as 15%, exceeding EU export standards. Against this backdrop, the development of ecologically safe biological control technologies has become an urgent need for the industry.

[0003] For the fungal disease of maize leaf spot, identifying and utilizing biocontrol strains with broad-spectrum resistance and significant growth-promoting functions is a good biocontrol strategy. Studies have shown that the fermentation broth of some biocontrol bacteria has a high control rate against maize leaf spot, and rhizosphere growth-promoting bacteria (PGPRs) can promote crop growth and improve crop resistance to maize leaf spot by secreting metabolites such as indoleacetic acid (IAA) and siderophores. In actual production, high rainfall and humidity are the most important environmental conditions for the development of maize leaf spot, and continuous rainy weather often accompanies the disease in maize-growing areas where leaf spot is prevalent. Therefore, the application of biocontrol bacteria to control maize leaf spot needs to take into account the colonization of biocontrol bacteria on leaves under rain washout conditions. Therefore, improving the rain washout resistance of biocontrol agents is key to ensuring the control of maize leaf spot by biocontrol agents. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis GL57, a rain-resistant inoculant, and its application, in order to solve the problems existing in the prior art. The GL57 strain and its inoculant provided by this invention have good application potential and prospects for the prevention and control of diseases of gramineous plants in areas where corn leaf blight is prevalent due to frequent rainfall.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a strain of Bacillus subtilis GL57, which was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35080. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0007] The present invention also provides the application of the aforementioned Bacillus subtilis GL57 in the preparation of rain-resistant bacterial agents.

[0008] The present invention also provides a rain-resistant bacterial agent comprising Bacillus subtilis GL57, xanthan gum, chitosan and diatomaceous earth.

[0009] This invention also provides a method for preparing the rain-resistant bactericidal agent, comprising the following steps:

[0010] The seed culture of Bacillus subtilis GL57 was inoculated into a fermentation medium and cultured to obtain the fermentation broth;

[0011] The fermentation broth is centrifuged and the supernatant is discarded to obtain bacterial sludge. After resuspending, xanthan gum and chitosan are added, followed by the addition of diatomaceous earth. The mixture is then dried to obtain the rain-resistant bacterial agent.

[0012] Preferably, the fermentation medium consists of: 20 g / L glucose, 15 g / L soybean meal, 2 g / L KH2PO4 and 0.5 g / L MgSO4·7H2O, with an initial pH of 7.2.

[0013] Preferably, the amount of xanthan gum and chitosan added is 0.1% w / v of the bacterial sludge;

[0014] The mass ratio of the diatomaceous earth to the bacterial sludge is 1:2.

[0015] Alternatively, the drying process may further include a step of passing the material through a 60-mesh sieve.

[0016] The present invention also provides the application of the aforementioned Bacillus subtilis GL57 or the aforementioned rain-resistant bacterial agent in promoting corn growth.

[0017] The present invention also provides the application of the aforementioned Bacillus subtilis GL57 or the aforementioned rain-resistant bacterial agent in the prevention and control of corn leaf blight.

[0018] The present invention also provides a method for preventing and controlling corn leaf spot, including the step of treating corn leaves with the aforementioned rain-resistant bactericidal agent.

[0019] Optionally, the method for treating corn leaves includes uniformly spraying the rain-resistant bactericidal agent onto both sides of the corn leaves.

[0020] The present invention discloses the following technical effects:

[0021] This invention screened and isolated a strain of Bacillus subtilis (B. subtilis) GL57, which was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35080. Verification showed that strain GL57 exhibited excellent inhibitory activity against *Helicobacter spp.* zeolites, indicating that strain GL57 possesses antibacterial activity. Its inoculum was obtained through a two-stage temperature control method combined with a carrier and a rain-resistant agent. Under field conditions, application of the GL57 inoculum to maize leaves showed significant biocontrol effects against maize leaf spot disease, demonstrating that the GL57 and its inoculum provided by this invention have good application potential and prospects for the control of gramineous plant diseases in areas where maize leaf spot disease is prevalent due to frequent rainfall. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the inhibitory effect of Bacillus subtilis GL57 on corn leaf blight pathogens; the left side is the Escherichia coli DH 5α control, and the right side is the GL57 strain.

[0024] Figure 2 Phylogenetic tree of Bacillus subtilis GL57;

[0025] Figure 3 The control effect of Bacillus subtilis GL57 inoculum on maize leaf spot disease caused by B. maydis (Figure A) and disease index (B);

[0026] Figure 4 A statistical graph showing the effects of Bacillus subtilis GL57 inoculum treatment on maize plant height (A), stem diameter (B), single ear grain weight (C), and grain yield (D). Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0033] Preparation of the culture medium used in the examples:

[0034] (1) Potato glucose agar medium (PDA medium): Cut 200 g of potatoes into small pieces and put them in a pot. Add 1000 mL of water and heat to boiling on a heater. Maintain for 30 min. Filter the hot liquid through two layers of gauze on a measuring cup and discard the residue. Add 20 g of glucose and 20 g of agar to the filtrate and add water to 1000 mL. Then put it into a high-temperature autoclave for sterilization. The sterilization program is 121℃ for 15 min.

[0035] (2) Luria-Bertani medium (LB medium): Weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride into a beaker or Erlenmeyer flask. Add approximately 800 mL of deionized water, stir magnetically or heat (≤60℃) until completely dissolved, then add water to 1 L and gently stir to mix. Dispense the medium into test tubes or Erlenmeyer flasks (liquid culture is usually filled to ≤1 / 3 of the container volume) and autoclave at 121℃ (20 min). Use after cooling to room temperature, or store at 4℃ for later use. The preparation steps are the same as for liquid LB, but agar powder (1.5% w / v) needs to be added before final volume adjustment.

[0036] Example 1: Isolation and Identification of Strains

[0037] 1. Soil sample collection and pretreatment

[0038] In my country's main maize-producing areas, continuously cropped maize fields were selected as sampling areas. The sampling sites were located in areas with a high incidence of maize leaf spot disease, and the soil types were brown soil and alluvial soil, representing typical characteristics. At each sampling point, 10 healthy maize plants with uniform growth were selected. Rhizosphere soil from 0-20 cm depth around the plant roots was collected using a sterile shovel. To minimize environmental disturbance, the top 5 cm of topsoil was removed, and the roots were gently brushed with a sterile brush. Approximately 50 g of soil tightly adhering to the root surface was collected, mixed, and placed in a sterile polyethylene bag, immediately transported to the laboratory in a 4°C ice box. The soil sample was evenly spread in a laminar flow hood, and visible plant debris and stones were removed. The sample was then passed through a 2 mm sterile sieve. 10 g of soil was weighed and added to 90 mL of sterile phosphate-buffered saline (PBS, pH 7.4), and vortexed at 3000 rpm for 30 min in an IKA Vortex Genius 3 oscillator to fully disperse soil aggregates. Then, serial dilutions were performed: 1 mL of suspension was added to 9 mL of PBS, and 10 mL of the solution was prepared sequentially. -1 Up to 10 -6 Diluent. Take 100 μL of each dilution and spread it evenly on LB agar plates (containing 100 μg / mL actinomycete ketone to inhibit fungal growth), and incubate at 30°C for 24–48 h. Observe colony morphology daily and record the time of single colony appearance and distribution density.

[0039] 2. Screening for antibacterial activity

[0040] Bipolaris maydis, the pathogen causing small leaf spot in maize (published in the literature Guichard B, Wu H, La Camera S, Hu R, Marivingt-Mounir C, Chollet JF. Synthesis, phloemmobility and induced plant resistance of synthetic salicylic acid amino acidor glucose conjugates. Pest Manag Sci. 2022 Nov;78(11):4913-4928. doi:10.1002 / ps.7112. Epub 2022 Aug 26. PMID: 36054797; PMCID: PMC9804902.), was inoculated onto PDA medium (potato dextrose agar) and cultured at 28°C for 5 days. Mycelial cakes with a diameter of 5 mm were collected using a sterile punch. Single colonies of the isolated bacteria were inoculated onto LB liquid medium and cultured at 37°C with shaking at 180 rpm for 18 h until OD (dose retardation). 600 =1.2. Take 5 μL of bacterial suspension and spot it onto one side of a PDA plate, 2.5 cm from the center; place the pathogenic bacterial pellet in the center, and place the negative control symmetrically on the other side. Use *Escherichia coli* DH 5α as the negative control, with three replicates per group. Incubate the plates in the dark at 28℃ for 5 days. Measure the maximum length from the edge of the pathogenic colony to the inhibition zone using calipers (accuracy 0.02 mm), and calculate the inhibition rate: IR (%) = (D0 - Dt) / D0 × 100% (D0 is the control diameter, Dt is the treatment group colony diameter). The inhibitory effect of strain GL57 on *Sclerotinia sclerotiorum* is as follows: Figure 1 As shown, strain GL57 has a strong antibacterial effect, with a calculated inhibition rate of 78% (P<0.01, one-way ANOVA).

[0041] 3. Molecular biological identification

[0042] One mL of GL57 cells in logarithmic growth phase was lysed using the TIANGEN Bacterial Genomic DNA Extraction Kit (DP302). After treatment with lysozyme (10 mg / mL) at 37°C for 30 minutes, proteinase K and buffer GB were added, and the mixture was incubated at 65°C for 30 minutes. The DNA was then purified using an adsorption column, eluted with 50 μL of TE buffer, and stored at -20°C. The 16S rRNA gene was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.2) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.3).

[0043] Reaction system (25 μL): 12.5 μL 2×Taq PCR Master Mix, 0.5 μM each of forward and reverse primers, 1 μL template DNA, and ddH2O to make up. Amplification conditions: 94℃ pre-denaturation for 5 min; 30 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 90 s; final extension at 72℃ for 10 min. The amplified products were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. An approximately 1000 bp 16S rRNA gene sequence (SEQ ID NO.1) was obtained. BLAST alignment using the EzBioCloud database showed a 99.7% similarity to Bacillus subtilis. Multiple sequence alignment (Clustal W algorithm) was performed using MEGA 11 software, and a phylogenetic tree was constructed using the Neighbor-Joining method (Bootstrap value 1000 replicates). The results showed that GL57 clustered with Bacillus subtilis in the same clade (Bootstrap support rate 98%), and showed significant differentiation from closely related species such as Bacillus subtilis. Figure 2 ).

[0044] SEQ ID NO.1:

[0045]

[0046] Therefore, the above-mentioned strain was identified as Bacillus subtilis and named Bacillus subtilis GL57. This strain was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 35080.

[0047] Example 2 Preparation of microbial agent

[0048] 1. Fermentation process optimization

[0049] GL57 bacterial culture was retrieved from a -80℃ glycerol storage tube and streaked onto LB agar plates, incubated at 37℃ for 24 h. Single colonies were picked and inoculated into 50 mL LB liquid medium (250 mL shake flask), and incubated at 37℃ with shaking at 180 rpm for 12 h until OD reached. 600 =1.5, used as the primary seed solution. The optimal combination of carbon and nitrogen sources was determined through single-factor experiments.

[0050] The single-factor experiments to screen the optimal carbon and nitrogen source combination followed the principle of optimization from carbon to nitrogen: using a basal culture medium (formulation: 1L of medium containing 5g peptone, 4.5g dipotassium hydrogen phosphate, 1.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 10mg ferric sulfate heptahydrate, 5mg manganese sulfate monohydrate, and 10mg calcium chloride; after preparation, the initial pH was adjusted to 7.0-7.4 and autoclaved at 121℃ for 20 minutes) as the base, the effects of adding 20 g / L glucose, sucrose, and soluble starch as single carbon sources on bacterial growth were tested. The results showed that when glucose was used as the carbon source, the bacterial biomass (OD) was significantly higher. 600 The OD50 of sucrose is the highest, reaching 1.50, therefore it was determined to be the optimal carbon source (OD50 of sucrose and soluble starch). 600 (1.13 and 1.25 respectively).

[0051] Subsequently, under the condition of fixing the optimal carbon source glucose (20 g / L), fermentation was compared by adding soybean meal, peptone, and ammonium sulfate as nitrogen sources of different properties at equal nitrogen amounts (equivalent to 0.8 g / L nitrogen element). The results showed that organic nitrogen sources were more conducive to cell growth, and the cell biomass (OD) was higher when soybean meal was used as the nitrogen source. 600 Both the growth rate (1.68%) and spore formation rate were significantly better than other groups, while the inorganic nitrogen source ammonium sulfate group had the worst growth (OD). 600 =0.85) (OD of peptone and ammonium sulfate) 600(The values ​​were 0.75 and 0.68 respectively). This ultimately determined that glucose and soybean meal were the optimal carbon and nitrogen source combination for this strain, laying the foundation for subsequent culture medium optimization.

[0052] Therefore, based on bacterial biomass (OD) 600 Based on the indicators of spore formation rate and fermentation cost and raw material availability, the optimal fermentation medium was selected: glucose 20 g / L, soybean meal powder 15 g / L, KH2PO4 2 g / L, MgSO4·7H2O 0.5 g / L, initial pH 7.2. Under this formula, the cell growth rate was 1.8 times higher than that of the basic LB medium.

[0053] Fermentation was controlled in two stages. The first stage was the cell proliferation period (0-18 h): the primary seed culture was inoculated into a 5 L fermenter (BIOTECH-5BG, Baoxing Co.) at a 2% inoculation rate, and the temperature was controlled at 37℃, the stirring speed at 200 rpm, the aeration rate at 1.0 vvm, and the dissolved oxygen (DO) was maintained above 30%. During this stage, the cells proliferated rapidly, and the viable cell count reached 8.6 × 10⁶ cells / year at 18 h. 9 CFU / mL. The second stage was the spore induction period (18-36 h): the temperature was lowered to 30℃, the rotation speed was adjusted to 150 rpm, and the aeration rate was reduced to 0.8 vvm. Spore formation was induced by limiting the carbon source (glucose concentration <2 g / L) and increasing the osmotic pressure (adding 0.3 mol / L NaCl). At 36 h, the spore formation rate was >95%, and the total viable count reached 1.2 × 10⁻⁶ CFU / mL. 10 CFU / mL. The fermentation broth was filtered and centrifuged (4000×g) for 10 minutes. The supernatant was discarded, and the bacterial sludge was resuspended in sterile physiological saline to 1 / 10 of its original volume. An anti-erosion agent and a carrier (0.1% xanthan gum + 0.1% chitosan as the anti-erosion agent, and diatomaceous earth as the carrier) were added, and the mixture was stirred thoroughly for 30 minutes to ensure uniform bacterial adsorption. The mixture was then transferred to a fluidized bed dryer (FLB-5, Changzhou), with the inlet air temperature set at 40℃ and the air velocity at 1.5 m / s, and dried until the moisture content was ≤8%. The dried granules were sieved through a 60-mesh sieve to obtain a free-flowing powdered bacterial agent. Testing showed that the initial viable count of the agent was 5.8 × 10⁻⁶. 8 The viable bacterial count (CFU / g) after 180 days of storage at 25°C in the dark was 4.3 × 10⁻⁶. 8 CFU / g (survival rate 74.1%).

[0054] 2. Screening of anti-erosion agents and carriers

[0055] Comparing different substances (combinations) that resist rain erosion, including sodium alginate, xanthan gum, carboxymethyl cellulose (CMC), starch derivatives, polyvinyl alcohol (PVA), polyacrylamide, and chitosan, it was found that the combination of xanthan gum and chitosan can significantly improve the rain erosion resistance of GL57 in maize leaves. Single-factor and multi-factor combination methods were used for screening; the specific screening procedures are as follows.

[0056] Different anti-rain-washing agents were prepared using single-component formulations of 0.2% sodium alginate, 0.2% xanthan gum, 0.2% carboxymethyl cellulose (CMC), 0.2% starch derivative, 0.2% polyvinyl alcohol (PVA), 0.2% polyacrylamide, and 0.2% chitosan, as well as combinations of two-factor formulations (considering cost, the concentration of each factor was reduced to half of the single-factor concentration). After spraying onto surface-disinfected corn leaves (soaked in 0.5% sodium hypochlorite solution for 8 min) for 1 h, simulated rainfall (30 mm / h, lasting 10 min) was applied, and the leaves were then covered with LB medium. Colony counts were calculated to test the bacterial retention rate. The results showed that when 0.1% xanthan gum and 0.1% chitosan were used in combination, the cell retention rate was significantly increased to 89.7%, which was significantly higher than any single component and also better than other binary combinations (Table 1). This proves that the excellent film-forming properties of xanthan gum and the good adhesion of chitosan produced a synergistic effect, thus providing the best anti-rain erosion performance.

[0057] Table 1 Screening of different rainwater erosion resistant agents

[0058] serial number formula concentration Retention rate (%) 1 Sodium alginate 0.2% 35.5 2 Xanthan Gum 0.2% 58.6 3 Carboxymethyl cellulose 0.2% 28.9 4 Starch derivatives 0.2% 19.6 5 Polyvinyl alcohol 0.2% 16.1 6 Polyacrylamide 0.2% 24.5 7 Chitosan 0.2% 46.2 8 Sodium alginate + xanthan gum 0.1% each 49.9 9 Sodium alginate + CMC 0.1% each 33.4 10 Sodium alginate + starch derivatives 0.1% each 25.5 11 Sodium alginate + PVA 0.1% each 35.4 12 Sodium alginate + polyacrylamide 0.1% each 26.2 13 Sodium alginate + chitosan 0.1% each 39.8 14 Xanthan Gum + CMC 0.1% each 67.3 15 Xanthan gum + starch derivatives 0.1% each 59.4 16 Xanthan gum + PVA 0.1% each 71.8 17 Xanthan gum + polyacrylamide 0.1% each 64.5 18 Xanthan gum + chitosan 0.1% each 89.7 19 CMC+ starch derivatives 0.1% each 26.6 20 CMC+PVA 0.1% each 29.1 21 CMC + Polyacrylamide 0.1% each 34.6 22 CMC + Chitosan 0.1% each 31.4 23 Starch derivatives + PVA 0.1% each 26.4 24 Starch derivatives + polyacrylamide 0.1% each 28.7 25 Starch derivatives + chitosan 0.1% each 25.3 26 PVA + Polyacrylamide 0.1% each 21.4 27 PVA + Chitosan 0.1% each 28.9 28 Polyacrylamide + chitosan 0.1% each 34.3

[0059] Note: Chitosan should be dissolved in 1% acetic acid before use.

[0060] By comparing the adsorption properties of carriers such as diatomaceous earth, bentonite, and humic acid, it was found that diatomaceous earth (particle size 80-120 mesh) had the highest adsorption rate (92.3%) for GL57 spores. The carrier selection process was accomplished by systematically comparing the adsorption capacity and survival rate of different materials on GL57 spores: equal amounts of diatomaceous earth (80-120 mesh), bentonite (200 mesh), humic acid (powder), and light calcium carbonate were weighed as candidate carriers and added to an equal volume of spore fermentation broth (1.0 × 10⁻⁶ m³). 10After adsorption of the adsorbed spores using a mixture of diatomaceous earth (CFU / mL) for 2 h, the adsorption rate was measured. The results showed that diatomaceous earth had the highest adsorption rate (92.3%), significantly better than bentonite (85.1%), humic acid (78.6%), and light calcium carbonate (70.2%). Further analysis of spore viability after storing the adsorbed carriers at 4℃ for 30 days revealed that the diatomaceous earth group maintained a viability rate of 88.5%, also higher than other carriers (80.1% for bentonite and 75.3% for humic acid). Considering both adsorption performance and spore preservation stability, diatomaceous earth was ultimately determined to be the optimal carrier.

[0061] Example 3 Rainwater erosion and prevention efficacy verification test

[0062] The soil type used in the field experiment was typical brown soil, with the following basic fertility: organic matter 1.35%, available nitrogen 78.4 mg / kg, available phosphorus 21.3 mg / kg, available potassium 145.6 mg / kg, and pH 6.8. The previous crop was maize, and the annual incidence of small leaf spot disease in the field was 35%-40%.

[0063] The maize variety planted was Zhengdan 958. Potted maize seedlings were treated with a microbial agent by foliar spraying after reaching the 5-6 leaf stage. A control group (CK, treated with a microbial agent without xanthan gum and chitosan) and a rain-resistant microbial agent treatment group (treated with the microbial agent prepared in Example 2) were set up. Each treatment was repeated three times, for a total of six plots, each 30 m² (6 m × 5 m), randomly arranged with a protective row around the perimeter.

[0064] The bacterial agent was evenly sprayed onto both sides of the leaves. After air-drying for 1 hour, a rain washout experiment was conducted. A spray device was used to simulate rainfall, spraying 500 mL of sterile water per plant from a height of 50 cm over 5 minutes to simulate high-intensity rain washout. After washing and air-drying for 1 hour, samples were taken. Standard leaf discs were collected using a leaf flushing device, and the bacterial cells on the leaf surface were eluted by vortexing with PBS solution containing Tween-80. The eluent was serially diluted and plated for culture, and the colony count at each dilution was recorded. A two-way ANOVA was used to test the interaction effect of adjuvant type and washout time on the residual rate, and multiple comparisons were performed using the Tukey method (significance level P < 0.05).

[0065] The results showed that the control group of 10 3 The average colony count on the 10-fold dilution plates was 5, and that of the treatment group was 10. 3 The average colony count on the diluted plates was 102, indicating that the colonization of the leaves in the rain-resistant bacterial agent treatment group was significantly higher than that in the control group after being washed by rain, with a 20-fold increase.

[0066] When maize leaf spot disease occurred, 10 plants were surveyed in each group, and the leaf lesion area was recorded by grade. The disease index grading standard for maize leaf spot disease adopted the internationally accepted percentage of lesion area method, with 6 grades: Grade 0 (no lesions on the entire leaf), Grade 1 (lesion area ≤ 5% of leaf area), Grade 3 (lesion area 6%–10% of leaf area), Grade 5 (lesion area 11%–20% of leaf area), Grade 7 (lesion area 21%–50% of leaf area), and Grade 9 (lesion area > 50% of leaf area). The disease index (DI) was calculated using the formula (Disease index, DI = Σ(disease grade × number of plants) / (highest disease grade × total number of plants) × 100).

[0067] Results: The disease index of the rain-resistant bacterial agent treatment group was 17.5±2.6, significantly lower than that of the CK group (38.5±3.4, P<0.01), with a relative control efficacy of 54.5%. Figure 3 ).

[0068] At harvest, 20 plants from each group were sampled to determine agronomic traits (plant height, stem diameter, single ear weight, and grain yield). Results are as follows: Figure 4 As shown, the plant height in the rain-resistant bacterial agent treatment group was 276.8±14.1 cm, the stem diameter was 2.9±0.3 cm, and the grain weight per ear was 190.0±21.1 g, which were 10.1%, 12.3%, and 13.5% higher than those in the control group (250.7±13.2 cm, 2.6±0.1 cm, and 167.4±23.2 g), respectively. The grain yield reached 795.1 kg / mu, an increase of 8.9% compared to the control group (730.1 kg / mu).

[0069] In summary, this invention screened and isolated a strain of Bacillus subtilis, GL57. Verification revealed that strain GL57 exhibits excellent inhibitory activity against *Helicobacter spp.* in maize, indicating its antibacterial activity. The bacterial agent was obtained through a two-stage temperature control method combined with a carrier and a rain-resistant agent. Under field conditions, application of the GL57 rain-resistant bacterial agent to maize leaves showed significant biocontrol effects against maize leaf spot, demonstrating that the GL57 and its bacterial agent provided by this invention have good application potential and prospects for the control of gramineous plant diseases in areas where maize leaf spot is prevalent due to frequent rainfall.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Bacillus subtilis GL57, characterized in that, It was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35080, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of Bacillus subtilis GL57 as described in claim 1 in the preparation of rain-resistant bacterial agents.

3. A rain-resistant bactericidal agent, characterized in that, It contains Bacillus subtilis GL57, xanthan gum, chitosan, and diatomaceous earth as described in claim 1.

4. The preparation method of the rain-resistant bacterial agent as described in claim 3, characterized in that, Includes the following steps: The seed culture of Bacillus subtilis GL57 was inoculated into a fermentation medium and cultured to obtain the fermentation broth; The fermentation broth is centrifuged and the supernatant is discarded to obtain bacterial sludge. After resuspending, xanthan gum and chitosan are added, followed by the addition of diatomaceous earth. The mixture is then dried to obtain the rain-resistant bacterial agent.

5. The preparation method according to claim 4, characterized in that, The fermentation medium consisted of: 20 g / L glucose, 15 g / L soybean meal, 2 g / L KH2PO4 and 0.5 g / L MgSO4·7H2O, with an initial pH of 7.

2.

6. The preparation method according to claim 4, characterized in that, The amount of xanthan gum and chitosan added is 0.1% w / v of the bacterial sludge; The mass ratio of the diatomaceous earth to the bacterial sludge is 1:

2.

7. The application of Bacillus subtilis GL57 as described in claim 1 or the rain-resistant bacterial agent as described in claim 3 in promoting maize growth.

8. The application of Bacillus subtilis GL57 as described in claim 1 or the rain-resistant bacterial agent as described in claim 3 in the control of corn leaf blight.

9. A method for controlling corn leaf blight, characterized in that, The step includes treating corn leaves with the rain-resistant bactericidal agent as described in claim 3.

10. The method as described in claim 9, characterized in that, The method for treating corn leaves includes uniformly spraying the rain-resistant bactericidal agent onto both sides of the corn leaves.

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