Bacillus amyloliquefaciens ba-6 and application thereof in prevention and treatment of corn leaf spot disease

Abstract

The application discloses a bacillus amyloliquefaciens BA-6 and application thereof in prevention and treatment of corn leaf spot disease, and relates to the technical field of screening of biocontrol bacterial strains for prevention and treatment of corn leaf spot disease. The BA-6 strain is preserved in the China General Microbiological Culture Collection Center on March 27, 2024, has a preservation number of CGMCC No. 30157, a preservation address of No. 3, Xibei Road, Beichen, Chaoyang District, Beijing, and is classified and named as bacillus amyloliquefaciens. The strain can effectively antagonize black ball bacteria, sorghum ball bacteria, planospore corn, alternaria alternata and large spot convex umbilical rot fungus, and pot experiment shows that the fermentation liquor inoculated with the strain can improve the prevention and treatment effect on the corn leaf spot disease, thereby providing a theoretical basis for subsequent development of biocontrol agents for the corn leaf spot disease.

Description

Technical Field

[0001] This invention relates to the field of screening biocontrol strains for the control of maize leaf spot disease, and more specifically to a Bacillus amyloliquefaciens BA-6 and its application in the control of maize leaf spot disease. Background Technology

[0002] Corn is an important food and feed crop in my country and the world, and it is also the crop with the highest total yield globally, second only to rice and wheat in terms of planting area. However, corn is often affected by significant diseases such as leaf spot. Corn leaf spot is mainly 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 frequently or intermittently, causing significant losses to corn production in my country. Leaf spot is very harmful to corn production; in normal years, yield reduction in affected fields is around 10%, while in years with epidemics of susceptible varieties, yield reduction can exceed 50%, and in severe cases, it can lead to total crop failure. Currently, the control of leaf spot still relies mainly on chemical control and resistant varieties. However, due to the complexity of the pathogen, the significant variation and differentiation of pathogenic bacteria, the diversity and differences among corn varieties, and improper cultivation due to climate and expanding demand, highly effective and targeted chemical agents and resistant varieties have not yet been developed. While disease-resistant varieties cannot meet production needs, chemical control can easily lead to problems such as pesticide residues and environmental pollution, and cause pathogens to develop drug resistance.

[0003] Biological control, as a field with great development potential, has received much attention in recent years. Current research indicates that antagonistic bacteria are highly effective in controlling maize leaf spot disease, and significant progress has been made in utilizing the active organic compounds produced by biocontrol bacteria to inhibit the pathogen of maize leaf spot. However, the available strain resources are still relatively limited, and research on effective antibacterial substances is lacking. Therefore, it is necessary to screen for highly effective biocontrol strains that antagonize maize leaf spot pathogens and explore the antibacterial active organic compounds in their metabolites.

[0004] Therefore, how to screen out a strain that can control maize leaf spot disease is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides Bacillus amyloliquefaciens BA-6 and its application in the prevention and control of maize leaf spot disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a strain of Bacillus amyloliquefaciens BA-6, which was deposited on March 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30157, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Bacillus amyloliquefaciens.

[0008] A second aspect of the present invention provides the use of Bacillus amyloliquefaciens BA-6, the use including:

[0009] 1) To control corn leaf spot disease and to prepare microbial agents for controlling corn leaf spot disease;

[0010] 2) Antagonizes the pathogens that cause corn leaf spot disease.

[0011] Preferably, the pathogenic bacteria are *Helicobacter niger*, *Helicobacter sorghum*, *Helicobacter sacchariformis*, *Alternaria alternata*, and *Helicobacter davidii*.

[0012] The third aspect of the present invention provides a sterile fermentation broth of Bacillus amyloliquefaciens BA-6: the initially screened strain BA-6 is inoculated into LB liquid medium, cultured at 28°C and 220 r / min for 3 days, then centrifuged at 10000 r / min and 4°C for 20 min, and filtered through a 0.22 μm filter membrane to obtain a sterile fermentation broth.

[0013] The fourth aspect of this invention provides a formulation for preventing and controlling maize leaf spot disease, comprising Bacillus amyloliquefaciens BA-6 bacterial agent or its sterile fermentation broth.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention has screened a new biocontrol strain BA-6 that can antagonize the pathogens related to maize leaf spot disease. After 16S rDNA amplification and comparison, it was determined to be Bacillus amyloliquefaciens. This strain can effectively antagonize Bacillus niger, Bacillus sorghum, Helicobacter zebrina, Alternaria alternata, and Helicobacter davidii. Moreover, pot experiments showed that the fermentation broth inoculated with this bacterium can improve the control effect against maize leaf spot disease, providing a theoretical basis for the subsequent development of biocontrol agents for maize leaf spot disease. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 The attached figures show the colony characteristics and antibacterial effects of each pathogen. In particular, A, from left to right, shows the colony characteristics of the pathogens *Helicobacter niger*, *Helicobacter sorghum*, *Helicobacter auricula-judae*, *Alternaria alternata*, and *Helicobacter auricula-judae*. B, from left to right, shows the antibacterial effect of strain BA-6 on the five pathogens *Helicobacter niger*, *Helicobacter sorghum*, *Helicobacter auricula-judae*, *Alternaria alternata*, and *Helicobacter auricula-judae*.

[0017] Figure 2 The attached image shows the morphological characteristics of strain BA-6.

[0018] Figure 3 The attached figure shows the phylogenetic tree of strain BA-6 constructed based on the 16S rDNA gene sequence neighbor-joining method.

[0019] Figure 4 The attached figure shows the inhibitory effect of BA-6 metabolites on four pathogens (5d); AD: pure cultures of *Helicobacter niger*, *Helicobacter zebrina*, *Alternaria alternata*, and *Helicobacter macrocarpa*; ad: plate culture containing BA-6 fermentation broth.

[0020] Figure 5 The attached figure shows the pot control efficacy experiment of strain BA-6 against maize leaf spot, where: CK1: 'Xinzhongyu 801' maize inoculated only with the pathogen, A1: 'Xinzhongyu 801' maize treated with prochloraz and the pathogen, B1: 'Xinzhongyu 801' maize treated with BA-6 and the pathogen; CK2: 'Mintian 986' maize inoculated only with the pathogen, A2: 'Mintian 986' maize treated with prochloraz and the pathogen, B2: 'Mintian 986' maize treated with BA-6 and the pathogen. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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"). The tested maize pathogens were five strains: *Epicoccumnigrum* (maize leaf spot pathogen), *Micrococcus sorghum* (maize leaf spot pathogen), *Alternaria alternata* (maize leaf spot pathogen), *Bipolarismaydis* (maize small leaf spot pathogen), and *Exs erohilum turcicum* (maize large leaf spot pathogen), provided by the Institute of Plant Protection, Fujian Academy of Agricultural Sciences.

[0023] Reagents and culture media

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

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

[0026] Bacteria in soil were isolated using a dilution-spreading method. After drying the soil sample, 1g of crushed soil was weighed and repeatedly diluted 10-fold to 100% of the original sample. -2 10 -3 and 10 -4 After thoroughly mixing the diluted solutions at each concentration, pipette 200 μL onto PDA medium and spread evenly using a flame-sterilized spreader. Each concentration was tested in triplicate. The medium was incubated at 28°C for 3 days before transfer purification.

[0027] The strain purification was carried out using the streak plate method. The antagonistic bacteria to be purified were picked up from the medium containing the antagonistic bacteria in the initial screening using a sterile pipette tip and streaked in parallel on the surface of a sterile NA medium plate. One strain was streaked on 3 plates.

[0028] Initial screening: The plate confrontation method was used. Target pathogens were inoculated at the center of the culture medium, and biocontrol bacteria were inoculated 2.5 cm away from the central pathogen. A total of four inoculation points were selected, with three replicates. Biocontrol bacterial strains with good antibacterial effects were screened.

[0029] Secondary screening: The initially screened strains were inoculated into LB liquid medium and cultured at 28℃ and 220 rpm for 2 days with shaking. After centrifugation at 10000 rpm and 4℃ for 20 minutes, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain sterile fermentation broth. The broth was then cultured on potato dextrose agar (PDA) plates containing bacteria (1 × 10⁻⁶ ppm per 100 mL). 7 Place 5 mL of a CFU / mL suspension of leaf spot pathogen spores into Oxford cups at appropriate intervals. Inject 100 μL of biocontrol bacteria filtrate into each Oxford cup. After standing for 30 min, incubate at 28℃ for 4 days. Measure the diameter of the inhibition zone and record the results. Repeat each treatment three times to screen strains with strong antibacterial activity and good growth for subsequent experiments.

[0030] Using five pathogens of maize leaf spot disease as target bacteria, the inhibitory effect of antagonistic strains on the five pathogens was determined by the confrontation culture method. Each treatment was repeated three times, and all plates were incubated in the dark at 28℃. After the control group colonies fully covered the plates, the inhibition bandwidth of the experimental group was measured. The colony diameter was also measured, and the colony growth inhibition rate was calculated. The inhibition bandwidth is the distance between the edges of the antagonistic and pathogen colonies.

[0031] The results showed that 15 single colonies were isolated from soil samples at three different gradients. Three strains with inhibition zones greater than 5 mm were obtained from the initial screening and used for secondary screening. After culturing the initially screened strains against the pathogens for 7 days, the inhibition zone diameter of strain BS-9 was found to be greater than 5 mm. An inhibition spectrum experiment was conducted using strain BA-6 against five maize leaf spot pathogens, and the results showed that BA-6 had excellent antibacterial effects. Figure 1 Finally, strain BA-6 was selected for further classification and identification. The inhibition width is shown in Table 1.

[0032] Table 1. Inhibition bandwidth (5 days) of strain BA-6 against five pathogens causing maize leaf spot disease.

[0033]

[0034] The results of the antibacterial spectrum assay are as follows: Figure 1 As shown in Table 2, strain BA-6 exhibits varying degrees of inhibitory effects against five common maize leaf spot pathogens. The most significant inhibitory effect is against *Helicobacter pylori*, with an inhibition band of 14.33 mm. It also shows good inhibitory effects against the other four pathogens, with inhibition bands ranging from 5.85 to 12.23 mm. Therefore, strain BA-6 demonstrates good inhibitory effects against multiple common maize leaf spot pathogens.

[0035] Identification of antagonistic strains

[0036] Morphological and physiological / biochemical identification: First, the strain was inoculated onto PDA medium and incubated at 28°C for 2 days. Colony morphology was observed to preliminarily identify the strain. Then, the strain was inoculated onto NA medium, and colony color, transparency, and edge characteristics were observed and recorded. Based on the preliminary morphological identification results, Gram staining was performed on the strain. VP reaction, glucose fermentation, lactose fermentation, and maltose fermentation reactions were measured. Referencing *Manual of Systematic Identification of Common Bacteria* and *Bergey's Manual of Bacteriological Identification*.

[0037] The colonies are pale yellow or milky white, with irregular edges, and their surfaces are not moist, dull, or transparent. Figure 2 The strain was Gram-positive, with rod-shaped cells measuring 0.6–0.8 μm × 2.0–4.0 μm.

[0038] The physiological and biochemical characteristics of strain BA-6 are shown in Table 2. It was positive for spore formation, VP and 7% NaCl growth tests, but negative for citrate utilization and methyl red tests. It could not produce nitrogen fixation, phosphorus solubilization and potassium solubilization.

[0039] Table 2 Physiological and biochemical characteristics of strain BA-6

[0040] 1 Sporulation test + 2 Citrate utilization test - 3 Methyl red test - 4 VP test + 5 7% NaCl growth + 6 Indole-3-acetic acid + 7 Nitrogen fixation - 8 Phosphate solubilization - 9 Potassium solubilization -

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

[0042] Molecular biological identification: Genomic DNA of the biocontrol bacteria was extracted using a bacterial genomic DNA extraction kit (OMEGA). The BA-6 strain was molecularly identified by 16S rDNA sequence analysis. The PCR amplification primers for 16S rDNA were the universal 16S primers for bacteria, 27-F (5′-AGAGTTTGATCCTGGC TCAG-3′) / 1541-R (5′-AAGGAGGTGATCCAGCCGCA-3′), to amplify its sequence.

[0043] The PCR reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 45 s, 55℃ annealing for 90 s, 72℃ extension for 2 min, 35 cycles, and a final extension at 72℃ for 5 min. The obtained PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The resulting sequences were then assembled and analyzed using BLAST in the NCBI database. The corresponding GenBank accession numbers were obtained from NCBI. Based on the alignment analysis results, the 16S rDNA sequences of the relevant strains were downloaded from GenBank. After BLAST homology alignment, a phylogenetic tree was constructed using MEGA 7.0 Neighbor-joining analysis.

[0044] The results showed that amplification of the 16S rDNA of strain BA-6 yielded a 1477 bp sequence fragment. Sequence alignment in GenBank revealed a 99% similarity to the type strain BA09 (accession number: MT250917.1) of *Bacillus amyloliquefaciens*. A phylogenetic tree was constructed using 16S rDNA gene sequences from closely related *Bacillus* strains selected from the GenBank database. Figure 3 The results showed that strain BA-6 clustered with Bacillus amyloliquefaciens in the phylogenetic tree based on 16S rDNA. Combining morphological and physiological-biochemical characteristics, strain BA-6 was finally identified as Bacillus amyloliquefaciens and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30157, deposit date March 27, 2024, address No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. It was classified as Bacillus amyloliquefaciens.

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

[0046] The initially screened strain BA-6 was inoculated into LB liquid medium and cultured at 28℃ and 220 rpm for 3 days with shaking. Afterward, it was centrifuged at 10000 rpm for 20 minutes at 4℃ and filtered through a 0.22 μm filter to obtain sterile fermentation broth. This broth was added in three gradients (1 mL, 3 mL, and 5 mL) to 95 mL of PDA, and the plates were inverted and allowed to solidify. Four common pathogens—*Helicobacter niger*, *Helicobacter aegyptium*, *Alternaria alternata*, and *Helicobacter macrocarpa*—were selected and inoculated onto the center of plates containing BA-6 metabolites. The plates were then incubated at 28℃, with each treatment repeated three times. Pathogens cultured without sterile fermentation broth served as a control. Records were taken at 3, 5, and 7 days of culture. After 7 days of culture, the colony diameter was measured using the cross-cross method, and the inhibition rate of the antagonistic strain's metabolites was calculated. Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - mycelial cake diameter) × 100.

[0047] The antibacterial effect of the metabolites of strain BA-6 against four common pathogens of maize leaf spot disease was determined as follows: Figure 4 As shown, after 5 days of constant temperature incubation, the metabolites of strain BA-6 showed significant inhibitory effects on four common pathogens causing maize leaf spot disease.

[0048] The colony diameters of the CK and test groups were measured using the cross-multiplication method, and the antibacterial rates of the BA-6 metabolites were calculated as shown in Table 3. It can be seen that the BA-6 metabolites had the highest inhibition rate against *Pseudomonas niger*, reaching 96.70%, while the inhibition rates against the other three pathogens ranged from 93.17% to 95.57%, all showing good inhibitory effects.

[0049] Table 3. Antibacterial rate of BA-6 strain metabolites

[0050]

[0051]

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

[0053] GC-MS analysis: The tested strain BA-6 was inoculated into a screw-top glass bottle (15 mL) containing an appropriate amount of NA, sealed tightly, and incubated at 28°C for 5 days. The bottle was then preheated in a 40°C water bath for 20 min. A 100 μm polydimethylsiloxane (PDMS) extraction head was inserted, and headspace extraction was performed at 50°C for 30 min to collect volatile organic compounds from the bottle.

[0054] GC-MS conditions: DB-5MS flexible quartz capillary column (30 mm × 0.25 mm × 0.25 μm). Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min. -1 No shunt, inlet temperature 250℃. Heating process: initial temperature 35℃, heating for 1 min, then at 5℃·min. -1 The temperature was increased to 250℃ at a rate of [missing value], and heated for 5 min. Mass analysis conditions: interface temperature 250℃, ion source temperature 250℃, electron energy 70 eV, scan rate 0.5 s·scan. -1 The peak surface normalization method (quadrupole mass spectrometer, NIST05 standard library) was used for relative quantitative analysis of volatile organic compounds. After GC-MS analysis, the data were first compared with the database of the National Institute of Standards and Technology (NIST), and components with a similarity greater than 80% were selected for further component analysis.

[0055] LC-MS analysis: The test strain BA-6 was inoculated into 1L of sterile LB liquid medium and cultured at 28℃ and 150rpm for 5 days. 50mL of fermentation broth was taken and centrifuged at 12000r / min for 5min. The supernatant was retained and filtered through a 0.22μL filter membrane. An equal volume of ethyl acetate was added to a separatory funnel, shaken to mix, and allowed to stand until the layers separated. Shaking was repeated 3 times. The upper organic phase was collected, and the lower aqueous phase was extracted with ethyl acetate for a total of 3 extractions. All the extracted organic phases were concentrated to dryness using a rotary evaporator and dissolved in 3mL of chromatographic grade methanol in three portions.

[0056] LC-MS conditions: Column: F5 (100mm × 2.1mm, 2.6μm); 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] Samples separated by high-performance liquid chromatography (HPLC) were analyzed by electrospray ionization (ESI) in both positive and negative ion modes using a Q-Exactive quadrupole-electrostatic field orbital trap high-resolution mass spectrometer. ESI source conditions were as follows: ion source gases 1 and 2 were 60 g / L, curtain gas was 30 g / L, source temperature was 320 °C, and ion source spray voltage was ±3.5 kV; the MS scan range was 80–1200 Da. Secondary mass spectra were obtained using a data-dependent scan in high-sensitivity mode, with a declustering potential of ±60 V and a collision energy of 35 ±15 eV. Initial data obtained from LC-MS analysis were interpreted and analyzed in detail using Qualitative Analysis B.05.00 software, and then compared with a database to obtain the main component information of the crude ethyl acetate extract of strain BA-6 fermentation broth.

[0058] The initial extract of volatile organic compounds produced by the metabolism of strain BA-6 was obtained by headspace solid phase microextraction. The volatile compounds produced during the fermentation of BA-6 were detected and analyzed by HS-SPME-GC-MS. A total of 9 components with similarity greater than 80% were detected, as shown in Table 4.

[0059] Table 4. GC-MS analysis of volatile organic compounds in strain BA-6

[0060]

[0061] Nine components with a similarity greater than 80% were identified in the volatile substances produced by BA-6 metabolism, mainly including esters, alkanes, and phenols. Among the volatile components of BA-6 metabolites identified in this study, 2,4-di-tert-butylphenol has been shown to have strong antibacterial activity against some plant pathogens.

[0062] Metabolite components analyzed by LC-MS

[0063] Non-volatile primary extracts produced by the metabolism of strain BA-6 were obtained by extraction with ethyl acetate solvent and rotary evaporation. LC-MS results showed that 13 known compounds were matched in the primary extract of BA-6 fermentation broth, all of which showed single peaks. The names of the compounds, their mass-to-charge ratios and types are detailed in Table 5.

[0064] Table 5. LC-MS analysis of organic bioactive substances in strain BA-6

[0065]

[0066] Thirteen components with a similarity greater than 90% were identified among the non-volatile substances produced by BA-6 metabolism. These mainly included acids, amines, esters, cyclic peptide antibiotics, and lipopeptides.

[0067] Example 4: Potted control efficacy of antagonistic strains

[0068] A pot experiment was conducted to determine the control efficacy of the disease-resistant maize variety 'Xinzhongyu 801' and the susceptible variety 'Mintian 986'. Healthy maize plants approximately 15cm in height were selected, and the experiment included three treatments (one variety, three treatments). Purified maize leaf spot pathogen colonies were collected and cultured in PDB at 28℃ and 220 rpm for 7 days with shaking. After filtering to remove mycelium, a concentration of 1×10⁻⁶ was prepared. 7 20 mL of pathogenic spore suspension with CFU / mL was inoculated, and 2 days later, 20 mL of sterile water (CK) and 1×10⁻⁶ CFU / mL were sprayed on the spores. 8 CFU / mL BA-6 fermentation broth and 1000-fold diluted 45% prochloraz solution were used in three replicates per treatment, with 30 plants per treatment. Routine management was maintained during the experiment. Disease index was calculated on days 7 and 14 post-inoculation to determine control efficacy. Disease grading criteria were based on the methods outlined in GB / T17980.107-2004, "Guidelines for Field Efficacy Trials of Pesticides (II), Part 107: Fungicides for Control of Maize Leaf Spot Disease".

[0069] Experimental data were entered using Excel 2019. One-way analysis was performed using DPS 7.05, and the Duncan method was used to analyze the significant differences in the inhibitory bandwidth and seedling-stage control efficacy of antagonistic strain BA-6 against different maize leaf spot pathogens. Disease index was calculated based on disease severity: Disease index (%) = [∑(number of disease spots × disease severity value)] / (number of inoculation points × highest severity value) × 100%; Control rate (%) = [(disease index of control group - disease index of treatment group)] / disease index of control group × 100%. The lesion grading standards were based on (Subramani and Aal bersberg 2012) with appropriate adjustments: Grade 0: lesion at puncture wounds; 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.

[0070] The results of a pot experiment (14 days) on the control efficacy of strain BA-6 against maize leaf spot are shown below. Figure 5 The results showed that the control group of corn had weak growth, with more yellow-brown or brown lesions on the leaves, indicating a more severe disease and wilting of the leaves. The corn plants treated with the pesticide imazalil had fewer lesions on their leaves. Compared with the control group and the pesticide-treated experimental group, the corn plants in the experimental group inoculated with the fermentation liquid of strain BA-6 were more robust, with better leaf growth, fewer lesions on the leaves, and a milder disease.

[0071] As shown in Table 6, 7 days after inoculation, the control efficacy of BA-6 fermentation broth against maize leaf spot reached 95.31% and 83.00% in the resistant variety 'Xinzhongyu 801' and the susceptible variety 'Mintian 986', respectively. On day 14 after inoculation, the control efficacy of BV-3 fermentation broth against maize leaf spot reached 73.26% and 55.20% in 'Xinzhongyu 801' and the susceptible variety 'Mintian 986', respectively. Compared with the control efficacy of 1000-fold dilution of 45% prochloraz, the BA-6 fermentation broth showed better control effect against maize leaf spot. These results indicate that this antagonistic bacterium has an inhibitory effect on maize leaf spot caused by *Trichophyton spp.*, demonstrating good potential for biocontrol applications.

[0072] Table 6. Pot control efficacy of strain BA-6 against maize leaf spot.

[0073]

[0074] Note: - indicates an empty string.

[0075] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0076] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BA-6, characterized in that, The BA-6 strain was deposited on March 27, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30157, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens .

2. The Bacillus amyloliquefaciens according to claim 1 ( Bacillus amyloliquefaciens The purpose of BA-6 is characterized by, The uses include: 1) To control corn leaf spot disease and prepare microbial agents for controlling corn leaf spot disease; 2) Antagonizes the pathogens that cause corn leaf spot disease.

3. The use according to claim 2, characterized in that, The pathogenic bacteria are *Helicobacter niger*, *Helicobacter sorghum*, *Helicobacter sacchariformis*, *Alternaria alternata*, and *Helicobacter davidii*.

4. A Bacillus amyloliquefaciens according to claim 1 ( Bacillus amyloliquefaciens The aseptic fermentation broth of BA-6 is characterized in that, The preparation process is as follows: the initial screening strain BA-6 is inoculated into LB liquid medium, cultured at 28℃ and 220 r / min for 3 days, then centrifuged at 4℃ and 10000 r / min for 20 min, and filtered through a 0.22 μm filter membrane to obtain sterile fermentation broth.

5. A preparation for controlling maize leaf spot disease, characterized in that, Includes Bacillus amyloliquefaciens as described in claim 1 ( Bacillus amyloliquefaciens )BA-6 prepared microbial agent or its sterile fermentation broth.

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