A strain of Bacillus erythropoietinus and its application

Abstract

This invention provides a strain of *Bacillus erythropoietinus* and its applications, belonging to the field of bioengineering technology. This *Bacillus erythropoietinus* is classified and named... Paenibacillus ehimensis WX01, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2026113, dated January 15, 2026, describes a strain of *Bacillus erinaceus* that can ferment and simultaneously produce 2-methylbutyric acid (2-methylbutyric acid) and 6-methyl-2-heptanone, showing promising applications in plant disease control. Further research revealed that the concentrations of 2-methylbutyric acid and 6-methyl-2-heptanone obtained through this strain can reach 4.85 g / L and 48.34 mg / L, respectively. The fermentation production of 2-methylbutyric acid and 6-methyl-2-heptanone using this strain is low-cost, high-yield, safe, and environmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a strain of Bacillus erythropoietinus and its applications. Background Technology

[0002] The genera *Bacillus*, *Cladosporium*, and *Pseudomonas* contain a variety of common biocontrol bacteria. These biocontrol bacteria can produce antimicrobial peptides or other chemical substances through metabolism, thereby killing pests or inhibiting the growth of other harmful pathogens, thus controlling agricultural plant diseases. Research has shown that biocontrol bacteria that control plant diseases include not only antimicrobial peptides produced by their metabolism, but also many natural chemical substances.

[0003] Common natural compounds used for plant disease control include 2-methylbutyric acid, 6-methyl-2-heptanone, methyl 2-ethylhexanoate, and hexanonitrile. 2-Methylbutyric acid is an organic acid naturally found in fruits and vegetables such as apples and strawberries. Studies have shown that 2-methylbutyric acid can inhibit the growth of plant pathogens such as *Aspergillus fumigatus*, *Cladosporium*, *Alternaria*, *Botrytis cinerea*, and *Penicillium expansum*, demonstrating its great potential as a fumigant in preventing plant diseases. 6-Methyl-2-heptanone is naturally found in tobacco leaves. Research indicates that 6-methyl-2-heptanone, as a fumigant, can effectively inhibit various pathogenic fungi, including *Coccidioidomyces chinensis*, *Rhizoctonia solani*, *Prunus cerevisiae*, *Verticillium dahliae*, and *Alternaria solanacea*. Its mechanism of action includes disrupting hyphal structure, increasing cell membrane permeability, and inducing ATP leakage, thus showing promising application prospects in the field of plant disease control.

[0004] The preparation methods for the aforementioned natural compounds generally employ chemical synthesis; the biological synthesis of 2-methylbutyric acid and 6-methyl-2-heptanone has not been reported. Currently, the chemical synthesis of 2-methylbutyric acid suffers from drawbacks such as expensive raw materials, complex operations, low yield, large wastewater volume, and poor safety. The chemical synthesis of 6-methyl-2-heptanone involves the catalytic hydrogenation of 6-methyl-3-hepten-2-one as a raw material; this process is cumbersome, costly, and generates significant pollution. To address the high cost and safety risks associated with the chemical synthesis of 2-methylbutyric acid and 6-methyl-2-heptanone, it is necessary to screen for a bacterial strain capable of producing these control compounds. This would enrich the production of these natural compounds, avoid the pollution and safety issues associated with chemical synthesis, and enable their application in plant disease control. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems by screening a strain capable of producing the above-mentioned preventive compounds; another purpose of this invention is to provide the application of this strain.

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

[0007] This invention isolates a strain of *Bacillus ehime* from the soil of a planting base of Suzhou Kanglai Ecological Agriculture Development Co., Ltd., located in Changshu City, Suzhou, Jiangsu Province, China. This *Bacillus ehime* is classified as... Paenibacillus ehimensis WX01 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M2026113, deposited on January 15, 2026, and identified as viable on January 22, 2026. The China Center for Type Culture Collection (CCTCC) is located at Wuhan University, Wuhan, China, 430072, China.

[0008] This strain has the following properties: it is a rod-shaped, Gram-positive bacterium, and it is positive for VP test, starch hydrolysis test, catalase test, gelatin liquefaction test and nitrate reduction test; it is negative for indole test, citrate test, H2S gas production test and methyl red test.

[0009] This invention, through research, discovered that this *Ehime-like* spore-forming bacterium... Paenibacillus ehimensis WX01 exhibits different properties compared to the commercially available Bacillus erythropoietinus strain with accession number CGMCC 1.3451. Paenibacillus ehimensis Fermentation of WX01 can simultaneously produce the natural compounds 2-methylbutyric acid and 6-methyl-2-heptanone, which can be used for plant disease control, while the aforementioned commercially available *Bacillus erythropoietinus* strain did not show any related compounds; therefore, this *Bacillus erythropoietinus* strain... Paenibacillus ehimensis WX01 has the potential to simultaneously produce 6-methyl-2-heptanone and 2-methylbutyric acid.

[0010] Based on this Ehime-like Bacillus Paenibacillus ehimensis Based on the characteristics of WX01, this invention provides the application of the aforementioned Ehime-like Bacillus in the prevention and control of plant diseases.

[0011] Based on this Ehime-like Bacillus Paenibacillus ehimensis Based on the characteristics of WX01, this invention provides the application of the above-mentioned Ehime-like Bacillus in the production of 2-methylbutyric acid or 6-methyl-2-heptanone.

[0012] Furthermore, the *Bacillus erythropoietinus* was inoculated into a fermentation medium and cultured aerobically to prepare 2-methylbutyric acid or 6-methyl-2-heptanone.

[0013] Furthermore, the fermentation medium comprises the following components: 10-60 g / L carbon source, 4-25 g / L nitrogen source, 0.5-1.0 g / L metal salt, pH 6.5-7.5, and water as solvent.

[0014] Furthermore, the nitrogen source is any one or a combination of several of the following: tryptone, beef extract, soybean powder, yeast powder, urea, ammonium chloride, sodium nitrate, ammonium sulfate, ammonium nitrate, and ammonium dihydrogen phosphate.

[0015] Furthermore, the carbon source is one or a combination of several of lactose, sucrose, glucose, fructose, and maltose.

[0016] Furthermore, the metal salt is one or a combination of several of zinc chloride, manganese sulfate, calcium chloride, magnesium sulfate, and ferrous sulfate.

[0017] Furthermore, the fermentation medium comprises the following components: sucrose 10-50 g / L, beef extract 4-20 g / L, ammonium sulfate 5-25 g / L, and magnesium sulfate 0.20-0.70 g / L. The initial pH of the fermentation broth is adjusted to 6.0-8.0 using ammonia water.

[0018] Furthermore, the aerobic culture conditions are as follows: pH 6.0-8.0, culture temperature 28-38℃; inoculum size 1-14% of fermentation medium volume, and culture time 10-48 h.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) This invention has discovered a strain of Ehime-like Bacillus. Paenibacillus ehimensis Compared to other strains of the same species, strain WX01 can ferment and produce 2-methylbutyric acid and 6-methyl-2-heptanone simultaneously, showing promising applications in plant disease control and the production of 2-methylbutyric acid and 6-methyl-2-heptanone.

[0021] (2) Through further research on the composition of its fermentation medium and fermentation conditions, the concentration of 2-methylbutyric acid accumulated in the medium can reach up to 4.85 g / L, and the concentration of 6-methyl-2-heptanone can reach up to 48.34 mg / L. Compared with traditional chemical synthesis, the production cost of 2-methylbutyric acid and 6-methyl-2-heptanone by this Bacillus erythropoietin is low, and it has the advantages of simple operation, strong safety and environmental friendliness, and has the prospect of industrial promotion and application. Attached Figure Description

[0022] Figure 1 Colony characteristics of Bacillus erythropoietinus WX01.

[0023] Figure 2 Phylogenetic tree of Bacillus erythrozoae WX01.

[0024] Figure 3 The effect of carbon source type on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0025] Figure 4 Effect of carbon source concentration on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0026] Figure 5 The effect of organic nitrogen source on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0027] Figure 6 Effects of organic nitrogen source concentration on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0028] Figure 7 The effect of inorganic nitrogen source type on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0029] Figure 8 Effect of inorganic nitrogen source concentration on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0030] Figure 9 The effect of metal ion type on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0031] Figure 10 Effects of metal ion concentration on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0032] Figure 11 Effect of temperature on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0033] Figure 12 The effect of pH on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by Bacillus erythropoietin WX01.

[0034] Figure 13 Progress curves for the batch feeding production of 2-methylbutyric acid and 6-methyl-2-heptanone in a 5 L fermenter.

[0035] Figure 14 The effects of Bacillus erythrophorus WX01 and Bacillus erythrophorus with the market accession number CGMCC 1.3451 on the growth of pathogens causing plant diseases.

[0036] Figure 15 The effects of fermentation broths of *Bacillus erythropoietinus* WX01 and *Bacillus erythropoietinus* with the market accession number CGMCC 1.3451 on the growth of pathogens causing plant diseases. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. 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 should fall within the scope of protection of the present invention.

[0038] Unless otherwise specified, all reagents or medicines used in the embodiments of this invention are commercially available.

[0039] Example 1

[0040] This embodiment focuses on the isolation, screening, morphological characteristics, and physiological and biochemical properties determination of Bacillus erythropoietinus, as detailed below:

[0041] (1) Experimental method: Ten soil samples were taken from different planting areas of the planting base of Suzhou Kanglai Ecological Agriculture Development Co., Ltd. in Changshu City, Suzhou, Jiangsu Province, China. Ten g of each sample was added to Erlenmeyer flasks containing 100 mL of sterile water and incubated at 36℃ and 200 rpm for 1 h. The Erlenmeyer flasks were then placed in a constant temperature water bath at 80℃ for 30 min. After the water bath, the Erlenmeyer flasks were left to stand in a clean bench for 10 min, and then serially diluted to a concentration of 10. -4 10 -5 and 10 -6 100 µL of each diluted soil solution was spread onto solid enrichment medium, and the petri dishes were incubated upside down in a 36℃ incubator for 48 h. After observation, single colonies were picked and entrained, then inoculated into fermentation medium and cultured for 20 h to test for strains that could produce 2-methylbutyric acid and 6-methyl-2-heptanone. The selected strains were then cultured, and their physiological and biochemical characteristics were observed and tested.

[0042] The solid enrichment medium consisted of 5 g / L yeast extract, 5 g / L tryptone, 5 g / L glucose, and 20 g / L agar, with water as the solvent and a natural pH. The seed culture medium consisted of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, with water as the solvent and a natural pH. The fermentation culture medium consisted of 5 g / L yeast extract, 10 g / L tryptone, and 10 g / L sodium chloride, with water as the solvent and a natural pH. Unless otherwise specified, these culture media were used in subsequent implementations.

[0043] (2) Experimental Results: Through the above screening experiments, a strain capable of simultaneously fermenting and producing 2-methylbutyric acid and 6-methyl-2-heptanone was obtained. The screened strain was cultured, and its colony morphology was observed and its physiological and biochemical characteristics were tested. The physiological and biochemical characteristics are shown in Table 1. Colony morphology is shown in Table 1. Figure 1 As shown, Figure 1 In the image, A represents the colony morphology under plate culture. Figure 1 B indicates the colony morphology of this strain under a microscope, which is... Figure 1 It can be seen that this strain is a Gram-positive bacterium, the bacteria are rod-shaped, the colony surface on LB medium is moist, slightly viscous, the edges are relatively neat, and the color is uniform.

[0044] Table 1. Colony morphological characteristics and physiological and biochemical properties

[0045]

[0046] Note: "+" indicates positive, and "-" indicates negative.

[0047] Example 2

[0048] This embodiment focuses on the identification and preservation of the screened strains, as detailed below:

[0049] (1) Experimental methods: Genomic DNA was extracted from the strains screened in the examples using a bacterial genomic DNA extraction kit. Gene amplification was performed using upstream primer 27F and downstream primer 1492R. PCR was performed using the extracted genomic DNA as a template. The PCR amplification products were purified by gel extraction and transferred to Suzhou Genewise Biotechnology Co., Ltd. for sequencing. The 16S rRNA sequence of the strains of this invention was compared with the 16S rRNA sequences of strains already included in the GenBank database for nucleotide sequence homology. A phylogenetic tree was constructed using MEGA 6.0 software, and the identified strains were deposited.

[0050] (2) Experimental results: The phylogenetic tree of this strain is as follows Figure 2 As shown, this strain is similar to Bacillus erythropoietinus. Paenibacillus ehimensis ZBP22 showed the highest homology similarity, reaching 94%. Based on morphological characteristics and the results of physiological and biochemical experiments, this strain was identified as... Paenibacillus ehimensis WX01 is a type of Bacillus erythropoietinus. Paenibacillus ehimensis It has been deposited at the China Center for Type Culture Collection, and the taxonomic name of this *Bacillus erythropoietinus* is... Paenibacillus ehimensisThe accession number is CCTCC NO: M2026113, the accession date is January 15, 2026, and the date of identification as viable is January 22, 2026. The China Center for Type Culture Collection (CCTCC) is located at Wuhan University, Wuhan, China, 430072, and this *Bacillus erythropoietinus* is abbreviated as WX01.

[0051] Example 3

[0052] This embodiment investigates the effect of carbon source type on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0053] (1) Experimental method: WX01 seed culture was inoculated at a rate of 2% (v / v) into fermentation medium containing 15 g / L sucrose, lactose, glucose, fructose and maltose respectively. The initial pH value was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The volume of fermentation medium was 100 mL / 500 mL Erlenmeyer flask. The fermentation culture was carried out for 20 h. After the culture was completed, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0054] (2) Experimental results: such as Figure 3 As shown, sucrose was used as the carbon source to obtain the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone, with measured contents of 2.4 g / L and 24.12 mg / L, respectively. Therefore, sucrose was chosen for subsequent fermentation.

[0055] Note: Examples 4-13 below were optimized using sucrose as the carbon source, and the experiments were conducted with 2-methylbutyric acid and 6-methyl-2-heptanone as the main products.

[0056] Example 4

[0057] This example investigates the effect of carbon source concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0058] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium with sucrose concentrations of 10 g / L, 20 g / L, 30 g / L, 40 g / L and 50 g / L at an inoculation rate of 2% (v / v). The initial pH value was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The fermentation medium was filled into 100 mL / 500 mL Erlenmeyer flasks. The fermentation was carried out for 20 h. After the culture was completed, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage were measured by mass spectrometry.

[0059] (2) Experimental results: such as Figure 4As shown, the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained using 30 g / L sucrose as the carbon source, with measured yields of 2.62 g / L and 31.43 mg / L, respectively.

[0060] Example 5

[0061] This embodiment investigates the effect of organic nitrogen source on the co-production of 2-methylbutyric acid and 6-methyl-2-heptanone by the screened strain WX01, as detailed below:

[0062] (1) Experimental method: WX01 seed culture was inoculated at a rate of 2% (v / v) into fermentation medium containing 12 g / L tryptone, beef extract, soybean powder, yeast powder and urea. The initial pH was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The volume of fermentation medium was 100 mL / 500 mL Erlenmeyer flask. The fermentation was carried out for 20 h. After the culture was completed, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0063] (2) Experimental results: such as Figure 5 As shown, beef extract yielded the highest amounts of 2-methylbutyric acid and 6-methyl-2-heptanone when used as an organic nitrogen source. The measured yields of 2-methylbutyric acid and 6-methyl-2-heptanone were 2.81 g / L and 33.27 mg / L, respectively. Therefore, beef extract was selected for subsequent fermentation.

[0064] Example 6

[0065] This example investigates the effect of beef extract concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0066] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium with organic nitrogen source concentrations of 4 g / L, 8 g / L, 12 g / L, 16 g / L and 20 g / L at an inoculation rate of 2% (v / v). The initial pH value was 6.5. The culture was carried out at 30℃ and 200 rpm with shaking. The fermentation medium was filled into 100 mL / 500 mL Erlenmeyer flasks. The fermentation was carried out for 20 h. After the culture was completed, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage were measured by mass spectrometry.

[0067] (2) Experimental results: such as Figure 6As shown, the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained when the beef extract concentration was 16 g / L, with yields of 2.92 g / L and 34.18 mg / L, respectively. Therefore, 16 g / L was selected as the optimal organic nitrogen source concentration for subsequent fermentation.

[0068] Example 7

[0069] This embodiment investigates the effect of inorganic nitrogen source on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0070] (1) Experimental method: 2% (v / v) of the inoculum was inoculated into fermentation medium containing 4 g / L ammonium chloride, sodium nitrate, ammonium sulfate, ammonium nitrate and ammonium dihydrogen phosphate. The initial pH was 6.5. The fermentation was carried out at 36℃ and 200 rpm with shaking. The volume of the fermentation medium was 100 mL / 500 mL Erlenmeyer flask. The fermentation was carried out for 20 h. After the culture was completed, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0071] (2) Experimental results: such as Figure 7 As shown, ammonium sulfate, as the inorganic nitrogen source, yielded the highest amounts of 2-methylbutyric acid and 6-methyl-2-heptanone. The measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.08 g / L and 35.42 mg / L, respectively. Therefore, ammonium sulfate was chosen for subsequent fermentation.

[0072] Example 8

[0073] This example investigates the effect of ammonium sulfate concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0074] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium with inorganic nitrogen source concentrations of 4 g / L, 8 g / L, 12 g / L, 16 g / L and 20 g / L at an inoculation rate of 2% (v / v). The initial pH value was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The fermentation medium was filled into 100 mL / 500 mL Erlenmeyer flasks. The fermentation was carried out for 20 h. After the culture was completed, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage were measured by mass spectrometry.

[0075] (2) Experimental results: such as Figure 8As shown, the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained when the ammonium sulfate concentration was 8 g / L. The measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.27 g / L and 36.23 mg / L, respectively. Therefore, 8 g / L ammonium sulfate was selected for subsequent fermentation.

[0076] Example 9

[0077] This embodiment investigates the effect of metal salt type on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0078] (1) Experimental method: WX01 seed culture was inoculated at a rate of 2% (v / v) into fermentation medium containing 0.35 g / L zinc chloride, manganese sulfate, calcium chloride, magnesium sulfate and ferrous sulfate. The initial pH was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The volume of fermentation medium was 100 mL / 500 mL Erlenmeyer flask. The fermentation was carried out for 20 h. After the culture was completed, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0079] (2) Experimental results: such as Figure 9 As shown, the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained when magnesium sulfate was used as the metal salt, with measured contents of 3.33 g / L and 36.79 mg / L, respectively. Therefore, magnesium sulfate was selected as the most suitable metal salt.

[0080] Example 10

[0081] This example investigates the effect of metal salt concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0082] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium with magnesium sulfate concentrations of 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L and 0.5 g / L at an inoculation rate of 2% (v / v). The initial pH value was 6.5. The culture was carried out at 36℃ and 200 rpm with shaking. The fermentation medium volume was 100 mL / 500 mL Erlenmeyer flask. The fermentation was carried out for 20 h. After the culture was completed, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage were measured by mass spectrometry.

[0083] (2) Experimental results: such as Figure 10As shown, the highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained when the magnesium sulfate concentration was 0.6 g / L, with measured contents of 3.42 g / L and 30.58 mg / L, respectively. Therefore, 0.6 g / L magnesium sulfate was selected as the optimal concentration of the metal salt.

[0084] Example 11

[0085] This example investigates the effect of temperature on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0086] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium at an inoculation rate of 2% (v / v), the initial pH value was 6.5, and the culture temperature was 28℃, 30℃, 32℃, 34℃, 36℃ and 38℃ respectively. The culture was shaken at 200 rpm. The volume of fermentation medium was 100 mL / 500 mL Erlenmeyer flask. Fermentation was carried out for 20 h. After the culture was completed, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0087] (2) Experimental results: such as Figure 11 As shown, the measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.62 g / L and 39.42 mg / L, respectively. The highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained at a temperature of 34℃. Therefore, 34℃ was selected as the optimal fermentation temperature.

[0088] Example 12

[0089] This example investigates the effect of pH on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as detailed below:

[0090] (1) Experimental method: WX01 seed culture was inoculated into fermentation medium at an inoculation rate of 2% (v / v). The pH values ​​were 6.0, 6.5, 7.0, 7.5 and 8.0, respectively. The culture was carried out at 34℃ and shaken at 200 rpm. The culture medium volume was 100 mL / 500 mL Erlenmeyer flask. The fermentation culture was carried out for 20 h. The yield of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry at each pH value.

[0091] (2) Experimental results: such as Figure 12 As shown, the measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.76 g / L and 41.03 mg / L, respectively. The highest yields of 2-methylbutyric acid and 6-methyl-2-heptanone were obtained at pH 7.0. Therefore, pH 7.0 was selected as the optimal fermentation pH.

[0092] Example 13

[0093] This embodiment investigates the effects of batch feeding in a 5 L fermenter on the products 2-methylbutyric acid and 6-methyl-2-heptanone, specifically targeting the screened strain WX01. Details are as follows:

[0094] (1) Experimental method: WX01 strain was inoculated onto a slant culture medium and incubated at 34℃ for 16 h. Single colonies were then picked and streaked onto the slant culture medium and incubated at 34℃ for 20 h to obtain activated strains for later use. The activated strains were inoculated aseptically into a shake flask containing seed culture medium and placed on a shaker at 200 rpm for 20 h at 34℃ to obtain fermentation seed liquid. The seed liquid was inoculated into a sterile fermentation culture medium at a volume ratio of 5%: sucrose 15 g / L, beef extract 16 g / L, ammonium sulfate 10 g / L, and magnesium sulfate 0.45 g / L. The initial pH of the fermentation liquid was adjusted to 7.0 using ammonia water. The total liquid volume of the fermenter was 3 L, the fermentation temperature was 34℃, the stirring speed was 200 rpm, and the aeration rate was 1.2. VVM was used for fermentation. The initial pH of the fermentation was 7.0. During fermentation, an automatic pH control device was activated, and the pH of the fermentation broth was maintained at around 7.0 using ammonia or hydrochloric acid. The fermentation time was 24 hours. Ten hours after the start of fermentation, concentrated feed medium (30 g / L sucrose) was added to the inoculation site under flame protection. Samples were taken every 2 hours to determine the concentrations of 2-methylbutyric acid (2-methylbutyric acid) and 6-methyl-2-heptanone. *Bacillus erythropoietinus* strain CGMCC 1.3451 (purchased from the market) was used as a control. Under the same culture conditions, without sucrose supplementation, the strain was cultured, and the contents of 2-methylbutyric acid (2-methylbutyric acid) and 6-methyl-2-heptanone were tested. Following the same method, only *Bacillus erythropoietinus* strain CGMCC 1.3451 was replaced with *Bacillus erythropoietinus* strain CGMCC 1.3451 (abbreviated as CGMCC 1.3451), and the contents of 2-methylbutyric acid (2-methylbutyric acid) and 6-methyl-2-heptanone were tested.

[0095] (2) Experimental results: such as Figure 13 As shown, in a batch feed experiment in a 5 L fermenter, WX01 produced final yields of 4.85 g / L of 2-methylbutyric acid and 48.34 mg / L of 6-methyl-2-heptanone. However, under the same culture conditions, the commercially available Bacillus erythropoietin strain CGMCC 1.3451 did not synthesize either 2-methylbutyric acid or 6-methyl-2-heptanone.

[0096] Example 14

[0097] This embodiment focuses on the selected strain WX01 and the commercially available Bacillus erythropoietinus with accession number CGMCC 1.3451, and investigates the effects of their respective fermentation broths on the growth of common plant disease pathogens, *Botrytis cinerea* and *Colletotrichum gloeosporioides*. *Botrytis cinerea* is a plant pathogenic fungus that can cause various tree and fruit tree cankers, such as poplar canker and apple ring rot; *Colletotrichum gloeosporioides* is a common pathogen that can cause anthracnose in plants.

[0098] (1) Experimental method: Potato Dextrose Aga (PDA) medium was prepared. The PDA medium was punched in the middle. The mycelial cakes of commercially available *Botrytis cinerea* and *Colletotrichum discus* were transferred into the middle well of the PDA medium and cultured at 30℃ for 5-7 days to obtain fresh plant pathogens for subsequent antibacterial experiments.

[0099] Fresh *Botrytis cinerea* and *Discocephala spp.* were inoculated separately into PDA medium at a distance of 2 cm from the center of the right-hand plate. Figure 14 As shown in Figure A, activated WX01 and commercially available Bacillus erythropoietinus strain CGMCC 1.3451 were streaked 4.5 cm away from the inoculation site. PDA medium inoculated with Staphylococcus aureus and Discocele discus were used as controls, respectively. The strains were incubated at 30℃ for 5 days, and their growth was observed.

[0100] Fresh *Botrytis cinerea* and *Discocephala spp.* cakes were placed in the center of a new PDA medium. 300 μL of fermentation broth from *Bacillus ehime* (WX01) and commercially available *Bacillus erythropoietinus* (CGMCC 1.3451) were evenly spread onto LB solid medium. The fermentation broth was prepared in batches using a 5 L fermenter as described in Example 13. The PDA medium inoculated with the pathogens was inverted onto the LB solid medium inoculated with the above fermentation broth, and sealed with sealing film. The blank LB medium served as a control group. The cultures were incubated at 30°C for 5 days, and the growth of the strains was observed.

[0101] (2) Experimental results: The effects of Bacillus erythropoietinus WX01 and the commercially available Bacillus erythropoietinus with accession number CGMCC 1.3451 on the growth of plant pathogens Staphylococcus aureus and Colletotrichum discoidus are as follows: Figure 14 China B and Figure 14 As shown in Figure C, compared with the commercially available Bacillus erythropoietinus with accession number CGMCC 1.3451, WX01 can inhibit the growth of Staphylococcus aureus and Colletotrichum discoidus, while the commercially available Bacillus erythropoietinus with accession number CGMCC 1.3451 does not have this inhibitory effect.

[0102] The effects of the fermentation broths of *Bacillus erythropoietinus* (WX01 and CGMCC 1.3451) on the growth of plant pathogens *Staphylococcus aureus* and *Colletotrichum discoidus* are as follows: Figure 15 China A and Figure 15 As shown in Figure B, compared with the fermentation broth of *Bacillus erythropoietinus* with the commercially available accession number CGMCC 1.3451, the fermentation broth of WX01 significantly inhibited the growth of *Staphylococcus aureus* and *Colletotrichum discoidus*, while the *Bacillus erythropoietinus* with the commercially available accession number CGMCC 1.3451 showed a significantly lower inhibitory effect on the growth of *Staphylococcus aureus* and *Colletotrichum discoidus*. Figure 15 China A and Figure 15 In the B-cell assay, the growth of pathogens Staphylococcus aureus and Colletotrichum discoidus is largely unaffected.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the solutions. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention based on the understanding of the present invention, without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A strain of *Bacillus ehime* WX01, characterized in that, The classification of the *Ehime* spore-forming bacteria is named as follows: Paenibacillus ehimensis It has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2026113, and the deposit date is January 15, 2026.

2. The application of the *Bacillus erythropoietinus* strain described in claim 1 in the prevention and control of plant diseases.

3. The use of the *Bacillus erythropoietinus* of claim 1 in the production of 2-methylbutyric acid or 6-methyl-2-heptanone.

4. The application according to claim 3, characterized in that, The *Bacillus erythropoietinus* was inoculated into a fermentation medium and cultured aerobically to prepare 2-methylbutyric acid or 6-methyl-2-heptanone.

5. The application according to claim 4, characterized in that, The fermentation medium comprises the following components: carbon source 10~60 g / L, nitrogen source 4~25 g / L, metal salt 0.5~1.0 g / L, pH value 6.5~7.5, and water as solvent.

6. The application according to claim 5, characterized in that, The nitrogen source is any one or a combination of several of the following: tryptone, beef extract, soybean powder, yeast powder, urea, ammonium chloride, sodium nitrate, ammonium sulfate, ammonium nitrate, and ammonium dihydrogen phosphate.

7. The application according to claim 5, characterized in that, The carbon source is one or a combination of several of lactose, sucrose, glucose, fructose and maltose.

8. The application according to claim 5, characterized in that, The metal salt is one or a combination of zinc chloride, manganese sulfate, calcium chloride, magnesium sulfate, and ferrous sulfate.

9. The application according to claim 5, characterized in that, The fermentation medium consists of the following components: sucrose 10-50 g / L, beef extract 4-20 g / L, ammonium sulfate 5-25 g / L and magnesium sulfate 0.20-0.70 g / L. The initial pH of the fermentation broth is adjusted to 6.0-8.0 using ammonia water.

10. The application according to claim 5, characterized in that, The aerobic culture conditions are as follows: pH 6.0-8.0, culture temperature 28-38℃; inoculum size 1-14% of fermentation medium volume, and culture time 10-48 h.

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