Petroyuan bacillus and application thereof

Abstract

The invention provides a strain of paenibacillus albidus and application thereof, and belongs to the technical field of bioengineering. The classification name of the yuan paenibacillus is Paenibacillus ehimensis WX01, the yuan paenibacillus is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M2026113, and the preservation date is January 15, 2026. The paenibacillus brevis can be fermented to produce compounds 2-methylbutyric acid and 6-methyl-2-heptanone at the same time, and has the application prospect of preventing and treating plant diseases; according to the invention, further research finds that the concentrations of 2-methylbutyric acid and 6-methyl-2-heptanone obtained by the production of the paenibacillus brevis can reach 4.85 g / L and 48.34 mg / l respectively; the method for producing the 2-methylbutyric acid and the 6-methyl-2-heptanone through fermentation of the paenibacillus brevis is low in production cost, high in yield, high in safety and environmentally friendly.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a Paenibacillus yunnanensis strain and application thereof. BACKGROUND

[0002] The groups of Bacillus, Clavibacter and Pseudomonas contain many common biocontrol agents. These biocontrol agents can produce antimicrobial peptides or other chemicals through metabolism, thereby killing pests or inhibiting the growth of other harmful pathogenic fungi, to achieve the control of plant diseases. Through research, in addition to the antimicrobial peptides produced by the biocontrol agents for plant disease control, many natural chemicals are also included.

[0003] Common natural compounds for plant disease control include 2-methylbutyric acid, 6-methyl-2-heptanone, 2-ethylhexyl methyl ester and hexanitrile, etc. 2-methylbutyric acid is an organic acid that naturally exists in fruits and vegetables such as apples and strawberries. Research has shown that 2-methylbutyric acid can inhibit the growth of plant pathogenic fungi such as Aspergillus fumigatus, Cladosporium, Alternaria alternata and Penicillium expansum. It has great potential as a fumigant in preventing plant diseases. 6-methyl-2-heptanone naturally exists in tobacco leaves. Studies have shown that 6-methyl-2-heptanone as a fumigant can effectively inhibit many pathogenic fungi such as Cercospora crassicauda, Rhizoctonia solani, Monilinia fructicola, Verticillium dahliae and Alternaria solani. Its mechanism of action includes disrupting the hyphal structure, increasing the cell membrane permeability and inducing ATP leakage, thus showing good application prospects in the field of plant disease control.

[0004] For the above-mentioned natural compounds, their preparation methods generally use chemical synthesis, and there is no report on the synthesis of 2-methylbutyric acid and 6-methyl-2-heptanone by biological methods. At present, chemical synthesis of 2-methylbutyric acid has the disadvantages of expensive raw materials, complicated operation, low yield, large amount of wastewater and poor safety. Chemical synthesis of 6-methyl-2-heptanone is to use 6-methyl-3-hepten-2-one as a raw material to prepare 6-methyl-2-heptanone by catalytic hydrogenation. The whole process is complicated, the production cost is high, and the production process produces serious pollution. In order to solve the problems of high cost and high safety risk in the chemical synthesis of the above-mentioned natural compounds 2-methylbutyric acid and 6-methyl-2-heptanone, it is necessary to screen a strain that can produce the above-mentioned control compounds to enrich the production of the above-mentioned natural compounds, avoid pollution and safety problems caused by chemical synthesis, and apply to plant disease control. SUMMARY

[0005] The purpose of the present application is to solve the above technical problems by screening a strain that can produce the above-mentioned control compounds. Another purpose of the present application is to provide the application of the strain.

[0006] To achieve the above object, the application adopts the following technical solutions:

[0007] The application screens and separates a strain of Paenibacillus elyi from the soil of a planting base of Suzhou Kangle Ecological Agricultural Development Co., Ltd. located in Changshu City, Suzhou City, Jiangsu Province, China. The strain of Paenibacillus elyi is classified and named as Paenibacillus ehimensis WX01, which has been preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M2026113, a preservation date of January 15, 2026, and a date of identification of survival of January 22, 2026. The China Center for Type Culture Collection is abbreviated as CCTCC, and is located at Wuhan University, Wuhan, China, with a postal code of 430072.

[0008] The strain has the following properties: the strain is rod-shaped and gram-positive, and the VP test, starch hydrolysis test, contact enzyme test, gelatin liquefaction test, and nitrate reduction test are all positive; the indole test, citrate test, H2S gas production test, and methyl red test are all negative.

[0009] The application finds that the Paenibacillus elyi Paenibacillus ehimensis WX01 has different properties compared with a commercially available Paenibacillus elyi strain with a preservation number of CGMCC 1.3451. The Paenibacillus elyi Paenibacillus ehimensis WX01 fermentation can simultaneously produce natural compounds 2-methylbutyric acid and 6-methyl-2-heptanone that can be used for plant disease control, while the above-mentioned commercially available Paenibacillus elyi strain does not detect related compounds; therefore, the Paenibacillus elyi Paenibacillus ehimensis WX01 has the application prospect of simultaneously producing 6-methyl-2-heptanone and 2-methylbutyric acid.

[0010] Based on the properties of the Paenibacillus elyi Paenibacillus ehimensis WX01, the application provides the application of the above-mentioned Paenibacillus elyi in plant disease control.

[0011] Based on the properties of the Paenibacillus elyi Paenibacillus ehimensis WX01, the application provides the application of the above-mentioned Paenibacillus elyi in the production of 2-methylbutyric acid or 6-methyl-2-heptanone.

[0012] Further, the Paenibacillus elyi is inoculated into a fermentation medium for aerobic culture to prepare 2-methylbutyric acid or 6-methyl-2-heptanone.

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

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

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

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

[0017] Further, the components of the fermentation medium are as follows: 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, and the initial pH of the fermentation broth is adjusted to 6.0-8.0 by using ammonia water.

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

[0019] The beneficial effects of the present application are as follows: (1) The present application discovers a strain of Paenibacillus iyoensis Paenibacillus ehimensis WX01, which can produce 2-methylbutyric acid and 6-methyl-2-heptanone simultaneously compared with the same strain, and has application prospects in plant disease control and production of 2-methylbutyric acid and 6-methyl-2-heptanone.

[0020] (2) Through further research on the composition of the fermentation medium and the fermentation conditions, the present application can accumulate 2-methylbutyric acid in the medium at a concentration of up to 4.85 g / L, and 6-methyl-2-heptanone at a concentration of up to 48.34 mg / L; compared with traditional chemical synthesis, the present Paenibacillus iyoensis produces 2-methylbutyric acid and 6-methyl-2-heptanone at low cost, and has the advantages of simple operation, high safety and environmental friendliness, and has the prospect of industrialization and application. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Colony characteristics of Paenibacillus iyoensis WX01.

[0022] Figure 2 Phylogenetic tree of Paenibacillus iyoensis WX01.

[0023] Figure 3 Effect of carbon source type on production of 2-methylbutyric acid and 6-methyl-2-heptanone by Paenibacillus iyoensis WX01.

[0024] Figure 4 Effect of carbon source concentration on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0025] Figure 5 Effect of organic nitrogen source type on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0026] Figure 6 Effect of organic nitrogen source concentration on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0027] Figure 7 Effect of inorganic nitrogen source type on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0028] Figure 8 Effect of inorganic nitrogen source concentration on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0029] Figure 9 Effect of metal ion type on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0030] Figure 10 Effect of metal ion concentration on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0031] Figure 11 Effect of temperature on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0032] Figure 12 Effect of pH on 2-methylbutyric acid and 6-methyl-2-heptanone production by Paenibacillus ehimensis WX01.

[0033] Figure 13 Fed-batch production of 2-methylbutyric acid and 6-methyl-2-heptanone in a 5 L fermenter.

[0034] Figure 14 Effect of Paenibacillus ehimensis WX01 and commercially available Paenibacillus ehimensis with accession number CGMCC 1.3451 on the growth of pathogenic fungi of plant diseases.

[0035] Figure 15 Effect of fermentation broth of Paenibacillus ehimensis WX01 and commercially available Paenibacillus ehimensis with accession number CGMCC 1.3451 on the growth of pathogenic fungi of plant diseases. DETAILED DESCRIPTION

[0036] In the following, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application, so that those skilled in the art can better understand the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work should fall within the protection scope of the present application.

[0037] The reagents or drugs in the embodiments of the present application are commercially available unless otherwise specified.

[0038] Example 1 This embodiment is directed to the isolation and screening of Paenibacillus elyakovii, and the determination of morphological characteristics and physiological and biochemical characteristics, which are specifically as follows. (1) Experimental method: 10 g of soil was taken from each of 10 different planting areas of the planting base of Suzhou Kanglai Ecological Agricultural Development Co., Ltd. in Changshu City, Suzhou City, Jiangsu Province, China, and was added into a triangular flask containing 100 mL of sterile water. The flask was cultured at 36°C and 200 rpm for 1 h. The flask was placed in a constant-temperature water bath at 80°C for 30 min of water bath. After the water bath, the flask was placed in an ultra-clean workbench for 10 min, and then gradient dilution was performed. 100 μL of soil diluent with concentrations of 10 -4 , 10 -5 and 10 -6 was respectively coated on a solid enrichment culture medium, and the culture dish was incubated in a 36°C constant-temperature incubator for 48 h. After observation, single colonies were picked and inoculated into a fermentation medium for 20 h. Strains producing 2-methylbutyric acid and 6-methyl-2-heptanone were tested, and the screened strains were cultured, observed and tested for physiological and biochemical characteristics.

[0039] The solid enrichment culture medium is composed of 5 g / L of yeast powder, 5 g / L of tryptone, 5 g / L of glucose and 20 g / L of agar, with water as the solvent and natural pH. The seed culture medium is composed of 5 g / L of yeast powder, 10 g / L of tryptone and 10 g / L of sodium chloride, with water as the solvent and natural pH. The fermentation medium is composed of 5 g / L of yeast powder, 10 g / L of tryptone and 10 g / L of sodium chloride, with water as the solvent and natural pH. In subsequent embodiments, this culture medium is still used unless otherwise specified.

[0040] (2) Experimental results: Through the above screening experiment, a strain capable of simultaneously producing 2-methylbutyric acid and 6-methyl-2-heptanone was screened. The screened strain was cultured, the colony morphology was observed, and the physiological and biochemical characteristics were tested. The physiological and biochemical characteristics are shown in Table 1. The colony morphology is shown in Table 2. Figure 1 Figure 1 ​Medium A represents the colony morphology under plate culture, Figure 1 Medium B represents the colony morphology under microscope, which is shown by Figure 1 It can be seen that the strain is a gram-positive bacterium, the bacteria are rod-shaped, the colony surface on LB medium is moist, slightly viscous, the edge is slightly neat, and the color is uniform.

[0041] Table 1: Colony morphological characteristics and physiological and biochemical characteristics

[0042] Note: "+" represents positive, and "-" represents negative.

[0043] Example 2 This example is directed to the identification and preservation of the screened strain, specifically as follows: (1) Experimental method: The genomic DNA of the strain screened in the example was extracted by using a bacterial genomic DNA extraction kit, and gene amplification was performed using upstream primer 27F and downstream primer 1492R. The extracted genomic DNA was used as a template for PCR, and the PCR amplified product was gel recovered and purified. The gel recovery and purification product was transferred to Suzhou Jinyuzhi Biological Technology Co., Ltd. for sequencing. The 16S rRNA sequence of the strain of the present application was compared with the 16S rRNA sequence of the strains already included in the GenBank database for nucleotide sequence homology, and a phylogenetic tree was drawn using software MEGA 6.0, and the identified strain was preserved.

[0044] (2) Experimental results: The phylogenetic tree of the strain is shown in Figure 2 The strain has the highest homology of 94% with Bacillus eurymetabola ZBP22. Combined with the results of morphological characteristics and physiological and biochemical experiments, it is determined that the strain Paenibacillus ehimensis WX01 is Bacillus eurymetabola. Paenibacillus ehimensis Paenibacillus ehimensis The Bacillus eurymetabola is classified as Paenibacillus ehimensis , with a preservation number of CCTCC NO: M2026113, a preservation date of January 15, 2026, and an identification survival date of January 22, 2026. The Chinese Typical Culture Collection Center is abbreviated as CCTCC, located at Wuhan University, Wuhan, China, with a postcode of 430072. The Bacillus eurymetabola is abbreviated as WX01.

[0045] Example 3 This example is directed to the screened strain WX01, and studies the effect of carbon source type on the production of 2-methylbutyric acid and 6-methyl-2-heptanone, specifically as follows: ​(1) Experimental method: The WX01 seed culture solution was inoculated into fermentation medium containing 15 g / L sucrose, lactose, glucose, fructose, and maltose at an inoculation amount of 2% (v / v), the initial pH value was 6.5, and the fermentation medium was cultured at 36°C with 200 rpm oscillation. The liquid volume of the fermentation medium was 100 mL / 500 mL in a triangular flask, and the fermentation culture was carried out for 20 h. After the culture was completed, the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0046] (2) Experimental results: As shown in Table 1, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone obtained by using sucrose as the carbon source was the highest, and the measured results of the contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 2.4 g / L and 24.12 mg / L, respectively. Therefore, sucrose was selected for subsequent fermentation. Figure 3

[0047] Note: The following examples 4-13 all use sucrose as the carbon source for optimization, and 2-methylbutyric acid and 6-methyl-2-heptanone are used as the main products for experiments.

[0048] Example 4 This example aims to study the influence of carbon source concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as follows: (1) Experimental method: The WX01 seed culture solution was inoculated into fermentation medium containing 15 g / L sucrose, lactose, glucose, fructose, and maltose at an inoculation amount of 2% (v / v), the initial pH value was 6.5, and the fermentation medium was cultured at 36°C with 200 rpm oscillation. The liquid volume of the fermentation medium was 100 mL / 500 mL in a triangular flask, and the fermentation culture was carried out for 20 h. After the culture was completed, the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0049] (2) Experimental results: As shown in Table 1, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone obtained by using sucrose as the carbon source was the highest, and the measured results of the contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 2.4 g / L and 24.12 mg / L, respectively. Therefore, sucrose was selected for subsequent fermentation. Figure 4

[0050] Example 5 This example aims to study the influence of the type of organic nitrogen source on the co-production of 2-methylbutyric acid and 6-methyl-2-heptanone of the screened strain WX01, as follows: ​​(1) Experimental method: The WX01 seed culture solution was inoculated into a fermentation medium containing 12 g / L of tryptone, beef extract, soybean powder, yeast powder, and urea at a 2% (v / v) inoculation amount, with an initial pH value of 6.5, and was cultured at 36°C with 200 rpm shaking. The fermentation medium was 100 mL / 500 mL in a triangular flask, and the fermentation culture was 20 h. After the culture ended, the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0051] (2) Experimental results: As shown in Table 1, the use of beef extract as an organic nitrogen source resulted in the highest yield of 2-methylbutyric acid and 6-methyl-2-heptanone, with measured yields of 2.81 g / L and 33.27 mg / L, respectively. Therefore, beef extract was selected for subsequent fermentation. Figure 5

[0052] Example 6 This example aimed to study the effect of beef extract concentration on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by the screened strain WX01, as follows: (1) Experimental method: The WX01 seed culture solution was inoculated into a fermentation medium containing 12 g / L of tryptone, beef extract, soybean powder, yeast powder, and urea at a 2% (v / v) inoculation amount, with an initial pH value of 6.5, and was cultured at 36°C with 200 rpm shaking. The fermentation medium was 100 mL / 500 mL in a triangular flask, and the fermentation culture was 20 h. After the culture ended, the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry.

[0053] (2) Experimental results: As shown in Table 2, when the beef extract concentration was 16 g / L, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone was the highest, 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. Figure 6

[0054] Example 7 This example aimed to study the effect of inorganic nitrogen source type on the production of 2-methylbutyric acid and 6-methyl-2-heptanone by the screened strain WX01, as follows: ​​(1) Experimental method: 2% (v / v) inoculation amount was inoculated into fermentation medium containing 4 g / L ammonium chloride, sodium nitrate, ammonium sulfate, ammonium nitrate and ammonium dihydrogen phosphate, the initial pH value was 6.5, and the fermentation medium was cultured at 36°C, 200 rpm, the liquid volume of the fermentation medium was 100 mL / 500 mL triangular flask, the fermentation culture was 20 h, and the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry after the culture was completed.

[0055] (2) Experimental results: as shown in Figure 7 , the yield of 2-methylbutyric acid and 6-methyl-2-heptanone obtained from ammonium sulfate is the highest, the measured results of 2-methylbutyric acid and 6-methyl-2-heptanone are 3.08 g / L and 35.42 mg / L respectively, so ammonium sulfate is selected for subsequent fermentation.

[0056] Example 8 This example is aimed at the screened strain WX01, and the effect of ammonium sulfate concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, which is as follows: (1) Experimental method: the seed culture of WX01 was inoculated into fermentation medium with inorganic nitrogen source concentration of 4 g / L, 8 g / L, 12 g / L, 16 g / L and 20 g / L, the initial pH value was 6.5, and the fermentation medium was cultured at 36°C, 200 rpm, the liquid volume of the fermentation medium was 100 mL / 500 mL triangular flask, the fermentation culture was 20 h, and the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage was measured by mass spectrometry after the culture was completed.

[0057] (2) Experimental results: as shown in Figure 8 , when the concentration of ammonium sulfate is 8 g / L, the yield of 2-methylbutyric acid and 6-methyl-2-heptanone is the highest, the measured results of 2-methylbutyric acid and 6-methyl-2-heptanone are 3.27 g / L and 36.23 mg / L respectively. Therefore, 8 g / L ammonium sulfate is selected for subsequent fermentation.

[0058] Example 9 This example is aimed at the screened strain WX01, and the effect of metal salt type on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, which is as follows: (1) Experimental method: The WX01 seed culture solution was inoculated into the fermentation medium containing 0.35 g / L zinc chloride, manganese sulfate, calcium chloride, magnesium sulfate and ferrous sulfate at an inoculation amount of 2% (v / v), the initial pH value was 6.5, and the fermentation medium was cultured at 36°C and 200 rpm, the liquid volume of the fermentation medium was 100 mL / 500 mL in a triangular flask, the fermentation culture was carried out for 20 h, and then the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0059] (2) Experimental results: As shown in Table 2, when magnesium sulfate was used as the metal salt, the highest yield of 2-methylbutyric acid and 6-methyl-2-heptanone was obtained, and the measured results of the contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.33 g / L and 36.79 mg / L. Therefore, magnesium sulfate was selected as the most suitable metal salt type. Figure 9

[0060] Example 10 This example is directed to the screened strain WX01, and the effect of metal salt concentration on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, which is as follows: (1) Experimental method: The WX01 seed culture solution was inoculated into the fermentation medium containing 0.35 g / L zinc chloride, manganese sulfate, calcium chloride, magnesium sulfate and ferrous sulfate at an inoculation amount of 2% (v / v), the initial pH value was 6.5, and the fermentation medium was cultured at 36°C and 200 rpm, the liquid volume of the fermentation medium was 100 mL / 500 mL in a triangular flask, the fermentation culture was carried out for 20 h, and then the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0061] (2) Experimental results: As shown in Table 2, when magnesium sulfate was used as the metal salt, the highest yield of 2-methylbutyric acid and 6-methyl-2-heptanone was obtained, and the measured results of the contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.33 g / L and 36.79 mg / L. Therefore, magnesium sulfate was selected as the most suitable metal salt type. Figure 10

[0062] Example 11 This example is directed to the screened strain WX01, and the effect of temperature on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, which is as follows: ​​(1) Experimental method: the WX01 seed culture was inoculated into the fermentation medium at an inoculation amount of 2% (v / v), the initial pH value was 6.5, the culture temperature was 28°C, 30°C, 32°C, 34°C, 36°C and 38°C respectively, and the fermentation culture was cultured at 200 rpm, the liquid volume of the fermentation medium was 100 mL / 500 mL in a triangular flask, the fermentation culture was cultured for 20 h, and then the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0063] (2) Experimental results: as shown in Table 2, the measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.62 g / L and 39.42 mg / L respectively. When the temperature was 34°C, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone were the highest. Therefore, 34°C was selected as the optimal fermentation temperature. Figure 11

[0064] Example 12 This example is directed to the screened strain WX01, and the effect of pH on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, as follows: (1) Experimental method: the WX01 seed culture was inoculated into the fermentation medium at an inoculation amount of 2% (v / v), the culture pH value was 6.0, 6.5, 7.0, 7.5 and 8.0 respectively, the culture was cultured at 34°C and 200 rpm, the liquid volume of the culture medium was 100 mL / 500 mL in a triangular flask, the fermentation culture was cultured for 20 h, and then the mass spectrometry method was used to measure the production of 2-methylbutyric acid and 6-methyl-2-heptanone in the production stage.

[0065] (2) Experimental results: as shown in Table 3, the measured contents of 2-methylbutyric acid and 6-methyl-2-heptanone were 3.76 g / L and 41.03 mg / L respectively. When the pH value was 7.0, the yields of 2-methylbutyric acid and 6-methyl-2-heptanone were the highest. Therefore, the pH value of 7.0 was selected as the optimal fermentation pH. Figure 12

[0066] Example 13 This example is directed to the screened strain WX01, and the effect of 5 L fermentation tank batch feeding on the products 2-methylbutyric acid and 6-methyl-2-heptanone is studied, as follows: ​​(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.

[0067] (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.

[0068] Example 14 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.

[0069] (1) Experimental method: prepare PDA solid medium, punch a hole in the middle of the PDA solid medium, respectively inoculate the purchased V. inaequalis and P. discolor into the hole, and cultivate at 30°C for 5-7 days to obtain fresh plant pathogenic fungi for subsequent antibacterial experiments.

[0070] Inoculate fresh V. inaequalis and P. discolor into the PDA medium, respectively, as shown in FIG. 2A, and inoculate the activated WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 at a distance of 4.5 cm from the inoculation site of the pathogenic fungi. Figure 14 Inoculate fresh V. inaequalis and P. discolor into the PDA medium, respectively, as shown in FIG. 2A, and inoculate the activated WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 at a distance of 4.5 cm from the inoculation site of the pathogenic fungi.

[0071] Inoculate fresh V. inaequalis and P. discolor into the PDA medium, respectively, as shown in FIG. 2A, and inoculate the activated WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 at a distance of 4.5 cm from the inoculation site of the pathogenic fungi.

[0072] Figure 14 (2) Experimental results: the effects of WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 on the growth of the plant pathogenic fungi V. inaequalis and P. discolor are shown in FIG. 2B and FIG. 2C, respectively. Figure 14 The effects of the fermentation liquor of WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 on the growth of the plant pathogenic fungi V. inaequalis and P. discolor are shown in FIG. 2A and FIG. 2B, respectively.

[0073] Figure 15 The effects of the fermentation liquor of WX01 and the purchased Bacillus eurycius with the preservation number CGMCC 1.3451 on the growth of the plant pathogenic fungi V. inaequalis and P. discolor are shown in FIG. 2A and FIG. 2B, respectively. Figure 15 ​​As shown in the middle B, compared with the fermentation broth of the commercially available Paenibacillus ehimensis with the preservation number of CGMCC 1.3451, the fermentation broth of WX01 can significantly inhibit the growth of Botryosphaeria dothidea and Physalospora discolor, while the inhibition effect of the commercially available Paenibacillus ehimensis with the preservation number of CGMCC 1.3451 on the growth of Botryosphaeria dothidea and Physalospora discolor is significantly lower, and the inhibition effect of the fermentation broth of WX01 on the growth of Botryosphaeria dothidea and Physalospora discolor is shown in the following table. Figure 15 The middle A and Figure 15 As shown in the middle B, the growth of the pathogenic bacteria Botryosphaeria dothidea and Physalospora discolor is basically not affected.

[0074] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit the solutions, although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents on the basis of understanding the solutions, without departing from the purpose and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A strain of *Bacillus erythropoietinus*, characterized in that, The classification of the *Ehime* spore-forming bacteria is named as follows: Paenibacillus ehimensis WX01 has been deposited at the China Center for Type Culture Collection (CCTCCNO: M2026113) on 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 several 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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