Paenibacillus polymyxa, volatile organic compounds produced by the same and applications thereof

Abstract

The application provides a Paenibacillus polymyxa and volatile organic compounds generated by the Paenibacillus polymyxa and application. The Paenibacillus polymyxa WB-126 of the application has a significant inhibiting effect on various plant pathogenic fungi; the Paenibacillus polymyxa WB-126 of the application can generate various volatile organic compounds, such as 2-methyl butyric acid, 2-propyl-1-pentanol and the like, and the volatile organic compounds have a good control effect on fungal diseases; wherein the compound 2-methyl butyric acid has a good control effect on fungal diseases caused by Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium pseudograminearum and Fusarium graminearum; 2-propyl-1-pentanol can completely inhibit the growth of Botrytis cinerea and Fusarium graminearum when the spatial concentration reaches 67 μL / L, and 2-methyl butyric acid can completely inhibit the growth of Botrytis cinerea and Sclerotinia sclerotiorum when the spatial concentration reaches 133 μL / L.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and in particular to a polymyxa bacillus, its volatile organic compounds, and their applications. Background Technology

[0002] Volatile organic compounds (VOCs) have small molecular weights and are generally liquids that readily evaporate into gases under normal temperature and pressure. VOCs can rapidly diffuse into the air and penetrate the air-filled pores of soil, exhibiting excellent cell membrane permeability and quickly exerting physiological activity. Research has found that many bacteria and fungi can produce various volatile substances that can inhibit the growth and reproduction of plant pathogens, thus playing a significant role in prevention and control. Bacillus, in particular, possesses characteristics such as heat resistance, rapid reactivation, and strong environmental adaptability, surviving under both aerobic and anaerobic conditions. It is an excellent biocontrol strain, and Bacillus species that produce VOCs are gradually becoming a research hotspot in the field of biocontrol.

[0003] Sclerotinia sclerotiorum rot, caused by *Scleropsis sclerotiorum* (Lib.) de Bary, is a fungal disease affecting rapeseed. It primarily damages the stems, leaves, flowers, pods, and seeds of rapeseed. A global disease, sclerotinia sclerotiorum rot not only reduces yield by 10-70% but also drastically reduces the oil content of seeds from infected plants, severely impacting both yield and quality. It is a soil-borne disease, with sclerotia oversummering (in winter rapeseed areas) and overwintering (in winter and spring rapeseed areas) in the soil, diseased plant debris, and seeds. Under suitable conditions, *Scleropsis sclerotiorum* germinates and produces ascospores, which are then dispersed by air currents. Due to the wide range of transmission routes of soil-borne diseases, conventional pesticides are insufficient to cover all transmission pathways, especially in the soil. Therefore, it is necessary to provide a formulation with better control efficacy against rapeseed sclerotinia sclerotiorum rot. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a *Paenibacillus* sp. strain WB-126, along with its volatile organic compounds and applications. During the screening of biocontrol strains to inhibit *Sclerotinia sclerotiorum*, this invention identified a strain exhibiting good inhibitory activity against *Sclerotinia sclerotiorum* and other fungi. Further research revealed that this strain produces volatile substances that inhibit *Sclerotinia sclerotiorum*, and also show good effects against *Botrytis cinerea*, *Fusarium graminearum*, and *Fusarium verticillatum*. GC-MS analysis of multiple volatile compounds, using the plate-discharging method, showed that 2-methylbutyric acid exhibited excellent inhibitory effects against *Sclerotinia sclerotiorum*, and 2-propyl-1-pentanol showed excellent inhibitory effects against *Botrytis cinerea* and *Fusarium graminearum*. These experiments demonstrate that WB-126 can control pathogenic fungi non-contactly, showing promising application prospects in soil-borne diseases and post-harvest fruit preservation.

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

[0006] In a first aspect, the present invention provides a polymyxin Bacillus, the polymyxin Bacillus being classified as: polymyxin Bacillus sp. WB-126, and the polymyxin Bacillus preservation number is CCTCCNO: M 20241922.

[0007] Secondly, the present invention also provides the use of Bacillus polymyxa or its fermentation broth in the preparation of drugs or preparations that inhibit fungal diseases that harm plant growth.

[0008] Preferably, the fungal diseases include those caused by at least one of the following: Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Sclerotinia sclerotiorum, Phytophthora litchii, Botrytis cinerea, Fusarium graminearum, Fusarium verticillata, Penicillium italicum, Penicillium fingernail, Colletotrichum gloeosporioides, Alternaria peariformis, Phytophthora capsici, Pseudomonas aeruginosa, and Phytophthora tumefaciens.

[0009] Preferably, the method for preparing the Bacillus polymyxa fermentation broth includes the following steps:

[0010] Polymyxin Bacillus was cultured in a primary seed culture medium to obtain a primary seed solution;

[0011] The primary seed culture was inoculated into a fermentation medium and cultured to obtain the fermentation broth;

[0012] The primary seed culture medium comprises the following components by mass fraction: 1-2% glucose, 2-3% beef extract, 1-2% peptone, 0.35-0.45% sodium chloride, and the remainder is water;

[0013] The fermentation medium comprises the following components by mass fraction: maltose 3.0-4.0%, soybean meal 3.0-4.0%, magnesium sulfate 0.3-0.4%, potassium dihydrogen phosphate 0.3-0.4%, dipotassium hydrogen phosphate 0.2-0.3%, ammonium chloride 0.2-0.3%, and calcium carbonate 0.2-0.3%.

[0014] Preferably, in the step of inoculating Bacillus polymyxa in a primary seed culture medium, the culture temperature is 28~32℃ and the time is 10~12h;

[0015] In the step of inoculating the primary seed culture into the fermentation medium, the culture temperature is 27~29℃ and the time is 20~60h.

[0016] Thirdly, the present invention also provides an application of the aforementioned polymyxa Bacillus in the production of volatile organic compounds, wherein the volatile organic compounds include at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 2,5-dimethyl-pyrazine, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0017] Preferably, the Bacillus polymyxa culture is inoculated into LB solid medium and cultured to produce volatile organic compounds.

[0018] Fourthly, the present invention also provides the application of volatile organic compounds in the preparation of drugs or preparations for inhibiting fungal diseases that harm plant growth;

[0019] The volatile organic compounds include at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0020] Preferably, the fungal diseases include those caused by at least one of *Sclerotinia sclerotiorum*, *Phytophthora capsici*, *Staphylococcus aureus*, *Fusarium graminearum*, and *Fusarium verticillatum*.

[0021] Fifthly, the present invention also provides an agent for preventing and controlling plant fungal diseases, comprising volatile organic compounds, said volatile organic compounds including at least one selected from 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0022] The polymyxa bacillus, its volatile organic compounds, and their applications of the present invention have the following advantages over the prior art:

[0023] 1. The present invention relates to Paenibacillus sp. WB-126, which has a significant inhibitory effect on a variety of plant pathogenic fungi. This strain can produce antifungal volatile substances such as 2-propyl-1-pentanol and 2-methylbutyric acid, which can produce a non-contact bactericidal effect. It can be used for the prevention and control of soil-borne diseases such as sclerotinia rot in rapeseed, stalk rot in corn, and fungal diseases caused by gray mold on fruits and vegetables. It can also be used for post-harvest preservation of fruits.

[0024] 2. The *Paenibaci* L. *poLymyxa* WB-126 of this invention can produce a variety of volatile organic compounds, such as 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol. These volatile organic compounds have good control effects on fungal diseases. Among them, the compound 2-methylbutyric acid has a good control effect on fungal diseases caused by *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Fusarium graminearum*, and *Fusarium verticillatum*. When the spatial concentration of 2-propyl-1-pentanol reaches 67 μL / L, it can completely inhibit the growth of *Sclerotinia sclerotiorum* and *Fusarium graminearum*. When the spatial concentration of 2-methylbutyric acid reaches 133 μL / L, it can completely inhibit the growth of *Sclerotinia sclerotiorum* and *Botrytis cinerea*. Attached Figure Description

[0025] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Gram staining results and colony morphology on LB agar plates for the present invention PaenibaciLLus poLymyxa WB-126;

[0027] Figure 2 This is a phylogenetic tree diagram of the polymyxin Bacillus (PaenibaciLLus poLymyxa) WB-126 of the present invention;

[0028] Figure 3 The gas chromatography-mass spectrometry (GC-MS) chromatograms and peak search results in Example 7 using LB solid medium as a blank control;

[0029] Figure 4 The results are gas chromatography-mass spectra and peak search results of volatile substances produced after the primary seed culture was inoculated into LB solid medium in Example 7. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of embodiments. Furthermore, in the description of this application, the term "comprising" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single digits within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any referenced number (fraction or integer) within the indicated range.

[0032] This application provides a polymyxin Bacillus sp. WB-126, which was deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2024. Its classification name is: Polymyxin Bacillus sp. WB-126; its accession number is CCTCC NO: M20241922; and its location is: Wuhan University, Wuhan, China.

[0033] Based on the same inventive concept, the present invention also provides the application of the above-mentioned Bacillus polymyxa or its fermentation broth in the preparation of drugs or preparations for inhibiting fungal diseases that harm plant growth.

[0034] In some embodiments, fungal diseases include those caused by at least one of the following: Rhizoctonia solani, Sclerotium truncatum, Fusarium oxysporum, Sclerotium sclerotiorum, Phytophthora litchii, Botrytis cinerea, Fusarium graminearum, Fusarium verticillata, Penicillium italicum, Penicillium fingernail, Colletotrichum gloeosporioides, Alternaria pearis, Phytophthora capsici, Pseudomonas aeruginosa, and Phytophthora tumefaciens.

[0035] In some embodiments, the preparation method of Bacillus polymyxa fermentation broth includes the following steps:

[0036] Polymyxin Bacillus was cultured in a primary seed culture medium to obtain a primary seed solution;

[0037] The primary seed culture was inoculated into a fermentation medium and cultured to obtain the fermentation broth;

[0038] The primary seed culture medium comprises the following components by mass fraction: glucose 1-2%, beef extract 2-3%, peptone 1-2%, sodium chloride 0.35-0.45%, with the remainder being water;

[0039] The fermentation medium comprises the following components by mass fraction: maltose 3.0-4.0%, soybean meal 3.0-4.0%, magnesium sulfate 0.3-0.4%, potassium dihydrogen phosphate 0.3-0.4%, dipotassium hydrogen phosphate 0.2-0.3%, ammonium chloride 0.2-0.3%, and calcium carbonate 0.2-0.3%.

[0040] In some embodiments, in the step of culturing Bacillus polymyxa in a primary seed culture medium, the culture temperature is 28-32°C and the time is 10-12 hours.

[0041] In the step of inoculating the primary seed culture into the fermentation medium, the culture temperature is 27~29℃ and the time is 20~60h.

[0042] Preferably, the preparation method of Bacillus polymyxa fermentation broth includes the following steps:

[0043] S1. Activation of bacterial strain: The Bacillus polymyxa strain WB-126, stored at -80℃, was inoculated onto LB agar slants for activation.

[0044] S2. Primary Seed Culture: Add 100-120 mL of primary seed culture medium to a 500 mL Erlenmeyer flask and sterilize at 120-125℃ for 30-40 min. Inoculate the activated WB-126 strain culture into the primary seed culture medium and incubate at 28-32℃ and 150-200 r / min for 10-12 h to obtain the primary seed solution. The raw materials and quantities of the culture medium used for primary seed culture are: 1% glucose, 2% beef extract, 1% peptone, 0.35% sodium chloride, and pH 7.0 (percentages are mass fractions, the same in the examples below, the remainder is water).

[0045] S3. Fermentation: Add 300-320L of fermentation medium and 300-320 mL of defoamer (specifically, defoamer GP330, purchased from Haian Petrochemical Plant, Jiangsu Province) to a 500L fermenter. Sterilize at 120-125℃ for 30 minutes. When the tank temperature drops to 32℃, inoculate with 300-320 mL of the primary seed culture from step S2; pressurize the tank to 0.5 × 10⁻⁶. 5 ~1×10 5Fermentation was carried out at a temperature of 27-29℃ for 36 hours. Samples were taken every 2 hours for microscopic examination. The fermentation endpoint was reached when 20% of the spores were detached and separated, at which point the fermentation was stopped, and the fermentation broth was obtained. The raw materials and amounts of the culture medium used for fermentation were as follows: maltose 3.0%, soybean meal 3.0%, magnesium sulfate 0.3%, potassium dihydrogen phosphate 0.3%, dipotassium hydrogen phosphate 0.2%, ammonium chloride 0.2%, calcium carbonate 0.2%, and the pH of the culture medium was 8.0 (the remainder of the culture medium was water).

[0046] The *Paenibacillus* sp. WB-126 and its fermentation broth of this invention are used for the control of fungal diseases. WB-126 and its fermentation broth can be used for the control of soil-borne diseases such as sclerotinia rot in rapeseed and fungal diseases caused by gray mold on fruits and vegetables, and can also be used for post-harvest preservation of fruits.

[0047] This invention provides a strain of Paenibacillus sp. WB-126, which has a significant inhibitory effect on a variety of plant pathogenic fungi. Furthermore, this strain can produce antifungal volatile substances such as 2-propyl-1-pentanol and 2-methylbutyric acid, which can produce a non-contact bactericidal effect. It can be used for the control of soil-borne diseases such as sclerotinia rot in rapeseed, corn stalk rot, and fungal diseases caused by gray mold on fruits and vegetables. It can also be used for post-harvest preservation of fruits.

[0048] Based on the same inventive concept, the present invention also provides an application of the above-mentioned polymyxa bacillus in the generation of volatile organic compounds, the volatile organic compounds including at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 2,5-dimethyl-pyrazine, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0049] In some embodiments, the fermentation broth of Paenibacillus sp. WB-126 is streaked onto the surface of LB solid medium and cultured statically at 28-30°C for 8-10 days to produce volatile organic compounds.

[0050] Based on the same inventive concept, the present invention also provides the application of volatile organic compounds in the preparation of drugs or preparations for inhibiting fungal diseases that harm plant growth;

[0051] Volatile organic compounds include at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0052] In some embodiments, fungal diseases include those caused by at least one of Sclerotinia sclerotiorum, Phytophthora capsici, Staphylococcus aureus, Fusarium graminearum, and Fusarium verticillatum.

[0053] Specifically, compound 2-propyl-1-pentanol has good control effects against fungal diseases caused by Botrytis cinerea and Fusarium graminearum, and can be used in postharvest preservation of fruits and vegetables and biological control of grain crops; compound 2-methylbutyric acid has good control effects against fungal diseases caused by Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum and Fusarium verticillatum; 2-propyl-1-pentanol can completely inhibit the growth of Botrytis cinerea and Fusarium graminearum when the space concentration reaches 67 μL / L, and 2-methylbutyric acid can completely inhibit the growth of Botrytis cinerea and Sclerotinia sclerotiorum when the space concentration reaches 133 μL / L.

[0054] Based on the same inventive concept, the present invention also provides an agent for preventing and controlling plant fungal diseases, comprising volatile organic compounds, including at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

[0055] The following specific embodiments further illustrate the *Bacillus polymyxa* strain of this application, its volatile organic compounds, and their applications. This section further explains the invention in conjunction with specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in the art.

[0056] Example 1

[0057] Isolation and identification of Paenibacillus sp. WB-126

[0058] Paenibacillus sp. WB-126 was isolated from the rhizosphere soil of healthy rapeseed plants in a severely diseased rapeseed field in Jiangxia District, Wuhan City, Hubei Province. Gram staining was performed on the cells of strain WB-126, and the results are shown below. Figure 1 , Figure 1 A shows the Gram staining results, and B shows the colony morphology on LB agar plates. When strain WB-126 is streaked onto LB agar plates, the colonies are round, milky white, with neat edges, a raised center, and a smooth surface. Figure 1The physiological and biochemical characteristics of strain WB-126 were determined according to the *Handbook of Systematic Classification and Identification of Common Bacteria and Archaea* (results are shown in Table 1). Strain WB-126 can utilize glucose, lactose, and maltose to produce acid and gas; it can hydrolyze starch, decompose casein and cellulose, liquefy gelatin, and produce catalase; it is positive for the Voges-Proskauer test (VP test) and methyl red staining, can reduce nitrate, and can produce IAA (indole-3-acetic acid). However, it cannot produce H2S and urease, and cannot utilize citrate. This strain can grow normally in a medium with 8% salt content, exhibiting good salt tolerance. The results indicate that strain WB-126 possesses the typical morphological and physiological and biochemical characteristics of *PaenibaciLLus* bacteria.

[0059] Table 1 - Physiological and biochemical characteristics of strain WB-126

[0060]

[0061] Note: In Table 1, "+" indicates a positive result; "-" indicates a negative result.

[0062] Total genomic DNA was extracted from the antagonistic strains. Using this as a template, PCR amplification of the 16S rDNA gene was performed using universal bacterial primers. The PCR product was purified and sequenced, and sent to the company for processing. The obtained 16S rDNA sequence (accession number: px964340) has been submitted to the NCBI public database. BLAST homology analysis was performed between this sequence and sequences in the GenBank database. A phylogenetic tree was constructed using the neighbor-joining method with MEGA 11.0 software to determine the phylogenetic position of the strains. Figure 2 As shown.

[0063] Based on Gram staining, morphological and physiological-biochemical characteristics analysis, and 16S rDNA sequence analysis, strain WB-126 isolated from rapeseed rhizosphere soil was identified as *Paenibacillus* sp. WB-126. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on September 5, 2024, with the following classification and name: *Paenibacillus* sp. WB-126; accession number: CCTCC NO: M 20241922; location: Wuhan University, Wuhan, China.

[0064] The 16S rDNA sequencing results of Bacillus polymyxa WB-126 are as follows:

[0065]

[0066] Example 2

[0067] The preparation method of Bacillus polymyxa WB-126 fermentation broth includes the following steps:

[0068] S1. Activation of the strain: The Bacillus polymyxa strain WB-126, which was stored at -80℃, was inoculated onto LB agar slant (provided by Beijing Naphthalene Biochemical Technology Co., Ltd., catalog number BNCC373277) and activated at 30℃ for 48h.

[0069] S2. Primary Seed Culture: Add 100 mL of primary seed culture medium to a 500 mL Erlenmeyer flask and sterilize at 121 °C for 30 min. Inoculate the activated WB-126 strain culture into the primary seed culture medium and incubate at 30 °C and 200 r / min for 10 h to obtain the primary seed solution. The raw materials and quantities of the culture medium used for primary seed culture are: 1% glucose, 2% beef extract, 1% peptone, 0.35% sodium chloride, and pH 7.0 (percentages are mass fractions, the same in this example, the remainder is water).

[0070] S3, Fermentation: Add 300L of fermentation medium and 300mL of defoamer (specifically, defoamer GP330, purchased from Haian Petrochemical Plant, Jiangsu Province) to a 500L fermenter. Sterilize at 121℃ for 30min. When the tank temperature drops to 32℃, inoculate with 300mL of the primary seed culture from step S2; pressurize the tank to 0.5×10⁻⁶. 5 The culture was carried out at 28℃ for 36 hours, and samples were taken every 2 hours for microscopic examination. The fermentation was stopped when 20% of the spores were detached. The raw materials and amounts of the culture medium used for fermentation were: maltose 3.0%, soybean meal 3.0%, magnesium sulfate 0.3%, potassium dihydrogen phosphate 0.3%, dipotassium hydrogen phosphate 0.2%, ammonium chloride 0.2%, calcium carbonate 0.2%, and the pH of the culture medium was 8.0 (the remainder of the culture medium was water).

[0071] S4. Fermentation ended when 20% of the spores had fallen off. The spore count was determined to be 45.7 × 10⁻⁶. 8 CFU / mL was used as the fermentation broth stock solution in the following examples.

[0072] Example 3

[0073] Antibacterial activity test

[0074] In vitro antibacterial experiments were conducted using the confrontation culture method. Four sterile filter paper discs were placed on the crosshairs of a PDA agar plate, approximately 30 mm from the center. Then, 10 μL of the WB-126 fermentation broth prepared in Example 2 was added. After 24 hours, the pathogens listed in Table 2 were inoculated using a 4 mm punch. Sterile water was used as a blank control, and each treatment was repeated in triplicate. After the control colonies fully colonized the plate, the colony diameters for each treatment and the control were recorded, and the inhibition rate was calculated using the following formula. The inhibition rates against the mycelial growth of different plant pathogenic fungi are shown in Table 2.

[0075]

[0076] Table 2 - Inhibition rate of WB-126 fermentation broth against tested plant pathogenic fungi

[0077]

[0078] As can be seen from Table 2 above, the WB-126 fermentation broth showed significant antibacterial activity against all 15 tested pathogenic fungi, covering multiple pathogenic groups such as fungi (Fusarium, Anthrax, Alternaria, etc.), oomycetes (Phytophthora), Sclerotinia, and Penicillium, demonstrating broad-spectrum antibacterial potential. Among them, *Phytophthora indicum* (81.79%), *Phytophthora capsici* (74.40%), and *Phytophthora tumefaciens* (74.88%) can be used for the biological control of soil-borne / airborne diseases in economic crops such as tea, chili peppers, and tobacco; *Phytophthora downyicicum* (73.84%), *Botrytis cinerea* (69.88%), *Penicillium italicum* (64.05%), *Penicillium fingeringum* (66.83%), *Colletotrichum gloeosporioides* (71.73%), and *Alternaria piriformis* (73.66%) can be used for the prevention and control of diseases in fruit cultivation and post-harvest preservation; *Rhizoctonia solani* (68.82%), *Fusarium graminearum* (72.61%), and *Fusarium verticillatum* (71.36%) can be used for the biological control of soil-borne diseases in grain crops such as wheat, corn, and rice.

[0079] Example 4

[0080] The efficacy of WB-126 in controlling sclerotinia stem rot in rapeseed (ex vivo leaf method)

[0081] The rapeseed variety used in the test was Zhongshuang 18. The third true leaf was picked from rapeseed plants cultivated for 30 days. After rinsing, the leaf surface was disinfected with a 2% sodium hypochlorite solution, rinsed clean, and air-dried. The leaves were then placed in petri dishes with moistened filter paper at the bottom. Different concentrations of diluted WB-126 fermentation broth prepared in Example 2 (diluted with water to the corresponding ratio, e.g., 10-fold dilution, i.e., WB-126 fermentation broth from Example 2 diluted 10 times with water) were sprayed onto the rapeseed leaves using a spraying method. Sterile water was used as a control. After the rapeseed leaves dried, 5 mm diameter Sclerotinia sclerotiorum mycelium was inoculated onto each leaf. Each treatment was repeated 5 times. The plants were placed in a constant temperature incubator at 20℃ with 16 h light / 8 h darkness. After 2 days, the diameter of the lesions was measured, and the relative control efficacy was calculated using the following formula: Relative control efficacy (%) = (Lesion area of ​​control group - Lesion area of ​​treatment group) / Lesion area of ​​control group × 100%. Experiments showed that WB-126 has a good control effect on sclerotinia stem rot in rapeseed, and the control effect of a 10-fold dilution can reach more than 90%. The specific results are shown in Table 3.

[0082] Table 3 - Control efficacy of WB-126 fermentation broth (external leaf method) against Sclerotinia stem rot in rapeseed

[0083]

[0084] Note: In Table 3, data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05), and the same applies below.

[0085] Example 5

[0086] Application of WB-126 in the prevention and control of gray mold

[0087] Tomatoes of uniform size were immersed in a 2% sodium hypochlorite solution for 2 minutes for surface disinfection, followed by rinsing three times with sterile distilled water. After drying under sterile conditions, uniform wounds were created by making cross-shaped incisions in the same locations on the tomatoes using a sterile scalpel. A suspension of gray mold spores was mixed with dilutions of different concentrations of the biocontrol strain WB-126 fermentation broth (diluted with water to the appropriate ratio) in a specific ratio to achieve the final test concentration (final gray mold spore concentration 1×10⁻⁶). 5 (Spores / mL), with sterile water as a control. 10 μL of the mixture was added dropwise to the wounds on the tomato plants. The plants were placed in a constant temperature incubator at 20℃ with 16 h light / 8 h darkness. The disease incidence on the tomato fruits was assessed after 2 days. Disease severity was graded using a 0-4 scale: Grade 0: No lesions on the fruit surface; Grade 1: Some lesions on the fruit surface; Grade 2: Lesions covering <20% of the fruit surface area; Grade 3: Lesions covering 20%–50% of the fruit surface area; Grade 4: Lesions covering >50% of the fruit surface area.

[0088] Disease index = ∑ (number of fruits at each level × level) / (total number of fruits surveyed × highest representative level) × 100.

[0089] Relative efficacy = (Control disease index - Treatment disease index) / Control disease index × 100%.

[0090] Fruit trials showed that WB-126 has good control efficacy against Grape Botrytis cinerea. The control efficacy was greater than 90% after 10-fold and 20-fold dilution of the fermentation broth. It can be used for post-harvest preservation of fresh fruit. The specific results are shown in Table 4.

[0091] Table 4 - Effects of WB-126 fermentation broth on the control of Grape gray mold

[0092]

[0093] Example 6

[0094] The antibacterial effect of WB-126's volatile substances

[0095] The inhibitory effect of volatile substances produced by the fungal strain on various plant pathogens was determined using the double-plate inverting method. Following the method in Example 2, a primary seed culture was obtained. 100 µL of the primary seed culture was evenly spread on a 90 mm diameter LB agar plate (specifically, LB nutrient agar plate, catalog number HBPM0129, purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.). The spread LB plate was then inverted onto a PDA plate (specifically, CP0010 potato dextrose agar (PDA) plate, purchased from Guangdong Huankai Microbial Technology Co., Ltd.) containing a 4 mm inoculated block of plant pathogenic fungus in the center. The plates were sealed with sealing film. A blank LB plate was used as a control. After the pathogenic fungus control had fully colonized the plate, the diameter of the lesions from each treatment was measured. The inhibition rate was calculated using the following formula: Inhibition rate = (R... 空白 -R 菌 ) / R 空白 ×100%, where R 空白 R represents the diameter of plant pathogens growing on a blank LB plate. 菌 The diameter of the plant pathogenic fungus after the addition of Bacillus is shown in Table 5. The inhibition rates of all volatile substances produced by Bacillus polymyxa WB-126 against various plant pathogenic fungi are shown in Table 5.

[0096] Table 5. Determination of the antagonistic effect of volatile substances produced by strain WB-126 on the tested plant pathogenic fungi.

[0097]

[0098] The results above suggest that when using WB-126 for field control, the bacteria do not need to be in direct contact to achieve a good effect in controlling the disease.

[0099] Example 7

[0100] Detection of volatile substances

[0101] VOCs produced by Bacillus were monitored using headspace solid-phase microextraction. Approximately 5 mL of LB solid culture medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., catalog number HB0129; 0.2 g of HB0129 dry powder was dissolved in 5 mL of deionized water at a ratio of 40.0 g / L) was poured into a headspace vial. After sealing and sterilization, 100 µL of primary seed culture prepared according to the method in Example 2 was inoculated and cultured at 28 °C for 8 days. An extraction head (divinyLbenzene (DVB)-carboxen (CAR)-PDMS SPME fiber (Supelco, BeLLefonte, 50 / 30 µm, 50 / 30 µm DVB-CAR-PDMS: a three-layer composite coated solid-phase microextraction (SPME) fiber head, with 50 µm polydimethylsiloxane (PDMS) as the substrate and 30 µm carbon molecular sieve (CAR) + divinylbenzene (DVB) as the composite adsorption layer, is a classic general-purpose coating for detecting trace volatile organic compounds (VOCs), and is a hybrid coated fiber head from the Supelco brand) was inserted into a headspace vial and placed in a 50 °C water bath for equilibration for 10 min. The fiber head was then removed and placed 1-2 cm away from the bacterial block. After adsorption for 50 min, it was removed and inserted into the gas chromatograph injection port for desorption for 5 min. An 8890-5977C GC-MS (Agilent Technologies Inc.) and an HP-5MS (30 µm) were used. A gas chromatographic column (Agilent Technologies Inc.) measuring 30 meters in length, 0.25 mm in inner diameter, and 0.25 μm in stationary phase film thickness was used. GC-MS conditions were set as follows: inlet temperature 250°C; helium carrier gas flow rate 1 mL / min; splitless injection. The temperature program started at 40°C (hold for 3 minutes), increased to 160°C at 3°C / min (hold for 2 minutes), and then increased to 220°C at 8°C / min (hold for 3 minutes). The ion source and quadrupole temperatures were 230°C and 150°C, respectively, with an electron ionization (EI) of 70 eV in full scan mode (50-550 amu). LB solid medium (without primary seed culture) was used as a blank control to detect VOCs produced by Bacillus. The experiment was performed in triplicate. The results were compared with a standard library (NIST). 20) Comparison and retrieval were performed to identify VOCs, and the content of substances was determined using the peak area normalization method. The comparison results and information on some compounds are shown in Table 6.

[0102] Table 6 - Identification results of VOC substances produced by Bacillus.

[0103]

[0104] Specifically, Figure 3 The gas chromatography-mass spectrometry (GC-MS) chromatograms and peak search results in Example 7 using LB solid medium as a blank control; Figure 4 The gas chromatography-mass spectra and peak search results of volatile substances produced after the primary seed culture was inoculated into LB solid medium in Example 7; Figures 3-4 In the figure, the horizontal axis represents retention time and the vertical axis represents abundance.

[0105] Figure 3 Peak 1 corresponds to 6-methylcyclotrisiloxane, peak 2 corresponds to benzaldehyde, peak 3 corresponds to 6-methylcyclotrisiloxane, peak 4 corresponds to β-myrcene, peak 5 corresponds to 8-methylcyclotetrasiloxane, peak 6 corresponds to D-limonene, peak 7 corresponds to eucalyptol, peak 8 corresponds to 10-methylcyclopentasiloxane, peak 9 corresponds to 8-methylcyclotetrasiloxane, and peak 10 corresponds to 10-methylcyclopentasiloxane. The peak corresponding to pentasiloxane is 3-tetramethylsilyl-5-amino-1-methylpyrazole-4-carboxamide, the peak corresponding to 12 is 10-methylcyclopentasiloxane, the peak corresponding to 13 is 10-methylcyclopentasiloxane, the peak corresponding to 14 is 12-methylcyclohexasiloxane, the peak corresponding to 15 is 14-methylcycloheptasiloxane, the peak corresponding to 16 is 16-methylcyclooctasiloxane, and the peak corresponding to 17 is 18-methylcyclononasiloxane.

[0106] Figure 4 Peak 1 corresponds to 6-methylcyclotrisiloxane, peak 2 corresponds to 5-methyl-2-hexanone, peak 3 corresponds to 2-methylbutyric acid, peak 4 corresponds to 2-heptanone, peak 5 corresponds to 2,5-dimethylpyrazine, peak 6 corresponds to 6-methyl-2-heptanone, peak 7 corresponds to benzaldehyde, peak 8 corresponds to 5-methyl-2-heptanone, peak 9 corresponds to trimethylsilylarsenite, peak 10 corresponds to 8-methylcyclotetrasiloxane, peak 11 corresponds to 2-propyl-1-pentanol, peak 12 corresponds to phellandrene, peak 13 corresponds to 10-methylcyclopentasiloxane, and peak 14 corresponds to 8-methylcyclotetrasiloxane.

[0107] The mass spectrometry results above indicate that Bacillus can produce a number of volatile substances, mainly including 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 2,5-dimethyl-pyrazine, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol, all of which are small molecules with a molecular weight of less than 200. Among them, the compounds with higher content are 2-heptanone and 2,5-dimethyl-pyrazine. The antibacterial activity of the detected compounds will be further tested.

[0108] Example 8

[0109] Antibacterial test of volatile substances

[0110] The inhibitory effects of compounds such as 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 2,5-dimethyl-pyrazine, 6-methyl-2-heptanone, and 2-propyl-1-pentanol on *Sclerotinia sclerotiorum* and *Staphylococcus aureus* were determined using the double-plate inverting method. PDA plates (specifically CP0010 potato dextrose agar (PDA) plates, purchased from Guangdong Huankai Microbial Technology Co., Ltd.), containing 4 mm inoculated blocks of plant pathogenic fungi, were inverted onto LB plates. Small circular filter paper discs were placed on each LB plate, and 40 μL, 20 μL, 10 μL, 5 μL, 2.5 μL, 1.25 μL, and 0.625 μL of the compound were added sequentially onto the filter paper discs. The plates were sealed with sealing film. LB plates containing 40 μL of water served as a control. After the pathogenic fungi had fully colonized the plates, the diameter of the lesions from each treatment was measured. The inhibition rate was calculated using the following formula: Inhibition rate = (R... 空白 -R 化合物 ) / R 空白 ×100%, where R 空白 R represents the diameter of plant pathogens growing on LB agar plates with added pure water. 化合物 The diameter of plant pathogens after the addition of different volumes of the compound is shown in Tables 7-8. The inhibitory effects of the compounds on various plant pathogenic fungi are shown in Tables 7-8.

[0111] Table 7 - Inhibitory effects of volatile substances on *Sclerotinia sclerotiorum*, *Staphylococcus aureus*, *Fusarium verticillatum*, and *Fusarium graminearum*.

[0112]

[0113] Table 8 - Inhibitory effects of volatile substances on *Sclerotinia sclerotiorum*, *Staphylococcus aureus*, *Fusarium verticillatum*, and *Fusarium graminearum*.

[0114]

[0115] In Tables 7 and 8, - indicates that no antibacterial activity was observed when the compound was added at a maximum volume of 40 μL; IC50 50 The volume added when the inhibition rate is 50%, IC95 The volume added when the test bacteria show no growth whatsoever.

[0116] The experimental results in Tables 7 and 8 show that, except for 2,5-dimethylpyrazine, all the tested compounds exhibited antifungal activity, with 2-methylbutyric acid and 2-propyl-1-pentanol showing the strongest activity. In double-sided plates, 1.2 μL and 2.4 μL of 2-methylbutyric acid and 2-propyl-1-pentanol significantly inhibited the growth of *Sclerotinia sclerotiorum*, respectively; 4.8 μL and 1.3 μL significantly inhibited the growth of *Staphylococcus aureus*; 4.1 μL and 1.9 μL significantly inhibited the growth of *Fusarium graminearum*; and 3.2 μL and 0.9 μL significantly inhibited the growth of *Fusarium graminearum*. Compound 2-methylbutyric acid completely inhibited the growth of *Sclerotinia sclerotiorum* and *Staphylococcus aureus* at a concentration of 133 μL / L (the space volume of the double-sided plate is approximately 0.075 L), and completely inhibited the growth of *Fusarium graminearum* and *Fusarium graminearum* at a concentration of 266 μL / L. 2-Propyl-1-pentanol, at a lower concentration of 67 μL / L, can completely inhibit the growth of *Botrytis cinerea* and *Fusarium graminearum*. In postharvest preservation, adding a small amount of 2-propyl-1-pentanol to storage containers can effectively extend the storage time of fruits and vegetables.

[0117] It is understood that the technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The above are merely preferred embodiments of this application, and only specifically describe the technical principles of this application. These descriptions are only for explaining the principles of this application and should not be construed as limiting the scope of protection of this application in any way. Based on this explanation, any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application, as well as other specific embodiments of this application that can be conceived by those skilled in the art without creative effort, should be included within the scope of protection of this application.

Claims

1. A polymyxa-like Bacillus, characterized in that, The classification name of the polymyxin Bacillus is: Polymyxin Bacillus sp. WB-126, and the preservation number of the polymyxin Bacillus is CCTCC NO: M20241922.

2. The use of the *Bacillus polymyxa* or its fermentation broth as described in claim 1 in the preparation of a drug or formulation for inhibiting fungal diseases that harm plant growth.

3. The application as described in claim 2, characterized in that, The fungal diseases mentioned include those caused by at least one of the following: Rhizoctonia solani, Sclerotium truncatum, Fusarium oxysporum, Sclerotium sclerotiorum, Phytophthora lichei, Botrytis cinerea, Fusarium graminearum, Fusarium verticillatum, Penicillium italicum, Penicillium fingernail, Colletotrichum anthracnose, Alternaria pearis, Phytophthora capsici, Pseudomonas aeruginosa, and Phytophthora tumefaciens.

4. The application as described in claim 2, characterized in that, The preparation method of the Bacillus polymyxa fermentation broth includes the following steps: Polymyxin Bacillus was cultured in a primary seed culture medium to obtain a primary seed solution; The primary seed culture was inoculated into a fermentation medium and cultured to obtain the fermentation broth; The primary seed culture medium comprises the following components by mass fraction: 1-2% glucose, 2-3% beef extract, 1-2% peptone, 0.35-0.45% sodium chloride, and the remainder is water; The fermentation medium comprises the following components by mass fraction: maltose 3.0-4.0%, soybean meal 3.0-4.0%, magnesium sulfate 0.3-0.4%, potassium dihydrogen phosphate 0.3-0.4%, dipotassium hydrogen phosphate 0.2-0.3%, ammonium chloride 0.2-0.3%, and calcium carbonate 0.2-0.3%.

5. The application as described in claim 4, characterized in that, In the step of inoculating Bacillus polymyxa in primary seed culture medium, the culture temperature is 28~32℃ and the time is 10~12h; In the step of inoculating the primary seed culture into the fermentation medium, the culture temperature is 27~29℃ and the time is 20~60h.

6. An application of the *Bacillus polymyxa* as described in claim 1 in the production of volatile organic compounds, said volatile organic compounds comprising at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 2,5-dimethylpyrazine, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

7. The application as described in claim 6, characterized in that, The primary seed culture is inoculated into LB solid medium and cultured to produce volatile organic compounds; the primary seed culture is prepared by inoculating Bacillus polymyxa in a primary seed medium and cultured according to the method of claim 4 or 5 to obtain the primary seed culture.

8. The use of a volatile organic compound in the preparation of a drug or formulation for inhibiting fungal diseases that harm plant growth; The volatile organic compounds include at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

9. The application as described in claim 6, characterized in that, The fungal diseases include those caused by at least one of Sclerotinia sclerotiorum, Phytophthora capsici, Staphylococcus aureus, Fusarium graminearum, and Fusarium verticillatum.

10. A preparation for controlling plant fungal diseases, characterized in that, It includes volatile organic compounds, said volatile organic compounds including at least one of 5-methyl-2-hexanone, 2-methylbutyric acid, 2-heptanone, 6-methyl-2-heptanone, 5-methyl-2-heptanone, and 2-propyl-1-pentanol.

Images