Methylotrophic bacillus JK21, biocontrol agent and application thereof

Abstract

This invention relates to the field of plant disease control technology, specifically disclosing a methylotrophic Bacillus JK21, a biocontrol agent, and its application. JK21 was deposited at the China Center for Type Culture Collection (CCTCC) on May 27, 2024, with accession number CCTCC NO: M 20241072, and is classified as follows: Bacillus methylotrophicus JK21. The methyltrophic Bacillus JK21 provided by this invention inhibits the growth of ginseng rust rot pathogens by producing JK21 extracellular secretions, thereby achieving the effect of preventing and controlling ginseng rust rot.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to a methyl-trophic Bacillus JK21 strain, a biocontrol agent, and its application. Background Technology

[0002] Ginseng, belonging to the genus Panax in the family Araliaceae, is a precious medicinal plant in China and a unique specialty resource of Jilin Province. Because ginseng prefers a cool, moist environment during its growth period, it is susceptible to diseases. Rust rot is one of the major diseases affecting ginseng, causing mass mortality, reduced yields, and significant economic losses. Chemical control methods may leave pesticide residues and cause secondary pollution to the environment, while biological control can control the disease while minimizing environmental impact, resulting in less pollution, lower pesticide residues, and a more environmentally friendly approach. Therefore, developing a biological product for controlling ginseng rust rot is of great significance. Summary of the Invention

[0003] To develop a biological product for preventing and controlling ginseng rust rot, this invention provides a methylotrophic Bacillus strain JK21, a biocontrol agent, and their applications. The methylotrophic Bacillus JK21 provided by this invention inhibits the growth of ginseng rust rot pathogens by producing JK21 extracellular secretions, thereby achieving the effect of preventing and controlling ginseng rust rot.

[0004] This invention provides a methyltrophic Bacillus strain ( Bacillus methylotrophicus The methyltrophic Bacillus JK21 was deposited at the China Center for Type Culture Collection (CCTCC) on May 27, 2024, with accession number CCTCC NO: M 20241072, and classified as follows: Bacillus methylotrophicus JK-21.

[0005] The methylotrophic Bacillus JK21 provided by this invention inhibits the growth of ginseng rust pathogens by producing JK21 extracellular secretions, thereby achieving the effect of preventing and controlling ginseng rust disease.

[0006] The present invention also provides a methyltrophic Bacillus bacterial culture, which is obtained by inoculating the methyltrophic Bacillus JK21 into a culture medium and culturing it.

[0007] Furthermore, the culture medium is tryptic soy peptone liquid medium.

[0008] The present invention also provides an extracellular secretion of JK21, which is obtained from the secretion of the methyltrophic Bacillus JK21.

[0009] Furthermore, the JK21 extracellular secretions are obtained by extracting the fermentation supernatant after liquid fermentation of the JK21 strain.

[0010] Furthermore, the JK21 extracellular secretions include any one or a combination of several of the following: crude ester peptides, crude protein, ethyl acetate, n-butanol, and petroleum ether secreted by the JK21 strain.

[0011] The present invention also provides a biocontrol agent, which is a compound of the JK21 extracellular secretion and the WY1 extracellular secretion;

[0012] The extracellular secretions of WY1 are produced by Neurospora crassa (… NeurosporacrassaStrain WY1 secretion acquisition;

[0013] The *Neurospora crassa* WY1 strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 21, 2019, with accession number CCTCC NO: M 2019845, and classified as follows: Neurospora crassa Strain WY1.

[0014] Furthermore, the extracellular secretions of WY1 include any one or a combination of several of the following: crude ester peptides, crude protein, ethyl acetate, n-butanol, and petroleum ether secreted by the WY1 strain.

[0015] Furthermore, the biocontrol agent is a compound of crude protein secreted by strain JK21 and ethyl acetate secreted by strain WY1 in a concentration ratio of 1-9:1-9.

[0016] Furthermore, the biocontrol agent is a mixture of crude protein secreted by strain JK21 and ethyl acetate secreted by strain WY1 in a concentration ratio of 6:4.

[0017] The present invention also provides the application of the aforementioned methylotrophic Bacillus JK21, the aforementioned methylotrophic Bacillus bacterial solution, the aforementioned JK21 extracellular secretion, or the aforementioned biocontrol agent in the prevention and control of ginseng rust disease, the application including inhibiting the germination of ginseng rust disease pathogenic fungal spores and inhibiting the growth of ginseng rust disease pathogenic fungal hyphae;

[0018] The pathogen causing ginseng rust is *Streptococcus destructis* (…). Cylindrocarpondestructans ).

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The methyltrophic Bacillus isolated from ginseng rhizosphere in this invention ( Bacillus methylotrophicus JK21 and Neurospora crassa ( NeurosporacrassaStrainWY1 has the function of inhibiting ginseng rust rot pathogen. Crude ester peptides, crude protein, ethyl acetate, n-butanol, and petroleum ether secreted by strains JK21 and WY1 also have the effect of inhibiting ginseng rust rot pathogen. This invention also obtained a biocontrol agent for inhibiting ginseng rust rot pathogen by compounding the crude protein secreted by strain JK21 and the ethyl acetate secreted by strain WY1.

[0021] This invention isolates and extracts the extracellular secretions of strains JK21 and WY1, clarifies the active composition of antibacterial active substances, explores the mechanism of action of ginseng rhizosphere strains against ginseng rust rot pathogens, and provides a theoretical basis and application foundation for the prevention and control of ginseng rust rot disease.

[0022] Information on the Preservation of Biological Materials

[0023] JK21, referred to in this invention as Methyltrophic Bacillus JK21, was deposited on May 27, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20241072. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072. Its classification is as follows: Bacillus methylotrophicus JK-21.

[0024] WY1, referred to in this invention as *Neurospora crassa* WY1, was deposited on October 21, 2019, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2019845. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072. It is classified as follows: Neurospora crassa Strain WY1. 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 The figure shows the plate confrontation test results of JK21 and WY1 bacteria obtained by the present invention against ginseng rust rot fungus. In the figure, CK is the control group inoculated only with ginseng rust rot fungus, JK21 represents the plate confrontation results of JK21 bacteria against ginseng rust rot fungus, and WY1 represents the plate confrontation results of WY1 bacteria against ginseng rust rot fungus.

[0027] Figure 2 This is a statistical chart showing the antibacterial rates of different active substances in the extracellular secretions of strains JK21 and WY1.

[0028] Figure 3The inhibitory effect of different concentrations of JK21 crude protein mixed with WY1 ethyl acetate on ginseng rust fungus was investigated.

[0029] Figure 4 The study investigated the inhibitory effect of different concentrations of ethyl acetate WY1 mixed with crude protein extract JK21 on ginseng rust fungus.

[0030] Figure 5 The inhibitory effect of a mixture of crude protein JK21 and ethyl acetate WY1 at a ratio of 6:4 on ginseng rust fungus at different concentrations was investigated.

[0031] Figure 6 The virulence equation for the inhibition of ginseng rust fungus at different concentrations of a mixture obtained by mixing JK21 crude protein and WY1 ethyl acetate at a ratio of 6:4.

[0032] Figure 7 The effect of mixed antibacterial active substances on the prevention and treatment of ginseng rust disease;

[0033] In the figure, a is the control group; b is the rust-rot fungus treatment; c is the treatment group treated with a mixture of JK21 crude protein and WY1 ethyl acetate in a ratio of 6:4 after inoculation with ginseng rust-rot fungus.

[0034] Figure 8 The effect of JK21 crude protein mixed with WY1 ethyl acetate on the hyphal morphology and structure of ginseng rust rot fungus;

[0035] In the figure, A is a micrograph of the mycelium of *Gynostemma pentaphyllum* treated with a mixture of crude protein JK21 and ethyl acetate WY1.

[0036] B is a micrograph of normal hyphae of the fungus causing ginseng rust. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0038] Example 1: Isolation and identification of ginseng rhizosphere antagonistic strains.

[0039] A total of 122 bacterial strains were isolated from the rhizosphere of ginseng. Biocontrol bacteria were screened using the plate confrontation method. A 7.0 mm diameter mycelial cake of *Ginseng rust rot* was inoculated into the center of sterilized PDA medium. CylindrocarpondestructansThe strain was purchased from the China Center for Type Culture Collection (CCTCC), strain accession number CCTCC NF20081871. Four 7.0 mm diameter filter paper discs were placed symmetrically at a distance of 20 mm from the center of the pathogenic fungal disc. 5 μL of bacterial suspension was added to each disc using a pipette. Plates inoculated only with the rust fungal disc served as a blank control. Each treatment was tested in triplicate. After inoculation, the plates were incubated at 28°C. After 10 days of incubation, the mycelial diameter of the treated and blank groups was observed and measured, and the inhibition rate was calculated.

[0040] The formula for calculating the antibacterial rate is:

[0041] Inhibition rate (%) = (control colony diameter - pathogen colony diameter) / control colony diameter × 100.

[0042] A total of 21 antagonistic strains were found, among which two strains had an inhibition rate greater than 70%, designated JK21 and WY1, respectively. Figure 1 .

[0043] Morphological and molecular biological identification of strains JK21 and WY1 indicates that JK21 is a methyltrophic Bacillus. Bacillus methylotrophicus WY1 is Neurospora crassa. NeurosporacrassaStrain .

[0044] Example 2: Composition and analysis of the antibacterial active ingredients of the strain.

[0045] 1. Isolation and extraction of active substances from extracellular secretions of strains JK21 and WY1

[0046] The methods for isolating and extracting active substances from the extracellular secretions of strains JK21 and WY1 are the same, as detailed below:

[0047] (1) Fermentation supernatant: The antagonistic strains JK21 and WY1 were inoculated into 200 mL of LB liquid medium and cultured at 37℃ and 180 rpm for 36 h to obtain the stock solution. 10 mL of the stock solution was inoculated into 200 mL of freshly prepared NB liquid medium for subculture and cultured at 37℃ and 180 rpm for 3 days to thicken the bacterial culture. The cultured fermentation broth was centrifuged at 10000 rpm for 30 min at 4℃. The supernatant was filtered through a 0.22 µm microporous membrane to remove bacteria. The sterilized solution was the fermentation supernatant. The fermentation supernatants obtained from strains JK21 and WY1 were respectively recorded as JK21 fermentation supernatant and WY1 fermentation supernatant.

[0048] (2) Crude lipopeptide: Take 20 mL of JK21 fermentation supernatant and transfer it to a centrifuge tube. Adjust the pH to 2.0 with HCl, let it stand at 4℃ for 12 h, and centrifuge at 10000 rpm for 25 min at 4℃. Take the supernatant and adjust the pH to 7.0 and store it at 4℃. Dissolve the precipitate after centrifugation with methanol and then filter it. Repeat the process twice. Combine the liquids from the two filtrations and concentrate the liquid to dryness using a rotary evaporator. Adjust the volume to 10 mL with PBS buffer (pH 7.2, concentration 0.02 mol / L). Sterilize the solution by passing it through a 0.22 µm filter membrane to obtain the crude lipopeptide of JK21. Obtain the crude lipopeptide of WY1 using the same method.

[0049] (3) Crude protein: 20 mL of JK21 fermentation supernatant was placed in a centrifuge tube. Ammonium sulfate particles were added to the centrifuge tube and shaken to make the supernatant saturation above 90%. The mixture was placed at 4℃ for 12 h and centrifuged at 10000 rpm for 25 min. PBS buffer with pH 7.2 and concentration of 0.02 mol / L was added to the precipitate to dissolve it. The dissolved solution was transferred to a dialysis bag with a molecular weight cutoff of 8 kD to 14 kD for desalting. Desalting was continued until no precipitate was formed in the external solution of 1% BaCl2 during dialysis. The resulting solution was then lyophilized. The lyophilized material was brought to a final volume of 10 mL with PBS buffer and sterilized by passing through a 0.22 µm filter membrane to obtain JK21 crude protein. WY1 crude protein was obtained using the same method.

[0050] (4) Ethyl acetate extraction: Take 20 mL of fermentation supernatant of JK21 and put it into a centrifuge tube. Use 20 mL of ethyl acetate to extract it in 3 times. Combine the extracted substances into the same centrifuge tube and evaporate the three organic solvents separately using a rotary evaporator. Take the prepared PBS buffer and make up to 10 mL. Sterilize the solution by filtering it through a 0.22 µm filter membrane to obtain ethyl acetate of JK21. Obtain ethyl acetate of WY1 using the same method.

[0051] (5) Water-saturated n-butanol extraction: Take 20 mL of JK21 fermentation supernatant and put it into a centrifuge tube. Use 20 mL of water-saturated n-butanol to extract the mixture in three portions. Combine the extracted substances into the same centrifuge tube and evaporate the three organic solvents to dryness using a rotary evaporator. Take the prepared PBS buffer and make up to 10 mL. Sterilize the solution by filtering it through a 0.22 µm filter to obtain JK21 n-butanol. Obtain WY1 n-butanol using the same method.

[0052] (6) Petroleum ether extraction: Take 20 mL of JK21 fermentation supernatant and put it into a centrifuge tube. Use 20 mL of petroleum ether to extract it in 3 times. Combine the extracted substances into the same centrifuge tube and evaporate the three organic solvents separately using a rotary evaporator. Take the prepared PBS buffer and make up to 10 mL. Sterilize the solution by filtering it through a 0.22 µm filter membrane to obtain JK21 petroleum ether. WY1 petroleum ether was obtained by the same method.

[0053] It should be noted that JK21 crude protein refers to the crude protein extract extracted from the supernatant of strain JK21, and WY1 crude protein refers to the crude protein extract extracted from the supernatant of strain WY1; JK21 ethyl acetate refers to the ethyl acetate extract extracted from the supernatant of strain JK21, and WY1 ethyl acetate refers to the ethyl acetate extract extracted from the supernatant of strain WY1; JK21 n-butanol refers to the water-saturated n-butanol extract extracted from the supernatant of strain JK21, and WY1 n-butanol refers to the water-saturated n-butanol extract extracted from the supernatant of strain WY1; JK21 petroleum ether refers to the petroleum ether extract extracted from the supernatant of strain JK21, and WY1 petroleum ether refers to the petroleum ether extract extracted from the supernatant of strain WY1.

[0054] 2. Antagonistic test of various antibacterial active substances in the extracellular secretions of strains JK21 and WY1 against rust fungus.

[0055] The antibacterial active substances obtained above, namely crude protein of JK21, crude protein of WY1, ethyl acetate of JK21, ethyl acetate of WY1, n-butanol of JK21, n-butanol of WY1, petroleum ether of JK21, and petroleum ether of WY1, were added to PDA medium at 45℃ at a volume ratio of 1:99. After mixing, the mixture was poured into plates and cooled to obtain solid plates containing different antibacterial active substances. A 7.0 mm diameter mycelium of *Ginseng rust rot* was inoculated onto the solid plates. Pure PDA medium without antibacterial active substances was used as a control. Three plates were prepared for each antibacterial active substance for three replicates. The plates were incubated at 25℃ and the growth was observed. After about 10 days, when the mycelium in the control medium was fully grown, the colony diameter of *Ginseng rust rot* was measured using a cross-sectional method on the solid plates containing different antibacterial active substances, and the inhibition rate was calculated.

[0056] The results are as follows Figure 2 As shown, the antibacterial rates of the antibacterial active substances secreted by strains JK21 and WY1, from highest to lowest, are: JK21 crude protein > WY1 ethyl acetate > WY1 crude protein > JK21 ethyl acetate > WY1 n-butanol > JK21 n-butanol > JK21 crude ester peptide > WY1 crude ester peptide > JK21 petroleum ether > WY1 petroleum ether. Among these, JK21 crude protein and WY1 ethyl acetate played the main antibacterial roles, with JK21 crude protein exhibiting an antibacterial rate of 81.54% and WY1 ethyl acetate an antibacterial rate of 78.12%.

[0057] Example 3: A biocontrol agent, its preparation method, and its application.

[0058] 1. Study on the antibacterial effect of a biocontrol agent obtained by mixing JK21 crude protein with WY1 ethyl acetate.

[0059] (1) Using different concentrations of JK21 crude protein as the drug-containing culture medium, plate confrontation was carried out with WY1 ethyl acetate.

[0060] Antibacterial tests were conducted using a plate-to-plate method with the presence of antibacterial active substances. JK21 crude protein was diluted with sterile water to prepare antibacterial activity solutions at concentrations of 0.5 mL / L, 1 mL / L, 2.5 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L. Different concentrations of JK21 crude protein antibacterial activity solutions were added to PDA medium to test the antibacterial effect. After the medium was prepared, it was poured into petri dishes to obtain plates containing the antibacterial active substances. A 7 mm ginseng rust fungus cake was inoculated into the center of each plate, and two 7.0 mm diameter filter paper discs were placed symmetrically at a distance of 20 mm from the center of the fungus. Finally, 5 μL of WY1 ethyl acetate was pipetted in to test the antibacterial effect. A plate containing only JK21 crude protein and a plate inoculated with WY1 ethyl acetate extract were used as positive controls, and pure PDA medium was used as a blank control. The inhibition rate could be calculated when the blank control was fully grown.

[0061] The results are as follows Figure 3 As shown, when using JK21 crude protein as the drug-containing culture medium and WY1 ethyl acetate as the plate for antibacterial experiments against antagonistic strains, the antibacterial rate of the mixture of JK21 crude protein and WY1 ethyl acetate was significantly higher than that of JK21 crude protein alone at the same concentration. The antibacterial rates at concentrations of 0.5 mL / L, 1 mL / L, 2.5 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L were 3.96%, 8.45%, 18.05%, 16.11%, 11.76%, and 10.76% higher than those of the single agent, respectively, with an average increase of 11.51%. When JK21 crude protein and WY1 ethyl acetate were mixed, the concentrations at 2.5 mL / L and 5 mL / L were higher than those at 5 mL / L and 10 mL / L for JK21 alone. Even with a reduced concentration, the mixture achieved the same antibacterial effect as the single agent. In tests using different concentrations of antibacterial agents, the antibacterial efficiency significantly increased with increasing concentration. Even at 0.5 mL / L, the combined use of these agents was superior to the use of WY1 ethyl acetate alone. This indicates that combined use is superior to single-agent use.

[0062] (2) Using different concentrations of WY1 ethyl acetate as the drug-containing culture medium, plate confrontation was carried out with JK21 crude protein.

[0063] The experimental method was the same as before. Ethyl WY1 was diluted with sterile water to prepare antibacterial activity solutions with concentrations of 0.5 mL / L, 1 mL / L, 2.5 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L. The antibacterial effect of adding different concentrations of ethyl WY1 to the culture medium was tested. After the culture medium was prepared, it was poured into petri dishes to obtain plates containing the antibacterial active substance. A 7 mm ginseng rust fungus cake was inoculated in the center of the plate containing the antibacterial active substance. Two 7.0 mm diameter filter paper discs were placed symmetrically at a distance of 20 mm from the center of the fungus. Then, 5 μL of JK21 crude protein was added using a pipette to test the antibacterial effect. The culture medium containing only ethyl WY1 and the rust fungus inoculated using the JK21 crude protein confrontation method served as positive controls, and pure PDA culture medium served as a blank control. The inhibition rate was calculated when the blank control reached confluence.

[0064] The results are as follows Figure 4 As shown, when WY1 ethyl acetate is used as the drug-containing culture medium and JK21 crude protein is used as the plate to confront the antagonistic strain, as... Figure 3 As shown, the antibacterial rate of WY1 ethyl acetate and JK21 crude protein was significantly higher when used in combination than when WY1 ethyl acetate was used alone at the same concentration. When used in combination, the antibacterial rates at concentrations of 0.5 mL / L, 1 mL / L, 2.5 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L were 8.15%, 9.15%, 9.13%, 7.11%, 9.85%, and 17.16% higher than those of the single agent, respectively. The average antibacterial rate was increased by 10.09%, and the antibacterial effect was significantly enhanced with increasing concentration at different concentrations.

[0065] 2. Screening for the optimal ratio of JK21 crude protein to WY1 ethyl acetate.

[0066] The antibacterial rates of JK21 crude protein and WY1 ethyl acetate were measured by mixing them at different ratios. A synergistic effect was observed when the virulence ratio was significantly greater than 1; an antagonistic effect when it was significantly less than 1; and an additive effect when the virulence ratio was around 1. The EC50 values ​​of JK21 crude protein and WY1 ethyl acetate were used as the concentrations for mixing. The concentration ratios of JK21 crude protein to WY1 ethyl acetate were set to 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10.

[0067] Expected antibacterial rate = (actual antibacterial rate of JK21 crude protein at EC50 dose × percentage ratio) + (actual antibacterial rate of WY1 ethyl acetate at EC50 dose × percentage ratio).

[0068] Virulence ratio = Actual inhibition rate / Expected inhibition rate.

[0069] Table 1. Toxicity ratio of JK21 crude protein to WY1 ethyl acetate mixture

[0070]

[0071] As shown in Table 1, when the concentration ratio of crude protein JK21 to ethyl acetate WY1 is 8:2 and 4:6, the toxicity ratio is close to 1, indicating that the two substances exhibit an additive effect at this ratio. At the ratios of 7:3, 6:4, and 5:5, the toxicity ratio is significantly greater than 1 at 10-12 days, indicating that the two antibacterial substances have a synergistic effect at these ratios. However, at other ratios, the toxicity ratio is less than 1, indicating that the two antibacterial substances exhibit antagonism at these ratios.

[0072] Table 2. Determination of the toxicity of the optimal ratio of crude protein (JK21) to ethyl acetate (YY15) on mycelial growth inhibition.

[0073]

[0074] The toxicity of the selected 7:3, 6:4, and 5:5 ratios of JK21 crude protein to WY1 ethyl acetate mixtures with synergistic effects was determined. Table 2 shows that the co-toxicity coefficients were all greater than 100 at the 7:3, 6:4, and 5:5 ratios, indicating a synergistic effect. The highest co-toxicity coefficient (243.67) was observed at the 6:4 ratio. Therefore, the optimal ratio of JK21 crude protein to YY15 ethyl acetate is 6:4. 50 It is 1.72 mL / L.

[0075] like Figure 5 As shown, the mixture of JK21 crude protein and WY1 ethyl acetate in a 6:4 ratio showed significant antibacterial effects against ginseng rust bacteria at different concentrations.

[0076] Based on the optimal ratio of JK21 crude protein to WY1 ethyl acetate (6:4), a virulence regression equation was established to investigate the inhibitory effect of different concentrations on ginseng rust fungus. Figure 6 As shown.

[0077] 3. Inhibitory effect of JK21 crude protein mixed with WY1 ethyl acetate on spore germination of ginseng rust fungus.

[0078] In the screening of the optimal ratio of JK21 crude protein to WY1 ethyl acetate for spore germination of *Pseudomonas aeruginosa* at different proportions, Table 3 shows that the toxicity ratios were greater than 1 at ratios of 7:3, 6:4, and 5:5, indicating a synergistic effect between the two antibacterial substances at these ratios. However, the toxicity ratios were less than 1 at other ratios, showing no synergistic effect.

[0079] Table 3 Screening of the optimal mixing ratio of JK21 crude protein and WY1 ethyl acetate

[0080]

[0081] Indoor toxicity testing:

[0082] The relevant formulas for toxicity testing are as follows:

[0083] X = Y / Z × 100;

[0084] Where X: Single-dose toxicity index, used to represent the comparison of the toxicity of the test drug relative to the standard drug; Y: Half-maximum effect concentration (EC) of the standard drug. 50 Value, i.e., the concentration of the standard substance required to produce a 50% maximum effect in the test subject; Z: the half-maximum effect concentration (EC) of the test reagent. 50 The value is the concentration required for the analyte to produce a 50% maximum effect in the test subject.

[0085] M = N / P × 100;

[0086] Wherein, M: Actual toxicity index of the mixture, used to measure the relative toxicity of "JK21 crude protein + WY1 ethyl acetate mixture" compared to the standard reagent; N: Half-maximum effect concentration (EC50) of the standard reagent. 50 Value, i.e., the concentration required for the standard substance to produce a 50% maximum effect in the test subject; P: the half-maximum effect concentration (Cg) of "JK21 crude protein + WY1 ethyl acetate mixture". 50 The value is the concentration required for the mixture to produce a 50% maximum effect in the test subject.

[0087] Q = R + S + T + U;

[0088] Wherein, Q: Theoretical toxicity index of the mixture, used to evaluate the theoretical comprehensive toxicity index of the mixture composed of JK21 crude protein and WY1 crude protein; R: Toxicity index of JK21 crude protein, that is, the comparison value of the toxicity of JK21 crude protein relative to the standard agent; S: Content of JK21 crude protein in the mixture, referring to the proportion or concentration of JK21 crude protein in the mixture; T: Toxicity index of WY1 crude protein, that is, the comparison value of the toxicity of WY1 crude protein relative to the standard agent; U: Content of WY1 crude protein in the mixture, referring to the proportion or concentration of WY1 crude protein in the mixture.

[0089] V = W / X × 100;

[0090] Wherein, V: Co-toxicity coefficient (CTC) of the mixture, used to measure the relationship between the actual toxicity and the theoretical toxicity of the mixture, reflecting the combined effect of the components in the mixture, such as synergistic, antagonistic or additive effects; W: Actual toxicity index of the mixture, that is, the comparison value of the actual toxicity of the mixture relative to the standard drug; X: Theoretical toxicity index of the mixture, that is, the theoretical comprehensive toxicity value calculated based on the toxicity index of each component in the mixture and its content in the mixture.

[0091] Based on the results of the ratio of crude protein JK21 to ethyl acetate WY1, a ratio with a toxicity ratio greater than 1 and a significant synergistic effect was selected for toxicity testing. Drug-containing culture media were prepared at concentrations of 0.5 mL / L, 1 mL / L, 2.5 mL / L, 5 mL / L, 10 mL / L, and 20 mL / L, and injected into the center of each drug-containing culture medium at different concentrations into the rust-rotten roots of ginseng. The inhibition rate was calculated. The co-toxicity coefficient (CTC) was also calculated. A CTC greater than 100 indicated a synergistic effect; less than 100 indicated an antagonistic effect; and close to 100 indicated an additive effect. The half-maximum effect concentration (EC) is abbreviated as EC. 50 value.

[0092] Toxicological tests were conducted on the selected synergistic ratios of JK21 crude protein and WY1 ethyl acetate (7:3, 6:4, and 5:5). Table 4 shows that the co-toxicity coefficients of these three ratios were 166.92, 195.75, and 164.80, respectively, all greater than 100. This indicates that these three ratios have a synergistic effect on inhibiting the germination of ginseng rust spores. The highest co-toxicity coefficient (195.75) was observed at a ratio of 6:4, indicating that the 6:4 ratio showed the best inhibitory effect on ginseng rust spore germination, demonstrating a significant synergistic effect.

[0093] Table 4. Toxicological assay of spore germination by mixing JK21 crude protein with WY1 ethyl acetate.

[0094]

[0095] 4. The effect of JK21 crude protein mixed with WY1 ethyl acetate on the prevention and control of rust disease.

[0096] Select healthy, disease-free, and uniformly sized fresh ginseng. Rinse them thoroughly. In a clean bench, soak the ginseng in sodium hypochlorite for 60 seconds, then in 75% ethanol for 60 seconds. Remove and wipe dry with sterile filter paper. A biocontrol agent, prepared by mixing JK21 crude protein and WY1 ethyl acetate at a concentration ratio of 6:4, was applied to ginseng detached roots 3 days after inoculation with *Ginseng rust fungus*. Sterile water was used as the control group. Figure 7 a; Treatment with ginseng rust fungus suspension as a positive control, such as... Figure 7 b. The result is as follows Figure 7 As shown in c, the appearance of ginseng did not change significantly with the increase of days, indicating that the biocontrol agent effectively inhibited the growth of ginseng rust bacteria.

[0097] 5. Effects of mixing JK21 crude protein with WY1 ethyl acetate on the morphology and structure of rust-causing fungal hyphae.

[0098] Microscopic observation was performed on the hyphal morphology and structure of *Ginseng rust rot fungus* treated with a mixture of JK21 crude protein and WY1 ethyl acetate, comparing it with that of normal hyphae. Results are as follows: Figure 8 As shown, the mixture of crude protein JK21 and ethyl acetate WY1 significantly inhibited the mycelial growth of *Gynostemma pentaphyllum*, the pathogen causing ginseng rust. The inhibited mycelia exhibited severe deformities, twisting, and thinning.

[0099] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0100] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A biocontrol agent, characterized in that, The biocontrol agent is a mixture of JK21 crude protein and WY1 ethyl acetate in a concentration ratio of 7:3, 6:4 or 5:

5. The crude protein extraction step of JK21 is as follows: preparation of methyltrophic Bacillus (… Bacillus methylotrophicus The fermentation supernatant of strain JK21 was saturated with ammonium sulfate granules to achieve a supernatant saturation of over 90%. The mixture was incubated at 4°C for 12 hours and centrifuged at 10,000 rpm for 25 minutes. PBS buffer was added to the precipitate to dissolve it. The dissolved solution was then transferred to a dialysis bag with a molecular weight cutoff of 8kD to 14kD for desalting. Desalting continued until no precipitate was formed in the external solution of 1% BaCl2 during dialysis. The resulting solution was then lyophilized. The lyophilized material was brought to a final volume of 10 mL with PBS buffer and sterilized by passing it through a 0.22µm filter membrane to obtain crude protein from JK21. The methyltrophic Bacillus JK21 was deposited at the China Center for Type Culture Collection (CCTCC) on May 27, 2024, with accession number CCTCC NO: M 20241072, and classified as follows: Bacillus methylotrophicus JK21; The WY1 ethyl acetate was processed by Neurospora roughensis ( Neurosporacrassa Strain The fermentation supernatant of strain WY1 was obtained by extraction with ethyl acetate; The *Neurospora crassa* WY1 strain was deposited at the China Center for Type Culture Collection (CCTCC) on October 21, 2019, with accession number CCTCC NO: M 2019845, and classified as follows: Neurosporacrassa Strain WY1.

2. The application of the biocontrol agent according to claim 1 in the prevention and control of ginseng rust disease, characterized in that, The application includes inhibiting the germination of spores of the ginseng rust rot pathogen and inhibiting the growth of mycelia of the ginseng rust rot pathogen; The pathogen causing ginseng rust is *Streptococcus destructis* (…). Cylindrocarpondestructans ).

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