Bacillus paralicheniformis 2-12, biocontrol inoculant and application of biocontrol inoculant

Abstract

The invention relates to the technical field of agricultural microorganisms, and particularly discloses bacillus paralicheniformis 2-12, a biocontrol inoculant and application of the biocontrol inoculant. The bacillus parlicheniformis 2-12 is preserved in the China Center for Type Culture Collection on December 19, 2023, the preservation number of the bacillus parlicheniformis 2-12 is CCTCC NO: M 20232598, and the bacillus parlicheniformis 2-12 is classified and named as bacillus parlicheniformis 2-12. The bacillus paralicheniformis 2-12 provided by the invention can generate antibacterial volatile organic compounds, plays a bacteriostatic role by destroying the form of pathogenic bacteria hyphae and reducing the content of ergosterol, can effectively replace chemical pesticides, and is used for biological prevention and control of plant root rot.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a strain of Bacillus paralicheniformis 2-12, a biocontrol agent, and its application. Background Technology

[0002] Astragalus is a species of Astragalus in the genus Astragali of the legume family (Astragalus membranaceus). Astragalus membranaceus var. mongholicus ) or Astragalus membranaceus ( Astragalus membranceus Astragalus root, also known as Astragalus membranaceus, has the effects of tonifying qi and strengthening the exterior, as well as promoting wound healing and tissue regeneration. It is a widely recognized major medicinal material in traditional Chinese medicine and is known as "eight out of ten medicines contain Astragalus membranaceus". Root rot, a devastating soil-borne disease, has been increasingly prevalent in Astragalus membranaceus cultivation in recent years.

[0003] Currently, the dominant fungal flora causing root rot in Astragalus membranaceus is Fusarium solani (… Fusarium solani ) and Fusarium oxysporum ( Fusarium oxysporum The most common method for controlling root rot in Astragalus membranaceus is chemical control, such as chemical pesticides like carbendazim, difenoconazole, and thiophanate-methyl. However, the excessive use of chemical pesticides can easily lead to soil, water, and air pollution, which in turn can affect human health.

[0004] Therefore, discovering new biocontrol bacteria that can prevent and control root rot pathogens is of great significance for Astragalus membranaceus cultivation. Summary of the Invention

[0005] To develop a biocontrol agent capable of preventing and controlling root rot pathogens, this invention provides a strain of *Bacillus paralichrysogenus* 2-12, a biocontrol agent, and its applications. The *Bacillus paralichrysogenus* 2-12 provided by this invention can produce antibacterial volatile organic compounds, exerting its antibacterial effect by disrupting the mycelial morphology of the pathogen and reducing ergosterol content. It can effectively replace chemical pesticides for the biological control of plant root rot.

[0006] This invention provides a Bacillus paralicheniformis ( Bacillus paralicheniformis 2-12, the aforementioned *Bacillus paralichrysogenus* 2-12 was deposited at the China Center for Type Culture Collection on December 19, 2023, with accession number CCTCCNO: M 20232598, and classified as *Bacillus paralichrysogenus* 2-12. Bacillus paralicheniformis 2-12.

[0007] The Bacillus paralichrysogenus 2-12 provided by this invention can produce antibacterial volatile organic compounds, which exert antibacterial effects by destroying the mycelial morphology of pathogens and reducing ergosterol content. It can effectively replace chemical pesticides and be used for the biological control of plant root rot.

[0008] The present invention also provides a biocontrol agent containing the aforementioned Bacillus paralichrysiformis 2-12, wherein the biocontrol agent contains live Bacillus paralichrysiformis 2-12 cells, its fermentation broth, or metabolites.

[0009] Furthermore, the metabolites of the aforementioned Bacillus paralichrysogenus 2-12 are volatile organic compounds.

[0010] Further, the volatile organic compound is any one or more of ethyl 2-methylbutyrate, 2,3,4-3-methylpentane, 2-methylundecane, N-acetyl-L-alanine, n-tetane, and alanine.

[0011] Furthermore, the ethyl 2-methylbutyrate and 2,3,4-3-methylpentane are used to inhibit the activity of Fusarium oxysporum.

[0012] This invention also provides the application of the aforementioned *Bacillus paralicheniformis* 2-12 or the aforementioned biocontrol agent in inhibiting plant pathogens, wherein the pathogen is *Fusarium solani* (…). Fusarium solani Fusarium equisetifolium ( Fusarium equiseti Fusarium argentis ( ), Fusarium acuminatum ), Fusarium verticillata ( Fusarium verticillioide Fusarium oxysporum ( Fusarium oxysporum ), large spot disease bulging umbelliferous worms ( Exserohilum turcicum ), Fusarium semi-nakedense ( Fusarium semitectum Fusarium graminearum ( ), Fusarium graminearum ( Fusarium graminearum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Fusarium solani ( ), Fusarium solani Fusarium avenaceum Alternaria ( Alternaria alternata ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides Any one or more of the following.

[0013] Furthermore, the Bacillus paralichrysiformis 2-12 or the biocontrol agent is used to disrupt the mycelial morphology of the pathogen and reduce the ergosterol content.

[0014] Furthermore, the plant in question is Astragalus membranaceus.

[0015] Furthermore, the aforementioned Bacillus paralicheniformis 2-12 or biocontrol agent is used to reduce the incidence of Astragalus root rot caused by Fusarium oxysporum and Fusarium solani.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The volatile organic compounds generated by the strain 2-12 provided by the application can cause the pathogenic fungus hypha to break, shrink, atrophy and the content to flow out, directly destroy the integrity of the fungus, and have a significant inhibitory effect on 16 kinds of plant pathogenic fungi, including the dominant pathogenic fungus of Astragalus root rot, Fusarium oxysporum (inhibition rate 60.41%), Fusarium solani (inhibition rate 75.89%) and Botrytis cinerea (inhibition rate 94.96%) and other high-risk pathogenic fungi. The volatile organic compounds can significantly reduce the content of ergosterol of Fusarium oxysporum (decrease by 46.93%), destroy the function of the cell membrane, and inhibit the growth of the pathogenic fungus, wherein the inhibition rate of 2-methyl butyric acid ethyl ester on Fusarium oxysporum is 100%, and the inhibition rate of 2,3,4-trimethyl pentane is 73.9%.

[0017] After the strain 2-12 provided by the application is treated for 7 days, the incidence of Astragalus root rot is reduced from 100% to 13.33%, and the lesion diameter is reduced from 2.20 cm to 0.20 cm; after 14 days of treatment, the incidence is maintained at 23.33%, and the lesion diameter is only 0.78 cm, which is significantly better than the control group (P<0.05). P <0.05>.

[0018] Biological material preservation information 2-12, referred to as B. paralicheniformis 2-12 in the present application, has been preserved in the China Center for Type Culture Collection on December 19, 2023, with a preservation number of CCTCC NO: M 20232598, and the address of the preservation unit is Wuhan, Wuhan University, China, with a postcode of 430072, and the classification and naming is B. paralicheniformis 2-12 Bacillus paralicheniformis 2-12. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creating laborious work.

[0020] Figure 1 Antagonistic effect of the strain 2-12 on Fusarium oxysporum and Fusarium solani.

[0021] Figure 2 Effect of the VOCs of the strain 2-12 on the growth of Fusarium oxysporum hypha.

[0022] Figure 3 Effect of the VOCs of the strain 2-12 on Fusarium oxysporum hypha; In the figure, A is the effect of the VOCs of the strain 2-12 on the wet weight of Fusarium oxysporum hypha; B. The effect of VOCs of strain 2-12 on the mycelial dry weight of F. oxysporum; Figure 4 A. The morphological characteristics of strain 2-12; B. The morphological characteristics of strain 2-12 under light microscope after Gram staining; C. The morphological characteristics of strain 2-12 under scanning electron microscope after Gram staining.

[0023] Figure 5 B. The effect of VOCs of strain 2-12 on the mycelial dry weight of F. oxysporum; gyrB Phylogenetic tree of strain 2-12 constructed based on

[0024] Figure 6 B. The effect of VOCs of strain 2-12 on the mycelial dry weight of F. oxysporum; rpoB Phylogenetic tree of strain 2-12 constructed based on

[0025] Figure 7 B. The effect of VOCs of strain 2-12 on the mycelial dry weight of F. oxysporum; purH Phylogenetic tree of strain 2-12 constructed based on

[0026] Figure 8 B. The effect of VOCs of strain 2-12 on the mycelial dry weight of F. oxysporum; A. The mycelial morphology of F. oxysporum in the control group (first repetition); B. The mycelial morphology of F. oxysporum in the non-control group (second repetition); C. The mycelial morphology of F. oxysporum in the non-control group (third repetition); D. The mycelial morphology of F. oxysporum after inoculation with strain 2-12 (first repetition); E. The mycelial morphology of F. oxysporum after inoculation with strain 2-12 (second repetition); F. The mycelial morphology of F. oxysporum after inoculation with strain 2-12 (third repetition).

[0027] Figure 9 B. The effect of VOCs of strain 2-12 on the ergosterol content of F. oxysporum;

[0028] Figure 10 B. The inhibitory effect of 2-methylbutyric acid ethyl ester (7452-79-1) on F. oxysporum; A. The blank control; B. The inhibitory effect of 2-methylbutyric acid ethyl ester (7452-79-1) on F. oxysporum; C. The inhibitory effect of 2,3,4-3-methylpentane (565-75-3) on F. oxysporum; ​D is the inhibition of 2-methylundecane (7045-71-8) on A. menziesii root rot fungus; E is the inhibition of N-acetyl-L-alanine (97-69-8) on A. menziesii root rot fungus; F is the inhibition of n-tridecane (629-50-5) on A. menziesii root rot fungus; G is the inhibition of propylglycine (2345-56-4) on A. menziesii root rot fungus.

[0029] Figure 11 The preventive effect of VOCs of strain 2-12 on A. menziesii root rot fungus; In the figure, A is the statistical analysis of the incidence of root rot; B is the statistical analysis of the lesion diameter of root rot. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods, and the materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0031] Example 1: Isolation, screening and identification of Bacillus subtilis 2-12.

[0032] I. Isolation of strains Put alcohol lamp, conical flask containing 30 mL of sterile water, and other equipment in the clean bench, sterilize for 30 min; take 3 g of healthy Astragalus root rhizosphere soil sample and add sterile water, culture in a 180 r / min shaking incubator for 30 min, and put culture dishes, coating rods, alcohol lamps, centrifuge tubes, and pipettes in the clean bench and sterilize for 30 min. Take 1 mL of soil suspension in 9 mL of sterile water, mix thoroughly and dilute to 1000 μg / mL and 100 μg / mL concentration gradients; mix the soil suspensions with concentrations of 1000 μg / mL and 100 μg / mL, then use a pipette to take 100 μL of the mixture and plate it on NA medium (3 repeats for each concentration), evenly spread it with a coating rod, seal it with a sealing film, and culture at 28℃ for 48 h. According to the size, color, shape, protrusion, edge, transparency and other characteristics of the colonies, single colonies are picked and streaked for purification culture.

[0033] Beef extract peptone medium (NA): peptone 10 g, beef extract 3 g, NaCl 5 g, agar powder 15 g, distilled water 1000 mL, pH 7.0.

[0034] II. Screening of bacterial strains Purified bacterial colonies were picked and cultured in LB medium with shaking at 180 r / min for 12 h. Fusarium oxysporum and Fusarium solani mycelia were inoculated into the center of PDA medium. Two symmetrical points were then taken 3 cm symmetrically on both sides of the plate and holes were punched. 50 μL of the cultured LB broth was inoculated into the holes, and the antibacterial effect was observed after incubation at 28℃ for 7 days. Strains with inhibition bands were preserved for later use, and the inhibition rate was measured. Inhibition rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter - 6)] × 100.

[0035] The results showed that a bacterial strain with significant inhibitory effects against Fusarium oxysporum and Fusarium solani was isolated from the soil sample and named strain 2-12. Figure 1 Further investigation was conducted to determine the antibacterial effect of volatile organic compounds (VOCs) produced by strain 2-12 on *Fusarium oxysporum*. LA plates inoculated with strain 2-12 were used as the treatment group, and blank LA plates were used as the control group. The results showed that after 7 days of cultivation, the VOCs from strain 2-12 significantly inhibited the mycelial growth of both *Fusarium oxysporum* and *Fusarium solani*. The calculated inhibition rates of strain 2-12 against *Fusarium oxysporum* and *Fusarium solani* were 60.41% and 75.89%, respectively. Figure 2 After 7 days of cultivation, the wet weight of *Fusarium oxysporum* mycelium in the control group was 0.0815 g, and the wet weight of mycelium in the treatment group was 0.0203 g. The dry weight of mycelium in the control group was 0.0800 g, and the dry weight of mycelium in the treatment group was 0.0124 g. Both the wet and dry weights of *Fusarium oxysporum* mycelium in the treatment group were lower than those in the control group. Figure 3 (A and B).

[0036] III. Identification of Strains 1. Morphological characteristics and physiological and biochemical properties of the strain Strain 2-12 was inoculated onto NA medium and cultured at 28℃ for 48 h. Colony morphology characteristics were analyzed, including size, gloss, transparency, protrusion, shape, and edge. Gram staining and scanning electron microscopy were also used to observe bacterial cell morphology. Results showed that strain 2-12 colonies on NA medium were oval, with neat edges, opaque, and milky white in color. Figure 4 A); Gram staining is blue-purple, indicating Gram-positive bacteria (A); Figure 4 (B); Under scanning electron microscopy, the bacteria were rod-shaped, measuring (1.5 μm to 3.0 μm) × (0.5 μm to 0.8 μm) (n=50) Figure 4 (C).

[0037] Table 1. Physiological and biochemical characteristics of strain 2-12 The physiological and biochemical test results are shown in Table 1. The contact enzyme test, nitrate reduction test, hydrogen sulfide test, VP test, methyl red test, amylase test, and 1% to 10% NaCl growth of strain 2-12 are all positive; the phenylalanine deaminase test and citrate test are negative, and it can grow in different carbon sources (glucose, starch, sucrose, lactose, maltose) and different nitrogen sources (peptone, tryptone, yeast powder, beef extract, urea). According to the morphological characteristics and physiological and biochemical test results, strain 2-12 is preliminarily identified as Bacillus sp. Bacillus

[0038] 2. Molecular identification of the strain Strain 2-12 was inoculated in LB liquid medium and cultured at 28°C and 180 r / min for 14 h, and the DNA of the antagonistic bacteria was extracted by using DNA-EZ Reagents V All-DNA-Fast-Out of Sangon Company.

[0039] The formula of the LB liquid medium is as follows: beef extract 5.0 g, peptone 10.0 g, NaCl 5.0 g, distilled water 1000 mL, and pH 7.0.

[0040] gyrB , rpoB and purH The 25 μL PCR reaction system was used for the genes: 1 μL of sequencing primer with a concentration of 10 mM, 1 μL of template DNA, 12.5 μL of Taq DNA polymerase, and 9.5 μL of ddH2O. The PCR reaction was pre-denaturation at 95°C for 4 min, and 34 cycles. The PCR amplification program was as follows: 98°C for 10 s, annealing temperature of 62°C ( gyrB ) or 51°C ( rpoB and purH ) for 1 min, 72°C for 2 min, and 72°C for 10 min. After purification of the PCR amplification product, sequencing was performed, and the sequence was compared in the NCBI database by Blast. After downloading the relevant homologous sequences, the MEGA 5.0 software was used to construct a phylogenetic tree. The homology comparison results show that: rpoB the sequence has a homology of more than 99% with LR134165.1 Bacillus paralicheniformis ; purH the sequence has a homology of more than 99% with CP020352.1 Bacillus paralicheniformis ; Bacillus paralicheniformis Phylogenetic analysis shows that strain 2-12 and strain are clustered in the same branch Figures 5-7 .

[0041] ​Example 2: Inhibition of VOCs produced by B. paralicheniformis 2-12 on different pathogenic fungi The LA medium formula is: beef extract 5.0 g, peptone 10.0 g, NaCl 5.0 g, agar 20 g, distilled water 1000 mL, pH 7.0.

[0042] The PDA medium formula is: potato 200 g, glucose 20 g, agar 15 g, distilled water 1000 mL, pH 7.0.

[0043] The strain 2-12 was inoculated in LB liquid medium and cultured at 28°C with 180 r / min shaking for 14 h. The LA medium and PDA medium were poured into plates respectively, and after cooling, 100 μL of the LB cultured strain was taken with a pipette gun and evenly coated on the LA plate with a coating rod. The 15 kinds of pathogenic fungi cultured for 5 d were punched into 6 mm diameter fungus cakes and inoculated on the surface of the PDA plate. The culture plates of the strain 2-12 and the pathogenic strain were paired and cultured at 25°C, and the test was repeated 3 times. A control group inoculated with pathogenic fungi only was set. The colony diameters of the control group and the treatment group were observed every day until the mycelium of the control group covered the plate, and the inhibition rate was determined. Inhibition rate (%) = [(control colony diameter-treatment colony diameter) / control colony diameter-0.6] x 100.

[0044] The results of the inhibition spectrum determination showed that the VOCs produced by B. paralicheniformis 2-12 had good inhibition effect on 15 other plant pathogenic fungi, with inhibition rates of 31.88% to 94.96% (Table 2).

[0045] Table 2 Inhibition rate of VOCs produced by strain 2-12 on different pathogenic fungi Example 3: Effect of VOCs of B. paralicheniformis 2-12 on the mycelial morphology of A. canadensis The strain was inoculated in LB liquid medium and cultured at 28°C with 180 r / min shaking for 14 h. The LA medium and PDA medium were poured into plates respectively, and after cooling, 100 μL of the LB cultured strain was taken with a pipette gun and evenly coated on the LA plate with a coating rod. The F. oxysporum and F. solani cultured for 5 d were punched into 6 mm diameter fungus cakes and inoculated on the surface of the PDA plate, and sterilized cover glasses were inserted at a distance of 1 cm from the fungus cakes on the surface of the PDA medium, with 3 pieces inserted in each plate. The culture plates of the strain and the pathogenic strain were paired and cultured as the treatment group at 25°C, and a control group inoculated with pathogenic fungi only was set. The test was repeated 3 times. The mycelial morphology of F. oxysporum was observed by scanning electron microscopy after the mycelium was evenly distributed on the glass.

[0046] The mycelium-containing slide was fixed with 2.5% glutaraldehyde for 16 h, the fixed sample was washed with an acetone solution for 3 times, 10 min each time, and then was fixed with 1% osmium tetroxide for 3 h, and then was dehydrated with an ethanol gradient of 30%, 50%, 70%, 80%, 90%, 95% and 100% by volume, 10 min each time, and finally was replaced with acetone for 3 times, 10 min each time. After replacement, the sample was dried, adhered and coated, and then was observed for mycelium morphology by using a scanning electron microscope.

[0047] The influence of VOCs produced by B. paralicheniformis 2-12 on the ultrastructure of root rot pathogen of Astragalus membranaceus was observed by using a scanning electron microscope, and the results showed that the mycelium of F. oxysporum in the control group was uniform in thickness, smooth in surface and full in shape, while the mycelium of F. oxysporum in the treatment group was obviously broken, shriveled, atrophied, ruptured and had internal substances flowing out (Fig. 2), indicating that the VOCs produced by strain 2-12 had a significant inhibitory effect on the mycelium of the root rot pathogen of Astragalus membranaceus. Figure 8

[0048] Example 4: Influence of VOCs of B. paralicheniformis 2-12 on the ergosterol content of root rot pathogen of Astragalus membranaceus The influence of VOCs of strain 2-12 on the ergosterol content of F. oxysporum was analyzed by using a double-dish pair coupling method. After 7 days of treatment, the mycelium was scraped with a sterile blade, and the wet weight of the mycelium was recorded. 5 mL of freshly prepared 25% ethanol / KOH solution was added to the mycelium, and the mixed sample was placed in a 85℃ constant temperature water bath for 4 h. After the sample was cooled to 23±2℃, 3 mL of n-heptane and 1 mL of sterile water were added, and the sample was vortexed for 15 min and then was placed for 1 h. Then, the heptane layer was transferred into a cuvette by using a pipette gun, and the characteristic wavelength was detected at 230 nm and 282 nm by using an ultraviolet spectrophotometer.

[0049] Ergosterol content (%) = A 282 / ((290 x wet weight of mycelium) - A 230 / (518 x wet weight of mycelium)).

[0050] The results showed that the ergosterol content of F. oxysporum was reduced after treatment with the VOCs of strain 2-12, and the treatment group was reduced by 46.93% compared with the control group (P<0.05). Figure 9

[0051] Example 5: Detection of VOC components of B. paralicheniformis 2-12 After strain 2-12 was activated and cultured for 48 h, a single colony was inoculated into LB liquid medium and was cultured for about 14 h. 6 mL of LA medium was added into a headspace bottle, and the bottle wall was blown to be free of water vapor in a clean bench. The OD 600 of the bacterial suspension was adjusted to 0.2-0.3, and the culture was incubated at 30℃, 200 rpm for 48 h.​​600 =1, 20 μL of bacterial solution was added, and the bacterial suspension was slowly added to the LA medium. After the entire medium surface was evenly spread, the polytetrafluoroethylene cap was tightly closed and sealed with a PE sealing film. The culture was incubated at 28°C in the dark for 5 days. The headspace bottle containing only LA medium and heptyl acetate was used as a control.

[0052] After equilibration at 40°C for 40 min, the SPME fiber was inserted into the vial and extracted at 40°C for 30 min with a 40 mm injection depth. The analytes were desorbed in a gas chromatograph injector at 270°C in splitless mode for 10 min.

[0053] Gas chromatography conditions: The chromatographic column was TR-5MS (30.0 m x 0.25 mm x 0.25 μm); the carrier gas was helium, and the TriPlus RSH autosampler separated different VOCs. The GC oven temperature was set to 40°C for 3 min, increased to 180°C at a frequency of 10°C / min, and then increased to 270°C at a frequency of 40°C / min, and held for 4 min.

[0054] Mass spectrometry conditions: The ion source was EI, the electron energy was 70 eV, and the ion source temperature and MS transfer line temperature were both set to 250°C; the mass-to-charge ratio scan range was 35 amu to 500 amu, and the scan interval time was 0.2 s.

[0055] Table 3. Types of VOCs produced by strain 2-12 Finally, the VOCs were identified by mass spectrometry, direct matching (SI), and reverse matching (RSI) with the NIST library. The results showed that: B. paralicheniformis Strain 2-12 can produce 17 types of VOCs, including 9 alkanes, 3 alcohols, 3 acids and esters, 1 aromatic compound, and 1 alkyne (Table 3).

[0056] Example 6: Determination of antibacterial VOCs of B. paralicheniformis 2-12 Six representative VOCs with high content in strain 2-12 were selected for antibacterial activity detection. PDA medium plates were prepared, and a 6 mm diameter Fusarium oxysporum fungus cake was inoculated in the center of the PDA plate. Ten mL of each of the six VOCs were placed in another culture dish, and the two dishes were quickly paired and sealed with a PE sealing film. A blank control was set up by inoculating only F. oxysporum on the PDA medium, and a background control was set up by inoculating 10 mL of dimethyl sulfoxide. The culture was incubated at 25°C in the dark for 7 days. The colony diameters of the blank control and different treatments were measured using the cross method, and the antibacterial rate was calculated.

[0057] The results showed that ethyl 2-methylbutyrate had a 100% inhibitory effect on Fusarium oxysporum; 2,3,4-methylpentane was the second most effective, with an inhibition rate of 73.9%; the other four substances had no inhibitory effect on Fusarium oxysporum. Figure 10 ).

[0058] Example 7: The effect of VCOs from Bacillus paralichrysiforme 2-12 on root rot of Astragalus membranaceus. Preparation of spore suspension: Place 10 mL of distilled water in a container, prepare two bottles, autoclave them, and pour them into PDA plates of Fusarium oxysporum and Fusarium solani that have been cultured for about 15 days. Use a spreading stick to completely dissolve the mycelium in the water to obtain spore suspensions of Fusarium oxysporum and Fusarium solani.

[0059] The strain was inoculated into LB medium and cultured at 28℃ and 180 r / min for 14 h with shaking. Healthy, uniformly sized Astragalus roots were soaked in 2% sodium hypochlorite solution for 2 min, rinsed three times with sterile water, and then air-dried. The dried healthy Astragalus roots were placed in petri dishes with moistened sterile filter paper underneath. Using a sterile inoculation needle, wounds approximately 3 mm (depth) × 2 mm (width) were made on the upper, middle, and lower surfaces of the Astragalus roots. 20 μL of spores of *Fusarium oxysporum* and *Fusarium solani*, the pathogens of Astragalus root rot, were inoculated at each wound. 100 μL of LB culture of strain 2-12 was pipetteted onto LA plates and spread evenly with a sterile spreader. This was inverted with the inoculated Astragalus roots at 25℃ as the treatment group; 100 μL of uninoculated LB culture served as the control group. The incidence rate and diameter of lesions at the Astragalus wound site were observed and recorded at 7 days and 14 days, respectively.

[0060] The biocontrol efficacy of VOCs was evaluated after 7 and 14 days of treatment. After 7 days of treatment, the incidence of Astragalus root rot in the treated groups inoculated with Fusarium oxysporum and Fusarium solani were 13.33% and 23.33%, respectively, while the control group was 100%. The lesion diameters in the treated groups were 0.20 cm and 0.34 cm, respectively, while the lesion diameter in the control group was 2.20 cm. After 14 days of treatment, the incidence of Astragalus root rot remained low after inoculation with Fusarium oxysporum and Fusarium solani (23.33% and 36.67%, respectively), with lesion diameters of 0.78 cm and 1.24 cm, respectively, showing a significant difference compared to the control group. P <0.05)( Figure 11 The above results indicate that the VCOs of Bacillus paralichrysiforme 2-12 can significantly inhibit the spread of lesions and reduce the incidence of root rot in Astragalus membranaceus.

[0061] 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.

[0062] Obviously, many modifications and changes can be made to the present application without departing from the spirit and scope of the application. It is not intended to limit the application to the exact construction and arrangements described and shown herein, and any suitable modification which does not depart from the spirit and scope of the application is intended to be within the scope of the application.

Claims

1. A type of Bacillus paralicheniformis ( Bacillus paralicheniformis )2-12, characterized in that, The *Bacillus paralichrysogenus* 2-12 was deposited at the China Center for Type Culture Collection (CCTCC) on December 19, 2023, with accession number CCTCCNO: M 20232598, and classified as *Bacillus paralichrysogenus* 2-12. Bacillus paralicheniformis 2-12.

2. A biocontrol agent containing *Bacillus paralichrysogenum* 2-12 as described in claim 1, characterized in that, The biocontrol agent contains 2-12 live Bacillus paralicheniformis cells, its fermentation broth, or metabolites.

3. The biocontrol agent containing Bacillus paralichrysogenum 2-12 according to claim 2, characterized in that, The metabolites of *Bacillus paralicheniformis* 2-12 are volatile organic compounds.

4. The biocontrol agent containing Bacillus paralichrysogenum 2-12 according to claim 3, characterized in that, The volatile organic compound is any one or more of ethyl 2-methylbutyrate, 2,3,4-3-methylpentane, 2-methylundecane, N-acetyl-L-alanine, n-tetane, and alanine.

5. The biocontrol agent containing Bacillus paralichrysogenum 2-12 according to claim 4, characterized in that, The ethyl 2-methylbutyrate and 2,3,4-3-methylpentane are used to inhibit Fusarium oxysporum activity.

6. The application of *Bacillus paralichrysogenum* 2-12 as described in claim 1 or the biocontrol agent as described in any one of claims 2-5 in inhibiting plant pathogens, characterized in that... The pathogen is *Fusarium solani* (…). Fusarium solani Fusarium equisetifolium ( Fusarium equiseti Fusarium argentis ( ), Fusarium acuminatum ), Fusarium verticillata ( Fusarium verticillioide Fusarium oxysporum ( Fusarium oxysporum ), large spot disease bulging umbelliferous worms ( Exserohilum turcicum ), Fusarium semi-nakedense ( Fusarium semitectum Fusarium graminearum ( ), Fusarium graminearum Fusarium graminearum Botrytis cinerea ( ), Botrytis cinerea ( Botrytis cinerea Fusarium solani ( ), Fusarium solani Fusarium avenaceum Alternaria ( Alternaria alternata ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides Any one or more of the following.

7. The application according to claim 6, characterized in that, The aforementioned Bacillus paralicheniformis 2-12 or biocontrol agent is used to disrupt the mycelial morphology of the pathogen and reduce the ergosterol content.

8. The application according to claim 6, characterized in that, The plant in question is Astragalus membranaceus.

9. The application according to claim 6, characterized in that, The aforementioned Bacillus paralicheniformis 2-12 or biocontrol agent is used to reduce the incidence of root rot in Astragalus membranaceus caused by Fusarium oxysporum and Fusarium solani.

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