Bacillus subtilis, fungicide, preparation method, prevention and control method and application

Abstract

The invention belongs to the technical field of microbial prevention and control, and particularly relates to bacillus subtilis, a microbial agent, a preparation method, a prevention and control method and application. The invention provides a strain of bacillus subtilis M4, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.35477. The bacillus subtilis M4 is used for preventing and treating root rot of cornutgionium axe. The bacillus subtilis M4 can improve the activity of disease-resistant defensin superoxide dismutase (SOD), the activity of peroxidase (POD) and the activity of catalase (CAT), reduce the content of malondialdehyde (MDA), finally reduce the morbidity and the morbidity index of plant root rot and promote plant growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial control technology, specifically relating to a strain of Bacillus subtilis, a bacterial agent, a preparation method, a control method, and its application. Background Technology

[0002] Desert mustard, a pioneer plant of the desert, is an annual or biennial herbaceous plant belonging to the genus *Vitex* of the Brassicaceae family. It is mainly distributed in deserts or grasslands in Gansu, Ningxia, Inner Mongolia, and other regions. *Vitex axe-wing* is a succulent plant with well-developed thin-walled tissues in its stems and leaves and little mechanical tissue. It is rich in sugars, proteins, fats, amino acids, and various trace elements needed by the human body, making it a low-sugar, high-protein wild vegetable.

[0003] In recent years, root rot in *Sinocyclocheilus serrata* has become increasingly serious, posing a significant obstacle to local agriculture. Chemical fungicides and crop rotation are the main control methods, but chemical fungicides easily cause environmental pollution and pathogen resistance, while crop rotation requires high capital investment and technical expertise, limiting its widespread application. Considering environmental pollution and agricultural product safety issues, biological control methods can be used to control the disease. Biological agents, as an important breakthrough in controlling crop diseases, show broad application prospects. Currently, there are no reports on the use of *Bacillus subtilis* to control root rot in *Sinocyclocheilus serrata*. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis, a fungal agent, a preparation method, a control method, and an application. The Bacillus subtilis M4 provided by this invention can reduce the incidence of root rot in *Strombus haematomarginatus*.

[0005] To address the aforementioned technical problems, the present invention proposes the following technical solution: This invention provides a Bacillus subtilis ( Bacillus subtilis Bacillus subtilis M4, whose preservation number is CGMCC No. 35477, is used to control root rot of Axillary sedge.

[0006] The present invention also provides a bacterial agent containing Bacillus subtilis M4 as described in the above-described scheme.

[0007] As a preferred embodiment, the bacterial agent comprises a bacterial suspension of Bacillus subtilis M4 and / or metabolites of Bacillus subtilis M4.

[0008] As a preferred embodiment, the concentration of Bacillus subtilis M4 in the bacterial agent is 10. 6 ~10 10 CFU / mL.

[0009] The present invention also provides a method for preparing a microbial agent, comprising the following steps: inoculating the Bacillus subtilis M4 described in the above scheme into a culture medium for fermentation culture to obtain a microbial agent.

[0010] As a preferred embodiment, the fermentation culture temperature is 26~32℃, the fermentation culture time is 55~65h, the fermentation rotation speed is 180~220rpm, the initial pH of the culture medium used for the fermentation culture is 7.5~8.5, and the volume of the culture medium occupies 38%~41% of the culture container volume.

[0011] As a preferred embodiment, the culture medium used for the fermentation culture comprises: 9-11 g / L sucrose, 9-11 g / L yeast extract, and 0.3-0.8 g / L magnesium sulfate.

[0012] The present invention also provides the application of Bacillus subtilis M4 as described in the above scheme, the inoculum agent described therein, or the inoculum agent prepared by the above preparation method in the control of plant root rot; The plant in question is *Strombus haematomarginatus*; the pathogen causing the root rot is *Fusarium oxysporum*. Fusarium oxysporum ).

[0013] The present invention also provides a method for controlling plant root rot, comprising the following steps: applying Bacillus subtilis M4 as described in the above scheme to the plant roots by drenching. The plant in question is *Strombus haematomarginatus*; the pathogen causing the root rot is *Fusarium oxysporum*. Fusarium oxysporum ).

[0014] The beneficial effects of this invention are as follows: This invention provides a strain of Bacillus subtilis M4, with the preservation number CGMCC No. 35477; the Bacillus subtilis M4 is used to control root rot in *Strombus haematomarginatus*. The Bacillus subtilis M4 of this invention can increase the activity of disease-resistant defense enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and reduce malondialdehyde (MDA) content, ultimately reducing the incidence and disease index of root rot in plants, and can reduce the rate of biomass decline in *Strombus haematomarginatus* caused by root rot. Attached Figure Description

[0015] Figure 1 The diagram shows the antagonistic activity of antagonistic bacteria M4 and N33 against the pathogen causing root rot of *Sinomenium acutum*. Figure 2 The images show the colony morphology and Gram staining of Bacillus subtilis M4; the left side shows the colony morphology, and the right side shows the Gram staining. Figure 3 A phylogenetic tree of strain M4 constructed based on the 16S rDNA sequence; Figure 4 Figure showing the effect of carbon source type on the growth of Bacillus subtilis M4; Figure 5 Figure showing the effect of sucrose concentration on the growth of Bacillus subtilis M4; Figure 6 Figure showing the effect of different nitrogen sources on the growth of Bacillus subtilis M4; Figure 7 Figure showing the effect of yeast powder concentration on the growth of Bacillus subtilis M4; Figure 8 Figure showing the effect of inorganic salt types on the growth of Bacillus subtilis M4; Figure 9 Figure showing the effect of magnesium sulfate concentration on the growth of Bacillus subtilis M4; Figure 10 This is a graph showing the change in bacterial concentration during the growth of Bacillus subtilis M4. Figure 11 Figure showing the effect of different liquid volumes on Bacillus subtilis M4; Figure 12 Figure showing the effect of different initial pH values ​​on Bacillus subtilis M4; Figure 13 Biocontrol efficacy of Bacillus subtilis M4 against root rot of Sinapis alba; Figure 14 Figure showing the effect of different treatments on SOD enzyme activity in potted *Strombax ceiba* leaves; Figure 15 Figure showing the effect of different treatments on POD enzyme activity in potted *Sinocyclocheilus roxburghii* leaves. Figure 16 Figure showing the effect of different treatments on CAT enzyme activity in potted *Strombus haematomarginatus* leaves; Figure 17 Figure showing the effect of different treatments on malondialdehyde content in the leaves of potted *Strombax ceiba*.

[0016] Biological Preservation Instructions Strain M4, classified as Bacillus subtilis ( Bacillus subtilis It was deposited on July 31, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35477. Detailed Implementation

[0017] This invention provides a Bacillus subtilis ( Bacillus subtilisThe Bacillus subtilis M4 strain, with preservation number CGMCC No. 35477, is used to control root rot in *Musa acutissima*. This *Bacillus subtilis* M4 strain was isolated from the *Musa acutissima* continuous cropping disease experimental field of the College of Horticulture and Plant Protection, Inner Mongolia Agricultural University. The colonies of the *Bacillus subtilis* M4 strain are small, milky white, opaque, with neat edges, dry, and not easily picked up.

[0018] The present invention also provides a bacterial agent containing *Bacillus subtilis* M4 as described in the above-described technical solution. The bacterial agent of the present invention preferably comprises a bacterial suspension of *Bacillus subtilis* M4 and / or metabolites of *Bacillus subtilis* M4, more preferably a bacterial suspension of *Bacillus subtilis* M4 or metabolites of *Bacillus subtilis* M4. The concentration of *Bacillus subtilis* M4 in the bacterial agent of the present invention is preferably 10. 6 ~10 10 CFU / mL.

[0019] The preferred method for preparing the Bacillus subtilis M4 bacterial suspension of the present invention includes: inoculating Bacillus subtilis M4 onto LB medium to obtain single colonies of Bacillus subtilis M4. The present invention involves culturing the single colonies of Bacillus subtilis M4 in a culture medium to obtain a bacterial suspension of Bacillus subtilis M4. The preferred culture temperature is 26-32°C, more preferably 28°C; the preferred culture time is 55-65 hours, more preferably 58-62 hours, and more preferably 60 hours. The metabolic products of Bacillus subtilis M4 of the present invention include a supernatant, and the preparation method of the supernatant preferably includes centrifuging the bacterial suspension to obtain the supernatant. The present invention does not have a particular limitation on the centrifugation method; conventional methods are acceptable.

[0020] This invention provides a method for preparing a microbial agent, comprising: inoculating the *Bacillus subtilis* M4 described in the above-mentioned technical solution into a culture medium for fermentation culture to obtain a microbial agent. Preferably, this invention involves inoculating a single colony of *Bacillus subtilis* M4 into a culture medium for fermentation culture to obtain a fermentation broth. The fermentation culture temperature is preferably 26-32℃, more preferably 28℃; the fermentation culture time is preferably 55-65 h, further preferably 58-62 h, and more preferably 60 h. The initial pH of the fermentation culture is preferably 7.5-8.5, more preferably 7.8-8.2, and more preferably 8.0.

[0021] The fermentation culture speed described in this invention is preferably 180-220 rpm, more preferably 190-210 rpm. The volume of the culture medium described in this invention accounts for 38%-41% of the volume of the culture flask, more preferably 40%. In a specific embodiment of this invention, 100 mL of culture medium is added to a 250 mL Erlenmeyer flask. The composition of the culture medium used for the fermentation culture described in this invention includes: 9-11 g / L sucrose, 9-11 g / L yeast extract, and 0.3-0.8 g / L magnesium sulfate. The culture medium described in this invention preferably includes 9-11 g / L sucrose, more preferably 10 g / L; the culture medium described in this invention preferably includes 9-11 g / L yeast extract, more preferably 10 g / L; the culture medium described in this invention preferably includes 0.3-0.8 g / L magnesium sulfate, more preferably 0.4-0.6 g / L, more preferably 0.5 g / L. The sucrose, yeast extract, and magnesium sulfate in the culture medium described in this invention can all promote the growth of Bacillus subtilis M4.

[0022] This invention also provides the application of Bacillus subtilis M4 as described in the above-mentioned technical solutions, or the inoculum agent described in the above-mentioned technical solutions, or the inoculum agent prepared by the above-mentioned technical methods, in the control of plant root rot. The plant described in this invention is *Strombus haematomarginatus*. The pathogen causing the root rot described in this invention is *Fusarium oxysporum* (…). Fusarium oxysporum The Bacillus subtilis M4 of this invention inhibits Fusarium oxysporum in plates. Fusarium oxysporum To promote the growth of *Aristolochia debilis*, the application of Bacillus subtilis M4 in potted plants can reduce the incidence of root rot in *Aristolochia debilis*.

[0023] The Bacillus subtilis M4 described in this invention can enhance the activity of disease-resistant and defensive enzymes in the leaves of Aristolochia debilis, such as increasing the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and can reduce the content of malondialdehyde (MDA), causing oxidative stress damage to cells, leading to the destruction of the pathogen's cell membrane, thereby inhibiting the growth of the pathogen and achieving the effect of resisting root rot.

[0024] This invention also provides a method for controlling plant root rot, comprising: drenching the plant with Bacillus subtilis M4 as described in the above technical solution; wherein the plant is *Strombus haematomarginatus*; and the pathogen of the root rot is *Fusarium oxysporum*. Fusarium oxysporum As an optional implementation method, the root drenching method described in this invention includes soil drenching and / or root-damage drenching. The application period described in this invention is the four-leaf-one-heart stage. When *Sinocyclocheilus* reaches the four-leaf-one-heart stage, the taproot has elongated and lateral roots have begun to sprout. Applying the inoculant at this time avoids both the problem of excessively short roots during the cotyledon stage and the defect of high lignification of roots in mature plants. Simultaneously, the small individual differences among *Sinocyclocheilus* plants at the four-leaf-one-heart stage ensure the reproducibility and reliability of experimental results. To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1: Screening and Identification of Bacillus mojaveensis 1. Isolation and screening of antagonistic bacteria In June 2024, soil samples were collected in the experimental field of *Vitex negundo* (a type of wild mustard) with continuous cropping disease at the College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, using a standardized rhizosphere soil collection method. Specifically, plants showing typical wilting symptoms were pulled up and the rhizosphere soil samples, 1-2 mm thick, were obtained by shaking the plants vertically three times. The samples were immediately placed in liquid nitrogen for rapid freezing and then transferred to an ultra-low temperature freezer at -80℃ (temperature fluctuation ±2℃) for long-term storage to ensure the originality of the microbial community structure.

[0026] Antagonistic bacterial strains were isolated from the collected soil using a dilution plating method. The isolated bacteria were then purified until single colonies were obtained. Ten antagonistic bacterial strains showing control effects against *Fusarium oxysporum*, the pathogen causing root rot in *Sinomenium acutum*, were obtained through plate confrontation. These ten bacterial strains were named N35, M3, L12, N33, W3, N3O, W2, L7, N23, and M4, respectively. The control (CK) was treated with sterile water. The inhibition rates of CK and the antagonistic strains are shown in Table 1. Among them, the antagonistic effects of M4 and N33 against *Fusarium oxysporum* are as follows: Figure 1 As shown.

[0027] Table 1 shows that the 12 biocontrol strains exhibited different inhibitory effects against the root rot pathogen. Strain M4 showed significantly higher inhibitory effects than the other strains; therefore, strain M4 was selected as the experimental biocontrol strain for further research. The formula for calculating the inhibition rate is shown below: .

[0028] Table 1. Antibacterial rate of antagonistic strains

[0029] 2. Identification of antagonistic bacteria 2.1 Morphological characteristics of antagonistic bacteria Antagonistic strain M4 was streaked in three zones on LB agar plates and incubated at 29°C. The results showed that M4 colonies were milky white, opaque, with neat edges, dry, and difficult to pick up. Figure 2 (B). Gram staining results of strain M4 indicate that this strain is a Gram-positive strain. Figure 2 (b)

[0030] Figure 2 In the text, B represents the colony morphology of M4; b represents the Gram staining of M4.

[0031] 2.2 Molecular biological identification of antagonistic bacteria Gene sequence alignment was performed using BLAST from NCBI, and a phylogenetic tree was constructed using MEGA. The results are shown in the figure for strain M4 and... Bacillus subtilis They share high gene homology and belong to the same branch, see... Figure 3 Based on morphological characteristics, strain M4 was identified as Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ).

[0032]

[0033] Example 2: Optimization of Bacillus subtilis M4 fermentation The LB liquid medium consists of 10g peptone, 5g yeast extract, 10g sodium chloride and 1L distilled water, pH 8.0.

[0034] 1. Carbon source optimization: 1.1 Using LB liquid medium as the base, five carbon source replacement media were designed: sucrose, maltose, xylose, glucose, and fructose were used to replace peptone in the basal medium in equal amounts (all other components remained unchanged). The specific compositions of the replaced media 1-5 are as follows: Culture medium 1 consists of: 10g sucrose, 5g yeast powder, 10g sodium chloride and 1L distilled water.

[0035] Culture medium 2 consists of 10g maltose, 5g yeast powder, 10g sodium chloride and 1L distilled water.

[0036] The culture medium consists of 10g xylose, 5g yeast extract, 10g sodium chloride, and 1L distilled water.

[0037] The culture medium consists of 4 components: 10g glucose, 5g yeast powder, 10g sodium chloride and 1L distilled water.

[0038] The culture medium consists of 10g fructose, 5g yeast extract, 10g sodium chloride and 1L distilled water.

[0039] The experiment employed a completely randomized block design, with three biological replicates for each carbon source treatment. Before inoculation, activated M4 seed culture was added at a volume ratio of 1% (v / v) to 250 mL Erlenmeyer flasks containing 100 mL of culture medium 1–5. The flasks were then incubated at 28 °C with shaking at 180 rpm for 24 h. After incubation, the OD values ​​of the culture media in each treatment group were measured using a UV spectrophotometer. 600 value.

[0040] See results Figure 4 It can be seen that the OD of the fermentation broth 600 The values ​​from left to right are 1.26, 1.11, 0.95, 0.8, and 0.84. Bacillus subtilis M4 grows best with sucrose as the carbon source, and the fermentation broth OD... 600 The value is 1.26, so sucrose is the optimal carbon source for the strain.

[0041] 1.2 Optimization Experiment of Sucrose Gradient Based on culture medium 1, sucrose concentrations were set at 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively. Culture media 1-1 to 1-6 were prepared with three replicates for each concentration. After inoculation with the activated strain, the cultures were incubated at 28℃ with shaking at 180 rpm for 24 h. OD was measured. 600 The growth status of the strain was assessed using the same inoculation method as in 1.1.

[0042] Culture medium 1-1 consists of 5g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0043] Culture medium 1-2 consists of 10g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0044] Culture media 1-3 consist of: 20g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0045] Culture media 1-4 consist of: 30g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0046] Culture media 1-5 consist of: 40g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0047] Culture media 1-6 consist of: 50g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0048] See results Figure 5 It can be seen that the OD of the fermentation broth 600 The values ​​from left to right are 1.12, 1.28, 1.21, 1.11, 1.12, and 1.04. At a sucrose concentration of 1% (10 g / L), the OD values ​​of the Bacillus subtilis M4 fermentation broth are... 600 The highest value is 1.28.

[0049] 2. Nitrogen source optimization: 2.1 Using LB liquid medium as the base, seven nitrogen source replacement media were designed. The specific composition of the replaced media (6-12) is as follows: The culture medium consists of 10g peptone, 5g yeast extract, 10g sodium chloride, and 1L distilled water.

[0050] The culture medium 7 consisted of 10g peptone, 5g beef extract, 10g sodium chloride, and 1L distilled water.

[0051] The culture medium consists of 10g peptone, 5g peptone, 10g sodium chloride and 1L distilled water.

[0052] The culture medium consists of 10g peptone, 5g glutamic acid, 10g sodium chloride and 1L distilled water.

[0053] The culture medium 10 consists of: 10g peptone, 5g ammonium sulfate, 10g sodium chloride and 1L distilled water.

[0054] Culture medium 11 consists of: 10g peptone, 5g ammonium chloride, 10g sodium chloride and 1L distilled water.

[0055] The culture medium 12 consists of: 10g peptone, 5g potassium nitrate, 10g sodium chloride and 1L distilled water.

[0056] Each nitrogen source treatment was performed in triplicate. M4 bacterial suspension was inoculated at a rate of 2% (v / v) into Erlenmeyer flasks (250 mL each), each containing 100 mL of culture medium 6–12. The flasks were then incubated at 28°C with shaking at 180 rpm for 24 h. After incubation, the absorbance of each treatment group's culture medium at 600 nm was measured using a UV spectrophotometer.

[0057] The results are as follows Figure 6 As shown, the fermentation broth OD 600 The values ​​from left to right are 1.51, 0.08, 0.08, 0.86, 0.87, 0.156, and 0.13. The OD values ​​of Bacillus subtilis M4 strain when using yeast extract as the nitrogen source are... 600 The value is the highest at 1.51, therefore yeast extract is chosen as the best nitrogen source for the strain.

[0058] 2.2 Yeast Powder Gradient Optimization Experiment Based on culture medium 6, yeast powder concentrations were set at 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively, and culture media 6-1 to 6-6 were prepared. Three replicates were performed for each concentration. After inoculation with activated strains, the cultures were incubated at 28℃ with shaking at 180 rpm for 24 h. OD was measured. 600 The growth status of the strain was assessed to determine the optimal concentration of each nutrient. The inoculation method was the same as in 2.1.

[0059] Culture medium 6-1 consists of: 10g peptone, 5g yeast extract, 10g sodium chloride and 1L distilled water.

[0060] The culture medium 6-2 consists of 10g peptone, 10g yeast extract, 10g sodium chloride and 1L distilled water.

[0061] The culture medium 6-3 consists of: 10g peptone, 20g yeast extract, 10g sodium chloride and 1L distilled water.

[0062] The culture medium 6-4 consists of: 10g peptone, 30g yeast extract, 10g sodium chloride and 1L distilled water.

[0063] Culture medium 6-5 consists of: 10g peptone, 40g yeast extract, 10g sodium chloride and 1L distilled water.

[0064] Culture medium 6-6 consists of: 10g peptone, 50g yeast extract, 10g sodium chloride and 1L distilled water.

[0065] See results Figure 7 It can be seen that as the concentration of yeast powder increases, the concentration of the bacterial culture first increases and then decreases, and the OD of the fermentation broth... 600 The values ​​from left to right are 1.18, 1.53, 1.00, 1.07, 1.07, and 0.89. The OD values ​​of the bacterial culture at a yeast powder concentration of 1% (10 g / L) are... 600 The highest value is 1.53.

[0066] 3. Inorganic salt optimization: 3.1 Using standard LB liquid medium as a baseline, seven representative inorganic salts—K₂HPO₄, KH₂PO₄, FeSO₄, ZnSO₄, MgSO₄, NaCl (control), and KCl—were selected to replace the NaCl component in the basic formulation (other components remained constant). Specifically, based on 1 L of medium, the macroelements K₂HPO₄, KH₂PO₄, NaCl, and KCl were added at 0.5% (w / v), and the microelements FeSO₄, ZnSO₄, and MgSO₄ were added at 0.05% (w / v). The specific composition of the replaced medium (13-19) is as follows: Culture medium 13 consists of: 10g peptone, 5g yeast extract, 5g K2HPO4 and 1L distilled water.

[0067] Culture medium 14 consists of: 10g peptone, 5g yeast extract, 5g KH2PO4 and 1L distilled water.

[0068] The culture medium 15 consisted of 10g peptone, 5g yeast extract, 0.5g FeSO4 and 1L distilled water.

[0069] The culture medium 16 consisted of 10g peptone, 5g yeast extract, 0.5g ZnSO4 and 1L distilled water.

[0070] Culture medium 17 consisted of 10g peptone, 5g yeast extract, 0.5g MgSO4 and 1L distilled water.

[0071] The culture medium 18 consisted of 10g peptone, 5g yeast extract, 5g NaCl, and 1L distilled water.

[0072] The culture medium 19 consisted of 10g peptone, 5g yeast extract, 5g KCl and 1L distilled water.

[0073] The activated M4 bacterial suspension was inoculated into culture media 13-19 and placed in a constant temperature shaking incubator. The culture was carried out continuously for 24 hours at 28℃ and a shaking speed of 180 rpm. OD was then measured. 600 The value is used to assess the growth status of bacteria, thereby screening for the most suitable inorganic salts.

[0074] See results Figure 8 ,according to Figure 8 It can be seen that the OD of the fermentation broth 600 The values ​​from left to right are 1.56, 0.15, 1.47, 0.77, 0.20, 1.32, and 1.16. When MgSO4 is used as the inorganic salt in the culture medium, the OD values ​​of the bacterial culture are... 600 The value is the highest.

[0075] 3.2 MgSO4 Gradient Optimization Experiment Based on medium 17, MgSO4 concentrations were set to 0.01%, 0.05%, 0.10%, 0.20%, 0.40%, and 0.50%, respectively, and mediums 17-1 to 17-6 were prepared. Three replicates were performed for each concentration. After inoculation with activated strain M4, the cultures were incubated at 28℃ with shaking at 180 rpm for 24 h. OD was measured. 600 The growth status of the strains was assessed to determine the optimal concentration of each nutrient. The inoculation method was the same as in 3.1.

[0076] The composition of culture medium 17-1 is: 10g peptone, 5g yeast extract, 0.1g MgSO4 and 1L distilled water.

[0077] The culture medium 17-2 consists of: 10g peptone, 5g yeast extract, 0.5g MgSO4 and 1L distilled water.

[0078] The culture medium 17-3 consists of: 10g peptone, 5g yeast extract, 1g MgSO4 and 1L distilled water.

[0079] The culture medium 17-4 consists of: 10g peptone, 5g yeast extract, 2g MgSO4 and 1L distilled water.

[0080] The culture medium 17-5 consists of: 10g peptone, 5g yeast extract, 4g MgSO4 and 1L distilled water.

[0081] The culture medium 17-6 consists of 10g peptone, 5g yeast extract, 5g MgSO4 and 1L distilled water.

[0082] See results Figure 9 ,according to Figure 9 It can be seen that, similarly, with the increase of MgSO4 concentration, the OD of the fermentation broth of strain M4 also increases. 600 The value first increases and then decreases; fermentation broth OD600 The values ​​from left to right are 1.22, 1.56, 1.34, 1.30, 1.30, and 1.28. When the MgSO4 concentration reaches 0.05% (0.5 g / L), the OD value is... 600 The maximum value is 1.56.

[0083] 4. Orthogonal experiment of culture medium components Based on the optimal types and concentration ranges of carbon source (A), organic nitrogen source (B), and inorganic salt (C) determined by previous single-factor optimization experiments, a three-factor, three-level L9 (3 3 An orthogonal experimental design was used to systematically optimize the ratio of key nutrients in the culture medium. The culture medium consisted of sucrose, yeast extract, KCl, and water. The levels of each factor were set as shown in Table 2: the optimal carbon source content (A1~A3), organic nitrogen source content (B1~B3), and inorganic salt content (C1~C3), thereby determining the optimal culture medium formulation.

[0084] Table 2 L9 (3) of Bacillus subtilis M4 3 Orthogonal experiment

[0085] Using sucrose (A), yeast powder (B), and MgSO4 (C) as factors, according to L9(3) 3 An orthogonal array design with 3 factors and 3 levels was used for an orthogonal experiment, with 3 biological replicates for each treatment. Before inoculation, the activated M4 bacterial suspension was inoculated into Erlenmeyer flasks (250 mL) at a rate of 1% (v / v), with each flask containing 100 mL of culture medium. The flasks were incubated at 28°C with shaking at 180 rpm for 24 h. The OD values ​​of Bacillus subtilis M4 after 24 h of fermentation were measured. 600 The optimal fermentation formula was determined by analyzing the values ​​of the three factors. Range analysis of the orthogonal experiment showed that the influence of the three factors on the growth of Bacillus subtilis was in the order B>C>A, and the optimal level combination was A²B²C² (Table 4). Analysis of variance showed that factor C (yeast extract) had a significant impact on the growth and development of Bacillus subtilis. Therefore, the optimal culture medium formula was determined to be: 1% sucrose, 1% yeast extract, and 0.05% MgSO₄.

[0086] Table 3 Results of the orthogonal experiment of Bacillus subtilis M4

[0087] Note: K1, K2, and K3 represent the different levels of OD for each factor. 600 The average value; R represents the range, the same below.

[0088] 5. Effect of culture time on strain growth The optimal culture medium for strain M4 consists of 10g sucrose, 10g yeast extract, 0.5g MgSO4, and 1L distilled water, with a pH of 8.0.

[0089] Strain M4 was inoculated into the optimal culture medium and cultured in batches at 28℃ and 180 rpm for 96 h, following the same inoculation method as in step 4. Samples were taken every 2 h during the rapid growth phase (0–12 h) and every 12 h during the stationary and decline phases (12–96 h). The absorbance of the culture medium at 600 nm was measured at each time point using a UV spectrophotometer. The experiment was conducted in triplicate, and the average value was calculated from the three replicate measurements. The OD value was plotted on the x-axis as culture time (h). 600 The values ​​are plotted on the ordinate to create a high-temporal-resolution growth curve. See the results below. Figure 10 ,according to Figure 10 It can be seen that the growth retardation phase of strain M4 at 28℃ is about 4 hours, and it enters the logarithmic growth phase at the 5th hour, where it grows most vigorously. Strain M4 reaches its highest OD at the 60th hour of culture. 600 The value was 1.58. The strain showed a decrease after 72 hours, then increased again, and finally entered the death phase.

[0090] 6. Effects of different liquid volumes on the growth of Bacillus subtilis M4 The optimal culture medium for strain M4 consists of 10g sucrose, 10g yeast extract, 0.5g MgSO4, and 1L distilled water, with a pH of 8.0.

[0091] Four different liquid volumes (50 mL, 75 mL, 100 mL, and 125 mL) were set up in 250 mL Erlenmeyer flasks to investigate the effect of different liquid volumes on the growth of strain M4. Each liquid volume treatment group was inoculated with 400 μL of activated M4 bacterial suspension, with 3 replicates. The inoculation method was the same as in step 4. After incubation at 28℃ and 180 rpm for 24 h, the absorbance of the culture medium at a wavelength of 600 nm was measured using a UV spectrophotometer.

[0092] Different amounts of culture medium were added to 150 mL Erlenmeyer flasks, and OD was measured after 24 h. 600 Values ​​such as Figure 11 As shown. According to Figure 11 It can be seen that the OD of the fermentation broth 600 The values ​​from left to right are 0.43, 0.88, 1.35, and 1.24. The strain exhibits the best cell growth at a solution volume of 100 mL. OD 600 Value 1.35.

[0093] 7. Effects of different initial pH values ​​on bacterial growth The optimal culture medium for strain M4 consists of: 10g sucrose, 10g yeast extract, 0.5g MgSO4, and 1L distilled water.

[0094] Based on the optimal culture medium, the effects of initial pH values ​​of 4, 5, 6, 7, 8, and 9 on the growth of strain M4 were investigated. Specifically, 400 μL of activated bacterial suspension was inoculated into 100 mL of culture medium at different pH treatments, with three replicates for each pH gradient. The inoculation method was the same as in step 4, and the cultures were incubated at 28℃ with shaking at 180 rpm for 24 h. The absorbance of the culture medium at 600 nm was measured using a UV spectrophotometer, and graphs were plotted.

[0095] The results are as follows Figure 12 As shown, the fermentation broth OD 600 The values ​​from left to right are 0.11, 0.23, 0.33, 0.93, 1.53, and 0.84. The OD values ​​of the bacterial culture at an initial pH of 8 are... 600 The maximum value is 1.53.

[0096] 8. Box-Behnken experimental design OD of Bacillus subtilis M4 fermentation broth under different culture conditions 600 The value was used as the response value. The results of three single-factor experiments (time, volume, and initial pH) were analyzed. Factors A (time, h), B (volume, mL), and C (initial pH) were selected as independent variables. The culture conditions were: 28℃, 180 rpm shaking culture. The OD value of the fermentation broth was used as the response value. 600 The values ​​were used as response values. A Box-Behnken central composite design was employed to optimize the parameters of the culture conditions using response surface methodology to obtain the optimal culture conditions. The experimental levels are shown in Table 4.

[0097] Table 4. Box-Behnken experimental design of Bacillus subtilis M4.

[0098] The Box-Behnken test results for Bacillus subtilis M4 are shown in Table 5. The OD values ​​of the fermentation broth were analyzed using Design-Expert 10 software. 600 The quadratic polynomial regression model of the value (Y) on time (A), liquid volume (B), and pH (C) is: Y = 1.62 + 0.0225A - 0.005B + 0.0225C + 0.16AB + 0.04AC - 0.055BC - 0.1785A 2 -0.2435B 2 -0.1235C 2 As shown in Table 5 of the regression model analysis, the Box-Behnken test model for strain M4 showed extremely significant regression (P<0.01), indicating that this model can be used to measure the OD of the strain's fermentation broth. 600 nmThe prediction of the F-value. The magnitude of the F-value represents the prediction of the strain's OD value. 600 nm The influence strength of the three factors on the bacterial OD value is shown in Table 6. 600 The order of influence of pH on the solution volume is: pH > volume > time. The significance test of the regression equation coefficients shows that A... 2 B 2 and C 2 The result was highly significant (P < 0.01). The lack-of-fit term in the equation was 0.0399, indicating that the Box-Behnken model for the strain was very stable and could make good predictions; the coefficient of determination R0 was [value missing]. 2 The value of 0.9913 indicates that the model has a good fit and can reflect the prediction well.

[0099] Table 5. Box-Behnken assay protocol and results for Bacillus subtilis M4

[0100] Table 6 Results of regression model analysis for Bacillus subtilis M4

[0101] Note: * indicates significant, ** indicates extremely significant.

[0102] Design-Expert 10 software was used to create 3D response surface methodology plots and corresponding 2D contour plots to investigate the relationships between time (A), liquid volume (B), and initial pH (C). The contour lines for time (A), liquid volume (B), liquid volume (B), pH (C), and time (A), initial pH (C) exhibit approximately elliptical shapes, indicating a significant relationship between these factors. Further analysis of the trends in the three 3D surface plots for time, liquid volume, and initial pH using Design-Expert 10 software revealed maximum values ​​for time, liquid volume, and pH within the experimental range. It was predicted that under these conditions, the OD value of Bacillus subtilis would be highest at 61.103 h, a liquid volume of 96.779 mL, and an initial pH of 7.89. 600 The predicted value was 1.57. For ease of operation and feasibility, the optimal fermentation conditions for the strain were changed to 61 h, 97 mL of liquid, and an initial pH of 7.9.

[0103] Example 3: Efficacy of Bacillus subtilis M4 in controlling root rot of potted *Sinomenium acutum* The pot experiment was conducted from August 2024 to January 2025 in the greenhouse of the Science and Technology Park of the College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, using *Sinomenium acutum* seedlings as experimental material. The microbial inoculant used in the experiment was prepared from *Bacillus subtilis* M4, with a bacterial concentration of 1×10⁻⁶. 6 CFU / mL. The pathogen used was a suspension of Fusarium oxysporum spores (concentration adjusted to 1×10⁻⁶). 6 (CFU / mL), recorded as pathogenic bacteria.

[0104] 1. Preparation of Bacillus subtilis M4 fermentation broth The culture medium consisted of 10g sucrose, 10g yeast extract, 0.5g magnesium sulfate, and 1L distilled water, with a pH of 8.0.

[0105] Bacillus subtilis M4 was inoculated into a culture medium with a volume of 100 mL / 250 mL Erlenmeyer flask, an initial pH of 8.0, and cultured for 60 h at a temperature of 28 °C and a rotation speed of 180 rpm to obtain the M4 fermentation broth.

[0106] 2. Preparation of fermentation broth for pathogenic bacteria The *Fusarium oxysporum* used in the experiment was provided by the Desert Vegetable Research Group of the College of Horticulture and Plant Protection, Inner Mongolia Agricultural University. Pathogen activation followed a standard procedure: in a clean bench, mycelial discs were obtained from the edge of pre-cultured PDA plates using a sterile punch (6 mm). Five discs were randomly selected and inoculated into 500 mL Erlenmeyer flasks containing 200 mL of PDA liquid medium. The flasks were then incubated in a constant temperature shaker (28℃, 180 rpm) in the dark for 7 days. After incubation, spores were counted using a hemocytometer (0.1 mm depth). The spores were then washed by centrifugation (4000 rpm, 10 min) and resuspended in sterile water to prepare a final concentration of 1×10⁻⁶. 8 A standardized spore suspension of 1 spore / mL was prepared and stored at 4°C for later use.

[0107] 3. Experimental Design This invention employs a single-factor randomized block design, selecting uniformly growing and robust potted *Strombus haematomarginatus* seedlings as experimental materials. Sterilized nutrient soil was filled into standard cultivation pots (15cm in diameter × 12cm in height), with two seedlings planted in each pot. The soil was compacted to ensure uniform root contact. All treatments began at the four-leaf stage of *Strombus haematomarginatus*, and the experiment consisted of four treatment groups, as detailed in Table 7.

[0108] Control group (CK): First, apply 50 mL of sterile water, and then apply 50 mL of sterile water again after 7 days. Biological prevention group (T2): First, inoculate with 50 mL of Bacillus subtilis M4 fermentation broth, and then inoculate with 50 mL of pathogenic bacteria fermentation broth 7 days later; Biological therapy group (T6): First, 50 mL of pathogenic bacteria fermentation broth was inoculated, and 50 mL of Bacillus subtilis M4 fermentation broth was inoculated 7 days later; Pathogen stress group (T9): 50 mL of pathogenic bacteria fermentation broth was inoculated first, and 50 mL of pathogenic bacteria fermentation broth was inoculated again 7 days later.

[0109] Each treatment consisted of 20 pots, with 3 biological replicates (60 pots / treatment in total). The root wound irrigation method was used for inoculation: three 1cm deep wounds were created at the root zone of the plant, and 50mL of either strain W1 fermentation broth or pathogenic bacteria fermentation broth was injected quantitatively. The control group was simultaneously treated with an equal volume of sterile water.

[0110] Table 7 Experimental Treatments

[0111] (1) The incidence of disease was statistically analyzed 30 days after the second vaccination. After the plants show symptoms of root rot, the pathogen is isolated from the diseased roots again and verified according to Koch's postulates. The disease grading criteria for *Strombus haematomarginatus* are based on the following standards: Grade 0: No root disease; Grade 1: Root disease incidence ≤20%, leaves are normal, roots have lesions but the area is small, and the plant is healthy and not wilted; Level 2: 20% ≤ root disease rate ≤ 40%, with lesions on the roots, but the plant is healthy and does not wilt; Grade 3: 40% ≤ root disease rate ≤ 60%, leaves are normal, roots and leaves are slightly wilted, and few lower leaves fall off; Grade 4: 60% ≤ root disease incidence ≤ 80%, root lesions reaching 1.0-2.0cm, seedling leaves obviously wilting or falling off, or the whole plant wilting; Level 5: Root disease incidence ≥80%, leaves wither, roots turn black or even the whole plant dies.

[0112] The following formulas are used to calculate the disease incidence (DI), disease severity index (DSI), and prevention and control effectiveness: ; ; .

[0113] As shown in Table 8 and Figure 13 Through pot experiments, the incidence rate, disease index, and relative control effect of each treatment were obtained after 30 days. The disease incidence index and relative control efficacy were compared. It was found that the disease incidence rate of *Sinomenium acutum* seedlings treated with pathogenic bacteria T9 was 80%, the disease index was 66%, and the control efficacy of T2 (*Bacillus subtilis*) was 66.36%, which showed better control efficacy. The results showed that the control efficacy of applying antagonistic bacteria first and then pathogenic bacteria was better than that of applying pathogenic bacteria first and then antagonistic bacteria.

[0114] Table 8. Control effects of different treatments on potted *Symplocos edulis*

[0115] Note: Different lowercase letters indicate significant differences between different treatment groups within the same column of data, and the same applies below.

[0116] (2) Determination of biomass indicators Thirty days after the second inoculation, samples of *Symplocos edulis* were collected for biomass determination: 1) The plants were separated into aboveground parts and roots using a sterile blade; 2) The fresh weight of each part was weighed immediately; 3) The parts were placed in an oven at 105℃ for 30 minutes, then the temperature was adjusted to 80℃ and dried until constant weight was achieved; 4) The dry weight was weighed using an analytical balance.

[0117] The results are shown in Table 9. After 30 days, it can be seen that the T2 treatment (application of antagonistic bacteria + pathogenic bacteria) can reduce the rate of biomass decline in *Symplocos edulis* caused by root rot. The T6 treatment (application of pathogenic bacteria + antagonistic bacteria) can also reduce the rate of biomass decline in *Symplocos edulis* caused by root rot, but the effect is not as good as T2 (antagonistic bacteria + pathogenic bacteria). Inoculation with pathogenic bacteria alone showed a significant decrease in biomass. This indicates that antagonistic bacteria can inhibit the growth of the pathogen causing root rot in *Symplocos edulis* and reduce the impact of the disease on *Symplocos edulis*.

[0118] Table 9. Effects of Bacillus subtilis M4 on the biomass of potted *Sinapis alba*.

[0119] (3) Antagonistic bacteria regulate the disease resistance and defense enzyme system of *Strombus haematomarginatus* seedlings This invention selects potted *Sinocyclocheilus roxburghii* plants and uses a systematic sampling method. Three representative plants are randomly selected from each treatment group, and three functional leaves are taken from each plant as test samples. A total of three biological replicates are set up for subsequent determination and analysis of physiological and biochemical indicators.

[0120] (1) SOD activity was determined using the nitroblue tetrazol method.

[0121] (2) POD activity was determined using the guaiacol method.

[0122] (3) The CAT activity was determined by ultraviolet absorption method.

[0123] (4) MDA (malondialdehyde) content was determined by spectrophotometry.

[0124] Different treatments had different effects on the SOD, POD, CAT enzyme activities and MDA content of *Strombus haematomarginatus* plants, as shown in the results. Figure 14~Figure 17 As shown.

[0125] The results showed that different treatments had different effects on the SOD, POD, CAT enzyme activities, and MDA content of *Strombax ceiba* plants. The results indicated that treatment T2 showed a significant increase in SOD activity (227.70 U / g), while treatment T6 showed little difference from the control (CK), and treatment T9 showed a decrease of 45.76 U / g compared to the CK. Figure 14 Regarding POD enzyme activity, the T6 treatment was similar to the CK treatment. Figure 15 The T2 treatment significantly increased CAT activity, and could improve CAT activity in *Sinomenium acutum* plants to a certain extent. Figure 16 Each treatment reduces the MDA content to some extent. Figure 17 Bacillus subtilis M4 fermentation broth can increase SOD enzyme activity, POD enzyme activity, CAT enzyme activity, and reduce MDA content.

[0126] The T2 treatment, by first applying Bacillus subtilis M4 fermentation broth, can activate the plant's defense mechanisms in advance, giving the plant stronger antioxidant capacity when encountering pathogens, thus more effectively reducing ROS accumulation and lowering MDA. Conversely, the T9 treatment, by first inoculating with pathogens, may lead to a large amount of ROS production. At this time, applying antagonistic bacteria fermentation broth may not be enough to effectively activate the defense system, resulting in a poorer reduction in MDA.

[0127] In summary, the Bacillus subtilis M4 of the present invention can reduce the incidence of root rot and promote plant growth.

[0128] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Bacillus subtilis ( Bacillus subtilis Bacillus subtilis M4, whose preservation number is CGMCC No. 35477, is used to control root rot of Axillary sedge.

2. An inoculum containing Bacillus subtilis M4 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent includes a bacterial suspension of Bacillus subtilis M4 and / or the metabolites of Bacillus subtilis M4.

4. The microbial agent according to claim 2 or 3, characterized in that, The concentration of Bacillus subtilis M4 in the bacterial agent is 10. 6 ~10 10 CFU / mL.

5. A method for preparing a microbial agent, characterized in that, The process includes the following steps: inoculating the Bacillus subtilis M4 of claim 1 into a culture medium for fermentation culture to obtain the bacterial agent.

6. The preparation method according to claim 5, characterized in that, The fermentation culture temperature is 26~32℃, the fermentation culture time is 55~65h, the fermentation rotation speed is 180~220rpm, the initial pH of the culture medium used for the fermentation culture is 7.5~8.5, and the volume of the culture medium occupies 38%~41% of the culture container volume.

7. The preparation method according to claim 5, characterized in that, The culture medium used for the fermentation culture comprises: 9-11 g / L sucrose, 9-11 g / L yeast extract, and 0.3-0.8 g / L magnesium sulfate.

8. The application of Bacillus subtilis M4 as described in claim 1, the inoculant as described in any one of claims 2 to 4, or the inoculant prepared by any one of claims 5 to 7 in the control of plant root rot; The plant in question is *Strombus haematomarginatus*; the pathogen causing the root rot is *Fusarium oxysporum*. Fusarium oxysporum ).

9. A method for controlling plant root rot, characterized in that, The procedure includes the following steps: applying the Bacillus subtilis M4 of claim 1 to the roots of the plant through a root irrigation treatment; The plant in question is *Strombus haematomarginatus*; the pathogen causing the root rot is *Fusarium oxysporum*. Fusarium oxysporum ).

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