Bacillus mojavensis, fungicide, preparation method, control method and application

Abstract

The invention belongs to the technical field of microbial control, and particularly relates to bacillus mojavensis, a microbial agent, a preparation method, a control method and application. The invention provides a bacillus mojavensis W1 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35479. The bacillus mojavensis W1 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] The application belongs to the technical field of microbial prevention and treatment, and particularly relates to a Bacillus mojavensis, a microbial agent, a preparation method, a prevention and treatment method and application. BACKGROUND

[0002] Root rot can harm various crops, trees and horticultural plants. Its pathogenesis is usually that pathogenic microorganisms invade through root wounds, natural openings or directly penetrate root epidermis, reproduce and secrete toxins in root tissues, destroy the normal physiological functions of roots, and cause root rot. The prevention and treatment methods mainly include chemical prevention and treatment methods and biological prevention and treatment methods. In the chemical prevention and treatment methods, frequent application of the same type of fungicides (such as carbendazim and hymexazol) can cause the pathogen of root rot (such as fusarium and pythium) to gradually develop drug resistance through gene mutation or metabolic pathway change.

[0003] In the biological prevention and treatment methods, Trichoderma, Paecilomyces lilacinus and Bacillus subtilis are commonly used to prevent and treat root rot, and the types of biological prevention and treatment bacteria are few. SUMMARY

[0004] The application aims to provide a Bacillus mojavensis, a microbial agent, a preparation method, a prevention and treatment method and application. The Bacillus mojavensis W1 provided by the application can reduce the incidence and incidence index of plant root rot and promote plant growth.

[0005] To solve the above technical problems, the application provides the following technical solutions. The application provides a Bacillus mojavensis (Bacillus mojavensis) W1, which is characterized in that the preservation number is CGMCC No.35479. Bacillus mojavensis )W1, which is characterized in that the preservation number is CGMCC No.35479.

[0006] The application further provides a microbial agent, which comprises the Bacillus mojavensis W1 described in the above scheme.

[0007] As a preferred scheme, the viable bacterial count of the Bacillus mojavensis W1 in the microbial agent is ≥10 8 CFU / mL or ≥10 8 CFU / g.

[0008] The application further provides a preparation method of the microbial agent, which comprises the following steps: inoculating the Bacillus mojavensis W1 described in the above scheme into a culture medium for fermentation culture to obtain the microbial agent.

[0009] As a preferred scheme, the fermentation culture temperature is 25-33 DEG C, the fermentation culture time is 55-65 h, and the fermentation culture rotation speed is 180-220 rpm.

[0010] As a preferred solution, the initial pH of the culture medium used in the fermentation culture is 7.0-8.0; the volume of the culture medium accounts for 19%-25% of the volume of the culture vessel.

[0011] As a preferred solution, the composition of the culture medium used in the fermentation culture includes: sucrose 9-11 g / L, yeast powder 9-11 g / L and KCl 9-11 g / L.

[0012] The application further provides application of the Bacillus mojavensis W1, the microbial agent or the microbial agent prepared by the preparation method in the above solutions in prevention and treatment of plant diseases and / or plant growth promotion.

[0013] As a preferred solution, the plant diseases include root rot; and the plants include P. saxicola.

[0014] The application further provides a method for preventing and treating plant diseases, which comprises: applying the Bacillus mojavensis W1 in the above solutions to plants or to soil in which plants are planted.

[0015] The application has the following beneficial effects: the application provides a Bacillus mojavensis W1 with a preservation number of CGMCC No. 35479. The Bacillus mojavensis W1 in the application can increase activities of disease resistance defense enzymes superoxide dismutase SOD, peroxidase POD and catalase CAT, reduce malondialdehyde MDA content, finally reduce incidence and incidence index of plant root rot, and promote plant growth. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure for antagonistic activity of antagonistic bacteria W1 and W2 on pathogenic bacteria of P. saxicola root rot; Figure 2 Figure for colony morphology and gram staining of Bacillus mojavensis W1; the left side A is a colony morphology figure; and the right side a is a gram staining figure; Figure 3 Figure for a phylogenetic tree of strain W1 constructed based on 16S rDNA sequences; Figure 4 Figure for influence of carbon source types on growth of Bacillus mojavensis W1; Figure 5 Figure for influence of sucrose concentration on growth of Bacillus mojavensis W1; Figure 6 Figure for influence of nitrogen source types on growth of Bacillus mojavensis W1; Figure 7 Figure for influence of yeast powder concentration on growth of Bacillus mojavensis W1; Figure 8 Figure for influence of inorganic salt types on growth of Bacillus mojavensis W1; Figure 9 Figure for the influence of potassium chloride concentration on the growth of Bacillus mojavensis W1; Figure 10 Figure for the concentration change curve of the bacterial liquid during the growth of Bacillus mojavensis W1; Figure 11 Figure for the influence of different liquid loading amounts on Bacillus mojavensis W1; Figure 12 Figure for the influence of different initial pH values on Bacillus mojavensis W1; Figure 13 Figure for the biocontrol effect of Bacillus mojavensis W1 on the root rot of Pteryxia hispidula; Figure 14 Figure for the influence of different treatments on the SOD enzyme activity of the leaves of potted Pteryxia hispidula; Figure 15 Figure for the influence of different treatments on the POD enzyme activity of the leaves of potted Pteryxia hispidula; Figure 16 Figure for the influence of different treatments on the CAT enzyme activity of the leaves of potted Pteryxia hispidula; Figure 17 Figure for the influence of different treatments on the content of malondialdehyde in the leaves of potted Pteryxia hispidula.

[0017] Biological preservation instructions Strain W1, classified as Bacillus mojavensis (Bacillus mojavensis) Bacillus mojavensis , was preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 31, 2025, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 35479. DETAILED DESCRIPTION

[0018] The present application provides a Bacillus mojavensis (Bacillus mojavensis) Bacillus mojavensis W1, with a preservation number of CGMCC No. 35479.

[0019] The Bacillus mojavensis W1 described in the present application is obtained by being isolated from the rhizosphere soil of healthy Pteryxia hispidula, W1 is a gram-positive bacterium, the colony of the strain is round, milky white, the colony edge is translucent, the colony surface is moist, the shape is irregular, the edge is irregular, and it is slightly protruding.

[0020] The 16S rDNA of the Bacillus mojavensis W1 is aligned using Blast, and a phylogenetic tree is constructed using MEGA as shown in Figure 3 , and it is found that the strain is Bacillus mojavensis (Bacillus mojavensis) Bacillus mojavensis .

[0021] The Bacillus mojavecifolius W1 of this invention can enhance the activity of plant disease-resistant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and reduce malondialdehyde (MDA) content, thereby reducing the incidence and disease index of plant root rot and promoting plant growth.

[0022] This invention provides a microbial agent comprising *Bacillus mojaverius* W1 as described in the above-mentioned technical solution. As an optional embodiment, the application form of *Bacillus mojaverius* W1 of this invention includes fermentation broth, wherein the viable cell count of the fermentation broth is ≥10⁻⁶. 8 CFU / mL or ≥10 8 CFU / g, more preferably 10 8 CFU / mL or 10 8 CFU / g. The preparation method of the bacterial agent of the present invention includes the following steps: inoculating the Mojave Bacillus W1 into a culture medium for fermentation culture to obtain the bacterial agent.

[0023] As an optional implementation, the present invention does not specifically limit the inoculation method; conventional methods can be used. As an optional implementation, the inoculation form of *Bacillus mojaverius* W1 according to the present invention includes single colony inoculation or seed culture.

[0024] As an optional implementation, the fermentation culture temperature of the present invention is 25~33℃, or 27~30℃, more preferably 28℃; in specific embodiments of the present invention, the fermentation culture temperature is 25, 26, 27, 29, 29, 30, 31, 32, or 33℃. The fermentation culture time of the present invention is 55~65h, or 58~62h, more preferably 60h. The fermentation culture rotation speed of the present invention is 180~220rpm, further preferably 190~210rpm, more preferably 200rpm. The initial pH of the culture medium used in the fermentation culture of the present invention is 8.0; the volume of the culture medium accounts for 19%~25% of the volume of the culture container; in specific embodiments of the present invention, the volume of the culture medium accounts for 19%, 19.5%, 20%, 20.8%, 21%, 22%, 23%, 24%, or 25% of the volume of the culture container. In specific embodiments of the present invention, the culture container is a culture flask. The culture medium used in the fermentation culture of the present invention comprises: 9-11 g / L sucrose, 9-11 g / L yeast powder, and 9-11 g / L KCl. The culture medium includes 9-11 g / L sucrose, more preferably 10 g / L; the culture medium includes 9-11 g / L yeast powder, more preferably 10 g / L; and the culture medium includes 9-11 g / L KCl, more preferably 10 g / L. The sucrose, yeast powder, and KCl in the culture medium can all promote the growth of Bacillus mojaveii W1 and increase the viable count of Bacillus mojaveii W1.

[0025] This invention provides the application of Bacillus mojaverifolium W1 as described in the above technical solution, or the inoculum prepared by the above technical solution or the preparation method described in the above technical solution, in the prevention and control of plant diseases.

[0026] As an optional implementation, the plant disease described in this invention includes root rot; the plant described in this invention includes *Strombus haematomarginatus*.

[0027] As an optional implementation, the pathogens causing the root rot include Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum ).

[0028] As an optional implementation method, the application of Bacillus mojaverifolium W1 according to the present invention includes root drenching, and the root drenching method includes root-damaging drenching; the application period is the four-leaf-one-heart stage. When Sand Mustard reaches the four-leaf-one-heart stage, the taproot has elongated and lateral roots have begun to sprout. Applying the inoculant at this time can avoid the problem of excessively short roots in the cotyledon stage, and also avoid the defect of high lignification of roots in the mature stage. At the same time, the Sand Mustard at the four-leaf-one-heart stage has small individual differences, which can ensure the reproducibility and reliability of experimental results.

[0029] The Mojave Bacillus W1 strain described in this invention can increase the activity of disease-resistant defense enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), while reducing malondialdehyde (MDA) content and decreasing the incidence and disease index of plant root rot.

[0030] This invention provides the application of Bacillus mojaverifolium W1 as described in the above technical solution, or the bacterial agent as described in the above technical solution, or the bacterial agent prepared by the preparation method described in the above technical solution, in plant growth promotion.

[0031] The technical features of the application of the plant and Bacillus mojavei W1 described in this invention have been discussed above and will not be repeated here.

[0032] As an optional implementation, the plant growth promotion method of the present invention includes increasing aboveground biomass; the aboveground biomass includes aboveground dry weight and / or fresh weight. In a specific embodiment of the present invention, treatment with Bacillus mojaverifolium W1 can reduce the rate of biomass decline in *Strombus haematomarginatus* caused by root rot.

[0033] This invention provides a method for controlling plant diseases, comprising: applying the *Bacillus mojaveniformis* W1 described in the above technical solution to plants or to the soil in which plants are grown. The technical features of the plants and the application of *Bacillus mojaveniformis* W1 described in this invention have been discussed above and will not be repeated here.

[0034] 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.

[0035] Example 1: Screening and Identification of Bacillus mojaveensis 1. Isolation and screening of antagonistic bacteria 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. Twelve antagonistic bacterial strains showing control effects against *Fusarium oxysporum*, the pathogen causing root rot in *Sinomenium acutum*, were obtained through plate confrontation. These 12 bacterial strains were named N35, M3, L12, N33, W3, N3O, W2, L7, N23, M4, L21, and W1, respectively. The control (CK) was treated with sterile water. The inhibition rates of CK and the antagonistic strains are shown in Table 1. The antagonistic effects of W1 and W2 against *Fusarium oxysporum* are as follows: Figure 1 As shown.

[0036] Table 1 shows that the 12 biocontrol strains exhibited different inhibitory effects against the root rot pathogen. Strains W1, M4, and L21 showed significantly higher inhibitory effects than the other nine strains; therefore, these three strains were selected as the biocontrol strains for subsequent experiments. The formula for calculating the inhibition rate is shown below: .

[0037] Table 1. Antibacterial rate of antagonistic strains

[0038] 2. Identification of antagonistic bacteria 2.1 Morphological characteristics of antagonistic bacteria Antagonistic strain W1 was streaked in three zones on LB agar plates and incubated at 29°C. Colony morphology was observed. W1 colonies were round, milky white, with translucent edges, a moist surface, irregular shape, and slightly raised, uneven edges. Figure 2 (A). Gram staining results of strain W1 indicate that this strain is a Gram-positive strain. Figure 2 (a) Figure 2 In the text, A represents the colony morphology of W1; a represents the Gram staining of W1.

[0039] 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. Bacillus mojavensis They share high gene homology and belong to the same branch, see... Figure 3 Based on morphological characteristics, strain W1 was identified as Bacillus mojaveli ( Bacillus mojavensis ).

[0040]

[0041] Example 2: Optimization of fermentation of Bacillus mojaverius strain W1 The LB liquid medium consists of 10g peptone, 5g yeast extract, 10g sodium chloride and 1L distilled water, pH 8.0.

[0042] 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.

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

[0044] The culture medium consists of 10g xylose, 5g yeast extract, 10g sodium chloride, and 1L distilled water.

[0045] The culture medium consists of 4 components: 10g glucose, 5g yeast powder, 10g sodium chloride and 1L distilled water.

[0046] The culture medium consists of 10g fructose, 5g yeast extract, 10g sodium chloride and 1L distilled water.

[0047] The experiment employed a completely randomized block design, with three biological replicates for each carbon source treatment. Before inoculation, activated W1 biocontrol bacterial suspension was inoculated into Erlenmeyer flasks (250 mL) at a rate of 1% (v / v). Each flask contained 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 absorbance of the culture medium in each treatment group was measured at 600 nm using a UV spectrophotometer.

[0048] See results Figure 4 It can be seen that Bacillus mojaverius W1 can grow on culture media with sucrose, glucose, maltose, fructose, and xylose as carbon sources, and the fermentation broth OD... 600 The values ​​from left to right are 1.17, 1.15, 0.58, 0.78, and 0.60. The strain showed the best growth when sucrose and glucose were used as carbon sources, and the OD of the fermentation broth was [value missing]. 600 The values ​​were 1.17 and 1.15, respectively, so sucrose was the optimal carbon source for the strain.

[0049] 1.2 Optimization Experiment of Sucrose Gradient Based on culture medium 1, the sucrose concentrations were set to 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively. Three replicates were set for each concentration in culture media 1-1 to 1-6. After inoculation with activated strains, the cultures were incubated at 28°C and 180 rpm for 24 h with shaking. The growth status of the strains was evaluated by measuring the OD600 value, thereby determining the optimal addition concentration of each nutrient. The inoculation method was the same as in 1.1.

[0050] Culture medium 1-1 consists of 5g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0051] Culture medium 1-2 consists of 10g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0052] Culture media 1-3 consist of: 20g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0053] Culture media 1-4 consist of: 30g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0054] Culture media 1-5 consist of: 40g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0055] Culture media 1-6 consist of: 50g sucrose, 5g yeast powder, 10g sodium chloride, and 1L distilled water.

[0056] See results Figure 5 It can be seen that, and with the increase of sucrose concentration, the OD of the fermentation broth of the strain... 600 The value showed a trend of first increasing and then decreasing, indicating that the OD value of the fermentation broth was... 600 The values ​​from left to right are 0.92, 1.18, 1.04, 1.00, 0.98, and 0.99. At a sucrose concentration of 1% (10 g / L), the OD value of the Bacillus mojaveniformis W1 fermentation broth... 600 The highest value is 1.18.

[0057] 2. Nitrogen source optimization 2.1 Using LB liquid medium as the base, seven nitrogen source replacement media were designed: yeast extract (10 g / L concentration, control), beef extract, peptone, glutamic acid, ammonium sulfate, ammonium chloride, and potassium nitrate were used to replace the yeast extract in the base medium in equal amounts, while keeping other media components unchanged. 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.

[0058] The culture medium 7 consisted of 10g peptone, 5g beef extract, 10g sodium chloride, and 1L distilled water.

[0059] The culture medium consists of 10g peptone, 5g peptone, 10g sodium chloride and 1L distilled water.

[0060] The culture medium consists of 10g peptone, 5g glutamic acid, 10g sodium chloride and 1L distilled water.

[0061] The culture medium 10 consists of: 10g peptone, 5g ammonium sulfate, 10g sodium chloride and 1L distilled water.

[0062] Culture medium 11 consists of: 10g peptone, 5g ammonium chloride, 10g sodium chloride and 1L distilled water.

[0063] The culture medium 12 consists of: 10g peptone, 5g potassium nitrate, 10g sodium chloride and 1L distilled water.

[0064] The experiment employed a completely randomized block design, with three biological replicates for each nitrogen source treatment. Before inoculation, activated W1 biocontrol bacterial suspension was inoculated into Erlenmeyer flasks (250 mL) at a rate of 2% (v / v), each flask containing 100 mL of culture medium (C1-C12). The flasks were then incubated at 28°C with shaking at 180 rpm for 24 h. After incubation, the absorbance of the culture medium in each treatment group was measured at 600 nm using a UV spectrophotometer.

[0065] The results are as follows Figure 6 As shown, Bacillus mojaveii W1 can grow on a culture medium with yeast extract, ammonium sulfate, glutamic acid, beef extract, peptone, ammonium chloride, and potassium nitrate as nitrogen sources. The fermentation broth OD... 600 The values ​​from left to right are 1.44, 0.11, 0.09, 0.76, 0.62, 0.10, and 0.12. The strain grew best when yeast extract was used as the nitrogen source; therefore, yeast extract was chosen as the optimal nitrogen source for the strain.

[0066] 2.2 Yeast Powder Gradient Optimization Experiment Based on culture medium 6, yeast extract 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.

[0067] Culture medium 6-1 consists of: 10g peptone, 5g yeast extract, 10g sodium chloride and 1L distilled water.

[0068] The culture medium 6-2 consists of 10g peptone, 10g yeast extract, 10g sodium chloride and 1L distilled water.

[0069] The culture medium 6-3 consists of: 10g peptone, 20g yeast extract, 10g sodium chloride and 1L distilled water.

[0070] The culture medium 6-4 consists of: 10g peptone, 30g yeast extract, 10g sodium chloride and 1L distilled water.

[0071] Culture medium 6-5 consists of: 10g peptone, 40g yeast extract, 10g sodium chloride and 1L distilled water.

[0072] Culture medium 6-6 consists of: 10g peptone, 50g yeast extract, 10g sodium chloride and 1L distilled water.

[0073] See results Figure 7 The results show that as the yeast powder concentration increases, the bacterial concentration of the strain first increases and then decreases, and the OD of the fermentation broth... 600 The values ​​from left to right are 1.14, 1.51, 1.08, 1.08, 1.09, and 0.77. The OD of the bacterial culture was [value missing] when the yeast powder concentration reached 1% (10 g / L). 600 The highest value is 1.51.

[0074] 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.

[0075] Culture medium 14 consists of: 10g peptone, 5g yeast extract, 5g KH2PO4 and 1L distilled water.

[0076] The culture medium 15 consisted of 10g peptone, 5g yeast extract, 0.5g FeSO4 and 1L distilled water.

[0077] The culture medium 16 consisted of 10g peptone, 5g yeast extract, 0.5g ZnSO4 and 1L distilled water.

[0078] Culture medium 17 consisted of 10g peptone, 5g yeast extract, 0.5g MgSO4 and 1L distilled water.

[0079] The culture medium 18 consisted of 10g peptone, 5g yeast extract, 5g NaCl, and 1L distilled water.

[0080] The culture medium 19 consisted of 10g peptone, 5g yeast extract, 5g KCl and 1L distilled water.

[0081] The experiment employed a completely randomized block design, with three biological replicates for each inorganic salt treatment. Before inoculation, activated W1 bacterial suspension was inoculated at a rate of 2% (v / v) into Erlenmeyer flasks (250 mL each), each containing 100 mL of culture medium (Vol. 13-Vol. 19). The flasks were placed in a constant-temperature shaking incubator and cultured continuously for 24 hours at 28°C and a shaking speed of 180 rpm. OD values ​​were then measured. 600 The value is used to assess the growth status of bacteria, thereby screening for the most suitable inorganic salts.

[0082] See results Figure 8 The results show that the OD of the fermentation broth... 600 The values ​​from left to right are 0.68, 0.15, 1.23, 1.37, 0.15, 1.24, and 1.15; when KCl is used as the inorganic salt in the culture medium, the OD of the bacterial solution... 600 The highest value is 1.37.

[0083] 3.2 KCl gradient optimization experiment Based on medium 19, KCl concentrations were set to 0.25%, 0.50%, 1%, 1.5%, 2%, and 3%, respectively, and mediums 19-1 to 19-6 were set up. Three replicates were performed 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 strains was assessed to determine the optimal concentration of each nutrient. The inoculation method was the same as in 3.1.

[0084] The culture medium 19-1 consists of: 10g peptone, 5g yeast extract, 2.5g KCl and 1L distilled water.

[0085] The culture medium 19-2 consists of 10g peptone, 5g yeast extract, 5g KCl and 1L distilled water.

[0086] The culture medium 19-3 consists of 10g peptone, 5g yeast extract, 10g KCl and 1L distilled water.

[0087] The culture medium 19-4 consists of 10g peptone, 5g yeast extract, 15g KCl and 1L distilled water.

[0088] The culture medium 19-5 consists of: 10g peptone, 5g yeast extract, 20g KCl and 1L distilled water.

[0089] The culture medium 19-6 consists of 10g peptone, 5g yeast extract, 30g KCl and 1L distilled water.

[0090] See results Figure 9 The results show that, similarly, with increasing KCl concentration, the OD of the fermentation broth of strain W1 also increases. 600 The value first increases and then decreases; fermentation broth OD 600 The values ​​from left to right are 1.36, 1.41, 1.48, 1.21, 1.20, and 0.91; when the KCl concentration reaches 1% (10 g / L), the OD... 600 The maximum value is 1.48.

[0091] 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.

[0092] Table 2 Mojavec W1 L9 (3 3 Orthogonal experiment

[0093] Using sucrose (A), yeast powder (B), and KCl (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 W1 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 mojaveniformis W1 after 24 h of fermentation were measured. 600 The results were used to determine the optimal fermentation formula. Range analysis of the orthogonal experiment showed that the influence of the three factors on the growth of the strain was C>A>B, and the combination most favorable to the growth of *Bacillus mojaverius* W1 was A2B2C2 (Table 3). Analysis of variance showed that KCl had the most significant effect on the growth of *Bacillus mojaverius* W1. Therefore, the optimal culture medium formula for the strain was determined to be: 1% sucrose, 1% yeast extract, and 1% KCl.

[0094] Table 3 Results of the orthogonal experiment of Bacillus mojavei W1

[0095] 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.

[0096] 5. Effect of culture time on strain growth The optimal culture medium for strain W1 consists of 10g sucrose, 10g yeast extract, 10g KCl, and 1L distilled water at pH 8.0.

[0097] Based on the optimal culture medium, the effect of different culture times on the growth of strain W1 was investigated. The experiment was conducted in triplicate, with batch culture at 28℃ and a rotation speed of 180 rpm for 96 hours, using the same inoculation method as in step 4. Samples were taken every 2 hours during the rapid growth phase (0–12 h) and every 12 hours during the stationary and decline phases (12–96 h). The absorbance of the culture medium at 600 nm was measured using a UV spectrophotometer at each time point, and the average value was obtained from 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 It can be seen that the bacterial strain experiences a slow growth phase of approximately 4 hours at 28℃, enters the logarithmic growth phase around the 5th hour, and grows most vigorously. As the bacterial cell count increases, nutrients are continuously consumed while toxic substances increase, leading to a balance between bacterial reproduction and death. The bacterial culture reaches its peak OD value around the 60th hour. 600 The value was 1.51, and the strain entered the death phase after 60 hours.

[0098] 6. Effects of different liquid volumes on the growth of Bacillus mojaverius W1 The optimal culture medium for strain W1 consists of 10 g / L sucrose, 10 g / L yeast extract, 10 g / L KCl, and 1 L distilled water, with a pH of 8.0.

[0099] Based on the optimal culture medium, an experiment was conducted to investigate the effect of different culture medium volumes on the growth of strain W1. Specifically, four different culture medium volumes (50 mL, 75 mL, 100 mL, and 125 mL) were set up to explore the effect of different volume volumes on the growth of the strain. The strain was cultured at a constant temperature of 28℃ and a rotation speed of 180 rpm, with the inoculation method the same as in step 4. Too much or too little culture medium will affect the oxygen content dissolved in the culture medium, thus affecting bacterial growth and development. Different volumes of culture medium were added to 250 mL Erlenmeyer flasks, and the OD was measured after 24 hours. 600 Values ​​such as Figure 11 As shown. Fermentation broth OD 600 The values ​​from left to right are 1.47, 1.41, 1.35, and 1.24, respectively. The strain showed optimal growth at a solution volume of 50 mL. OD 600The values ​​were 1.47, indicating that increasing the liquid volume affected the bacterial cell count. As the liquid volume increased, the growth of antagonistic bacteria gradually decreased.

[0100] 7. Effects of different initial pH values ​​on bacterial growth The optimal culture medium for strain W1 consists of 10 g / L sucrose, 10 g / L yeast extract, 10 g / L KCl, and 1 L distilled water.

[0101] Based on the optimal culture medium, the effect of initial pH values ​​(4, 5, 6, 7, 8, 9, with a gradient interval of 1.0) on the growth of strain W1 was investigated. Specifically, 400 μL of activated bacterial suspension was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of culture medium at different pH treatments, with three replicates for each pH gradient. Culture conditions: 28℃, 180 rpm shaking, for 24 h; inoculation method was the same as in step 4. The absorbance of the culture medium at 600 nm was measured using a UV spectrophotometer, and the results were plotted.

[0102] The results are as follows Figure 12 Both excessively high and low pH values ​​can affect the OD of Bacillus mojaverifolium W1 fermentation broth. 600 The growth rate of the strain showed a slow upward trend with increasing pH, and the OD value of the fermentation broth increased. 600 The values ​​from left to right are 0.11, 0.32, 0.51, 1.46, 1.53, and 1.40, representing the OD values ​​of the bacterial culture at an initial pH of 8. 600 The maximum value is 1.53.

[0103] 8. Box-Behnken experimental design OD of Bacillus mojaveniformis W1 fermentation broth under different culture conditions 600 The value was used as the response value. The results of three single-factor experiments (time, liquid volume, and initial pH) were analyzed. Factors A (time), B (liquid volume), 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.

[0104] Table 4. Experimental factor level design for Bacillus mojavesiculus W1Box-Behnken.

[0105] The results of the W1 Box-Behnken assay for Bacillus mojaverii are shown in Table 5. The OD values ​​of the fermentation broth were analyzed using Design-Expert 10 software. 600The quadratic polynomial regression model of the value (Y) against time (A), liquid volume (B), and initial pH (C) is: Y = 1.53 - 0.0538A + 0.0375B + 0.0163C - 0.0275AB + 0.00AC + 0.0725BC - 0.28A 2 -0.1475B 2 -0.2250C 2 As shown in Table 6 of the regression model analysis, the regression of the Box-Behnken experimental model for strain Box-Behnken was highly significant (P<0.01), indicating that this model can be used to measure the OD of the fermentation broth of strain Box-Behnken. 600 The F-value is a prediction of the OD value of the strain. 600 The influence strength of the three factors on the OD value of Bacillus mojaveniformis W1 can be seen from the data in Table 6. 600 The influence of pH on the concentration was in the order of pH > time > volume. The significance tests of the regression equation coefficients showed that A2, B2, and C2 were highly significant (P < 0.01). The lack-of-fit term was 0.0291, 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 0.9147 indicates that the model has a good fit and can reflect the prediction well.

[0106] Table 5. Box-Behnken assay protocol and results for Bacillus mojavesiculus W1.

[0107] Table 6 Results of W1 regression model analysis for Bacillus mojavei.

[0108] Note: * indicates significant, ** indicates highly significant.

[0109] 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), volume (B), and pH (C). The contour lines for time (A) and volume (B), volume (B) and pH (C), and time (A) and pH (C) exhibited approximately elliptical shapes, indicating significant relationships between these relationships. Further analysis of the trends in the three 3D surface plots for time, volume, and pH using Design-Expert 10 software revealed maximum values ​​for time, volume, and pH within the experimental range. It was predicted that at a time of 58.584 h, a volume of 58.009 mL, and a pH of 8.156, the optimal conditions for *Bacillus mojaveniformis* W1OD were met. 600The predicted value was 1.54. For ease of operation and feasibility, the optimal fermentation conditions for the strain were changed to 59 h, 58 mL of liquid, and pH 8.2.

[0110] Example 3: Efficacy of Bacillus mojavecifolius W1 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 mojaverifolium* W1, with a bacterial concentration of 1×10⁻⁶. 8 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.

[0111] 1. Preparation of Bacillus mojaverius W1 fermentation broth The culture medium consisted of 10g sucrose, 10g yeast extract, 10g KCl and 1L distilled water, with a pH of 8.0.

[0112] Mojave Bacillus W1 was inoculated into a culture medium with a volume of 50 mL / 250 mL Erlenmeyer flask, an initial pH of 8.0, and cultured at 28 °C and 180 rpm for 60 h to obtain the W1 fermentation broth.

[0113] 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.

[0114] 3. Experimental Design This invention employs a single-factor randomized block design, selecting uniformly growing and robust potted *Gnaphalium affine* seedlings as experimental materials. High-temperature and high-pressure 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 *Gnaphalium affine*, and the experiment consisted of four treatment groups, as detailed in Table 7. 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 (T1): First, inoculate with 50 mL of fermentation broth of antagonistic bacterium Bacillus mojaveii W1, and then inoculate with 50 mL of fermentation broth of pathogenic bacteria 7 days later. Biological therapy group (T5): First, 50 mL of pathogenic bacteria fermentation broth was inoculated, and 100 mL of the corresponding antagonistic bacterium Bacillus mojaveii W1 bacterial solution 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.

[0115] 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.

[0116] Table 7 Experimental Treatments

[0117] (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.

[0118] The following formulas are used to calculate the disease incidence (DI), disease severity index (DSI), and prevention and control effectiveness: ; ; .

[0119] The results are shown in Table 8 andFigure 13 As shown, pot experiments were conducted, and the incidence rate, disease index, and relative control effect of each treatment were obtained after 30 days. It was found that the incidence rate of *Strombus haematomarginatus* seedlings in treatment T9 (inoculated with the pathogen) was 80%, and the disease index was 66.76%. In contrast, the incidence rate of *Strombus haematomarginatus* seedlings in treatment T1 was 35%, and the disease index was 20.44%, with a relative control effect of 69.61%, indicating better control efficacy. The results showed that the control effect of treatment T1 (antagonist bacteria + pathogen) was superior to that of treatment T5 (pathogen + antagonist bacteria). This may be because treatment T1 involves inoculating with antagonist bacteria followed by pathogen, which increases the activity of defensive enzymes in the plant compared to inoculating with antagonist bacteria first, leading to better defense against pathogen inoculation. In contrast, treatment T5 involves inoculating with antagonist bacteria after pathogen inoculation. The antagonist bacteria cannot promptly regulate enzyme activity, resulting in rapid infection of the plant by the pathogen after inoculation, thus lower control efficacy than treatment T1.

[0120] Table 8. Control effects of different treatments on potted *Symplocos edulis*

[0121] Note: Different lowercase letters indicate significant differences between different treatment groups within the same column of data, and the same applies below.

[0122] (2) Determination of biomass indicators Thirty days after the second inoculation, samples of *Syngonium sibiricum* 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 plants 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. The results are shown in Table 9.

[0123] Table 9 shows that, in terms of aboveground fresh weight, treatment T1 increased the aboveground fresh weight compared to the control (CK), with T1 having a greater effect on increasing aboveground fresh weight, while T9 showed a greater decrease. Regarding aboveground dry weight, treatment T1 increased aboveground dry weight, while T9 had the lowest average aboveground dry weight. In terms of underground fresh weight, treatment T1 increased underground fresh weight, but the effect was not significant. Treatment T9 showed a decrease in underground fresh weight, underground dry weight, aboveground fresh weight, and aboveground dry weight. After 30 days, it can be seen that treatment T1 (applying antagonist W1 + pathogen) can reduce the rate of biomass decline caused by root rot in *Strombus haematomarginatus*, and treatment T5 (applying pathogen + antagonist W1) can also reduce the rate of biomass decline caused by root rot in *Strombus haematomarginatus*, but the effect is not as good as treatment T1 (antagonist W1 + pathogen). Treatment with only pathogen (T9) showed a significant decrease in biomass. This indicates that antagonistic bacteria W1 can inhibit the growth of the pathogen causing root rot in *Sinocyclocheilus serratus*, thus reducing the impact of the disease on *Sinocyclocheilus serratus*.

[0124] Table 9. Effects of Bacillus mojavesiculus W1 on biomass of potted *Sinapis alba*.

[0125] (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.

[0126] (1) SOD activity was determined using the nitroblue tetrazol method.

[0127] (2) POD activity was determined using the guaiacol method.

[0128] (3) The CAT activity was determined by ultraviolet absorption method.

[0129] (4) MDA (malondialdehyde) content was determined by spectrophotometry.

[0130] 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.

[0131] The results show that treatment T1 significantly increased SOD activity by 228.43 U / g, while treatment T9 reduced it by 45.76 U / g compared to the control (CK). Figure 14 The T1 treatment significantly increased POD activity by 288.21 U / g, while the T9 treatment decreased it by 60.02 U / g compared to the control (CK). Figure 15 The T1 treatment significantly increased CAT activity, raising it to a certain extent in *Strombus haematomarginatus* plants. The T9 treatment, however, reduced CAT activity in *Strombus haematomarginatus* plants to a level of 158 U / g. Figure 16 It can be seen that applying the fermentation broth of Bacillus mojaveii W1 before the fermentation broth of the pathogen may enhance the plant's defense system before it even encounters the pathogen. The fermentation broth of Bacillus mojaveii W1 may activate the plant's systemic resistance (ISR), allowing antioxidant enzymes to prepare in advance and respond more quickly to invasion, clearing more ROS, thus resulting in a more significant increase in SOD activity. Conversely, if the fermentation broth of the pathogen is applied first, followed by the fermentation broth of Bacillus mojaveii W1, the plant may already be damaged, and the cells may be compromised. Applying the fermentation broth of Bacillus mojaveii W1 at this point may have limited repair effects, resulting in a less significant increase in activity compared to the former. Bacillus mojaveii W1 has a certain impact on malondialdehyde (MDA) content; compared to treatment T9, which only inoculated with the pathogen, the other treatments all reduced MDA content to some extent. Figure 17Applying Mojave Bacillus W1 fermentation broth beforehand may activate the plant's defense mechanisms in advance, giving the plant a stronger antioxidant capacity when encountering pathogens, thereby more effectively reducing ROS accumulation and lowering MDA.

[0132] In summary, the Mojave Bacillus W1 of this invention can reduce the incidence of root rot and promote plant growth.

[0133] 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 strain of Bacillus mojaveli ( Bacillus mojavensis W1, characterized in that, The accession number is CGMCCNo.35479.

2. A microbial agent, characterized in that, Includes the Mojave Bacillus W1 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of Bacillus mojaverifolium W1 in the bacterial agent is ≥10. 8 CFU / mL or ≥10 8 CFU / g.

4. A method for preparing a microbial agent, characterized in that, The process includes the following steps: inoculating the Bacillus mojavei W1 of claim 1 into a culture medium for fermentation culture to obtain the bacterial agent.

5. The preparation method according to claim 4, characterized in that, The fermentation temperature is 25~33℃; the fermentation time is 55~65h; and the fermentation speed is 180~220rpm.

6. The preparation method according to claim 5, characterized in that, The initial pH of the culture medium used for the fermentation culture is 7.0 to 8.0; the volume of the culture medium accounts for 19% to 25% of the volume of the culture container.

7. The preparation method according to claim 4, characterized in that, The culture medium used for the fermentation culture comprises: 9-11 g / L sucrose, 9-11 g / L yeast extract, and 9-11 g / L KCl.

8. The application of the Bacillus mojaveii W1 as described in claim 1, the microbial agent as described in claim 2 or 3, or the microbial agent prepared by any one of the preparation methods described in claims 4 to 7 in the prevention and control of plant diseases and / or the promotion of plant growth; The plant disease is root rot; the pathogen causing the root rot is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum ).

9. The application according to claim 8, characterized in that, The plant mentioned includes *Axewing Sand Mustard*.

10. A method for controlling plant diseases, characterized in that, include: Apply the Mojave Bacillus W1 of claim 1 to plants or to the soil in which plants are grown; The plant disease is root rot; the pathogen causing the root rot is Fusarium oxysporum.

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