Salt-tolerant bacillus, fungicide, preparation method, control method and application

Abstract

The invention belongs to the technical field of microbial control, and particularly relates to a salt-tolerant bacillus, a microbial agent, a preparation method, a control method and application. The invention provides a salt-tolerant bacillus L21 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35478. The salt-tolerant bacillus L21 can activate the defense mechanism of plants, improve the activity of defensive enzymes in the plants, and enable the plants to have higher oxidation resistance when encountering pathogenic bacteria, so that ROS accumulation is more effectively reduced, MDA is reduced, and the morbidity of root rot is reduced. The result of the embodiment shows that the bacillus halotolerant L21 can improve the activity of superoxide dismutase (SOD), the activity of peroxidase (POD) and the activity of catalase (CAT), reduce the content of malondialdehyde (MDA) and reduce the morbidity of root rot of cornutgionium axataenense.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial prevention and treatment, and particularly relates to a salt-tolerant Bacillus, a bacterial agent, a preparation method, a prevention and treatment method and application. BACKGROUND

[0002] Root rot is a kind of disease that can cause harm to crops, trees and horticultural plants and various plants. The pathogenesis is generally as follows: the pathogenic microorganism invades the plant body by means of root wounds, natural orifices or directly penetrating the root epidermis, and then continuously reproduces in the root tissue while secreting toxins, which can destroy the normal physiological functions of the root system, and eventually cause the root to rot.

[0003] Desert pioneer plant Arabis alpina is an annual or biennial herbaceous plant of the Brassicaceae Arabis genus, mainly distributed in deserts or grassland sandy land in Gansu, Ningxia, Inner Mongolia and other places.

[0004] In recent years, the root rot of Arabis alpina has caused great obstacles to local planting industry. Fusarium oxysporum is one of the main pathogenic bacteria causing root rot, which seriously reduces the content of active ingredients and medicinal value of Arabis alpina. At present, chemical fungicides and crop rotation are the main control methods, but chemical fungicides can easily cause environmental pollution and pathogenic bacteria resistance, and crop rotation requires high capital investment and technical requirements, which limits its wide application. Therefore, it is very important to strengthen the research and development of biological control methods. Microbial strains are an important breakthrough for biological control, At present, Trichoderma harzianum, Bacillus subtilis and Bacillus amyloliquefaciens are commonly used in biological control methods, so it is necessary to strengthen the research of microbial strains for preventing and treating root rot. SUMMARY

[0005] The application aims to provide a salt-tolerant Bacillus, a bacterial agent, a preparation method, a prevention and treatment method and application, and the salt-tolerant Bacillus L21 provided by the application can reduce the incidence of root rot.

[0006] To solve the above technical problems, the application provides the following technical solutions: The application provides a salt-tolerant Bacillus (Bacillus halodurans) L21 with a preservation number of CGMCC No.35478. Bacillus halotolerans The salt-tolerant Bacillus L21 is used for preventing and treating Arabis alpina root rot.

[0007] The application further provides a biocontrol agent comprising the salt-tolerant Bacillus L21 described in the above solution.

[0008] As a preferred solution, the number of viable bacteria of the salt-tolerant Bacillus L21 in the biocontrol agent is ≥10 8 CFU / mL or ≥108 CFU / g.

[0009] The present invention also provides a method for preparing a biocontrol agent, comprising the following steps: inoculating the salt-tolerant Bacillus L21 described in the above scheme into a culture medium for fermentation culture to obtain a biocontrol agent.

[0010] As a preferred embodiment, the fermentation culture temperature is 25~35℃, the fermentation culture time is 55~65h, the fermentation culture speed is 170~230rpm, the initial pH of the culture medium used for the fermentation culture is 7.0~7.5, and the volume of the culture medium occupies 16%~21% 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 0.3-1.0 g / L magnesium sulfate.

[0012] The present invention also provides the application of the salt-tolerant Bacillus L21 described in the above-described scheme, the biocontrol agent described therein, or the biocontrol agent prepared by the above-described preparation method in the control of plant diseases; The plant disease is root rot; the pathogen of root rot is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum The plant in question is *Axypsis cuspidatum*.

[0013] The present invention also provides a method for the prevention and control of plant diseases, comprising the following steps: applying the halophilic Bacillus L21 described in the above scheme to the soil in which the plants are planted; The plant disease is root rot; the pathogen of root rot is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum The plant in question is *Axypsis cuspidatum*.

[0014] As a preferred embodiment, the application of the halophilic Bacillus L21 includes an inoculum, and the application method includes soil drenching and / or root-damaged drenching; the viable count of halophilic Bacillus L21 in the inoculum is ≥10. 8 CFU / mL or ≥10 8 CFU / g.

[0015] The present application provides a salt-tolerant Bacillus L21, with a preservation number of CGMCC No. 35478, and the salt-tolerant Bacillus L21 is used for preventing and treating root rot of Prenanthes alpina Maxim. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure is an antagonistic activity diagram of salt-tolerant Bacillus L21 and L12 against Prenanthes alpina Maxim root rot pathogen; Figure 2 Figure is a colony morphology diagram and a gram staining diagram of salt-tolerant Bacillus L21; the left side is a colony morphology diagram, and the right side is a gram staining diagram; Figure 3 Figure is a phylogenetic tree of salt-tolerant Bacillus L21 constructed based on 16S rDNA sequences; Figure 4 Figure is a diagram of the influence of different carbon sources on the growth of salt-tolerant Bacillus L21; Figure 5 Figure is a diagram of the influence of different sucrose concentrations on the growth of salt-tolerant Bacillus L21; Figure 6 Figure is a diagram of the influence of different nitrogen sources on the growth of salt-tolerant Bacillus L21; Figure 7 Figure is a diagram of the influence of different yeast powder concentrations on the growth of salt-tolerant Bacillus L21; Figure 8 Figure is a diagram of the influence of different inorganic salts on the growth of salt-tolerant Bacillus L21; Figure 9 Figure is a diagram of the influence of different magnesium sulfates on the growth of salt-tolerant Bacillus L21; Figure 10 Figure is a growth curve diagram of salt-tolerant Bacillus L21; Figure 11 Figure is a diagram of the influence of different liquid loading amounts on the growth of salt-tolerant Bacillus L21; Figure 12 Figure is a diagram of the influence of different initial pH values on the growth of salt-tolerant Bacillus L21; Figure 13 Figure is a biocontrol effect diagram of salt-tolerant Bacillus L21 on Prenanthes alpina Maxim root rot; Figure 14 Figure is a diagram of the influence of different treatments on the SOD enzyme activity of potted Prenanthes alpina Maxim leaves; Figure 15 Figure for the effect of different treatments on the POD enzyme activity of the leaves of the potted Peltaria Figure 16 Figure for the effect of different treatments on the CAT enzyme activity of the leaves of the potted Peltaria Figure 17 Figure for the effect of different treatments on the MDA content of the leaves of the potted Peltaria

[0017] Biological preservation instructions The strain L21, which is classified as Bacillus halodurans, Bacillus halotolerans was preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 31, 2025, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 35478. DETAILED DESCRIPTION

[0018] The present application provides a Bacillus halodurans Bacillus halotolerans L21, with a preservation number of CGMCC No. 35478; the Bacillus halodurans L21 is used for preventing and treating Peltaria root rot.

[0019] The Bacillus halodurans L21 in the present application is obtained by being isolated from the rhizosphere soil of healthy Peltaria, and the strain L21 is a gram-positive bacterium. The colony of the strain L21 is opaque and milky white, with wrinkles at the edge and a moist surface. The 16S rDNA of the Bacillus halodurans L21 is aligned using Blast, and a phylogenetic tree is constructed using MEGA, as shown in Figure 3 , it can be known that the strain L21 is Bacillus halodurans Bacillus halotolerans .

[0020] The Bacillus halodurans L21 involved in the present application has a significant anti-root rot effect, which specifically manifests as: it can effectively improve the activity of disease-resistant enzymes such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and reduce the content of malondialdehyde (MDA), and exhibits good effects in the prevention and treatment of root rot.

[0021] The present application provides a microbial agent, which comprises the Bacillus halodurans L21 in the above technical solution. In the present application, the viable count of the Bacillus halodurans L21 in the microbial agent needs to reach ≥10 8CFU / mL. As an alternative embodiment, the bacterial agent of the present application comprises the fermentation broth as a component thereof. The method for preparing the fermentation broth of the present application comprises: inoculating the Bacillus halodurans L21 into a culture medium for fermentation culture to obtain the fermentation broth. As an alternative embodiment, the method for inoculation of the present application is not particularly limited and the conventional method can be used. The form of inoculation of the Bacillus halodurans L21 of the present application comprises a single colony.

[0022] As an alternative embodiment, the fermentation culture of the present application is carried out at a temperature of 25-35℃, which can also be 27-30℃, and more preferably 28℃; in the specific embodiments of the present application, the fermentation culture is carried out at a temperature of 25, 26, 27, 29, 29, 30, 31, 32, 34 or 35℃. The fermentation culture of the present application is carried out for a time period of 55-65h, which can also be 58-62h, and more preferably 60h. The fermentation culture of the present application is carried out at a rotation speed of 170-230rpm, and more preferably 200-210rpm. The culture medium used in the fermentation culture of the present application comprises: sucrose 9-11g / L, yeast powder 9-11g / L and magnesium sulfate 0.3-1.0g / L. The initial pH of the culture medium used in the fermentation culture of the present application is 7.0-7.5, which can also be 7.0-7.3, and more preferably 7.1. The volume of the culture medium occupies 16%-21% of the volume of the culture vessel, which can also be 16.8%-20%. In the specific embodiments of the present application, the culture vessel is a culture bottle. In the specific embodiments of the present application, 50mL of the culture medium is added to each 250mL conical flask.

[0023] As an alternative embodiment, the culture medium used in the fermentation culture of the present application comprises sucrose 9-11g / L, which can also be 9.5-10.5g / L, and more preferably 10g / L. The culture medium of the present application comprises yeast powder 9-11g / L, which can also be 9.8-10.8g / L, and more preferably 10g / L. The culture medium of the present application comprises magnesium sulfate 0.3-1.0g / L, which can also be 0.4-0.8g / L, and more preferably 0.5g / L. The sucrose, yeast powder and magnesium sulfate in the culture medium of the present application can promote the growth of the Bacillus halodurans L21 and increase the growth density of the Bacillus halodurans L21. As an alternative embodiment, the initial pH of the culture medium of the present application is 7.0-7.5, which can also be 7.0-7.3, and more preferably 7.1.

[0024] The present application provides the use of the Bacillus halodurans L21, the bacterial agent or the bacterial agent prepared by the method of the present application in the prevention and treatment of plant diseases. The plant disease is root rot. The pathogenic fungus of the root rot is Fusarium oxysporum. Fusarium oxysporum) ; the plant is P. axillaris. In a specific embodiment of the present application, the salt-tolerant Bacillus L21 is applied to the P. axillaris in a pot, and the incidence of the root rot of the P. axillaris is significantly reduced. As an alternative embodiment, the pathogenic bacteria of the root rot include Fusarium oxysporum ( Fusarium oxysporum ).

[0025] As an alternative embodiment, the application method of the salt-tolerant Bacillus L21 includes soil root irrigation and / or injured root irrigation, and the application time is the four-leaf-one-heart stage. The main root of the P. axillaris has elongated, and the lateral root has started to germinate at the four-leaf-one-heart stage. Therefore, the application of the bacterial agent at this stage can avoid the problem of short root at the cotyledon stage and the defect of high lignification of the root at the adult stage. At the same time, the P. axillaris at the four-leaf-one-heart stage has small individual differences, which can ensure the repeatability and reliability of the experimental results.

[0026] The present application provides a method for preventing and treating plant diseases, wherein the salt-tolerant Bacillus L21 described in the above technical solution is applied to the plant; the plant disease is root rot; the pathogenic bacteria of the root rot are Fusarium oxysporum ( Fusarium oxysporum ) ; the plant is P. axillaris. As an alternative embodiment, the application method described in the present application includes soil root irrigation and / or injured root irrigation. The technical features of the application of the salt-tolerant Bacillus L21 have been discussed above, and will not be repeated here.

[0027] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0028] Example 1: Screening and identification of salt-tolerant Bacillus 1. Isolation and screening of salt-tolerant Bacillus L21 The collected soil was subjected to dilution and coating to isolate the salt-tolerant Bacillus L21 strain. The isolated bacteria were further purified until single colonies were obtained. Ten strains of bacteria that had a control effect on Fusarium oxysporum, the pathogenic bacteria of the root rot of P. axillaris, were obtained by plate confrontation, and the ten strains of bacteria were named as N35, M3, L12, N33, W3, N3O, W2, L7, N23 and L21, respectively. CK was treated with sterile water. The inhibition rates of CK and the antagonistic bacterial strains are shown in Table 1. Among them, the antagonistic effects of L21 and L12 on Fusarium oxysporum are shown in Table 1. According to Table 1, the inhibition effect of strain L21 is good. The calculation formula of the inhibition rate is as follows: Figure 1 .

[0029] Table 1: Inhibition rate of salt-tolerant Bacillus L21 strain ​

[0030] 2. Identification of halophilic Bacillus L21 2.1 Morphological characteristics of halophilic Bacillus L21 Strawberry strain L21 was inoculated into three zones on LB agar plates and incubated at 29°C. Colony morphology was observed. It was found that colonies of strain L21 were opaque, milky white, with wrinkled edges and a moist surface (see...). Figure 2 (Left image). Gram staining results for strain L21 indicate that this strain is Gram-positive (see image). Figure 2 (Right image in the middle)

[0031] 2.2 Molecular biological identification The 16S rDNA sequence of strain L21 was aligned to the BLAST gene sequence in NCBI, and a phylogenetic tree was constructed using MEGA. The results are as follows: Figure 3 As shown, strain L21 and Bacillus halotolerans The strains showed high genetic homology, belonging to the same branch. Combined with morphological characteristics, strain L21 was identified as a halophilic Bacillus. Bacillus halotolerans ), namely, salt-tolerant Bacillus L21.

[0032]

[0033] Example 2 Fermentation optimization of salt-tolerant Bacillus L21 strain The composition of LB liquid medium is: 10 g of proteose peptone, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water, pH 7.0.

[0034] 1. Carbon source optimization 1.1 Using LB liquid medium as the basic medium, replace the proteose peptone in the basic medium with sucrose, maltose, xylose, glucose and fructose at a concentration of 10 g / L respectively (the rest of the components remain unchanged). The composition of the replaced medium 1-5 is as follows: The composition of medium 1 is: 10 g of sucrose, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

[0035] The composition of medium 2 is: 10 g of maltose, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

[0036] The composition of medium 3 is: 10 g of xylose, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

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

[0038] The composition of medium 5 is: 10 g of fructose, 5 g of yeast powder, 10 g of sodium chloride and 1 L of distilled water.

[0039] The experiment adopts a completely randomized block design, and 3 biological replicates are set for each carbon source treatment. Before inoculation, the activated L21 biocontrol bacterial suspension is inoculated into a 250 mL conical flask at a inoculation amount of 1% (v / v), and the conical flask contains 100 mL of medium 1-5, and then it is cultured in a constant temperature shaker at 28°C with a rotation speed of 180 rpm for 24 h. After the culture is completed, the absorbance value of each treatment group at 600 nm wavelength is measured using a UV spectrophotometer, i.e. OD 600 value.

[0040] The results are shown in Figure 4 , which shows that when salt-tolerant Bacillus L21 uses sucrose, glucose, maltose, fructose and xylose as carbon source, the OD 600 value of the fermentation broth obtained at the end of the culture is 1.49, 1.27, 0.82, 0.86, 0.77 respectively, so sucrose is the most beneficial to the growth of salt-tolerant Bacillus L21.

[0041] 1.2 Sucrose gradient optimization experiment On the basis of the culture medium 1, the sucrose concentration was set to 0.5%, 1%, 2%, 3%, 4%, 5% respectively, 3 replicates for each concentration, after inoculating salt-tolerant Bacillus L21, it was cultured at 28℃, 180rpm for 24h, the growth condition of the strain was evaluated by measuring OD 600 value, so as to determine the optimal addition concentration of each nutrient component, the inoculation method was the same as 1.1.

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

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

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

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

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

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

[0048] The results are shown in Figure 5 , and with the increase of sucrose concentration, the OD 600 value of the fermentation broth of the strain showed a trend of first rising and then falling, and the OD 600 value of the obtained fermentation broth from left to right was 1.21, 1.50, 1.42, 1.32, 1.35 and 1.25. When the sucrose concentration reached 1% (10g / L), the OD 600 value of the fermentation broth of the salt-tolerant Bacillus L21 was the highest, which was 1.50.

[0049] 2. Nitrogen source optimization: 2.1 Using LB liquid medium as the basis, 7 kinds of nitrogen source replacement medium were designed, and the composition of the replaced medium 6~12 was as follows: Culture medium 6 consisted of 10g proteose peptone, 5g yeast powder, 10g sodium chloride and 1L distilled water.

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

[0051] Culture medium 8 consisted of 10g proteose peptone, 5g proteose 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] The experiment employed a completely randomized block design, with three biological replicates for each nitrogen source treatment. Before inoculation, activated L21 biocontrol bacterial suspension was inoculated into 250 mL Erlenmeyer flasks at a rate of 2% (v / v), each flask containing 100 mL of culture medium (C12–C2). 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.

[0057] The results are as follows Figure 6 As shown, the fermentation broth OD 600 The values ​​from left to right are 1.61, 0.09, 0.07, 0.88, 0.70, 0.06, and 0.07. The OD values ​​of halophilic Bacillus L21 in the bacterial culture using yeast extract as the nitrogen source are... 600 The value is 1.61, which is the highest. Therefore, yeast extract is selected 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] Medium 6-4 consists of 10 g of peptone, 30 g of yeast extract, 10 g of sodium chloride and 1 L of distilled water.

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

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

[0065] The results are shown in Table 2. Figure 7 As can be seen from Table 2, with the increase of the concentration of yeast extract, the concentration of the bacterial solution first increased and then decreased, and the OD value of the fermentation solution first increased and then decreased. 600 The OD values from left to right are 1.21, 1.63, 1.25, 1.17, 1.10 and 0.94. When the concentration of yeast extract reached 1% (10 g / L), the OD value of the bacterial solution was the highest, reaching 1.64. 600 The OD values from left to right are 1.21, 1.63, 1.25, 1.17, 1.10 and 0.94. When the concentration of yeast extract reached 1% (10 g / L), the OD value of the bacterial solution was the highest, reaching 1.64.

[0066] 3. Optimization of inorganic salts 3.1. Based on the standard LB liquid medium, seven representative inorganic salts, K2HPO4, KH2PO4, FeSO4, ZnSO4, MgSO4, NaCl (control) and KCl, were selected to replace the NaCl component in the basic formula (other components remained constant). The addition amount of K2HPO4, KH2PO4, NaCl and KCl macroelements was 0.5% (w / v), and the addition amount of FeSO4, ZnSO4 and MgSO4 microelements was 0.05% (w / v) in 1 L of medium. The compositions of the replaced media 13-19 are as follows: Medium 13 consists of 10 g of peptone, 5 g of yeast extract, 5 g of K2HPO4 and 1 L of distilled water.

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

[0068] Medium 15 consists of 10 g of peptone, 5 g of yeast extract, 0.5 g of FeSO4 and 1 L of distilled water.

[0069] Medium 16 consists of 10 g of peptone, 5 g of yeast extract, 0.5 g of ZnSO4 and 1 L of distilled water.

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

[0071] Medium 18 consists of 10 g of peptone, 5 g of yeast extract, 5 g of NaCl and 1 L of distilled water.

[0072] Medium 19 consists of 10 g peptone, 5 g yeast extract, 5 g KCl and 1 L distilled water.

[0073] The experiment adopts a completely randomized block design, and 3 biological replicates are set for each nitrogen source treatment. The activated L21 bacterial suspension is inoculated into 250 mL conical flasks at an inoculation amount of 1% (v / v), and the conical flasks contain 100 mL of culture medium 13-19 respectively. The conical flasks are placed in a constant temperature shaking incubator, and continuous culture is carried out at a culture temperature of 28°C and a shaking speed of 180 rpm for 24 h. The OD 600 value is measured to evaluate the growth status of the bacterial cells, so as to screen the most suitable inorganic salt type.

[0074] The results are shown in Table 1. Figure 8 According to the results, the OD 600 values of the fermentation broth from left to right are 1.40, 0.16, 0.13, 1.33, 0.15, 1.23 and 1.16. When MgSO4 is used as the inorganic salt of the culture medium, the OD 600 value of the bacterial solution is the highest.

[0075] 3.2 MgSO4 Gradient Optimization Experiment On the basis of Medium 17, the concentrations of MgSO4 are set to be 0.01%, 0.05%, 0.10%, 0.20%, 0.40% and 0.50%, and Medium 17-1 to 17-6 are set. Each concentration is set with 3 repeated experiments. After the strain L21 is activated, it is inoculated into the aforementioned culture medium, and is cultured at 28°C and 180 rpm for 24 h. The OD 600 value is measured to evaluate the growth status of the strain L21, so as to determine the optimal addition concentration of each nutrient component. The inoculation method is the same as that in 3.1.

[0076] Medium 17-1 consists of 10 g peptone, 5 g yeast extract, 0.1 g MgSO4 and 1 L distilled water.

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

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

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

[0080] Medium 17-5 consists of 10 g peptone, 5 g yeast extract, 4 g MgSO4 and 1 L 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 The results show that the OD of the fermentation broth... 600 The values ​​from left to right are 1.05, 1.39, 1.31, 1.27, 1.14, and 1.09. When the MgSO4 concentration reaches 0.05% (0.5 g / L), the OD... 600 The value is the largest, at 1.39.

[0083] 4. Orthogonal experiment of culture medium components Based on the results of the single-factor experiments, three factors were selected: carbon source (sucrose), nitrogen source (yeast powder), and inorganic salt (MgSO4). Three levels were set, employing a three-factor, three-level L9 (3...) model. 3 Orthogonal experimental design was used. The culture medium consisted of sucrose, yeast extract, KCl, and water. The experimental design levels are shown in Table 2, and the results are shown in Table 3. Each treatment had three biological replicates. The activated L21 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. After inoculation, the flasks were incubated at 28°C with shaking at 180 rpm for 24 h. OD was measured. 600 Values ​​were used to assess the growth status of strain L21.

[0084] Table 2. Salt-tolerant Bacillus L21L9 (3 3 Orthogonal experiment

[0085] Table 3 Results of the orthogonal experiment on halophilic Bacillus L21

[0086] 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.

[0087] Table 3 shows that the influence of the three factors on the growth of Bacillus subtilis is B>C>A, and the optimal combination of levels is A2B2C2 (Table 3). Analysis of variance indicates that factor B (yeast extract) has a significant impact on the growth and development of the strain. Therefore, the optimal culture medium formulation is determined to be: 1% sucrose, 1% yeast extract, and 0.05% MgSO4.

[0088] 5. Effect of culture time on strain growth The optimal culture medium for strain L21 consists of 10g sucrose, 10g yeast extract, 0.5g MgSO4, and 1L distilled water, with a pH of 7.0.

[0089] Based on the optimal medium, the effect of different culture time on the growth of strain L21 was studied. Each treatment was set with 3 parallel repeats. The activated L21 bacterial suspension was inoculated into a conical flask (250 mL) at an inoculation amount of 1% (v / v), and the conical flask contained 100 mL of medium. The batch culture was carried out at 28°C under constant temperature conditions at a rotation speed of 180 rpm for 96 h, and the inoculation method was the same as step 4. In the rapid growth period (0-12 h), sampling was carried out every 2 h, and in the stable and decline periods (12-96 h), sampling was carried out every 12 h. The absorbance of the culture solution at 600 nm wavelength was measured using a UV spectrophotometer, and the average value was obtained by three repeated measurements. The growth curve with high time resolution was plotted with culture time (h) as the abscissa and OD 600 value as the ordinate. The results are shown in Figure 10 , which shows that the lag phase of strain L21 at 30°C is about 4 h, and it enters the logarithmic growth phase at the 5th hour, and the growth is vigorous. When the bacterial cells increase continuously, the nutrients are consumed and the toxic substances increase, and the reproduction and death of bacteria reach a balance, and the salt-tolerant Bacillus L21 reaches the highest OD 600 value of 1.61 at the 60th hour; after 60 h, the strain enters the decline phase.

[0090] 6、Different liquid loading amounts affect the growth of salt-tolerant Bacillus L21 The composition of the optimal medium for strain L21 is: sucrose 10 g, yeast powder 10 g, 0.5 g MgSO4 and 1 L distilled water, pH 7.0.

[0091] Based on the optimal medium, the effect of different liquid loading amounts on the growth of strain L21 was studied. Specifically: Four different liquid loading amounts were set: 50 mL of medium was added to a 250 mL conical flask, 75 mL of medium was added to a 250 mL conical flask, 100 mL of medium was added to a 250 mL conical flask, and 125 mL of medium was added to a 250 mL conical flask. Then 400 μL of activated bacterial solution was inoculated into each treatment group, and 3 repeats were set. The inoculation method was the same as step 4, and the culture was carried out at 28°C and 180 rpm for 24 h. The absorbance of the culture solution at 600 nm wavelength was measured using a UV spectrophotometer. It can be seen that too much or too little liquid loading will affect the oxygen content dissolved in the culture solution, which will affect the growth and development of bacteria. The results are shown in Figure 11 . According to Figure 11 , the OD 600 values of the fermentation broth from left to right are 1.32, 1.23, 0.87 and 0.82, and the OD 600 value of the strain is the highest at 1.32 when the liquid loading amount is 50 mL.

[0092] 7、Different initial pH affects the growth of the strain The optimal medium for strain L21 was composed of 10 g sucrose, 10 g yeast powder, 0.5 g MgSO4 and 1 L distilled water.

[0093] Based on the optimal medium, the initial pH values were set to 4, 5, 6, 7, 8 and 9, respectively. 400 μL of activated bacterial suspension was inoculated into 100 mL of medium treated with different initial pH values, with 3 replicates for each pH gradient. The activated L21 bacterial suspension was inoculated into a conical flask (250 mL) at an inoculation amount of 1% (v / v), and the conical flask contained 100 mL of medium. After inoculation, the culture was incubated at 28°C with 180 rpm shaking for 24 h. The absorbance of the culture at 600 nm was measured by ultraviolet spectrophotometry, and a graph was plotted.

[0094] The results are shown in Table 2. Figure 12 As shown in Table 2, the OD 600 value of the fermentation broth of salt-tolerant Bacillus L21 was affected by excessively high or low initial pH. The growth of the strain showed a slow upward trend with the increase of pH, and the OD 600 values of the fermentation broth from left to right were 0.10, 0.26, 0.35, 1.29, 1.17 and 0.95, respectively. The OD 600 value of the broth was the largest when the initial pH value was 7, which was 1.29.

[0095] 8. Box-Behnken experimental design The optimal medium for strain L21 was composed of 10 g sucrose, 10 g yeast powder, 0.5 g MgSO4 and 1 L distilled water. The OD 600 value of the fermentation broth of salt-tolerant Bacillus L21 under different culture conditions was used as the response value. The culture conditions were 28°C and 180 rpm shaking. Based on the analysis of the results of three single-factor experiments (time, liquid volume and initial pH), the parameters of the culture conditions were optimized by Box-Behnken central composite experimental design to obtain the optimal culture conditions. The experimental levels are shown in Table 4.

[0096] Table 4 Box-Behnken experimental factor level design of salt-tolerant Bacillus L21

[0097] The results of the Box-Behnken experiment of salt-tolerant Bacillus L21 are shown in Table 5. The results were analyzed by Design-Expert 10 software, and the quadratic multinomial regression equation model of the OD 600 value (Y) of the fermentation broth of the strain against time (A), liquid volume (B) and initial pH (C) was obtained as follows: Y = 1.69 + 0.0188A + 0.0113B + 0.0025C + 0.055AB - 0.0275AC + 0.1125BC - 0.14A 2 - 0.18B 2 - 0.2075C 2 .

[0098] From the regression model analysis of Table 6, the Box-Behnken test model of the strain was extremely significant (P < 0.01), indicating that the model could be used to predict the OD 600 value of the strain fermentation broth. The size of F value represented the influence intensity on the OD 600 value of the strain. From the data in Table 6, the influence size of the three factors on the OD 600 value of the strain L21 was in the order of liquid volume > pH > time. From the regression equation coefficient significance test, A 2 , B 2 and C 2 were extremely significant (P < 0.01). The equation misfit term was 0.0125, indicating that the Box-Behnken model of the strain was very stable and could be well predicted. The determination coefficient R 2 was 0.9420, indicating that the model fitting degree was good and could better reflect the prediction.

[0099] Table 5 Box-Behnken test scheme and results of the strain L21

[0100] Table 6 Regression model analysis results of the strain L21

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

[0102] The 3D response surface analysis chart and the corresponding 2D contour chart were drawn using Design-Expert 10 software to explore the mutual relationship of time (A), liquid loading (B) and initial pH (C). Among them, the time (A) and pH (C) contour presented a near circular shape, indicating that the mutual relationship of time (A) and initial pH (C) was not significant. The time (A) and liquid loading (B), liquid loading (B) and initial pH (C) contour presented a near elliptical shape, indicating that the mutual relationship of time (A) and liquid loading (B), liquid loading (B) and initial pH (C) was significant. Similarly, through the change trend of the three 3D surface charts of time, liquid loading and initial pH, further analysis was carried out using Design-Expert 10 software, and it was found that there was a maximum point of time, liquid loading and pH in the test range. It was predicted that when the time was 62.009 h, the liquid loading was 41.659 mL, and the initial pH was 7.159, the OD 600 of the strain L21 was 1.67 under the above conditions. In order to simplify the operation feasibility, the optimal fermentation conditions of the strain were as follows: fermentation time was 62 h, 250 conical flask was loaded with 42 mL of culture medium, and initial pH was 7.1.

[0103] Example 3: Effect of salt-tolerant Bacillus L21 on root rot of potted P. saxicola The pot experiment was carried out in the greenhouse of the Science and Technology Park of the College of Horticulture and Plant Protection of Inner Mongolia Agricultural University from August 2024 to January 2025, using P. saxicola seedlings as experimental materials.

[0104] 1. Preparation of salt-tolerant Bacillus L21 fermentation broth The composition of the culture medium was: sucrose 10 g, yeast powder 10 g, 0.5 g MgSO4 and 1 L distilled water, pH 7.0.

[0105] After the salt-tolerant Bacillus L21 was activated on the LB plate, a single colony was picked and inoculated into the culture medium. The liquid loading of the culture medium was: 50 mL of culture medium was loaded into a 250 mL conical flask, the initial pH of the culture medium was 7.0, and the culture was carried out at 28°C, 180 rpm for 60 h. The L21 fermentation broth was obtained, and the concentration of the bacterial solution was 1×10 8 CFU / mL.

[0106] 2. Preparation of pathogenic bacteria fermentation broth The test used Fusarium oxysporum was provided by the xerophytic vegetable research group of the College of Horticulture and Plant Protection, Inner Mongolia Agricultural University. The standard process was used for pathogen activation: in a clean bench, a sterile punch (6 mm) was used to obtain a bacterial cake from the edge of a pre-cultured PDA plate, and 5 bacterial cakes were randomly selected to inoculate a 500 mL conical flask containing 200 mL PDA liquid medium, which was placed in a constant temperature shaker (28°C, 180 rpm) for 7 days in the dark. After the culture ended, a hemocytometer (0.1 mm depth) was used for spore counting, and the final standardized spore suspension with a concentration of 1×10 8 was prepared by centrifugal washing (4000 rpm, 10 min) and resuspension in sterile water, and stored at 4°C for standby use.

[0107] 3. Test design The present application adopts single factor randomized block design, and selects uniform and robust potted Pterocypsela scabiosoides seedlings as test materials. The high-temperature and high-pressure sterilized nutrient soil is loaded into standard cultivation pots (diameter 15 cm x height 12 cm), and 2 seedlings per pot are planted, and the soil is compacted to ensure uniform root contact. All treatments start from the four-leaf one-heart stage of Pterocypsela scabiosoides, and the test sets 4 treatment groups, the specific design is as follows, see Table 7.

[0108] Control group (CK): First, irrigate with equal amount of sterile water 50 mL, and then irrigate with sterile water 50 mL after 7 days; Biological prevention group (T3): First, inoculate with salt-tolerant Bacillus L21 fermentation broth 50 mL, and then inoculate with pathogenic bacteria fermentation broth 50 mL after 7 days; Biological treatment group (T7): First, inoculate with pathogenic bacteria fermentation broth 50 mL, and then inoculate with corresponding salt-tolerant Bacillus L21 fermentation broth 50 mL after 7 days; Pathogenic stress group (T9): First, inoculate with pathogenic bacteria fermentation broth 50 mL, and then inoculate with pathogenic bacteria fermentation broth 50 mL again after 7 days.

[0109] Each treatment has 20 pots, 3 biological repeats, and a total of 60 pots per treatment. The root injury irrigation method is used for inoculation: 3 1 cm deep wounds are made in the rhizosphere of the plant, and 50 mL of fermentation broth or pathogenic bacteria fermentation broth is injected quantitatively, and the control group is treated with equal amount of sterile water at the same time.

[0110] Table 7 Test treatment

[0111] (1) After the second inoculation 30 days, the incidence of disease was counted After the plant shows root rot symptoms, the pathogen is isolated again from the diseased root system, and verified according to Koch's rule. The disease grading standard of Pterocypsela scabiosoides is counted according to the following standard: 0 level: no disease on root system; Grade 1: root disease incidence ≤20%, normal leaves, small root lesions, healthy plants; Grade 2: 20%≤root disease incidence≤40%, root lesions, healthy plants; Grade 3: 40%≤root disease incidence≤60%, normal leaves, slightly wilted root and leaf, a few lower leaves dropped; Grade 4: 60%≤root disease incidence≤80%, root lesions up to 1.0-2.0 cm, seedling leaves wilted or dropped or the whole plant wilted; Grade 5: root disease incidence≥80%, leaf wilted, root blackened or even the whole plant died.

[0112] Disease incidence (DI) and disease severity index (DSI) and control effect were calculated by the following formula: ; ; .

[0113] As shown in Tables 8 and Figure 13 , through pot experiment, the disease incidence of each treatment was obtained after 30 days, it was found that the disease incidence of the P. axialis seedlings of the treatment T9 inoculated with the pathogenic bacteria was 80%, the disease severity index was 66%, and the control effect of T3 (salt-tolerant Bacillus) was 59.68%. The control effect of T3 (first applying salt-tolerant Bacillus L21 and then applying pathogenic bacteria, i.e. salt-tolerant Bacillus L21+pathogenic bacteria) was better than that of T7 (first applying pathogenic bacteria and then applying salt-tolerant Bacillus L21, i.e. pathogenic bacteria+salt-tolerant Bacillus L21). The reason was analyzed as follows: in the treatment T3, the inoculation of salt-tolerant Bacillus L21 first could improve the defense enzyme activity in the plant body, and the inoculation of pathogenic bacteria could better defend. In the treatment T7, the inoculation of salt-tolerant Bacillus L21 was after the inoculation of pathogenic bacteria, and the salt-tolerant Bacillus L21 could not timely regulate the enzyme activity in the body, so that the subsequent inoculation of pathogenic bacteria led to the rapid infection of the plant by the pathogenic bacteria, and thus the control effect was lower than that of the treatment T3.

[0114] Table 8 Control effect of different treatments on pot P. axialis

[0115] Note: different lowercase letters represent significant differences between different treatment groups in the same column, and the same below.

[0116] (2) Determination of biomass index After the second inoculation for 30 days, the samples of P. saxatile were collected for biomass determination: 1) the plants were separated into aboveground and root system using sterilized blades; 2) the fresh weight of each part was immediately weighed; 3) after being placed in a 105℃ oven for 30 min, the temperature was adjusted to 80℃ and the drying was continued until the constant weight was reached; 4) the dry weight was weighed using an analytical balance.

[0117] The results are shown in Table 9. In terms of biomass, the decline rate of T9 was large, and the rest of the treatments were lower than CK. After 30 days, it can be seen that the T3 (Bacillus halotolerans L21 + pathogen) treatment can reduce the decline rate of P. saxatile biomass caused by root rot, indicating that Bacillus halotolerans L21 can inhibit the growth of P. saxatile root rot pathogen and reduce the impact of disease on P. saxatile.

[0118] Table 9 Effect of different treatments on the biomass of P. saxatile in pots

[0119] (3) Bacillus halotolerans L21 regulates the disease defense enzyme system of P. saxatile seedlings In the present application, the P. saxatile plants cultivated in pots are selected, and the system sampling method is used. Three representative plants are randomly selected in each treatment group, and three functional leaves are taken from each plant as test samples. Three biological replicates are set to determine and analyze the subsequent physiological and biochemical indicators.

[0120] (1) The SOD activity is determined by using the nitro blue tetrazolium method.

[0121] (2) The POD activity is determined by using the guaiacol method.

[0122] (3) The CAT activity is determined by using the ultraviolet absorption method.

[0123] (4) The MDA (malondialdehyde) content is determined by using the spectrophotometric method.

[0124] Different treatments have different effects on the SOD, POD, CAT enzyme activities and MDA content of P. saxatile plants, and the results are shown in Table 9. Figure 14~Figure 17

[0125] According to the results, the SOD enzyme activity of T3 treatment is 213.45 U / g, and the T9 treatment is reduced by 45.76 U / g compared with CK. Figure 14 The POD enzyme activity of T7 treatment is close to CK, and the T9 treatment is reduced by 60.02 U / g compared with CK. Figure 15 The CAT activity of T9 treatment is reduced to a certain extent, which is 158 U / g, and the rest of the treatments are close to CK. Figure 16 ​It can be seen that the application of Bacillus halodurans L21 fermentation liquor first and then the pathogenic bacteria fermentation liquor can enhance the defense system of the plant before it encounters the pathogenic bacteria. The Bacillus halodurans L21 fermentation liquor can activate the systemic resistance (ISR) of the plant, so that the antioxidant enzyme is prepared in advance, and when the pathogenic bacteria invade, it can respond more quickly and clear more ROS, so that the SOD activity is improved more obviously. On the contrary, if the pathogenic bacteria fermentation liquor is applied first and then the Bacillus halodurans L21 fermentation liquor is applied, the plant may have been invaded by the pathogenic bacteria, and the cells are damaged, at this time, the repair effect of the Bacillus halodurans L21 fermentation liquor may be limited, or the ROS accumulates too much, which exceeds the regulation capacity of the Bacillus halodurans L21 fermentation liquor, so that the activity is not improved as obviously as the former. Each treatment has a certain influence on the content of malondialdehyde (MDA), compared with the treatment T9 of only inoculating the pathogenic bacteria, the content of MDA of the rest of the treatments is improved to a certain extent Figure 17 Therefore, the application of the Bacillus halodurans L21 fermentation liquor first can activate the defense mechanism of the plant in advance, so that the plant has stronger antioxidant capacity when it encounters the pathogenic bacteria, thereby more effectively reducing the accumulation of ROS and reducing MDA. On the contrary, inoculation of the pathogenic bacteria first can cause a large amount of ROS to be generated, at this time, the application of the Bacillus halodurans L21 fermentation liquor can not effectively activate the defense system, so that the MDA is reduced with poor effect.

[0126] In summary, the Bacillus halodurans L21 of the application can reduce the incidence of root rot.

[0127] Although the above embodiment describes the application in detail, it is only a part of the embodiments of the application, but not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which all belong to the protection scope of the application.

Claims

1. A strain of salt-tolerant Bacillus ( Bacillus halotolerans L21, characterized in that, The preservation number is CGMCCNo.35478; the salt-tolerant Bacillus L21 is used to control root rot of Acer buergerianum.

2. A biocontrol agent, characterized in that, Includes the salt-tolerant Bacillus L21 as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The viable count of halophilic Bacillus L21 in the biocontrol agent is ≥10. 8 CFU / mL or ≥10 8 CFU / g.

4. A method for preparing a biocontrol agent, characterized in that, The process includes the following steps: inoculating the salt-tolerant Bacillus L21 of claim 1 into a culture medium for fermentation culture to obtain a biocontrol agent.

5. The preparation method according to claim 4, characterized in that, The fermentation culture temperature is 25~35℃, the fermentation culture time is 55~65h, the fermentation rotation speed is 170~230rpm, the initial pH of the culture medium used for the fermentation culture is 7.0~7.5, and the volume of the culture medium occupies 16%~21% of the culture container volume.

6. 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 0.3-1.0 g / L magnesium sulfate.

7. The application of the salt-tolerant Bacillus L21 according to claim 1, the biocontrol agent according to claim 2 or 3, or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 6 in the control of plant diseases; The plant disease is root rot; the pathogen causing the root rot is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum The plant in question is *Axypsis cuspidatum*.

8. A method for controlling plant diseases, characterized in that, The procedure includes the following steps: applying the salt-tolerant Bacillus L21 of claim 1 to the soil in which the plants are grown; The plant disease is root rot; the pathogen causing the root rot is Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum The plant in question is *Axypsis cuspidatum*.

9. The prevention and control method according to claim 8, characterized in that, The application of the halophilic Bacillus L21 includes a microbial agent, and the application method includes soil drenching and / or root-damaged drenching; the viable count of halophilic Bacillus L21 in the microbial agent is ≥10. 8 CFU / mL or ≥10 8 CFU / g.

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