Bacillus subtilis, biocontrol inoculant and application of biocontrol inoculant

Abstract

The bacillus subtilis is named as bacillus subtilis z373 and is preserved in the China General Microbiological Culture Collection Center on March 31, 2025, and the preservation number is CGMCC No.34034. Effective components of the biocontrol microbial agent comprise a bacterial liquid and / or a fermentation liquid of bacillus subtilis, and the bacterial liquid and / or the fermentation liquid of bacillus subtilis are / is preserved in the China General Microbiological Culture Collection Center on March 31, 2025. The soybean rust disease prevention and treatment effect stably reaches 80% or above, the soybean rust disease can be effectively prevented and treated, and the composition is non-toxic and safe to people and livestock and environmentally friendly, meets the requirement for medication safety and has important application significance and value.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural biological control, and particularly relates to a Bacillus subtilis, a biocontrol agent and their applications. Background Art

[0002] Soybean rust is an air-borne and obligate parasitic disease, and is one of the most devastating diseases to soybean production worldwide. This disease can cause premature leaf fall of soybeans in the field within just a few days and may even lead to complete crop failure.

[0003] Currently, the methods for controlling soybean rust mainly rely on chemical agents. However, over-frequent and excessive use of chemical agents is likely to cause problems such as environmental pollution and the generation of resistance. Biological control can effectively avoid these series of problems brought by chemical control, and has the characteristics of safety, effectiveness and durability compared with it. Biocontrol agents have good control effects on diseases, are non-toxic to humans and livestock, do not pollute the environment, and have no residues; they have strong specificity in killing pests and diseases, do not harm natural enemies and beneficial organisms, and can maintain ecological balance; the production raw materials and active ingredients are natural products and are easily degradable, can return to nature, and ensure sustainable development.

[0004] In view of the current situation that the chemical control of soybean rust with chemical agents is likely to cause environmental pollution and generate resistance, and there is a lack of biocontrol agents for highly efficient control of soybean rust, there is an urgent need to provide a safe and highly efficient biocontrol agent. Summary of the Invention

[0005] The purpose of the present invention is to address at least one deficiency in the prior art, and provide a Bacillus subtilis, a biocontrol agent and their applications, so as to solve the problems existing in the related technologies that the chemical control of soybean rust with chemical agents is likely to cause environmental pollution and generate resistance, and there is a lack of biocontrol agents for highly efficient control of soybean rust, and to provide a safe and highly efficient biocontrol agent.

[0006] To achieve the above object of the present invention, the following technical solutions are adopted: The first aspect of the present invention is to provide a Bacillus subtilis, and the Bacillus subtilis ( Bacillus subtilis ) is named Bacillus subtilis z373, and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 31, 2025, with the deposit number: CGMCC No. 34034.

[0007] Furthermore, the 16srDNA sequence of the Bacillus subtilis is as shown in SEQ ID NO: 1.

[0008] The second aspect of the present invention is to provide a biocontrol agent, and the active ingredient of the biocontrol agent comprises the bacterial liquid and / or fermentation broth of the Bacillus subtilis as described in the first aspect.

[0009] Furthermore, the viable cell concentration of Bacillus subtilis in the biocontrol agent is 1×10 7 ~1×10 10 CFU / mL.

[0010] Furthermore, the viable cell concentration of Bacillus subtilis in the biocontrol agent is 4×10 7 ~8×10 7 CFU / mL.

[0011] Furthermore, the biocontrol agent includes a liquid preparation.

[0012] Furthermore, the biocontrol agent can promote the growth of soybeans.

[0013] The third aspect of the present invention is to provide a preparation method of the biocontrol agent as described in the second aspect, including the following steps: After activating the Bacillus subtilis, inoculate it into the LB medium and shake culture until the bacterial liquid concentration is 2×10 8 CFU / mL~4×10 8 CFU / mL, and dilute it 5 to 100 times to obtain the biocontrol agent; preferably, dilute it 5 times to obtain the biocontrol agent.

[0014] Furthermore, the shake culture is carried out at 28 °C and 180 r / min.

[0015] The fourth aspect of the present invention is to provide a method for controlling soybean rust, using the biocontrol agent as described in the second aspect or the biocontrol agent prepared by the preparation method as described in the third aspect, and spraying it onto the surface of soybean leaves.

[0016] Furthermore, spray it onto the leaf surface at the two-leaf stage.

[0017] The fifth aspect of the present invention is to provide an application of the biocontrol agent as described in the second aspect or the biocontrol agent prepared by the preparation method as described in the third aspect or the control method as described in the fourth aspect, and the application includes at least one of the following applications: application in controlling soybean rust, application in preparing a preparation for controlling soybean rust. In the application of controlling soybean rust, the control rate > 80%.

[0018] The present invention adopts the above technical solutions, compared with the prior art, has the following technical effects: The biocontrol agent provided by the present invention uses Bacillus subtilis z373 as the active ingredient, wherein the viable cell concentration is 4×10 7 ~8×10 7When it is at [CFU / mL], spraying it on the leaf surface of plants can effectively prevent and control the occurrence of soybean rust, and the control effect on soybean rust is stably above 80%. Compared with the chemical agents in the prior art, the biocontrol agent of the present application is non-toxic and safe for humans and livestock, environmentally friendly, meets the requirements of drug safety, and its high efficiency in preventing and controlling soybean rust has important application values in preventing and controlling plant diseases, preparing preparations for preventing and controlling plant diseases, and preparing preparations for promoting plant growth.

[0019] A Bacillus subtilis involved in the present invention is classified and named as Bacillus subtilis Bacillus subtilis , hereinafter simply referred to as Bacillus subtilis z373, was deposited with the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 31, 2025, and the deposit number is: CGMCC No. 34034. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings described herein are used to provide a further understanding of the present invention and form a part of the present invention. The schematic embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 is the control effect diagram of the isolated strain on soybean rust in an embodiment of the present invention; Figure 2 is the enzyme activity effect diagram of the biocontrol agent in an embodiment of the present invention; Figure 3 is the diagram showing the influence of the biocontrol agent diluted by different multiples on the germination of rust spores in an embodiment of the present invention; Figure 4 is the pot control effect diagram of the biocontrol agent on soybean rust in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually determined according to national standards. The experimental materials not indicated the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is all conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standard, conventional conditions, or the conditions recommended by the manufacturer. Unless otherwise stated, all parts are by weight and all percentages are by mass percentage. Unless otherwise defined or explained, all the professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the method of the present invention.

[0022] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but it is not a limitation of the present invention.

[0023] For other manufacturers not specially marked in the embodiments of the present invention, they can be obtained conventionally through commercial purchase.

[0024] The present invention will be exemplarily described below.

[0025] Example 1 - Isolation, purification, screening and identification of Bacillus subtilis ( Bacillus subtilis ) z373 The Bacillus subtilis ( Bacillus subtilis ) involved in this example was isolated from the leaves of healthy plants in the diseased field of the soybean planting base in Zigong City, Sichuan Province in 2023, named Bacillus subtilis z373, and was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 31, 2025, with the deposit number: CGMCC No. 34034. Its 16srDNA sequence is as shown in SEQ ID NO: 1, specifically as follows: The soybean variety involved in this invention is Williams 82, a disease-susceptible soybean variety, provided by the Oomycete and Fungal Molecular Biology Laboratory of the College of Plant Protection, Nanjing Agricultural University. Vermiculite was stirred with water in advance and waited until it was completely wet, then filled into seedling trays, and the seeds were sown into the trays. The seedling trays were placed in a climate chamber at 25 °C with a photoperiod of 16 h light and 8 h darkness for cultivation. When the seeds grew to 14 days and had two true leaves, they were reserved for use. Phakopsora pachyrhizi Phakopsora pachyrhizi SS4, provided by the Oomycete and Fungal Molecular Biology Laboratory of the College of Plant Protection, Nanjing Agricultural University, was stored in a 2 mL EP tube in an -80 °C refrigerator.

[0026] 1. Isolation Using the five-point sampling method, soybean leaves were taken and put into sterilized sealed plastic bags, and immediately taken back to the laboratory. The leaves were cut into small pieces and added to 45 mL of sterile water. After culturing on a shaker at 200 r / min for 1 h, the suspension was filtered through sterile gauze to obtain a suspension, and a ten-fold serial dilution method was used to obtain suspensions with three gradients of 10 -4 、10 -5 、10 -6 . 100 μL of the suspensions with these three gradients were pipetted onto LB plates. After the plates were cultured at 28 °C for 48 h, single colonies with different colors, glossiness, sizes and types on the plates were picked, purified, counted, marked, and then transferred into an LB slant medium, stored with 80% sterilized glycerol, and stored at -80 °C for standby.

[0027] 2. Screening (1) Select the isolated strains with strong growth and shake them in an LB medium for 24 h (OD 600=1 ), to obtain a bacterial solution.

[0028] (2) Dilute the bacterial solution five times and spray it onto the surface of soybean leaves at the two-leaf stage, and culture at 25 °C for 24 h.

[0029] (3) Take out the 2 mL EP tube containing spores from the -80 °C refrigerator, put it into a 42 °C water bath for heat shock for 5 min. Add 1 mL of 1 / 1000 Tween-20 prepared in advance to wash the spores, centrifuge at 12000 rpm at room temperature for 3 min, and discard the waste liquid. Add ddH2O to make a spore suspension with a concentration of 1×10 5 spores / mL, and evenly spray it onto the leaf surface. After dark treatment for 24 h, transfer it to a 25 °C light incubator.

[0030] (4) After 14 d, observe the disease incidence of the leaves, investigate the disease index, and repeat the experiment three times. The results are as Figure 1 shown.

[0031] The incidence degree of soybean rust is graded as follows: Grade 0: No disease is observed on the leaves; Grade 1: Lesions are observed on the leaves, and the proportion of the lesion area to the leaf area ≤ 1%; Grade 3: The spore heaps on the back of the leaves are sporadically distributed, and 1% < the proportion of the lesion area to the leaf area ≤ 10%; Grade 5: The leaves are severely diseased, and 10% < the proportion of the lesion area to the leaf area ≤ 25%; Grade 7: The diseased area of the leaves is relatively large, and 25% < the proportion of the lesion area to the leaf area ≤ 70%; Grade 9: The leaves are severely diseased, and the proportion of the lesion area to the leaf area > 70%.

[0032] The disease index and control effect are calculated according to the following formula: Disease index = ∑(number of diseased leaves at each level × representative value of each disease level) × 100 / representative value of the highest level × total number of leaves surveyed.

[0033] Control effect (%) = (disease index of the blank control group - disease index of the medicated treatment group) / disease index of the blank control group × 100%.

[0034] The control effect statistics are shown in Table 1. It can be seen from Table 1 that the control effect of Bacillus subtilis z373 is stable and significant, reaching more than 83.33%.

[0035] Table 1 Control effect of soybean disease index 3. Identification (1) Select the vigorously growing isolated strains and shake them in LB culture medium for 24 h (OD 600=1 ).

[0036] (2) Use 16srDNA conventional primers to perform PCR amplification according to the reaction system in Table 2 and the amplification conditions in Table 3.

[0037] (3) After the reaction is completed, take out the reaction product from the PCR amplifier, perform agarose gel electrophoresis to verify and evaluate the length of the amplified DNA fragment, and then send it to a sequencing company for further sequencing.

[0038] (4) Compare the sequencing results on the NCBI website, and the results show that it belongs to the genus Bacillus subtilis.

[0039] Table 2 Reaction system for colony PCR verification Table 3 Amplification conditions for colony PCR verification Example 2 - Preparation of biocontrol agent This example relates to a specific preparation method of a biocontrol agent, and a biocontrol agent is prepared based on the Bacillus subtilis z373 isolated in Example 1.

[0040] 1. Medium preparation: (1) LB liquid medium (for preparation of bacterial solution): Yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, made up to 1 L with ultrapure water, adjusted to pH 7.2, and sterilized at 1×10 5 Pa for 30 min.

[0041] (2) LB solid medium (for activation and preservation of biocontrol strains): Yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L, made up to 1 L with ultrapure water, adjusted to pH 7.2, and sterilized at 1×10 5 Pa for 30 min.

[0042] 2. Preparation of biocontrol agent: Activate the Bacillus subtilis z373 strain, inoculate the z373 strain into the LB medium, and shake-culture at 28°C and 180 r / min until the OD of the bacterial solution 600=1 , and the concentration of the bacterial solution is about 2×10 8 ~4×10 8 CFU / mL; then dilute the bacterial solution 5 times, 10 times, and 100 times respectively, and the viable bacterial concentrations after dilution are 4×10 7 ~8×10 7 CFU / mL, 2×10 7 ~4×10 7 CFU / mL, 2×10 6 ~4×10 6 CFU / mL, which are the biocontrol agents.

[0043] Example 3 - Enzyme activity test In this example, the Bacillus subtilis z373 isolated in Example 1 is used as the experimental object for enzyme activity test, which specifically includes the following steps: 1. Medium preparation (1) Protease activity detection medium formula: Solution A: 8 g of skim milk powder, made up to 300 mL with deionized water, sterilized at 121°C for 10 min.

[0044] Solution B: 8 g of agar, made up to 300 mL with deionized water, sterilized at 121°C for 20 min.

[0045] When in use, mix Solution A and Solution B and pour plates.

[0046] (2)Detection medium formula for siderophore-producing enzyme activity: Solution C: Dissolve 60.5 mg of CAS (Chrome Azurol S) in 50 mL of deionized water; prepare 10 mL of ferric ion solution (1 mM FeCl3·6H2O, with 10 mM hydrochloric acid as the solvent); dissolve 72.9 mg of HDTMA in 40 mL of deionized water. Then mix the above three solutions and make up the volume to 100 mL, adjust the pH to neutral, and sterilize at 121 °C for 20 min.

[0047] Solution D: Add 30.24 g of Pipes to 900 mL of WA medium, adjust the pH to 6.8, and sterilize at 121 °C for 20 min.

[0048] When in use, mix Solution C and Solution D and pour plates.

[0049] (3)Detection medium formula for cellulase activity: 10 g of peptone, 10 g of yeast extract, 10 g of sodium carboxymethyl cellulose, 5 g of sodium chloride, 1 g of potassium dihydrogen phosphate, 18 g of agar, make up the volume to 1000 mL, pH 7.0.

[0050] 2. Inspection: Activate the z373 strain, pick single colonies with strong vitality and place them on the protease activity detection medium for 2 - 3 d, on the siderophore-producing enzyme activity detection medium for 5 d, and on the cellulase activity detection medium for 3 - 4 d (add 8 ml of 1 g / L congo red solution to the plate, let it stand for 1 h for staining, then add 15 ml of 1 M NaCl solution and let it stand for 1 h to wash off the floating color), observe the presence or absence of clear zones, and record the size of the clear zones. The results are as Figure 2 shown.

[0051] 3. Result analysis: As Figure 2 can be seen, the diameter of the clear zone formed by the z373 strain on the protease activity detection medium is 36.35 ± 0.03 mm; the diameter of the clear zone formed by the z373 strain on the siderophore-producing enzyme activity detection medium is 15.37 ± 0.14 mm; the diameter of the clear zone formed by the z373 strain on the cellulase activity detection medium is 31.87 ± 0.09 mm, indicating that the z373 strain exhibits relatively high protease, siderophore-producing enzyme, and cellulase activities.

[0052] Example 4 - Effect on the germination of rust spores In this example, the Bacillus subtilis z373 isolated in Example 1 was used as the experimental object, and the biocontrol agent was prepared by the preparation method of Example 1 for exploration.

[0053] The z373 strain was activated and shaken in LB culture medium for 24 h (OD 600= 1), the concentration of the bacterial solution was approximately 2×10 8 ~4×10 8 CFU / mL; the bacterial solution was diluted 5-fold, 10-fold, and 100-fold to make its concentrations 4×10 7 ~8×10 7 CFU / mL, 2×10 7 ~4×10 7 CFU / mL, 2×10 7 ~4×10 6 CFU / mL respectively. Then, the urediniospores of the same concentration were treated. After dilution, the volume ratio of the bacterial solution to the urediniospores of soybean rust was 1:1; after placing it in an incubator at 28 °C for 36 h, 10 μL of the diluted solution of each treatment was placed on a hemocytometer, six fields of view were randomly selected, the total number of spores and the number of germinated spores were counted, and their germination rates were calculated. Each treatment was counted three times repetitively.

[0054] The results were as Figure 3 shown. The microscopic observation results showed that the germination rate of the urediniospores treated with the bacterial solution of z373 decreased significantly, and the situation of spore rupture and the outflow of intracellular substances was significantly more than that of the water treatment control group.

[0055] Example 5 - Determination of the greenhouse control effect on soybean rust In this example, Bacillus subtilis z373 isolated in Example 1 was used, and a biocontrol agent was prepared by the preparation method of Example 1 to explore the effect of the biocontrol agent on soybean rust.

[0056] One pot of soybean plants (the soybean plants prepared in Example 1) was taken for both the control group and the treatment group, and 12 - 15 soybean plants were planted in each pot to ensure the repeatability of the experiment.

[0057] Treatment group B: The strain z373 was inoculated into LB medium and cultured with shaking at 28 °C and 180 r / min until the OD of the bacterial solution 600 = 1. After dilution five-fold, the z373 biocontrol agent was obtained and sprayed onto the leaf surface of the second leaf stage of the soybean plants in the treatment group; the control group A was not sprayed with the biocontrol agent.

[0058] One day later, the urediniospores of soybean rust stored in advance in a refrigerator at -80 °C were taken out, heat shocked in a water bath at 42 °C for 5 min, then treated with 0.1% Tween - 20, centrifuged at 12000 rpm for 3 min, the Tween solution was discarded, and sterile water was added to make 1.0×10 5The spore suspension at a concentration of Figure 4 per mL was placed in a suitable sprayer and sprayed on the leaves of the well-grown soybean plants. When the soybean leaves were in a wet state, the soybeans were then placed in an incubator at 25 °C with a photoperiod of 16 h of light and 8 h of darkness and high humidity for cultivation. During this period, moisture was maintained in a timely manner. After two weeks, the control effect of the biocontrol agent z373 on soybean rust in the greenhouse was observed, and the results were as

[0059] shown. The severity of soybean rust was classified as follows: Grade 0: No disease was observed on the leaves; Grade 1: Lesions were observed on the leaves, and the proportion of the lesion area to the leaf area was ≤1%; Grade 3: The spore masses on the back of the leaves were sporadically distributed, and 1% < the proportion of the lesion area to the leaf area ≤10%; Grade 5: The leaves were severely diseased, and 10% < the proportion of the lesion area to the leaf area ≤25%; Grade 7: The diseased area of the leaves was relatively large, and 25% < the proportion of the lesion area to the leaf area ≤70%; Grade 9: The leaves were severely diseased, and the proportion of the lesion area to the leaf area >70%.

[0060] The disease index and control effect were calculated according to the following formulas: Disease index = ∑(number of diseased leaves at each level × representative value of each disease level) × 100 / representative value of the highest level × total number of leaves surveyed.

[0061] Control effect (%) = (disease index of the blank control group - disease index of the treatment group with the drug) / disease index of the blank control group × 100%.

[0062] The control effect calculated according to the above formula is shown in Table 4.

[0063] Table 4 Greenhouse control effect of the biocontrol agent on soybean rust From Figure 4 and Table 4, it can be seen that the biocontrol agent prepared in this example can effectively control soybean rust, and the greenhouse control effect on soybean rust is 83.33%.

[0064] In summary, the biocontrol agent of the present application uses Bacillus subtilis z373 as the active ingredient, with a viable cell concentration of 4×10 7 ~8×10 7Obtained by CFU / mL preparation; the biocontrol agent of the present application is mainly sprayed on the leaves, and the method of screening bacteria directly according to the control effect can simulate the field effect, effectively prevent and control the occurrence of soybean rust, and its control effect on soybean rust is stable, reaching 83.33%. Moreover, it is non-toxic and safe for humans and animals, friendly to the environment, meets the requirements of drug safety, can be applied to field control, and has important application significance and value in preventing and controlling soybean rust, preparing preparations for preventing and controlling soybean rust, and promoting plant growth.

[0065] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention accordingly. For those skilled in the art, it should be able to realize that all the equivalent replacements and obvious changes made by using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis, characterized in that The Bacillus subtilis ( Bacillus subtilis ) was named Bacillus subtilis z373, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 31, 2025, with the deposit number: CGMCC No.34034. The 16srDNA sequence of the Bacillus subtilis is shown in SEQ ID NO: 1, and the Bacillus subtilis exhibits protease, siderophilase, and cellulase activities.

2. A biocontrol agent, characterized in that: The active ingredient of the biocontrol agent comprises the bacterial liquid and / or fermentation liquid of Bacillus subtilis according to claim 1.

3. The biocontrol agent according to claim 2, characterized in that The concentration of live bacteria of Bacillus subtilis in the biocontrol agent is 4×10 7 ~8×10 7 CFU / mL.

4. A method for preparing a biocontrol agent as claimed in any one of claims 2 to 3, characterized in that: The following steps are involved: The activated Bacillus subtilis was inoculated into LB medium and cultured with shaking until the bacterial solution concentration was 2×10 8 CFU / mL~4×10 8 CFU / mL, and the biocontrol agent can be obtained by diluting 5 to 100 times.

5. The preparation method according to claim 4, characterized in that: The shaking culture is carried out at 28° C. and 180 r / min.

6. A method for preventing and controlling soybean rust, characterized in that: The biocontrol agent according to any one of claims 2 to 3 or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 5 is sprayed onto the surface of soybean leaves.

7. Use of the biocontrol agent according to any one of claims 2 to 3, or the biocontrol agent prepared by the preparation method according to any one of claims 4 to 5, or the control method according to claim 6, characterized in that: The application includes at least one of the following applications: application in preventing and controlling soybean rust, application in preparing a preparation for preventing and controlling soybean rust, wherein in the application in preventing and controlling soybean rust, the prevention rate is greater than 80%.

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