Bacteriostatic compound derived from bacillus velezensis and application thereof

Abstract

The invention discloses an antibacterial compound derived from bacillus velezensis and application of the antibacterial compound, and belongs to the technical field of agricultural microbial biocontrol. The compound with the activity of inhibiting fusarium and pythium pathogenic fungi is identified from bacillus velezensis metabolites, and the compound has a good application prospect in the aspect of developing and preventing agents. The biocontrol nutrient solution formed by the antibacterial compound provided by the invention can inhibit pathogenic fungi of fusarium and pythium at the same time, is practically applied to a field for planting potatoes, and improves the yield and quality of the potatoes by reducing the incidence rate of crops.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial biocontrol technology, specifically relating to an antibacterial compound derived from Bacillus belye and its application. Background Technology

[0002] Potatoes, as a high-yield tuber crop, possess multiple values, including food security, nutritional benefits, and industrial raw materials. However, they are severely threatened by soil-borne root rot diseases, necessitating the development of highly effective biocontrol agents to achieve green control and sustainable production. Potato root rot is mainly caused by pathogens of the genus *Fusarium* (*Fusarium*). Fusarium spp.) and Pythium ( Pythium Caused by pathogenic fungi (spp.), these soil-borne pathogens can survive in the soil for extended periods due to continuous cropping, infecting roots and causing yield reductions of 20%–70%. Currently, control still relies on chemical fungicides (such as tebuconazole and azoxystrobin), but long-term use has led to fungal resistance in pathogens (e.g., ...). Fusarium oxysporum The detection rate of benzimidazole-resistant strains reached 38%, and there was a risk of pesticide residues.

[0003] Currently, biological control is an important measure for controlling soil-borne diseases. Biocontrol agents prepared using antagonistic bacteria are not only environmentally friendly and inhibit the growth of pathogens, but also have the ability to promote crop growth, making them the best alternative to chemical agents. Summary of the Invention

[0004] This application provides an antibacterial compound derived from Bacillus belye and its application, in order to overcome the defects and shortcomings of existing chemical pesticides for the control of root rot in tuber crops such as potatoes.

[0005] To achieve the above objectives, the present invention is implemented through the following solution: An antibacterial compound derived from Bacillus belye has the molecular formula C7H9NO and a molecular weight of 123. Its structural formula is shown in formula (1) below: Equation (1).

[0006] The antibacterial compound has an inhibitory effect on both Fusarium and Pythium genera that cause potato root rot.

[0007] The antibacterial compound can be used in the preparation of agents that inhibit pathogenic fungi of the Fusarium and Pythium genera.

[0008] The application of the antibacterial compound in the preparation of drugs for the prevention and treatment of potato root rot.

[0009] A biocontrol nutrient solution includes the aforementioned antibacterial compound; it also includes nutrient elements such as urea, superphosphate, potassium sulfate, copper sulfate, manganese sulfate, boron, and zinc sulfate.

[0010] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention identifies a compound from Bacillus belye metabolites that exhibits activity against pathogenic fungi of the genera *Fusarium* and *Pythium*, showing promising application potential in the development of biocontrol agents. The invention also provides a biocontrol nutrient solution formed from this antibacterial compound, which simultaneously inhibits pathogenic fungi of the genera *Fusarium* and *Pythium*, and has been practically applied in potato fields, improving potato yield and quality by reducing crop disease incidence. Attached Figure Description

[0011] Figure 1 Morphological and scanning electron microscope images of Bacillus belyss strain.

[0012] Figure 2 This is the change in potato yield after 140 days of using microbial inoculants in Example 1.

[0013] Figure 3 Example 1 shows the change in the rot rate of potato tubers after 140 days of using microbial inoculants.

[0014] Figure 4 Example 2 illustrates the inhibitory effect of the Bacillus belyss metabolite 2-propionylpyrrole on Pythium and Fusarium species.

[0015] Figure 5 Example 3 illustrates the effect of nutrient solution on potato disease incidence.

[0016] Figure 6 Example 3 illustrates the effect of nutrient solution on increasing potato yield.

[0017] Figure 7 The plant height changes in Example 3 after 14, 28, and 42 days of using nutrient solution.

[0018] Figure 8 The stem diameter changes in Example 3 after 14, 28, and 42 days of using nutrient solution.

[0019] Figure 9 The leaf length changes in Example 3 after 14, 28, and 42 days of using nutrient solution.

[0020] Figure 10 The leaf width changes in Example 3 after 14, 28, and 42 days of using nutrient solution.

[0021] Figure 11 The SPAD values ​​of Example 3 were observed after 14, 28, and 42 days of using the nutrient solution.

[0022] Figure 12 This is Example 3, showing the changes in soil pH after 28 and 140 days of using the nutrient solution.

[0023] Figure 13 This is Example 3, showing the changes in soil organic matter content after 28 and 140 days of using nutrient solution.

[0024] Figure 14 This is Example 3, showing the changes in available nitrogen content in the soil after 28 and 140 days of using nutrient solution.

[0025] Figure 15 This is Example 3, showing the changes in available phosphorus content in the soil after 28 and 140 days of using nutrient solution.

[0026] Figure 16 This is Example 3, showing the changes in urease activity in the soil after 28 and 140 days of using nutrient solution.

[0027] Figure 17 This is Example 3, showing the changes in sucrase activity in the soil after 28 and 140 days of using nutrient solution.

[0028] Figure 18 This is Example 3, showing the changes in acid phosphatase activity in the soil after 28 and 140 days of using nutrient solution.

[0029] Figure 19 This is Example 3, showing the changes in catalase activity in the soil after 28 and 140 days of using nutrient solution.

[0030] Figure 20 This is Example 3, showing the changes in peroxidase activity in the soil after 28 and 140 days of using nutrient solution.

[0031] Figure 21 This is Example 3, showing the change in vitamin C content in potato tubers after 140 days of using nutrient solution.

[0032] Figure 22 This is Example 3, showing the change in dry matter content in potato tubers after 140 days of using nutrient solution.

[0033] Figure 23 Example 3 shows the change in starch content in potato tubers after 140 days of using nutrient solution.

[0034] Figure 24 Example 3 shows the change in nitrite content in potato tubers after 140 days of using nutrient solution.

[0035] Figure 25 Example 3 shows the change in total protein content in potato tubers after 140 days of using nutrient solution.

[0036] Figure 26 Example 3 shows the change in soluble sugar content in potato tubers after 140 days of using nutrient solution.

[0037] Appendix Figure 2-26 In this study, CK was the control group; BLS was the microbial agent treatment group; and BF was the nutrient solution treatment group. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field; the reagents and materials used in the following embodiments are all commercially available.

[0039] Example 1: In this example, potatoes were grown using Bacillus vesicles in a demonstration experiment.

[0040] 1. Preparation method of microbial inoculants (1) Strain activation: Use an inoculation loop to pick up Bacillus belye from the preservation tube (strain morphology and scanning electron micrographs are shown in the figure). Figure 1 (As shown) Inoculated onto Luria-Bertani (LB) solid medium and cultured at 37℃ for 24h. After activation, single colonies of each strain were picked and placed into Erlenmeyer flasks containing 200mL LB liquid medium. The culture was then shaken at 150rpm~200rpm and 28℃~37℃ for 20h~30h to obtain seed culture.

[0041] (2) Fermentation: The obtained seed liquid is inoculated into the fermentation medium at an inoculation rate of 1%, and fermented at 37 ℃ for 24 h ~ 96 h to obtain microbial inoculum.

[0042] The LB solid culture medium consisted of 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, 20 g / L agar powder, and 1 L distilled water, and was sterilized at 121°C for 30 min before use.

[0043] The LB liquid culture medium consisted of 10 g / L tryptone, 10 g / L sodium chloride, 5 g / L yeast extract, and 1 L distilled water, sterilized at 121°C for 30 min before use.

[0044] The fermentation medium is brown sugar water, with a brown sugar addition ratio of 2% to 8%, and is used after sterilization at 121 ℃ for 30 min.

[0045] The viable count of Bacillus vesiculosus in the microbial agent is 1 billion to 5 billion / mL.

[0046] 3. In this example, the potato variety used was "Atlantic," and the application rate was 1 L / acre, applied once every 7 days for a total of 4 applications. Yield and rot rate were measured at harvest, and the results are shown in the attached figure. Figure 2As shown in Figure 3, the microbial inoculant significantly increased potato yield by 61.40%. Meanwhile, the rot rate of potatoes treated with the microbial inoculant was only 1.3%, compared to 26.00% in the control group.

[0047] Example 2: This embodiment screens and identifies a compound with inhibitory activity against pathogenic fungi from the metabolites of Bacillus belysae.

[0048] 1. Bacillus belye was cultured in 200 mL LB liquid medium in Erlenmeyer flasks, incubated at 150 rpm to 200 rpm and 28°C to 37°C for 20 to 30 hours with constant shaking, followed by centrifugation (5000 rpm to 10000 rpm for 5 to 10 minutes). The supernatant was then used for non-target metabolomics analysis.

[0049] 2. Comparison with the HMDB database yielded 433 secondary metabolites, including 2 organic acids and their derivatives, 38 phenylpropanoids and polyketides, 18 organosulfur compounds, 101 organic heterocyclic compounds, 2 organohalides, 27 organic oxygen-containing compounds, 22 organic nitrogen-containing compounds, 48 ​​organic acids and their derivatives, 108 lipids and lipid molecules, 3 lignans, neolignans and related compounds, 2 hydrocarbons, 48 ​​benzene ring compounds, and 14 alkaloids and their derivatives. The top 10 most abundant metabolites included (±)-erythro-isoleucine, phenylalanine, palmitic acid, p-methoxyaniline, 5-aminovaleric acid, choline, 2-propionylpyrrole, and D-proline.

[0050] 3. The top 10 most abundant metabolites were added to solid potato dextrose medium, and pathogenic fungi were inoculated in the center of the medium. The results were observed after 3 to 10 days.

[0051] The amount of the metabolite added is 0.01 ~ 0.1 g / L.

[0052] The solid potato glucose culture medium consists of 6 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, and 1 L distilled water, and is used after sterilization at 121 °C for 15-20 min.

[0053] The pathogenic fungi are from the genera *Fusarium* and *Pythium*.

[0054] 4. Experimental Results and Analysis Among the top 10 most abundant metabolites, 2-propionylpyrrole inhibited the growth of *Fusarium* and *Pythium* genera. Results are attached. Figure 4 .

[0055] Example 3: In this example, potatoes were grown using a biocontrol nutrient solution containing 2-propionylpyrrole in a demonstration experiment.

[0056] 1. Nutrient solution: 4-8 parts urea, 1-5 parts superphosphate, 2-6 parts potassium sulfate, 2.5-5.0 parts copper sulfate, 10-20 parts manganese sulfate, 10-20 parts boron, and 10-20 parts zinc sulfate. The preparation method is to add the above materials to 1 L of water according to the specified proportions.

[0057] 2. Add 2-propionylpyrrole to the above nutrient solution to prepare a bioprotective nutrient solution.

[0058] The amount of 2-propionylpyrrole added is 0.01~0.1g / L.

[0059] The potato variety used was "Atlantic," and the biocontrol nutrient solution was applied at a rate of 1-5 L / mu. Nutrient solution was applied by irrigation on days 14, 28, and 42 after sowing. Changes in plant height, stem diameter, leaf length, leaf width, and SPAD value were randomly investigated. Results are attached. Figure 7 ~ 11. On day 28 of nutrient solution application and at harvest (day 140), large soil samples were collected around the potato roots to determine nutrient composition and enzyme activity. The results are shown in the appendix. Figure 12 ~ 20. At harvest, yield and disease rate were measured, and potato tubers were collected for quality index determination. Results are attached. Figure 5 , 6 And 21 ~ 26.

[0060] 3. Results of Case Study 3: (1) Nutrient solution significantly increased potato yield by 16.21% and reduced potato disease incidence to 0.20%. During the potato growth period, compared with the control group, the average plant height of the treated potato plants increased by 6.13% to 15.85%, the average stem diameter increased by 20.29% to 40.57%, the average leaf length increased by 5.04% to 77.39%, the average leaf width increased by 11.39% to 80.70%, and the average SPAD value increased by 5.15% to 21.75%.

[0061] (2) Nutrient solution can significantly increase the content of nutrients and enzyme activity in soil. Compared with the control group, the organic matter content in the soil of the treatment group increased by an average of 62.44%, the alkaline nitrogen content increased by an average of 14.87% to 22.48%, the available phosphorus content increased by an average of 23.44%, and the soil pH increased by an average of 6.19% to 10.25%.

[0062] (3) Nutrient solution can significantly improve the quality of potatoes. Compared with the control group, the average vitamin C content in potato tubers of the treatment group increased by 1.31%, the average dry matter content increased by 5.04% to 77.39%, the average starch content increased by 63.81%, the average nitrite content decreased by 3.21%, the average total protein content increased by 78.14%, and the average soluble sugar content increased by 58.85%.

[0063] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A bacteriostatic compound derived from Bacillus velezensis, characterized in that, The bacteriostatic compound has a molecular formula of C7H9NO and a molecular weight of 123, and its structural formula is shown in the following formula (1). Formula (1).

2. Use of the bacteriostatic compound derived from Bacillus velezensis in claim 1 in the preparation of a preparation for inhibiting Fusarium and Pythium pathogenic fungi.

3. Use of the bacteriostatic compound derived from Bacillus velezensis in claim 1 in the preparation of a medicine for preventing and treating potato root rot.

4. A biocontrol nutrient solution, characterized by, The biocontrol nutrient solution comprises the bacteriostatic compound derived from Bacillus velezensis in claim 1.

5. The biocontrol nutrient solution according to claim 4, wherein The biocontrol nutrient solution further comprises urea, superphosphate, potassium sulfate, copper sulfate, manganese sulfate, boron, and zinc sulfate.

6. The biocontrol nutrient solution of claim 4, wherein, Urea 4-8 parts, superphosphate 1-5 parts, potassium sulfate 2-6 parts, copper sulfate 2.5-5.0 parts, manganese sulfate 10-20 parts, boron 10-20 parts, and zinc sulfate 10-20 parts; the bacteriostatic compound derived from Bacillus velezensis is added in an amount of 0.01-0.1 g / L.

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