Bacillus subtilis L32 and application thereof
Through the application of Bacillus subtilis L32, the problem of poor prevention and control of banana blight has been solved, biological antagonism and nitrogen and phosphorus nutrition have been achieved, and an environmentally friendly and stable biological control method is provided, suitable for field applications.
Patent Information
- Application Number
- CN202411532322.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-30
- Publication Date
- 2025-01-07
AI Technical Summary
The prior art has poor results in preventing and treating banana blight. Antagonist bacteria are difficult to adapt to the banana growth climate and soil environment. The long-term use of chemical pesticides poses a threat to the environment and human health, and lacks effective biological control methods.
Bacillus subtilis L32 (Bacillus subtilis) is provided, which is isolated from the rhizosphere soil of bananas, has the ability to significantly inhibit Fusarium and promotes the absorption of nitrogen and phosphorus by bananas. It is used for preparation of bacterial agents and fermentation. Products, as biological antagonists and fertilizer additives, are used to inhibit banana blight and promote growth.
Bacillus subtilis L32 significantly reduces the incidence and incidence of banana blight, improves the nitrogen and phosphorus nutrition and growth of bananas, and is pollution-free and residue-free. It is suitable for field applications and has environmentally friendly biological control effects.
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Figure CN119265076A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microbiology and plant nutrition, and in particular to a Bacillus subtilis L32 capable of preventing and treating banana wilt and promoting nitrogen and phosphorus nutrient absorption, and application thereof. Background Art
[0002] Banana wilt is caused by Fusarium oxysporum Cuban type ( Fusarium oxysporum f.sp.cubense Fusarium wilt (hereinafter referred to as Fusarium) is a soil-borne disease caused by infection, which is a devastating disease in banana production. The pathogen invades from the roots, spreads to the pseudostem and leaves through the vascular bundle, blocks the xylem vessels, and finally the plant withers and dies. Banana wilt pathogen is a soil-dwelling fungus, and its thick-walled spores can survive in the soil for 8 to 10 years. At present, the main methods for the prevention and control of banana wilt are biological breeding, chemical disinfection and biological control. In the reclamation of banana plantations in severely diseased areas, the planting of disease-resistant varieties has been popularized. However, disease-resistant varieties require 1 to 3 years to establish resistance, and the disease resistance performance is poor in the first two years of planting. At the same time, the growth period of disease-resistant varieties is longer than that of ordinary varieties, which increases production costs and management difficulties. Chemical disinfection refers to the use of chemical pesticides or disinfectants to disinfect soil and plants. However, there is no effective chemical pesticide, and the long-term use of chemical pesticides poses a serious threat to the environment and human health. Compared with chemical control methods, the use of antagonistic bacteria to control banana wilt has the advantages of being environmentally friendly, low cost, and pathogens are not likely to develop drug resistance. It is the direction of future disease control.
[0003] Although there have been a lot of studies and applications of antagonistic microorganisms, the application effect in the prevention and control of banana Fusarium wilt is not very ideal. Most antagonistic bacteria that perform well in the laboratory do not perform well in field applications. This may be because the strains are difficult to adapt to the climate, soil and rhizosphere environment where bananas grow, making it difficult to colonize effectively. Or they are greatly affected by field production management and it is difficult to exert the effect of antagonizing Fusarium wilt. Therefore, it is of great significance to find microorganisms that are not affected by the natural conditions of farmland and have stable antagonistic effects for the biological control of banana Fusarium wilt.
[0004] In view of this, the present invention is proposed. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a Bacillus subtilis L32 and its application. The Bacillus subtilis L32 has the functions of preventing and controlling banana wilt and promoting nitrogen and phosphorus nutrient absorption, has efficient and stable antagonism, and is suitable for field application.
[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a Bacillus subtilis ( Bacillus subtilis ) L32, its deposit number is CGMCC No. 31255.
[0007] In a second aspect, the present invention provides a bacterial agent containing the Bacillus subtilis L32.
[0008] Preferably, the bacterial agent is a wilt disease antagonist and / or a nitrogen and phosphorus nutrient absorption promoter.
[0009] Preferably, the effective viable bacteria count of Bacillus subtilis L32 contained in the bacterial agent is not less than 1×10 9 CFU / mL.
[0010] In a third aspect, the present invention provides a fermentation product obtained by fermenting the Bacillus subtilis L32 or the bacterial agent.
[0011] In a fourth aspect, the present invention provides a fertilizer or a fertilizer additive, the main active ingredient of which is the Bacillus subtilis L32 and / or the fermentation product.
[0012] In a fifth aspect, the present invention provides the use of the Bacillus subtilis L32, the bacterial agent, the fermentation product, or the fertilizer or fertilizer additive in inhibiting banana wilt.
[0013] In a sixth aspect, the present invention provides the use of the Bacillus subtilis L32, the bacterial agent, the fermentation product, or the fertilizer or fertilizer additive in improving the nitrogen and phosphorus nutrition of bananas and promoting the growth of bananas.
[0014] Beneficial effects: The present invention provides a Bacillus subtilis ( Bacillus subtilis ) L32, whose deposit number is CGMCC No. 31255. The present invention isolates the above-mentioned Bacillus subtilis ( Bacillus subtilis )L32 strain, which has the ability to effectively inhibit the growth and reproduction of Fusarium in the soil. Experiments have found that this Bacillus subtilis can promote the absorption of nitrogen and phosphorus by bananas, increase the activity of stress-resistant enzymes such as catalase and phenylalanine ammonia lyase, and promote the growth of bananas. During the application process, there is no pollution, no residue, and biological environmental protection. It is an antagonistic strain with good application prospects in the field of plant biocontrol. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.
[0016] Figure 1 For strains ( Bacillus subtilis ) Plate confrontation results of L32 against Fusarium.
[0017] Figure 2 To apply the strain ( Bacillus subtilis)Effect of L32 fermented bacteria on the occurrence of Fusarium wilt in potted banana. DETAILED DESCRIPTION
[0018] The invention provides a Bacillus subtilis which can effectively prevent and control banana wilt in the field and promote the absorption of nitrogen and phosphorus nutrients of banana and application thereof.
[0019] Specifically, the Bacillus subtilis provided by the present invention is Bacillus subtilis ( Bacillus subtilis ) L32, which was isolated from banana rhizosphere soil and deposited on July 10, 2024 in the General Microbiology Center of China Microbiological Culture Collection (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Bacillus subtilis Bacillus subtilis , the deposit number is CGMCC No. 31255.
[0020] The present invention has found that Bacillus subtilis ( Bacillus subtilis )L32 has the functions of significantly inhibiting the occurrence of banana wilt, improving banana nitrogen and phosphorus nutrition, and promoting banana growth. Specifically, its microbial agent can significantly reduce the content of Fusarium in bananas, increase the activity of banana catalase, increase the concentration of nitrogen and phosphorus nutrients in banana leaves, and promote banana growth.
[0021] The invention provides a microbial agent containing the Bacillus subtilis L32.
[0022] Preferably, the bacterial agent contains Bacillus subtilis ( Bacillus subtilis )The effective viable count of L32 is not less than 1×10 9 CFU / mL.
[0023] In a more specific embodiment of the present invention, the Bacillus subtilis ( Bacillus subtle ) The preparation method of the bacterial agent of L32 or its fermentation product is as follows: the L32 strain is inoculated into the LB liquid culture medium, and the activation is completed by shaking and culturing at 30°C overnight. The activated functional strain is inoculated into the liquid LB culture medium at a 1% inoculation amount, and the fermentation bacterial agent of the strain is obtained after culturing at a constant temperature of 30°C and a rotation speed of 200 rpm for 24 hours.
[0024] In a more specific embodiment of the present invention, the culture medium used for fermentation is LB culture medium, and its preparation method is as follows: accurately weigh the required amount of tryptone, yeast extract and sodium chloride, and place them in a beaker. Add 2 / 3 of the required amount of distilled water to the beaker, stir with a glass rod to dissolve it completely. Use 1mol / L NaOH solution to adjust the pH value of the solution to ensure that the microorganisms can grow in the best environment. Pour the solution into a measuring cylinder and add water to the required volume. If it is necessary to make a solid culture medium, add an appropriate amount of agar powder and heat it to melt to make up for the loss of water. Finally, the liquid culture medium is divided into test tubes or culture dishes, plugged, bandaged, and sterilized by a high-pressure steam sterilizer.
[0025] The bacterial agent provided by the present invention can be directly used as a wilt disease antagonist and a nitrogen and phosphorus nutrient absorption promoter, and can also be used as the main active ingredient of a wilt disease antagonist and a nitrogen and phosphorus nutrient absorption promoter, and can be matched with auxiliary materials and / or carrier accessories to form a mature commercial product.
[0026] Based on the Bacillus subtilis ( Bacillus subtilis ) L32 functional properties, the present invention also provides Bacillus subtilis ( Bacillus subtilis )L32 fermentation agent is used to inhibit banana wilt and promote the absorption of nitrogen and phosphorus nutrients in banana.
[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0028] The endpoints and any values of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0029] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
[0030] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.
[0031] Unless otherwise specified, the culture medium used in the following examples has the following formula: LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g NaCl, 1000 ml distilled water, adjust pH to 7.0-7.2.
[0032] PDA solid culture medium: 200 g potato, 20 g glucose, 15 g agar, 1000 ml distilled water, adjust pH to 7.0-7.2.
[0033] All culture media were sterilized at high temperature and high pressure under the conditions of 0.1 MPa, 120°C, 30 min.
[0034] Example 1
[0035] This example provides strains ( Bacillus subtilis ) Plate confrontation test of L32 against Fusarium.
[0036] 1. Test materials: functional strains ( Bacillus subtilis ) L32, Fusarium oxysporum Cuban species 4 (Foc4), PDA solid medium.
[0037] 2. Test methods: Using the plate confrontation growth method, the strain to be tested that was activated and cultured for 24 hours was inoculated with an inoculation loop at 2 cm from the edge of a 9 cm diameter PDA plate, and the bacterial cake of banana wilt pathogen that was cultured for 4 days and punched with a diameter of 5 mm was inoculated at the center of the circle. The plate was cultured in a 28°C constant temperature incubator for 7 days, and the colony diameter was measured with a ruler using the "cross" measurement method to calculate the inhibition rate. The uninoculated functional bacteria to be tested were used as the control, and each treatment was repeated 3 times.
[0038] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter) × 100%
[0039] 3. Test results: The diameter of the bacterial circle of banana wilt pathogen Foc4 was measured, and according to the calculation formula, it was known that the inhibition rate of Bacillus subtilis L32 against banana wilt under plate conditions was 46.32%, which proved the effectiveness of this functional bacteria in inhibiting banana wilt pathogen.
[0040] Example 2
[0041] This example provides strains ( Bacillus subtilis ) Pot experiment on inhibition of banana wilt by L32 fermented bacteria.
[0042] 1. Test materials: functional strains ( Bacillus subtilis )L32 fermentation agent, Fusarium oxysporum Cuban species 4 (Foc4), susceptible variety Williams banana seedlings, disease-resistant variety Baodao banana seedlings, banana garden soil in the seriously diseased area of Longan County, Nanning City, Guangxi Zhuang Autonomous Region (22°59′40″N, 107°52′16″E).
[0043] 2. Test methods: The blank LB medium was used as the blank control treatment (CK), and the functional bacteria L32 agent was used for the experimental treatment, and each treatment was repeated 5 times. The purchased 5-6 leaf banana seedlings were transplanted into potted plants, and each pot was filled with 3.5 kg of sterilized soil. Different bacterial liquids were used for treatment on the same day, and wilt spore suspension was applied to each pot after 3 days. After that, the bacterial liquid was cultivated once a week for irrigation, and 200 mL of the agent was applied to each pot and irrigated to the rhizosphere of the banana seedlings each time, and the whole experiment was applied 8 times.
[0044] 3. Test results: The blank control CK had the most serious disease, with leaves turning yellow and drooping ( Figure 2 ). Among them, the susceptible variety Williams had the highest incidence rate, reaching 60.0%. After applying the functional bacterial solution, the incidence and severity of banana wilt disease were reduced for both the susceptible variety Williams and the disease-resistant variety Baodaojiao (Table 1). The application of functional bacterial solution has a significant promoting effect on the overall growth of different varieties of banana plants. There were significant differences in plant height, number of green leaves, and SPAD values of different varieties of bananas after treatment with each strain (Table 2). The application of functional bacterial agents can promote the absorption of nitrogen and phosphorus by different varieties of bananas. The difference between the bacterial agent treatment and the blank control was significant (Table 3). The application of functional bacterial agents significantly improved the stress resistance of bananas. The effects of different treatments on the plant-related resistance enzyme activities of catalase, phenylalanine ammonia lyase, and superoxide dismutase (SOD) of bananas were significantly different (Table 4).
[0045]
[0046] Example 3
[0047] This example provides strains ( Bacillus subtilis )Field trial of L32 fermented bacteria agent in inhibiting banana wilt disease.
[0048] 1. Test materials: functional strains ( Bacillus subtilis ) L32 fermentation bacteria agent, disease-resistant variety Baodao Banana No. 1.
[0049] 2. Test methods: A typical diseased plot with a high hazard index of banana wilt disease was selected, and the first generation of Baodao banana plants were selected as the test objects. Different strains were treated in the experiment, and clean water was used as the blank control. There were three treatments in total. 12 bananas with consistent growth were selected in each treatment. 400 ml of bacterial solution was applied to each banana along with water, and the application frequency was 14 days / time.
[0050] 3. Test results: The test results showed that the application of functional bacteria significantly reduced the incidence and severity of banana wilt (Table 5). The application of functional bacteria enhanced the robustness of bananas and significantly increased the stem girth and root vitality of bananas (Table 6). The application of functional bacteria significantly promoted the absorption of nitrogen and phosphorus nutrients by bananas, and the nitrogen and phosphorus contents of banana leaves were significantly different (Table 7).
[0051]
[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Bacillus subtilis ( Bacillus subtilis ) L32, its deposit number is CGMCC No. 31255.
2. A bacterial agent containing the Bacillus subtilis L32 according to claim 1.
3. The bacterial agent according to claim 2, characterized in that: It is a wilt disease antagonist and / or a nitrogen and phosphorus nutrient absorption enhancer.
4. The bacterial agent according to claim 2 or 3, characterized in that: The effective viable bacteria count of Bacillus subtilis L32 is not less than 1×10 9 CFU / mL.
5. A fermentation product, characterized in that The invention is prepared by fermenting the Bacillus subtilis L32 described in claim 1 or the bacterial agent described in any one of claims 2 to 4.
6. A fertilizer or a fertilizer additive, characterized in that The main active ingredient is the Bacillus subtilis L32 described in claim 1 and / or the fermentation product described in claim 5.
7. Use of the Bacillus subtilis L32 according to claim 1, the bacterial agent according to any one of claims 2 to 4, the fermentation product according to claim 5, or the fertilizer or fertilizer additive according to claim 6 in inhibiting banana wilt.
8. Use of the Bacillus subtilis L32 according to claim 1, the bacterial agent according to any one of claims 2 to 4, the fermentation product according to claim 5, or the fertilizer or fertilizer additive according to claim 6 in improving nitrogen and phosphorus nutrition of banana and promoting banana growth.