Pure bacillus simplex TWS22 and application thereof
By screening and applying simple pure Bacillus TWS22 grown in the range of 10-35°C, the problem that existing antagonistic microorganisms are similar to that of plant pathogens at growth temperatures, and effective inhibition and disease resistance of various plant pathogens under lower temperature conditions is achieved.
Patent Information
- Application Number
- CN202510092585.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-30
AI Technical Summary
Existing antagonist microorganisms such as Bacillus subtilis have similar growth temperatures to plant pathogens, and the application time is often in the early stages of pathogenic outbreaks. The population advantage is not obvious and it is difficult to effectively resist the disease.
A simple pure Bacillus TWS22 was screened and applied. This strain grew normally within 10-35℃, which has an inhibitory effect on a variety of plant pathogens such as rice trench blight and wheat gibberelliae. It can occupy the population advantage at 10-20℃, enhancing the disease resistance effect.
By applying the TWS22 strain under lower temperature conditions, it can gain an advantage in the soil microecological environment, significantly enhance the resistance to rice trench blight and wheat gibberellia, and provide better disease prevention and control effects.
Smart Images
Figure CN120060004A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of agricultural microorganisms, and specifically to a Peribacillus simplex TWS22 and its application. Background Art
[0002] The growth temperature range of plant pathogens is usually between 20 - 35°C, and the specific temperature varies depending on the type of pathogen. The optimal growth temperature for most plant pathogens is 25 - 30°C, which is why plant diseases mostly occur in summer and autumn. Conventional antagonistic microorganisms such as Bacillus subtilis have a growth temperature similar to that of plant pathogens, and their application time or reproduction and growth time is often at the initial stage of the outbreak of plant pathogens, and the population advantage is not obvious. A Peribacillus simplex TWS22 screened in the present invention can grow normally at 10 - 35°C, has an inhibitory effect on various pathogens such as rice sheath blight and wheat scab, and can be applied or reproduced and grown before the outbreak of pathogens at a temperature of 10 - 20°C, occupying the population advantage and having a better disease resistance effect. Summary of the Invention
[0003] The present invention discloses a Peribacillus simplex TWS22 and its application to solve the deficiencies of the prior art.
[0004] To achieve the above invention objectives, the technical solution adopted by the present invention is:
[0005] A Peribacillus simplex TWS22, the Peribacillus simplex TWS22 has been deposited in the General Microbiology Center of the China Microbial Culture Collection Center on October 25, 2024, with the deposit number CGMCC No. 32346.
[0006] The cells of Peribacillus simplex TWS22 prepared by the Peribacillus simplex TWS22.
[0007] A method for preparing the cells of Peribacillus simplex TWS22, inoculating the Peribacillus simplex TWS22 into an LB liquid medium, and the liquid fermentation conditions are: the fermentation temperature is 28 - 30°C, the rotation speed is 170 - 190 r / min, and the fermentation time is 24 - 48 h. After fermentation, centrifuge to obtain the cells of Peribacillus simplex TWS22.
[0008] Furthermore, the cells of Peribacillus simplex TWS22 are resuspended with sterile water to obtain a cell suspension, that is, the microbial inoculant, and the effective viable count ≥ 0.2×10 8 cfu / ml.
[0009] Furthermore, the microbial inoculant is a solid inoculant, biological organic fertilizer or compound microbial fertilizer prepared by adsorption, mixing and solid fermentation, namely the microbial fertilizer.
[0010] Furthermore, the application of the microbial inoculant in antagonizing the pathogen of rice sheath blight under the condition of 10 - 35°C.
[0011] Furthermore, the application of the simple pure Bacillus sp. TWS22 (Peribacillus simplex) and / or the cells of the simple pure Bacillus sp. TWS22 and / or the microbial inoculant and / or the microbial fertilizer in antagonizing the pathogen of wheat head blight under the condition of 10 - 35°C.
[0012] Furthermore, the best use effect is achieved when the air temperature is 10 - 20°C.
[0013] Furthermore, the application method of the simple pure Bacillus sp. TWS22 (Peribacillus simplex) includes irrigation of roots and / or direct spraying and / or flushing on the plants / leaves and / or soil with the simple pure Bacillus sp. TWS22 (Peribacillus simplex) and / or the cells of the simple pure Bacillus sp. TWS22 and / or the microbial inoculant and / or the microbial fertilizer.
[0014] Beneficial effects of the present invention: The present invention screens out a simple pure Bacillus sp. TWS22 with the function of antagonizing the pathogens of rice sheath blight and wheat head blight within a relatively wide temperature range. Utilizing its characteristic of being able to grow and reproduce normally at a relatively low temperature, when applied at an air temperature of 10 - 20°C, its competitiveness in the soil micro - ecological environment is relatively strong, which is conducive to its colonization and forming a growth advantage over the pathogens, and can more effectively exert its disease - resistant ability. Description of the Drawings
[0015] Figure 1 It is the colony morphology of strain TWS22 on LB medium;
[0016] Figure 2 It is strain TWS22 antagonizing the pathogen of rice sheath blight;
[0017] Figure 3 It is strain TWS22 antagonizing the pathogen of wheat head blight;
[0018] Figure 4 It is the 16S rRNA phylogenetic tree of strain TWS22;
[0019] Figure 5 It is the biocontrol test effect of strain TWS22 in rice planting.
[0020] Biological Deposit Information
[0021] TWS22, classified and named as Peribacillus simplex, was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 25, 2024, and the deposit number is CGMCC NO. 32346. Detailed implementation manners
[0022] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.
[0023] It should be noted that the details not elaborated in the present invention are well-known to those skilled in the art. For the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. For the experimental steps not detailed, refer to pathophysiological experiments, online databases, etc.
[0024] Unless otherwise specified, the raw materials used in the following examples are as follows:
[0025] LB liquid medium (1L): 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, made up to 1L with deionized water, sterilized at 121 °C for 30 min.
[0026] LB solid medium (1L): 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, 15 g of agar, made up to 1L with deionized water, sterilized at 121 °C for 30 min.
[0027] PDA solid medium (1L): 200 g of potato, 20 g of glucose, 15 g of agar, dissolved in water and finally made up to 1L, pH 7.0 - 7.2, sterilized at 121 °C for 30 min.
[0028] Example 1
[0029] A strain of Peribacillus simplex TWS22, the Peribacillus simplex TWS22 has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 25, 2024, with the deposit number of CGMCC No. 32346.
[0030] Isolation, screening and identification of Peribacillus simplex TWS22:
[0031] (1) Isolation of low-temperature resistant strains
[0032] Soil samples were collected from alpine mountains in Changdu City, Tibet Autonomous Region. 10 g of soil samples were added to a triangular flask containing 100 ml of sterile normal saline. After standing for 20 min, it was shaken on a shaker at 30 °C and 180 rpm for 30 min. 1 ml of the sample was added to 9 ml of sterile normal saline, and then diluted successively by 10 2 , 10 3 , 10 4 times. 100 μl of the above soil suspensions were respectively taken and evenly spread on the plates of LB medium (5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, 15 g / L agar). They were cultured in an incubator at 10 °C for 48 h. After picking single bacterial colonies with a sterile toothpick and purifying them by streaking, they were stored for later use.
[0033] (2) Screening of low-temperature-tolerant antagonistic strains
[0034] Mycelial blocks of the pathogens of rice sheath blight, wheat scab, pepper anthracnose, and tobacco black shank were taken with a sterilized punch with a diameter of 2 mm and placed in the center of a PDA solid medium plate. It was placed in an incubator at 28 °C for cultivation. After 1 d, TWS22 was inoculated at 4 points 2 cm away from the center of the pathogen, and then placed in an incubator at 28 °C for 5 - 7 d to observe the antibacterial situation of the test strain, and the size of the inhibition zone was recorded. As Figure 2 , Figure 3 shown, TWS22 has a good inhibitory effect on the pathogens of rice sheath blight and wheat scab.
[0035] (3) Strain identification
[0036] After the strain TWS22 was cultured on an LB solid medium at 30 °C for 48 h, its colonies were nearly circular, milky white and opaque, with neat edges and slightly raised in the middle. The results of phylogenetic tree alignment analysis constructed with the 16S rRNA gene sequence showed that the strain TWS22 had the highest homology with Peribacillus simplex( Figure 4 ). Combining the results of phylogenetic tree alignment analysis with colony morphological characteristics and the 16S rRNA gene sequence, the strain TWS22 was identified as Peribacillus simplex.
[0037] The cells of Peribacillus simplex TWS22 prepared by the Peribacillus simplex TWS22, the preparation method of the cells of Peribacillus simplex TWS22. The Peribacillus simplex TWS22 is inoculated into an LB liquid medium, and the liquid fermentation conditions are: the fermentation temperature is 28 °C, the rotation speed is 170 r / min, and the fermentation time is 24 h. After fermentation, the cells of Peribacillus simplex TWS22 are obtained by centrifugation.
[0038] The microbial inoculant is prepared by resuspending the cells of Peribacillus simplex TWS22 with sterile water, that is, the effective viable count of the microbial inoculant is ≥ 0.2×10 8 cfu / ml.
[0039] The solid inoculant, bio-organic fertilizer or compound microbial fertilizer prepared by the microbial inoculant through adsorption, mixing and solid fermentation, that is, the microbial fertilizer.
[0040] The application of the microbial inoculant in antagonizing the pathogen of rice sheath blight under the condition of 10 - 35 °C. The best use effect is achieved when the temperature is 10 °C. The microbial inoculant is applied by irrigation of roots and / or spraying directly on the plants / leaves and / or soil and / or flushing.
[0041] The application of the Peribacillus simplex TWS22 and / or the cells of Peribacillus simplex TWS22 and / or the microbial inoculant and / or the microbial fertilizer in antagonizing the pathogen of wheat head blight under the condition of 10 - 35 °C. The best use effect is achieved when the temperature is 10 °C.
[0042] Example 2
[0043] This example is basically the same as Example 1, and the difference lies in the preparation method of obtaining the cells of Peribacillus simplex TWS22 by the Peribacillus simplex TWS22.
[0044] The cells of Peribacillus simplex TWS22 prepared by the Peribacillus simplex TWS22, the preparation method of the cells of Peribacillus simplex TWS22. The Peribacillus simplex TWS22 is inoculated into an LB liquid medium, and the liquid fermentation conditions are: the fermentation temperature is 29 °C, the rotation speed is 180 r / min, and the fermentation time is 36 h. After fermentation, the cells of Peribacillus simplex TWS22 are obtained by centrifugation.
[0045] The microbial inoculant is prepared by resuspending the cells of Peribacillus simplex TWS22 in sterile water, and the effective viable count of the microbial inoculant is ≥ 0.2×10 8 cfu / ml.
[0046] The solid microbial fertilizer, bio-organic fertilizer or compound microbial fertilizer prepared by adsorbing, mixing and solid fermentation of the microbial inoculant is the microbial fertilizer.
[0047] Application of the microbial inoculant in antagonizing the pathogen of rice sheath blight under the condition of 10-35°C. The best application effect is achieved when the temperature is 15°C. The microbial inoculant is applied by irrigation of roots and / or spraying directly on the plants / leaves and / or soil and / or flushing.
[0048] Application of Peribacillus simplex TWS22 and / or the cells of Peribacillus simplex TWS22 and / or the microbial inoculant and / or the microbial fertilizer in antagonizing the pathogen of wheat head blight under the condition of 10-35°C. The best application effect is achieved when the temperature is 15°C.
[0049] Example 3
[0050] This example is basically the same as Example 1, except for the preparation method of obtaining the cells of Peribacillus simplex TWS22 by using Peribacillus simplex TWS22.
[0051] The cells of Peribacillus simplex TWS22 are prepared by using Peribacillus simplex TWS22. The preparation method of the cells of Peribacillus simplex TWS22 is to inoculate Peribacillus simplex TWS22 into LB liquid medium, and the liquid fermentation conditions are: fermentation temperature is 30°C, rotation speed is 190 r / min, fermentation time is 48 h. After fermentation, the cells of Peribacillus simplex TWS22 are obtained by centrifugation.
[0052] The microbial inoculant is prepared by resuspending the cells of Peribacillus simplex TWS22 in sterile water, and the effective viable count of the microbial inoculant is ≥ 0.2×10 8 cfu / ml.
[0053] The solid microbial fertilizer, bio-organic fertilizer or compound microbial fertilizer prepared by adsorbing, mixing and solid fermentation of the microbial inoculant is the microbial fertilizer.
[0054] Application of the microbial inoculant in antagonizing the pathogen of rice sheath blight under the condition of 10-35°C. The best application effect is achieved when the temperature is 20°C. The microbial inoculant is applied by root irrigation and / or spraying directly on the plant / leaf surface and / or soil and / or flushing application.
[0055] Application of the Peribacillus simplex TWS22 and / or the cells of Peribacillus simplex TWS22 and / or the microbial inoculant and / or the microbial fertilizer in antagonizing the pathogen of wheat head blight under the condition of 10-35°C. The best application effect is achieved when the temperature is 20°C.
[0056] The following is the biocontrol effect test of the microbial inoculant on rice:
[0057] The test site is Wuyang Town, Suining County, Hunan Province. Three treatments are set in the experiment, namely control treatment (CK), treatment with inoculant applied in winter (T1), treatment with inoculant applied in spring (T2), and pesticide treatment (T3). Each treatment has 5 replicates, and the distance between each treatment is more than 500 m. In November 2023, the inoculant was applied into the field of treatment T1 at a rate of 1 L / acre. In May 2024, the inoculant was applied into the field of treatment T2 at a rate of 1 L / acre. In treatment T3, 55 ml of 10% hexaconazole was used per acre at the late tillering stage of rice, that is, after the rice was closed. After the rice was planted, unified management was carried out, and the incidence of rice sheath blight was investigated during the rapid development period of sheath blight. As Figure 5 shown, applying the TWS22 inoculant can effectively prevent the occurrence of rice sheath blight, and applying in winter is better than applying in spring.
[0058] It can be seen from this that adding the microbial inoculant can effectively prevent the occurrence of rice sheath blight, and applying in winter is better than applying in spring.
[0059] Based on the comprehensive analysis of the above examples and the above test results, the strain TWS22 has an antagonistic effect on the pathogens of rice sheath blight and wheat head blight, and applying in the low-temperature season can effectively control rice sheath blight.
[0060] The components not detailed in this article are prior art.
[0061] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention, and the modifications or deformations without creative labor are still within the protection scope of the present invention.
Claims
1. A simple pure Bacillus strain TWS22, characterized in that: The simple pure Bacillus TWS22 (Peribacillus simplex) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32346.
2. The simple pure Bacillus TWS22 bacterial cell prepared according to the simple pure Bacillus TWS22 (Peribacillus simplex) according to claim 1.
3. The method for preparing a simple pure Bacillus TWS22 cell according to claim 2, characterized in that: The simple pure Bacillus TWS22 (Peribacillus simplex) was inoculated into LB liquid culture medium. The liquid fermentation conditions were as follows: fermentation temperature was 28-30°C, rotation speed was 170-190r / min, and fermentation time was 24-48h. After the fermentation was completed, the simple pure Bacillus TWS22 cells were obtained by centrifugation.
4. A microbial agent containing the simple pure Bacillus TWS22 bacterial cell according to claim 2 or 3, characterized in that: The simple pure Bacillus TWS22 bacteria are resuspended in sterile water to prepare a bacterial suspension, i.e., the microbial agent, with an effective viable bacterial count of ≥ 0.2×10 8 cfu / ml.
5. A microbial fertilizer containing the microbial agent according to claim 4, characterized in that: The microbial agent is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by the microbial agent through adsorption, mixing and solid fermentation, that is, the microbial fertilizer.
6. Use of the microbial agent according to claim 4 in antagonizing rice sheath blight pathogens at 10-35°C.
7. Use of the simple pure Bacillus TWS22 (Peribacillus simplex) according to claim 1 and / or the simple pure Bacillus TWS22 cells according to claim 2 and / or the microbial agent according to claim 4 and / or the microbial fertilizer according to claim 5 in antagonizing wheat fusarium pathogens under the condition of 10-35°C.
8. The use according to claim 6 or 7, characterized in that: The best effect is achieved when the temperature is 10-20°C.
9. A method for using simple pure bacillus TWS22 (Peribacillus simplex), characterized in that: The method comprises irrigating the roots and / or directly spraying and / or flushing the simple pure Bacillus TWS22 (Peribacillus simplex) according to claim 1 and / or the simple pure Bacillus TWS22 bacteria according to claim 2 and / or the microbial agent according to claim 4 and / or the microbial fertilizer according to claim 5 onto the plants / leaves and / or soil.