Bacillus and application thereof
By using Bacillus A1, the problem of cotton Verticillium wilt control has been solved, achieving highly efficient antagonism against Verticillium dahliae and promoting cotton growth. This provides a green microbial inoculant product suitable for Xinjiang, improving cotton yield and quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-06
- Publication Date
- 2026-04-17
AI Technical Summary
Existing technologies are insufficient to effectively control cotton Verticillium wilt, and there is a lack of multifunctional strains with highly efficient antibacterial activity and growth-promoting effects in biological control.
Bacillus sp. A1 was used. This strain was isolated from the rhizosphere soil of cotton and has a strong ability to antagonize Verticillium dahliae and promote healthy growth of cotton. It is used to prepare inoculants to inhibit fungi, improve soil and enhance plant disease resistance.
It significantly inhibits the growth and reproduction of Verticillium dahliae, reduces the risk of cotton Verticillium wilt, improves cotton yield and quality, adapts to the environment of Xinjiang, and provides a green and environmentally friendly microbial fertilizer solution.
Smart Images
Figure CN121874041A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biotechnology, specifically to a Bacillus species and its applications. Background Technology
[0002] Cotton belongs to the genus *Gossypium* in the family Malvaceae. Common types include upland cotton and sea island cotton, with upland cotton being more widely cultivated throughout my country due to its greater adaptability. As the world's largest cotton producer, my country's cotton has a wide range of uses, primarily in textile production. Common everyday items like cotton yarn, cotton cloth, and various cotton fabrics are mostly made from cotton. Besides textiles, cotton is also used to make filling materials (such as cushion and pillow stuffing), hygiene products (such as cotton wipes and sanitary napkins), and even plays a role in tire production. For Xinjiang, the cotton industry is not only a pillar industry of the local economy but also a unique and advantageous industry. Its stable development is of great significance to the stable economic operation of border regions and the maintenance of national unity.
[0003] Thanks to its unique climate (such as abundant sunshine and a large diurnal temperature range) and geographical conditions, Xinjiang has long held an absolute advantage in cotton production nationwide. It is also my country's largest high-quality commercial cotton production base and the only base in the country capable of producing long-staple cotton. Therefore, the healthy and orderly development of the cotton industry plays an irreplaceable and crucial role in ensuring the economic and social stability of Xinjiang.
[0004] Verticillium dahliae, the causal agent of cotton wilt, is a devastating disease that severely damages cotton and is often referred to as the "cancer" of cotton in agricultural production. This disease seriously restricts the sustainable cultivation of cotton and other crops. The key issue lies in the extremely wide host range of Verticillium dahliae; studies have found that it can infect more than 600 plant species, all of which can cause Verticillium wilt. Verticillium dahliae-induced crop wilt not only directly causes a significant reduction in crop yield but also adversely affects the fiber quality of cotton—such as reducing fiber length and weakening fiber strength. This decline in fiber quality further has a cascading negative impact on the downstream cotton textile industry, affecting the overall profitability of the industry. Even more challenging is the difficulty in controlling cotton Verticillium wilt, as the pathogenic Verticillium dahliae can survive in the soil for extended periods, remaining active even without a host. When conditions are suitable, it invades the cotton plant through the roots, gradually disrupting its normal physiological functions, ultimately causing yellowing and wilting of the leaves, and in severe cases, the entire plant dies. According to estimates, in my country alone, the direct economic losses caused by this single disease reach as high as 20 billion yuan annually, placing a heavy economic burden on the cotton industry.
[0005] Cotton Verticillium wilt, a typical soil-borne disease, significantly restricts cotton yield and quality, making it one of the most pressing problems in cotton production. Currently, agricultural control methods for this disease mainly fall into three categories: agricultural control, chemical control, and biological control. Agricultural control methods commonly include rational crop rotation, cultivating disease-resistant cotton varieties, and strengthening field water, fertilizer, and sanitation management. However, these methods have significant limitations: cultivating disease-resistant varieties often requires several years or even longer, and the resulting varieties may conflict with high-yield requirements; crop rotation patterns are limited by regional planting structures and land planning, making it difficult to implement flexibly on a large scale. Chemical control has the advantage of rapid effectiveness and the ability to handle outbreaks, but long-term reliance on chemical agents brings many problems: it easily leads to resistance in Verticillium dahliae, reducing subsequent control effectiveness, and it also pollutes the soil, water sources, and other ecological environments. Furthermore, the efficacy of chemical agents is significantly affected by environmental conditions such as rainfall and temperature. In contrast, biological control, with its environmentally friendly nature and ability to play a long-term and sustainable role, has gradually become a key research direction in the field of green control of cotton Verticillium wilt.
[0006] In the technical system of biological control, the screening and practical application of highly effective biocontrol strains are core components. Ideal biocontrol strains should not only exhibit strong antagonistic effects against *Verticillium dahliae*—such as inhibiting its mycelial growth or spore germination—but also possess the comprehensive functions of promoting cotton root development, enhancing plant resistance to drought or salinity, and simultaneously contributing to increased cotton yield. However, current research shows that multifunctional strains possessing both highly effective antibacterial activity and significant growth-promoting effects are relatively scarce. Therefore, targeted screening and development of such microbial strains that can both suppress the activity of *Verticillium wilt* pathogens and promote healthy cotton growth will provide a new solution for building a more efficient integrated control system for cotton *Verticillium wilt*. Summary of the Invention
[0007] In view of the above-mentioned technical limitations, this application proposes a Bacillus species and its application; which overcomes the deficiencies and defects mentioned in the background art.
[0008] To achieve the above objectives, this application adopts the following technical solution: The inventive point of this application is to provide a Bacillus species, namely Bacillus sp. A1, with accession number CCTCC M 20252592, accession date November 19, 2025, and depositary institution China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.
[0009] The second inventive point of this application is to provide any one of the following applications of the above-mentioned Bacillus, the metabolites of the above-mentioned Bacillus, and / or the fermentation products of the above-mentioned Bacillus: A1) Its application in inhibiting fungi or preparing fungal inhibitors; A2) Application in soil improvement or preparation of soil conditioners; A3) Application in improving plant disease resistance or in the preparation of fertilizers that improve plant disease resistance.
[0010] Optionally, in the above application, the fungus is Verticillium dahliae.
[0011] Optionally, in the above application, improving plant disease resistance means improving the plant's resistance to Verticillium wilt.
[0012] The third inventive point of this application is to provide a microbial agent product containing the aforementioned Bacillus, the metabolites of the aforementioned Bacillus, and / or the fermentation products of the aforementioned Bacillus.
[0013] Optionally, the above-mentioned microbial agent product can be any one of the following microbial agent products: B1) Inoculant products that inhibit fungi; B2) Soil-improving microbial products; B3) Microbial agents that enhance plant disease resistance.
[0014] The fourth inventive point of this application is to provide any one of the following applications of the above-mentioned microbial agent product: C1) The application of the aforementioned fungal agent in inhibiting fungi; C2) Application of the microbial agent product in soil improvement; C3) The application of the microbial agent product in improving plant disease resistance.
[0015] The fifth inventive point of this application is to provide a culture of Bacillus, which is a fermentation product obtained by fermenting the Bacillus of claim 1 in a microbial culture medium.
[0016] The sixth inventive point of this application is to provide a method for culturing the above-mentioned Bacillus, including the step of culturing the Bacillus amyloliquefaciens in a culture medium.
[0017] The seventh inventive point of this application is to provide a method for preparing the above-mentioned microbial agent product, comprising the step of preparing the microbial agent product by taking the Bacillus spp. A1, the metabolites of the Bacillus spp. A1 and / or the fermentation products of the Bacillus spp. A1 as components of the microbial agent product.
[0018] Compared with the prior art, this application has the following advantages: This application discloses for the first time a Bacillus sp. A1 strain with antagonistic activity that can effectively inhibit Verticillium wilt in cotton. This strain was isolated from cotton rhizosphere soil. Experimental studies have confirmed that it has a strong antagonistic effect on Verticillium dahliae, the pathogen causing Verticillium wilt in cotton. It not only significantly inhibits the growth and reproduction of Verticillium dahliae but also effectively reduces the risk of Verticillium wilt in cotton, demonstrating good application potential in the preparation of microbial fertilizers and functional inoculants. Furthermore, the characteristics of Bacillus sp. A1 are highly compatible with the environmental conditions of Xinjiang, providing core strain support for the development of locally adapted, high-efficiency, and environmentally friendly microbial fertilizers. Attached Figure Description
[0019] Figure 1 The image shows the plate confrontation method test results of the antagonistic effect of Bacillus A1 against Verticillium dahliae in one embodiment of this application.
[0020] Figure 2 The image shown is an embodiment of this application, illustrating the phenotypic observation results of a potted plant infected with Verticillium dahliae for 30 days.
[0021] Figure 3 The image shows the statistical results of the disease index of Verticillium wilt during a pot experiment, as described in one embodiment of this application.
[0022] Figure 4 The image shows the colonization detection results of wild-type Verticillium dahliae Kleb. V592-G in one embodiment of this application.
[0023] Figure 5 The phylogenetic tree of Bacillus A1 is shown in one embodiment of this application. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.
[0026] The plant is: Gossypium hirsutum L., an annual herb or subshrub belonging to the genus Gossypium in the Malvaceae family.
[0027] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments. Example 1
[0028] Bacillus sp. A1 isolated from soil, with accession number CCTCC M 20252592, deposited on November 19, 2025, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Bayi Road, Hongshan District, Wuhan, Hubei Province, 430072, China.
[0029] This bacterium is an antagonistic strain of Verticillium dahliae, the pathogen of Verticillium wilt in cotton, and its use can effectively control Verticillium wilt in cotton.
[0030] The screening, identification, analysis and verification of Bacillus A1 are shown below. 1. Rhizosphere soil and soil mass collection:
[0031] Soil: 149th Regiment, Shihezi City, Eighth Division, Xinjiang (Latitude: 45°01′02.02″; Longitude: 86°10′31.75″); Cotton rhizosphere soil: The five-point sampling method was used to collect cotton rhizosphere soil from the cotton fields of the 14th Company of the 149th Regiment of the 8th Division.
[0032] 2. Isolation and purification of strains
[0033] The initial screening of the strains was performed using the rhizosphere shaking method. 5 g of cotton rhizosphere soil was weighed and placed in a 250 mL Erlenmeyer flask containing 45 mL of sterile water. A soil suspension was prepared by shaking at 28 ℃ and 180 r·min⁻¹ for 30 min. The suspension was then diluted sequentially to a concentration of 10. -4 10 -5 10 -6 Gradients were prepared by spreading 100 μL of each culture onto inorganic phosphorus solid plates and incubating upside down at 28°C for 5 days. Single colonies were selected based on phenotypic differences such as colony morphology, size, and color. After three streak purifications, the culture was preserved in 50% glycerol and stored at −80°C for later use.
[0034] 3. Screening of antagonistic strains
[0035] The target biocontrol strains obtained from the previous screening were aseptically inoculated into LB liquid medium. The inoculated medium was placed in a constant temperature shaking incubator at 37℃ and a shaking rate of 150 r / min for 16 h. After the strain entered the logarithmic growth phase, the fermentation broth of the biocontrol strain was collected and temporarily stored in a 4℃ refrigerator for later use. The stored Verticillium dahliae strain was inoculated into liquid Caspase medium and placed in a constant temperature shaking incubator at 28℃ and 150 r / min for 5 days to promote the large-scale sporulation of the strain. 100 μL of Verticillium dahliae spore suspension was taken from the culture broth and evenly spread on the surface of PDA solid medium using a gradient spread method. The plates were incubated upside down in a 28℃ constant temperature incubator for 3 days to complete the activation culture of the pathogen.
[0036] The antagonistic activity of antagonistic strains was screened using the plate confrontation method. Using a sterile punch with a diameter of 1 cm, a piece of culture medium containing the bacteria (approximately 0.3 cm in thickness) was cut from an activated *Verticillium dahliae* PDA plate and inoculated upside down into the center of freshly prepared PDA medium. A cross-shaped mark was made on the bottom of the PDA medium, centered on the bacterial piece. Along the mark, two symmetrical sites were selected 2.5 cm from the center of the medium as inoculation points for the antagonistic bacterial suspension. 2 μL of activated target bacterial suspension was added to each site using a sterile pipette. A blank control group was set up, where only *Verticillium dahliae* bacterial pieces were inoculated under the same operating conditions, without the addition of bacterial suspension. Each experiment was performed in triplicate to ensure the reliability of the results. All treated petri dishes were placed upside down in a 28 ℃ constant temperature incubator and cultured in the dark for 5-7 days. After the culture was completed, the diameter of the inhibition zone of each treatment group was measured using a vernier caliper (accuracy 0.01 mm), and the mean and standard deviation were calculated as the core indicators for evaluating the antagonistic activity of the antagonistic strains.
[0037] Eighteen bacterial strains were isolated from the rhizosphere soil of cotton. The antagonistic function of these 18 strains against Verticillium dahliae was tested using the plate confrontation method. Among them, Bacillus A1 strain showed a significant antagonistic effect. Figure 1 Therefore, strain A1 was chosen as the target for subsequent research.
[0038] 4. Molecular biological identification of the strain:
[0039] After PCR amplification of the bacterial strain gene using universal primers for bacterial 16S rDNA, the product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The obtained sequence was compared with known strain sequences in the NCBI database using BLAST homology analysis. Then, a phylogenetic tree of the strain was constructed using MEGA11 software to determine its taxonomic position.
[0040] 16S rDNA sequence of strain A1 (SEQ ID No. 1): >A1
[0041] The specific steps are as follows: Fresh bacterial samples were sent to Shanghai Sangon Biotech Co., Ltd. Bacterial DNA was extracted using the Ezup column-based bacterial genomic DNA extraction kit (SK8255). PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3'). The PCR reaction program and PCR amplification system are shown in Tables 1 and 2. Electrophoresis was performed on 1% agarose gel at 150V, 100mA for 20 min. DNA fragments were then recovered from the agarose gel using the SanPrep column-based DNA gel recovery kit and finally sequenced. The assembled sequences were uploaded to the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov) database for sequence alignment. Sequences with high similarity were selected from the database and a phylogenetic tree was constructed using the Neighbor Joining Method with MEGA 11.0 software. Finally, the 16S rDNA sequence of the antagonistic bacteria was uploaded to the NCBI database to obtain a sequence accession number.
[0042] Table 1
[0043] Table 2
[0044] The phylogenetic tree of this Bacillus strain A1 is as follows: Figure 5 As shown.
[0045] 5. Physiological and biochemical characteristics analysis: A series of physiological and biochemical experiments were performed on the antagonistic bacterium KRS010, referring to the "Handbook of Systematic Identification of Common Bacteria". These included: indole test, amylase test, tryptophan deaminase test, urease test, catalase test, methyl red test, and VP test. The results are shown in Table 3.
[0046] Table 3
[0047] +: Positive reaction; -: Negative reaction.
[0048] 6. Pot experiment on the antagonistic effect of cotton: 6.1 Production of Microbial Fertilizer
[0049] (1) Preparation of bacterial agent: Take out the resistant strain (Bacillus A1 strain) from the -80℃ freezer, streak the strain on LB solid medium plate for activation, and inoculate the activated single colony strain into LB liquid medium at 180 r.min. -1 Shake the bacteria on a shaker at 28°C until the bacterial concentration reaches 10. 8 -10 9 Cfu / mL (OD600=1). 3500 r.min -1 Centrifuge for 5 minutes, discard the supernatant, and resuspend in sterile water to 10⁻⁶. 8 -10 9 Cfu / mL.
[0050] (2) Preparation of microbial fertilizer: Add 50 mL of microbial agent to 400-500 g of carrier to obtain a solution containing 10 7 -10 8 Cfu / g strain microbial fertilizer.
[0051] 6.2 Pot Experiment
[0052] Mix 5g of microbial fertilizer with 100mL of sterile water to form a microbial fertilizer solution. When the cotton plants have 1-2 true leaves, apply 100mL of the microbial fertilizer solution to each pot. The control group receives an equal volume of sterile carrier liquid. One week later, inoculate with Verticillium dahliae filtrate, and adjust the spore concentration to 1×10⁻⁶ using a hemocytometer. 7 Apply 50ml per pot, per cell / mL.
[0053] 6.3 Determination of agronomic traits
[0054] After inoculation, potted plants were photographed twice a week to observe cotton phenotype. On days 7, 14, 21, and 28 post-inoculation, the roots of cotton seedlings removed in the above steps were thoroughly rinsed. Three to five seedlings were randomly selected from each treatment group and the control group. Plant height, root hair number, leaf number, and root length were recorded. Partial biomass (plant height, leaf number, stem diameter) was measured directly in the experimental area, while the remaining biomass (above-ground fresh weight, root fresh weight, root length) was measured and photographed in the laboratory. Collected cotton samples were stored at -80°C.
[0055] Phenotypic observation of Verticillium dahliae infection 30 days later Figure 2 As shown.
[0056] 6.4 Incidence survey and disease index statistics
[0057] After inoculation with the pathogen, the disease incidence of cotton was recorded daily, and a disease survey was conducted, with classification as follows: Grade 0 - Healthy cotton plants, no diseased leaves, and normal growth; Grade 1 – Less than a quarter of the cotton plant's leaves are affected, turning yellow and wilting; Grade 2 - More than one-quarter but less than one-half of the cotton plant's leaves are affected, turning yellow and wilting; Grade 3 - More than half and less than three-quarters of the cotton plant's leaves are affected, turning yellow and wilting; Level 4 - More than three-quarters of the cotton plant's leaves are infected, or the cotton plant dies. Based on the number of infected plants, the disease incidence rate is calculated according to the formula. The disease index is calculated using the recorded disease level according to formula (2), and the control effect is calculated using formula (3).
[0058] Formula (1): Incidence rate (%) = Number of infected plants / Total number of plants surveyed × 100%; Formula (2): DI = [Σ(number of diseased plants at each level × corresponding disease level) / total number of plants surveyed × highest disease level] × 100; Formula (3): Prevention and control effect = [(disease index of control group - disease index of treatment group) / disease index of control group] × 100%.
[0059] The results of the disease index statistics are as follows: Figure 3 As shown.
[0060] 6.5 Observation of mycelial morphology
[0061] Cotton leaves were taken at 7, 14, 21 and 28 days after inoculation with Verticillium dahliae and perforated with a round hole punch to make temporary specimens. The growth and number of mycelia and spores were observed under a fluorescence microscope.
[0062] The results of V592-G mycelial colonization observation are as follows: Figure 4 As shown.
[0063] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A Bacillus characterized in that, The Bacillus species is Bacillus sp. A1, with accession number CCTCC M 20252592.
2. Any one of the following applications of the Bacillus of claim 1, the metabolites of the Bacillus of claim 1, and / or the fermentation products of the Bacillus of claim 1: A1) Its application in inhibiting fungi or preparing fungal inhibitors; A2) Application in soil improvement or preparation of soil conditioners; A3) Application in improving plant disease resistance or in the preparation of fertilizers that improve plant disease resistance.
3. The application according to claim 2, characterized in that, The fungus is Verticillium dahliae.
4. The application according to claim 2, characterized in that, The improvement of plant disease resistance refers to improving the plant's resistance to Verticillium wilt.
5. A microbial agent product, characterized in that, The microbial agent product contains the Bacillus of claim 1, the metabolites of the Bacillus of claim 1, and / or the fermentation products of the Bacillus of claim 1.
6. The microbial agent product according to claim 5, characterized in that, It can be any one of the following microbial agents: B1) Inoculant products that inhibit fungi; B2) Soil-improving microbial products; B3) Microbial agents that enhance plant disease resistance.
7. Any one of the following applications of the microbial agent product according to claim 5 or 6: C1) The application of the aforementioned fungal agent in inhibiting fungi; C2) Application of the microbial agent product in soil improvement; C3) The application of the microbial agent product in improving plant disease resistance.
8. A culture of Bacillus, characterized in that, The culture is a fermentation product obtained by fermenting the Bacillus of claim 1 in a microbial culture medium.
9. A method for culturing Bacillus as described in claim 1, characterized in that, This includes the step of culturing the Bacillus amyloliquefaciens in a culture medium.
10. A method for preparing the microbial agent product according to any one of claims 5 or 6, characterized in that, The step includes preparing the microbial agent product by taking the Bacillus genus A1, the metabolites of the Bacillus genus A1, and / or the fermentation products of the Bacillus genus A1 as components of the microbial agent product.