Bacillus subtilis JTFB-01 and application thereof

Abstract

This application discloses a Bacillus subtilis strain JTFB-01 and its applications, belonging to the field of microbiology. The Bacillus subtilis strain JTFB-01 has the accession number CGMCC No. 37199 and is deposited at the China General Microbiological Culture Collection Center on December 25, 2025. Field trials show that this inoculant, applied through seed treatment, achieves control effects of 68.20%, 71.73%, and 72.15% against wheat take-all, sheath blight, and root rot, respectively, and significantly increases wheat plant height and yield. It also shows a field control effect of over 50% against cotton Verticillium wilt, with a yield increase of over 17%. This inoculant effectively controls soil-borne diseases and overcomes crop replanting obstacles through multiple mechanisms, including inhibiting pathogens, colonizing the rhizosphere, and promoting growth, thus possessing significant agricultural application value.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a Bacillus subtilis JTFB-01 and its applications. Background Technology

[0002] With the increasing trend of desertification and land degradation globally, arable land resources are continuously shrinking. The contradiction of a large population and limited land in my country is particularly prominent, making it an urgent need to improve the output efficiency per unit area of ​​land to ensure food security. Against this backdrop, increasing the multiple cropping index and implementing continuous cropping have become realistic choices for expanding production in many regions. However, long-term continuous cropping leads to soil ecological imbalance, specifically manifested in the large-scale accumulation and reproduction of certain soil-borne pathogenic fungi (such as Fusarium, Verticillium, and Rhizoctonia) and bacteria in the soil. These pathogenic microorganisms continuously infect crop roots, causing various diseases caused by continuous cropping, such as wilt, root rot, Verticillium wilt, and sheath blight, resulting in poor crop growth and widespread plant death. Generally, the incidence rate in continuously cropped fields is between 10% and 30%, often leading to missing seedlings and gaps in rows. In severe cases, the incidence rate can reach as high as 80% to 90%, causing large-scale crop yield reduction or even crop failure, seriously restricting the efficient and sustainable development of agricultural production.

[0003] Currently, the control of soil-borne diseases mainly relies on chemical agents, such as triazole fungicides. However, long-term and excessive use of chemical pesticides not only easily leads to drug resistance in pathogens, resulting in reduced efficacy, but also may cause pesticide residues, environmental pollution, and damage to beneficial microbial communities in the soil, further exacerbating soil ecological deterioration and failing to fundamentally solve the problem of continuous cropping obstacles. Therefore, an efficient, environmentally friendly, and sustainable solution is urgently needed in agricultural production. Summary of the Invention

[0004] This application provides a Bacillus subtilis JTFB-01 strain and its application to offer a biological solution that can effectively overcome crop replanting obstacles. Therefore, this application provides a microbial strain and its preparation, which can efficiently inhibit or kill various soil-borne pathogenic fungi that cause replanting diseases. Simultaneously, it can successfully colonize the crop rhizosphere. Through multiple synergistic mechanisms, it directly controls diseases while restoring the imbalanced soil microecology, improving soil physicochemical properties, and promoting crop growth. This systematically and sustainably solves the problem of crop yield reduction and even crop failure caused by continuous cropping, overcoming the limitations of chemical control methods.

[0005] In the first aspect, this application provides a Bacillus subtilis JTFB-01, which has the accession number CGMCC No. 37199 and is deposited at the China General Microbiological Culture Collection Center on December 25, 2025.

[0006] Optionally, the 16S rDNA sequence of Bacillus subtilis JTFB-01 is shown in SEQ ID NO: 1.

[0007] Secondly, this application provides a microbial agent comprising at least one of Bacillus subtilis JTFB-01, Bacillus subtilis JTFB-01 fermentation product, or Bacillus subtilis JTFB-01 culture as described in the first aspect.

[0008] Optionally, the microbial agent is a solid or liquid agent, wherein the viable count of Bacillus subtilis JTFB-01 in the solid agent is not less than 1.5 × 10⁻⁶. 11 CFU / g.

[0009] Thirdly, this application provides the application of Bacillus subtilis JTFB-01 as described in the first aspect in the prevention and control of soil-borne fungal diseases of plants, including: wheat take-all disease, wheat sheath blight, wheat root rot, or cotton verticillium wilt.

[0010] Fourthly, this application provides the application of Bacillus subtilis JTFB-01 as described in the first aspect in promoting plant growth, wherein the promotion of plant growth is manifested in increasing at least one of plant height, number of grains per spike, thousand-grain weight, or yield.

[0011] Fifthly, this application provides the application of Bacillus subtilis JTFB-01 as described in the first aspect in improving the soil microecological environment or improving soil structure.

[0012] In a sixth aspect, this application provides a method for preventing and controlling soil-borne plant diseases and / or promoting plant growth by applying the microbial agent described in the second aspect to plant seeds, roots, or soil.

[0013] Optionally, the application method is seed dressing, root irrigation, or soil treatment, and the seed dressing dosage is 5-15 g of microbial agent / kg of seeds.

[0014] The technical solutions provided in this application have the following advantages compared with the prior art:

[0015] (1) Effectively kills soil-borne infectious bacteria: Bacillus subtilis strain JTFB-01 can secrete a large amount of antimicrobial peptides, which can effectively kill a variety of soil-borne pathogens, regulate the soil micro-ecological environment, and reduce the occurrence of soil-borne diseases. It has significant effects on the prevention and control of root rot in vegetables such as tomatoes, peppers, cucumbers, and strawberries, fruit trees such as apples, pears, grapes, peaches, dates, citrus, and lychees, as well as wilt disease in watermelons and melons, Verticillium wilt in cotton, take-all disease in wheat, root rot in soybeans, and soil-borne diseases and replanting diseases in potatoes and sugar beets.

[0016] (2) Formation of biological protection barrier: Bacillus subtilis JTFB-01 can grow and reproduce rapidly in the roots of crops, forming a dominant beneficial bacterial community and biological protection barrier, purifying the soil environment.

[0017] (3) Improve nutrition level: Bacillus subtilis JTFB-01 can effectively promote the release of various micronutrients such as phosphorus, potassium, silicon, iron, boron, and molybdenum in the soil and their absorption by crops during its growth and reproduction.

[0018] (4) Produces a variety of active substances: The metabolic activities of Bacillus subtilis JTFB-01 can produce a variety of bioactive substances, which can effectively stimulate crops to develop strong roots, promote plant growth, protect flowers and fruits, and enhance the crop's resistance to cold, drought and lodging.

[0019] (5) Improve soil structure: The activity of Bacillus subtilis JTFB-01 can effectively improve soil aggregate structure, eliminate soil compaction, enhance soil permeability, retain fertilizer and water, reduce salt and alkali damage, and improve the soil environment for crop growth.

[0020] (6) Increase production: Grain crops will increase by about 10%, economic oil crops by more than 15%, and fruits, vegetables and other crops by more than 20%. Attached Figure Description

[0021] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Image of Bacillus subtilis strain JTFB-01 provided in Example 1 of this application cultured on LB medium;

[0024] Figure 2 A pot experiment was conducted to test the control effect of the antagonistic bacterial strain provided in Example 5 of this application on cotton Verticillium wilt (A. JTFB-01 strain; B. culture medium control; C. pathogen control). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0026] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0027] This application provides a Bacillus subtilis JTFB-01, which has the accession number CGMCC No. 37199 and is deposited at the China General Microbiological Culture Collection Center on December 25, 2025.

[0028] In some embodiments, the 16S rDNA sequence of the Bacillus subtilis JTFB-01 is shown in SEQ ID NO: 1.

[0029] Based on a general inventive concept, this application provides a microbial agent comprising at least one of Bacillus subtilis JTFB-01, Bacillus subtilis JTFB-01 fermentation product, or Bacillus subtilis JTFB-01 culture as described in the first aspect.

[0030] In some embodiments, the microbial agent is a solid or liquid agent, wherein the viable count of Bacillus subtilis JTFB-01 in the solid agent is not less than 1.5 × 10⁻⁶. 11 CFU / g.

[0031] Based on a general inventive concept, this application provides an application of the above-mentioned Bacillus subtilis JTFB-01 in the prevention and control of soil-borne fungal diseases of plants, including: wheat take-all disease, wheat sheath blight, wheat root rot or cotton verticillium wilt.

[0032] In controlling soil-borne fungal diseases, the core mechanism of JTFB-01 lies in its strong antagonistic ability. During its growth and reproduction, this strain secretes various antibacterial active substances, such as lipopeptide antibiotics (e.g., surfactants, iturobrine) and antibacterial proteins. These substances can directly inhibit or kill pathogenic fungi in the soil, such as those causing wheat take-all, sheath blight, root rot, and cotton verticillium wilt, thereby directly reducing the number of pathogens in the soil. Simultaneously, when applied through seed treatment, root drenching, or other methods, JTFB-01 can rapidly colonize and reproduce in the plant rhizosphere, forming a "biological protective barrier" composed of dominant beneficial microorganisms. This barrier effectively blocks pathogen infection of the roots through space occupation and nutrient competition, significantly reducing disease occurrence. Experimental data show that its control effect on major soil-borne diseases of wheat and cotton is superior to that of the chemical agent triadimefon, thanks to its sustained biological control effect.

[0033] Based on a general inventive concept, this application provides an application of the above-described Bacillus subtilis JTFB-01 in promoting plant growth, wherein the promotion of plant growth is manifested in increasing at least one of plant height, number of grains per spike, thousand-grain weight, or yield.

[0034] In promoting plant growth, JTFB-01 works through both direct and indirect pathways. The direct pathway involves the strain's own metabolism producing plant growth regulators such as indoleacetic acid (IAA) and cytokinins. These substances stimulate plant cell division and elongation, promoting root development (manifested as strong root growth), thereby enhancing the plant's ability to absorb water and nutrients, ultimately resulting in improved agronomic traits such as plant height, number of grains per ear, and thousand-grain weight, and a significant increase in yield. The indirect pathway is achieved by improving the plant's nutritional status. JTFB-01 activates fixed mineral elements in the soil, for example, by secreting organic acids to dissolve phosphates, promoting the release of phosphorus, potassium, and various micronutrients, converting them into forms that plants can absorb and utilize. Furthermore, healthy roots and an improved soil environment create better conditions for nutrient absorption. The robust growth, dark green leaves, and increased yield observed in the treatment groups during field trials are a comprehensive manifestation of these growth-promoting effects.

[0035] Based on a general inventive concept, this application provides an application of Bacillus subtilis JTFB-01 described above in improving the soil microecological environment or improving soil structure.

[0036] In improving the soil environment, JTFB-01's effects are mainly reflected in two aspects: physical structure and microecology. In terms of physical structure, the strain and its metabolites (such as polysaccharides and organic acids) can promote soil particle aggregation, increase soil aggregate structure, thereby reducing soil bulk density, improving soil aeration and permeability, alleviating soil compaction, and enhancing the soil's water and fertilizer retention capacity. At the microecological level, as an introduction of beneficial microorganisms, JTFB-01 can regulate the balance of the rhizosphere microbiota, inhibit the development of pathogenic bacteria, enrich beneficial microorganisms, and establish a healthier and more resilient soil microecological system. This improved soil environment not only benefits the growth of seasonal crops but also lays the foundation for sustainable agricultural production.

[0037] Based on a general inventive concept, this application provides a method for preventing and controlling soil-borne plant diseases and / or promoting plant growth by applying the aforementioned microbial agents to plant seeds, roots, or soil.

[0038] In some embodiments, the application method is seed dressing, root irrigation, or soil treatment, and the amount of seed dressing is 5-15 g of microbial agent / kg of seeds.

[0039] The Bacillus subtilis JTFB-01 of this application effectively solves the complex agricultural problem of continuous cropping through a multi-dimensional and systematic biological mechanism. Its solution does not rely on a single method, but rather fundamentally improves the deteriorating soil environment caused by continuous cropping through the synergistic effects of multiple effects such as pathogen elimination, root protection, soil repair, and growth promotion.

[0040] Its primary function is to directly eliminate and inhibit pathogenic fungi accumulated in the soil. The core of replant disease is the continuous accumulation of soil-borne pathogens such as Fusarium, Verticillium, and Rhizoctonia. JTFB-01, through metabolism, secretes lipopeptide antibiotics and antimicrobial proteins, directly inhibiting or killing key pathogens causing wheat take-all, root rot, sheath blight, and cotton Verticillium wilt. Through seed treatment or soil application, high concentrations of live bacteria and their metabolites can rapidly reduce the pathogen population in the soil, curbing disease outbreaks at their source. This has been directly confirmed by experimental data; for example, its control effect on wheat root rot can reach 72.15%, and its potted control effect on cotton Verticillium wilt reaches 76.05%.

[0041] While actively attacking the root system, this strain rapidly establishes a biological defense system. It exhibits excellent rhizosphere adaptability, quickly multiplying around crop roots and forming a dominant microbial community, thus constructing a biological protective barrier. This barrier effectively crowds out subsequent pathogens through space occupation and nutrient competition, preventing them from infecting the roots and creating a safe rhizosphere microenvironment for the crop.

[0042] JTFB-01 plays a crucial role in addressing systemic degradation caused by continuous cropping, such as soil compaction, nutrient imbalance, and deterioration of the microbial community. It promotes soil particle aggregation, improves aggregate structure, increases porosity, alleviates compaction, and enhances soil aeration and water and fertilizer retention capacity, optimizing physical conditions for root growth. Simultaneously, its metabolic activities activate and release phosphorus, potassium, and micronutrients fixed in the soil, converting them into forms absorbable by plants and alleviating nutrient absorption barriers. More importantly, by introducing and establishing a dominant population of beneficial bacteria, it inhibits pathogens and promotes the growth of beneficial microorganisms, gradually guiding the pathogen-dominated, diseased soil microbial community towards a healthy and balanced state.

[0043] Furthermore, this strain can directly enhance the vitality and resistance of crops. It can secrete plant growth hormones such as indoleacetic acid, which strongly stimulate root development, forming a robust root system and enhancing the absorption capacity of water and nutrients. Through the combined effects of various bioactive substances, it can also enhance crop cell vitality and improve its resistance to adverse conditions such as drought and cold damage, as well as disease infestation.

[0044] In summary, Bacillus subtilis JTFB-01's approach to replanting problems embodies a comprehensive and synergistic systemic logic. At the symptomatic level, it rapidly controls seasonal diseases by directly killing pathogens and through rhizosphere competition, ensuring crop survival and growth. At the fundamental level, it gradually restores degraded soil ecosystems by activating nutrients, improving structure, and regulating microbial communities, breaking the vicious cycle caused by replanting. Simultaneously, it strengthens crop adaptability by promoting root development and enhancing plant resistance, creating a positive cycle between growth and the environment. Ultimately, these synergistic effects lead to a significant reduction in crop disease incidence, robust plant growth, and restored and improved yield and quality, thus providing a more sustainable biological solution than simple chemical control.

[0045] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0046] Example 1: Isolation, screening and identification of strain JTFB-01

[0047] Sample collection and pretreatment: Rhizosphere soil samples were collected from greenhouses where cucumbers had been continuously planted in the same location for a long time and where diseases were severe. After the samples were mixed evenly, 10 g of the samples were placed in 90 mL of sterile physiological saline and shaken in a shaker for 30 minutes to prepare a soil suspension.

[0048] Strain isolation: The soil suspension was serially diluted (10⁻⁶) -1 Up to 10 -6 Take 100 μL of each dilution of suspension and spread it onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, pH 7.2). Invert the plates and incubate at 37°C for 24-48 hours.

[0049] Functional screening (antagonistic screening): Single colonies from the plate were picked and inoculated into the center of a PDA plate using the spot inoculation method. Simultaneously, important soil-borne pathogens such as *Verticillium dahliae* V190 (cotton wilt) and *Rhizoctonia graminearum* (wheat sheath blight) were inoculated at equal intervals around the perimeter of the plate. After incubation at 28℃ for 5-7 days, the diameter of the inhibition zone was observed and measured. Strains that produced clear inhibition zones against multiple pathogens were selected as candidate strains.

[0050] Strain purification and preservation: Candidate strains with strong antagonistic effects were repeatedly streaked to obtain pure cultures. The purified strains were inoculated into LB slant medium and cultured at 37°C for 24 hours. After short-term preservation at 4°C, glycerol tubes (final concentration 20%) were prepared and stored at -80°C for long-term preservation.

[0051] Strain identification: Molecular biological identification: Genomic DNA was extracted from candidate strains and amplified by PCR using universal primers 27F and 1492R for bacterial 16S rDNA. The 16S rDNA sequence (SEQ ID NO: 1, >2931-XM-25-0343-KC2-14_TSS20251217-0571-08708-BSN) of the screened Bacillus subtilis strain JTFB-01 was 1371 bp in length. Homology was compared with all previously identified 16S rDNA sequences of prokaryotes in GenBank. Bacillus subtilis JTFB-01 showed 100.00% similarity to NR113265 Bacillus subtilis. Their cell morphology (e.g., ...) was also similar. Figure 1 The image shown conforms to the characteristics of Bacillus subtilis and can be identified as Bacillus subtilis.

[0052] Preservation: Bacillus subtilis JTFB-01 was sent to the China General Microbiological Culture Collection Center (CGMCC) for preservation and obtained the preservation number CGMCC No. 37199.

[0053] Example 2: Fermentation culture of Bacillus subtilis JTFB-01

[0054] Seed culture preparation: Pick a single colony of Bacillus subtilis JTFB-01 from an LB slant or glycerol tube and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2). Incubate at 37℃ and 200 rpm for 12-14 hours until the culture becomes turbid (OD600 approximately 1.0-1.5) to obtain primary seed culture.

[0055] Fermentation scale-up: Transfer the primary seed culture at an inoculum rate of 5-10% to a 5 L fermenter containing sterilized fermentation medium. The fermentation medium consists of: 20 g / L soybean meal powder, 15 g / L glucose, 10 g / L corn steep liquor powder, 5 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, and natural pH (adjusted to 7.0 before sterilization). Fermentation conditions are controlled as follows: temperature 35-37℃, stirring speed 300-400 rpm, aeration rate 1.0-1.5 vvm, and pH maintained at 6.8-7.2 during fermentation by adding ammonia.

[0056] Fermentation process monitoring: Regular sampling was conducted to monitor OD600, pH, and residual sugar content. Microscopic observation of cell morphology and spore formation was also performed. After approximately 24 hours of fermentation, the vegetative cells entered a stable growth phase and began to form spores. Fermentation was continued until approximately 36-48 hours had passed. Microscopic examination revealed a spore formation rate exceeding 90%, with most cells having formed mature spores. Fermentation was then terminated at this point.

[0057] Fermentation broth treatment: After fermentation, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 15 minutes, and the cell pellet was collected. The cell pellet was washed twice with sterile physiological saline to obtain a high-concentration spore suspension.

[0058] Example 3: Preparation of Bacillus subtilis JTFB-01 inoculum

[0059] Preparation of solid adsorbent: The high-concentration spore suspension obtained in Example 2 is mixed with sterilized and dried peat (or diatomaceous earth, attapulgite) carrier in a certain ratio (e.g., 1:1 to 1:2, volume / mass ratio) under sterile conditions.

[0060] Place the mixture in a sterile tray or fluidized bed dryer and air dry at 35-40°C until the moisture content is below 10%.

[0061] The dried product was pulverized using a sterile pulverizer and passed through an 80-mesh sieve to obtain Bacillus subtilis JTFB-01 solid powder bacterial agent.

[0062] Quality testing: The viable bacteria content of the finished bacterial agent was determined using the dilution plating count method. Results showed that the solid bacterial agent prepared according to the method described in this embodiment had a stable viable spore count of Bacillus subtilis JTFB-01 at 1.5 × 10⁻⁶. 11 The concentration of CFU / g is above the standard, which meets the product standards.

[0063] Example 4: Control effect on wheat root fungal diseases

[0064] Extensive trials and investigations were conducted using the produced Bacillus solid formulation to control wheat root fungal diseases, providing a preliminary understanding of its application effects. To obtain more refined and accurate experimental data, a large-scale application demonstration was conducted, selecting a plot of land with a severe and uniform disease incidence in the previous year, covering an area of ​​approximately 666.7 m². 2 It is divided into 3 small pieces, each approximately 222.2 m². 2 Three groups were selected: a control group, a pesticide group, and a fungicide group. The pesticide group used 15% triadimefon wettable powder. Following the instructions, 3 g / kg of seeds were added to the pesticide solution. The seeds and pesticide were placed in a sturdy plastic bag, the bag was tied tightly, and the bag was inverted several times until the pesticide and wheat seeds were thoroughly mixed. The fungicide group used seeds with a spore content of 1.5 × 10⁻⁶. 11 Treat seeds with a solid inoculum agent containing CFU / g, at an inoculation rate of 10 g / kg of seeds. Sow mechanically, and manage the seeds as usual in a field.

[0065] A five-point sampling method was used during the wheat jointing stage, with 100 plants selected at each point, for a total of 500 plants. During sampling, the entire plant was dug up with a shovel, the soil was gently shaken off, and any plants with severed roots were removed. The roots were then thoroughly cleaned with water. Disease prevalence was assessed, and the disease index and control efficacy were calculated. The results are shown in Table 1, which illustrates the control efficacy of the inoculant against root fungal diseases during the wheat jointing stage.

[0066] Table 1. Control efficacy of inoculants against root fungal diseases in wheat during the jointing stage.

[0067]

[0068] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level as determined by the LSD test.

[0069] During the milk stage of wheat, a 5-point sampling method was used, with 3 consecutive rows sampled at each point, and each row measuring 0.4 m. Disease incidence and disease index were investigated, and the disease control effect was calculated. The disease index and control effect were investigated using the 5-point method. Yield was assessed by recording plant height and average number of grains per ear (30 plants per random sampling point, 150 plants per group). After drying the seeds, the thousand-grain weight was measured, and yield per square meter was determined by individual harvesting. The results are shown in Table 2, which presents the control effect of the inoculant on root fungal diseases during the wheat milk stage.

[0070] Table 2. Control efficacy against root fungal diseases during the milk stage of wheat.

[0071]

[0072] The data in the table are mean ± standard deviation. Different letters after the data in the same column indicate significant differences at the P < 0.05 level as determined by the LSD test.

[0073] The efficacy of inoculants in controlling root fungal diseases in wheat was investigated. Wheat seedlings began to emerge 7 days after sowing, and almost all seedlings emerged after 10 days. The results of seedling emergence surveys in the experimental plots showed that the inoculant and pesticide groups had better emergence, with dark green leaves and robust seedlings. In the control plot, the wheat seedlings were sparse and short, with yellowing leaf tips. After winter greening, the wheat plants in the inoculant and pesticide groups showed better growth and green leaves, while the control group had sparser, weaker plants with yellowing leaf tips. During the jointing stage, the wheat seedlings in the inoculant group showed better growth, with robust, dense plants, dark green and thick leaves, and significantly increased plant height compared to the control group. The disease index of wheat take-all disease treated with biocontrol bacteria was significantly reduced to 6.03, a significant decrease from 18.96 in the control group, with a control effect of 68.20%, which was better than the 55.85% of the pesticide group. The disease index of wheat sheath blight in the biocontrol bacteria treatment group was 5.69, while the disease index in untreated diseased areas was 20.13, with a control effect of 71.73%, which was better than the 61.25% of the pesticide group. The disease index of wheat root rot treated with biocontrol bacteria was 6.98, while the disease index in untreated diseased areas was 25.06, with a control effect of 72.15%, which was better than the 58.90% of the pesticide group (Table 1). A survey of the control effect of biocontrol bacteria on take-all disease at the milk stage of wheat showed that, compared with the control group, the plant height and yield of the inoculum group increased significantly, with wheat yield increasing from 657.53 g / m². 2 Increased to 790.46 g / m 2 The effect was significant; a survey on the control effect of the fungicide showed that the plant height and yield of the treatment group increased significantly, with the yield increasing from 653.61 g / m² in the control group of the diseased field. 2 Increased to 791.30 g / m³ in the inoculant treatment group 2The effect was significant; a survey on the control effect of wheat sheath blight showed that the plant height and yield of the inoculant-treated group increased significantly, with the yield increasing from 679.78 g / m² in the control group of the diseased field. 2 Increased to 779.93 g / m² in the inoculant treatment group 2 The effect was significant (Table 2).

[0074] Example 5: Control effect on cotton Verticillium wilt

[0075] Studies have shown that the vast majority of pathogens causing soil-borne plant diseases are fungi, and cotton Verticillium wilt is a typical soil-borne fungal disease. Soil fungalization may be one of the factors hindering crop growth. Richard J. Ellis points out that culturable bacteria in the soil are perhaps the group that contributes the most to the soil ecosystem. They are more susceptible to changes in the soil ecosystem than the entire microbial community and can be used as indicators related to pollution impacts. The strain is a wild-type strain isolated and purified from nature, with strong resistance to harsh environments. It is a Bacillus strain with strong heat and stress resistance, and can be developed into a biocontrol agent to control cotton Verticillium wilt. Pot experiments were conducted on antagonistic bacteria to test the control effect of cotton Verticillium wilt antagonistic bacteria on cotton Verticillium wilt, providing a basis for the development of biocontrol agents with biological control functions.

[0076] 1. Materials and Methods

[0077] 1.1 Materials

[0078] Verticillium dahliae strain V190 was preserved in the laboratory. The tested cotton variety was upland cotton, the Verticillium wilt-susceptible cultivar Han 208.

[0079] 1.2 Methods

[0080] 1.2.1 Cotton Seedling Cultivation

[0081] Soak sulfuric acid-delinted cotton seeds in water at room temperature for 12-14 hours. Place them on a porcelain dish lined with two layers of damp gauze, then cover with several more layers of damp gauze. Germinate at 25-30℃, spraying water to keep the gauze moist during this time. When the sprouts reach 0.5-1.0 cm in length, sort the germinated seeds and plant 3 seeds in each seedling pot. After planting, fill the seedling pots with vermiculite and spray with water. Cultivate the cotton seedlings at room temperature.

[0082] 1.2.2 Pathogen culture and preparation of spore suspension

[0083] The cryopreserved pathogens were transferred to PDA agar medium and activated at 25°C for 7–10 days. Then, they were transferred to Erlenmeyer flasks containing 150 mL of PDA culture medium and cultured with shaking at 25°C for 8–10 days. The bacterial culture was then rinsed with sterile water, filtered through two layers of gauze, and prepared to a concentration of 0.94–1.00 × 10⁻⁶. 7 A spore suspension of 1 spore / mL should be prepared as needed.

[0084] 1.2.3 Determination of the effect of antagonistic bacteria in potted plants on controlling cotton Verticillium wilt

[0085] Plastic flowerpots with a mouth diameter of 9 cm, a bottom diameter of 6 cm, a height of 7 cm, and 6 round holes at the bottom were selected. Sterilized vermiculite was filled into the flowerpots to prepare nutrient pots. The vermiculite surface of the nutrient pots was flattened and placed approximately 1 cm below the top edge of the nutrient pot. Each nutrient pot was then watered with 15 mL of a prepared pathogen spore suspension. Pretreated cotton seeds were planted into the nutrient pots, and 15 mL of a bacterial suspension with a concentration on the order of 10⁹ CFU / mL (incubated at 37℃ for 24 h) was added to the nutrient pots. Two control groups were set up: one without antagonistic bacteria, one with only pathogen, and one without antagonistic bacteria or pathogen, but only with culture medium. Ten pots were added to each control group. The plants were incubated at room temperature, and Hoagland nutrient solution and water were added periodically. Disease incidence and disease index of Verticillium wilt were recorded for each treatment, and the relative disease control effect of the biocontrol bacteria against Verticillium wilt in cotton was calculated. Statistical analysis of relevant data was performed using SPSS 17.0.

[0086] 1.2.4 Calculation method for disease prevention effect

[0087] The occurrence of Verticillium wilt was recorded according to a 5-level classification standard (Level 0, disease-free plants; Level 1, plants with 25% of leaves infected; Level 2, plants with 25%–50% of leaves infected; Level 3, plants with 50%–75% of leaves infected; Level 4, plants with more than 75% of leaves infected). The disease index was recorded and calculated, and the relative disease control effect of the tested bacteria against Verticillium wilt in cotton was calculated.

[0088] Disease index = ∑ (level value × number of plants) × 100 / (4 × total number of plants)

[0089] Prevention and control efficacy (%) = (Control disease index - Treatment disease index) × 100 / Control disease index

[0090] 2 Results and Analysis

[0091] The results of the potted plant control experiment are shown in Table 3 and Figure 2As shown, the results indicate that when there are no pathogens in the nutrient pots, the incidence of cotton Verticillium wilt is extremely low, at 8.21%. When the nutrient pots are inoculated with pathogens, compared with the control without antagonistic bacteria treatment, the application of the strain significantly reduces the disease index, and the control effect on cotton Verticillium wilt reaches 76.05%.

[0092] Table 3. Potted plant effects of antagonistic bacterial strains on control of cotton Verticillium wilt.

[0093]

[0094] Note: In the same column, different lowercase letters in the superscript indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01).

[0095] Example 6: Control effect on cotton Verticillium wilt

[0096] 1. Materials and Methods

[0097] 1.1 Materials

[0098] The inoculant was produced in a pilot-scale production line. The cotton seed variety was DP99B, purchased commercially.

[0099] 1.2 Methods

[0100] 1.2.1 Sowing Method

[0101] Sowing methods, timing, and cotton field management were the same as in local practices. Before sowing cotton in the field, the inoculant powder (10¹⁰ CFU / g) was mixed with cotton seeds at a ratio of 1:20 (w / w, inoculant / seed weight): the cotton seeds were moistened with water, the powder was sprinkled into the seeds, mixed well, dried, and then sown. Seeds without any treatment served as a blank control. Each treatment area was 666.6 m².

[0102] 1.2.2 Disease Investigation and Yield Estimation Methods

[0103] During the peak period of cotton Verticillium wilt in the local area, a 5-point sampling method (30 plants / point) was used to investigate the disease situation and calculate the disease index and control effect. Before cotton harvest, a 5-point sampling method (20 plants / point) was used to investigate the boll setting and density of individual plants to estimate cotton yield.

[0104] Yield (kg / hm) 2 = Density × Boll weight per plant × 0.004 (where the coefficient 0.004 is the weight of a single boll of the DP99B variety, in kg)

[0105] 1.2.3 Data Statistical Analysis

[0106] The statistical analysis of the data in this paper was performed using SPSS version 13.0 software.

[0107] 2 Results

[0108] Table 4 Field control effect survey of microbial agents

[0109]

[0110] Note: In the same column, different lowercase letters in the superscript indicate significant differences (P<0.05), and different uppercase letters indicate extremely significant differences (P<0.01).

[0111] A survey of cotton Verticillium wilt disease was conducted during the peak period of the disease. The results showed that the disease index of the fungal powder treatment was significantly lower than that of the control group, with disease control effects of 50.33% in 2020 and 55.24% in 2021. Simultaneously, it significantly increased cotton yield, with an increase of 18.56% in 2020 and 17.08% in 2021 (Table 4).

[0112] As can be seen from the experimental results in Tables 1 to 4, the Bacillus subtilis JTFB-01 and its microbial agents described in this application effectively solve the problems of disease spread and yield reduction caused by continuous cropping by efficiently controlling a variety of typical soil-borne diseases caused by continuous cropping and significantly promoting crop growth.

[0113] Specifically, the JTFB-01 inoculant demonstrated excellent control efficacy against key pathogenic fungi accumulated in continuously cropped soils, including wheat take-all, sheath blight, root rot, and cotton Verticillium wilt. As shown in Table 1, at the wheat jointing stage, the inoculant treatment achieved control efficacy of 68.20%, 71.73%, and 72.15% against these three diseases, respectively, significantly outperforming conventional chemical agents such as triadimefon. This highly efficient disease control directly reduced the damage to the root system caused by pathogens, laying the foundation for normal crop growth. Further field results (Tables 2 and 4) show that effective disease control directly translates into considerable agronomic and economic benefits. At the wheat milk stage, the inoculant treatment not only significantly increased plant height but also substantially increased yield compared to the disease control group; for example, the yield in wheat take-all disease fields increased from 657.53 g / m². 2 Increased to 790.46 g / m 2 Similarly, in a two-year field trial on cotton (Table 4), the fungal treatment significantly reduced the Verticillium wilt disease index while increasing cotton yield by 17.08% to 18.56%.

[0114] These data collectively demonstrate that JTFB-01 inoculant is not merely a single disease inhibitor. Through its synergistic biological action, it effectively curbs the outbreak of replanting diseases while simultaneously enhancing crop growth and vitality, thus breaking the vicious cycle of "replanting → aggravated soil-borne diseases → poor crop growth and reduced yield → further deterioration of the soil environment." Therefore, the technical solution provided in this application offers an efficient, environmentally friendly, and sustainable biological control approach to address the problem of soil-borne diseases exacerbated by increased multiple cropping index (replanting tillage). It significantly restores and increases yields in replanted land while ensuring crop survival and health, fundamentally addressing the production challenges posed by replanting obstacles.

[0115] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0116] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0117] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A Bacillus subtilis JTFB-01, characterized in that, The Bacillus subtilis JTFB-01 has the accession number CGMCC No. 37199 and is deposited at the China General Microbiological Culture Collection Center on December 25, 2025.

2. The Bacillus subtilis JTFB-01 according to claim 1, characterized in that, The 16S rDNA sequence of Bacillus subtilis JTFB-01 is shown in SEQ ID NO:

1.

3. A microbial inoculant, characterized in that, The microbial agent comprises at least one of Bacillus subtilis JTFB-01, Bacillus subtilis JTFB-01 fermentation product, or Bacillus subtilis JTFB-01 culture as described in claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is a solid or liquid agent, and the viable count of Bacillus subtilis JTFB-01 in the solid agent is not less than 1.5 × 10⁻⁶. 11 CFU / g.

5. The application of Bacillus subtilis JTFB-01 as described in claim 1 or 2 in the control of soil-borne fungal diseases of plants, wherein the soil-borne fungal diseases of plants include: Take-all disease of wheat, sheath blight of wheat, root rot of wheat, or Verticillium wilt of cotton.

6. The application of Bacillus subtilis JTFB-01 as described in claim 1 or 2 in promoting plant growth, wherein the promotion of plant growth is manifested in increasing at least one of plant height, number of grains per spike, thousand-grain weight, or yield.

7. The application of Bacillus subtilis JTFB-01 as described in claim 1 or 2 in improving soil microecological environment or improving soil structure.

8. A method for preventing and controlling soil-borne plant diseases and / or promoting plant growth, characterized in that, The microbial agent described in claim 3 or 4 is applied to plant seeds, roots or soil.

9. The method according to claim 8, characterized in that, The application methods are seed dressing, root irrigation, or soil treatment. The dosage for seed dressing is 5-15 g of microbial agent per kg of seeds.

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