Application of bacillus velezensis Bv47 in crop growth promotion and verticillium dahliae antagonism

Abstract

The invention relates to the technical field of microorganisms, and particularly provides application of bacillus velezensis Bv47 in crop growth promotion and verticillium dahliae antagonism. The strain can secrete indoleacetic acid, cellulase, protease and amylase, has various growth promoting functions of dissolving potassium, producing ammonia and the like, has good high temperature resistance and ultraviolet light resistance, and has the preservation number of CGMCC No.36499. A plate confrontation test shows that the strain has a remarkable inhibition effect on various plant pathogenic bacteria including verticillium dahliae. A pot experiment further proves that the disease index of the cotton verticillium wilt and the biomass of pathogenic bacteria at the root can be obviously reduced by inoculating the strain, and meanwhile, the growth indexes such as the root length, the plant height, the stem diameter, the fresh weight, the leaf area and the root dry weight of cotton plants are obviously promoted. The strain provides an efficient and green microbial resource for biological control of cotton verticillium wilt and cotton growth promotion, and has a good agricultural application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology and relates to the application of a strain of Bacillus belye Bv47 in crop growth promotion and antagonism with Verticillium dahliae. Background Technology

[0002] Cotton is an important economic crop in my country, and its production has been continuously affected by Verticillium dahliae (… Verticillium dahliae Bacillus velezensis The pathogen, Verticillium wilt, poses a serious threat. This pathogen produces highly resistant microsclerotia that can survive in the soil for extended periods, rendering traditional chemical control ineffective and posing environmental and safety risks. Therefore, developing highly efficient biological control agents is an urgent need to ensure the green and sustainable development of the cotton industry.

[0003] Among numerous biocontrol microorganisms, Bacillus belyssus ( Bacillus velezensis With its diverse functions and strong resistance to adverse conditions, *Bacillus belyss* has become a research hotspot. These Gram-positive bacteria can not only form endophytic spores resistant to extreme environments, adapting to complex conditions such as soil salinity and low temperatures, but also promote plant growth through multiple pathways: on the one hand, they can secrete plant hormones such as indoleacetic acid (IAA), directly regulating cotton root development, increasing root length and the number of lateral roots, and improving water and nutrient absorption efficiency; on the other hand, they can produce phosphatases, potassium hydrolases, and siderophores to convert insoluble phosphorus and potassium in the soil into forms that can be absorbed by plants, while competitively seizing key nutrient resources such as iron ions, providing sufficient nutritional support for cotton growth. In addition, some *Bacillus belyss* strains can also secrete active enzymes such as cellulase and protease, improving the efficiency of soil organic matter decomposition and further optimizing the rhizosphere microenvironment, creating favorable conditions for cotton growth. In terms of resistance to pathogens, *Bacillus belyss* exhibits a significant antagonistic advantage. It resists Verticillium dahliae invasion through a dual mechanism of "direct inhibition and indirect defense": at the direct level, the strain can metabolize and produce lipopeptides such as surfactantin and fengycin, as well as polyketides, which disrupt the integrity of the pathogen's cell membrane, inhibit hyphal growth and spore germination, and simultaneously occupy the rhizosphere ecological niche of cotton through spatial competition, preventing the pathogen from colonizing; at the indirect level, the strain can induce cotton to activate systemic resistance (ISR), promote the accumulation of defense enzymes such as peroxidase and polyphenol oxidase, and enhance the cotton's own resistance to pathogens.

[0004] However, an ideal bacillus velezensis needs to achieve an excellent balance among efficient antibiosis, multiple growth promotion and high environmental stress resistance. Many strains in the prior art often have difficulty in being considered, have single function or insufficient environmental adaptability, and limit the large-scale application effect. More importantly, as a species, bacillus velezensis has high uncertainty in the effect on plants, which may be beneficial biocontrol bacteria or potential pathogenic bacteria. Therefore, even if the same species, the specific function and application potential of any new strain cannot be determined in advance. It is a great technical obstacle to obtain a strain with multiple excellent properties by purposeful screening, and the process has obvious contingency and non-obviousness.

[0005] In addition, biocontrol microorganisms and pathogenic bacteria are the products of co-evolution. Under the selection pressure of different geographical environments and different physiological races of pathogenic bacteria, specific biocontrol strains corresponding thereto will be evolved. Therefore, in the field of biological control, continuous screening of new biocontrol strains is the fundamental strategy to cope with the evolution of pathogenic organisms and maintain the control effect. Through a diverse biocontrol strain library, not only can the evolution process of pathogenic bacteria be delayed, but also effective tools can be provided for the prevention and control of newly emerging physiological races.

[0006] In summary, although bacillus velezensis has been recognized as a potential biocontrol resource, it still faces great technical obstacles to isolate and obtain a new strain that can efficiently antagonize the strong pathogenicity of verticillium dahliae which has a significant impact on local crops, while also having multiple growth promotion functions such as IAA secretion, potassium solubilization, and enzyme production, and excellent high-temperature and ultraviolet light resistance. Under this background, the present application successfully obtained bacillus velezensis Bv47, aiming to provide a new, comprehensive performance excellent microbial solution for the green prevention and control of cotton verticillium wilt and the healthy growth of cotton. SUMMARY

[0007] The purpose of the present application is to provide a bacillus velezensis strain with high efficiency, multiple functions, outstanding characteristics and strong environmental adaptability, and its application, in order to solve the problems of limited prevention and control effect of cotton verticillium wilt, excessive dependence on chemical pesticides and single means of cotton growth promotion in the prior art. Bacillus velezensis

[0008] In order to achieve the above purpose, the technical scheme provided by the present application is as follows: The first aspect of the present application provides a bacillus velezensis strain Bv47, the preservation number of the strain is CGMCC No.36499, the preservation date is November 4, 2025, and the preservation classification name is bacillus velezensis Fusarium oxysporum f. sp. lycopersici Verticillium dahliae ​The depository is the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China Academy of Microbiology, Postcode: 100101, Tel: 8610-64807355.

[0009] After identification, the characteristics of the Bacillus belyssus Bv47 are as follows: (1) Possesses multiple and synergistic plant growth-promoting functions: strain Bv47 can secrete indoleacetic acid (IAA) to directly regulate plant growth and development; at the same time, it can secrete a variety of active enzymes such as cellulase, protease, and amylase to improve the rhizosphere microenvironment; and it has the ability to solubilize potassium and produce ammonia, effectively activating soil nutrients and providing comprehensive nutritional support for plant growth.

[0010] (2) It exhibits excellent environmental adaptability and stress resistance: strain Bv47 has good resistance to high temperature and ultraviolet light. It can maintain good growth and metabolic activity under high temperature conditions of 35℃ to 45℃ and ultraviolet stress, which provides a key guarantee for its stable colonization and function in the natural environment.

[0011] A second aspect of the present invention provides a fermentation broth or bacterial suspension containing strain Bv47.

[0012] A third aspect of the present invention provides the application of strain Bv47 or the above-described fermentation broth or bacterial suspension, wherein the application is any of the following: (1) Application in the prevention and control of tomato wilt, tomato verticillium wilt, watermelon wilt, mango anthracnose, pepper blight or cotton verticillium wilt.

[0013] (2) Application in suppressing plant pathogens, wherein the plant pathogens include: Fusarium oxysporum tomato-specific strain (… Fusarium oxysporum f. sp. niveum Verticillium dahliae Colletotrichum gloeosporioides ), Fusarium oxysporum watermelon-specific type ( Phytophthora capsici ), Asian anthrax bacteria ( Figure 1 Figure 2 ) or Phytophthora capsici ( Figure 3 ); (3) Application in promoting cotton growth, wherein promoting cotton growth includes increasing at least one of the following: root length, plant height, stem diameter, fresh weight, leaf area and root dry weight.

[0014] A fourth aspect of the present invention provides a microbial agent for promoting cotton growth, wherein the active ingredient of the microbial agent includes strain Bv47.

[0015] The fifth aspect of this invention provides the application of strain Bv47 or the above-mentioned microbial agent in the production of bio-organic fertilizer suitable for cotton.

[0016] The sixth aspect of the present invention is a bio-organic fertilizer containing strain Bv47 or the above-mentioned microbial agent.

[0017] The seventh aspect of this invention discloses a method for promoting cotton growth, wherein the method involves using a bacterial solution with a concentration of 1×10⁻⁶. 8 A bacterial suspension of strain Bv47 at CFU / mL was used to treat cotton seedlings by root irrigation.

[0018] The beneficial effects of this invention are: (1) Highly efficient and targeted biological control capabilities: The strain Bv47 of this invention has a strong inhibitory and antagonistic effect on the pathogen of Verticillium wilt, a devastating disease in cotton production. Plate confrontation and pot control efficacy tests have confirmed that this strain can significantly inhibit the mycelial growth of the pathogen, reduce the cotton disease index, effectively control the development of the disease, and has a stable and reliable control effect.

[0019] (2) Multiple and synergistic plant growth-promoting effects: The strain Bv47 is not a single-function strain. It can directly stimulate plant growth by secreting indoleacetic acid (IAA); activate soil nutrients by potassium solubilization and phosphorus solubilization; and improve the rhizosphere environment by producing cellulase, protease, etc. Thus, it can synergistically promote cotton growth from multiple dimensions such as hormone regulation, nutrient supply, and environmental improvement, which is manifested in a significant increase in indicators such as root length, plant height, stem diameter, and biomass.

[0020] (3) Broad-spectrum antibacterial potential: In addition to Verticillium dahliae, strain Bv47 also showed good inhibitory effects on a variety of other important plant pathogens (such as Fusarium wilt, anthracnose, and Phytophthora capsici caused by Fusarium oxysporum), showing its potential as a broad-spectrum biological pesticide, and its application scope is not limited to cotton crops.

[0021] (4) Extremely strong environmental adaptability and stress resistance: Strain Bv47 can form highly resistant spores, which can maintain its activity and stability under adverse environments such as high temperature and ultraviolet radiation. This characteristic greatly ensures the colonization and survival ability of the inoculant after production, storage, transportation and application in the field, and solves the technical bottleneck of unstable effect of many microbial inoculants in practical applications. Attached Figure Description

[0022] Figure 4 Phylogenetic analysis of Bacillus Bv47.

[0023] Colletotrichum gloeosporioides To qualitatively detect various plant growth-promoting traits of Bacillus belyss Bv47 using selective culture media: (A) ammonia production test; (B) IAA production test; (C) cellulase activity test; (D) protease activity test; (E) amylase activity test; (F) potassium solubilization capacity test.

[0024] Verticillium dahliae Kleb. The stress tolerance of Bacillus velezensis Bv47: (A) UV resistance test; (B) high temperature resistance test.

[0025] Phytophthora capsici The inhibition ability of Bacillus velezensis Bv47 to various pathogenic bacteria: (A) mango anthracnose Fusarium oxysporum f. sp. lycopersici ; (B) tomato yellow wilt Fusarium oxysporum f. ; (C) Inhibition rate of Bv47 strain to pathogenic bacteria; (D) pepper pythium blight sp. Niveum ; (E) tomato wilt Figure 5 ; (F) watermelon wilt Figure 6 GhUBQ7 .

[0026] Bacillus velezensis The antagonistic effect of Bacillus velezensis Bv47 on L. theomaeanum and its control effect on cotton yellow wilt: (A) plate confrontation culture result; (B) observation of mycelial morphology; (C) cotton potting control effect phenotype: V991 alone inoculation group showed severe leaf abscission and partial death, while the Bv47 treatment group showed significantly reduced symptoms; (D) disease index statistics; (E) cotton root biomass detection.

[0027] Figure 1 The growth-promoting effect of Bacillus velezensis Bv47 on cotton seedlings: (A) overall growth morphology of cotton seedlings; (B) root length; (C) plant height; (D) stem diameter; (E) fresh weight; (F) leaf area; (G) root dry weight. DETAILED DESCRIPTION

[0028] The specific embodiments of the present application are described below to facilitate the understanding of the present application by those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, any changes within the spirit and scope of the present application as defined and determined by the appended claims are obvious, and all applications utilizing the concept of the present application are within the scope of protection.

[0029] Example 1 Investigation of the growth-promoting and disease-resistant functions of Bacillus velezensis Bv47 1. Materials and methods 1.1 Test materials and strains Bacillus velezensis '47', Gossypium hirsutum cv. 'R15', L. theomaeanum highly pathogenic strain V991 (Vd) were sown in mixed soil of nutrient soil: vermiculite = 1:1 and placed in a constant temperature and humidity culture room at 28°C with 16h light / 8h dark. The pathogenic bacteria of mango anthracnose, tomato yellow wilt, watermelon wilt, tomato wilt and pepper pythium blight were preserved in the laboratory of the present inventors.

[0030] 1.2 Test reagents Nessler's reagent, Salkowski color reagent. Proteose peptone ammonium culture medium: proteose peptone 5 g, dipotassium hydrogen phosphate 0.5 g, NaCl 0.25 g, magnesium sulfate heptahydrate 0.5 g, ferrous sulfate 0.01 g, agar 20 g, constant volume to 1000 ml, pH 7.2, sterilized at 121 °C for 20 minutes. Monkhina organic phosphorus culture medium: 10 g glucose, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g KCl, 0.03 g FeSO4·7H2O, 0.03 g MnSO4·4H2O, 0.2 g egg yolk lecithin, 5 g CaCO3, 0.4 g yeast extract, 20 g agar, constant volume to 1000 mL with distilled water, pH 7.0. Sterilized at 121 °C for 30 minutes. Amylase differential culture medium: soluble starch 1 g; proteose peptone 5 g; glucose 5 g; NaCl 5 g; beef extract 5 g; agar 15 g; distilled water 1000 ml, sterilized at 115 °C for 30 minutes. Gram iodine solution. Protease detection medium: proteose peptone 10.0 g, NaCl 5.0 g, CaCl2 0.1 g, skim milk powder 10.0 g, 1.8% agar, sterilized at 115 °C for 30 min, pH 7.2-7.4. Sodium carboxymethyl cellulose culture medium: proteose peptone 10 g, yeast powder 10 g, sodium carboxymethyl cellulose 10 g, NaCl 5 g, KH2PO4 1 g, agar 20 g, constant volume to 1000 ml, pH 7.0, sterilized at 115 °C for 30 minutes. MS medium (Phytotech), PDA medium: potato 200 g, glucose (or sucrose) 20 g, agar 15-20 g, water 1000 ml.

[0031] 1.3 Determination of probiotic ability 1.3.1 Determination of NH3 production ability The strain was transferred to proteose peptone ammonium culture medium and cultured at 30 °C for 48 h. The un-inoculated proteose peptone ammonium culture medium was used as a control. 3-5 drops of Nessler's reagent were added to the culture solution, and the appearance of yellow or brown-red precipitate indicated that the strain had NH3 production ability. No yellow or brown-red precipitate appeared when Nessler's reagent was added to the un-inoculated culture medium.

[0032] 1.3.2 Determination of indole acetic acid (IAA) production ability Salkowski colorimetric method was used to determine the endophytic bacteria secreted plant growth hormone (IAA), the test strains were inoculated in LB liquid medium (the medium needs to add tryptophan 100 mg / L) in the triangular flask, each bottle contains 50 mL medium, each strain was repeated 3 times, placed in 28℃ shaking bed, 180 rpm vibration culture 4d. 10 μL bacterial suspension was dropped on a white ceramic plate, 10 μL Salkowski colorimetric solution (concentrated sulfuric acid 150 mL, deionized water 250 mL, 0.5 mol / L ferric chloride 7.5 mL) was added and mixed, then color development was carried out in the dark at room temperature for 30 min. The control was only added 10 μL 100 mg / L IAA in the colorimetric solution. The white ceramic plate was placed in the dark at room temperature for 30 min, and then observed. The color changed to red, indicating that it could secrete IAA.

[0033] 1.3.3 Phosphorus dissolving test Microorganisms can dissolve calcium, phosphorus, iron and aluminum compounds in soil by producing organic acids and other substances, so as to promote the dissolution and utilization of soil ineffective phosphorus. The phosphorus-dissolving microorganisms were cultured in Mengjina organic phosphorus medium, and transparent circles were formed. The activated strains were inoculated on the Mengjina organic phosphorus medium, and each strain was repeated 3 times. After 30℃ culture for about one week, the colony diameter d1 and the phosphorus-dissolving circle diameter d2 were measured. The ratio of the phosphorus-dissolving circle diameter d2 to the colony diameter d1 was used as the standard for evaluating the phosphorus-dissolving capacity. The larger the ratio, the stronger the phosphorus-dissolving capacity.

[0034] 1.3.4 Amylolytic ability Each strain was inoculated on starch amylase identification medium plate and cultured for 2-4d, then poured into Gram iodine solution to cover the medium, and after 2 min, the Gram iodine solution was poured out to observe whether transparent circle was produced around the colony. If transparent circle was produced, it indicated that the strain had the ability to secrete amylase.

[0035] 1.3.5 Protease production ability detection The transparent circle method was used for determination. The test strains were inoculated in protease detection medium and cultured at 30℃ for 2d. The size of transparent circle around the colony was observed to determine the proteolytic ability of the strain.

[0036] 1.3.6 Cellulase production Each strain was inoculated on carboxymethyl cellulose sodium medium plate and cultured for 2-4d. After dyeing with sufficient 1g / l Congo red solution for 1h, the Congo red solution was poured out, and then sufficient 1 mol / l solution was added for rinsing. After 1h, the NaCl solution was poured out, and transparent circle around the colony was observed. If transparent circle was produced, it indicated that the strain could secrete cellulase.

[0037] 1.3.7 High temperature resistance determination Take 2 μl of seed liquid of the strain in a 50 mL centrifuge tube, add 5 mL of LB liquid medium, and cultivate at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C, respectively, at 180 r / min on a shaker for 30 h. After centrifugation, pour off the supernatant and resuspend with 5 mL ddH2O to measure OD 600 absorbance value.

[0038] 1.3.8 Determination of UV resistance After sterilization of the prepared LB liquid medium, 7 mL was taken in a 10 mL centrifuge tube, and a single colony was inoculated in the centrifuge tube and mixed. 1 mL of the mixed bacterial liquid was taken in a conical flask, and the conical flask was cultivated at 28°C, 180 r / min, in the dark and at 28°C, 180 r / min, under UV 254 illumination until the logarithmic growth phase. At 0, 0.5, 2, 4, 8, 11, 24, and 29 h, samples were taken, and the OD 600 value of the bacterial liquid was measured. The growth curve was plotted with time as the abscissa and the absorbance value as the ordinate.

[0039] 1.4 Plate confrontation experiment Bacillus sp. Bv47 was cultivated in LB liquid medium at 37°C and 180 r / min until the logarithmic growth phase, and the concentration was adjusted to 1×10 8 CFU / mL. L. rosyum Vd was cultivated in CM liquid medium at 28°C and 180 r / min until the logarithmic growth phase, and the concentration was adjusted to 1×10 8 CFU / mL. With the plate center as the reference, 10 μL of Bacillus sp. Bv47 bacterial suspension and L. rosyum Vd spore suspension were added at symmetrical positions (distance between two points ≥3 cm, distance from plate edge ≥1.5 cm). After the bacterial liquid was absorbed, the plate was inverted and cultured in a 28°C constant temperature incubator in the dark. The plate was taken out after about 7 days, and the growth state of the two microorganisms, the antibacterial circle in the confrontation area, and the mycelial inhibition were observed and recorded. Each treatment had 3 biological replicates, and the PDA plate inoculated with only L. rosyum Vd was used as the blank control.

[0040] 1.6 Pot experiment for prevention effect Take 1 mL of V991 bacterial liquid and add it to CM medium (6 g / L yeast extract, 6 g / L acid hydrolyzed casein, and 10 g / L sucrose) containing kanamycin and carbenicillin antibiotics, and cultivate at 28°C and 220 rpm. After 4-5 days (d) of cultivation, observe the spores under a microscope and count the spore concentration using a hemocytometer. When the spore concentration reaches 10 7CFU / mL, and the mycelium was filtered out with 4 layers of gauze, and the spore suspension was collected. When the cotton grew to the "two-leaf-one-heart" stage, the roots were soaked in the V991 spore suspension for 5 min to complete inoculation, and then the cotton was replanted in the nutrient soil; another bacillus strain Bv47 was adjusted to a concentration of 5 x 10 5 CFU / mL, and the mycelium was filtered out with 4 layers of gauze, and the spore suspension was collected. When the cotton grew to the "two-leaf-one-heart" stage, the roots were soaked in the V991 spore suspension for 5 min to complete inoculation, and then the cotton was replanted in the nutrient soil; another bacillus strain Bv47 was adjusted to a concentration of 5 x 10 ×4 )] x 100. The cotton genomic DNA was extracted, and the fungal biomass was detected in the ribosomal RNA gene ITS1 and ITS2 region (Z29511) in the genome DNA of L. rostrate, and the primers Vd-ITS-F / R were used, and the internal reference gene was B. velezensis (Table 1). The qRT-PCR reaction was performed on an ABI7500 Fast instrument, and the data was analyzed by 2 -ΔΔCt Method.

[0041] Table 1 Primers used in the present application

[0042] 2 Results and analysis 2.1 Phylogenetic analysis of bacillus strain Bv47 The extracted bacterial DNA was used as a template, and the universal primer 16S rDNA sequence was sequenced, and the sequence was searched and analyzed by NCBI Blast, and a phylogenetic tree was constructed based on the obtained gene sequence. It was found that Bv47 and MK367786 Figure 2 strain IICE-1 were in the same branch ( Figure 2 ), and the strain was preliminarily identified as Bacillus velezensis Figure 2 .

[0043] 2.2 Phenotype analysis of bacillus velezensis Bv47 on culture medium plate A series of plant growth-promoting traits were qualitatively tested by using a selective culture medium. On the NH3-producing determination medium, a obvious yellow color (A in Figure 2 ) was produced, indicating that the strain had ammonia-producing ability. After Salkowski's colorimetric method was used for determination, the reaction solution turned pink (B in Figure 2The presence of B in the colony indicates that strain Bv47 possesses the ability to produce indoleacetic acid. Strain Bv47 was inoculated onto potassium-solubilizing, cellulase-producing, protease-producing, amylase-producing, and potassium-solubilizing test media, respectively. The formation of a clear halo around the colony was observed to determine its enzyme-producing activity. The detection of a clear hydrolysis halo around the colony indicates that strain Bv47 produces cellulase (…). Figure 2 C in the protein), protease ( Figure 3 D), amylase ( Figure 3 The ability of strain Bv47 to produce a clear zone (E) after inoculation on potassium-solubilizing assay medium. Figure 4 The presence of F in the figure indicates that Bv47 has a certain potassium solubilizing ability.

[0044] 2.3 Determination and analysis of the UV resistance and high temperature resistance of strain Bv47 Growth curves reflect the growth patterns and reproductive characteristics of bacteria under different culture conditions. The effect of ultraviolet light on the growth changes of strain Bv47 is as follows: Fusarium oxysporum f. sp. As shown in Figure A, strain Bv47 maintained stable growth even after UV irradiation under culture conditions of 28℃ and 180 r / min. No growth stagnation or significant biomass reduction due to UV stress was observed, fully demonstrating the strain's good tolerance to UV radiation. lycopersici As shown in Figure B, strain Bv47 exhibits the characteristic of "better growth at 35℃ than at 30℃". At 25℃, the strain grows more slowly, with a lower OD... 600 The OD value was 1.6-2.1; growth improved at 30℃, but biomass remained below that at 35℃. The optimal growth range for this strain was 30℃-45℃, with the growth rate gradually increasing, and biomass reaching its peak at 35℃. 600 The value was 2.4-2.8. Growth of the strain began to be inhibited above 35℃, and the OD value reached 40℃. 600 The value can reach 1.7-2.2, indicating that Bv47 has good tolerance to high temperatures.

[0045] 2.4 Strain Bv47 has inhibitory effects on a variety of pathogens.

[0046] The results of the plate confrontation experiment show that ( Verticillium dahliae Bv47 showed significant inhibitory effects against the pathogens of mango anthracnose, tomato verticillium wilt, watermelon wilt, tomato wilt, and pepper blight, demonstrating broad-spectrum antibacterial potential. Specifically, this strain showed significant inhibitory effects against the tomato wilt pathogen (Fusarium oxysporum tomato-specific strain). Fusarium oxysporum f. sp. niveum Colletotrichum gloeosporioides ) and the pathogen of tomato Verticillium wilt (Verticillium dahliae, PhytophthoraThe inhibition rates of the two soil-borne pathogens were 62.2% and 31.8%, respectively, which significantly slowed down the growth rate of the two soil-borne pathogens; even against the watermelon wilt pathogen (Fusarium oxysporum watermelon-specific strain), capsici This strain also exhibits a certain antagonistic effect, with an inhibition rate of 40.0%. It is effective against the anthracnose pathogen of mango (Anthracnose asiaticus). Figure 5 The inhibition rate of the complex group was 61.3%, even against the pathogen of Phytophthora capsici (Phytophthora capsici). Figure 5 Figure 5 This strain can also exert a certain antagonistic effect against oomycete pathogens, with an inhibition rate of 32.1%.

[0047] 2.5 Bacillus belye Bv47 inhibits the growth of Verticillium dahliae. Bacillus belye Bv47 was cultured in LB liquid medium at 37°C with shaking at 180 r / min until the logarithmic growth phase, and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL, *Verticillium dahliae* V991 was cultured in CM liquid medium at 28℃ with shaking at 180 r / min until the logarithmic growth phase, and the concentration was adjusted to 1×10⁻⁶. 8 The CFU / mL concentration was used to determine the degree of antagonism between strain Bv47 and Verticillium dahliae using the plate confrontation method. The results are as follows: Figure 5 Studies have shown that strain Bv47 has a strong inhibitory effect on the growth of the pathogenic fungus *Verticillium dahliae*. Compared with the control group inoculated only with *Verticillium dahliae* V991, the growth of *Verticillium dahliae* on plates inoculated with both Bv47 and *Verticillium dahliae* V991 was significantly higher. Figure 5 In section A), the growth of *Verticillium dahliae* was significantly inhibited. Looking at the overall plate condition, *Verticillium dahliae* V991 colonies grew more vigorously in the control group, while the colony area of ​​*Verticillium dahliae* V991 in the experimental group was relatively smaller, indicating that *Bacillus belye* Bv47 has a significant inhibitory effect on the growth of *Verticillium dahliae*; this suggests that *Bacillus belye* Bv47 can produce certain substances that inhibit the growth and spread of *Verticillium dahliae*; further observation of hyphae ( Figure 6 In the control group (B), the mycelium of *Verticillium dahliae* was denser and grew better, while in the experimental group, the mycelium was sparse and its growth was affected. This indicates that *Bacillus belye* Bv47 has a significant antagonistic effect on *Verticillium dahliae*. Phenotypic observations from the potted plant control experiment showed that cotton plants inoculated only with *Verticillium dahliae* exhibited obvious wilting and yellowing symptoms, with some plants even dying completely. Figure 6 (B in the text); the corresponding disease index statistics and cotton root fungal biomass detection results show ( Figure 6Groups C and D showed the highest disease index and pathogen biomass, indicating an extremely high incidence of Verticillium wilt. However, cotton plants inoculated with Bacillus Bv47 showed significantly reduced leaf wilting, a significantly lower disease index compared to the V991-only inoculation group, and a marked decrease in pathogen biomass within the cotton plants. These results indicate that Bacillus Bv47 can significantly weaken the infectivity and colonization efficiency of Verticillium dahliae in cotton plants, thereby significantly inhibiting Verticillium wilt. In conclusion, it can be inferred that Bacillus Bv47 plays a crucial role in controlling Verticillium dahliae infection in cotton and mitigating the damage caused by Verticillium wilt.

[0048] 2.6 Phenotypic Analysis of the Cotton Growth-Promoting Ability of Bacillus belye Bv47 To investigate the growth-promoting effect of Bacillus belye Bv47 on cotton, two-week-old cotton seedlings with uniform growth were selected, transplanted, and cultured until growth was stable. Sterile water treatment was used as a control (CK). 100 mL of a 1×10⁻⁶ solution was used. 8 The roots of cotton treated with a Bacillus Bv47 suspension at CFU / mL were irrigated with 15 replicates. Plant growth indicators were measured after 18 days. The results showed that cotton treated with Bacillus Bv47 exhibited phenotypic differences ( Figure 6 In sample A), the results were significantly better than the control, with more robust and vigorous plants. Specifically, root length (…) was the most significant indicator. Figure 6 B), plant height ( Figure 6 C in the middle), stem thickness ( Figure 6 D) Fresh weight ( ​ E in the middle), leaf area ( ​ F in the middle) and root dry weight ( ​ The values ​​of G in the control group were significantly higher than those in the control group (**** indicates P<0.0001), indicating that Bacillus Bv47 can effectively promote cotton root development, plant height increase and biomass accumulation, showing good potential for potted plant growth promotion, and providing data support for its application in the growth regulation of cotton and other crops in agricultural production.

[0049] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A Bacillus velezensis strain Bacillus velezensis Strain Bv47, characterized in that, The strain Bv47 is classified as Bacillus velezensis Bacillus velezensis and was preserved in China General Microbiological Culture Collection Center on November 4, 2025 with the preservation number of CGMCC No. 36499.

2. A fermentation broth or bacterial suspension containing the bacterial strain Bv47 of claim 1.

3. Use of the bacterial strain Bv47 of claim 1 or the fermentation broth or bacterial suspension of claim 2 in controlling tomato fusarium wilt, tomato verticillium wilt, watermelon fusarium wilt, mango anthracnose, pepper late blight, or cotton verticillium wilt.

4. Use of the bacterial strain Bv47 according to claim 1 or the fermentation broth or bacterial suspension according to claim 2 for inhibiting plant pathogenic bacteria, including: fusarium oxysporum f. sp. lycopersici Fusarium oxysporum f. sp. lycopersici verticillium dahliae Verticillium dahliae fusarium oxysporum f. sp. niveum Fusarium oxysporum f. sp. niveum colletotrichum gloeosporioides Colletotrichum gloeosporioides or phytophthora capsici Phytophthora capsici .

5. Use of the bacterial strain Bv47 of claim 1 or the fermentation broth or bacterial suspension of claim 2 in promoting cotton growth.

6. The use of claim 5, wherein the promoting cotton growth comprises increasing at least one of root length, plant height, stem diameter, fresh weight, leaf area, and root dry weight of the cotton.

7. A microbial inoculum for promoting cotton growth, characterized in that, The active ingredient of the bacterial agent comprises the bacterial strain Bv47 of claim 1.

8. Use of the bacterial strain Bv47 of claim 1 or the bacterial agent of claim 7 in producing a bio-organic fertilizer suitable for cotton.

9. A bio-organic fertilizer, characterized by, The bio-organic fertilizer contains the bacterial strain Bv47 of claim 1 or the bacterial agent of claim 7.

10. A method of promoting cotton growth, characterized by, The bacterial suspension of the strain Bv47 according to claim 1 was used for the root drenching of cotton seedlings at a concentration of 1 x 10 8 CFU / mL.

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