Bacillus safensis endophyte hmb39572 and application thereof

Abstract

This invention provides a strain of cotton endophytic Bacillus saforticus HMB39572 and its applications, belonging to the field of agricultural microbiology technology. This strain, isolated from healthy cotton stems from cotton fields where broccoli residues were returned to the field, can tolerate high concentrations of salt and a wide pH range, and exhibits significant antagonistic effects against Verticillium dahliae and Rhizoctonia solani. Seed coating with the dry powder of this strain shows good control effects against cotton Verticillium wilt and damping-off, with control efficacies reaching 79.41% and 76.23%, respectively. Simultaneously, this strain can promote the growth of cotton plants in terms of height, stem diameter, fresh weight, and dry weight, increase leaf area and chlorophyll content, and significantly improve yield traits such as boll weight and lint percentage, as well as quality indicators such as fiber length and strength. The strain of this invention has strong environmental adaptability and outstanding disease prevention and growth-promoting effects, making it suitable for green control and quality improvement of cotton soil-borne diseases.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a cotton endophytic Bacillus sabensis strain HMB39572 and its applications. Background Technology

[0002] Cotton is an important economic crop in my country, and cotton production occupies a vital position in the national economy. Cotton Verticillium wilt, a typical soil-borne vascular disease caused by *Verticillium dahliae*, is characterized by its wide spread, high pathogenicity, and difficulty in control, and has become a key bottleneck restricting the sustainable development of the cotton industry. This pathogen can survive in the soil for a long time as microsclerotia, invading the vascular bundles of the plant through the roots, leading to wilting, yellowing, and shedding of cotton leaves. In severe cases, the entire plant dies, significantly reducing cotton yield and fiber quality.

[0003] Current methods for controlling cotton Verticillium wilt have significant limitations. Traditional soil fumigants, such as methyl bromide, while highly effective against soil-borne pathogens, have been banned globally due to their severe environmental damage and threats to human and animal health. Chemical control remains the primary method for controlling cotton Verticillium wilt in production. However, the number of chemical agents registered in my country for cotton Verticillium wilt control is limited. In production, fungicides such as carbendazim, triadimefon, and thiophanate-methyl are commonly used for soil treatment or seed coating. But long-term, large-scale, and singular use of chemical pesticides easily leads to soil residues and environmental pollution, disrupts the soil microbial community structure and rhizosphere microecological balance, reduces soil fertility, and induces drug resistance in pathogens, resulting in a gradual decline in control effectiveness and making long-term, stable control difficult.

[0004] Agricultural control measures such as crop rotation, breeding of disease-resistant varieties, and returning cruciferous crop residues to the field can, to some extent, reduce the occurrence of soil-borne diseases in cotton. Rotating gramineous crops with cotton can optimize the soil microbial community and reduce the initial number of pathogens. Returning cruciferous crop residues, such as broccoli, to the field can release antibacterial active substances, increase the abundance of beneficial microorganisms such as Bacillus in the soil, and inhibit the reproduction and infection of Verticillium dahliae. However, crop rotation is difficult to promote on a large scale due to limitations in arable land resources and planting structure. Cotton varieties resistant to Verticillium wilt are scarce and have long breeding cycles. The effectiveness of returning crop residues to the field is greatly affected by climate, soil conditions, and the method of return, and its application alone is insufficient to meet production needs.

[0005] Biological control, with its advantages of being environmentally friendly, having strong persistence, and being less prone to developing resistance, has become an important direction for the green control of soil-borne diseases in cotton. Endophytic Bacillus bacteria can colonize within plants and exert their disease-preventing and growth-promoting effects through multiple mechanisms, including antagonizing pathogens, inducing systemic resistance in the host plant, and promoting plant growth. They are biocontrol resources with great application potential. However, existing biocontrol strains show inconsistent efficacy against cotton Verticillium wilt and damping-off, and have weak environmental adaptability, making it difficult to stably colonize and function under complex field conditions such as salinity, alkalinity, and acid-base stress. Therefore, the targeted screening of stress-resistant, highly effective, and growth-promoting / quality-improving endophytic Bacillus bacteria from healthy cotton plants with returned plant residues to the field, and the development of highly efficient microbial agents, is of great significance for constructing a green control technology system for soil-borne diseases in cotton and ensuring the high-quality development of the cotton industry. Summary of the Invention

[0006] In view of this, the present invention provides a cotton endophytic Bacillus sabensis strain HMB39572 and its applications to solve the above problems.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a cotton endophytic Bacillus safensis strain HMB39572, which was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.37014.

[0009] The present invention also provides a microbial agent comprising the aforementioned Bacillus salsa HMB39572.

[0010] Preferably, the formulation of the microbial agent includes liquid and dry powder.

[0011] Preferably, the microbial agent is a dry powder, and the preparation method of the dry powder is as follows: centrifuging the bacterial culture of Bacillus sabolicii HMB39572, discarding the supernatant, and obtaining a concentrated fermentation broth of the strain; mixing the concentrated fermentation broth of the strain with talc powder at a mass ratio of 0.5~1:1, and drying, to obtain the final product; the content of Bacillus sabolicii HMB39572 in the seed coating dry powder is 1.0~2.0×10⁻⁶. 9 CFU / g.

[0012] This invention provides the application of the aforementioned Bacillus salsa HMB39572 in any one or more of the following:

[0013] (1) Application in the prevention and control of pathogens;

[0014] (2) Application in the prevention and control of cotton diseases;

[0015] (3) Application in promoting cotton plant growth;

[0016] (4) Application in improving cotton yield and quality;

[0017] (5) Application in increasing the content of defense enzymes in cotton plants;

[0018] (6) Application in the preparation of agents for the prevention and control of pathogenic bacteria;

[0019] (7) Application in the preparation of fungal agents for the prevention and control of cotton diseases;

[0020] (8) Application in the preparation of microbial agents that promote the growth of cotton plants;

[0021] (9) Application in the preparation of microbial agents to improve cotton yield and quality;

[0022] (10) Application in the preparation of microbial agents that increase the content of defense enzymes in cotton plants.

[0023] Preferably, the pathogens include Verticillium dahliae, Rhizoctonia solani, Fusarium oxysporum (cotton-specific), Fusarium oxysporum (cucumber-specific), Fusarium oxysporum (tomato-specific), Fusarium pseudoverticum, Fusarium graminearum, and / or Fusarium pseudograminearum.

[0024] Preferably, the cotton diseases include damping-off and / or Verticillium wilt.

[0025] Preferably, the cotton plant defense enzymes include peroxidase, polyphenol oxidase, and / or superoxide dismutase.

[0026] The present invention also provides a method for preventing and controlling cotton diseases, wherein the cotton seeds are coated with the seed coating powder, wherein the mass of the seed coating powder is 5-10% of the seed mass.

[0027] Preferably, the cotton diseases include damping-off and / or Verticillium wilt.

[0028] By adopting the above technical solution, the present invention has the following beneficial effects:

[0029] 1. The biocontrol bacterium *Bacillus safras* provided by this invention is a plant endophyte that has excellent antagonistic activity against both *Verticillium dahliae* and *Rhizoctonia solani*.

[0030] 2. The biocontrol bacterium *Bacillus safranin* provided by this invention can increase cotton plant height, fresh weight, and dry weight, and has a significant growth-promoting effect on cotton plants.

[0031] 3. The biocontrol bacterium *Bacillus safras* provided by this invention can tolerate high concentrations of salt and a wide range of acids and alkalis.

[0032] 4. The biocontrol bacterium *Bacillus safranin* provided by this invention can increase the leaf area and chlorophyll content of cotton leaves.

[0033] 5. The biocontrol bacterium Bacillus salsa HMB39572 provided by the present invention can effectively induce the enhancement of the activity of defense-related enzymes POD and PPO and the increase of PRO content, while reducing the MDA content of cotton plants.

[0034] 6. The seed treatment dry powder prepared by the biocontrol bacterium Bacillus safoetida HMB39572 provided by the present invention achieved a control effect of 76.23% and 79.41% on cotton damping-off and Verticillium wilt, respectively, and can significantly reduce the severity of cotton Verticillium wilt.

[0035] 7. The seed treatment dry powder prepared by the biocontrol bacterium Bacillus salsa HMB39572 provided by the present invention can improve the yield traits (single boll weight, lint percentage and seed index) and quality traits (average length of upper half, uniformity index and breaking strength) of cotton. Attached Figure Description

[0036] Figure 1 The graph shows the inhibitory effect of strain HMB39572 on different pathogens.

[0037] Figure 2 To establish a phylogenetic tree for strain HMB39572.

[0038] Figure 3 The figure shows the effect of strain HMB39572 on the proline content in cotton leaves.

[0039] Figure 4 The figure shows the effect of strain HMB39572 on the malondialdehyde content in cotton leaves.

[0040] Figure 5 The figure shows the effect of strain HMB39572 on the superoxide dismutase activity of cotton plants.

[0041] Figure 6 The figure shows the effect of strain HMB39572 on the activity of polyphenol oxidase in cotton leaves.

[0042] Figure 7 The figure shows the effect of strain HMB39572 on the peroxidase activity of cotton leaves.

[0043] Biological Preservation Instructions

[0044] The taxonomic name of the Bacillus safensis strain HMB39572 of this invention is Bacillus safensis. This strain was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.37014. Detailed Implementation

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Example 1. Isolation and Identification of Strain HMB39572

[0047] The strain HMB39572 of Bacillus sabinatus was isolated from healthy cotton stems after broccoli residues were returned to the field by the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences.

[0048] Healthy cotton samples were collected from cotton fields treated with broccoli residues in Quzhou County, Handan City, Hebei Province. After being brought back to the laboratory, endophytic Bacillus bacteria were isolated from the plants using high-temperature heating and gradient dilution methods. The specific steps are as follows:

[0049] (1) Take healthy cotton plants collected from the field and treated with broccoli residue, rinse the plant roots and stems with tap water, cut them into 1-2 cm long sections, disinfect them with 75% alcohol for 3 min, rinse them with sterile water, dry the surface moisture with sterile filter paper, peel off the outer skin of the plant and cut them into 1 mm thick slices for later use.

[0050] (2) Place several samples prepared in step (1) into LB culture medium and shake for 12 h. Take 10 mL of culture medium into a sterile test tube, place it in an 80℃ water bath for 10 min, take it out and cool it to room temperature to obtain an endophytic bacterial suspension.

[0051] (3) Take 1 mL of endophytic bacterial suspension and dilute it serially. Take 10 mL of the solution. -2 10 -3 and 10 -4 Three concentrations of dilution were spread at 100 μL onto LB agar plates and incubated at 30°C for 12 h. Colonies with vigorous growth and different morphologies and colors were selected for staining and microscopic examination. Bacillus strains were screened, transferred, purified, and stored at -20°C for long-term preservation.

[0052] (4) Antibacterial experiment: *Verticillium dahliae*, the pathogen causing cotton Verticillium wilt; *Rhizoctonia solani*, the pathogen causing cotton damping-off; *Fusarium oxysporum f. sp. vasinfectum*, the pathogen causing cucumber wilt; *Fusarium oxysporum f. sp. cucumerinum*, the pathogen causing tomato wilt; *Fusarium oxysporum f. sp. lycopersici*, the pathogen causing maize stalk rot; *Fusarium graminearum*, the pathogen causing wheat stem rot; and *Fusarium pseudograminearum*, a fungus preserved in the Plant Disease Biological Control Laboratory of the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. Using *Pseudomonas pseudograminearum* as the target bacterium, the pathogen was pre-inoculated onto PDA plates and incubated at 25°C until the mycelium completely covered the plate. A 5 mm diameter mycelial cake was then punched and inoculated into the center of a new 9 cm diameter PDA plate. Purified *Bacillus* was then inoculated using a cross-hatching method 3 cm from the periphery of the mycelial cake. Plates inoculated only with the pathogen served as a control. All plates were incubated at 25°C. After the control colonies had fully colonized the plate, the diameter of the inhibition zone, the diameter of the antagonistic bacteria, and the diameter of the pathogen colony were measured, and the inhibition rate was calculated. The results are shown in Table 1 and [Table data missing]. Figure 1 As shown.

[0053] Inhibition rate (%) = (Coronavirus diameter of control group - Coronavirus diameter of treatment group) / Coronavirus diameter of control group × 100.

[0054] Table 1. Inhibitory effects of strain HMB39572 against different pathogens.

[0055]

[0056] 2. Identification of strain HMB39572

[0057] DNA was extracted from strain HMB39572 using a modified CTAB method, and the bacterial whole genome was sequenced by Shanghai Meiji Biotechnology Co., Ltd. The resulting series was then analyzed by selecting 19 strains that were most closely related at the species level based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf). A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software for evolutionary analysis. Figure 2 The results showed that strain HMB39572 and strain B. safensis belong to the same branch, with a Bootstrops value of 0.904, indicating a close phylogenetic relationship. Therefore, strain HMB39572 was identified as Bacillus safensis.

[0058] Example 2. Determination of pH and salt tolerance of strain HMB39572

[0059] 1. Determination of the optimal pH for strain HMB39572

[0060] The strain HMB39572 from Example 1 was inoculated into 5 mL of liquid LB medium and cultured at 180 r / min and 37℃ for 48 h with shaking to obtain fermentation broth. The fermentation broth was inoculated into LB liquid medium of different pH (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) at a volume ratio of 1:20, with conventional LB medium at pH=7 as the control. The culture was carried out at 30℃ and 180 r / min for 24 h with shaking, and the absorbance at 600 nm was measured. The strain was serially diluted with sterile water. According to the growth of the strain, 100 µL of the appropriate concentration gradient was evenly spread on LB solid plates and incubated at 30℃ for 12 h. The number of colonies grown on the plates was counted. Each concentration was repeated 3 times to compare the growth of the strain at different pH concentrations. The results are shown in Table 2.

[0061] Table 2. Determination of the optimal pH for strain HMB39572

[0062]

[0063] Note: In the same column, different letters indicate significant differences between groups (p < 0.05), and the same applies below.

[0064] The results in Table 2 show that strain HMB39572 can grow well in a pH range of 4 to 12, indicating that it can tolerate a wide range of acid and alkali conditions.

[0065] 2. Salt tolerance test of strain HMB39572

[0066] The strain HMB39572 from Example 1 was inoculated into 5 mL of liquid LB medium and cultured at 180 r / min and 37℃ for 48 h with shaking to obtain the fermentation broth. The fermentation broth of strain HMB39572 was inoculated into LB liquid medium with different NaCl concentrations (2%, 5%, 7%, 10%, 15%, and 20%) at a volume ratio of 1:20, with conventional LB medium (NaCl concentration of 0.5%) as a control. The culture was carried out at 30℃ and 180 r / min with shaking for 24 h, and the absorbance at 600 nm was measured. The strain was serially diluted with sterile water. According to the growth of the strain, 100 µL of the appropriate concentration gradient was evenly spread on LB solid plates and incubated at 30℃ for 12 h. The number of colonies growing on the plates was counted. Each concentration was repeated 3 times to compare the effect of different NaCl concentrations on the growth of the strain. The results are shown in Table 3.

[0067] Table 3 Salt tolerance test of strain HMB39572

[0068] NaCl content <![CDATA[OD 600 ]]> Lg(CFU) Normal LB (0.5%) 1.71±0.04a 7.81±0.04c 2% 1.63±0.03b 8.99±0.10a 5% 1.59±0.03c 8.58±0.20b 7% 1.38±0.01d 6.70±0.04d 10% 0.83±0.01e 6.56±0.05d 15% 0.24±0.01f 1.80±0.18e 20% 0.23±0.01f 1.52±0.20f

[0069] The results in Table 3 show that the strain HMB39572 can grow in a range of 2%-5% NaCl content, indicating that it has significant salt tolerance.

[0070] Example 3. Preparation of HMB39572 seed coating powder

[0071] Strain HMB39572 was inoculated into 5 mL of liquid LB medium and cultured at 180 r / min and 37℃ for 24 h with shaking to obtain seed fermentation broth. This seed fermentation broth was then transferred to fresh LB medium and cultured at 180 r / min and 37℃ for 48 h with shaking. The broth was then concentrated by centrifugation at 10000 rpm. The concentrated fermentation broth was then mixed with talc powder at a 1:1 mass ratio, air-dried, ground, and sieved to prepare bacterial powder for later use. The concentration of Bacillus in the bacterial powder was between 1.0 × 10⁻⁶. 9 CFU / g ~2.0×10 9 Between CFU / g.

[0072] Example 4. Evaluation of the control efficacy of strain HMB39572 against cotton damping-off in greenhouse pot culture

[0073] 1. Preparation of Rhizoctonia solani spore suspension

[0074] After activating and culturing *Rhizoctonia solani* on PDA plates at 25°C for 7 days, the culture was inoculated into PDB medium and cultured at 25°C with shaking at 180 r / min for 5 days. The mycelia in the culture medium were filtered out using four layers of sterile gauze. The culture was centrifuged at 8000 r / min for 20 min, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in sterile water to obtain a spore suspension. The number of spores was counted under an optical microscope, and the concentration of the spore suspension was determined to be 1.0 × 10⁻⁶. 9 CFU / mL.

[0075] 2. Pot control experiment of strain HMB39572 against cotton damping-off.

[0076] (1) Preparation of culture medium containing damping-off pathogen: Sand, substrate and vermiculite were mixed in a volume ratio of 1:1:1 to make a mixed soil, which was thoroughly dried, and then mixed according to the following ratio: 6.0 × 10⁻⁶ g / g dry soil. 6 The concentration of each spore is determined by mixing the prepared Rhizoctonia solani spore suspension with the mixed soil to prepare a culture medium containing the pathogen.

[0077] (2) Seed coating: Select high-quality cotton seeds, disinfect with 3% sodium hypochlorite for 1 min, rinse several times with sterile water, prepare a 5% (m / v) solution of sodium carboxymethyl cellulose film-forming agent with water, coat cotton seeds (Ejing No. 1) with the seed coating dry powder of strain HMB39561 prepared in Example 3 at 5% and 10% of seed weight, add a small amount of the prepared 5% sodium carboxymethyl cellulose solution to moisten the seeds, shake until the bacterial powder is evenly coated on the seed surface, spread it out and air dry to complete the seed coating.

[0078] (3) Sowing and survey: Coated cotton seeds were sown into flowerpots containing a culture medium containing pathogens, 20 seeds per pot, and covered with about 2 cm of fine soil, ensuring complete coverage. After sowing, the number of seedlings was surveyed every 3 days until no more seedlings emerged. The number of diseased seedlings and the total number of seedlings were surveyed every 3 days until no more seedlings died in each treatment. Each treatment was repeated 4 times. The emergence rate, disease incidence, and control effect were calculated, and the results are shown in Table 4.

[0079] Incidence rate (%) = Number of diseased seedlings / Total number of seedlings × 100;

[0080] Prevention and control effect (%) = (incidence rate of control group - incidence rate of treatment group) / incidence rate of control group × 100.

[0081] Emergence rate (%) = (Number of seedlings / Number of seeds sown) × 100

[0082] Table 4. Control efficacy of biocontrol agents against cotton damping-off.

[0083]

[0084] The results of the greenhouse pot experiment showed that the incidence of disease was significantly reduced by the HMB39572 inoculant treatment. The inoculant was used at 5% and 10% of the seed weight to control cotton damping-off disease at 65.83% and 74.29% respectively.

[0085] Example 5. Evaluation of the control efficacy of strain HMB39572 against cotton Verticillium wilt in greenhouse pot culture.

[0086] 1. Preparation of Verticillium dahliae spore suspension

[0087] The preserved *Verticillium dahliae* Vd991 spore suspension was inoculated into CM culture medium and cultured at 25℃ and 180 r / min with shaking for 5 days. The mycelia in the culture medium were filtered out using four layers of sterile gauze. The suspension was centrifuged at 8000 r / min for 20 min, the supernatant was discarded, and the bacterial cells were collected and resuspended in sterile water to obtain a spore suspension. The number of spores was counted under an optical microscope, and the concentration of the spore suspension was found to be 1.0 × 10⁻⁶. 9 CFU / g.

[0088] 2. Pot control experiment of strain HMB39572 against cotton Verticillium wilt

[0089] (1) Preparation of culture medium containing Verticillium wilt pathogen: Sand, substrate and vermiculite were mixed in a volume ratio of 1:1:1 to make a mixed soil, which was thoroughly dried, and then mixed according to the following ratio: 6.0 × 10⁻⁶ g / g dry soil. 6 The concentration of each spore is determined by mixing the prepared Verticillium dahliae spore suspension with the mixed soil to prepare the diseased soil.

[0090] (2) Seed coating: Cotton seeds were coated with the HMB39561 seed coating powder prepared in Example 3 at 5% and 10% of the seed weight, respectively. The coating method was the same as in Example 4.

[0091] (3) Sowing and survey: Coated cotton seeds were sown into flowerpots containing a culture medium containing pathogens, 20 seeds per pot, and covered with about 2 cm of fine soil, so as to completely cover the cotton seeds. Fifty days after sowing, the incidence of Verticillium wilt in greenhouse cotton was investigated according to the disease survey standards (the disease classification standards for cotton plants are: level 0: healthy plants; level 1: less than 25% of leaves are diseased; level 2: 25%~50% of leaves are diseased; level 3: 51%~75% of leaves are diseased; level 4: more than 75% of leaves are diseased). The disease index and control efficacy were calculated, and the results are shown in Table 5.

[0092] Disease index = Σ (number of diseased plants at each level × number of each level) / (total number of plants surveyed × representative value of the highest disease level) × 100;

[0093] Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0094] Table 5. Biocontrol effect of strain HMB39572 against cotton Verticillium wilt.

[0095]

[0096] The results of the greenhouse pot experiment showed that the disease index of the HMB39572 strain was significantly reduced, and the control effect of the inoculant on cotton Verticillium wilt was 58.99% and 79.40% when applied at 5% and 10% of the seed weight, respectively.

[0097] Example 6. Growth-promoting effect of strain HMB39572 on cotton plants

[0098] The seed coating powder of strain HMB39572, prepared according to the method of Example 3, was used to coat 10% of the seed weight, with uncoated seeds as a control. The coating method and greenhouse pot cultivation conditions were the same as in Example 4. After 50 days of cultivation, the growth-promoting effect of strain HMB39572 on cotton plants was investigated, and the results are shown in Table 6.

[0099] Table 6. Effects of biocontrol bacteria on cotton growth

[0100] deal with Stem diameter / mm Total fresh weight / g Fresh weight on the ground / g underground fresh weight / g Total dry weight / g Dry weight on the ground / g underground dry weight / mg HMB39572 Coating Processing 2.71±0.01 a 2.21±0.03 a 1.91±0.04a 0.30±0.05a 0.51±0.02a 0.37±0.04a 135±43.59a CK 2.42±0.01 b 1.63±0.04 b 1.43±0.02b 0.20±0.05a 0.34±0.01b 0.25±0.02b 90±14.14a

[0101] The results in Table 6 show that when the inoculant HMB39572 was applied at 10% of the seed weight, the cotton plant stem diameter, fresh weight (above ground and below ground), and dry weight (above ground and below ground) were all higher than those of the blank control treatment. The growth promotion rates of stem diameter, total fresh weight, above ground fresh weight, below ground fresh weight, total dry weight, and above ground dry weight were 11.98%, 35.69%, 33.68%, 50%, 47.79%, 47%, and 50%, respectively.

[0102] Example 7. Effects of strain HMB39572 on leaf area and chlorophyll content of cotton leaves

[0103] Seed coating powder of strain HMB39572, prepared according to the method in Example 3, was used to coat 10% of the seeds by weight, with uncoated seeds serving as a control. The coating method and greenhouse pot cultivation conditions were the same as in Example 4. After 50 days of cultivation, the leaf area of ​​leaves collected from the same position at the top of cotton plants in different treatment groups was measured using a leaf area meter (Zhejiang Top Cloud Agriculture Technology Co., Ltd.). The results showed that treatment with strain HMB39572 increased the leaf area of ​​cotton leaves by 10.15%. The chlorophyll content of leaves collected from the same position at the top of cotton plants in different treatment groups was measured using a chlorophyll content meter (Shijiazhuang Shiya Technology Co., Ltd.), and the results are shown in Table 7.

[0104] Table 7 Effects of biocontrol bacteria on leaf area and chlorophyll content of cotton leaves

[0105] deal with <![CDATA[Leaf area (mm 2 )]]> Chlorophyll content (SPAD) HMB39572 Coating Processing 2049.08±26.47 a 59.88±1.37 a CK 1860.33±34.54 b 48.81±1.52 b

[0106] The results in Table 7 show that treatment with strain HMB39572 increased the chlorophyll content of cotton by 22.68%.

[0107] Example 8. Effect of strain HMB39572 on proline content in cotton leaves

[0108] The seed coating powder of strain HMB39572, prepared according to the method in Example 3, was used to coat 10% of the seeds by weight, with uncoated seeds serving as a control. The coating method and greenhouse pot cultivation conditions were the same as in Example 4. After 50 days of cultivation, the proline (PRO) content was determined using the acidic ninhydrin method. The specific operating steps are as follows:

[0109] Weigh 1 g of fresh cotton leaves, cut the sample into small pieces, place them in a mortar, add 3 mL of 80% ethanol, grind and homogenize, pour the resulting liquid into a test tube, seal and extract for 60 min, then add activated carbon powder at a ratio of 0.025 g per mL of extract, shake thoroughly, filter out the activated carbon powder with filter paper, and rinse the test tube, residue, and filter paper with 80% ethanol at least three times. Evaporate the added ethanol from the obtained filtrate in an 80℃ water bath, and make up to 10 mL to obtain the test solution. Add 1 g of artificial zeolite to the test solution, shake for 10 min, filter with filter paper, take 5 mL of filtrate, add 5 mL of glacial acetic acid and 5 mL of acidic ninhydrin, mix well, and use distilled water as a blank control. Keep in a 100℃ water bath for 60 min, cool to room temperature, transfer to a separatory funnel, add 5 mL of benzene, and perform an extraction reaction. Measure the absorbance of the red reaction product at 515 nm. Plot the concentration of proline on the x-axis and the OD value on the y-axis. 515 A standard curve was established with the ordinate as the vertical axis. The proline content was calculated using the standard curve, and the results are as follows: Figure 3 As shown.

[0110] Figure 3 The results showed that treatment with strain HMB39572 significantly increased the proline content in cotton leaves, with an increase of 36.05%. This indicates that treatment with the strain enhanced the plant's proline content, improved its adaptability to abiotic stress induced by Verticillium wilt, and reduced physiological damage caused by stress.

[0111] Example 9. Effect of strain HMB39572 on malondialdehyde content in cotton leaves

[0112] Seed-coating powder of strain HMB39572 prepared according to the method in Example 3 was used to coat 10% of the seeds, with uncoated seeds as a control. The coating method and greenhouse pot cultivation conditions were the same as in Example 4. After 50 days of cultivation, the MDA content in cotton leaves of different treatments was determined using the thiobarbituric acid method. The specific operation steps are as follows: Weigh 0.5 g of cotton leaves, cut them into small pieces, place them in a mortar, add 5 mL of 50 mmol / L phosphate buffer, and grind them into a homogenate in an ice bath. Transfer the resulting mixture to a centrifuge tube and centrifuge at 10000 r / min at 4℃ for 10 min. Take the supernatant as the crude enzyme extract of the plant sample. Add 2 mL each of the supernatant and 0.5% thiobarbituric acid to a 5 mL centrifuge tube, boil in a water bath for 20 min, and then immediately cool to room temperature in an ice box. Centrifuge again at 5000 r / min for 15 min, collect the supernatant and measure its volume. Use 0.5% thiobarbituric acid solution as a blank control to measure OD. 534 and OD 600 The result is as follows Figure 4 As shown.

[0113] Malondialdehyde (MDA) content C = (ΔOD × V) / 0.15d × W. Where C: MDA content, in μmol / g; d: cuvette path length; ΔOD: OD0. 534 -OD 600 V: Total volume of supernatant; W: Sample mass (g).

[0114] Figure 4 The results showed that treatment with strain HMB39572 significantly reduced the malondialdehyde (MDA) content in cotton leaves, with a reduction of 28.43%. This indicates that the strain treatment inhibited lipid peroxidation, reduced cell membrane damage, and maintained cell structural stability.

[0115] Example 10. Effects of strain HMB39572 on the activity of defense enzymes in cotton leaves

[0116] Seed coating powder of strain HMB39572 prepared according to the method of Example 3 was used to coat 10% of the seeds. The coating method and greenhouse pot cultivation conditions were the same as in Example 4, with uncoated seeds as a control. After 50 days of cultivation, the activity of defense enzymes in cotton leaves under different treatments was measured. The specific operation steps are as follows: 0.5 g of cotton leaves were weighed, chopped, and placed in a mortar. 5 mL of 50 mmol / L phosphate buffer was added, and the mixture was ground into a homogenate in an ice bath. The resulting mixture was then centrifuged at 10,000 r / min and 4℃ for 10 min. The supernatant was taken as the crude enzyme extract of the plant sample. Peroxidase (POD) activity was determined by the guaiacol colorimetric method, polyphenol oxidase (PPO) activity was determined by the catechol method, and superoxide dismutase (SOD) activity was determined by the nitroblue tetrazolium (NBT) photochemical reduction method.

[0117] The results showed that treatment with strain HMB39572 significantly increased the activity of superoxide dismutase in cotton leaves, with an increase of 30.59%. Figure 5 Treatment with strain HMB39572 significantly increased the activity of polyphenol oxidase in cotton leaves, with an increase of 33.31%. Figure 6 Treatment with strain HMB39572 significantly increased the activity of peroxidase in cotton leaves, with an increase of 18.34%. Figure 7 ).

[0118] Example 11. Field control efficacy evaluation of strain HMB39572 against cotton damping-off and Verticillium wilt.

[0119] Using "Ejing No. 1" as the test cotton variety, the seed coating powder of strain HMB39572 was prepared according to the method of Example 3. The seed coating powder was used to coat 10% of the seeds. Uncoated seeds were used as a control. The coating method was the same as in Example 4.

[0120] The field efficacy of strain HMB39572 against different cotton diseases was evaluated in cotton fields with severe cotton disease outbreaks in Dingxing County, Baoding City, Hebei Province. Talc-coated seeds served as a control, and each treatment was replicated four times. Before seedling establishment, the incidence of cotton damping-off was investigated. The grading criteria for damping-off were: 0: no lesions at the stem base; 1: lesions at the stem base covering less than 1 / 3 of the total stem circumference; 3: lesions at the stem base covering 1 / 3 to 1 / 2 of the total stem circumference; 5: lesions at the stem base covering 1 / 2 to 3 / 4 of the total stem circumference; 7: lesions at the stem base covering more than 4 / 3 of the total stem circumference; 9: diseased and dead seedlings. The damping-off disease severity index and control efficacy were calculated.

[0121] The occurrence of Verticillium wilt in cotton was investigated during the flowering and boll-forming stage. The grading criteria for Verticillium wilt during this stage were as follows: Grade 0: Healthy plants with no leaf lesions; Grade 1: Plants with 25% leaf lesions; Grade 2: Plants with 26%–50% leaf lesions; Grade 3: Plants with 51%–75% leaf lesions; Grade 4: Plants with 75%–100% leaf lesions. The disease index and control efficacy were calculated. After cotton harvest, the effects on yield traits (single boll weight, lint percentage, seed index) and quality traits (average upper boll length, uniformity index, breaking strength, micronaire value) were evaluated. The results are shown in Tables 8 and 9.

[0122] Table 8. Field control efficacy of biocontrol agents against cotton damping-off and Verticillium wilt.

[0123]

[0124] The experimental results in Table 8 show that when the inoculant HMB39572 was applied at 10% of the seed weight, the disease index of cotton damping-off and Verticillium wilt was significantly reduced compared with the control (CK), and the field control efficacy was 76.23% and 79.41%, respectively.

[0125] Table 9. Effects of biocontrol agents on cotton yield and quality.

[0126]

[0127] The results in Table 9 show that the HMB39572 inoculant treatment group significantly improved boll weight, lint percentage and seed index, and significantly improved the average length, uniformity and breaking strength of the upper half of the cotton.

[0128] As can be seen from the above embodiments, the present invention provides a cotton endophytic Bacillus spp. HMB39572 and its application. The strain of the present invention has a good inhibitory effect on a variety of plant pathogens, and can significantly promote cotton growth and improve yield and quality.

[0129] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus safranin ( Bacillus safensis HMB39572, characterized in that, This strain was deposited on December 17, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 17, 2025, and the accession number is CGMCC No. 37014.

2. A microbial inoculant, characterized in that, The bacterial agent comprises Bacillus salsa HMB39572 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The formulations of the microbial agent include liquid and dry powder.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is a dry powder, and the preparation method of the dry powder is as follows: centrifuge the bacterial culture of Bacillus sabolicii HMB39572, discard the supernatant, and obtain a concentrated fermentation broth of the strain; mix the concentrated fermentation broth of the strain with talc powder at a mass ratio of 0.5~1:1, and dry to obtain the final product; the content of Bacillus sabolicii HMB39572 in the seed coating dry powder is 1.0~2.0×10⁻⁶. 9 CFU / g.

5. The use of Bacillus salsa HMB39572 as described in claim 1 in any one or more of the following: (1) Application in the prevention and control of pathogens; (2) Application in the prevention and control of cotton diseases; (3) Application in promoting cotton plant growth; (4) Application in improving cotton yield and quality; (5) Application in increasing the content of defense enzymes in cotton plants; (6) Application in the preparation of agents for the prevention and control of pathogenic bacteria; (7) Application in the preparation of fungal agents for the prevention and control of cotton diseases; (8) Application in the preparation of microbial agents that promote the growth of cotton plants; (9) Application in the preparation of microbial agents to improve cotton yield and quality; (10) Application in the preparation of microbial agents that increase the content of defense enzymes in cotton plants.

6. The application according to claim 5, characterized in that, The pathogens include Verticillium dahliae, Rhizoctonia solani, Fusarium oxysporum (cotton-specific), Fusarium oxysporum (cucumber-specific), Fusarium oxysporum (tomato-specific), Fusarium pseudoverticum, Fusarium graminearum, and / or Fusarium pseudograminearum.

7. The application according to claim 5, characterized in that, The cotton diseases mentioned include damping-off and / or Verticillium wilt.

8. The application according to claim 5, characterized in that, The cotton plant's defensive enzymes include peroxidase, polyphenol oxidase, and / or superoxide dismutase.

9. A method for controlling cotton diseases, characterized in that, The seed coating powder of claim 4 is used to coat cotton seeds, wherein the mass of the seed coating powder is 5-10% of the seed mass.

10. The method according to claim 9, characterized in that, The cotton diseases mentioned include damping-off and / or Verticillium wilt.

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