Bacillus velezensis for soil fumigation and application thereof

Abstract

The present application belongs to the field of microbial inoculants, and relates to a bacillus velezensis for soil fumigation and application thereof. In order to overcome the defects such as poor tolerance and low colonization rate of the existing microbial inoculants after soil fumigation, the present application obtains a strain bacillus velezensis A15 with a very high tolerance threshold to multiple fumigants through systematic screening of soil after multiple fumigations, and the preservation number is CGMCC No. 37759. The strain can still grow rapidly and stably colonize under the pressure of fumigant residues, and show strong growth promoting ability. At the same time, the strain shows excellent antagonistic inhibition effect on typical soil-borne pathogens, and the field application effect shows that the plant height, stem diameter and chlorophyll content of crops can be significantly improved, and the problem of slow recovery of soil productivity after fumigation is solved.

Description

Technical Field

[0001] This invention belongs to the field of microbial agents, specifically relating to a strain of Bacillus belye for soil fumigation and its application. Background Technology

[0002] Crop growth is highly dependent on a healthy soil environment. However, with the increasing intensification of agriculture and the continuous cropping of high-value-added cash crops, the outbreaks of soil-borne diseases (such as wilt, root rot, bacterial wilt, and root-knot nematode disease) are becoming increasingly serious. These pathogens rapidly multiply and multiply in the soil, leading to a sharp decline in soil biodiversity, ecological imbalance, and a significant decrease in arable land quality. Traditional control methods for these diseases often involve root irrigation with pesticides, but due to their limited spectrum of control and the inability of pesticides to migrate, the control effect is poor, especially in effectively controlling harmful organisms in deeper soil layers.

[0003] Soil fumigation, as one of the most effective and stable technologies for controlling soil-borne diseases, has been widely used in greenhouse vegetables, Panax notoginseng, ginseng, strawberries, and other crops to overcome continuous cropping obstacles, with an application area exceeding 300,000 mu (approximately 20,000 hectares). However, fumigants have broad-spectrum bactericidal activity, which, while eliminating harmful organisms, also significantly interferes with the non-target microbial community in the soil. Studies have found that the structure of soil bacterial and fungal communities fluctuates drastically after fumigation. A brief "vacuum period" occurs in the soil microbial community after fumigation, characterized by: 1) simplified community diversity and composition; and 2) nutrient pulses stimulated by cell lysis. During this time, if no colonizing and competitive strains quickly occupy niches to guide the community towards health, reinvasion by pathogens can easily trigger a secondary outbreak of diseases. To regulate the microbial community after fumigation, microbial agents or fertilizers are often applied in production to restore soil function and build a rhizosphere immune barrier. However, commercially available microbial agents (such as the common Bacillus subtilis) generally suffer from weak resistance to fumigation residues, resulting in low colonization rates of functional bacteria and an inability to exert their functions. These agents exhibit low survival rates and poor colonization capabilities under the high-pressure stress environment following fumigation, making it difficult to rapidly replenish and stably activate ecological functions. Therefore, it is urgent to screen and discover a functional strain that can tolerate fumigation stress (i.e., strong resistance) and possesses high competitiveness (rapid colonization rate) to rapidly reconstruct the microbial community and guide it towards a healthy state, thereby building an immune barrier. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing microbial agents in soil fumigation applications, such as poor tolerance, low colonization rate, and unstable growth-promoting effects, and to provide a Bacillus velezensis A15 specifically for soil fumigation and its application. The specific technical problems to be solved by this invention include:

[0005] (1) Solving the problem of bacterial agents failing to colonize under the stress of fumigant residue: This invention obtained a strain A15 with an extremely high tolerance threshold to fumigants by systematically screening the soil after multiple fumigations, ensuring that it can still grow rapidly and colonize stably under the pressure of agent residue after fumigation.

[0006] (2) Solve the problem of slow recovery of soil productivity after fumigation: Utilize the strong growth-promoting ability of A15 to specifically fill the ecological niche vacuum after fumigation, significantly accelerate the construction of the rhizosphere immune barrier of crops, show excellent antagonistic and inhibitory effects on pathogens, and at the same time significantly increase the plant height, stem diameter and chlorophyll content of crops, thereby ensuring the healthy and rapid growth of crops after fumigation.

[0007] This invention first provides a strain of Bacillus velezensis, A15, with accession number CGMCC No. 37759, which was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), and is classified and named as Bacillus velezensis.

[0008] The present invention also provides a soil remediation microbial agent, wherein the soil remediation microbial agent comprises the aforementioned Bacillus belesii.

[0009] The present invention also provides the use of Bacillus berleis as described above in the preparation of microbial agents for soil remediation.

[0010] As described above, the soil remediation includes remediation after soil fumigation treatment. Preferably, the fumigation treatment includes, for example, horseradish fumigation treatment with horseradish, dazomet, thiamethoxam, or chloropicrin.

[0011] The present invention also provides the application of Bacillus berberis or the soil remediation agent described above in crop cultivation.

[0012] The present invention also provides the application of Bacillus berreatus or the soil remediation agent described above in promoting crop growth, improving crop quality, shortening the seedling establishment period, and constructing a rhizosphere immune barrier.

[0013] As described above, the crops are preferably, for example, vegetables, medicinal herbs, and fruit trees.

[0014] The present invention also provides a method for applying Bacillus berberis or the soil remediation agent as described above, after the soil fumigation and ventilation are completed.

[0015] The application method described above involves inoculating the Bacillus or the soil remediation bacterial strain by root irrigation, irrigation with water, or mixing with the soil.

[0016] As described above, in the inoculum, the concentration of Bacillus velezensis A15 is OD600 = 0.4-2.0, preferably OD600 0.8-1.0, and the application dosage is 0.5-2.0 kg / mu, preferably 1 kg / mu.

[0017] The technical effects achieved by the technical solution of this invention are as follows:

[0018] The Bacillus belye A15 obtained by screening in this invention has shown outstanding technical effects in remediating fumigation-damaged soil and promoting crop productivity:

[0019] (1) Strong tolerance to fumigants: The toxicity test results showed that the EC50 value of strain A15 to horseradish extract (AITC) fumigant was as high as 47.46 mg / L, and the tolerance threshold was 5-36.1 times that of soil-borne pathogens. At the same time, it was significantly better than conventional commercially available Bacillus strains (with tolerance increased by about 3.7-4.8 times). Under high concentration fumigation stress, the growth rate of A15 was no different from that of the control group, showing extremely strong environmental adaptability.

[0020] (2) Strong antibacterial activity: In the antagonistic experiment against Fusarium oxysporum, A15 showed an inhibition rate of up to 89%, which was significantly better than other treatment groups screened at the same time, and could effectively prevent reinfection of pathogens after fumigation.

[0021] (3) Significant growth-promoting effect: Pot experiment data showed that cucumbers inoculated with A15 after fumigation had the best growth-promoting effect. Their plant height (33.87cm) increased by 126.3% compared with the control group, and was significantly better than commercially available Bacillus. Field application also confirmed that the application of A15 after fumigation with dazomet and chloropicrin had a significantly higher growth-promoting effect on lily and apple seedlings than commercially available Bacillus. Attached Figure Description

[0022] Figure 1 Results of the confrontation experiment between the isolated strain and Fusarium oxysporum.

[0023] Figure 2 Phylogenetic tree.

[0024] Figure 3 Growth rate curves. A: Ralstonia solanacearum; B: Strain A8; C: Strain A15; D: Strain A22; E: Strain A23.

[0025] Information on the preservation of biological materials:

[0026] The Bacillus velezensis strain A15 of this invention was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China), with accession number CGMCC No. 37759 and classification name: Bacillus velezensis. Detailed Implementation

[0027] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0028] 1. Strains Isolation

[0029] (1) Methods for strain isolation

[0030] To encompass a diverse microbiome background, this study selected five typical soil types: Beijing alluvial soil, Harbin black soil, Yunnan red soil, paddy field soil, and greenhouse chili pepper soil. After removing stones and plant debris, the soil was sieved through a 2mm sieve and mixed thoroughly. The soil was pre-incubated at 25℃ for 7 days to stabilize activity, and the moisture content was adjusted to 21% (w / w) before treatment. 150g of soil was placed in a 500mL glass bottle. The treatment group was treated with 94% allyl isothiocyanate (AITC) at a dose of 60mg / kg, while the control group received an equal volume of sterile water. The mixture was sealed and fumigated at 25℃ for 7 days, followed by 24 hours of ventilation. The moisture content was then readjusted, and the above steps were repeated for a total of three consecutive fumigations. Oneg of fresh soil sample from the three fumigations was serially diluted, spread on NA agar plates, and incubated at 30℃ for 48 hours. Pure cultures of bacteria were obtained after 3-4 rounds of purification. Genomic DNA was extracted from the strain using the M5 Hiper kit. The 16S rDNA fragment was amplified using the universal bacterial primer pair 338F / 806R and sent to Shanghai Sangon Biotech for sequencing. The sequence results were compared with NCBI BLAST to determine the taxonomic position of the strain.

[0031] (2) Results of strain isolation

[0032] Thirty bacterial strains were isolated from soil after three AITC fumigation cycles. DNA gene sequences amplified using primers 338F / 806R were compared with known sequences in NCBI. Based on colony culture characteristics, the 30 strains were preliminarily identified as belonging to the phylum Actinobacteria and Firmicutes, as shown in Table 1. After identification, there were 3 strains of Microbacterium sp. YJN-G, 5 strains of Bacillus sp., 6 strains of Arthrobacter sp., 11 strains of Pseudarthrobacter phenanthrenivorans Sphe3, 1 strain of Sinomonas sp., and 1 strain of Rhodococcus ruber. Micrococci were the most abundant order.

[0033] Table 1. Strains isolated from soil after three fumigation cycles

[0034] ;

[0035] 2. Determination of antibacterial activity of bacterial strains

[0036] (1) Antibacterial activity test method

[0037] The plate confrontation method was used. Identified bacterial strains were inoculated into LB liquid medium and incubated at 30℃ and 180 rpm for 48 h. 15 mL of bacterial suspension was added to 50 mL centrifuge tubes and centrifuged at 6000 rpm for 10 min. The centrifuged bacterial pellet was washed 2–3 times with sterile water and mixed thoroughly. The OD600 was adjusted to 1 to obtain a bacterial suspension. Using a 5 mm punch, activated Fusarium mycelium pellets were inoculated into the center of a 9 cm diameter PDA plate. 2 μL of bacterial suspension was added dropwise at four mutually perpendicular directions, 25 mm away from the mycelium pellet. After standing for 5 min, the plates were incubated at 27℃. An equal volume of sterile water was added as a control. Antagonism was observed, and colony diameters were recorded and measured.

[0038] The method for calculating the growth inhibition rate of Fusarium is as follows:

[0039] ;

[0040] Y represents the fungal mycelial growth inhibition rate (%), d0 represents the diameter of the fungal cake, d1 represents the diameter of the colony in the drug-treated group, and d2 represents the diameter of the colony in the blank control group.

[0041] (2) Results of antibacterial test

[0042] Using Fusarium oxysporum as the target bacterium, 30 bacterial strains were subjected to initial and secondary antagonistic screening. The results showed that 4 strains isolated from soil after 3 AITC fumigation cycles exhibited antagonistic activity against Fusarium oxysporum. Figure 1 Of the four strains, A8 showed an inhibition rate of 79% against Fusarium, A15 89%, A22 72%, and A23 74%. Among the four strains, A15 exhibited the strongest direct inhibitory effect against Fusarium. The bacteria were further identified, and a phylogenetic tree was constructed. Figure 2 Four strains were found to be Bacillus species, including A15 Bacillus velezensis, A8 Bacillus siamensis, A22 Bacillus amyloliquefaciens, and A23 Bacillus subtilis.

[0043] 3. Strain tolerance test

[0044] (1) Test method for virulence determination of AITC strains

[0045] The virulence of AITC fumigation against four strains (A8, A15, A22, and A23) exhibiting high antibacterial activity was determined using the bacterial growth rate method. Commercially available Bacillus amyloliquefaciens, Trichoderma polysporum, Ralstonia solanacearum, and Fusarium oxysporum were selected as controls. The procedure for the bacteria was as follows: 200 μL of bacterial suspension was added to a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Different volumes of 10000 mg / L AITC stock solution (prepared from 20% AITC and sterile water) were transferred onto filter paper suspended below the rubber stopper to create a series of concentration gradients. The Erlenmeyer flask was then quickly sealed (ensuring the filter paper did not come into contact with the bacterial suspension). For the blank control, sterile water was added to the filter paper. Each treatment group was set up in triplicate. The conical flasks were incubated for 24 hours in the dark at 180 rpm on a shaker at 28°C. Then, 1 mL of bacterial suspension was aspirated using a sterile pipette tip, and the absorbance (OD600) at 600 nm was measured using a microplate reader. The absorbance of different bacterial suspensions was measured using OD600 to evaluate the antibacterial effect of different AITC concentrations against pathogenic bacteria and the four isolated bacterial strains. The AITC concentration gradients are shown in Table 2 below.

[0046] Table 2 AITC Concentration Gradient

[0047] ;

[0048] The procedure for fungal infections was as follows: Take an activated, uncontaminated agar plate containing the tested fungus. Collect a 5mm diameter mycelial cake from the edge of the colony and transfer it to the center of a freshly prepared sterile PDA agar plate. Invert the plate and quickly add different volumes of AITC solution onto sterile filter paper inside the plate lid to create a series of concentration gradients. Seal the plate immediately. The blank control group received only 10μL of sterile water. All procedures were performed under aseptic conditions. Each treatment was replicated three times. All treatments were incubated at 25℃ in the dark for 2–6 days. The AITC concentration gradients are shown in Table 2. When the colonies in the blank control group covered two-thirds of the plate area, the desiccator was opened, and the colony diameter was measured using the cross-sectional method.

[0049] The method for calculating the bacterial growth inhibition rate is as follows:

[0050] ;

[0051] Y represents the bacterial growth inhibition rate (%), Au represents the absorbance OD600 of the blank control group, and At represents the absorbance OD600 of the drug-treated group.

[0052] The method for calculating the growth inhibition rate of fungi is as follows:

[0053] ;

[0054] Y represents the fungal mycelial growth inhibition rate (%), d0 represents the diameter of the fungal cake, d1 represents the diameter of the colony in the drug-treated group, and d2 represents the diameter of the colony in the blank control group.

[0055] (2) Results of AITC virulence against the strain

[0056] The virulence test results (Table 3) show that AITC's EC50 against the typical soil-borne pathogen *Ralstonia solanacearum* was only 1.28 mg / L, and against *Fusarium oxysporum* it was 7.87 mg / L. However, the EC50 of the four isolated strains ranged from 26.94 to 47.46 mg / L, significantly higher than that of the pathogen and commercially available Bacillus (8.22 mg / L) and *Trichoderma* (10.01 mg / L). Strain A15 had the highest EC50 (47.46 mg / L), which was 5.0-36.1 times that of the pathogen and 3.7-4.8 times that of conventional fungicides (*Bacillus belyssus* and *Trichoderma harzianum*).

[0057] Table 3 Comparison of toxicity data

[0058] ;

[0059] 4. Determination of bacterial growth rate after fumigation stress

[0060] (1) Method for determining the growth rate of bacterial strains

[0061] To further investigate the growth of the above-mentioned strains after fumigation stress, we used the bacterial suspension growth rate method to determine the changes in the growth rate of strains A8, A15, A22, and A23 after AITC fumigation, while Ralstonia solanacearum was selected as a control. 200 μL of bacterial suspension was added to a 100 mL Erlenmeyer flask containing 50 mL of LB liquid medium. After shaking and incubating for 24 h at 180 rpm, 28 °C, and in the dark, different volumes of 10000 mg / L AITC stock solution (prepared from 20% AITC formulation and sterile water) were transferred onto filter paper suspended below the rubber stopper, forming a concentration gradient of 8 mg / L, 16 mg / L, and 32 mg / L. The Erlenmeyer flask was then quickly sealed (ensuring the filter paper did not come into contact with the bacterial suspension). For the blank control, sterile water was added to the filter paper. Each treatment group was set up in triplicate. After the conical flasks were statically cultured at 28°C in the dark for 24 h, 100 μL of bacterial suspension was added to a new sterilized 150 mL conical flask containing 50 mL of LB liquid medium. The conical flasks were then shaken and cultured at 180 r / min at 28°C in the dark for 24 h. At 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 52 h, 56 h, 60 h, 64 h, and 68 h, 1 mL of bacterial suspension was taken and the absorbance OD600 of the bacterial suspension at 600 nm was measured using an ELISA reader. The bacterial growth rate curve was plotted based on the measured absorbance OD600.

[0062] The bacterial growth rate is calculated as follows:

[0063] ;

[0064] μ represents the bacterial growth rate, t1-t2 is determined to be the logarithmic growth phase, and OD1 and OD2 are the corresponding OD600 values.

[0065] (2) Results of strain growth rate

[0066] Changes in bacterial growth rate after AITC fumigation treatment are as follows: Figure 3 As shown, at a high concentration of 32 mg / L, the logarithmic growth phase of Ralstonia solanacearum is 24-56 hours, with a growth rate of only 0.019 h. -1 At a medium concentration of 16 mg / L, the logarithmic growth phase is 24-48 hours, and the growth rate is 0.038 h / min. -1 At a low concentration of 8 mg / L, the logarithmic growth phase was 24-48 hours, and the growth rate was 0.053 h / min. -1 The growth rate was similar to that of the control treatment (0.055 h). -1The results showed that at a low concentration of 8 mg / L, AITC fumigation did not affect the growth of Ralstonia solanacearum, but fumigation at concentrations of 16-32 mg / L significantly inhibited the growth rate of Ralstonia solanacearum. Figure 3 (A)

[0067] The logarithmic growth phase of strain A15 was 20-40 hours, while the growth rate of the control group was 0.069 hours. -1 The growth rate of A8 after treatment with various concentrations of AITC was approximately 0.065-0.079 h⁻¹. -1 There was no significant difference compared to the control group. Figure 3 (B) The logarithmic growth phase of strain A8 was 16-44 h, while the growth rate of the control group was 0.060 h. -1 The growth rate of A8 after treatment with various concentrations of AITC was approximately 0.061-0.070 h⁻¹. -1 There was no significant difference compared to the control group. Figure 3 (C). The logarithmic growth phase of strain A22 was 16-48 h, while the growth rate of the control group was 0.060 h. -1 The growth rate of A22 after treatment with various concentrations of AITC was approximately 0.056-0.071 h. -1 The growth rate of the strains under each solubility treatment was not significantly different from that of the control group. Figure 3 (D). The logarithmic growth phase of strain A23 was 20-40 h, while the growth rate of the control group was 0.064 h. -1 The growth rate of A23 after treatment with various concentrations of AITC was approximately 0.064-0.070 h⁻¹. -1 The growth rate of the strains under each solubility treatment was not significantly different from that of the control group. Figure 3 (E). The results showed that fumigation stress with high concentrations of AITC (32 mg / L) had no significant impact on the growth of the four strains A8, A15, A22, and A23, indicating that they have high tolerance to AITC. Moreover, among the four strains A8, A15, A22, and A23, A15 exhibited faster growth.

[0068] 5. Tolerance test of strain A15 to other fumigants

[0069] The tolerance of strain A15 to other fumigants was determined using the bacterial growth rate method. Commercially available Bacillus velezensis, Bacillus subtilis, and Bacillus amyloliquefaciens were selected as controls. The procedure was as follows: 200 μL of bacterial suspension was added to a 150 mL Erlenmeyer flask containing 50 mL of LB liquid medium. Different volumes of 10000 mg / L fumigant stock solution were transferred onto filter paper suspended below the rubber stopper to create a series of concentration gradients (Table 4). The Erlenmeyer flask was then quickly sealed. For the blank control, sterile water was added to the filter paper. Each treatment was repeated three times. After incubating the Erlenmeyer flasks at 180 rpm and 28 °C in the dark for 24 h, 1 mL of bacterial suspension was aspirated with a sterile pipette tip, and the absorbance (OD600) of the bacterial suspension at 600 nm was measured using a microplate reader. The absorbance of different bacterial solutions was measured using OD600 to evaluate the tolerance of strain A15 to different fumigants. The concentration gradients of the fumigants are shown in Table 4 below.

[0070] Table 4 Fumigant Concentration Gradient

[0071] ;

[0072] (2) Results of strain A15's tolerance to different fumigants

[0073] The tolerance test results (Table 5) show that A15 exhibits strong tolerance to all three tested fumigants. The EC50 values ​​of chloropicrin, MITC, and AITC for strain A15 were 56.62 mg / L, 66.94 mg / L, and 49.35 mg / L, respectively, significantly higher than the EC50 values ​​of the three commercially available Bacillus species. For example, the EC50 values ​​of the three fumigants for Bacillus subtilis were 14.42-16.55 mg / L, for Bacillus amyloliquefaciens were 17.55-19.46 mg / L, and for Bacillus belyssus were 11.45-15.16 mg / L. This demonstrates that common commercially available Bacillus species are relatively sensitive to fumigants, while A15, as discovered in this study, exhibits high tolerance to all three typical fumigants, with a tolerance level 2.44-3.31 times higher than that of commercially available Bacillus species.

[0074] Table 5. Tolerance of strain A15 to different fumigants

[0075] ;

[0076] 6. Effects of tolerant strain A15 on crop growth

[0077] (1) Pot test method

[0078] Soil samples were collected from cucumber greenhouses for indoor fumigation treatment. After one week of AITC fumigation (20 kg / mu), different bacterial suspensions were sprayed. Five treatment groups were set up: strain A15, S1 (commercially available Bacillus subtilis), S2 (commercially available Bacillus amyloliquefaciens), S3 (commercially available Bacillus vesiculosus), and a water control. The cell density of the bacterial suspension was adjusted to OD600 = 0.8-1.0, and applied at a rate of 1 kg / mu. After spraying, cucumbers with 1-2 true leaves were transplanted into the soil of the above treatment groups. One month later, plant height, stem diameter, and chlorophyll content were measured in each treatment group to evaluate the growth-promoting potential of each strain.

[0079] (2) Cucumber growth

[0080] The results of the pot experiment (Table 6) showed that although commercially available Bacillus strains (S1-S3) had a growth-promoting effect on cucumbers (plant height was significantly different from the control group CK), the strain A15 screened in this study showed excellent growth-promoting potential. Data analysis showed that after inoculation with A15, the cucumber plant height reached 33.87 cm, which was significantly increased by about 126.3% compared with the control group (14.97 cm) (P<0.05), and increased by 58.49%-70.81% compared with commercially available Bacillus strains. In terms of stem diameter, the A15 treatment group reached 6.89 mm (significantly higher than CK 5.94 mm), and significantly higher than the three commercially available Bacillus strains. In addition, the chlorophyll content of the A15 treatment group also increased to 43.33 SPAD, significantly higher than the control and the three commercially available Bacillus strains, indicating that this strain can not only promote the accumulation of plant biomass, but also enhance the physiological vitality of the plant by increasing the content of photosynthetic pigments. In summary, the application of strain A15 after fumigation was significantly superior to the three commercially available conventional Bacillus strains in improving cucumber growth indicators.

[0081] Table 6. Cucumber growth indicators after inoculation with strains

[0082] ;

[0083] 7. Field application effects of strain A15

[0084] (1) Lily

[0085] In May 2025, soil in a lily-growing area of ​​Yongjing County, Gansu Province, was fumigated with dazomet (30 kg / mu). After fumigation and removal of the film, the bacterial solution was used for irrigation to test the application potential of strain A15. Commercially available Bacillus subtilis (S1), Bacillus amyloliquefaciens (S2), and Bacillus belyssus (S3) were selected as positive controls. Before irrigation, the cell density of the A15 bacterial solution was adjusted to OD600 = 0.8-1.0, and the application rate was 1 kg / mu, diluted with water and applied to the soil surface. Commercially available Bacillus (S3) was directly diluted with water and applied at a rate of 1 kg / mu. One month after lily germination, the stem diameter and chlorophyll content of lilies in each treatment group were measured to evaluate the growth-promoting potential of strain A15.

[0086] The results of lily growth index measurements (Table 7) show that the application of strain A15 after dazomet fumigation significantly promoted lily growth. Regarding stem diameter, the A15 treatment increased by 26.45% compared to the control (p<0.05), and was also 15.98%-30.44% higher than the three commercially available Bacillus strains (p<0.05). Regarding chlorophyll content, the A15 treatment increased by 62.69% compared to the control (p<0.05), and was also 39.57%-43.29% higher than the three commercially available Bacillus strains (p<0.05). Therefore, the application of A15 after dazomet fumigation has a significant growth-promoting effect on lilies, and is significantly superior to conventional Bacillus strains.

[0087] Table 7 Lily Growth Indicators

[0088] ;

[0089] (2) Apple seedlings

[0090] In March 2025, in apple-growing areas of Qianyang County, Shaanxi Province, soil fumigation (20 kg / mu) was conducted on nurseries experiencing chloropicrin-induced continuous cropping obstacles. After one month of fumigation, the film was removed and the area was ventilated. Different bacterial solutions were then applied to test the application potential of strain A15. Commercially available Bacillus subtilis (S1), Bacillus amyloliquefaciens (S2), and Bacillus belyssus (A11S3) were selected as positive controls. Before irrigation, the cell density of the A15 bacterial solution was adjusted to OD600 = 0.8-1.0, and the application dosage was 1 kg / mu. Commercially available Bacillus subtilis (A11S3) was directly diluted with water and sprayed at a dosage of 1 kg / mu. One month after transplanting, the plant height and stem diameter of apple seedlings in each treatment group were measured to evaluate the growth-promoting potential of strain A15.

[0091] The analysis of apple seedling growth indicators (Table 8) further confirmed the growth-promoting potential of strain A15 after fumigation. Regarding stem diameter, the A15 treatment increased by 29.47% compared to the control (p<0.05), and was 15.63%-20.73% higher than the three commercially available Bacillus strains (p<0.05). Regarding plant height, the A15 treatment increased by 33.32% compared to the control (p<0.05), and was also 11.22%-12.52% higher than the three commercially available Bacillus strains (p<0.05). Therefore, the application of A15 after chloropicrin fumigation has a significant growth-promoting effect on apple seedlings, and is significantly superior to conventional Bacillus strains.

[0092] Table 8 Apple seedling growth indicators

[0093] ;

Claims

1. A strain of Bacillus velezensis, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37759.

2. A soil remediation microbial agent, characterized in that, The soil remediation microbial agent comprises Bacillus berleis as described in claim 1.

3. The Bacillus belye as described in claim 1 is used for soil remediation after soil fumigation.

4. The application according to claim 3, characterized in that, The soil remediation specifically refers to the restoration of soil function after soil fumigation treatment, which includes fumigation treatment with horseradish, dazomet, thiamethoxam, and chloropicrin.

5. The application of Bacillus berreatus as described in claim 1 or the soil remediation agent as described in claim 2 after soil fumigation, or in the suppression of soil-borne diseases, promotion of crop growth, and construction of rhizosphere immune barriers.

6. The application according to claim 5, characterized in that, The crops mentioned are vegetables, medicinal herbs, and fruit trees.

7. The method of applying the Bacillus berberis of claim 1 or the soil remediation microbial agent of claim 2, characterized in that, Apply at the beginning or end of soil fumigation.

8. The application method according to claim 7, characterized in that, The *Bacillus berreatus* or the soil remediation agent strain can be inoculated by root irrigation, irrigation with water, or mixing with the soil.

9. The application method according to claim 8, characterized in that, The inoculum contains Bacillus belyi at an OD600 of 0.4-2.0 and is applied at a rate of 0.5-2.0 kg / mu.

10. The application method according to claim 9, characterized in that, The inoculum contains Bacillus belye at an OD600 of 0.8-1.0 and is applied at a rate of 1 kg / acre.

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