Bacillus sp. sneb2538 from marine sediment, fermentation broth and application thereof

Abstract

The present application relates to the technical field of plant disease control, and specifically discloses a strain of marine sediment bacillus Sneb2538, a fermentation liquor thereof and application, wherein the marine sediment bacillus Sneb2538 was preserved in the China General Microbiological Culture Collection Center on August 12, 2024, with a preservation number of CGMCC No. 31569 and a classification name of Cytobacillus oceanisediminis. The marine sediment bacillus Sneb2538 provided by the present application can effectively control cucumber fusarium wilt and promote the growth of cucumber plants.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to a marine sediment Bacillus spp. Sneb2538, its fermentation broth, and its applications. Background Technology

[0002] With the continuous development of large-scale, specialized, and production-oriented cucumber farming, and changes in the ecological environment, the occurrence and severity of cucumber diseases are becoming increasingly serious. Among them, cucumber wilt caused by *Fusarium oxysporum* f. sp. *cucumerinum* Owen is a serious soil-borne fungal disease in cucumber production. This pathogen survives easily under natural conditions and is mainly spread through soil, seeds, and fertilizers. Cucumber wilt is characterized by a short onset time, wide affected area, high degree of damage, and high difficulty in control. The incidence rate of cucumber wilt is generally 20%–50%, but can reach over 80% in severe cases.

[0003] Fusarium wilt is the main cause of death in cucumbers grown in greenhouses under continuous cropping, and it can occur throughout the entire growth cycle of cucumbers. The fusarium wilt pathogen primarily infects the vascular bundles of the cucumber stem, entering the plant through root hairs or root wounds. The spread and expansion of the disease accelerates the death of the entire plant. In cucumber seedlings, the base of the stem turns yellowish-brown, the cotyledons wither, and in severe cases, the stem base shrinks, leading to seedling death. Fusarium wilt is particularly severe during the flowering and fruiting stages. In mature plants, the leaves gradually wither from top to bottom. The base of the main vine of diseased plants shrinks, and longitudinal cracks are often observed. A longitudinal cut of the diseased stem reveals browning of the vascular bundles. Under high humidity conditions, white or pinkish mold appears on the surface of the affected areas, eventually leading to root rot and making the plant easy to uproot.

[0004] For a long time, the control of cucumber wilt disease has mainly relied on chemical pesticides. Although the application of chemical pesticides has achieved certain results, their harm to human and animal health and the ecological environment has become increasingly serious and has attracted widespread attention. Biological control is favored because of its good environmental compatibility, long duration, and lack of pollution. Studies have shown that the imbalance of soil microbial community structure is the main cause of cucumber wilt disease. Therefore, in continuous cropping production models, using biocontrol microorganisms to regulate the rhizosphere soil microbial community structure and create a healthy soil microbial environment is a safe and effective strategy for solving cucumber wilt disease. Therefore, isolating and screening microorganisms from nature for the control of cucumber wilt disease is of great significance. Summary of the Invention

[0005] To provide a microorganism for controlling cucumber wilt disease, this invention provides a strain of marine sediment Bacillus spp. Sneb2538, its fermentation broth, and its application. The marine sediment Bacillus spp. Sneb2538 provided by this invention can effectively control cucumber wilt disease and promote the growth of cucumber plants.

[0006] This invention provides a strain of marine sediment Bacillus (Cytobacillus oceanisediminis) Sneb2538, which was deposited on August 12, 2024 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31569 and classified as marine sediment Bacillus (Cytobacillus oceanisediminis).

[0007] This invention isolates a strain of marine sediment Bacillus Sneb2538 from nature. Experimental verification shows that this strain can effectively prevent and control cucumber wilt and promote the growth of cucumber plants, laying a practical foundation for cucumber cultivation.

[0008] The present invention also provides a fermentation broth obtained by fermentation of the aforementioned marine sediment Bacillus Sneb2538, wherein the fermentation broth is prepared by inoculating marine sediment Bacillus Sneb2538 into a liquid culture medium and culturing the strain to a concentration of 1×10⁻⁶. 7 CFU / mL ~ 1×10 8 The result was obtained from CFU / mL.

[0009] The present invention also provides a method for preparing the fermentation broth, wherein the preparation steps are as follows: activating *Sneb. 2538*, a marine sediment bacterium, and inoculating it into a liquid fermentation medium, incubating at a temperature of 26℃~28℃ and 150 r·min. -1 ~200r·min -1 Shake culture for 1.5 to 2 days to obtain fermentation broth.

[0010] Furthermore, the liquid fermentation medium is LB broth medium.

[0011] Further, the preparation steps of the fermentation broth are as follows: activating *Sneb. 2538*, a marine sediment bacterium, and inoculating it into a liquid fermentation medium at a temperature of 28°C and 200 rpm. -1 Shake culture for 2 days to obtain fermentation broth.

[0012] The present invention also provides an application of the aforementioned marine sediment Bacillus Sneb2538 or the aforementioned fermentation broth in cucumber cultivation, wherein the marine sediment Bacillus Sneb2538 or the fermentation broth is used to prevent and control cucumber wilt disease and promote the growth of cucumber plants.

[0013] Furthermore, the marine sediment Bacillus Sneb2538 is used to inhibit the cucumber-specific Fusarium oxysporum f.sp.cucumerinum Owen, the pathogen causing cucumber wilt.

[0014] Furthermore, the marine sediment Bacillus Sneb2538 is used to inhibit the germination of Fusarium oxysporum spores.

[0015] Furthermore, the fermentation broth of the marine sediment Bacillus Sneb2538 was used to treat cucumber seedlings with root irrigation to prevent cucumber wilt disease.

[0016] Furthermore, promoting the growth of cucumber plants includes increasing the plant height, fresh weight per plant, and fresh weight of roots.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The marine sediment Bacillus Sneb2538 isolated by this invention can effectively prevent and control cucumber wilt and promote the growth of cucumber plants. Marine sediment Bacillus Sneb2538 can act on cucumber plants in multiple ways, thereby preventing and controlling cucumber wilt. For example, marine sediment Bacillus Sneb2538 can be made into pure bacterial powder, freeze-dried powder and bacterial agent, etc., and applied to cucumber plants to achieve the purpose of preventing and controlling cucumber wilt while promoting the growth of cucumber plants.

[0019] In indoor pot experiments and field experiments of this invention, the fermentation broth prepared by marine sediment Bacillus Sneb2538 can effectively prevent and control wilt disease when applied to the roots of cucumber seedlings and promote plant growth.

[0020] Information on the Preservation of Biological Materials

[0021] Sneb2538, referred to in this application as *Cytobacillus oceanisediminis*, was deposited on August 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31569. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The classification name is *Cytobacillus oceanisediminis*. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Figure 1 shows the results of a plate confrontation experiment between marine sediment Bacillus Sneb2538 and Fusarium oxysporum; in the figure, A represents Fusarium oxysporum; B represents the confrontation between Sneb2538 and Fusarium oxysporum. Detailed Implementation

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0025] Example 1: Isolation of a marine sediment Bacillus Sneb2538 and preparation of its fermentation broth.

[0026] I. Isolation of Bacillus spp. Sneb2538 from Marine Sediments

[0027] Soil was collected from a cucumber field in Yuhong District, Shenyang City, Liaoning Province (123.162° longitude, 41.949° latitude), and a strain of marine sediment Bacillus Sneb2538 was isolated and purified from it.

[0028] II. Preparation of fermentation broth of Bacillus spp. Sneb2538 from marine sediments

[0029] (1) The purified marine sediment Bacillus Sneb2538 was activated on LB solid medium for 12 h.

[0030] The solid culture medium (LB medium) consists of 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1.0 g glucose, and 15-20 g agar, dissolved in 1.0 L of distilled water, pH 7.0 ± 0.2, and sterilized by moist heat at 121 °C for 30 min.

[0031] (2) Select a single colony with good activation and growth from LB solid medium and inoculate it into 150 mL of liquid fermentation medium for shake fermentation. The fermentation temperature is 28℃ and the shaker speed is 200 r·min. -1 Fermentation time was 2 days to obtain fermentation broth.

[0032] The liquid fermentation medium (LB broth) consisted of 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, and 1.0 g glucose, with a pH of 7.0 ± 0.2. It was dissolved in 1.0 L of distilled water and sterilized by moist heat at 121 °C for 30 min.

[0033] Example 2: Antagonistic effect of marine sediment Bacillus Sneb2538 fermentation broth on Fusarium oxysporum and its inhibitory effect on spore germination.

[0034] I. Experimental Methods

[0035] 1. Study on the antagonistic effect of fermentation broth on Fusarium oxysporum

[0036] The strain *Fusarium oxysporum* f.sp. *cucumerinum* Owen, a cucumber-specific strain, was donated by Professor Liu Zhiheng of the Fungal Laboratory, College of Plant Protection, Shenyang Agricultural University. This strain was recorded in the Chinese Journal of Biological Control, in an article titled: "Optimization of Fermentation Conditions for Streptomyces Synthesis ST-2 and Its Control Effect on Cucumber Fusarium Wilt".

[0037] The *Fusarium oxysporum* f. sp. *cucumerinum* Owen strain was activated on PDA plates and incubated upside down at 28°C for 4 days. A mycelial cake was created at the edge of the *Fusarium oxysporum* Owen colony using a punch (d = 9 mm), and placed in the center of a new PDA medium plate. Two μL of *Bacillus spp.* Sneb2538 fermentation broth was symmetrically dropped 2.0 cm away from the mycelial cake. Plates inoculated only with the pathogenic mycelial cake and not with the fermentation broth served as controls. The plates were incubated at 28°C for 4 days until the control pathogenic colonies completely covered the plates. The colony radius was observed and recorded, and the inhibition rate was calculated.

[0038] Potato glucose agar (PDA) medium consists of 200g of peeled potato, 20g of glucose, and 15g of agar, dissolved in 1.0L of distilled water. The pH is adjusted to 7.2–7.5, and the medium is sterilized by moist heat at 121°C for 30 minutes.

[0039] Potato glucose liquid culture medium (PD) consists of 200g of peeled potato and 20g of glucose dissolved in 1.0L of distilled water. The pH is adjusted to 7.2-7.5, and the medium is sterilized by moist heat at 121℃ for 30min.

[0040]

[0041] 2. Inhibitory effect of fermentation broth on the germination of Fusarium oxysporum spores

[0042] Fusarium oxysporum mycelia were inoculated onto PDA solid medium and cultured at 28°C for 7 days until the culture dishes were confluent with the pathogen. Under aseptic conditions, 5 mL of sterile water was added to the confluent PDA plates. The fungal mycelia were scraped off with a disposable inoculation loop and filtered through three layers of sterile gauze to obtain a Fusarium oxysporum spore suspension. 40 μL of fermentation broth and 40 μL of Fusarium oxysporum spore suspension were mixed 1:1 and transferred to sterile glass slides, which were then incubated at 28°C for 12 hours. Control treatments were prepared by adding equal volumes of sterile water and LB liquid medium to the pathogen spore suspension, respectively. After incubation, the germination of Fusarium oxysporum spores was observed under a microscope, and the number of germinating spores and the total number of examined spores were recorded. The spore germination rate and relative inhibition rate were calculated.

[0043]

[0044]

[0045] II. Experimental Results

[0046] The plate confrontation test showed that the colony radius of the control Fusarium oxysporum was 3.92 cm, while the colony radius of Fusarium oxysporum after treatment with the fermentation filtrate of strain Sneb2538 was 1.42 cm, with an inhibition rate of 63.78% against Fusarium oxysporum. This indicates that strain Sneb2538 can effectively antagonize the growth of Fusarium oxysporum.

[0047] The spore germination inhibition test showed that after 12 hours of treatment, the spore germination rates of sterile water and PD liquid medium were 100.000% and 100.00%, respectively. After treatment with fermentation filtrate of Sneb2538 strain, the spore germination rate was 43.22%, which was significantly lower than that of the control, and the relative inhibition rate of spore germination was 56.78%. This indicates that Bacillus cereus of marine sediments Sneb2538 significantly inhibits the germination of Fusarium oxysporum spores.

[0048] Example 3: Effects of marine sediment Bacillus Sneb2538 fermentation broth on cucumber seed germination and growth.

[0049] I. Experimental Methods

[0050] 1. Experimental materials

[0051] The cucumber variety is Zhongnong No. 6; the seeds were disinfected using the hot water soaking method (soaking at 55℃ for 30 minutes) and are ready for use.

[0052] The fermentation broth prepared in Example 1.

[0053] Instruments and equipment: steam sterilizer, electric thermostatic drying oven, electric thermostatic incubator, clean workbench, disposable petri dishes, shaking incubator, seedling trays.

[0054] 2. Experimental Procedure

[0055] After sterilization, the seeds were soaked in the bacterial fermentation broth for 3 minutes for coating. They were then placed in sterile Petri dishes (d = 90 mm) lined with sterile filter paper. Each treatment was repeated three times, with 10 seeds per replicate. Controls were prepared by treating the seeds with equal volumes of sterile water and culture medium. The Petri dishes were incubated in a constant temperature and humidity incubator at 25°C for 24 hours. Germination was then counted daily for 7 days. Germination index and germination rate were calculated, and shoot and root lengths were measured to determine seed vigor index.

[0056]

[0057]

[0058] Seed vigor index = seedling length × germination index.

[0059] II. Experimental Results

[0060] Table 1. Effects of fermentation broth on cucumber seed germination and growth.

[0061] deal with Germination rate / % Germination Index Seed Vigor Index Root length / cm <![CDATA[ddH2O]]> 76.67±5.77a 0.96±0.14a 2.38±0.07b 2.52±0.28a LB 73.33±5.77a 0.90±0.06a 2.25±0.33b 2.49±0.45a Sneb2538 83.33±5.77a 1.03±0.22a 3.90±0.65a 3.99±1.37a

[0062] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P<0.05 level according to Duncan's test.

[0063] Table 1 shows that after treatment of cucumber seeds with the fermentation broth of strain Sneb2538, the germination rate of cucumber seeds was 83.33%, the germination index was 1.0, the seed vigor index was 3.90, and the root length was 3.99 cm. Compared with the control, the seed vigor index of cucumber seeds was significantly increased, and the germination rate, germination index, and root length were all increased. This indicates that the seed soaking and coating treatment of cucumber seeds with the fermentation broth of Bacillus subtilis 'Sneb2538' not only did not inhibit cucumber seed germination and plant growth, but also significantly increased the seed vigor index.

[0064] Example 4: Study on the efficacy of marine sediment Bacillus Sneb2538 fermentation broth against cucumber wilt disease.

[0065] I. Experimental Methods

[0066] 1. Experimental materials

[0067] The fermentation broth prepared in Example 1.

[0068] Prepare a spore suspension according to the aforementioned method for culturing Fusarium oxysporum, and set aside for later use.

[0069] The cucumber variety is Zhongnong 6.

[0070] 2. Experimental Procedure

[0071] Cucumber seeds were sterilized using a warm water soaking method (55℃ for 30 minutes). After sterilization, the seeds were sown in seedling trays containing nutrient soil, which was a 1:1 volume mixture of sand and seedling substrate. Before sowing, the nutrient soil was sterilized at 160℃ for 180 minutes and then placed in a greenhouse at 25℃ with 16 hours of light and 8 hours of darkness for approximately two weeks. Once the seedlings reached the two-leaf-one-heart stage, they were transplanted into 9.5cm × 11.5cm pots, one seedling per pot, for later use.

[0072] The seedling substrate (mainly peat moss) was purchased from Pinstop Horticulture Ltd. in Denmark. The brand name is Pinstop Substrate, the product number is msh-1013, the product is 10-30mm, and the Pinstop peat moss is 80L.

[0073] The experiment was designed with the following treatments: A blank control (ddH2O) was created by drenching the roots with sterile water and inoculating with *Fusarium oxysporum* fermentation broth; controls were created by drenching the roots with sterile water and inoculating with *Fusarium oxysporum* fermentation broth (ddH2O + F.), drenching the roots with LB medium and inoculating with *Fusarium oxysporum* fermentation broth (LB + F.), and drenching the roots with a fungicide (1.8% methyl thiophanate) and inoculating with *Fusarium oxysporum* fermentation broth (1.8% methyl thiophanate + F.). A treatment was created by drenching the roots with the *Fusarium oxysporum* strain fermentation broth and inoculating with *Sneb2538* fermentation broth (Sneb2538 + F.). 10 mL of *Fusarium oxysporum* fermentation broth (concentration approximately 1 × 10⁻⁶) was added to each seedling. 8 CFU / mL), each seedling was treated with 10 mL of the bacterial fermentation broth (concentration approximately 1×10⁻⁶ CFU / mL) by root irrigation. 8 The control group received an equal volume of sterile water or culture medium (LB broth) or a fungicide (1.8% methyl thiophanate) per seedling. Each treatment was replicated in triplicate, with five seedlings per replicate, and all seedlings were randomly placed. After 30 days of culture, plant disease incidence was observed, and the disease index and control efficacy were calculated.

[0074] Grading standards for cucumber wilt: Grade 0, no disease; Grade 1, wilting area of ​​leaves or stems accounts for 1 / 4 or less of the whole plant; Grade 2, wilting area of ​​leaves or stems accounts for 1 / 2 of the whole plant; Grade 3, wilting area of ​​leaves or stems accounts for 3 / 4 of the whole plant; Grade 4, whole plant wilting; Grade 5, complete death.

[0075]

[0076]

[0077] II. Experimental Results

[0078] Table 2. Effects of fermentation broth root irrigation on the prevention of cucumber wilt disease.

[0079]

[0080]

[0081] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0082] Table 2 shows that, under pot cultivation conditions, 30 days after inoculation, the disease indices of cucumber plants treated with sterile water, LB, and 1.8% thiophanate-methyl were 62.22, 55.56, and 44.44, respectively. After root irrigation with the fermentation broth of Sneb2538, the disease index of cucumber seedlings was significantly lower than the control, with a control efficacy of 42.86%. Therefore, under pot cultivation conditions, *Bacillus subtilis* Sneb2538 can effectively control cucumber wilt.

[0083] Example 5: Effects of marine sediment Bacillus spp. Sneb2538 fermentation broth on cucumber plant growth.

[0084] I. Experimental Methods

[0085] Prepare the fermentation broth of Bacillus cereus Sneb2538 from marine sediments using the method described above, and set aside for later use.

[0086] The cucumber variety used was Zhongnong No. 6. The seeds were sterilized using a warm water soaking method (soaking at 55℃ for 30 minutes). After sterilization, the seeds were sown in seedling trays containing nutrient soil (sand volume: seedling substrate volume = 1:1). Before sowing, the nutrient soil was sterilized at 160℃ for 180 minutes. The trays were then placed in a greenhouse at 25℃ with 16 hours of light and 8 hours of darkness for about two weeks. After the seedlings reached the two-leaf and one-heart stage, the etiolated seedlings were transplanted into flowerpots (9.5cm × 11.5cm), one seedling per container, for later use.

[0087] The experiment was designed with the following treatments: 10 mL of sterile water or culture medium (LB broth) was used as a control for root irrigation per seedling; 10 mL of the bacterial fermentation broth (concentration approximately 1×10⁻⁶) was used as a control for root irrigation per seedling. 8 The treatment consisted of three replicates per treatment, with three seedlings per replicate, placed completely randomly. After 30 days of culture, the plant height, fresh weight, root length, and fresh weight of the cucumber plants were assessed.

[0088] II. Experimental Results

[0089] Table 3. Effects of root irrigation with Bacillus spp. Sneb2538 from marine sediments on cucumber plant growth.

[0090] deal with Plant height / cm Fresh weight of plants / g Root length / cm Fresh root weight / g <![CDATA[ddH2O]]> 20.86±0.98b 8.55±0.50b 15.48±0.70b 2.39±0.17a LB 18.75±0.96c 5.41±0.64c 18.37±0.93a 2.11±0.33b Sneb2538 25.32±1.48a 15.29±0.86a 17.67±0.79a 2.64±0.20a

[0091] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0092] The results are shown in Table 3. Under pot conditions, after root irrigation with Sneb2538 fermentation broth, cucumber seedlings showed a plant height of 25.32 cm, a plant fresh weight of 15.29 g, a root length of 17.67 cm, and a root fresh weight of 2.64 g. Compared with the LB control, plant height, plant fresh weight, and root fresh weight were all significantly increased, while root length showed no significant difference. Therefore, under pot conditions, *Bacillus subtilis* Sneb2538 effectively promotes the growth of cucumber plants.

[0093] Example 6: Field efficacy experiment of fermentation broth of marine sediment Bacillus spp. Sneb2538.

[0094] I. Experimental Methods

[0095] Prepare the fermentation broth of Bacillus cereus Sneb2538 from marine sediments using the method described above, and set aside for later use.

[0096] The cucumber variety was Zhongnong 6. The seeds were sterilized using a hot water soaking method (soaking at 55℃ for 30 minutes). After sterilization, the seeds were sown in seedling trays containing nutrient soil (sand volume: seedling substrate volume = 1:1). Before sowing, the nutrient soil was sterilized at 160℃ for 180 minutes. The trays were then placed in a greenhouse at 25℃ with 16 hours of light and 8 hours of darkness for about two weeks. After reaching the two-leaf and one-heart stage, the seeds were transplanted to a field where cucumber wilt disease was present.

[0097] The experiment was designed with the following treatments: At transplanting, root irrigation with sterile water, culture medium, and a fungicide (1.8% methyl thiophanate) served as controls. Each seedling was irrigated with 10 mL of sterile water, culture medium (LB broth), or the fungicide (1.8% methyl thiophanate). Root irrigation with bacterial fermentation broth was the second treatment, with each seedling irrigated with 10 mL of the bacterial fermentation broth (concentration approximately 1×10⁻⁶). 8 (CFU / mL). After 30 days of culture, physiological indicators such as plant height, root length, root fresh weight, and plant fresh weight of cucumber plants were measured. The disease index was investigated and the control efficacy was calculated. Disease index grading standards: Grade 0, no disease; Grade 1, wilting area of ​​leaves or stems accounts for 1 / 4 or less of the whole plant; Grade 2, wilting area of ​​leaves or stems accounts for 1 / 2 of the whole plant; Grade 3, wilting area of ​​leaves or stems accounts for 3 / 4 of the whole plant; Grade 4, whole plant wilting; Grade 5, complete death.

[0098]

[0099]

[0100] II. Experimental Results

[0101] Table 4. Field control efficacy of marine sediment Bacillus Sneb2538 fermentation broth against cucumber wilt.

[0102] deal with Root length / cm Fresh root weight / g Fresh weight of plants / g Plant height / cm Disease index efficacy / % <![CDATA[ddH2O]]> 8.49±0.83b 2.24±0.49a 150.50±11.26c 139.17±3.2c 93.06±6.36a LB 9.04±0.38b 2.82±0.09a 165.67±7.45b 159.00±6.03b 87.50±4.17a 5.97 1.8% methyl thiophanate 8.52±0.32b 2.53±0.16a 160.33±8.66bc 146.33±4.63bc 58.33±7.22b 37.31 Sneb2538 15.30±0.80a 2.66±0.74a 234.83±13.11a 230.50±19.76a 40.28±2.41c 56.72

[0103] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level according to Duncan's test.

[0104] As shown in Table 4, under field conditions, the disease index of cucumber plants treated with sterile water and LB broth was 93.06 and 87.50, respectively. After root irrigation of cucumber seedlings with methyl thiophanate and Sneb2538 fermentation broth, the disease index was 58.33 and 40.28, respectively, with control efficacies of 37.31% and 56.72%. The disease index and control efficacy after Sneb2538 fermentation broth treatment were significantly lower than those after sterile water, LB broth treatment, and methyl thiophanate treatment, indicating that *Bacillus cereus* Sneb2538 effectively controls cucumber wilt. Simultaneously, compared with sterile water, LB broth, and methyl thiophanate treatment, root length, fresh weight, and plant height of cucumber seedlings treated with Sneb2538 fermentation broth were significantly increased, while there was no significant difference in fresh weight, indicating that *Bacillus cereus* Sneb2538 effectively promotes plant growth.

[0105] Therefore, under field conditions, Bacillus spp. Sneb2538 from marine sediments can effectively control cucumber wilt and promote plant growth.

[0106] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0107] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of marine sediment Bacillus ( Cytobacillusoceanisediminis Sneb2538, characterized in that, The marine sediment Bacillus Sneb2538 was deposited on August 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31569, and classified as *Bacillus marine sediment*. Cytobacillusoceanisediminis .

2. A fermentation broth obtained by fermenting *Sneb. 2538*, a marine sediment bacterium, as described in claim 1, characterized in that... The fermentation broth was inoculated with marine sediment Bacillus Sneb2538 into liquid culture medium and cultured until the bacterial concentration reached 1×10⁻⁶. 7 CFU / mL ~ 1×10 8 The result was obtained from CFU / mL.

3. A method for preparing the fermentation broth according to claim 2, characterized in that, The preparation steps of the fermentation broth are as follows: Activated marine sediment Bacillus Sneb2538 is inoculated into liquid fermentation medium and fermented at a temperature of 26℃~28℃ and 150 r·min. -1 ~200 r·min -1 Shake culture for 1.5 to 2 days to obtain fermentation broth.

4. The method for preparing fermentation broth according to claim 3, characterized in that, The liquid fermentation medium is LB broth medium.

5. The method for preparing fermentation broth according to claim 3, characterized in that, The preparation steps of the fermentation broth are as follows: Activated marine sediment Bacillus Sneb2538 is inoculated into liquid fermentation medium and fermented at 28℃ and 200 r·min. -1 Shake culture for 2 days to obtain fermentation broth.

6. The application of the marine sediment Bacillus Sneb2538 of claim 1 or the fermentation broth of claim 2 in cucumber cultivation, characterized in that, The fermentation broth of *Bacillus cereus* Sneb2538 or *Bacillus cereus* Sneb2538 is used to prevent and control cucumber wilt and promote the growth of cucumber plants.

7. The application according to claim 6, characterized in that, The marine sediment Bacillus Sneb2538 was used to inhibit the cucumber-specific strain of Fusarium oxysporum, the pathogen causing cucumber wilt (Fusarium oxysporum). Fusarium oxysporum f.sp. cucumerinum Owen).

8. The application according to claim 6, characterized in that, The marine sediment Bacillus Sneb2538 is used to inhibit the germination of Fusarium oxysporum spores.

9. The application according to claim 6, characterized in that, The fermentation broth of the marine sediment Bacillus Sneb2538 was used to control cucumber wilt disease by drenching cucumber seedlings.

10. The application according to claim 6, characterized in that, The promotion of cucumber plant growth includes increasing plant height and fresh weight.

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