Halophilic bacillus HMF09 and application thereof

Abstract

This invention relates to a halophilic Bacillus ( Halobacillus sp HMF09, with accession number CGMCC No. 30475, is an antagonistic strain against *Fusarium oxysporum* tomato-specific strain. It exhibits high salt and alkali tolerance and efficient cadmium ion fixation. Application of this strain can reduce the ionic activity of cadmium in soil, alleviate salt and alkali stress on crops, and promote crop growth.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a halophilic Bacillus strain HMF09 and its applications. Background Technology

[0002] Heavy metal contaminated soil refers to the phenomenon where heavy metals are introduced into the soil due to human activities, resulting in a significantly higher level of heavy metals than before and causing ecological degradation. Cadmium (Cd) is a highly chemically active, mobile, and persistently toxic heavy metal that easily accumulates in the food chain. It can affect normal physiological activities such as crop enzyme activity systems, cell and organelle structure, and metabolism, posing a potential threat to human health.

[0003] Saline-alkali soil is a general term for saline soil and alkaline soil. Saline soil mainly refers to saline soil with high chloride or sulfate content, which is alkaline, but the pH is not necessarily high. Alkaline soil refers to soil containing carbonate or diphosphate, which has a higher pH and is alkaline. Saline-alkali soil has low organic matter content, low soil fertility, poor physical and chemical properties, and contains more anions and cations that are harmful to crops, making it difficult for crops to germinate.

[0004] In some parts of my country, soils suffer from both heavy metal pollution and salinity, which has a dual impact on plants and greatly affects their growth and development.

[0005] Addressing soil heavy metal pollution and salinization is an urgent task. Studies have shown that some functional microorganisms can produce substances such as carbonates, hydrogen sulfide, and phosphates to form precipitates with heavy metals, thereby reducing their mobility and bioavailability in the soil. This has led to their widespread application in the remediation of heavy metal-contaminated soils. For cadmium pollution in soil, microbial adsorption and degradation is a low-cost, environmentally friendly, and efficient method for treating cadmium-contaminated soil.

[0006] However, there are currently few strains that possess both high salt and alkali tolerance and efficient cadmium fixation capabilities. Therefore, screening for strains and inoculants that can promote plant growth and reduce the activity of heavy metal ions in the soil (i.e., reduce the content of available cadmium in the soil) and reduce the stress of salt and alkali on crops is of great significance for the sustainable agricultural development and efficient utilization of resources in saline-alkali land.

[0007] Fusarium wilt of tomatoes is a soil-borne disease caused by fungi. It is mainly spread through the soil and mainly damages the root and stem parts and vascular system of tomatoes. The development and application of fungal agents to control Fusarium wilt of tomatoes is of great significance in production. Summary of the Invention

[0008] The purpose of this invention is to provide a highly salt-tolerant, highly efficient cadmium ion-fixing bacterium, and antagonistic strain of *Fusarium oxysporum* (tomato-specific type) *Halophilus* HMF09. Applying this strain can reduce the ionic activity of cadmium in the soil, reduce the stress of salt and alkali on crops, prevent tomato wilt, and promote crop growth.

[0009] The present invention adopts the following technical solution:

[0010] A halophilic Bacillus ( Halobacillus sp HMF09 was deposited on April 30, 2024, at the China General Microbiological Culture Collection Center, Beijing, China, with accession number CGMCC No. 30475.

[0011] Furthermore, the halophilic Bacillus HMF09 is tolerant to salt and alkali conditions.

[0012] Furthermore, the halophilic Bacillus HMF09 is tolerant to the heavy metal cadmium.

[0013] Furthermore, the halophilic Bacillus HMF09 fixes or adsorbs the heavy metal cadmium.

[0014] Furthermore, the halophilic Bacillus HMF09 can antagonize Fusarium oxysporum tomato-specific strain.

[0015] Furthermore, the halophilic Bacillus HMF09 can promote plant growth.

[0016] Application of the aforementioned halophilic Bacillus HMF09 in the remediation of cadmium-contaminated soil.

[0017] Application of the above-mentioned halophilic Bacillus HMF09 in the treatment of saline-alkali soil.

[0018] Application of the above-mentioned halophilic Bacillus HMF09 in the remediation of cadmium-contaminated saline-alkali soil.

[0019] Application of the above-mentioned halophilic Bacillus HMF09 in promoting crop growth in cadmium-contaminated soil environment.

[0020] Application of the above-mentioned halophilic Bacillus HMF09 in the control of tomato wilt disease.

[0021] A microbial inoculant containing the aforementioned Halophilic Bacillus HMF09.

[0022] The beneficial effects of this invention are as follows: the halophilic Bacillus strain HMF09 exhibits excellent salt and alkali tolerance and the ability to reduce cadmium content, enabling it to grow normally in high-salt-alkali culture media and cadmium-containing environments. Simultaneously, the halophilic Bacillus strain HMF09 effectively alleviates the inhibition of plant vegetative growth under salt and alkali stress, improves the plant's salt and alkali tolerance, and promotes plant growth.

[0023] Fusarium wilt of tomato is a soil-borne vascular disease caused by Fusarium oxysporum, primarily affecting the root and stem regions of tomatoes, often leading to systemic wilting and death of plants during the flowering and fruiting stages. The halophilic Bacillus HMF09 strain of this invention can antagonize the tomato-specific Fusarium oxysporum. Applying this strain can effectively control tomato wilt and also induce tomato plants to increase the activity of tomato defense enzymes, thereby enhancing disease resistance.

[0024] This invention proposes new methods and ideas for the utilization of saline-alkali land and the prevention and utilization of cadmium-contaminated soil. It has obtained excellent microbial resources with better salt and alkali tolerance, life-promoting properties, and the ability to be used for the prevention and control of heavy metal-contaminated soil and resistance to plant diseases, for production selection. Attached Figure Description

[0025] Figure 1 The colony morphology of halophilic Bacillus HMF09.

[0026] Figure 2 The cell morphology of Bacillus halophilus HMF09.

[0027] Figure 3 A phylogenetic tree for halophilic Bacillus HMF09 constructed based on 16S rDNA.

[0028] Figure 4 This image shows a confrontation between Halophilic Bacillus HMF09 and Fusarium oxysporum. Detailed Implementation

[0029] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples were all purchased from conventional biochemical reagent stores.

[0031] Example 1 Screening of strain HMF09

[0032] (1) Isolation of strains

[0033] Soil samples were collected from a wheat experimental field (saline-alkali soil) in Emin County, Tacheng Prefecture, Xinjiang Uygur Autonomous Region, China. 5g of soil sample was added to a conical flask containing 100mL of sterile water and shaken at 180rpm for 30min to ensure thorough dispersion of microorganisms in the soil sample, yielding a soil mixture. The soil mixture was then diluted using a gradient of 10-1. -2 10 -3 10 -4 10 -5 10 -6 A soil mixture was spread, and 100 μL of the bacterial suspension was spread onto 10% sodium chloride LB solid medium for isolating salt-tolerant microorganisms. The plates were then inverted and incubated at 30°C for 48–96 hours.

[0034] (2) Purification of strains

[0035] Based on differences in colony morphology, color, and size, individual microbial colonies were selected and purified multiple times until monoclonal strains were obtained. The purified strains were stored in a -80°C freezer with 30% glycerol. Based on colony morphological characteristics, six salt-tolerant strains tolerant to 10% sodium chloride were isolated and purified, and numbered HH1, HH2, HH3, HH4, HH5, and HH6 in the order of purification.

[0036] (3) Discovery and initial screening of cadmium-resistant strains

[0037] Prepare PDA medium (200g potato, 20g glucose, 1000mL ultrapure water). Inoculate strains numbered HH1, HH2, HH3, HH4, HH5, and HH6 onto solid plates with a cadmium chloride concentration of 25 mmol / L for screening and culture. Set up 3 replicates for each concentration. Incubate at 28℃ for 4-5 days and observe and record the characteristics of single colonies.

[0038] (4) Re-screening of cadmium-resistant strains

[0039] Under aseptic conditions, single colonies from a 25 mmol / L PDA solid medium were picked up with an inoculation needle and streaked onto PDA solid medium plates with cadmium chloride concentration gradients of 10 mmol / L, 25 mmol / L, and 50 mmol / L. A strain with good growth and full colonies was obtained on the 25 mmol / L plate. After three purifications, the strain was numbered HH4.

[0040] (5) Test of HH4 strain's tolerance to cadmium-containing culture medium

[0041] 1) Preparation of cadmium-containing culture medium

[0042] LB solid cadmium-containing medium: 0.0816 g cadmium chloride, 5 g yeast extract, 10 g peptone, 10 g NaCl, 20 g agar, add distilled water to a final volume of 1 L, sterilize at 121 °C for 30 min to prepare a medium with a cadmium ion concentration of 50 mg / L, for later use.

[0043] 2) Test treatment

[0044] After appropriately diluting the seed culture of pure strain HH4 in a clean bench, take 200 μL and spread it evenly on an LB solid cadmium-containing plate using a spreader. After it is completely air-dried, seal the plate with sealing film and place it in a constant temperature shaker at 35℃ for incubation. After 72 hours, the bacterial concentration in the liquid culture medium is measured.

[0045] 3) Results Analysis

[0046] As shown in Table 1, after 72 hours of culture, the cell concentration of strain HH4 decreased from OD0.05. 600 =0.039 increased to OD 600 =1.676, proving that strain HH4 can grow normally in an environment with a cadmium concentration of 50 mg / L, demonstrating not only strong salt tolerance but also good survival in cadmium-contaminated environments. Strain HH4 was named HMF09.

[0047] Table 1. Results of cadmium resistance test for strain HH4

[0048] .

[0049] Example 2 Identification of strain HMF09

[0050] (1) Morphological characteristics

[0051] The colonies are round, orange-yellow, with smooth, even edges and no bumps. Figure 1 As shown. The bacteria are rod-shaped, have spores, and are Gram-positive, as indicated. Figure 2 As shown.

[0052] (2) Physiological and biochemical characteristics

[0053] The physiological and biochemical characteristics of strain HMF09 were identified, and the results are shown in Table 2.

[0054] Table 2. Physiological and biochemical characteristics of strain HMF09

[0055] .

[0056] (3) Molecular biological characteristics

[0057] Genomic DNA was extracted from strain HMF09. Using it as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced, yielding a 1500 bp DNA sequence (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST alignment analysis. Comparison with the 16S rDNA sequence in the NCBI database revealed that HMF09 shares 99% identity with *Bacillus halophilicus*. The phylogenetic tree was constructed as follows: Figure 3 As shown. Based on the morphological, sequencing analysis, and physiological and biochemical detection results, HMF09 was identified as a halophilic Bacillus (Bacillus). Halobacillus sp .).

[0058] Halophilic Bacillus HMF09 was deposited on April 30, 2024, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 30475.

[0059] Example 3: Determination of salt and alkali tolerance of halophilic Bacillus HMF09

[0060] (1) Determination of salt tolerance

[0061] The activated strain HMF09 was inoculated into 100 mL of LB liquid medium with NaCl concentrations of 10 g / L, 50 g / L, 100 g / L, 150 g / L, and 200 g / L. The medium was then incubated at 35 °C for 3 days. The growth of the bacteria was observed, and the bacterial concentration in the medium was measured. The results are shown in Table 3.

[0062] Table 3. Salt tolerance test results of strain HMF09

[0063] .

[0064] The strain HMF09 still exhibits good activity at a salt concentration of 150 g / L, indicating that the strain can tolerate high-salt environments.

[0065] (2) Determination of alkali resistance

[0066] The activated bacterial strains were inoculated into 100 mL of LB liquid medium with pH values ​​of 7, 8, 9, 10, and 11, and then placed in an incubator at 35°C for 3 days. The bacterial growth was observed, and the bacterial concentration in the liquid medium was measured. The results are shown in Table 4.

[0067] Table 4. Results of alkali resistance test for strains

[0068] .

[0069] The strain HMF09 still exhibits good activity at pH 10, indicating that the strain can tolerate alkaline environments.

[0070] Example 4: Test on the curing effect of strain HMF09 on cadmium ions

[0071] (1) Preparation of cadmium-containing culture medium

[0072] LB liquid cadmium-containing medium: 15 mg cadmium chloride (99.99%), 5 g yeast extract, 10 g peptone, 10 g NaCl, add sterile water to a final volume of 1 L, and sterilize at 121 °C for 15 min.

[0073] (2) Preparation of HMF09 bacterial suspension

[0074] The strain stored at -80℃ was activated by streaking on LB solid medium. Two colonies were picked up with an inoculation loop and inoculated into an Erlenmeyer flask containing 50 mL of LB liquid medium. The culture was incubated at 35℃ for 48 h to obtain the fermentation broth of HMF09. The fermentation broth was centrifuged at high speed (10000 r / min), and the supernatant was discarded to obtain bacterial sludge. This sludge was then diluted with an appropriate amount of sterile water to a viable count of 1 × 10⁻⁶ cells / mL. 8 The concentration of cfu / mL was used to obtain an HMF09 bacterial suspension.

[0075] LB liquid culture medium preparation method: 5g yeast extract, 10g peptone, 10g NaCl, 1000mL water, pH 7.2, sterilize at 121℃ for 30min.

[0076] (3) Experimental treatment

[0077] HMF09 bacterial suspension was added at a 10% (v / v) inoculation rate to three Erlenmeyer flasks containing 50 mL and 250 mL LB liquid cadmium-containing medium, respectively. The flasks were incubated at 30℃ and 180 rpm for 48 h, 72 h, and 96 h, respectively, to obtain the treatment solutions of strain HMF09. The three treatment solutions were centrifuged at 10000 rpm, and the supernatant was filtered through a 0.22 mm filter membrane before determining the cadmium ion content. The experiment was repeated three times, with LB liquid cadmium-containing medium as a control, and the percentage of cadmium ion solidification was calculated.

[0078] (4) Results Analysis

[0079] The results are shown in Table 5. The cadmium ion concentration in the control group was 9.20 mg / L, while after treatment with strain HMF09 for 48 h, 72 h, and 96 h, the cadmium ion concentrations in the solution were 8.42 mg / L, 5.28 mg / L, and 2.78 mg / L, respectively. After 96 h, the cadmium ion content in the solution decreased by 69.78%, demonstrating that strain HMF09 has a significant and effective cadmium ion fixation effect.

[0080] Table 5. Immobilization effect of strain HMF09 on cadmium ions in liquid cadmium-containing culture medium.

[0081] .

[0082] Example 5: Test of cadmium ions in soil stabilized by strain HMF09

[0083] (1) Preparation of bacterial suspension: The preparation method is the same as in Example 4, and the viable count is 1×10⁻⁶. 8 cfu / mL.

[0084] (2) First, put Cd 2+ Soil with a cadmium content of 50 mg / kg was dried at 80℃ for 5 hours, then placed at room temperature. A bacterial suspension (10 ml of bacterial suspension for every 20 g of soil) was added to the sterilized soil, mixed thoroughly, and left at room temperature for 3 days. A portion of the soil was then air-dried, and the available cadmium content was determined using the DTPA extraction method (GB / T23739-2009). The treatment without the bacterial suspension served as the control group, and each treatment was replicated three times.

[0085] As shown in Table 6, under the action of strain HMF09, the cadmium ion content in the treatment group decreased by 29.12% compared with the control group, indicating that Bacillus halophilus HMF09 has a significant immobilization effect on heavy metal cadmium ions in the soil.

[0086] Table 6. Immobilization effect of strain HMF09 on cadmium in soil (unit: mg / kg)

[0087] .

[0088] Example 6: Experiment on the growth-promoting effect of strain HMF09 on crops

[0089] (a) Wheat germination test

[0090] (1) Seed disinfection: Select wheat seeds of uniform size and full grains, soak them in 70% alcohol for 1 minute, then soak them in 3% NaClO for 10 minutes, then rinse them with sterile water 4 times and air dry.

[0091] (2) Preparation of HMF09 bacterial suspension: The preparation method is the same as in Example 4, and the viable count is 1×10⁻⁶. 8 cfu / mL.

[0092] (3) Germination test: First use a viable count of 1×10 8Wheat seeds were soaked in CFU / mL bacterial suspension (T1-T4) for 12 h in the dark, then drained. The control group (CK1-CK4) soaked their seeds in sterile water. NaCl solutions of 0 mmol / L, 100 mmol / L, 150 mmol / L, and 200 mmol / L were prepared, and sterile filter paper was moistened with each solution. Two petri dishes were placed on each concentration, one for treatment and one for control. The treated seeds were then arranged on the moistened filter paper, 50 seeds per dish, with each treatment repeated three times. The petri dishes were placed flat in an incubator and incubated at 26℃. Seed germination was recorded daily, and the results are shown in Table 7.

[0093] Table 7. Statistical results of the experiment on promoting wheat germination with Bacillus halophilus HMF09

[0094] .

[0095] As shown in Table 7, under the same salt stress concentration, the germination rate of wheat seeds treated with HMF09 bacterial suspension was higher than that treated with sterile water soaking. In the range of NaCl solution concentration from 0 to 200 mmol / L, the increase in germination rate was greater with the increase in salt concentration, further indicating that strain HMF09 can improve the salt stress tolerance of wheat seeds and increase their germination rate.

[0096] (II) Pot experiment on the promotion of wheat growth by strain HMF09

[0097] Wheat seeds with uniform germination potential were selected from CK1 and transplanted into flowerpots (18cm high, 16cm in diameter) filled with agronomic soil. The soil for this experiment was a mixture of nutrient soil, vermiculite and perlite in a ratio of 3:1:1.

[0098] The pot experiment consisted of two groups and four treatments, as shown in Table 8. The first group (T5, CK5) was filled with farmyard soil and then watered with 270 mL of deionized water, serving as the salt-free stress group. For T5, 2 mL of bacterial suspension was applied to the seedling roots every 7 days, while for CK5, an equal amount of water was applied as a control. The second group (T6, CK6) was filled with farmyard soil and then watered with 270 mL of 100 mmol / L NaCl solution, serving as the salt stress group. For T6, 2 mL of bacterial suspension was applied to the seedling roots every 7 days, while for CK6, an equal amount of water was applied as a control. Ten wheat seedlings were transplanted into each pot, with three pots for each treatment. The plants were cultured in a light incubator (26℃, 16 h / d). 100 mL of water was applied every 2 days, and seedling growth was observed regularly. Plant height and root length were measured after 40 days.

[0099] Table 8. Statistical results of the wheat growth promotion experiment using Bacillus halophilus HMF09

[0100] .

[0101] The above results indicate that, under both salt-free and salt-stressed conditions, the plant height and root length of wheat in the treatment group were significantly higher than those in the control group. Furthermore, under salt stress, the plant height and root length increased even more than those in the control group, further demonstrating that Bacillus halophilus HMF09 can promote wheat growth.

[0102] Example 7: Confrontation test between Halophilic Bacillus HMF09 and Fusarium oxysporum

[0103] The pathogen causing tomato wilt is Fusarium oxysporum tomato-specific strain (Fusarium oxysporum tomatoense). Fusarium oxysporum f. sp. lycopersici The pathogens used in this embodiment were provided by the Microbiology Laboratory of Hebei University of Science and Technology.

[0104] The *Fusarium oxysporum* strain, a tomato-specific pathogen, was activated on a PDA plate and incubated upside down at 28°C for 4 days. A mycelial cake was created at the edge of the *Fusarium oxysporum* colony using a sterile punch and placed in the center of a new PDA plate. Sterile filter paper discs were placed symmetrically at a distance of 2.0 cm from the mycelial cake, and 2 μL of the *Bacillus halophilus* HMF09 suspension prepared in Example 4 was dropped onto each filter paper disc. Plates inoculated only with pathogenic mycelial cakes and not with HMF09 suspension served as controls. The plates were incubated at 28°C for approximately 4 days until the control pathogenic colonies completely covered the plates. The pathogenic colonies and inhibition zones were then observed.

[0105] The results are as follows Figure 4 The results showed that inhibition zones were observed around Bacillus halophilus HMF09, indicating that strain HMF09 has a strong antagonistic ability against Fusarium oxysporum tomato-specific strain.

[0106] Example 8 Preparation of Bacillus halophilus HMF09 bacterial powder

[0107] (1) Preparation of LB liquid culture medium: Same as in Example 4.

[0108] (2) Activation of strain: Pick one loop of halophilic Bacillus HMF09 colony, inoculate it into a test tube containing 10 mL LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 35 ℃ for 24 h.

[0109] (3) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of LB liquid culture medium. Incubate at 160 rpm and 35 °C for 24 h to obtain seed culture.

[0110] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.6 L of LB liquid medium and cultured at 160 rpm and 35 °C for 48 h with constant temperature shaking to obtain the fermentation broth of Bacillus halophilus HMF09, with an effective viable count of 3.31 × 10⁻⁶ cells / mL. 9 cfu / mL.

[0111] (5) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by freeze dryer to obtain Bacillus halophilicus HMF09 powder. The effective viable count was tested to be 3.15 × 10⁻⁶. 10 cfu / g.

[0112] Example 9: Pot experiment of Bacillus halophilus HMF09 inoculum on cadmium-containing saline-alkali soil

[0113] Preparation of cadmium-containing saline-alkali soil: Soil taken from Huanghua area of ​​Cangzhou (salt content of 12.2g / kg, pH of 8.12) was prepared with cadmium chloride reagent to form saline-alkali soil with cadmium ion content of 20mg / kg. The soil was sterilized at 128℃ for 30min and left to stand for 1 day before use.

[0114] Seed treatment: Select uniform and plump corn seeds, soak them in 50ppm sodium hypochlorite solution for 1 hour, then wash the seeds 3 times with deionized water, and germinate them at 25℃ until they show white sprouts.

[0115] Preparation of HMF09 bacterial suspension: Take 1g of HMF09 powdered bacterial agent prepared in Example 7, add it to 1L of deionized water, stir to dissolve and set aside.

[0116] The pot experiment consisted of two treatments: an experimental group and a control group, with 10 pots in each treatment. Each pot (18 cm high and 16 cm in diameter) contained 1.5 kg of cadmium-containing saline-alkali soil and planted one maize seed at a depth of approximately 5 cm. After sowing, the experimental group was watered with 270 mL of bacterial suspension until the soil was completely moistened without seeping from the bottom of the pot, while the control group was watered with 270 mL of deionized water. The pots were then placed in a tissue culture room at 25°C and incubated. 100 mL of deionized water was applied every 4 days to maintain soil moisture. Once the maize plants had two true leaves, the experimental group was watered with 90 mL of the prepared bacterial suspension, while the control group was watered with 90 mL of deionized water. After 20 days of continued incubation, plant height, stem diameter, taproot length, and cadmium ion content in the aboveground parts and roots of the maize plants were measured to investigate the effects of the Bacillus halophilus HMF09 inoculant on maize biomass and cadmium ion content in maize plants.

[0117] Method for detecting cadmium ion content in maize plants: Take 100g samples from both the aboveground and root parts of maize plants. First, wash them with clean water, then rinse them three times each with EDTA solution and deionized water. Wipe the surface of the plants dry with a towel and dry them in an oven at 105℃ until constant weight. Ash the aboveground and root samples separately in a muffle furnace. Determine the cadmium content of the ash samples using atomic absorption spectrophotometry, and then calculate the cadmium content of the aboveground and root parts of the maize plants.

[0118] As shown in Table 9, under the condition of cadmium ion content of 20 mg / kg in saline-alkali soil, the maize plants treated with Bacillus halophilus HMF09 inoculant showed significantly better plant height, stem diameter, and taproot length than the control group, indicating that the HMF09 inoculant can effectively promote maize growth and inhibit the toxic effects of cadmium ions in the soil. Simultaneously, the cadmium content in the aboveground parts and roots of maize plants treated with HMF09 inoculant was significantly reduced by 20.76% and 31.44%, respectively, indicating that HMF09 inoculant has a good immobilization effect on the heavy metal cadmium in the soil, reducing the absorption of cadmium ions by roots and their translocation to the aboveground parts, and significantly reducing the content of harmful cadmium ions in maize plants.

[0119] The experimental results show that the application of HMF09 microbial agent is beneficial to the safe planting and development of farmland soil contaminated with heavy metal cadmium.

[0120] Table 9. Effects of strain HMF09 on maize plant biomass and cadmium ion content.

[0121] .

[0122] Example 10: Field efficacy test of Bacillus halophilus HMF09 inoculant for controlling tomato wilt disease.

[0123] The field efficacy trial was conducted in a plot of land in Xinjizhen, Changli County, Hebei Province, where tomato wilt disease has been a persistent problem. Tomato seedlings were transplanted on June 3, 2024, with a plant spacing of 30 cm and a row spacing of 60 cm. The tomato variety was Jiafen 15.

[0124] Preparation of HMF09 bacterial suspension: Following the preparation method described in Example 4, the viable bacterial count was 1 × 10⁻⁶. 8 cfu / mL.

[0125] The experiment included three treatments: one with a viable bacterial count of 1×10⁻⁶. 8HMF09 bacterial suspension (cfu / mL), 30% Mancozeb·Oxythiamethoxam aqueous solution (Changchun Changshuang Pesticide Co., Ltd.) diluted 2000 times, and water control were used. Each treatment was replicated three times, corresponding to three plots, with 120 seedlings in each plot, and the plots were randomly arranged. Root irrigation treatments were performed on June 3, 2024 (transplanting), June 15, 2024 (end of seedling establishment), and July 5, 2024 (first fruit cluster flowering), with 100 mL applied to each seedling.

[0126] Tomato Fusarium wilt disease severity grading standard:

[0127] Level 0: Healthy plant with no symptoms;

[0128] Grade 1: One or two true leaves wilt and droop or turn yellow noticeably, eventually falling off;

[0129] Grade 2: 3 or 4 true leaves turn yellow or the whole plant turns yellow, and the leaves wilt and droop.

[0130] Grade 3: Five or six true leaves are obviously wilted and drooping or the true leaves are severely yellowed, and the plant growth is inhibited and stunted;

[0131] Level 4: The entire plant is severely wilted and eventually dies.

[0132] Disease index = ∑(number of diseased plants at each level × corresponding level) / (total number of plants investigated × maximum level) × 100.

[0133] Prevention and control effect (%) = [(blank control disease index - treatment disease index) / blank control disease index] × 100.

[0134] Investigation time and method: On July 5, 2024 (when the first fruit cluster flowered), 10 and 20 days after root irrigation treatment, 100 plants in the plot were investigated to count the disease incidence and calculate the control efficacy. Another 20 seedlings in the plot were used to collect fresh roots and measure the activity of plant root defense enzymes. The control effect of tomato wilt is shown in Table 10.

[0135] Table 10 Control efficacy against tomato wilt

[0136] .

[0137] Experimental results show that the use of Bacillus halophilus HMF09 inoculant can reduce the damage of tomato wilt disease, with a control effect of over 75%.

[0138] Five days after the root irrigation treatment on July 5, 2024 (when the first fruit spike flowered), 10 tomato plants were selected from each plot, and their roots were collected, quickly washed, and rinsed three times with distilled water. The activity of the tomato root defense enzyme, peroxidase (POD), was determined using the guaiacol method; the determination of phenylalanine ammonia-lyase activity was based on the method of Ji Mingshan ("The Effect of Chitosan Seed Soaking on the Activity of Several Defense Enzymes in the Root System of Maize Seedlings," author Ji Mingshan, Shenyang Agricultural University), and also referenced Wang Bin's "Induction Effect of Arbuscular Mycorrhizal Fungi on Tomato Signaling Substances," 15 July 2010, 29(4): 561-568. The test results are shown in Table 11.

[0139] Table 11 Activity detection of POD and PAL

[0140] .

[0141] As shown in Table 11, the POD activities of tomato roots treated with water and with methyl thiamethoxam·oxadixyl water-soluble agent were 23.5 U·g. -1 FW·min -1 and 20.1 U·g -1 FW·min -1 The PAL activity values ​​were 24.3 U·g. -1 FW·h -1 20.2 U·g -1 FW·h -1 The POD activity of tomato roots treated with HMF09 bacterial suspension reached as high as 60.7 U·g. -1 FW·min -1 The POD activity was increased by 37.2 U·g compared to that of water treated with clean water. -1 FW·min -1 The PAL activity reached 51.6 U·g. -1 FW·h -1 It improves efficiency by 27.3 U·g compared to clean water treatment. -1 FW·h -1 .

[0142] In summary, the above results indicate that the mechanism of action of HMF09 biocontrol bacteria in controlling tomato wilt is as follows: firstly, halophilic Bacillus HMF09 can antagonize Fusarium oxysporum (Example 7 confrontation experiment); secondly, HMF09 can also induce resistance in tomato plants by increasing the activity of tomato defense enzymes and enhancing disease resistance, thereby resisting the infection of the pathogen Fusarium oxysporum.

[0143] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A halophilic Bacillus ( Halobacillus sp. HMF09, characterized in that, The accession number is CGMCCNo.30475.

2. The application of the halophilic Bacillus HMF09 as described in claim 1 in the remediation of cadmium-contaminated soil.

3. The application of the halophilic Bacillus HMF09 as described in claim 1 in the remediation of cadmium-contaminated saline-alkali soil.

4. The application of the halophilic Bacillus HMF09 as described in claim 1 in promoting maize growth in cadmium-contaminated soil.

5. The application of the halophilic Bacillus HMF09 as described in claim 1 in the control of tomato wilt disease.

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