Bacillus velezensis bvx001, compound microbial inoculum and application

Abstract

This application belongs to the field of microbial technology and specifically relates to a Bacillus velezensis bvx001, a compound microbial inoculum, and their applications. The said Bacillus velezensis ( Bacillus velezensis ) bvx001 was deposited at the General Microbiology Center of the China Microbial Culture Collection Center on January 3, 2025, at the address No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33291. The present invention provides a compound microbial inoculum that has the abilities of silicon solubilization, phosphorus solubilization, and nitrogen fixation, with comprehensive functions. It can increase the contents of silicon, phosphorus, and nitrogen elements in the soil, enhance the stress resistance of plants, and promote plant growth, and can be used for the development and utilization of saline-alkali land.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and specifically relates to a Bacillus velezensis bvx001, a compound microbial agent, and applications thereof. Background Art

[0002] Saline-alkali soil contains a large amount of salt ions, resulting in low soil moisture content and poor air permeability; high-concentration salt ions disrupt the osmotic balance and water balance of plants, reduce the photosynthetic rate of plants and limit the absorption of water by plants, thereby causing difficulties in plant budding and yield loss. In addition, the soil contains a large amount of nutrient elements, but due to problems such as low solubility of nutrient elements caused by saline-alkali stress, the absorption and utilization by plants are greatly hindered, mainly including elements such as silicon, phosphorus, and nitrogen, and these elements all play important roles in the growth and development of plants. Among them, nitrogen element is a component of important biological macromolecules such as proteins and nucleic acids, directly affecting the growth, development, and metabolism of crops; phosphorus element plays an irreplaceable role in key physiological processes such as energy transfer and photosynthesis; and silicon element can not only promote the formation and development of plant organs, but also play a more important role in improving the resistance of plants to biotic and abiotic stresses. At present, many studies have shown that silicon element can improve the resistance of plants in adversity and promote plant growth. Therefore, improving the biological utilization rate of silicon by plants in the soil has great research significance for the development and utilization of saline-alkali land. However, most of the silicon in the soil cannot be directly utilized by plants. Currently, the more common method is to apply silicon fertilizer additionally, but the silicon fertilizer is easily lost after being applied to the soil, the fertilizer effect is slow, the utilization rate is low, and due to the small amount of silicon fertilizer and the lack of a unified standard, the quality of silicon fertilizer on the market varies; at the same time, most silicon fertilizers are relatively expensive, resulting in high economic costs and little benefit. Therefore, there is an urgent need to provide a new solution. Summary of the Invention

[0003] The object of the present invention is to solve the deficiencies of the prior art and provide a Bacillus velezensis bvx001, a compound microbial agent, and applications thereof. The following specific technical solutions are adopted:

[0004] In the first aspect, the present invention provides a strain of Bacillus velezensis bvx001, and the Bacillus velezensis ( Bacillus velezensis ) bvx001 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 3, 2025, at the address of No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number of CGMCC No. 33291.

[0005] The gene sequence of the 16S rDNA of the above-mentioned Bacillus velezensis bvx001 is as shown in SEQ ID No.1.

[0006] SEQ ID No.1:

[0007]

[0008] The silicon-solubilizing bacteria provided by the present invention can convert insoluble silicate into silicon (i.e., monosilicic acid) that can be absorbed by plants through a series of biochemical reactions. Moreover, the strain can colonize around the roots of plants in the soil, form a symbiotic relationship with plants, and continuously play a role, thereby alleviating the damage of salt to plants and promoting plant growth.

[0009] In a second aspect, the present invention provides a compound microbial inoculant, and the microbial inoculant includes the above-mentioned compound microbial inoculant.

[0010] As a further preferred embodiment, the compound microbial inoculant further includes Acinetobacter acin003; the Acinetobacter ( Acinetobacter sp. ) acin003 was deposited at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on January 3, 2025, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33292.

[0011] The gene sequence of the 16S rDNA of the above-mentioned Acinetobacter acin003 is shown in SEQ ID No. 2.

[0012] SEQ ID No. 2:

[0013]

[0014] As a further preferred embodiment, the composite microbial inoculum further comprises Exiguobacterium exio007; the Exiguobacterium ( Exiguobacterium sp. ) exio007 was deposited on January 3, 2025 at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33293.

[0015] The gene sequence of the 16S rDNA of the above Exiguobacterium exio007 is shown in SEQ ID No. 3.

[0016] SEQ ID No. 3:

[0017]

[0018] The present invention provides a compound microbial inoculant. All three strains in the compound microbial inoculant have certain salt and alkali tolerance capabilities and plant growth promotion capabilities. The compound microbial inoculant simultaneously has the capabilities of silicon solubilization, phosphorus solubilization, and nitrogen fixation. According to the saline-alkali soil experiment of the present invention, the strains were compounded into the BAE inoculant and inoculated into Chinese cabbages to study its effect on the growth of Chinese cabbages. The results show that: compared with single strains, the compound microbial inoculant BAE can more significantly increase the silicon content in the leaves of Chinese cabbages, increase the leaf area, fresh weight, and chlorophyll content of Chinese cabbages, improve the growth condition of Chinese cabbages under saline conditions, and has high practical application value.

[0019] In a third aspect, the present invention provides the application of the above-mentioned Bacillus velezensis bvx001 or the above-mentioned compound microbial inoculant in a product for promoting plant growth, and the product is a product that simultaneously has the functions of silicon solubilization, phosphorus solubilization, and nitrogen fixation.

[0020] In a fourth aspect, the present invention provides the application of the above-mentioned Bacillus velezensis bvx001 or the above-mentioned compound microbial inoculant in promoting the germination of Chinese cabbage seeds and / or preparing a Chinese cabbage seed germination promoter.

[0021] In a fifth aspect, the present invention provides a Chinese cabbage seed germination promoter, and the Chinese cabbage seed germination promoter includes the above-mentioned Bacillus velezensis bvx001 or the above-mentioned compound microbial inoculant.

[0022] In a sixth aspect, the present invention provides the application of the above-mentioned Bacillus velezensis bvx001 or the above-mentioned compound microbial inoculant in inhibiting plant pathogenic bacteria and / or preparing a plant pathogenic bacteria inhibitor.

[0023] As a further preferred embodiment, the plant pathogenic bacteria include Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. tritici, Fusarium culmorum, Fusarium oxysporum, Fusarium graminearum, and Fusarium moniliforme.

[0024] In a seventh aspect, the present invention provides a plant pathogenic bacteria inhibitor, and the plant pathogenic bacteria inhibitor includes the above-mentioned Bacillus velezensis bvx001 or the above-mentioned compound microbial inoculant.

[0025] The beneficial effects of the present invention are as follows:

[0026] The present invention provides three strains of salt- and alkali-tolerant strains with excellent silicon solubilization capabilities, which have certain plant growth promotion capabilities, can secrete auxin, siderophores, etc., and can promote the growth of crops when applied in saline-alkali land. It also provides a compound microbial inoculant containing these three strains and several salt- and alkali-tolerant silicon-solubilizing bacteria. The compound microbial inoculant simultaneously has the capabilities of silicon solubilization, phosphorus solubilization, and nitrogen fixation, and has comprehensive functions. It can increase the contents of silicon, phosphorus, and nitrogen elements in the soil, enhance the stress resistance of plants, and promote plant growth, and can be used for the development and utilization of saline-alkali land. Brief Description of the Drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Shown are the colony morphologies and Gram stainings of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 on LB plates, with a scale bar of 1 μm;

[0029] Figure 2 Shown is the phylogenetic tree map of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007;

[0030] Figure 3 Shown is the silicon-degrading hydrolysis zone of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 on the silicon-degrading plate;

[0031] Figure 4 Shown is the determination of the silicon-degrading ability of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 in liquid;

[0032] Figure 5 Shown is the growth of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 in LB liquid medium supplemented with different concentrations of NaCl;

[0033] Figure 6 Shown is the growth of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 in LB liquid medium with different pH values;

[0034] Figure 7 Shown is the phosphorus-degrading hydrolysis zone of Acinetobacter acin003 and Exiguobacterium exio007;

[0035] Figure 8 Shown is the nitrogen-fixing hydrolysis zone of Acinetobacter acin003;

[0036] Figure 9 Shown is the size of the silicon-degrading hydrolysis zone of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 under different salt conditions;

[0037] Figure 10The figure shows the effects of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 on the growth of Chinese cabbage under salt conditions;

[0038] Figure 11 The figure shows the effects of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 on the physiological parameters of Chinese cabbage under salt conditions;

[0039] Figure 12 The figure shows the antagonistic effect of Bacillus velezensis bvx001 against pathogenic fungi. In the figure, A is Gaeumannomyces graminis var. avenae ( Gaeumonnomyces graminis var avenae ); B in the figure is Gaeumannomyces graminis var. tritici ( Gaeumonnomyces graminis ); C in the figure is Fusarium culmorum ( Fusariumgraminearum culmorum ); D in the figure is Fusarium oxysporum ( Fusariumgraminearum oxysporum ); E in the figure is Fusarium graminearum ( Fusariumgraminearum ); F in the figure is Fusarium moniliforme ( Fusarium verticillioides ). Detailed implementation manners

[0040] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without creative efforts shall fall within the protection scope of the present application.

[0041] Example 1

[0042] Screening of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007

[0043] 1. Preparation of soil suspension: Weigh 10.0 g of soil sample and add it to a sterilized 250 mL shaking flask containing 90 mL of sterile water. Place it on a shaker and shake (200 rpm, 30 °C, 2 h - 3 h) to obtain a 10 -1 g / mL soil suspension; Dilute the soil suspension to 10 -2 g / mL, 10 -3 g / mL, 10 -4 g / mL, 10 -5 g / mL, 10 -6 g / mL using the 10-fold dilution method;

[0044] 2. Purification of strains: Take 10 -4 g / mL, 10 -5 g / mL, 10 -6100 μL of the suspension with dilution gradients of g / mL was evenly coated on the Alexandrov solid medium (5 g / L of sucrose, 2 g / L of Na2HPO4, 0.5 g / L of MgSO4·7H2O, 0.005 g / L of FeCl3·6H2O, 0.1 g / L of CaCO3, 2.5 g / L of magnesium silicate, 20 g / L of agar) plates, and placed in a constant temperature incubator at 30 °C for 2 d to 3 d. Multiple single colonies with characteristics related to silicon-dissolving microorganisms were picked and purified by streaking on the Alexandrov solid plates, repeating 2 to 3 times.

[0045] 3. Strain identification:

[0046] (1)Observation of strain morphology and Gram staining

[0047] The single strain was screened out by the dilution coating plate method and named: bvx001, acin003, exio007. It was streaked on the LB solid plate (10 g / L of NaCl, 10 g / L of peptone, 5 g / L of yeast powder, 1.5 g / L of agar) and cultured in an incubator at 30 °C for 24 h to observe the colony morphology. It was inoculated into the liquid LB medium (10 g / L of NaCl, 10 g / L of peptone, 5 g / L of yeast powder). 10 μL of the bacterial liquid was dropped on a sterile glass slide, and the strain was slightly heated on the alcohol lamp flame to fix it. After the glass slide cooled down, crystal violet solution was dropped until the colony was covered, and stained for about 1 min; the stain was gently rinsed off with deionized water and dried with absorbent paper; iodine solution was dropped until the colony was covered, and stained for about 1 min; the stain was rinsed and dried in the same way; 95% absolute ethanol was dropped for decolorization until the colony became colorless, washed with water; safranin solution was dropped until the colony was covered, and stained for about 1 min, rinsed and dried in the same way, and then observed under the microscope.

[0048] The results are as Figure 1 shown: The single colony of strain bvx001 on the LB solid plate was milky white, smooth on the surface, spherical, viscous in texture and neat at the edge. Under the microscope, the bacteria were rod-shaped, and the Gram staining result was blue-violet, belonging to Gram-positive bacteria.

[0049] The colony of strain acin003 on the LB solid plate was grayish white, spherical, smooth on the surface, and neat at the edge. Under the microscope, the bacteria were coccobacilli, often arranged in pairs, and the Gram staining result was purplish red, belonging to Gram-negative bacteria.

[0050] The colony of strain exio007 on the LB solid plate was orange-yellow, round, and moist on the surface. Under the microscope, the bacteria were short rods, arranged singly or in pairs, and the Gram staining result was blue-violet, belonging to Gram-positive bacteria.

[0051] (2)Molecular biology identification

[0052] Extract the genomes of three strains of bacteria using a bacterial DNA extraction kit and use them as templates. Using the 16S rDNA universal primer sequences 27F (SEQ ID No.4) 5’-AGAGTTTGATCCTGGCTCAG-3’ and 1492R (SEQ ID No.5) 5’-GGTTACCTTGTTACGACTT-3’, perform PCR amplification on the screened strains. After completion, send them to Shanghai Jieli Biotechnology Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3.

[0053] Table 1 PCR reaction system

[0054]

[0055] Submit the obtained sequences on NCBI for Blast sequence alignment, and construct a phylogenetic tree using the mega11 software (Molecular Evolutionary Genetics Analysis).

[0056] The results show that:

[0057] The phylogenetic trees of the three strains of bacteria are as Figure 2 shown. bvx001 was identified as Bacillus velezensis; acin003 was identified as Acinetobacter; exio007 was identified as Exiguobacterium.

[0058] Example 2

[0059] Detection of silicon-solubilizing ability of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007

[0060] (1) Qualitative detection by plate method

[0061] Take 10 μL of the bacterial solution respectively on the magnesium silicate plate (glucose 10 g / L, magnesium silicate 2.5 g / L, agar 1.5 g / L) and observe the size of the hydrolysis zone. The results are as Figure 3 shown: Hydrolysis zones of silicon solubilization can appear on the magnesium silicate plate for the three strains of bacteria, indicating that all three strains have the ability to dissolve silicon.

[0062] (2) Quantitative detection by liquid method

[0063] Inoculate into the Alexandrov liquid medium at an inoculation amount of 1%, culture at 30 °C and 200 rpm for 5 days, centrifuge the culture solution at 8000 rpm for 15 min, take the supernatant and filter it, and detect the soluble silicon content in the supernatant by inductively coupled plasma-mass spectrometry (ICP-MS).

[0064] The experimental results are as Figure 4 shown: Compared with the blank control CK without adding bacteria, after inoculating the three strains of bacteria bvx001, exio007 and acin003, the soluble silicon content in the culture solution has increased to a certain extent. Among them, the group adding the bvx001 strain has the most significant change, followed by exio007 and acin003.

[0065] Example 3

[0066] Detection of salt and alkali tolerance and growth promotion ability of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007

[0067] (1) Salt and alkali tolerance

[0068] Pick single colonies into LB liquid medium overnight respectively, and inoculate them into liquid LB containing 1% NaCl (w / v), 3% NaCl (w / v), 5% NaCl (w / v), 6% NaCl (w / v), 7% NaCl (w / v), 8% NaCl (w / v), 9% NaCl (w / v), 10% NaCl (w / v), 11% NaCl (w / v) and liquid LB medium with pH = 7, 8, 9, 10, 11 at an inoculation amount of 1%, culture in a constant temperature shaker at 30 °C and 200 rpm for 24 h, and use a spectrophotometer to measure the OD600nm of the bacterial solution.

[0069] The results are as Figure 5 shown: The strain with the strongest salt tolerance is exio007. When the NaCl concentration is 7% (w / v), about 50% of the strains can grow and reproduce; followed by the strain bvx001. Compared with 1% NaCl (w / v), under the condition of 7% NaCl (w / v), about 30% of the strains can survive; finally, it is acin003. Although its salt tolerance is not as strong as that of the two strains, it also has a certain salt tolerance and can tolerate up to 6% NaCl (w / v) at most.

[0070] The results are as Figure 6 shown: The three strains all have strong alkali tolerance. Among them, acin003 and exio007 can tolerate the alkaline environment with pH = 10 at most, and bvx001 can tolerate the alkaline environment with pH = 9 at most.

[0071] (2)Determination method of auxin (IAA)

[0072] Pick single colonies into LB liquid medium overnight; inoculate into LB liquid medium containing L-tryptophan (200 mg / L) at an inoculation amount of 1%, and culture on a shaker at 180 r / min and 30 °C for 36 h; pipette 2 mL of the bacterial liquid into a centrifuge tube, centrifuge at 10000 r / min for 10 min, take 1.5 mL of the supernatant and add an equal volume of Salkowski colorimetric solution (50 mL of 35% HClO4 + 1 mL of 0.5 mol / L FeCl3) into a 5 mL centrifuge tube, let stand in the dark for 30 min, and measure its OD530 nm value. After making a standard solution with analytical pure indoleacetic acid and performing the Salkowski colorimetric reaction, obtain a standard curve. Calculate the content of IAA in the fermentation broth per unit volume by referring to the standard curve.

[0073] The results are shown in Table 2, and the results indicate that: all three strains can secrete auxin, among which the yield of bvx001 is the highest, up to 54.4 ± 0.22 mg / L, compared with only 8.0 mg / L of the negative control Escherichia coli DH5-α.

[0074] Table 2 Auxin production by different strains

[0075]

[0076] (3)Determination method of siderophore

[0077] Pick single colonies into LB liquid medium overnight; inoculate into MKB (full name) liquid medium (acid-hydrolyzed casein 5 g / L, MgSO4·7H2O 2.5 g / L, glycerol 15 mL / L, K2HPO4·3H2O 3.28 g / L) and culture at a constant temperature of 30 °C on a shaker for 36 h; take 1 mL of the bacterial liquid and centrifuge at 12000 rpm for 10 min, take 100 μL of the supernatant and mix it with 100 μL of chromazurol S (CAS) quantitative detection solution, perform the same operation with sterile ddH2O as the control, after reacting at room temperature for 1 h, record the OD630nm values of the sample (As) and the control (Ar) respectively, and calculate the siderophore yield using the following formula:

[0078] Siderophore unit (%) = 100×(Ar—As) / Ar

[0079] The results are shown in Table 3, and the results indicate that: all three strains can secrete siderophores, indicating that they all have a certain growth-promoting ability. Among them, the amount of siderophores secreted by bvx001 is as high as 87%, and exio007 is as high as 73%, belonging to strains with high-efficiency secretion of siderophores.

[0080] Table 3 Siderophore secretion of different strains

[0081]

[0082] (4)Determination of ACC deaminase production ability

[0083] a. Pick a single colony and inoculate it into 30 mL of DF (KH2PO4 4 g / L, Na2HPO4 6 g / L, MgSO4·7H2O 0.2 g / L, glucose 2 g / L, gluconic acid 2 g / L, citric acid 2 g / L, H3BO3 0.01 mg / L, MnSO4·H2O 0.01 mg / L, ZnSO4·7H2O 0.078 mg / L, CuSO4·5H2O 0.05 mg / L, MoO3 0.01 mg / L, FeSO4·7H2O 1 mg / L) liquid medium, and culture it in the dark with shaking at 30 °C and 200 r / min for 12 h;

[0084] b. Then centrifuge the bacterial solution at 4 °C and 8000 r / min for 10 min, collect the bacterial suspension, and resuspend the precipitate in 30 mL of ADF (DF medium supplemented with 5 mmol / L ACC solution) liquid medium again, and culture it in the dark with shaking at 30 °C and 200 r / min for 24 h;

[0085] c. Centrifuge again at 4 °C and 8000 r / min for 10 min, resuspend with 5 mL of 0.1 moL / L Tris-HCl buffer (pH 7.6) and wash twice; transfer to a 1.5 mL centrifuge tube, resuspend the bacterial cells in 600 μL of 0.1 moL / L Tris-HCl buffer (pH = 8.5) to dissolve the bacterial cells; add 30 μL of toluene and vortex for 30 s to break the bacterial cells;

[0086] d. Enzyme activity determination: Pipette 200 μL of the broken cell bacterial suspension, add 20 μL of 0.5 moL / L ACC solution, mix well and keep in a water bath at 30 °C for 15 min;

[0087] e. Then add 1 mL of 0.56 moL / L HCl solution and shake well, centrifuge at 10000 r / min for 10 min;

[0088] f. Take 1 mL of the above centrifuged supernatant, add 800 μL of 0.56 moL / L HCl solution, mix well and then add 300 μL of 2 g / L 2,4-dinitrophenylhydrazine solution, and keep in a water bath at 30 °C for 30 min;

[0089] g. Add 2 mL of 2 moL / L NaOH solution for color development and measure the OD540 value.

[0090] After deducting the spontaneous products in the control samples for each strain, calculate the OD540 value of the sample and substitute it into the standard curve to calculate the corresponding α-ketobutyric acid content. Define the amount of 1 μmoL α-ketobutyric acid formed per minute as 1 enzyme activity unit.

[0091] (6)Determination of cellular protein concentration

[0092] Take 10 μL of toluene-treated bacterial cells and dilute them 10-fold with 1×PBS; dilute the 1 mg / mL BSA protein standard solution with 1×PBS to obtain protein concentration gradients of 20 μg·mL -1 , 40 μg·mL -1 , 60 μg·mL -1 , 80 μg·mL -1 , 100 μg·mL -1 . Use 1×PBS as the blank control, with 3 replicates for each concentration; measure the protein concentration using the Bradford protein kit. After reacting for 5 min, measure the OD595 value of each well with an enzyme-linked immunosorbent assay reader. Use the average OD595 value as the ordinate and the corresponding protein concentration as the abscissa to plot the standard curve; determine the protein concentration of the sample dilution based on the average OD595 value of the sample dilution on the standard curve.

[0093]

[0094] The results are shown in Table 4: Bacillus velezensis bvx001 can produce ACC deaminase, and the enzyme activity can reach 2.733 ± 0.55 U / mg. Using Escherichia coli DH5-α as the negative control, the enzyme activity produced by Escherichia coli DH5-α is 0.0073 U / mg.

[0095] Table 4 ACC deaminase activities of different strains

[0096]

[0097] (5)Determination of phosphate solubilization ability

[0098] Take 10 μL of the bacterial solution and place it on the Meng Jina inorganic phosphorus medium (PKO) (10 g / L of glucose, 0.5 g / L of (NH4)2SO4, 0.3 g / L of KCl, 0.3 g / L of NaCl, 0.03 g / L of FeSO4·7H2O, 0.3 g / L of MgSO4·7H2O, 0.03 g / L of MnSO4·4H2O, 10 g / L of Ca3(PO4)2, 15 g / L of agar) and culture it in an incubator at 30 °C for 5 to 7 days. If a clear hydrolysis zone appears, it indicates the ability to dissolve phosphorus.

[0099] The results are as Figure 7 shown: On the PKO plate, clear hydrolysis zones appeared around the colonies of acin003 and exio007, indicating that the strains have the ability to dissolve phosphorus.

[0100] (6)Determination of nitrogen fixation ability

[0101] Take 10 μL of the bacterial solution and place it on the Ashby nitrogen-free medium plate (0.2 g / L of KH2PO4, 0.2 g / L of NaCl, 0.2 g / L of MgSO4, 5 g / L of CaCO3, 0.1 g / L of CaSO4, 10 g / L of glucose, 20 g / L of agar powder) and culture it in an incubator at 30 °C for 5 to 7 days. If a clear hydrolysis zone appears, it indicates the ability to fix nitrogen.

[0102] The results are as Figure 8 shown: On the Ashby nitrogen-free medium plate, a clear hydrolysis zone appeared around the colony of acin003, indicating that the strain has the ability to fix nitrogen.

[0103] Example 4

[0104] Determination of the silicon-dissolving ability of Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007 under salt conditions

[0105] Take 10 μL of the bacterial solution and place it on the magnesium silicate plates with different concentrations of NaCl. The NaCl concentrations are set to 3% NaCl (w / v) and 6% NaCl (w / v). Subsequently, observe and record the size of the hydrolysis zone.

[0106] The experimental results are as Figure 9As shown: On the two plates with additional NaCl, the three strains still have the ability to degrade silicon, and under the condition of 3% NaCl (w / v) medium, the silicon degrading efficiency (D / d) of aci003 and exio007 is higher than that under normal conditions, but as the salt concentration continues to increase, the hydrolysis circle formed by the strains' silicon degrading shrinks again, indicating that the high-salt environment will inhibit the strains' silicon degrading ability. In addition, the three strains still have the ability to degrade silicon on the plates with added NaCl, which shows that the composite bacterial agent has potential application value in the improvement of saline-alkali land.

[0107] Table 5 Silicon-degrading ability of strains at different salinities

[0108]

[0109] Example 5

[0110] To explore the growth-promoting effect of a composite microbial agent consisting of Bacillus velez bvx001, Acinetobacter acin003 and Exiguobacterium exio007 on Chinese cabbage

[0111] The pots used in the experiment had a capacity of about 5.5 L, and each pot contained 2.0 kg of soil. The soil was irrigated with different concentrations of sodium chloride solution (3.0 g / kg and 4.5 g / kg) in advance and stirred evenly. It was placed for 1 to 2 days to make it closest to the natural state. Cabbage seeds with full grains were selected and sown in a triangular pattern, with 3 seeds per pot. The bacterial suspension was inoculated into the soil around the cabbage seeds in each pot at a 10% inoculation rate. The blank control group was replaced with an equal amount of clean water and placed in a natural environment for growth. After that, tap water was used for irrigation, and insoluble magnesium silicate was added to the water for irrigation, with an additional amount of 2 g / kg. Harvest after about 50 days.

[0112] Table 6 Potted plant experimental groups

[0113]

[0114] It should be noted that in the above table, BE represents a composite microbial agent of Bacillus belezii bvx001 and Exiguobacterium exio007; AE represents a composite microbial agent of Acinetobacter acin003 and Exiguobacterium exio007; AB represents a composite microbial agent of Bacillus belezii bvx001 and Acinetobacter acin003; BAE represents a composite microbial agent of Bacillus belezii bvx001, Acinetobacter acin003 and Exiguobacterium exio007.

[0115] After 50 days of cultivation, the physiological conditions of each group of cabbage plants were recorded, and their physical indicators were measured: root weight and leaf area, etc.; biochemical indicators: silicon content, total chlorophyll and malondialdehyde content in leaves.

[0116] The experimental results are as follows Figure 10 - Figure 11 : In the pot experiment, salt stress significantly inhibited the normal growth and development of plants, resulting in plant dwarfing, withered and yellowed leaves and other characteristics. After adding different bacterial suspensions under salt stress, the growth state of Chinese cabbage was significantly better than that of plants treated with salt stress alone, and the same salt-tolerant strains had different alleviating effects. For example, after adding only the bvx001 strain, the leaf areas of Chinese cabbage under the two salt gradients increased by 68% and 46% respectively, the root weights increased by 61% and 120% respectively, and the fresh weights increased by 30% and 65% respectively, showing a relatively obvious growth-promoting effect. At the same time, it can be observed that the effect of adding the composite bacterial agent group was the most significant. Under the environments of 3.0 g / kg and 4.5 g / kg NaCl, the leaf areas increased by 173% and 186% respectively; the fresh weights increased by 140% and 300% respectively, even higher than those of Chinese cabbage in the blank control group; at the same time, under the condition of 4.5 g / kg NaCl, the root weight of Chinese cabbage was most significantly inhibited by salt stress, significantly lower than that of the blank control, while the root weight increased significantly after inoculating the composite microbial agent, with an increase of 200%.

[0117] It was found by measuring the change of silicon content in leaves that salt stress affected the absorption of nutrient elements including silicon in the soil by plants, resulting in the characteristics of yellowing and dwarfing of leaves, and thus the silicon content in leaves was also low. Under the condition of adding insoluble magnesium silicate to each pot, the silicon content in the leaves of Chinese cabbage inoculated with silicon-dissolving bacteria increased to a certain extent compared with the salt control group. Especially for the Chinese cabbage inoculated with the compound bacterium agent group, there was a significant difference compared with the control group. In addition, salt stress would inhibit the photosynthesis of plants, resulting in a decrease in the total chlorophyll content of plants. Compared with CK, the photosynthetic pigment content in the Chinese cabbage plants under the two salt stress treatments decreased. Under the 3.0 g / kg salt treatment, after adding the single strain bvx001 and the compound bacterium agent, the total chlorophyll content increased by 40% and 137% respectively; when the salt concentration was increased to 4.5 g / kg, the total chlorophyll of the two groups increased by 17% and 67% respectively. In addition, the strain could also reduce the content of malondialdehyde (MDA) in plants, and MDA is a commonly used index. When plant organs age or are damaged under adversity, lipid peroxidation often occurs. MDA is the final decomposition product of lipid peroxidation, and its content can reflect the degree of adversity damage suffered by plants. After inoculating bvx001, MDA decreased by 8% and 34% respectively under the two salt treatments; when the BAE compound bacterium agent was inoculated into the environment, under the two salt treatment conditions, MDA decreased by 58% and 68% respectively, showing a significant difference. To sum up, the compound bacterium agent BAE in this patent significantly promoted the growth and development process of Chinese cabbage in the salt stress environment, effectively alleviated the physiological damage caused by salt to Chinese cabbage, thus enhancing the stress resistance of Chinese cabbage, and showing high application value in the improvement of saline-alkali soil and the agricultural production practice under salt stress.

[0118] Example 6

[0119] Experiment on the inhibition of pathogenic bacteria by Bacillus velezensis bvx001, Acinetobacter acin003, and Exiguobacterium exio007

[0120] Use a sterile toothpick to pick up a fungal block about 2 cm × 2 cm and place it upside down in the center of a PDA solid plate. Drop the same OD of equal amounts of bacterial liquid at three corners of the plate. There are three strains in the experiment: bvx001, acin003, and exio007. Observe the antibacterial results after culturing in an incubator at 26°C for 5 days. The pathogenic bacteria are shown in Table 7.

[0121] Table 7 List of pathogenic bacteria

[0122]

[0123] The experimental results showed that: in the experiment of testing 8 strains of bacteria, only bvx001 had an inhibitory effect on pathogenic fungi. As Figure 12The shown bvx001 has an obvious inhibitory effect on all six strains. From the comprehensive antibacterial results of bvx001, the strains can mainly inhibit fungi of the genus Fusarium, which mainly cause plant root rot, stem rot, and basal stem rot, such as wheat scab, wheat take-all, and corn stalk rot. Thus, it can be seen that the strain bvx001 has potential biological control ability. When applied to the preparation of compound microbial agents, on the one hand, it can promote the growth of plants in a salt stress environment, and on the other hand, it can effectively inhibit the growth and spread of specific pathogenic bacteria, having high application value in the field of agricultural production.

[0124] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples are used in this article to elaborate on the principle and implementation manner of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are merely illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. A Bacillus velezensis bvx001, characterized in that, The Bacillus velezensis ( Bacillus velezensis ) bvx001 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 3, 2025. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33291.

2. A composite microbial inoculant, characterized in that, The compound microbial inoculum includes Bacillus velezensis bvx001 described in claim 1.

3. The composite microbial inoculum according to claim 2, wherein The composite microbial inoculum further includes Acinetobacter acin003; the Acinetobacter ( Acinetobacter sp. ) acin003 was deposited at the General Microbiological Center of the China National Center for Culture Collection of Microorganisms on January 3, 2025. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33292.

4. The composite microbial inoculant according to any one of claims 2-3, characterized in that, The composite microbial inoculum further includes Exiguobacterium exio007; the Exiguobacterium ( Exiguobacterium sp. ) exio007 was deposited at the General Microbiological Center of the China National Center for Biotechnology Development on January 3, 2025. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33293.

5. Use of the composite microbial inoculant according to any one of claims 3-4 in a product for promoting the growth of Chinese cabbage, characterized in that, The product is a product that simultaneously has the functions of silicon dissolution, phosphorus dissolution and nitrogen fixation.

6. Use of Bacillus velezensis bvx001 described in claim 1 or the compound microbial inoculum described in any one of claims 2-4 in promoting the growth of Chinese cabbage and / or preparing a Chinese cabbage growth promoter.

7. A Chinese cabbage growth promoter, characterized in that, The Chinese cabbage growth promoter includes Bacillus velezensis bvx001 described in claim 1 or the compound microbial inoculum described in any one of claims 2-4.

8. Use of Bacillus velezensis bvx001 as claimed in claim 1 or the compound microbial inoculum as claimed in any one of claims 2-4 in inhibiting plant pathogenic bacteria and / or preparing an inhibitor of plant pathogenic bacteria, characterized in that The plant pathogenic bacteria include Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, Fusarium culmorum, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme.

9. A plant pathogen inhibitor, characterized in that, The plant pathogenic bacteria inhibitor includes Bacillus velezensis bvx001 described in claim 1 or the compound microbial inoculum described in any one of claims 2-4; the plant pathogenic bacteria include Gaeumannomyces graminis var. avenae, Gaeumannomyces graminis var. graminis, Fusarium culmorum, Fusarium oxysporum, Fusarium graminearum, Fusarium moniliforme.

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