Heavy metal-tolerant and growth-promoting bacillus velezensis strain fsuxf-717 and application thereof

Abstract

The present application discloses a heavy metal tolerant growth promoting bacillus velezensis FSUXF-717 and its application, and belongs to the field of agricultural microbial technology. The bacillus velezensis FSUXF-717 has the following characteristics: (1) multi-functional metabolic characteristics: it can utilize various carbon sources such as sucrose, raffinose and esculin, and has the functions of producing auxin, dissolving phosphorus, secreting ACC deaminase and forming efficient biofilm; (2) broad spectrum adaptability: it has strong tolerance to heavy metal pollution (Cd / Pb / Cr / Ni), drought and broad spectrum acid-base stress (pH 4.5-9.5); under the condition of low bacterial concentration, it can still stably synthesize auxin in the heavy metal-acid-base combined stress environment; (3) it can enhance the activity of soil sucrase and soil phosphatase. The mining of the strain can provide important germplasm resource basis for the development and utilization of new efficient microbial fertilizer, and lay the foundation for the green and high-quality development of agriculture.

Description

Technical Field

[0001] This invention relates to a growth-promoting Bacillus FSUXF-717 strain that can tolerate heavy metals and its applications, belonging to the field of agricultural microbial technology. Background Technology

[0002] In the development of modern agriculture, growth-promoting bacteria have functions such as activating nutrients in the crop root zone, promoting plant growth, enhancing plant resistance to stress, and improving soil structure. Simultaneously, microorganisms with growth-promoting functions are also important components of microbial fertilizers and microbial pesticides. Studies have shown that *Bacillus belye* can produce various organic matter-degrading enzymes, plant growth hormones, and lipopeptide antibacterial substances, thus promoting plant growth and preventing various plant diseases. It is a key research focus in the field of agricultural functional microorganisms. Several strains of *Bacillus belye* (…) have been identified. Bacilus velezensis While some microbial agents have been successfully developed into commercial microbial fertilizers, different strains possess varying growth-promoting characteristics and tolerance to adverse environments. This limits the universal control effectiveness and environmental adaptability of these strains and corresponding microbial fertilizers, further restricting the development and application of highly efficient growth-promoting fertilizers. Therefore, identifying growth-promoting bacteria that possess multiple growth-promoting characteristics and strong stress resistance can not only enrich microbial germplasm resources but also provide an important germplasm resource foundation for the development and utilization of novel, highly efficient microbial fertilizers, laying the foundation for green and high-quality agricultural development. Summary of the Invention

[0003] The *Bacillus belyssus* FSUXF-717 discovered in this invention not only possesses indoleacetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) activity and the ability to dissolve inorganic phosphorus, but also can be produced at a relatively low cell concentration (cell concentration: 1 x 10⁻⁶). 7 It exhibits a high CFU / mL tolerance to acid, alkali, salt, heavy metal, and drought conditions, and retains some growth-promoting ability even under adverse conditions. Furthermore, Bacillus belye FSUXF-717 possesses strong film-forming ability and a high capacity for root colonization. In practical applications, this bacterium can enhance soil sucrase and phosphatase activity, thereby improving soil vitality.

[0004] This invention provides a strain of Bacillus belye that can secrete auxin ( Bacillus velezensis FSUXF-717 and its applications, wherein Bacillus belyes FSUXF-717 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 8, 2023, with accession number GDMCC No: 64133.

[0005] The *Bacillus belyssiensis* strain FSUXF-717 isolated in this invention exhibits the following typical characteristics: the colonies are milky white and translucent with irregularly rounded edges; it is Gram-positive after staining, and microscopic observation shows that the cells have a typical purple rod-shaped structure, mostly existing in single-cell form. This strain possesses diverse metabolic characteristics, can utilize carbon sources such as glucose, mannitol, sucrose, and raffinose, and has aescin hydrolysis function and exhibits amylase activity, capable of degrading starch polysaccharides.

[0006] The present invention also provides a microbial inoculum containing the aforementioned Bacillus belyssus FSUXF-717.

[0007] In one embodiment, the microbial agent contains live cells, inactivated cells, fermentation broth, lysate, or metabolites of the Bacillus vesiculosus FSUXF-717.

[0008] In one embodiment, the dosage form of the microbial agent includes liquid agent and powdered agent.

[0009] The present invention also provides products containing the aforementioned Bacillus belyssus FSUXF-717, or products containing the aforementioned microbial agent.

[0010] In one embodiment, the product includes biopesticides, biofertilizers, phosphorus activators, or plant growth promoters.

[0011] In one embodiment, when the product contains cells, the number of cells in the product is not less than 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0012] The present invention also provides the application of the Bacillus berberis FSUXF-717, or the microbial agent, in the preparation of biofertilizer.

[0013] In one embodiment, the bio-fertilizer has at least one of the following functions:

[0014] (1) Produces auxin, which promotes plant growth; said auxin includes indoleacetic acid;

[0015] (2) Phosphorus dissolution;

[0016] (3) Increased the activity of 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase);

[0017] (4) It improves the plant's resistance to environmental stress; the environmental stress includes heavy metal stress, acid-base stress, and drought stress; the heavy metals include chromium, cadmium, lead, and nickel;

[0018] (5) It increased the activity of soil sucrase and soil phosphatase in the soil.

[0019] The present invention also provides the application of the Bacillus belyssus FSUXF-717, or the microbial agent, in promoting plant growth.

[0020] Beneficial effects:

[0021] The *Bacillus belyceae* FSUXF-717 provided by this invention can utilize glucose, mannitol, sucrose, and raffinose as carbon sources, and possesses the ability to hydrolyze aescin and starch polysaccharides. The *Bacillus belyceae* FSUXF-717 discovered in this invention not only possesses the activity of producing indoleacetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) and the ability to dissolve inorganic phosphorus, but also can be produced at relatively low cell concentrations (cell concentration: 10). 7 (CFU / mL), it is tolerant of acidic, alkaline, saline, heavy metal, and drought conditions, and still has a certain ability to promote growth under adverse conditions. In addition, Bacillus belye FSUXF-717 has a strong film-forming ability and a strong ability to colonize plant roots. In practical applications, this bacterium can enhance the activity of soil sucrase and soil phosphatase, increasing their enzyme activities by 72.52%±6.87% and 13.17%±6.09%, respectively, demonstrating its ability to improve soil vitality.

[0022] Preservation of biological materials

[0023] A strain of Bacillus belye ( Bacillus velezensis FSUXF-717, classified as Bacillus velezensis It was deposited on December 8, 2023 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCCNo: 64133, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0024] Figure 1 The image shows the chromogenic pattern of cloning agents produced by Bacillus FSUXF-717 of the present invention.

[0025] Figure 2 This is a diagram of the phosphate lysing zone of Bacillus FSUXF-717 of the present invention.

[0026] Figure 3 This is a colony morphology diagram of Bacillus belyssus FSUXF-717 of the present invention.

[0027] Figure 4 This is a Gram staining image of Bacillus FSUXF-717 of the present invention.

[0028] Figure 5This invention presents a phylogenetic tree of Bacillus belyssus FSUXF-717 based on 16S rRNA.

[0029] Figure 6 The present invention is based on Bacillus belyssus FSUXF-717. gyrB The phylogenetic tree.

[0030] Figure 7 This invention relates to the production of auxin by Bacillus belyssus FSUXF-717 under heavy metal stress; A: Different Ni 2+ The effect of FSUXF-717 concentration on IAA yield; B: Different Pb concentrations 2+ Effect of FSUXF-717 concentration on IAA yield; C: different Cr 3+ Effect of FSUXF-717 concentration on IAA yield; D: Different Cd concentrations 2+ The effect of FSUXF-717 concentration on IAA yield; different lowercase letters in the figure indicate significant differences ( p <0.05).

[0031] Figure 8 This is a biofilm formation of Bacillus FSUXF-717 of the present invention.

[0032] Figure 9 To investigate the effects of Bacillus vesicles FSUXF-717 fermentation broth on the agronomic traits of Chinese cabbage.

[0033] Figure 10 To investigate the effects of Bacillus vesiculosus FSUXF-717 fermentation broth on the agronomic traits of tomatoes.

[0034] Figure 11 To investigate the effects of Bacillus vesiculosus FSUXF-717 fermentation broth on the agronomic traits of sweet corn.

[0035] Figure 12 This study aims to screen for a single protectant for the Bacillus FSUXF-717 bacterial agent of the present invention.

[0036] Figure 13 The state of Bacillus Fischer-F-717 inoculum after vacuum freezing.

[0037] Figure 14 To investigate the effects of Bacillus vesicularis FSUXF-717 inoculum on the agronomic traits of Chinese cabbage.

[0038] Figure 15 To investigate the effects of Bacillus vesiculosus FSUXF-717 inoculant on agronomic traits of tomatoes.

[0039] Figure 16To investigate the effects of Bacillus vesiculosus FSUXF-717 inoculant on the agronomic traits of sweet corn. Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments.

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0042] The LB liquid culture medium formulations involved in the following examples are as follows (g / L): 10.0 g peptone, 5.0 g sodium chloride, 1.0 g glucose, 5.0 g yeast extract, and 1000 mL distilled water.

[0043] The following examples involve Chinese cabbage seeds from Liuzhou Xinfengda Seed Industry Co., Ltd., tomato seeds from Qingxian Xingyun Seed Industry Co., Ltd., and sweet corn seeds from the sweet corn research team of Foshan University.

[0044] Example 1: Determination and identification of the growth-promoting characteristics of the strain

[0045] 1. Screening and identification of strains

[0046] (1) Screening of growth-promoting strains:

[0047] Fermented glutinous rice wine from a winery in Guangdong Province was selected as a sample. After pretreatment, 0.5 mL of the sample was mixed and added to 4.5 mL of 0.9% physiological saline for serial dilution. The appropriate serial dilution was spread on LB solid medium and incubated at 37℃ for 48 h. Colonies were picked and streaked onto LB plates for purification. Single colonies were transferred to liquid LB medium for enrichment and preserved in 50% glycerol. 72 isolated and purified strains were obtained. The 72 strains were then screened for growth-promoting abilities, including the production of glutathione (IAA), phosphate solubilization, potassium solubilization, nitrogen fixation, iron production, and ACC deaminase production. Finally, the strain with the best growth-promoting characteristics, FSUXF-717, was selected.

[0048] (2) Identification of growth-promoting strains:

[0049] Physiological and biochemical identification was performed with reference to Bergey's Manual of Bacteriological Identification and appropriate modifications. The results are as follows:

[0050] Colony, cell morphology, and physiological and biochemical characteristics: The colonies of the strain FSUXF-717 are milky white, semi-transparent, and irregularly shaped. Figure 3 The bacteria are purple rod-shaped, arranged singly or in pairs. Figure 4 This strain can utilize glucose, mannitol, sucrose, and raffinose as carbon sources and has the ability to hydrolyze aescin; it also exhibits amylase activity, which can degrade starch polysaccharides.

[0051] Molecular biological identification: DNA was extracted from Bacillus belyssus and used as a template. The 16S rRNA gene of the strain to be identified and... gyrB Gene sequences were analyzed, and sequence homology was performed using the BLAST program in EzbiocLoud. The results showed that the 16S rRNA gene of the screened strains and... gyrB The gene sequence is most similar to Bacillus belesii, with homology exceeding 99%, and a phylogenetic tree was constructed ( Figure 5 , Figure 6 The bacterium was identified as Bacillus belesii and named Bacillus belesii (B. belesii). Bacillus velezensis FSUXF-717, classified as Bacillus velezensis It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 8, 2023, with accession number GDMCC No: 64133.

[0052] 2. Determination of strain characteristics

[0053] Bacillus belye FSUXF-717, preserved in a laboratory freezer at -80℃, was thawed at 4℃. 200 μL of the bacterial culture was then added to 5 mL of LB liquid medium in a sterile operating table and cultured at 37℃ with shaking for 12 h to obtain the first-generation activated strain. Another 200 μL of the first-generation activated bacterial culture was transferred to 5 mL of LB liquid medium and cultured at 37℃ with shaking until OD (dose elapsed). 600nm =0.6~0.8, which is the second-generation activated bacterial solution, used to screen strains for growth-promoting properties, with a viable count of 1×10⁻⁶. 7 CFU / mL.

[0054] (1) Determination of indoleacetic acid (IAA) content: The second-generation activated bacterial suspension was inoculated into LB liquid medium containing a final concentration of 0.5 mg / mL tryptophan and cultured continuously in a constant temperature shaking incubator at 37℃ for 48 h. After the culture was completed, the bacterial suspension was centrifuged for 10 min, and 2 mL of the supernatant was accurately transferred and mixed with an equal volume of Salkowski colorimetric solution (at a volume ratio of 1:1). After thorough shaking and mixing, the mixture was allowed to stand in the dark for 30 min for color development. The absorbance value was measured at a wavelength of 530 nm using a UV-Vis spectrophotometer. A blank medium without bacterial suspension was set up as a negative control throughout the experiment. At the same time, a standard curve was established using IAA standards with gradient concentrations, and the actual yield (mg / L) of indoleacetic acid in the sample was calculated by linear regression equation.

[0055] The fermentation supernatant of Bacillus belye FSUXF-717 reacts with Salkowski colorimetric solution to produce a pink color ( Figure 1 The strain produced 21.96±0.24 mg / L of IAA.

[0056] (2) Determination of dissolved inorganic phosphorus content: 2 mL of second-generation activated bacterial suspension was inoculated into 100 mL of LB liquid medium and cultured with shaking at 37℃ and 160 rpm until the logarithmic growth phase. 2.5 μL of fermentation broth was evenly spotted onto NBRIP inorganic phosphorus selective solid plates and incubated upside down in a 37℃ constant temperature incubator for 4 days. The phosphorus solubility activity of strain FSUXF-717 was determined to be: clear zone diameter 9.76±0.35 mm, colony diameter 4.19±0.04 mm, and phosphorus solubility index (D / d) reaching 2.33±0.07 ( ). Figure 2 Two mL of the second-generation activated bacterial suspension was inoculated into 50 mL of Monkina inorganic phosphorus liquid medium (containing 0.015% w / v available phosphorus, with Ca3(PO4)2 as the sole phosphorus source) and cultured continuously with shaking at 30℃ and 160 rpm for 72 h. The fermentation broth was centrifuged at low temperature and high speed for 10 min, and 2.5 mL of the supernatant was accurately transferred to a 25 mL volumetric flask. 2-3 drops of 2,6-dinitrophenol indicator (0.1% w / v) were added, and 10% (w / v) Na2CO3 solution was added dropwise to adjust the pH to a slightly yellow endpoint. 5 mL of molybdenum antimony anti-chromic agent was accurately added, and the mixture was brought to a final volume with ultrapure water and developed at room temperature in the dark for 30 min. The absorbance was measured at 700 nm using a UV-Vis spectrophotometer, and the soluble phosphorus concentration was calculated using a phosphorus standard curve. The experiment was performed in triplicate, with an equal volume of sterile distilled water used as a blank control instead of the bacterial suspension.

[0057] The phosphorus solubility of Bacillus belyss FSUXF-717 was 22.51 ± 0.26 mg / L.

[0058] (3) Determination of ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity: First, the bacterial cells were activated and cultured. The target strain was inoculated into DF medium without (NH4)2SO4 for pre-culture. The cells were collected by low-temperature centrifugation for 10 min and washed three times with pre-cooled DF medium. The initial viable cell concentration was adjusted to 1×10⁻⁶. 7 After CFU / mL concentration, the bacterial cells were resuspended in ADF medium and cultured with shaking at 30℃ and 160 rpm for 24 h to complete the secondary culture. The cells were then collected again by low-temperature centrifugation and purified by three washes with Tris-HCl buffer (50 mM, pH 7.5). The bacterial suspension was then mixed with 200 μL of toluene and sonicated on ice to obtain the crude enzyme solution. Reaction reagents were added, and the reaction was precisely incubated at 30℃ for 30 min, followed by boiling to terminate the reaction. The OD was measured using a spectrophotometer. 540nm The product concentration was calculated by converting the α-butanone standard curve. Enzyme activity was defined as the amount of enzyme producing 1 μmol of α-butanone per minute. Protein content was determined using the Coomassie Brilliant Blue G-250 method, and specific activity (U / mg protein) was calculated. Distilled water blank control, standard curve, and three biological replicates were included throughout the experiment. All operations were performed in a biosafety cabinet to ensure data reliability.

[0059] The enzyme activity of ACC deaminase produced by Bacillus belyssus FSUXF-717 is 0.2640±0.0079 U / mg.

[0060] Example 2: Heavy Metal Tolerance Test of Strains

[0061] The second-generation activated bacterial solution prepared in Example 1 was used to adjust the bacterial cell concentration to OD using a centrifugal concentration method. 600 nm After the inoculum concentration was set within the range of 0.6–0.8, the cultures were transferred to LB liquid medium with different heavy metal ion concentrations at an inoculum rate of 2%. The heavy metal concentrations were set according to the risk screening values ​​for agricultural land soil pollution in the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard (Trial)". After inoculation, the culture systems were cultured at 37℃ in a shaker at 160 r / min for 24 h. The absorbance of each group of bacterial solutions at 600 nm was then measured spectrophotometrically. By comparing the biomass growth levels of the experimental groups, the tolerance of the target strain to heavy metal stress was quantitatively assessed using a specific calculation formula, as follows:

[0062] Relative growth rate (%) = (OD of different heavy metal bacterial solutions) 600 nm / Control group bacterial solution OD 600 nm ) × 100%.

[0063] Table 1. Effects of different heavy metal concentrations at different pH levels on the growth rate of the strain.

[0064]

[0065] The heavy metal tolerance results of Bacillus belyssus FSUXF-717 are shown in Table 1. The optimal growth conditions for Bacillus belyssus FSUXF-717 to different heavy metals showed significant differences. p <0.05: This strain exhibited optimal growth activity under Cd (2.0 mg / kg, pH 6) and Pb (500 mg / kg, pH 6) contamination conditions, while the tolerance threshold for Ni appeared under acidic conditions (300 mg / kg, pH 5). Notably, this strain showed extremely low tolerance to Cr, with a growth rate of only 42.38% ± 3.25% under alkaline conditions (1300 mg / kg, pH 8). Based on the inhibition intensity analysis under high concentration exposure, the toxicity effects of the four heavy metals were ranked as Cr > Ni > Cd > Pb. Furthermore, pH environment had a significant regulatory effect on tolerance; neutral conditions (pH: 6-7) were more favorable for Cd and Pb tolerance, while the tolerance mechanisms for Cr and Ni showed stronger adaptability under acidic conditions (pH 5).

[0066] Example 3: Assay for the production of auxin by strains under heavy metal stress

[0067] The method is the same as in Example 2, using an inoculum size of 2% (initial bacterial concentration of 1×10⁻⁶). 7 CFU / mL were transferred to LB liquid medium containing different concentrations of heavy metal ions. The heavy metal concentrations in the experimental groups were set according to the risk screening values ​​for agricultural land soil pollution in the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard (Trial Implementation)". A blank control was also set up with no added heavy metal ions. After inoculation, the medium was incubated at 37℃ and 160 r / min for 24 h with shaking before OD was measured. 600 nm The heavy metal tolerance of the strain was evaluated by the auxin content of the strain under different heavy metal concentrations.

[0068] Table 2. Growth hormones produced by strains at different pH levels and heavy metal concentrations.

[0069]

[0070] The results of the content of growth factor produced by Bacillus belyssus FSUXF-717 under heavy metal stress are shown in Table 2 and Figure 7As shown in the figure, for the Cd-treated group, the auxin concentration increased from 6.43±0.26 mg / L to 8.71±0.35 mg / L with increasing pH (5→8), indicating that high pH may enhance the promoting effect of Cd; while the Pb group showed the opposite trend, with the concentration decreasing from 6.56±0.49 mg / L (pH 5) to 3.97±0.18 mg / L (pH 8). The effects of Cr and Ni were more complex: the Cr group had the highest concentration at pH 8 (6.38±0.30 mg / L), while the Ni group showed an abnormally high concentration at pH 5 (18.10±1.94 mg / L), possibly suggesting that Ni has a specific interference with auxin metabolism under low pH conditions. Based on Bacillus belye FSUXF-717 as a whole, the effect of heavy metals on auxin has a significant pH dependence, and under heavy metal stress, its ability to produce auxin is enhanced to varying degrees compared to the control group.

[0071] Example 4: Assay for the production of gluten by strains under acid-base stress

[0072] Take the second-generation activated bacterial culture in the logarithmic growth phase and adjust its cell concentration to 1×10⁻⁶. 7 CFU / mL was inoculated at a 2% inoculum into LB liquid medium at different pH values ​​(pH adjusted to 2.5, 3.5, 4.5, 6, 7, 9.5, 10.5, and 11.5 using 1 mol / L NaOH and 1 mol / L HCl, respectively), and cultured at 37℃ with shaking at 160 r / min. The absorbance of each group of bacterial suspensions at 600 nm was measured every 12 h using spectrophotometry. The tolerance of the target strain to acid and alkali stress was evaluated by comparing the biomass growth levels of the experimental groups.

[0073] Table 3. Growth factor produced by strains at different pH levels

[0074]

[0075] The auxin content of *Bacillus belyssiensis* FSUXF-717 under acid-base stress is shown in Table 3. At pH 4.5 and 9.5, the auxin concentrations were 4.35±0.3176 mg / L and 5.29±0.0999 mg / L, respectively, both significantly higher than other pH conditions, indicating that extreme pH environments may be more conducive to cell growth or auxin accumulation. Comparing neutral conditions (pH: 6-7) with the blank control group, it was found that the OD value and auxin concentration were lower than other acid-base conditions, suggesting that neutral conditions inhibited the relevant metabolic processes of the strain. Therefore, it can be concluded that pH has a significant regulatory effect on the growth and auxin synthesis of *Bacillus belyssiensis* FSUXF-717, and extreme acid-base conditions are more conducive to the improvement of related indicators.

[0076] Example 5: Determination of the biofilm formation ability of the strain

[0077] The biofilm-forming ability of the strain was determined by crystallization staining. 160 μL of 1×10⁻⁶ L / L liquid medium was added to each well of a 24-well cell culture plate, and 40 μL of 1×10⁻⁶ L of the medium was inoculated initially. 7 CFU / mL bacterial suspension (final volume 200 μL / well) was prepared, and pure LB medium without bacterial suspension was set up as a blank control. Each treatment had three biological replicates. After incubation at 37℃ for 24 h, the liquid and free bacterial cells in the wells were gently aspirated using a pipette, and stained with 0.1% (w / v) crystal violet solution for 10 min. After staining, the plates were rinsed three times with sterile deionized water to remove free dye. 200 μL of 30% (v / v) glacial acetic acid solution was added to each well for desorption at room temperature for 30 min. The absorbance at OD600 nm was quantitatively detected using a UV-Vis spectrophotometer. The biofilm-forming ability of different isolates was qualitatively and quantitatively analyzed by comparing the differences between the bacterial groups and the control group. The OD600 nm absorbance of the negative control group was... 600 nm The average value plus three times its standard deviation is used as the critical value for bacterial biofilm formation, and the OD values ​​are adjusted according to different strains. 600 nm The value of OD forms the following "film-forming ability judgment criteria" for its biofilm-forming ability: 600 nm ≤OD is judged as "non-film-forming strain"; OD <OD 600 nm ≤2OD is classified as "weak film-forming ability strain"; 2OD <OD 600 nm ≤4 OD is classified as "moderate film-forming ability strain"; OD 600 nm >4OD was identified as a "strong film-forming ability strain".

[0078] Table 4 Biofilm formation ability of strains

[0079]

[0080] Biofilms, as an important survival strategy for bacteria to adapt to complex environments, are essentially structured aggregates formed by microbial communities through self-secreted extracellular matrix. This structure not only endows microbial communities with stronger pollutant tolerance and environmental adaptability, but its unique decomposition and metabolic mechanisms can also effectively degrade a variety of stubborn pollutants, thereby significantly improving bioremediation efficiency (Pooja Sharma, Role and significance of biofilm-forming microbes inphytoremediation - A review[J]. Environmental Technology & Innovation, 2022, 102182). Experimental observations have confirmed ( Figure 8The culture medium of *Bacillus belye* FSUXF-717 treated group exhibited typical turbidity, and a uniform and dense biofilm structure formed at the solid-liquid interface. Furthermore, quantitative determination using crystal violet showed that the biofilm-forming capacity of this strain reached 0.244 ± 0.0311 (Table 4), significantly higher than the baseline value of the control group. p <0.05).

[0081] Example 6: Drought tolerance test of strains

[0082] Take the second-generation activated bacterial culture in the logarithmic growth phase and adjust its cell concentration to 1×10⁻⁶. 7 CFU / mL, at an inoculum rate of 2%, was transferred to LB liquid medium containing PEG-6000 (PEG-6000 mass concentrations of 5%, 10%, 20%, 30%, and 40%), and incubated at 37℃ with shaking at 160 r / min for 48 h before measuring OD. 600 nm , with OD 600nm The value indicates the growth and reproduction status under different drought levels. Before the measurement, the instrument is zeroed with the corresponding PEG-6000 culture medium. The optical density (OD) value of drought-resistant strains is used for classification: highly sensitive: OD < 0.3; sensitive: 0.3 ≤ OD ≤ 0.39; tolerant: 0.4 ≤ OD ≤ 0.5; completely tolerant: OD > 0.5.

[0083] Table 5. Results of drought tolerance of strains

[0084]

[0085] Note: Different lowercase letters indicate significant differences. p <0.05)

[0086] The drought tolerance results of Bacillus belyssus FSUXF-717 are shown in Table 5. It showed complete tolerance to 5%, 10%, 20% and 30% concentrations of PEG-6000, and tolerance to 40% concentration of PEG-6000.

[0087] Example 7: Antimicrobial Resistance Test of Strains

[0088] Second-generation activated bacterial suspension in the logarithmic growth phase was inoculated into 100 mL of LB broth and incubated at 37°C and 160 r / min for 24 h. The bacterial suspension was then spread onto LB agar plates using sterile swabs. Staphylococcus aureus was used as a control. Antibiotic susceptibility testing strips for cefazolin, cefoperazone, chloramphenicol, gentamicin, trimethoprim-sulfamethoxazole, and amikacin were respectively affixed to the plate surface. After incubation at 37°C for 12 h, the diameter of the inhibition zone was observed and recorded. Following the standards of the Clinical Laboratory Standards Committee (CLCSC), the antibiotic susceptibility of the strain was determined by the size of the inhibition zone produced by the antibiotic susceptibility testing strips on the plate.

[0089] Table 6 Results of Drug Resistance Tests

[0090]

[0091] Note: S indicates sensitive, I indicates moderately sensitive, and R indicates resistant.

[0092] The drug resistance test results of Bacillus belyssus FSUXF-717 are shown in Table 6. It is sensitive to cefazolin, cefoperazone, gentamicin and trimethoprim-sulfamethoxazole, and moderately sensitive to streptomycin and amikacin.

[0093] Example 8: Effects of fermentation broth of bacterial strains on agronomic traits of vegetables

[0094] (1) Preparation of fermentation broth:

[0095] After activating the strain obtained in Example 1, the second-generation bacterial suspension was inoculated into liquid LB medium supplemented with 0.5 mg / mL tryptophan and cultured in a shaker at 37°C for 48 h. The bacterial suspension was then centrifuged at 5000 r / min for 10 min. The supernatant was discarded, and the bacteria at the bottom of the centrifuge tube were repeatedly washed with sterile water. The suspension was then resuspended in sterile water, and the OD of the bacterial suspension was measured using a turbidimetric method. 600 nm To modulate, modulate the OD of bacterial suspension 600 A value between 0.8 and 1.0 indicates the presence of viable cells in the fermentation broth, with a viable cell count of 1 × 10⁻⁶. 7 CFU / mL.

[0096] (2) Vegetable growth experiment:

[0097] Selected seeds of Chinese cabbage, tomato, and sweet corn were carefully chosen, ensuring uniform seed size and endosperm plumpness. The seeds were treated with 75% alcohol for 10 minutes, rinsed twice with sterile water, then treated with 10% sodium hypochlorite for 10 minutes, and rinsed five times with sterile water. The resulting sterile seeds were then soaked in sterile water for 12 hours. The treated seeds were then placed on agar plates for germination. After 5 days, germinated seeds with similar growth were selected and transferred to pots containing 250 g of soil. Six seeds of Chinese cabbage, tomato, and sweet corn were transferred to each pot, and 30 mL of sterile water was added to each pot. After 6-8 hours, the experimental groups were treated with 20 mL of the corresponding bacterial suspension (OD200). 600nm The experimental group was treated with bacterial suspension (0.8~1.0 g / L), while the control group was treated with the same amount of sterile water. The cultures were incubated at room temperature for 7 days, with appropriate irrigation with sterile water to maintain adequate moisture. On day 7, the experimental and control groups were treated again with bacterial suspension and sterile water, respectively, and the solution was evenly applied to the soil around the roots. After approximately four weeks of incubation at 28℃ and 16 / 8 h light, the plant height, root length, above-ground dry and fresh weight, and underground dry and fresh weight of the seedlings of Chinese cabbage, tomato, and sweet corn were measured.

[0098] The effects of the bacterial strain fermentation broth on the agronomic traits of Chinese cabbage are shown in Table 7 and... Figure 9 As shown, the plant height, root length, above-ground fresh weight, below-ground fresh weight, above-ground dry weight, and below-ground dry weight of Chinese cabbage seedlings treated with Bacillus vesicularis FSUXF-717 increased by 10.71%±3.26%, 33.94%±8.51%, 121.22%±21.26%, 181.16%±124.42%, 189.70%±27.19%, and 131.71%±92.67%, respectively, compared with the control group.

[0099] Table 7 Effects of fermentation broth from bacterial strains on agronomic traits of Chinese cabbage.

[0100]

[0101] Note: Different lowercase letters indicate significant differences. p <0.05).

[0102] The effects of the bacterial fermentation broth on the agronomic traits of tomatoes are shown in Table 8 and... Figure 10 As shown, tomato seedlings treated with Bacillus vesiculosus FSUXF-717 showed increases in plant height, root length, and above-ground fresh weight of 45.84%±2.66%, 76.99%±3.63%, and 57.10%±21.38%, respectively, compared to the control group.

[0103] Table 8 Effects of fermentation broth from strains on agronomic traits of tomatoes

[0104]

[0105] Note: Different lowercase letters indicate significant differences. p <0.05).

[0106] The effects of the fermentation broth of the strain on the agronomic traits of sweet corn are shown in Table 9 and... Figure 11 As shown, the plant height, root length, above-ground fresh weight, below-ground fresh weight, above-ground dry weight, and below-ground dry weight of sweet corn seedlings treated with Bacillus vesiculosus FSUXF-717 increased by 37.59%±0.99%, 61.82%±1.26%, 117.27%±13.24%, 293.89%±28.96%, 590.37%±54.25%, and 318.63%±15.84%, respectively, compared with the control group.

[0107] Table 9 Effects of fermentation broth from strains on agronomic traits of sweet corn

[0108]

[0109] Note: Different lowercase letters indicate significant differences. p <0.05).

[0110] Example 9 Preparation of microbial agent

[0111] Trehalose, sucrose, sorbitol, mannitol, skim milk powder, and dextran solutions with mass concentrations of 5%, 7.5%, 10%, 12.5%, and 15% were selected as single freeze-drying protectants. They were added to the bacterial cells at a mass ratio of 1:3, with an equal amount of sterile water added as a control group. After pre-freezing, the cells were subjected to vacuum freeze-drying. The freeze-drying survival rate was used as an indicator to screen for protectants with better freeze-drying protection effects against Bacillus belyssus.

[0112] When 7.5% trehalose was chosen as the preservative, the freeze-dried form was good. Figure 13 The freeze-dried product survival rate reached 93.28%. Figure 12 Therefore, 7.5% trehalose was chosen as the microbial agent protectant.

[0113] Example 10 Effects of microbial agents on agronomic traits of vegetables

[0114] (1) Detection of agronomic traits of vegetables:

[0115] The method was the same as the growth experiments of Chinese cabbage, tomato, and sweet corn in Example 6. The bacterial agent was reconstituted with sterile distilled water. The experimental groups used 20 mL of the corresponding reconstituted bacterial agent (with a viable count of 1 × 10⁻⁶). 7 The experimental, control, and blank groups were treated with a mixture of CFU / mL of commercially available Bacillus vesiculosus CY30 suspension (equal volume and viable count) and sterile water, respectively. All groups were incubated at room temperature for 7 days, with adequate moisture maintained by irrigation with sterile water. On day 7, the experimental, control, and blank groups were treated again with the reconstituted bacterial agent, Bacillus vesiculosus CY30, and sterile water, respectively, and the solution was evenly applied to the soil around the roots. After approximately four weeks of incubation at 28℃ and 16 / 8 h light, the plant height, root length, above-ground dry and fresh weight, and underground dry and fresh weight of Chinese cabbage, tomato, and sweet corn seedlings were measured.

[0116] The effects of microbial agents on the agronomic traits of Chinese cabbage are shown in Table 10 and... Figure 14 As shown, the root length, above-ground fresh weight, underground fresh weight, and above-ground dry weight of Chinese cabbage treated with the fungal agent FSUXF-717 were all significantly increased compared with the control group and the blank group. p <0.05, which increased the yields by 20.86%±5.17%, 64.11%±8.86%, 47.92%±29.77%, 49.89%±7.57%, 58.28%±11.74%, and 28.89%±33.23%, respectively. However, there was no significant difference in the underground dry weight of Chinese cabbage after treatment with the fungal agent FSUXF-717 compared to the control group. p >0.05).

[0117] Table 10 Effects of microbial inoculants on agronomic traits of Chinese cabbage

[0118]

[0119] Note: Different lowercase letters indicate significant differences. p <0.05).

[0120] The effects of microbial agents on the agronomic traits of tomatoes are shown in Table 11 and Figure 15 As shown, the plant height, root length, above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight of tomatoes treated with the inoculant FSUXF-717 were all significantly increased compared with the control group and the blank group. p <0.05), which increased by 23.50%±1.21%, 34.01%±7.57%, 48.81%±9.37%, 101.99%±24.38%, 63.45%±8.48%, and 132.88%±12.12%, respectively.

[0121] Table 11 Effects of microbial inoculants on agronomic traits of tomatoes

[0122]

[0123] Note: Different lowercase letters indicate significant differences. p <0.05).

[0124] The effects of microbial agents on the agronomic traits of sweet corn are shown in Table 12 and... Figure 16 As shown, the plant height, root length, above-ground fresh weight, underground fresh weight, above-ground dry weight, and underground dry weight of sweet corn treated with the inoculant FSUXF-717 were all significantly increased compared with the control group and the blank group. p <0.05), which increased by 87.57%±7.16%, 92.48%±10.38%, 234.83%±19.51%, 378.81%±56.72%, 261.01%±10.67%, and 425.86%±35.09%, respectively.

[0125] Table 12 Effects of microbial inoculants on agronomic traits of sweet corn

[0126]

[0127] Note: Different lowercase letters indicate significant differences. p <0.05).

[0128] Example 11 Effect of microbial agents on soil enzyme activity

[0129] Plant rhizosphere soil samples were collected 28 days after treatment with inoculant using the root shaking method. The collected soil samples were dried in an oven at 37°C or air-dried naturally, and then sieved. The sieved soil samples were used for enzyme activity determination.

[0130] Soil catalase (CAT), soil sucrase (SC), and soil phosphatase (ACP) were all measured using a kit method (Isehisa (Jiangsu Lianyungang) Biotechnology Co., Ltd.), following the instructions in the kit's manual.

[0131] The results are shown in Table 13. The effect of the inoculant FSUXF-717 on soil CAT activity was not significantly different from that of the control group. p >0.05), but significantly increased soil SC enzyme activity and ACP enzyme activity compared to the control group ( p <0.05), their enzyme activities increased by 72.52%±6.87% and 13.17%±6.09%, respectively.

[0132] Table 13 Effects of microbial agents on soil enzyme activity

[0133]

[0134] Note: Different lowercase letters indicate significant differences. p <0.05).

[0135] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis FSUXF-717, characterized in that, The Bacillus belyssus FSUXF-717 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 8, 2023, with accession number GDMCC No: 64133.

2. A microbial inoculum containing Bacillus belyssus FSUXF-717 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent contains live cells of the Bacillus berberis FSUXF-717.

4. The microbial agent as described in claim 3, characterized in that, The formulations of the microbial agents include liquid agents and powdered agents.

5. A product containing Bacillus belyssus FSUXF-717 as described in claim 1 or containing any of the microbial agents described in claims 2 to 4.

6. The product as described in claim 5, characterized in that, The products include bio-fertilizers.

7. The product as described in claim 5, characterized in that, The product includes a phosphorus activator.

8. The product as described in claim 5, characterized in that, The product includes plant growth promoters.

9. The product as described in any one of claims 5 to 8, characterized in that, The product contains at least 1 × 10⁻⁶ cells. 7 CFU / mL or 1× 10 7 CFU / g.

10. The use of Bacillus berreatus FSUXF-717 as described in claim 1 or any of the microbial agents described in claims 2 to 4 in the preparation of biofertilizers.

11. The application as described in claim 10, characterized in that, The bio-fertilizer has at least one of the following functions: (1) It produces indoleacetic acid, which promotes plant growth; (2) Dissolve inorganic phosphorus; (3) Production of 1-aminocyclopropane-1-carboxylic acid deaminase; (4) Increase the activity of soil sucrase and soil phosphatase in the soil.

12. The use of Bacillus belyssus FSUXF-717 as described in claim 1 or any of the microbial agents described in claims 2 to 4 in promoting plant growth.

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