Growth-promoting bacillus velezensis FSUXF-717 capable of tolerating heavy metals and application of growth-promoting bacillus velezensis FSUXF-717

Abstract

The invention discloses a growth-promoting bacillus velezensis FSUXF-717 capable of tolerating heavy metals and application of the growth-promoting bacillus velezensis FSUXF-717, and belongs to the technical field of agricultural microorganisms. The bacillus velezensis FSUXF-717 has the following advantages: (1) the bacillus velezensis FSUXF-717 has multifunctional metabolic characteristics: various carbon sources such as cane sugar, raffinose and aesculin can be utilized, and the bacillus velezensis FSUXF-717 has the functions of producing long-chain hormone, dissolving phosphorus, secreting ACC deaminase, efficiently forming a biological membrane and the like; (2) broad-spectrum adaptability: the strain 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 relatively low bacterium concentration, the auxin can be stably synthesized in a heavy metal-acid-base composite stress environment. And (3) the activity of soil sucrase and soil phosphatase is enhanced. Excavation of the strain can provide an important germplasm resource basis for development and utilization of novel efficient microbial fertilizers and lay a foundation for green and high-quality development of agriculture.

Description

Technical Field

[0001] The invention relates to a growth-promoting Bacillus Velez subtilis FSUXF-717 capable of tolerating heavy metals and application thereof, belonging to the technical field of agricultural microorganisms. Background Art

[0002] In the process of modern agricultural development, growth-promoting bacteria have the functions of activating nutrients in the root zone of crops, promoting plant growth, enhancing plant resistance, and improving soil structure. At the same time, microorganisms with growth-promoting functions are also important components of microbial fertilizers and microbial pesticides. Studies have shown that Bacillus velez can produce a variety of organic matter degrading enzymes, plant growth hormones, and lipopeptide antibacterial substances, which can promote plant growth and prevent and control a variety of plant diseases. It is the focus of research in the field of agricultural functional microorganisms. There are currently many strains of Bacillus velez ( Bacilus velezensis ) have been successfully developed into commercial microbial fertilizers, but different strains have different growth-promoting properties and different tolerance to adverse environments. This has limited the universal prevention and control effects of the strains and corresponding microbial fertilizers, and their poor environmental adaptability, further restricting the development and application of high-efficiency growth-promoting fertilizers. Therefore, exploring growth-promoting bacteria with multiple growth-promoting properties and strong stress resistance can not only enrich microbial germplasm resources, but also provide an important germplasm resource base for the development and utilization of new and efficient microbial fertilizers, laying the foundation for the green and high-quality development of agriculture. Summary of the Invention

[0003] The Bacillus velez FSUXF-717 discovered in the present invention not only has the ability to produce indoleacetic acid IAA, 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) activity, and dissolve inorganic phosphorus, but also can produce 1-aminocyclopropane-1-carboxylic acid deaminase at a low bacterial concentration (bacterial concentration: 1x10 7 CFU / mL), is tolerant to acid, alkali, salt, heavy metal environments, and drought, and maintains a certain growth-promoting ability even under adverse conditions. Furthermore, Bacillus velezinis FSUXF-717 has strong film-forming ability and is highly capable of colonizing plant roots. In practical applications, it has been shown to enhance soil sucrase and phosphatase activities, improving soil vitality.

[0004] The present invention provides a strain of Bacillus velezine ( Bacillus velezensis ) FSUXF-717 and its application, the Bacillus velezensis FSUXF-717 was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 8, 2023, with the deposit number GDMCC No: 64133.

[0005] The isolated Bacillus velezensis FSUXF-717 strain exhibits the following typical characteristics: its colonies are milky white and translucent with irregularly rounded edges; it is Gram-positive by staining, and microscopic observation reveals that the bacteria are typically purple, rod-shaped structures, mostly existing as single cells. This strain possesses diverse metabolic characteristics, capable of utilizing carbon sources such as glucose, mannitol, sucrose, and raffinose. It also has the ability to hydrolyze esculin and exhibits amylase activity, enabling it to degrade starch polysaccharides.

[0006] The present invention also provides a microbial agent containing the Bacillus Velez subtilis FSUXF-717.

[0007] In one embodiment, the microbial agent contains living cells, inactivated cells, fermentation broth, lysate or metabolites of the Bacillus Velez subtilis 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 a product containing the Bacillus Velez subtilis FSUXF-717 or the microbial agent.

[0010] In one embodiment, the product comprises a biopesticide, a biofertilizer, a phosphorus activator, or a plant growth promoter.

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

[0012] The present invention also provides the use of the Bacillus Velezii FSUXF-717 or the microbial agent in preparing biological fertilizers.

[0013] In one embodiment, the biofertilizer has at least one of the following functions: (1) Producing auxin to promote plant growth; the auxin includes indoleacetic acid; (2) Phosphate dissolution; (3) Increased the activity of 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase); (4) Improved plant 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; (5) Increased the activities of soil sucrase and soil phosphatase in the soil.

[0014] The application also provides application of the Bacillus velezensis FSUXF-717 or the microbial inoculant in promoting plant growth.

[0015] Beneficial effects: The Bacillus velezensis FSUXF-717 provided by the application can utilize glucose, mannitol, sucrose and raffinose as carbon sources, and has esculin and starch polysaccharide hydrolysis functions. The Bacillus velezensis FSUXF-717 mined in the application not only has indole acetic acid IAA production, 1-aminocyclopropane-1-carboxylic acid deaminase (ACC deaminase) activity and inorganic phosphorus solubilization ability, but also has a certain growth promoting ability under adverse conditions at a low bacterial concentration (bacterial concentration: 10 7 CFU / mL). In addition, the Bacillus velezensis FSUXF-717 has strong film forming ability and strong colonization ability in plant roots. In practical application, the bacteria can enhance the activities of soil sucrose and soil phosphatase, and the enzyme activities are increased by 72.52%±6.87% and 13.17%±6.09% respectively, and have the ability to improve soil activity.

[0016] Biological material preservation A Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) FSUXF-717, which is classified and named as Bacillus velezensis , was preserved in the Guangdong Microbial Culture Collection Center on December 8, 2023, with a preservation number of GDMCC No: 64133 and a preservation location of No. 59 Building, 5th Floor, 100 Middle Martyrs Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a long color picture of the auxin produced by the Bacillus velezensis FSUXF-717 of the application.

[0018] Figure 2 It is a phosphorus solubilization ring picture of the Bacillus velezensis FSUXF-717 of the application.

[0019] Figure 3 It is a colony morphology picture of the Bacillus velezensis FSUXF-717 of the application.

[0020] Figure 4 It is a gram staining picture of the Bacillus velezensis FSUXF-717 of the application.

[0021] Figure 5 It is a phylogenetic tree of the Bacillus velezensis FSUXF-717 of the application based on 16S rRNA.

[0022] Figure 6The present invention is based on the Bacillus velezensis FSUXF-717 gyrB Phylogenetic tree of .

[0023] Figure 7 The results of the present invention show that the Bacillus Velezii FSUXF-717 produces auxin under heavy metal stress; A: different Ni 2+ Effect of FSUXF-717 on IAA production at different concentrations; B: different Pb 2+ Effects of FSUXF-717 on IAA production at different concentrations; C: different Cr 3+ Effects of FSUXF-717 on IAA production at different concentrations; D: different Cd 2+ Effects of FSUXF-717 on IAA production at different concentrations; different lowercase letters in the figure indicate significant differences ( p <0.05).

[0024] Figure 8 This is the biofilm formation situation of Bacillus Velez FSUXF-717 of the present invention.

[0025] Figure 9 To investigate the effects of fermentation broth of Bacillus velez FSUXF-717 on agronomic traits of Chinese cabbage.

[0026] Figure 10 To investigate the effects of Bacillus Velez FSUXF-717 fermentation broth on agronomic traits of tomatoes.

[0027] Figure 11 To investigate the effects of fermentation broth of Bacillus velez FSUXF-717 on agronomic traits of sweet corn.

[0028] Figure 12 This is the screening of a single protective agent for the Bacillus Velez FSUXF-717 bacterial agent of the present invention.

[0029] Figure 13 The invention discloses a state of the Bacillus Velez FSUXF-717 bacterial agent after vacuum freezing.

[0030] Figure 14 To investigate the effects of the Bacillus Velez FSUXF-717 inoculant on the agronomic traits of Chinese cabbage.

[0031] Figure 15 To investigate the effects of Bacillus Velez FSUXF-717 on agronomic traits of tomatoes.

[0032] Figure 16 To investigate the effect of Bacillus Velez FSUXF-717 on the agronomic traits of sweet corn. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to specific embodiments. The following will be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] The formula of the LB liquid culture medium involved in the following examples is as follows (g / L): peptone 10.0 g, sodium chloride 5.0 g, glucose 1.0 g, yeast extract powder 5.0 g, and distilled water 1000 mL.

[0035] The Chinese cabbage seeds involved in the following examples were sourced from Liuzhou Xinfeida Seed Co., Ltd., the tomato seeds were sourced from Qingxian Xingyun Seed Co., Ltd., and the sweet corn seeds were donated by the sweet corn research team of Foshan University.

[0036] Example 1 Determination and identification of growth-promoting properties of strains 1. Screening and identification of strains (1) Screening of strains with growth-promoting properties: Glutinous rice wine fermentation broth from a winery in Guangdong was selected as the sample. After pretreatment, the sample was mixed and 0.5 mL of the sample was pipetted into 4.5 mL of 0.9% saline for gradient dilution. Appropriate gradient dilutions were selected and spread on LB solid medium. The plate was incubated at 37°C for 48 h, and colonies were picked and streaked onto LB plates for purification. Single colonies were picked and transferred to liquid LB liquid medium for enrichment and preserved in 50% glycerol. A total of 72 isolated and purified strains were obtained. The 72 strains screened were screened for their growth-promoting abilities, including the production of inducing glutinin (IAA), solubilizing phosphate, solubilizing potassium, fixing nitrogen, producing iron, and producing ACC deaminase. Finally, the strain FSUXF-717 with the best growth-promoting properties was obtained.

[0037] (2) Identification of growth-promoting strains: Physiological and biochemical identification was performed with reference to the Bergey's Manual of Bacterial Identification and with appropriate modifications. The results are as follows: Colony, bacterial morphology and physiological and biochemical characteristics: The colony of the strain FSUXF-717 is milky white, translucent and irregularly round ( Figure 3 ), the bacteria are purple rod-shaped, arranged singly or in pairs ( Figure 4 The strain can utilize glucose, mannitol, sucrose, and raffinose as carbon sources, and has the ability to hydrolyze aesculin; it also exhibits amylase activity and can degrade starch polysaccharides.

[0038] Molecular biological identification: DNA of Bacillus velez was extracted and used as a template to identify the 16SrRNA gene and gyrB The gene sequences were compared using the BLAST program in EzbiocLoud. The results showed that the 16S rRNA gene and gyrB The gene sequence has the highest similarity with Bacillus velezinsis, with the homology exceeding 99%. The phylogenetic tree was constructed ( Figure 5 、 Figure 6 ), the bacteria was identified as Bacillus Velezii and named as Bacillus Velezii ( Bacillus velezensis ) FSUXF-717, classified as Bacillus velezensis , and was deposited in Guangdong Provincial Microbiological Culture Collection on December 8, 2023, with the deposit number GDMCC No: 64133.

[0039] 2. Determination of strain characteristics Thaw the Bacillus velezensis FSUXF-717 stored in the -80℃ refrigerator in the laboratory in a 4℃ refrigerator. Take 200 μL of the bacterial solution and add it to 5 mL of LB liquid medium in a sterile operating table. Place it in a 37℃ shaker and shake for 12 hours to obtain the first generation activated strain. Take 200 μL of the first generation activated bacterial solution and transfer it to 5 mL of LB liquid medium again. Place it in a 37℃ shaker and shake and culture until the OD 600nm =0.6~0.8, which is the second generation of activated bacterial solution, used for subsequent screening of the growth-promoting properties of the strains, and its viable bacterial count reaches 1×10 7 CFU / mL.

[0040] (1) Determination of the content of indoleacetic acid (IAA): The second-generation activated bacterial suspension was inoculated into LB liquid medium containing a final concentration of 0.5 mg / mL tryptophan and placed in a constant temperature shaking incubator at 37°C for continuous culture 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 (1:1 by volume). After thorough oscillation and mixing, the suspension was kept in the dark for 30 min for color development. The absorbance value was measured at a wavelength of 530 nm using a UV-visible spectrophotometer. A blank culture medium without inoculation of bacterial solution was set as a negative control throughout the experiment. At the same time, a standard curve was established using gradient concentrations of IAA standards. The actual yield of indoleacetic acid (mg / L) in the sample was calculated using a linear regression equation.

[0041] The fermentation supernatant of Bacillus velezensis FSUXF-717 reacts with SaLkowski colorimetric solution to produce a pink color ( Figure 1 ), the IAA content of the strain was 21.96±0.24 mg / L.

[0042] (2) Determination of dissolved inorganic phosphorus content: 2 mL of the second-generation activated bacterial suspension was inoculated into 100 mL of LB liquid culture medium and cultured at 37°C and 160 rpm with shaking until the logarithmic growth phase. 2.5 μL of the fermentation liquid was evenly spotted on the NBRIP inorganic phosphorus selective solid plate, which was inverted and incubated in a 37°C constant temperature incubator for 4 days. The phosphate solubilization activity of the strain FSUXF-717 was as follows: the diameter of the transparent zone was 9.76±0.35 mm, the diameter of the colony was 4.19±0.04 mm, and the phosphate solubilization index (D / d) was 2.33±0.07 ( Figure 2 2 mL of the second-generation activated bacterial suspension was inoculated into 50 mL of Montgena inorganic phosphate liquid medium (containing 0.015% w / v available phosphorus, with Ca₃(PO₄)₂ as the sole phosphorus source) and incubated at 30°C and 160 rpm with continuous shaking for 72 h. The fermentation broth was centrifuged at low temperature for 10 min, and 2.5 mL of the supernatant was accurately pipetted into a 25 mL volumetric flask. Two to three drops of 0.1% w / v 2,6-dinitrophenol indicator were added. The pH was adjusted to a slightly yellow endpoint by dropwise addition of 10% (w / v) Na₂CO₃ solution. 5 mL of molybdenum antimony colorimetric reagent was accurately added, and the volume was adjusted to volume with ultrapure water. The color was then developed at room temperature in the dark for 30 min. The absorbance was measured at 700 nm using a UV-visible spectrophotometer, and the soluble phosphorus concentration was calculated using a phosphorus standard curve. Three biological replicates were performed, and an equal volume of sterile distilled water was used instead of the bacterial suspension as a blank control.

[0043] The phosphorus solubilization capacity of Bacillus velez FSUXF-717 was 22.51±0.26 mg / L.

[0044] (3) Determination of ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity: First, activate the bacteria and inoculate the target strain into DF medium without (NH4)2SO4 for pre-culture. Collect the bacteria by centrifugation at low temperature for 10 min and wash them three times with pre-cooled DF medium. Adjust the initial viable bacterial concentration to 1×10 7 After the cells reached 400 CFU / mL, the cells were suspended in ADF medium and cultured at 30°C and 160 rpm for 24 h. After the secondary culture was completed, the cells were collected again by low-temperature centrifugation and purified by washing three times with Tris-HCl buffer (50 mM, pH 7.5). The cell suspension was then mixed with 200 μL of toluene and ultrasonically disrupted in an ice bath to obtain a crude enzyme solution. The reaction reagents were added and the reaction was accurately reacted in a 30°C water bath for 30 min. The reaction was terminated in a boiling water bath and the OD was measured using a spectrophotometer. 540nmThe product concentration was calculated using an α-ketobutyric acid standard curve. Enzyme activity was defined as the amount of enzyme required to generate 1 μmol of α-ketobutyric acid per minute. Protein content was determined using the Coomassie Brilliant Blue G-250 assay, and specific activity was calculated (U / mg protein). A distilled water blank control, a standard curve, and three biological replicates were included throughout the experiment. All operations were performed in a biosafety cabinet to ensure data reliability.

[0045] The enzyme activity of ACC deaminase produced by Bacillus velezensis FSUXF-717 was 0.2640±0.0079 U / mg.

[0046] Example 2 Heavy metal tolerance test of strains The second generation activated bacterial solution prepared in Example 1 was taken and the bacterial concentration was adjusted to OD 600 nm After the inoculation volume was in the range of 0.6-0.8, the inoculation volume was transferred to LB liquid culture medium with different concentrations of heavy metal ions at a 2% inoculum. The heavy metal concentration was set with reference to the agricultural land soil pollution risk screening value in the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard (Trial)". After the inoculated culture system was cultured in a constant temperature shaker at 37°C at an oscillation speed of 160 r / min for 24 hours, the absorbance value of each group of bacterial liquid at a wavelength of 600 nm was measured by spectrophotometry. By comparing the biomass growth levels of the experimental groups, the tolerance of the target strain to heavy metal stress was quantitatively evaluated according to a specific calculation formula, which is as follows: Relative growth rate (%) = (OD of different heavy metal bacterial solutions) 600 nm / control group bacterial solution OD 600 nm ) × 100%.

[0047] Table 1 Effects of different heavy metal concentrations on strain growth rate at different pH values

[0048] The results of heavy metal tolerance of Bacillus velez FSUXF-717 are shown in Table 1. The optimal growth conditions of Bacillus velez FSUXF-717 for different heavy metals are significantly different ( p<0.05: It exhibited optimal growth activity under Cd (2.0 mg / kg, pH 6) and Pb (500 mg / kg, pH 6) contamination, while its tolerance threshold for Ni occurred at acidic conditions (300 mg / kg, pH 5). Notably, the strain exhibited extremely low tolerance to Cr, with its growth rate reaching only 42.38%±3.25% under alkaline conditions (1300 mg / kg, pH 8). Based on inhibitory intensity analysis under high-concentration exposure, the toxic effects of the four heavy metals ranked as Cr > Ni > Cd > Pb. Furthermore, pH significantly modulated tolerance, with neutral conditions (pH 6-7) favoring Cd and Pb tolerance, while Cr and Ni tolerance mechanisms exhibited greater adaptability under acidic conditions (pH 5).

[0049] Example 3 Experiment on the production of elongation factor by strains under heavy metal stress The method was the same as in Example 2 with a 2% inoculation amount (initial bacterial concentration was 1×10 7 CFU / mL) were transferred to LB liquid culture medium containing different concentrations of heavy metal ions. The heavy metal concentrations in the experimental groups were set based on the agricultural soil pollution risk screening values ​​in the "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard (Trial)". A medium without heavy metal ions was also set as a blank control. After inoculation, the culture was shaken at 37°C and 160 rpm for 24 hours, and the 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.

[0050] Table 2 Growth hormones produced by strains at different pH and heavy metal concentrations

[0051] The results of the content of longin produced by Bacillus velez FSUXF-717 under heavy metal stress are shown in Table 2 and Figure 7As shown in the figure, in the Cd-treated group, auxin concentration increased from 6.43±0.26 mg / L to 8.71±0.35 mg / L with increasing pH (5→8), suggesting that high pH may enhance the promoting effect of Cd. The opposite trend was observed in the Pb-treated group, with concentrations 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 (6.38±0.30 mg / L) at pH 8, while the Ni group showed an abnormal increase in concentration to 18.10±1.94 mg / L at pH 5, possibly suggesting a specific interference of Ni with auxin metabolism under low pH conditions. Overall, the effects of heavy metals on auxin in Bacillus velezensis FSUXF-717 were significantly pH-dependent, and under heavy metal stress, the production of auxin was enhanced to varying degrees compared to the blank control.

[0052] Example 4 Experiment on the production of elongation factor by strains under acid-base stress Take the second generation activated bacterial solution in the logarithmic growth phase and adjust its bacterial concentration to 1×10 7 CFU / mL, the inoculum was transferred to LB liquid culture 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). Culture was incubated at 37°C with shaking at 160 rpm. The absorbance of each bacterial broth at 600 nm was measured spectrophotometrically every 12 hours. The tolerance of the target strain to acid-base stress was assessed by comparing the biomass growth levels of the experimental groups.

[0053] Table 3 Growth hormones produced by strains at different pH

[0054] Table 3 shows the auxin production levels of Bacillus velez FSUXF-717 under acid-base stress. At pH 4.5 and 9.5, auxin concentrations were 4.35 ± 0.3176 mg / L and 5.29 ± 0.0999 mg / L, respectively, significantly higher than those under other pH conditions, suggesting 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 revealed lower OD values ​​and auxin concentrations than those under other pH conditions, suggesting that neutral conditions inhibited the strain's metabolic processes. This suggests that pH significantly regulates the growth and auxin synthesis of Bacillus velez FSUXF-717, with extreme acid-base conditions favoring improvements in these parameters.

[0055] Example 5 Determination of biofilm formation ability of strains The biofilm formation ability of the strains was determined by crystal staining method. 160 μL of LB liquid medium was added to 24-well cell culture plates, and 40 μL of the initial concentration of 1×10 7 CFU / mL bacterial suspension (final volume 200 μL / well), and pure LB culture medium without bacterial suspension was set up as a blank control. Three biological replicates were set up for each treatment. After the culture plate was placed in a 37°C constant temperature incubator for 24 hours, the liquid and free bacteria in the well were gently aspirated with a pipette, and stained with 0.1% (w / v) crystal violet solution for 10 minutes. After staining, the free dye was removed by rinsing three times with sterile deionized water, and 200 μL of 30% (v / v) glacial acetic acid solution was added to each well for desorption at room temperature for 30 minutes. The absorbance at a wavelength of OD600 nm was quantitatively detected by a UV-visible spectrophotometer. By comparing the differences between the strain group and the control group, the biofilm formation ability of different isolates was qualitatively and quantitatively analyzed. The OD 600 nm The mean value plus 3 times the standard deviation was used as the critical value for bacterial biofilm formation, and the OD values ​​of different strains were calculated. 600 nm The value of OD forms the following "film-forming ability judgment standard" for its biofilm-forming ability: 600 nm OD ≤ was judged as “no film-forming ability strain”; OD <OD 600 nm ≤2OD was judged as “weak film-forming ability strain”; 2OD <OD 600 nm OD ≤4 was judged as “medium film-forming ability strain”; OD 600 nm >4OD was judged as "strong film-forming ability strain".

[0056] Table 4 Biofilm formation ability of strains

[0057] Biofilm is an important survival strategy for bacteria to adapt to complex environments. Its essence is a structured aggregate formed by microbial communities through the self-secreted extracellular matrix. This structure not only gives the microbial community stronger pollutant tolerance and environmental adaptability, but its unique catabolic mechanism can also effectively degrade a variety of stubborn pollutants, thereby significantly improving the efficiency of bioremediation (Pooja Sharma, Role and significance of biofilm-forming microbes inphytoremediation - A review[J]. Environmental Technology&Innovation, 2022,102182). Experimental observations have confirmed ( Figure 8), the culture solution of Bacillus velezensis FSUXF-717 showed typical turbidity, and a uniform and dense biofilm structure was formed at the solid-liquid interface. And the determination by crystal violet quantitative method showed that the biofilm formation ability of the strain reached 0.244±0.0311 (Table 4), which was significantly higher than the reference value of the control group (0.1±0.0151) p <0.05).

[0058] Example 6: Drought tolerance test of the strain The second generation of activated bacteria liquid in the logarithmic growth phase was taken, and the bacterial concentration was adjusted to 1×10 7 CFU / mL, and was inoculated into LB liquid medium containing PEG-6000 (the mass concentration of PEG-6000 was 5%, 10%, 20%, 30%, and 40%, respectively) at an inoculation amount of 2%, and was placed in a 37°C, 160 r / min shaking culture for 48 h. The OD 600 nm was measured, and the growth and reproduction status under each drought level was represented by the size of OD 600nm . The instrument was zeroed with the corresponding PEG-6000 culture solution before determination, and the optical density (OD) value of the drought-tolerant strain was 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.

[0059] Table 5: Drought tolerance results of the strain

[0060] Note: Different lowercase letters indicate significant differences (p<0.05) p <0.05) The drought tolerance results of Bacillus velezensis FSUXF-717 are shown in Table 5. It showed complete tolerance to PEG-6000 at concentrations of 5%, 10%, 20%, and 30%, and tolerance to PEG-6000 at a concentration of 40%.

[0061] Example 7: Drug resistance test of the strain The second generation of activated bacteria liquid in the logarithmic growth phase was taken, and was inoculated into 100 mL LB culture solution, which was cultured at 37°C and 160 r / min for 24 h. The bacteria liquid was spread on the LB solid plate with a sterile cotton swab, and Staphylococcus aureus was used as a control. Cefazolin, cefoperazone, chloramphenicol, gentamicin, compound sulfamethoxazole, and amikacin were respectively attached to the surface of the plate, and after 12 h of culture at 37°C, the diameter of the inhibition zone was observed and recorded. According to the standard of the American Clinical Laboratory Standardization Committee, the sensitivity of the strain to antibiotics was determined by the diameter of the inhibition zone produced by the drug sensitivity test paper on the plate.

[0062] Table 6: Results of drug resistance test

[0063] Note: S stands for sensitive, I stands for intermediately sensitive, and R stands for resistant.

[0064] The drug resistance test results of Bacillus velez FSUXF-717 are shown in Table 6. It is sensitive to cefazolin, cefoperazone, gentamicin and cotrimoxazole, and moderately sensitive to streptomycin and amikacin.

[0065] Example 8 Effects of strain fermentation broth on agronomic traits of vegetables (1) Preparation of fermentation broth: After the strain obtained in Example 1 was activated, the second generation bacterial suspension was inoculated into liquid LB medium supplemented with 0.5 mg / mL tryptophan and cultured at 37°C on a shaker for 48 h. The bacterial suspension was then centrifuged at 5000 r / min for 10 min, the supernatant was discarded, and the bacterial cells at the bottom of the centrifuge tube were repeatedly washed with sterile water and resuspended in sterile water. The OD value of the bacterial suspension was determined by turbidimetry. 600 nm To modulate, adjust the bacterial suspension OD 600 The value is between 0.8 and 1.0, which is the fermentation broth of the strain, in which the number of viable bacteria is 1×10 7 CFU / mL.

[0066] (2) Vegetable growth test: Selected Chinese cabbage, tomato, and sweet corn seeds were used to ensure uniform particle size and endosperm plumpness. The seeds were then treated with 75% alcohol for 10 minutes, washed twice with sterile water, and then treated with 10% sodium hypochlorite for 10 minutes. After that, they were washed five times with sterile water. The sterile seeds were then soaked in sterile water for 12 hours. The treated seeds were placed on a plate containing water agar for germination. After 5 days, seeds that had germinated and grown at the same rate were selected and transferred to pots containing 250 g of soil. Six seeds of Chinese cabbage, tomato, and sweet corn seeds were transferred to each pot, and 30 mL of sterile water was added to each pot. After 6-8 hours, 20 mL of the corresponding bacterial suspension (OD 600nm =0.8-1.0) and a blank control treated with an equal amount of sterile water. The seedlings were incubated at room temperature for 7 days, with adequate watering during this period. On the 7th day, the experimental and blank controls were treated again with the bacterial suspension and sterile water, respectively, and the soil surrounding the roots was evenly irrigated. After approximately four weeks of incubation at 28°C and 16 / 8 h light intensity, plant height, root length, and aboveground and underground dry and fresh weights of Chinese cabbage, tomato, and sweet corn seedlings were measured.

[0067] The results of the effect of the fermentation liquid of the strain on the agronomic traits of Chinese cabbage are shown in Table 7 and Figure 9As shown in Table 7, the plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of the Brassica chinensis seedlings treated with Bacillus velezensis 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% compared with the blank group, respectively.

[0068] Table 7 Effect of strain fermentation liquor on the agronomic traits of Brassica chinensis

[0069] Note: Different lowercase letters indicate significant differences (P < 0.05). p <0.05).

[0070] The results of the effect of strain fermentation liquor on the agronomic traits of tomato are shown in Table 8 and Figure 10 As shown in Table 8, the plant height, root length, and aboveground fresh weight of the tomato seedlings treated with Bacillus velezensis FSUXF-717 increased by 45.84%±2.66%, 76.99%±3.63%, and 57.10%±21.38% compared with the blank group, respectively.

[0071] Table 8 Effect of strain fermentation liquor on the agronomic traits of tomato

[0072] Note: Different lowercase letters indicate significant differences (P < 0.05). p <0.05).

[0073] The results of the effect of strain fermentation liquor on the agronomic traits of sweet corn are shown in Table 9 and Figure 11 As shown in Table 9, the plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight, and underground dry weight of the sweet corn seedlings treated with Bacillus velezensis 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% compared with the blank group, respectively.

[0074] Table 9 Effect of strain fermentation liquor on the agronomic traits of sweet corn

[0075] Note: Different lowercase letters indicate significant differences (P < 0.05). p <0.05).

[0076] Example 9 Preparation of microbial agent Trehalose, sucrose, sorbitol, mannitol, skim milk powder and dextran solution with mass concentrations of 5%, 7.5%, 10%, 12.5% ​​and 15% were selected as single lyophilization protectants and added to the bacteria at a mass ratio of 1:3. An equal amount of sterile water was added as the control group. After pre-freezing, vacuum freeze-drying was performed. The freeze-drying survival rate was used as an indicator to screen the protectants with better freeze-drying protection effect on Bacillus velezensis.

[0077] When 7.5% trehalose was selected as the protective agent, the freeze-dried morphology was good ( Figure 13 ), the freeze-dried survival rate reached 93.28% ( Figure 12 ), so 7.5% trehalose was selected as the protective agent for the bacterial agent.

[0078] Example 10 Effects of microbial agents on agronomic traits of vegetables (1) Vegetable agronomic traits testing: The method was the same as the Chinese cabbage, tomato, and sweet corn growth test in Example 6. The bacterial agent was reconstituted with sterile distilled water. The experimental group used 20 mL of the corresponding reconstituted bacterial agent (the viable cell count was 1 × 10 7 CFU / mL). The control group received an equal volume of a commercial suspension of Bacillus Velez CY30 containing the same viable count. The blank group received an equal volume of sterile water. The seedlings were incubated at room temperature for 7 days, with adequate watering. On the 7th day, the experimental group, control group, and blank group were treated again with the reconstituted inoculum, Bacillus Velez CY30, and sterile water, respectively, and the soil surrounding the roots was evenly irrigated. After approximately four weeks of incubation at 28°C and 16 / 8 h light intensity, the plant height, root length, and aboveground and belowground dry and fresh weights of Chinese cabbage, tomato, and sweet corn seedlings were measured.

[0079] The results of the effect of the microbial agent on the agronomic traits of Chinese cabbage are shown in Table 10 and Figure 14 As shown in the figure, the root length, aboveground fresh weight, underground fresh weight and aboveground dry weight of Chinese cabbage treated with the fungus FSUXF-717 were significantly improved compared with the control group and the blank group ( p <0.05), increased 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 fungus FSUXF-717 compared with the control group ( p >0.05).

[0080] Table 10 Effects of microbial agents on agronomic traits of Chinese cabbage

[0081] Note: Different lowercase letters indicate significant differences (p <0.05).

[0082] The effects of the microbial agent on the agronomic traits of tomatoes are shown in Table 11 and Figure 15 As shown in the figure, plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of tomatoes treated with the fungus FSUXF-717 were significantly improved 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.

[0083] Table 11 Effects of microbial agents on agronomic traits of tomatoes

[0084] Note: Different lowercase letters indicate significant differences ( p <0.05).

[0085] The effects of microbial agents on agronomic traits of sweet corn are shown in Table 12 and Figure 16 As shown, the plant height, root length, aboveground fresh weight, underground fresh weight, aboveground dry weight and underground dry weight of sweet corn treated with the fungus FSUXF-717 were significantly improved 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.

[0086] Table 12 Effects of microbial agents on agronomic traits of sweet corn

[0087] Note: Different lowercase letters indicate significant differences ( p <0.05).

[0088] Example 11 Effects of bacterial agents on soil enzyme activities The root shaking method was used to collect rhizosphere soil samples of plants 28 days after the inoculant treatment. 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.

[0089] Soil catalase (CAT), soil sucrase (SC), and soil phosphatase (ACP) were determined using kits (Yisejiu (Jiangsu Lianyungang) Biotechnology Co., Ltd.) according to the kit instructions.

[0090] The results are shown in Table 13. The effect of the bacterial agent 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 with the control group ( p <0.05), and their enzyme activities increased by 72.52%±6.87% and 13.17%±6.09%, respectively.

[0091] Table 13 Effects of microbial agents on soil enzyme activities

[0092] Note: Different lowercase letters indicate significant differences ( p <0.05).

[0093] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

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

2. A microbial agent containing the Bacillus Velez subtilis FSUXF-717 according to claim 1.

3. The microbial agent according to claim 2, wherein The microbial agent contains living cells, inactivated cells, fermentation liquid, lysate or metabolites of the Bacillus Velezii FSUXF-717.

4. The microbial agent according to claim 3, wherein The dosage forms of the microbial agent include liquid agent and powdered agent.

5. A product containing the Bacillus Velez subtilis FSUXF-717 according to claim 1 or the microbial agent according to any one of claims 2 to 4.

6. The product according to claim 5, characterized in that The products include biopesticides, biofertilizers, phosphorus activators or plant growth promoters.

7. The product according to claim 6, characterized in that When the product contains cells, the number of cells in the product is not less than 1 × 10 7 CFU / mL or 1× 10 7 CFU / g.

8. Use of the Bacillus Velez subtilis FSUXF-717 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in the preparation of biofertilizer.

9. The use according to claim 8, characterized in that The biological fertilizer has at least one of the following functions: (1) Producing auxin to promote plant growth; the auxin includes indoleacetic acid; (2) Phosphate dissolution; (3) Increase the activity of 1-aminocyclopropane-1-carboxylic acid deaminase; (4) Improving the resistance of plants 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; (5) Increase the activity of soil sucrase and soil phosphatase in the soil.

10. Use of the Bacillus velezensis FSUXF-717 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in promoting plant growth.

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