Bacillus amyloliquefaciens and application thereof

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a bacillus amyloliquefaciens and application thereof, the strain exhibits significant rhizosphere growth promoting and biocontrol functions. Experiments show that the bacillus amyloliquefaciens M2-1 can efficiently decompose potassium feldspar and calcium phosphate, and can secrete auxin, gibberellin and abscisic acid to promote plant growth. In addition, the inhibition rate of 10 times dilution of the bacillus amyloliquefaciens M2-1 fermentation liquor on Sclerotium rolfsii and Valsa sordida is 100%, and the inhibition rate on Fusarium solani is 34.6%, and the antibacterial effect is stable with the concentration gradient change. Combined with physiological and biochemical and 16S rDNA identification, the strain has the functions of potassium dissolution, phosphorus dissolution, hormone production and broad-spectrum antibacterial capacity, and has the potential to be developed as a multifunctional biological agent in green agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus amyloliquefaciens and its applications. Background Technology

[0002] The active growth and reproduction of soil microorganisms are closely related to sustainable agricultural production. Soil is one of the important material foundations for agricultural production, and the positive role of plant rhizosphere microorganisms in soil contributes to soil formation and can significantly improve soil fertility. Plant rhizosphere microorganisms generally occupy a certain ecological niche in the soil. In the process of continuous evolution, rhizosphere microorganisms have gradually formed a symbiotic relationship with plants with diverse functions. They are currently recognized as a promising second genome for plant development in green agriculture. In the complex material cycle and energy flow of soil, plant rhizosphere microorganisms directly or indirectly participate and have an important impact. Among these microorganisms, those that can colonize extensively in the plant rhizosphere, effectively promote plant growth and development, promote the absorption and utilization of mineral nutrients by the plant itself, and inhibit the growth of pathogens through various mechanisms are currently referred to as plant rhizosphere growth-promoting bacteria. The rhizosphere growth-promoting bacteria discovered so far are mainly bacteria and some fungi. They mainly include Bacillus and Pseudomonas. Trichoderma harzianum is currently the most widely used fungal growth-promoting bacterium.

[0003] Studies have shown that there is significant strain diversity among the currently identified Bacillus subtilis strains, and their biocontrol efficacy against various plant pathogens varies considerably. For example, strain B29 can effectively inhibit the occurrence of cucumber wilt; strain XG-1 can disrupt the mycelial morphology of watermelon wilt fungus and significantly reduce its spore germination rate; while strain R-11 shows good control effects against Rhizoctonia graminearum, the pathogen of wheat sheath blight.

[0004] Given the specificity of different Bacillus subtilis strains in terms of control spectrum and efficacy, it is necessary to conduct systematic strain evaluation to screen out superior strains with broad-spectrum disease resistance characteristics in order to enhance their application value in the field of biological control. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bacillus amyloliquefaciens that, when screened in the plant rhizosphere, exhibits excellent crop growth-promoting functions such as potassium and phosphorus solubilization, while also showing some inhibitory effects against certain pathogens. This invention further aims to develop green and pollution-free microbial fertilizers and inoculants for agricultural production, thereby replacing some pesticides and chemical fertilizers in agricultural production without reducing crop yields.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a strain of Bacillus amyloliquefaciens, classified as Bacillus amyloliquefaciens M2-1, which was deposited at the China Center for Type Culture Collection on December 30, 2024, with accession number CCTCC NO:M20242929.

[0008] This invention screened a strain of *Bacillus amyloliquefaciens* that exhibited significant rhizosphere growth-promoting and biocontrol functions. Experiments showed that *Bacillus amyloliquefaciens* M2-1 efficiently decomposes potassium feldspar and calcium phosphate, with soluble potassium and phosphorus contents in its fermentation broth reaching 65.67 mg / L and 153.67 mg / L, respectively, significantly higher than the control group. Simultaneously, it secreted auxin (0.531 μg / mL), gibberellin (2.130 μg / mL), and abscisic acid (0.588 μg / mL), promoting plant growth. Furthermore, *Bacillus amyloliquefaciens* M2-1 showed a 100% inhibition rate against *Sclerotium arachnoides* and *Phyllostachys edulis*, and a 34.6% inhibition rate against *Fusarium oxysporum*, with the antibacterial effect remaining stable with varying fermentation broth concentration gradients. Combined with physiological and biochemical analysis and 16S rDNA identification, this strain possesses potassium-solubilizing, phosphorus-solubilizing, hormone-producing, and broad-spectrum antibacterial abilities, demonstrating potential for development as a multifunctional biological agent in green agriculture.

[0009] A second aspect of the present invention provides a microbial inoculant, comprising the above-mentioned Bacillus amyloliquefaciens and its metabolites.

[0010] Furthermore, the microbial agent is in liquid form, including bacterial liquid or fermentation broth; or, the microbial agent is in solid form, including freeze-dried bacterial powder, wettable powder or carrier adsorbent, wherein the carrier includes at least one of diatomaceous earth, bentonite, wheat bran or straw powder.

[0011] A third aspect of the present invention provides a method for culturing the above-mentioned Bacillus amyloliquefaciens, comprising inoculating Bacillus amyloliquefaciens into a culture medium for culturing to obtain a culture containing Bacillus amyloliquefaciens.

[0012] A fourth aspect of the present invention provides a bio-organic fertilizer, such as the microbial agent described above.

[0013] The fifth aspect of the present invention provides the application of the above-mentioned Bacillus amyloliquefaciens or the above-mentioned microbial agent in promoting plant growth.

[0014] The sixth aspect of the present invention provides the application of the above-mentioned Bacillus amyloliquefaciens or the above-mentioned microbial agent in potassium solubilization and phosphorus solubilization.

[0015] The seventh aspect of the present invention provides the application of the above-mentioned Bacillus amyloliquefaciens or the above-mentioned microbial agent in inhibiting plant pathogens, wherein the plant pathogens are at least one of Peanut White Silkworm, Apple Rot Bacterium, and Potato Fusarium.

[0016] The eighth aspect of the present invention provides the application of the above-mentioned Bacillus amyloliquefaciens or the above-mentioned microbial agent in the secretion of plant hormones.

[0017] Furthermore, the plant hormones are auxins, gibberellins, and abscisic acid.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] This invention screened a strain of *Bacillus amyloliquefaciens* that exhibited significant rhizosphere growth-promoting and biocontrol functions. Experiments showed that *Bacillus amyloliquefaciens* M2-1 efficiently decomposes potassium feldspar, calcium phosphate, and calcium phytate. The soluble potassium and phosphorus contents in its fermentation broth reached 65.67 mg / L and 153.67 mg / L, respectively, significantly higher than the control group. Simultaneously, it secreted auxin (0.531 μg / mL), gibberellin (2.130 μg / mL), and abscisic acid (0.588 μg / mL), promoting plant growth. Furthermore, a 10-fold dilution of the fermentation broth of *Bacillus amyloliquefaciens* M2-1 showed a 100% inhibition rate against *Sclerotium arachnoideum* and *Fungiella asiatica*, and a 34.6% inhibition rate against *Fusarium oxysporum*, with the antibacterial effect remaining stable with varying fermentation broth concentration gradients. This strain possesses potassium-solubilizing, phosphorus-solubilizing, hormone-producing, and broad-spectrum antibacterial abilities, showing potential for development as a multifunctional biological agent in green agriculture. Attached Figure Description

[0020] Figure 1 The graph shows the potassium content test results in the fermentation broth of the strain after 7 days.

[0021] Figure 2 The images show the phosphate-solubilizing zones on M2-1 medium (left: inorganic phosphorus medium; right: organic phosphorus medium).

[0022] Figure 3 The graph shows the quantitative test results of phosphorus solubilization by growth-promoting bacteria.

[0023] Figure 4 The results of tests on the ability of growth-promoting bacteria to secrete auxins, gibberellins, and abscisic acid are shown in the figure.

[0024] Figure 5 The graph shows the antagonistic effect of the fermentation broth of strain M2-1 on three pathogens (a, b, and c are normally grown Fusarium oxysporum, Sclerotium arachnoides, and Pseudomonas aeruginosa (control), respectively; d, e, and f are the corresponding pathogens above, with sterile fermentation broth of M2-1 added to the culture medium).

[0025] Figure 6 This is a graph showing the antagonistic effect of fermentation broth of strain M2-1 at different concentrations on two pathogens (top layer: white mold of peanut; bottom layer: apple rot pathogen).

[0026] Figure 7This is a diagram showing the morphology of streaked bacterial colonies.

[0027] Figure 8 Electrophoresis diagram of rDNA amplification in strain M2-116S.

[0028] Figure 9 Phylogenetic tree diagram of strain M2-116S rDNA. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings.

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] Example 1

[0032] 1. Test materials

[0033] 1.1 Soil Collection

[0034] Soil samples were collected from the rhizosphere of different crops at a depth of 10-20 cm. The rhizosphere soil samples collected included those from apple, pea, wild pea, sorrel, alfalfa, peony, rockery weeds, corn, and cotton.

[0035] 1.2 Culture medium and some reagents

[0036] (1) Potassium-solubilizing bacteria isolation medium (sucrose 5.0g, sodium dihydrogen phosphate dodecahydrate 2.0g, magnesium sulfate heptahydrate 0.5g, calcium carbonate 0.5g, agar 20g, primary water 1000mL, pH=7.0~7.5)

[0037] (2) Silicate solid culture medium (sucrose 5.0g, ammonium sulfate 0.5g, tryptone 0.5g, magnesium sulfate heptahydrate 0.3g, disodium hydrogen phosphate 2g, ferric sulfate heptahydrate 0.03g, manganese sulfate monohydrate 0.03g, potassium feldspar powder K2O·Al2O3 (K[AlSi3O8] 2g, agar 15g, primary water 1000mL)

[0038] (3) Starch-dissolving medium (10g peptone, 3g beef extract, 5g sodium chloride, 17g agar, 0.2% soluble starch, 1000mL primary water, pH=7.2)

[0039] (4) Lugol's iodine solution (1g iodine tablets, 2g potassium iodide, 300ml)

[0040] (5) Peptone aqueous culture medium (10g peptone, 5g sodium chloride, 1000ml distilled water)

[0041] (6) Ehrlich reagent (4g of p-dimethylaminobenzaldehyde, 380ml of 95% ethanol, 80ml of concentrated hydrochloric acid)

[0042] (7) Nitrate liquid culture medium (5.0 g peptone, 0.2 g potassium nitrate, 1000 mL primary water, pH = 7.4)

[0043] (8) LB solid medium (5g yeast extract, 10g tryptone, 10g sodium chloride, 15g agar powder, 1000mL primary water)

[0044] (9) Citrate medium (sodium citrate 2g, dipotassium hydrogen phosphate 1g, diammonium hydrogen phosphate 1g, sodium chloride 5g, magnesium sulfate 0.2g, agar 2g, 1% bromothymol blue ethanol solution 10ml, primary water 1000ml)

[0045] (10) Liquid seed culture medium (starch 5g, magnesium sulfate heptahydrate 0.2g, calcium sulfate 0.1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 0.2g, sodium chloride 0.2g, calcium carbonate 0.5g, sucrose 5g, primary water 1000mL)

[0046] (11) Liquid seed fermentation broth (0.5g disodium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1g potassium feldspar K2O·Al2O3 (K[AlSi3O8], a few drops of FeCl3 (1%), 5g sucrose, 1000mL primary water, pH=7.2~7.4)

[0047] (12) PDA medium (potato 200g, glucose 20g, agar 20g, primary water 1000mL)

[0048] (13) Inorganic phosphorus culture medium (10.0g glucose, 5.0g calcium phosphate, 5.0g magnesium chloride hexahydrate, 0.25g magnesium sulfate heptahydrate, 0.2g potassium chloride, 0.1g ammonium sulfate, 20.0g agar, 1000mL primary water)

[0049] (14) Organic phosphorus culture medium (10.0g glucose, 5.0g calcium phytate, 5.0g magnesium chloride hexahydrate, 0.25g magnesium sulfate heptahydrate, 0.2g potassium chloride, 0.1g ammonium sulfate, 20.0g agar, 1000mL primary water)

[0050] (15) Silicate solid culture medium (sucrose 5.0g, ammonium sulfate 0.5g, tryptone 0.5g, magnesium sulfate heptahydrate 0.3g, disodium hydrogen phosphate 2g, ferric sulfate heptahydrate 0.03g, manganese sulfate monohydrate 0.03g, potassium feldspar K2O·Al2O3 (K[AlSi3O8] 2g, agar 15g, primary water 1000mL)

[0051] 1.3 Main Instruments

[0052] Erlenmeyer flasks, pipettes, spreading rods, inoculation loops, glass and plastic petri dishes, test tubes, funnels, glass rods, beakers, graduated cylinders, centrifuge tubes, centrifuges, Shimadzu UV spectrophotometers, clean benches, balances, autoclaves, shakers, PCR amplifiers, incubators, electrophoresis apparatus, and high-performance liquid chromatographs.

[0053] 2. Test methods

[0054] 2.1 Soil Sample Collection

[0055] (1) Soil samples were collected from the root zone of peony, corn and cotton at a depth of 10cm to 20cm in a field; soil samples were also collected from apple, alfalfa, pea, wild pea and sorrel.

[0056] (2) Place the collected soil samples in a cool place to air dry, mark them and store them.

[0057] 2.2 Isolation of potassium-solubilizing bacteria

[0058] Prepare a potassium-solubilizing bacteria isolation medium according to the formula. Separately, add 200 mL of primary water to a 250 mL Erlenmeyer flask and autoclave for later use. Accurately weigh 10 g of soil sample collected outdoors and place it in the 250 mL Erlenmeyer flask. Add 90 mL of sterilized primary water to the Erlenmeyer flask, seal with newspaper, and fix it on a shaker. Set the shaker speed to 180 rpm and the temperature to 25℃, and incubate for 30 minutes. After incubation, remove the flask and use a 1 mL pipette to take 1 mL of the shaken soil suspension under a clean bench. Dilute it to 10⁻⁶ ppm using a stepwise dilution method. -1 10 -3 10 -5 Three concentrations were prepared. The prepared potassium-solubilizing bacteria isolation medium was melted in a microwave oven and cooled to 60°C to form plates. 0.1 ml of soil suspension from each concentration was then transferred and evenly spread onto the potassium-solubilizing bacteria isolation medium using a spreader until the surface was free of watermarks. Three replicates were performed for each concentration. The plates were incubated at 37°C for 24–48 hours. LB slants were prepared. Strains exhibiting good growth on the potassium-solubilizing bacteria isolation medium were selected and inoculated onto silicate solid medium. Strains showing good growth on this medium were then transferred to LB slants using the stab method and stored at 4°C.

[0059] 2.3 Potassium-solubilizing bacteria fermentation

[0060] (1) Prepare liquid seed culture medium. Prepare 250mL Erlenmeyer flasks, add 50mL of liquid seed culture medium to each Erlenmeyer flask, and sterilize in an autoclave for later use.

[0061] (2) On a clean bench, use an inoculation loop to pick up the growth-promoting bacteria strain and inoculate it into the liquid seed culture medium, labeling it with the strain number. Set the shaker to 28℃ and 180r / min and incubate for 72h to prepare the primary seed culture.

[0062] (3) In a clean bench, use a pipette to transfer 5 mL of the primary seed culture of potassium-solubilizing bacteria and inoculate it again into the same liquid seed culture medium. Culture under the same conditions for another 3 days to obtain the secondary seed culture. Repeat this step with the secondary seed culture to obtain the tertiary seed culture medium.

[0063] (4) Prepare liquid seed fermentation broth. Each conical flask contains 100 mL of liquid. Under sterile conditions, add 5 mL of tertiary seed culture medium to the liquid seed fermentation broth. Set the temperature of the shaker to 30℃ and the rotation speed to 200 r / min. Cultivate for 7 days. Use the fermentation medium with 5 mL of seed culture medium as a blank control.

[0064] 2.4 Determination of potassium-solubilizing activity of potassium-solubilizing bacteria

[0065] The soluble potassium content in the fermentation broth was determined using the sodium tetraphenylborate turbidimetric method.

[0066] (1) Preparation of reagents: 60% sodium tetraphenylborate solution: Weigh 3.0g of analytical grade sodium tetraphenylborate, dissolve in 50ml of water, adjust the pH to approximately 8-9 with 0.5N sodium hydroxide solution (20g of sodium hydroxide dissolved in 1000ml of water), stir for 10-15min, let stand for 24h, filter into a brown reagent bottle and store. 5% EDTA solution: Weigh 5g of disodium ethylenediaminetetraacetate (analytical grade) and 2g of sodium hydroxide (analytical grade), dissolve in 100ml of water, store in a plastic container at 4℃.

[0067] (2) To prepare the standard curve, take a certain amount of KCl (analytical grade), dry it, and regularly weigh 0.4767 g of the dried KCl. Dissolve it in water and dilute it to 500 mL in a volumetric flask to obtain the 500 ppm potassium standard solution stock solution. Prepare eight 50 mL Erlenmeyer flasks, labeled and cleaned, and dried for later use. Using a pipette, transfer 0.5, 1.25, 2.5, 3.75, 5.0, 6.25, 7.5, and 8.75 mL of the stock solution into the Erlenmeyer flasks according to their numbers, and dilute them to 50 mL with primary water. This yields a series of standard solutions with concentrations of 5.0, 12.5, 25, 37.5, 50, 62.5, 75, and 87.5 ppm, each in 50 mL.

[0068] (3) Determination of standard curve

[0069] Prepare nine 25mL test tubes, clearly label them with concentration numbers, and rinse them with primary water. Use a pipette to transfer 1mL of each concentration standard solution into the corresponding test tube. Then, add 2mL of the prepared 5% EDTA solution and 1mL of 37% formaldehyde solution to each test tube, and shake well. Next, add 1mL of 60% sodium tetraphenylborate solution as a turbidity test solution to each tube, shake well, let stand for 10 minutes, and then add primary water to bring the volume to 25mL. Use a UV spectrophotometer set to a wavelength of 440nm for turbidity measurement, completing the measurement within 20–100 minutes. Plot a standard curve with potassium ion concentration on the x-axis and OD value on the y-axis.

[0070] (4) Determination of bacterial strain samples. Take 1.5 mL of potassium-solubilizing fermentation broth from each strain, centrifuge at 4500 r / min for 20 min, and take 1 mL of the supernatant as the sample to be tested for determining the soluble potassium content in the fermentation broth. Use the supernatant instead of potassium chloride standard solution to add reagents according to the standard curve determination order to complete the sample determination.

[0071] Potassium solubility = K in bacterial suspension + Content - K in sterile fermentation broth + content.

[0072] 2.5 Determination of the phosphorus solubility zone

[0073] Inorganic phosphorus and organic phosphorus solid culture media were prepared separately, and plates were fabricated under aseptic conditions in a laminar flow hood. The bacterial strain was inoculated into the center of each plate using an inoculation loop, and the plates were sealed with sealing film. The plates were incubated at 30°C for 5 days. The presence of phosphate-solubilizing zones was observed.

[0074] 2.6 Determination of inorganic phosphorus soluble activity

[0075] LB liquid culture medium was prepared for inoculation of the bacterial strain, and cultured at 30℃ and 180 rpm for 24 h. Inorganic phosphorus liquid culture medium was prepared by sterilizing 100 mL of the medium in 250 mL Erlenmeyer flasks. Under aseptic conditions in a clean bench, 5 mL of the prepared LB bacterial culture was inoculated into the inorganic phosphorus liquid culture medium and cultured on a shaker at 180 rpm for 7 days. The inorganic phosphorus content was determined using a soil total phosphorus / organic phosphorus / inorganic phosphorus content assay kit.

[0076] 2.7 Determination of Organophosphorus Phosphate-Solubilizing Activity

[0077] (1) Preparation of organophosphorus fermentation broth: Prepare organophosphorus liquid culture medium by sterilizing 100 mL of the medium in a 250 mL Erlenmeyer flask. Inoculate the test bacterial suspension, adjusted to an OD value of 1 at 600 nm, into 1% (V / V) Erlenmeyer flasks containing 100 mL of organophosphorus liquid culture medium at a rate of 1%. Incubate at 30 °C and 180 r / min for 72 h on a shaker.

[0078] (2) Standard Curve Determination: Accurately pipette 5 mL of phosphorus standard stock solution and dilute to 100 mL in a volumetric flask. Prepare a 5 mg / L phosphorus standard solution. Prepare six 50 mL test tubes and label them with concentration numbers. Test tube #1 is a pure water control. Add 1, 2, 3, 4, and 5 mL of phosphorus standard solution to each tube respectively. Dilute the six test tubes to 30 mL with primary water. Pipette 2 drops of dinitrophenol indicator to each test tube. If the solution is colorless, add 4 mol / L NaOH solution dropwise until the liquid in the volumetric flask turns slightly yellow. Then add 1 mol / L sulfuric acid dropwise until the yellow color just disappears. If the solution is yellow from the beginning, add 1 mol / L sulfuric acid dropwise directly until the yellow color just disappears. Then add 5 mL of molybdenum antimony anti-color developing agent and dilute to 50 mL with distilled water. Develop the color at room temperature for 30 min and measure the absorbance value on a UV spectrophotometer with the wavelength set to 700 nm. Plot a phosphorus standard curve with phosphorus concentration on the x-axis and absorbance on the y-axis.

[0079] (3) Determination of organophosphorus phosphorus solubility: Pipette 20 ml of bacterial fermentation broth from organophosphorus liquid culture medium (after 72 h of shaking) into a centrifuge tube and centrifuge at 8000 r / min for 10 min; transfer 5 mL of supernatant to a 50 mL volumetric flask and add distilled water to bring the volume to 30 mL. Continue the operation according to the above standard curve preparation method, measure the OD value, and substitute it into the standard curve to calculate the corresponding available phosphorus content.

[0080] Phosphorus dissolved amount = P in bacterial suspension + Content - P in sterile fermentation broth + content.

[0081] 2.8 Determination of the plant hormone production capacity of the strain

[0082] Plant hormone test: Approximately 1.5 mL of the supernatant from the centrifuged fermentation broth was filtered through a 0.45 μm disposable microporous filter and injected into a 2 mL brown sample vial for high-performance liquid chromatography (HPLC) analysis. The absorption peaks for auxin and gibberellin were measured at 276 nm and 253 nm, respectively, and the abscisic acid concentration was determined at 260 nm.

[0083] 2.9 Determination of bacterial antagonism

[0084] ① Standoff culture method: Activate pathogenic bacterial strains (Fusarium oxysporum, Sclerotium arachidii) stored at 4℃. ② Group the isolated potassium-solubilizing bacteria by number, and test each group of four strains sequentially. Prepare PDA plates in 9mm petri dishes under aseptic conditions. Inoculate the target pathogenic bacteria into the center of the culture medium using a punch, and then inoculate the potassium-solubilizing bacteria (numbers 1-4) around the center. Continue this process for subsequent strains. Incubate at 30℃ for 4 days, observe the results, and determine the size of the inhibition zone. For strains with larger inhibition zones, perform a separate streak standoff test.

[0085] ② Aseptic Fermentation Broth Method: LB medium was used, with 1 mL of potassium-solubilizing bacteria seed culture added. The mixture was fermented at 30°C and 180 rpm for 72 hours on a shaker. After 72 hours, the fermentation broth was aliquoted into centrifuge tubes and centrifuged at 8000 rpm for 25 minutes. The bottom precipitate was removed, and the supernatant was collected. The supernatant was filtered twice using a bacterial filter and diluted tenfold with thawed PDA medium cooled to approximately 60°C to prepare agar plates. The pathogenic bacteria to be tested were inoculated into the center of the PDA medium containing the aseptic fermentation broth using a 5 mm punch, and simultaneously inoculated into a normal PDA plate. The growth of pathogenic fungi on both plates was observed. The colony diameter was measured by cross-sectioning to calculate the inhibition rate, confirming the inhibitory effect of the potassium-solubilizing bacteria aseptic fermentation broth on the growth and spread of pathogenic bacteria.

[0086] 2.10 Physiological and biochemical identification of the strain

[0087] (1) Starch hydrolysis test: Some large organic molecules, such as starch, fats, and proteins, cannot be directly utilized by bacteria. They can only be broken down by extracellular enzymes. During this process, the substrate changes. For starch, iodine can be used to verify this. After dissolving the starch culture medium, cool it to about 45°C and prepare plates aseptically. Inoculate the cultured strain into the center of the plate using an inoculation loop and incubate at 30°C for 24 hours. Add Lugol's iodine solution to the plate. If a colorless transparent zone appears around the colony, it indicates that the starch has been hydrolyzed and the strain has the ability to hydrolyze starch, which is a positive reaction. If there is no blue reaction, it is a negative reaction.

[0088] (2) Indole production test: Some bacteria containing tryptophanase can decompose tryptophan in their growth environment to produce a type of indole matrix, namely colorless indole. Colorless indole reacts with Ehrlich's reagent to form a red compound—rose indole. This phenomenon can be used to verify whether bacteria can produce indole-like substances. The bacterial strain is inoculated into peptone water medium and cultured at 37°C for 2–3 days. After culture, 0.5–1 mL of Ehrlich's reagent is added along the tube wall to the surface of the culture medium. The results are observed after 1 minute: the appearance of a rose-red ring at the interface indicates a positive indole test, meaning the strain has the ability to produce indole-like substances; conversely, the absence of a red ring indicates a negative indole production test.

[0089] (3) Citrate Test: The ability of bacteria to use citrate as a carbon source is a differential test. Bromothymol blue is used as the environmental pH indicator. If bacteria can use citric acid as a carbon source to decompose it, free sodium ions will appear in the culture medium, causing the overall pH of the solution to rise, and the culture medium will change from yellow to green. Prepare citrate culture medium, fabricate plates under sterile conditions, and streak the bacterial strain. After 72 hours of incubation, observe the phenomenon: a change in the plate from yellow-green to dark green indicates that the strain has the ability to utilize citrate, which is a positive reaction; otherwise, it is a negative reaction.

[0090] (4) Methyl red test: The methyl red test is used to detect the ability of bacteria to break down glucose to produce pyruvate, and further break down pyruvate into other acidic substances. Methyl red is yellow under alkaline conditions and red under acidic conditions. The color change can verify whether the bacteria have broken down glucose. The strain is inoculated into glucose peptone water medium and cultured at 37°C for 48 hours. The prepared methyl red reagent is added to the medium. If a red color appears, it indicates that the strain breaks down glucose to produce pyruvate, which is then further broken down into formic acid, acetic acid, etc., which is a positive reaction. Conversely, if the amount of acid produced is small or if the acid is further broken down into alcohols, phenols, etc., a yellow color appears, which is a negative reaction.

[0091] (5) Nitrate reduction test: Prepare nitrate liquid culture medium, inoculate the strain under sterile conditions, and incubate at 35℃ for 3 days. Mix the prepared solution A and solution B and add them to the culture medium. If the culture medium shows a red reaction, it indicates that the nitrate has been reduced by the strain to a positive result. If the culture medium does not show a color reaction, it indicates that the nitrate has not been reduced to a negative result or has been reduced to ammonia and ammonia gas, which is a false negative result.

[0092] (6) Catalase test: The catalase test is used to distinguish between aerobic bacteria, facultative anaerobic bacteria, and anaerobic bacteria. Generally, both aerobic and facultative anaerobic bacteria have the ability to produce catalase, which produces bubbles when exposed to hydrogen peroxide. The cultured bacterial strain is smeared onto a glass slide, and 3% hydrogen peroxide is added to the bacterial cells. Observe for 1 minute. If bubbles are produced, it indicates that the strain is an aerobic or facultative anaerobic bacterium, which is a positive reaction; otherwise, it is a negative reaction.

[0093] (7) Gram staining: Add physiological saline to a glass slide, apply the bacterial cells with an inoculation loop to form a bacterial film, dry, and pass the slide over an alcohol lamp three times until it is no longer hot to the touch. Add crystal violet staining solution to the bacterial film and stain for 1 minute, then wash away the excess stain with a wash bottle. Add an appropriate amount of iodine solution to the bacterial film and stain for 1 minute, then wash with water and blot dry with absorbent paper. Decolorize with 95% alcohol for 30 seconds, rinse with water and blot dry with absorbent paper. Stain with safranin reagent for 2 minutes, wash away the excess stain with water, and blot dry with absorbent paper. Observe under a microscope; if the staining result is blue-purple, it is a positive reaction; if it is red or purple-reddish, it is a negative reaction.

[0094] (8) Identification of 16S rDNA in the strain: LB strain fermentation broth was prepared and cultured overnight. The fermented broth was then used to extract strain DNA using a plasmid mini-extraction kit (from Beijing Zhuangmeng International Biotechnology Co., Ltd.) following the manufacturer's instructions. PCR amplification was performed on the extracted biocontrol bacterial DNA template using 16S rDNA primers: universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'). After PCR amplification, the PCR products were examined by agarose gel electrophoresis. The remaining products were directly sent to Qingke Biotechnology Co., Ltd. for sequencing. The obtained sequences were assembled using DNAMAN, and sequence homology analysis was performed using the BLAST program in NCBI, and a phylogenetic tree was constructed.

[0095] Example 2

[0096] 1. Screening results of potassium-solubilizing bacteria

[0097] Sixteen potassium-solubilizing and growth-promoting bacterial strains that grew normally on silicate solid culture medium were screened from the collected rhizosphere soil. Strain M2-1, which showed the best growth, was selected for subsequent experiments.

[0098] 2. Results of potassium-solubilizing and phosphorus-solubilizing activity assays of the screened rhizosphere growth-promoting strains

[0099] 2.1 Quantitative determination results of potassium solubilization activity

[0100] Using potassium feldspar powder as the sole potassium source, the fermentation broth of the strain was cultured on a shaker at 200 r / min for 7 days. The soluble potassium content in the fermentation broth was determined using the sodium tetraphenylborate turbidimetric method. The soluble potassium concentration in strain M2-1 was approximately 65.67 ± 0.16 mg / L, while the soluble potassium content in the sterile control fermentation broth was only 2.6 ± 0.003 mg / L. Figure 1 ).

[0101] 2.2 Quantitative determination results of phosphorus solubility activity

[0102] (1) Observation of the lysing zone

[0103] Strain M2-1 was inoculated onto organic phosphorus (calcium phytate) and inorganic phosphorus (calcium phosphate) media, respectively, and cultured at 30℃ for 5 days. A clear zone appeared on the culture medium. Figure 2 As shown.

[0104] (2) Quantitative determination results of phosphorus solubility

[0105] Phosphate-soluble fermentation broths were prepared using calcium phytate and tricalcium phosphate as the sole phosphorus source, respectively. The decomposition capacity of the growth-promoting bacteria for both organic and inorganic phosphorus forms was determined. In the organic phosphorus fermentation assay, after 3 days of shaking culture, the soluble phosphorus content of the fermentation broth of strain M2-1 was 385.236±11.93 mg / L, while the soluble phosphorus content of the sterile fermentation broth control was 259.232±12.87 mg / L. In the inorganic phosphorus fermentation assay, the soluble phosphorus concentration in the control (CK) was 0.228 mg / L, as determined by a soil total phosphorus / organic phosphorus / inorganic phosphorus content assay kit, while the soluble phosphorus concentration in the fermentation broth of strain M2-1 was 153.670±0.45 mg / L. Figure 3 ).

[0106] 2.3 Results of the assay for the plant hormone production capacity of the strain

[0107] The concentrations of three plant hormones—auxin, gibberellin, and abscisic acid—in the fermentation broth were determined using high-performance liquid chromatography (HPLC). Aseptic fermentation broth served as a control, with all hormone concentrations in the control group being 0 μg / mL. The concentrations of the three hormones were: auxin 0.531 μg / mL, gibberellin 2.130 μg / mL, and abscisic acid concentration of 0.588 μg / mL for strain M2-1. Figure 4 ).

[0108] 2.4 Inhibitory effect of strains on plant pathogens

[0109] Inhibitory effect of fermentation broth of strain M2-1 against plant pathogens: The inhibitory effect of fermentation broth of strain M2-1 against three pathogens, including *Sclerotium arachnoideum*, was determined using the growth rate method. (Table 1 and...) Figure 5 The experimental results are presented. The fermentation broth method showed that the fermentation broth of strain M2-1 diluted 10 times exhibited 100% inhibition against *Sclerotium arachnoides* and *Fungiformis chinensis*, and 34.6% inhibition against *Fusarium oxysporum*. This indicates that the active substances in the fermentation broth have high toxicity against *Fungiformis chinensis* and *Sclerotium arachnoides*.

[0110] Table 1. Antagonistic effect of fermentation broth of strain M2-1 diluted tenfold.

[0111]

[0112]

[0113] Antagonistic experiments were conducted on fermentation broths at three concentrations (10x, 20x, and 40x) against both apple rot pathogen and peanut white mold. The results are shown in Table 2 and... Figure 6After three days of cultivation, the fermentation broth of strain M2-1 at all three concentrations showed a 100% inhibitory effect on apple rot pathogens. Regarding *Sclerotium arachidii*, at a 10-fold dilution, the pathogen could not grow normally on the agar plate, and aerial mycelia grew on the inoculated fungal cake, indicating that the antibacterial substances in the fermentation broth failed to diffuse into the fungal cake. At 20-fold and 40-fold dilutions, the inhibition rate of the fermentation broth against *Sclerotium arachidii* decreased to 67.7% and 43.7%, respectively.

[0114] Table 2. Antagonistic effects of different concentrations of fermentation broth from strain M2-1

[0115]

[0116] 2.5. Strain classification and identification

[0117] 2.51. Morphological identification of strains

[0118] The strain was inoculated into LB medium using a streak method with serial dilutions. In LB medium, strain M2-1 colonies had a rough, wrinkled surface, were slightly raised with a central depression, and had irregular edges. The colonies were generally grayish-white and opaque, and when picked up with a toothpick, they were quite viscous. The streak colony morphology is as follows: Figure 7 As shown.

[0119] 2.52. Results of physiological and biochemical characterization of the strain

[0120] Physiological and biochemical identification results showed that strain M2-1 was Gram-positive and showed positive reactions in starch solubility test, nitrate test, indole production test and catalase test, while it was negative in methyl red test and citrate test.

[0121] like Figure 8 The 16S rDNA amplified fragment of strain M2-1 shown is approximately 1500 bp in size, consistent with the typical size of bacterial 16S rDNA. Blast comparison and phylogenetic tree analysis revealed that this strain belongs to the same clade as *Bacillus amyloliquefaciens* MN-JXJ:6, with a homology of up to 98%. The resulting phylogenetic tree is shown below. Figure 9 Based on the colony morphology and physiological and biochemical characteristics of strain M2-1, it was preliminarily identified as Bacillus amyloliquefaciens.

[0122] The strain was classified and named Bacillus amyloliquefaciens M2-1, and was deposited at the China Center for Type Culture Collection on December 30, 2024, with accession number CCTCC NO:M 20242929.

[0123] The 16S rDNA sequence of Bacillus amyloliquefaciens M2-1 strain is as follows:

[0124]

[0125] This invention screened a strain of *Bacillus amyloliquefaciens* that exhibited significant rhizosphere growth-promoting and biocontrol functions. Experiments showed that *Bacillus amyloliquefaciens* M2-1 efficiently decomposes potassium feldspar and calcium phosphate, with soluble potassium and phosphorus contents in its fermentation broth reaching 65.67 mg / L and 153.67 mg / L, respectively, significantly higher than the control group. Simultaneously, it secreted auxin (0.531 μg / mL), gibberellin (2.130 μg / mL), and abscisic acid (0.588 μg / mL), promoting plant growth. Furthermore, *Bacillus amyloliquefaciens* M2-1 showed a 100% inhibition rate against *Sclerotium arachnoides* and *Phyllostachys edulis*, and a 34.6% inhibition rate against *Fusarium oxysporum*, with the antibacterial effect remaining stable with varying fermentation broth concentration gradients. Combined with physiological and biochemical analysis and 16S rDNA identification, this strain possesses potassium-solubilizing, phosphorus-solubilizing, hormone-producing, and broad-spectrum antibacterial abilities, demonstrating potential for development as a multifunctional biological agent in green agriculture.

[0126] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), characterized in that, Strain naming Bacillus amyloliquefaciens M2-1 was deposited at the China Center for Type Culture Collection on December 30, 2024, with accession number CCTCC NO: M 20242929.

2. A microbial inoculant, characterized in that, Includes Bacillus amyloliquefaciens as described in claim 1, and its metabolites.

3. The microbial agent according to claim 2, characterized in that, The microbial inoculant is in liquid form, including bacterial liquid or fermentation broth; or, the microbial inoculant is in solid form, including freeze-dried bacterial powder, wettable powder or carrier adsorbent, wherein the carrier includes at least one of diatomaceous earth, bentonite, wheat bran or straw powder.

4. A method for culturing Bacillus amyloliquefaciens as described in claim 1, characterized in that, This includes inoculating Bacillus amyloliquefaciens into a culture medium and culturing it to obtain a culture containing Bacillus amyloliquefaciens.

5. A bio-organic fertilizer, characterized in that, The microbial agent as described in claim 2 or 3.

6. The application of Bacillus amyloliquefaciens as described in claim 1 or the microbial agent as described in claim 2 in promoting plant growth.

7. The application of Bacillus amyloliquefaciens as described in claim 1 or the microbial agent as described in claim 2 in potassium solubilization and phosphorus solubilization; wherein the potassium solubilization is potassium-solubilizing feldspar, and the phosphorus solubilization is phytate-soluble calcium and calcium phosphate.

8. The application of *Bacillus amyloliquefaciens* as described in claim 1 or the microbial agent as described in claim 2 in inhibiting plant pathogens, characterized in that... The plant pathogen is at least one of the following: peanut white mold, apple rot fungus, and potato fusarium.

9. The application of Bacillus amyloliquefaciens as described in claim 1 or the microbial agent as described in claim 2 in the secretion of plant hormones; wherein the plant hormones are auxins, gibberellins, and abscisic acid.

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