Acid fast-growing bacillus strain and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an acid fast-growing bacillus strain and application thereof. The invention provides a strain of bacillus acidogenes C7, which is preserved in the China General Microbiological Culture Collection Center on September 10, 2024, the preservation date is September 10, 2024, and the preservation number is CGMCC No.31934. The invention also provides a method for preparing the bacillus acidogenes C7. The bacillus acidogensis C7 has a strong function of producing indoleacetic acid and can increase the content of indoleacetic acid in plants, so that the stress resistance of the plants is improved; and dissolution of insoluble phosphorus and potassium in soil can be promoted, soil nutrients are improved, plant growth is promoted, and plant quality is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a strain of Bacillus acidiceler and its application. Background Art

[0002] Yellow-brown soil refers to a weakly ferralitic soil with strong leaching, strong acidic reaction, and base unsaturation under the deciduous and evergreen broad-leaved forests in the north subtropical zone, and can be used for growing economic crops such as tobacco. However, due to continuous cropping obstacles, the physical and chemical properties of tobacco-growing soil deteriorate, the yield and quality of tobacco leaves decline, and the high-quality tobacco-growing areas are shrinking continuously. Alleviating the continuous cropping obstacles in yellow-brown soil tobacco fields helps to improve the quality and yield of tobacco leaves, maintain soil health, improve resource utilization efficiency, and promote the development of sustainable agriculture.

[0003] Plant growth-promoting rhizobacteria (PGPR) can synthesize substances that have a direct effect on plant growth and development, such as plant hormones to promote plant growth; they can also change the forms of some ineffective elements in the soil, such as promoting the dissolution of insoluble phosphorus and potassium in the soil to make them available for plant absorption. However, there are few reports on multifunctional PGPR with both phosphorus-solubilizing and potassium-solubilizing, hormone-producing, and disease-resistant functions at present. There is an urgent need for a multifunctional PGPR with both phosphorus-solubilizing and potassium-solubilizing and hormone-producing functions to improve soil properties. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Bacillus acidiceler and its application. The Bacillus acidiceler C7 of the present invention has a strong indole-3-acetic acid-producing function, can promote the dissolution of insoluble phosphorus and potassium in the soil, improve soil nutrients, promote plant growth, and improve plant quality.

[0005] The present invention provides a strain of Bacillus acidiceler C7, which is deposited in the China General Microbiological Culture Collection Center, with the deposition date of September 10, 2024, and the deposition number of CGMCC No. 31934.

[0006] The present invention also provides a bacterial agent, and the active ingredient of the bacterial agent includes the Bacillus acidiceler C7 described in the above solution.

[0007] As a preferred scheme, the effective viable count of Bacillus acidiceler C7 in the bacterial agent ≥ 1×10 11 CFU / g.

[0008] The present invention also provides the application of the Bacillus acidiceler C7 described in the above solution or the bacterial agent described in the above solution, and the application includes at least one of the following 1) to 4):

[0009] 1) Increasing the indole-3-acetic acid content of plants;

[0010] 2) Promote plant growth;

[0011] 3) Improve soil nutrients;

[0012] 4) Improve plant quality.

[0013] As a preferred embodiment, the promoting plant growth includes at least one of promoting root growth, increasing plant height, and increasing leaf area.

[0014] As a preferred embodiment, the plant includes at least one of tobacco, cotton, fruit trees, pakchoi, tomatoes, wheat, millet, corn, and sorghum.

[0015] As a preferred embodiment, the improving soil nutrients includes promoting the dissolution of insoluble phosphorus and / or potassium in the soil.

[0016] As a preferred embodiment, the improving plant quality includes at least one of increasing the total sugar, reducing sugar, and potassium content of the plant.

[0017] As a preferred embodiment, the application includes the following steps: applying the Bacillus acidiceler C7 described in the above solution or the microbial agent described in the above solution into the soil where the plant is planted.

[0018] As a preferred embodiment, the application amount of the microbial agent is 35 - 45 kg / hm 2 .

[0019] Beneficial effects:

[0020] The present invention provides a multifunctional growth-promoting Bacillus acidiceler C7, which is deposited in the China General Microbiological Culture Collection Center. The deposit date is September 10, 2024, and the deposit number is CGMCC No. 31934. The concentration of indole-3-acetic acid produced by the Bacillus acidiceler C7 of the present invention reaches more than 58.56 mg / L, which can directly promote the elongation of plant roots and increase the chance of contact with nutrients in the soil; and can increase the content of endogenous IAA in plants, induce the expression of plant defense genes, and improve the disease resistance, drought resistance and other stress resistances of plants. At the same time, the Bacillus acidiceler C7 has the ability to dissolve phosphorus, and the concentration of dissolved insoluble inorganic phosphorus reaches more than 428.85 mg / L, which can convert the difficult-to-use phosphorus into available phosphorus and increase the content of available phosphorus in the soil. The Bacillus acidiceler C7 of the present invention also has the ability to release potassium, and the solubility of dissolved insoluble potassium reaches more than 198.15 mg / L, which can improve the utilization rate of fertilizers, promote the growth and development of plants and the absorption of fertilizers.

[0021] The Bacillus acidiceler C7 of the present invention can promote plant growth. The results of the examples show that when Bacillus acidiceler C7 is applied to tobacco, pakchoi, and wheat potted plants, it can promote the increase of IAA content and nutrient content in the soil. At the same time, it promotes the roots of crops to be thicker, longer, and have a larger surface area, which is beneficial to the absorption of water and nutrients by crop roots, making the plant height, dry weight, and fresh weight of the above-ground parts of the crops significantly higher than those of the control treatment. In the field experiment of tobacco, compared with the control, the treatment with Bacillus acidiceler C7 makes the tobacco have a higher plant height and leaf area, promoting the increase of tobacco yield. The contents of total sugar and reducing sugar in the cured tobacco are increased by 12.12% and 13.44% respectively compared with the control treatment. At the same time, the potassium content in the cured tobacco leaves is significantly increased by 20.86% compared with the control treatment. Bacillus acidiceler C7 can improve the yield and quality of tobacco. Brief Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0023] Figure 1 It is the colony map of Bacillus acidiceler C7 of the present invention;

[0024] Figure 2 It is the phylogenetic tree of Bacillus acidiceler C7 of the present invention.

[0025] Biological Deposit Description

[0026] Bacillus acidiceler C7 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024. The unit abbreviation is CGMCC, and the address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 31934, and the strain number is C7. Detailed Embodiments

[0027] The present invention provides a strain of Bacillus acidiceler C7, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024, and the deposit number is CGMCC No. 31934.

[0028] The Bacillus subtilis C7 of the present invention is isolated from yellow brown soil. The morphological characteristics of the Bacillus subtilis C7 are as follows: colonies of 1-2 mm can be formed after 24 h of cultivation. The colony morphology is that the colonies are gray and translucent, round, with a smooth surface, regular edges, no halo, and flat. The physiological and biochemical characteristics of the strain C7 are: Gram-positive, facultative anaerobic, negative for catalase, positive for M.R. test, positive for V.P. test, positive for starch hydrolysis, positive for gelatin liquefaction, positive for nitrate reduction, and positive for citrate utilization.

[0029] In the present invention, the Bacillus subtilis C7 has the ability to secrete IAA and the ability to promote the dissolution of insoluble phosphorus and potassium in the soil; the strain can secrete more than 58.56 mg / L of IAA, the ability to dissolve inorganic phosphorus reaches more than 428.85 mg / L, and the ability to dissolve insoluble potassium reaches more than 198.15 mg / L.

[0030] The present invention also provides a bacterial agent, and the active ingredient of the bacterial agent includes the Bacillus subtilis C7 described in the above scheme. As an embodiment, the effective viable count of the Bacillus subtilis C7 in the bacterial agent is ≥1×10 11 CFU / g. In the present invention, the bacterial agent includes the Bacillus subtilis C7 and a carrier; the present invention has no special limitation on the type of the carrier, and the carriers well-known in the art can be used; in the embodiments of the present invention, the type of the carrier is bone meal. The present invention has no special limitation on the preparation method of the bacterial agent, and the preparation methods well-known in the art can be used.

[0031] The present invention also provides the application of the Bacillus subtilis C7 described in the above scheme or the bacterial agent described in the above scheme, including at least one of the following 1)-4): 1) increasing the indoleacetic acid content of plants; 2) promoting plant growth; 3) improving soil nutrients; 4) improving plant quality.

[0032] The Bacillus subtilis C7 of the present invention has the ability to secrete IAA, reaching 58.56 mg / L, which is significantly higher than 17.98 μg / mL secreted by the plant growth-promoting rhizobacterium YC5 in tobacco rhizosphere disclosed in the prior art (Chinese Patent 201410761450.2). The Bacillus subtilis C7 of the present invention can increase the content of endogenous IAA in plants, induce the expression of plant defense genes, and improve the disease resistance, drought resistance and other stress resistances of plants.

[0033] As an implementation mode, the promotion of plant growth includes at least one of promoting root growth, increasing plant height, and increasing leaf area. As an implementation mode, the plant includes at least one of tobacco, cotton, fruit trees, pakchoi, tomatoes, wheat, millet, corn, and sorghum. The indoleacetic acid produced by the Bacillus subtilis C7 of the present invention can directly promote the growth of plant roots, making the roots thicker, longer, and having a larger surface area, thereby increasing the chance of contact between the plant and nutrients in the soil, and increasing the plant height, leaf area, and dry and fresh weights of the above-ground part of the plant.

[0034] As an implementation mode, the improvement of soil nutrients includes promoting the dissolution of insoluble phosphorus and / or potassium in the soil. The concentration of insoluble inorganic phosphorus dissolved by the Bacillus subtilis C7 of the present invention reaches more than 428.85 mg / L, which can convert the difficult-to-use phosphorus into available phosphorus and increase the content of available phosphorus in the soil; and this strain also has the ability to decompose potassium, and the solubility of insoluble potassium reaches more than 198.15 mg / L, thereby improving the utilization rate of fertilizers, promoting the growth and development of plants, and the absorption of fertilizers.

[0035] As an implementation mode, the improvement of plant quality includes at least one of increasing the total sugar, reducing sugar, and potassium content of the plant. In the present invention, the Bacillus subtilis C7 can increase the total sugar, reducing sugar, and potassium content in flue-cured tobacco, thereby increasing the yield and quality of tobacco.

[0036] As an implementation mode, the application includes the following steps: applying the Bacillus subtilis C7 described in the above solution or the microbial agent described in the above solution into the soil planted with plants. As an implementation mode, the water content of the soil is 50% - 60% of the maximum field water holding capacity.

[0037] As an implementation method, before applying the Bacillus subtilis C7 to the soil colonized with plants, it further includes: expanding the culture of Bacillus subtilis C7 to obtain a bacterial suspension. In the present invention, the pH of the medium for the expanded culture is 6-8; the medium for the expanded culture includes an inorganic salt medium; the inorganic salts include one or more of sodium chloride, potassium chloride, tricalcium phosphate, ammonium sulfate, magnesium sulfate heptahydrate, manganese sulfate, and ferrous sulfate heptahydrate. As an implementation method, each 1000 mL of the inorganic salt medium of the present invention further includes the following components in mass percentage: 1% carbon source and 0.05%-0.1% nitrogen source; as an implementation method, the carbon source includes one or more of glucose, sucrose, and maltose; the nitrogen source includes one or more of urea, peptone, and yeast powder. The present invention has no special restrictions on the sources of the carbon source, nitrogen source, and inorganic salts, and the above-mentioned sources of the carbon source, nitrogen source, and inorganic salts well-known to those skilled in the art can be used. As an implementation method, the time for the expanded culture is 68 h. In the present invention, the expanded culture is an oscillating culture, and the rotation speed of the oscillating culture is 180 rpm; the volume of the medium filled during the oscillating culture is 30-70 mL / 250 mL conical flask.

[0038] As an implementation method, the application amount of the Bacillus subtilis C7 is 1-9×10 8 CFU / g soil. In the specific implementation method of the present invention, the application amount of the Bacillus subtilis C7 can be any value within 1-9×10 8 CFU / g soil. As an implementation method, the application amount of the bacterial agent is 35-45 kg / hm 2 , in the specific embodiment of the present invention, the application amount of the bacterial agent can be any value within 35-45 kg / hm 2 . As an implementation method, the effective viable count of Bacillus subtilis C7 in the bacterial agent is 1-9×10 11 CFU / g. In the specific implementation method of the present invention, the effective viable count of Bacillus subtilis C7 in the bacterial agent can be any value within 1-9×10 11 CFU / g.

[0039] To further illustrate the present invention, the following describes in detail a strain of Bacillus subtilis and its application provided by the present invention with reference to the drawings and examples, but they cannot be understood as limiting the protection scope of the present invention.

[0040] Example 1

[0041] (1) Preparation of the experimental medium.

[0042] LB medium: 10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, 20 g of agar and 1000 mL of distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 20 min. On this basis, without adding agar, it is LB liquid medium.

[0043] Inorganic phosphate bacteria medium (PKO medium): 5 g of tricalcium phosphate, 10 g of glucose, 0.5 g of ammonium sulfate, 0.3 g of sodium chloride, 0.3 g of magnesium sulfate heptahydrate, 0.3 g of potassium chloride, 0.03 g of manganese sulfate, 0.03 g of ferrous sulfate heptahydrate and 1000 mL of distilled water, pH 7.0 - 7.2, sterilized at 121 °C for 20 min.

[0044] Liquid potassium - solubilizing bacteria medium: 10.0 g of sucrose, 0.5 g of yeast extract, 1.0 g of ammonium sulfate, 2.0 g of sodium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 1.0 g of CaCO 3 1.0 g, 1.0 g of potassium feldspar powder and 1000 mL of distilled water, sterilized at 121 °C for 20 min.

[0045] Inorganic salt medium: 2.0 g of ammonium sulfate; 0.5 g of sodium dihydrogen phosphate; 0.5 g of dipotassium hydrogen phosphate; 0.2 g of magnesium sulfate heptahydrate; 0.1 g of calcium chloride dihydrate and 1000 mL of distilled water, pH 7.0, sterilized at 121 °C for 20 min.

[0046] (2) Isolate bacteria.

[0047] A. Collect 10 g of tobacco - growing soil under natural conditions in Luoshan County, Xinyang City, Henan Province. The soil is yellow - brown soil, and its basic physical and chemical properties are shown in Table 1. B. Place the soil collected in step A into a 250 - mL Erlenmeyer flask containing 100 mL of sterilized water, and shake it in a shaker at 30 °C and 150 rpm for 20 min, then let it stand for 10 min to obtain a soil bacteria suspension. C. The soil bacteria suspension obtained in step B contains several plant - growth - promoting rhizobacteria. After dilution by the dilution method, it is spread on the LB medium. Invert the petri dish and culture it in an incubator at 30 °C for 24 h. Then pick different types of typical single colonies, after purification on the plate, store them at 4 °C on the LB slant, and prepare a seed solution with an OD 600 of 1 for use, and screen out plant - growth - promoting bacteria that can secrete indole - 3 - acetic acid through qualitative and quantitative determinations.

[0048] Table 1 Basic physical and chemical properties of the tested soil

[0049]

[0050] (3) Qualitative determination

[0051] A. In step (2), the purified bacteria were respectively inoculated into LB liquid medium containing L-tryptophan (100 mg / L) at an inoculation amount of 1% with the seed liquid, and cultured on a shaker at 30 °C and 180 rpm for 1 day. B. After the culture was completed, 50 μL of the bacterial suspension in step A was dropped onto a white ceramic plate, and at the same time, 50 μL of Salkowski colorimetric solution (50 mL of 35% HClO 4 + 1 mL of 0.5 M FeCl 3 ) was added. C. The positive control group was: 50 μL of 50 mg / L indoleacetic acid and 50 μL of Salkowski colorimetric solution were dropped onto the white ceramic plate. D. The white ceramic plate was placed in the dark at room temperature for 30 min and then observed. Those with a red color change indicated the ability to secrete indoleacetic acid.

[0052] (4) Quantitative determination

[0053] A. For the bacteria that secrete IAA obtained by the preliminary screening in the qualitative determination in step (3), a quantitative determination was carried out, and the culture conditions were the same as those in A in step (3). B. The culture solution in step A was centrifuged at 10000 rpm for 10 min, and the supernatant was taken and an equal volume of Salkowski colorimetric solution was added, and it was allowed to stand in the dark for 30 min, and its OD 530 value was measured. A standard curve was drawn using an analytically pure indoleacetic acid gradient dilution solution, the content of IAA in each liter of fermentation broth was detected, and the ability of the strain to produce IAA was determined. The results are shown in Table 2.

[0054] (5) Screening of bacteria with better IAA production ability for phosphorus solubilizing ability determination

[0055] A. The seed liquid of the isolated strain was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of inorganic phosphorus medium at an inoculation amount of 1%, placed in a shaker, the shaker temperature was set at 30 °C, and the rotation speed was 180 rpm, and cultured for 3 days. B. After the culture was completed, the culture solution was centrifuged at 10000 rpm for 5 min using a 2 mL centrifuge tube, the centrifuge tube was taken out, the supernatant was transferred out, and the available phosphorus content was measured by the molybdenum blue colorimetric method. The phosphorus solubilizing ability of the strain was determined by the available phosphorus content in the supernatant. The results are shown in Table 2.

[0056] (6) Screening of bacteria with better IAA production ability for potassium solubilizing ability determination.

[0057] A. Inoculate the seed solution of the isolated strain into a 250 mL Erlenmeyer flask containing 50 mL of liquid potassium-solubilizing bacteria medium at an inoculation amount of 1%, place it in a shaker, set the shaker temperature to 30 °C, the rotation speed to 180 rpm, and culture for 3 days. B. After the culture is completed, centrifuge the culture solution in a 10 mL centrifuge tube at 10,000 rpm for 10 minutes, take out the centrifuge tube, transfer the supernatant, and measure the potassium content with a flame spectrophotometer. The potassium content in the supernatant is used to measure the potassium-solubilizing ability of the strain. The results are shown in Table 2.

[0058] Through the above measurements, strains with high indole acetic acid production, strong phosphorus-solubilizing and potassium-solubilizing abilities can be screened out, named C7, and the colony diagram is as Figure 1 shown.

[0059] Table 2 Growth-promoting ability of strain C7

[0060] Test strains IAA production capacity (mg / L) Inorganic phosphorus solubilization capacity (mg / L) Potassium solubilization capacity (mg / L) C7 58.56 428.85 198.15

[0061] According to Table 2, the isolated strain has the ability to secrete IAA, reaching 58.56 mg / L; and strain C7 has the ability to dissolve inorganic phosphorus, reaching 428.85 mg / L; and has the ability to dissolve potassium, reaching 198.15 mg / L.

[0062] (7) Aerobicity test

[0063] Pour the sterilized LB medium into 3 sterilized test tubes, about 2 / 3 full. On the sterile operating table, use an inoculation needle to pick the strain C7 cultured on the slant and stab-inoculate it into the above medium (it must be stabbed to the bottom of the tube). Culture at 30 °C and observe the results at 3 - 7 days respectively.

[0064] Those growing on the surface of the agar column are aerobic bacteria, and those growing along the stab line are anaerobic or facultative anaerobic bacteria. The test results show that the colonies of strain C7 grow along the surface of the agar column and there are colonies growing inside the stab line, indicating it is facultative anaerobic.

[0065] (8) Determination of catalase

[0066] Drop 1 drop of 3% H 2 O 2 on a clean glass slide. Use an inoculation loop to pick 1 loop of the strain C7 culture grown on the LB slant for 18 - 24 hours and smear it in the H 2 O 2 . If bubbles are generated, it is positive, otherwise it is negative. The test results are shown in Table 3, and the results show that strain C7 is catalase negative.

[0067] (9) Methyl red test (M.R. test)

[0068] A. Preparation of culture medium: 5 g of peptone, 5 g of glucose, 5 g of sodium chloride and 1000 mL of distilled water. Adjust the pH to 7.0 - 7.2, dispense into test tubes, 4 - 5 mL per tube, and sterilize at 121 °C for 20 min. Required reagents: 0.1 g of methyl red, 300 mL of 95% ethanol, and 200 mL of distilled water.

[0069] B. Bacterial strain culture and result observation: Inoculate strain C7 into the above culture medium and culture at 30 °C for 1 - 2 d. Add a few drops of methyl red reagent to the culture medium. If the culture medium turns red, it is methyl red positive; if it turns yellow, it is negative (methyl red color change range: 4.4 red - 6.0 yellow). The test results are shown in Table 3, indicating that strain C7 is methyl red positive.

[0070] (10) Acetylmethylcarbinol test (VP test)

[0071] A. The culture medium is prepared in the same way as in step (9) methyl red test.

[0072] B. Bacterial strain culture and result observation: Inoculation and culture are the same as in step (9) methyl red test. When performing the VP test, take the culture medium (about 2 mL) and mix it with an equal volume of 40% NaOH, add a small amount of creatine, and shake well for 2 - 5 min. If the culture medium turns red, it is VP positive. The test results are shown in Table 3, indicating that strain C7 is VP positive.

[0073] (11) Starch hydrolysis test

[0074] A. ① Preparation of culture medium: Add 0.2% soluble starch to nutrient broth agar, dispense into Erlenmeyer flasks, and sterilize at 121 °C for 20 min for standby. ② Lugol's iodine solution: 1 g of iodine tablets, 2 g of potassium iodide. First, dissolve potassium iodide with a small amount (3 - 5 mL) of distilled water, then add iodine tablets. After the iodine is completely dissolved, dilute with water to 300 mL.

[0075] B. Bacterial strain culture and result observation: Inoculate strain C7 onto the plate and culture at 30 °C for 2 - 4 d. After colonies are formed, add Lugol's iodine solution dropwise onto the plate until it covers the area around the colonies. If the plate turns blue and there is a colorless transparent circle around the colonies, it indicates that the starch has been hydrolyzed. The size of the transparent circle generally indicates the ability to hydrolyze starch. The test results are shown in Table 3, indicating that strain C7 is positive for starch hydrolysis.

[0076] (12) Gelatin hydrolysis test

[0077] A. Preparation of culture medium: 5 g of peptone, 120 g of gelatin, and 1000 mL of distilled water. Adjust the pH to 7.2 - 7.4, dispense into test tubes, and the height of the culture medium is about 4 - 5 cm. Sterilize at 121 °C for 20 min.

[0078] B. Bacterial strain culture and result observation: Inoculate strain C7 in the center of the test tube by the stab inoculation method. Culture it in an incubator at 30°C for one month and observe whether the gelatin is liquefied. The test results are shown in Table 3, and the results show that strain C7 is positive for gelatin hydrolysis.

[0079] (13) Nitrate reduction test

[0080] A. ① Preparation of nitrate liquid medium: 10 g of peptone, 1 g of potassium nitrate, and 1000 mL of distilled water, pH 7.0 - 7.4. ② Griess reagent: Solution A: 0.5 g of sulfanilic acid, 150 mL of dilute acetic acid (about 10%); Solution B: 0.1 g of naphthylamine, 20 mL of distilled water, 150 mL of dilute acetic acid (about 10%). ③ Diphenylamine reagent: 0.5 g of diphenylamine is dissolved in 100 mL of concentrated sulfuric acid and diluted with 20 mL of distilled water.

[0081] B. Bacterial strain culture and result observation: Inoculate strain C7 into the nitrate liquid medium and culture it at 30°C for 1, 3, and 5 days. Pour a little culture solution into the small holes of a white porcelain plate, and then drop 1 drop of reagent A and solution B into it respectively. When the culture solution turns pink, rose red, orange, or brown, etc., it indicates the presence of nitrite and is positive for nitrate reduction, otherwise it is negative. The test results are shown in Table 3, and the results show that strain C7 is positive for nitrate reduction.

[0082] (14) Utilization of citrate

[0083] A. Medium preparation: 2 g of sodium citrate, 5 g of sodium chloride, 0.2 g of magnesium sulfate heptahydrate, 1 g of diammonium hydrogen phosphate, 10 mL of 1% bromothymol blue aqueous solution, 20 g of agar, and 1000 mL of distilled water, pH 6.8 - 7.0, sterilize at 121°C for 20 min.

[0084] B. Bacterial strain culture and result observation: Inoculate fresh strain C7 on the slant and culture it at 30°C for 3 - 7 days. If the medium turns alkaline (blue), it is a positive reaction, and if it remains unchanged, it is negative. The test results are shown in Table 3, and the results show that strain C7 is positive.

[0085] Table 3 Physiological and biochemical characteristics of strain C7

[0086] Item Result Item Result Gram staining + Starch hydrolysis + Aerobic test Facultative anaerobic Gelatin liquefaction + Catalase test - Nitrate reduction + Methyl red (M.R) reaction + Citrate utilization + V-P test + / /

[0087] Note: +: Positive reaction; -: Negative reaction

[0088] The strain C7 isolated by the above method was sequenced by Shanghai Yingjun Biotechnology Co., Ltd. According to the sequencing results of 16S rDNA, it was queried and analyzed online at http: / / www.ncbi.nlm.nih.gov. The Blast software was used to compare the homology with other 16S rDNA sequences in GenBank. The sequences similar to that of strain C7 were selected to construct the 16S rDNA phylogenetic tree of strain C7 using the MEGAversion3 software, as shown in Figure 2 shown. The sequence information of the 16S rDNA of strain C7 is shown in SEQ ID NO.1. According to the physiological and biochemical characteristics of this strain, it was identified as Bacillus acidiceler. This strain was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 10, 2024, with the deposit number CGMCC No.31934.

[0089] The cell morphology of strain C7 is as follows: After culturing for 24 h, colonies of 1 - 2 mm can be formed. The colony morphology is that the colonies are grayish translucent, round, with a smooth surface, regular edges, no halo, and are flat. The physiological and biochemical characteristics of strain C7 are: Gram - positive, facultative anaerobic, negative for catalase, positive for M.R. test, positive for V.P. test, positive for starch hydrolysis, positive for gelatin liquefaction, positive for nitrate reduction, and positive for citrate utilization.

[0090] Example 2

[0091] (1) In order to further verify the indole - 3 - acetic acid (IAA) production ability and the optimal conditions of the plant - growth - promoting rhizobacterium C7 obtained in Example 1, the effects of different pH values, liquid volumes, different carbon sources, and different nitrogen sources on the IAA production were explored below.

[0092] Four factors, namely carbon source, nitrogen source, pH, and liquid volume, were selected for an orthogonal experiment. The level settings of each factor are shown in Table 4. The inorganic salt medium containing 100 mg / L of L - tryptophan was adjusted respectively. Among them, the carbon and nitrogen sources were added in amounts of 1% and 0.1% (M / V) respectively. The selected strain was inoculated into a 250 - mL Erlenmeyer flask and cultured at 30 °C and 180 rpm for 1 d. The Salkowski colorimetric method was used to measure the amount of IAA produced, and the results are shown in Table 5.

[0093] Table 4 Orthogonal table for optimizing the plant - growth - promoting function of strain C7

[0094] Different conditions Carbon source Nitrogen source pH Liquid loading volume (mL / 250 mL) 1 Maltose Urea 6 30 2 Maltose Yeast powder 7 50 3 Maltose Peptone 8 70 4 Glucose Urea 7 70 5 Glucose Yeast powder 8 30 6 Glucose Peptone 6 50 7 Sucrose Urea 8 50 8 Sucrose Yeast powder 6 70 9 Sucrose Peptone 7 30

[0095] Table 5 Results of the orthogonal experiment on the IAA production ability of strain C7

[0096]

[0097] Note: K1, K2, and K3 represent the average values of each factor, and R represents the maximum range among K1, K2, and K3.

[0098] As can be seen from Table 5, the order of the influence of each factor on the IAA-producing ability of strain C7 is carbon source > pH > nitrogen source > liquid loading amount. Through comprehensive comparison of the means of the four factors and intuitive analysis, the optimal condition combination for strain C7 to produce IAA is sucrose as the carbon source, peptone as the nitrogen source, pH of 6, and liquid loading amount of 50 mL / 250 mL.

[0099] (2) In order to further verify the inorganic phosphorus-solubilizing ability and the optimal conditions of the plant growth-promoting rhizobacterium C7 obtained in Example 1, the effects of different pH values, liquid loading amounts, different carbon sources, and different nitrogen sources on the inorganic phosphorus-solubilizing ability were explored below.

[0100] Four factors, namely carbon source, nitrogen source, pH, and liquid loading amount, were selected for an orthogonal experiment. The level settings of each factor are shown in Table 4, and the inorganic phosphorus bacteria medium was adjusted respectively. Among them, the carbon and nitrogen sources were added in amounts of 1% and 0.1% (M / V) respectively. The screened strain was inoculated into a 250 mL Erlenmeyer flask and cultured at 30 °C and 180 rpm for 3 days. The phosphorus content in the supernatant was measured by the molybdenum blue colorimetric method, and the results are shown in Table 6.

[0101] Table 6 Results of the orthogonal experiment on the inorganic phosphorus-solubilizing ability of strain C7

[0102]

[0103] Note: K1, K2, and K3 represent the average values of each factor, and R represents the maximum range among K1, K2, and K3.

[0104] As can be seen from Table 6, the order of the influence of each factor on the inorganic phosphorus-solubilizing ability of strain C7 is nitrogen source > liquid loading amount > carbon source > pH. Through comprehensive comparison of the means of the four factors and intuitive analysis, the optimal condition combination for the inorganic phosphorus-solubilizing ability of strain C7 is maltose as the carbon source, yeast powder as the nitrogen source, pH of 7, and liquid loading amount of 30 mL / 250 mL.

[0105] (3) In order to further verify the optimal conditions for the potassium-solubilizing ability of the plant growth-promoting rhizobacterium C7 obtained in Example 1, the effects of different pH values, liquid loading amounts, different carbon sources, and different nitrogen sources on the potassium-solubilizing ability were explored below.

[0106] Four factors, namely carbon source, nitrogen source, pH, and liquid loading amount, were selected for an orthogonal experiment. The level settings of each factor are shown in Table 4, and the liquid potassium-solubilizing bacteria medium was adjusted respectively. Among them, the carbon and nitrogen sources were added in amounts of 1% and 0.1% (M / V) respectively. The screened strain was inoculated into a 250 mL Erlenmeyer flask and continuously cultured at 30 °C and 180 rpm for 3 days. The supernatant was centrifuged and the available potassium content was measured by a flame spectrophotometer, and the results are shown in Table 7.

[0107] Orthogonal test results of potassium-solubilizing ability of strain C7 in Table 7

[0108]

[0109] Note: K1, K2, and K3 represent the average values of each factor, and R represents the maximum range among K1, K2, and K3.

[0110] It can be seen from Table 7 that the order of the influence of each factor on the potassium-solubilizing ability of strain C7 is nitrogen source > pH > carbon source > liquid loading. Through comprehensive comparison of the means of the four factors and intuitive analysis, the optimal condition combination for the potassium-solubilizing ability of C7 is sucrose as the carbon source, yeast powder as the nitrogen source, pH of 7, and liquid loading of 50 mL / 250 mL.

[0111] Example 3

[0112] (1) Preparation of bacterial suspension: Inoculate C7 isolated in Example 1 into LB liquid medium, culture at 30 °C and 180 rpm on a shaker until the bacteria grow to the logarithmic growth phase, then centrifuge the bacterial solution at 3000 rpm for 10 min, and resuspend it with sterile water. Centrifuge three times in the same way to prepare the bacterial suspension.

[0113] (2) Preparation of test soil: Collect fresh soil from the 0-20 cm soil layer of tobacco-growing soil under natural conditions in Luoshan County, Xinyang City, Henan Province, sieve it through a 5 mm sieve, fill 700 g of soil into each pot, adjust the water content to 60% of the maximum field water holding capacity, and randomly divide it into an experimental group (C7) and a control group (CK).

[0114] (3) Treatment of seeds: Disinfect tobacco seeds with 20% hydrogen peroxide on the surface for 20 min, rinse with sterile water multiple times, and germinate for 2 d; select tobacco seedlings with consistent germination and inoculate them into the soil of the experimental group and the control group in step (2) respectively.

[0115] (4) Experimental group: According to the inoculation amount of 10 8 CFU / g of soil, 24 h before transplanting the seedlings, inoculate it into the soil (that is, inoculate 10 8 CFU / g of Acid-fast growing Bacillus C7 per gram of dry soil).

[0116] Control group: As a control, the soil is not sprayed with C7 bacterial solution, and an equal amount of sterile water is added.

[0117] There are 4 replicates for each treatment. Place the potted plants in a light incubator, sample after 30 days, use HPLC method to measure the IAA content in the soil, and measure the soil pH, organic matter, alkaline hydrolyzable nitrogen, available phosphorus, available potassium contents, fresh weight of plants, plant height, and total nitrogen, total phosphorus, and total potassium contents. The results are shown in Table 8 and Table 9.

[0118] Table 8 Effects of Strain C7 on the Physical and Chemical Properties of Tobacco Potting Soil

[0119]

[0120] Note: "*" indicates significant differences between treatments (p < 0.05), and "**" indicates extremely significant differences between treatments (p < 0.01).

[0121] Table 9 Effects of Strain C7 on the Biological Traits of Tobacco

[0122]

[0123] Note: "*" indicates significant differences between treatments (p < 0.05), and "**" indicates extremely significant differences between treatments (p < 0.01).

[0124] According to Table 8, after inoculating with Acidiphilium sp. C7, compared with the CK treatment, the contents of soil IAA, available phosphorus, and available potassium increased significantly by 77.78%, 25.37%, and 10.01% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of tobacco. According to Table 9, after inoculating with Acidiphilium sp., compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of tobacco seedlings increased significantly by 15.16%, 11.17%, 29.36%, 7.66%, 14.54%, and 27.46% respectively.

[0125] Based on the above results, it can be seen that the multifunctional plant growth-promoting rhizobacterium Acidiphilium sp. C7 of the present invention can effectively increase the soil nutrient content, has an obvious effect on promoting the growth and development of tobacco roots, and can effectively promote the growth of tobacco.

[0126] Example 4

[0127] (1) Preparation of bacterial suspension: Operate according to step (1) in Example 3 to prepare the Acidiphilium sp. C7 bacterial suspension.

[0128] (2) Preparation of test soil: Operate according to step (2) in Example 3 to collect soil and randomly divide it into an experimental group (C7) and a control group (CK).

[0129] (3) Treatment of seeds: Surface sterilize the pakchoi seeds with 20% hydrogen peroxide for 20 min, rinse them with sterile water multiple times, and germinate them for 2 d; select pakchoi seedlings with consistent germination and inoculate them into the soils of the experimental group and the control group in step (2) respectively.

[0130] (4) Experimental group: The treatments of the experimental group and the control group are carried out according to step (4) in Example 3.

[0131] Each treatment had 4 replicates. The potted plants were placed in a light incubator and sampled after 30 days. The content of soil IAA was determined by HPLC method, and the available phosphorus, available potassium contents in the soil, fresh weight of the plants, plant height, and total nitrogen, total phosphorus, and total potassium contents were also measured. The results are shown in Tables 10 and 11.

[0132] Table 10 Effects of Strain C7 on Physicochemical Properties of Pakchoi Potted Soil

[0133]

[0134] Note: "*" indicates significant difference between treatments (p < 0.05), and "**" indicates extremely significant difference between treatments (p < 0.01).

[0135] Table 11 Effects of Strain C7 on Biological Traits of Pakchoi

[0136]

[0137] Note: "*" indicates significant difference between treatments (p < 0.05), and "**" indicates extremely significant difference between treatments (p < 0.01).

[0138] According to Table 10, after inoculating with Bacillus acidotrophicus C7, compared with the CK treatment, the contents of soil IAA, available phosphorus, and available potassium increased significantly by 77.14%, 22.85%, and 14.39% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of pakchoi. According to Table 11, after inoculating with Bacillus acidotrophicus C7, compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of pakchoi seedlings increased significantly by 13.11%, 23.20%, 60.00%, 8.94%, 17.39%, and 28.00% respectively.

[0139] Based on the above results, it can be seen that the multifunctional plant growth-promoting rhizobacterium Bacillus acidotrophicus C7 of the present invention can effectively increase the soil nutrient content, has an obvious effect on promoting the growth and development of pakchoi roots, and can effectively promote the growth of pakchoi.

[0140] Example 5

[0141] (1) Preparation of bacterial suspension: Operate according to step (1) in Example 3 to prepare the bacterial suspension of Bacillus acidotrophicus C7.

[0142] (2) Preparation of test soil: Operate according to step (2) in Example 3 to collect the soil and randomly divide it into an experimental group (C7) and a control group (CK).

[0143] (3) Treat the seeds: Disinfect the wheat seeds on the surface with 20% hydrogen peroxide for 20 min, rinse them with sterile water multiple times, and germinate them for 2 days; Select wheat seedlings with consistent germination and inoculate them into the soil of the experimental group and the control group in step (2) respectively.

[0144] (4) The treatments of the experimental group and the control group were carried out according to the operation in step (4) of Example 3.

[0145] There were 4 replicates for each treatment. The potted plants were placed in a light incubator. After 30 days, samples were taken. The content of soil IAA was determined by HPLC method, and the content of available phosphorus, available potassium in the soil, fresh weight of plants, plant height, and the content of total nitrogen, total phosphorus and total potassium were measured. The results are shown in Tables 12 and 13.

[0146] Table 12 Effects of strain C7 on the physical and chemical properties of wheat potted soil Note: "*" indicates significant difference between treatments (p < 0.05), and "**" indicates extremely significant difference between treatments (p < 0.01).

[0147] Table 13 Effects of strain C7 on the biological traits of wheat Note: "*" indicates significant difference between treatments (p < 0.05), and "**" indicates extremely significant difference between treatments (p < 0.01).

[0148] According to Table 12, after inoculating with Bacillus acidotrophicus C7, compared with the CK treatment, the contents of soil IAA, available phosphorus, and available potassium increased significantly by 90.91%, 31.18%, and 14.56% respectively, indicating that this strain can effectively increase the nutrient content in the soil to promote the growth of wheat. According to Table 13, after inoculating with Bacillus acidotrophicus C7, compared with the CK treatment, the plant height, fresh weight, dry weight, total root length, root surface area, and root volume of wheat seedlings increased significantly by 15.16%, 11.17%, 29.36%, 3.63%, 18.13%, and 28.17% respectively.

[0149] Based on the above results, it can be seen that the multifunctional rhizosphere growth-promoting bacterium Bacillus acidotrophicus C7 of the present invention can effectively increase the soil nutrient content, has an obvious effect on promoting the growth and development of wheat roots, and can effectively promote the growth of wheat.

[0150] Example 6

[0151] The Bacillus acidotrophicus C7 isolated in Example 1 was made into a microbial inoculum with bone meal as the carrier. The obtained microbial inoculum contained viable bacteria count of 10 11 CFU / g. Food crops wheat and corn, oil crop peanut, and cash crop tobacco were planted at the appropriate planting time for various crops respectively to conduct a field experiment to verify the efficacy of C7.

[0152] Taking tobacco as an example to illustrate the efficacy of Bacillus fastidiosus C7. Tobacco was planted in the experimental field of Luoshan County, Xinyang City, Henan Province on April 18, 2022. The altitude of the area where the experimental field is located is 55.8m (N32.1924, E114.3305), the annual average temperature is 15.3°C, and the annual average precipitation is 1050mm. The basic physical and chemical properties of the soil in the experimental field are pH 5.34, organic matter 14.9g / kg, total nitrogen 1.12g / kg, available phosphorus 10.22mg / kg, and available potassium 104.37mg / kg.

[0153] All experiments were set with 3 replicates, randomly arranged in blocks, and the plot area was 6×3m 2 , with 80 tobacco plants planted in each plot, row spacing 120cm, and plant spacing 50cm. All treatments were applied with 15-15-15 compound fertilizer as basal fertilizer, with an application rate of 600kg·hm -2 , and at the same time, 40kg·hm of C7 microbial inoculant was applied -2 . The control treatment was applied with an equal amount of bone meal. The yield and corresponding quality indicators were measured at the harvest stage, and the results are shown in Tables 14 and 15.

[0154] Table 14 Effects of strain C7 on the biological traits of tobacco Note: "*" indicates a significant difference between treatments (p < 0.05), and "**" indicates a highly significant difference between treatments (p < 0.01).

[0155] Table 15 Effects of strain C7 on the chemical components of flue-cured tobacco

[0156] Treatment Total sugar (%) Reducing sugar (%) Nicotine (%) Total nitrogen (%) Potassium (%) Chlorine (%) CK 25.50±1.12 19.87±0.84 2.63±0.08 1.95±0.14 1.63±0.09 0.41±0.02 C7 28.59±0.42* 22.54±0.62* 2.53±0.21 2.05±0.07 1.97±0.04** 0.37±0.05

[0157] Note: "*" indicates a significant difference between treatments (p < 0.05), and "**" indicates a highly significant difference between treatments (p < 0.01).

[0158] According to Table 14, compared with the control, the treatment with Bacillus fastidiosus C7 significantly increased the plant height of tobacco by 4.97% and the maximum leaf area by 38.09%, indicating that this strain can increase the yield of tobacco in the field environment. According to Table 15, at the same time, Bacillus fastidiosus C7 can also improve the quality of tobacco. After inoculating this strain, compared with the CK treatment, the total sugar, reducing sugar, and potassium content of flue-cured tobacco leaves increased significantly by 12.12%, 13.44%, and 20.86% respectively.

[0159] From the above examples, it can be seen that the Bacillus fastidiosus provided by the present invention has a strong IAA-producing function, can also produce substances that promote the dissolution of insoluble phosphorus and potassium in the soil, can significantly improve the growth rate of plants, and improve the yield and quality of agricultural products.

[0160] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus acidiceler C7, characterized in that: It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is September 10, 2024, and the deposit number is CGMCC No.31934.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the acid-fast-growing Bacillus C7 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The effective viable bacteria count of acid-fast Bacillus C7 in the bacterial agent is ≥1×10 11 CFU / g.

4. The use of the acid-fast Bacillus C7 according to claim 1 or the bacterial agent according to claim 2 or 3, characterized in that: The application includes at least one of the following 1) to 4): 1) Increase the indoleacetic acid content of plants; 2) Promote plant growth; 3) Improve soil nutrients; 4) Improve plant quality.

5. The use according to claim 4, characterized in that: The promoting plant growth comprises at least one of promoting root growth, increasing plant height and increasing leaf area.

6. The use according to claim 4 or 5, characterized in that: The plant comprises at least one of tobacco, cotton, fruit trees, Chinese cabbage, tomatoes, wheat, millet, corn and sorghum.

7. The use according to claim 4, characterized in that: Improving soil nutrients includes promoting the dissolution of sparingly soluble phosphorus and / or potassium in the soil.

8. The use according to claim 4, characterized in that: The improving of plant quality comprises increasing at least one of the content of total sugar, reducing sugar and potassium in the plant.

9. The use according to claim 4, characterized in that: The application The method comprises the following steps: applying the acid-fast-growing Bacillus C7 according to claim 1 or the bacterial agent according to claim 2 or 3 into soil in which plants are planted.

10. The use according to claim 9, characterized in that: The amount of the microbial agent applied is 35-45 kg / hm 2 .

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