Soil acid-controlling and acid-inhibiting bacteria and application thereof

Abstract

This invention discloses a soil acid-controlling and acid-suppressing bacterium and its application, belonging to the field of microbial screening and isolation technology, including at least one of strains A and B, wherein strain A is... Neobacillus massiliamazoniensis Its accession number is CCTCC No. M 20241830, the accession date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection; the strain B is... Ralstonia pickettii Its accession number is CCTCC No.M 20241831, the deposit date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening and isolation technology, specifically relating to a soil acid-controlling and acid-suppressing bacterium and its application. Background Technology

[0002] Soil acidification has become one of the major challenges facing agriculture in recent years. It severely impacts agricultural production, directly affecting nutrient availability, leading to significant loss or fixation of essential elements like calcium, magnesium, potassium, and phosphorus, resulting in nutrient imbalance and consequently affecting crop growth. Furthermore, soil acidification alters the entire soil microenvironment, causing imbalances in the soil microecological environment and community structure, greatly increasing the risk of soil-borne diseases in crops. In addition, soil acidification affects the activity of heavy metals in the soil, significantly impacting the quality and safety of agricultural products and posing a potential threat to human health.

[0003] Internationally, methods for inhibiting and reducing soil acidification mainly include controlling nitrogen fertilizer application, applying soil conditioners, and optimizing fertilization regimes and farming practices. However, with ongoing research, the drawbacks of traditional chemical soil conditioners have become increasingly apparent. Long-term application may lead to soil surface compaction, damage soil structure, and consequently, nutrient imbalance, weakening the soil's buffering capacity and potentially exacerbating soil acidification. Microorganisms play a crucial role in soil nutrient cycling, influencing soil physicochemical properties and enhancing plant nutrient absorption. Therefore, the use of microorganisms to remediate acidified soils has gained increasing attention. Currently, there are many microbial fertilizers and agents available on the market. Common soil-improving microorganisms include Bacillus and Trichoderma. However, research on the application of microorganisms in improving soil acidification remains insufficient, and their effects on soil acidification or acid reduction are still uncertain. The specific effects are also influenced by soil and environmental factors. Therefore, in practical applications, it is necessary to comprehensively consider the influence of different factors, screen suitable microorganisms, provide more microbial resources for soil acidification improvement, and conduct scientific and rational microbial regulation tailored to specific soil environmental conditions. Summary of the Invention

[0004] The purpose of this invention is to provide a new type of soil acid-controlling and acid-suppressing bacteria, which increases the variety of soil acid-controlling and acid-suppressing bacteria species, provides more choices for soil acid-controlling and acid-suppressing microbial agents, and reduces the cost of microbial soil acidification remediation.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A soil acid-controlling and acid-suppressing bacterium, comprising at least one of strains A and B, wherein strain A is... Neobacillus massiliamazoniensisIts accession number is CCTCC No. M 20241830, the accession date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection; the strain B is... Ralstonia pickettii Its accession number is CCTCC No. M 20241831, the deposit date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection.

[0007] The application of the aforementioned soil acid-controlling and acid-suppressing bacteria as acid-controlling and acid-suppressing agents in acidic soil amendments.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The soil acid-controlling and acid-suppressing bacteria provided by this invention include at least one of strains A and B, with strain A alone ( Neobacillus massiliamazoniensis B () Ralstonia pickettii The present invention and its combination have the effect of improving the acidity of acidic soils, and can significantly increase the pH value of acidic soil suspensions, providing more options for soil acid control and inhibition microbial agents; and according to the research on the growth of Chinese cabbage in acidic soil, it can be found that the soil acid control and inhibition bacteria provided by the present invention can be effectively applied in the field and have a significant positive promoting effect on the growth of Chinese cabbage. Attached Figure Description

[0010] Figure 1 Gram staining microscopic images of strains A and B (a: strain A; b: strain B).

[0011] Figure 2 The effects of different inoculation amounts of single and mixed strains on pH enhancement in acidic soils;

[0012] Figure 3 The effects of adding strains on the biomass and soil pH of Chinese cabbage grown in two acidic soils. Detailed Implementation

[0013] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0014] Unless otherwise specified, all reagents and materials used in the following examples are commercially available. Example

[0015] The strains A and B provided in this embodiment were isolated from the purple soil of the Jiaguan Formation in Guanyin Town, Xuzhou District, Yibin City, Sichuan Province. The isolation process was as follows:

[0016] (1) Take 10 g of soil sample and place it in an Erlenmeyer flask containing 90 mL of sterile water. Incubate at 170 rpm and 30℃ for 3 h.

[0017] (2) Remove and let stand until separation occurs. Take 0.1 mL of the supernatant and mix it with 0.9 mL of sterile water. This is 10 -1 Dilute the liquid, and repeat the steps to obtain 10. -2 10 -3 10 -4 10 -5 Diluent.

[0018] (3) Take 0.1 mL of each of the 10 mL aliquots. -3 ~10 -5 The diluted solution was added dropwise to GB agar plates at pH 4.5, and spread evenly on the plates using a glass triangular rod. The plates were then incubated upside down in a 30°C incubator for 3–4 days until colonies formed. Six parallel experiments were performed for each concentration, with an uninoculated blank medium as a control.

[0019] (4) Pick up bacterial cells with an inoculation loop and streak them onto GB agar plates, covering the entire medium as much as possible. The entire process should be performed aseptically in a laminar flow hood. After streaking, invert the plates and incubate them in a 30°C incubator for 3–4 days. Once colonies have formed, observe their morphology and purify them further using the method described above until the colony morphology in the solid medium is consistent and free of contaminants. This completes the purification process.

[0020] (5) Inoculate a single colony into GB medium at pH 4.5 and incubate overnight at 30 °C and 170 r / min with shaking. Then, inoculate the culture medium at a rate of 1% (v / v) into GB medium at pH 4.5 containing 1% (v / v) methyl red indicator. After culturing for 48 h, observe the color change of the culture medium and determine the pH. The strain that can grow normally in an environment of pH 4.5 and can increase the environmental pH is the target strain.

[0021] Further identification of strains A and B obtained from the above isolation steps:

[0022] I. Morphological observation of the strain

[0023] The obtained strains A and B were inoculated into GB medium at pH 4.5 and streaked for 3–4 days. The growth of colonies on each medium was then observed, and the results are shown in Table 1.

[0024] Table 1. Colony morphology observation and molecular biological identification of each strain

[0025]

[0026] The isolated and screened strains were inoculated onto GB medium at pH 4.5 and incubated at 30 ℃ for 3–4 days. After obvious colony growth on the medium, single colonies were picked and purified using the streak plate method to obtain pure bacteria. The colony morphology is as follows: Figure 1 As shown.

[0027] II. Molecular Identification of the Strains

[0028] BLAST alignment analysis was performed on the 16S rRNA gene fragments of strains A and B. Strains A and B... Neobacillus massiliamazoniensis The highest similarity was observed, with a homology of 98.17%; strain B and... Ralstonia pickettii The similarity is the highest, with a homology of 99.66%, as shown in Table 1.

[0029] III. Determination of the physicochemical properties of the strain

[0030] Strains A and B were inoculated into GB medium at pH 4.5 and cultured overnight with shaking for 24 hours. The resulting culture solution was then inoculated into GB medium at pH 4.5 at an inoculum rate of 1% (volume fraction). The pH of the culture solution was measured after 48 hours, and the results are shown in Table 2.

[0031] Table 2. Strains culture and pH

[0032]

[0033] When OD 600 When the coefficient of performance is 0.8, the number of colonies of strain A is 7.7 × 10⁸. 8 The colony count of strain B was 1.52 × 10⁻⁶. 8 .

[0034] Physiological and biochemical assays were performed on the strains using Gram staining, oxidase, catalase, glucose oxidative fermentation, starch hydrolysis, methyl red, acetylmethylethanol, indole, and gelatin liquefaction tests. The results showed that strain A, after staining, appeared purple, indicating it was a Gram-positive bacterium. It was positive for oxidase, catalase, and gelatin liquefaction tests, but negative for MR, indole, VP, and starch hydrolysis tests, making it a glucose-oxidizing strain. Strain B, also appearing purple, was a Gram-positive bacterium. It was positive for catalase and gelatin liquefaction tests, but negative for oxidase, MR, indole, VP, and starch hydrolysis tests, making it a glucose-fermenting strain.

[0035] IV. Microbial Preservation

[0036] Strains A and B were deposited at the China Center for Type Culture Collection (CCTCC) on August 21, 2024, with accession numbers CCTCC No. M 20241830 and CCTCC No. M 20241831, respectively.

[0037] V. Experiment on the pH-raising effect of bacterial strains on acidic soil

[0038] 1. Experimental subjects

[0039] This experiment used acidic soil suspension as the experimental subject. The acidic soil was taken from Guanyin Town, Xuzhou District, Yibin City, Sichuan Province. The preparation process of the acidic soil suspension was as follows: the obtained acidic soil was dispersed in deionized water to form an acidic soil suspension.

[0040] 2. Experiment on the pH-raising effect of a single strain on acidic soil

[0041] The 24-hour culture broths of strains A and B were inoculated into acidic soil suspension at inoculum concentrations of 1%, 2%, and 3% (v / v), respectively. The pH values ​​of the acidic soil suspensions were measured at 48, 72, and 96 hours. The results are as follows: Figure 2 As shown in the figure, strains A and B both have a certain effect on increasing the pH of acidic soil, and the effect on increasing the pH of acidic soil increases with the increase of inoculation concentration. At a dosage of 3%, strains A and B increased the pH of acidic soil by 0.89 and 1.00 units, respectively, after 72 hours.

[0042] 3. Experiment on the pH-raising effect of mixed bacterial strains on acidic soil

[0043] The 24-hour culture broths of strains A and B were inoculated into acidic soil suspension at inoculum concentrations of 0.5% ± 0.5%, 1% ± 1%, and 1.5% ± 1.5% (volume fraction), respectively. The pH values ​​of the acidic soil suspensions were measured at 48 h, 72 h, and 96 h. The results are as follows: Figure 2 As shown, the mixed bacterial solution has a significant effect on increasing the pH of acidic soil. With an addition of 1.5% + 1.5%, the mixed bacterial solution increased the pH of acidic soil by 0.92 units after 72 hours.

[0044] VI. Effects of bacterial strain addition on Chinese cabbage biomass and soil pH in acidic soil

[0045] 1. Experimental subjects

[0046] The soils used in this experiment were purple soil from the Jiaguan Formation in Yibin City, Sichuan Province (pH=3.99) and purple soil from the Shaximiao Formation in Guang'an City, Sichuan Province (pH=6.04).

[0047] 2. Effects of bacterial strains on the biomass of Chinese cabbage grown in acidic soil

[0048] In this experiment, 1-gallon flowerpots were used, each containing 1.3 kg of soil. Compound fertilizer (N:P₂O₅:K₂O = 15:15:15, application rate: 60 kg / mu) was added to the soil as base fertilizer and mixed thoroughly. Microbial fertilizer (6% by volume) was applied as a single base fertilizer application. After harvesting, the entire plant was dug up, and biomass was measured. The results are as follows: Figure 3 As shown, the strains significantly increased the biomass of Chinese cabbage. Compared with the control group (CK), the addition of strains A, B, and the mixed bacterial solution increased the soil biomass of Chinese cabbage in the Jiaguan group by 5.34, 11.23, and 1.86 g / m³, respectively. 2 The concentrations in the Shaximiao Formation increased by 61.00, 60.78, and 86.67 g / m³, respectively. 2 .

[0049] 3. Effects of bacterial strains on soil pH

[0050] pH was measured using rhizosphere soil samples from potted Chinese cabbage plants, and the results are as follows: Figure 3 As shown, the strains significantly increased the pH of the soil in potted Chinese cabbage. Compared with the control group (CK), the soil pH of the Jiaguan group increased by 0.25, 0.23, and 0.27 units, respectively, after the addition of strains A, B, and the mixed bacterial solution, while the pH of the Shaximiao group increased by 0.58, 0.37, and 0.26 units, respectively.

[0051] The above embodiments are merely one of the preferred embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but solve the same technical problem as the present invention, should be included within the scope of protection of the present invention.

Claims

1. A soil acid-controlling and acid-suppressing bacterium, characterized in that, Includes at least one of strains A and B, where strain A is... Neobacillus massiliamazoniensis Its accession number is CCTCC No: M 20241830, the accession date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection; strain B is *Rowstoneella pylori*. (Ralstonia) pickettii) Its accession number is CCTCC No: M 20241831, the deposit date is August 21, 2024, and the depositary institution is the China Center for Type Culture Collection.

2. The application of the soil acid-controlling and acid-suppressing bacteria as described in claim 1 in the preparation of acidic soil amendments.

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