Paenibacillus sp. 52-6 with phosphorus solubilizing function and application thereof

Abstract

This invention discloses a *Bacillus subtilis* strain 52-6 with phosphate-solubilizing function and its applications, belonging to the field of microbial technology. This invention provides a *Bacillus subtilis* strain, classified and named *Bacillus subtilis*. Mesobacillus subterraneus Its accession number is CGMCC No. 32719. The colonies of the *Bacillus subtilis* 52-6 are round, light coral red, translucent, with a smooth surface and neat edges. The optimal growth temperature is 35-38℃. It has a short lag phase, grows rapidly, and is easy to cultivate. *Bacillus subtilis* 52-6 can efficiently dissolve insoluble phosphates in soil, promoting the absorption of phosphorus by plants, thus promoting plant growth, effectively improving soil fertility, and reducing the use of chemical fertilizers. *Bacillus subtilis* 52-6 has great potential in promoting plant growth and improving soil fertility.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a subterranean genus *Bacillus subtilis* 52-6 with phosphate-solubilizing function and its applications. Background Technology

[0002] Phosphorus is an essential nutrient element for plant growth and development. It can promote root growth, increase yield, and improve crop resistance to stress. Soil contains organic phosphorus, such as nucleic acids, phytic acid, and lecithin, which cannot be directly utilized by plants. Inorganic phosphorus-containing substances in the soil, such as magnesium phosphate, calcium phosphate, and apatite, are mostly in the form of insoluble phosphates, which are difficult for plants to absorb directly.

[0003] In traditional agricultural production, the phosphorus requirement for plant life activities is mainly met by applying phosphate fertilizer to the soil. However, the utilization rate of phosphate fertilizer in the current season is generally only 5%-20%, and even with the aftereffects of crops, it generally does not exceed 25%. Most of the phosphorus and potassium applied to the soil are lost with rainwater and surface water, resulting in resource waste and environmental pollution. Long-term and excessive application leads to soil compaction and decreased fertility. At the same time, some heavy metal elements carried by agricultural phosphate fertilizer also enter the soil, causing soil heavy metal pollution. Therefore, improving the utilization rate of phosphate fertilizer in the soil plays an important role in promoting agricultural development.

[0004] Besides methods such as altering soil structure and adjusting fertilization ratios, increasing the content of phosphate-solubilizing microorganisms in the soil is one of the most important and environmentally friendly ways to improve phosphate fertilizer utilization. Phosphate-solubilizing microorganisms can not only convert insoluble phosphorus in the soil into available phosphorus that is easily absorbed and utilized by plants, thus activating soil nutrients and improving phosphate fertilizer utilization, but also increase the absorption of other micronutrients (such as zinc and copper) by plant roots, enhance plant resistance to diseases, and promote plant growth. Therefore, the discovery of phosphate-solubilizing microorganisms and related preparations is of great significance for achieving fertilizer reduction and efficiency improvement in agriculture and promoting green and sustainable agricultural development. Summary of the Invention

[0005] The present invention provides a subterranean *Bacillus subtilis* 52-6 with phosphorus-solubilizing function and its application. The subterranean *Bacillus subtilis* can effectively dissolve inorganic insoluble phosphorus, increase the content of available phosphorus in the soil, and thus promote plant growth.

[0006] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a *Bacillus subtilis* strain 52-6 with phosphorus-solubilizing function, which is classified and named *Bacillus subtilis*. Mesobacillus subterraneus It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32719.

[0008] Furthermore, after being cultured on LB plates at 37°C for 2 days, the colonies of the aforementioned underground Bacillus subtilis 52-6 were round, light coral red, translucent, with a smooth surface and neat edges.

[0009] Furthermore, the 16S rDNA sequence of the underground genus *Bacillus subtilis* 52-6 is shown in SEQ ID No. 1.

[0010] Furthermore, the optimal growth temperature for the underground genus *Bacillus subtilis* 52-6 is 35-38℃.

[0011] Furthermore, the growth curve of the underground genus *Bacillus subtilis* 52-6 is S-shaped, with a lag phase within 6 hours, a logarithmic growth phase after 6 hours, and a plateau phase after 16 hours. It grows rapidly and is easy to culture.

[0012] Furthermore, the aforementioned underground genus *Bacillus subtilis* 52-6 has the function of dissolving insoluble inorganic phosphates.

[0013] Furthermore, the phosphorus solubility of the aforementioned *Bacillus subtilis* 52-6 was 22-30 mg / L after 2 days of culture in inorganic phosphorus medium, 50-60 mg / L after 5 days, and 70-80 mg / L after 8 days.

[0014] Furthermore, the phosphorus solubility of *Bacillus subtilis* 52-6 was 25.69 mg / L after 2 days of culture, 52.65 mg / L after 5 days, and 72.06 mg / L after 8 days.

[0015] The present invention also provides the application of Bacillus subtilis 52-6 in the preparation of biological agents that promote plant growth.

[0016] Furthermore, the underground Bacillus subtilis 52-6 can increase the fresh and / or dry weight of plants above and / or below ground.

[0017] Furthermore, the aforementioned underground Bacillus subtilis 52-6 can improve the stem diameter and plant height growth characteristics of plants.

[0018] Furthermore, the plant in question is alfalfa.

[0019] This invention also provides the application of underground Bacillus subtilis 52-6 in improving soil fertility.

[0020] Furthermore, the aforementioned underground Bacillus subtilis 52-6 has the function of dissolving insoluble phosphates in soil.

[0021] Compared with the prior art, the advantages and beneficial technical effects of the present invention are:

[0022] 1. This invention provides a subterranean Bacillus 52-6 with phosphorus solubilization function. The colonies of strain 52-6 are round, light coral red, translucent, with a smooth surface and neat edges. The suitable growth temperature is 35-38℃. It has a short lag period, grows rapidly, and is easy to cultivate.

[0023] 2. The *Bacillus subtilis* 52-6 described in this invention has the function of dissolving insoluble inorganic phosphates. After 2 days of culture, the phosphorus solubility is 25.69 mg / L, after 5 days it is 52.65 mg / L, and after 8 days it is 72.06 mg / L. It can be used to improve soil fertility and provides a theoretical basis for subsequent related research, which has reference value.

[0024] 3. The underground Bacillus 52-6 described in this invention has the effect of promoting plant growth, significantly increasing the dry and wet weight of plants above and below ground, and also significantly increasing the stem diameter and plant height, especially the plant height, which is nearly 3 times higher than the control group, further demonstrating that underground Bacillus 52-6 is of great significance in promoting plant growth. Attached Figure Description

[0025] Figure 1 The colony characteristics of Bacillus subtilis 52-6 on LB solid medium.

[0026] Figure 2 Phylogenetic tree of the underground genus *Bacillus* 52-6.

[0027] Figure 3 The growth curve of Bacillus subtilis 52-6 is shown.

[0028] Figure 4 The results are from a pot experiment on alfalfa with Bacillus subtilis 52-6. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the specific accompanying drawings and embodiments. The description is for explanation and not limitation of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores. The quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0030] The components and culture conditions of the culture medium involved in this invention are as follows:

[0031] 1. Inorganic phosphorus medium: 10 g glucose, 0.5 g ammonium sulfate, 0.3 g sodium chloride, 0.3 g magnesium sulfate heptahydrate, 0.03 g manganese sulfate, 0.3 g potassium chloride, 0.03 g ferrous sulfate heptahydrate, 5 g tricalcium phosphate, 0.5 g yeast extract. Add deionized water to a final volume of 1 L, pH 7.0-7.2, and sterilize at 121℃ for 20 min. Add agar powder to the inorganic phosphorus solid medium at a ratio of 15 g / L. After sterilization, pour into plates.

[0032] 2. LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, bring to a final volume of 1 L with deionized water, pH 7.2-7.4, sterilize at 121℃ for 20 min, add agar powder to LB solid medium at a ratio of 15 g / L, and pour into plates after sterilization.

[0033] Example 1: Isolation, purification, morphology, identification and preservation of strains

[0034] 1. Strains Isolation and Screening

[0035] Using mud and water collected from the bottom of a sea cucumber farm in Qingdao as the isolation sample, the sample was serially diluted and spread on inorganic phosphorus solid medium at an appropriate gradient. After incubation at 37°C for 3 days, single colonies with phosphate-solubilizing zones were picked and purified by streak plating on LB solid medium to obtain Bacillus subtilis 52-6.

[0036] 2. Strain identification

[0037] (1) Morphological identification: After the strain 52-6 was cultured on LB agar plates at 37°C for 2 days, the colonies were round, light coral red, translucent, with a smooth surface and neat edges.

[0038] (2) Gene identification: DNA was extracted from strain 52-6, and its 16S rDNA sequence was amplified. Universal bacterial primers 27F and 1492R were used as primers for PCR amplification of the extracted DNA. The PCR reaction system consisted of 2 μL DNA template, 2 μL each primer, and 44 μL PCR SuperMix. The PCR amplification program was: 94℃ pre-denaturation for 5 min; 94℃ for 1 min, 55℃ for 1 min, and 72℃ for 2 min, for a total of 30 cycles. Sequencing was performed, and a phylogenetic tree was constructed using the Neighbor-Jionig method. The results are shown below. Figure 2 As shown, it was identified as a subterranean *Bacillus* species, with the taxonomic name [missing information]. Mesobacillus subterraneus .

[0039] 3. Strain preservation:

[0040] The selected *Bacillus subtilis* 52-6 was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on November 20, 2024. Mesobacillus subterraneus The accession number is CGMCC No. 32719.

[0041] Example 2: Growth curve determination of *Bacillus subtilis* 52-6

[0042] A single colony of strain 52-6 was inoculated into LB liquid medium and incubated in a shaker at 37°C and 180 rpm, with three replicates. OD values ​​were measured every 2 hours. 600 Numerical values ​​are recorded and growth curves are plotted.

[0043] The results show that ( Figure 3 The growth curve of strain 52-6 is S-shaped, with a lag phase within 6 hours, followed by a logarithmic growth phase after 6 hours, and a plateau phase after 16 hours. This strain has a short lag phase, grows rapidly, and is easy to culture.

[0044] Example 3: Determination of phosphorus solubilization capacity of Bacillus subtilis 52-6

[0045] Preparation of phosphorus standard curve: Take 0, 2.5, 5, 7.5, 10, 12.5, and 15 mL of 5 mg / L phosphorus standard solution into 50 mL volumetric flasks, respectively. Dilute with water to 20 mL, add 2 drops of 2,4-dinitrophenol indicator, and then add 5 mL of molybdenum antimony colorimetric reagent to each flask. Shake well and dilute to 50 mL to obtain phosphorus standard gradient solutions of 0, 0.25, 0.5, 0.75, 1, 1.25, and 1.5 mg / L. Measure the OD using a spectrophotometer. 700 The absorbance values ​​are used to plot a standard curve.

[0046] Bacillus subtilis 52-6 was inoculated into inorganic phosphorus liquid medium, with the stock medium as a negative control. Both were incubated in a shaker at 37°C and 180 rpm. Samples were taken after 2, 5, and 8 days of incubation, and the OD of the culture supernatant was determined using the molybdenum antimony colorimetric method. 700 The absorbance value is used to calculate the content of soluble phosphorus.

[0047] The results are shown in Table 1. The phosphorus solubility of Bacillus subtilis 52-6 was 25.69 mg / L after 2 days of culture, 52.65 mg / L after 5 days, and 72.06 mg / L after 8 days.

[0048] Table 1. Results of phosphorus solubilization capacity determination of *Bacillus subtilis* 52-6

[0049]

[0050] Example 4: Potted plant growth promotion experiment of Bacillus subtilis 52-6

[0051] Alfalfa was used for pot experiments. Three alfalfa seeds were sown in each pot, with five pots planted in each of the experimental and control groups. The potting soil was washed and sterilized, with 0.2 g of tricalcium phosphate mixed in each pot. Bacillus subtilis 52-6 was inoculated into LB liquid medium and cultured in a shaker at 37℃ and 180 rpm for 24 h. The bacterial solution was diluted 1000 times to obtain the reaction solution. On the 7th and 15th day after germination, the experimental group was soaked in the reaction solution, while the blank control group was soaked in deionized water for 10 min. All alfalfa plants were placed in a light incubator for cultivation, and the growth of alfalfa was observed. The plants were removed and measured after 45 days.

[0052] Experimental results show that ( Figure 4 According to Table 2), compared with the control group, the experimental group showed significant increases in above-ground fresh weight, below-ground fresh weight, above-ground dry weight, below-ground dry weight, stem diameter, and plant height. The fermentation broth of strain 52-6 had a significant growth-promoting effect on alfalfa.

[0053] Table 2. Results of alfalfa pot experiment

[0054]

[0055] Although the embodiments of the present invention have been described above in conjunction with the examples, it should be noted that the described embodiments are only a part of the specific implementation of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent substitutions or improvements made within the scope of the technical concept and technical method of the present invention shall fall within the scope of protection and disclosure of the present invention.

Claims

1. A subterranean *Bacillus mesenchyme* strain 52-6 with phosphorus-solubilizing function, characterized in that... Its classification name is *Bacillus subtilis*. Mesobacillus subterraneus The sample is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32719; the described underground genus *Bacillus mesenchyme* 52-6 has the function of dissolving insoluble inorganic phosphates.

2. The *Bacillus subtilis* 52-6 according to claim 1, characterized in that, The colonies of the underground genus *Bacillus subtilis* 52-6 are round, light coral red, translucent, with a smooth surface and neat edges.

3. The *Bacillus subtilis* 52-6 according to claim 1, characterized in that, The optimal growth temperature for the underground genus *Bacillus subtilis* 52-6 is 35-38℃.

4. The use of *Bacillus subtilis* 52-6 as described in any one of claims 1-3 in the preparation of biological agents that promote plant growth, characterized in that... The underground genus *Bacillus subtilis* 52-6 can increase the stem diameter and plant height of alfalfa.

5. The application according to claim 4, characterized in that, The aforementioned underground genus *Bacillus* 52-6 can increase the fresh and / or dry weight of plants above and / or below ground.

6. The application of *Bacillus subtilis* 52-6 as described in any one of claims 1-3 in improving soil fertility, characterized in that... The improvement of soil fertility is achieved by using Bacillus subtilis 52-6, which has the function of dissolving insoluble phosphates in the soil.

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