A compound microbial phosphate-solubilizing agent and its application

Abstract

This invention discloses a compound microbial phosphate-solubilizing agent and its application, relating to the field of microbial technology. The phosphate-solubilizing agent includes the following strain: *Bacillus belye* C1 (… Bacillus velezensis ), Salt-resistant Bacillus F3 ( Bacillus halotolerant ) and Laryn F4 ( Rahnella sp. This invention also discloses the application of this composite microbial phosphate-solubilizing agent in the transformation of poorly soluble inorganic phosphorus in soil. This composite microbial phosphate-solubilizing agent can convert phosphorus in soil that is difficult for crops to absorb and utilize into phosphorus that crops can absorb and utilize, thus having high application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound microbial phosphate-solubilizing agent and its application. Background Technology

[0002] Phosphorus in soil is one of the key nutrients essential for crop growth. However, under natural conditions, phosphorus in soil mainly exists in the form of insoluble inorganic phosphorus (such as calcium phosphate, iron phosphate, and aluminum phosphate) and organic phosphorus (such as phytic acid and nucleic acids), with extremely low bioavailability, making it difficult for plants to directly absorb and utilize it. Statistics show that approximately 95% of the phosphorus in my country's arable land soil is fixed or passivated and cannot be absorbed by crops, leading to the necessity of applying large amounts of phosphate fertilizer to meet crop needs in agricultural production. However, the utilization rate of phosphate fertilizer is usually less than 20%, and long-term excessive application not only increases production costs but also causes environmental problems such as soil compaction and eutrophication of water bodies.

[0003] Phosphate-solubilizing bacteria are a class of functional microorganisms that can convert insoluble phosphorus into soluble phosphates by secreting metabolic products such as organic acids, enzymes, and protons. Studies have shown that the application of phosphate-solubilizing bacteria can significantly increase the available phosphorus content in the soil, promote crop phosphorus absorption, and reduce the amount of phosphate fertilizer applied. Compared with traditional chemical phosphate fertilizers, phosphate-solubilizing bacteria have advantages such as environmental friendliness and strong sustainability, but in practical applications, they still face problems such as poor strain adaptability and unstable phosphate-solubilizing efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a compound microbial phosphorus-solubilizing agent and its application. This compound microbial phosphorus-solubilizing agent can convert phosphorus in the soil, which is difficult for crops to absorb and utilize, into phosphorus that can be absorbed and utilized by crops, thus having high application value.

[0005] The technical solution of this invention to solve the above-mentioned technical problems is as follows: A compound microbial phosphate-solubilizing agent is provided, comprising the following strains: Bacillus belye C1 (… Bacillus velezensis ), Salt-resistant Bacillus F3 ( Bacillus halotolerans ) and Laryn F4 ( Rahnella sp. ).

[0006] Furthermore, the accession number of Bacillus belyss C1 is CGMCC No: 35705, the depository name is China General Microbiological Culture Collection Center, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025.

[0007] Furthermore, the accession number of halophilic Bacillus F3 is CGMCC No: 35703, the depository is China General Microbiological Culture Collection Center, the depository address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025.

[0008] Furthermore, the accession number of *Laenia* F4 is CGMCC No: 35704, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025.

[0009] Furthermore, the bacterial count ratio of Bacillus belyss C1, halophilic Bacillus F3, and Laenella F4 was 1:1-3:1-3.

[0010] Furthermore, the bacterial count ratio of Bacillus belye C1, halophilic Bacillus F3, and Laenella F4 was 1:1:1.

[0011] This invention provides the application of the above-mentioned composite microbial phosphorus-solubilizing agent in the transformation of poorly soluble inorganic phosphorus in soil.

[0012] Furthermore, when using the compound microbial phosphate-solubilizing agent, adjust the total bacterial OD of the culture solution. 600 Up to 0.6-0.9.

[0013] Further, adjust the total bacterial culture OD 600 Up to 0.8.

[0014] Furthermore, the dosage of the above-mentioned bacterial solution is 100 L / mu.

[0015] The present invention has the following beneficial effects:

[0016] The highly efficient compound phosphate-solubilizing bacterial preparation developed in this invention broadens environmental adaptability and enhances phosphate-solubilizing efficiency through the synergistic effect of multiple bacterial strains, which is of great significance for promoting green agricultural development and efficient utilization of soil phosphorus resources. Attached Figure Description

[0017] Figure 1 Phylogenetic tree diagram of Bacillus belyss C1;

[0018] Figure 2 Phylogenetic tree diagram of halophilic Bacillus F3;

[0019] Figure 3 Phylogenetic tree diagram of Larn bacterium F4;

[0020] Figure 4 Diagrams of the phosphate-solubilizing zones of various bacteria;

[0021] Figure 5A comparison chart showing the phosphorus-solubilizing effects of different phosphorus-solubilizing bacteria;

[0022] Figure 6 A comparison chart showing the phosphorus-solubilizing effects of different combinations of phosphorus-solubilizing bacteria. Detailed Implementation

[0023] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0024] The culture medium formulations for the following examples are as follows:

[0025] Inorganic phosphorus culture medium: 10 g glucose, 0.5 g yeast extract, 0.3 g NaCl, 0.3 g KCl, 0.5 g (NH4)2SO4, 0.3 g MgSO4∙7H2O, 0.03 g FeSO4∙7H2O, 0.03 g MnSO4, 2.5 g Ca3(PO4), 1000 mL distilled water, pH 7.0-7.5.

[0026] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, distilled at 1000 mL, pH 7.0-7.5.

[0027] Beef extract peptone medium: 5 g beef extract, 10 g tryptone, 5 g NaCl, 1000 mL distilled water, pH 7.0-7.5.

[0028] All of the above culture media are liquid culture media. Adding 15 g of agar to these media will produce the corresponding solid culture media. Sterilize at 121℃ for 30 min.

[0029] Example 1

[0030] (1) Strains Isolation

[0031] Weigh 10 g of soil sample (from Jiuwanxi Citrus Orchard and Tea Factory in Zigui County, Hubei Province) and add it to an Erlenmeyer flask containing 90 mL of sterile water. Shake at 28℃ and 150 r / min for 1 h to form a soil suspension. Prepare 10-fold dilutions using the 10-fold dilution method. -1 -10 -7Diluted solutions were prepared by spreading 100 μL onto inorganic phosphorus solid medium, with each dilution repeated three times. The cultures were incubated upside down at 28°C for 2-5 days, observing for colonies exhibiting phosphate-solubilizing zones. The presence or absence of phosphate-solubilizing zones was used to preliminarily determine whether the strain possessed phosphate-solubilizing ability. Healthy single colonies were picked up with an inoculation stick and streaked onto inorganic phosphorus solid medium, incubated upside down at 28°C for 2-5 days, and purified three times. The purified cultures were then stored as slant agar.

[0032] (2) Identification of microorganisms

[0033] Morphological identification: The strain was streaked for 2 days and the morphology of single colonies was observed. The results showed that the colonies were round, milky white, smooth and moist, and opaque.

[0034] Physiological and biochemical identification: The strain was subjected to physiological and biochemical tests such as Gram staining, glucose fermentation, and starch hydrolysis. The results are shown in Table 1.

[0035] Table 1. Physiological and biochemical identification results of the strains

[0036]

[0037] (3) Molecular biological identification

[0038] DNA was extracted from the strain and 16S rDNA sequencing was performed. The sequences are shown in SEQ ID Nos. 1-3. The sequencing results were uploaded to NCBI for BLAST analysis, and a phylogenetic tree was constructed using MEGA11 software, as shown in the figures below. Figure 1-3 As shown, based on plate morphology and physiological and biochemical identification, strains C1, F3 and F4 were preliminarily identified as *Bacillus belyssae*, *Bacillus halophilus*, and *Lahnella*.

[0039] Example 2 Phosphorus solubilization capacity

[0040] (1) Qualitative phosphorus solubility of the phosphorus-solubilizing zone

[0041] Three wells were punched in an inorganic phosphorus medium, and 30 μL of bacterial suspension was inoculated into each well. Each plate was replicated three times. The plates were incubated at 28°C for 3 days, and the diameter of the transparent zone was observed and measured. The results are as follows: Figure 4 As shown, (a)-(c) are images of the phosphate-solubilizing zones of strains F3, C1, and F4, respectively. It can be preliminarily determined that strains C1, F3, and F4 all possess a certain degree of phosphate-solubilizing ability.

[0042] (2) Molybdenum-antimony resistance to quantitative phosphorus solubility by colorimetric method

[0043] Plotting the phosphorus standard curve:

[0044] Pipettes 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of 5 mg / L phosphorus standard solution into 25 mL volumetric flasks, respectively. Dilute with water to approximately 15 mL, then add 2 drops of dinitrophenol indicator, followed by 1 mol / L H₂SO₄ until the yellow color of the solution just disappears. Add 2.5 mL of molybdenum antimony colorimetric reagent, shake well, and dilute to volume to obtain a series of phosphorus standard solutions with concentrations of 0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, and 1.2 mg / L. After the colorimetric system has stood for 30 min, dilute with 0 mg / L... -1 The phosphorus standard solution was used as a control solution, and the absorbance of the other phosphorus standard solutions was measured. A working curve was plotted with the measured absorbance as the ordinate and the phosphorus concentration (mg / L) as the abscissa.

[0045] Determination of phosphorus solubility: The preliminarily screened phosphorus-solubilizing strains were cultured in Erlenmeyer flasks containing 50 mL / 100 mL of LB liquid medium to prepare a bacterial suspension. The culture conditions were 28℃, 180 r / min, and 3 days. The absorbance value at 600 nm was adjusted (OD). 600 ) to OD 600 =0.6-0.7.

[0046] The bacterial suspension (at a 1% inoculum) was added to an Erlenmeyer flask containing 50 mL / 100 mL of inorganic phosphorus liquid culture medium. An equal volume of sterile water was used as a blank control. Each treatment was repeated three times. The flasks were incubated at 28°C and 180 rpm for 7 days. 2 mL of the fermented bacterial solution (sampled every other day) was centrifuged at 10000 rpm for 10 min. The supernatant was collected, and the available phosphorus content was determined using the molybdenum antimony colorimetric method.

[0047] bacterial solution at 10000 r / min -1 After centrifugation for 10 min, take 0.5 mL of the supernatant and determine its soluble phosphorus content using the molybdenum-antimony colorimetric method. Transfer the supernatant to a 25 mL volumetric flask, add 10 mL of distilled water and 2 drops of 2,4-dinitrophenol indicator solution. The solution should be slightly yellow. If colorless, adjust the pH with dilute sodium hydroxide solution until the solution is just slightly yellow, then adjust with dilute sulfuric acid solution until colorless. Finally, add 2.5 mL of molybdenum-antimony colorimetric reagent, shake well, and dilute to the mark of the volumetric flask. For the blank test, do not add bacterial suspension but follow the same treatment. Allow the colorimetric system to develop fully for approximately 30 min. Using the blank test as a control, zero the sample. Take 3-4 mL of the colorimetric solution and measure the absorbance at 700 nm using a spectrophotometer. Read the absorbance value and find the soluble phosphorus concentration on the working curve to represent the phosphorus solubility of the strain.

[0048] Experiments were conducted on the ability of different phosphorus bacteria and combinations to dissolve tricalcium phosphate. The specific treatment combinations are shown in Table 2 (F1, F2, and SZ3 are other strains screened).

[0049] Table 2 Treatment Combinations for Phosphate-Soluble Bacteria

[0050]

[0051] Add 50 mL of sterilized phosphorus-containing bacterial fermentation medium to a 100 mL Erlenmeyer flask. Different phosphorus-containing bacteria and their combinations are inoculated into the fermentation liquid medium at an inoculation rate of 1%. A no-inoculation treatment is also included. Each treatment is repeated three times. The mixture is incubated at 28℃ and 180 r / min in a constant temperature shaking incubator for 7 days. Samples are taken every other day to continuously monitor the phosphorus dissolution effect. The phosphorus dissolution effect is as follows: Figure 5 As shown.

[0052] Depend on Figure 5 It can be seen that the phosphorus solubility of strain F4 reached its highest level on the first day, and then decreased sharply; the phosphorus solubility of strains C1 and F3 remained relatively stable over these 6 days, fluctuating around 100 mg / L.

[0053] Based on the results of single-strain culture, three strains were selected for mixed culture, and the phosphate-solubilizing effect was as follows: Figure 6 As shown in the figure. The results indicate that the simultaneous mixing of the three strains yielded the best phosphate-solubilizing effect, and soil culture experiments will be conducted on their combination.

[0054] Example 3

[0055] The basic physicochemical properties of the conventional soil were as follows: available phosphorus 6.53 mg / kg. Two treatments were established: uninoculated conventional soil (CK) and inoculated conventional soil, with three replicates for each treatment. 150 g of the conventional soil was weighed into a 250 mL beaker, which was shielded from light by aluminum foil. The bacterial suspension was added to the soil (diluted with sterile water to OD). 600 =0.8) 30 mL, mix thoroughly, and use an equal volume of sterile water as a control. Incubate at 28℃, and after 10 days, take samples to determine the available phosphorus content in the soil according to the sodium bicarbonate method.

[0056] The results showed that the available phosphorus content in the uninoculated soil was 6.73 mg / kg, while the available phosphorus content in the inoculated soil was 10.41 mg / kg. Compared with the control (CK), the phosphorus-solubilizing bacteria increased the available phosphorus content in the soil by 54.68%.

[0057] 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 or 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 compound microbial phosphate-solubilizing agent, characterized in that, Composed of the following strains: Bacillus belyssus C1 ( Bacillus velezensis ), Salt-resistant Bacillus F3 ( Bacillus halotolerans ) and Laryn F4 ( Rahnella sp .); The Bacillus belyss C1 has the accession number CGMCC No: 35705, the depository is the China General Microbiological Culture Collection Center, the depository address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025. The preservation number of the salt-tolerant Bacillus F3 is CGMCC No: 35703, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025. The accession number of the *Laenia* F4 strain is CGMCC No: 35704, the depositary institution is the China General Microbiological Culture Collection Center, the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025. The bacterial count ratio of Bacillus belyss C1, halophilic Bacillus F3, and Laenella F4 is 1:1-3:1-3.

2. The application of the compound microbial phosphorus-solubilizing agent according to claim 1 in the transformation of poorly soluble inorganic phosphorus in soil.

3. The application as described in claim 2, characterized in that, When using the compound microbial phosphate-solubilizing agent, adjust the total bacterial OD of the culture solution. 600 Up to 0.6-0.

9.

4. The application as described in claim 3, characterized in that, Adjusting the total bacterial culture OD 600 Up to 0.8.

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