Composite microbial phosphate-solubilizing bacterial agent and application thereof

Abstract

The invention discloses a compound microorganism phosphate solubilizing bacterium agent and application thereof, and relates to the technical field of microorganisms, the phosphate solubilizing bacterium agent comprises the following strains: bacillus velezensis C1, bacillus halotolans F3 and rahnella sp., and the compound microorganism phosphate solubilizing bacterium agent comprises the following strains: bacillus velezensis C1, bacillus halotolans F3 and rahnella sp. The invention also discloses an application of the composite microbial phosphate-solubilizing bacterial agent in conversion of insoluble inorganic phosphorus in soil. The compound microorganism phosphate-solubilizing bacterium agent can convert phosphorus elements which are difficult to absorb and utilize by crops in soil into phosphorus elements which can be absorbed and utilized by the crops, and the application value is high.

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] Further, the preservation number of the salt-tolerant Bacillus F3 is CGMCC No: 35703, the preservation unit name is China General Microbiological Culture Collection Center, the preservation address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation date is August 21, 2025.

[0008] Further, the preservation number of the Lassenia F4 is CGMCC No: 35704, the preservation unit name is China General Microbiological Culture Collection Center, the preservation address is No. 1, Beichen West Road, Haidian District, Beijing, and the preservation date is August 21, 2025.

[0009] Further, the ratio of the bacterial amount of the Bacillus velezensis C1, the salt-tolerant Bacillus F3 and the Lassenia F4 is 1:1-3:1-3.

[0010] Further, the ratio of the bacterial amount of the Bacillus velezensis C1, the salt-tolerant Bacillus F3 and the Lassenia F4 is 1:1:1.

[0011] The application provides application of the above-mentioned complex microbial phosphorus-solubilizing agent in transformation of difficult-to-dissolve inorganic phosphorus in soil.

[0012] Further, when the complex microbial phosphorus-solubilizing agent is used, the OD 600 of the total bacterial liquid is adjusted to 0.6-0.9.

[0013] Further, the OD 600 of the total bacterial liquid is adjusted to 0.8.

[0014] Further, the above-mentioned bacterial liquid dosage is 100 L / acre.

[0015] The application has the following beneficial effects: The efficient complex phosphorus-solubilizing bacterial preparation developed by the application has the advantages that the environmental adaptability is widened and the phosphorus-solubilizing efficiency is improved through the synergistic effect of multiple strains, and the efficient complex phosphorus-solubilizing bacterial preparation has important significance for promoting agricultural green development and efficient utilization of soil phosphorus resources. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is a phylogenetic tree of the Bacillus velezensis C1; Figure 2 It is a phylogenetic tree of the salt-tolerant Bacillus F3; Figure 3 It is a phylogenetic tree of the Lassenia F4; Figure 4 It is a phosphorus-solubilizing circle diagram of each bacterium; Figure 5 It is a phosphorus-solubilizing effect comparison diagram of different phosphorus-solubilizing bacteria; Figure 6 It is a phosphorus-solubilizing effect comparison diagram of different phosphorus-solubilizing bacteria combinations. DETAILED DESCRIPTION

[0017] The principles and features of the present application are described below, and the examples are used to explain the present application, but are not intended to limit the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained by commercial purchase. The following example medium formula is as follows: Inorganic phosphorus medium: glucose 10 g, yeast powder 0.5 g, NaCl 0.3 g, KCl 0.3 g, (NH4)2SO4 0.5 g, MgSO4·7H2O 0.3 g, FeSO4·7H2O 0.03 g, MnSO4 0.03 g, Ca3(PO4) 2.5 g, distilled water 1000 mL, pH 7.0-7.5.

[0018] LB medium: tryptone 10 g, yeast powder 5 g, NaCl 10 g, distilled water 1000 mL, pH 7.0-7.5.

[0019] Beef extract tryptone medium: beef extract 5 g, tryptone 10 g, NaCl 5 g, distilled water 1000 mL, pH 7.0-7.5.

[0020] The above-mentioned media are liquid media, and 15 g of agar is added to the above-mentioned media to obtain the corresponding solid medium, which is sterilized at 121°C for 30 min.

[0021] Example 1

[0022] (1) Strain isolation 10 g of soil samples (from citrus orchards and tea factories in Jiuyanxi, Zigui, Hubei Province) were weighed and added to a triangular flask containing 90 mL of sterile water, and shaken at 150 r / min for 1 h at 28°C to form a soil suspension. The 10-fold dilution method was used to prepare dilutions with concentrations of 10 -1 -10 -7 μL of each dilution was applied to the inorganic phosphorus solid medium, and each dilution was repeated 3 times. The samples were incubated at 28°C in an inverted biochemical incubator for 2-5 days, and colonies with phosphorus-dissolving rings were observed. The presence or absence of phosphorus-dissolving rings was used to preliminarily determine whether the strains had phosphorus-dissolving ability. The single colonies with good growth were picked with an inoculation stick and streaked on the inorganic phosphorus solid medium, and incubated at 28°C for 2-5 days. The purification was repeated 3 times, and the slant was stored.

[0023] (2) Microbial identification Morphological identification: the strain was streaked and cultured for 2 days, and the single colony morphology was observed. The results showed that the colony was round, milky white, smooth and moist, and the colony was not transparent.

[0024] Physiological and biochemical identification: physiological and biochemical tests such as gram staining, glucose fermentation and starch hydrolysis were performed on the strain, and the results are shown in Table 1.

[0025] Table 1 Physiological and biochemical identification results of the strain

[0026] (3) Molecular biology identification The DNA of the strain was extracted, and 16S rDNA sequencing was performed. The sequences are shown in SEQ ID No. 1-3, respectively. The sequencing results were uploaded to NCBI for BLAST, and the phylogenetic tree was established by MEGA11 software, as shown in Figures 1-3 respectively. Combined with plate morphology and physiological and biochemical identification, the strains C1, F3 and F4 were preliminarily identified as B. velezensis, B. halodurans and Raenella sp.

[0027] Example 2 Phosphorus solubilizing ability (1) Solubilization of phosphorus circle qualitative phosphorus solubilizing ability Three holes were punched on the inorganic phosphorus medium with a puncher, and 30 μL of bacterial liquid was inoculated in each hole, and each plate was repeated three times. The plates were cultured at 28°C for 3 days, and the diameter of the transparent circle was observed and measured. The results are shown in Figure 4 , wherein (a)-(c) are the phosphorus solubilization circle pictures of strains F3, C1 and F4, respectively. It can be preliminarily judged that strains C1, F3 and F4 all have certain phosphorus solubilizing ability.

[0028] (2) Molybdenum antimony anti-colorimetric method quantitative phosphorus solubilizing ability Preparation of phosphorus standard curve: 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL of 5 mg / L phosphorus standard solution were respectively taken into a 25 mL volumetric flask, and water was added to dilute to about 15 mL. Then 2 drops of nitrophenol indicator were added, and 1 mol / L H2SO4 was added to the solution until the yellow color just disappeared. 2.5 mL of molybdenum antimony color developing agent was added, shaken and diluted to the mark. 0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L phosphorus standard series solution was obtained. The color developing system was placed for 30 min, and the absorbance was measured at 420 nm with 0 mg / L phosphorus standard solution as the control solution. -1 The absorbance of the rest of the phosphorus standard solution was measured. The measured absorbance was taken as the vertical coordinate, and the phosphorus concentration (mg / L) was taken as the horizontal coordinate to draw the working curve.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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).

[0033] Table 2 Treatment Combinations for Phosphate-Soluble Bacteria

[0034] 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.

[0035] Depend onFigure 5 It can be seen that the F4 strain reached the highest amount of dissolved phosphorus on the first day, and then the amount of dissolved phosphorus decreased sharply; the amounts of dissolved phosphorus of C1 and F3 were relatively stable within 6 days, and fluctuated around 100 mg / L.

[0036] According to the results of single bacterial culture, three strains were selected for mixed culture, and the phosphorus dissolution effect was as shown in Figure 6 The results showed that the three strains had the best effect when mixed for phosphorus dissolution, and soil culture experiments would be performed on the combinations.

[0037] Example 3

[0038] The basic physicochemical properties of the conventional soil were as follows: the effective phosphorus was 6.53 mg / kg. The conventional soil without bacteria (CK) and the conventional soil with bacteria were set up, and each treatment was set up in triplicate. 150 g of the conventional soil was weighed into a 250 mL beaker, the beaker was covered with tin foil paper to block light, 30 mL of bacterial suspension (the bacterial suspension was diluted with sterile water to OD 600 =O.8) was added to the soil, and the soil was mixed thoroughly. The same amount of sterile water was used as a control. The soil was cultured at 28°C, and after 10 days of culture, the effective phosphorus content in the soil was determined according to the sodium bicarbonate method.

[0039] The results showed that the effective phosphorus content in the soil without bacteria was 6.73 mg / kg, and the effective phosphorus content in the soil with bacteria was 10.41 mg / kg. Compared with the CK, the phosphorus-dissolving bacteria increased the effective phosphorus content in the soil by 54.68%.

[0040] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A compound microbial phosphate-solubilizing agent, characterized in that, Including the following strains: Bacillus belyssus C1 ( Bacillus velezensis ), Salt-resistant Bacillus F3 ( Bacillus halotolerans ) and Laryn F4 ( Rahnella sp .).

2. The compound microbial phosphate-solubilizing agent as described in claim 1, characterized in that, 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.

3. The compound microbial phosphate-solubilizing agent as described in claim 1, characterized in that, The preservation number of the salt-tolerant 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.

4. The compound microbial phosphate-solubilizing agent as described in claim 1, characterized in that, 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, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit date is August 21, 2025.

5. The compound microbial phosphate-solubilizing agent as described in claim 1, characterized in that, The bacterial count ratio of Bacillus belyss C1, halophilic Bacillus F3, and Laenella F4 is 1:1-3:1-3.

6. The application of the compound microbial phosphorus-solubilizing agent according to any one of claims 1-5 in the transformation of poorly soluble inorganic phosphorus in soil.

7. The application as described in claim 6, 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.

8. The application as described in claim 7, characterized in that, Adjusting the total bacterial culture OD 600 Up to 0.8.

Images