Bacillus for decomposing insoluble calcium phosphate and lecithin in acid soil and application thereof

Abstract

The invention discloses bacillus for decomposing insoluble calcium phosphate and lecithin in acid soil and application of the bacillus, and relates to the technical field of soil remediation and biologication.The bacillus is bacillus thuringiensis MSSW004 and is delivered to the China Center for Type Culture Collection on June 21, 2024 to be preserved, and the preservation number of the bacillus thuringiensis is CCTCC NO: M20241338. The strain provided by the invention enriches genetic resources of wild phosphate solubilizing bacteria, enlarges a backup library for whole genome breeding of the phosphate solubilizing bacteria, can improve the content of available phosphorus in acid soil, improve the utilization efficiency of phosphate fertilizer and increase the yield of crops, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the fields of soil remediation and biotechnology, and specifically to a Bacillus bacterium capable of decomposing insoluble calcium phosphate and lecithin in acidic soil and its application. Background Art

[0002] Phosphorus is a major nutrient element essential for the growth and development of all organisms. In plants, the phosphorus content accounts for approximately 0.2% - 1.1% of the total biomass. Phosphorus participates in many important physiological and biochemical processes in plants. Approximately 67% of the arable land globally lacks phosphorus. Among them, weathered alkaline soils are rich in oxides such as calcium, and acidic soils are rich in aluminum and iron oxides. Phosphorus in the soil is easily fixed, and the available phosphorus is very low. When phosphate fertilizers are applied to farmland soils, they are easily fixed by the soil, and the phosphorus utilization efficiency for the growth of the current-season crops is less than 20%, and most of it is fixed by the soil and remains in the soil. Therefore, how to use phosphate fertilizers more effectively, reduce the waste and loss of phosphorus during the crop life cycle, and improve the utilization efficiency of phosphorus in the soil has become a research hotspot in the field of plant nutrition and resource utilization.

[0003] There is a kind of microorganism in the soil that can convert insoluble combined phosphorus in the soil into available phosphorus. Microorganisms with this function are called phosphorus-solubilizing bacteria, and phosphorus-solubilizing bacteria play an important role in promoting crop growth and improving the utilization efficiency of insoluble phosphorus in the soil. Phosphorus-solubilizing bacteria include phosphate-dissolving bacteria, phosphate-dissolving fungi, and actinomycetes. So far, the research reports on phosphate-dissolving bacteria are the most, and the proportion is the highest. Phosphate-dissolving bacteria are mainly Bacillus, Klebsiella, Pseudomonas, Escherichia, etc. As a kind of Bacillus, Bacillus thuringiensis is currently mainly reported for its insecticidal function, and there are few reports on its ability to dissolve insoluble phosphorus and organic phosphorus in acidic soil. Summary of the Invention

[0004] The purpose of the present invention is to provide a Bacillus bacterium capable of decomposing insoluble calcium phosphate and lecithin in acidic soil and its application, so as to solve the problems raised in the above background art.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A Bacillus bacterium capable of decomposing insoluble calcium phosphate and lecithin in acidic soil, the Bacillus bacterium is Bacillus thuringiensis MSSW004, which was deposited with the China Center for Type Culture Collection on June 21, 2024. The address of the depository unit is: Wuhan University, Wuhan, China, and its deposit number is: CCTCC NO: M20241338.

[0006] Another object of the present invention is to provide an application of Bacillus in decomposing insoluble calcium phosphate and lecithin in acidic soil.

[0007] Preferably, the decomposition of insoluble calcium phosphate and lecithin in acidic soil comprises the following steps: using the Bacillus described in claim 1, obtaining a spore suspension after amplification culture, and then inoculating the suspension into acidic soil.

[0008] Preferably, the culture medium involved in the amplification culture includes NA liquid medium, insoluble calcium phosphate solid medium, insoluble calcium phosphate liquid medium, lecithin liquid medium, and lecithin solid medium.

[0009] Preferably, the formula of the NA liquid medium is: 3 g of beef extract, 5 g of peptone, 5 g of NaCl, and 1000 mL of distilled water, and the pH of the distilled water is 7.2 - 7.4.

[0010] Preferably, the formula of the insoluble calcium phosphate solid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H 2 O, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO 4 ·7H 2 O, 0.03 g of MnSO4·H2O, 5 g of Ca3(PO4)2, 20 g of agar, 1000 mL of distilled water, and the pH of the distilled water is 7.0 - 7.2.

[0011] Preferably, the formula of the insoluble calcium phosphate liquid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H 2 O, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO 4 ·7H 2 O, 0.03 g of MnSO 4 ·H 2 O, 5 g of Ca 3 (PO 4 )2, 1000 mL of distilled water, and the pH of the distilled water is 7.0 - 7.2.

[0012] Preferably, the formula of the lecithin liquid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H2 0.3 g of glucose, 0.3 g of NaCl, 0.3 g of KCl, FeSO 4 ·7H 2 O 0.03 g, MnSO 4 ·H 2 O 0.03 g, 2.8 g of lecithin, 1000 mL of distilled water, and the pH of the distilled water is 7.0 - 7.2.

[0013] Preferably, the formulation of the lecithin solid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H 2 O, 0.3 g of NaCl, 0.3 g of KCl, FeSO 4 ·7H 2 O 0.03 g, MnSO 4 ·H 2 O 0.03 g, 2.8 g of lecithin, 1000 mL of distilled water, the pH of the distilled water is 7.0 - 7.2, and 20 g of agar.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The strains provided by the present invention enrich the genetic resources of wild phosphate-solubilizing bacteria and expand the reserve library for the whole-genome breeding of phosphate-solubilizing bacteria. This bacterium can activate the insoluble inorganic phosphorus and organic phosphorus in the soil, increase the content of available phosphorus in the soil, improve the utilization efficiency of phosphate fertilizers and the yield of crops, and has broad application prospects. Description of the Drawings

[0016] Figure 1 It is a comparison diagram of the maximum phosphate-solubilizing amount of strains JL1 - 4 in 7 days in the examples of the present invention;

[0017] Figure 2 It is a comparison diagram of the influence of different temperatures on the phosphate-solubilizing ability of strain JL1 in the present invention;

[0018] Figure 3 It is a comparison diagram of the influence of different initial pH values on the phosphate-solubilizing ability of strain JL1 in the present invention;

[0019] Figure 4 It is an effect diagram of the phosphate-solubilizing bacteria provided in the examples of the present invention alleviating the low-phosphate stress of rapeseed seedlings under low-phosphate conditions in a pot experiment. Detailed Embodiments

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] The present invention provides a technical solution: a Bacillus that decomposes insoluble calcium phosphate and lecithin in acidic soil. The Bacillus was deposited with the China Center for Type Culture Collection on May 12, 2024, and its deposit number is: CCTCC M20241338.

[0022] The decomposition of insoluble calcium phosphate and lecithin in acidic soil includes the following steps: using the Bacillus in claim 1, amplifying and culturing to obtain a spore suspension, and then inoculating the suspension into acidic soil;

[0023] Among them, the culture media involved in the amplification culture include NA liquid medium, insoluble calcium phosphate solid medium, insoluble calcium phosphate liquid medium, lecithin liquid medium, and lecithin solid medium;

[0024] The formula of the NA liquid medium is: 3 g of beef extract, 5 g of peptone, 5 g of NaCl, and 1000 mL of distilled water, and the pH of the distilled water is 7.2 - 7.4;

[0025] The formula of the insoluble calcium phosphate solid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H 2 O, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO 4 ·7H 2 O, 0.03 g of MnSO4·H2O, 5 g of Ca3(PO4)2, 20 g of agar, 1000 mL of distilled water, and the pH of the distilled water is 7.0 - 7.2;

[0026] The formula of the insoluble calcium phosphate liquid medium is: 10 g of glucose, (NH 4 ) 2 SO 4 0.5 g, 0.3 g of MgSO 4 ·7H 2 O, 0.3 g of NaCl, 0.3 g of KCl, 0.03 g of FeSO 4 ·7H 2 O, 0.03 g of MnSO 4 ·H 2 O, Ca3 (PO 4 ) 25 g, distilled water 1000 mL, pH of distilled water 7.0 - 7.2;

[0027] The formula of the lecithin liquid medium is: glucose 10 g, (NH 4 ) 2 SO 4 0.5 g, MgSO 4 ·7H 2 O 0.3 g, NaCl 0.3 g, KCl 0.3 g, FeSO 4 ·7H 2 O 0.03 g, MnSO 4 ·H 2 O 0.03 g, lecithin 2.8 g, distilled water 1000 mL, pH of distilled water 7.0 - 7.2;

[0028] The formula of the lecithin solid medium is: glucose 10 g, (NH 4 ) 2 SO 4 0.5 g, MgSO 4 ·7H 2 O 0.3 g, NaCl 0.3 g, KCl 0.3 g, FeSO 4 ·7H 2 O 0.03 g, MnSO 4 ·H 2 O 0.03 g, lecithin 2.8 g, distilled water 1000 mL, pH of distilled water 7.0 - 7.2, agar 20 g.

[0029] Another object of the present invention is to provide an application of Bacillus in decomposing insoluble calcium phosphate and lecithin in acidic soil.

[0030] Example 1

[0031] Preliminary screening of phosphorus-solubilizing microorganisms: Weigh 5 g of soil sample and add it to a triangular flask containing 45 mL of sterile water and 10 sterile glass beads. Oscillate for about 2 h to completely mix the soil sample with water. Use a pipette to aspirate 1 mL of the soil suspension and add it to 100 mL of NA liquid medium. Incubate at 28 °C and 200 rpm for 24 h. Use a pipette to aspirate 1 mL of the suspension and add it to a test tube containing 9 mL of sterile water and mix well to prepare a solution with a dilution factor of 10 -1 . Then perform gradient dilution according to the 10-fold dilution method to obtain soil solutions with different dilution factors of 10 -2 - 10 -7 . Take the soil solutions with dilution factors of 10 -4 , 10 -5 , 10 -6 , 10 -7100 μL of the soil suspension of each was evenly spread on the cooled inorganic phosphorus and organic phosphorus selective medium plates, with 3 replicates for each concentration. After spreading, it was left to stand at room temperature for 5 - 10 min to allow the bacterial suspension to adsorb into the medium. Then, it was sealed and inverted, and cultured at a constant temperature of 28 °C for 3 d. Observe the colony growth and the formation of the phosphorus-dissolving circle in the plate and record. Select the strains with obvious color changes and transparent circles around the colonies, and sequentially number them as JL1, JL2, JL3, and JL4 according to the serial numbers.

[0032] Example 2

[0033] Rescreening of phosphorus-solubilizing microorganisms: The strains obtained from the primary screening were respectively inoculated into 250 mL conical flasks containing 100 mL of inorganic phosphorus selective medium and organic phosphorus medium. After shaking well, they were placed on a shaker at 28 °C and 180 rpm for 7 days. At the same time, a liquid medium without any bacteria was used as a control group. After seven days, 1 mL of the culture solution was taken from it, centrifuged at 5000 r / min for 10 min, and the supernatant was taken. The soluble phosphorus concentration was measured by the molybdenum antimony anti-colorimetric method. The results are as Figure 1 shown. The above 4 strains screened by the present invention all have a certain phosphorus-solubilizing ability and can decompose the insoluble phosphorus (Ca 3 (PO 4 ) 2 ) and organic phosphorus (lecithin) in the medium into soluble phosphorus. Among them, the strain JL1 has the highest phosphorus-solubilizing efficiency. The content of soluble phosphorus in the inorganic phosphorus medium is as high as 264.05 mg / L, and the content of soluble phosphorus in the organic phosphorus medium reaches 21.90 mg / L. Therefore, this strain can be made into a phosphorus-solubilizing microbial agent and can be widely used in agricultural production.

[0034] Example 3

[0035] Identification of strain JL1:

[0036] (1) Colony morphology of strain JL1

[0037] The colonies are round or oval, light yellow in color, with irregular edges, opaque, slightly raised and waxy;

[0038] (2) Molecular biological identification of strain JL1

[0039] The strain screened above was identified by molecular biology methods. The 16S rDNA sequence of the strain was amplified by PCR to obtain an amplification product with a length of about 1500 bp. The amplification product was sequenced by a sequencing company, and the sequenced sequence was compared with the sequences in the GenBank database by BLAST. The results showed that the similarity of the strain to Bacillus thuringiensis was more than 99%. Combining the morphological characteristics, cultural characteristics and 16S rDNA sequence analysis, the strain was identified as Bacillus thuringiensis

[0040] Example 4

[0041] Effect of different culture temperatures on the phosphate-solubilizing ability of the strain:

[0042] The above strain was inoculated into NA liquid medium and cultured in a shaker at 28 °C and 180 rpm for one day. 1 mL of the bacterial solution was inoculated into 250 mL conical flasks containing 100 mL of inorganic phosphate liquid medium and organic phosphate liquid medium, and the addition of the same volume of liquid medium without inoculation was used as a control (CK). The cultures were shaken at 180 r / min at seven temperatures of 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C and 45 °C respectively. Each treatment had four replicates. After culturing for seven days, samples were taken to measure the soluble phosphorus content in the medium. The results are as Figure 2 shown

[0043] It can be seen from Figure 2 that the strain has a strong phosphate-solubilizing ability for inorganic phosphorus between 30 and 40 °C, and the phosphate-solubilizing effect is the best at 35 °C, with the maximum phosphorus solubilization amount of 230.46 mg / L. The order of the maximum phosphorus solubilization amounts is 35 °C > 30 °C > 40 °C > 25 °C > 45 °C > 20 °C > 15 °C. The strain has a strong phosphate-solubilizing ability for organic phosphorus between 25 and 35 °C, and the phosphate-solubilizing effect is the best at 30 °C, with the maximum phosphorus solubilization amount of 18.13 mg / L. The order of the maximum phosphorus solubilization amounts is 30 °C > 35 °C > 25 °C > 40 °C > 20 °C > 45 °C > 15 °C

[0044] Example 5

[0045] Effect of different initial pH values on the phosphate-solubilizing ability of the strain:

[0046] The experimental method was the same as that in Example 5. The initial pH values of the culture solutions were adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 respectively, and the cultures were shaken at 28 °C and 180 r / min. Each treatment had four replicates. After culturing for seven days, samples were taken to measure the soluble phosphorus content in the medium. The results are as Figure 3 shown

[0047] It can be seen from Figure 3It can be seen that strain JL1 has strong adaptability in the pH range of 4 - 8 and has a strong phosphorus-solubilizing ability. When the pH value is 6, the phosphorus-solubilizing amounts of JL1 for calcium phosphate and lecithin are both the highest. The initial pH values of the culture medium that affect the maximum phosphorus-solubilizing amount are in the order of pH6 > pH5 > pH7 > pH8 > pH9 > pH10 > pH4. This shows that strain PA01 can well adapt to the acidic environment. If it is inoculated in acidic soil in the future, it will have a good ability to dissolve insoluble phosphorus.

[0048] Application Example

[0049] Verification of the effect of strain JL1 on alleviating low-phosphorus stress in rapeseed seedlings under low-phosphorus conditions in pot experiments:

[0050] Take the bacterial liquid of strain JL1 in the above example and verify its alleviating effect on low-phosphorus stress of rapeseed seedlings under low-phosphorus conditions in pot experiments. The experiment is divided into a strain-added group (inoculating 100 mL of JL1 bacterial liquid in the pot) and a CK group (blank control group, inoculating 100 mL of inactivated JL1 bacterial liquid in the pot). Each group repeats 4 pots, and 8 germinated rapeseed seeds are sown in each pot. Each pot is fertilized with 0.2 g / kg soil of (NH 4 ) 2 SO 4 、0.2 g / kg soil of KCl, 0.02 g / kg soil of P 2 O 5 、1 mL of EDTA-Fe mother liquor (200×, Hoagland formula) and 1 mL of Arnon mother liquor (1000×, Hoagland formula). After emergence, thin out the seedlings, and finally keep 4 plants in each pot. Regularly water each pot with an equal amount of deionized water and continuously culture for 90 days.

[0051] The growth status of rapeseed is as Figure 4 shown. It can be seen from the figure that after adding strain JL1 under low-phosphorus conditions, the growth of rapeseed is significantly better than that of the control group. The effects of phosphorus-solubilizing bacteria JL1 on the shoot dry weight, root dry weight, shoot phosphorus content, root phosphorus content, and net photosynthesis of rapeseed seedlings under low-phosphorus conditions are shown in Table 1. It can be seen from the table that after inoculating phosphorus-solubilizing bacteria JL1 under low-phosphorus conditions, the shoot dry weight of rapeseed seedlings increased by 164.91%, the root dry weight increased by 55.56%, the shoot phosphorus content increased by 54.55%, the root phosphorus content increased by 41.43%, and the net photosynthesis increased by 69.26%.

[0052] Table 1 Effects of phosphorus-solubilizing bacteria JL1 on the growth of rapeseed seedlings under low-phosphorus conditions

[0053]

[0054] Note: The data in Table 1 represent the average value ± standard deviation of 3 repeated experiments. Different letters after the data indicate significant differences between the data.

[0055] This application example shows that after the fermentation broth of phosphate-solubilizing bacterium JL1 is applied, the insoluble phosphorus in the soil is converted into soluble phosphorus, increasing the content of available phosphorus in the soil, reducing the amount of chemical fertilizers used, and promoting the growth and development of crops. It has important significance and application value in cultivating and exerting soil ecological fertility and maintaining agricultural ecological balance.

[0056] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil, characterized in that: The Bacillus is Bacillus thuringiensis MSSW004, which was sent to the China Center for Type Culture Collection for preservation on June 21, 2024, and its preservation number is: CCTCC NO:M20241338.

2. Use of the bacillus according to claim 1 in decomposing poorly soluble calcium phosphate and lecithin in acidic soil.

3. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 1, characterized in that: The method for decomposing the insoluble calcium phosphate and lecithin in acidic soil comprises the following steps: using the Bacillus described in claim 1, amplifying and culturing to obtain a spore liquid, and then inoculating the bacterial liquid into the acidic soil.

4. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 3, characterized in that: The culture medium involved in the amplification culture includes NA liquid culture medium, insoluble calcium phosphate solid culture medium, insoluble calcium phosphate liquid culture medium, lecithin liquid culture medium and lecithin solid culture medium.

5. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 4, characterized in that: The formula of the NA liquid culture medium is: 3g beef extract, 5g peptone, 5g NaCl and 1000mL distilled water, and the pH value of the distilled water is 7.2-7.

4.

6. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 4, characterized in that: The formula of the insoluble calcium phosphate solid culture medium is: 10g of glucose, 0.5g of (NH4)2SO4, 0.3g of MgSO4·7H2O, 0.3g of NaCl, 0.3g of KCl, 0.03g of FeSO4·7H2O, 0.03g of MnSO4·H2O, 25g of Ca3(PO4), 20g of agar, 1000mL of distilled water, and the pH value of the distilled water is 7.0-7.

2.

7. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 4, characterized in that: The formula of the insoluble calcium phosphate liquid culture medium is: 10g glucose, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.3g NaCl, 0.3g KCl, 0.03g FeSO4·7H2O, 0.03g MnSO4·H2O, 25g Ca3(PO4), 1000mL distilled water, and the pH value of the distilled water is 7.0-7.

2.

8. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 4, characterized in that: The formula of the lecithin liquid culture medium is: 10g of glucose, 0.5g of (NH4)2SO4, 0.3g of MgSO4·7H2O, 0.3g of NaCl, 0.3g of KCl, 0.03g of FeSO4·7H2O, 0.03g of MnSO4·H2O, 2.8g of lecithin, 1000mL of distilled water, and pH value of distilled water is 7.0-7.

2.

9. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 4, characterized in that: The formula of the lecithin solid culture medium is: 10g of glucose, 0.5g of (NH4)2SO4, 0.3g of MgSO4·7H2O, 0.3g of NaCl, 0.3g of KCl, 0.03g of FeSO4·7H2O, 0.03g of MnSO4·H2O, 2.8g of lecithin, 1000mL of distilled water, pH value of distilled water 7.0-7.2, and 20g of agar.

10. The Bacillus for decomposing poorly soluble calcium phosphate and lecithin in acidic soil according to claim 1, characterized in that: Also provided is a microbial agent for decomposing insoluble calcium phosphate and lecithin in acidic soil, wherein the active ingredient is the bacillus described in claim 1.

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