Application of paeonia suffruticosa rhizosphere Pantoea cypripedii in soil phosphate solubilization and plant growth promotion

Abstract

The invention discloses an application of Pantoea cypripedii in soil phosphate solubilization and plant growth promotion, and belongs to the technical field of microorganisms. The Pantoea cypripedii provided by the invention can be used for effectively converting insoluble inorganic phosphorus into soluble inorganic phosphorus, so that the utilization of phosphorus is promoted. The strain can also adapt to the soil environment, can improve the utilization efficiency of phosphorus in soil and promote the growth of plants when being added into the soil, can also improve the nutrient condition of the soil, and has important agricultural and environmental values.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to the application of peony rhizosphere Pantoea cypripedii in soil phosphorus solubilization and plant growth promotion, and specifically relates to a strain of Pantoea cypripedii and the application of the strain in soil phosphorus solubilization and plant growth promotion. Background Art

[0002] In my country, about two-thirds of cultivated land faces serious phosphorus deficiency problems. In agricultural production practices, large amounts of phosphorus fertilizers are usually applied to meet the plant's demand for phosphorus. However, most of the phosphorus fertilizers applied to the soil will be fixed by the soil and converted into a form that is difficult for plants to directly absorb and utilize. Given that the production of phosphorus fertilizers is heavily dependent on phosphate rock, a non-renewable resource, its production cost is relatively high. Therefore, using phosphate-solubilizing bacteria to convert phosphorus forms in the soil and increase the effective phosphorus content in the soil has become an effective strategy to alleviate the problem of phosphorus deficiency. Summary of the invention

[0003] The present invention provides a phosphate-solubilizing bacterium Pantoea cypripedii, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 12, 2024, with a deposit number of CGMCC No.33054.

[0004] The present invention provides a bacterial agent containing the Pantoea cypripedii described above.

[0005] In some embodiments, the content of Pantoea cypripedii in the bacterial agent is not less than 10 6 cfu / mL or 10 6 cfu / g.

[0006] The present invention provides a method for converting insoluble inorganic phosphorus into soluble inorganic phosphorus, wherein the method comprises adding the Pantoea cypripedii described above or the bacterial agent described in claim 2 or 3 to a system containing insoluble inorganic phosphorus.

[0007] In some embodiments, the poorly soluble inorganic phosphorus includes calcium phosphate, iron phosphate, and aluminum phosphate.

[0008] The present invention provides a method for improving soil nutrient status, which comprises adding the above-mentioned Pantoeacypripedii or the above-mentioned bacterial agent into the soil.

[0009] 7. The method according to claim 6, wherein the improving soil nutrient status comprises at least one of (a) to (c):

[0010] (a) Increase the content of fast-acting potassium;

[0011] (b) increasing the content of available phosphorus;

[0012] (c) Increase the content of organic matter.

[0013] The present invention provides a method for promoting plant growth, comprising adding the Pantoea cypripedii or the bacterial agent mentioned above into a plant cultivation system.

[0014] In some embodiments, the plant comprises tomato, wheat, rice, corn, cotton, rapeseed, soybean, or pea.

[0015] In some embodiments, the promoting plant growth comprises at least one of (a) to (c):

[0016] (a) Increase plant height;

[0017] (b) increasing the circumference of the plant stem;

[0018] (c) Increase the fresh weight of the plant.

[0019] Beneficial effects:

[0020] 1. The present invention provides a strain of Pantoea cypripedii, which can form an obvious phosphate-solubilizing circle on an inorganic phosphorus solid culture medium, and its diameter ratio (D / d) is 2.87±0.08, indicating that it has a high phosphate-solubilizing ability, and the soluble phosphorus content in the supernatant of the culture medium fermentation broth reaches 589.64 mg / L.

[0021] 2. The present invention provides a method for promoting plant growth. After adding Pantoea cypripedii, the plant height and above-ground and underground biomass of crops can be increased.

[0022] 3. The present invention provides a method for improving soil nutrient status. After adding Pantoea cypripedii, the available phosphorus, available potassium and organic matter in the rhizosphere soil of crops can be increased.

[0023] Biomaterial Deposit

[0024] The PoPSB-2 bacteria provided by the present invention are classified and named Pantoea cypripedii, and have been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 12, 2024, with a deposit number of CGMCC No.33054, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is the colony morphology of strain PoPSB-2.

[0026] Figure 2 This is the phylogenetic tree of the 16S rRNA gene of strain PoPSB-2. DETAILED DESCRIPTION

[0027] The invention screened a phosphate-solubilizing bacterial strain PoPSB-2 which can convert insoluble phosphorus into soluble phosphorus from the rhizosphere soil of peony 'Fengdan'.

[0028] Since tomato is a plant that needs soluble phosphorus to support its growth and development, it is highly dependent on soluble phosphorus. Soluble phosphorus is a key factor in biological processes such as photosynthesis, energy metabolism and signal transduction in tomato plants. Soluble phosphorus in the soil can be absorbed by the tomato root system and converted into phosphorus compounds required by the plant, promoting the formation and ripening of the fruit. Therefore, in some exemplary embodiments of the present invention, the screened phosphate-solubilizing strain PoPSB-2 is added to the soil for culturing tomatoes. The results show that after adding the phosphate-solubilizing strain PoPSB-2 to cultivate tomatoes, the growth state of tomatoes is better, and the organic matter content in the soil is also higher. Therefore, the present invention also provides the use of the phosphate-solubilizing strain PoPSB-2 in improving soil nutrient conditions.

[0029] In this article, "alkaline nitrogen" refers to the form of nitrogen produced by the decomposition of organic matter in an alkaline environment. It mainly exists in soil organic matter and is one of the main forms of nitrogen in the soil. The release rate of alkaline nitrogen is slow and it needs to be decomposed by microorganisms to be converted into a form that can be absorbed by plants.

[0030] In this article, "available potassium" refers to potassium in the soil that is easily absorbed and utilized by crops, including soil solution potassium and soil exchangeable potassium. Available potassium accounts for 0.1% to 2% of the total potassium in the soil, of which soil solution potassium accounts for 1% to 2% of available potassium. Due to its low proportion, it is often included in exchangeable potassium. The content of available potassium is one of the important indicators to characterize the supply of soil potassium.

[0031] In this article, "available phosphorus", also known as fast-acting phosphorus, is the phosphorus component in the soil that can be absorbed by plants. It includes all water-soluble phosphorus, some adsorbed phosphorus and organic phosphorus, and some soils also include some precipitated phosphorus.

[0032] In this article, "organic matter" refers to substances in the soil that come from life, including soil microorganisms and soil animals and their secretions, as well as plant residues and plant secretions in the soil.

[0033] Example 1

[0034] 1.Prepare liquid and solid culture media containing insoluble inorganic phosphorus for the isolation and screening of phosphate-solubilizing bacteria.

[0035] PVF inorganic phosphorus liquid culture medium: glucose 10.0g, (NH4)2SO4 0.5g, NaCl 0.3g, MgSO4·7H2O0.3g, MnSO4·H2O 0.03g, KCl 0.3g, FeSO4·7H2O 0.03g, Ca3(PO4)2 5.0g, distilled water 1000mL, pH 6.8-7.2, sterilization at 121℃ for 20min.

[0036] PVF inorganic phosphate solid medium: Add 15.0 g agar to PVF inorganic phosphate liquid medium.

[0037] 2. The soil bacteria suspension was spread on PVF inorganic phosphorus solid culture medium by plate spreading method, and cultured in an incubator at 30°C until colonies grew. Colonies with phosphate-solubilizing circles were selected for purification to obtain strain PoPSB-2.

[0038] 3. Determination of the phosphate solubilization ability of strain PoPSB-2

[0039] After the strain PoPSB-2 was inoculated into PVF inorganic phosphorus solid medium, obvious phosphate solubilization circles appeared around the colonies after 1 to 2 days of cultivation, and these phosphate solubilization circles gradually expanded with the extension of the culture time, and basically stabilized on the seventh day. The phosphate solubilization ability of phosphate solubilizing bacteria was qualitatively analyzed by the diameter ratio (D / d) of the phosphate solubilization circle (D) and the single colony (d) on PVF inorganic phosphorus solid medium (Table 1).

[0040] Table 1

[0041] Strain name Diameter ratio (D / d) PoPSB-2 2.87±0.08

[0042] Take OD 600 =1.0, was inoculated into PVF inorganic phosphorus liquid culture medium at an inoculum size of 2% (v / v), and an equal volume of sterilized LB liquid culture medium was inoculated as a control. After constant temperature culture at 30°C for 7 days, the supernatant was collected after centrifugation, and the soluble inorganic phosphorus content in the supernatant was determined by molybdenum antimony antimicrobial spectrophotometry.

[0043] The results showed that the soluble inorganic phosphorus content in the fermentation supernatant of strain PoPSB-2 reached 589.64 mg / L, while the soluble inorganic phosphorus content of the control group was less than 10 mg / L.

[0044] 4. 16S rDNA sequencing and physiological and biochemical identification of PoPSB-2 strain

[0045] The efficient phosphate-solubilizing bacteria PoPSB-2 strain is rod-shaped and is a Gram-negative bacterium. On a solid medium containing soluble inorganic phosphorus, its colonies are round, milky white, flat in the middle, moist on the surface, with neat and transparent edges ( Figure 1 ).

[0046] The selected PoPSB-2 strain was cultured and sent to Beijing Qingke Biotechnology Co., Ltd. for 16S rDNA sequencing. After blast comparison, the sequence similarity was 96%, which was a known strain. The 16S rRNA gene phylogenetic tree was constructed using MEGA7.0 software, and it had a high homology with Pantoea cypripedii of the genus Pantoea ( Figure 2 ).

[0047] Refer to the Manual of Identification of Common Bacterial Systems for physiological and biochemical identification of bacteria, including catalase test, oxidase test, methyl red test, VP test, indole test, starch hydrolysis, glucose oxidation, sucrose fermentation, and gelatin liquefaction.

[0048] The results are shown in Table 2. The strain PoPSB-2 was catalase positive, and the other indicators were negative.

[0049] Thus, PoPSB-2 was identified as Pantoea cypripedii of the genus Pantoea.

[0050] Table 2

[0051] index PoPSB-2 index PoPSB-2 Catalase + glucose - Oxidase - sucrose + Methyl red + Starch hydrolysis - VP - Gelatin liquefaction + Indole - -

[0052] Example 2

[0053] The PoPSB-2 strain was inoculated into LB liquid culture medium to prepare a bacterial suspension for plant growth promotion experiments.

[0054] The selected phosphate-solubilizing bacteria PoPSB-2 were inoculated into 40 mL LB liquid medium and cultured overnight at 180 r / min and 30 °C. After 18 h, the bacterial solution was collected by centrifugation. 40 mL of sterile water was added to the bacterial solution to wash the bacteria twice. Finally, sterile water was added to adjust the concentration of the bacterial solution by 10 8 cfu / mL for future use.

[0055] Another strain of Bacillus subtilis WB800 (commonly used commercial phosphate-dissolving bacteria) was purchased for control, and the WB800 bacterial suspension was prepared using the same procedure.

[0056] Each pot was filled with 1.0 kg of test soil, and 30 tomato seedlings with basically the same growth were selected and planted in it. 40 mL of bacterial suspension (bacterial concentration 10 8cfu / mL), with 40mL sterile water or 40mL WB800 bacterial suspension (bacterial concentration 10 8 cfu / mL) was used as the control. Six replicates were set for both the treatment group and the control group. The root irrigation method was used for inoculation, and the inoculation was placed in an artificial climate incubator for culture. After 25 days of culture, samples were taken to determine the growth index of tomato seedlings and the nutrient content of the soil. The determination of alkaline nitrogen, available potassium, available phosphorus, and organic matter was referred to "LY / T 1228-2015", "NY / T 889-2004", "NY / T 1121.7-2014", and "NY / T1121.6-2006", respectively. The results are shown in Tables 3 and 4: Tomatoes treated with strain PoPSB-2 can grow better, and the organic matter content in the soil where tomatoes are cultured is higher.

[0057] Table 3 Tomato growth indicators:

[0058]

[0059] Table 4 Soil nutrient content:

[0060]

[0061] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of phosphate-solubilizing bacteria, Pantoea cypripedii, was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 12, 2024, with the deposit number CGMCC No.33054.

2. A bacterial agent, characterized in that Containing the Pantoea cypripedii according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The content of Pantoeacypripedii described in claim 1 in the bacterial agent is not less than 10 6 cfu / mL or 10 6 cfu / g.

4. A method for converting sparingly soluble inorganic phosphorus into soluble inorganic phosphorus, characterized in that: Pantoea cypripedii according to claim 1 or the bacterial agent according to claim 2 or 3 is added to a system containing poorly soluble inorganic phosphorus.

5. The method according to claim 4, characterized in that The poorly soluble inorganic phosphorus includes calcium phosphate, iron phosphate and aluminum phosphate.

6. A method for improving soil nutrient status, characterized in that: Pantoea cypripedii according to claim 1 or the bacterial agent according to claim 2 or 3 is added to the soil.

7. The method according to claim 6, characterized in that The improvement of soil nutrient status includes at least one of (a) to (c): (a) Increase the content of fast-acting potassium; (b) increasing the content of available phosphorus; (c) Increase the content of organic matter.

8. A method for promoting plant growth, characterized in that: The Pantoea cypripedii described in claim 1 or the bacterial agent described in claim 2 or 3 is added to a plant cultivation system.

9. The method according to claim 8, characterized in that The plants include tomato, wheat, rice, corn, cotton, rapeseed, soybean and pea.

10. The method according to claim 8 or 9, characterized in that: The plant growth promotion comprises at least one of (a) to (c): (a) Increase plant height; (b) increasing the circumference of the plant stem; (c) Increase the fresh weight of the plant.

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