Application of Pantoea cypripedii rhizosphere in soil phosphorus solubilization and plant growth promotion

Abstract

This invention discloses the application of *Pantoea cypripedii* in the rhizosphere of peony in soil phosphorus solubilization and plant growth promotion, belonging to the field of microbial technology. The *Pantoea cypripedii* provided by this invention can effectively convert insoluble inorganic phosphorus into soluble inorganic phosphorus, promoting phosphorus utilization. Furthermore, this strain can adapt to the soil environment; adding it to the soil can improve phosphorus utilization efficiency and promote plant growth, while also improving soil nutrient status, thus possessing significant agricultural and environmental value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology. It relates to the application of *Pantoea cypripedii* in the rhizosphere of peony in soil phosphorus solubilization and plant growth promotion, specifically involving a *Pantoea cypripedii* strain and its application in soil phosphorus solubilization and plant growth promotion. Background Technology

[0002] In agricultural production, large amounts of phosphate fertilizer are typically applied to meet plants' phosphorus requirements. However, most of the phosphate fertilizer applied to the soil is fixed and transformed into forms that plants cannot directly absorb and utilize. Given that phosphate fertilizer production heavily relies on phosphate rock, a non-renewable resource, its production costs are relatively high. Therefore, utilizing phosphate-solubilizing bacteria to transform the forms of phosphorus in the soil and increase the available phosphorus content has become an effective strategy for alleviating phosphorus deficiency. Summary of the Invention

[0003] This invention provides a phosphate-solubilizing bacterium, Pantoea cypripedii, which was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33054.

[0004] This invention provides a fungal agent containing the aforementioned Pantoea cypripedii.

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

[0006] This invention provides a method for converting poorly soluble inorganic phosphorus into soluble inorganic phosphorus, wherein the method involves adding Pantoea cypripedii as described herein or the bacterial agent as described in claim 2 or 3 to a system containing poorly soluble inorganic phosphorus.

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

[0008] This invention provides a method for improving soil nutrient status by adding Pantotheacypripedii or the microbial agent described above to the soil.

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

[0010] (a) Increase the content of readily available potassium;

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

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

[0013] This invention provides a method for promoting plant growth by adding Pantoea cypripedii or the above-described microbial agent to the plant cultivation system.

[0014] In some embodiments, the plants include tomatoes, wheat, rice, corn, cotton, rapeseed, soybeans, and peas.

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

[0016] (a) Increase plant height;

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

[0018] (c) Increase plant fresh weight.

[0019] Beneficial effects:

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

[0021] 2. This invention provides a method for promoting plant growth. Adding Pantoea cypripedii can increase the plant height and above-ground and below-ground biomass of crops.

[0022] 3. This invention provides a method for improving soil nutrient status. Adding Pantoea cypripedii can increase the available phosphorus, available potassium, and organic matter in the rhizosphere soil of crops.

[0023] Preservation of biological materials

[0024] The PoPSB-2 bacterium provided by this invention, classified as Pantoea cypripedii, was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33054. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

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

[0026] Figure 2 Phylogenetic tree of the 16S rRNA gene of strain PoPSB-2. Detailed Implementation

[0027] This invention screened a phosphorus-solubilizing strain, PoPSB-2, from the rhizosphere soil of peony 'Fengdan' that can convert insoluble phosphorus into soluble phosphorus.

[0028] Tomatoes are plants that require soluble phosphorus to support their growth and development, and are highly dependent on it. Soluble phosphorus is a key factor in the biological processes of tomato plants, such as photosynthesis, energy metabolism, and signal transduction. Soluble phosphorus in the soil can be absorbed by the tomato roots and converted into phosphorus compounds needed by the plant, promoting fruit formation and ripening. Therefore, in some exemplary embodiments of the present invention, the screened phosphorus-solubilizing strain PoPSB-2 was added to the soil in which tomatoes were cultivated. The results showed that after adding the phosphorus-solubilizing strain PoPSB-2 to cultivate tomatoes, the tomatoes grew better, and the organic matter content in the soil was also higher. Therefore, the present invention also provides the use of the phosphorus-solubilizing strain PoPSB-2 in improving soil nutrient status.

[0029] In this article, "alkaline-available nitrogen" refers to the form of nitrogen produced by the decomposition of organic matter under alkaline conditions. It is mainly found in soil organic matter and is one of the main forms of nitrogen in the soil. The release rate of alkaline-available nitrogen is relatively slow, requiring microbial decomposition 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%. Due to its very low proportion, it is often included in exchangeable potassium. The content of available potassium is one of the important indicators characterizing the potassium supply status of the soil.

[0031] In this article, "available phosphorus," also known as readily available phosphorus, refers to the phosphorus components in the soil that can be absorbed by plants. It includes all water-soluble phosphorus, some adsorbed phosphorus, and organic phosphorus; some soils also contain certain precipitated phosphorus.

[0032] In this article, "organic matter" refers to substances in the soil that originate 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, MgSO4·7H2O 0.3g, MnSO4·H2O 0.03g, KCl 0.3g, FeSO4·7H2O 0.03g, Ca3(PO4)2 5.0g, Distilled Water 1000mL, pH 6.8-7.2, Sterilize at 121℃ for 20min.

[0036] PVF Inorganic Phosphorus Solid Medium: Add 15.0g of agar to PVF Inorganic Phosphorus Liquid Medium.

[0037] 2. Using the plate coating method, the soil bacterial suspension was spread on PVF inorganic phosphorus solid medium and cultured at 30°C in an incubator until colonies grew. Colonies with phosphate-solubilizing zones were selected for purification to obtain strain PoPSB-2.

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

[0039] After inoculating strain PoPSB-2 onto PVF inorganic phosphorus solid medium, distinct phosphate-solubilizing zones appeared around the colonies after 1 to 2 days of cultivation. These zones gradually expanded with increasing cultivation time, stabilizing by the seventh day. The phosphate-solubilizing ability of the phosphate-solubilizing bacteria was qualitatively analyzed using the ratio of the diameter of the phosphate-solubilizing zone (D) to the diameter of a single colony (d) on the PVF inorganic phosphorus solid medium (D / d) (Table 1).

[0040] Table 1

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

[0042] Take OD 600 Fresh PoPSB-2 bacterial suspension with a concentration of 1.0 was inoculated into PVF inorganic phosphorus liquid medium at an inoculum volume of 2% (v / v), with an equal volume of sterilized LB liquid medium as a control. After incubation at 30℃ for 7 days, the supernatant was collected after centrifugation, and the soluble inorganic phosphorus content in the supernatant was determined by molybdenum antimony 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 in the control group was less than 10 mg / L.

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

[0045] The highly efficient phosphate-solubilizing bacterium PoPSB-2 is rod-shaped and Gram-negative. On soluble inorganic phosphorus solid medium, its colonies are characterized by being round, milky white, flat in the middle, moist on the surface, and 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. BLAST comparison showed a 96% sequence similarity, confirming it as a known strain. A phylogenetic tree of the 16S rRNA gene was constructed using MEGA 7.0 software, showing high homology with *Pantoea cypripedii* of the *Pantoea* genus. Figure 2 ).

[0047] Physiological and biochemical identification of bacteria was performed in accordance with the "Handbook for Systematic Identification of Common 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 positive for catalase, while all other indicators were negative.

[0049] Therefore, 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 medium to prepare a bacterial suspension, which was used for plant growth promotion experiments.

[0054] The selected phosphate-solubilizing bacterium PoPSB-2 was inoculated into 40 mL of LB liquid medium and cultured overnight at 30 °C with a speed of 180 rpm. After 18 h, the bacterial culture was collected by centrifugation. The bacterial cells were washed twice with 40 mL of sterile water. Finally, sterile water was added to adjust the bacterial concentration to 10. 8 CFU / mL available for use.

[0055] Another control strain of Bacillus subtilis WB800 (a commonly used commercial phosphorus-dissolving bacterium) was purchased, and a WB800 bacterial suspension was prepared using the same steps.

[0056] Each pot was filled with 1.0 kg of test soil, and 30 tomato seedlings of similar growth were planted in it. The treatment group received 40 mL of bacterial suspension (bacterial concentration 10). 8 cfu / mL), with 40 mL of sterile water or 40 mL of WB800 bacterial suspension (bacterial concentration 10). 8The control group (cfu / mL) was used as the reference. Both the treatment and control groups had six replicates. Inoculation was performed using the root drenching method, and the plants were then placed in an artificial climate incubator. After 25 days of culture, samples were taken to determine tomato seedling growth indicators and soil nutrient content. The determination of available nitrogen, available potassium, available phosphorus, and organic matter followed the standards 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 grew better, and the soil in which the tomatoes were cultured had a higher organic matter content.

[0057] Table 3 Tomato growth indicators:

[0058]

[0059] Table 4 Soil nutrient content:

[0060]

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A phosphate-solubilizing bacterium, Pantoea cypripedii, was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33054.

2. A microbial agent, characterized in that, Contains Pantoea cypripedii as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The content of Pantothecia pripedii as described in claim 1 in the fungal 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, Add the Pantoea cypripedii of claim 1 or the fungal agent of claim 2 or 3 to a system containing poorly soluble inorganic phosphorus.

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

6. A method for improving soil nutrient status, characterized in that, Add Pantoea cypripedii as described in claim 1 or the microbial agent as described in claim 2 or 3 to the soil; the improvement of soil nutrient status is at least one of the following (a) to (c): (a) Increase the content of readily available potassium; (b) Increase the content of available phosphorus; (c) Increase the content of organic matter.

7. A method for promoting plant growth, characterized in that, The plant culture system is supplemented with the Pantoea cypripedii of claim 1 or the microbial agent of claim 2 or 3; the plant is tomato; the promotion of plant growth is to increase plant height and / or increase plant fresh weight.

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