Phosphorus-solubilizing bacteria as well as fungicide and application thereof

Abstract

The invention provides a phosphate solubilizing bacterium as well as a fungicide and application thereof. The phosphate solubilizing bacteria are pantoea septicum, and the preservation number of the phosphate solubilizing bacteria is CGMCC (China General Microbiological Culture Collection Center) The pantoea septicum can significantly improve the plant yield, biomass and phosphorus absorption amount. The pantoea septicum has the advantages of being easy to breed, high in phosphate solubilizing capacity and the like, and has a good application prospect.

Description

[0001] The present invention claims the priority of a prior application with patent application number 202510008652.8 filed with the State Intellectual Property Office of China on January 3, 2025, and invention title “A Phosphate-Solubilizing Bacteria and Its Bacterial Agent and Application”. The entire text of the prior application is incorporated into the present invention by reference. Technical Field

[0002] The invention relates to the technical field of bacteria, and in particular to a phosphate-solubilizing bacterium and a bacterial agent and application thereof. Background Art

[0003] Phosphorus is the second largest nutrient required for crop growth. Phosphorus is involved in all important metabolic processes of plants. However, most of the phosphorus in the soil cannot be absorbed and utilized by crops. Phosphorus combines with calcium, iron, aluminum and other ions in the soil to form insoluble phosphates that cannot be absorbed and utilized by plants. The utilization rate of phosphorus fertilizer in the season is generally 5% to 15%. Excessive application of phosphorus fertilizer not only affects soil health and the stability of underground ecosystems, but also causes serious environmental problems. How to convert phosphorus in the soil that is difficult for plants to absorb and utilize into an absorbable and utilizeable form of phosphorus has become an important issue that needs to be urgently solved in current agricultural production practices.

[0004] Phosphate-solubilizing microorganisms can secrete small molecular weight organic acids, which can increase the pH value of the soil, promote the dissolution of insoluble phosphates, and improve soil phosphorus utilization. Therefore, using the microbial action of phosphate-solubilizing bacteria to improve phosphorus utilization is a hot topic in current research.

[0005] However, the efficiency of phosphate-solubilizing bacteria in soil is generally low, especially when the insoluble phosphate (such as apatite) is high, and it is unable to quickly release enough phosphorus to meet the needs of crop growth. In addition, phosphate-solubilizing bacteria compete with local microorganisms in the soil for nutrients and living space in the natural environment, and their colonization ability is weak, and they are easily replaced by other indigenous microbial communities. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a phosphate-solubilizing bacterium and a bacterial agent thereof and application thereof, which can be used to activate insoluble phosphorus in soil and promote plant growth.

[0007] In one aspect, the present invention provides a Pantoea septicagensis, whose deposit number is CGMCC No.33333.

[0008] According to an embodiment of the present invention, the 16S rDNA sequence of Pantoea septica is shown as SEQ ID NO:1.

[0009] According to an embodiment of the present invention, the Pantoea septica has the ability to solubilize phosphate. Further preferably, the Pantoea septica has the ability to solubilize poorly soluble phosphorus.

[0010] According to an embodiment of the present invention, the Pantoea septica can increase the yield, biomass and phosphorus absorption of plants. In one embodiment of the present invention, the plants are cotton and corn.

[0011] The present invention screened out a highly efficient phosphate-solubilizing strain from the rhizosphere soil of the saltwort salsa plants in the saline-alkali land of Manas County, and identified it as Pantoea septica. The strain was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee CGMCC on January 8, 2025, with a deposit number of CGMCC No.33333.

[0012] According to an implementation plan of the present invention, the geographical location of the saline-alkali land in Manas County is: 43°09′ north latitude, 86°15′ east longitude.

[0013] The second aspect of the present invention provides a bacterial agent and / or biological fertilizer, including the above-mentioned Pantoea septicagensis.

[0014] According to an embodiment of the present invention, the effective viable bacteria count of Pantoea septica in the bacterial agent and / or biological fertilizer is approximately 1×10 4 ~1×10 12 CFU / mL. Preferably, the effective viable count of Pantoea septica in the bacterial agent and / or biological fertilizer reaches 1.2×10 9 CFU / mL.

[0015] According to an embodiment of the present invention, the bacterial agent and / or biological fertilizer includes active or inactivated Pantoea septicagensis.

[0016] According to an embodiment of the present invention, the bacterial agent and / or biofertilizer includes one or more of a suspension, a culture solution and bacterial cells of Pantoea septicagensis.

[0017] According to an embodiment of the present invention, the bacterial agent and / or biological fertilizer further comprises a cell-free culture filtrate of Pantoea septica.

[0018] According to an embodiment of the present invention, the bacterial agent and / or biofertilizer further comprises a derivative of Pantoea septica. In one embodiment of the present invention, the derivative is selected from metabolites, enzymes, cell structure components (e.g., cell walls or components thereof), exopolysaccharides, bacteriocins or any combination thereof.

[0019] According to an embodiment of the present invention, the microbial agent and / or biofertilizer further comprises pharmaceutically acceptable adjuvants. Preferably, the adjuvants comprise film formers, pesticides, dispersants, protective agents, colorants, nutrients, preservatives, antioxidants and any combination thereof.

[0020] According to an embodiment of the present invention, the film-forming agent includes but is not limited to polyvinyl alcohol (PVA), gelatin, hydroxypropyl methylcellulose (HPMC), sodium alginate, chitosan. The insecticide includes but is not limited to thiamethoxam (Imidacloprid), bifenthrin (Deltamethrin), pyrethrins (Pyrethrins), chlorantraniliprole (Chlorantraniliprole). The dispersant includes but is not limited to polyvinyl pyrrolidone (PVP), sodium dodecylbenzenesulfonate (Sodium dodecylbenzenesulfonate), xanthan gum (Xanthan gum). The protective agent includes but is not limited to glycerol, propylene glycol (Propylene Glycol), vitamin C (ascorbic acid). The colorant includes but is not limited to plant dyes (such as anthocyanins), artificial pigments (such as amaranth, brilliant blue, etc.). The nutrient includes but is not limited to amino acids, vitamins, minerals (such as calcium, phosphorus, potassium, etc.), and sugars. The preservative includes but is not limited to benzoic acid, propionic acid, potassium sorbate. The antioxidant includes but is not limited to vitamin E (tocopherol), ascorbic acid, sodium thiosulfate, etc.

[0021] According to an embodiment of the present invention, the microbial agent and / or biological fertilizer can be in any dosage form, including emulsion, pill, tablet, powder, liquid or gel form. In a specific embodiment of the present invention, the microbial agent and / or biological fertilizer is an emulsion or a powder.

[0022] According to a third aspect of the present invention, a method for preparing a bacterial agent and / or a bio-fertilizer is provided, wherein the above-mentioned Pantoea septica is inoculated into a culture medium for culturing to obtain a culture solution, and the culture solution is centrifuged and then resuspended to obtain the bacterial agent and / or the bio-fertilizer.

[0023] According to an embodiment of the present invention, the culture temperature is 28-30°C; the culture is carried out until the logarithmic growth phase of Pantoea septica; and the culture pH is 6.8-7.2.

[0024] According to an embodiment of the present invention, the culture medium comprises LB culture medium. In one embodiment of the present invention, the LB culture medium comprises 10 g of peptone, 10 g of sodium chloride, 5 g of yeast extract powder, and water is added to make the volume to 1000 ml, and the pH value is adjusted to 7.0. In one embodiment of the present invention, 20 g of agar is added per liter of LB liquid culture medium to prepare LB solid culture medium.

[0025] In a fourth aspect, the present invention provides the use of the above-mentioned Pantoea septica, the bacterial agent and / or the biological fertilizer in one or more of the following 1) to 6);

[0026] 1) Increase the effective phosphorus content of the planting medium;

[0027] 2) Increase plant yield;

[0028] 3) Increase the aboveground biomass of plants;

[0029] 4) Improve plant phosphorus absorption;

[0030] 5) Promote plant growth;

[0031] 6) Degrade the insoluble phosphorus in the planting medium.

[0032] According to an embodiment of the present invention, the poorly soluble phosphorus includes, but is not limited to, one or more of tricalcium phosphate, iron phosphate, aluminum phosphate, lecithin and calcium phytate.

[0033] According to an embodiment of the present invention, the application includes mixing the Pantoea septica, the bacterial agent and / or the biological fertilizer with a planting matrix; or the application includes root irrigation and / or foliar spraying.

[0034] A fifth aspect of the present invention provides a method for increasing the available phosphorus content in soil, comprising the following steps: applying the above-mentioned Pantoea septicagen to the soil.

[0035] According to an embodiment of the present invention, the method further comprises the following steps: applying the above-mentioned Pantoea septica, a carbon source and a nitrogen source into the soil, wherein the nitrogen source comprises urea; and the carbon source comprises one or more of lactose, xylose and glucose.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention can not only promote the release of soil phosphorus by adding phosphate-solubilizing bacteria to glucose, but also improve soil quality, improve soil fertility, and promote plant growth. By applying it to cotton and corn through field experiments, the yield, biomass and phosphorus absorption of cotton and corn can be significantly increased. The septicacid Pantoea in the present invention has the advantages of easy reproduction and strong phosphate-solubilizing ability, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the colony morphology of Pantoea septica in LB medium plates.

[0039] Figure 2 This is the formation of the phosphate-dissolving circle of Pantoea septica. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0041] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0042] Phosphate solubilizing ability refers to the ability of a strain to convert insoluble phosphorus into soluble phosphorus. In one embodiment of the present invention, a solid culture medium containing insoluble phosphorus (such as tricalcium phosphate, aluminum phosphate, iron phosphate, etc.) is used to observe whether a transparent circle appears, and the size of the phosphate solubilizing ability is calculated by measuring the diameter of the transparent circle and the diameter of the colony. The calculation formula is: E=D1 / D2 (E: phosphate solubilizing ability, D1: transparent circle diameter, D2: strain diameter). The phosphate solubilizing medium used is PVK medium (Pikovskaya's Medium), which contains tricalcium phosphate as a source of insoluble phosphorus. The formula example (per liter) is shown below. Inoculate Pantoea septicagen into the solid culture medium. Culture at a suitable temperature (such as 28-30°C) for 3-7 days.

[0043] Aboveground biomass refers to the total mass of the plant body above the soil surface, usually including stems, leaves, branches, flowers, fruits and other components. Its quantification involves the dry weight of the plant, often per unit area (such as per hectare or per square meter), characterizing the biomass stock of the plant community. It reflects the productivity and health of the plant community, and is of great significance for evaluating the growth status of plants, the carbon storage capacity of the ecosystem and productivity.

[0044] The culture medium and culture method used by phosphate-solubilizing bacteria of the present invention are as follows:

[0045] LB liquid medium: peptone 10g, sodium chloride 10g, yeast extract powder 5g, add water to 1000ml, adjust the pH value to 7.0. LB solid medium is composed of the following mass fraction ratios: add 20g agar per liter of LB liquid medium.

[0046] Pikovasky's phosphate solubilizing medium:

[0047] Glucose 10g, (NH4)2SO4 0.5g, MgSO4·7H2O 0.1g, yeast extract 0.5g, KCl 0.2g, NaCl0.2g, FeSO4·7H2O 0.002g, MnSO4·7H2O 0.002g, Ca3(PO4)25.0g, bromophenol blue 0.025g, agar 20g, distilled water 1L, pH 7.0, sterilization for 15min, 121℃.

[0048] Preparation of phosphate-solubilizing bacteria fermentation broth:

[0049] Pick a single colony of purified phosphate-solubilizing bacteria and inoculate it into a 250mL Erlenmeyer flask containing 100mL LB culture medium, and culture it at 150r / min and 30℃ for 24h. Then, aspirate 2mL of culture medium and transfer it to a 500mL Erlenmeyer flask containing 200mL LB culture medium, and culture it at 150r / min and 30℃ for 48h.

[0050] Example 1 Screening, separation and purification of phosphate-solubilizing strains

[0051] 1. Screening of phosphate-solubilizing strains

[0052] Weigh 1g of rhizosphere soil sample of Suaeda salsa in saline-alkali land of Manas County, Xinjiang into a sterilized conical bottle, add 99ml of sterile water, and prepare 10 -2 The suspension was placed in a shaker at 25°C and 180 r / min, shaken for 30 min, taken out, sprayed with 75% alcohol for disinfection, placed in a clean bench, and diluted step by step to 10 -3 , 10 -4 , 10 -5 Suspension, take 100 μL of bacterial solution of different concentrations, add it to the insoluble inorganic phosphorus solid culture medium, and evenly spread it on the inorganic phosphorus solid culture medium with a triangular coating stick. Make three parallels for each concentration, place it in a 30℃ constant temperature incubator and culture it for 5-7 days, observe the growth of the colony, streak the colony with the phosphate-soluble ring on the LB solid culture medium again to purify it to obtain a single strain, and store the purified strain in LB liquid culture medium containing 30% glycerol and store it at -80℃;

[0053] 2. Identification of phosphate-solubilizing strains

[0054] (1) Morphological identification

[0055] Strain morphology reference Figure 1 The characteristics of the colonies are as follows: round, opaque yellow, smooth and moist, without halo, small and convex.

[0056] (2) 16S rDNA molecule determination (SEQ ID NO: 1)

[0057] Identification of phosphate-solubilizing bacteria: The activated strains were sent to Beijing Novogene Technology Co., Ltd. for sequencing analysis. The sequence results are as follows:

[0058]

[0059] Through 16S rDNA sequence analysis and comparison with the NCBI database, the strain was identified as Pantoeaseptica and numbered PS-1. Pantoea septica was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration CGMCC on January 8, 2025, with the deposit number: CGMCC No.33333.

[0060] Example 2 Phosphate-solubilizing ability detection of phosphate-solubilizing strains

[0061] The strain was inoculated on Pikovasky's phosphate solubilizing medium plate, and then cultured at 30℃ for 7 days to observe whether a transparent circle appeared. If a transparent circle appeared, it was a phosphate-soluble strain. The diameter of the transparent circle and the colony diameter were measured, and the phosphate solubilizing ability was calculated: E = D1 / D2 (E: phosphate solubilizing ability, D1: transparent circle diameter, D2: strain diameter). Each strain was repeated 3 times, and the average value of the phosphate solubilizing ability was used as an indicator of the phosphate solubilizing ability. The results of phosphate solubilization are shown in Figure 2 The solubility of the strains for insoluble phosphorus is shown in Table 1.

[0062] Table 1 Analysis of phosphate solubilization characteristics of strain Pantoea septica

[0063] D1 / cm D2 / cm E Calcium phosphate 3.50 0.80 4.38

[0064] It can be seen that this strain of Pantoea septica has a significant phosphate-solubilizing effect.

[0065] Example 3 Application of phosphate-solubilizing strains

[0066] The bacterial solution of Pantoea septica was prepared according to the above method and applied to cotton and corn at 8.33 mL / m 2 on the 56th, 63rd, 70th, 80th and 94th day after cotton sowing and on the 44th, 51st, 58th, 68th and 82nd day after corn sowing. 2 .

[0067] The results show that the phosphate-solubilizing bacteria in the invention can significantly increase the yield, aboveground biomass, and phosphorus absorption of cotton and corn, as follows:

[0068] Table 2 Experimental treatment methods

[0069]

[0070] Table 3 Effects of phosphate-solubilizing bacteria on the growth and yield of cotton in the field

[0071]

[0072]

[0073] Table 4 Effects of phosphate-solubilizing bacteria on growth and yield of corn in the field

[0074]

[0075] It can be seen that after applying the phosphate-solubilizing bacteria of the present invention, the yield of cotton and corn increased by 9.82% and 13.54% respectively, the aboveground biomass of cotton and corn increased by 32.97% and 14.63% respectively, and the phosphorus absorption of cotton and corn increased by 23.21% and 23.93% respectively compared with not applying the phosphate-solubilizing bacteria.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A Pantoea septicagensis, characterized in that: Its deposit number is CGMCC No.33333.

2. The Pantoea septica according to claim 1, characterized in that: The 16S rDNA sequence of Pantoea septica is shown in SEQ ID NO:

1.

3. The Pantoea septica according to claim 1, characterized in that: The septicacid Pantoea has one or more of the following characteristics: 1) Phosphate solubilization ability. More preferably, the Pantoea septicagensis has the ability to solubilize poorly soluble phosphorus; 2) Increase plant yield; 3) Increase plant biomass; 4) Improve the phosphorus absorption of plants.

4. A bacterial agent and / or biological fertilizer, characterized in that: The invention comprises the Pantoea septica according to any one of claims 1 to 3.

5. A bacterial agent and / or biological fertilizer according to claim 4, characterized in that: The effective viable bacteria count of Pantoea septica in the bacterial agent and / or biological fertilizer is about 1×10 4 ~1×10 12 / dose. Further preferably, the therapeutically effective amount of Pantoea septica in the bacterial agent is approximately 1.2×10 9 / dose.

6. A bacterial agent and / or biological fertilizer according to claim 4, characterized in that: The bacterial agent and / or biological fertilizer includes active or inactivated Pantoea septica; Preferably, the bacterial agent and / or biological fertilizer comprises one or more of a suspension, a culture solution and bacterial cells of Pantoea septica; according to an embodiment of the present invention, the bacterial agent and / or biological fertilizer further comprises a cell-free culture filtrate of Pantoea septica; Preferably, the bacterial agent and / or biofertilizer further comprises a derivative of Pantoea septica; more preferably, the derivative is selected from metabolites, enzymes, cell structure components, exopolysaccharides, bacteriocins or any combination thereof; Preferably, the microbial agent and / or biofertilizer further comprises pharmaceutically acceptable adjuvants, more preferably, the adjuvants comprise film formers, pesticides, dispersants, protective agents, colorants, nutrients, preservatives, antioxidants and any combination thereof.

7. A method for preparing a microbial agent and / or a biofertilizer, characterized in that: Inoculating the Pantoea septica according to any one of claims 1 to 3 into a culture medium for culturing to obtain a culture solution, and resuspending the culture solution after centrifugation to obtain a bacterial agent and / or a biofertilizer; Preferably, the culture temperature is 28-30°C; the culture pH is 6.8-7.2; Preferably, the culture medium comprises LB medium.

8. Use of the Pantoea septica according to any one of claims 1 to 3, the bacterial agent and / or the biofertilizer according to any one of claims 4 to 6 in one or more of the following 1) to 6); 1) Increase the effective phosphorus content of the planting medium; 2) Increase plant yield; 3) Increase the aboveground biomass of plants; 4) Improve plant phosphorus absorption; 5) Promote plant growth; 6) Degrade the insoluble phosphorus in the planting medium.

9. The use according to claim 8, characterized in that: The insoluble phosphorus includes but is not limited to one or more of tricalcium phosphate, iron phosphate, aluminum phosphate, lecithin and calcium phytate; Preferably, the application includes mixing the Pantoea septica, the bacterial agent and / or the biological fertilizer with a planting matrix; or the application includes root irrigation and / or foliar spraying.

10. A method for increasing the available phosphorus content in soil, characterized in that: The following steps are involved: The above-mentioned Pantoea septicagen was applied to the soil.

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