Application of IAA-producing Klebsiella variicola in plants
By isolating and identifying high yield indole acetic acid and habitat Klebsiella with phosphorus-soluble and nitrogen-fixing functions, the problems of soil phosphorus deficiency and fertility reduction are solved, and the efficient utilization of phosphorus in the soil and the promotion of plant growth are achieved.
Patent Information
- Application Number
- CN202311496340.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-10
- Publication Date
- 2025-05-13
AI Technical Summary
The existing technology is difficult to effectively utilize the phosphorus in the soil, which leads to serious problems in soil phosphorus deficiency and affects plant growth. Long-term use of phosphorus fertilizer will lead to soil slab formation, acidification and fertility reduction.
A high yield of indoleacetic acid and high indoliacetic acid and phosphorus-soluble and nitrogen-fixing function was isolated and identified. This strain was able to survive in a high salt drought environment and significantly promote wheat growth.
This strain can not only increase the phosphorus utilization rate in the soil and promote plant growth, but also fix nitrogen, improve soil fertility, and reduce soil pollution.
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Abstract
Description
Technical Field
[0001] The invention relates to the fields of agricultural planting and microbiology, and in particular to an indoleacetic acid-producing and phosphate-solubilizing Klebsiella allochthonus and an application thereof. Background Art
[0002] Indoleacetic acid belongs to the indole class of compounds and is an endogenous auxin that is widely found in plants. It is also known as auxin and growth hormone. Auxin works from seedling to fruit maturity. Auxin controls the reversible red light inhibition of mesocotyl elongation in seedlings; when indoleacetic acid is transferred to the lower side of the branch, the branch becomes geotropic; when indoleacetic acid is transferred to the backlit side of the branch, the branch becomes phototropic; indoleacetic acid causes apical dominance; delays leaf senescence; auxin applied to leaves inhibits abscission, while auxin applied to the adaxial end of the abscission layer promotes abscission; auxin promotes flowering, induces the development of parthenocarpic fruits, and delays fruit maturity.
[0003] Phosphorus is an essential element for plant growth and development. It is not only a component of important chemical substances in plants, but also has the ability to improve plant resistance and adapt to the external environment. If there is a lack of phosphorus in the soil, it will seriously affect the life activities of plants. Therefore, phosphorus is the most important nutrient that restricts plant growth besides nitrogen. It is reported that about 74% of the cultivated land in my country is phosphorus-deficient, and the effective inorganic phosphorus available to plants accounts for only 2% to 3% of the total phosphorus in the soil. In order to ensure agricultural production, a large amount of phosphorus fertilizer needs to be invested. However, studies have shown that the utilization rate of phosphorus fertilizer in the season is only 10% to 20%, and the total utilization rate does not exceed 25%. The reason is that phosphorus applied to the soil is easily fixed by soil minerals in an adsorbed or exchanged state, forming insoluble phosphates and enriched in the soil. Long-term use of phosphorus fertilizers causes soil compaction, acidification and reduced fertility, resulting in serious water pollution and soil pollution. Therefore, reducing the use of chemical fertilizers and improving the utilization of soil phosphorus are of great significance to agricultural production and sustainable development.
[0004] Phosphate-solubilizing microorganisms can convert insoluble phosphorus in the soil into effective phosphorus that can be absorbed and utilized by plants. They exist in large quantities in the rhizosphere of crops and coexist with plants in a mutually beneficial way. They can secrete growth hormones, promote the development of plant roots, facilitate plant growth, and improve plant disease resistance. Therefore, the application of phosphate-solubilizing microorganisms can be an effective way to alleviate soil phosphorus deficiency. Summary of the invention
[0005] In order to overcome the problems and shortcomings of the prior art, the present invention provides a strain of Klebsiella variicola that has high indoleacetic acid production, phosphorus solubilization and nitrogen fixation. The present invention isolates a strain of high indoleacetic acid production from the rhizosphere soil of plants in the Gobi Desert of Xinjiang, which can survive in a high-salt and drought environment, and can colonize in the soil, significantly promoting wheat growth.
[0006] The indoleacetic acid-producing bacteria HHXJ2T-1 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2022, with the address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City. The deposit number is GDMCC No: 62759, and the classification name is Klebsiella variicola. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Figure 1 These are the results of a colorimetric test to identify the IAA production function of the HHXJ2T-1 strain.
[0008] Figure 2 This is a picture of the HHXJ2T-1 inorganic phosphorus dissolution functional flat plate test.
[0009] Figure 3 To identify the nitrogen-fixing function of the HHXJ2T-1 strain, a picture of the colony morphology on a solid Ashby nitrogen-free medium plate.
[0010] Figure 4 This is a comparison chart between wheat seed treated with HHXJ2T-1 strain and wheat in the control group. Implementation
[0011] The present invention is further described in detail below with reference to specific examples, which are provided only for explaining the present invention and do not limit the scope of use of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0012] LB medium: 10 g tryptone, 5 g yeast extract powder, 10 g sodium chloride.
[0013] Indoleacetic acid production medium: sucrose 5g, tryptone 5g, yeast powder 2.5g, sodium chloride 2.5, tryptophan 500mg.
[0014] Liquid inorganic phosphorus screening medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, tricalcium phosphate 5.0g, MgSO4·7H2O 0.3g, FeSO4·7H2O 30mg, MnSO4·H2O 30mg, water 1000mL, pH 7.0-7.5.
[0015] Solid inorganic phosphorus screening medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, tricalcium phosphate 5.0g, MgSO4·7H2O 0.3g, FeSO4·7H2O 30mg, MnSO4·H2O 30mg, water 1000mL, pH 7.0-7.5, agar powder 20g.
[0016] Ashby's nitrogen-free medium: mannitol 10 g, KH2PO4 0.2 g, NaCl 0.2 g, MgSO4 ·7H2O 0.2 g, CaSO 4 0.1 g, CaCO3 5.0 g, agar 20 g, water 1000 mL.
[0017] Salkowski colorimetric solution: 50ml 35% HClO4, 1ml 0.5mol / l FeCl3.
[0018] Example 1 Isolation and Screening of Klebsiella variicola 1. Initial screening of strains Soil samples were collected from rhizosphere soil of plants in the Gobi Desert of Xinjiang, placed in sterile bags and brought back to the laboratory, and stored at 4°C. Weigh 10 g of fresh soil sample and add it to 90 mL of sterile saline, shake at 180 r / min for 30 min, and place in a 28°C incubator for 24 h. After enrichment, take the supernatant and dilute it to 10 -3 , 10 -4 , 10 -5 Three gradients, take 100 μL of each dilution liquid and spread it on LB solid medium, Ashby's nitrogen-free solid medium and inorganic phosphorus screening solid medium, 3 parallels per group. Invert and culture in a 30-35℃ incubator for 2-5 days, observe the good growth and large colonies on the LB solid medium plate, observe the good growth and large colonies on the Ashby's nitrogen-free solid medium plate, and observe the transparent circle on the inorganic phosphorus screening solid medium plate. Pick the colonies with good observation results and transparent circles in the above culture media and repeatedly streak and purify them. After purification, pick a single colony and transfer it to the LB medium slant at 4℃ for storage.
[0019] Example 2 Determination of indoleacetic acid production capacity The Klebsiella exogena strain stored at 4°C in Example 1 was inoculated with 1-2 loops into LB medium, cultured at 32°C for 24h to obtain a primary seed solution, inoculated into a high-yield indoleacetic acid liquid medium at a 2% inoculation amount, set 3 replicates, and fermented on a shaking table at 35°C and a rotation speed of 200r / min for 48h to obtain an indoleacetic acid-producing fermentation liquid, centrifuged the fermentation liquid at 10000r / min for 5min, took the supernatant, added an equal volume of Salkowski colorimetric solution, and stood for 30min in the dark. The color was pink for positive, and indoleacetic acid could be secreted. The redder the color, the more secretion, and vice versa. The exogena Klebsiella was preliminarily screened to secrete indoleacetic acid, and the supernatant of the fermentation liquid was further subjected to liquid phase content determination to obtain a fermentation liquid with an indoleacetic acid content of 409mg / l.
[0020] Example 3 Determination of the ability to dissolve inorganic phosphorus The Klebsiella exogena preserved in Example 1 was inoculated into LB liquid medium, cultured in a shaking incubator at 35°C and 200 r / min for 24 h, 10 μL of the seed solution was inoculated onto a solid inorganic phosphorus screening medium plate by spot inoculation, and cultured in a 28°C incubator for 5 days. Figure 1 It can be observed that Klebsiella exogenous produces phosphate-dissolving circles on solid inorganic phosphorus screening culture medium plates, indicating that it has the ability to dissolve inorganic phosphorus.
[0021] Example 4 Determination of nitrogen fixation ability The Klebsiella exogena preserved in Example 1 was inoculated into LB liquid medium, cultured in a shaking incubator at 35°C and 200 r / min for 24 h, 10 μL of the seed solution was inoculated onto an Ashby nitrogen-free selection medium plate by spot inoculation, and cultured in a 28°C incubator for 5 days. Figure 2 It can be observed that Klebsiella exogena grows well on solid Ashby's nitrogen-free selective medium plates, indicating that it has nitrogen fixing ability.
[0022] Example 5 Plant growth promotion experiment In the experiment of promoting germination by mixing seeds with bacterial solution of Klebsiella in exogenous habitat, four treatments were set up, each treatment had four replicates, each replicate had 100 seeds, and the number of seedlings in each replicate was counted on the 10th day; the fresh weight of the aboveground part of each replicate was counted on the 10th day.
[0023] 1.1 Experimental treatment Table 1. Experimental treatments 2. Test results and analysis 2.1 Effects of different treatments on germination rate Table 2 lists the different repeated germination rates of each treatment. It can be seen from Table 2 that: The germination rates of the treatments with bacterial solution ratio of 1:2000 increased by 4.99%~6.65% compared with the control, and the germination rates of the treatments with bacterial solution ratio of 1:1000 and 1:500 increased by 5.54%~6.65% compared with the control, and the differences reached an extremely significant level.
[0024] Table 2. Effects of different treatments on germination rate (unit: 1) 3 Effects of different treatments on the fresh weight of aboveground parts Table 3 lists the fresh weight of the aboveground part of different replicates of each treatment. It can be seen from Table 3 that the fresh weight of the aboveground part of each bacterial solution treatment increased by 34.56%~56.01% compared with the control, the fresh weight of the aboveground part of the bacterial solution ratio of 1:2000 increased by 34.56% compared with the control, reaching a significant level, and the fresh weight of the aboveground part of the bacterial solution ratio of 1:1000 and 1:500 increased by 41.48%~56.01% compared with the control, reaching an extremely significant level.
[0025] Table 3. Effects of different treatments on the fresh weight of aboveground parts (unit: g) The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.
Claims
1. A strain of Klebsiella sp. HHXJ2T-1, which produces indoleacetic acid and solubilizes phosphate, characterized in that: It was isolated from the rhizosphere soil of plants in the Gobi Desert of Xinjiang, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 2, 2022. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The collection number is: GDMCC No. 62759, and the classification name is Enterobacter ludwigii strain.
2. An indoleacetic acid producing and phosphate solubilizing bacterium Klebsiella allochthonii HHXJ2T-1 according to claim 1, wherein the indoleacetic acid production reaches 409 mg / l.
3. The indoleacetic acid-producing and phosphate-solubilizing bacterium Klebsiella aquaticus HHXJ2T-1 according to claim 1 has the ability to dissolve inorganic phosphorus.
4. The indoleacetic acid-producing and phosphate-solubilizing bacterium Klebsiella allochthonii HHXJ2T-1 according to claim 1 has nitrogen fixing ability.
5. A use of the indoleacetic acid-producing and phosphate-solubilizing bacterium Klebsiella allochthonii HHXJ2T-1 according to claim 1 in promoting plant growth, characterized in that: The seeds are mixed with the bacterial solution of Klebsiella exogena to promote seed germination and seedling growth.