Soybean rhizosphere growth promoting strain and application thereof

Abstract

The application provides a soybean rhizosphere growth promoting strain, named Acinetobacter sp. A11, which is preserved in the China Center for Type Culture Collection, has a preservation number of CCTCC NO: M20242725, and a preservation date of December 4, 2024; and a nucleotide sequence of 16S rRNA is shown as SEQ ID NO. 1. The application also provides application of the soybean rhizosphere growth promoting strain, which is used for nitrogen fixation, organic phosphorus dissolution, inorganic phosphorus dissolution and potassium dissolution; and can also be used for promoting soybean growth. The strain has the functions of strong nitrogen fixation, organic phosphorus dissolution, inorganic phosphorus dissolution and potassium dissolution, and provides high-quality strain resources for development of microbial fertilizer and microbial agent, and has important significance for soil improvement, crop yield improvement and realization of sustainable development of ecological environment.

Description

Technical Field

[0001] This invention belongs to the field of soybean rhizosphere growth-promoting bacteria technology, specifically relating to a soybean rhizosphere growth-promoting strain and its application. Background Technology

[0002] Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that live in the soil near or on plant roots, significantly promoting plant growth and development, enhancing their absorption and utilization of mineral nutrients, alleviating environmental stress, and improving their resistance to pests and diseases. The rhizosphere, as the direct interface for material and energy exchange between plants and soil, is the most active region for microbial activity. In recent years, the enormous potential of the rhizosphere microbiome has been confirmed in related studies. The most significant characteristic of these microorganisms is their functional diversity; they not only promote plant growth and development directly or indirectly by promoting nutrient cycling, synthesizing and regulating plant hormones, and alleviating environmental stress, but also maintain soil stability and improve the rhizosphere microbial network. With the development of modern agriculture, ecological crop cultivation has gradually gained attention. In modern agricultural practice, the long-term use of chemical fertilizers and pesticides has led to increasingly serious problems such as soil compaction, soil salinization, heavy metal pollution, and the destruction of soil microbial diversity. Microbial fertilizers, as a new type of fertilizer, have rhizosphere growth-promoting bacteria as their main functional component. These bacteria can fix nitrogen in the atmosphere and release fixed phosphorus and potassium elements in the soil, allowing the nutrient potential of the environment to be fully utilized and creating a good soil microbial environment for crop growth. They are of great significance in reducing the amount of chemical fertilizers used, reducing environmental pollution, and improving the quality of crops. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a multifunctional soybean rhizosphere growth-promoting strain and its application, which addresses the shortcomings of the prior art. This strain has strong nitrogen fixation, organic and inorganic phosphorus solubilization, and potassium solubilization functions. It not only enriches the local microbial growth-promoting strain resource bank in Xinjiang, but also lays an important research foundation for the development and application of microbial fertilizers and agents related to soybean planting in Xinjiang. It is of great significance for soil improvement, increasing crop yield, and achieving sustainable development of the ecological environment.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a soybean rhizosphere growth-promoting strain, wherein the soybean rhizosphere growth-promoting strain is named Acinetobactersp. A11 is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M20242725 and deposit date of December 4, 2024; the nucleotide sequence of the 16S rRNA of the soybean rhizosphere growth-promoting strain is shown in SEQ ID NO. 1.

[0005] The application of the above-mentioned soybean rhizosphere growth-promoting strain of the present invention is as follows: the soybean rhizosphere growth-promoting strain is used for nitrogen fixation, dissolution of organic phosphorus, dissolution of inorganic phosphorus, and potassium solubilization; the soybean rhizosphere growth-promoting strain is used to promote soybean growth.

[0006] Preferably, the soybean rhizosphere growth-promoting strain is used to improve the germination rate of soybean seeds and increase the length of soybean embryos.

[0007] Compared with the prior art, the present invention has the following advantages: This invention relates to a soybean rhizosphere growth-promoting strain developed from indigenous rhizosphere growth-promoting bacteria found in the arid environment of Xinjiang. This not only enriches the local microbial growth-promoting bacteria resource bank in Xinjiang but also lays an important research foundation for the development and application of microbial fertilizers and inoculants related to soybean cultivation in Xinjiang. The application of this strain provides important scientific evidence for alleviating plant environmental stress, promoting plant growth and development, improving crop quality, maintaining soil stability, improving the soil rhizosphere environment, reducing fertilizer use, and mitigating environmental pollution.

[0008] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0009] Figure 1 This is a determination of the nitrogen fixation function of strain A11 in Example 1 of the present invention.

[0010] Figure 2 This is a determination of the organophosphate dissolution ability of strain A11 in Example 1 of the present invention.

[0011] Figure 3 This is a determination of the inorganic phosphorus solubility of strain A11 in Example 1 of the present invention.

[0012] Figure 4 This refers to the determination of the potassium solubilizing ability of strain A11 in Example 1 of the present invention.

[0013] Figure 5 This is the colony morphology of strain A11 in Example 1 of the present invention on LB solid medium.

[0014] Figure 6 This is a growth curve of strain A11 in Example 1 of the present invention.

[0015] Figure 7This is the Gram staining result of strain A11 under an optical microscope in Example 1 of the present invention.

[0016] Figure 8 This is a hemolysis test diagram of strain A11 in Example 1 of the present invention.

[0017] Figure 9 This is a phylogenetic tree diagram of strain A11 based on 16S rRNA in Example 1 of the present invention.

[0018] Figure 10 This is a diagram of the soybean seed germination rate test using strain A11 in Example 1 of the present invention. Detailed Implementation Example 1

[0019] The formulations of the culture media used in this embodiment are as follows: LB medium: tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar 18 g / L, pH 7.0-7.2.

[0020] Ashby's nitrogen-free medium: mannitol 10.0 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaCO3 5 g / L, CaSO4 0.1 g / L, pH 6.8-7.0.

[0021] Monkina Organic Phosphorus Medium (for functional verification of organic phosphorus dissolution): MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, CaCO3 5.0 g / L, glucose 10.0 g / L, (NH4)2SO4 0.5 g / L, lecithin 0.2 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, yeast extract 0.4 g / L, agar 18 g / L, pH 7.0-7.2.

[0022] Inorganic phosphorus medium: glucose 10.0 g / L, MgCl2·6H2O 5.0 g / L, MgSO4·7H2O 0.25 g / L, KCl 0.2 g / L, (NH4)2SO4 0.1 g / L, Ca3(PO4)2 5.0 g / L, pH 6.8-7.0.

[0023] Potassium-solubilizing medium: glucose 10 g / L, Na2HPO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4 0.2 g / L, CaCO3 5 g / L, potassium feldspar 2.5 g / L, agar 18 g / L, pH 7.0-7.2.

[0024] Acinetobacter The isolation, purification, and identification of strain sp. A11 are as follows: (a) Isolation and purification of strains Soil samples for bacterial strain isolation were collected from wild soybean plant distribution areas in Burqin County, Altay Prefecture, Xinjiang Uygur Autonomous Region (E 87.29818675, N 47.81434647). This planting area is characterized by sandy loam soil with poor texture and very low organic matter and nutrient content. Rhizosphere soil (including nodule soil) was collected and diluted with sterile water to a concentration of 10. -4 Spread the culture onto LB medium. Incubate at 28 °C, pick single colonies of different colors and morphologies, and repeatedly streak to isolate until purified.

[0025] (II) Determination of the growth-promoting function of the strain The isolated strains (A-5-1, A-5-2, A-5-3, A-5-4, A9, A11, A-11-1-2) were inoculated onto different functional media. The specific determination methods are as shown in (1)-(4). A strain A11 with nitrogen fixation, organic phosphorus solubilization, and potassium solubilization functions was obtained. The diameter of its organic phosphorus solubilization transparent zone was 0.5 mm, and the diameter of its potassium solubilization transparent zone was 1.3 mm. The specific results are shown in Table 1. Figure 1-4 As shown.

[0026] (1) Determination of nitrogen fixation function of bacterial strains: The nitrogen fixation capacity of bacterial isolates was determined using Ashby nitrogen-free medium. The purified strains were inoculated onto Ashby nitrogen-free medium and cultured at 28 °C for 7 days. The ability of the bacteria to fix nitrogen was determined by whether they could grow normally on the Ashby nitrogen-free medium, and the nitrogen fixation capacity was judged based on the colony diameter. The results of the nitrogen fixation experiment are as follows: Figure 1 As shown, strain A11 can grow normally on Ashby nitrogen-free medium after being cultured at a constant temperature, indicating that strain A11 has nitrogen-fixing function.

[0027] (2) Determination of Organophosphate Solubility: Qualitative determination of organophosphate solubility: The purified strain was inoculated onto Monkina organophosphate medium and cultured in a constant temperature incubator at 28 ℃ for 5-7 days. The diameter of the solubilizing zone (D) and the colony diameter (d) were measured. During growth, the phosphatase secreted by the strain with phosphate solubilizing function can decompose organophosphate compounds such as lecithin into phosphate and other products, making the organophosphates in the medium soluble, thus forming a transparent area around the colony. The size of the transparent zone directly reflects the bacteria's ability to decompose organophosphates and the activity of phosphatase. Therefore, the larger the ratio of the diameter of the solubilizing zone (D) to the colony diameter (d) of the organophosphate degrading bacteria, the better the organophosphate degradation effect.

[0028] Quantitative experiment on dissolved organophosphorus compounds: The bacterial suspension was inoculated at a rate of 1% (v / v) into 50 mL of organophosphorus liquid culture medium. After incubation at 30 °C and 180 r / min for 3 days, the culture was centrifuged at 10,000 r / min for 15 min. The supernatant was then collected, and the available phosphorus content in the supernatant was determined using the molybdenum blue colorimetric method. Figure 2 As shown, qualitative experiments revealed that the organic phosphorus solubility index of strain A11 was 1.7; quantitative experiments showed that strain A11 converted 3.96 mg / L of lecithin after 3 days of culture.

[0029] (3) Determination of Inorganic Phosphorus Solubility: Qualitative determination of inorganic phosphorus solubility: The purified strain was inoculated on inorganic phosphorus medium and cultured in a constant temperature incubator at 28 ℃ for 5-7 days. The diameter of the phosphorus-solubilizing zone (D) and the colony diameter (d) were measured. The formation of the phosphorus-solubilizing transparent zone is due to the conversion of insoluble phosphorus in the medium into soluble phosphorus through acidification, chelation, exchange reaction and polymer formation by phosphorus-solubilizing bacteria, which allows the phosphate in the medium to be released and diffused, forming a transparent zone. Therefore, the larger the ratio of the diameter of the phosphorus-solubilizing zone (D) to the colony diameter (d) of inorganic phosphorus-degrading bacteria, the better the inorganic phosphorus degradation effect. Quantitative experiment of inorganic phosphorus solubility: The bacterial suspension was inoculated into 50 mL of inorganic phosphorus liquid medium at an inoculation rate of 1% (v / v). After culturing at 30 ℃ and 180 r / min for 3 days, the supernatant was collected after centrifugation at 10,000 r / min for 15 min. The effective phosphorus content in the supernatant was determined by the molybdenum blue colorimetric method. The results of the inorganic phosphorus function determination are as follows: Figure 3 As shown, the qualitative test results indicate that the inorganic phosphorus solubility index of strain A11 is 1.8; the quantitative test results indicate that the calcium phosphate conversion capacity of strain A11 reaches 32.96 mg / L after 3 days of culture.

[0030] (4) Screening of potassium-solubilizing strains Qualitative experiment: The purified strain was inoculated onto potassium-solubilizing medium and cultured in a 28 ℃ incubator for 5-7 days. The diameter of the clear zone (D) and the colony diameter (d) were measured. Potassium-solubilizing bacteria can dissolve insoluble potassium minerals in the culture medium by secreting organic acids. The organic acids diffuse in the medium and form a clear zone. The larger the ratio of the potassium-solubilizing zone diameter (D) to the colony diameter (d), the more potassium ions the strain can release at the same growth scale, showing a stronger potassium-solubilizing ability. Therefore, the larger the D / d value, the better the potassium-solubilizing effect of the strain.

[0031] Quantitative experiment: The prepared bacterial suspension was inoculated into 50 mL of potassium-solubilizing liquid medium without agar at an inoculation rate of 1% (v / v). After incubation at 30 °C and 180 r / min for 3 days, the culture was centrifuged at 6000 r / min for 20 min. The supernatant was collected and the potassium content in the supernatant was determined by flame spectrophotometer.

[0032] The results of the potassium solubilization function test are as follows: Figure 4 As shown, the qualitative test results indicate that the potassium solubility index of strain A11 is 2.17; the quantitative test results indicate that the conversion capacity of potassium feldspar powder by strain A11 reaches 5.91 mg / L after 3 days of culture.

[0033] Table 1. Results of functional assays for different strains

[0034] Note: "+" indicates that the strain has the corresponding function, and the more "+" signs, the stronger the function; "-" indicates that the strain does not have the corresponding function. "D" represents the diameter of the lysozyme zone or the clear zone, and "d" represents the colony diameter. The larger the D / d ratio, the stronger the corresponding function.

[0035] The isolated strain was inoculated onto different functional culture media, and a strain A11 with nitrogen fixation, organophosphorus solubilization, and potassium solubilization functions was screened. After 7 days of culture, its organophosphorus solubilization index was 1.7, and the conversion of lecithin reached 3.96 mg / L; its inorganic phosphorus solubilization index was 1.8, and the conversion of calcium phosphate reached 32.96 mg / L; and its potassium solubilization index was 2.17, and the conversion of potassium feldspar powder reached 5.91 mg / L.

[0036] (III) Morphological and physiological-biochemical identification of the strain Morphological identification: Once the bacterial strain is clearly visible and growing well on the culture medium, observe its morphological characteristics, such as colony color, shape, and transparency, referring to Bergey's Manual of Bacteriological Identification. Take a small amount of the strain and streak it onto LB agar plates. Pick a purified single colony for Gram staining.

[0037] Salt tolerance test: LB medium with pH=7.0 and NaCl content of 1%, 1.5%, 2%, 3%, 4%, and 5% was prepared. The activated strain was streaked onto LB medium with different NaCl concentration gradients and cultured at 28 ℃ for 3 days. The tolerance of the strain to NaCl concentration was determined by whether the strain could grow normally on the medium.

[0038] Alkali tolerance test: The bacterial suspension was inoculated at a rate of 1% into LB liquid medium with a NaCl concentration of 1% and different pH gradients (pH=5, 6, 7, 8, 9). The shaking conditions were set at 30 ℃ and 180 r / min. The OD was measured after 7 days. 600 The value was used to determine the acid and alkali tolerance of the strain.

[0039] Determination of bacterial growth curve: Single colonies of strain A11, streaked on LB agar plates, were picked and placed in a 500 mL Erlenmeyer flask containing 350 mL of LB liquid medium. The flask was then incubated on a shaker at 30 °C and 180 r / min for 3 days. The bacterial cells were centrifuged at 10000 r / min at 4 °C for 10 min, and the collected cells were diluted with sterile water to prepare an OD curve. 600 =0.5% bacterial suspension, and then transferred to the same volume of culture medium at an inoculum rate of 5%. Sampling was performed at time points of 0, 12, 24, 36, 48, 72, 96, and 120 h, and the cell concentration was measured at 600 nm using a UV spectrophotometer.

[0040] Catalase test: Use a sterilized inoculation loop to take an appropriate amount of activated bacterial cells and spread them on a glass slide. Add 2-3 drops of 3% hydrogen peroxide solution to perform the catalase test. Observe whether bubbles are produced. If bubbles are produced, it proves that the catalase reaction is positive; otherwise, it is negative.

[0041] like Figure 5-7 As shown, the colony morphology of the target strain A11 in LB solid medium is as follows. Figure 5 As shown, the color is pale yellow, the colonies are round, the surface is smooth and moist, and the edges are relatively neat. Figure 6 The growth curve shows that strain A11 reaches the late logarithmic growth phase around 72 hours of growth. Microscopic observation of the bacterial cells shows... Figure 7As shown, Gram staining was negative, the bacterial cell morphology was rod-shaped, and there were no spores. Salt tolerance tests showed that strain A11 could tolerate 1.5% (w / v) NaCl concentration and grew well in the pH range of 5 to 9, with a cell concentration OD600 above 1.0. It grew best in 1% (w / v) NaCl medium at pH 7. The catalase assay result was positive. (IV) Hemolysis Test: The purified A11 strain was inoculated onto TSA medium containing 5% sheep blood and cultured in a 28 ℃ incubator for 3 days. The presence or absence of a clear zone was observed. A clear zone was considered positive, while the absence of a clear zone was considered negative. Specific results are shown below. Figure 8 As shown. The hemolysis test results show that strain A11 does not produce a clear zone on the plate, indicating that the hemolysis reaction of the strain is negative. (V) Molecular identification: A single colony of the purified strain was picked with a sterile inoculation loop and 200 L of 5% (w / v) BT-chelex 100 resin (prepared with distilled water and sterilized at 121 ℃ for 30 min) was added. The mixture was boiled in a water bath for 15 min, then quickly frozen at -20 ℃ and thawed at room temperature. It was centrifuged at 4 ℃ and 6000 r·min-1 for 3 min, and 2 L of the supernatant was used as template DNA. The template DNA was amplified using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene.

[0042] The total volume of the PCR reaction was 25 mL, including 2.0 mL of DNA template, 0.5 mL each of forward and reverse primers, 9.5 mL of ddH2O, and 12.5 mL of Green tapMix enzyme. The PCR program was as follows: 95 °C, 5 min pre-denaturation; 94 °C, 1 min denaturation, 55 °C, 1 min annealing, 72 °C, 1.5 min extension, 35 cycles; 72 °C, 10 min extension, and storage at 4 °C. After detection by 1.0% agarose gel electrophoresis, the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared and analyzed in the bacterial and archaea 16S rRNA gene database EZBioCloud (https: / / www.ezbiocloud.net / ), and strain A11 was found to be similar to *Acinetobacter pittii* CIP 70.29. T The similarity was 99.65%. A phylogenetic tree was constructed using MEGA 11.0 software.

[0043] Sequencing revealed that the 16S rRNA gene sequence of strain A11 was 1409 bp. Homology analysis of the strain's sequence was performed on EZBioCloud. Based on the homology analysis results, a phylogenetic tree was constructed using MEGA 11.0, and the results are as follows. Figure 9 As shown in the phylogenetic tree based on the 16S rRNA gene, strain A11 belongs to the Acinetobacter strains, and is related to... Acinetobacter pittii CIP 70.29 T (APQP01000001) Acinetobacter geminorum J00019 T (MT380471) belongs to the same branch. Comparative analysis using the EzBioCloud database revealed that strains closely related to A11 were mostly medically relevant isolates, significantly different from the A11 isolate obtained from the rhizosphere of soybean in arid regions in this study. Furthermore, A11 exhibits good growth-promoting functions and shows no hemolytic reaction, suggesting it may be a new species or subspecies developed during strain evolution. This result aligns with previous studies that, through phylogenetic analysis of prokaryotic strain genotypes to explore phosphate-solubilizing microorganisms, *Acinetobacter* was identified as one of the six most promising phosphate-solubilizing bacteria.

[0044] (vi) Growth promotion test of the strain Preparation of bacterial suspension: After activating the test strain, inoculate it into a 250 mL Erlenmeyer flask containing 150 mL of LB liquid medium and incubate at 30 ℃ and 180 r / min for 72 h with shaking. Centrifuge at 8000 r / min for 10 min, discard the supernatant, collect the bacterial cells, wash the bacterial cells twice with sterile water, and resuspend them in sterile water. Adjust the OD600 of the bacterial suspension to 0.5 and set aside for later use.

[0045] Seed germination experiment: Plump soybean seeds of the Heinong 87 variety with uniform size were selected and surface-sterilized (immersed in 70% alcohol for 10 min, rinsed 3 times with sterile water). Two treatments were included: CK (blank control group) and A11 (inoculated with A11 bacterial suspension), with 3 replicates per treatment and 20 seeds per replicate. Two layers of sterilized filter paper were placed in each sterile petri dish. The sterilized and air-dried soybean seeds were spread evenly on the filter paper. 5 mL of sterile water was added to each petri dish for the CK (control group), and 5 mL of the prepared bacterial suspension was added to each petri dish for the treatment groups. The seeds were incubated at 28 ℃ in the dark for 3 days. Sterile water was added as needed to keep the filter paper moist during the incubation period. Germination rate and sprout length were then calculated. Specific results are shown in Table 2. Figure 10 As shown.

[0046] Table 2 Seed germination rate test

[0047] Seed germination test results showed that, compared with CK, the germination rate of seeds inoculated with A11 increased by 28.6%, and the plumule length increased by 49.5%, demonstrating a significant growth-promoting effect.

[0048] The soybean rhizosphere growth-promoting strain was named Acinetobacter sp. A11, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M20242725, and deposited on December 4, 2024; the nucleotide sequence of the 16S rRNA of the soybean rhizosphere growth-promoting strain is shown in SEQ ID NO. 1.

[0049] The soybean rhizosphere growth-promoting strain of the present invention can be used for nitrogen fixation, dissolution of organic phosphorus, dissolution of inorganic phosphorus, potassium solubilization, and has the effect of promoting soybean growth, improving soybean seed germination rate, and increasing embryo length.

[0050] This invention relates to a soybean rhizosphere growth-promoting strain developed from indigenous rhizosphere growth-promoting bacteria found in the arid environment of Xinjiang. It possesses multiple growth-promoting functions, including strong nitrogen fixation, dissolution of organic and inorganic phosphorus, and potassium solubilization. This not only enriches the local microbial growth-promoting bacteria resource bank in Xinjiang but also provides high-quality strains for the development of microbial fertilizers and inoculants. Furthermore, the strain's multiple growth-promoting functions help plants decompose and utilize insoluble inorganic silicate minerals such as phosphorus and potassium feldspar in the soil, converting them into soluble nutrients. This effectively improves soil fertility and provides important scientific evidence for alleviating plant environmental stress, promoting plant growth and development, improving crop quality, maintaining soil stability, improving the rhizosphere environment, reducing fertilizer use, and mitigating environmental pollution.

[0051] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A soybean rhizosphere growth-promoting bacterial strain, characterized in that, The soybean rhizosphere growth-promoting strain was named Acinetobacter sp. A11, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20242725, deposited on December 4, 2024; the nucleotide sequence of the 16S rRNA of the soybean rhizosphere growth-promoting strain is shown in SEQ ID NO.

1.

2. The application of a soybean rhizosphere growth-promoting strain as described in claim 1, characterized in that, The soybean rhizosphere growth-promoting strains are used for nitrogen fixation, dissolution of organic phosphorus, dissolution of inorganic phosphorus, and potassium solubilization; the soybean rhizosphere growth-promoting strains are used to promote soybean growth.

3. According to claim 2, the soybean rhizosphere growth-promoting strain is used to improve the germination rate of soybean seeds and increase the length of soybean embryos.

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