Phosphorus-solubilizing bacterium suitable for alkaline soil and application of phosphate-solubilizing bacterium

Abstract

The invention provides a phosphate solubilizing bacterium suitable for alkaline soil and application of the phosphate solubilizing bacterium, and belongs to the technical field of beneficial microorganisms. The invention provides a strain of acinetobacter calcoaceticus PT14-1. The strain can normally grow under the condition that the concentration of NaCl is 1%-7%, and has better salt tolerance; the strain can grow in a pH range of 5.0-9.0, has good acid and alkali resistance, also has IAA and protease production capability, and has significant nitrogen fixation capability. According to the embodiment of the invention, the acinetobacter calcoaceticus PT14-1 has the capability of decomposing and releasing inorganic phosphorus and organic phosphorus. In addition, in the embodiment of the invention, the growth promoting capacity of the strain PT14-1 is verified by utilizing rape seeds, and the PT14-1 strain has a remarkable promoting effect on the germination rate, the germination potential, the germination index, the rooting amount and the like of rape.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of beneficial microorganisms, and particularly relates to a phosphorus-solubilizing bacterium suitable for alkaline soil and application thereof. BACKGROUND

[0002] Phosphorus is one of the essential nutrients for plant growth and development, and is widely involved in key physiological processes such as energy metabolism, photosynthesis, genetic information transmission and biological membrane construction. However, phosphorus in soil is easily combined with metal ions to form insoluble inorganic phosphate, or combined with organic matter to form organic phosphorus such as phytate, resulting in a significant reduction in effective utilization rate, and the actual utilization of effective phosphorus by plants accounts for only 2-3% of the total phosphorus, far lower than the required level for growth and development.

[0003] To improve crop yield, a large amount of chemical phosphorus fertilizer is generally applied in agricultural production. However, the utilization rate of conventional phosphorus fertilizer in soil is generally only 10-20% in the season, and most of the phosphorus is converted into fixed state phosphorus that is difficult for plants to absorb, not only causing waste of phosphorus resources and significantly increasing production costs, but also more seriously, the phosphorus remaining in the farmland soil will flow into water bodies through soil erosion, groundwater leaching and other ways, causing environmental pollution problems such as water body eutrophication, and posing a serious threat to water ecological system safety.

[0004] Phosphorus-solubilizing microorganisms can decompose and convert insoluble phosphorus in soil into effective phosphorus that can be absorbed by plants, thereby reducing the amount of phosphorus fertilizer and improving the utilization rate of phosphorus. The application of phosphorus-solubilizing microorganisms to develop biological fertilizers has become an important way to achieve agricultural fertilizer saving and efficiency increasing and green development. Therefore, it is of great significance to separate and screen new strains with high-efficiency phosphorus-solubilizing ability, multiple growth-promoting characteristics and strong adaptability for developing a new generation of microbial fertilizers and promoting sustainable agricultural development. SUMMARY

[0005] The application provides a phosphorus-solubilizing bacterium suitable for alkaline soil and application thereof, and the bacterium has high-efficiency phosphorus-solubilizing function and multiple growth-promoting characteristics.

[0006] The application provides a strain of Acinetobacter calcoaceticus (A. Acinetobacter calcoaceticus ) PT14-1, and the preservation number of the Acinetobacter calcoaceticus PT14-1 is CCTCC M 20251082.

[0007] The application also provides application of the above-mentioned Acinetobacter calcoaceticus PT14-1 in soil phosphorus solubilization.

[0008] In a preferred mode of the application, the types of soil phosphorus solubilization include organic phosphorus and inorganic phosphorus.

[0009] The application also provides application of the above-mentioned Acinetobacter calcoaceticus PT14-1 in crop growth promotion.

[0010] In a preferred mode of the present application, the type of the crop includes monocotyledonous crops and / or dicotyledonous crops.

[0011] The present application also provides a phosphorus-dissolving and growth-promoting bacterial agent, and the active ingredient includes the bacterial body of the above-mentioned Acinetobacter calcoaceticus PT14-1.

[0012] In a preferred mode of the present application, the number of living Acinetobacter calcoaceticus PT14-1 in the bacterial agent is not less than 1×10 12 CFU / mL.

[0013] The present application also provides a preparation method of the above-mentioned bacterial agent, which includes the following steps: inoculating the above-mentioned Acinetobacter calcoaceticus PT14-1 into a liquid culture medium for oscillation culture to prepare a seed liquid; inoculating the seed liquid into a fermentation liquid culture medium for oscillation culture to obtain a PT14-1 fermentation liquid; and preparing the bacterial agent by using the PT14-1 fermentation liquid.

[0014] The present application also provides a soil improvement method, which includes inoculating the above-mentioned Acinetobacter calcoaceticus PT14-1 into soil or applying the above-mentioned bacterial agent.

[0015] The present application also provides a method for promoting plant growth, which includes soaking crop seeds with a dilute solution of the above-mentioned bacterial agent before sowing.

[0016] Beneficial effects: the present application provides an Acinetobacter calcoaceticus PT14-1 strain, which can grow normally under a NaCl concentration of 1%-7% and has good salt tolerance; can grow in a pH range of 5.0-9.0 and has good acid and alkali tolerance; has the ability to produce IAA and protease and has significant nitrogen fixation ability. It is verified in the examples of the present application that the Acinetobacter calcoaceticus PT14-1 has the ability to decompose and release inorganic phosphorus and organic phosphorus. In the examples of the present application, the growth-promoting ability of the strain PT14-1 is also verified by using rape seeds, and the strain PT14-1 has a significant promoting effect on the germination rate, germination potential, germination index and root amount of rape.

[0017] Biological preservation information Acinetobacter calcoaceticus (A. Acinetobacter calcoaceticus ) PT14-1, which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC M 20251082 and a preservation date of May 15, 2025. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The colony morphology and gram staining of the strain PT14-1 on an LB plate; Figure 2 The phylogenetic tree of the strain PT14-1; Figure 3 Figure 4 is a growth curve of the strain PT14-1 under different salt concentrations; Figure 4 Figure 5 is a growth curve of the strain PT14-1 under different pH conditions; Figure 5 Figure 6 is a curve for determining the IAA production ability of the strain PT14-1; Figure 6 Figure 7 is a curve for determining the protease production ability of the strain PT14-1; Figure 7 Figure 8 is a curve for determining the nitrogen fixation ability of the strain PT14-1; Figure 8 Figure 9 is a curve for determining the phosphorus solubilization ability of the strain PT14-1 on inorganic phosphorus and organic phosphorus plates; Figure 9 Figure 10 is a curve for determining the root growth of rape seeds after treatment of the fermentation liquid of the strain PT14-1. DETAILED DESCRIPTION

[0019] The application provides a strain of Acinetobacter calcoaceticus (A. Acinetobacter calcoaceticus ) PT14-1, and the preservation number of the Acinetobacter calcoaceticus PT14-1 is CCTCC M 20251082.

[0020] The strain PT14-1 is a gram-negative bacterium, and the colony of the strain PT14-1 on an LB culture medium is light yellow, round, smooth, has a neat edge and is moist and easy to pick up. The nucleotide sequence of 16S rDNA of the strain PT14-1 after sequencing is shown as SEQ ID No. 1.

[0021] The strain can secrete indole acetic acid (IAA), produce protease, has nitrogen fixation ability and phosphorus solubilization ability, and is resistant to salt, acid and alkali.

[0022] The application further provides application of the above-mentioned Acinetobacter calcoaceticus PT14-1 in soil phosphorus solubilization.

[0023] The type of soil phosphorus solubilization includes organic phosphorus and inorganic phosphorus, and the diameter of the transparent circle of the organic phosphorus is 12.20 mm.

[0024] The application further provides application of the above-mentioned Acinetobacter calcoaceticus PT14-1 in crop growth promotion.

[0025] The strain PT14-1 described in this invention can secrete IAA, produce proteases, and has nitrogen-fixing capabilities. It can also degrade insoluble organic and / or inorganic phosphorus in the soil, thereby increasing the soluble phosphorus content in the soil, improving phosphorus utilization, reducing fertilizer usage, and promoting crop growth. The types of crops include monocotyledonous and / or dicotyledonous crops. Rapeseed is used as an example in this embodiment, but it should not be considered as representing the entire scope of protection of this invention.

[0026] The present invention also provides a phosphorus-solubilizing and growth-promoting agent, the active ingredient of which includes the above-mentioned Acinetobacter calcitrate PT14-1 bacterial cells.

[0027] The viable count of Acinetobacter calcifera PT14-1 in the bacterial agent of this invention is not less than 1×10⁻⁶. 12 CFU / mL.

[0028] The present invention also provides a method for preparing the above-mentioned microbial agent, comprising the following steps: inoculating the above-mentioned Acinetobacter calcitriol PT14-1 into a liquid culture medium and performing shaking culture to obtain a seed liquid; inoculating the seed liquid into a fermentation liquid culture medium and performing shaking culture to obtain a PT14-1 fermentation broth; and using the PT14-1 fermentation broth to prepare the microbial agent.

[0029] In this invention, the strain PT14-1 was inoculated into LB liquid medium and cultured with shaking to obtain a seed culture. The shaking culture temperature was 30℃, the shaking frequency was 180 r / min, and the shaking culture time was 24h to obtain the seed culture.

[0030] In this invention, the seed culture is inoculated into a fermentation liquid culture medium and shaken to obtain the PT14-1 fermentation broth. The shaking culture temperature is 30℃, the shaking frequency is 200 r / min, and the shaking culture time is 24h to obtain the PT14-1 fermentation broth.

[0031] The present invention uses the PT14-1 fermentation broth as the raw material for preparing the bacterial agent, and the suspension of the bacterial cells in the fermentation broth can be used as the bacterial agent.

[0032] The present invention also provides a method for soil improvement, comprising inoculating the soil with the above-mentioned Acinetobacter calcitrate PT14-1, or applying the above-mentioned bacterial agent.

[0033] The present invention also provides a method for promoting plant growth, comprising soaking crop seeds in a diluted solution of the above-mentioned microbial agent before sowing.

[0034] In one embodiment of the present invention, crop seeds are soaked in a diluted solution of the bacterial agent to promote seed germination rate, increase germination potential, and increase root quantity and root length.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a phosphate-solubilizing bacterium adapted to alkaline soil and its application, should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1: Screening of Acinetobacter calciacetate PT14-1 Soil sample source: Xinyu Grape Base, Liuhudi Town, Manas County, Changji, Xinjiang. The soil has a pH of 8.17 and is slightly alkaline.

[0037] Strain screening: Weigh 5g of soil sample and place it in an Erlenmeyer flask containing 45mL of sterile water and glass beads. Shake at 30℃ and 180r / min for 30min to prepare a soil suspension. Take the soil suspension and divide it into 10... -1 10 -2 10 -3 10 -4 The culture was serially diluted, and 100 μL of each culture was spread onto inorganic phosphorus solid medium plates and incubated at 30°C for 5-7 days. After the colonies grew, strains with a clear zone around a single colony were picked and repeatedly streaked to purify the cultured strain PT14-1.

[0038] The inorganic phosphorus solid culture medium is composed of the following: 10.0g glucose, 0.5g ammonium sulfate, 0.5g magnesium sulfate, 0.5g sodium chloride, 0.5g potassium chloride, 0.03g ferrous sulfate heptahydrate, 0.03g manganese sulfate, 5.0g tricalcium phosphate, 15.0g agar, and 1L distilled water.

[0039] Example 2: Identification of strain PT14-1 (1) Observation of colony morphology The selected strain PT14-1 was inoculated into LB solid medium and cultured at 30℃ for 48 h. The colony morphology of the strain was observed, and then Gram staining was performed to observe the cell morphology under a microscope.

[0040] like Figure 1 As shown, strain PT14-1 colonies on LB medium are pale yellow, round, smooth, with regular edges, moist, and easily picked up. Gram staining is negative.

[0041] (2) Physiological and biochemical characteristics analysis The physiological and biochemical characteristics of Acinetobacter calciacetate PT14-1 were determined according to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press, 2011.).

[0042] Table 1 Physiological and biochemical characteristics of strain PT14-1

[0043] Note: "+" indicates positive; "-" indicates negative.

[0044] (3) Molecular biological identification of PT14-1 Genomic DNA was extracted from strain PT14-1. Using the genomic DNA as a template, PCR amplification was performed using 16S rDNA bacterial universal primers. The amplified product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the obtained sequence is shown in SEQ ID NO.1. The sequence was BLAST aligned in GenBank, and a phylogenetic tree was constructed using MEGA 7.0 software, as shown below. Figure 2 As shown.

[0045] 27F (SEQ ID No. 2): 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R (SEQ ID No. 3): 5'-GGTTACCTTGTTACGACTT-3'.

[0046] The results showed that strain PT14-1 belonged to the same branch as Acinetobacter calcium acetate, and its homology with Acinetobacter calcium acetate was 99.93%. Based on the colony morphology and physiological characteristics, it was identified as Acinetobacter calcium acetate.

[0047] Example 3: Determination of salt tolerance, acid and alkali tolerance, and functional characteristics of strain PT14-1 (1) Salt tolerance test The PT14-1 strain was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain seed culture. LB liquid media with different NaCl concentrations (1%, 2%, 5%, 7%, 10%) were prepared, and 1 mL of seed culture was added to each medium. The cultures were then cultured at 30℃ and 180 rpm for 48 h. The growth of the strain was observed, and the OD was measured using a spectrophotometer. 600 value.

[0048] like Figure 3 As shown, this bacterium grows normally at NaCl concentrations of 1-7%, but cannot grow at a NaCl concentration of 10%, exhibiting good salt tolerance.

[0049] (2) Determination of acid and alkali resistance The PT14-1 strain was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain seed culture. LB liquid medium with different pH values ​​(5.0, 6.0, 7.0, 8.0, 9.0) was prepared, and 1 mL of seed culture was added to each medium. The cultures were then cultured at 30℃ and 180 rpm for 48 h. The growth of the strain was observed, and the OD600 value was measured using a spectrophotometer.

[0050] likeFigure 4 As shown, the strain can grow in a pH range of 5.0 to 9.0 and has good acid and alkali resistance.

[0051] (3) IAA production capacity determination The PT14-1 strain was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain a seed culture. 1 mL of the seed culture was inoculated into LB liquid medium containing 1% tryptophan and cultured at 30℃ and 180 rpm for 48 h. The culture was then centrifuged at 10000 rpm for 5 min, and the supernatant was collected. 2 mL of the supernatant was added to 4 mL of Salkowaski reagent (15 mL of 0.5 mol / L FeCl3, 300 mL of concentrated sulfuric acid, and 500 mL of distilled water), and reacted at 28℃ for 2 h. If the mixture turned red, it indicated that the strain could secrete IAA.

[0052] The results are as follows Figure 5 As shown, the mixture inoculated with PT14-1 turned red, while the control group did not, indicating that strain PT14-1 has the ability to produce IAA.

[0053] (4) Determination of protease production capacity The activated strain PT14-1 was spotted in the center of a protease medium plate and incubated at 30°C for 3-7 days. The presence of a clear zone was observed; if present, it indicated that the strain had the ability to produce protease.

[0054] The results are as follows Figure 6 As shown, a clear zone with a diameter of 14.40 mm was produced near strain PT14-1, indicating that strain PT14-1 has the ability to produce protease.

[0055] (5) Nitrogen fixation capacity determination The activated strain PT14-1 was spotted in the center of a nitrogen-free medium plate and incubated at 30°C for 3-7 days. The presence of a clear zone was observed; if present, it indicated that the strain had nitrogen-fixing ability.

[0056] The results are as follows Figure 7 As shown, a clear zone with a diameter of 11.08 mm was generated near strain PT14-1, indicating that strain PT14-1 has nitrogen-fixing ability.

[0057] Example 4: Determination of phosphorus release capacity of strain PT14-1 (1) Qualitative determination The strain PT14-1 was inoculated onto LB medium plates for activation. The activated strain was then inoculated onto inorganic phosphorus and organic phosphorus solid medium plates and cultured at 30°C for 7 days. The diameter of the phosphorus lysing zone was measured using vernier calipers.

[0058] The inorganic phosphorus solid culture medium consists of the following components: 10.0 g glucose, 5.0 g Ca3(PO4)2, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, 0.3 g K2SO4, 0.03 g FeSO4·4H2O, 15.0 g agar, and 1000 mL distilled water.

[0059] The organic phosphorus solid culture medium consists of the following components: 10.0 g glucose, 2.0 g calcium phytate, 0.5 g (NH4)2SO4, 0.3 g NaCl, 0.3 g MgSO4·7H2O, 0.03 g MnSO4·4H2O, 0.3 g K2SO4, 0.03 g FeSO4·4H2O, 15.0 g agar, and 1000 mL distilled water.

[0060] The results are as follows Figure 8 As shown, strain PT14-1 has the ability to decompose and release inorganic and organic phosphorus, and the diameter of its organic phosphorus transparent zone is 12.20 mm.

[0061] (2) Quantitative determination Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain seed culture. 1 mL of seed culture was inoculated into inorganic phosphorus and organic phosphorus liquid media and cultured at 30℃ and 180 rpm for 7 days. The culture was centrifuged at 4000 rpm for 20 min, and the supernatant was collected. A culture medium with an equal volume of sterile water was used as a blank control. The available phosphorus content in the fermentation supernatant was determined using the molybdenum-antimony colorimetric method.

[0062] The results are shown in Table 2. The amount of inorganic phosphorus released by strain PT14-1 was 24.04 mg / L, and the amount of organic phosphorus released was 20.05 mg / L.

[0063] Table 2. Phosphorus release capacity of strain PT14-1

[0064] Example 5: Optimization of fermentation conditions for strain PT14-1 (1) Prepare the initial fermentation medium Initial fermentation medium formula: 10.0g yeast extract, 4.0g peptone, 5.0g NaCl, 1.0g calcium carbonate, 1L distilled water.

[0065] (2) Determination of optimal growth temperature The strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. 1 mL of seed culture was inoculated into an Erlenmeyer flask containing 50 mL of LB liquid medium, and cultured at different temperatures of 24, 26, 28, 30, 32, and 34℃ for 24 h at 180 rpm. The bacterial count was then determined.

[0066] The results are shown in Table 3. The OD of strain PT14-1 after culturing at different temperatures for 24 hours was measured. 600 The values ​​show that the strain grows the most at 30℃, indicating that the optimal growth temperature for PT14-1 is 30℃.

[0067] Table 3. Growth of PT14-1 at different temperatures

[0068] (3) Determination of optimal growth speed Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. 1 mL of seed culture was inoculated into an Erlenmeyer flask containing 50 mL of LB liquid medium, and the culture was incubated at 30℃ for 24 h at rotation speeds of 140, 160, 180, 200, and 220 r / min, respectively, and the bacterial count was determined.

[0069] The results are shown in Table 4. The OD of strain PT14-1 after culturing at different rotation speeds for 24 hours was measured. 600 The values ​​show that the strain grows the most at a rotation speed of 200 r / min, indicating that the optimal growth speed for PT14-1 is 200 r / min.

[0070] Table 4. Growth of PT14-1 at different rotation speeds

[0071] (4) Screening of the optimal carbon source Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. The seed culture was inoculated into the initial fermentation medium at a rate of 1 mL. Lactose, glucose, sucrose, mannitol, maltodextrin, and yeast extract were used to replace the carbon source in the initial fermentation medium, respectively. The culture was carried out at 30℃ and 180 r / min for 24 h, and the bacterial count was measured to select the optimal carbon source.

[0072] The results are shown in Table 5. By measuring the viable count of strain PT14-1 under different carbon sources, it was found that the effective viable count in the fermentation broth was the highest when yeast extract was used as the carbon source, reaching 9.88 × 10⁻⁶. 12 With a CFU / mL concentration, yeast extract was chosen as the optimal carbon source.

[0073] Table 5. Growth of PT14-1 under different carbon sources

[0074] (5) Screening of the optimal nitrogen source Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. The seed culture was inoculated into the initial fermentation medium at a rate of 1 mL. The nitrogen source in the initial fermentation medium was replaced by corn flour, peptone, potassium nitrate, ammonium sulfate, ammonium chloride, and diammonium hydrogen phosphate, respectively. The culture was carried out at 30℃ and 180 r / min for 24 h, and the bacterial count was measured to select the optimal nitrogen source.

[0075] The results are shown in Table 6. By measuring the viable count of strain PT14-1 under different nitrogen sources, it was found that the effective viable count in the fermentation broth was the highest when diammonium hydrogen phosphate was used as the nitrogen source, reaching 3.70 × 10⁻⁶. 11 With a concentration of CFU / mL, diammonium hydrogen phosphate was chosen as the optimal nitrogen source.

[0076] Table 6. Growth of PT14-1 under different nitrogen sources.

[0077] (6) Optimal inorganic salt screening Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. The seed culture was then inoculated into the initial fermentation medium at a rate of 1 mL. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous sulfate, calcium carbonate, and magnesium sulfate were used to replace the inorganic salts in the initial fermentation medium, respectively. The culture was carried out at 30℃ and 180 r / min for 24 h, and the bacterial count was measured to select the optimal inorganic salts.

[0078] The results are shown in Table 7. By measuring the viable count of strain PT14-1 under different inorganic salt conditions, it was found that the highest number of viable bacteria in the fermentation broth was 4.50 × 10⁻⁶ when dipotassium hydrogen phosphate was used as the inorganic salt. 9 Therefore, dipotassium hydrogen phosphate was chosen as the optimal inorganic salt, given the CFU / mL concentration.

[0079] Table 7. Growth of PT14-1 under different inorganic salts

[0080] (7) Screening of optimal carbon source concentration Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. Fermentation media with carbon source concentrations of 5, 10, 15, 20, and 25 g / L were prepared respectively. The seed culture was inoculated into the above media at an inoculation volume of 1 mL and cultured at 30℃ and 180 r / min for 24 h. The bacterial content was then measured.

[0081] The results are shown in Table 8. By measuring the viable count of strain PT14-1 under different carbon source concentrations, it was found that the highest number of effective viable bacteria in the fermentation broth was 5.20 × 10⁻⁶ when the yeast powder concentration was 10 g / L. 12 Therefore, the optimal concentration of yeast powder is 10 g / L, calculated using CFU / mL.

[0082] Table 8. Growth of PT14-1 under different carbon source concentrations

[0083] (8) Screening of optimal nitrogen source concentration Strain strain PT14-1 was inoculated into LB liquid medium and cultured at 30℃ for 24 h to obtain seed culture. Fermentation media with nitrogen source concentrations of 4, 6, 8, 10, and 12 g / L were prepared respectively. The seed culture was inoculated into the above media at an inoculation volume of 1 mL and cultured at 30℃ and 180 r / min for 24 h. The bacterial content was then measured.

[0084] The results are shown in Table 9. By measuring the viable count of strain PT14-1 under different nitrogen source concentrations, it was found that the highest number of viable bacteria in the fermentation broth was 2.30 × 10⁻⁶ when the concentration of diammonium hydrogen phosphate was 6 g / L. 10 Therefore, the optimal concentration is 6 g / L diammonium hydrogen phosphate, calculated using the formula CFU / mL.

[0085] Table 9. Growth of PT14-1 under different nitrogen source concentrations.

[0086] (9) Cultivation under optimal fermentation conditions The above experimental results indicate that the optimal fermentation conditions for strain PT14-1 are as follows: PT14-1 is inoculated into LB liquid medium and cultured at 30℃ and 180 rpm for 24 h to obtain the seed culture. The seed culture is then inoculated into the optimized fermentation medium at a rate of 1 mL and cultured at 30℃ and 200 rpm with shaking for 24 h to obtain the PT14-1 fermentation broth. The bacterial count of strain PT14-1 is then determined after culturing under the optimal fermentation conditions.

[0087] Optimized fermentation medium formula: 10.0g yeast powder, 6.0g diammonium hydrogen phosphate, 5.0g sodium chloride, 1.0g dipotassium hydrogen phosphate, and 1L distilled water.

[0088] The results are shown in Table 10. Under the optimal fermentation conditions, the viable count of strain PT14-1 was 1.51 × 10⁻⁶. 12 CFU / mL.

[0089] Table 10 Growth of PT14-1 under optimal fermentation conditions.

[0090] Example 6: Growth-promoting effect of Acinetobacter Calcium Acetate PT14-1 on rapeseed seeds Preparation of PT14-1 fermentation broth: A single colony of PT14-1 was picked up with an inoculation loop and inoculated into LB liquid medium. The culture was then incubated at 30℃ and 180 rpm for 24 h to obtain the seed culture. The seed culture was then inoculated into the fermentation optimization medium and incubated at 30℃ and 200 rpm for 24 h to obtain the fermentation broth.

[0091] Fermentation broth was diluted 250, 500, and 1000 times, designated as treatments 1, 2, and 3, with water as the control (CK). Rapeseed seeds were treated in a 55℃ water bath for 20 min, disinfected with 75% ethanol for 1 min, rinsed three times with sterile water, and finally disinfected with sodium hypochlorite for 30 s, followed by three rinses with sterile water. The disinfected rapeseed seeds were then soaked in fermentation broths of different dilution gradients, with water serving as the control, for 3 h. After soaking, seeds of uniform size were neatly arranged in germination bags, and 15 ml of purified water was added to each bag using a syringe. The bags were then cultured in a 25℃ artificial climate chamber, with shading treatment applied before germination. Germination rates were recorded at 24 h, 48 h, and 72 h, and the germination index was calculated on day 3. After 7 days of culture, the taproot length and the number of lateral roots were measured. Each treatment was replicated three times, with 20 seeds per replicate.

[0092] ; ; Among them, D t G represents the number of days since germination. t To be with D t The corresponding number of seeds that germinate each day.

[0093] As shown in Table 11, the germination rate of rapeseed seeds treated with PT14-1 fermentation broth diluted 1000 times reached over 95.00% on the second day and 100.00% on the third day, which was significantly higher than that of the control group.

[0094] As shown in Table 12, the germination potential of each treatment reached its highest point on the first day and then showed a downward trend. Among them, the 1000-fold dilution of PT14-1 fermentation broth showed the best germination potential and germination index, indicating that its seed germination speed, vigor, and adaptability were relatively strong.

[0095] As shown in Table 13, the PT14-1 fermentation broth, when diluted 1000 times, had the longest taproot, the most fibrous roots, and the longest hypocotyl.

[0096] Based on the above data on germination rate, germination potential, germination index, and rooting of each treatment, it is shown that a 1000-fold dilution of PT14-1 fermentation broth is most beneficial for rapeseed germination and root growth.

[0097] Table 11 Germination rate of rapeseed under different treatments

[0098] Table 12 Germination potential and germination index of rapeseed under different treatments

[0099] Table 13 Rooting data of rapeseed seeds under different treatments

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Acinetobacter calciacetate ( Acinetobacter calcoaceticus PT14-1, characterized in that, The preservation number of Acinetobacter calcitrate PT14-1 is CCTCC M 20251082.

2. The application of Acinetobacter calcitriol PT14-1 as described in claim 1 in soil phosphorus solubilization.

3. The application according to claim 2, characterized in that, The types of phosphorus that can be solubilized in the soil include organic phosphorus and inorganic phosphorus.

4. The application of Acinetobacter calcium acetate PT14-1 as described in claim 1 in crop growth promotion.

5. The application according to claim 4, characterized in that, The types of crops include monocotyledonous crops and / or dicotyledonous crops.

6. A phosphorus-solubilizing and growth-promoting bacterial agent, characterized in that, The active ingredient includes the cells of Acinetobacter calciacetate PT14-1 as described in claim 1.

7. The microbial agent according to claim 6, characterized in that, The viable count of Acinetobacter calciacetate PT14-1 in the bacterial agent is not less than 1×10⁻⁶. 12 CFU / mL.

8. The method for preparing the microbial agent according to claim 6 or 7, characterized in that, The process includes the following steps: inoculating the Acinetobacter calcitriol PT14-1 of claim 1 into a liquid culture medium and performing shaking culture to obtain a seed culture; The seed culture was inoculated into a fermentation liquid culture medium and cultured with shaking to obtain PT14-1 fermentation broth; the bacterial agent was prepared using the PT14-1 fermentation broth.

9. A method for soil improvement, characterized in that, This includes inoculating the soil with the Acinetobacter calcium acetate PT14-1 as described in claim 1, or applying the bacterial agent as described in claim 6 or 7.

10. A method for promoting plant growth, characterized in that, This includes soaking crop seeds in a diluted solution of the microbial agent described in claim 6 or 7 before sowing.

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