Azotobacter sp. i102 and application thereof

Abstract

The application relates to a nitrogen-fixing spirillum (Azospirillum sp.) I102 with a preservation number of CGMCC No. 34685. Azospirillum The strain is a new species of the Azospirillum genus, can produce indole-3-acetic acid at a high yield, and can grow under the conditions of pH 5-9, NaCl concentration 0-4%, and temperature 20-42 DEG C. The strain has the abilities of nitrogen fixation, iron carrier production and urease production, and can resist heavy metals such as cadmium, copper and lead. The application also relates to application of the strain in promoting seed germination and plant growth, and the strain has a good application prospect in soil improvement, development of new biological organic fertilizer and the like.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, specifically relating to a nitrogen-fixing spirochete I102 and its applications. Background Technology

[0002] With the development of green and ecological agriculture, new types of bio-fertilizers, such as microbial agents and microbial fertilizers mainly composed of plant growth-promoting bacteria, are gradually being applied to crop cultivation. The use of bio-fertilizers has increased crop yields by 25% and significantly reduced agriculture's demand for inorganic fertilizers. To date, an increasing number of microorganisms have achieved pure culture, but the number of effective microbial agents remains far below market demand.

[0003] Indole-3-acetic acid (IAA) is one of the earliest discovered plant hormones. It plays a regulatory role in the growth and development of plants. The rational use of IAA-producing inoculants can not only reduce the use of chemical fertilizers and environmental pollution, but also promote the absorption and utilization of nutrients by crops, thereby reducing costs and increasing yields.

[0004] Therefore, screening plant growth-promoting bacteria that are safe, environmentally friendly, and produce high levels of indoleacetic acid, and developing microbial agents with plant growth-promoting properties as the main active ingredient, to reduce the use of pesticides and fertilizers, is of great significance to the development of ecological agriculture. Summary of the Invention

[0005] The purpose of this invention is to provide a nitrogen-fixing spirochete I102 that can produce high levels of indole-3-acetic acid and has plant growth-promoting functions, and its applications.

[0006] The present invention adopts the following technical solution: A type of nitrogen-fixing spirochete ( Azospirillum sp.) I102 is deposited at the China General Microbiological Culture Collection Center, Beijing, China, with accession number CGMCC No. 34685, on May 27, 2025.

[0007] Furthermore, the nitrogen-fixing spirochete I102 is capable of nitrogen fixation, production of indole-3-acetic acid, production of siderophores, and urease.

[0008] Furthermore, the nitrogen-fixing spirochete I102 is able to produce high levels of indole-3-acetic acid in the presence of tryptophan.

[0009] Furthermore, the nitrogen-fixing spirochete I102 can grow under conditions of 20℃~42℃, pH 5~9, and NaCl concentration of 0~4%.

[0010] Furthermore, the *Azotrophus* I102 exhibits good tolerance to metals such as cadmium, copper, lead, and manganese, and can grow in LB medium containing 2 mM cadmium, 2 mM copper, 1.5 mM lead, and 10 mM manganese, respectively.

[0011] Furthermore, the nitrogen-fixing spirochete I102 can improve the germination rate, germination potential, germination index, and vigor index of plant seeds.

[0012] Furthermore, the nitrogen-fixing spirochete I102 can promote plant growth.

[0013] In particular, the nitrogen-fixing spirochete I102 can promote root growth, leaf length and fresh weight of bok choy and cucumber.

[0014] Application of the aforementioned nitrogen-fixing spirochete I102 in promoting plant growth and improving soil quality.

[0015] Application of the above-mentioned nitrogen-fixing spirochete I102 in the preparation of bio-organic fertilizer.

[0016] The application of the above-mentioned nitrogen-fixing spirochete I102 in the preparation of soil conditioner.

[0017] An application of the aforementioned azotoxin-fixing spirochete I102 in the preparation of artificial substrates.

[0018] The beneficial effects of this invention are as follows: (1) In this invention, a high-yielding strain of indole-3-acetic acid, I102, was screened in an alfalfa plantation near Yutian Dabeinong Breeding Farm in Tangshan City, Hebei Province. After whole genome comparison and physiological and biochemical experiments, it was found that the strain does not belong to the known species of the genus Azotospira, and it was identified as a new species under the genus Azotospira.

[0019] (2) The nitrogen-fixing spirochete I102 of the present invention can produce indole-3-acetic acid and can produce high levels of indole-3-acetic acid in the presence of tryptophan.

[0020] (3) The nitrogen-fixing spirochete I102 of the present invention can be applied to the soaking of plant seeds to improve the germination rate, germination potential, germination index and vigor index of the seeds.

[0021] (4) The nitrogen-fixing spirochete I102 of the present invention can tolerate heavy metal ions and can be applied to the soil remediation process in areas contaminated by heavy metals.

[0022] (5) This invention can provide excellent strains for the development of new bio-organic fertilizers, soil conditioners, artificial substrates, etc., and provide high-quality strain resources for the development of functional microorganisms. It has practical economic and social benefits and broad application prospects. Attached Figure Description

[0023] Figure 1A diagram showing the average nucleotide identity (ANI) between the genome sequence of Azospirobacter I102 and that of other Azospirobacter species.

[0024] Figure 1 The species represented by the numbers are as follows: 1. A. canadense strain DS2; 2. A. baldaniorum strain Sp245; 3. A. brasilense strain Sp 7; 4 A. baldaniorum strain BR12001; 5. A. brasilense strain Cd; 6. A. brasilense strain ATCC 29145; 7. A. brasilense strain BR 11796; 8. A. formosense strain CC-NFb-7; 9. A. rugosum strainIMMIB AFH-6; 10 A. formosense strain Nfb7; 11, A. rugosum strain DSM 19657; 12, A. isscasi strain C340-1; 13, A. formosense strain JCM 17639; 14 A. formosense strainUWAZO-1; 15, A. rugosum strain CCUG 53966; 16, Strain I102.

[0025] Figure 2 The effects of culture medium temperature, pH and NaCl concentration on the growth of *Azotrophus nigra* I102 were investigated.

[0026] Figure 3 Effects of different culture times on the IAA production capacity of Azotospirobacter I102 Figure 4 The effects of different culture temperatures and tryptophan addition levels on IAA production by Azotospirobacter I102.

[0027] Figure 5 The effects of different concentrations of different metal ions on the growth of strain I102.

[0028] Figure 6 The effects of applying nitrogen-fixing spirochete I102 on cucumber root length, leaf area, and fresh weight were investigated.

[0029] Figure 7 The effects of applying nitrogen-fixing spirochete I102 on root length, leaf length, and fresh weight of Chinese cabbage were investigated. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention.

[0031] Example 1: Isolation, identification and preservation of *Azotrophus* I102 Rhizosphere soil samples were obtained from alfalfa plantations near the Dabeinong Livestock Farm in Yutian County, Tangshan City, Hebei Province. 10g of alfalfa rhizosphere soil sample was weighed, and 100mL of sterile water and 5g of sterilized glass beads were added. The mixture was shaken at room temperature for 30 min. The supernatant was serially diluted and spread onto Ashby nitrogen-free solid medium, and incubated at 30℃ for 3 days. Colonies with differences in morphology, color, and size were streaked multiple times on nitrogen-fixing medium to obtain single colonies. The activated bacterial strain was inoculated at a 1% (v / v) inoculum into LB liquid medium containing 0.1 g / L tryptophan and incubated at 30℃ and 180 rpm for 5 days. 2 mL of the bacterial culture was centrifuged at 8000 rpm for 10 min, and the supernatant was used to determine the indole-3-acetic acid (IAA) content.

[0032] Ashby's nitrogen-free medium: 10 g mannitol, 0.2 g magnesium sulfate, 0.2 g sodium chloride, 5 g calcium carbonate, 0.1 g calcium sulfate, 1000 mL water, pH 7~7.1. For solid medium, add 15~20 g agar.

[0033] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 1000 mL water, pH 7.2~7.5. For solid medium, add 15~20 g agar.

[0034] Indole-3-acetic acid (IAA) determination method: Prepare IAA standard sample, add equal volume of Salkowski colorimetric reagent (1 mL 0.5 mol / L ferric chloride solution mixed with 50 mL 35% perchloric acid solution), develop color in the dark for 30 min, and measure the OD of the colorimetric solution. 540 To determine the OD value, plot a standard curve. Take 2 mL of the test bacterial culture, centrifuge at 8000 rpm for 10 min, collect the supernatant, add the same volume of Salkowski reagent, and incubate in the dark for 30 min. Measure the OD value of the solution. 540 The value is then substituted into the standard curve to calculate the IAA content.

[0035] A total of 38 nitrogen-fixing bacteria were obtained after multiple streaking on Ashby nitrogen-free medium. After primary and secondary screening, a high-IAA-producing strain, I102, was obtained. After culturing in LB medium supplemented with 0.1 g / L tryptophan for 3 days, it produced approximately 51 mg / L of IAA.

[0036] Colonies formed by strain I102 on LB solid medium are round, opaque, pink, with a rough, dry, and wrinkled surface. Physiological and biochemical identification shows that strain I102 is a Gram-negative bacterium capable of producing urease, indole, and siderophores; it cannot produce ammonia, hydrolyze starch or cellulose, and its MR and VP tests are negative. It can utilize arabinose, galactose, glycerol, fructose, and rhamnose, but cannot utilize glucose, mannitol, sorbitol, maltose, or lactose.

[0037] The genome sequence of strain I102 was sequenced, and the genomes of *Azospirillum* species were selected for average nucleotide identity (ANI) analysis. The results showed that the ANI value of strain I102 was less than 94% compared to other strains in the genus (generally, an ANI > 95% indicates the same species). Figure 1 The physiological and biochemical characteristics of strain I102 and two model strains of *Azospirillum* were compared, and the results are shown in Table 1.

[0038] Table 1 Physiological and biochemical characteristics of strain I102 .

[0039] Based on comprehensive morphological, physiological and biochemical characteristics and genome sequence analysis, strain I102 was identified as a new species of the genus *Azospirillum*. Azospirillum This strain was deposited on May 27, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 34685.

[0040] Example 2: Growth characteristics experiment of *Azotospirobacter I102* The growth characteristics of strain I102 were investigated based on its growth under different temperatures, pH values, and NaCl concentrations. Specifically, a seed culture of strain I102 was prepared, with temperatures ranging from 15℃ to 42℃, pH values ​​from 5 to 10, and NaCl concentrations from 0% to 6%. A 1% inoculum of the strain I102 seed culture was inoculated into 5 mL LB liquid test tubes and cultured on a shaker at 30℃ and 180 r / min for 24 h. The OD values ​​of the culture medium were measured. 600Numerical values ​​were used to determine the growth status of the bacteria. The preparation method for the seed culture of strain I102 was as follows: a single colony of strain I102 was picked and inoculated into 5 mL of liquid LB medium, and cultured at 30℃ and 180 rpm for 24 hours.

[0041] Experimental results showed that strain I102 could grow in the range of 20℃ to 42℃, and grew well in the range of 30℃ to 37℃, with the optimal growth temperature being 37℃. Figure 2 A). Strain I102 can grow in a pH range of 5–9, and grows well in a pH range of 6–9, with the best growth at pH 6. Figure 2 B). Neutral to slightly acidic soils are suitable for most grain crops, vegetables, and legumes, and strain I102 exhibits good growth as an inoculum. Strain I102 can grow in the range of 0-4% NaCl, and grows well in the range of 0-2% NaCl, with the best growth observed at a NaCl concentration of 0%. Figure 2 C). This indicates that strain I102 does not require additional NaCl supplementation during growth and fermentation, and has good application prospects.

[0042] Example 3: Ability of *Azotrophic spirochete* I102 to produce indole-3-acetic acid (IAA) The effects of different culture times, temperatures, and tryptophan additions on IAA production by strain I102 were investigated.

[0043] (A) Effect of culture time on IAA production by strain I102.

[0044] Seed culture of strain I102 (prepared as in Example 2) was inoculated at a 1% inoculum into LB medium containing 0.1 g / L tryptophan and cultured at 30°C with shaking at 180 rpm. Samples were taken once a day for 10 consecutive days to investigate the effect of different culture times on IAA yield. The method for IAA determination was the same as in Example 1.

[0045] The experimental results showed that the IAA content in the culture system increased rapidly in the first two days of culture; the IAA content reached its highest level of 72.45 mg / L on the 6th day of culture, and thereafter the IAA content accumulated in the culture medium gradually decreased. Figure 3 During their growth, microorganisms continuously convert tryptophan into IAA, and IAA accumulates in the culture medium as the culture time progresses. In the later stages of culture, due to nutrient scarcity, microorganisms prioritize maintaining their own metabolic processes and cease producing IAA. The IAA in the culture medium may then be further utilized by the microorganisms.

[0046] (B) Effect of culture temperature on IAA production by strain I102.

[0047] Three temperatures were set: 25℃, 30℃, and 37℃. The seed culture of strain I102 (prepared in the same way as in Example 2) was inoculated into LB medium containing 0.1 g / L tryptophan at an inoculation rate of 1%. After culturing at 180 rpm for 6 days, samples were taken to investigate the effect of different culture temperatures on IAA yield.

[0048] The results showed that different culture temperatures had a significant effect on IAA production by strain I102. The concentration of IAA in strain I102 at 37℃ was 84.72 mg / L, which was significantly higher than that at 20℃ and 30℃. p <0.05)( Figure 4 A). Strain I102 grows better at 37°C than at 20°C and 30°C. The metabolic activity of the microorganism is higher at 37°C, thus producing more IAA.

[0049] (C) Effect of tryptophan addition on IAA production by strain I102: Seed culture of strain I102 (prepared by the same method as in Example 2) was inoculated into LB medium containing 0, 0.1 and 0.5 g / L tryptophan at an inoculation rate of 1%, respectively. The culture was carried out at 30°C and 180 rpm. Samples were taken every 24 h to investigate the effect of tryptophan addition on IAA production.

[0050] The results showed that the initial tryptophan content in the culture medium had a certain impact on the yield of IAA. In the experimental group without tryptophan, the IAA content in the culture medium was 24.30 mg / L after 6 days of cultivation, indicating that strain I102 can produce IAA even without tryptophan. This may be through the cladonic acid pathway to form anthranilic acid, which further produces indoleglycerol phosphate, and then synthesizes IAA. In the experimental groups with tryptophan contents of 0.1 g / L and 0.5 g / L, the IAA contents were 72.45 mg / L and 100.40 mg / L, respectively. Figure 4 B). This indicates a positive correlation between IAA production and tryptophan content in the culture medium.

[0051] Example 4: Tolerance of Azotospirobacter I102 to Different Heavy Metals Different heavy metal ion stock solutions were prepared, such as 500 mM cadmium chloride, 500 mM copper sulfate, 250 mM lead acetate, and 500 mM manganese sulfate, and sterilized by filtration. After autoclaving liquid LB medium, liquid culture medium containing heavy metal ions was prepared by adding the stock solutions containing heavy metal ions. At an inoculation rate of 1%, the seed culture of strain I102 (prepared as in Example 2) was inoculated into culture media containing different concentrations of heavy metals and cultured on a shaker at 30°C and 180 rpm for 24 h. The turbidity (OD) of the culture medium was measured. 600The tolerance of strain I102 to heavy metals was investigated. Cadmium ions (Cd) in the culture medium were set at a concentration of cadmium (Cd). 2+ The contents of lead ions (Pb) were: 0 mM, 0.1 mM, 0.2 mM and 0.3 mM. 2+ The contents of ) were 0 mM, 0.5 mM, 0.75 mM, 1 mM, 1.25 mM and 1.5 mM; copper ions (Cu 2+ The contents of manganese ions (Mn) were 0 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM and 3 mM; 2+ The concentrations are 0 mM, 2 mM, 4 mM, 6 mM, 8 mM and 10 mM.

[0052] Experimental results showed that strain I102 could tolerate 0.2 mM (22.48 mg / L) cadmium ions and 1.5 mM (310.8 mg / L) lead ions, far exceeding the maximum allowable discharge concentrations of cadmium and lead in the Wastewater Discharge Standard (GB8978-1996) and the maximum risk values ​​specified in the Agricultural Land Soil Pollution Risk Control Standard (GB15618-2018). Strain I102 could tolerate 2 mM (127.1 mg / L) copper ions, far exceeding the maximum allowable discharge concentration of copper in the Wastewater Discharge Standard (GB8978-1996). The Agricultural Land Soil Pollution Risk Control Standard (GB15618-2018) specifies that the risk screening value range for copper ions in orchard soil is 150~200 mg / L, but the risk screening value range for other soils is 50~100 mg / L. In addition, strain I102 could tolerate 10 mM of metallic manganese ions (… Figure 5 Therefore, strain I102 has good plant growth-promoting effects and a certain degree of heavy metal tolerance.

[0053] Example 5: Effect of *Azotobacter nigra* I102 on seed germination of *Pakchoi*. At an inoculation rate of 1%, the seed culture of strain I102 (prepared as in Example 2) was inoculated into 50 mL of LB medium (100 mL shake flask) and cultured on a shaker at 30℃ and 180 rpm for 16 h. The bacterial suspension was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were resuspended in 50 mL of sterile water. The original bacterial suspension was designated as group T0, and the 10-fold, 100-fold, and 500-fold dilutions were labeled as groups T1, T2, and T3, respectively. 100 μL of both the original and diluted bacterial suspensions were spread onto LB solid medium, and the bacteria in the suspensions were counted using the colony counting method. Uniformly sized pak choi seeds (“Koukouxiang Cream No. 2” pak choi) were selected, disinfected by soaking in 0.1% potassium permanganate for 30 min, washed three times with sterile water, and then soaked in 25 mL of bacterial suspensions of different concentrations for 5 h. After soaking, the seeds were air-dried on filter paper. 25 seeds and 5 mL of sterile water were placed in each petri dish (90 cm in diameter), with 6 replicates per group. Sterile water was used as the control group. Seed germination was recorded daily.

[0054] The experimental results showed that the germination rate of the control group of pakchoi was 49%, lower than the theoretical germination rate (85%). This may be because the pakchoi seeds used in the experiment were produced in September 2023, while the experiment was conducted in June 2025. Under the same treatment conditions, after soaking the seeds in the stock solution (T0), 10-fold dilution (T1), 100-fold dilution (T2), and 500-fold dilution (T3), the germination rates were 61%, 65%, 66%, and 54.5%, respectively. The germination rates of pakchoi in groups T0, T1, and T2 were significantly higher than those in the control group (Table 2). The germination potential of the seeds in groups T0, T1, and T2 were significantly higher than that in the control group, and the germination index of all four experimental groups was significantly higher than that of the control group. The vigor index of the seeds in groups T1, T2, and T3 was significantly higher than that of the control group. The experimental results indicate that strain I102 can improve the germination rate and promote seed germination through soaking or other methods, and has good application prospects.

[0055] Table 2. Effects of different concentrations of strain I102 on the germination of Chinese cabbage seeds. .

[0056] Example 6: Effects of Azotospirobacter I102 on Plant Growth The seed culture of strain I102 (prepared using the same method as in Example 2) was inoculated into LB liquid medium at a 1% inoculum and cultured at 30°C and 180 rpm for 24 h to obtain the I102 bacterial culture. The experimental group consisted of 100 μL of I102 bacterial culture (approximately 5 × 10⁻⁶ cells per gram of soil substrate) applied as irrigation. 6(CFU / mL), mix thoroughly. Equal volumes of LB medium and sterile water were added to the soil substrate as control groups. Mature, plump, and uniformly sized seeds of Chinese cabbage and cucumber were selected, and the seed surfaces were disinfected with a 0.1% potassium permanganate solution for 30 min. The seeds were rinsed three times with distilled water, air-dried, and then sown. Plants were grown in 10×10 cm square pots, with 2-3 seeds planted in each pot. Thinning was done after two true leaves emerged. Five replicates were set up for each group. The bacterial solution was applied twice, on days 7 and 14, with 25 mL each time. After 30 days of culture, plant growth indicators, such as root length, leaf length, leaf area, and fresh weight, were measured.

[0057] Thirty days after planting, the root length of the cucumbers in the experimental group was 15.23 ± 0.23 cm, which was 69.2% and 66.4% higher than that of the group treated with culture medium (9 ± 0.95 cm) and water (9.15 ± 1.38 cm), respectively; the leaf area of ​​the cucumbers in the experimental group was 67.11 ± 3.75 cm². 2 The values ​​were significantly higher than those obtained with the applied culture medium (45.8 ± 7.10 cm). 2 ) and water (43.9 ± 3.90 cm) 2 The experimental group consisted of a control group and a control group. The average fresh weight of cucumbers in the experimental group was 4.0 ± 0.28 g, which was 125.9% and 120.9% higher than that of the control group treated with culture medium (1.77 ± 0.14 g) and water (1.81 ± 0.30 g), respectively. Figure 6 Strain I102 significantly promoted the increase of cucumber leaf length, leaf area, and fresh weight. The experimental results indicate that strain I102 can be used as a biological inoculant to promote the growth of cucumber seedlings.

[0058] Thirty days after planting, the root length of the experimental group (irrigated with I102 bacterial solution) of pakchoi was 9.91 ± 2.25 cm, significantly higher than that of the control group (irrigated with water, 5.9 ± 1.17 cm, and culture medium, 4.7 ± 0.87 cm). The leaf length of the experimental group was 9.78 ± 0.27 cm, which was 40.3% and 82.8% higher than that of the control group (irrigated with water, 6.97 ± 1.21 cm, and culture medium, 5.35 ± 0.45 cm, respectively). The average fresh weight of the pakchoi in the experimental group was 2.68 ± 0.11 g, which was 185.1% and 312.3% higher than that of the control group (irrigated with water, 0.94 ± 0.17 g, and culture medium, 0.65 ± 0.21 g, respectively). Figure 7 Using strain I102 as a biological inoculant can significantly promote the root length, leaf length, and fresh weight of Chinese cabbage, and has a significant promoting effect on the growth of Chinese cabbage seedlings, showing good application prospects.

[0059] The present invention has been described in detail with reference to the above embodiments. It should be noted that the above embodiments are merely illustrative examples. Without departing from the spirit and essence of the present invention, those skilled in the art can devise various alternatives and improvements to the present invention, all of which should be understood to be within the scope of protection of the present invention.

Claims

1. A type of nitrogen-fixing spirochete ( Azospirillum sp.)I102, characterized in that, Its accession number is CGMCCNo.34685.

2. The *Azotoxinus* I102 according to claim 1, characterized in that, It can fix nitrogen, produce indole-3-acetic acid, produce siderophores and urease.

3. The *Azotoxinus* I102 according to claim 1, characterized in that, It can grow under conditions of 20℃~42℃, pH 5~9, and NaCl concentration of 0~4%.

4. The *Azotoxinus* I102 according to claim 1, characterized in that, It has good tolerance to metals such as cadmium, copper, lead, and manganese.

5. The *Azotoxinus* I102 according to claim 1, characterized in that, It can improve the germination rate, germination potential, germination index and vigor index of plant seeds, and promote plant growth.

6. The application of the nitrogen-fixing spirochete I102 as described in claim 1 in promoting plant growth and improving soil quality.

7. The application of the nitrogen-fixing spirochete I102 as described in claim 1 in the preparation of bio-organic fertilizer.

8. The application of the nitrogen-fixing spirochete I102 as described in claim 1 in the preparation of a soil conditioner.

9. The application of the nitrogen-fixing spirochete I102 as described in claim 1 in the preparation of artificial substrates.

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