Low-temperature-resistant azotobacter pseudomonad ZJY-Z1 and application thereof

Abstract

The invention relates to a low-temperature-resistant azotobacter pseudomonad ZJY-Z1 and application thereof, and belongs to the technical field of microbial agents. The low-temperature-resistant azotobacter pseudomonad ZJY-Z1 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center on February 26, 2025, and the preservation number is CGMCC NO.33670; the low temperature resistant azotobacter pseudomonad ZJY-Z1 has the Latin name of Pseudomonas sp. ZJY-Z1, and the Latin name of the low temperature resistant azotobacter pseudomonad ZJY-Z1 is Pseudomonas sp. ZJY-Z1. The nitrogen-fixing bacterium provided by the invention is resistant to low temperature, has nitrogen fixation capability, can effectively decompose insoluble phosphate, also has certain siderophore production capability and plant growth hormone IAA production capability, and creates more favorable conditions for growth and development of plants.

Description

Technical Field

[0001] The invention relates to low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 and application thereof, belonging to the technical field of bacterial agents. Background Art

[0002] The Tibetan Plateau has a wide distribution area of ​​natural grasslands. The main vegetation type in this area is alpine meadows, which account for about 46.7% of the grassland area of ​​the Qinghai-Tibet Plateau. They have extreme environmental characteristics such as low annual average temperature, low temperature, strong radiation, and frequent seasonal freeze-thaw. The soil in the alpine meadow area of ​​the Qinghai-Tibet Plateau is poor, mainly composed of a thin layer of soil formed by alpine weathering, with low organic matter content and lack of necessary minerals and nutrients. In addition, the cold climate conditions limit the activity of microorganisms, which seriously hinders the nitrogen cycle in the soil. The lack of nitrogen in the soil is one of the key factors limiting plant growth and grassland productivity. Traditional fertilization methods have limited effects in alpine areas and are prone to cause environmental pollution and soil degradation. Therefore, finding a microbial strain that can survive in a low temperature environment and has an efficient nitrogen fixation function has become the key to solving the problems of poor soil and blocked nitrogen cycle in the alpine meadows of the Qinghai-Tibet Plateau.

[0003] Nitrogen-fixing bacteria are a type of microorganism that can absorb nitrogen from the air and convert it into ammonia or amino acids that can be used by plants. Compared with industrial nitrogen fixation, biological nitrogen fixation can be carried out at room temperature and pressure and is one of the main nitrogen sources in the nitrogen cycle in the biosphere. However, traditional nitrogen-fixing bacteria are mostly adapted to warm and humid environments and are difficult to meet the extreme conditions of low temperature and drought in the alpine meadows of the Qinghai-Tibet Plateau. Therefore, it is urgent to screen and cultivate a type of microbial strain that can survive at low temperatures and maintain efficient nitrogen fixation capabilities, so as to improve the soil fertility of the alpine meadows of the Qinghai-Tibet Plateau, promote plant growth, and improve grassland productivity and ecosystem stability.

[0004] Not only is nitrogen a macronutrient that is essential to organisms, but phosphorus is also a macronutrient that is essential to organisms. The nitrogen and phosphorus in the soil that can be used by crops are limited, and the content is usually only maintained in the range of 25-30 mg / kg, which is far from meeting the basic needs of healthy crop growth. Long-term reliance on chemical fertilizer supplementation is not only inefficient, but may also cause a series of negative effects such as soil compaction and acidification, which seriously threatens soil health.

[0005] Existing nitrogen-fixing strains mostly focus on room temperature or mild low-temperature environments (≥10°C). For example, although Citrobacter freundii MBC7 (CGMCC No.10823) is stress-resistant, its nitrogen-fixing activity decreases by more than 50% at 5°C, which is difficult to meet the needs of high-altitude cold regions; although Bacillus siamensis (CGMCC No.16411) has both phosphorus solubilization and nitrogen fixation functions, its optimal growth temperature is 25-30°C, and its metabolic rate is significantly reduced in low-temperature environments.

[0006] In addition, the existing strains have poor single functions and ecological adaptability. Most bacterial agents are limited to a single function (such as nitrogen fixation or phosphorus solubilization), and there are no strains suitable for the soil characteristics of alpine meadows. For example, efficient nitrogen-fixing bacteria in water bodies are cultivated through gamma-ray mutagenesis, but the target application is sewage treatment and lacks soil colonization ability; although Nostoc (CCTCC M2023744) improves the nitrogen fertilizer utilization rate of rice, it is not adaptable enough to the low temperature and low organic matter environment of alpine meadows.

[0007] Existing low-temperature bacterial agents mostly rely on encapsulation or adsorption immobilization (such as sodium alginate carriers). Although this improves the survival rate of bacteria, the carriers are easily affected by pH fluctuations and microbial competition in the soil, resulting in functional attenuation.

[0008] In view of the above challenges, we urgently need to find a type of microbial species that can survive at low temperatures and have the function of nitrogen fixation. They can build a symbiotic system with crops and use their unique nitrogenase conversion mechanism to convert nitrogen in the atmosphere into ammonia nitrogen or nitrate nitrogen that can be directly used by plants, thereby effectively alleviating the problem of soil nitrogen deficiency. Summary of the invention

[0009] The first object of the present invention is to provide a low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1.

[0010] In order to achieve the first purpose of the present invention, the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 of the present invention was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on February 26, 2025, with a preservation number of CGMCCNO.33670; the Latin name of the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Pseudomonas sp.ZJY-Z1.

[0011] In a specific embodiment, the gene sequence of 16s rDNA of the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is sequence number (ID): 1.

[0012] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Gram-negative, catalase-negative, and nitrate-reducing-ability-positive.

[0013] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to fix nitrogen.

[0014] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to produce the plant growth hormone IAA.

[0015] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to solubilize phosphate; preferably, it also has the ability to produce siderophores.

[0016] The second object of the present invention is to provide a bacterial agent.

[0017] To achieve the second object of the present invention, the active ingredient of the bacterial agent comprises the above-mentioned low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1.

[0018] The third object of the present invention is to provide an application of the above-mentioned low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 in improving soil nutrients.

[0019] To achieve the third purpose of the present invention, the improvement includes nitrogen fixation, phosphorus solubilization, and increasing the plant growth hormone IAA, and the application includes inoculating the above-mentioned low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 into the soil.

[0020] In a specific embodiment, the application temperature is 5°C to 35°C, and the promoting plant growth preferably includes promoting the increase of plant biomass and / or the development of root system.

[0021] In a specific embodiment, the plant is a plateau plant; preferably includes Elymus dahliae, Stipa grass, and Lolium perenne.

[0022] Beneficial effects:

[0023] 1. The nitrogen-fixing bacteria provided by the present invention have excellent low-temperature resistance, which enables them to maintain activity in cold environments.

[0024] 2. The low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 of the present invention can effectively decompose insoluble phosphates, exhibit strong phosphorus-solubilizing ability, promote the conversion of insoluble phosphorus nutrients into soluble nutrients, thereby increasing the content of fast-acting nutrients in the soil, helping to reduce the accumulation of chemical fertilizers in the soil, and also has a certain ability to produce iron carriers, thereby improving the soil structure and creating more favorable conditions for plant growth and development.

[0025] 3. The low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 of the present invention has the ability to produce plant growth hormone IAA and has the function of promoting plant growth.

[0026] 4. The low-temperature-resistant Pseudomonas of the present invention not only broadens the application scope of microbial agents, especially in low-temperature ecosystems, but also provides powerful biotechnological support for sustainable agricultural practices. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the pure culture colony image of strain ZJY-Z1 on the culture medium;

[0028] Figure 2 is the 16S rRNA phylogenetic tree of strain ZJY-Z1;

[0029] Figure 3 is a picture of strain ZJY-Z1 on organophosphate medium;

[0030] Figure 4 is a picture of strain ZJY-Z1 on potassium-dissolving solid medium;

[0031] Figure 5 The IAA standard curve is plotted with IAA concentration as the abscissa and absorbance as the ordinate;

[0032] Figure 6 This graph shows the effect of strain ZJY-Z1 on the biomass of Elymus dahliae under different temperature conditions.

[0033] Figure 7 This is a graph showing the effect of strain ZJY-Z1 on the total nitrogen content of Elymus dahliae plants. DETAILED DESCRIPTION

[0034] In order to achieve the first purpose of the present invention, the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 of the present invention was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on February 26, 2025, with a preservation number of CGMCCNO.33670; the Latin name of the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Pseudomonas sp.ZJY-Z1.

[0035] The storage address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0036] The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is isolated from alpine meadow soil in Qamdo, Tibet. The isolation and culture method comprises: diluting a portion of alpine meadow soil collected in Qamdo, Tibet and applying the soil on a solid nitrogen-free screening medium for screening and culture to obtain a strain; then transferring a single colony to a liquid nitrogen-free screening medium for culture; the liquid nitrogen-free screening medium comprises the following base materials in parts by weight: malic acid; acid) 5.000g, potassium hydroxide (KOH) 4.500g, potassium dihydrogen phosphate (K2HPO4) 0.500g, calcium chloride dihydrate (CaCl2·2H2O) 0.020g, sodium chloride (NaCl) 0.100g, sodium molybdate dihydrate (Na2MoO4·2H2O) 0.002g, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.002g, biotin (Biotine) 10μg, 5% bromothymol blue (BTB) 5.000mL, pH 7.0-8.0. Agar is added to the formula of the solid nitrogen-free screening medium at a mass volume ratio of 2%.

[0037] Furthermore, the culture temperature on the solid screening medium is 26° C. for 5 to 7 days; the fermentation culture temperature is 30° C., pH=7.0 to 7.6, and is carried out under aerobic conditions.

[0038] A culture of the above-mentioned Pseudomonas sp. is obtained by culturing the above-mentioned ZJY-Z1 strain in a solid culture medium.

[0039] The biological characteristics of the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 are as follows: after culturing on a solid nitrogen-free screening medium at 26°C for 24 hours, the colonies are spherical, white in color, transparent and moist on the surface, and about 2 mm in diameter. They are Gram-negative, catalase-negative, and positive in the nitrate reduction test.

[0040] In a specific embodiment, the gene sequence of 16s rDNA of the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is sequence number (ID): 1.

[0041] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Gram-negative, catalase-negative, and nitrate-reducing-ability-positive.

[0042] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to fix nitrogen.

[0043] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to produce the plant growth hormone IAA.

[0044] In a specific embodiment, the low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to solubilize phosphate; preferably, it also has the ability to produce siderophores.

[0045] To achieve the second object of the present invention, the active ingredient of the bacterial agent comprises the above-mentioned low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1.

[0046] To achieve the third purpose of the present invention, the improvement includes nitrogen fixation, phosphorus solubilization, and increasing the plant growth hormone IAA, and the application includes inoculating the above-mentioned low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 into the soil.

[0047] In a specific embodiment, the application temperature is 5°C to 35°C, and the promoting plant growth preferably includes promoting the increase of plant biomass and / or the development of root system.

[0048] In a specific embodiment, the plant is a plateau plant; preferably includes Elymus dahliae, Stipa grass, and Lolium perenne.

[0049] The specific implementation modes of the present invention are further described below in conjunction with embodiments, but the present invention is not limited to the scope of the embodiments.

[0050] Example 1

[0051] Isolation, Purification and Identification of Pseudomonas ZJY-Z1

[0052] 1. Preparation of Culture Medium

[0053] Nitrogen-free bacteria screening medium (NFM medium): malic acid (Malic acid) 5.000g, potassium hydroxide (KOH) 4.500g, potassium dihydrogen phosphate (K2HPO4) 0.500g, calcium chloride dihydrate (CaCl2·2H2O) 0.020g, sodium chloride (NaCl) 0.100g, sodium molybdate dihydrate (Na2MoO4·2H2O) 0.002g, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.002g, biotin (Biotine) 10μg, 5% bromothymol blue (BTB) 5.000mL, agar 2% (2% agar is added to the solid culture medium, and no agar is added to the liquid medium), pH 7.0-8.0.

[0054] LB medium: tryptone 10 g, yeast extract 5 g, NaCl 5 g, distilled water 1000 mL, pH 7.0.

[0055] 2. Sample collection and separation and purification

[0056] In 2022, a piece of meadow soil was collected near the Guangzhao Township, Chaya County, Qamdo City, Tibet Autonomous Region. 10 g of the sample was weighed and added to a 250 mL conical flask. 100 mL of sterile water was poured in to fully dissolve it. Then it was placed in a shaker at 30 °C and 180 r / min for 30 min. The soil suspension of the enriched culture was diluted in a gradient to prepare 10 -5 -10 -9 Suspensions of different gradients were spread on solid nitrogen-free culture medium, and three parallels were made for each concentration. The suspensions were inverted and placed in a 28°C constant temperature incubator for culture. They were observed every day and single colonies with good growth and clear morphology were selected on the 7th day and transferred to liquid nitrogen-free culture medium for culture. The purification was repeated three times to obtain single colonies. The obtained strain was recorded as strain ZJY-Z1.

[0057] 3. Identification of strain ZJY-Z1

[0058] (1) Characteristics of strain ZJY-Z1. Strain ZJY-Z1 was inoculated into LB medium and cultured at 30°C for 24 h. The colonies were white, transparent, moist, and spherical. The colony morphology was shown in Figure 1(2) Physiological and biochemical experiments. The colony morphology was observed with the naked eye, including the shape, smoothness, texture, color, etc. The results are shown in Table 1.

[0059] Table 1 Morphological characteristics of ZJY-Z1 colonies

[0060] type color form Wet and dry Diameter(mm) texture ZJY-Z1 white spherical wet 2 Oil

[0061] At the same time, the strains were subjected to Gram staining and spore staining experiments to observe their morphological characteristics under a microscope. Physiological and biochemical identification mainly includes Gram staining, spore staining, nitrate reduction test, etc., and the identification was carried out according to the Manual of Identification of Common Bacterial Systems. The results are shown in Table 2.

[0062] Table 2 Physiological and biochemical identification results of ZJY-Z1 strain

[0063] type MR VP catalyst Catalase Nitrate oxidation Gram stain Spore staining ZJY-Z1 Negative Negative Positive Negative Positive Negative Not observed

[0064] The morphological and physiological and biochemical characteristics of strain ZJY-Z1 were studied and analyzed. The sequencing results were compared with the NCBI database using the Blastn method (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and a phylogenetic tree was constructed in combination with 16sRNA homology analysis. Figure 2 ), and was named Pseudomonas sp. ZJY-Z1.

[0065] Example 2

[0066] Test of nitrogen fixation ability of Pseudomonas ZJY-Z1

[0067] The ethylene peak area was detected by gas chromatography (reference: Wang Huiqiao, Chen Weifeng, et al. 2018. Identification of two autogenous nitrogen-fixing bacteria and basic research on their application [J]. Acta Agriculturae Boreali-Sinica, 33(4): 211-216. The acetylene reduction method was used to detect the amount of ethylene produced, which indirectly reflects the activity of nitrogenase). The single colony to be tested was inoculated into nitrogen-free liquid NFM medium and cultured at 15℃, 20℃, 25℃, 30℃, and 35℃ respectively. After 1d, 3d, and 5d of culture, 20ml of liquid was taken and injected into a sterilized conical flask (volume 70ml), and the sealing film was replaced with a rubber stopper on the sterile bench, and 5ml of gas was extracted with a sterile syringe, and then 5ml of acetylene was injected, sealed, and then cultured at 15℃, 20℃, 25℃, 30℃, and 35℃ for 24h.

[0068] Referring to Table 3, a series of temperature gradients were set up, with the nitrogen-free liquid culture medium without inoculation of bacterial agent as the blank control (CK), and the common nitrogen-fixing Bacillus subtilis as the control (KC), and the nitrogen content in the liquid culture medium was determined by spectrophotometry.

[0069] Through continuous 5-day interval monitoring, the nitrogenase activity of ZJY-Z1 strain was the highest at 30℃, which was 579.07nmol / mL·h, indicating that it has good nitrogen fixation ability. And under low temperature conditions, the nitrogenase activity of ZJY-Z1 strain was slightly higher than that of KC strain. The results are shown in Table 3.

[0070] Table 3 Nitrogenase activity of ZJY-Z1 and KC strains

[0071] Nitrogenase activity (nmol / mL·h) 1d 3d 5d ZJY-Z1(15℃) 18.33 155.69 0.31 ZJY-Z1(25℃) 46.67 719.54 0.96 ZJY-Z1(30℃) 17.70 579.07 1.34 ZJY-Z1(35℃) 10.72 112.19 0.68 Nitrogen-fixing Bacillus subtilis as control KC (15℃) 8.19 99.70 0.31 KC(25℃) 14.55 489.06 0.89 KC(30℃) 52.19 881.86 1.22 KC(35℃) 33.51 279.93 0.56

[0072] At the same time, the nitrogen content in the liquid culture medium at 15°C was measured by spectrophotometry and monitored at continuous 5D intervals. The results are shown in Table 4.

[0073] The nitrogen content of the blank group did not change significantly, but the nitrogen content of the ZJY-Z1 group increased significantly at 3 days and was greater than that of the control group, indicating that it had a good nitrogen fixation ability. It decreased significantly at 5 days, but was slightly higher than that at 1 day. The microorganism fixed nitrogen from the air into the culture medium, and consumed some of the nitrogen as the culture time increased.

[0074] Table 4 Total nitrogen content in each NFM liquid culture medium

[0075] Total nitrogen content in liquid culture medium 1d Nitrogen content 3d Nitrogen Content 5d Nitrogen content CK 0.4732±0.0032 0.4727±0.0182 0.4712±0.0143 ZJY-Z1 0.4652±0.0428b 0.5159±0.02549a 0.4751±0.01527b KC 0.4612±0.0095c 0.5021±0.0118b 0.5214±0.0156a

[0076] Note: Different lowercase letters indicate significant differences at the p < 0.05 level.

[0077] Example 3

[0078] Routine performance testing of strains

[0079] 1. Phosphorus dissolution experiment of nitrogen-fixing bacteria

[0080] After the test strain is activated, it is inoculated into the organic phosphorus-dissolving medium and placed in a 37°C constant temperature incubator for 5 days. Observe whether there is a transparent ring formed on the medium and its size. Use a ruler to measure the diameter of the colony and the hydrolysis circle. The relative size of the strain's phosphorus-dissolving ability can be judged based on the diameter ratio of the hydrolysis circle to the colony circle. Figure 3

[0081] The experiment showed that the ratio of ZJY-Z1 was 7.21, which showed a strong ability to dephosphorize phosphorus.

[0082] 2. Experiment on potassium dissolution by nitrogen-fixing bacteria

[0083] After the test strain is activated, it is inoculated on a potassium-dissolving solid medium and placed in a 37°C constant temperature incubator for 5 days. Observe whether a transparent ring is formed on the medium and its size. Use a ruler to measure the hydrolysis circle and colony diameter. The relative size of the strain's potassium-dissolving ability can be judged based on the diameter ratio of the hydrolysis circle to the colony circle. Figure 4

[0084] The experiment showed that ZJY-Z1 does not have the ability to dissolve potassium.

[0085] 3. Experiment on IAA production by nitrogen-fixing bacteria

[0086] After the test strain is activated, it is inoculated into LB liquid medium containing L-tryptophan and cultured at 30°C for 48 hours. A mixture of distilled water and Salkowski colorimetric reagent is used as a blank control, and the absorbance of each concentration is measured at OD530 using a UV spectrophotometer. The IAA standard curve is drawn with IAA concentration as the horizontal axis and absorbance as the vertical axis.

[0087] The experiment showed that the IAA content of ZJY-Z1 was significantly greater than 10 mg / L (P<0.05), which had a positive effect on plant growth.

[0088] Results Figure 5

[0089] 4. Experiment on Siderophore of Nitrogen-Fixing Bacteria

[0090] After the test strain was activated, it was inoculated into LB medium and placed in a 30°C shaker at 150 r / min for 48 h. The fermentation broth was centrifuged at 6000 r / min for 10 min. 2 mL of the supernatant was added with an equal amount of CAS detection solution and mixed thoroughly. After 1 h, the absorbance of the strain was measured using an OD630 UV spectrophotometer.

[0091] Take another 2 mL of CAS test solution and mix it with 2 mL of sterile LB liquid culture medium. Determine the absorbance value as the reference value using the same method and observe the color change.

[0092] Experiments show that the iron production capacity of ZJY-Z1 is weak.

[0093] Example 4

[0094] Effects of Pseudomonas ZJY-Z1 on Soil and Plant Growth

[0095] In this experiment, Elymus dahliae was selected as the experimental variety. The seeds were sterilized with 10% sodium hypochlorite solution for 5 minutes, then washed with sterile water before sowing.

[0096] The culture matrix consists of sieved (2 mm pore size) waste, 1% humic acid modified bentonite and 1% organic fertilizer. The seedling raising process is carried out in a solar greenhouse to ensure sufficient natural light. The environmental conditions of the greenhouse are set at a temperature of 20 to 25°C and a relative humidity of 60% to 80%.

[0097] When the Elymus dactylis seedlings grow to 30 days, select those with good growth and transplant them into plastic pots for cultivation.

[0098] The experiment set up 6 treatment groups, including plants inoculated and cultured with ZJY-Z1 strain at 25℃ and 5℃, and plants inoculated with commercial bacteria (KC). A control group (CK) without inoculation of bacteria was also set up. Figure 6 The experiment showed that ZJY-Z1 had little effect on the biomass of Elymus dahliae under different temperature conditions, and its biological activity was less affected by temperature and had a certain stability.

[0099] At the same time, under the same temperature conditions, the plants in the ZJY-Z1 group grew well, the biomass of Elymus dahliae was significantly higher than that of the commercial fungus (KC), and the plant root system was well developed, which was beneficial to soil improvement. Figure 6 .

[0100] Table 5 Total biomass of plants at 5℃ and 25℃

[0101] temperature Z1 Total biomass CK total biomass Total KC biomass 5℃ 0.0957 0.0546 0.0767 25℃ 0.1133 0.0867 0.1050

[0102] In terms of total nitrogen content of Elymus dahliae plants, the ZJY-Z1 bacterial agent group was superior to other groups. At 25°C, the nitrogen content was increased by 21.55% compared with the untreated group (CK); at 5°C, the nitrogen content of plants was increased by 38.71% compared with the commercial bacteria (KC). Figure 7 .

[0103] Table 6 Total biomass of plants at 5℃ and 25℃

[0104] Z1 Plant Nitrogen Content CK Plant Nitrogen Content KC Plant Nitrogen Content 5℃ 11.4151 9.3988 9.2001 25℃ 14.2943 11.0152 12.7032

[0105] In summary, the ZJY-Z1 strain has a significant promoting effect on the biomass of Elymus dahliae, and its effect is not affected by temperature. It has a strong biological fertilizer effect. At the same time, ZJY-Z1 has a good nitrogen-raising effect under different temperature conditions, which is beneficial to the growth of plants.

Claims

1. Low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1, characterized in that: The low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on February 26, 2025, with a preservation number of CGMCCNO.33670; the Latin name of the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Pseudomonassp.ZJY-Z1.

2. The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 according to claim 1, characterized in that: The gene sequence of 16s rDNA of the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is sequence number (ID):

1.

3. The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 according to claim 1 or 2, characterized in that: The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 is Gram-negative, catalase-negative, and nitrate-reducing-ability-positive.

4. The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 according to claim 1 or 2, characterized in that: The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to fix nitrogen.

5. The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 according to claim 1 or 2, characterized in that: The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to produce the plant growth hormone IAA.

6. The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 according to claim 1 or 2, characterized in that: The low-temperature resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 has the ability to solubilize phosphate; preferably, it also has the ability to produce siderophores.

7. A bacterial agent, characterized in that The active ingredient of the bacterial agent comprises the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 described in any one of claims 1 to 6.

8. Application of the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 in improving soil environment and promoting plant growth, wherein the improvement includes nitrogen fixation, phosphorus solubilization, and increasing plant growth hormone IAA, and the application includes inoculating the low-temperature-resistant Pseudomonas nitrogen-fixing bacteria ZJY-Z1 described in any one of claims 1 to 6 into the soil.

9. The use according to claim 8, characterized in that: The application temperature is 5° C. to 35° C., and the promoting plant growth preferably includes promoting the increase of plant biomass and / or the development of root system.

10. The use according to claim 9, characterized in that: The plants are plateau plants; preferably include Elymus dahliae, Stipa grass and Lolium perenne.

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