Preparation method and application of low-temperature-resistant growth-promoting complex microbial inoculant

Abstract

The invention discloses a preparation method and application of a low-temperature-resistant growth-promoting complex microbial inoculant, and relates to the technical field of microorganisms. The method comprises the following steps: mixing a bacterial solution of Enterobacter sp. DNCS-S1 (P1), a bacterial solution of Enterobacter sp. DNCS-S2 (P2) and a bacterial solution of Pseudomonas sp. DNLK-S1 (K5), and inoculating the mixture into an LB liquid culture medium with the volume fraction of the inoculation amount being 1%; shaking and culturing at 4-6 DEG C for 3-4 days to obtain the low-temperature-resistant growth-promoting complex microbial inoculant. The low-temperature-resistant growth-promoting complex microbial inoculant is applied to plant growth promotion under low-temperature stress. The invention can obtain the preparation method and the application of the low-temperature-resistant growth-promoting complex microbial inoculant.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for preparing and applying a low-temperature resistant growth-promoting compound bacterial agent. Background Technology

[0002] As a globally crucial crop used for food, feed, and industrial raw materials, the safety of maize production directly impacts national food security and agricultural economic stability. my country's annual maize planting area exceeds 600 million mu (approximately 40 million hectares), making it a core raw material in the feed industry (accounting for over 60% of feed formulations) and widely used in deep processing fields such as starch and fuel ethanol. However, low-temperature stress is a key abiotic factor restricting maize yield and quality. As a warm-season crop, maize's optimal growth temperature is 25-30℃. Below 10℃, root activity decreases by more than 50%, and the photosynthetic rate drops sharply. Temperatures below 5℃ during the seedling stage lead to leaf yellowing and increased cell membrane permeability, while low temperatures during the grain-filling stage reduce the thousand-grain weight by 10-20%, resulting in quality deterioration.

[0003] Currently, measures to alleviate crop low-temperature stress mainly rely on chemical regulation (such as spraying antifreeze agents) and genetic improvement. However, chemical reagents easily cause soil pollution, and the breeding cycle for cold-resistant varieties is long and costly. Microbial agents, as a green and environmentally friendly biological regulation method, currently available single-strain agents have limited stress resistance effects and insufficient low-temperature tolerance, making it difficult to meet actual production needs. Therefore, screening compound low-temperature resistant growth-promoting microbial agents with synergistic effects has become key to solving the problem of low-temperature yield reduction in maize. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned technical problems by providing a method for preparing and applying a low-temperature resistant growth-promoting compound microbial agent.

[0005] A method for preparing a low-temperature resistant growth-promoting compound microbial agent comprises the following steps:

[0006] Enterobacter sp. DNCS-S1 (P1) bacterial suspension, Enterobacter sp. DNCS-S2 (P2) bacterial suspension and Pseudomonas sp. DNLK-S1 (K5) bacterial suspension were mixed and inoculated into LB liquid medium at a volume fraction of 1%; after shaking culture at 4-6℃ for 3-4 days, a low-temperature resistant growth-promoting compound bacterial agent was obtained.

[0007] The volume ratio of Enterobacter sp. DNCS-S1 (P1) bacterial suspension, Enterobacter sp. DNCS-S2 (P2) bacterial suspension and Pseudomonas sp. DNLK-S1 (K5) bacterial suspension is (2.7~3):(1.8~2):(0.9~1);

[0008] The Enterobacter sp. DNCS-S1 (P1) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025853 on April 22, 2025.

[0009] The Enterobacter sp. DNCS-S2 (P2) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025854 on April 22, 2025.

[0010] The Pseudomonas sp. DNLK-S1 (K5) strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025855 on April 22, 2025.

[0011] Application of a low-temperature resistant growth-promoting compound microbial agent, wherein the low-temperature resistant growth-promoting compound microbial agent is used to promote plant growth under low-temperature stress.

[0012] The beneficial effects of this invention are:

[0013] Synergistic effects of the strains: The three strains, when combined, possess the ability to fix nitrogen, solubilize phosphorus, solubilize potassium, produce IAA, produce siderophores, and produce protease (specific quantitative data: nitrogen fixation increases ammonium nitrogen in the culture medium by 7.2~12.6 mg / L; phosphorus solubilization by 149~307 mg / L; potassium solubilization by 7.1~12.5 mg / L; IAA production by 12.3~28.6 mg / L; siderophore activity by 16.2%~30.7%; and protease production by 75.3~104.3 U / mL), thus alleviating low-temperature stress damage in maize from multiple dimensions.

[0014] Significant growth-promoting effects: When foliar sprayed, corn root length increased by 64.24% compared to the control group (CK), plant height increased by 31.23%, and net photosynthetic rate increased by 13.79 μmol. The nitrogen, phosphorus, and potassium contents in the aboveground parts increased by 22.1%, 37.3%, and 32.8%, respectively. During root irrigation, the nitrogen content in the underground parts of maize increased by 50.3% compared to the control (CK), the potassium content increased by 41.7%, and the chlorophyll content increased by 3.27 mg. (P<0.05).

[0015] Environmentally friendly: It does not damage the soil ecosystem after application and meets the needs of sustainable agricultural development.

[0016] This invention obtained Enterobacter DNCS-S1 (P1), Enterobacter DNCS-S2 (P2), and Pseudomonas DNLK-S1 (K5) by collecting maize root soil and using low-temperature targeted screening technology. Experiments showed that the low-temperature resistant growth-promoting bacterial agent obtained by combining the above three strains has the ability to fix nitrogen, solubilize phosphorus, solubilize potassium, produce IAA, produce siderophores, and produce proteases. It can also significantly improve the resistance of maize to low-temperature stress, providing new microbial resources for improving maize's cold resistance, and providing strain resources and theoretical support for sustainable agricultural development.

[0017] This invention provides a compound microbial agent obtained through microbial compounding and inter-strain interactions. The application of this compound microbial agent in promoting maize growth under low-temperature stress significantly increases maize plant height, root length, dry weight, and fresh weight. Furthermore, under low-temperature stress, it increases the nitrogen, phosphorus, and potassium content and photosynthetic intensity of both above-ground and below-ground parts of maize, with chlorophyll content significantly higher than the control group. This indicates that the compound low-temperature resistant growth-promoting microbial agent provided in this application plays a protective and growth-promoting role in maize's resistance to low-temperature stress.

[0018] By applying the product through root irrigation or foliar spraying, it can significantly increase the plant height, root length, fresh weight, and dry weight of maize under low temperature stress, as well as enhance photosynthetic intensity, chlorophyll content, and the absorption of nitrogen, phosphorus, and potassium nutrients, providing an efficient microbial solution for low-temperature resistant maize cultivation.

[0019] This invention provides a method for preparing and applying a low-temperature resistant growth-promoting compound microbial agent. Attached Figure Description

[0020] Figure 1 The figures show the morphology of the three strains in this invention on LB medium. Figure a represents DNCS-S1 (P1), Figure b represents DNCS-S2 (P2), and Figure c represents DNLK-S1 (K5).

[0021] Figure 2 This represents a phylogenetic tree of Enterobacter sp. DNCS-S1 (P1) constructed based on 16sDNA;

[0022] Figure 3 This represents a phylogenetic tree of Enterobacter sp. DNCS-S2 (P2) constructed based on 16sDNA;

[0023] Figure 4This represents a phylogenetic tree of Pseudomonas sp. DNLK-S1 (K5) constructed based on 16sDNA;

[0024] Figure 5 A diagram showing the antagonistic relationship between the three strains in this invention;

[0025] Figure 6 The diagram shows the regulatory effect of the low-temperature resistant growth-promoting compound microbial agent of the present invention on maize seedlings. CK represents the control group, T1 represents the sprayed microbial agent group, and T2 represents the root-drenched microbial agent group.

[0026] Figure 7 The diagram shows the effect of the low-temperature resistant growth-promoting compound microbial agent of the present invention on the growth promotion of maize seedlings. CK represents the control group, T1 represents the sprayed microbial agent group, and T2 represents the root-drenched microbial agent group.

[0027] Figure 8 The diagram shows the regulatory effect of the low-temperature resistant growth-promoting compound microbial agent of the present invention on the photosynthesis and chlorophyll content of corn. CK represents the control group, T1 represents the sprayed microbial agent group, and T2 represents the root-drenched microbial agent group.

[0028] Figure 9 The graph shows the regulation of nitrogen, phosphorus and potassium content in corn by the low-temperature resistant growth-promoting compound microbial agent of the present invention. CK represents the control group, T1 represents the foliar spraying group, and T2 represents the root irrigation group. Detailed Implementation

[0029] Specific Implementation Method 1: This implementation method describes a method for preparing a low-temperature resistant growth-promoting compound microbial agent, which is carried out according to the following steps:

[0030] Enterobacter sp. DNCS-S1 (P1) bacterial suspension, Enterobacter sp. DNCS-S2 (P2) bacterial suspension and Pseudomonas sp. DNLK-S1 (K5) bacterial suspension were mixed and inoculated into LB liquid medium at a volume fraction of 1%; after shaking culture at 4-6℃ for 3-4 days, a low-temperature resistant growth-promoting compound bacterial agent was obtained.

[0031] The volume ratio of Enterobacter sp. DNCS-S1 (P1) bacterial suspension, Enterobacter sp. DNCS-S2 (P2) bacterial suspension and Pseudomonas sp. DNLK-S1 (K5) bacterial suspension is (2.7~3):(1.8~2):(0.9~1);

[0032] The Enterobacter sp. DNCS-S1 (P1) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025853 on April 22, 2025.

[0033] The Enterobacter sp. DNCS-S2 (P2) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025854 on April 22, 2025.

[0034] The Pseudomonas sp. DNLK-S1 (K5) strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025855 on April 22, 2025.

[0035] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that the rotation speed of the oscillation culture is 140~160 rpm.

[0036] The other steps are the same as in Specific Implementation Method 1.

[0037] Specific Implementation Method 3: The difference between this implementation method and Specific Implementation Method 1 or 2 is that the optical density OD600 of the low-temperature resistant growth-promoting compound microbial agent is 0.89~0.98.

[0038] The other steps are the same as in Specific Implementation Method 1 or 2.

[0039] Specific Implementation Method Four: The difference between this implementation method and Specific Implementation Methods One to Three is that the viable count of the Enterobacter sp. DNCS-S1 (P1) bacterial solution is 2 × 10⁻⁶. 8 CFU / mL ~5×10 8 CFU / mL.

[0040] The other steps are the same as those in Specific Implementation Methods One to Three.

[0041] Specific Implementation Method Five: The difference between this implementation method and Specific Implementation Methods One to Four is that the viable count of the Enterobacter sp. DNCS-S2 (P2) bacterial suspension is 2 × 10⁻⁶. 8 CFU / mL ~2×10 9 CFU / mL.

[0042] The other steps are the same as those in Specific Implementation Methods One through Four.

[0043] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Methods One to Five is that the viable count of the Pseudomonas sp. DNLK-S1 (K5) bacterial suspension is 2 × 10⁻⁶. 8 CFU / mL ~8×10 9 CFU / mL.

[0044] The other steps are the same as those in Specific Implementation Methods 1 to 5.

[0045] Specific Implementation Method Seven: This implementation method describes the application of a low-temperature resistant growth-promoting compound microbial agent, specifically its application in promoting plant growth under low-temperature stress.

[0046] Specific Implementation Method Eight: The difference between this implementation method and Specific Implementation Method Seven is that the low-temperature resistant growth-promoting compound bacterial agent is used as one or more of the following: root irrigation agent, seed soaking agent, and foliar spray agent.

[0047] The other steps are the same as in Specific Implementation Method Seven.

[0048] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Seven or Eight in that: promoting plant growth includes increasing the fresh weight of the plant, increasing the dry weight of the plant, increasing the plant height, and increasing the root length of the plant, or one or more of these.

[0049] The other steps are the same as in specific implementation methods seven or eight.

[0050] Specific Implementation Method 10: The difference between this implementation method and Specific Implementation Methods 7 to 9 is that the plant mentioned is corn.

[0051] The other steps are the same as those in Specific Implementation Methods 7 to 9.

[0052] The aforementioned low-temperature resistant growth-promoting compound microbial agent is used as a root irrigation agent for corn seedlings to increase the dry weight, fresh weight, and plant height of corn seedlings under low-temperature stress, as well as to increase the nitrogen, phosphorus, and potassium nutrient content in the above-ground and underground parts of corn seedlings under low-temperature stress, and to increase the photosynthetic intensity and chlorophyll content of corn seedlings under low-temperature stress.

[0053] The aforementioned low-temperature resistant growth-promoting compound microbial agent is used as a foliar spray for corn seedlings to increase the dry weight, fresh weight, and plant height of corn seedlings under low-temperature stress, as well as to increase the nitrogen, phosphorus, and potassium nutrient content in the above-ground and underground parts of corn seedlings under low-temperature stress, and to increase the photosynthetic intensity and chlorophyll content of corn seedlings under low-temperature stress.

[0054] The beneficial effects of the present invention are verified using the following embodiments:

[0055] Example 1:

[0056] The low-temperature resistant growth-promoting compound bacterial agent in this embodiment includes Enterobacter sp. DNCS-S1 (P1) bacterial solution, Enterobacter sp. DNCS-S2 (P2) bacterial solution and Pseudomonas sp. DNLK-S1 (K5) bacterial solution.

[0057] The Enterobacter sp. DNCS-S1 (P1) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025853, deposited on April 22, 2025, at Wuhan University, Wuhan, China.

[0058] The Enterobacter sp. DNCS-S2 (P2) is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025854, deposited on April 22, 2025, at Wuhan University, Wuhan, China.

[0059] The Pseudomonas sp. DNLK-S1 (K5) strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: 2025855, deposited on April 22, 2025, at Wuhan University, Wuhan, China.

[0060] The composition of the culture medium used is shown below:

[0061] LB medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 20 g agar, 1000 mL distilled water, adjust pH to 7.0. If agar is omitted, it is LB liquid medium.

[0062] Ashby's nitrogen-free medium: mannitol (or glycerol) 10.0 g, KH2PO4 0.2 g, 0.2g, NaCl 0.2g, 5.0 g CaCO3, 20 g agar, 10 mL 0.5% Congo red solution, 1000 mL distilled water, adjust pH to 7.2 ± 0.2.

[0063] Modified Alexandrov medium: sucrose 5.0 g, Na₂HPO₄ 2.0 g, FeCl₃ 0.005 g, 0.5 g, CaCO3 0.1 g, potassium feldspar powder (washed 5 times) 1.0 g, bromothymol blue (BTB) 0.1 g, agar 18~20 g, distilled water 1000 mL, adjust pH to 7.0.

[0064] NBRIP phosphorus-solubilizing medium: Solid medium: Ca3(PO4)2 5.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3 g, 0.3 g, 0.003 g yeast extract, 0.5 g glucose, 10 g distilled water, 1000 mL, adjust pH to 7.0-7.5. Liquid culture medium: Prepare according to the above formula, but without agar.

[0065] 1. Screening and identification of strains:

[0066] Corn root soil was collected and analyzed using a gradient dilution method (10... -4 ~10 -6 The isolated strains were initially screened for low-temperature tolerance by culturing at 5℃ for 7 days. Target strains were then screened by combining Ashby nitrogen-fixing medium (to observe nitrogen-fixing ability), NBRIP phosphorus-solubilizing medium (to determine phosphorus-solubilizing ability using the clear zone method), modified Alexandrov potassium-solubilizing medium (to assess potassium-solubilizing ability), and functional verification of IAA, siderophores, and proteases. The strains were then preserved in glycerol tubes at -80℃.

[0067] Functional verification method: The strain was activated and propagated using LB liquid medium. Then, the strain was inoculated onto Ashby nitrogen-fixing medium, NBRIP phosphorus-solubilizing medium and modified Alexandrov potassium-solubilizing medium, respectively. The growth of the cultured strain was observed, and the method of judgment was whether a clear zone was formed on the medium.

[0068] IAA production capacity: After culturing in King medium at 5°C with shaking for 5 days, the Salkowski colorimetric method was used for detection, with absorbance measured at 530 nm and quantification performed using a standard curve.

[0069] Siderophore production capacity: cultured in MKB medium at 5℃ for 5 days, color development was performed using CAS detection solution, and absorbance was measured at 630 nm. The calculation formula was: siderophore activity (%) = (A0-A1) / A0×100 (A0 is the absorbance of the control, and A1 is the absorbance of the fermentation broth).

[0070] Protease production capacity: According to GB / T 23527-2009, the enzyme activity was determined by the casein degradation method, with absorbance measured at 660 nm, and the enzyme activity was quantified by a standard curve.

[0071] Antagonistic effect of strains: Three strains were streaked crosswise onto LB agar and incubated at 5°C for 5 days. Colony growth in the cross-strain area was observed. The results showed no antagonistic effect (colonies in the cross-strain area grew normally). Figure 5 As shown.

[0072] Strain identification: 16S rDNA sequencing (performed by Shanghai Ling'en Biotechnology Co., Ltd.) and phylogenetic tree construction using MEGA 7.0 were performed. The DNA sequence of DNCS-S1 (P1) was compared with that of Enterobacter sp., with accession number NO.2025853. The DNA sequence of DNCS-S2 (P2) was compared with that of Enterobacter sp., with accession number NO.2025854. The DNA sequence of DNLK-S1 (K5) was compared with that of Pseudomonas sp., with accession number NO.2025855.

[0073] The NCBI sequence number of Enterobacter sp. DNCS-S1 (P1) is OR335099, the NCBI sequence number of Enterobacter sp. DNCS-S2 (P2) is OR335204, and the NCBI sequence number of Pseudomonas sp. DNLK-S1 (K5) is PP077109.

[0074] Strain morphology: The three strains were activated and cultured in LB solid medium, and their morphology on the medium was as follows. Figure 1 As shown. DNCS-1 strain has a nearly circular shape from the inside out, with relatively regular edges, and appears yellow. DNCS-2 shows that this strain gradually forms nearly circular colonies at the beginning of cultivation, with a smooth surface, irregular edges, and a milky white color, approximately 0.5 × 0.43 µm in size. After 24 h of cultivation, the colony volume gradually increases, white wrinkles begin to appear, it becomes somewhat sticky, and the shape no longer approaches circularity. DNLK-S1 (K5) has an elliptical morphology, appears yellow, and has spiral patterns. The results are as follows. Figure 1 As shown.

[0075] 2. Cultivation and preparation of compound microbial agents:

[0076] Strain activation: The three strains were cultured in LB liquid medium at 5℃ and 150 rpm for 3 days. The concentrations of the activated bacterial solutions were: DNCS-S1(P1) 2×10⁻⁶. 8 ~5×10 8 DNCS-S2(P2)2×10 8 ~2×10 9 DNLK-S1(K5)2×10 8 ~8×10 9 .

[0077] Compound fermentation: Inoculate the bacterial culture into LB liquid medium at a volume ratio of 3:2:1 (1% inoculum), and incubate at 5℃ and 150 rpm with shaking for 3 days. Detect the viable cell count ≥ OD 600 The value is 0.89, which yields the compound bacterial agent.

[0078] 3. Verification of the growth-promoting effect of compound microbial agents on maize seedlings:

[0079] Test variety: Pioneer 1216 (purchased from Heilongjiang Academy of Agricultural Sciences);

[0080] Test soil: Sampled from farmland in Heilongjiang Province. The basic physicochemical properties of the farmland soil are as follows:

[0081] pH=6.87, organic matter Total nitrogen Total phosphorus Total potassium fast-acting phosphorus Quick-acting potassium .

[0082] Treatment groups: control group (CK, sprayed with an equal volume of sterile water), foliar spraying group (T1, spraying rate of bacterial agent 500 mL / min), root irrigation group (T2), with 5 replicates for each group;

[0083] Environmental conditions: 5℃ during the day, 14 hours of light, 10℃ at night, 10 hours of light, humidity 60%~70%, indicators were measured after 28 days of treatment.

[0084] Measurement indicators and methods:

[0085] Growth indicators: plant height, root length (measured with a ruler), fresh weight, dry weight (weighed after drying at 80℃ to constant weight);

[0086] Photosynthetic indicators: Net photosynthetic rate (Pn), stomatal conductance (Gs), etc. were measured by the Li-6400 portable photosynthesis meter, and chlorophyll content was measured by the SPAD-502 Plus.

[0087] Nutrient content: Nitrogen, phosphorus and potassium content were determined using a flow analyzer (model AA3, SEAL GmbH, Germany) in accordance with NY / T 2017-2011.

[0088] Results analysis:

[0089] One-way ANOVA was performed using SPSS 22.0 software, and Duncan's test was used to test for statistical significance (P<0.05).

[0090] The synergistic effect of three strains of Enterobacter sp. DNCS-S1 (P1), Enterobacter sp. DNCS-S2 (P2), and Pseudomonas sp. DNLK-S1 (K5) is the core reason why the compound microbial agent improves the growth, photosynthesis, and nutrient indicators of maize. The synergistic principle can be summarized as follows: the three strains form a highly efficient synergistic system through functional complementarity, metabolic synergy, and microenvironment adaptation. DNCS-S1 (P1) and DNCS-S2 (P2) mainly control nitrogen fixation and IAA (12.3~28.6 mg / L) synthesis, while DNLK-S1 (K5) mainly controls phosphorus solubilization (149~307 mg / L), potassium solubilization (7.1~12.5 mg / L), and siderophore secretion (activity 16.2%~30.7%). DNCS-S1 (P1) and DNCS-S2 (P2)... The production of protease (75.3~104.3 U / mL) also assists in nutrient release. These three functions cover nutrient activation, growth regulation, and photosynthetic enhancement, precisely compensating for problems such as decreased root vigor, insufficient nutrient absorption, and inhibited hormone synthesis in maize under low temperatures. Simultaneously, the organic acids produced by DNLK-S1 (K5) provide a suitable growth environment for DNCS-S1 (P1) and DNCS-S2 (P2). The small-molecule nutrients produced by the degradation of organic nitrogen by DNCS-S1 (P1) and DNCS-S2 (P2) feed back to DNLK-S1 (K5), forming a metabolic cycle. Furthermore, the extracellular polysaccharides and antifreeze protein analogs secreted after the combination enhance low-temperature tolerance and maintain a high viable cell count (≥2×10⁻⁶). 9 In addition, the three synergistically inhibited harmful rhizosphere microorganisms, optimized soil structure, and created a microenvironment conducive to maize's stress resistance. Ultimately, through multi-dimensional synergy, growth indicators were achieved: root length of T1 group was 23.27 cm (64.24% higher than CK), and plant height of T2 group was 89.63 cm (34.78% higher than CK).

[0091] Photosynthetic index: Net photosynthetic rate of group T1 (Compared to CK +13.79), chlorophyll content in group T2 (Compared to CK +3.27).

[0092] Nutrient indicators: Phosphorus content in the aboveground parts of group T1 (Compared to CK +37.3%), potassium content in the underground part of group T2 (Compared to CK +41.7%).

Claims

1. A preparation method of a low-temperature-resistant growth-promoting composite microbial inoculant, characterized in that, The preparation method is carried out according to the following steps: After mixing Enterobacter sp. DNCS-S1 (P1) bacterial solution, Enterobacter sp. DNCS-S2 (P2) bacterial solution and Pseudomonas sp. DNLK-S1 (K5) bacterial solution, inoculate into LB liquid medium, and the volume fraction of the inoculation amount is 1%; oscillation culture at 4~6℃ for 3~4d, to obtain low-temperature-resistant growth-promoting composite microbial inoculant; The volume ratio of the Enterobacter sp. DNCS-S1 (P1) bacterial solution, the Enterobacter sp. DNCS-S2 (P2) bacterial solution and the Pseudomonas sp. DNLK-S1 (K5) bacterial solution is (2.7~3):(1.8~2):(0.9~1); The Enterobacter sp. DNCS-S1 (P1) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025853, and the preservation time is April 22, 2025; The Enterobacter sp. DNCS-S2 (P2) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025854, and the preservation time is April 22, 2025; The Pseudomonas sp. DNLK-S1 (K5) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025855, and the preservation time is April 22, 2025.

2. The preparation method of the low-temperature-resistant growth-promoting composite microbial agent according to claim 1, characterized in that, The rotation speed of oscillation culture is 140~160 rpm.

3. The preparation method of the low-temperature-resistant growth-promoting composite microbial agent according to claim 1, characterized in that, The optical density OD600 of the low-temperature-resistant growth-promoting composite microbial inoculant is 0.89~0.

98.

4. The preparation method of the low-temperature-resistant growth-promoting composite microbial agent according to claim 1, characterized in that, The viable cell count of the Enterobacter sp. DNCS-S1 (P1) bacterial solution is 2 x 10 8 CFU / mL~5 x 10 8 CFU / mL.

5. The preparation method of the low-temperature-resistant growth-promoting composite microbial agent according to claim 1, characterized in that, The viable cell count of the Enterobacter sp. DNCS-S2 (P2) bacterial solution is 2 x 10 8 CFU / mL~2 x 10 9 CFU / mL.

6. The preparation method of the low-temperature resistant growth promoting microbial agent according to claim 1, characterized in that, The live bacteria amount of the Pseudomonas sp. DNLK-S1 (K5) bacterial solution is 2x10 8 CFU / mL~8x10 9 CFU / mL.

7. The application of low-temperature-resistant growth-promoting microbial agent prepared by the method of any one of claims 1-6, characterized in that, The application of the low-temperature-resistant growth-promoting composite microbial inoculant in promoting plant growth under low-temperature stress.

8. The application of the low-temperature resistant growth-promoting compound microbial inoculant according to claim 7, characterized in that, The low-temperature-resistant growth-promoting composite microbial inoculant is used as one or several of root irrigation agent, seed soaking agent and foliar spraying agent.

9. The application of the low-temperature resistant growth-promoting composite microbial agent according to claim 7, characterized in that, Promoting plant growth includes one or several of increasing fresh weight of plants, increasing dry weight of plants, increasing plant height and increasing root length of plants.

10. The application of the low-temperature resistant growth-promoting compound microbial agent according to claim 7 or 9, characterized in that, The plant is corn. The preparation method is carried out according to the following steps: After mixing Enterobacter sp. DNCS-S1 (P1) bacterial solution, Enterobacter sp. DNCS-S2 (P2) bacterial solution and Pseudomonas sp. DNLK-S1 (K5) bacterial solution, inoculate into LB liquid medium, and the volume fraction of the inoculation amount is 1%; oscillation culture at 4~6℃ for 3~4d, to obtain low-temperature-resistant growth-promoting composite microbial inoculant; The volume ratio of the Enterobacter sp. DNCS-S1 (P1) bacterial solution, the Enterobacter sp. DNCS-S2 (P2) bacterial solution and the Pseudomonas sp. DNLK-S1 (K5) bacterial solution is (2.7~3):(1.8~2):(0.9~1); The Enterobacter sp. DNCS-S1 (P1) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025853, and the preservation time is April 22, 2025; The Enterobacter sp. DNCS-S2 (P2) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025854, and the preservation time is April 22, 2025; The Pseudomonas sp. DNLK-S1 (K5) is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO:2025855, and the preservation time is April 22, 2025. The rotation speed of oscillation culture is 140~160 rpm. The optical density OD600 of the low-temperature-resistant growth-promoting composite microbial inoculant is 0.89~0.

98. The application of the low-temperature-resistant growth-promoting composite microbial inoculant in promoting plant growth under low-temperature stress. The low-temperature-resistant growth-promoting composite microbial inoculant is used as one or several of root irrigation agent, seed soaking agent and foliar spraying agent. Promoting plant growth includes one or several of increasing fresh weight of plants, increasing dry weight of plants, increasing plant height and increasing root length of plants. The plant is corn.

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