Composite microbial inoculant prepared from salt-tolerant strain Y3 and application thereof

Abstract

The application discloses a composite microbial inoculant prepared from a salt and alkali resistant strain Y3, wherein the composite microbial inoculant comprises bacteria and fungi; the bacteria are the salt and alkali resistant strain Y3 with a preservation number of CCTCC NO: M 20251595; and the fungi are a strain NJAU4742 with a preservation number of CGMCC NO.12166. The application also discloses application of the composite microbial inoculant in promoting growth of crops in a salt and alkali resistant manner. The salt and alkali resistant strain Y3 screened by the application has good salt and alkali resistance, IAA production and other functions; the Y3 single microbial inoculant prepared from the strain Y3 has better growth promotion capacity on crops under a salt and alkali condition; and the composite microbial inoculant prepared from the strain Y3 and the strain NJAU4742 has better growth promotion capacity on crops under a salt and alkali condition. The application provides a strain resource and technical support for promoting growth of crops in a salt and alkali area.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound bacterial agent prepared from a salt-tolerant bacterial strain Y3 and its application. Background Technology

[0002] Soil salinization is a prominent problem in soil degradation. Compared with other improvement measures, using microbial agents prepared from functional microorganisms to improve saline-alkali land can achieve better improvement results while having the advantages of being ecologically friendly, pollution-free, and having a long-lasting effect. Currently, the application of microbial agents prepared from functional microorganisms has become one of the important measures for the improvement of saline-alkali land.

[0003] Bacillus is a type of Gram-positive bacterium that is aerobic or facultative anaerobic and produces spores. Bacillus is widely studied and applied as a model species, becoming one of the most extensively researched and widely used crop growth-promoting and biocontrol bacteria in the world. While there are many types of Bacillus, the main species used in agriculture and forestry include Bacillus subtilis (…). Bacillus subtilis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus pumilus ( Bacillus pumilus ), Bacillus megaterium ( Bacillus megaterium ) etc. Ji Yan et al. [1] Studies have shown that Bacillus subtilis enhances maize growth and promotes dry matter accumulation in maize under saline-alkali conditions by improving root morphology and stress-resistant enzyme activity; Lü Zhengyang et al. [2] Studies have shown that *Bacillus amyloliquefaciens* PT6-1 increased the plant height, total fresh weight, total photosynthetic pigment content, peroxidase activity, and catalase activity of *Giant Napier* under saline-alkali conditions; Chen Lu et al. [3] Studies have shown that Bacillus pumilus JT8 promotes alfalfa seed germination and seedling growth at a saline solution concentration of 100 mmol / L; Li Qingqing et al. [4] Studies have shown that *Bacillus megaterium* possesses strong salt tolerance and phosphorus-solubilizing effects, and can effectively promote soybean seed germination. Meanwhile, with ongoing research on *Bacillus*, more and more new strains with good biocontrol and growth-promoting effects have been discovered, such as *Bacillus oryzae*. In recent years, some studies have reported that *Bacillus oryzae* promotes crop growth, but most of these promotions are indirect, achieved through antagonistic effects against pathogens. For example, Xu Lingna et al. [5] The study reported the inhibitory effect of volatile substances produced by Bacillus cereus 12a on peach brown rot; Liu Zhihui et al. [6] The inhibitory effect of Bacillus cereus 265ZY1 on Fusarium wilt of potato was reported; Zhang Guoyi et al. [7]The inhibitory effect of Bacillus oryzae HJ-5 on Verticillium wilt of cotton was reported; Zhang Meng et al. [8] The inhibitory effects of Bacillus oryzae wm005 on various pathogens, including Fusarium wilt of watermelon, Sclerotinia sclerotiorum of rapeseed, and Rhizoctonia solani of cucumber, were reported.

[0004] However, there are still shortcomings in the exploration of Bacillus resources in saline-alkali environments. By screening for functions such as salt tolerance, nutrient activation, and growth promotion, it is hoped that new strains with strong adaptability to high saline-alkali environments, the ability to improve saline-alkali soils, regulate crop resistance under saline-alkali soil conditions, and promote crop growth can be discovered. At the same time, based on this, other strains can be combined to achieve better crop salt tolerance and growth promotion functions.

[0005] [1] Ji Yan, Zhang Wenming, Liu Jizhi, et al. Effects of five nitrogen-fixing bacteria on maize growth and stress-resistance enzyme activity in different saline-alkali soils [J]. Chinese Agricultural Science Bulletin, 2024, 40(18):14-21.

[0006] [2] Lü Zhengyang, Shao Dengke, Zhang Chunyuan, et al. Screening and identification of a salt-tolerant bacterium and its growth-promoting effect on plants [J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2023, 52(01):41-47. DOI:10.13323 / j.cnki.j.fafu(nat.sci.).2023.01.007.

[0007] [3] Chen Lu, Zhang Yanrui, Sun Huifu, et al. Application research of microbial inoculants in improving saline-alkali land [J]. Grassland Science, 2023, (05): 1-10.

[0008] [4] Li Qingqing, Zhang Rui, Gao Yanting, et al. Phosphorus-solubilizing effect of salt-tolerant phosphate-solubilizing bacteria and its influence on soybean germination [J]. Bulletin of Microbiology, 2024, 51(11):4574-4589. DOI:10.13344 / j.microbiol.china.240190.

[0009] [5] Xu Lingna, Xu Xinyue, Dong Zheng. Inhibitory effect of volatile substances produced by Bacillus cereus on brown rot of peach [J]. Agricultural Engineering Technology, 2022, 42(08):20-22. DOI:10.16815 / j.cnki.11-5436 / s.2022.08.012.

[0010] [6] Liu Zhihui, Hao Rongrong, Xu Yongfeng, et al. Screening, identification and efficacy of biocontrol strains against Fusarium solani dry rot of potato [J]. Journal of Zhejiang Agricultural Sciences, 2019, 31(07):1105-1111.

[0011] [7] Zhang Guoyi, Cheng Lin, Huang Liying, et al. Synergistic effect of mycorrhizal fungi and Bacillus thuringiensis to inhibit Verticillium wilt in cotton [J]. Journal of Zhejiang Agricultural Sciences, 2018, 30(06):1008-1015.

[0012] [8] Zhang Meng, Wang Qiong, Wan Dongguang, et al. Isolation, identification and control effect of a strain of Bacillus cereus on watermelon wilt [J]. Jiangsu Agricultural Sciences, 2017, 45(08):97-100. DOI:10.15889 / j.issn.1002-1302.2017.08.027. Summary of the Invention

[0013] The purpose of this invention is to provide a compound bacterial agent prepared from salt-tolerant strain Y3 and its application, in order to overcome the shortcomings of the prior art.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] The first aspect of this invention provides a compound bacterial agent prepared from a salt-tolerant bacterial strain Y3, the compound bacterial agent comprising bacteria and fungi, wherein the bacteria is a salt-tolerant bacterial strain Y3, which is classified and named as follows: Peribacillus frigoritolerans Y3, deposited at the China Center for Type Culture Collection (CCTCC) on July 14, 2025, with accession number CCTCC NO: M20251595; the fungus described is strain NJAU4742, taxonomically named *Trichoderma guiyuanense*. Trichoderma guizhouense It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC NO. 12166.

[0016] Further, the compound microbial agent is prepared by the following steps: mixing Y3 bacterial powder and NJAU4742 bacterial powder in water to obtain the compound microbial agent; wherein, the Y3 bacterial powder is a bacterial powder obtained from the salt-tolerant strain Y3, and the effective viable count is 1×10⁻⁶. 9 CFU / g or higher; NJAU4742 mycelial powder is made from strain NJAU4742, with an effective viable count (i.e., spore count) of 1×10⁻⁶. 9 The mass ratio of Y3 bacterial powder to NJAU4742 bacterial powder is 2-3:1, and the total mass of Y3 bacterial powder and NJAU4742 bacterial powder is 1.5-2% of the mass of water.

[0017] The second aspect of the present invention provides the application of the above-mentioned salt-tolerant strain Y3 in promoting salt-tolerant growth of crops, wherein the crop is maize.

[0018] The beneficial effects of this invention are:

[0019] This invention screened a salt- and alkali-tolerant bacterial strain Y3, which exhibits good salt and alkali tolerance, and produces indole-3-acetic acid (IAA). A single-strain inoculant prepared from Y3 shows superior growth-promoting ability in crops under saline- and alkali conditions; a compound inoculant prepared from Y3 and strain NJAU4742 shows even better growth-promoting ability in crops under saline- and alkali conditions. This invention provides bacterial strain resources and technical support for promoting crop growth in saline- and alkali-tolerant land. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain Y3.

[0021] Figure 2 This is a phylogenetic tree of strain Y3 constructed based on the 16S rRNA gene sequence.

[0022] Figure 3 This is the growth curve of strain Y3.

[0023] Figure 4 This is the IAA standard curve graph.

[0024] Figure 5 The images show the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions, and the phenotypic images of Chinese cabbage under -2‰ NaCl salt stress.

[0025] Figure 6 The images show the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions, and the phenotypic images of Chinese cabbage under -4‰ NaCl salt stress.

[0026] Figure 7 These are images showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions, and images of the phenotype of Chinese cabbage under -5‰ NaCl salt stress.

[0027] Figure 8 This image shows the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - a phenotypic image of Chinese cabbage under pH 8.5 alkaline stress.

[0028] Figure 9 This image shows the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - a phenotypic image of Chinese cabbage under pH 9 alkaline stress.

[0029] Figure 10 These are images showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions: -4‰ NaCl + pH 8.5.

[0030] Figure 11 The images show the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions: -5‰ NaCl + pH 9.

[0031] Figure 12This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The plant height of Chinese cabbage under -4‰ NaCl salt stress is also shown.

[0032] Figure 13 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The stem diameter of Chinese cabbage plants under -4‰ NaCl salt stress is also shown.

[0033] Figure 14 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The aboveground fresh weight of Chinese cabbage plants under -4‰ NaCl salt stress.

[0034] Figure 15 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The SPAD value of Chinese cabbage plants under -4‰ NaCl salt stress is also shown.

[0035] Figure 16 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - plant height of Chinese cabbage under pH 8.5 alkali stress.

[0036] Figure 17 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - stem diameter of Chinese cabbage plants under pH 8.5 alkali stress.

[0037] Figure 18 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - the fresh weight of the aboveground parts of Chinese cabbage plants under pH 8.5 alkali stress.

[0038] Figure 19 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted Chinese cabbage under saline-alkali conditions - SPAD value of Chinese cabbage plants under pH 8.5 alkali stress.

[0039] Figure 20 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The plant height of Chinese cabbage under salt-alkali stress is shown in the figure: -4‰ NaCl + pH 8.5.

[0040] Figure 21 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The stem diameter of Chinese cabbage plants under salt-alkali stress is shown in the figure: -4‰ NaCl + pH 8.5.

[0041] Figure 22 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The aboveground fresh weight of Chinese cabbage plants under saline-alkali stress was -4‰ NaCl + pH 8.5.

[0042] Figure 23This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted Chinese cabbage under saline-alkali conditions. The SPAD value of Chinese cabbage plants under salt-alkali stress is -4‰ NaCl + pH 8.5.

[0043] Figure 24 This image shows the effect of strain Y3 on the salt and alkali tolerance of the aboveground parts of potted maize under saline-alkali conditions, along with phenotypic images of maize under -4‰ NaCl salt stress.

[0044] Figure 25 This image shows the effect of strain Y3 on the salt and alkali tolerance of the aboveground parts of potted maize under saline-alkali conditions - a phenotypic image of maize under pH 8.5 alkali stress.

[0045] Figure 26 These are images showing the effect of strain Y3 on the salt and alkali tolerance of the aboveground parts of potted maize under saline-alkali conditions, and phenotypic images of maize under -4‰ NaCl + pH 8.5 salt and alkali stress.

[0046] Figure 27 This image shows the effect of strain Y3 on the salt and alkali tolerance of the aboveground parts of potted maize under saline-alkali conditions: -5‰ NaCl + pH 9.

[0047] Figure 28 This image shows the effect of strain Y3 on the salt and alkali tolerance of the underground parts of potted maize under saline-alkali conditions. It is also a phenotypic image of maize roots under -4‰ NaCl salt stress.

[0048] Figure 29 This image shows the effect of strain Y3 on the salt and alkali tolerance of the underground parts of potted maize under saline-alkali conditions - a phenotypic image of maize roots under pH 8.5 alkali stress.

[0049] Figure 30 These are images showing the salt tolerance effect of strain Y3 on the underground parts of potted maize under saline-alkali conditions, specifically images of maize root phenotypes under -4‰ NaCl + pH 8.5 saline-alkali stress.

[0050] Figure 31 This image shows the effect of strain Y3 on the salt and alkali tolerance of the underground parts of potted maize under saline-alkali conditions. It is a phenotypic image of maize roots under salt and alkali stress of -5‰ NaCl + pH9.

[0051] Figure 32 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The plant height of maize under -4‰ NaCl salt stress is also shown.

[0052] Figure 33 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted maize under saline-alkali conditions - plant height of maize plants under pH 8.5 alkali stress.

[0053] Figure 34This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The plant height of maize under -4‰ NaCl + pH 8.5 salt-alkali stress is also shown.

[0054] Figure 35 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The plant height of maize under -5‰ NaCl + pH 9 salt-alkali stress is also shown.

[0055] Figure 36 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The fresh weight of maize plants under -4‰ NaCl salt stress is also shown.

[0056] Figure 37 This is a statistical bar chart showing the effect of strain Y3 on the salt and alkali tolerance of potted maize under saline-alkali conditions - fresh weight of maize plants under pH 8.5 alkali stress.

[0057] Figure 38 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The fresh weight of maize plants under salt-alkali stress was -4‰ NaCl + pH 8.5.

[0058] Figure 39 This is a statistical bar chart showing the effect of strain Y3 on the salt tolerance of potted maize under saline-alkali conditions. The fresh weight of maize plants under salt-alkali stress was -5‰ NaCl + pH 9.

[0059] Figure 40 This is a graph showing the effect of strain Y3+ strain NJAU4742 on salt-alkali tolerance and growth promotion in field maize.

[0060] Figure 41 This is a statistical bar chart showing the effect of strain Y3+ strain NJAU4742 on salt-alkali tolerance and growth promotion of maize in the field - maize plant height.

[0061] Figure 42 This is a statistical bar chart showing the effect of strain Y3+ strain NJAU4742 on salt tolerance and growth promotion of maize in the field - stem diameter of maize plants.

[0062] Figure 43 This is a statistical bar chart showing the effect of strain Y3+ strain NJAU4742 on salt-alkali tolerance and growth promotion of maize in the field - fresh weight of maize plants (above ground).

[0063] Figure 44 This is a statistical bar chart showing the salt tolerance and growth-promoting effects of strain Y3+ NJAU4742 on field maize – SPAD values ​​of maize plants.

[0064] Information on the preservation of biological materials

[0065] Y3, categorized as Peribacillus frigoritolerans Y3, Latin name Peribacillus frigoritolerans It is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 14, 2025, with accession number CCTCC NO: M 20251595.

[0066] NJAU4742, classified as *Trichoderma guiyuan*. Trichoderma guizhouense Latin name Trichoderma guizhouense It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 11, 2016, with accession number CGMCC NO.12166. Detailed Implementation

[0067] The technical solutions of the present invention will be further described below with reference to the accompanying drawings in the embodiments of the present invention. However, the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0068] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0069] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0070] The culture medium formulations involved in the following examples are as follows:

[0071] LB solid medium (1L): 5g yeast extract, 5g sodium chloride, 10g peptone, 20g agar, pH 7.

[0072] LB liquid medium (1L): 5 g yeast extract, 5 g sodium chloride, 10 g peptone, pH 7.

[0073] Unless otherwise specified, the petri dishes (plates) used in the following examples are all 90 mm in diameter.

[0074] Example 1: Isolation and screening of salt-tolerant bacterial strains

[0075] (1) Isolation of salt-tolerant strains: Rhizosphere soil from healthy maize grown in saline-alkali soil in Zhenlai County, Baicheng City, Jilin Province (this saline-alkali soil is mainly composed of soda-type saline-alkali soil, with sodium carbonate and sodium bicarbonate as the main salts, characterized by high pH and high alkalinity, compact soil structure, and poor permeability, which is extremely unfavorable for crop growth) was selected as soil samples. 5g of soil sample was added to a centrifuge tube containing 45mL of sterile water, vortexed for 2min to mix thoroughly, and then allowed to stand for 5min to form a soil suspension. 1mL of the soil suspension was added to 9mL of sterile water, vortexed for 2min to mix thoroughly, and then allowed to stand for 5min to obtain 10 -1 Dilute the soil suspension in a gradient, diluting it sequentially to obtain 10... -2 10 -3 10 -4 10 -5 Soil suspensions were diluted in a gradient. 100 μL of soil suspension from each dilution gradient was spread onto LB solid medium (pH 7) containing 6.0% (w / v) NaCl using a sterile spreader. Each dilution gradient was repeated three times. The culture was incubated at 30 °C. Single colonies were picked and streaked three times to purify the strains.

[0076] (2) Screening of salt-tolerant strains: While keeping the pH of LB solid medium constant at 7, a salt concentration gradient medium was set up (the NaCl content was increased by 1% (w / v) each time), that is, the NaCl content was 6.0% (w / v), 7.0% (w / v), 8.0% (w / v), 9.0% (w / v), and 10.0% (w / v). The pure culture strains obtained in step (1) were first inoculated into LB solid medium plates containing 6.0% (w / v) NaCl and cultured at 28°C for 72 h. The growth was observed. The strains that could grow on the medium plates of this salt concentration were then inoculated into LB solid medium plates containing 7.0% (w / v) NaCl and cultured at 28°C for 72 h. The growth was observed. The strains that could grow on the medium plates of this salt concentration were then inoculated into LB solid medium plates containing 8.0% (w / v) NaCl, and so on. Finally, strains Y1, Y2, A2, A5, S1, S3, C1-1 and Y3 were selected, which can grow on media with a maximum salt concentration of 10.0% (w / v).

[0077] (3) Screening of alkali-tolerant strains: While maintaining the NaCl concentration of 0.5% (w / v) in the LB solid medium, a pH gradient medium was set up (the pH value was increased by 1 unit each time), with pH values ​​of 7, 8, 9, 10 and 11 (the pH value was adjusted with 1M NaOH). The pure culture strains obtained in step (1) were first inoculated onto LB solid medium plates with a pH value of 7 and cultured at 28℃ for 72 h. The growth was then observed. The strains that could grow on the medium plates with this pH value were then inoculated onto LB solid medium plates with a pH value of 8 and cultured at 28℃ for 72 h. The growth was then observed. The strains that could grow on the medium plates with this pH value were then inoculated onto LB solid medium plates with a pH value of 9, and so on. Finally, strains SK3, SK4, A2, A5, S1, S3, C1-1 and Y3 were screened. These strains could grow on a medium with a maximum pH value of 11.

[0078] In summary, strains A2, A5, S1, S3, C1-1, and Y3 were selected for subsequent experiments.

[0079] Example 2 Identification of strain Y3

[0080] (1) Morphological identification of strain Y3: Strain Y3 was inoculated on LB solid medium and cultured at 28℃ for 2 days. The morphology of single colonies was observed, and the results are as follows. Figure 1 As shown, the colonies are round, light yellow, with smooth and neat edges, and the surface of the colonies is moist and raised.

[0081] (2) Molecular biological identification of strain Y3: Genomic DNA of strain Y3 was extracted using a DNA extraction kit from Beijing Polymer Biotechnology Co., Ltd. The 16S rRNA gene was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', as shown in SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', as shown in SEQ ID NO.2). The obtained 16S rRNA gene sequence of strain Y3 (as shown in SEQ ID NO.3) was compared with the GenBank database using the NCBI BLAST tool for homology analysis, and a phylogenetic tree was constructed using MEGA 11.0 software (…). Figure 2 The results showed that strains Y3 and NR_117474 Peribacillus frigoritolerans strain DSM 8801 belongs to the same branch, with a homology of 99%.

[0082] Based on the colony morphology characteristics of strain Y3 and the phylogenetic tree comparison analysis constructed using the 16S rRNA gene sequence, strain Y3 was identified as... Peribacillus frigoritoleransStrain Y3 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251595.

[0083] Example 3: Growth curve determination of strain Y3

[0084] Single colonies of strain Y3 were picked and cultured in LB broth at 30°C and 180 rpm on a constant temperature shaking incubator. The absorbance (OD) of the bacterial culture at 600 nm was measured using a UV-Vis spectrophotometer. 600 ), until the OD of the bacterial solution 600 Stop culturing when the value reaches 0.6; change the OD value. 600 Y3 bacterial suspension with a pH of 0.6 was inoculated into LB liquid medium at a 2% (v / v) inoculation rate and cultured on a constant temperature shaking incubator at 30℃ and 180 rpm. The absorbance of the bacterial suspension at 600 nm was measured every 2 hours using a UV-Vis spectrophotometer. An uninoculated LB liquid medium was used as a control group. Growth curves were plotted. Figure 3 As shown, strain Y3 exhibits a logarithmic growth phase of 8-14 hours, providing reference data for the further application and large-scale production of this strain.

[0085] Example 4: IAA production capacity of strain Y3

[0086] (1) Qualitative detection of IAA production: L-tryptophan was added to LB liquid medium (200 μL of L-tryptophan stock solution was added per mL of LB liquid medium, with a concentration of 1 mg / mL), and dispensed into sterile test tubes. Strain Y3 was inoculated into the test tubes in a clean bench. After incubation at 30°C and 180 r / min for 4 days in a constant temperature shaking incubator, 50 μL of bacterial culture was placed in a blank white porcelain plate, and 50 μL of Salkowski colorimetric solution was added. 50 μL of Salkowski colorimetric solution was added to a white porcelain plate containing 50 μL of 50 μg / mL IAA solution (the 50 μg / mL IAA solution was prepared as follows: weigh 10 mg of IAA, dissolve it in a small amount of anhydrous ethanol, then dilute to 100 mL with distilled water to prepare a 100 μg / mL IAA stock solution, and then dilute with distilled water to a 50 μg / mL IAA solution) as a positive control. All white porcelain plates were stored in the dark and reacted at room temperature for 30 minutes. Color changes were observed afterward. If the strain was capable of producing IAA, it would react with the Salkowski sample to produce a red color. Three replicates were performed. The results showed that strain Y3 was capable of producing IAA.

[0087] (2) Construction of the IAA standard curve: Weigh 10 mg of IAA, dissolve it in a small amount of anhydrous ethanol, and then dilute to 100 mL with distilled water to prepare a 100 μg / mL IAA stock solution. Then dilute with distilled water to prepare IAA standard solutions of 20, 40, 60, 80, and 100 μg / mL respectively. Take 6 clean test tubes and add 2 mL of IAA standard solution and 2 mL of Salkowski colorimetric solution to each tube. Place them in the dark and react at room temperature for 30 min. Measure and record the absorbance of A450 using a UV-Vis spectrophotometer. Set up 3 replicates. Organize the data and plot the standard curve with the absorbance of A450 as the X-axis and the IAA concentration (μg / mL) as the Y-axis, as shown below. Figure 4 As shown, the calculated univariate linear regression equation for the standard curve is y = 86.614x - 8.5045 (R²). 2 =0.9911).

[0088] (3) Quantitative determination of IAA production: A single colony of strain Y3 was picked and inoculated into a sterile test tube containing 5 mL of LB liquid medium. After incubation at 30℃ and 180 r / min for 1 day on a constant temperature shaking incubator, 2 mL of bacterial solution was transferred to a centrifuge tube and centrifuged at 14000 rcf for 10 min. The supernatant was transferred to a clean test tube and an equal volume of Salkowski colorimetric solution was added. The tube was placed in the dark and reacted at room temperature for 30 min. The absorbance at A450 was measured and recorded using a UV-Vis spectrophotometer. Three replicates were set. The data were processed and the univariate linear regression equation of the standard curve was y = 86.614x - 8.5045 (R² - 2π) was used. 2 The concentration of IAA produced by strain Y3 was calculated using the formula (=0.9911). The results showed that the IAA production of strain Y3 was 87.01 μg / mL.

[0089] Example 5: Effects of strain Y3 on the growth of potted Chinese cabbage under saline-alkali conditions.

[0090] Preparation of Y3 bacterial inoculum: Inoculate a single colony of strain Y3 into a sterile test tube containing 5 mL of LB liquid medium and incubate on a constant temperature shaking incubator at 30°C and 180 rpm for 12 h to obtain the seed culture. Inoculate 5 mL of the seed culture into a 2 L shake flask containing 500 mL of LB liquid medium at a 1% (v / v, seed culture volume / LB liquid medium volume) and incubate on a constant temperature shaking incubator at 30°C and 180 rpm for 12 h. The bacterial concentration is 1 × 10⁻⁶. 8 After centrifuging at 10000 rcf for 10 min with CFU / mL or higher, discard the supernatant and resuspend the bacterial cells in an equal volume of sterile physiological saline to a bacterial concentration of 1×10⁻⁶. 8CFU / mL or higher. The preparation methods for A2, A5, S1, S3, and C1-1 inoculants are the same as for Y3 inoculant. For each strain (Y3, A2, A5, S1, S3, C1-1), the bacterial concentration of the inoculant should be uniformly adjusted to 1×10⁻⁶ using sterile water. 7 CFU / mL. NJAU4742 inoculum is produced from NJAU4742 bacterial powder (purchased from Jiangsu Sinong High-Tech Co., Ltd.). It is NJAU4742 spore powder, obtained from strain NJAU4742 (preservation number CGMCC NO.12166), through solid-state shallow-tray fermentation technology. The effective viable cell count, i.e., the number of spores, is 1×10⁻⁶. 9 The product (with a count of 1 or more per gram) is prepared by diluting NJAU4742 bacterial powder with sterile water at a ratio of 1g:100g.

[0091] Treatment of cabbage seeds: Select whole, plump and uniform cabbage seeds (Yiheqiu variety), disinfect them with 75% (v / v) alcohol for 0.5-1 min, then soak them in 0.1% (m / v) sodium hypochlorite solution for 15 min; then wash the cabbage seeds 4 times with sterile water, and finally put them in a beaker and add sterile water to submerge the cabbage seeds, and germinate them overnight at room temperature and in the dark.

[0092] Soil treatment for pot experiments: To more realistically reproduce the state of saline-alkali soil under natural conditions, yellow-brown soil taken from the Baima Teaching and Research Base of Nanjing Agricultural University was naturally air-dried, passed through a 20-mesh sieve, and then mixed evenly with vermiculite at a volume ratio of 1:1 to obtain a mixed soil for pot experiments. Alkaline solutions with pH 8.5 and pH 9 were prepared using two alkaline salts, NaHCO3 and Na2CO3. Salt solutions containing 2‰ NaCl, 4‰ NaCl, and 5‰ NaCl (i.e., 2g NaCl, 4g NaCl, and 5g NaCl added to 1L of distilled water, respectively) were prepared using the two alkaline salts NaHCO3 and Na2CO3 and the neutral salt NaCl. A 4‰ NaCl + pH 8.5 saline-alkali solution and a 5‰ NaCl + pH 9 saline-alkali solution were prepared using the two alkaline salts NaHCO3 and Na2CO3 and the neutral salt NaCl. The mixed soil was thoroughly soaked with different concentrations of alkaline solutions, salt solutions, and saline-alkali solutions, and then naturally air-dried for later use (i.e., the mixed soil was submerged in different concentrations of alkaline solutions, salt solutions, and saline-alkali solutions and then naturally air-dried). Seven soil treatments were included in the experiment: three saline soil treatments (2‰ NaCl, 4‰ NaCl, and 5‰ NaCl), two alkaline soil treatments (pH 8.5 and pH 9), and two saline-alkaline treatments (4‰ NaCl + pH 8.5 and 5‰ NaCl + pH 9). Germinated Chinese cabbage seeds (selected from seeds with uniform growth) were sown in each experimental soil in 32-cell trays, with 50g of soil per cell and 3 seeds per cell. After emergence, 2 seedlings were thinned per cell. The pot experiment was conducted in the greenhouse of the Resources and Environment Building at Nanjing Agricultural University, with a 12h / 12h photoperiod, daytime temperature of 25℃, nighttime temperature of 17℃, and relative humidity of 60%. Each cell was watered with 15mL of water every two days.

[0093] Five days after sowing, each hole was drenched with 10 mL of the inoculant, while the control group (CK) received an equal volume of sterile water. Eight treatments were set up: control (CK, drenched with sterile water), A2 (drenched with A2 inoculant), A5 (drenched with A5 inoculant), S1 (drenched with S1 inoculant), S3 (drenched with S3 inoculant), NJAU4742 (drenched with NJAU4742 inoculant), C1-1 (drenched with C1-1 inoculant), and Y3 (drenched with Y3 inoculant). Each treatment had 10 replicates.

[0094] On day 35 after sowing, the plant height, stem diameter, aboveground fresh weight and SPAD value of each treatment were measured (eight healthy cabbage plants were selected for each treatment to measure each index) to analyze the growth-promoting ability of different inoculants on cabbage under saline-alkali conditions.

[0095] Plant height measurement: Use a ruler to measure the vertical distance from the root collar of the cabbage plant to the tip of the highest leaf in the center of the rosette when it is in a naturally extended state.

[0096] Stem diameter measurement: The diameter of the root collar of the cabbage plant was measured using an electronic vernier caliper.

[0097] Determination of fresh weight of above-ground parts: Cut the cabbage plant at the root neck, take the above-ground parts of the cabbage plant, and weigh them using an electronic balance to obtain the fresh weight of the above-ground parts.

[0098] SPAD value determination: The SPAD value of the largest leaf of the Chinese cabbage plant was determined using a handheld chlorophyll meter (SPAD-502PIU).

[0099] The results are as follows Figures 5-11 and Figure 12-23 As shown, inoculation with inoculants Y3 and C1-1 significantly promoted the growth of Chinese cabbage. Among these, the 4‰ NaCl saline soil treatment, pH 8.5 alkaline soil treatment, and 4‰ NaCl + pH 8.5 saline-alkali soil treatment showed the most significant differences in plant phenotypes among the saline soil treatment, alkaline soil treatment, and saline-alkali soil treatment. Compared with the control, under 4‰ NaCl salt stress, inoculation with inoculant C1-1 significantly promoted plant height, stem diameter, and aboveground fresh weight of Chinese cabbage plants, while inoculation with inoculant Y3 significantly promoted plant height and stem diameter. Under pH 8.5 alkaline stress, inoculation with inoculants C1-1 and Y3 significantly promoted plant height, stem diameter, aboveground fresh weight, and SPAD value of Chinese cabbage plants. Under 4‰ NaCl + pH 8.5 saline-alkali stress, inoculation with inoculants C1-1 and Y3 significantly promoted plant height, stem diameter, aboveground fresh weight, and SPAD value of Chinese cabbage plants.

[0100] Example 6: Effects of strain Y3 on the growth of potted maize under saline-alkali conditions

[0101] As shown in Example 5, among the screened strains, strains C1-1 and Y3 showed better growth-promoting effects on Chinese cabbage. This example uses strains C1-1 and Y3 for testing, with existing Bacillus SQR9 (preservation number CGMCCNO.5808) as a positive control and sterile water as a negative control. Y3, C1-1, and SQR9 inoculants were prepared, with the preparation methods for Y3 and C1-1 inoculants the same as in Example 5. The preparation of the SQR9 inoculant followed the preparation method for the Y3 inoculant in Example 5. The bacterial concentration of each inoculant was uniformly adjusted to 1×10⁻⁶. 7 CFU / mL.

[0102] Treatment of corn seeds: Select whole, plump and uniform corn seeds (Tianyu 108 variety), disinfect with 75% (v / v) alcohol for 0.5-1 min, then soak in 0.1% (m / v) sodium hypochlorite solution for 15 min; then wash the corn seeds 4 times with sterile water, and finally put them in a beaker and add sterile water to submerge the corn seeds, and germinate overnight at room temperature and in the dark.

[0103] Soil treatment for pot experiments: To more realistically reproduce the state of saline-alkali soil under natural conditions, yellow-brown soil taken from the Baima Teaching and Research Base of Nanjing Agricultural University was naturally air-dried, passed through a 20-mesh sieve, and then mixed evenly with vermiculite at a volume ratio of 1:1 to obtain a mixed soil for pot experiments. Alkaline solutions with pH 8.5 and pH 9 were prepared using two alkaline salts, NaHCO3 and Na2CO3. Salt solutions containing 4‰ NaCl and 5‰ NaCl (i.e., 4g NaCl and 5g NaCl respectively added to 1L of distilled water) were prepared using the two alkaline salts NaHCO3 and Na2CO3 and the neutral salt NaCl. A 4‰ NaCl + pH 8.5 saline-alkali solution and a 5‰ NaCl + pH 9 saline-alkali solution were prepared using the two alkaline salts NaHCO3 and Na2CO3 and the neutral salt NaCl. The mixed soil was thoroughly soaked with different concentrations of alkaline solutions, salt solutions, and saline-alkali solutions, and then naturally air-dried for later use (i.e., the mixed soil was submerged in different concentrations of alkaline solutions, salt solutions, and saline-alkali solutions and then naturally air-dried). Four soil treatments were included in the experiment: a 4‰ NaCl saline soil treatment, a pH 8.5 alkaline soil treatment, and two saline-alkaline soil treatments: 4‰ NaCl + pH 8.5 and 5‰ NaCl + pH 9. Germinated maize seeds were sown in each soil treatment (seeds with uniform growth were selected for the experiment). Pot experiments were conducted using 7.7cm diameter (mouth diameter), 9cm height, and 5.7cm base diameter pots, with 50g of experimental soil per pot and one maize seed sown per pot. The pot experiments were conducted in the greenhouse of the Resources and Environment Building at Nanjing Agricultural University, with a 12h / 12h photoperiod, daytime temperature of 25℃, nighttime temperature of 17℃, and relative humidity of 60%. Each pot was watered with 15mL of water every two days.

[0104] Five days after sowing, each pot was treated with 10 mL of the inoculant by root drenching. The control group (CK) was treated with an equal volume of sterile water. Four treatments were set up: control (CK, drenched with sterile water), SQR9 (drenched with SQR9 inoculant), C1-1 (drenched with C1-1 inoculant), and Y3 (drenched with Y3 inoculant). Each treatment had six replicates.

[0105] The plant height of maize was measured on days 8, 12, 16, and 20 after sowing. The aboveground and underground fresh weights of maize were measured on day 20 after sowing (four maize plants with good growth were selected for each treatment to measure each index). The growth-promoting ability of different inoculants on maize under saline-alkali conditions was analyzed.

[0106] Plant height measurement: Use a ruler to measure the vertical distance from the root collar of the corn plant to the top of the tallest leaf in its naturally extended state.

[0107] Determination of aboveground and underground fresh weight: The corn plant was cut off from the root neck, and the aboveground and underground parts of the corn plant were taken separately. The aboveground fresh weight of the corn plant was obtained by weighing it with an electronic balance. The underground part of the corn plant was washed with clean water and dried with absorbent paper, and then weighed with an electronic balance to obtain the underground fresh weight.

[0108] The results are as follows Figure 24-31 and Figure 32-39 As shown, inoculant Y3 has a significant growth-promoting effect on both the aboveground and underground parts of maize plants. Under different alkaline, salt, and saline-alkali stress conditions, inoculation with inoculant Y3 promoted the growth of maize plants to varying degrees. Specifically, under 4‰ NaCl salt stress conditions, inoculation with inoculant Y3 resulted in increases of 38.86%, 68.77%, and 169.32% in plant height, aboveground fresh weight, and underground fresh weight at 20 days compared to the control, respectively. Under pH 8.5 alkaline stress conditions, inoculation with inoculant Y3 resulted in increases of 37.55% in plant height, aboveground fresh weight, and underground fresh weight at 20 days compared to the control, respectively. The growth rates of maize plants inoculated with Y3 microbial agent were 41.01% and 130.16% respectively. Under saline-alkali stress conditions of 4‰ NaCl + pH 8.5, the 20-day plant height, aboveground fresh weight, and underground fresh weight of maize plants increased by 91.71%, 181.21%, and 316.05% respectively compared with the control. Under saline-alkali stress conditions of 5‰ NaCl + pH 8.5, the 20-day plant height, aboveground fresh weight, and underground fresh weight of maize plants inoculated with Y3 microbial agent increased by 22.13%, 68.86%, and 169.61% respectively compared with the control. Compared with other microbial agents, maize plants inoculated with Y3 microbial agent also showed certain advantages in plant height, aboveground fresh weight, and underground fresh weight under different alkaline stress, salt stress, and saline-alkali stress conditions.

[0109] Example 7: The effect of a compound inoculant of strain Y3 and Trichoderma NJAU4742 on salt-alkali tolerance and growth promotion of maize in the field.

[0110] NJAU4742 inoculant is produced from NJAU4742 bacterial powder (purchased from Jiangsu Sinong High-Tech Co., Ltd.). It is NJAU4742 spore powder, obtained from strain NJAU4742 (preservation number CGMCC NO.12166), through solid-state shallow-tray fermentation technology. The effective viable cell count, i.e., the number of spores, is 1×10⁻⁶. 9 It is prepared by diluting water (more than 1 / g) in a certain proportion.

[0111] Y3 bacterial agent is prepared by Jiangsu Sinong High-Tech Co., Ltd. (Y3 bacterial powder is prepared by centrifuging the culture solution of strain Y3, discarding the supernatant, resuspending the bacterial cells, and then freeze-drying. The effective viable count is 1×10⁻⁶). 9It is prepared by diluting water with a ratio of CFU / g or higher.

[0112] On April 27, 2025, a field experiment was conducted in Zhenlai County, Baicheng City, Jilin Province, under natural climatic conditions (the field used in the experiment was a typical soda-type saline-alkali land, with sodium carbonate and sodium bicarbonate as the main salts, the total salt content of the field was 0.68 g / kg, and the pH value was 8.8) to investigate the effect of compound microbial agents on the salt-alkali tolerant growth of maize in the field.

[0113] This experiment included 8 treatments:

[0114] The CK group served as the control group, receiving an equal volume of water with a fungicide but no fertilizer.

[0115] Group CF was treated with an equal volume of inoculant and conventional fertilization, which included basal fertilizer and topdressing. Basal fertilizer was applied at sowing time with potassium sulfate compound fertilizer (nitrogen-phosphorus-potassium ratio of 15-15-15) at a rate of 10 kg / mu. Topdressing was applied at the large trumpet stage with urea (46% nitrogen content) at a rate of 8 kg / mu. The following procedures are the same as conventional fertilization.

[0116] The HY3+CF group consists of Y3 bacterial agent after high-temperature inactivation, with a dosage of 2 kg Y3 bacterial powder (high-temperature inactivated) per acre. When applying, dilute the Y3 bacterial powder (high-temperature inactivated) with water at a ratio of 2 kg Y3 bacterial powder (high-temperature inactivated): 200 kg water, and apply fertilizer as usual at the same time.

[0117] The HN+CF group was formed by applying NJAU4742 inoculant after high-temperature inactivation. The dosage was 2 kg of NJAU4742 inoculant powder (high-temperature inactivated) per acre. When applying, the NJAU4742 inoculant powder (high-temperature inactivated) was diluted with water at a ratio of 2 kg of NJAU4742 inoculant powder (high-temperature inactivated) to 200 kg of water. At the same time, conventional fertilization was carried out.

[0118] The HY3+HN+CF group consists of Y3 inoculant after high-temperature inactivation, applied at a rate of 1 kg Y3 inoculant powder / acre, and NJAU4742 inoculant after high-temperature inactivation, applied at a rate of 1 kg NJAU4742 inoculant powder / acre. When applying, dilute the Y3 inoculant powder (high-temperature inactivated) and NJAU4742 inoculant powder (high-temperature inactivated) with water at a ratio of (1 kg Y3 inoculant powder (high-temperature inactivated) + 1 kg NJAU4742 inoculant powder (high-temperature inactivated)): 200 kg of water, and apply fertilizer as usual at the same time.

[0119] The Y3+CF group involves applying Y3 microbial agent at a dosage of 2 kg Y3 microbial powder per acre. When applying, the Y3 microbial powder is diluted with water at a ratio of 2 kg Y3 microbial powder to 200 kg water, and conventional fertilization is carried out simultaneously.

[0120] The N+CF group was treated with NJAU4742 microbial agent at a dosage of 2 kg NJAU4742 microbial powder per acre. When applying, the NJAU4742 microbial powder was diluted with water at a ratio of 2 kg NJAU4742 microbial powder to 200 kg water, and conventional fertilization was carried out at the same time.

[0121] The Y3+N+CF group involves applying Y3 microbial agent at a dosage of 1 kg Y3 microbial powder per acre and NJAU4742 microbial agent at a dosage of 1 kg NJAU4742 microbial powder per acre. When applying, dilute the Y3 microbial powder and NJAU4742 microbial powder with water at a ratio of (1 kg Y3 microbial powder + 1 kg NJAU4742 microbial powder): 200 kg water, and apply conventional fertilizer at the same time.

[0122] The experiment employed a randomized block design, with each treatment replicated three times. Each replicate was a separate plot with a plot size of 1200 m². 2 Corn (variety Tianyu 108) was planted using the hill-sowing method, with one seed per hill, row spacing of 60cm, and plant spacing of 22cm. Inoculant was applied by root drenching during the seedling stage. Field management was consistent with that of local farmers (except for the use of drones to spray herbicides at the three-leaf stage for each treatment; no other pesticides were applied).

[0123] At the large trumpet stage (10 days after topdressing), maize plant growth indicators were measured (15 representative maize plants were selected from each plot). Plant height (vertical distance from the root collar to the highest point of the plant under natural conditions) was measured using a ruler. Stem diameter (thickness of the root collar) was measured using electronic calipers. Fresh weight (above-ground fresh weight) was measured using an electronic balance. SPAD values ​​were measured using a handheld chlorophyll meter (SPAD-502PIU) on the ear-side leaves. Figure 40 and Figures 41-44As shown, treatment with live bacteria Y3+NJAU4742 significantly promoted salt and alkali tolerance and growth in maize. Compared with the control, the plant height, stem diameter, fresh weight (aboveground parts), and SPAD value of maize plants treated with live bacteria Y3+NJAU4742 increased by 57.30%, 53.81%, 247.50%, and 27.2%, respectively. Compared with inactivated Y3, inactivated NJAU4742, and inactivated Y3+NJAU4742 treatments, the plant height, stem diameter, fresh weight (aboveground parts), and SPAD value of maize plants treated with live bacteria Y3+NJAU4742 were all significantly improved. Compared with live bacteria treatment with Y3, the plant height, stem diameter, fresh weight (aboveground parts), and SPAD value of maize plants treated with live bacteria Y3+NJAU4742 were all significantly improved, increasing by 22.8%, 20.6%, 26.4%, and 18.9%, respectively. Compared with the NJAU4742 live bacteria treatment, the Y3+NJAU4742 live bacteria treatment significantly increased the plant height, stem diameter and fresh weight (above ground) of maize plants, by 21.2%, 17.6% and 25.2%, respectively.

Claims

1. A composite microbial inoculant prepared from a saline-alkali tolerant bacterial strain Y3, characterized in that, The compound microbial agent comprises bacteria and fungi, wherein the bacteria are salt-tolerant strain Y3, and its classification name is... Peribacillus frigoritolerans Y3, deposited at the China Center for Type Culture Collection (CCTCC) on July 14, 2025, with accession number CCTCC NO: M 20251595; the fungus described is strain NJAU4742, taxonomically named *Trichoderma guizhouense*. Trichoderma guizhouense It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 11, 2016, with accession number CGMCC NO.12166; The compound microbial agent is prepared by the following steps: mixing Y3 bacterial powder and NJAU4742 bacterial powder in water to obtain the compound microbial agent; wherein, the Y3 bacterial powder is a bacterial powder obtained from the salt-tolerant strain Y3, and the effective viable count is 1×10⁻⁶. 9 CFU / g or higher; NJAU4742 mycelial powder is a mycelial powder obtained from strain NJAU4742, with a spore count of 1×10⁻⁶. 9 The mass ratio of Y3 bacterial powder to NJAU4742 bacterial powder is 1:1, and the total mass of Y3 bacterial powder and NJAU4742 bacterial powder is 1% of the mass of water.

2. The application of the complex microbial agent in claim 1 in crop salt-tolerant and alkali-promoting growth, characterized in that, The crop is corn.

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