Complex microbial inoculant prepared from saline-alkaline tolerant strain Y3 and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant prepared from a saline-alkaline tolerant strain Y3, the complex microbial inoculant comprises bacteria and fungi, the bacteria are the saline-alkaline tolerant strain Y3, and the preservation number is CCTCC NO: M 20251595; the fungus is a strain NJAU4742, and the preservation number of the fungus is CGMCC (China General Microbiological Culture Collection Center) NO. 12166. The invention also discloses application of the prepared compound bacterium agent in saline-alkaline tolerance and growth promotion of crops. The saline-alkaline tolerant strain Y3 screened by the invention has good functions of salt resistance, alkali resistance, IAA production and the like; the prepared Y3 single-bacterium microbial agent has better growth promoting capability on crops under saline-alkali conditions; and a complex microbial inoculant prepared from the strain NJAU4742 and the strain NJAU4742 has better growth promoting capability on crops under saline and alkaline conditions. The invention provides strain resources and technical support for promoting the growth of crops in saline-alkali soil.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to a composite microbial inoculant prepared from a salt-tolerant bacterial strain Y3 and application thereof. BACKGROUND

[0002] Soil salinization is a prominent problem of soil degradation. Compared with other improvement measures, using microbial inoculants prepared from functional microorganisms to improve saline-alkali soil can achieve good improvement effect while having the advantages of ecological environmental protection, no pollution, long duration of improvement effect, etc. At present, applying microbial inoculants prepared from functional microorganisms has become one of the important measures for improving saline-alkali soil.

[0003] Bacillus is a kind of aerobic or facultative anaerobic, spore-forming gram-positive bacteria. Bacillus is widely studied and applied as a model species, and has become one of the most widely used crop growth-promoting and biocontrol bacteria in the world. There are many species of Bacillus, but the main species used in agriculture and forestry are Bacillus subtilis (Bacillus subtilis) ( Bacillus subtilis ), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ( Bacillus amyloliquefaciens ), Bacillus licheniformis (Bacillus licheniformis) ( Bacillus licheniformis ), Bacillus pumilus (Bacillus pumilus) ( Bacillus pumilus ), Bacillus megaterium (Bacillus megaterium) ( Bacillus megaterium ), etc. Ji Yan et al. [1] research shows that Bacillus subtilis enhances the growth of corn by improving its root morphology and stress resistance enzyme activity, and promotes the accumulation of dry matter of corn under saline-alkali conditions; Lv Zhengyang et al. [2] research shows that Bacillus amyloliquefaciens PT6-1 improves the height, total fresh weight, total photosynthetic pigment content, peroxidase activity and catalase activity of Eupatorium odoratum under saline-alkali conditions; Chen Lu et al. [3] research shows that Bacillus pumilus JT8 promotes the germination of alfalfa seeds and the growth of seedlings under the concentration of 100 mmol / L of saline-alkali solution; Li Qingqing et al. [4] research shows that Bacillus megaterium has strong salt tolerance and phosphorus dissolution effect, and can better promote the seed germination of soybeans. At the same time, with the development of Bacillus research, more and more new species of Bacillus with good biocontrol and growth-promoting effect have been discovered, such as Bacillus vallismortis, etc. In recent years, some research reports that Bacillus vallismortis promotes crop growth, but mostly indirectly promotes crop growth through antagonistic effect on pathogenic bacteria. For example, Xu Lingna et al. [5] reported the fungistatic effect of volatile substances produced by Bacillus vallismortis 12a on peach brown rot; Liu Zhihui et al. [6] reported the fungistatic effect of Bacillus vallismortis 265ZY1 on potato fusarium dry rot; Zhang Guoyi et al. [7]The inhibitory effect of Bacillus amyloliquefaciens HJ-5 on cotton verticillium wilt was reported; Zhang Meng et al. [8] The inhibitory effect of Bacillus amyloliquefaciens wm005 on watermelon fusarium wilt, oilseed rape sclerotinia rot, cucumber fusarium wilt and other pathogens was reported.

[0004] However, there is still a lack of exploration of new bacillus resources in saline-alkali environments. Through the screening of salt-tolerant ability, nutrient activation, and growth promotion, it is expected to discover new bacterial resources that have strong adaptability to high-salt-alkali environments, can improve saline-alkali soils, regulate crop resistance in saline-alkali soil conditions, and promote crop growth. At the same time, based on this, other strains can be combined to achieve better crop salt-tolerant and growth-promoting functions.

[0005] [1] Ji Y, Zhang W, Liu J, 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. [2] Lv Z, Shao D, Zhang C, et al. Screening and identification of a salt-tolerant bacterial strain 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. [3] Chen L, Zhang Y, Sun H, et al. Application of microbial fertilizer in improving saline-alkali soil [J]. Grass Science, 2023, (05): 1-10. [4] Li QQ, Zhang R, Gao YT, et al. Phosphorus solubilization effect of salt-tolerant phosphorus-solubilizing bacteria and its influence on soybean germination [J]. Microbiology Bulletin, 2024, 51(11): 4574-4589. DOI: 10.13344 / j.microbiol.china.240190. [5] Xu LN, Xu XM, Dong Z. Inhibitory effect of volatile substances produced by Bacillus amyloliquefaciens on peach brown rot [J]. Agricultural Engineering Technology, 2022, 42(08): 20-22. DOI: 10.16815 / j.cnki.11-5436 / s.2022.08.012. [6] Liu Z, Hao R, Xu Y, et al. Screening and identification of a biocontrol strain against potato dry rot caused by Fusarium solani and its control effect [J]. Zhejiang Journal of Agricultural Sciences, 2019, 31(07): 1105-1111. [7] Zhang GY, Cheng L, Huang LY, et al. Inhibition of cotton verticillium wilt by mycorrhizal fungi in cooperation with Bacillus amyloliquefaciens [J]. Zhejiang Journal of Agricultural Sciences, 2018, 30(06): 1008-1015. [8]Zhang M, Wang Q, Wan D, et al. Isolation and identification of a Bacillus subtilis strain and its effect on Fusarium wilt of watermelon [J]. Jiangsu Agricultural Sciences, 2017, 45(08): 97-100. DOI:10.15889 / j.issn.1002-1302.2017.08.027. SUMMARY

[0006] The application aims to provide a composite microbial inoculant prepared from a saline-alkaline tolerant strain Y3 and application thereof, so as to solve the problems in the prior art.

[0007] To achieve the above-mentioned object, the application adopts the following technical scheme: The application provides a composite microbial inoculant prepared from a saline-alkaline tolerant strain Y3 in a first aspect, wherein the composite microbial inoculant comprises bacteria and fungi; the bacteria are the saline-alkaline tolerant strain Y3, which is classified and named as Peribacillus frigoritolerans Y3, and is preserved in the China Center for Type Culture Collection on July 14, 2025, with a preservation number of CCTCC NO: M20251595; and the fungi are strain NJAU4742, which is classified and named as Guizhou Trichoderma Trichoderma guizhouense , and is preserved in the China General Microbiological Culture Collection Center on April 11, 2016, with a preservation number of CGMCC NO.12166.

[0008] Further, the composite microbial inoculant is prepared by the following steps: mixing Y3 bacterial powder and NJAU4742 bacterial powder in water to obtain the composite microbial inoculant; wherein the Y3 bacterial powder is prepared from the saline-alkaline tolerant strain Y3, and has an effective viable bacterial count of 1×10 9 CFU / g or above; the NJAU4742 bacterial powder is prepared from the strain NJAU4742, and has a spore count of 1×10 9 CFU / g or above; the mass ratio of the Y3 bacterial powder to the NJAU4742 bacterial powder is 2-3:1, and the total mass of the Y3 bacterial powder and the NJAU4742 bacterial powder is 1.5-2% of the mass of the water.

[0009] The application provides application of the saline-alkaline tolerant strain Y3 in crop saline-alkaline tolerance and growth promotion in a second aspect, and the crop is corn.

[0010] The application has the following beneficial effects: The application screens a saline-alkaline tolerant strain Y3, which has good salt tolerance, alkali tolerance and indole-3-acetic acid (IAA) production function; the Y3 single bacterial inoculant prepared therefrom has good crop growth promotion ability under saline-alkaline conditions; and the composite microbial inoculant prepared from the strain Y3 and the strain NJAU4742 has better crop growth promotion ability under saline-alkaline conditions. The application provides a strain resource and technical support for promoting crop growth in saline-alkaline soil. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a colony morphology chart of strain Y3.

[0012] Figure 2 is a phylogenetic tree constructed based on 16S rRNA gene sequences of strain Y3.

[0013] Figure 3 is a growth curve chart of strain Y3.

[0014] Figure 4 is a standard curve chart of IAA.

[0015] Figure 5 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under 2‰ NaCl salt stress.

[0016] Figure 6 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under 4‰ NaCl salt stress.

[0017] Figure 7 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under 5‰ NaCl salt stress.

[0018] Figure 8 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under pH 8.5 alkali stress.

[0019] Figure 9 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under pH 9 alkali stress.

[0020] Figure 10 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under 4‰ NaCl + pH 8.5 salt-alkali stress.

[0021] Figure 11 is a picture of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - a picture of the phenotype of Chinese cabbage under 5‰ NaCl + pH 9 salt-alkali stress.

[0022] Figure 12 is a statistical column chart of the results of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - the plant height of Chinese cabbage under 4‰ NaCl salt stress.

[0023] Figure 13 is a statistical column chart of the results of the effect of strain Y3 on the salt-tolerant effect of potted Chinese cabbage under saline conditions - the stem diameter of Chinese cabbage under 4‰ NaCl salt stress.

[0024] Figure 14 Figure 11 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the plant height of Chinese cabbage under 4‰ NaCl salt stress.

[0025] Figure 15 Figure 12 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the stem diameter of Chinese cabbage under 4‰ NaCl salt stress.

[0026] Figure 16 Figure 13 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the fresh weight of the aboveground part of Chinese cabbage under pH 8.5 alkali stress.

[0027] Figure 17 Figure 14 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the stem diameter of Chinese cabbage under pH 8.5 alkali stress.

[0028] Figure 18 Figure 15 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the fresh weight of the aboveground part of Chinese cabbage under pH 8.5 alkali stress.

[0029] Figure 19 Figure 16 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the SPAD value of Chinese cabbage under pH 8.5 alkali stress.

[0030] Figure 20 Figure 17 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the plant height of Chinese cabbage under 4‰ NaCl + pH 8.5 salt-alkali stress.

[0031] Figure 21 Figure 18 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the stem diameter of Chinese cabbage under 4‰ NaCl + pH 8.5 salt-alkali stress.

[0032] Figure 22 Figure 19 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the fresh weight of the aboveground part of Chinese cabbage under 4‰ NaCl + pH 8.5 salt-alkali stress.

[0033] Figure 23 Figure 20 is a statistical histogram of the results of the effect of strain Y3 on the salt-alkali tolerance of potted Chinese cabbage under salt-alkali conditions - the SPAD value of Chinese cabbage under 4‰ NaCl + pH 8.5 salt-alkali stress.

[0034] Figure 24 Figure 21 is a picture of the salt-alkali tolerance effect of strain Y3 on the aboveground part of potted corn under salt-alkali conditions - the phenotype of corn under 4‰ NaCl salt stress.

[0035] Figure 25 Figure 3 is a picture of the phenotype of corn under alkaline stress at pH 8.5 under the effect of strain Y3 on the aboveground parts of potted corn.

[0036] Figure 26 Figure 4 is a picture of the phenotype of corn under salt-alkaline stress at 4‰ NaCl + pH 8.5 under the effect of strain Y3 on the aboveground parts of potted corn.

[0037] Figure 27 Figure 5 is a picture of the phenotype of corn under salt-alkaline stress at 5‰ NaCl + pH 9 under the effect of strain Y3 on the aboveground parts of potted corn.

[0038] Figure 28 Figure 6 is a picture of the phenotype of corn roots under salt stress at 4‰ NaCl under the effect of strain Y3 on the underground parts of potted corn.

[0039] Figure 29 Figure 7 is a picture of the phenotype of corn roots under alkaline stress at pH 8.5 under the effect of strain Y3 on the underground parts of potted corn.

[0040] Figure 30 Figure 8 is a picture of the phenotype of corn roots under salt-alkaline stress at 4‰ NaCl + pH 8.5 under the effect of strain Y3 on the underground parts of potted corn.

[0041] Figure 31 Figure 9 is a picture of the phenotype of corn roots under salt-alkaline stress at 5‰ NaCl + pH 9 under the effect of strain Y3 on the underground parts of potted corn.

[0042] Figure 32 Figure 10 is a bar graph of the results of the effect of strain Y3 on the salt-alkaline tolerance of potted corn under salt stress at 4‰ NaCl on the plant height of corn.

[0043] Figure 33 Figure 11 is a bar graph of the results of the effect of strain Y3 on the salt-alkaline tolerance of potted corn under alkaline stress at pH 8.5 on the plant height of corn.

[0044] Figure 34 Figure 12 is a bar graph of the results of the effect of strain Y3 on the salt-alkaline tolerance of potted corn under salt-alkaline stress at 4‰ NaCl + pH 8.5 on the plant height of corn.

[0045] Figure 35 Figure 13 is a bar graph of the results of the effect of strain Y3 on the salt-alkaline tolerance of potted corn under salt-alkaline stress at 5‰ NaCl + pH 9 on the plant height of corn.

[0046] Figure 36Figure 4 is a statistical column chart of the results of the effect of strain Y3 on the saline-alkali tolerance of potted corn under saline-alkali conditions - fresh weight of corn plants under 4‰ NaCl salt stress.

[0047] Figure 37 Figure 5 is a statistical column chart of the results of the effect of strain Y3 on the saline-alkali tolerance of potted corn under saline-alkali conditions - fresh weight of corn plants under pH 8.5 alkali stress.

[0048] Figure 38 Figure 6 is a statistical column chart of the results of the effect of strain Y3 on the saline-alkali tolerance of potted corn under saline-alkali conditions - fresh weight of corn plants under 4‰ NaCl + pH 8.5 saline-alkali stress.

[0049] Figure 39 Figure 7 is a statistical column chart of the results of the effect of strain Y3 on the saline-alkali tolerance of potted corn under saline-alkali conditions - fresh weight of corn plants under 5‰ NaCl + pH 9 saline-alkali stress.

[0050] Figure 40 Figure 8 is a picture of the growth-promoting effect of strain Y3 + strain NJAU4742 on field corn.

[0051] Figure 41 Figure 9 is a statistical column chart of the results of the growth-promoting effect of strain Y3 + strain NJAU4742 on field corn - plant height of corn plants.

[0052] Figure 42 Figure 10 is a statistical column chart of the results of the growth-promoting effect of strain Y3 + strain NJAU4742 on field corn - stem diameter of corn plants.

[0053] Figure 43 Figure 11 is a statistical column chart of the results of the growth-promoting effect of strain Y3 + strain NJAU4742 on field corn - fresh weight (aboveground part) of corn plants.

[0054] Peribacillus frigoritolerans Figure 12 is a statistical column chart of the results of the growth-promoting effect of strain Y3 + strain NJAU4742 on field corn - SPAD value of corn plants. Biological material preservation information

[0055] Y3, classified as Trichoderma sp. Peribacillus Y3, Latin name as Trichoderma sp. frigoritolerans Trichoderma guizhouense , preserved in the China Center for Type Culture Collection, located in Wuhan, Wuhan University, China, on July 14, 2025, with the preservation number CCTCC NO: M 20251595.

[0056] NJAU4742, classified as Trichoderma guizhouense Trichoderma guizhouense , Latin name as Trichoderma guizhouense Figure 1, and was preserved in China General Microbiological Culture Collection Center, located at No. 1, Huayuancun, Beijing Economic-Technological Development Area, Beijing, China, on April 11, 2016, with a preservation number of CGMCC NO. 12166. DETAILED DESCRIPTION

[0057] The technical solutions in the embodiments of the present application will be further described below with reference to the drawings in the embodiments of the present application. However, the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.

[0058] The test methods used in the following examples are conventional methods unless otherwise specified.

[0059] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0060] The culture medium formula involved in the following examples is as follows: LB solid culture medium (1L): yeast extract 5g, sodium chloride 5g, peptone 10g, agar 20g, pH value is 7.

[0061] LB liquid culture medium (1L): yeast extract 5g, sodium chloride 5g, peptone 10g, pH value is 7.

[0062] The petri dishes (plates) involved in the following examples are 90mm in diameter unless otherwise specified.

[0063] Example 1 Isolation and screening of salt-tolerant and alkali-tolerant bacterial strains (1) Isolation of salt-tolerant and alkali-tolerant bacterial strains: The rhizosphere soil of healthy corn growing in a saline-alkali land in Zhenlai County, Baicheng City, Jilin Province (the saline-alkali land is mainly soda type saline-alkali soil, the salt is mainly sodium carbonate and sodium bicarbonate, the soil has the characteristics of high pH value and high alkalization degree, the soil structure is compact, the water permeability is poor, and it is extremely unfavorable for crop growth) was selected as the soil sample, 5g of the soil sample was added into a centrifuge tube containing 45mL of sterile water, and vortexed and shaken for 2min to fully mix and then left for 5min to form a soil suspension. 1mL of the soil suspension was taken and added into 9mL of sterile water, vortexed and shaken for 2min to fully mix and then left for 5min to obtain a 10 -1 The soil suspension was diluted in gradient, and was diluted in turn to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5Dilution gradient soil suspension, respectively, each dilution gradient of soil suspension 100 μL, with a sterile coating rod coated on the LB solid medium containing 6.0% (w / v) NaCl (pH value is 7), each dilution gradient repeated three times; culture at 30℃; pick single colony for three times, purified to obtain pure culture strains.

[0064] (2) screening of salt-tolerant strains: in the case of keeping the pH value of LB solid medium as 7, set the salt concentration gradient medium (NaCl content increased by 1% (w / v) each time), namely NaCl content is 6.0% (w / v), 7.0% (w / v), 8.0% (w / v), 9.0% (w / v), 10.0% (w / v). The pure culture strains obtained in step (1) were inoculated into LB solid medium containing 6.0% (w / v) NaCl, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the salt concentration were inoculated into LB solid medium containing 7.0% (w / v) NaCl, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the salt concentration were inoculated into LB solid medium containing 8.0% (w / v) NaCl, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the salt concentration were inoculated into LB solid medium containing 9.0% (w / v) NaCl, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the salt concentration were inoculated into LB solid medium containing 10.0% (w / v) NaCl, respectively, and cultured at 28℃ for 72h, then the growth was observed. Finally, strains Y1, Y2, A2, A5, S1, S3, C1-1 and Y3 were screened, which could grow in the medium with the highest salt concentration of 10.0% (w / v).

[0065] (3) screening of alkali-tolerant strains: in the case of keeping the NaCl content of LB solid medium as 0.5% (w / v) salt concentration, set the pH value gradient medium (pH value increased by 1 unit each time), namely pH value is 7, 8, 9, 10 and 11 (pH value is adjusted by 1M NaOH). The pure culture strains obtained in step (1) were inoculated into LB solid medium with pH value of 7, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the pH value were inoculated into LB solid medium with pH value of 8, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the pH value were inoculated into LB solid medium with pH value of 9, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the pH value were inoculated into LB solid medium with pH value of 10, respectively, and cultured at 28℃ for 72h, then the growth was observed. The strains which could grow on the medium with the pH value were inoculated into LB solid medium with pH value of 11, respectively, and cultured at 28℃ for 72h, then the growth was observed. Finally, strains SK3, SK4, A2, A5, S1, S3, C1-1 and Y3 were screened, which could grow in the medium with the highest pH value of 11.

[0066] In summary, strains A2, A5, S1, S3, C1-1 and Y3 were selected for subsequent experiments.

[0067] Example 2 Identification of strain Y3 (1) Morphological identification of strain Y3: The strain Y3 was inoculated on LB solid medium and cultured at 28°C for 2d, and the single colony was observed for morphology. As shown in Table 1, the colony was round, light yellow, smooth and neat in edge, and the surface of the colony was moist and convex. Figure 2

[0068] (2) Molecular biological identification of strain Y3: The genomic DNA of strain Y3 was extracted using the DNA extraction kit of Beijing Joinn Biotechnology Co., Ltd., and 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 subjected to homology comparison with the GenBank database by NCBI BLAST tool, and a phylogenetic tree was constructed using MEGA 11.0 software (as shown in FIG. 2). Peribacillus frigoritolerans Peribacillus frigoritolerans The results showed that strain Y3 and NR_117474 strain DSM 8801 were in the same branch, with a homology of 99%.

[0069] Combined with the colony morphological characteristics and the phylogenetic tree constructed by the 16S rRNA gene sequence of strain Y3, strain Y3 was identified as Figure 3 Y3. Strain Y3 has been preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: M 20251595.

[0070] Example 3 Determination of growth curve of strain Y3 A single colony of strain Y3 was picked into LB liquid medium and cultured in a 30°C, 180r / min constant temperature shaking incubator. The absorbance value (OD 600 ) of the bacterial solution at 600nm was measured by ultraviolet-visible spectrophotometer until the OD 600 value of the bacterial solution was 0.6; the Y3 bacterial solution with OD 600 value of 0.6 was inoculated into LB liquid medium at an inoculation amount of 2% (v / v), and cultured in a 30°C, 180r / min constant temperature shaking incubator. The absorbance value of the bacterial solution at 600nm was measured by ultraviolet-visible spectrophotometer every 2h, and the LB liquid medium without inoculation was used as a control group, and the growth curve was drawn as shown in FIG. 3. Strain Y3 was in logarithmic growth phase at 8-14h, which provided reference data for further application and large-scale production of the strain. Figure 4

[0071] Example 4 Ability of strain Y3 to produce IAA​​ (1) IAA production qualitative detection: L-tryptophan was added to LB liquid medium (200 μL L-tryptophan stock solution was added to each mL of LB liquid medium, and the concentration of L-tryptophan stock solution was 1 mg / mL), and the mixture was dispensed into sterile test tubes. In the clean bench, strain Y3 was inoculated into the test tubes. After being cultured in a constant temperature shaking incubator at 30°C and 180 r / min for 4 days, 50 μL of the bacterial solution was taken and placed in a blank white porcelain plate, and 50 μL of Salkowski color developing solution was added. 50 μL of Salkowski color developing solution was added to a white porcelain plate containing 50 μg / mL IAA solution (50 μg / mL IAA solution was prepared as follows: 10 mg of IAA was weighed, dissolved with a small amount of anhydrous ethanol, and then dissolved in 100 mL of distilled water to prepare a 100 μg / mL IAA stock solution, and then diluted with distilled water to prepare a 50 μg / mL IAA solution) as a positive control. All white porcelain plates were stored in the dark, and the color change was observed after 30 min of reaction at room temperature. If the strain has the ability to produce IAA, it will produce red color with Salkowski. Three replicates were set. The results showed that strain Y3 has the ability to produce IAA.

[0072] (2) IAA standard curve: 10 mg of IAA was weighed, dissolved with a small amount of anhydrous ethanol, and then dissolved in 100 mL of distilled water to prepare a 100 μg / mL IAA stock solution, and then diluted with distilled water to prepare IAA standard solutions of 20, 40, 60, 80, and 100 μg / mL in proportion. Six clean test tubes were taken, and 2 mL of IAA standard solution and 2 mL of Salkowski color developing solution were added in turn. The mixture was placed in the dark, and the absorbance at A450 was measured and recorded using a UV-visible spectrophotometer after 30 min of reaction at room temperature. Three replicates were set. The data were arranged, the absorbance at A450 was taken as the X-axis, and the concentration of IAA (μg / mL) was taken as the Y-axis, and the standard curve was drawn as shown in Figures 5-11 The monadic linear regression equation of the standard curve was calculated as y = 86.614x - 8.5045 (R 2 = 0.9911).

[0073] (3) IAA production quantitative determination: a single colony of Y3 strain was picked and inoculated into a sterile test tube containing 5 mL of LB liquid medium, and the mixture was cultured in a constant temperature shaking incubator at 30°C and 180 r / min for 1 day. Then, 2 mL of the bacterial solution was taken and centrifuged in a centrifuge tube at 14000 rcf for 10 min, and the supernatant was transferred to a clean test tube. An equal volume of Salkowski color developing solution was added. The mixture was placed in the dark, and the absorbance at A450 was measured and recorded using a UV-visible spectrophotometer after 30 min of reaction at room temperature. Three replicates were set. The data were arranged, and the monadic linear regression equation of the standard curve y = 86.614x - 8.5045 (R2 The concentration of IAA produced by strain Y3 was calculated according to the formula: =0.9911). The results showed that the yield of IAA synthesized by strain Y3 was 87.01 μg / mL.

[0074] Example 5 Effect of strain Y3 on the growth of potted Chinese cabbage under saline-alkaline conditions Preparation of Y3 inoculant: a single colony of strain Y3 was inoculated into a sterile test tube containing 5 mL of LB liquid medium, and then placed in a constant temperature shaking incubator at 30°C and 180 r / min for 12 h as seed liquid. According to the inoculation amount of 1% (v / v, seed liquid volume / LB liquid medium volume), 5 mL of seed liquid was taken and inoculated into a 2 L large flask containing 500 mL of LB liquid medium, and then placed in a constant temperature shaking incubator at 30°C and 180 r / min for 12 h. The concentration of the bacterial liquid was more than 1×10 8 CFU / mL, and after centrifugation at 10,000 rcf for 10 min, the supernatant was discarded, and the bacterial body was resuspended with an equal volume of sterile normal saline, and the concentration of the bacterial liquid was more than 1×10 8 CFU / mL. The preparation methods of A2 inoculant, A5 inoculant, S1 inoculant, S3 inoculant, and C1-1 inoculant were the same as those of Y3 inoculant. The bacterial liquid concentrations of each strain (Y3, A2, A5, S1, S3, and C1-1) inoculant were uniformly adjusted to 1×10 7 CFU / mL with sterile water. The NJAU4742 inoculant was prepared by diluting NJAU4742 bacterial powder (NJAU4742 bacterial powder was purchased from Jiangsu Sinong High-tech Co., Ltd., which was NJAU4742 spore powder prepared by strain NJAU4742 (preserved number CGMCC NO.12166) through solid shallow plate fermentation technology, and the effective viable bacterial number, i.e., the spore number, was more than 1×10 9 CFU / g) with sterile water at a ratio of NJAU4742 bacterial powder:sterile water=1 g:100 g.

[0075] Treatment of Chinese cabbage seeds: select intact and full Chinese cabbage seeds (variety Yihexiu), 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 Chinese cabbage seeds with sterile water for 4 times, and finally immerse the Chinese cabbage seeds in a beaker with sterile water, and germinate overnight at room temperature in the dark.

[0076] Treatment of soil for pot experiment: In order to more truly restore the state of saline-alkali soil in natural conditions, the natural air-dried loess soil taken from the Baima teaching and scientific research base of Nanjing Agricultural University was mixed with vermiculite in a volume ratio of 1:1 after being sieved through a 20-mesh sieve to obtain mixed soil for pot experiment. Two kinds of basic salts, NaHCO3 and Na2CO3, were used to prepare basic solutions with pH 8.5 and pH 9. Neutral salt NaCl was used to prepare salt solutions containing 2‰ NaCl, 4‰ NaCl, and 5‰ NaCl (i.e., 1 L of distilled water containing 2 g of NaCl, 4 g of NaCl, or 5 g of NaCl). Two kinds of basic salts, NaHCO3 and Na2CO3, and neutral salt NaCl were used to prepare salt-alkali solutions of 4‰ NaCl + pH 8.5 and 5‰ NaCl + pH 9. The mixed soil was soaked with different concentrations of basic solutions, salt solutions, and salt-alkali solutions, and then naturally air-dried for standby use (i.e., the mixed soil was naturally air-dried after being soaked with different concentrations of basic solutions, salt solutions, and salt-alkali solutions). The experimental soil was divided into 7 treatments: 2‰ NaCl, 4‰ NaCl, and 5‰ NaCl salt soil treatments, pH 8.5 and pH 9 alkali soil treatments, and 4‰ NaCl + pH 8.5 and 5‰ NaCl + pH 9 salt-alkali treatments. After the seeds of Chinese cabbage (selected seeds with uniform growth for the experiment) were sown and germinated, they were sown in 32-hole plug trays, with 50 g of experimental soil per hole and 3 seeds of Chinese cabbage per hole. After germination, the seedlings were thinned to 2 plants per hole. The pot experiment was conducted in the greenhouse of Nanjing Agricultural University, with a light cycle of 12 h / 12 h, a daytime temperature of 25℃, a nighttime temperature of 17℃, and a relative humidity of 60%. Each hole was watered with 15 mL of water every two days.

[0077] After sowing, 10 mL of bacterial agent was used for root irrigation treatment every hole, and the control group (CK) used the same volume of sterile water instead. A total of 8 treatments were set: control (CK, irrigation with sterile water), A2 (irrigation with A2 bacterial agent), A5 (irrigation with A5 bacterial agent), S1 (irrigation with S1 bacterial agent), S3 (irrigation with S3 bacterial agent), NJAU4742 (irrigation with NJAU4742 bacterial agent), C1-1 (irrigation with C1-1 bacterial agent), and Y3 (irrigation with Y3 bacterial agent). Each treatment had 10 replicates.

[0078] On the 35th day after sowing, the plant height, stem diameter, aboveground fresh weight, and SPAD value of Chinese cabbage plants in each treatment were measured (8 healthy Chinese cabbage plants were selected for each index measurement), and the growth-promoting ability of different bacterial agents under saline-alkali conditions was analyzed.

[0079] Plant height measurement: The vertical distance from the root neck of the Chinese cabbage plant to the tip of the highest leaf in the natural stretching state of the Chinese cabbage plant was measured using a ruler.

[0080] Stem diameter measurement: The thickness of the root neck of the Chinese cabbage plant was measured using an electronic vernier caliper.

[0081] 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.

[0082] SPAD value determination: The SPAD value of the largest leaf of the Chinese cabbage plant was determined using a handheld chlorophyll meter (SPAD-502PIU).

[0083] The results are as follows Figures 12-23 and Figures 24-31 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.

[0084] Example 6: Effects of strain Y3 on the growth of potted maize under saline-alkali conditions 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.

[0085] 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.

[0086] Treatment of soil for pot experiment: In order to more truly restore the state of saline-alkali soil in natural conditions, the natural air-dried loess soil taken from the Baima teaching and scientific research base of Nanjing Agricultural University was mixed with vermiculite in a volume ratio of 1:1 after being sieved through a 20-mesh sieve to obtain mixed soil for pot experiment. Two kinds of basic salts, NaHCO3 and Na2CO3, were used to prepare basic solutions with pH 8.5 and pH 9. Neutral salt NaCl was used to prepare salt solutions containing 4‰ NaCl and 5‰ NaCl (i.e., 4 g and 5 g of NaCl were added to 1 L of distilled water, respectively). Two kinds of basic salts, NaHCO3 and Na2CO3, and neutral salt NaCl were used to prepare 4‰ NaCl + pH 8.5 saline-alkali solution and 5‰ NaCl + pH 9 saline-alkali solution. The mixed soil was soaked with different concentrations of basic solutions, salt solutions, and saline-alkali solutions, and then naturally air-dried for standby use (i.e., the mixed soil was naturally air-dried after being soaked with different concentrations of basic solutions, salt solutions, and saline-alkali solutions). The experimental soil was divided into four treatments: 4‰ NaCl salt soil treatment, pH 8.5 alkali soil treatment, and 4‰ NaCl + pH 8.5 and 5‰ NaCl + pH 9 saline-alkali soil treatments. Corn seeds were sown and germinated in each experimental soil (corn seeds with consistent growth were selected for the experiment). A 7.7 cm diameter, 9 cm high, and 5.7 cm bottom diameter Wanxiang pot was used for the pot experiment, 50 g of experimental soil was placed in each pot, and one corn seed was sown in each pot. The pot experiment was conducted in the greenhouse of the Resource and Environment Building of Nanjing Agricultural University, with a light cycle of 12 h / 12 h, a daytime temperature of 25℃, a nighttime temperature of 17℃, and a relative humidity of 60%. Each pot was watered with 15 mL of water every two days.

[0087] After sowing, 10 mL of bacterial agent was used for root irrigation treatment in each pot, and the control group (CK) used the same volume of sterile water instead. A total of four treatments were set up: control (CK, watering with sterile water), SQR9 (watering with SQR9 bacterial agent), C1-1 (watering with C1-1 bacterial agent), and Y3 (watering with Y3 bacterial agent). Each treatment had six replicates.

[0088] The plant height of corn was measured on the 8th, 12th, 16th, and 20th days after sowing, and the aboveground and underground fresh weights of corn were measured on the 20th day after sowing (four corn plants with good growth were selected for each treatment to measure each index). The growth-promoting ability of different bacterial agents under saline-alkali conditions was analyzed.

[0089] Plant height measurement: The vertical distance from the root neck of the corn plant to the top of the highest leaf of the corn plant in its natural stretched state was measured using a ruler.

[0090] 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.

[0091] The results are as follows Figures 32-39 and Figure 40 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.

[0092] 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. 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.

[0093] 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). 9CFU / g or more) is prepared by diluting with water in proportion.

[0094] On April 27, 2025, a field test was conducted under natural climatic conditions in Zhenlai County, Baicheng City, Jilin Province (the field used for the test is a typical soda-type saline-alkali soil, with sodium carbonate and sodium bicarbonate as the main salt, the total salt content of the field is 0.68 g / kg, and the pH value is 8.8). The effect of compound microbial agent on the growth of field corn under saline-alkali conditions was explored.

[0095] This test sets up 8 treatments: The CK group is the control group, and the microbial agent is diluted with water in the same volume, without fertilization; The CF group is to apply microbial agents with the same volume of water, and regular fertilization, which includes basal fertilization and topdressing. The basal fertilization is to apply potassium sulfate compound fertilizer (nitrogen, phosphorus and potassium ratio is 15-15-15) at the time of sowing, with a dosage of 10 kg / mu, and the topdressing is to apply urea (nitrogen content 46%) at the large trumpet period, with a dosage of 8 kg / mu; the same operation is involved in the following regular fertilization; The HY3+CF group is to apply Y3 microbial agent after high-temperature inactivation, with a dosage of 2 kg Y3 microbial powder (high-temperature inactivation) per mu, and to dilute Y3 microbial powder (high-temperature inactivation) with water in the ratio of 2 kg Y3 microbial powder (high-temperature inactivation):200 kg water at the time of application, while regular fertilization is applied; The HN+CF group is to apply NJAU4742 microbial agent after high-temperature inactivation, with a dosage of 2 kg NJAU4742 microbial powder (high-temperature inactivation) per mu, and to dilute NJAU4742 microbial powder (high-temperature inactivation) with water in the ratio of 2 kg NJAU4742 microbial powder (high-temperature inactivation):200 kg water at the time of application, while regular fertilization is applied; The HY3+HN+CF group is to apply Y3 microbial agent after high-temperature inactivation, with a dosage of 1 kg Y3 microbial powder per mu, and NJAU4742 microbial agent after high-temperature inactivation, with a dosage of 1 kg NJAU4742 microbial powder per mu, and to dilute Y3 microbial powder (high-temperature inactivation) and NJAU4742 microbial powder (high-temperature inactivation) with water in the ratio of (1 kg Y3 microbial powder (high-temperature inactivation) +1 kg NJAU4742 microbial powder (high-temperature inactivation)):200 kg water at the time of application, while regular fertilization is applied; The Y3+CF group is to apply Y3 microbial agent, with a dosage of 2 kg Y3 microbial powder per mu, and to dilute Y3 microbial powder with water in the ratio of 2 kg Y3 microbial powder:200 kg water at the time of application, while regular fertilization is applied; The N+CF group is to apply NJAU4742 microbial agent, with a dosage of 2 kg NJAU4742 microbial powder per mu, and to dilute NJAU4742 microbial powder with water in the ratio of 2 kg NJAU4742 microbial powder:200 kg water at the time of application, while regular fertilization is applied; Y3+N+CF group is applied Y3 fungicide, the amount is 1 kg Y3 fungus powder per mu and NJAU4742 fungicide, the amount is 1 kg NJAU4742 fungus powder per mu, when applying, Y3 fungus powder and NJAU4742 fungus powder are diluted with water according to the ratio of (1 kg Y3 fungus powder + 1 kg NJAU4742 fungus powder): 200 kg water, and normal fertilization is carried out at the same time.

[0096] The test adopts randomized block design, each treatment is repeated 3 times, each repetition is an independent plot, and the plot area is 1200 m 2 . Corn (variety is Tianyu 108) is planted by hole planting, 1 grain per hole, row spacing is 60 cm, and plant spacing is 22 cm. The fungicide is applied at the seedling stage. The field management is consistent with that of local farmers (among them, herbicides are sprayed by unmanned aerial vehicles at the three-leaf stage of each treatment, and no other agents are applied except this).

[0097] At the large trumpet mouth stage (10 days after topdressing), the growth indexes of corn plants are determined (15 corn plants which can represent the whole plot are selected in each plot to determine the growth indexes), the plant height of corn is determined by ruler (the vertical distance from the root neck of corn plant to the highest point of corn plant in natural state), the stem diameter of corn is determined by electronic vernier caliper (the thickness of root neck of corn plant), the fresh weight of corn plant is determined by electronic balance (the above-ground fresh weight of corn plant is weighed after cutting the corn plant from the root neck), and the SPAD value of corn plant is determined by handheld chlorophyll meter (SPAD-502PIU). As shown in Figures 41-44 and ​ Y3+NJAU4742 live bacteria treatment has a significant salt-tolerant and growth-promoting effect on corn. Compared with the control, the plant height, stem diameter, fresh weight (above-ground part) and SPAD value of corn plants treated by Y3+NJAU4742 live bacteria are increased by 57.30%, 53.81%, 247.50% and 27.2% respectively. Compared with Y3 inactivation, NJAU4742 inactivation and Y3+NJAU4742 inactivation treatment, the plant height, stem diameter, fresh weight (above-ground part) and SPAD value of corn plants treated by Y3+NJAU4742 live bacteria are significantly improved. Compared with Y3 live bacteria treatment, the plant height, stem diameter, fresh weight (above-ground part) and SPAD value of corn plants treated by Y3+NJAU4742 live bacteria are significantly improved, which are increased by 22.8%, 20.6%, 26.4% and 18.9% respectively. Compared with NJAU4742 live bacteria treatment, the plant height, stem diameter and fresh weight (above-ground part) of corn plants treated by Y3+NJAU4742 live bacteria are significantly improved, which are increased by 21.2%, 17.6% and 25.2% respectively.

Claims

1. A compound bacterial agent prepared from a salt-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 CGMCCNO 12166.

2. The compound microbial agent according to claim 1, characterized in that, 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.

3. The application of the compound microbial agent according to any one of claims 1-2 in promoting crop salt-alkali tolerance, characterized in that, The crop in question is corn.

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