A growth-promoting microbial preparation for alleviating saline-alkali stress and application thereof

Abstract

The application provides a growth promoting microbial preparation for relieving saline-alkali stress and application thereof, and belongs to the technical field of agricultural microorganisms. The microbial preparation provided by the application can significantly relieve the adverse effect of saline-alkali stress on the growth and development of corn, and can stably improve the growth indexes of corn such as plant height, stem diameter, fresh weight, dry weight, leaf area index and chlorophyll content in different saline-alkali degree plots, and continuously promotes the growth in the whole growth period of corn, especially promotes the grain fresh weight in the late growth period, which is beneficial to yield formation. Meanwhile, the different strains in the compound microbial preparation have good interaction ability, can play the synergistic effect of multiple bacteria and functional complementary effect, and further improve the adaptability and growth promoting effect of the microbial preparation in the complex soil environment. The application has the advantages of environmental friendliness, low cost, difficulty in producing drug resistance and the like, and has a good application prospect in the fields of agricultural yield increase in saline-alkali land and development of microbial fertilizers.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a growth-promoting microbial preparation for alleviating salt and alkali stress and its application. Background Technology

[0002] Salt-alkali stress is one of the major environmental factors restricting global agricultural production. High concentrations of soluble salts and alkaline conditions can lead to osmotic stress, ion toxicity, nutrient imbalance, and oxidative damage in crops, thereby inhibiting seed germination, slowing growth and development, and reducing crop yield. my country has a wide distribution of saline-alkali land, and how to effectively alleviate salt-alkali stress and improve crop productivity under adverse conditions is a pressing technical problem that needs to be solved for sustainable agricultural development.

[0003] Currently, the main technical means to alleviate salt-alkali stress and promote crop growth include physical improvement, chemical conditioning, agronomic regulation, and biotechnology. Physical improvement measures, such as deep plowing, topsoil application, and laying salt-barrier layers, involve large-scale engineering projects, are costly, and their effects are difficult to sustain. Chemical conditioning methods, such as applying gypsum, organic acids, and polymeric soil conditioners, can improve soil physicochemical properties in the short term, but improper application may cause secondary pollution, and their adaptability varies greatly among different types of saline-alkali soils. Agronomic regulation includes salt-tolerant variety breeding, rational irrigation, and mulch cultivation. Among these, salt-tolerant variety breeding has a long cycle and limited genetic resources, making rapid promotion and application difficult.

[0004] Microbial inoculants, as an environmentally friendly biological input, have received widespread attention in recent years for promoting crop growth and enhancing stress resistance. In existing technologies, single-function microbial inoculants (such as nitrogen-fixing bacteria, phosphate-solubilizing bacteria, and plant hormone-producing bacteria) have been tested for use in saline-alkali land crop cultivation. These inoculants can alleviate saline-alkali stress to some extent by secreting plant hormones, producing siderophores, and improving the rhizosphere microenvironment. However, in actual field applications, single inoculants often face problems such as weak colonization ability, unstable functional expression, and susceptibility to competition from native microorganisms, leading to significant fluctuations in their growth-promoting effects in complex saline-alkali soil environments, making it difficult to guarantee a sustained and stable yield-increasing effect. Furthermore, the application methods of existing microbial inoculants are relatively simple, lacking differentiated application strategies for different salinity levels, further limiting their widespread adoption in saline-alkali land agriculture.

[0005] Therefore, developing microbial inoculants with stable growth-promoting effects and strong environmental adaptability, along with their supporting application technologies, is of great significance for improving crop productivity in saline-alkali land. Summary of the Invention

[0006] The purpose of this invention is to provide a growth-promoting microbial preparation that alleviates salt and alkali stress, and solves the technical problems of unstable growth-promoting effect of existing microbial agents under salt and alkali stress conditions, single agent with limited function and poor colonization ability, and imperfect construction of multi-microbial synergistic system.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a growth-promoting bacterial agent for alleviating salt-alkali stress. The bacterial agent includes at least one of *Pseudomonas aeruginosa*, *Enterobacter aeruginosa*, and *Bacillus amyloliquefaciens*. The preservation number of *Pseudomonas aeruginosa* is CGMCC NO. 30005, the preservation number of *Enterobacter aeruginosa* is CGMCC NO. 34206, and the preservation number of *Bacillus amyloliquefaciens* is CGMCC NO. 13267.

[0008] Preferably, the bacterial agent is in the form of a liquid bacterial suspension, and its OD... 600 The value is 0.5 to 1.0.

[0009] Preferably, when the bacterial agent simultaneously contains Pseudomonas aeruginosa, Enterobacter aeruginosa, and Bacillus amyloliquefaciens, the ratio of the viable counts of the three is 0.5~1.5:0.5~1.5:0.5~1.5.

[0010] The present invention also provides the application of the above-mentioned microbial agent in the preparation of biofertilizers for alleviating salt and alkali stress and / or promoting crop growth.

[0011] Preferably, the salt-alkali stress includes mild salt-alkali stress and severe salt-alkali stress.

[0012] Preferably, the crop is corn, and promoting crop growth includes improving one or more of the following indicators: plant height, stem diameter, root length, fresh weight, dry weight, leaf area index, chlorophyll content, and grain fresh weight.

[0013] The present invention also provides a method for alleviating salt and alkali stress and promoting maize growth, comprising applying the above-mentioned microbial agent to the rhizosphere of maize plants at the 2-3 leaf stage.

[0014] Preferably, in the method, after the first application, the application is repeated every 20 to 40 days.

[0015] Preferably, in the method, the application rate of the microbial agent is OD per corn plant. 600 1-10 ml of bacterial suspension with a value of 0.5-1.0.

[0016] Preferably, the method can promote maize growth in non-saline-alkali, slightly saline-alkali, and severely saline-alkali plots.

[0017] The beneficial effects of this invention are: The microbial agent provided by this invention can significantly alleviate the adverse effects of saline-alkali stress on maize growth and development. It can stably increase maize plant height, stem diameter, fresh weight, dry weight, leaf area index, and chlorophyll content in plots with varying salinity levels, exhibiting a continuous growth-promoting effect throughout the entire maize growth cycle, especially promoting grain fresh weight in the later stages of growth, thus contributing to yield formation. Simultaneously, the different strains in the compound microbial agent exhibit good interaction capabilities, demonstrating synergistic and complementary effects, further enhancing the agent's adaptability and growth-promoting effect in complex soil environments. This invention has advantages such as being environmentally friendly, low-cost, and unlikely to induce resistance, showing promising application prospects in increasing agricultural yields in saline-alkali land and developing microbial fertilizers. Attached Figure Description

[0018] Figure 1 A phylogenetic tree of the strains was constructed. Figure 2 The diagram shows the results of the strain interaction assay. From left to right, the diagram shows the interaction processes of strains FZB42 with T8, FZB42 with JT421, JT421 with T8, and FZB42, T8, and JT421.

[0019] Figure 3 The results of the strain promoting maize growth in the early seedling stage are shown in the figure. Figure 4 Figure showing the growth-promoting effects of bacterial strains on maize during the jointing and early stages; Figure 5 Figure showing the growth-promoting effects of strains on corn silking and late jointing stages; Figure 6 Figure 1 shows the growth-promoting effect of the strains during the corn kernel formation stage and the late silking stage. Figure 7 This figure shows the growth-promoting effect of strains during the milk stage of maize. Biological Preservation

[0020] The biological material samples involved in this invention have been deposited with an international depository for biological material samples recognized by the State Intellectual Property Office of China prior to the application date. The specific deposit information is as follows: Pseudomonas aeruginosa ( Pseudomonas chlororaphis ): The strain was classified as *Pseudomonas aeruginosa*. Pseudomonas chlororaphis The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is March 11, 2024, and the deposit number is CGMCC NO.30005.

[0021] Enterobacter aeruginosa ( Enterobacter asburiae ): The strain is classified as Enterobacter auriculi. Enterobacter asburiae The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is April 15, 2025, and the deposit number is CGMCC NO.34206.

[0022] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ): The strain is classified as Bacillus amyloliquefaciens. Bacillus amyloliquefaciens The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit date is November 14, 2016, and the deposit number is CGMCC NO.13267. Detailed Implementation

[0023] This invention provides a growth-promoting bacterial agent to alleviate salt-alkali stress. The agent comprises at least one of *Pseudomonas aeruginosa*, *Enterobacter aeruginosa*, and *Bacillus amyloliquefaciens*. The *Pseudomonas aeruginosa* has the accession number CGMCC NO. 30005, the *Enterobacter aeruginosa* has the accession number CGMCC NO. 34206, and the *Bacillus amyloliquefaciens* has the accession number CGMCC NO. 13267. In this invention, the *Pseudomonas aeruginosa* strain used is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 30005. This strain was isolated from potato tuber tissue and exhibits good environmental adaptability and metabolic activity. The *Enterobacter aeruginosa* strain used in this invention has the accession number CGMCC NO. 34206 and was isolated from the rhizosphere of maize, showing good affinity with the maize host. The *Bacillus amyloliquefaciens* strain used in this invention has the accession number CGMCC NO. 13267 and was isolated from seabed silt, exhibiting unique metabolic characteristics. The microbial agent of this invention can contain any one, any two, or all three of the above-mentioned strains; that is, it can be a single-strain agent or a compound-strain agent, specifically selected according to the salinity and alkalinity of the target plot and the needs of the crop. When the microbial agent contains multiple strains, the strains can form functional complementarity and niche synergy, thereby improving the stability and promoting efficacy of the microbial agent in complex soil environments.

[0024] Preferably, the bacterial agent is in the form of a liquid bacterial suspension, and its OD... 600 The value is 0.5~1.0. In this invention, the bacterial agent can be prepared as a liquid bacterial suspension, or further prepared into a dry powder formulation by freeze-drying or spray drying, but the liquid bacterial suspension form is preferred because it has high bacterial cell activity, is convenient to apply, and does not require rehydration or activation. OD 600 The absorbance value (OD) is a commonly used optical indicator for measuring the cell concentration of a bacterial suspension, specifically the absorbance of the bacterial solution at a wavelength of 600 nm. The OD value described in this invention... 600 The value is 0.5~1.0, for example, it can be 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0, preferably 0.8~1.0, more preferably 0.9~1.0, and most preferably 1.0. In actual preparation, the bacterial cells can be collected by centrifugation, resuspended in sterile water, and the concentration adjusted to the target OD. 600The culture medium used to cultivate the strain can be LB liquid medium, with the following conventional formula: NaCl 10.0 g, yeast extract 5.0 g, acetyl peptone 10.0 g, distilled water 1 L, pH adjusted to 7.0-7.4. The culture conditions are typically 37℃, 200 r / min shaking culture for 12-14 h. After preparation, the bacterial agent should be stored at 4℃ for a short period, or for long-term storage with the addition of a protectant. Commonly used protectants include glycerol (final concentration 15%-30%), trehalose (5%-10%), and skim milk powder (5%-10%), to maintain cell survival and activity.

[0025] Preferably, when the microbial agent simultaneously contains *Pseudomonas aeruginosa*, *Enterobacter aeruginosa*, and *Bacillus amyloliquefaciens*, the ratio of the viable counts of the three strains is 0.5~1.5:0.5~1.5:0.5~1.5. In this invention, the ratio of the viable counts of the three strains in the compound microbial agent can be adjusted within a certain range to adapt to different application scenarios and soil conditions. For example, the ratio of viable counts can be 0.5:0.5:0.5, 1:1:1, or 1.5:1.5:1.5, preferably 0.8:0.8:0.8~1.2:1.2:1.2, more preferably 0.9:0.9:0.9~1.1:1.1:1.1, and most preferably 1:1:1. The above ratio refers to the ratio between the viable counts of the three strains (usually expressed as CFU / mL or CFU / g), not an absolute value. The total viable cell concentration can be adjusted according to actual needs. Generally, the total viable cell concentration is in the range of 10^8~10^9 CFU / mL when preparing the bacterial suspension. Mixing the three strains in equal proportions can maximize their respective functional strengths. *Pseudomonas aeruginosa* excels at secreting antibacterial substances and plant hormones, *Enterobacter aeruginosa* has nitrogen-fixing and phosphorus-solubilizing abilities, and *Bacillus amyloliquefaciens* has strong stress resistance and enzyme production capabilities. Combining the three in equal proportions can achieve functional complementarity and synergistic effects. Before mixing, each strain should be cultured separately in liquid fermentation to the logarithmic growth phase, and the OD value should be measured. 600 Determine the viable count using a plate count or plating, then mix thoroughly according to the target ratio. The mixed compound bacterial agent should be used within 24 hours, or stored at low temperature after adding a protectant to ensure the activity and proportional stability of each strain.

[0026] This invention also provides the application of the above-mentioned microbial agent in the preparation of bio-fertilizers for alleviating salt-alkali stress and / or promoting crop growth. In this invention, bio-fertilizer refers to a type of product containing specific living microorganisms, which improve plant nutrition conditions, regulate plant growth, and enhance plant resistance through the life activities of microorganisms, thereby achieving the purpose of increasing yield and efficiency. Compared with traditional chemical fertilizers, bio-fertilizers have advantages such as being environmentally friendly, resource-saving, not producing drug resistance, and having strong sustainability. The microbial agent of this invention can be added to bio-fertilizer products as a core functional strain. Bio-fertilizers can also contain carrier substances and excipients, such as peat moss, vermiculite, diatomaceous earth, bentonite, humic acid, alginic acid, chitosan, etc., as carriers, and adhesives, dispersants, stabilizers, etc. When preparing bio-fertilizers, the microbial suspension of this invention can be mixed with the sterilized carrier in a certain proportion, dried at low temperature, and then made into powder or granules, or directly packaged as liquid microbial fertilizer. The viable bacteria content of biofertilizers is typically no less than 1×10^8 CFU / g or CFU / mL to ensure effective field application. The microbial agent of this invention is particularly suitable for crop-growing areas under saline-alkali stress, significantly alleviating the inhibitory effect of salinity on crop growth and improving crop yield and quality.

[0027] Preferably, the saline-alkali stress includes mild saline-alkali stress and severe saline-alkali stress. In this invention, saline-alkali stress refers to the adverse effects on plant growth caused by excessively high soluble salt content and / or high pH value in the soil. Based on soil electrical conductivity (EC value) and pH value, saline-alkali land can be classified into different types and degrees. Typically, mild saline-alkali stress has a soil electrical conductivity of approximately 2-4 dS / m and a pH value of approximately 7.5-8.5; severe saline-alkali stress has a soil electrical conductivity greater than 4 dS / m and a pH value greater than 8.5. The specific classification of mild and severe saline-alkali land plots in this invention can be carried out with reference to relevant agricultural standards or conventional methods in the field. For example, non-saline-alkali land plots have a total salt content of <0.1%, mild saline-alkali land plots have a total salt content of 0.1%-0.3%, and severe saline-alkali land plots have a total salt content >0.3%. The microbial agent of this invention can effectively promote crop root development and nutrient absorption under mild saline-alkali stress, and significantly enhance crop salt tolerance under severe saline-alkali stress by improving the rhizosphere microenvironment and regulating plant osmotic balance. Therefore, the microbial agent of this invention has broad saline-alkali adaptability and can be applied to agricultural production in saline-alkali land of different types and degrees.

[0028] Preferably, the crop is maize, and promoting crop growth includes increasing one or more of the following indicators: plant height, stem diameter, root length, fresh weight, dry weight, leaf area index, chlorophyll content, and kernel fresh weight. In this invention, maize (Zea mays L.) is a globally important food, feed, and economic crop, and is relatively sensitive to salt-alkali stress, making it one of the main target crops for the improvement and utilization of saline-alkali land in my country. Plant height refers to the vertical height from the ground to the highest leaf or tassel tip of the maize plant, and is a basic indicator for measuring vegetative growth. Stem diameter usually refers to the diameter of the second internode at the base of the maize plant, reflecting the plant's mechanical strength and support capacity. Root length refers to the length from the root base to the root tip. For maize, the degree of root development directly affects the absorption capacity of water and nutrients, especially under salt-alkali stress; promoting root elongation helps the crop avoid the surface salt accumulation zone. Fresh weight and dry weight refer to the fresh sample weight of the aboveground and underground parts of the plant after removing impurities, and the weight after drying to constant weight, respectively, and are direct indicators for measuring biomass. Leaf area index (LAI) is the ratio of the total leaf area of ​​plants per unit land area to the land area, reflecting canopy structure and light energy utilization efficiency. A higher LAI indicates more vigorous photosynthesis. Chlorophyll content (usually expressed as SPAD value) is closely related to photosynthetic rate. Salt and alkali stress often leads to chlorophyll degradation or inhibited synthesis; an increase in chlorophyll content signifies a recovery in photosynthetic capacity. Grain fresh weight is an important indicator of maize yield and directly relates to economic benefits. After application of the microbial agent of this invention, varying degrees of improvement in the above indicators can be observed at different growth stages of maize (such as seedling stage, jointing stage, silking stage, grain formation stage, and milk stage). The overall effect is manifested in robust plants, increased biomass accumulation, enhanced stress resistance, and ultimately, increased yield.

[0029] This invention also provides a method for alleviating salt-alkali stress and promoting maize growth, comprising applying the aforementioned microbial agent to the rhizosphere of maize plants during the 2-3 leaf stage. In this invention, the 2-3 leaf stage is a critical period for maize seedlings to transition from heterotrophic to autotrophic growth. At this time, the maize endosperm nutrients are largely depleted, and the roots begin to absorb soil nutrients, making it easier for exogenous beneficial microorganisms to colonize and exert their effects in the rhizosphere. Applying the microbial agent for the first time during the 2-3 leaf stage helps the strains occupy the rhizosphere niche as early as possible, forming a dominant microbial community, thereby continuously exerting a growth-promoting effect during subsequent growth. The preferred application method is rhizosphere irrigation, that is, directly pouring the microbial suspension into the soil around the base of the maize plant, ensuring full contact between the microbial solution and the root zone. Irrigation is more conducive to the survival and reproduction of microorganisms in the rhizosphere than spraying or seed dressing, and it is simple to operate and suitable for field application. Before irrigation, the microbial suspension should be appropriately diluted or used directly to ensure uniform distribution of the microbial solution. The application time should preferably be chosen on cloudy days or in the early morning or late evening of sunny days to avoid high temperatures and strong sunlight causing microbial inactivation. If heavy rain occurs after application, reapplication should be made promptly to prevent the bacterial solution from being washed away. Throughout the corn growing season, multiple applications can be made as needed, depending on weather and soil conditions, to maintain the population density and activity of beneficial rhizosphere bacteria.

[0030] Preferably, in the method, after the first application, the application is repeated every 20-40 days. In this invention, the interval between repeated applications can be adjusted appropriately according to the growth cycle of maize, soil environmental conditions, and the colonization capacity of the bacterial strain. For example, the interval can be 20 days, 25 days, 30 days, 35 days, or 40 days, preferably 28-32 days, and more preferably 30 days. Too short an interval will increase labor costs and the amount of bacterial agent used, while too long an interval may lead to a decrease in the number of beneficial bacteria in the rhizosphere, affecting the sustained growth-promoting effect. For spring maize, the entire growth period is usually 100-120 days. After the first application at the 2-3 leaf stage, it can be applied again at the jointing stage (about 30 days later) and the tasseling stage (about 60 days later), that is, 2-3 applications throughout the entire growth period. For plots with a long growing season or severe saline-alkali stress, the number of applications can be appropriately increased (e.g., 4 times), with the interval between each application still within the range of 20-40 days. When applying the fungicide repeatedly, the concentration and dosage can be the same as the first time, or the dosage can be increased appropriately according to the size of the plant. It is worth noting that repeated applications should avoid simultaneous use with chemical fungicides to prevent affecting the activity of the fungi. If chemical agents must be used, an interval of more than 7 days should be observed.

[0031] Preferably, in the method, the application rate of the microbial agent is OD per corn plant. 600The amount of bacterial suspension applied is 1-10 ml with a concentration of 0.5-1.0. In this invention, the application rate of the bacterial suspension can be adjusted reasonably according to the planting density of maize, plant size, and soil moisture content. For example, the application rate per plant can be 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, or 10 ml, preferably 3-7 ml, and more preferably 5 ml. For field maize, the planting density is usually 45,000-60,000 plants per hectare. Based on an application rate of 5 ml per plant, the total application rate per hectare is 225-300 liters, which is economically and technically feasible. For smaller seedlings, the application rate can be set at the lower limit (1-5 ml), gradually increasing to the upper limit (5-10 ml) as the plant grows. If the bacterial suspension concentration is high (e.g., OD...), the application rate can be adjusted accordingly. 600 If the concentration is close to 1.0, the dosage per plant can be appropriately reduced; conversely, if the concentration is low (e.g., OD400), the dosage per plant can be reduced. 600 If the concentration is close to 0.5, the dosage per plant can be increased appropriately to ensure that each corn plant receives a sufficient number of effective live bacteria. Before application, gently shake the bacterial suspension to ensure even distribution of the bacteria. When applying, fill a sprayer or irrigation can with the bacterial solution and water evenly around the base of the corn plant. If dry weather occurs after application, supplement with water to promote bacterial migration and colonization in the rhizosphere.

[0032] Preferably, the method can promote maize growth in non-saline-alkali, slightly saline-alkali, and severely saline-alkali soils. In this invention, non-saline-alkali soils refer to normal farmland with a total soil salt content below 0.1% where crop growth is not inhibited by salt; slightly saline-alkali soils refer to soils with a total soil salt content between 0.1% and 0.3%, where crop growth is somewhat inhibited but can still complete its life cycle; severely saline-alkali soils refer to soils with a total soil salt content above 0.3%, where crop growth is severely inhibited, and emergence and yield are significantly reduced. The method of this invention can achieve different degrees of growth-promoting effects in all three types of soils. In non-saline-alkali soils, the microbial agent mainly promotes maize growth through mechanisms such as secreting plant hormones and improving nutrient utilization, resulting in robust plants, dark green leaves, and increased biomass. In mildly saline-alkali soils, in addition to the aforementioned growth-promoting effects, the microbial agent can alleviate saline-alkali stress by improving the rhizosphere microenvironment, reducing direct salt damage to the roots, and regulating the expression of plant stress-resistance-related genes, thus bringing maize growth indicators closer to those under non-saline-alkali conditions. In severely saline-alkali soils, while the method of this invention cannot completely eliminate the harm of salinity, it can significantly improve maize emergence rate, survival rate, and biomass, mitigate growth delays caused by salinity, and promote an increase in grain fresh weight during the milk stage, thereby achieving a certain economic yield. Therefore, the method of this invention has good universality and promotional value, and is particularly suitable for application in saline-alkali maize planting areas in northern my country.

[0033] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example

[0034] This embodiment provides a microbial inoculum containing: *Pseudomonas aeruginosa* EZ199 (referred to as T8 in this document), deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 11, 2024; accession number: CGMCC NO.30005; *Enterobacter aestivus* JT421, deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 15, 2025; accession number: CGMCC NO.34206; and *Bacillus amyloliquefaciens* 11B91 (referred to as FZB42 in this document), deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2016; accession number: CGMCC NO.13267. The microbial compound agent consists of two strains of Pseudomonas aeruginosa EZ199(T8), Enterobacter aeruginosa JT421 and Bacillus amyloliquefaciens 11B91(FZB42) mixed in a 1:1:1 ratio.

[0035] Culture conditions: Inoculate strains FZB42, T8 and JT421 into 5 mL of LB liquid medium and culture at 37℃ with shaking at 200 r·min-1 for 12~14 h. Take 100 uL of bacterial solution and spread it on LB solid medium and incubate in a constant temperature incubator for 12 h.

[0036] Survival testing conditions: Culture medium composition (LB liquid medium: NaCl 10.0 g, yeast extract 5.0 g, acetyl peptone 10.0 g, distilled water 1 L; LB solid medium requires the addition of 15-18 g agar powder to the above materials), pH 7.0-7.4, temperature 37℃.

[0037] The sequencing results of the three strains were as follows: EZ199(T8): EZ199_Pseudomonas_chlororaphis

[0038] JT421

[0039] 11B91(FZB42) 11B91_Bacillus_amyloliquefaciens

[0040] This example demonstrates the identification of three bacterial strains, determination of their colony-organism interaction capabilities, and evaluation of their maize-promoting performance under different saline-alkali stress conditions. (1) Strain identification Identification methods: ① Extraction of genomic DNA Fresh cultures of strains FZB42, T8, and JT421 were taken respectively, and their genomic DNA was extracted using conventional methods to obtain template DNA for subsequent amplification.

[0041] ②16S rDNA gene amplification Using extracted genomic DNA as a template, PCR amplification was performed using universal primers, wherein: The forward primer is 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′, as shown in SEQ ID NO.4; The reverse primer is 1492R: 5′-GGYTACCTTGTTACGACTT-3′, as shown in SEQ ID NO.5.

[0042] The PCR reaction conditions were set as follows: pre-denaturation at 95 ℃ for 4 min; followed by cycling: denaturation at 94 ℃ for 1 min, annealing at 50 ℃ for 1 min, extension at 72 ℃ for 2 min, for a total of 34 cycles; and finally extension at 72 ℃ for 10 min.

[0043] ③ Detection and sequencing of amplified products The PCR amplification products were detected by agarose gel electrophoresis. After confirming that the amplification band size met the expectations, the amplification products were recovered, purified, and sequenced for analysis.

[0044] ④ Sequence splicing and alignment analysis The sequences obtained from sequencing were spliced ​​and quality-corrected to obtain complete 16S rDNA sequences. Homology alignment analysis was performed on the obtained sequences in nucleic acid sequence databases to determine their phylogenetic classification and taxonomic position.

[0045] ⑤ Phylogenetic analysis Based on the alignment results, similar reference sequences were selected, and a phylogenetic tree was constructed using bioinformatics software to further verify the taxonomic status of strains FZB42, T8, and JT421.

[0046] Test results: strain T8 and Pseudomonas chlororaphis AFS 089126 Pseudomonas chlororaphis ATCC 13985Pseudomonas chlororaphis ST-1 and Pseudomonas chlororaphis The 16S rDNA gene sequences of AFS 076060 are similar and cluster into the same branch, indicating that strain T8 belongs to the same family. Pseudomonas chlororaphis .

[0047] strain JT421 and Enterobacter asburiae J95 Enterobacter asburiae 14A and Enterobacter asburiae The 16S rDNA gene sequences of AF503338 are similar, and they clustered into the same branch in phylogenetic analysis, indicating that strain JT421 belongs to the same family. [[ID= .

[0048] strain FZB42 and ​ HYM25 ​ ​ ST-24 ​ SH207 and ​ ​ The 16S rDNA gene sequences of NB1_KX75 are similar and cluster into the same branch, indicating that strain FZB42 belongs to the same family. ​ .

[0049] (2) Determination of colony interaction ability Test method: The test strains FZB42, T8, and JT421 were selected. Single colonies were picked from fresh plates and inoculated into LB liquid medium. The medium was then cultured with shaking at 28 °C until the bacterial culture reached the logarithmic growth phase (OD50). 600 = 0.5).

[0050] Take bacterial suspensions of the above-mentioned strains and conduct pairwise or multi-strain interaction experiments according to the preset combinations. Under aseptic conditions, mark the inoculation sites on the surface of LB solid medium (LB agar plates). Using a micropipette, aspirate 5 μL of bacterial suspension of each strain and spot-pipette them on the same plate surface in a "V" shape or equidistant arrangement from far to near, maintaining a certain initial distance between different strains to observe their interaction process.

[0051] After inoculation, seal the petri dishes to prevent moisture evaporation and external contamination. Incubate the plates at 28 ℃ for 2–4 days.

[0052] After the culture was completed, the colony growth morphology, boundary changes, inhibition zone formation and mutual influence were observed, and the experimental results were recorded by taking pictures.

[0053] Experimental results: The strains have good interaction capabilities. (3) The growth-promoting effects of different microbial inoculants on maize under different salt and alkali stresses Test method: Preparation of bacterial suspension: The bacterial strain was activated in LB liquid medium for 12-14 h at 37 ℃ and 180 r / min. The suspension was then transferred to 50 mL centrifuge tubes and centrifuged at 8000 r / min for 10 min at 10 ℃. The supernatant was discarded, and the bacterial cells were washed three times with sterile water. An appropriate amount of sterile water was added, and the suspension was vortexed to prepare a suspension. OD was measured using LB liquid as a control. 600 The absorbance value was adjusted to the OD value. 600 =0.5 and OD 600 =1.0, spare.

[0054] Corn planting and microbial agent application: Select plump and uniformly sized corn seeds and sow them directly in field plots with different salinity gradients. The experimental plots were divided into non-saline-alkali plots (N), slightly saline-alkali plots (L), and severely saline-alkali plots (S) according to soil salinity. Microbial agent treatment was used as another experimental factor, setting up a control without microbial agent application (CK), a compound microbial agent treatment (CB), and a single microbial agent treatment (SB). The compound microbial agent included FZB42, T8, and JT421.

[0055] The experiment employed a randomized block design, with three replicates per treatment and each replicate consisting of an independent plot. Sowing time, sowing density, plant spacing, row spacing, and fertilizer and water management practices were kept consistent. After maize emergence, microbial treatments were applied at the 2-3 leaf stage. The FZB42 treatment group received a single-agent FZB42 solution as a rhizosphere drenching, while the compound microbial treatment group received a compound microbial solution containing FZB42, T8, and JT421 as a rhizosphere drenching. The control group received no microbial treatment. Depending on maize growth, the treatment could be repeated once after a 30-day interval.

[0056] At different growth stages of maize, the emergence rate, plant height, stem diameter, root length, aboveground fresh weight, underground fresh weight, and dry matter content of each treatment were measured. A control (CK) treatment under the same saline-alkali gradient was used to calculate the promoting effect of different microbial agents on maize growth. The results were used to evaluate the effects of single-acid agent FZB42 and compound microbial agents on improving maize growth under different saline-alkali stress conditions.

[0057] Experimental results: (1) Measurement of early growth indicators of maize seedlings During the early growth stage of maize seedlings, measurements revealed no significant difference in emergence rate. Compared to the control (CK), stem diameter showed significant differences in both non-saline-alkali and saline-alkali soils when single-strain (SB) and compound-strain (CB) were applied. Plant height also showed significant differences compared to the control, except for single-strain application under severe saline-alkali conditions. Fresh weight showed no significant difference under non-saline-alkali conditions, but significant differences were observed under mild and severe saline-alkali stress. This suggests that the application of microbial fertilizers may play a crucial role in the early growth and development of plants. ​ ).

[0058] (2) Measurement of relevant growth indicators of maize during and before jointing stage Field observations revealed that after more than a month, maize under non-salt-alkali stress entered the jointing stage, while under salt-alkali stress, regardless of whether the stress was mild or severe, maize remained in the pre-jointing stage, indicating that salt-alkali stress has a certain impact on the maize growth cycle. Measurements showed differences in various growth indicators among different treatments. Compared to the control (CK), under normal conditions (N), the application of compound bacteria (CB) and single bacteria (SB) significantly increased plant height, stem diameter, and above-ground and below-ground dry weight, except under severe stress. Leaf area index and chlorophyll content under salt-alkali stress also showed significant differences compared to the control, indicating that the microbial agents significantly promoted maize jointing and the pre-jointing stage. ​ ).

[0059] (3) Measurement of relevant growth indicators of maize during silking and jointing stages One month later, field observations revealed that under non-salt-alkali stress, maize entered the silking stage, while under salt-alkali stress, regardless of whether it was mild or severe, maize was in the late jointing stage, indicating that salt-alkali stress has a certain impact on the maize growth cycle. Measurements showed differences in various growth indicators among different treatments. Compared to the control (N), under mild salt-alkali stress, the application of compound bacteria (CB) and single bacteria (SB) resulted in significant differences in plant height, while the differences were not significant under non-salt-alkali and severe salt-alkali stress. However, stem diameter and leaf area index showed significant differences, indicating that the microbial agents have a certain alleviating effect on maize under salt-alkali stress, making their application particularly important at this stage. ​ ).

[0060] (4) Measurement of relevant growth indicators during the kernel formation period and late silking period of maize A month-long observation revealed that under non-salt-alkali stress, maize entered the grain-forming stage. Under salt-alkali stress, regardless of whether the stress was mild or severe, maize was in the late silking stage, indicating a sustained impact of salt-alkali stress on the maize growth cycle. Measurements showed differences in various growth indicators among different treatments. Under non-salt-alkali conditions, the application of compound microbial agents significantly increased maize plant height, while single-agent agents showed no significant difference. Under salt-alkali stress, the application of microbial agents significantly increased plant height, and compared to stem diameter, the application of microbial agents under salt-alkali stress showed significant differences, with increased chlorophyll content. This indicates that the application of microbial agents not only improved the phenotypic growth of maize at this stage but also further promoted chlorophyll content, thus having a certain promoting effect on maize photosynthesis. ​ ).

[0061] (5) Measurement of relevant growth indicators of maize at milk stage After one month, field observations revealed that all corn plants, regardless of whether they were in non-saline-alkali, slightly saline-alkali, or severely saline-alkali conditions, entered the milk stage simultaneously, with their growth periods synchronized. Furthermore, measurements of relevant indicators showed that the application of microbial agents, whether single-strain or compound-strain agents, significantly promoted corn growth under field experimental conditions. Corn plant height increased under both saline-alkali and non-saline-alkali conditions. Significant differences were also observed in stem diameter, chlorophyll content, and above-ground fresh weight. Most importantly, the application of these microbial agents ultimately promoted the fresh weight of corn kernels, indicating that the application of these microbial agents had a very good effect on corn yield. ​ ).

[0062] As shown in the above examples, this invention provides three bacterial strains (Bacillus amyloliquefaciens FZB42, Pseudomonas aeruginosa T8, and Enterobacter aeruginosa JT421) and their single-strain and compound-strain agents. The taxonomic positions of each strain were confirmed by 16S rDNA identification, and colony interaction assays showed good compatibility and synergistic growth among the three strains. Under field conditions, three gradients were set up: non-salt-alkali, slightly saline-alkali, and severely saline-alkali. Single-strain and compound-strain agents were applied respectively, with no agent application serving as a control. Growth indicators of maize at different growth stages (seedling stage, jointing stage, silking stage, grain formation stage, and milk stage) were measured. The results showed that, compared with the control, the application of the agents increased maize plant height, stem diameter, root length, fresh weight, dry weight, leaf area index, and chlorophyll content to varying degrees under all three saline-alkali gradients. The compound-strain agent was generally more effective than the single-strain agent. Especially under severe saline-alkali stress, the microbial agent treatment significantly alleviated the delayed effect of salinity on the maize growth cycle and promoted an increase in grain fresh weight during the milk stage. These results demonstrate that the microbial agent and its application method provided by this invention can effectively alleviate saline-alkali stress, promote maize vegetative growth and yield formation, and have broad soil adaptability.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A growth-promoting bacterial agent for alleviating salt-alkali stress, characterized in that, The bacterial agent includes at least one of *Pseudomonas aeruginosa*, *Enterobacter aeruginosa*, and *Bacillus amyloliquefaciens*, wherein the preservation number of *Pseudomonas aeruginosa* is CGMCC NO.30005, the preservation number of *Enterobacter aeruginosa* is CGMCC NO.34206, and the preservation number of *Bacillus amyloliquefaciens* is CGMCC NO.13267.

2. The microbial agent according to claim 1, characterized in that, The bacterial agent is in the form of a liquid bacterial suspension, and its OD... 600 The value is 0.5 to 1.

0.

3. The microbial agent according to claim 1, characterized in that, When the bacterial agent contains Pseudomonas aeruginosa, Enterobacter aeruginosa, and Bacillus amyloliquefaciens, the ratio of the number of viable bacteria of the three is 0.5~1.5:0.5~1.5:0.5~1.

5.

4. The use of the microbial agent according to any one of claims 1 to 3 in the preparation of biofertilizers for alleviating salt-alkali stress and / or promoting crop growth.

5. The application according to claim 4, characterized in that, The salt-alkali stress includes mild salt-alkali stress and severe salt-alkali stress.

6. The application according to claim 4, characterized in that, The crop is corn, and promoting crop growth includes improving one or more of the following indicators: plant height, stem diameter, root length, fresh weight, dry weight, leaf area index, chlorophyll content, and grain fresh weight.

7. A method for alleviating salt-alkali stress and promoting maize growth, characterized in that, This includes applying the microbial agent according to any one of claims 1 to 3 to the rhizosphere of the maize plant during the 2-3 leaf stage.

8. The method according to claim 7, characterized in that, After the first application, repeat the application every 20 to 40 days.

9. The method according to claim 7, characterized in that, The application rate of the microbial agent is OD per corn plant. 600 1-10 ml of bacterial suspension with a value of 0.5-1.

0.

10. The method according to claim 7, characterized in that, The method can promote maize growth in non-saline-alkali, slightly saline-alkali, and severely saline-alkali soils.

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