A microbial agent for enhancing wheat salt tolerance and its preparation method

Abstract

This invention relates to the field of microbiology, specifically disclosing a microbial agent for enhancing the salt tolerance of wheat and its preparation method. The agent comprises a cold-resistant *Peribacillus frigoritolerans* GZ1 strain, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33784. This invention isolates and cultivates salt-tolerant growth-promoting functional strains from the rhizosphere soil of salt-tolerant wheat. The growth curve, biofilm formation, ACC deaminase, and IAA synthesis of the strains were measured. The growth-promoting ability of the strains was verified by inoculating them into the soils where salt-sensitive and salt-tolerant wheat varieties are grown. This invention solves the problem that some exogenous strains are difficult to survive under severe salt stress, or even if they survive, they are unlikely to produce stable beneficial effects on crops. A functional strain capable of stably increasing the biomass of both salt-sensitive and salt-tolerant wheat under severe salt stress has been discovered.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically a microbial agent for enhancing the salt tolerance of wheat and its preparation method. Background Technology

[0002] Soil salinization reduces arable land and crop yields, hindering agricultural development worldwide. For most crop varieties, including wheat, rice, sorghum, corn, and potatoes, moderate salinization (soil solution ECe 4–8 dS / m) can reduce yields by more than 50%. Unlike traditional engineering measures and chemical amendments that target soil improvement, microbial inoculants directly target the crop itself, enhancing its salt tolerance. Furthermore, microbial inoculants offer advantages such as minimal environmental disturbance, long-lasting effects, low cost, and ease of implementation.

[0003] Microbial agents used in saline-alkali soils not only need to generate biofilms to ensure their own stress resistance, but also need to synthesize and secrete metabolites such as ACC deaminase and IAA (auxin) to act on crops and help them enhance their salt tolerance. Currently, most microbial resource development is based on the analysis results of high-throughput sequencing data, which suffers from problems such as poor microbial stress resistance, unstable functional traits, and insignificant growth-promoting effects. This invention isolates a cold-resistant *Peribacillus frigoritolerans* GZ1 strain with salt-tolerant growth-promoting capabilities from the rhizosphere soil of salt-tolerant wheat varieties, and it also shows outstanding performance in ACC deaminase activity and IAA. Therefore, this invention can provide resources for improving crop productivity in saline-alkali soils. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a microbial agent to enhance wheat salt tolerance. This addresses the issue that the development of most microbial resources in the prior art is based on the analysis results of high-throughput sequencing data, which results in poor microbial stress resistance, unstable functional traits, and insignificant growth-promoting effects.

[0005] A microbial agent for enhancing wheat salt tolerance comprises a cold-resistant *Peribacillus frigoritolerans* GZ1 strain deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33784, wherein the strain possesses at least one of the following characteristics:

[0006] (a) After culturing in LB liquid medium containing 1%~7% NaCl for 70 hours, the OD600 absorbance value is ≥1.12;

[0007] (b) The biofilm formed in TSB medium containing 2% NaCl has an OD590 value ≥2.13;

[0008] (c) In DF-ACC medium containing 2% NaCl, the ACC deaminase activity is ≥0.2 mM;

[0009] (d) After culturing in LB medium containing 2% NaCl, the amount of IAA synthesized was ≥5.4 mg / L.

[0010] Preferably, the strain has an OD600 absorbance value ≥1.25 after being cultured in LB liquid medium containing 5% NaCl for 70 hours.

[0011] Preferably, the 16S rRNA gene sequence of the strain has ≥99% homology with the typical strain of Peribacillus frigoritolerans GZ1.

[0012] A method for preparing the microbial inoculant as described above includes the following steps:

[0013] (1) Isolation of salt-tolerant wheat rhizosphere microorganisms from saline soil: Dilute salt-tolerant wheat rhizosphere soil samples and spread them on LB solid medium containing 2% NaCl. Incubate at 26°C for 48 hours and screen for single colonies.

[0014] (2) Purification and identification: The strain was purified by streak plating, and PCR amplification was performed using primers 27F and 1492R. Sequencing comparison confirmed that the strain was Peribacillus frigoritolerans GZ1.

[0015] (3) Functional verification: The growth curve, biofilm formation, ACC deaminase activity and IAA synthesis of the strain were measured, and strains that meet any of (a) to (d) were screened.

[0016] (4) Preparation of bacterial agent: The selected strains were inoculated into liquid LB medium and cultured at 26°C with shaking until OD600=0.6. After centrifugation, the strains were resuspended in sterile water to obtain bacterial agents with a concentration of 1×10^8~1×10^9 CFU / mL.

[0017] Preferably, the saline soil described in step (1) is sterilized by γ-rays and then used for pot experiments, and the rhizosphere soil is collected after wheat seedlings have grown under 250 mM NaCl stress for four weeks.

[0018] Preferably, the determination of biofilm formation in step (3) includes: diluting the bacterial solution to OD590=0.15, allowing it to stand for 48 hours, staining with crystal violet, and measuring the OD590 value.

[0019] Preferably, the determination of ACC deaminase activity in step (3) includes: calculating the ACC deaminase activity in the bacterial extract by means of the α-ketobutyrate standard curve, and the determination conditions are induced culture in DF-ACC medium containing 2% NaCl for 12 hours.

[0020] Preferably, the determination of IAA synthesis amount in step (3) includes: co-culturing the bacterial culture with LB medium containing 500 mg / L L-tryptophan for 72 hours, measuring the OD530 value after color development by Salkowski reagent, and calculating the synthesis amount based on the IAA standard curve.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention first used slightly saline soil to set up a pot experiment, planting salt-tolerant winter wheat varieties and subjecting them to salt stress treatment with NaCl solution. After four weeks of treatment, microorganisms in the rhizosphere soil of the salt-tolerant wheat were isolated and purified. The growth curves, biofilm formation, ACC deaminase, and IAA synthesis of each strain were measured, and the growth-promoting effects of single strains on salt-sensitive and salt-tolerant wheat varieties were verified through strain cross-inoculation experiments.

[0023] Key Inventions:

[0024] 1) Isolate and culture salt-tolerant and growth-promoting strains from the rhizosphere soil of salt-tolerant wheat;

[0025] 2) The growth curve of the strain, biofilm formation, ACC deaminase, and IAA synthesis were determined;

[0026] 3) The growth-promoting ability of the strain was verified by inoculating it into the soil in which salt-sensitive and salt-tolerant wheat varieties grow.

[0027] This invention addresses the problem that some exogenous bacterial strains struggle to survive under severe salt stress, or even if they do survive, they fail to produce stable beneficial effects on crops. Functional bacterial strains capable of stably increasing the biomass of both salt-sensitive and salt-tolerant wheat under severe salt stress have been identified. Attached Figure Description

[0028] Figure 1 The growth curves of strain GZ1 in LB medium with different salt concentrations are shown.

[0029] Figure 2 The effect of GZ1 bacterial inoculation on aboveground wheat biomass under salt stress;

[0030] Figure 3 The effect of GZ1 bacterial inoculation on wheat root biomass under salt stress;

[0031] Figure 4To investigate the effect of GZ1 bacterial inoculation on the total biomass of wheat plants under salt stress. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example:

[0034] 1. Pot experiment

[0035] Soil from saline-alkali farmland was collected, sieved, and used for all subsequent greenhouse pot experiments. First, to screen for bacteria recruited by salt-tolerant wheat, the soil was sterilized with gamma rays before being used in pot experiments with salt-tolerant winter wheat varieties. Wheat seeds were germinated in a constant-temperature incubator, and then two seedlings of uniform growth were transplanted into pots. When the seedlings developed two true leaves, they were treated with 250 mM NaCl solution, with four replicates. Rhizosphere soil samples were collected four weeks after the NaCl treatment.

[0036] 2. Strains isolation, purification, and identification

[0037] After thoroughly mixing the rhizosphere soil from 8 wheat plants (4 replicates), 10g of soil was added to 90mL of sterile water and shaken at 180 rpm for 30 min to obtain a soil suspension. The soil suspension was then divided into 10... -2 10 -3 10 -4 10 -5 10 -6 After gradient dilution, 200 μL of each dilution was plated onto LB agar containing 2% NaCl. After incubation at 26°C for 48 h, single colonies were picked for streak purification, and the purified strains were stored at -80°C. Strains were then transferred from the plates to LB liquid medium and incubated with shaking at 180 rpm for 48 h. The bacterial culture was then aspirated, and the strain sequence was PCR-sected using primers 27F and 1492R. The product was sent to Qingke Biotechnology Co., Ltd. for Sanger sequencing, and the sequence was identified as Peribacillus frigoritolerans GZ1 by BLAST alignment.

[0038] 3. Functional assay of the strain

[0039] 3.1 Growth curve monitoring

[0040] Liquid LB medium containing 1%, 3%, 5%, and 7% NaCl was prepared respectively. 200 μL of GZ1 bacterial suspension with OD600=0.6 was inoculated into different mediums, and the growth curves were monitored using a microbial growth curve analyzer.

[0041] The experimental results are shown in Figure 1 The GZ1 strain survived in LB media containing 1%, 3%, 5%, and 7% NaCl. After 70 hours of cultivation, the OD600 absorbance values ​​were 1.3466, 1.128, 1.2534, and 1.1986, respectively. In LB media containing 1%, 5%, and 7% NaCl, the OD600 values ​​continued to increase until 70 hours, indicating that the growth of the GZ1 strain was not limited by salt stress from 1% to 7% NaCl.

[0042] 3.2 Biofilm formation

[0043] The GZ1 strain was inoculated into TSB medium containing 2% NaCl and cultured at 26°C and 180 rpm for 48 h. The culture was then diluted with TSB medium containing 2% NaCl to an OD590 of 0.15. 200 μL of the bacterial culture was transferred to a 96-well plate, sealed with Parafilm membrane, and incubated statically at 20°C for 48 h. Unfixed cells were washed away with 200 μL of phosphate-buffered saline. The biofilm on the 96-well plate was then stained with 1% (w / v) crystal violet solution (20 min). The plate was then washed three times with PBS and incubated with 96% ethanol to release the crystal violet absorbed by the biofilm. The OD590 of the ethanol solution of crystal violet was measured using a spectrophotometer to quantify the amount of biofilm formed.

[0044] The experimental results are shown in Table 1. After staining the biofilm of strain GZ1 in a 2% NaCl environment, its OD590=2.13.

[0045] Table 1 Biofilm formation by strains

[0046] deal with Biofilm formation rate OD590 GZ1 2.1300 CK 0.4298

[0047] 3.3 ACC deaminase activity

[0048] Add 7.5 mL of LB medium containing 2% NaCl to a 10 mL centrifuge tube, and add 10 µL of GZ1 bacterial culture. Incubate at 26°C and 180 rpm for 48 h. Centrifuge the bacterial culture at 4°C and 12000 rpm for 10 min, and discard the supernatant. Resuspend the bacterial cells in 5 mL of DF nitrogen-free medium, centrifuge at 4°C and 12000 rpm for 10 min, and discard the supernatant. Add 7.5 mL of DF-ACC medium containing 2% NaCl, resuspend, and induce culture at 30°C and 180 rpm for 12 h. Centrifuge at 4°C and 12000 rpm for 10 min, remove the supernatant, add 5 mL of 0.1 mol / L Tris-HCl (pH 7.6) to resuspend the precipitate, centrifuge at 4°C and 12000 rpm for 10 min, and discard the supernatant. Repeat the resuspension and washing step, centrifuge, and then add 1 mL of 0.1 mol / L Tris-HCl (pH 7.6) to resuspend the precipitate in a 2 mL centrifuge tube. After centrifugation at 12000 rpm for 5 min, 600 µL of 0.1 mol / L pH 8.5 Tris-HCl was added to suspend the precipitate. 30 µL of toluene was added, and the mixture was shaken at high speed for 30 s. Two 200 µL aliquots of the lysed solution were added to 1.5 mL centrifuge tubes, with only one aliquot containing 20 µL of 0.5 mol / L LaCCC solution. The mixture was reacted at 30 °C for 15 min, then 1 mL of 0.56 mol / L HCl was added and mixed. After centrifugation at 12000 rpm for 5 min, 800 µL of the supernatant from each aliquot was added to 2 mL centrifuge tubes, and 800 µL of 0.56 mol / L HCl was added and mixed. 300 µL of 2,4-dinitrophenylhydrazine reaction solution was added, and the mixture was reacted at 30 °C for 30 min. 2 mL of 2 mol / L NaOH was added and mixed, and the absorbance was measured at 540 nm.

[0049] Standard curve: Dilute 0.1 mol / L α-ketobutyric acid stock solution with 0.1 mol / L pH 8.5 Tris-HCl to prepare α-ketobutyric acid standards of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mmol / L. Use 0.1 mol / L pH 8.5 Tris-HCl as a blank control. Add 200 µL of each standard to a 2 mL centrifuge tube. Add 1.4 mL of 0.56 mol / L HCl and mix well. Add 300 µL of 2,4-dinitrophenylhydrazine reaction solution and react for 30 min. Measure OD540.

[0050] The concentration of α-ketobutyric acid (α-ketobutyric acid) with the addition of ACC is calculated as OD (α-ketobutyric acid + bacterial extract), while the concentration without ACC is calculated as OD (bacterial extract). A standard curve is plotted based on the OD values ​​of the α-ketobutyric acid standard sample. The concentration of α-ketobutyric acid, i.e., the ACC deaminase activity, is calculated using the formula ODα-ketobutyric acid = OD (α-ketobutyric acid + bacterial extract) – OD (bacterial extract).

[0051] The experimental results are shown in Table 2. The ACC deaminase activity of strain GZ1 in a 2% NaCl environment was 0.2 mM.

[0052] Table 2. ACC deaminase activity of strains

[0053] deal with ACC deaminase activity mM GZ1 0.2 CK 0

[0054] 3.4 IAA Synthesis Amount

[0055] Transfer 50 μL of GZ1 bacterial culture to a 5 mL LB (2% NaCl) test tube containing 500 mg / L L-tryptophan and incubate in the dark at 26°C and 180 rpm for 72 h. After incubation, transfer 1600 μL of the bacterial culture to a sterile 2 mL centrifuge tube, centrifuge at 12000 rpm for 10 min, transfer 800 μL of the supernatant to a 2 mL centrifuge tube, add an equal volume of Salkowski's reagent, and mix thoroughly by inverting. After standing in the dark for 1 h, observe the color change and measure the OD530 absorbance.

[0056] The standard curve was prepared by diluting pure IAA gradients (0, 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, 25.0 mg / L) with an equal volume of Salkowski reagent, with a blank matrix control.

[0057] The experimental results are shown in Table 3. The amount of IAA synthesized by strain GZ1 in a 2% NaCl environment was 5.4 mg / L.

[0058] Table 3. IAA synthesis amount of strains

[0059] deal with IAA synthesis amount (mg / L) GZ1 5.4 CK 0

[0060] 4. Verification of the strain's ability to promote wheat growth

[0061] The GZ1 strain was inoculated into liquid LB medium and cultured at 26°C and 180 rpm for 48 h. After measuring the OD600 absorbance, the culture was diluted with liquid LB medium to OD600 = 0.6. The bacterial culture was centrifuged at 8000 rpm and washed with PBS, repeated 4 times. Finally, it was dissolved in sterile water and applied to the potting soil, 10 mL per pot.

[0062] After sterilizing the soil with gamma rays, pot experiments were conducted, planting salt-tolerant and salt-sensitive winter wheat varieties. Four treatments were established: salt-tolerant wheat with sterile water, salt-tolerant wheat with GZ1 bacterial solution, salt-sensitive wheat with sterile water, and salt-sensitive wheat with GZ1 bacterial solution, with each treatment replicated four times. Wheat seeds were germinated in a constant-temperature incubator, and then two seedlings of uniform growth were transplanted into pots. Bacterial solution or sterile water was inoculated into the soil around the seedling roots. When the seedlings developed two true leaves, they were treated with 250 mM NaCl solution. After four weeks of NaCl treatment, aerial and root samples were collected, cleaned, dried in the laboratory, and weighed.

[0063] The experimental results are shown in Figure 2 Inoculation with GZ1 bacterial solution significantly increased the aboveground biomass of salt-tolerant and salt-sensitive wheat under salt stress. Compared with the treatment of salt-tolerant wheat without inoculation, the treatment of salt-tolerant wheat with GZ1 bacterial solution increased the aboveground biomass by 32.45%; compared with the treatment of salt-sensitive wheat without inoculation, the treatment of salt-sensitive wheat with GZ1 bacterial solution increased the aboveground biomass by 28.65%.

[0064] Inoculation with GZ1 bacterial solution did not significantly affect wheat root biomass, such as Figure 3 As shown, compared with the treatment of salt-tolerant wheat without inoculation, the treatment of salt-tolerant wheat with GZ1 bacterial solution increased root biomass by 25.52%; compared with the treatment of salt-sensitive wheat without inoculation, the treatment of salt-sensitive wheat with GZ1 bacterial solution decreased root biomass by 7.73%.

[0065] In summary, inoculation with GZ1 bacterial suspension can significantly increase the biomass of both salt-tolerant and salt-sensitive wheat varieties. Figure 4 As shown, compared with the treatment of salt-tolerant wheat without inoculation, the treatment of salt-tolerant wheat with GZ1 bacterial solution increased the total biomass of the plants by 30.95%; compared with the treatment of salt-sensitive wheat without inoculation, the treatment of salt-sensitive wheat with GZ1 bacterial solution increased the total biomass of the plants by 18.62%.

[0066] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0067] The accompanying drawings of the embodiments disclosed in this invention only involve structures relevant to the embodiments disclosed in this invention. Other structures can be referred to with common designs. Unless otherwise specified, the same embodiment and different embodiments of this invention can be combined with each other.

[0068] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microbial inoculant for enhancing salt tolerance in wheat, characterized in that, The application relates to a cold-tolerant Peribacillus frigoritolerans GZ1, which is preserved in the China General Microbiological Culture Collection Center on March 11, 2025, and has the following characteristics: (a) the OD600 absorbance is greater than or equal to 1.12 after the strain is cultured in LB liquid medium containing 1%-7% NaCl for 70 hours; (b) the OD590 value of a biofilm formed in TSB medium containing 2% NaCl is greater than or equal to 2.13; (c) the ACC deaminase activity is greater than or equal to 0.2 mM in DF-ACC medium containing 2% NaCl; (d) the IAA synthesis amount is greater than or equal to 5.4 mg / L after the strain is cultured in LB medium containing 2% NaCl.

2. The microbial inoculant for enhancing salt tolerance in wheat as claimed in claim 1, wherein, The OD600 absorbance of the strain is greater than or equal to 1.25 after the strain is cultured in LB liquid medium containing 5% NaCl for 70 hours.

3. The microbial inoculant for enhancing salt tolerance in wheat as claimed in claim 1, wherein, The 16S rRNA gene sequence of the strain has a homology of greater than or equal to 99% with that of a typical strain of Peribacillus frigoritolerans GZ1.

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