Psychrophilic bacillus XYR12-4 and its application in saline-alkali land improvement and plant growth promotion

Abstract

The present application relates to a psychrophilic bacillus XYR12-4 and its application in saline-alkali soil improvement and plant growth promotion, and belongs to the technical field of microorganisms. The psychrophilic bacillus XYR12-4 was preserved in the China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC NO.34360. The psychrophilic bacillus XYR12-4 has salt-tolerant and low-temperature capabilities, strong adaptability, can survive in nutrient-poor saline-alkali soil, and can secrete more exopolysaccharides and indole acetic acid, thereby promoting the growth of plants and improving saline-alkali soil.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a psychrophilic bacillus XYR12-4 and its application in saline-alkali land improvement and plant growth promotion. Background Technology

[0002] my country has a large population and scarce arable land resources, and the future situation regarding food security and ecological environment security remains very serious. Currently, saline-alkali land accounts for about 10% of China's total land area. Therefore, in-depth efforts to improve soil salinity are of significant theoretical and practical value for alleviating land resource shortages, ensuring food security, and achieving sustainable and healthy regional land development. Saline-alkali land improvement measures mainly include physical improvement methods, chemical improvement methods, biological improvement methods, and comprehensive improvement methods. In recent years, with the deepening understanding of the functions of microorganisms in soil, the role of microorganisms in saline-alkali land improvement has gradually gained attention.

[0003] Microorganisms improve saline-alkali land primarily through various life activities that alter the soil's physicochemical properties. For example, microorganisms can lower soil pH by secreting organic acids; certain Bacillus species can produce macromolecular enzymes, and the extracellular polysaccharides (EPS) secreted by some microorganisms, such as Bacillus polymyxa, can promote soil aggregate formation, reduce soil salinity, and simultaneously form a protective film to mitigate the damage of salt ions to plant roots; some microorganisms can also promote crop growth by activating soil nutrients and producing plant hormones. In practical applications, commonly used microbial agents for saline-alkali land improvement often suffer from poor colonization and unstable improvement effects due to various environmental factors. Furthermore, while some microbial agents used for saline-alkali land improvement have the ability to produce indoleacetic acid (IAA), their yield is relatively low.

[0004] Therefore, how to provide a strain of bacteria that is both salt-tolerant and capable of producing high levels of growth regulators to improve saline-alkali land and promote plant growth is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a psychrophilic bacillus XYR12-4 and its application in saline-alkali soil improvement and plant growth promotion. This psychrophilic bacillus XYR12-4 has the ability to tolerate salinity and low temperatures, is highly adaptable, and can survive in nutrient-deficient saline-alkali soil. Furthermore, it can secrete a large amount of extracellular polysaccharides and indoleacetic acid, thereby promoting plant growth and improving saline-alkali soil.

[0006] This invention provides a psychrophilic bacillus XYR12-4, which was deposited on April 25, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.34360.

[0007] In some embodiments, the nucleotide sequence of the psychrophilic bacillus XYR12-4 is shown in SEQ ID NO:1.

[0008] In some embodiments, the psychrophilic bacillus XYR12-4 has the ability to produce indoleacetic acid and extracellular polysaccharides.

[0009] In some embodiments, the psychrophilic bacillus XYR12-4 is salt-tolerant and can grow normally at pH 6-10 and at NaCl content of 5%-15%.

[0010] In some embodiments, the psychrophilic bacillus XYR12-4 has low-temperature resistance and can grow normally at temperatures ranging from 10°C to 40°C.

[0011] The present invention also provides the application of the psychrophilic bacillus XYR12-4 described in the above technical solution in promoting plant growth.

[0012] In some embodiments, the plants are treated with a bacterial solution of *Bacillus psychrophilus* XYR12-4 via root irrigation. The OD value of the bacterial solution of *Bacillus psychrophilus* XYR12-4 is... 600 The value is 1, and the dosage is at least 100 mL / kg of soil.

[0013] The present invention further provides the application of the psychrophilic bacillus XYR12-4 described in the above technical solution in the improvement of saline-alkali soil.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0015] 1. This invention isolates and identifies a psychrophilic bacillus XYR12-4 from soil along the Bohai Sea coast. It has the ability to tolerate salt and alkali and low temperature. It can still grow well in a high-salt environment with a NaCl content of 15% and a strong alkaline environment with pH=10. Its minimum tolerance temperature can reach 10℃, which can better adapt to harsh environments, thereby ensuring the stable performance of its function in promoting growth and improving saline-alkali soil.

[0016] 2. The psychrophilic bacillus XYR12-4 provided by this invention can secrete more extracellular polysaccharides and indoleacetic acid, which can effectively promote plant growth and significantly reduce soil salinity, thus improving saline-alkali soil. Attached Figure Description

[0017] Figure 1 The growth of psychrophilic bacillus XYR12-4 provided in Example 1 of this invention under different NaCl contents;

[0018] Figure 2The growth of psychrophilic bacillus XYR12-4 provided in Example 1 of this invention at different pH values;

[0019] Figure 3 The growth of psychrophilic bacillus XYR12-4 provided in Example 1 of the present invention at different temperatures;

[0020] Figure 4 These are photos of the qualitative determination experiment of the IAA production capacity of Psychrophilic bacillus XYR12-4 provided in Example 1 of this invention;

[0021] Figure 5 The IAA standard curve plotted for Embodiment 1 of the present invention;

[0022] Figure 6 The glucose standard curve plotted for Example 1 of the present invention;

[0023] Figure 7 The image shows the colony morphology of Psychrophilic Bacillus XYR12-4 provided in Example 2 of this invention, where (a) is a complete colony photograph and (b) is a magnified view of a partial colony.

[0024] Figure 8 A phylogenetic tree was constructed based on the 16S rRNA gene sequence of Psychrophilic bacillus XYR12-4 provided in Example 2 of this invention;

[0025] Figure 9 The images are of chili seedlings and their root systems in a pot experiment provided in Embodiment 3 of the present invention, wherein (a) is a photo of chili seedlings in the control group, (b) is a photo of chili seedlings in the experimental group, and (c) is a comparison photo of the root systems of chili seedlings in the control group and the experimental group.

[0026] Figure 10 The figures show the changes in pH and EC of the soil in the control group and the experimental group after the pot experiment provided in Example 3 of the present invention. (a) is a figure showing the change in soil pH, and (b) is a figure showing the change in soil EC. Detailed Implementation

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0028] Example 1: Screening and functional determination of psychrophilic bacillus XYR12-4

[0029] Preparation of culture media and reagents

[0030] LB solid medium: 10g peptone, 5g yeast extract, 10g NaCl, 20g agar, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 20min.

[0031] LB liquid medium: 10g peptone, 5g yeast extract, 10g NaCl, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 20min.

[0032] IAA fermentation medium: 10g peptone, 5g yeast extract, 10g NaCl, 0.2g tryptophan, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 20min.

[0033] Salkowski colorimetric solution: Mix 50 mL of 35% HClO4 solution with 1 mL of 0.5 mol / L FeCl3 solution until homogeneous.

[0034] EPS solid culture medium: KH2PO4 0.2g, NaCl 0.2g, CaCO3 5.0g, CaSO4·2H2O 0.1g, MgSO4·7H2O 0.2g, glucose 10.0g, agar powder 20.0g, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 30min.

[0035] EPS liquid culture medium: KH2PO4 0.2g, NaCl 0.2g, CaCO3 5.0g, CaSO4·2H2O 0.1g, MgSO4·7H2O 0.2g, glucose 10.0g, 1L distilled water, pH adjusted to 7.0, sterilized at 121℃ for 30min.

[0036] Isolation and screening of strains

[0037] The isolated samples were soils from the Bohai Sea coast of Dongying City. 5g of sample was placed in an Erlenmeyer flask containing 45mL of sterile water and shaken at 180r / min for 30min to obtain a soil suspension. The soil suspension was then serially diluted with sterile water at a concentration gradient of 10. -3 10 -4 10 -5 50 μL of each soil dilution solution was spread onto LB solid medium, with 3 plates for each gradient, and incubated upside down in a 28℃ incubator for 48 h. Single colonies with different morphologies and colors were selected and streaked multiple times on LB solid medium for purification until a single colony grew, resulting in psychrophilic bacillus XYR12-4, which was then stored for later use.

[0038] The pure strains obtained above were inoculated into LB liquid medium and fermented for 48 hours to prepare seed culture for later use.

[0039] Salt and alkali resistance test of strains

[0040] (1) Salt tolerance test

[0041] LB liquid cultures with NaCl concentrations of 5%, 10%, 15%, 20%, and 25% were prepared in Erlenmeyer flasks. The OD values ​​of the above seed cultures were measured. 600 The value was adjusted to around 0.8, and inoculated into the above-mentioned media with different NaCl contents at an inoculation amount of 2% of the culture medium volume. The media were then incubated at 28℃ and 180 rpm with shaking. After 48 hours, the OD values ​​were measured. 600 Value, result as Figure 1 As shown.

[0042] (2) Alkali resistance test

[0043] The OD of the above seed liquid 600 The pH was adjusted to approximately 0.8. Inoculation was performed at 2% of the culture medium volume into LB liquid medium at pH 6, 7, 8, 9, 10, and 11. The medium was incubated at 28°C with shaking at 180 rpm for 48 hours, and the OD was measured afterward. 600 Value, result as Figure 2 As shown.

[0044] Depend on Figure 1 It is evident that *Psychrophilic bacillus* XYR12-4 can grow normally at NaCl concentrations of 5%–15%. Figure 2 It is evident that *Psychrophilus xYR12-4* can grow normally at pH 6–10. This demonstrates that *Psychrophilus xYR12-4* can still grow and reproduce well in a high-salt environment with 15% NaCl and a strongly alkaline environment with pH 10, indicating that *Psychrophilus xYR12-4* has a strong adaptability to saline-alkali environments, which is a fundamental guarantee for its role in improving saline-alkali soil.

[0045] Low temperature resistance test of strains

[0046] The OD of the above seed liquid 600 The pH was adjusted to approximately 0.8. Inoculation was performed at 2% of the culture medium volume in 150 mL Erlenmeyer flasks containing 50 mL of LB liquid medium. The flasks were then incubated at 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, and 40℃ with shaking at 180 rpm for 48 h. OD values ​​were measured at each incubation period. 600 Values, with 3 replicates at each temperature, results are as follows Figure 3 As shown.

[0047] Depend on Figure 3It can be seen that psychrophilic bacillus XYR12-4 can grow normally at temperatures ranging from 10℃ to 40℃, with its optimal growth temperature around 30℃ and its minimum tolerance temperature reaching 10℃, demonstrating excellent low-temperature resistance.

[0048] IAA production capacity test of strain

[0049] (1) Qualitative determination

[0050] The above seed culture was inoculated into IAA fermentation medium at an inoculation rate of 2% of the culture medium volume and cultured on a shaker at 28℃ and 180 rpm for 4 days. 50 μL of bacterial suspension was dropped onto a white ceramic plate, and 50 μL of Salkowski colorimetric solution was added, serving as the experimental group; 50 μL of 50 mg / L IAA was dropped onto a white ceramic plate, and 50 μL of Salkowski colorimetric solution was added, serving as the positive control group; the white ceramic plate was stored in the dark for 30 min, and it was observed that both the experimental group and the positive control group turned red (e.g., ...). Figure 4 As shown in the figure, this indicates that psychrophilic bacillus XYR12-4 has the ability to produce IAA.

[0051] (2) Quantitative determination

[0052] Construction of IAA standard curve: Accurately weigh 10.0 mg of IAA, dissolve it in a small amount of ethanol, and then dilute to 100 mL with distilled water (i.e., IAA concentration of 100 μg / mL) as a stock solution. Then, use the stock solution to prepare a series of standard solutions with concentrations of 0 (blank), 0.5, 1.0, 5.0, 10.0, 15.0, 20.0, and 25.0 μg / mL as working solutions (prepare fresh before use). Take 8 clean, dry test tubes (numbered 0-7), add 2 mL of the IAA working solution of each concentration sequentially, and add an equal volume of Salkowski colorimetric solution to each. Incubate in the dark at 40℃ for 30 min to accelerate the colorimetric reaction. Measure the absorbance at 530 nm. Plot the IAA standard curve based on the absorbance and the concentrations of the standard solutions. Figure 5 As shown, the fitted equation for the IAA standard curve is as follows:

[0053] , (1)

[0054] In equation (1), The absorbance at 530 nm This refers to the IAA concentration.

[0055] The OD of the above seed liquid 600Adjust the value to approximately 0.8, and inoculate 2% of the culture medium volume into IAA fermentation medium. Incubate at 28℃ and 180 rpm for 4 days using a shaker. Perform three parallel experiments. Take 2 mL of the 4-day bacterial suspension into a centrifuge tube, centrifuge at 10000 rpm for 10 min, and add the supernatant to a test tube. Add an equal volume of Salkowski colorimetric solution, incubate in the dark for 30 min to allow color development, and measure the OD value. 530 Values. OD values ​​corresponding to three parallel experiments. 530 The average value was 0.606±0.015. The concentration of IAA in a unit volume of fermentation broth was calculated to be 18.76 μg / mL using the IAA standard curve equation. This indicates that psychrophilic bacillus XYR12-4 can produce high levels of indoleacetic acid, which is beneficial for promoting plant growth.

[0056] EPS production capacity test of strain

[0057] (1) Qualitative determination

[0058] The test strain, Psychrophilic Bacillus XYR12-4, was inoculated onto EPS solid medium and incubated upside down in a 28°C incubator for 3 days. The strain was observed to have a moist surface, smooth protrusions, and mucus, indicating that Psychrophilic Bacillus XYR12-4 has the ability to produce EPS.

[0059] (2) Quantitative determination

[0060] Crude polysaccharide extraction: The OD of the above seed liquid was... 600 The value was adjusted to around 0.8, and 2% of the culture medium volume was inoculated into EPS liquid medium. The medium was then placed in a 28℃ constant temperature shaking incubator and cultured for 48 hours to obtain the fermentation broth. 10 mL of the fermentation broth was centrifuged at 8000 r / min for 10 min, and 5 mL of the supernatant was taken. 15 mL of anhydrous ethanol was added, and the mixture was stirred evenly with a glass rod until a flocculent precipitate appeared. The precipitate was allowed to stand in a 4℃ refrigerator for 24 hours to precipitate. Centrifugation at 10000 r / min for 15 min yielded the polysaccharide precipitate. The precipitate was washed several times with anhydrous ethanol, dried at 60℃ to constant weight, weighed, and then pulverized to obtain the crude polysaccharide.

[0061] Construction of the glucose standard curve: Dry glucose to constant weight at 60℃. Accurately weigh 0.25g of glucose and dissolve it in 50mL of distilled water to obtain a glucose standard solution with a concentration of 5mg / mL. Take 1mL of the glucose standard solution and dilute to 50mL to obtain a glucose dilution solution with a concentration of 0.1mg / mL. Take 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2mL of the glucose dilution solution and place them in 20mL graduated test tubes, and dilute to 2mL with distilled water to obtain a series of standard solutions. Then add 1mL of redistilled 5% phenol, followed by 5mL of concentrated sulfuric acid. Shake thoroughly to mix the liquids in the test tubes. Then heat in a boiling water bath for 20min. To prevent water evaporation, the test tubes can be stoppered. After removing the test tubes, cool them to room temperature with tap water. Use a UV spectrophotometer to measure the absorbance at 490nm. Plot the glucose standard curve based on the absorbance and the concentrations of the series of standard solutions. Figure 6 As shown, the equation for the glucose standard curve obtained by fitting is as follows:

[0062] , (2)

[0063] In equation (2), The absorbance at 490 nm This refers to the sugar concentration.

[0064] 20 mL of distilled water was added to dissolve the crude polysaccharide extracted from the seed culture of *Bacillus psychrophilus* XYR12-4. 1 mL of this solution was diluted to 10 mL to obtain the test sample solution. Three parallel experiments were performed. In each experiment, 1 mL of the test sample solution was placed in a test tube, and 1 mL of distilled water, 1 mL of 5% phenol, and 5 mL of concentrated sulfuric acid were added. The mixture was thoroughly shaken to ensure homogeneity, and then heated in a boiling water bath for 20 min. To prevent water evaporation, the test tubes were stoppered. After removing the test tubes, they were cooled to room temperature with tap water, and the absorbance at 490 nm was measured using a UV spectrophotometer. The average absorbance at 490 nm for the three parallel experiments was 0.332 ± 0.013. Using the glucose standard curve equation, the EPS content in the fermentation broth of *Bacillus psychrophilus* XYR12-4 was calculated to be 0.619 mg / mL, indicating that *Bacillus psychrophilus* XYR12-4 can produce high levels of extracellular polysaccharides.

[0065] Example 2 Identification of Psychrophilic Bacillus XYR12-4

[0066] (1) Morphological identification

[0067] The strain XYR12-4 isolated and screened in Example 1 was inoculated onto LB solid medium and incubated upside down in a 28°C incubator for 48 hours. Colony growth and morphological characteristics were observed, and the colony morphology is shown in the figure below. Figure 7 As shown.

[0068] Depend on Figure 7 As can be seen, the colonies of strain XYR12-4 are round, pale yellow, opaque, glossy, with a raised center and smooth edges.

[0069] (2) Molecular identification

[0070] The strain XYR12-4, isolated and screened in Example 1, was inoculated into LB liquid medium and cultured for 48 h. Genomic DNA was extracted, and its 16S rRNA was amplified by PCR using the genomic DNA as a template. The forward primer was 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO:2), and the reverse primer was 1492R: 5′-TACGGCTACCTTGTTACGACTT-3′ (SEQ ID NO:3). After agarose gel electrophoresis, the amplified products were sequenced by Ruiboxingke Biotechnology Co., Ltd. The obtained nucleotide sequence is shown in SEQ ID NO:1. The obtained gene sequence was submitted to the NCBI database for BLAST comparison, and similar sequences were downloaded. A phylogenetic tree of strain XYR12-4 was constructed using Mega11 software as shown below. Figure 8 As shown.

[0071] Based on morphological identification and phylogenetic tree comparison, strain XYR12-4 can be identified as a psychrophilic bacillus. Psychrobacter sp. It was deposited at the China General Microbiological Culture Collection Center on April 25, 2025, with accession number CGMCC NO.34360.

[0072] Example 3: Experiment on the growth-promoting effect of psychrophilic bacillus XYR12-4 on pepper seedlings in saline-alkali soil.

[0073] Pepper seedlings of uniform size and at the 4-leaf stage were selected and transplanted into flowerpots containing 1 kg of sterilized soil. Seven days after transplanting, the experimental group (referred to as XYR12-4) was treated with 100 mL of psychrophilic bacillus XYR12-4 fermentation broth (OD). 600 =1.00) was used for root irrigation treatment, while the control group (CK) received the same treatment with an equal volume of sterile water. Each group had 5 replicates, with 2 plants per pot. 28 days after root irrigation, the roots of the pepper seedlings were rinsed and excess water was absorbed. Plant height, root length, and fresh weight of the above-ground and underground parts were measured. The plant samples were then dried, and the dry weight of the above-ground and underground parts was measured. The results are shown in Table 1. Photos of the experimental and control groups and their root systems are shown below. Figure 9 As shown.

[0074] Table 1 Results of pot experiments with chili seedlings

[0075]

[0076] As shown in Table 1, root irrigation with *Bacillus psychrophilus* XYR12-4 effectively promoted the growth of pepper seedlings, increasing the aboveground and underground fresh weight by 20.51% and 19.04%, respectively, while increasing the aboveground and underground dry weight by 20.95% and 23.56%, respectively. Figure 9 It is evident that psychrophilic bacillus XYR12-4 has a significant growth-promoting effect on pepper seedlings.

[0077] After the pot experiment, soil was collected, air-dried, and sieved through a 2mm sieve. The soil was mixed with deionized water at a ratio of 1:5, shaken at room temperature for 30 minutes, centrifuged, and the supernatant was used to determine its pH and EC values. The results are as follows: Figure 10 As shown. By Figure 10 It can be seen that, compared with the control group, the application of psychrophilic bacillus XYR12-4 can reduce soil pH by 0.12 and EC value by 17.92%. Considering the short pot experiment period, the effect of psychrophilic bacillus XYR12-4 on soil pH is limited. However, psychrophilic bacillus XYR12-4 shows great potential in reducing soil salinity. It can be seen that psychrophilic bacillus XYR12-4 has the effect of improving saline-alkali soil.

Claims

1. Psychrobacter sp. XYR12-4, characterized in that, It was preserved in China General Microbiological Culture Collection Center on April 25, 2025, and the preservation number is CGMCC NO.34360.

2. Psychrobacter xyli XYR12-4 according to claim 1, characterized in that, The nucleotide sequence of the psychrophilic bacillus XYR12-4 is shown as SEQ ID NO:

1.

3. The Psychrobacillus sp. XYR12-4 according to claim 1, characterized in that, The psychrophilic bacillus XYR12-4 has the ability to produce indole acetic acid and exopolysaccharide.

4. The Psychrobacillus sp. XYR12-4 according to claim 1, characterized by, The psychrophilic bacillus XYR12-4 has the ability to resist salt and alkali, and can grow normally under the condition that the pH is 6-10 and the NaCl content is 5%-15%.

5. The Psychrobacillus sp. XYR12-4 according to claim 1, characterized in that, The psychrophilic bacillus XYR12-4 has the ability to resist low temperature, and can grow normally under the condition that the temperature is 10℃-40℃.

6. The psychrophilic bacillus XYR12-4 of claim 1 is applied to promote plant growth.

7. Use of Psychrobacter xylo lyticus XYR12-4 according to claim 6 for promoting plant growth, characterized in that, The plant is treated by root irrigation with the bacterial liquid of the psychrophilic bacillus XYR12-4, the OD value of the bacterial liquid of the psychrophilic bacillus XYR12-4 being 1, and the dosage being at least 100 mL / kg of soil. 600 value of 1, and the dosage being at least 100 mL / kg of soil.

8. The psychrophilic bacillus XYR12-4 of claim 1 is applied to improve saline-alkali soil.

Images