A halotolerant and cold-tolerant paenibacillus and application thereof

Abstract

The application belongs to the technical field of microbial application, and specifically provides a cold-tolerant Paenibacillus polymyxa (Y5R4-9). Peribacillus frigoritolerans Under the condition of saline-alkali stress, inoculation of the strain Y5R4-9 can promote the growth of forage oats in saline-alkali soil by improving the leaf antioxidant enzyme activity and proline content, reducing the MDA content, improving the soil enzyme activity and soil organic matter, increasing the plant height of forage oats, and improving the root morphology, so as to efficiently relieve the stress of saline-alkali on the growth of forage oats.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, specifically relating to a cold-resistant strain of Bacillus subtilis and its application. Background Technology

[0002] Saline-alkali land is a product of soil salinization, mainly formed by the accumulation of salt in the topsoil due to strong natural evaporation, high groundwater levels, or improper irrigation. Salinization has become a significant environmental problem affecting food security and ecological security. High salt content in the soil directly poisons crop roots, causing difficulties in seedling emergence and weakened growth, leading to significant reductions in crop yields or even crop failure. Moreover, it causes continuous damage to the ecological environment, resulting in sparse surface vegetation, a significant decline in biodiversity, and further triggering desertification and exacerbating ecological risks such as sandstorms.

[0003] Currently, there are physical, chemical, and biological measures for improving saline-alkali land. Physical improvement (such as topsoil replacement, land leveling, and ground cover) mainly uses engineering methods to regulate water and salt transport and rapidly reduce topsoil salinity. While it can quickly alleviate soil salinization symptoms in the short term, it suffers from high costs, short-lived effects that are prone to rebound, and negative impacts on the soil ecosystem. Chemical improvement (such as the application of gypsum and phosphogypsum) reduces soil alkalinity and improves structure through ion exchange. It also suffers from short-lived effects, strong dependence on chemical amendments, and risks of secondary pollution and soil degradation. In recent years, biological improvement has increasingly become a focus of research and application. By planting salt-tolerant plants and utilizing microbial-plant interactions to enhance soil fertility and strengthen system stability, it is considered a more sustainable remediation approach.

[0004] Bacteroides spp. ( Peribacillus *Bacillus simplex* is a rod-shaped, Gram-positive bacterium that forms endospores and belongs to the family Bacillusaceae. Currently, 22 species in this genus have been published and correctly named, many of which originate from soil or plants and can promote plant growth, such as *Bacillus simplex*. Peribacillus simplex ), cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans (etc.) Chinese patents CN120173800A and CN 118773075A both disclose a plant. Peribacillus frigoritolerans The patent claims that it can improve the salt and alkali tolerance of plants, but the patent does not disclose the specific salt and alkali stress parameters in the plant growth promotion experiment or only discloses the salt stress concentration. Therefore, it is not clear in the prior art whether the strain can tolerate high salt and high alkali stress at the same time in practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a novel salt- and alkali-tolerant and cold-resistant Bacillus subtilis strain (Y5R4-9) and its applications, providing a safe, efficient and easily promoted microbial technology for forage production in saline-alkali land.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a cold-resistant strain of *Bacillus subtilis* (…). Peribacillus frigoritolerans Y5R4-9. This strain was deposited on October 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 36186. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The above-mentioned cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans The 16S rRNA gene sequence of Y5R4-9 is shown in SEQ ID NO.1.

[0008] This invention also provides the above-mentioned cold-resistant *Bacillus subtilis* (… Peribacillus frigoritolerans Y5R4-9 is applied in the following aspects: (1) Improve the salt and alkali tolerance and ACC deaminase production of feed oats; (2) Promotes the germination of feed oats under salt and alkali stress; (3) Promotes the growth of feed oats under salt and alkali stress; (4) Increase the activity of S-UE, S-β-GC and S-AKP enzymes in soil under saline-alkali stress.

[0009] Furthermore, the improvement of salt and alkali tolerance of feed oats includes increasing the activity of SOD, POD, and CAT enzymes in the flag leaves of feed oats under salt and alkali stress.

[0010] Furthermore, promoting the growth of feed oats includes increasing the plant height, root length, and root surface area of ​​feed oats.

[0011] Furthermore, the above applications include the use of cold-resistant *Bacillus subtilis* (…). Peribacillus frigoritolerans Y5R4-9 liquid inoculant was used for root irrigation treatment of plants.

[0012] Furthermore, the liquid bacterial agent contains *Bacillus subtilis* (…). Peribacillus frigoritolerans The viable count of Y5R4-9 was 1×10⁻⁶. 7 CFU / mL.

[0013] The present invention also provides a strain containing the above-mentioned cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans Y5R4-9 microbial inoculant.

[0014] The beneficial effects of this invention are as follows: This invention provides a cold-resistant strain of *Bacillus subtilis* (…). Peribacillus frigoritoleransY5R4-9. Under saline-alkali stress conditions, inoculation with strain Y5R4-9 can increase the plant height and improve root morphology of forage oats by increasing leaf antioxidant enzyme activity and proline content, reducing MDA content, and increasing soil enzyme activity and soil organic matter. This effectively promotes the growth of forage oats planted in saline-alkali land and alleviates the stress of saline-alkali on forage oat growth. Attached Figure Description

[0015] Figure 1 Phylogenetic tree of the 16S rRNA gene of strain Y5R4-9.

[0016] Figure 2 The effect of strain Y5R4-9 on the relative germination rate of feed oats (Note: different lowercase letters indicate significant differences (p<0.05)).

[0017] Figure 3 The results of the hemolysis experiment for strain Y5R4-9 (cultured at 28℃ for 48 h).

[0018] Figure 4 The effect of cold-resistant Bacillus subtilis Y5R4-9 on the plant height of feed oats.

[0019] Figure 5 The effects of cold-resistant Bacillus subtilis Y5R4-9 on the root morphology of feed oats were investigated. Figure (a) shows the total root length; Figure (b) shows the root surface area; Figure (c) shows the average diameter; and Figure (d) shows the root volume.

[0020] Figure 6 The effects of cold-resistant Bacillus subtilis Y5R4-9 on the enzyme activity of flag leaves of feed oats were investigated. Figure (a) shows SOD activity; Figure (b) shows POD activity; and Figure (c) shows CAT activity.

[0021] Figure 7 The effect of cold-resistant Bacillus subtilis Y5R4-9 on the (a) MDA and (b) Pro content of feed oat flag leaves.

[0022] Figure 8 The effects of cold-resistant Bacillus subtilis Y5R4-9 on soil enzyme activity were investigated. Figure (a) shows soil S-POD activity; Figure (b) shows soil S-UE activity; Figure (c) shows soil S-β-GC activity; and Figure (d) shows soil S-AKP activity. Different lowercase letters indicate significant differences (p<0.05).

[0023] Figure 9 The effect of cold-resistant Bacillus subtilis Y5R4-9 on soil organic matter (Note: different lowercase letters indicate significant differences (p<0.05)).

[0024] Figure 10The effect of cold-resistant Bacillus subtilis Y5R4-9 on soil ammonia nitrogen content (Note: different lowercase letters indicate significant differences (p<0.05)).

[0025] Figure 11 The effect of cold-resistant Bacillus subtilis Y5R4-9 on soil available phosphorus content (Note: different lowercase letters indicate significant differences (p<0.05)). Detailed Implementation

[0026] Example 1: Isolation and screening of cold-resistant Bacillus repens Y5R4-9 Rhizosphere soil (EC) of Leymus chinensis was collected from saline-alkali land in Cangzhou City, Hebei Province. 1:5 With a pH of 2.02 ds / m and a pH of 8.3, rhizosphere soil was added to an Erlenmeyer flask containing 15 glass beads and sterile water. The soil sample was shaken to prepare a soil suspension. After boiling the soil suspension in a water bath for 5 minutes, it was serially diluted. 10 μL of each sample was taken... -4 10 -5 and 10 -6 Add 0.1 mL of soil suspension at three dilutions to the surface of LB agar plates, spread evenly, and let stand for 5 min. Invert the petri dishes for incubation at 28℃. Observe the growth of isolated microbial colonies in the soil over an incubation period of 16-72 h. Select well-grown single colonies and preserve them in LB slant agar.

[0027] The preserved soil-isolated bacterial strains were inoculated into LB liquid medium and cultured for 2 days to obtain seed culture. The OD of the seed culture was then measured. 600 The pH was adjusted to 1, and 1% inoculum was inoculated into LB liquid medium containing 2% NaCl, 4% NaCl, 6% NaCl, 8% NaCl, 10% NaCl, and 12% NaCl (pH=7, 8, 9, 10), respectively. The salt and alkali tolerance of the strain was determined, and the results are shown in Table 1. A strain with excellent salt and alkali tolerance was obtained through screening and designated Y5R4-9. This strain can grow in LB medium containing 10% NaCl at pH 9.

[0028] Table 1. Growth status of Y5R4-9 under saline-alkali conditions.

[0029] Note: + indicates OD 600 0-0.5, ++ indicates OD 600 0.5-1, +++ indicates OD 600 1-1.5, ++++ indicates OD 600 It is 1.5-2.

[0030] The ability of the obtained salt-tolerant strain Y5R4-9 to synthesize ACC deaminase was further investigated. Y5R4-9 was inoculated into ADF medium (pH 7.2) with ammonium sulfate as the sole nitrogen source and cultured at 28°C and 180 rpm for 3 days. After washing and centrifuging three times with DF medium, the washed cells were resuspended in ADF medium with ACC as the sole nitrogen source and cultured at 28°C and 180 rpm for 5 days. The ACC deaminase activity of the culture was then measured. The results showed that strain Y5R4-9 could secrete ACC deaminase, with an ACC deaminase activity of 447.537 nmol / min mg in the fermentation broth. -1 It still exhibits ACC deaminase activity in ADF medium containing 3% NaCl at pH 8 or 1% NaCl at pH 9.

[0031] Example 2 Identification of cold-resistant Bacillus repens Y5R4-9 Genomic DNA of the target strain Y5R4-9 was obtained using a bacterial genomic DNA extraction kit. PCR amplification of 16S rRNA was performed using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The reaction mixture (25 μL) contained 12 μL of 2×Taq PCR Master Mix, 1 μL each of 10 μmol / L forward and reverse primers, 1 μL of template DNA, and ddH2O to bring the final volume to the limit. The amplification program was: 95℃ pre-denaturation for 5 min; 35 cycles (94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1.5 min); final extension at 72℃ for 10 min. The amplified products were verified by 1% agarose gel electrophoresis and then sequenced by Shanghai Sangon Biotech Co., Ltd. A phylogenetic tree of the 16S rRNA gene of the target strain was constructed based on the sequencing results. Figure 1 Its sequence is shown in SEQ ID No. 1. The target strain Y5R4-9 was found to be similar to *Bacillus subtilis* (a cold-resistant bacterium). Peribacillus frigoritolerans DSM 8801 is the most closely related species with 100% sequence homology, and it has been identified as a cold-resistant spore-forming bacterium.

[0032] Example 3: Effect of cold-resistant Bacillus subtilis Y5R4-9 on germination rate of forage oat seeds This embodiment includes a control group and a treatment group inoculated with strain Y5R4-9. Mature, plump, and uniform feed oats (… Avena sativa L The seeds were soaked in a 1% NaClO solution for 10 minutes, then washed three times with sterile water until the NaClO solution was completely removed. The pretreated oat seeds were then immersed in a 1×10⁻⁶ NaClO solution. 7The seeds were soaked in a Y5R4-9 bacterial suspension at CFU / mL at 25℃ and shaken at 80 rpm for 2 h. The control group seeds were treated in the same way with sterile water. After drying, the seeds were transferred to petri dishes lined with sterile filter paper with NaCl concentrations of 0%, 0.4%, 0.6%, and 0.8% and a pH of 8.5 for germination. Each petri dish contained 50 seeds, and each treatment was repeated in triplicate.

[0033] like Figure 2 As shown, the germination rate of oat seeds in the uninoculated salt-free group was taken as 100%, and the germination rate of each treatment group was measured. The results showed that under salt-alkali stress, the germination rate of uninoculated oat seeds decreased significantly. When the NaCl concentration was 0.4%, 0.6%, and 0.8%, the relative germination rate of oat seeds decreased to 90.3%, 87.1%, and 83.9%, respectively. The germination rate of oat seeds in the group inoculated with strain Y5R4-9 was significantly increased. p <0.05): When the NaCl concentrations were 0%, 0.4%, 0.6% and 0.8%, the germination rates of oat seeds in the inoculated groups reached 135.5%, 122.6%, 119.4% and 116.1%, respectively. This result indicates that strain Y5R4-9 can effectively alleviate salt damage.

[0034] Example 4 Hemolysis experiment of cold-resistant Bacillus repens Y5R4-9 Take activated strain Y5R4-9, inoculate it in the center of a Columbia blood agar plate, and incubate at 28 ℃ for 48 h. Figure 3 As shown, no hemolytic zone appears around the Y5R4-9 colony, indicating that strain Y5R4-9 is non-hemolytic, meets the standards for microbial fertilizer, and has application potential.

[0035] Example 5: Application of cold-resistant *Bacillus subtilis* Y5R4-9 in alleviating salt and alkali stress in feed oats. The test soil consisted of a mixed substrate of nutrient soil, vermiculite, and perlite in a ratio of 5:1:1, with an additional 0.6% NaCl added. The soil electrical conductivity (EC) was measured. 1:5 The value was 1994 μs / cm, and the pH was 8.5.

[0036] Select a number of mature, plump, and uniformly sized forage oat seeds and plant them in plastic flowerpots (flowerpot specifications: upper opening length and width both 8 cm, lower bottom length and width both 5.3 cm, height 11 cm), with 270 g of soil per pot and 6 seedlings per pot. This experiment included a control group (CK2) and a Y5R4-9 inoculation treatment group (M0). The M0 group underwent root drenching treatment, i.e., the inoculum suspension was applied 7 days after sowing, with an inoculation rate of 1.5 × 10⁻⁶. 11CFU / kg seeds, CK2 group was treated with an equal volume of deionized water. Each treatment had 9 replicates.

[0037] After 30 days of growth, the plant height of the oats was measured, and samples of flag leaf, rhizosphere soil, and roots were collected for relevant index determination. Specific indexes measured included: superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities in the flag leaf, as well as malondialdehyde (MDA) and proline (Pro) content. All these indicators were measured using a Solarbio reagent kit. Rhizosphere soil activities were measured for peroxidase (S-POD), urease (S-UE), β-glucosidase (S-β-GC), and alkaline phosphatase (S-AKP) activities, and soil organic matter content was determined using the potassium dichromate method. Additionally, the seedling roots were morphologically scanned to obtain root morphological parameters such as total root length, root surface area, average diameter, and root volume.

[0038] (1) Effect of cold-resistant Bacillus repens Y5R4-9 on plant height of forage oats like Figure 4 As shown, there was a significant difference in plant height between the M0 group and the CK2 group. The average plant height of the M0 group was 24.43 cm, which was 22.5% higher than that of the CK2 group.

[0039] (2) Effects of cold-resistant Bacillus subtilis Y5R4-9 on root morphology of feed oats The effects of strain Y5R4-9 on the root morphology of feed oats, such as Figure 5 As shown in the figure, significant differences were observed between the M0 group and the CK2 group in terms of total root length, root surface area, average diameter, and root volume. Specifically, compared with the CK2 group, the total root length of the M0 group increased by 67.3%, reaching an average of 303.81 cm; the root surface area increased by 241.4%, averaging 52.83 cm². 2 The average diameter increased by 60.0%, averaging 0.40 mm; the root volume increased by 629.1%, averaging 0.78 cm. 3 .

[0040] (3) Effects of cold-resistant Bacillus subtilis Y5R4-9 on enzyme activity in flag leaves of feed oats Effects of strain Y5R4-9 on flag leaf enzyme activity in feed oats, such as Figure 6 As shown in the figure, significant differences were observed between the M0 group and the CK2 group in terms of enzyme activities such as SOD, POD, and CAT. Specifically, compared with the CK2 group, the M0 group showed an average SOD activity of 1097.30 U / g, an increase of 49.7%; an average POD activity of 21898.31 U / g, an increase of 56.6%; and an average CAT activity of 694.95 U / g, an increase of 74.5%.

[0041] (4) Effect of cold-resistant Bacillus subtilis Y5R4-9 on the content of substances in flag leaves of feed oats The effect of strain Y5R4-9 on the content of flag leaf substances in feed oats, such as Figure 7 As shown in the figure, significant differences were observed between the M0 group and the CK2 group in terms of MDA and Pro. Specifically, compared with the CK2 group, the MDA content in the M0 group averaged 25.08 nmol / g, a decrease of 24.1%; the Pro content averaged 96.59 μg / g, an increase of 73.9%.

[0042] (5) Effects of cold-resistant Bacillus subtilis Y5R4-9 on soil enzyme activity The effects of cold-resistant Bacillus subtilis Y5R4-9 on soil enzyme activity, such as Figure 8 As shown in the figure, significant differences were observed between the M0 and CK2 groups in the activities of soil S-POD, soil S-UE, soil S-β-GC, and soil S-AKP. Specifically, compared with the CK2 group, the average soil S-POD activity in the M0 group was 21.07 mg / mL, an increase of 45.6%; the average soil S-UE activity was 791.46 μg / mL, an increase of 27.5%; the average soil S-β-GC activity was 4.62 μmol / L, an increase of 20.3%; and the average soil S-AKP activity was 26055.56 U / g, an increase of 14.4%.

[0043] (6) Effects of cold-resistant Bacillus subtilis Y5R4-9 on soil organic matter The effects of cold-resistant Bacillus subtilis Y5R4-9 on soil organic matter, such as Figure 9 As shown, there was a significant difference between the CK2 group and the M0 group. Compared with the CK2 group, the average soil organic matter content in the M0 group reached 2.32 mg / kg, which increased by 7.7%.

[0044] (7) Effects of cold-resistant Bacillus subtilis Y5R4-9 on soil ammonium nitrogen and available phosphorus content The effects of cold-resistant Bacillus subtilis Y5R4-9 on soil ammonium nitrogen, such as Figure 10 As shown, there was a significant difference between the CK2 group and the M0 group. Compared with the CK2 group, the average soil ammonium nitrogen content in the M0 group reached 56.57 mg / kg, an increase of 31.70%.

[0045] The effects of cold-resistant Bacillus subtilis Y5R4-9 on available phosphorus in soil, such as Figure 11 As shown, there was a significant difference between the CK2 group and the M0 group. Compared with the CK2 group, the average available phosphorus content in the soil of the M0 group reached 80.00 mg / kg, which was reduced by 23.08%.

Claims

1. A cold-resistant strain of *Bacillus subtilis* ( Peribacillus frigoritolerans Y5R4-9, its accession number is: CGMCC No. 36186.

2. The cold-resistant *Bacillus subtilis* as described in claim 1 (… Peribacillus frigoritolerans Y5R4-9, whose 16S rRNA gene sequence is shown in SEQ ID NO.

1.

3. The cold-resistant *Bacillus subtilis* as described in claim 1 (… Peribacillus frigoritolerans Application of Y5R4-9 in improving the salt and alkali tolerance and ACC deaminase production of feed oats.

4. The application according to claim 3, characterized in that, The improvement of salt and alkali tolerance in feed oats includes increasing the activity of SOD, POD, and CAT enzymes in the flag leaves of feed oats under salt and alkali stress.

5. The cold-resistant *Bacillus subtilis* as described in claim 1 (… Peribacillus frigoritolerans Application of Y5R4-9 in promoting germination and growth of feed oats under salt and alkali stress.

6. The application according to claim 5, characterized in that, The promotion of feed oat growth includes increasing the plant height, root length, and root surface area of ​​feed oats.

7. The cold-resistant *Bacillus subtilis* as described in claim 1 (… Peribacillus frigoritolerans Application of Y5R4-9 in improving the activity of S-UE, S-β-GC and S-AKP enzymes in soil under saline-alkali stress.

8. The application as described in any one of claims 3 to 7, characterized in that, The application includes the use of *Bacillus subtilis* (a cold-resistant bacterium). Peribacillus frigoritolerans Y5R4-9 liquid inoculant was used for root irrigation treatment of plants.

9. The application according to claim 8, characterized in that, The liquid bacterial agent contains cold-resistant Bacillus subtilis ( Peribacillus frigoritolerans The viable count of Y5R4-9 was 1×10⁻⁶. 7 CFU / mL.

10. A strain containing the cold-resistant *Bacillus subtilis* as described in claim 1 (… Peribacillus frigoritolerans Y5R4-9 microbial inoculant.

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