Bacillus safensis hmc19 and application thereof

Abstract

This invention relates to a type of Bacillus safranin (Sarfus). Bacillus safensis HMC19, with accession number CGMCC No. 35493, can stably colonize in high saline-alkali environments, regulate soil pH, and secrete the plant growth regulator phenylacetic acid, which delays chlorophyll degradation and effectively alleviates premature aging of plants under saline-alkali stress. It achieves synergistic effects of saline-alkali land improvement and crop quality and yield enhancement. It can also effectively degrade soil autotoxic substances such as p-hydroxybenzoic acid and ferulic acid, improve the soil microenvironment, and prevent continuous cropping obstacles.

Description

Technical Field

[0001] This invention belongs to the field of soil microbial technology, specifically relating to Bacillus sabolicii HMC19 and its applications. Background Technology

[0002] Saline-alkali soils are a major bottleneck in global agricultural production, and their rational improvement is of strategic importance for ensuring food security and expanding arable land. Current methods for saline-alkali soil improvement include engineering, chemical, and biological methods. Among these, biological improvement has become a research hotspot in recent years due to its advantages such as low cost, long-lasting ecological benefits, and no secondary pollution. Microbial remediation, as a core direction of biological improvement, involves screening functional microorganisms with salt-alkali tolerance to regulate soil pH, improve soil structure, enhance soil fertility, and promote plant growth.

[0003] Premature senescence in plants is a key issue affecting crop yield and quality. Under saline-alkali stress, plants are prone to premature senescence due to factors such as osmotic pressure imbalance, nutrient absorption disorders, and the accumulation of reactive oxygen species. This manifests as leaf yellowing, decreased photosynthetic efficiency, and reduced seed setting rate, severely restricting crop productivity in saline-alkali land. Current technologies often alleviate premature senescence by applying chemical regulators or common microbial agents. However, chemical regulators are prone to leaving environmental residues, and common microbial agents generally have drawbacks such as weak salt and alkali tolerance, limited anti-premature senescence function, and poor colonization ability in saline-alkali environments, making it difficult to simultaneously meet the dual needs of saline-alkali land improvement and crop anti-premature senescence. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus sabolicii strain HMC19 with both strong salt and alkali resistance and anti-premature aging function, and its application.

[0005] The present invention adopts the following technical solution: A type of Bacillus safranin ( Bacillus safensis HMC19, with accession number CGMCC No. 35493, was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center, located in Beijing, China.

[0006] Furthermore, the *Bacillus sarcodactylis* HMC19 is capable of secreting phenylacetic acid.

[0007] Furthermore, the *Bacillus sarcodactylis* HMC19 is capable of colonizing in saline-alkali soils.

[0008] Furthermore, the *Bacillus sarcodactylis* HMC19 is capable of degrading soil autotoxic substances p-hydroxybenzoic acid and ferulic acid.

[0009] Furthermore, the *Bacillus safranin* HMC19 can alleviate premature aging of crops in saline-alkali soil and increase crop yield.

[0010] A microbial agent for the above-mentioned Bacillus sarcodactylis HMC19.

[0011] A method for preparing the above-mentioned microbial inoculant includes the following steps: (1) Pick a loopful of Bacillus sabolicii HMC19 colonies, inoculate them into NA liquid medium, and activate them by constant temperature shaking at 180 r / min and 37℃ for 24 hours; (2) Take the activated bacterial solution and inoculate it into NA liquid medium. Incubate at 180 r / min and 37℃ for 24 hours to obtain seed culture. (4) Take the prepared seed culture into NA liquid medium and culture it at 180 r / min and 37℃ for 48 hours to obtain Bacillus sabophore HMC19 fermentation broth; (5) Mix the fermentation broth of Bacillus salsa HMC19 with soluble starch at a mass ratio of 10:1, and spray the mixture through a freeze dryer to obtain the final product.

[0012] An application of the above-mentioned Bacillus salsa HMC19 in the remediation of saline-alkali soil.

[0013] Application of the above-mentioned Bacillus salsa HMC19 in alleviating premature aging of wheat in saline-alkali land.

[0014] An application of the above-mentioned Bacillus salsa HMC19 in increasing wheat yield in saline-alkali land.

[0015] An application of the above-mentioned Bacillus sarcodactylis HMC19 in the degradation of soil autotoxic substances p-hydroxybenzoic acid and ferulic acid.

[0016] The beneficial effects of this invention are as follows: The *Bacillus saeformis* HMC19 of this invention can stably colonize in high-salt-alkali environments, regulate soil pH, and secrete the plant growth regulator phenylacetic acid (PAA), delaying chlorophyll degradation and effectively alleviating premature plant aging under salt-alkali stress. This achieves a synergistic effect between saline-alkali land improvement and crop quality and yield enhancement. Furthermore, it can effectively degrade soil autotoxic substances such as p-hydroxybenzoic acid and ferulic acid, improving the soil microenvironment and preventing continuous cropping obstacles. The *Bacillus saeformis* HMC19 of this invention has significant practical importance and application value in saline-alkali land agricultural production. Attached Figure Description

[0017] Figure 1 The colony morphology of Bacillus salsa HMC19 of this invention after 24 hours of culture on NA solid medium.

[0018] Figure 2 The image shows the morphology of Bacillus salsa HMC19 after Gram staining under an optical microscope.

[0019] Figure 3 This is a phylogenetic tree of the 16S rDNA of Bacillus salsa HMC19 of this invention.

[0020] Figure 4 This is a picture of the growth of Bacillus sarcodactylis HMC19 on p-hydroxybenzoic acid selective medium.

[0021] Figure 5 This is a description of the growth of Bacillus sarcodactylis HMC19 on ferulic acid selective medium according to the present invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples were all purchased from conventional biochemical reagent stores.

[0024] Example 1: Isolation and Screening of Strains HMC19 (1) Sample collection Fresh, healthy wheat plant samples were collected from a drought-resistant wheat planting base in Huanghua City, Hebei Province. The samples were placed in sterile resealable bags and transported to the laboratory at 4°C.

[0025] (2) Culture of strains Select wheat stems and cut them into 1cm sections. Wash them with sterile water, soak them in 75% alcohol for 0.5-1 minute, then wash them with sterile water 3-4 times. Next, soak them in sodium hypochlorite solution with an effective chlorine content of 5% for 5 minutes, then soak them in sodium thiosulfate solution for 10 minutes. Finally, wash them with sterile water 4-5 times and retain the water used to rinse the wheat stems after the last rinse.

[0026] As a sample group, sterilized wheat stem segments were ground into a homogenate in a sterile mortar and then serially diluted to 10 with sterile water. -1 10 -2 10 -3 The samples were spread onto NA solid medium plates at different dilutions, with three replicates for each dilution, and incubated at 35°C for 1–3 days.

[0027] As a verification group, take an appropriate amount of the washing water after the last rinse of wheat stem segments, use a sterile pipette to draw it up and spread it evenly on NA solid culture medium plates, place the plates in a 35℃ constant temperature incubator, and culture them synchronously with the samples for 1~3 days.

[0028] If no colonies grow on the validation plate, it indicates that the wheat stem surface was thoroughly disinfected and free of contaminants. Subsequent colonies cultured will all originate from within the wheat stem (i.e., endophytes), making the experiment valid. The sample group can continue with subsequent experiments. If colonies grow on the validation plate, it indicates that the wheat stem surface was not thoroughly disinfected and contaminants remain. Continuing the experiment will result in subsequent colonies being mixed with surface contaminants. Wheat stems must be selected again, and the surface disinfection steps repeated until the disinfection validation is successful.

[0029] (3) Strains Isolation Based on the morphology, color, and size of the colonies growing on the plate, select different single colonies and repeatedly streak them on solid NA medium until a single strain is isolated.

[0030] (4) Function Filtering The purified strains were inoculated into NA liquid medium containing 2%, 5%, 7%, and 10% NaCl, with pH values ​​of 5.0, 7.0, 9.0, and 10.0, respectively, and cultured at 37°C and 180 rpm for 48 h. OD values ​​were then measured. 600 Three strains with the strongest salt and alkali tolerance were screened out. Further analysis of the phenylacetic acid (PAA) content in the fermentation broth of these three strains revealed that one strain had a PAA content of 25 μg / mL, significantly higher than the other strains. After purification, the PAA was stored in 15% glycerol at -20℃ and -80℃, and named HMC19.

[0031] Example 2 Identification of strain HMC19 (1) Morphological characteristics Colony morphology: After incubation at 37°C for 48 hours on solid NA medium, strain HMC19 colonies were milky white, viscous, smooth and moist, with neat edges, as shown in the image. Figure 1 As shown.

[0032] Bacterial morphology: Gram-positive bacteria, rod-shaped, with spores, such as... Figure 2 As shown.

[0033] (2) Physiological and biochemical characteristics Physiological and biochemical characteristics of strain HMC19 were tested, and the results are shown in Table 1.

[0034] Table 1. Experimental results of physiological and biochemical characteristics of strain HMC19 .

[0035] (3) Molecular biological characteristics Genomic DNA was extracted from strain HMC19. Using it as a template, PCR amplification was performed using universal primers for bacterial 16S rDNA. The amplified product was recovered and sequenced, yielding a DNA sequence containing 1485 bp (as shown in SEQ ID No. 1). The sequencing results were entered into the GeneBank database for BLAST alignment analysis. Comparison with the 16S rDNA sequence in the NCBI database revealed that HMC19 and Bacillus safranin showed 99% identity. The phylogenetic tree was constructed as follows: Figure 3 As shown. Based on the morphological, sequencing analysis, and physiological and biochemical test results, HMC19 was identified as *Bacillus safranin* (…). Bacillus safensis ).

[0036] Bacillus safortus HMC19 was deposited on August 1, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 35493.

[0037] Example 3: Degradation of p-hydroxybenzoic acid (PHBA) and ferulic acid by Bacillus salsa HMC19 An inorganic salt solid culture medium was prepared using p-hydroxybenzoic acid or ferulic acid as the sole carbon source; only strains that can degrade and utilize this acid can grow and form colonies, while non-degrading bacteria cannot grow.

[0038] p-Hydroxybenzoic acid (PHBA) selective medium: KH2PO4 3.0g, Na2HPO4 5.0g, MgSO4·5H2O 0.13g, NH4Cl 1.0g, PHBA 0.5g, agar 20g, distilled water 1000ml, pH 7.0.

[0039] Ferulic acid selective medium: KH2PO4 3.0g, Na2HPO4 5.0g, MgSO4·5H2O 0.13g, NH4Cl 1.0g, ferulic acid 0.5g, agar 20g, distilled water 1000ml, pH 7.0.

[0040] Preparation of bacterial suspension: Pick one loopful of Bacillus sabinatus HMC19 colonies and inoculate them into a 150 mL Erlenmeyer flask containing 50 mL of NA liquid medium. Incubate at 180 r / min and 37 °C for 24 hours with constant temperature shaking.

[0041] Use a pipette to draw up the cultured bacterial suspension, dilute it to an appropriate ratio, and spread it evenly on p-hydroxybenzoic acid (PHBA) selective medium and ferulic acid selective medium, respectively. Incubate in an inverted state at 37°C for 2-3 days and observe whether HMC19 colonies grow.

[0042] The results are as follows Figure 4 and Figure 5 As shown, Bacillus salsa HMC19 can grow on both p-hydroxybenzoic acid selective medium and ferulic acid selective medium, indicating that Bacillus salsa HMC19 can utilize and degrade p-hydroxybenzoic acid and ferulic acid.

[0043] Example 4: Detection of colonization ability of Bacillus salsa HMC19 in soils with different salinity contents (1) Bacillus salsa HMC19 bacterial culture: Bacillus salsa HMC19 was inoculated into NA liquid medium and cultured at 37℃ with shaking at 180 r / min for 24 h. The bacterial concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL, for later use.

[0044] (2) Test soils: Test soil 1 was taken from the applicant's test site. It was moist brown soil, which was air-dried and sterilized at high temperature to make sterile soil 1. The soil salt content was 0.05%; Test soil 2 was taken from saline-alkali soil in Huanghua City, Hebei Province. The soil salt content was 0.23%. It was air-dried and sterilized at high temperature to make sterile soil 2; Test soil 3 was taken from saline-alkali soil in Huanghua City, Hebei Province. The soil salt content was 0.35%. It was air-dried and sterilized at high temperature to make sterile soil 3.

[0045] (3) Take 100g of soil at each salt concentration after treatment, put it into a sterile Erlenmeyer flask, add the above Bacillus sabolicii HMC19 bacterial suspension at an inoculation amount of 10% by weight, and shake well; add an equal volume of sterile water to the blank control and shake well. Place all Erlenmeyer flasks in a constant temperature incubator at 28℃ and incubate in the dark, maintaining the soil moisture content at 60% throughout the process (weigh and replenish water periodically).

[0046] (4) After 15 days, samples were taken to detect the number of viable bacteria. For each treatment, 5g of soil sample was taken (five-point sampling method, mixed evenly). Detection method: The soil sample was diluted and spread on NA solid medium. After incubation at 37℃ for 48 hours, the colony count was counted.

[0047] Colonization rate (%) = number of colonies ÷ number of inoculated plants × 100.

[0048] The colonization number and colonization rate of Bacillus sabinatus HMC19 in soils with different salt contents were statistically analyzed. The results are shown in Table 2.

[0049] Table 2. Colonization rate detection results of strains .

[0050] As shown in Table 2, although the colonization rate of strain HMC19 in saline-alkali soils with salt contents of 0.05%, 0.23%, and 0.35% showed a decreasing trend, the colonization rate of the strain exceeded 100% at each salt content, indicating that strain HMC19 has a good colonization ability in moderately to slightly saline-alkali soils.

[0051] Example 5: Preparation of Bacillus salsa HMC19 inoculum (1) Preparation of NA liquid culture medium: 10.0g peptone, 3.0g beef extract, 5.0g sodium chloride, distilled water to a final volume of 1 liter, pH adjusted to 7.2~7.5, sterilized at 121℃ for 20 minutes; (2) Activation of strain: Pick one loop of Bacillus sabinatus HMC19 colony, inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of NA liquid medium, and activate it by constant temperature shaking at 180 r / min and 37 °C for 24 hours.

[0052] (3) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of NA liquid culture medium. Incubate at 180 r / min and 37 °C for 24 hours to obtain seed culture.

[0053] (4) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of NA liquid medium. The culture was incubated at 37 °C with constant temperature shaking at 180 r / min for 48 hours to obtain the Bacillus sabovellae HMC19 fermentation broth. The viable cell count was approximately 3.51 × 10⁻⁶. 9 CFU / mL.

[0054] (5) Preparation of powdered inoculum: The fermentation broth and the auxiliary material (soluble starch) were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by a freeze dryer to obtain Bacillus sabolicii HMC19 powder. The effective viable count was tested to be 6.65 × 10⁻⁶. 10 CFU / g.

[0055] Example 6: Field application effect of Bacillus salsa HMC19 inoculant The experiment was conducted at a dryland saline-alkali wheat planting base in Huanghua City, Hebei Province. Before planting, soil samples were taken and tested; the pH was 8.29 and the salinity was 0.35%, classifying it as moderately saline-alkali land. Wheat was sown on October 14, 2024, at a seeding rate of 15 kg / mu, with 20 kg / mu of basal fertilizer applied (NPK: 18-20-5). The experiment consisted of two treatment groups, each with three plots, each plot measuring 20 m². 2 The wheat variety planted was "Bomai 7". Treatment Group 1: Before wheat sowing, the microbial agent prepared in Example 5 was sprayed on the ground at a rate of 100g / mu (500 times dilution). After spraying, it was incorporated into the soil. On November 2nd, after wheat emergence, the microbial agent prepared in Example 5 was applied as a foliar spray at a rate of 100g / mu (500 times dilution). The same spraying method was used on March 18th, 2025, during the wheat's greening-up period. Treatment Group 2 (control group): No microbial agents were applied; water was used instead. Only base fertilizer was applied. Other field management measures were the same.

[0056] (1) Survey of agronomic traits On April 25, 2025, 30 healthy wheat plants with uniform growth, free from pests and diseases and mechanical damage were randomly selected from each treatment as survey samples.

[0057] Plant height: A ruler was used to measure the vertical height from the soil surface to the top growing point of the wheat plant. Data for each plant was recorded individually, and the average of the data from 30 plants was calculated to an accuracy of 0.1 cm.

[0058] Leaf area per plant: All fully expanded functional leaves of a single plant were picked, and the leaf area was measured leaf by leaf using a leaf area meter. The total leaf area of ​​a single plant was obtained by accumulating the total leaf area of ​​all leaves. After all samples were measured, the average leaf area of ​​each treatment was calculated.

[0059] Secondary root count per plant: Carefully dig out the entire wheat root system, clean the soil attached to the roots (avoid damaging the roots), count the number of all secondary roots that sprouted from each plant, record the count for each plant, and finally calculate the average number of secondary roots for each treatment.

[0060] Leaf chlorophyll content: The first fully expanded leaf below the ear was used to measure the chlorophyll content using a chlorophyll meter. Three different sites were measured on each leaf, and the average value of a single leaf was recorded. Finally, the data of 30 leaves were summarized, and the average chlorophyll content of each treatment was calculated.

[0061] A handheld chlorophyll meter was used, and the results were expressed as relative chlorophyll SPAD values. The first fully expanded functional leaf below the panicle was selected for each treatment, and three different sites were evenly selected on each leaf for measurement. The average SPAD value of a single leaf was taken. A total of 30 functional leaves were measured for each treatment, and the mean chlorophyll SPAD value of each treatment was finally calculated.

[0062] Percentage of withered leaves: Count the number of normal leaves and withered leaves, and calculate the percentage of withered leaves. Normal leaves: The main body of the leaf is green, and the area of ​​yellowing or drying of the leaf is less than 1 / 3 of the total leaf area, indicating normal photosynthetic function; Withered leaves: The area of ​​yellowing, fading green, or drying of the leaf is ≥1 / 3 of the total leaf area, indicating a decline in photosynthetic function.

[0063] The percentage of senescent leaves = (total number of senescent leaves ÷ total number of leaves of the plant) × 100%.

[0064] (2) Production survey On June 2, 2025, at maturity, the number of ears per mu, the number of grains per ear, the thousand-grain weight, and the yield of wheat were measured, and the soil pH and salinity were tested.

[0065] (3) Results statistics Table 3 Statistical Table of Wheat Agronomic Traits .

[0066] Table 4. Statistical table of soil salinity, pH, and wheat yield. .

[0067] The results showed that the chlorophyll content of wheat leaves in the treatment group increased by 29.88% compared with the control group, the proportion of prematurely senescent leaves decreased by 17.36%, the soil pH decreased by 0.48 compared with before planting, the soil salt content decreased by 0.14%, and the yield increased by 33.7% compared with the control group. The *Bacillus safranin* HMC19 of this invention has significant effects in alleviating soil salinization in saline-alkali land, preventing premature senescence of wheat in saline-alkali land, and increasing yield.

[0068] Example 7: Field application effect of Bacillus salsa HMC19 in degrading soil autotoxins (1) Experimental materials: Bacillus sabensis HMC19 inoculum prepared in Example 5, blank control group (water); cucumber variety was Green Island No. 5, the experimental site was a greenhouse where cucumbers had been continuously planted for 8 years, the soil was compacted and soil-borne diseases were frequent.

[0069] (2) Experimental method: Two treatment groups were set up, with three replicates for each group, and the plot area was 20 m². 2 Before transplanting, apply the microbial agent at a rate of 2.5 kg per mu. After transplanting, irrigate the roots once 7 days after the seedlings have recovered. Dilute the microbial agent 600 times and apply 1.2 kg per mu. The blank control group was given an equal amount of water, and the other field management measures were completely the same.

[0070] (3) Measurement indicators and results: The content of soil autotoxic substances before planting and the content of soil autotoxic substances 60 days after the application of microbial agents were measured respectively. The results are shown in Table 5.

[0071] Table 5. Soil autotoxic substance content .

[0072] As shown in Table 5, Bacillus saffron HMC19 can significantly degrade the content of p-hydroxybenzoic acid and ferulic acid in soil from continuous cropping of cucumbers in greenhouses.

Claims

1. Bacillus safensis (B. safensis) HMC19, characterized in that, Bacillus safensis The preservation number of which is CGMCC No. 35493. ​ 2. Bacillus safensis HMC19 according to claim 1, characterized in that, It can secrete phenylacetic acid.

3. Bacillus safensis HMC19 according to claim 1, characterized in that, It can stably colonize in light and moderate saline-alkali soil.

4. The Bacillus safensis HMC19 of claim 1, wherein, It can relieve the premature senescence of crops in saline-alkali soil and improve the yield of crops.

5. The Bacillus safensis HMC19 of claim 1, wherein, It can degrade soil autotoxic substances p-hydroxybenzoic acid and / or ferulic acid.

6. A microbial inoculant comprising the Bacillus safensis HMC19 of claim 1.

7. A method for preparing the microbial inoculant of claim 6, characterized by, It comprises the following steps: (1) picking a colony of Bacillus safensis HMC19, inoculating in NA liquid medium, and culturing at 180 r / min and 37°C constant temperature oscillation for 24 hours for activation; (2) taking the activated bacterial liquid to inoculate in NA liquid medium, and culturing at 180 r / min and 37°C constant temperature oscillation for 24 hours to obtain a seed liquid; (4) taking the prepared seed liquid to inoculate in NA liquid medium, and culturing at 180 r / min and 37°C constant temperature oscillation for 48 hours to prepare a Bacillus safensis HMC19 fermentation liquor; (5) mixing the Bacillus safensis HMC19 fermentation liquor with soluble starch at a mass ratio of 10:1, uniformly, and spraying through a freeze-drying machine to obtain the product.

8. The Bacillus safensis HMC19 of claim 1 in the application of saline-alkali soil remediation.

9. The Bacillus safensis HMC19 of claim 1 in the application of relieving the premature senescence of wheat in saline-alkali soil and improving the yield.

10. The Bacillus safensis HMC19 of claim 1 in the application of degrading soil autotoxic substances p-hydroxybenzoic acid and / or ferulic acid.

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