Bacillus megaterium strain and application thereof

Abstract

The invention discloses a bacillus megaterium strain and application thereof, relates to the technical field of microorganisms, and aims to solve the problem of lack of functional bacteria capable of resisting salt, reducing soil salt content and promoting plant growth in the prior art. According to the technical key points, the bacillus megaterium DX-1 is provided and is preserved in the China Center for Type Culture Collection on August 19, 2024, the preservation address is Wuhan University, China, the preservation number is CCTCC NO: M 20241771, and the bacillus megaterium DX-1 is relatively high in salt tolerance; the invention further provides a microbial agent containing the bacillus megaterium DX-1 and a soil remediation agent, and the microbial agent and the soil remediation agent have remarkable effects on improving the physicochemical property of saline-alkali soil and improving the growth state of crops in the saline-alkali soil.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Bacillus megaterium and its applications. Background Technology

[0002] The total area of ​​saline-alkali land in China has reached 99.13 million hectares. 2 China ranks third globally in terms of saline-alkali land area, accounting for one-tenth of the world's total. Currently, approximately 50% of China's saline-alkali land has the potential for agricultural use; however, the area available for development is gradually decreasing. With continuous social and scientific progress, saline-alkali land, as a valuable reserve of arable land, needs to be improved and utilized to better address China's food security and arable land security issues.

[0003] There are three main methods for remediating saline-alkali land: chemical remediation, engineering remediation, and bioremediation. Chemical remediation uses chemical reagents to alter the physical and chemical properties of saline-alkali soil, including the use of chemical amendments and the addition of chemical substances. Its main advantages are rapid remediation speed and wide coverage; however, its disadvantages include damaging the original soil structure, causing soil compaction, affecting crop growth, high cost, and potential soil pollution and secondary salinization. Engineering remediation refers to altering the salt content or composition of saline-alkali soil using engineering techniques without damaging the original land environment to achieve the goal of saline-alkali land management. It is suitable for large areas of saline-alkali land. Its advantage is the stable remediation capability, but the remediation cycle is long and the cost is relatively high, thus limiting its application. Bioremediation technology uses biological resources to improve saline-alkali land. It is characterized by low pollution, high ecological benefits, and long-lasting improvement effects, and can maintain the stability of the soil ecosystem by increasing soil microbial activity. However, current technologies lack functional bacteria that are salt-tolerant, reduce soil salinity, and promote plant growth. Summary of the Invention

[0004] The technical problem to be solved by this invention is:

[0005] Existing technologies lack functional bacteria that can tolerate salt, reduce soil salinity, and promote plant growth.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] This invention provides a strain of Bacillus megaterium, namely Bacillus megaterium DX-1, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 12, 2024, at Wuhan University, China, with accession number CCTCC NO: M 20241771.

[0008] This invention provides the application of the aforementioned Bacillus megaterium DX-1 in the preparation of microbial agents that reduce salt content and promote plant growth.

[0009] The present invention provides a microbial inoculant containing the aforementioned Bacillus megaterium DX-1.

[0010] Furthermore, the microbial agent is in the form of a dry powder, which is obtained through the following steps:

[0011] The Bacillus megaterium DX-1 was cultured on a large scale, and the obtained fermentation product was spray-dried, pulverized and diluted to obtain the microbial agent.

[0012] Furthermore, the culture medium for the scale-up culture comprises, by mass parts: 1-2 parts glucose, 0.1-1 parts peptone, 1-5 parts soybean meal, 1-5 parts corn starch, 0.1-1 parts sodium acetate, 0.1-1 parts calcium carbonate, 0.1-1 parts dipotassium hydrogen phosphate, and 0.01-0.1 parts magnesium sulfate.

[0013] Furthermore, the microbial agent contains 20 billion CFU / g of effective viable Bacillus megaterium DX-1.

[0014] The present invention provides a soil remediation agent comprising the above-mentioned Bacillus megaterium DX-1 and / or the biological agent as described in claims 3-6.

[0015] Furthermore, the soil remediation agent also includes an auxiliary microbial agent, which includes at least one of Bacillus subtilis, Bacillus licheniformis, Penicillium, or Trichoderma longifolia.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] This invention obtains a salt-tolerant Bacillus megaterium strain through domestication in a saline-alkali environment. This strain has strong salt tolerance and can grow in high-salinity soils. It can be used in the preparation of microbial agents and soil remediation agents for saline-alkali lands. It has significant effects on improving the physicochemical properties of saline-alkali soils and the growth status of crops in saline-alkali lands.

[0018] The soil conditioner provided by this invention, by introducing auxiliary microbial agents, is low in cost, has significant effects, and has good application prospects. Attached Figure Description

[0019] Figure 1 The strain is Bacillus megaterium DX-1, as described in this embodiment of the invention.

[0020] Figure 2This is a diagram showing the sodium chloride tolerance results of Bacillus megaterium DX-1 in an embodiment of the present invention. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, exemplary embodiments or examples of the present invention will be described below in conjunction with the accompanying drawings. Obviously, the described embodiments or examples are merely some, not all, of the embodiments or examples of the present invention. All other embodiments or examples obtained by those skilled in the art based on the embodiments or examples of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] In one typical embodiment, the present invention provides a strain of Bacillus megaterium, namely Bacillus megaterium DX-1, which was deposited at the China Center for Type Culture Collection (CCTCC) at Wuhan University, China on August 12, 2024, with accession number CCTCC NO: M 20241771. The provided Bacillus megaterium strain can grow in high-salinity soils and can be used in the preparation of soil remediation agents. In saline-alkali soil environments, it can reduce the salt content in the soil and promote plant growth, which is of great significance for the application of microbial fertilizers in saline-alkali land. The preferred plant is sunflower.

[0024] The Bacillus megaterium DX-1 provided by this invention has been identified as Gram-positive, exhibiting nearly circular colonies with a smooth surface, regular edges, pale yellow color, opaque appearance, and a glossy sheen. The Bacillus megaterium DX-1 possesses the 16S rDNA sequence shown in SEQ IN NO:1.

[0025] In one typical embodiment, the present invention provides the application of Bacillus megaterium DX-1 in the preparation of microbial agents that reduce salt content and promote plant growth as described above.

[0026] In one typical embodiment, the present invention also provides a microbial agent comprising Bacillus megaterium DX-1 as described in the above embodiments.

[0027] In a typical embodiment, preferably, the microbial agent is in the form of a dry powder, and the microbial agent is obtained through the following steps:

[0028] The Bacillus megaterium DX-1 was cultured on a large scale, and the obtained fermentation product was spray-dried, pulverized and diluted to obtain the microbial agent.

[0029] In a typical embodiment, preferably, the culture medium for scale-up culture comprises, by weight, 1-2 parts glucose, 0.1-1 parts peptone, 1-5 parts soybean meal powder, 1-5 parts corn starch, 0.1-1 parts sodium acetate, 0.1-1 parts calcium carbonate, 0.1-1 parts dipotassium hydrogen phosphate, and 0.01-0.1 parts magnesium sulfate. The culture medium used for scale-up culture includes, but is not limited to, the types of culture media described above; other culture media capable of scaling up the fermentation culture of the microbial agent can also be used in this invention.

[0030] In a typical embodiment, preferably, the microbial agent contains 20 billion CFU / g of Bacillus megaterium DX-1.

[0031] In one typical embodiment, the present invention provides a soil remediation agent comprising Bacillus megaterium DX-1 as described in the above embodiments, and / or the biological agent as described in the above embodiments.

[0032] In a typical embodiment, preferably, the soil remediation agent further includes an auxiliary microbial agent, which includes at least one of Bacillus subtilis, Bacillus licheniformis, Penicillium, or Trichoderma longifolia.

[0033] In one typical embodiment, the present invention also provides a method for remediating saline-alkali land, wherein the above-mentioned soil remediation agent is evenly spread on the saline-alkali land at a spreading density of 1.5 kg / m³. 2 Use soil turning equipment to turn over the soil layer above 20cm in depth in saline-alkali land, mix the turned-over soil with the remediation agent evenly and cover it in situ, and spray clean water regularly to keep the surface soil moist.

[0034] Example 1: Isolation and Identification of Bacillus megaterium

[0035] (1) Strain isolation and screening

[0036] 1) Isolation and screening of strains

[0037] Take 5g of soil sample from saline-alkali land in Kashgar region, Xinjiang, and set up 10 -2 10 -3 10 -4Soil suspensions were prepared at three different dilutions, with three replicates for each dilution. The suspensions were added to 100 mL of enrichment medium containing: 100 g NaCl, 5 g KCl, 1.0 g NH₄Cl, 0.5 g Na₂SO₄, 0.1 g MgSO₄, 0.5 g K₂HPO₄, 0.1 g CaCl₂, 0.5 g peptone, and 0.5 g yeast extract, dissolved in 1 L of distilled water, and the pH was adjusted to 7.0-7.2. The suspensions were incubated at 37°C for 3 days with a shaker speed of 100 rpm. The bacterial suspensions were then plated on LB agar plates. Based on the growth rate and morphological characteristics of the colonies, the strain with the highest colony count was selected for streaking isolation until a pure culture was obtained. These isolated and screened strains were individually labeled.

[0038] (2) Biochemical identification of strains

[0039] The morphology of the bacteria was observed using both Gram staining and spore staining. Biochemical identification strips for Bacillus were used to identify the biochemical reactions of the isolated strains. These results were compared with those in Bergey's Manual of Systematic Bacteriology. The experiments showed that the strains were Gram-positive, forming nearly circular colonies with smooth surfaces, regular edges, pale yellow color, opacity, and a glossy appearance (as shown in the image). Figure 1 (as shown); its biochemical characteristics are consistent with those of Bacillus megaterium.

[0040] Table 1. Biochemical test results of Bacillus megaterium

[0041]

[0042] "+" indicates a positive reaction, and "-" indicates a negative reaction.

[0043] (3) 16S rRNA gene sequence analysis of the strain

[0044] Bacterial DNA was extracted using a boiling method and used as a template for PCR amplification. Bacterial nucleic acid probes were prepared using a combination of boiling and biotinylation. The amplification system included: 10 μL Premix Taq, 0.5 μL each of upstream and downstream primers, 5 μL genomic DNA, and 4 μL ddH2O. PCR reaction conditions included: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 57°C annealing for 30 seconds, and 72°C extension for 30 seconds, for a total of 30 cycles. The final extension at 72°C was 5 minutes. The amplified products were stored at 4°C. Specific fragments were isolated. The PCR amplification products were detected by 1% agarose gel electrophoresis, and sequencing analysis was performed, as shown in SEQ.ID.NO.1. The sequencing results showed 100% homology with *Bacillus megaterium*.

[0045] (4) Mutagenesis treatment of strains

[0046] Strain samples were collected and subjected to radiation mutagenesis using the proton irradiation breeding platform at the Institute of Modern Agriculture, Harbin Institute of Technology. The mutagenesis dose was 100 Gy, and the mutagenesis was completed. The mutagenic bacterial solution was diluted and spread onto LB agar plates and incubated overnight at 37°C. Colony morphology was observed, and larger colonies were selected and streaked onto LB agar plates for purification and culture to obtain the mutagenic strain, named DX-1.

[0047] (5) Salt tolerance analysis of strains

[0048] Sodium chloride tolerance analysis of Bacillus megaterium DX-1 yielded the following results: Figure 2 As shown, the strain is highly tolerant to salt. Its growth is gradually inhibited as the sodium chloride concentration increases in solutions ranging from 2% to 14%, but it can still grow in a 14% sodium chloride solution.

[0049] (5) Preservation

[0050] On August 19, 2024, Bacillus megaterium DX-1 was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M 20241771.

[0051] Example 2: Preparation of microbial inoculants

[0052] Bacillus megaterium DX-1 was cultured into a bacterial suspension and inoculated into a fermenter containing 4000L of fermentation broth at a 5% inoculum size. The culture medium consisted of: 1.5% glucose, 0.3% peptone, 2.6% soybean meal, 2.4% corn starch, 0.4% sodium acetate, 0.5% calcium carbonate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, with the remainder being water. The total volume was 4000L. The fermentation parameters were as follows: inoculum size of 6%, aeration rate of 16 m³ / h, pressure maintained at 0.06 MPa, pH of the fermentation broth maintained at 6.0-6.2, fermentation temperature of 37°C, rotation speed of 100 r / min, and fermentation time of 20 hours. The bacterial slurry was obtained by centrifugation, and then water (50% by weight of the bacterial slurry) was added and stirred to form a bacterial slurry. An auxiliary material, consisting of a mixture of maltodextrin and corn starch in a 1:2 mass ratio, was added at 15% (w / w) of the bacterial slurry. This mixture was then subjected to colloid milling and spray drying. The spray drying parameters were: inlet air temperature set at 145°C, and feed flow rate at 15.50 mL / min, to obtain *Bacillus megaterium* powder. The powder was then counted and diluted with maltodextrin to a concentration of 20 billion CFU / g to obtain a *Bacillus megaterium* inoculum.

[0053] Example 3: Preparation of Soil Remediation Agent

[0054] Rice husk powder and dried chicken manure are pulverized to 50 mesh and mixed evenly according to the ratio of bacterial agent: rice husk powder: chicken manure 1:89:10 to obtain Bacillus megaterium soil remediation agent, which can promote and improve the fertility of saline-alkali land.

[0055] Example 4: Saline-alkali land restoration capacity test

[0056] Soil samples were taken from the saline-alkali land of Kashgar, Xinjiang, and the following experimental groups were set up:

[0057] A: Mix 3 grams of Bacillus megater soil remediation agent with 5 kg of soil;

[0058] B: Mix 5 kg of soil with 3 g of Bacillus megaterium soil remediation agent + 0.03 g of Bacillus licheniformis + 0.03 g of Bacillus subtilis;

[0059] C: 5 kg of soil mixed with 3 g Bacillus megaterium soil remediation agent + 0.03 g Bacillus licheniformis + 0.03 g Bacillus subtilis + 0.03 g Penicillium + 0.03 g Trichoderma longifolia;

[0060] CK: 5 kg of soil.

[0061] Each group of soil was placed in a cylindrical flowerpot and compacted to a thickness of 20 cm. A certain amount of water was sprayed daily to keep the surface moist. On the 30th and 60th days, 50 grams of soil from a depth of 10 cm were taken for testing. The test results are shown in Table 2.

[0062] Table 2 Soil Indicators for Each Experimental Group

[0063]

[0064] As shown in Table 1, the soil remediation agent of this invention effectively reduced soil salinity and pH. After 30 and 60 days of treatment, the soil salinity in group A decreased by 16.9% and 16.2% compared to group CK, respectively; the pH decreased from 8.75 and 8.73 to 7.33 and 7.22, respectively. Group C showed significant improvement in saline-alkali soil indicators. After 60 days of application, the soil salinity decreased by 21.0% compared to the control group, the pH decreased from 8.73 to 7.06, the available phosphorus content increased by 28.74%, the available nitrogen content increased by 70.8%, the available potassium content increased by 52.38%, and the porosity increased by 47.63%. The soil remediation agent helped improve the porosity of saline-alkali soil; after 30 and 60 days of treatment, the porosity of group C increased by 44.90% and 47.63%, respectively.

[0065] Example 5: Test on the growth-promoting ability of soil remediation agents in saline-alkali land

[0066] The experiment was conducted on a saline-alkali plot near Shengli Farm in Kashgar, Xinjiang, from May of the previous year to September of the following year. The selected land was characterized by its compatibility with the previous crop soil, gentle terrain, balanced fertility, and convenient irrigation and drainage. The soil types in the experimental plots were moderately saline-alkali and severely saline-alkali, and all soils were clay.

[0067] The experiment designed two different treatments to avoid duplicate planting. Planting was carried out in a north-south direction, with each treatment comprising half the area. The area was divided into two parts, S and CK, using soil slabs. Each part was 10 meters by 10 meters in area, and the slabs were 0.3 meters wide. 1 kg / 667m² of fertilizer was evenly spread on top of the soil layer in part S. 2 Soil remediation agent (Soil Remediation Agent C) was applied, and then a soil turner was used to turn over the soil to a depth of 20 cm and evenly compact it. Block CK was used as a baseline, without the remediation agent, and all other operating steps were consistent with block S. All crops used in the trials were edible sunflowers, variety number 9021. A double-row planting pattern was selected for water management throughout the growth period, with the wide rows being 1 m wide and irrigated using an alternating wet and dry irrigation method. The narrow rows were 0.5 m wide, with a spacing of 0.55 m between plants, and a theoretical plant count of 360 plants / 100 m². 2 All other field management methods are the same. Fertilization and mulching are carried out simultaneously on May 1st, and watering is carried out simultaneously on May 10th, with the water volume being consistent. Sowing begins on May 22nd, and water and fertilizer management is carried out after emergence, using a uniform manual sowing method. Before sowing, the soil is tilled and fertilized. When the sunflowers enter the bud stage, urea 1.5 kg / 100 m² is added in conjunction with irrigation. 2 After the sunflowers bloomed, sex pheromones and frequency-vibrating insecticidal lamps were used to control the sunflower borer, and yield and harvest tests were conducted in September of the following year.

[0068] Sampling and yield measurement methods

[0069] Five sampling points (top left, bottom left, middle, top right, and bottom right) were taken from each treatment plot. Each sampling point was a 2m x 2m square area. The number of plants was counted, the sunflower heads were numbered, and the actual density was calculated. The sunflower heads were threshed and mixed on-site. The sunflower index results for each experimental group are shown in Table 3.

[0070] Table 3 Sunflower Indicators for Each Experimental Group

[0071]

[0072] Compared to the control group (CK), the group treated with the soil remediation agent (S group) had a 37.7% higher sunflower survival rate, a 25.1% larger sunflower head diameter, a 33.4% higher average number of seeds per sunflower head, a 3.15% higher average weight per 100 seeds, and an 89.73% higher yield of sunflower seeds per mu (unit of land area). This demonstrates that the soil remediation agent of this invention has a significant effect on promoting plant growth.

[0073] Example 6: Effect on the emergence rate of cotton seedlings in Xinjiang

[0074] The experiment was conducted in Bachu County, Xinjiang, and the experimental field was a water and fertilizer integration experimental field. All demonstration sites used machine-harvested cotton with a 1-film, 6-row, 3-pipe (66+10) cm plant spacing configuration, theoretically resulting in 16,800 plants per 667m². 2 Sowing (drip irrigation for seedling emergence), topping, harvesting, and irrigation nine times throughout the growth period. The demonstration field followed local field production management practices. Using a field demonstration method, three treatment groups and one control group were set up. The treatment groups were: soil remediation agent (soil remediation agent C), market product 1 (Fulong microbial agent), and market product 2 (Huanong microbial agent) 800g / 667m². 2 +For conventional drip irrigation fertilization, the dosage of each irrigation for the first and second waterings of the microbial agents "experimental inoculants, market product 1 (Fulong inoculant), and market product 2 (Huanong inoculant)" is 200 g / 667m². 2 Dosage for each of the 3rd to 6th water treatments: 100 g / 667m³ 2 Control group (CK) received conventional drip irrigation locally. Fertilizer application in both control and experimental groups: 50 kg urea / 667 m². 2 +Monammonium phosphate 30 kg / 667m 2 + Potassium sulfate 25kg / 667m 2 The group leaders are shown in Table 4.

[0075] Table 4

[0076]

[0077] The experimental inoculant significantly promoted the emergence rate of cotton seedlings and was significantly better than market product 1 and market product 2, with an emergence rate of 72%, which was 53.18% higher than the 18.82% emergence rate of the control group. The main root length of the experimental inoculant group was the highest among the experimental groups of the same batch, which was 6.14% higher than the control group. The number of lateral roots in the experimental inoculant group was the highest, which was 29.54% more than the control group.

[0078] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention, and all such changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A strain of Bacillus megaterium, characterized in that, The strain is Bacillus megaterium DX-1, which was deposited at the China Center for Type Culture Collection (CCTCC) on August 12, 2024, at Wuhan University, China, with accession number CCTCC NO:M 20241771.

2. The application of Bacillus megaterium DX-1 as described in claim 1 in the preparation of microbial agents that reduce salt content and promote plant growth.

3. A microbial inoculant, characterized in that, The microbial agent contains Bacillus megaterium DX-1 as described in claim 1.

4. The microbial agent according to claim 3, characterized in that, The microbial agent is in the form of a dry powder, which is obtained through the following steps: The Bacillus megaterium DX-1 was cultured on a large scale, and the obtained fermentation product was spray-dried, pulverized and diluted to obtain the microbial agent.

5. The microbial agent according to claim 4, characterized in that, The culture medium for the scale-up culture comprises, by mass parts: 1-2 parts glucose, 0.1-1 parts peptone, 1-5 parts soybean meal, 1-5 parts corn starch, 0.1-1 parts sodium acetate, 0.1-1 parts calcium carbonate, 0.1-1 parts dipotassium hydrogen phosphate, and 0.01-0.1 parts magnesium sulfate.

6. The microbial agent according to claim 5, characterized in that, The microbial agent contains 20 billion CFU / g of Bacillus megaterium DX-1.

7. A soil remediation agent, characterized in that, The soil remediation agent includes Bacillus megaterium DX-1 as described in claim 1, and / or the biological agents as described in claims 3-6.

8. The soil remediation agent according to claim 7, characterized in that, The soil remediation agent also includes auxiliary microbial agents, which include at least one of Bacillus subtilis, Bacillus licheniformis, Penicillium, or Trichoderma longifolia.

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