Bacillus strain m04 in south china sea, bacterial manure, fermentation agent and application thereof

Abstract

The application provides a bacillus australimaris strain M04, a bacterial fertilizer, a fermentation agent and application thereof, and belongs to the technical field of microorganisms.The application provides a bacillus australimaris strain M04 with a preservation number of CGMCC No.30262.It is proved by experiments that the bacillus australimaris strain M04 has the ability of calcium dissolution, can improve the content of effective calcium in soil, improve the soil environment, can also produce auxin to promote plant growth; in addition, the bacillus australimaris strain M04 has good cellulase activity and the characteristics of salt and alkali tolerance, can effectively promote the rotting of organic fertilizer such as straw in a high-salt environment, has a positive effect on accelerating the conversion and utilization of straw returned to the field, and has a wide application prospect in the preparation of salt and alkali tolerant multifunctional biological organic fertilizer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus spp. M04 from the South China Sea, microbial fertilizer, fermentation agent, and their applications. Background Technology

[0002] Calcium is an essential nutrient for plant growth and development. It stimulates the growth of roots and stems, while also enhancing the absorption and utilization of other nutrients, thus promoting overall plant growth and development. The availability of calcium is also crucial for ensuring the sustainable use of farmland and forest land. However, the vast majority of calcium in the soil exists in mineral form within the mineral crystal lattice, accounting for 40-90% of the total calcium. Plants have difficulty absorbing and utilizing this calcium. Furthermore, due to the long-term and excessive application of chemical fertilizers and unbalanced fertilization practices, calcium can exhibit antagonistic effects with other elements, leading to frequent calcium deficiency in plants.

[0003] Currently, calcium supplementation is mainly achieved through soil application of calcium fertilizers, including lime, calcium nitrate, calcium chloride, calcium carbonate, superphosphate, and calcium magnesium phosphate. However, the application of traditional calcium fertilizers can alter soil pH, affect the soil microenvironment, and cause soil salinization. While some microorganisms promote the absorption of minerals in the soil, few are known to promote soil calcium dissolution. Therefore, screening for a strain that promotes soil calcium dissolution is crucial for improving calcium supply to plants and promoting plant growth. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide a Bacillus strain M04 from the South China Sea that can promote soil calcium dissolution, while also having strong salt and alkali tolerance, and can promote plant growth and organic fertilizer decomposition.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a strain of Bacillus australimaris M04, which has the accession number CGMCC No. 30262.

[0007] The present invention provides a fermentation method for the Bacillus strain M04 from the South China Sea, comprising the following steps: inoculating the seed culture of Bacillus strain M04 from the South China Sea into a fermentation medium for fermentation culture to obtain a fermentation broth;

[0008] The carbon source of the fermentation medium includes xylose, and the nitrogen source includes yeast extract and / or ammonium sulfate. The pH value of the fermentation medium is 6 to 9.

[0009] The fermentation culture temperature is 20–40°C.

[0010] This invention provides a microbial fertilizer, comprising the aforementioned Bacillus subtilis strain M04 from the South China Sea and auxiliary materials.

[0011] This invention provides an application of the Bacillus subtilis strain M04 from the South China Sea, the fermented bacterial broth prepared by the fermentation method, or the bacterial fertilizer in plant cultivation.

[0012] Preferably, the plant cultivation includes promoting soil calcium dissolution and / or promoting plant growth.

[0013] Preferably, the promotion of plant growth includes promoting seed germination and / or promoting plant growth.

[0014] Preferably, the plants include food crops and / or cash crops.

[0015] The present invention provides a fermentation agent comprising the aforementioned Bacillus subtilis strain M04 from the South China Sea and excipients.

[0016] This invention provides an application of the Bacillus subtilis strain M04 from the South China Sea, the fermentation broth prepared by the fermentation method, or the fermentation agent in the composting of organic fertilizer.

[0017] Preferably, the raw materials for the organic fertilizer include agricultural waste.

[0018] Beneficial effects:

[0019] This invention provides a strain of Bacillus australimaris from the South China Sea, strain M04, with accession number CGMCC No. 30262. This strain M04 promotes soil calcium dissolution, plant growth, and organic fertilizer decomposition, and is tolerant of high salinity and alkalinity environments. Experiments show that strain M04 possesses calcium dissolution capabilities, increasing the content of available calcium in the soil and promoting plant calcium absorption and utilization. It also produces auxin (IAA), promoting seed germination and plant growth. Furthermore, strain M04 exhibits good cellulase activity and salt tolerance, effectively promoting the decomposition of organic fertilizers such as straw even in high salinity environments. This has a positive effect on accelerating the conversion and utilization of straw returned to the field. The preparation of salt- and alkali-tolerant multifunctional bio-organic fertilizer using this strain has broad application prospects. Attached Figure Description

[0020] Figure 1 The graph shows the cellulase production capacity of different strains M01, M03, M04 and M08.

[0021] Figure 2 Diagram of the calcium dissociation zone of Bacillus strain M04 from the South China Sea;

[0022] Figure 3 This is a colony morphology diagram of Bacillus strain M04 from the South China Sea.

[0023] Figure 4 Phylogenetic tree diagram of Bacillus strain M04 from the South China Sea, constructed based on the 16S rDNA sequence;

[0024] Figure 5 Figure showing the effect of different carbon sources on the growth of Bacillus strain M04 from the South China Sea.

[0025] Figure 6 Figure showing the effect of different nitrogen sources on the growth of Bacillus strain M04 from the South China Sea.

[0026] Figure 7 Figure showing the effect of different pH values ​​on the growth of Bacillus strain M04 from the South China Sea.

[0027] Figure 8 Figure showing the effect of different temperatures on the growth of Bacillus strain M04 from the South China Sea.

[0028] Figure 9 The effect of different ventilation rates on the growth of Bacillus strain M04 from the South China Sea.

[0029] Information on the preservation of biological materials

[0030] Bacillus australimaris was deposited on April 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 30262 and the strain number is M04. Detailed Implementation

[0031] This invention provides a strain of Bacillus australimaris M04, which has the accession number CGMCC No. 30262.

[0032] The *Bacillus spp.* strain M04 from the South China Sea described in this invention was isolated from greenhouse soil and deposited at the China General Microbiological Culture Collection Center on April 3, 2024. The colony morphology of *Bacillus spp.* M04 is as follows: Figure 3As shown. The colonies of *Bacillus spp.* M04 from the South China Sea on LB medium are yellow, round, opaque, with neat edges, a moist and smooth surface, and are easily picked up. The *Bacillus spp.* M04 described in this invention is a Gram-positive bacterium; it is an aerobic bacterium, capable of producing catalase (catalase-positive), unable to hydrolyze starch, capable of utilizing citrate, negative for methyl red, capable of producing gelatinase, positive for VP test, capable of producing IAA, capable of producing cellulase, and possesses calcium-solubilizing ability and salt-alkali tolerance. The 16S rDNA sequence of the *Bacillus spp.* strain M04 from the South China Sea is shown in SEQ ID NO: 1.

[0033]

[0034] The 16S rDNA sequence of strain M04 was obtained and compared with sequences in the GenBank database using BLAST analysis. A phylogenetic tree of strain M04 was constructed, as shown below. Figure 4 As shown. Based on phylogenetic tree analysis, morphological analysis, and physiological and biochemical characteristics of the strain, strain M04 was identified as Bacillus australimaris.

[0035] The present invention provides a fermentation method for the Bacillus strain M04 from the South China Sea, comprising the following steps: inoculating the seed culture of Bacillus strain M04 from the South China Sea into a fermentation medium for fermentation culture to obtain a fermentation broth;

[0036] The carbon source of the fermentation medium includes xylose, and the nitrogen source includes yeast extract and / or ammonium sulfate. The pH value of the fermentation medium is 6 to 9.

[0037] The fermentation culture temperature is 20–40°C.

[0038] In this invention, the method for preparing the seed culture of *Bacillus spp.* strain M04 from the South China Sea preferably includes the following steps: inoculating activated *Bacillus spp.* strain M04 from the South China Sea into a seed culture medium and culturing it to obtain the seed culture of *Bacillus spp.* strain M04 from the South China Sea. The culturing time is preferably 14–28 h, more preferably 24 h; the culturing temperature is preferably 26–36 °C, more preferably 28 °C. The seed culture medium is preferably LB medium. The pH of the seed culture medium is preferably 7–9, more preferably 8. The culturing is preferably aerobic. The aerobic culturing is preferably performed on a shaker. The shaking speed is preferably 140–200 rpm, more preferably 160–180 rpm.

[0039] In this invention, the fermentation medium, in addition to the basic medium, includes the following additional components: 0.1% carbon source and 1% nitrogen source by mass. The basic medium preferably includes any one of the following: TSB medium, LB medium, liquid fermentation medium, aniline blue medium, phosphorus-solubilizing fermentation medium, and cellulose enrichment medium, more preferably LB medium. The pH of the fermentation medium is more preferably 8. In an embodiment of this invention, the fermentation medium is based on LB medium and further includes the following components: 0.1% xylose by mass and 1% yeast extract by mass. This invention does not impose any special limitations on the preparation method of the fermentation medium; any method well-known in the art can be used. During fermentation, the inoculation amount of the seed culture is preferably 0.5% to 5% of the fermentation culture volume, more preferably 1%. The fermentation temperature is preferably 24 to 38°C, more preferably 36°C. The fermentation time is preferably 6 to 24 hours, more preferably 10 to 20 hours, more preferably 18 hours. Aerobic fermentation is preferred. The aerobic culture is preferably carried out in a shaker. The rotation speed of the shaker is preferably 140-180 rpm, more preferably 180 rpm.

[0040] This invention provides a microbial fertilizer, comprising the aforementioned Bacillus subtilis strain M04 from the South China Sea and auxiliary materials.

[0041] In this invention, the effective viable count of Bacillus strain M04 from the South China Sea in the microbial fertilizer is preferably not less than 1×10⁻⁴. 8 CFU / g. The microbial fertilizer is suitable for high-salt environments and / or normal-salt environments. The preferred salinity is a sodium chloride solution, with a concentration preferably 1%–12%, more preferably 5%–12%, and most preferably 10%. The soil pH for which the microbial fertilizer is suitable is preferably 6–9, more preferably 7–8, and most preferably 8. The *Bacillus spp.* strain M04 from the South China Sea has calcium-solubilizing ability, effectively increasing the content of available calcium in the soil, and also secretes auxin (IAA), which efficiently promotes plant growth. The preparation method of the microbial fertilizer preferably includes the following steps: activating and propagating the *Bacillus spp.* strain M04 from the South China Sea, and then mixing it with acceptable excipients. This invention does not specifically limit the composition of the excipients; any microbial fertilizer excipients well-known in the art can be used, such as macroelements, gravimetric elements, microelements, and components that promote soil aggregate formation and reduce soil compaction.

[0042] This invention provides an application of the Bacillus subtilis strain M04 from the South China Sea, the fermented bacterial broth prepared by the fermentation method, or the bacterial fertilizer in plant cultivation.

[0043] In this invention, the plant cultivation preferably includes promoting soil calcium dissolution and / or promoting plant growth. Promoting plant growth preferably includes promoting seed germination and / or promoting plant growth. The pH of the soil in which the plants are cultivated is preferably 6-9, more preferably 7-8, and most preferably 8. The salinity of the soil preferably includes 0.5%-1.2%, more preferably 0.7%-1.1%, and most preferably 1%. The plants preferably include food crops and / or cash crops. The food crops preferably include any one of the following: wheat, rice, corn, oats, rye, barley, millet, sorghum, and highland barley; the cash crops preferably include any one of the following: vegetable crops, fiber crops, oil crops, sugar crops, beverage crops, and medicinal crops, more preferably tomatoes.

[0044] In this invention, the preferred method for promoting soil calcium dissolution using the Bacillus spp. M04 strain from the South China Sea is to apply the Bacillus spp. M04 strain from the South China Sea into the soil.

[0045] In this invention, the Bacillus strain M04 from the South China Sea includes various life cycle morphologies, such as spore dormancy period and reproductive growth period.

[0046] In this invention, the method for promoting seed germination using Bacillus strain M04 from the South China Sea preferably includes the following steps:

[0047] Plant seeds are soaked, disinfected, and germinated sequentially to obtain pretreated seeds;

[0048] Pretreated plant seeds were treated with Bacillus subtilis strain M04 from the South China Sea to obtain bacteria-treated seeds.

[0049] Plant seeds treated with bacteria are cultured.

[0050] In this invention, the seeds are preferably plant seeds that are uniform in size, mature and plump, and free from mold. The plants preferably include food crops and / or cash crops. The food crops preferably include any one of the following: wheat, rice, corn, oats, rye, barley, millet, sorghum, and highland barley; the cash crops preferably include any one of the following: vegetable crops, fiber crops, oil crops, sugar crops, beverage crops, and medicinal crops, more preferably tomatoes. After selecting healthy seeds, they are soaked. Tomato seeds that can germinate are initially screened by observing whether they float on the water surface. This invention does not have a specific limitation on the soaking method; conventional soaking methods in the art are acceptable. The preferred soaking method in this invention is to soak the seeds in water. This invention does not have a specific limitation on the amount of water used; the water should just cover the seeds. The preferred soaking time in this invention is 30–45 minutes, more preferably 30 minutes. After soaking, this invention preferably disinfects the seeds. The disinfection method described in this invention is not particularly limited; any conventional disinfection method in the art can be used. For example, disinfection can be performed first using an ethanol-water solution, followed by disinfection using a sodium hypochlorite-water solution. The volume percentage of ethanol in the ethanol-water solution is preferably 70%–75%, more preferably 75%; the disinfection time of the ethanol-water solution is preferably 5–10 minutes, more preferably 5 minutes. The volume percentage of sodium hypochlorite in the sodium hypochlorite-water solution is preferably 0.5%–1%, more preferably 1%. The disinfection time using the sodium hypochlorite-water solution is preferably 5–10 minutes, more preferably 5 minutes. After disinfection, the disinfected seeds are preferably washed with sterile water. The washing is preferably performed 3–5 times, more preferably 5 times. After washing, the obtained seeds are preferably germinated to obtain pretreated seeds. The germination method is not particularly limited; any conventional germination method in the art can be used. The preferred germination method is to immerse the washed seeds in sterile water. The germination temperature is preferably 28–34°C, more preferably 32°C; the germination time is preferably 18–26 hours, more preferably 24 hours.

[0051] After obtaining the pretreated seeds, the present invention treats the pretreated seeds with the above-mentioned Bacillus subtilis strain M04 from the South China Sea to obtain bacteria-treated seeds.

[0052] In this invention, the preferred treatment method is to soak or spray the seeds with a suspension or fermentation broth of *Bacillus spp.* strain M04 from the South China Sea. The preferred concentration of the suspension or fermentation broth is 1 × 10⁻⁶. 6 ~1×10 8 CFU / mL, further preferably 1×10⁻⁶ 7 ~1×10 8 CFU / mL, more preferably 1×10⁻⁶8 CFU / mL. The soaking time described in this invention is preferably 20–40 min, more preferably 30 min. The soaking temperature is preferably 26–36°C, more preferably 28–32°C, and most preferably 28°C.

[0053] After obtaining the seed treated with the fungus, the present invention cultivates the seed treated with the fungus.

[0054] This invention does not specifically limit the cultivation method; any conventional cultivation method in the art can be used. The cultivation method is preferably carried out in a petri dish; the petri dish is preferably lined with sterile filter paper. The cultivation temperature is preferably 24–36°C, more preferably 28°C; the light-to-dark ratio is preferably (12–16):(12–8), more preferably 16:8; the cultivation humidity is preferably 60%–75%, more preferably 70%. The cultivation humidity of this invention is preferably achieved by adding water to gauze. During the cultivation process, it is preferable to check whether the gauze has dried out every 4 hours and add water as needed. After cultivation, sowing can be carried out using conventional sowing methods in the art. In this invention, sowing is preferably done in greenhouse pots. After sowing the seeds, this invention does not specifically limit the cultivation method for the plants; any conventional management method in the art can be used.

[0055] In this invention, the method for promoting plant growth using the Bacillus subtilis strain M04 from the South China Sea preferably includes employing the above-described method for promoting seed germination or applying the Bacillus subtilis strain M04 from the South China Sea to the plant roots. The effective viable count of the Bacillus subtilis strain M04 from the South China Sea is preferably not less than 1 × 10⁻⁶. 8 CFU / mL. When applying Bacillus subtilis strain M04 from the South China Sea to the roots of plants, it is preferred to use one or more of the following methods: splashing, dripping, dipping, drenching, and hole application; the amount of splashing, dripping, dipping, drenching, and hole application is preferably 10-100 mL / plant, more preferably 20-60 mL / plant, and even more preferably 40 mL / plant.

[0056] In this embodiment of the invention, taking tomato as an example, the effect of Bacillus subtilis strain M04 from the South China Sea on promoting plant growth is illustrated. This invention uses the aforementioned Bacillus subtilis strain M04 to soak tomato seeds, which can effectively improve the germination rate of tomato seeds and promote the growth of tomato buds and roots. After soaking seeds with Bacillus subtilis strain M04, the germination rate of tomato seeds increased by 11.72% compared to the control group, the bud length increased by 34.69% compared to the control group, and the root length increased by 21.34% compared to the control group. Furthermore, considering the salt and alkali tolerance of Bacillus subtilis strain M04, it can be used for plant cultivation in saline-alkali environments, solving the problem of low strain activity in saline soils.

[0057] The present invention provides a fermentation agent comprising the aforementioned Bacillus subtilis strain M04 from the South China Sea and excipients.

[0058] In this invention, the effective viable count of Bacillus strain M04 from the South China Sea in the fermentation agent is preferably not less than 1×10⁻⁴. 8 CFU / g. The Bacillus spp. M04 strain from the South China Sea can produce cellulase, which efficiently promotes the degradation of organic fertilizer raw materials, thereby improving the efficiency of returning organic fertilizer raw materials to the field. The preparation method of the fermentation agent preferably includes the following steps: activating and propagating the Bacillus spp. M04 strain from the South China Sea, and then mixing it with acceptable excipients. The present invention does not specifically limit the composition of the excipients; fermentation agent excipients well known in the art can be used.

[0059] This invention provides the application of the Bacillus subtilis strain M04 from the South China Sea, the fermentation broth prepared by the fermentation method, or the fermentation agent in the composting of organic fertilizer.

[0060] The fermentation agent of this invention is preferably suitable for environments including high-salt concentration environments and / or normal-salt concentration environments. The salt environment is preferably a sodium chloride solution, and the concentration of the sodium chloride solution is preferably 1%–12%, more preferably 6%–12%, and most preferably 10%. The pH of the environment suitable for the fermentation agent is preferably 6–9, more preferably 7–8, and most preferably 8. A method for composting organic fertilizer using the South China Sea Bacillus strain M04 or the fermentation liquid prepared by the fermentation method or the fermentation agent includes the following steps: mixing pulverized organic fertilizer with water and yeast powder to obtain organic fertilizer material; inoculating the South China Sea Bacillus strain M04 fermentation liquid into the organic fertilizer material for degradation. The concentration of the South China Sea Bacillus strain M04 fermentation liquid is preferably 1×10⁶–1×10⁶. 8 CFU / mL, more preferably 1×10⁻⁶ 8CFU / mL. The inoculation amount of the fermentation broth of Bacillus subtilis strain M04 from the South China Sea to the straw is preferably (5-10) mL:(5-20) g, more preferably 10 mL:5 g. This invention does not have a specific limitation on the method of straw crushing; any conventional crushing method in the art can be used. After crushing the organic fertilizer material, this invention preferably sieves it, and the material passing through the sieve is taken to obtain the crushed organic fertilizer material. The sieve aperture of this invention is preferably 20-60 mesh, more preferably 20 mesh. The mass-to-volume ratio of the crushed organic fertilizer material to water is preferably (5-20) g:(10-30) mL, more preferably 5 g:30 mL. In this invention, the mass ratio of the crushed organic fertilizer material to yeast powder is preferably (5-20):(2-5), more preferably 5:2. This invention does not have a specific limitation on the mixing method of the crushed organic fertilizer material with water and yeast powder; any conventional mixing method in the art can be used. The purpose of adding yeast powder to the crushed organic fertilizer material in this invention is to provide a nitrogen source for microorganisms and accelerate decomposition. The preferred temperature for the degradation of organic fertilizer materials in this invention is 24–38°C, more preferably 36°C. During the composting process, aerobic cultivation is preferred. This aerobic cultivation preferably includes shaking culture or composting culture. This invention does not have a specific limitation on the degradation time of the organic fertilizer materials, as long as the degradation is complete. The preferred degradation time for the organic fertilizer materials in this invention is ≥20 days, more preferably 20–35 days, and most preferably 25 days.

[0061] In this invention, the raw materials for the organic fertilizer preferably include agricultural waste, such as straw and agricultural product residues. The straw is preferably any one of the following: wheat straw, corn straw, peanut straw, rice straw, and soybean straw, more preferably wheat straw. The agricultural product residues are preferably any one of the following: fruit peels, fruit pulp, vegetable leaves, wheat bran, and rice bran.

[0062] The organic fertilizer composting method provided by this invention can significantly improve the degradation rate of organic fertilizer raw materials under high salt concentration environments. Using the organic fertilizer composting method provided by this invention, wheat straw was degraded under both 10% salt concentration and normal salt concentration environments. After 20 days of degradation, the degradation rate of wheat straw under high salt concentration reached 15.33%, an increase of 28.05% compared to the control group; the degradation rate of wheat straw under normal salt concentration reached 15.62%, an increase of 29.39% compared to the control group. This demonstrates that the Bacillus subtilis strain M04 from the South China Sea can be used for organic fertilizer composting under both high and normal salt concentration environments.

[0063] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0064] Culture medium composition and preparation instructions

[0065] LB medium: 25g LB broth powder, 1000mL distilled water, sterilize at 121℃ for 25min (for solid medium, add 20g agar to this formula).

[0066] LB medium with salt concentrations of 4%, 5%, 6%, 8%, 10%, and 12%: 40g, 50g, 60g, 80g, 100g, and 120g of sodium chloride solid were added to the LB medium, respectively.

[0067] Liquid fermentation medium: 2.5g sodium nitrate, 1.0g potassium dihydrogen phosphate, 1.0g magnesium sulfate heptahydrate, 1.0g sodium chloride, 0.5g calcium chloride, 70mL trace element solution, 20g straw, 1000mL distilled water, sterilized at 121℃ for 20min.

[0068] Trace element solution in liquid fermentation medium: ferric chloride hexahydrate: 0.16g, zinc sulfate heptahydrate: 1.5g, cobalt chloride hexahydrate: 0.16g, copper sulfate pentahydrate: 0.15g, manganese sulfate monohydrate: 1.5g, boric acid: 0.3g, sodium molybdate crystals: 0.1g, distilled water 1000mL.

[0069] Cellulose enrichment medium: 20g sodium carboxymethyl cellulose, 5g microcrystalline cellulose, 5g cellulose powder, 1g dipotassium hydrogen phosphate, 1g nitric acid, 0.2g magnesium sulfate heptahydrate, 0.1g copper chloride dihydrate, 0.02g ferric chloride, and 1000mL distilled water.

[0070] Carboxymethyl cellulose medium: 15g sodium carboxymethyl cellulose, 1g ammonium nitrate, 1g yeast extract, 0.5g magnesium sulfate heptahydrate, 1g potassium dihydrogen phosphate, 15g agar and 1000mL distilled water.

[0071] CMC-Na Congo Red Solid Medium: Sodium nitrate 1.0g, disodium hydrogen phosphate 1.2g, dipotassium hydrogen phosphate, magnesium sulfate 0.5g, potassium chloride 0.5g, yeast extract powder 0.5g, acid-hydrolyzed casein 0.5g, Congo red 0.2g, cellulose powder 5.0g, agar 15.0g, distilled water 1000mL.

[0072] NA medium: 3g beef extract, 10g peptone, 5g sodium chloride, 1000mL distilled water, sterilized at 121℃ for 20min (for solid medium, add 20g agar to this formula).

[0073] TSB medium: 30g of commercially available TSB powder (17g / L tryptone; 3g / L plant peptone; 5g / L sodium chloride; 2.5g / L dipotassium hydrogen phosphate; 2.5g / L glucose) and 1000mL distilled water, sterilized at 121℃ for 20min.

[0074] Calcium-solubilizing medium: 0.5g ammonium sulfate, 0.5g yeast extract, 0.3g magnesium sulfate, 0.03g ferrous sulfate, 0.03g manganese sulfate, 2.5g calcium carbonate, 15g agar, 10g glucose, 0.3g sodium chloride, 0.39g potassium chloride, and 1000mL distilled water.

[0075] Example 1

[0076] 1. Strain screening

[0077] (1) The site is located in Dashu Town, Quanjiao County, Chuzhou City, Anhui Province. Soil samples were collected for microbial screening.

[0078] A 10g sample of soil collected from Dashu Town, Quanjiao County, Chuzhou City, Anhui Province was inoculated into 90mL of sterile water and shaken at 28℃ and 150rpm for 30min to obtain a bacterial suspension.

[0079] Take 1 mL of bacterial suspension and serially dilute it to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 Take the gradient as 10 -4 10 -5 10 -6 100 μL of the diluted solution was spread onto carboxymethyl cellulose agar plates and incubated at 36°C for 3 days. Single colonies from the plates were picked and streaked onto LB agar plates and incubated at 36°C for an extended period. Finally, single colony strains were selected from the plates.

[0080] The preliminarily selected strains were cultured on LB solid medium plates. Mycelial cakes were made using a 5 mm diameter punch and inoculated onto CMC-Na Congo red solid medium and cultured for 3 days. The transparent hydrolysis zone of cellulose bacteria was observed, and the diameter of the transparent hydrolysis zone (D) and the colony diameter (d) of each strain were measured using the cross-cross method. The ratio was calculated, and strains with larger D / d ratios were selected. Four strains, M01, M03, M04, and M08, were screened and found to have the ability to degrade cellulose.

[0081] (2) Cellulase activity assay of strains M04, M01, M03 and M08

[0082] Cellulase activity assays can further determine the enzyme production capacity of the strains. Strains M04, M01, M03, and M08 were individually inoculated into liquid fermentation medium and cultured in a constant temperature shaking incubator at 28°C and 180 rpm for 48 hours to prepare bacterial suspensions. These suspensions were then placed in 10 mL centrifuge tubes and centrifuged at 10000 rpm for 10 minutes at 4°C. The bacterial pellets were collected, and the samples were sent to Suzhou Keming Biotechnology Co., Ltd. for cellulase activity assays.

[0083] Enzyme activity assay results as follows Figure 1 As shown.

[0084] Depend on Figure 1 It can be seen that among the screened strains, M04, M03 and M08 have strong cellulose degradation ability. The cellulase activity of strain M04 is 222.76 U / g, while the cellulase activity of strain M01 is the lowest, at 86.46 U / g.

[0085] 2. Performance determination of strain M04

[0086] (1) Determination of IAA secretion capacity

[0087] 1) Plotting the IAA standard curve

[0088] Weigh 50 mg of IAA on a 0.01 g balance, dissolve it in tertiary water in a 500 mL volumetric flask, add water to make up to the mark, mix well, and the concentration of IAA in the solution is 100 mg / L, which is the standard solution. Then, pipette 0.00 mL, 0.15 mL, 0.30 mL, 0.45 mL, 0.60 mL, 0.75 mL, 0.90 mL, 1.05 mL, 1.25 mL, 1.35 mL, and 1.50 mL of IAA standard solution into clean test tubes, add distilled water to make up to 2 mL, then add 4 mL of Sahlkawski's reaction solution, mix well, and you will get a series of IAA standard solutions with final concentrations of 0.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 7.5 μg / mL, 10.0 μg / mL, 12.5 μg / mL, 15.0 μg / mL, 17.5 μg / mL, 20.0 μg / mL, 22.5 μg / mL, and 25.0 μg / mL. Mix well and react at room temperature in the dark for 30 min, then measure the absorbance at 530 nm. A standard curve for IAA was plotted with the measured absorbance on the ordinate and the IAA concentration (mg / L) on the abscissa. The relevant data for the standard curve are shown in Table 1.

[0089] Table 1. Relevant data for the IAA standard curve.

[0090]

[0091] According to Table 1, the standard curve equation for the IAA is y = 0.1242x - 0.183, RA 2 =0.9991.

[0092] 2) IAA production assay of the strain

[0093] IAA production assay of strain M04

[0094] Strain M04 was inoculated into a solution containing 5.0 mmol / L -1 L-tryptophan was cultured in TSB medium at 28°C and 180 rpm for 48 h. The bacterial suspension was centrifuged to remove cells, and 1.0 mL of sterile filtrate was collected. Then, 2.0 mL of Salkawski's chromogenic reagent (50 mL 35% HClO4, 1 mL 0.5 mol·L⁻¹) was added. -1 FeCl3 (stored in the dark), and observed after being placed in the dark for 30 minutes.

[0095] After being placed in the dark for 30 minutes, the absorbance at 530 nm was measured. A blank culture medium was used as a control. The amount of IAA secreted was calculated using an IAA standard curve.

[0096] Substituting the measured OD=530 value of 0.131 into the IAA standard curve equation, we can obtain that the IAA secretion of strain M04 is 2.53 mg / L.

[0097] (2) Determination of salt tolerance

[0098] Strain strain M04 was inoculated into LB medium with different salt concentrations (4%, 5%, 6%, 8%, 10%, and 12%). The plates were inverted and incubated in a 28°C incubator. The culture medium was observed after 6 days of incubation. Three replicates of the strain were set up, and the salt tolerance of the strains was preliminarily compared by observing the colony growth on the plates.

[0099] The plate experiment results showed that M04 could grow normally in media with different salt concentrations (4%, 5%, 6%, 8%, 10% and 12%), while the growth of M03 and M08 was severely inhibited when the salt concentration was higher than 8%, and M01 could not grow normally when the salt concentration was higher than 8%. Therefore, M04 had the best salt tolerance.

[0100] (3) Determination of calcium solubility

[0101] The appearance of a clear zone during the culture of a strain on a calcium-solubilizing solid medium indicates that the strain has the ability to dissolve calcium; the larger the clear zone, the stronger the calcium-solubilizing ability.

[0102] Qualitative detection: Purified strains M01, M03, M04 and M08 were picked up with an inoculation loop and inoculated into LB medium. They were then cultured at 28℃ and 180 r / min for 24 h with shaking to obtain the seed culture for the experiment.

[0103] Using an inoculation loop, the seed culture was inoculated into the center of a plate of calcium-solidifying solid medium. After drying, the plate was inverted and incubated at 28°C. After 6 days of incubation, the calcium-solidifying solid medium was observed, and the calcium-solidifying rings on the plates were measured. Three replicates of the strain were set up. The calcium-solidifying ability of the strains was preliminarily compared by measuring the diameter of the calcium-solidifying rings on the plates. The results are shown below. Figure 2 .

[0104] Depend on Figure 2 It can be seen that strain M04 can produce a calcium-dissolving zone, and the ratio of the diameter of the transparent zone to the diameter of the colony is 2.09±0.14. This shows that strain M04 has a strong calcium-dissolving ability, while strains M01, M03 and M08 cannot produce a calcium-dissolving zone and do not have the ability to dissolve calcium.

[0105] 3. Strain identification

[0106] (1) Observation of colony morphology

[0107] Strawberry strain M04 was streaked onto LB agar and incubated at 28°C for 48 hours. Single colony morphology was observed and recorded. Figure 3 As shown.

[0108] Depend on Figure 3 It can be seen that the colonies of strain M04 are yellow, round, opaque, with neat edges, rough and dry surface, and easy to pick up.

[0109] (2) Gram staining

[0110] After being stained with Gram stain and observed under an oil immersion microscope, strain M04 was found to be a Gram-positive bacterium.

[0111] (3) Physiological and biochemical characteristics

[0112] The physiological and biochemical characteristics of strain M04 were determined. The results are shown in Table 2.

[0113] Table 2 Physiological and Biochemical Characteristics of M04

[0114] project result project result Starch hydrolysis - Citrate utilization test + VP test + Gelatin liquefaction test + Methyl red test - Gram staining + Catalase test + Aerobic test +

[0115] Note: + indicates a positive reaction, - indicates a negative reaction.

[0116] As shown in Table 2, strain M04 is an aerobic bacterium that can produce catalase (catalase positive), cannot hydrolyze starch, can utilize citrate, is negative for methyl red, can produce gelatinase, and is positive for VP test.

[0117] (4) Molecular identification

[0118] DNA was extracted from strain M04, and after PCR, it was sequenced by Shanghai Meiji Biotechnology Co., Ltd. Based on the obtained 16S rDNA sequence results, alignment was performed in the GenBank database, and homologous sequences were searched using BLAST. A phylogenetic tree was constructed using the Neighbour-Joining method with MEGA-X software. Figure 4 As shown.

[0119] Based on morphological analysis and physiological and biochemical characteristics, this strain was identified as *Bacillus australimaris*. *Bacillus australimaris* M04 was deposited on April 3, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 30262. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0120] Example 2

[0121] 1. Effects of different carbon sources (maltose, sucrose, lactose, xylose, glucose, mannitol) on the growth of the strain

[0122] Strain strain M04 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm to prepare a seed culture.

[0123] LB broth was prepared separately, and different carbon sources (0.1% by weight) were added to the LB broth. 0.5 mL of seed culture was inoculated into each LB broth, and the mixture was incubated at 28°C and 180 rpm for 48 h. Uninoculated LB broth served as a control. Each treatment was replicated in triplicate. The fermentation broth was then analyzed at OD0.05. 600 OD was measured at a wavelength of nm. 600 A higher value indicates a higher bacterial concentration and better growth.

[0124] The growth of the strains cultured for 24 hours under different carbon source conditions was detected as follows: Figure 5 As shown.

[0125] The results are as follows Figure 5 As shown: when xylose is used as the carbon source, the absorbance of strain M04 reaches its maximum, OD 600 It is 2.490.

[0126] 2. Effects of different nitrogen sources (urea, yeast extract, glutamic acid, potassium nitrate, peptone, ammonium sulfate) on the growth of the strain

[0127] Strain strain M04 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm to prepare a seed culture.

[0128] LB broth was prepared separately, and different amounts of nitrogen source (1% by weight) were added to it. 0.5 mL of seed culture was inoculated into each LB broth, and the mixture was incubated at 28°C and 180 rpm for 48 h. Uninoculated LB broth served as a control. Each treatment was replicated in triplicate. The fermentation broth was then analyzed at OD0.05. 600 Ultraviolet absorbance was measured at a wavelength of nm.

[0129] The growth of the strains cultured for 24 hours under different nitrogen source conditions was detected as follows: Figure 6 As shown.

[0130] The results are as follows Figure 6 As shown: When yeast extract is used as the nitrogen source, strain M04 reaches its maximum absorbance and OD. 600 It is 2.463.

[0131] 3. Effects of different pH values ​​on bacterial growth

[0132] Strain strain M04 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm to prepare a seed culture.

[0133] LB culture medium was prepared separately, and its pH was adjusted to 4, 5, 6, 7, 8, 9, and 10. 0.5 mL of seed culture was added to 50 mL of LB culture medium, and each culture was incubated at 28℃ and 180 rpm for 24 h in a shaker. Uninoculated LB culture medium was used as a reference. Each treatment was performed in triplicate. The OD of the bacterial culture was measured. 600 value.

[0134] The growth of the strains cultured for 24 hours under different pH conditions was detected as follows: Figure 7 As shown.

[0135] The results are as follows Figure 7 As shown: the absorbance of strain M04 reached its maximum at pH 8, and the OD... 600 It is 2.569.

[0136] 5. Effects of different temperatures on strain growth

[0137] Strain strain M04 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm to prepare a seed culture.

[0138] A separate 50 mL LB culture medium was prepared, and 0.5 mL of seed culture was inoculated into it. The cultures were then incubated for 24 h at 20℃, 24℃, 28℃, 32℃, 36℃, and 40℃ using a shaker at 180 rpm. Uninoculated LB culture medium was used as a control. Each treatment was replicated in triplicate. The OD of the bacterial culture was measured. 600 value.

[0139] The growth of the strains cultured for 24 hours under different temperature conditions was detected as follows: Figure 8 As shown.

[0140] The results are as follows Figure 8 As shown: the absorbance of strain M04 reaches its maximum at a temperature of 32℃, and the OD... 600 It is 2.740.

[0141] 6. Effects of different ventilation rates on bacterial growth

[0142] Strain M15C3 was inoculated into LB liquid medium and cultured overnight at 28°C and 180 rpm to prepare a seed culture.

[0143] In a separate 250ml Erlenmeyer flask, 25, 50, 75, 100, 125, and 150mL of LB culture medium were added, respectively, and 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50mL of seed culture were inoculated into the flasks. The flasks were incubated at 28℃ and 180rpm for 24h, with uninoculated LB culture medium serving as a reference. Each treatment was performed in triplicate. The OD of the bacterial culture was measured. 600 value.

[0144] The growth of the strains cultured for 24 hours under different aeration conditions was detected as follows: Figure 9 As shown.

[0145] The results are as follows Figure 9 As shown: the absorbance of strain M15C3 reached its maximum when the aeration rate was 50 mL, and the OD... 600 It is 2.464.

[0146] Example 3

[0147] 1. Dynamic monitoring experiment on straw decomposition promotion using strain M04 as an inoculant

[0148] Preparation of M04 bacterial culture: Inoculate M04 into LB culture medium and incubate in a constant temperature shaking incubator at 28℃ and 180r / min for 18h. Remove the cultured bacterial culture and dilute it with sterile water to a concentration of 1×10⁻⁶. 8 CFU·mL -1 .

[0149] Normal salt environment test procedure: Weigh 5g of crushed wheat straw powder (passed through a 20-mesh sieve) into a 250mL Erlenmeyer flask, add 30mL of water, 2g of yeast powder, and 10mL of a 10% salt concentration. 8 CFU·mL -1 The bacterial culture was cultured in a constant temperature shaking incubator at 28℃ and 160 rpm for approximately 20 days. On day 20 of culture, the Erlenmeyer flasks were removed, and the straw in the flasks was filtered through a funnel made of three layers of gauze. The filtrate was discarded, and the straw on the gauze was collected. The filtered straw on the gauze was then dried in an oven at 80℃ until constant weight. Sterile water was used as a control, and all other steps were the same. Each treatment was repeated in triplicate.

[0150] High-salt environment test procedure: The test procedure is the same as the normal salt environment test procedure described above, except that the water is replaced with a 10% sodium chloride solution.

[0151] The formula for straw decomposition rate uses the weight loss rate method, and the formula is as follows.

[0152] Straw degradation rate formula

[0153]

[0154] In the formula: W0 represents the dry weight of wheat straw in the liquid culture medium before inoculation, and W1 represents the dry weight of wheat straw after the culture is completed.

[0155] The dynamic detection results of the degradation of wheat straw by strain M04 as a microbial agent are shown in Table 3.

[0156] Table 3. Results of straw degradation dynamics detection by strain M04

[0157]

[0158]

[0159] Note: * indicates significant difference (P<0.05) in the table.

[0160] Table 3 shows that after 20 days of degradation, the M04 bacterial solution achieved a decomposition rate of 15.62% for wheat straw under normal salt concentration, which was 29.39% higher than the control group. Under high salt concentration, the decomposition rate of wheat straw treated with the M04 bacterial solution was 15.33%, which was not significantly different from the decomposition rate under normal salt concentration, but was 28.05% higher than the control group. This indicates that strain M04 has good salt tolerance and can still effectively decompose organic fertilizer materials such as straw under a high salt environment of 10%.

[0161] 2. Growth-promoting test of strain M04 as an inoculant on tomatoes

[0162] Seeds used in the experiment: Shanghai Hezuo 903 tomato

[0163] Preparation of M04 bacterial culture: Inoculate M04 into LB culture medium and incubate in a constant temperature shaking incubator at 28℃ and 180r / min for 18h. Remove the cultured bacterial culture and dilute it with sterile water to a concentration of 1×10⁻⁶. 8 CFU / mL.

[0164] After selecting uniformly sized, mature, plump, and mold-free tomato seeds, soak them.

[0165] Soaking seeds: Pour an appropriate amount of tomato seeds into a small beaker, add pure water to the beaker (enough to cover the seeds), soak for 30 minutes, and remove the seeds that float to the top.

[0166] Disinfection: Disinfect the surface with 75% alcohol for 5 minutes, soak in 1% sodium hypochlorite aqueous solution for 5 minutes, and then rinse 5 times with sterile water.

[0167] Germination: Soak the cleaned seeds in a small beaker containing sterile water and incubate them in a 28°C incubator for 24 hours.

[0168] Bacterial solution soaking: Soak the germinated seeds in a solution with a bacterial activity of 1×10⁻⁶. 8 The bacterial solution with CFU / mL was soaked at 28°C for 12 hours.

[0169] Tomato seeds were soaked, disinfected, germinated, and soaked in bacterial solution before being evenly spread in sterile filter paper petri dishes. Sterile water soaking served as a control. Each group had three replicates. Each petri dish contained 55 seeds. The dishes were placed in a light incubator with conditions of 28℃, a light-to-dark ratio of 16:8, and 70% humidity. During cultivation, the filter paper was checked every 2 hours to ensure it was not dry, and water was added as needed. After 5 days of cultivation, germination rate, shoot length, and root length were observed.

[0170] The effects of inoculant M04 on tomato germination rate, shoot length, and root length are shown in Table 4.

[0171] Table 4. Effects of inoculant M04 on rice germination rate, shoot length, and root length.

[0172]

[0173]

[0174] The results are shown in Table 4. Strain M04 promoted plant growth. In this experiment, after inoculation, the germination rate, shoot length, and root length of tomato plants were all improved. Specifically, the germination rate, shoot length, and root length increased by 11.72%, 34.69%, and 21.34% respectively compared to the control treatment. Strain M04 can promote plant growth.

[0175] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis from the South China Sea ( Bacillus australimaris strain M04, characterized in that, The preservation number of the Bacillus strain M04 from the South China Sea is CGMCC No. 30262.

2. A fermentation method for the Bacillus strain M04 from the South China Sea as described in claim 1, characterized in that, Includes the following steps: The seed culture of Bacillus strain M04 from the South China Sea was inoculated into a fermentation medium for fermentation culture to obtain the fermentation broth; The carbon source of the fermentation medium includes xylose, and the nitrogen source includes yeast extract and / or ammonium sulfate. The pH value of the fermentation medium is 6 to 9. The fermentation culture temperature is 20–40 °C.

3. A microbial fertilizer, characterized in that, Includes the Bacillus subtilis strain M04 from the South China Sea as described in claim 1 and excipients.

4. The application of the Bacillus subtilis strain M04 from the South China Sea as described in claim 1, the fermented bacterial liquid prepared by the fermentation method described in claim 2, or the bacterial fertilizer described in claim 3 in tomato cultivation; The tomato cultivation is intended to promote soil calcium dissolution and / or promote tomato growth.

5. The application according to claim 4, characterized in that, Promoting tomato growth includes promoting seed germination.

6. A fermenting agent, characterized in that, Includes the Bacillus subtilis strain M04 from the South China Sea as described in claim 1 and excipients.

7. The application of the Bacillus spp. M04 from the South China Sea as described in claim 1, the fermentation broth prepared by the fermentation method described in claim 2, or the fermentation agent described in claim 6 in the composting of wheat straw.

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