Bacillus licheniformis mutant strain for promoting crop growth and relieving saline-alkali stress and application thereof

Abstract

The present application discloses a bacillus licheniformis mutant strain for promoting crop growth and relieving saline-alkali stress and application thereof. The present application provides a bacillus licheniformis or its offspring, the bacillus licheniformis being bacillus licheniformis (DD1-33) with a registration number of CGMCC No. 38141 in China General Microbiological Culture Collection Center. The bacillus licheniformis (DD1-33) has obvious effects in relieving plant saline-alkali stress and promoting plant growth, and can be used as a microbial organic fertilizer to improve the saline-alkali adaptability of crops. Bacillus licheniformis )DD1-33, with a registration number of CGMCC No. 38141 in China General Microbiological Culture Collection Center. The bacillus licheniformis (DD1-33) has obvious effects in relieving plant saline-alkali stress and promoting plant growth, and can be used as a microbial organic fertilizer to improve the saline-alkali adaptability of crops. Bacillus licheniformis )DD1-33 has obvious effects in relieving plant saline-alkali stress and promoting plant growth, and can be used as a microbial organic fertilizer to improve the saline-alkali adaptability of crops.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a Bacillus licheniformis mutant strain for promoting crop growth and mitigating salt-alkali stress, and its application. Background Technology

[0002] Bacillus licheniformis ( Bacillus licheniformis Bacillus licheniformis is a Gram-positive bacterium widely distributed in nature. In recent years, research on Bacillus licheniformis has become increasingly extensive, encompassing numerous fields such as antibiotics, traditional fermentation, biofertilizers, biopesticides, modern food industry, and synthetic biology (Chen Yukun. Adaptive Evolution and High-Throughput Screening of Acid-Producing α-Amylase-Generating Bacillus licheniformis [D]. Jiangnan University, 2021.). It can produce a variety of enzymes, such as amylase, β-mannanase, pectinase, pentosanase, rennet, and chitinase, and its products have high biosafety, making it highly favored in the food processing and fermentation industries. Bacillus licheniformis is also an important germplasm resource for microbial fertilizers. It can secrete plant hormones such as IAA, cytosine, and gibberellin, which participate in regulating plant growth and enhancing crop resistance to abiotic stress conditions. Studies have found that under drought stress conditions, Bacillus licheniformis FMC001 can significantly promote the development of maize roots and stems (Akhtar SS, Amby DB, Hegelund JN, Fimognari L, Großkinsky DK, Westergaard JC, Müller R, Moelbak L, Liu F, Roitsch T). Bacillus licheniformis FMCH001 increases water useefficiency via growth stimulation in both normal and drought conditions [J]. Front Plant Sci, 2020, 11: 297.).

[0003] However, existing technologies for Bacillus licheniformis still have certain limitations in promoting crop growth and mitigating salt-alkali stress. For example, the growth-promoting function and stress resistance of Bacillus licheniformis in its natural state are limited, failing to fully meet the needs of modern agriculture to improve crop yield and quality in harsh environments such as saline-alkali land. Under salt-alkali stress, crop growth is often severely inhibited, and yields drop significantly. Therefore, developing Bacillus licheniformis strains with stronger growth-promoting and salt-alkali resistance capabilities is of significant practical importance. Summary of the Invention

[0004] The present invention aims to address the shortcomings of existing Bacillus licheniformis strains in promoting crop growth and mitigating salt and alkali stress, and to provide a Bacillus licheniformis mutant strain with excellent growth-promoting properties and stronger salt and alkali tolerance, as well as its applications.

[0005] In a first aspect, the present invention claims protection for a Bacillus licheniformis or its descendants.

[0006] The Bacillus licheniformis claimed in this invention is Bacillus licheniformis ( Bacillus licheniformis DD1-33, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 38141. Bacillus licheniformis ( Bacillus licheniformis DD1-33 is a high-performance mutant strain obtained by space mutagenesis of the original strain DY.

[0007] Secondly, the present invention claims protection for compositions containing Bacillus licheniformis or its progeny as described in the first aspect above.

[0008] Further, the composition may be a culture, which is a substance obtained by culturing Bacillus licheniformis or its progeny as described in the first aspect above in a microbial culture medium (all substances in the culture container, i.e., fermentation products, such as fermentation broth containing Bacillus licheniformis or its progeny and substances secreted into the liquid culture medium, or solid fermentation product containing Bacillus licheniformis or its progeny and substances secreted into the solid culture medium).

[0009] The microbial culture medium may be a bacterial culture medium. The bacterial culture medium may be a solid culture medium or a liquid culture medium.

[0010] In the above-mentioned culture, the substances include Bacillus licheniformis or its progeny (the bacterial cells themselves) and / or its metabolites as described in the first aspect above.

[0011] The term "metabolite" refers to the primary and / or secondary metabolites produced during microbial metabolism. Primary metabolism refers to the process by which microorganisms absorb various nutrients from the external environment and, through catabolism and anabolism, generate substances and energy to sustain life activities. The products of primary metabolism are called primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids, and various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, and lipids. Secondary metabolism refers to the process by which microorganisms, at a certain growth stage, use primary metabolites as precursors to synthesize substances that have no clearly defined function for their life activities. The products of secondary metabolism are called secondary metabolites, and are mostly compounds with relatively complex molecular structures. Based on their functions, they can be classified into types such as antibiotics, hormones, alkaloids, and toxins.

[0012] The term "culture" refers to any liquid or solid culture medium that has grown a microbial community after artificial inoculation and cultivation. It is the product obtained by growing and / or amplifying microorganisms; it can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites, or other components produced during the cultivation process. The term "culture" also includes passaged cultures obtained by subculturing microorganisms; these can be cultures of a single generation or mixtures of several generations.

[0013] Furthermore, the composition may be a microbial agent, a microecological preparation, or a bio-fertilizer.

[0014] In the aforementioned microbial agents, microecological preparations, or biofertilizers, the active ingredients may be *Bacillus licheniformis* or its descendants, metabolites of *Bacillus licheniformis* or its descendants, and / or cultures of *Bacillus licheniformis* or its descendants, as described in the first aspect above. The active ingredients may also contain other biological and / or non-biological components. Other active ingredients in the microbial agents, microecological preparations, or biofertilizers can be determined by those skilled in the art based on the desired effects.

[0015] In addition to the active ingredients, the aforementioned microbial agents, microecological preparations, or biofertilizers may also contain a carrier. The carrier may be a biologically inert carrier commonly used in the pesticide field. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, plant material, or polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of wheat flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane.

[0016] In the above-mentioned microbial agents, microecological preparations, or bio-fertilizers, the dosage form of the microbial agents, microecological preparations, or bio-fertilizers can be various, such as liquid, emulsion, suspension, powder, granules, wettable powder, or water-dispersible granules.

[0017] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the microbial agents, microecological preparations, or bio-fertilizers.

[0018] In the bacterial agent, microecological preparation, or bio-fertilizer, the Bacillus licheniformis or its descendants and / or the metabolites of the Bacillus licheniformis or its descendants may be present in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0019] In this document, the metabolites of *Bacillus licheniformis* or its progeny can be obtained from the fermentation broth of *Bacillus licheniformis* or its progeny. The metabolites of *Bacillus licheniformis* or its progeny can be either sterile metabolites of *Bacillus licheniformis* or its progeny, or microbial metabolites of *Bacillus licheniformis* or its progeny. Specifically, the sterile metabolites of *Bacillus licheniformis* or its progeny (sterile fermentation filtrate) can be prepared as follows: *Bacillus licheniformis* or its progeny are cultured in a liquid culture medium, and the *Bacillus licheniformis* or its progeny are removed from the liquid culture (fermentation broth) by filtration, thus obtaining the sterile metabolites of *Bacillus licheniformis* or its progeny. Specifically, the microbial metabolites of *Bacillus licheniformis* or its progeny can be prepared as follows: *Bacillus licheniformis* or its progeny are cultured in a liquid fermentation medium, and the fermentation broth—containing *Bacillus licheniformis* or its progeny and substances secreted into the liquid culture medium—is collected; this fermentation broth is the microbial metabolites of *Bacillus licheniformis* or its progeny.

[0020] Furthermore, the composition may have at least one of the following properties: A1) Amylase production A2) Produces cellulase; A3) Phosphorus solubilization; A4) Potassium dissolution; A5) Relieves salt and alkali stress in plants; A6) Promotes plant growth; A7) Promotes increased plant height; A8) Promotes an increase in the dry weight of plant roots; A9) Promotes increased stem diameter in plants; A10) promotes an increase in plant root volume.

[0021] Thirdly, the present invention claims the use of Bacillus licheniformis or its descendants as described in the first aspect above, or the composition described in the second aspect above, wherein the use is any of the following: B1) Application in the production of amylase or in the preparation of products for the production of amylase; B2) Application in the production of cellulase or in the preparation of products for the production of cellulase; B3) Applications in phosphorus solubilization or in the preparation of products for phosphorus solubilization; B4) Applications in potassium solubilization or in the preparation of products for potassium solubilization; B5) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; B6) Use in promoting plant growth or in the preparation of products for promoting plant growth; B7) Application in promoting plant height increase or in the preparation of products for promoting plant height increase; B8) Application in promoting an increase in the dry weight of plant roots or in the preparation of products for promoting an increase in the dry weight of plant roots; B9) Application in promoting the increase of plant stem diameter or in the preparation of products for promoting the increase of plant stem diameter; B10) Application in promoting the increase of plant root volume or in the preparation of products for promoting the increase of plant root volume.

[0022] In some embodiments of the present invention, promoting plant growth refers to promoting plant growth under salt-alkali stress conditions. Promoting increased plant height refers to promoting increased plant height under salt-alkali stress conditions. Promoting increased plant root dry weight refers to promoting increased plant root dry weight under salt-alkali stress conditions. Promoting increased plant stem diameter refers to promoting increased plant stem diameter under salt-alkali stress conditions. Promoting increased plant root volume refers to promoting increased plant root volume under salt-alkali stress conditions.

[0023] Fourthly, the present invention claims a method for alleviating salt and alkali stress in plants.

[0024] The method for alleviating salt-alkali stress in plants claimed in this invention comprises: applying Bacillus licheniformis or its progeny, as described in the first aspect above, or the composition described in the second aspect above, to the plant and / or its cultivation substrate to alleviate the salt-alkali stress in the plant.

[0025] Fifthly, the present invention claims a method for promoting plant growth.

[0026] The method for promoting plant growth claimed in this invention may include: applying Bacillus licheniformis or its progeny, as described in the first aspect above, or the composition described in the second aspect above, to a plant and / or its cultivation substrate to promote the growth of the plant.

[0027] In some embodiments of the present invention, the promotion of plant growth refers to promoting plant growth under salt-alkali stress conditions.

[0028] In both methods described above, the *Bacillus licheniformis* or its progeny, or the composition thereof, can be applied to plant seeds, seedlings, roots, rhizosphere soil, or cultivation substrates. The application methods can specifically include root irrigation, root dipping, seed soaking, seed coating, soil mixing, hole application, fertigation, drip irrigation, etc. The application time of the *Bacillus licheniformis* or its progeny, or the composition thereof, can specifically be before sowing, at sowing time, during transplanting, or at the final planting time. In some embodiments of the present invention, specifically, the bacterial solution (OD) of the *Bacillus licheniformis* is used before sowing. 600 Soak the seeds for 30 minutes (approximately 0.7 g / L).

[0029] In the aforementioned aspects, the promotion of plant growth can specifically be manifested in all or part of the following: promoting increased plant height, promoting increased plant root dry weight, promoting increased plant stem diameter, and promoting increased plant root volume.

[0030] In the aforementioned aspects, the salt-alkali stress refers to salt stress and / or alkali stress. In an embodiment of the present invention, the salt-alkali stress is simulated by mixing soil with sodium chloride, sodium carbonate, and sodium bicarbonate. Specifically, 800 mL of alkaline salt solution (formulation: 120 mM sodium chloride, 20 mM sodium carbonate, and 80 mM sodium bicarbonate) is added to every 1 kg of substrate soil.

[0031] In the aforementioned relevant aspects, the plant may be any of the following: C1) Angiosperms; C2) Monocotyledons; C3) Plants of the order Poales; C4) Gramineae plants; C5) Plants of the genus *Zea*; C6) Corn.

[0032] The Bacillus licheniformis provided by this invention ( Bacillus licheniformis DD1-33 is a high-performance mutant strain obtained by space mutagenesis of the original bacterium DY. Testing showed that this mutant strain possesses the ability to produce amylase, protease, cellulase, and to solubilize potassium and phosphorus. Pot experiments on maize showed that, under saline-alkali stress, compared with the group treated with the fermentation broth of the original bacterium DY, the experimental group treated with the DD1-33 mutant strain exhibited significantly improved plant height, root dry weight, stem diameter, and root volume growth indicators. This indicates that the DD1-33 mutant strain plays an important role in enhancing the tolerance of maize plants to saline-alkali soil environments and promoting maize seedling growth. This demonstrates that the Bacillus licheniformis (B. licheniformis) provided in this invention… Bacillus licheniformis DD1-33 can be used as a microbial organic fertilizer to improve soil fertility, alleviate crop saline-alkali stress, and enhance crop adaptability to saline-alkali environments. This invention is of great significance in solving the current problems of saline-alkali land in agricultural production and provides a new solution for the sustainable development of agriculture in saline-alkali land.

[0033] Preservation Instructions 1 Suggested classification and nomenclature: Bacillus licheniformis ( Bacillus licheniformis ); Reference biological material (strain): DD1-33; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: March 24, 2026; Registered with the China National Collection Center (CGMCC) No. 38141.

[0034] Preservation Instructions 2 Suggested classification and nomenclature: Bacillus licheniformis ( Bacillus licheniformis ); Referenced biological material (strain): DY; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: March 24, 2026; Registered with the China National Collection Center (CGMCC) No. 38142. Attached Figure Description

[0035] Figure 1 A phylogenetic tree for strain DD1-33 was constructed based on the 16S rRNA gene sequence.

[0036] Figure 2 The first image shows the screening results of strain DD1-33. The left image shows the verification results of the amylase mutant strain (DD1-33 is in the 3 green circles), and the right image shows the purification plate of DD1-33.

[0037] Figure 3 The first image shows the screening results for strain DD1-33. The top image shows the results of the phosphorus solubilization function test (DD1-33 is in the two red circles), and the bottom image shows the results of the cellulase production capacity test (DD1-33 is in the two yellow circles).

[0038] Figure 4 To quantitatively detect the amylase activity of the mutant strain and the original strain DY, the differences in amylase activity between the two strains were visually presented. Blank represents starch medium without any strain inoculated, DY represents starch medium inoculated with the original strain DY, DD1-33 represents starch medium inoculated with the mutant strain DD1-33, and DBD-1 represents starch medium inoculated with the mutant strain DBD-1. ns indicates no significant difference, **** indicates extremely significant difference, P < 0.0001.

[0039] Figure 5 To illustrate the growth of maize seedlings in each treatment group and compare their growth under different treatments. 0%: Salt-free control group; CK0: Salt-alkali treatment control group; DY: Original strain DY treatment group under salt-alkali conditions; DD1-33: Mutant strain DD1-33 treatment group under salt-alkali conditions.

[0040] Figure 6 The results of a pot experiment on maize under saline-alkali soil conditions are shown. In the figures, A represents the root dry weight of maize seedlings in each treatment group; B represents the root volume of maize seedlings in each treatment group; C represents the stem diameter of maize seedlings in each treatment group; and D represents the plant height of maize seedlings in each treatment group. 0%: No saline-alkali control group; CK0: Saline-alkali treatment control group; DY: Original strain DY treatment group under saline-alkali conditions; DD1-33: DD1-33 mutant strain treatment group under saline-alkali conditions. ns indicates no significant difference; * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01); *** indicates extremely significant difference (P<0.001); **** indicates extremely significant difference (P<0.0001). Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] Example 1, Bacillus licheniformis ( Bacillus licheniformis Acquisition and identification of DD1-33 I. Obtaining strain DY Severely saline-alkali soil was collected from Yuli County, Xinjiang, and transported back to the laboratory at 4°C using an ice box. 1g of the soil was resuspended in 10mL of sterile water for serial dilution. 100μL of each dilution was spread onto TSA plates and incubated upside down at 30°C for one week. Based on physiological morphological characteristics, single colonies were picked with a bamboo stick and inoculated onto plates for purification. After confirming pure culture, the culture was transferred to slant culture for short-term storage at 4°C, and then transferred to 20% glycerol tubes for long-term storage at -80°C. Following extensive screening and validation, a strain DY was discovered that can promote crop growth and alleviate saline-alkali stress. This strain was identified as *Bacillus licheniformis* (B. licheniformis). Bacillus licheniformis The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2026, with the registration number CGMCC No. 38142. Hereinafter referred to as strain DY or original strain DY.

[0044] II. Bacillus licheniformis ( Bacillus licheniformis Screening and identification of DD1-33 In September 2024, strain DY was carried aboard the microbial breeding satellite "Shijian-19" to complete a 14-day space mutagenesis experiment. After screening for functional mutant strains, one of the mutant strains was obtained and named DD1-33.

[0045] 1. Colony morphology characteristics The colonies of strain DD1-33 are opaque, dark brown, and have a rough surface; they are Gram-positive, with oval spores and non-swollen sporangia.

[0046] 2. Physiological and biochemical characteristics The physicochemical characteristics of strain DD1-33 were detected using API 50CH reagent strips. Positive results were found for glucose, fructose, mannose, mannitol, L-arabinose, cellobiose, maltose, sucrose, trehalose, ribose, sorbitol, α-methyl-D-glucoside, N-acetylglucosamine, amygdalin, arbutin, aesculin, salinomyol, and D-merinobisaccharide. Negative results included D-arabinose, D-xylose, L-xylose, sorbitol, rhamnose, lactose, melibiose, inulin, and xylitol. Overall, the results were consistent with the metabolic characteristics of Bacillus spp.

[0047] 3. Molecular identification DNA was extracted from strain DD1-33, and a draft sequence was obtained by the company. The extracted 16S rRNA gene (containing the nucleotide sequence shown in SEQ ID NO: 1) was then used to construct a phylogenetic tree. Figure 1 The results showed that strain DD1-33 was similar to the Bacillus licheniformis type strain. Bacillus licheniformis ATCC 14580 clusters within the same evolutionary branch, indicating a close phylogenetic relationship, suggesting that strain DD1-33 belongs to the Bacillus licheniformis group. The obtained genome size is 4.18 M, with a G+C content of 46.3%, placing it within the Bacillus genus (…). Bacillus The G+C% range was determined; ANI values ​​of the strains were analyzed using the JSpeciesWS website (http: / / jspecies.ribohost.com / jspeciesws / #home) and compared with known species. Bacillus licheniformis ATCC 14580 T The ANI value was 99.8%; analysis using GGDC (Genome-to-Genome Distance Calculator 2.1, http: / / ggdc.dsmz.de / ggdc.php#) showed that this strain was similar to known species. Bacillus licheniformis The DDH value for genome sequence comparison was 98.1%. The DDH (DNA-DNA hybridization) value among members of the same species was ≥70%, and the ANI value was ≥95%; therefore, at the molecular biological level, this strain can be identified as belonging to *Bacillus licheniformis*. Bacillus licheniformis ).

[0048] Based on the above results, strain DD1-33 can be identified as Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 24, 2026, with the registration number CGMCC No. 38141. Hereinafter referred to as strain DD1-33 or mutant strain DD1-33.

[0049] Example 2: Evaluation of the growth-promoting function of the space-induced mutant strain of the original bacterium DY Test strains: Space-induced mutant strains derived from strain DY, including strain DD1-33.

[0050] 1. Qualitative evaluation of the growth-promoting function of the strain (1) Preparation of culture medium Inorganic phosphorus culture medium: 10g glucose, 0.2g potassium chloride, 0.5g ammonium sulfate, 0.2g sodium chloride, 5.0g calcium phosphate, 0.003g ferrous sulfate, 0.03g magnesium sulfate heptahydrate, 0.03g manganese sulfate, 0.5g yeast extract, 20g agar, 1000mL distilled water. Adjust pH to 6.8-7.0 and sterilize at 121℃ for 30 minutes.

[0051] Potassium-solubilizing medium: 5g sucrose, 0.5g magnesium sulfate heptahydrate, 0.005g ferric chloride, 0.1g calcium carbonate, 2g potassium feldspar, 1.2% agar, 1000mL distilled water.

[0052] Protease detection medium: 10.0 g peptone, 5.0 g sodium chloride, 0.1 g calcium chloride, 10.0 mL skim milk (add milk before pouring the plates, when the medium is not hot to the touch), adjust the pH to 7.2-7.4, add 1.2% agar, and sterilize at 115℃ for 30 minutes.

[0053] Amylase identification medium: 1g soluble starch, 5g peptone, 5g glucose, 5g sodium chloride, 5g beef extract, 1.2% agar, 1000 mL distilled water, sterilized at 115℃ for 30 minutes.

[0054] Sodium carboxymethyl cellulose medium: 10.0g peptone, 10g yeast extract, 10g sodium carboxymethyl cellulose, 1g sodium dihydrogen phosphate, 15g sodium chloride, 1.2% agar, 1000mL distilled water, sterilized at 115℃ for 30 minutes.

[0055] (2) Preparation and inoculation of bacterial suspension The bacterial growth on the slant culture medium was washed three times with 1 ml of sterile water to obtain a bacterial suspension, which was then diluted with 9 ml of sterile water to obtain a concentration of 10. -1The bacterial suspension was diluted with sterile water in a 10-fold volume gradient to obtain 10... -4 A dilution of the concentration. Take 10... -3 10 -4 100 μL suspension of each concentration was spread and inoculated onto various functional culture medium plates prepared in step (1). Three replicates were set for each concentration. The plates were incubated upside down in a 30℃ constant temperature incubator for 3-5 days to observe the growth of strain DD1-33.

[0056] (3) Result judgment If colonies on a potassium-solubilizing medium plate appear as oil droplets, it indicates that the strain has potassium-solubilizing ability. If colonies on an inorganic phosphorus medium plate produce a clear zone around them due to phosphorus solubilization, it indicates that the strain has phosphorus-solubilizing ability. If carboxymethyl cellulose sodium medium is stained with sufficient 1 g / L Congo red solution for 1 h, and then rinsed with 1 mol / L NaCl solution, and a clear zone appears around the colonies, it indicates that the strain can secrete cellulase. If a clear zone appears around the colonies when Gram's iodine solution is used to treat amylase identification medium, it indicates that the strain can secrete amylase. If a clear zone appears around the colonies on a protease detection medium, it indicates that the strain has protease production ability.

[0057] Figure 2 The image shows partial screening results for strain DD1-33. The left image shows the validation results of the amylase mutant (DD1-33 is represented by three green circles), and the right image shows the purification plate of DD1-33. The mutant strain DD1-33 was obtained from a space-induced mutant strain derived from strain DY. This strain simultaneously possesses amylase, cellulase, and phosphate-solubilizing growth-promoting functions. Figure 3 ).

[0058] 2. Quantitative detection of the amylase-producing ability of mutant strains (1) Drawing the standard curve Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose standard solution (1 mg / ml) into 15 ml test tubes, and bring the volume to 1.0 ml with distilled water. Accurately add 2 ml of DNS reagent to each tube, heat in a boiling water bath for 2 min, cool under running water, and bring the volume to 15 ml with water. Measure the absorbance at 540 nm; express the result as OD. 540 Plot a standard curve with the vertical axis as the ordinate and the glucose concentration (mg / ml) as the horizontal axis.

[0059] (2) Preparation of starch culture medium 10.0 g soluble starch, 3.0 g yeast extract, 5.0 g casein peptone, 3.0 g sodium chloride, 0.5 g magnesium sulfate heptahydrate, 3.0 g calcium chloride, 1000 mL distilled water, adjust pH to 5.5, sterilize at 115℃ for 30 minutes.

[0060] (3) Determination of amylase activity The strain was inoculated into starch medium at a 5% inoculum and cultured at a constant temperature and shaking at 120 r / min for 120 h. The fermentation broth was centrifuged at 4000 r / min for 10 min at 4℃, and the supernatant was collected as crude enzyme solution. 0.5 g of soluble starch was mixed with phosphate buffer and boiled until completely dissolved to obtain the reaction system. 1 ml of crude enzyme solution was added to 19 mL of the reaction system and reacted at 37℃ for 2 h. The reaction was terminated with 0.5 ml of 4% NaOH solution. The reaction solution was centrifuged at 4000 r / min for 5 min. The reducing sugar content in the supernatant was determined using the DNS reagent and DNS method provided by Beijing Solarbio Science & Technology Co., Ltd. The amount of glucose in the hydrolysis product was obtained by referring to the glucose standard curve, thereby determining the enzyme activity of amylase.

[0061] The results are as follows Figure 4 As shown in the figure, the amylase activity of the mutant strain DD1-33 and the original strain DY was quantitatively detected. The reducing sugar content in the reaction system of mutant strain DD1-33 reached 3.125 mg / ml (the reducing sugar content in the reaction system of mutant strain DBD-1 was 0.90 mg / ml), while the reducing sugar content in the reaction system of original strain DY was 0.98 mg / ml. The amylase activity of DD1-33 was increased by 218.9% compared with that of the original strain.

[0062] Example 3: Pot Experiment of Maize under Saline-Alkali Conditions Tested strains: strain DD1-33 and original strain DY.

[0063] 1. Seed treatment The selected maize germplasm resource was Zhengdan 958. The maize seeds were first soaked in 75% ethanol for 5 minutes to wash away the seed coat, then rinsed 8 times with clean water, and then soaked in 10% hypochlorous acid for 10 minutes for disinfection. After disinfection, they were rinsed 5-6 times with clean water. After treatment, the maize seeds were soaked in sterile water and covered with plastic wrap.

[0064] 2. Inoculum soaking treatment One to two days in advance, prepare an appropriate amount of fermentation broth and sterile water for the test strain, and repeatedly dilute with sterile water to the OD value. 600 The concentration is around 0.7. Take about 50 corn seeds and soak them in the diluted bacterial solution for 30 minutes.

[0065] 3. Soil and solution preparation When preparing a soil environment simulating saline-alkali stress, use vermiculite and potting soil in a 1:1 ratio to mix the substrate soil. Add 800 mL of alkaline salt solution (formula: sodium chloride 120 mM, sodium carbonate 20 mM, sodium bicarbonate 80 mM) to every 1 kg of substrate soil to simulate moderately saline-alkali soil. Fill each flowerpot with the mixed soil mixture, using 230 g per pot.

[0066] 4. Experimental Setup Configure different processing groups: (1) Under saline-alkali conditions, and with sterile solution treatment (i.e., saline-alkali treatment control group), it is denoted as CK0; (2) Under saline-alkali conditions, the original bacteria DY were used for treatment, and the result is denoted as DY; (3) Under saline-alkali conditions, the mutant strain DD1-33 was used for treatment, and it was denoted as DD1-33; (4) No salt or alkali treatment and no sterile solution treatment (i.e., no salt or alkali control group) is recorded as 0%.

[0067] Each treatment consisted of 5 pots, with 5 corn plants per pot after thinning. The bacterial strain treatment group underwent seed soaking treatment according to step 2 before sowing, while the control group was treated with an equal volume of sterile water.

[0068] The entire growth cycle was 28 days. Then, the plant height, root dry weight, stem diameter, and root volume of the corn seedlings in each group were recorded. The average of the results was taken.

[0069] The results are as follows Figure 5 and Figure 6 As shown, under saline-alkali stress conditions, compared with the original strain DY fermentation broth treatment group, the experimental group treated with mutant strain DD1-33 showed significantly improved growth indicators in maize plants, including plant height, root dry weight, stem diameter, and root volume. This indicates that mutant strain DD1-33 plays an important role in enhancing maize plants' tolerance to saline-alkali soil environments and promoting maize seedling growth. It is inferred that in the early stages of maize seed germination, the DD1-33 treatment group utilizes bacterial α-amylase to more fully break down and utilize the starch stored within the seed, hydrolyzing it into glucose, maltose, etc., which can be directly utilized by the embryo, thus promoting seed germination and seedling growth, improving the plant's carbon source utilization efficiency, and consequently promoting crop biomass accumulation.

[0070] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Bacillus licheniformis or its progeny, characterized in that: The Bacillus licheniformis is Bacillus licheniformis ( Bacillus licheniformis DD1-33, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.38141.

2. A composition containing the Bacillus licheniformis of claim 1 or its progeny.

3. The composition according to claim 2, characterized in that: The composition is a culture, which is a substance obtained by culturing the Bacillus licheniformis or its progeny in a microbial culture medium.

4. The composition according to claim 2 or 3, characterized in that: The composition is a microbial agent, a microecological preparation, or a bio-fertilizer.

5. The composition according to any one of claims 2-4, characterized in that: The composition has at least one of the following properties: A1) Amylase production A2) Produces cellulase; A3) Phosphorus solubilization; A4) Potassium dissolution; A5) Relieves salt and alkali stress in plants; A6) Promotes plant growth; A7) Promotes increased plant height; A8) Promotes an increase in the dry weight of plant roots; A9) Promotes increased stem diameter in plants; A10) promotes an increase in plant root volume.

6. The use of the Bacillus licheniformis of claim 1 or its descendants, or the composition of any one of claims 2-5, wherein the use is any one of the following: B1) Application in the production of amylase or in the preparation of products for the production of amylase; B2) Application in the production of cellulase or in the preparation of products for the production of cellulase; B3) Applications in phosphorus solubilization or in the preparation of products for phosphorus solubilization; B4) Applications in potassium solubilization or in the preparation of products for potassium solubilization; B5) Applications in alleviating salt and alkali stress in plants or in the preparation of products for alleviating salt and alkali stress in plants; B6) Use in promoting plant growth or in the preparation of products for promoting plant growth; B7) Application in promoting plant height increase or in the preparation of products for promoting plant height increase; B8) Application in promoting an increase in the dry weight of plant roots or in the preparation of products for promoting an increase in the dry weight of plant roots; B9) Application in promoting the increase of plant stem diameter or in the preparation of products for promoting the increase of plant stem diameter; B10) Application in promoting the increase of plant root volume or in the preparation of products for promoting the increase of plant root volume.

7. The application according to claim 6, characterized in that: The promotion of plant growth is to promote plant growth under salt-alkali stress conditions; and / or, the promotion of increased plant height is to promote increased plant height under salt-alkali stress conditions; and / or, the promotion of increased plant root dry weight is to promote increased plant root dry weight under salt-alkali stress conditions; and / or, the promotion of increased plant stem diameter is to promote increased plant stem diameter under salt-alkali stress conditions; and / or, the promotion of increased plant root volume is to promote increased plant root volume under salt-alkali stress conditions.

8. A method for alleviating salt and alkali stress in plants, comprising: Applying the Bacillus licheniformis of claim 1 or its progeny, or the composition of any one of claims 2-5, to the plant and / or its cultivation substrate to alleviate the salt stress of the plant.

9. A method for promoting plant growth, comprising: Applying the Bacillus licheniformis of claim 1 or its progeny, or the composition of any one of claims 2-5, to plant plants and / or their cultivation substrate to promote the growth of the plants.

10. The composition, application, or method according to any one of claims 5-9, characterized in that: The plant is any one of the following: C1) Angiosperms; C2) Monocotyledons; C3) Plants of the order Poales; C4) Gramineae plants; C5) Plants of the genus *Zea*; C6) Corn.

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