A halophilic bacillus, a microbial inoculant and use thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a halophilic bacillus, a bacterial agent and application thereof. The application provides a halophilic bacillus Halobacillus andaensis, and the preservation number is CCTCC M 20232394. The application also provides a bacterial agent containing the halophilic bacillus. The application also provides application of the halophilic bacillus or the bacterial agent in promoting plant growth under saline-alkali stress, improving plant stress resistance, improving soil, and crop production. The halophilic bacillus provided by the application has excellent saline-alkali tolerance, and can only grow under saline-alkali conditions. Meanwhile, the halophilic bacillus has growth-promoting characteristics such as salt reduction, acid production for alkali reduction, nitrogen fixation, and the like, can reduce salt damage, and improve crop yield and quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to a halophilic bacillus, a microbial agent and application thereof. BACKGROUND

[0002] With the increasing demand for food, the main agricultural land is degraded and functionless. According to the incomplete statistics of the United Nations Educational, Scientific and Cultural Organization and the Food and Agriculture Organization, the area of saline-alkali land in the world is 954 million hectares, which is widely distributed in arid, semi-arid and semi-humid regions of all continents. China is vast in territory and diverse in climate, and the total area of saline-alkali land is about 1.5 billion mu, of which about 550 million mu can be used, accounting for about 5% of the land available in China. Strengthening the comprehensive utilization of saline-alkali land is an important aspect of farmland protection and improvement, and is an important measure to protect the red line of cultivated land, ensure food security and improve the comprehensive production capacity of agriculture. Therefore, it is urgent to promote the soil improvement and upgrading of saline-alkali land through ecological, green and efficient ways, so as to realize the comprehensive development and sustainable utilization of saline-alkali land, which is also the necessity to expand the space of agricultural production and ensure national food security.

[0003] The main feature of saline-alkali land is that it contains a lot of water-soluble salt or alkaline substances. Due to the high salt content and alkalinity, the humus in the soil is leached, the soil structure is destroyed, and the growth rate, biomass and root growth of plants are affected, and in severe cases, plants will wilt, be poisoned and die with rotten roots, so soil improvement must be carried out on saline-alkali land. Different effective improvement methods can be formed for specific saline-alkali land, which can be summarized into four categories, namely water conservancy improvement, physical regulation, chemical improvement and biological improvement. Among them, biological improvement of saline-alkali land is currently recognized as the most effective, economic and ecological measure. Certain microbial populations can occupy dominant positions under specific conditions such as salt and alkali, drought, etc., and form enrichment communities, so functional strains that are tolerant / halophilic to salt and alkali can usually be screened from saline-alkali soil. Microorganisms and their metabolites can quickly affect the physical and chemical properties of saline-alkali soil, or secrete nutrients for plants or other microorganisms to use, or produce organic acids, proteins and polysaccharides and other extracellular secretions, which directly affect the structure, physical and chemical properties and fertility of the soil. Although salt-tolerant microorganisms have outstanding stress resistance in the laboratory, the complex and harsh natural environment still has a significant inhibitory effect on them, and most of them die when the conditions are not suitable, and only a small part can form dormant bodies, which will greatly limit the improvement effect of salt-tolerant microorganisms on soil. Therefore, it is necessary to develop a high-efficiency, stable, green and environmentally friendly salt-tolerant microbial fertilizer.

[0004] Halophilic bacteria are a class of extremophiles that thrive only at specific salt concentrations. The genus *Halobacillus*, belonging to the family Bacillaceae, was established by Spring et al. in 1996 and is primarily isolated from high-salt environments such as salt lakes, saline soils, and salt fields. In high-salt environments, moderately halophilic bacteria typically maintain their intracellular osmotic and turgor pressure by accumulating compatible solutes. *Halobacillus* can secrete various salt-tolerant enzymes (such as amylase and lipase), showing great potential for application in industrial microbiology. There are also reports that *Halobacillus* can synthesize antibacterial and antitumor active substances, attracting widespread attention from pharmaceutical researchers. Currently, research and applications regarding the promotion of plant growth and development by *Halobacillus* microorganisms under salt-alkali stress are limited. Therefore, existing technologies require further improvement. Summary of the Invention

[0005] Based on the deficiencies of the existing technology, this application has screened and isolated a salt-tolerant and growth-promoting halophilic Bacillus, Halobacillus andaensis, which has excellent salt tolerance and also has growth-promoting characteristics such as salt reduction, acid production and alkali reduction, and nitrogen fixation. It can be used for saline-alkali land improvement and to promote crop production.

[0006] The first aspect of this application provides a halophilic Bacillus halobacillus andaensis, which has the accession number CCTCC M 20232394.

[0007] The second aspect of this application provides a microbial agent comprising the halophilic Bacillus anandaensis described in the first aspect above.

[0008] The third aspect of this application provides a method for preparing the bacterial agent described in the second aspect, comprising inoculating a pure strain of Halobacillus andaensis as described in the first aspect into a culture medium, culturing it, and harvesting the bacterial solution.

[0009] The fourth aspect of this application provides the use of Halobacillus andaensis as described in the first aspect above, or the inoculant as described in the second aspect above, wherein the use is selected from: 1) promoting plant growth under salt-alkali stress; 2) improving plant resistance; 3) improving soil; 4) crop production.

[0010] The fifth aspect of this application provides a method for promoting plant growth under salt-alkali stress, comprising applying to the plant or soil a halophilic Bacillus andaensis as described in the first aspect above, or a microbial agent as described in the second aspect above.

[0011] The sixth aspect of this application provides a method for improving plant stress resistance, comprising applying to plants or soil a halophilic Bacillus andaensis as described in the first aspect above, or a microbial agent as described in the second aspect above.

[0012] The seventh aspect of this application provides a method for improving soil, comprising applying to the soil a halophilic Bacillus andaensis as described in the first aspect above, or a microbial agent as described in the second aspect above.

[0013] The beneficial effects of this application are as follows:

[0014] This application provides a salt-tolerant and growth-promoting halophilic Bacillus strain and its applications, named *Halobacillus andaensis* nx-9. This strain is a highly efficient, stable, and multifunctional strain with excellent salt-alkali tolerance, growing only under saline-alkali conditions. It also possesses growth-promoting properties such as salt reduction, acid production and alkali reduction, and nitrogen fixation. Microbial agents prepared using this strain can significantly improve plant stress resistance under salt-alkali stress, alleviate salt damage, and improve crop yield and quality. In conclusion, the *Halobacillus* strain and its microbial agents provided in this application have broad application prospects and market development potential. Attached Figure Description

[0015] Figure 1 This is the colony morphology of nx-9 in Example 2 of this application.

[0016] Figure 2 This is a scanning electron microscope image of nx-9 in Embodiment 2 of this application.

[0017] Figure 3 The images show the cell morphology of nx-9 cells under medium (pH 8.5 and 8% NaCl) and high (16% NaCl and pH 10.0) salinity and alkalinity conditions observed by cryo-electron microscopy and field emission scanning electron microscopy in Example 3 of this application; A is cryo-electron microscopy, medium salinity and alkalinity; B is cryo-electron microscopy, high salinity and alkalinity; C is field emission scanning electron microscopy, medium salinity and alkalinity; D is field emission scanning electron microscopy, high salinity and alkalinity.

[0018] Figure 4 This is a phylogenetic tree constructed based on the 16S rDNA gene sequence of nx-9 in Example 2 of this application.

[0019] Figure 5 This shows the growth of nx-9 in Ashube nitrogen-free medium in Example 4 of this application. Detailed Implementation

[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0021] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0022] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0023] This application has isolated and screened a halophilic Bacillus halobacillus anensis with excellent salt and alkali tolerance, and found that it has multiple growth-promoting characteristics such as salt reduction, acid and alkali production, and nitrogen fixation.

[0024] Therefore, this application first provides a halophilic Bacillus halobacillus andaensis, with accession number CCTCC M20232394, accession date November 30, 2023, and accession address China Center for Type Culture Collection (CCTCC), Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0025] In a specific embodiment of this application, the halophilic Bacillus halobacillus andaensis is named halophilic Bacillus halobacillus andaensis nx-9.

[0026] The halophilic Bacillus halobacillus andaensis nx-9 belongs to the phylum Firmicutes, class Bacilli, order Bacillales, family Bacillaceae, genus Halobacillus, and species Andaensis.

[0027] The morphological characteristics of Halobacillus andaensis nx-9 are as follows: the colonies are moist and smooth, opaque, with regular edges, and are milky yellow in color. Under scanning electron microscopy, they appear as rod-shaped bacteria, about 2-3 μm long and about 0.5-0.8 μm wide.

[0028] The physiological and biochemical identification results of Halobacillus andaensis nx-9 were as follows: positive for alkaline phosphatase, β-galactosidase, α-glucosidase, esterase (C4), lipoesterase (C8), and naphthol-AS-BI-phosphohydrolase; positive for galactose, D-glucose, D-fructose, D-mannitol, melibiose, sucrose, and trehalose.

[0029] The 16S rDNA sequence of the halophilic Bacillus halobacillus andaensis nx-9 is shown in SEQ ID NO.1.

[0030] 16S rDNA (16S ribosomal DNA) is part of the small subunit (30S subunit) of the ribosome in bacteria and archaea. It is a highly conserved gene with high sequence similarity in different bacteria. It can be used as a molecular marker for bacterial classification and identification. By comparing the similarity of 16S rDNA sequences of different bacteria, their phylogenetic relationships can be inferred and phylogenetic trees can be constructed.

[0031] SEQ ID NO.1:

[0032]

[0033] Due to the high conservation of 16S rDNA, universal primers can be designed to amplify it by PCR for bacterial detection and identification.

[0034] For example, in a specific embodiment of this application, the universal primer 27F / 1492R is used to amplify the 16S rDNA of the strain.

[0035] 27F primer: AGAGTTTGATCMTGGCTCAG (SEQ ID NO.2)

[0036] 1492R primer: GGTTACCTTGTTACGACTT (SEQ ID NO.3)

[0037] The halophilic Bacillus and aensis nx-9 has salt and alkali tolerance.

[0038] Specifically, the halophilic Bacillus andaensis can grow under conditions of 10-18% NaCl concentration, or pH 9.0-12.0, or 10-16% NaCl concentration and pH 8.5-10.0.

[0039] The NaCl concentration is expressed as a mass percentage, meaning the mass of NaCl accounts for the percentage of the mass of the culture medium.

[0040] In some embodiments of this application, Halobacillus andaensis nx-9 can grow under conditions of pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, and pH 12.0.

[0041] In other embodiments of this application, Halobacillus andaensis nx-9 can grow under conditions with NaCl concentrations of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, and 18%.

[0042] In some other embodiments of this application, Halobacillus andaensis nx-9 can grow under conditions of 10-16% NaCl concentration and pH 8.5-10.0, specifically 10% NaCl and pH 8.5, 10% NaCl and pH 9.5, 14% NaCl and pH 9.0, 14% NaCl and pH 10.0, 16% NaCl and pH 9.0, and 16% NaCl and pH 10.0.

[0043] In some embodiments of this application, the Halobacillus andaensis nx-9 is cultured for less than or equal to 4 days under conditions of 16% NaCl concentration and pH 10.0.

[0044] In some other embodiments of this application, the Halobacillus andaensis nx-9 is cultured for less than or equal to 2 days under conditions of 14% NaCl concentration and pH 10.0.

[0045] In some other embodiments of this application, the Halobacillus andaensis nx-9 is cultured for less than or equal to 1 day under conditions of 10% NaCl concentration and pH 9.5.

[0046] Specifically, the culture medium used for the salt and alkali tolerance test is LB medium with the above conditions, the culture temperature is 30℃, and the rotation speed is 150 rpm.

[0047] The cultivation time refers to the OD (October of the culture medium to the bacterial culture) from inoculation to the time from culture medium to bacterial suspension. 600 >0.5 of the time.

[0048] The halophilic Bacillus halobacillus andaensis has the ability to produce acid and reduce alkali and salt.

[0049] The ability to produce acid and reduce alkali refers to the ability to reduce the alkalinity (pH value) of the environment or solution by producing acidic substances. In this application, it is evaluated by the formula: Acid production and alkali reduction rate = (initial pH - pH after fermentation) / initial pH × 100%.

[0050] The salt reduction capacity refers to the ability to reduce or lower the salt content in the environment. In this application, it is evaluated using the formula: Salt reduction rate = (Salinity before cultivation - Salinity after cultivation) / (Salinity before cultivation) × 100%. The salinity before and after cultivation is evaluated by measuring changes in conductivity.

[0051] In some embodiments of this application, the halophilic Bacillus halobacillus andaensis nx-9 exhibits an acid production and alkali reduction rate of 12.17% and a salt reduction rate of 11.46% under conditions of 8% NaCl concentration and pH 8.5.

[0052] In other embodiments of this application, the halophilic Bacillus halobacillus andaensis nx-9 exhibited an acid production and alkali reduction rate of 16.53% and a salt reduction rate of 4.40% under conditions of 16% NaCl concentration and pH 10.0.

[0053] Specifically, the culture medium used for determining the salt reduction rate and acid and alkali reduction rate was LB medium with the above conditions, the culture temperature was 30℃, the rotation speed was 150 rpm, and the culture time was 4 days.

[0054] In a specific embodiment of this application, the medium salinity and alkalinity conditions are 8% NaCl and pH 8.5; the medium salinity and alkalinity culture medium refers to LB liquid culture medium with 8% NaCl and pH 8.5.

[0055] In a specific embodiment of this application, the high salinity / alkalinity condition is 16% NaCl concentration and pH 10.0; the high salinity / alkalinity culture medium refers to LB liquid culture medium with 16% NaCl concentration and pH 10.0.

[0056] Based on the Halobacillus andaensis nx-9 isolated and screened in this application, this application may also provide a bacterial agent containing the aforementioned Halobacillus andaensis nx-9.

[0057] The microbial agent refers to a preparation containing specific microorganisms, wherein the microbial agent contains an effective amount of the aforementioned halophilic Bacillus andaensis nx-9.

[0058] The effective amount refers to the number of active microorganisms in the bacterial agent that can play the expected role. In the specific embodiments of this application, the effective amount can be, for example, 10^9 to 10^10 cfu / mL.

[0059] The microbial agent may also include a microbial carrier and a stabilizer; the microbial carrier is used to provide the nutrients and environment required for the growth and reproduction of microorganisms; the microbial carrier may specifically be natural organic matter (plant residues such as rice husks and wheat straw, animal manure, seaweed powder), agricultural by-products (such as soybean meal, corn gluten meal, molasses), industrial by-products (such as distiller's grains, yeast sludge), soil conditioners (such as humus, bentonite, vermiculite, perlite and other minerals), synthetic organic matter (such as polymers such as polylactic acid and polyvinyl alcohol, synthetic nutrients), and microbial growth factors. Fermentation products (such as fermentation broth and fermentation residue), special carriers (such as microcapsules, agar, gelatin, etc.), inorganic substances (such as calcium carbonate and phosphate), and biodegradable materials (such as starch-based materials such as corn starch and tapioca starch, and protein-based materials such as soybean protein and whey protein); stabilizers are used to maintain the stability and activity of the microbial agent during storage and transportation. Stabilizers may include: antioxidants, such as ascorbic acid, lipoic acid, and ethylenediaminetetraacetic acid, to prevent microbial inactivation due to oxidation; and humectants, such as glycerin, propylene glycol, and sorbitol. Alcohols are used to keep the bacterial agent moist and prevent it from drying out; pH adjusters, such as citric acid, phosphate buffer, and sodium bicarbonate, are used to maintain the appropriate pH of the bacterial agent to preserve the activity of microorganisms; preservatives, such as sodium benzoate, potassium sorbate, and parabens, are used to prevent excessive growth or contamination of microorganisms in the bacterial agent; surfactants, such as nonionic surfactants (Tween) and cationic surfactants (such as ammonium dodecyl sulfate), are used to improve the dispersibility and adhesion of the bacterial agent; protectants, such as xanthan gum and carboxymethyl cellulose, are used to protect microorganisms from external factors such as ultraviolet radiation; nutrient enhancers, such as amino acids, vitamins, and minerals, are used to provide additional nutrition; gelling agents, such as agar, gelatin, and sodium carboxymethyl cellulose, are used to form gel-like substances that help fix and protect microorganisms; antifreeze agents, such as propylene glycol and glycerin, prevent the bacterial agent from freezing at low temperatures; antistatic agents, such as quaternary ammonium compounds, are used to reduce the accumulation of static electricity in the bacterial agent during storage and transportation; chelating agents, such as ethylenediaminetetraacetic acid, are used to chelate metal ions to prevent their adverse effects on microorganisms.

[0060] This application also provides a method for preparing the above-mentioned bacterial agent, which includes inoculating the pure strain of the above-mentioned halophilic Bacillus halobacillus andaensis into a culture medium for culture and collecting the bacterial solution.

[0061] The inoculation method involves inoculating the seed culture at an inoculation amount of 0.5% to 5%, for example, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0%. The seed culture can be obtained through conventional activation culture methods.

[0062] The culture temperature can be 28 to 37°C, for example, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.

[0063] The rotation speed of the culture can be 150-200 rpm, for example, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, or 200 rpm.

[0064] The culture time can be 36 to 60 hours, for example, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, or 60 hours.

[0065] When the viable bacterial count reaches 10^9 to 10^10 cfu / mL, the microbial inoculum stock solution is obtained.

[0066] The preparation method further includes centrifuging or allowing natural precipitation of the microbial agent mother liquor to obtain bacterial precipitate, and resuspending the bacterial precipitate to obtain the agent.

[0067] The centrifugation can be performed using conventional methods. For example, conventional centrifugation parameters for separating bacteria from fermentation broth can be a centrifugal force of 3000–5000 × g, a centrifugation time of 5–20 minutes, and a temperature of 4°C, and can be adjusted according to the actual situation.

[0068] In a specific embodiment of this application, the preparation method comprises the following steps: activating the bacterial strain in test tube culture medium to prepare a seed culture. Inoculating the seed culture into a conical flask containing a medium with moderate salinity at an inoculation rate of 0.5%–5%, and fermenting at 28–37°C and 150–200 rpm for 36–60 hours until the viable cell count reaches 10^9–10^10 CFU / mL, thus obtaining a microbial inoculum stock solution. After removing the supernatant, the bacterial cells are resuspended in ultrapure water to prepare a bacterial suspension for later use.

[0069] This application also provides the uses of the above-mentioned halophilic Bacillus andaensis, or the above-mentioned inoculant, wherein the uses are selected from: 1) promoting plant growth under salt and alkali stress; 2) improving plant resistance; 3) improving soil; 4) crop production.

[0070] This application also provides a method for promoting plant growth under salt-alkali stress, including applying the above-mentioned halophilic Bacillus halobacillus andaensis, or the above-mentioned inoculant, to the plant or soil.

[0071] This application also provides a method for improving plant stress resistance, including applying the above-mentioned halophilic Bacillus andaensis, or the above-mentioned inoculant, to the plant or soil.

[0072] This application also provides a method for improving soil, including applying the above-mentioned halophilic Bacillus andaensis, or the above-mentioned inoculant, to the soil.

[0073] Salt-alkali stress refers to abnormal salinity and pH in the soil solution of the plant growth environment, usually manifested as high salinity and high pH (alkalinity), which has an adverse effect on plant growth and development.

[0074] The plant may include the plant's propagules and the plant itself, such as seeds, seedlings, and mature plants.

[0075] The plant growth process includes seed germination, seedling growth, vegetative growth, and reproductive growth.

[0076] In a specific embodiment of this application, after plant seeds are treated with the halophilic Bacillus andaensis nx-9 or its inoculum, the germination rate and rooting rate of the plant seeds in a saline-alkali environment are both improved compared with the control group that is not treated with the halophilic Bacillus andaensis nx-9 or its inoculum.

[0077] In a specific embodiment of this application, after the plant plants were treated with the halophilic Bacillus andaensis nx-9 or the inoculum, compared with the control group that was not treated with the halophilic Bacillus andaensis nx-9 or the inoculum, the plant height, aboveground fresh weight, and biomass per plant were all increased in the saline-alkali environment.

[0078] Plant stress resistance refers to a plant's ability to resist and adapt to adverse environmental conditions (such as drought, low temperature, high temperature, salinity, pests and diseases).

[0079] In a specific embodiment of this application, after the plant was treated with the halophilic Bacillus andaensis nx-9 or its inoculum, the proline content and catalase activity in the plant were both increased compared with the control group that was not treated with the halophilic Bacillus andaensis nx-9 or its inoculum.

[0080] Soil improvement refers to the process of improving the physical, chemical, and biological properties of soil through various methods to enhance soil fertility, increase crop yield, improve crop quality, and promote healthy plant growth. Examples of methods to increase soil fertility include adjusting soil pH and improving soil biological activity.

[0081] In a specific embodiment of this application, the halophilic Bacillus andaensis nx-9 or its inoculum can grow on Assoube nitrogen-free medium plates. Therefore, the halophilic Bacillus andaensis nx-9 or its inoculum can improve soil fertility and promote plant growth through nitrogen fixation.

[0082] The term "crop growth" refers to the continuous process of increasing crop biomass, morphogenesis, and physiological function development from sowing to harvest.

[0083] The present application is further illustrated below by way of examples, but these examples do not limit the scope of the application. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention employ conventional techniques in molecular biology, biochemistry, chromosome structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature. Unless otherwise specified, the instruments, reagents, and materials used in the examples are obtained through conventional means.

[0084] Example 1: Separation and Screening of nx-9

[0085] Initial screening: 10.0g of saline-alkali soil from a wolfberry planting base in Ningxia was weighed and added to 100mL of sterile water, then allowed to stand for 20min. The mixture was then shaken thoroughly for 30min to prepare a basic bacterial suspension. After serial dilution, the suspension was added to LB agar plates with a medium salinity (pH 8.5 and 8% NaCl), spread evenly with a sterile spreader, and incubated at 30℃ for 3–7 days. Single colonies were isolated and numbered. Each single colony was streaked into fresh LB agar with a medium salinity (pH 8.5 and 8% NaCl), and purified using a serial dilution method. The purified cultures were then propagated, and the morphology, color, and transparency of the colonies were observed. Single colonies were selected and preserved for later use. Twelve strains were obtained from the initial screening and numbered nx-1 to nx-12.

[0086] Secondary screening: By continuously increasing the salinity and alkalinity of LB medium and culturing at 150 rpm for 4 days, the growth behavior of the strains was used to determine their tolerance to the current concentrations of salt and alkalinity. The strain with the best salt and alkali tolerance, nx-9, was selected from these strains. The conditions for secondary screening are shown in Table 1 below.

[0087] Characteristics of Ningxia saline-alkali soil: pH 8.98, organic matter 12.61 g·kg⁻¹ -1 Alkaline nitrogen uptake: 44.1 mg / kg -1 Available phosphorus 118.18 mg·kg -1 Available potassium 782.5 mg·kg -1The soil in this area is alkaline, with a pH value as high as 9.0 in the soil layer at a depth of 40-60 cm. The content of available phosphorus and organic matter decreases with increasing soil depth.

[0088] Table 1. Rescreening Conditions

[0089]

[0090]

[0091] Note: + indicates slow growth at this concentration (2d < culture time ≤ 4d); ++ indicates relatively fast growth at this concentration (1d < culture time ≤ 2d); +++ indicates vigorous growth at this concentration (culture time ≤ 1d); - indicates no growth at this salt concentration.

[0092] Example 2: Identification of strain nx-9

[0093] The morphological characteristics and physiological and biochemical properties of the strain were analyzed according to the *Handbook of Systematic Identification of Common Bacteria*. Strain NX-9 colonies on LB agar were moist, smooth, opaque, with regular edges, and a creamy-yellow color (see...). Figure 1 Scanning electron microscopy revealed it to be a rod-shaped bacterium, approximately 2–5 μm long and 0.5–0.8 μm wide (see...). Figure 2 ).

[0094] The results of physiological and biochemical identification are shown in Tables 2 and 3.

[0095] Table 2 Enzyme activity characteristics of strain nx-9

[0096]

[0097] Note: + positive; - negative; weak positive.

[0098] Table 3 Substrate utilization characteristics of strain NX-9

[0099]

[0100]

[0101] Note: + positive; - negative; weak positive.

[0102] Genomic DNA was extracted from the strain using a bacterial genomic DNA extraction kit. The 16S rDNA of the strain was amplified using universal primers 27F / 1492R. The PCR products were detected by 1% agarose gel electrophoresis, and after gel recovery, were sent to Shanghai Qingke Biotechnology Co., Ltd. for purification and sequencing. The sequencing results (SEQ ID NO.1) were compared with the NCBI database using BLAST. The purified and sequenced PCR product of strain nx-9 yielded a 1460bp sequence. BLAST comparison showed that the 16S rDNA sequence of nx-9 shared 99.72% homology with Halobacillus andaensis NEAU-ST10-40 (NR135887.1). Furthermore, a phylogenetic tree was constructed using the Neighbor-Joining Method, showing that nx-9 and Halobacillus andaensis NEAU-ST10-40 (NR135887.1) were genetically closest. Figure 4 Based on comprehensive analysis, strain nx-9 is identified as Halobacillus anandaensis.

[0103] Example 3: Salt and Alkali Tolerance of Strain NX-9

[0104] The salt and alkalinity tolerance of the strain was assessed by continuously increasing the salinity and alkalinity of the LB medium. While maintaining the pH of the LB medium at 7.0, the salinity was increased by 2% sequentially, resulting in NaCl concentrations of 10%, 12%, 14%, 16%, 18%, and 20% by mass. While maintaining the NaCl concentration at 1%, the pH was increased by one unit, resulting in pH values ​​of 9, 10, 11, 12, and 13. The strains were cultured at 30°C and 150 rpm for 4 days, and their growth was used to determine their tolerance to the current salt and alkalinity concentrations, as shown in Table 4. The strains were then inoculated into LB liquid medium with both high salinity and alkalinity (NaCl concentration and pH adjusted) to analyze their simultaneous tolerance to both. Results were obtained from cryo-scanning electron microscopy (CSE) and field emission scanning electron microscopy (FET) images. Figure 3 It can be seen that under high salinity and alkalinity conditions (16% NaCl and pH 10.0), fine particles are enriched on the surface of nx-9 cells, and the cells are mostly deformed and have a rough surface, which may be related to their strong tolerance to high salinity and alkalinity stress.

[0105] Table 4 Salt and alkali tolerance of strain nx-9

[0106]

[0107] Note: + indicates slow growth at this concentration (2d < culture time ≤ 4d); ++ indicates relatively fast growth at this concentration (1d < culture time ≤ 2d); +++ indicates vigorous growth at this concentration (culture time ≤ 1d); - indicates no growth at this salt concentration.

[0108] The strain nx-9 can tolerate a maximum NaCl concentration of 18% and a maximum pH of 12.0. Furthermore, nx-9 can grow under conditions with both high salinity and alkalinity (NaCl concentration >10% and pH >9), and grows well under saline-alkaline conditions of 16% NaCl and pH 10.0.

[0109] Example 4: The salt reduction, acid production and alkali reduction, and nitrogen fixation capabilities of strain nx-9

[0110] To investigate the salt-lowering, acid-lowering, and alkali-lowering abilities of strain NX-9, LB liquid medium with different salt and alkali concentrations was prepared. After culturing at 30℃ and 150 rpm for 4 days, the pH and conductivity of the medium were measured. The acid-lowering and alkali-lowering rates and the salt-lowering rate of the strain were analyzed: Acid-lowering and alkali-lowering rate = (initial pH - post-fermentation pH) / initial pH × 100%; Salt-lowering rate = (pre-culture salinity - post-culture salinity) / pre-culture salinity × 100%. Colonies were picked up with sterile toothpicks and inoculated onto Assoupy nitrogen-free agar plates, which were then incubated at 30℃. The growth on the medium was observed. Figure 5 ).

[0111] The specific procedure for determining salinity changes using the conductivity method is as follows:

[0112] Dilute the supernatant before and after culture 100 times, measure the conductivity of the diluted solution and calculate the salinity (S).

[0113] Salinity calculation formula: S=0.008×EC / (1+0.0325×T)

[0114] S indicates salinity expressed in practical salinity units (PSU);

[0115] EC represents conductivity in Siemens units per meter (S / m);

[0116] T represents temperature, expressed in degrees Celsius (°C). (The temperature at the time of measurement was 22.4°C.)

[0117] Table 5. Salt reduction, acid production, and alkali reduction capabilities of strain nx-9 under medium salinity and alkalinity (8% NaCl concentration and pH 8.5).

[0118]

[0119]

[0120] Table 6. Salt reduction, acid production, and alkali reduction capabilities of strain nx-9 under high salinity and alkalinity (16% NaCl concentration and pH 10.0).

[0121]

[0122] The results showed that, as shown in Tables 5 and 6, strain nx-9 has certain characteristics of reducing salt content, producing acid and reducing alkali content, and fixing nitrogen. It can effectively improve the soil properties of saline-alkali land, reduce its salt and alkali stress, and improve soil fertility through nitrogen fixation. This lays the foundation for the application of this strain in the colonization and growth promotion of saline-alkali land.

[0123] Example 5: Effects of nx-9 on seed germination of Chinese cabbage under salt-alkali stress

[0124] (1) Preparation of bacterial suspension. Single colonies were picked from the plate and inoculated into fresh liquid culture medium for activation culture to prepare seed culture. The seed culture was then inoculated at a rate of 0.5%–5% into Erlenmeyer flasks containing medium with moderate salinity. Fermentation was carried out at 28–37°C and 150–200 rpm for 36–60 h until the viable cell count reached 10^9–10^10 cfu / mL, yielding the microbial inoculum stock solution. After centrifugation to remove the supernatant, the bacterial cells were resuspended in ultrapure water to prepare a bacterial suspension for later use.

[0125] (2) Seed disinfection. Select a number of “Xin Ai Qing” cabbage seeds, soak them in 75% ethanol for 10 minutes for disinfection and sterilization, and rinse them with ultrapure water 3-4 times.

[0126] (3) Seed germination test. Disinfected seeds were soaked in a bacterial suspension for 4 hours and then cultured in petri dishes containing a 20 mM saline-alkali mixture (NaCl, Na₂SO₄, NaHCO₃, Na₂CO₃ = 9:1:1:9). The control group (CK) had its disinfected seeds soaked in sterile water for 4 hours, with identical culture conditions in the petri dishes. Each treatment contained 25 seeds, with three replicates. Seeds were incubated at 25℃ in the dark for 7 days. Germination rate and rooting rate were calculated: Germination rate = (Number of germinated seeds / Number of tested seeds) × 100%; Rooting rate = (Number of rooted seeds / Number of tested seeds) × 100%.

[0127] Table 7. Effects of strain NX-9 on seed germination of Chinese cabbage.

[0128]

[0129] Note: Mean (root length ≤ 1cm) represents the average proportion of roots with a length ≤ 1cm.

[0130] The results, as shown in Table 7, indicate that compared with the control group that was not treated with the inoculant, the germination rate of the seeds treated with the inoculant increased by 4 percentage points and the rooting rate increased by 8 percentage points. This suggests that nx-9 has a certain promoting effect on the germination and rooting of Chinese cabbage seeds under low salinity stress, especially on the rooting rate.

[0131] Example 6: Effects of nx-9 on the growth of Chinese cabbage seedlings under salt-alkali stress

[0132] (1) Set salinity and alkalinity levels of 0, 20, 40, 60, 80, and 100 mM (NaCl, Na2SO4, NaHCO3, Na2CO3 = 9:1:1:9) and observe the growth of Chinese cabbage seedlings under different salinity and alkalinity stresses. Based on the experimental results, 60 mM was finally determined as the salinity and alkalinity level to be used in subsequent experiments.

[0133] (2) Preparation of bacterial suspension. The bacterial strain was activated and cultured in a medium with moderate salinity in test tubes to prepare a seed culture. Inoculation was carried out at a rate of 0.5%–5% in Erlenmeyer flasks containing medium with moderate salinity. Fermentation was conducted at 28–37°C and 150–200 rpm for 36–60 h until the viable cell count reached 10^9–10^10 CFU / mL, yielding a microbial inoculum stock solution. After centrifugation to remove the supernatant, the bacterial cells were resuspended in ultrapure water to prepare a bacterial suspension for later use.

[0134] (3) Different experimental treatments. A 60 mM saline-alkali mixture was added to the soil of the treatment group to simulate saline-alkali stress, while an equal amount of tap water was added to the soil of the control group and mixed thoroughly. Two plump, uniformly sized cabbage seedlings were evenly sown in each pot, with 10 replicates per treatment. After the seedlings had 2-3 true leaves, 5 mL of bacterial suspension was applied to the treatment group, and 5 mL of sterile water was applied to the control group. Subsequent management measures were the same. After 15 days of cultivation, the cabbage seedlings were harvested and relevant indicators were measured.

[0135] Table 8. Effects of strain NX-9 on the growth of Chinese cabbage seedlings

[0136]

[0137] Note: ** indicates a significant difference at the p<0.01 level; *** indicates a significant difference at the p<0.001 level.

[0138] The results, as shown in Table 8, indicated that the application of NX-9 significantly increased the seedling height (2.39 cm), aboveground fresh weight (0.26 g), and biomass per plant (0.04 g) of Chinese cabbage seedlings, representing increases of 60.40%, 73.33%, and 100.00%, respectively, compared to the control (CK) treatment. Furthermore, it enhanced plant stress resistance, specifically by increasing proline content and catalase activity by 244.86% and 40.03%, respectively. Therefore, strain NX-9 can significantly alleviate the effects of salt-alkali stress on plant growth and promote plant growth.

[0139] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A halophilic Bacillus Halobacillus andaensis Its accession number is CCTCC M 20232394.

2. The halophilic Bacillus according to claim 1, characterized in that, The halophilic Bacillus Halobacillus andaensis The 16S rDNA sequence is shown in SEQ ID NO.

1.

3. The halophilic Bacillus according to claim 1, characterized in that, The halophilic Bacillus Halobacillus andaensis It can grow under conditions of 10-18% NaCl concentration, or pH 9.0-12.0, or 8-16% NaCl concentration and pH 8.5-10.0; And / or, the halophilic Bacillus Halobacillus andaensis It has the ability to reduce salt content, produce acid and reduce alkali, and fix nitrogen.

4. The halophilic Bacillus according to claim 3 Halobacillus andaensis Its characteristics are, The halophilic Bacillus Halobacillus andaensis It has one or more of the following characteristics: 1) The incubation time under conditions of 16% NaCl concentration and pH 10.0 is less than or equal to 4 days; 2) Under conditions of 8% NaCl concentration and pH 8.5, the acid production and alkali reduction rate was 12.17%, and the salt reduction rate was 11.46%. 3) Under the conditions of 16% NaCl concentration and pH 10.0, the acid production and alkali reduction rate was 16.53%, and the salt reduction rate was 4.40%.

5. A microbial agent comprising the halophilic Bacillus as described in any one of claims 1 to 4. Halobacillus andaensis .

6. A method for preparing the bacterial agent as described in claim 5, comprising: [using] the *Haloxylon ammodendron* as described in any one of claims 1 to 4. Halobacillus andaensis The pure bacterial strain was inoculated into the culture medium and cultured, and the bacterial solution was collected.

7. The halophilic Bacillus as described in any one of claims 1 to 4 Halobacillus andaensis Or the use of the microbial agent as described in claim 5, wherein the use is selected from one or more of the following: 1) promoting plant growth under salt and alkali stress; 2) improving plant resistance; 3) improving soil; 4) crop production.

8. A method for promoting plant growth under salt-alkali stress, comprising applying to the plant or soil the halophilic Bacillus as described in any one of claims 1 to 4. Halobacillus andaensis Or the microbial agent as described in claim 5.

9. A method for improving plant stress resistance, comprising applying to plants or soil the halophilic Bacillus as described in any one of claims 1 to 4. Halobacillus andaensis Or the microbial agent as described in claim 5.

10. A method for improving soil, comprising applying to the soil the halophilic Bacillus as described in any one of claims 1 to 4. Halobacillus andaensis Or the microbial agent as described in claim 5.

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