Halophilous bacillus, microbial inoculum and application of halophilous bacillus

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to halophilous bacillus, a microbial agent and application of the halophilous bacillus. The invention provides bacillus halophilous andaensis, and the preservation number of the bacillus halophilous andaensis is CCTCC (China Center for Type Culture Collection) M 20232394. The invention further provides a microbial inoculum which comprises the halophilous bacillus. The invention further provides application of the halophilous bacillus or the microbial inoculum to promotion of plant growth under saline-alkali stress, improvement of plant stress resistance, soil improvement and crop production. The halophilous bacillus provided by the invention has excellent saline-alkaline tolerance, can only grow under saline-alkaline conditions, also has the growth promoting characteristics of reducing salt, producing acid, reducing alkali, fixing nitrogen and the like, and can relieve the salt damage phenomenon and improve the crop yield and quality.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a halophilic bacillus, a bacterial agent and uses thereof. Background Art

[0002] As food demand continues to increase, major agricultural lands are degraded and lack functionality. According to incomplete statistics from UNESCO and FAO, the area of ​​saline-alkali land in the world is 954 million hectares, covering arid, semi-arid and semi-humid areas on all continents. my country has a vast territory and diverse climates. The total area of ​​saline-alkali land is about 1.5 billion mu, of which about 550 million mu are usable, accounting for about 5% of the country's usable land. Strengthening the comprehensive utilization of saline-alkali land is an important aspect of farmland protection and improvement, and an important measure to keep the red line of farmland, ensure food security, and improve agricultural comprehensive production capacity. Therefore, it is urgent to promote the transformation and improvement of saline-alkali land soil through ecological, green and efficient methods, so as to achieve comprehensive development and sustainable utilization of saline-alkali land, which is also the due meaning of expanding agricultural production space and ensuring national food security.

[0003] The main feature of saline-alkali land is that it contains more water-soluble salts or alkaline substances. Due to the high salt content and high alkalinity, soil humus is leached, soil structure is damaged, and plant growth rate, biomass and root growth are affected. In severe cases, plants will wilt, be poisoned and die from root rot. Therefore, soil improvement of saline-alkali land is necessary. Different effective improvement methods can be formed for specific saline-alkali land, which can be summarized into four categories, namely water conservancy project improvement, physical regulation, chemical improvement and biological improvement. Among them, biological improvement of saline-alkali land is currently recognized as the most effective, economical and ecological measure. Certain microbial populations can occupy a dominant position under specific conditions such as salinity and drought to form enriched communities. Therefore, functional strains that are resistant to / loving salt and alkali can usually be screened from saline-alkali soil. Microorganisms themselves 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 extracellular secretions such as organic acids, proteins and polysaccharides, which directly affect the structure, physical and chemical properties and fertility of the soil. Although salt-alkali tolerant microorganisms have outstanding resistance to saline-alkali environments in the laboratory, the complex and harsh natural environment still has a significant inhibitory effect on them. When the conditions are not suitable for survival, most microorganisms die, and a small number can form dormant bodies, which will greatly limit the improvement effect of salt-alkali tolerant microorganisms on the soil. Therefore, it is necessary to develop an efficient, stable, green and environmentally friendly salt-alkali tolerant growth-promoting microbial fertilizer.

[0004] Halophiles are a type of extreme environment microorganisms that can only grow well under certain salt concentrations. The genus Halobacillus belongs to the Bacillaceae family and was founded by Spring et al. in 1996. It is mostly isolated from high-salt environments such as salt lakes, saline soils, and salt fields. In a high-salt-alkali environment, moderately halophilic bacteria generally maintain their intracellular osmotic pressure and turgor pressure by accumulating compatible solutes. Halobacillus can secrete a variety of salt-tolerant enzymes (such as amylase and lipase), and has great application potential in the field of industrial microorganisms. It has been reported that Halobacillus can also synthesize antibacterial and anti-tumor active substances, which has attracted widespread attention from pharmaceutical workers. At present, there are few studies and applications on the promotion of plant growth and development by Halobacillus microorganisms under saline-alkali stress. Therefore, the existing technology needs to be further improved. Summary of the invention

[0005] Based on the above-mentioned defects of the prior art, the present application screened and isolated a salt- and alkali-tolerant and growth-promoting halobacillus Halobacillus andaensis, which has excellent salt- and alkali-tolerant ability, and also has growth-promoting properties such as salt reduction, acid and alkali reduction, and nitrogen fixation, and can be used to improve saline-alkali land and promote crop production.

[0006] In a first aspect, the present application provides a halophilic bacillus Halobacillus andaensis, whose deposit number is CCTCC M 20232394.

[0007] The second aspect of the present application provides a bacterial agent, comprising the Halobacillus andaensis of the first aspect.

[0008] The third aspect of the present application provides a method for preparing the bacterial agent of the second aspect, comprising inoculating a pure strain of Halobacillus andaensis of the first aspect into a culture medium for cultivation, and harvesting the bacterial liquid.

[0009] The fourth aspect of the present application provides the use of the Halobacillus andaensis of the first aspect or the bacterial agent of the second aspect, wherein the use is selected from: 1) promoting plant growth under saline-alkali stress; 2) improving plant stress resistance; 3) improving soil; 4) crop production.

[0010] In a fifth aspect, the present application provides a method for promoting plant growth under saline-alkali stress, comprising applying the Halobacillus andaensis of the first aspect or the bacterial agent of the second aspect to plants or soil.

[0011] A sixth aspect of the present application provides a method for improving plant stress resistance, comprising applying the Halobacillus andaensis of the first aspect or the bacterial agent of the second aspect to plants or soil.

[0012] A seventh aspect of the present application provides a method for improving soil, comprising applying the Halobacillus andaensis of the first aspect or the bacterial agent of the second aspect to the soil.

[0013] The beneficial effects of this application are:

[0014] The present application provides a salt-tolerant and growth-promoting halobacillus microorganism and its application. The bacterium is named Halobacillus andaensis nx-9. The strain is an efficient, stable, multifunctional strain with excellent salt-tolerant ability. It can only grow under saline-alkali conditions. It also has growth-promoting properties such as salt reduction, acid and alkali production, and nitrogen fixation. The microbial agent prepared using the bacterium can significantly improve the plant's resistance to stress under saline-alkali stress, reduce salt damage, and improve crop yield and quality. In summary, the halophilic Bacillus strain and its microbial agent provided in the present application have broad application prospects and market development potential. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 This is a scanning electron microscope image of nx-9 in Example 2 of the present application.

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

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

[0019] Figure 5 This is the growth condition of nx-9 in Example 4 of the present application in the nitrogen-free culture medium of Ashubei. DETAILED DESCRIPTION

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

[0021] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0022] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0023] The present application isolates and screens a halophilic Bacillus Halobacillusandaensis with excellent salt-alkali tolerance, and finds that it has multiple growth-promoting properties such as salt reduction, acid and alkali reduction, and nitrogen fixation.

[0024] Therefore, the present application first provides a halophilic Bacillus Halobacillus andaensis, whose preservation number is CCTCC M20232394, the preservation date is November 30, 2023, and the preservation address is China Type Culture Collection Center (CCTCC) at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0025] In a specific embodiment of the present application, the Halobacillus andaensis is named as Halobacillus andaensis nx-9.

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

[0027] The morphological characteristics of Halobacillus andaensis nx-9 are as follows: the colonies are moist and smooth, opaque, with neat edges and a milky yellow color. Under a scanning electron microscope, they appear as rod-shaped bacteria with a length of about 2 to 3 μm and a width of about 0.5 to 0.8 μm.

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

[0029] The 16S rDNA sequence of the halophilic bacillus Halobacillus andaensis nx-9 is shown as SEQ ID NO.1.

[0030] 16S rDNA (16S ribosomal DNA) is part of the small subunit (30S subunit) of bacterial and archaeal ribosomes. 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, we can infer their relationship and construct a phylogenetic tree.

[0031] SEQ ID NO.1:

[0032]

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

[0034] For example, in a specific embodiment of the present application, the universal primers 27F / 1492R are 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 Halobacillus andaensis nx-9 has salt-alkali resistance.

[0038] Specifically, the Halobacillus andaensis can grow under the 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 a mass percentage, which refers to the percentage of the mass of NaCl to the mass of the culture medium.

[0040] In some embodiments of the present application, Halobacillus andaensis nx-9 can grow under the 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 the present application, Halobacillus andaensis nx-9 can grow under the conditions of NaCl concentration of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, and 18%.

[0042] In some other embodiments of the present application, Halobacillus andaensis nx-9 can grow under the 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, 16% NaCl and pH 10.0.

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

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

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

[0046] Specifically, the culture medium for determining the salt-alkali tolerance is the LB culture medium with the above conditions, the culture temperature is 30° C., and the rotation speed during culture is 150 rpm.

[0047] The culture time refers to the time from the inoculation of bacteria into the culture medium to the OD of the bacterial solution. 600 >0.5 time.

[0048] The halophilic bacillus Halobacillus andaensis has the ability to produce acid, reduce alkali and reduce 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 the present 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, and is evaluated in this application by the formula: salt reduction rate = salinity before culture - salinity after culture) / salinity before culture × 100%. The salinity before culture and the salinity after culture are evaluated by measuring the change in conductivity.

[0051] In some embodiments of the present application, the acid-reducing rate of Halobacillus andaensis nx-9 under the conditions of 8% NaCl concentration and pH 8.5 is 12.17%, and the salt-reducing rate is 11.46%.

[0052] In other embodiments of the present application, the acid-reducing rate of Halobacillus andaensis nx-9 under the conditions of 16% NaCl concentration and pH 10.0 is 16.53%, and the salt-reducing rate is 4.40%.

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

[0054] In a specific embodiment of the present application, the medium salinity is a condition of NaCl 8% and pH 8.5; the medium salinity culture medium refers to an LB liquid culture medium with NaCl 8% and pH 8.5.

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

[0056] Based on the halophilic Bacillus Halobacillus andaensis nx-9 isolated and screened in the present application, the present application can also provide a bacterial agent comprising the above-mentioned halophilic Bacillus Halobacillus andaensis nx-9.

[0057] The bacterial agent refers to a preparation containing specific microorganisms, and the bacterial agent contains an effective amount of the above-mentioned Halobacillus 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 a specific embodiment of the present 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 feces, seaweed powder), agricultural by-products (such as soybean meal, corn protein powder, molasses), industrial by-products (such as distiller's grains, yeast mud), 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 nutrient salts), microbial growth factors (such as organic matter, ... Fermentation products (such as fermentation liquid, fermentation residues), special carriers (such as microcapsules, agar, gelatin and other gels), inorganic substances (such as calcium carbonate, phosphates), biodegradable materials (such as corn starch, cassava starch and other starch-based materials, soy protein, whey protein and other protein-based materials; stabilizers are used to maintain the stability and activity of the bacterial agent during storage and transportation. Stabilizers can include: antioxidants, such as ascorbic acid, lipoic acid, ethylenediaminetetraacetic acid, used to prevent the inactivation of microorganisms due to oxidation; humectants, such as glycerol, propylene glycol, sorbitan Alcohol is used to keep the inoculum 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 inoculum to maintain the activity of the microorganisms. Preservatives, such as sodium benzoate, potassium sorbate, and parabens, are used to prevent excessive growth or contamination of microorganisms in the inoculum. Surfactants, such as nonionic surfactants (Tween) and cationic surfactants (such as ammonium dodecyl sulfate), are used to improve the dispersibility and adhesion of the inoculum. Protective agents, such as xanthan gum and carboxymethyl cellulose, are used to protect microorganisms from external factors such as ultraviolet rays. Nutrition 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 a gel-like substance to help fix and protect microorganisms. Antifreeze agents, such as propylene glycol and glycerol, prevent the inoculum from freezing at low temperatures. Antistatic agents, such as quaternary ammonium salt compounds, are used to reduce the accumulation of static electricity in the inoculum during storage and transportation. Chelating agents, such as ethylenediaminetetraacetic acid, are used to chelate metal ions to prevent their adverse effects on microorganisms.

[0060] The present application also provides a method for preparing the above-mentioned bacterial agent, comprising inoculating the above-mentioned pure strain of Halobacillus andaensis into a culture medium for cultivation, and collecting the bacterial liquid.

[0061] The inoculation method is to inoculate the seed solution according to the inoculation amount, the inoculation amount is 0.5-5%, for example, it can be 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%. The seed solution can be obtained by a conventional activation culture method.

[0062] The culture temperature can be 28-37°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 culture rotation speed may 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 10^9~10^10 cfu / mL, the microbial agent mother solution is obtained.

[0066] The preparation method further comprises centrifuging or naturally settling the mother solution of the microbial agent to obtain bacterial precipitate, and resuspending the bacterial body to obtain the bacterial agent.

[0067] The centrifugation can be conventional, for example, conventional centrifugation parameters for separating bacteria from fermentation broth may be a centrifugal force of 3000-5000×g, a centrifugation time of 5-20 minutes, and a temperature of 4° C., and may be adjusted according to actual conditions.

[0068] In a specific embodiment of the present application, the steps of the preparation method are specifically as follows: activating and culturing the strain in a test tube culture medium to prepare a seed solution. Inoculating into a triangular flask containing a medium salinity culture medium at an inoculum of 0.5% to 5%, fermenting and culturing for 36 to 60 hours at 28 to 37°C and a rotation speed of 150 to 200 rpm, so that the number of viable bacteria reaches 10^9 to 10^10 cfu / mL, and preparing a microbial inoculant mother solution. After removing the supernatant, the bacteria are resuspended in ultrapure water to prepare a bacterial suspension for standby use.

[0069] The present application also provides uses of the above-mentioned Halobacillus andaensis or the above-mentioned bacterial agent, wherein the uses are selected from: 1) promoting plant growth under saline-alkali stress; 2) improving plant stress resistance; 3) improving soil; 4) crop production.

[0070] The present application also provides a method for promoting plant growth under saline-alkali stress, comprising applying the above-mentioned Halobacillus andaensis or the above-mentioned bacterial agent to plants or soil.

[0071] The present application also provides a method for improving plant stress resistance, comprising applying the above-mentioned Halobacillus andaensis or the above-mentioned bacterial agent to plants or soil.

[0072] The present application also provides a method for improving soil, comprising applying the above-mentioned Halobacillus andaensis or the above-mentioned bacterial agent to the soil.

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

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

[0075] The plant growth includes processes such as seed germination, seedling growth, vegetative growth, and reproductive growth.

[0076] In a specific embodiment of the present application, after the plant seeds are treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent, the germination rate and rooting rate of the plant seeds in a saline-alkali environment are improved compared with the control group not treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent.

[0077] In a specific embodiment of the present application, after the plant plants are treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent, the plant height, aboveground fresh weight and single plant biomass of the plant plants in a saline-alkali environment are increased compared with the control group not treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent.

[0078] The plant stress resistance refers to the ability of plants to resist and adapt to adverse environmental conditions (such as drought, low temperature, high temperature, salinity, pests and diseases, etc.).

[0079] In a specific embodiment of the present application, after the plant plants are treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent, the proline content and catalase activity in the plant plants are increased compared with the control group not treated with the halophilic Bacillus Halobacillus andaensis nx-9 or the bacterial agent.

[0080] Soil improvement refers to the process of improving the physical, chemical and biological properties of the soil through various methods to increase soil fertility, increase crop yields, improve crop quality and promote healthy plant growth, such as increasing soil fertility: regulating soil pH and improving soil biological activity.

[0081] In a specific embodiment of the present application, the halophilic bacillus Halobacillus andaensis nx-9 or bacterial agent can grow on an Axubei nitrogen-free culture medium plate, so the halophilic bacillus Halobacillus andaensis nx-9 or bacterial agent can improve soil fertility and promote plant growth through nitrogen fixation.

[0082] The crop growth refers to the continuous process of crop biomass increase, morphological construction and physiological function development from sowing to harvesting.

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

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

[0085] Initial screening: Weigh 10.0g of saline-alkali soil from a wolfberry planting base in Ningxia, add 100mL of sterile water, and let stand for 20min. Fully oscillate on an oscillator for 30min to make a basic bacterial suspension. After gradient dilution, the bacterial suspension is added to a medium-salinity (pH8.5 and NaCl 8%) LB medium plate, evenly spread with a sterile coating rod, cultured at 30°C for 3-7d, and single colonies are isolated and numbered. Each single colony is inoculated into a new medium-salinity (pH8.5 and NaCl 8%) LB medium by re-streaking, and the pure culture is separated again by gradient dilution. Each pure culture strain is cultured, the morphology, color, transparency and other characteristics of the colony are observed, and a single colony is picked and stored for later use. Twelve strains were obtained in the initial screening, numbered nx-1 to nx-12.

[0086] Rescreening: The salinity and alkalinity of the LB medium were continuously increased, and cultured at 150 rpm for 4 days. The strain's tolerance to the current concentration of salt and alkalinity was determined by growth, and the strain nx-9 with the best salt and alkalinity tolerance was selected. The rescreening conditions are shown in Table 1.

[0087] Characteristics of Ningxia saline-alkali soil: pH 8.98, organic matter 12.61 g kg -1 , alkaline nitrogen 44.1mg·kg -1 , available phosphorus 118.18 mg kg -1 , fast-acting potassium 782.5 mg kg -1The soil in this area is alkaline, with a pH value of up to 9.0 at a depth of 40 to 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 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 strain morphology and physiological and biochemical characteristics were analyzed by referring to the Manual of Identification of Common Bacterial Systems. The colonies of strain nx-9 on the LB plate were moist and smooth, opaque, with neat edges and a creamy yellow color (see Figure 1 ). Scanning electron microscopy shows that it is a rod-shaped bacterium, about 2 to 5 μm long and about 0.5 to 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] The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit, and the 16S rDNA of the strain was amplified using the universal primers 27F / 1492R. The PCR product was detected by 1% agarose gel electrophoresis, and after gel recovery, it was sent to Shanghai Qingke Biotechnology Co., Ltd. for purification and sequencing. The sequencing result (SEQ ID NO.1) was compared by BLAST in the NCBI database. After purification and sequencing of the PCR product of the nx-9 strain, a 1460bp sequence was obtained. BLAST comparison showed that the homology between the 16S rDNA sequence of nx-9 and Halobacillus andaensis NEAU-ST10-40 (NR135887.1) reached 99.72%. In addition, an evolutionary tree was constructed using the Neighbor-Joining Method, which showed that the genetic distance between nx-9 and Halobacillus andaensis NEAU-ST10-40 (NR135887.1) was the closest ( Figure 4 ). Comprehensive analysis showed that strain nx-9 was Halobacillus andaensis.

[0103] Example 3 Salt and alkali tolerance of strain nx-9

[0104] The salt-alkali tolerance of the strain was evaluated by continuously increasing the salinity and alkalinity of the LB medium. Keeping the pH of the LB medium at 7.0, the salinity of the medium was increased by 2% in sequence, that is, the mass fraction of NaCl was 10%, 12%, 14%, 16%, 18%, and 20%, respectively; while keeping the salinity of the LB medium at 1% NaCl, the pH value was increased by 1 unit, that is, the pH values ​​were 9, 10, 11, 12, and 13, respectively. Cultured at 30°C and 150rpm for 4 days, the strain's tolerance to the current concentration of salt and alkalinity was judged by the growth situation, as shown in Table 4. The strain was inoculated into an LB liquid medium with high salinity and alkalinity (NaCl concentration and pH value were adjusted) to analyze the strain's simultaneous tolerance to salinity and alkalinity. From the cryo-scanning electron microscopy images and field emission scanning electron microscopy results ( Figure 3 ) showed that under high salinity and alkalinity conditions (16% NaCl and pH 10.0), the surface of nx-9 bacteria was enriched with small particles, and the bacteria were mostly deformed and had a rough surface, which may be related to its 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 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. In addition, nx-9 can grow under conditions of both high salinity and alkali concentrations (NaCl concentration>10% and pH>9), and grows well under salinity conditions of 16% NaCl and pH 10.0.

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

[0110] In order to study the salt reduction, acid and alkali reduction ability of the nx-9 strain, LB liquid culture medium with different salt and alkali concentrations was prepared. After culturing at 30℃ and 150rpm for 4 days, the pH and conductivity in the culture medium were measured, and the acid and alkali reduction rate and salt reduction rate of the strain were analyzed: acid and alkali reduction rate = (initial pH-pH after fermentation) / initial pH×100%; salt reduction rate = (salinity before culture-salinity after culture) / salinity before culture×100%. A sterile toothpick was used to pick the colony and inoculate it on the Axubei nitrogen-free culture medium plate, which was placed in a 30℃ incubator for culture, and the growth on the culture medium was observed ( Figure 5 ).

[0111] The specific operation of determining salinity change by conductivity method is:

[0112] The supernatant before and after the culture was diluted 100 times, the conductivity of the dilution was measured and the salinity (S) was calculated.

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

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

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

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

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

[0118]

[0119]

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

[0121]

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

[0123] Example 5: Effect of nx-9 on seed germination of green vegetables under saline-alkali stress

[0124] (1) Preparation of bacterial suspension. Pick a single colony from the plate and transfer it to a fresh liquid culture medium for activation culture to prepare a seed solution. Inoculate it into a triangular flask containing a medium-salinity culture medium at a 0.5% to 5% inoculation rate, and ferment and culture it at 28 to 37°C and 150 to 200 rpm for 36 to 60 hours until the number of viable bacteria reaches 10^9 to 10^10 cfu / mL to obtain a mother solution of microbial inoculant. After centrifugation to remove the supernatant, resuspend the bacteria in ultrapure water to prepare a bacterial suspension for use.

[0125] (2) Seed disinfection: Select a number of "New Aiqing" cabbage seeds, soak them in 75% ethanol for 10 minutes for disinfection and sterilization, and rinse them with ultrapure water 3 to 4 times.

[0126] (3) Seed germination test. The sterilized seeds were soaked in bacterial suspension for 4 h and placed in a culture dish containing 20 mM saline-alkali mixture (NaCl, Na2SO4, NaHCO3, Na2CO3 = 9:1:1:9). For the control group CK, the sterilized seeds were soaked in sterile water for 4 h, and the culture conditions of the culture dish were the same. 25 seeds were treated for each treatment, and repeated 3 times. The seeds were placed in a constant temperature incubator at 25°C and protected from light for 7 days. The germination rate and rooting rate were calculated: germination rate = number of germinated seeds / number of test seeds × 100%; rooting rate = number of rooted seeds / number of test seeds × 100%.

[0127] Table 7 Effect of strain nx-9 on germination of cabbage seeds

[0128]

[0129] Note: Mean(root length ≤ 1 cm) means the mean proportion of roots with length ≤ 1 cm.

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

[0131] Example 6 Effect of nx-9 on the growth of Chinese cabbage seedlings under saline-alkali stress

[0132] (1) Set salinity of 0, 20, 40, 60, 80, and 100 mM (NaCl, Na2SO4, NaHCO3, Na2CO3 = 9:1:1:9) to observe the growth of Chinese cabbage seedlings under different salinity stresses. According to the test results, 60 mM was finally determined as the salinity to be selected in the future.

[0133] (2) Preparation of bacterial suspension. Activate and culture the strain in a medium-salinity alkalinity culture medium in a test tube to prepare a seed solution. Inoculate into a conical flask containing a medium-salinity alkalinity culture medium at a 0.5% to 5% inoculation rate, and ferment and culture at 28 to 37°C and 150 to 200 rpm for 36 to 60 hours until the number of viable bacteria reaches 10^9 to 10^10 cfu / mL to obtain a microbial agent mother solution. After centrifugation to remove the supernatant, resuspend the bacteria in ultrapure water to prepare a bacterial suspension for 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, and an equal amount of tap water was added to the soil of the control group and stirred evenly. Two green vegetable seedlings with full seeds of the same size were evenly sown in the pot, with 10 replicates per treatment. After the seedlings grew 2 to 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. Other subsequent management measures were the same. After 15 days of cultivation, the green vegetable seedlings were harvested and relevant indicators were measured.

[0135] Table 8 Effect of strain nx-9 on the growth of Chinese cabbage seedlings

[0136]

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

[0138] The results showed that, as shown in Table 8, the application of nx-9 bacteria significantly increased the plant height (2.39 cm), aboveground fresh weight (0.26 g) and single plant biomass (0.04 g) of Chinese cabbage seedlings, which were increased by 60.40%, 73.33% and 100.00% respectively compared with the CK treatment. In addition, the stress resistance of plants was also improved, specifically, the proline content and catalase activity increased by 244.86% and 40.03% respectively. Therefore, the strain nx-9 can significantly alleviate the effects of saline-alkali stress on plant growth and promote plant growth.

[0139] The above embodiments are merely illustrative of the principles and effects of the present application and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed in the present application shall still be covered by the claims of the present application.

Claims

1. A halophilic bacillus Halobacillus andaensis, whose deposit number is CCTCC M 20232394.

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

1.

3. The halophilic bacillus according to claim 1, characterized in that The halophilic bacillus Halobacillus andaensis can grow under the 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 has the ability to reduce salt, produce acid and reduce alkali, and fix nitrogen.

4. The Halobacillus andaensis according to claim 3, characterized in that The halophilic bacillus Halobacillus andaensis has any one or more of the following characteristics: 1) The culture time under the conditions of 16% NaCl concentration and pH 10.0 is less than or equal to 4 days; 2) Under the conditions of 8% NaCl concentration and pH 8.5, the acid-reducing rate was 12.17% and the salt-reducing rate was 11.46%; 3) Under the conditions of 16% NaCl concentration and pH 10.0, the acid-reducing rate was 16.53%, and the salt-reducing rate was 4.40%.

5. A bacterial agent comprising the Halobacillus andaensis according to any one of claims 1 to 4.

6. A method for preparing the bacterial agent as claimed in claim 5, comprising inoculating the pure strain of Halobacillus andaensis as claimed in any one of claims 1 to 4 into a culture medium for cultivation, and collecting the bacterial liquid.

7. Use of the Halobacillus andaensis according to any one of claims 1 to 4 or the microbial agent according to claim 5, wherein the use is selected from: 1) promoting plant growth under saline-alkali stress; 2) improving plant stress resistance; 3) improving soil; 4) crop production.

8. A method for promoting plant growth under saline-alkali stress, comprising applying the Halobacillus andaensis according to any one of claims 1 to 4 or the bacterial agent according to claim 5 to plants or soil.

9. A method for improving plant stress resistance, comprising applying the Halobacillus andaensis according to any one of claims 1 to 4 or the bacterial agent according to claim 5 to plants or soil.

10. A method for improving soil, comprising applying the Halobacillus andaensis according to any one of claims 1 to 4 or the bacterial agent according to claim 5 to the soil.

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