Pristia megaterium M23 and application thereof

Abstract

The invention provides a strain of Priestia megaterium M23 and application thereof, belongs to the technical field of agricultural microbial strains, and particularly provides a strain of Priestia megaterium M23 with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M 20232384, and a strain of Priestia megaterium M23 with the preservation number of CCTCC NO: M 20232384 and a strain of Priestia megaterium M23 with the preservation number of CCTCC NO: M 20232384. The strain Pristia megaterium M23 provided by the invention has high salt tolerance and drought tolerance, has the function of promoting plant growth, improves the salt tolerance and drought tolerance of plants, can improve the soluble sugar content of the plants, increases the abundance of soil microorganisms, increases the urease activity of the soil, improves the quality of crop seeds and the like, and has important application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of agricultural microbial strains and specifically relates to a strain Priestia megaterium M23 and its applications. Background Art

[0002] Abiotic stresses such as drought and high salinity are the primary natural disasters affecting crop growth and development, and have become one of the most significant factors limiting global crop production. In my country, approximately half of its territory lies in arid and semi-arid zones. Therefore, drought is the primary factor limiting corn production. As global temperatures rise, water resources in my country are becoming increasingly scarce, and the impact of drought on corn yields will only grow. According to data from the Food and Agriculture Organization of the United Nations, over one billion hectares of saline soil worldwide are too saline to be effectively utilized due to excessive salinity. Furthermore, the area of ​​saline-alkali land is expanding due to a combination of factors, including climate change, frequent extreme weather events, and inefficient farming systems. Soil salinization has become a global challenge. my country also has significant saline-alkali land. Arable land is a primary factor in ensuring food production, so tapping the potential of saline-alkali land and developing its comprehensive utilization are crucial for food security. Furthermore, with climate change, high temperatures are leading to water shortages. This is particularly true in saline-alkali land, where high salinity and drought stress often coexist. Therefore, improving crop tolerance to drought and salinity is crucial. For sustainable agriculture in saline soils, extensive development of salt-tolerant and drought-tolerant plant growth-promoting bacteria and other symbiotic bacteria is needed.

[0003] Soils with excessively high salt content (i.e., electrical conductivity (EC) > 4dS / M) are classified as saline soils. Salinized areas are rapidly increasing due to various factors, including low precipitation, high surface evaporation rates, weathering of local rocks, saline irrigation, and poor cultural practices. Salt stress severely impacts plant growth and causes significant losses to agricultural production. Therefore, for sustainable agricultural development, a range of mitigation strategies are needed to address soil salinization. Plant growth-promoting microorganisms are a natural tool for combating soil salt stress.

[0004] Drought is also the most important natural disaster affecting crop growth and development, and has become one of the main factors limiting global crop production. Drought not only affects crop growth and development and the structure of rhizosphere microbial communities, but also increases soil heterogeneity, restricts nutrient migration, and increases soil oxygen content, leading to a significant reduction in soil microbial biomass. Drought can significantly alter the structure of bacterial and fungal communities in the soil and promote the enrichment of drought-tolerant microorganisms. Therefore, it is of great significance to develop microbial agents that can resist drought stress and allow them to colonize in arid soils, thereby promoting plant growth.

[0005] Therefore, developing and researching bacterial strains that can be used in both saline-alkali soils and arid soils has important application value. Most plant growth-promoting bacteria discovered at home and abroad are unable to colonize in extremely arid soils, so developing microbial agents that can withstand hypertonic environments is of great significance. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a Priestia megaterium M23 bacteria and its applications.

[0007] The present invention isolates a bacterium from the rhizosphere soil of the halophyte Suaeda salsa, which grows in saline-alkali soil in Dongying, Shandong Province. The inventors found that in addition to producing auxin and being salt- and drought-resistant, the bacterium also has the functions of nitrogen fixation, phosphorus solubilization, ammonia production, and extracellular polysaccharide production. As a functional strain, it can promote plant growth under normal conditions and improve the salt and drought resistance of plants, and therefore has important application value.

[0008] This study found Priestia megaterium M23 has been identified as a plant growth-promoting bacterium that can significantly improve plant salt resistance and drought resistance, and has the characteristics of high salt tolerance and high drought resistance. It will provide a valuable source of strains for enriching the diversity of salt-tolerant plant growth-promoting bacteria.

[0009] The technical solutions of the present invention are as follows: One Priestia megaterium M23 bacteria were deposited in the China Center for Type Culture Collection on November 29, 2023, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20232384.

[0010] The above strains Priestia megaterium The method for culturing M23 comprises the following steps: The strain Priestia megaterium M23 was inoculated on a solid culture medium and cultured at 30-37°C for 1-2 days. Then, a colony was picked and inoculated into a liquid culture medium and cultured at 35-37°C and 150-200 rpm for 16-20 hours to obtain a fermentation broth of strain M23.

[0011] According to a preferred embodiment of the present invention, in the culture method, each liter of the solid culture medium contains 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 20 g / L of agar, and the balance of water; Each liter of the liquid culture medium contains the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the balance water.

[0012] The above strains Priestia megaterium Application of M23 in the production of biological agents.

[0013] Preferably according to the present invention, the biological preparation is a liquid preparation.

[0014] More preferably, the biological preparation contains the strain Priestia megaterium The effective viable count of M23 is 10 8 pcs / ml or more.

[0015] The above strains Priestia megaterium Application of M23 in plant cultivation.

[0016] According to the present invention, preferably, the application includes any one of the following: ① strain Priestia megaterium M23 is used to increase the soluble sugar content in plants; ② strain Priestia megaterium M23 is used to increase plant biomass; ③ strain Priestia megaterium M23 is used to increase VC in plant seeds 、 β-glucan and soluble protein content; ④ Priestia megaterium M23 strain absorbs Na in the environment under salt stress conditions + And accumulate K + To balance the K + / Na + ; ⑤ strain Priestia megaterium M23 is used to increase the salt tolerance of plants; ⑥ strain Priestia megaterium M23 can reduce Na in plant leaves under salt stress conditions. + content, increasing K in leaves + content; ⑦ strain Priestia megaterium M23 is used to increase the drought resistance of plants.

[0017] According to a preferred embodiment of the present invention, the plant is a crop.

[0018] Further preferably, in the application, the crops are corn and wheat.

[0019] The above strains Priestia megaterium Application of M23 in the production of one or more of glutamic acid (Glu), glutamine, proline, lysine, tyrosine, and phenylalanine.

[0020] The above strains Priestia megaterium Application of M23 in soil improvement.

[0021] According to the present invention, preferably, the application, strain Priestia megaterium The application of M23 in increasing soil microbial abundance and soil urease, dehydrogenase and alkaline phosphatase activities.

[0022] Beneficial effects of the present invention The strain provided by the present invention Priestia megaterium M23 has high salt and drought tolerance, can produce auxin, can fix nitrogen, solubilize phosphate, produce ammonia, produce extracellular polysaccharides, produce glutamic acid (Glu), glutamine, proline, lysine, Tyrosine and Phenylalanine, and has the functions of promoting plant growth, improving plant salt tolerance, drought resistance, biomass, increasing plant soluble sugar content, increasing soil microbial abundance, increasing soil urease activity, and improving crop seed quality. It has many functions and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a salt tolerance test diagram of the salt-tolerant bacteria isolated in Example 1; In the figure: A. Priestia megaterium Colony morphology of M23; B. Priestia megaterium The nitrogen fixation ability of M23 on Axubei culture medium; C. Priestia megaterium The performance of M23 in dissolving phosphate on NBRIP agar medium; D. Priestia megaterium M23 ammonia production performance; E, Priestia megaterium M23 produces long hormone expression.

[0024] Figure 2 for Priestia megaterium The production of elongatin by M23 under different NaCl concentration conditions.

[0025] Figure 3 for Priestia megaterium Exopolysaccharide production of M23; In the figure: Blank: blank control; Pm M23: Priestia megaterium M23; ** Indicates extremely significant difference P≤0.01.

[0026] Figure 4 Under different NaCl concentration conditions, Priestia megaterium OD value determination results of M23 after 12h, 24h, 36h and 48h of growth.

[0027] Figure 5 Under different concentrations of PEG6000, Priestia megaterium OD value determination results of M23 after 12h, 24h, 36h, and 48h of growth.

[0028] Figure 6 Under different NaCl concentration conditions, Priestia megaterium After M23 was grown for 0 h, 24 h, 48 h, and 72 h, the Na + , K + Content determination; In the figure: A is Na + Content determination; B is K + Content determination.

[0029] Figure 7 Under normal conditions and salt stress conditions, and without Priestia megaterium Effects of M23 on corn growth.

[0030] Figure 8 Under normal conditions and salt stress conditions, and without Priestia megaterium Effect of M23 on fresh weight of corn.

[0031] Figure 9 Under normal conditions and salt stress conditions, and without Priestia megaterium Results of SOD, CAT, and POD enzyme activity determination in corn leaves after M23.

[0032] Figure 10 Under normal conditions and salt stress conditions, and without Priestia megaterium Determination results of soluble sugar content in corn leaves after M23.

[0033] Figure 11 The results are as follows: normal conditions and drought stress conditions with or without water application Priestia megaterium Effects of M23 on corn growth.

[0034] Figure 12 The results are as follows: normal conditions and drought stress conditions with or without water application Priestia megaterium Effects of M23 on proline content (A) and ABA content (B) in maize leaves.

[0035] Figures 13 - 15 Under normal growth conditions and salt stress conditions, and without Priestia megaterium Effects of M23 on the microbial community in the maize rhizosphere soil; In the figure: CK: maize rhizosphere soil under normal conditions, CK-D: maize rhizosphere soil under drought stress conditions, PmM23: added Priestia megaterium Corn rhizosphere soil under normal growth conditions of M23, Pm M23-D: added Priestia megaterium M23 maize rhizosphere soil under drought stress conditions; Figure 13 For species composition analysis; Figure 14 for β-diversity analysis (PCoA analysis); Figure 15 Results of LefSe analysis.

[0036] Figure 16 Heat maps of the top 30 phyla (A) and genera (B) in the bacterial rhizosphere soil of maize. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below with reference to the Examples, but the scope of the present invention is not limited thereto. Where specific conditions are not specified in the Examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used without manufacturer's indication are conventional products that can be purchased commercially.

[0038] Biological sample deposit information One Priestia megaterium M23 bacteria were deposited in the China Center for Type Culture Collection on November 29, 2023, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20232384.

[0039] Example 1 Isolation of salt-tolerant plant growth-promoting bacteria The salt-alkali tolerant plant Suaeda salsa (S. Suaeda salas ) were collected from the rhizosphere of a 1.5-μm lysate (0.5 g sample in 1 ml 1× PBS) and plated on LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, balance water) containing 5% NaCl. A single colony was inoculated into liquid LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, balance water) and incubated at 30°C with shaking overnight. For bacterial identification, genomic DNA was extracted from individual isolates and sent to Pasino Biotech for PCR amplification of the 16S ribosomal RNA (rRNA) gene. The resulting sequence was used to identify the genus and species using a BLAST search against the NIH / NCBI bacterial database.

[0040] The inventors isolated a highly salt-tolerant bacterium that can produce longin ( Figure 1 、 Figure 2 ). Its salt tolerance was verified, see Figure 4 It was found that it could tolerate up to 13% NaCl. Priestia megaterium , named Priestia megateriumM23 (abbreviated as Pm M23, M23).

[0041] strain Priestia megaterium M23 was deposited in the China Center for Type Culture Collection on November 29, 2023, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: 20232384.

[0042] Example 2 strain Priestia megaterium M23 activation and bacterial suspension preparation The strain Priestia megaterium Inoculate an M23 slant onto solid culture medium and incubate at 37°C for 24 hours. Select plump, sticky M23 colonies and inoculate into liquid culture medium. Incubate at 37°C with shaking at 150 rpm for 20 hours. Transfer the fermentation broth to a sterile centrifuge bottle and centrifuge at 5000 rpm for 5 minutes to collect the cells. Wash with sterile deionized water and resuspend to a cell count of at least 500 million CFU / mL.

[0043] The solid culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, and the balance is water; the liquid culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the balance is water.

[0044] Example 3 strain Priestia megaterium Determination of salt and drought tolerance of M23 Pick a single colony and activate it on LB medium Priestia megaterium M23 was cultured at 37℃ for 24h before use. Then LB medium containing different concentrations of NaCl (0%, 3%, 5%, 10%, 13%, 14%, 15%) and LB medium containing different concentrations of PEG6000 (0%, 15%, 25%, 30%, 40%) were prepared. Then, 10µL of the activated Priestia megaterium The M23 bacterial solution was then cultured at 37°C in a shaking incubator, and samples were taken after 12h, 24h, 36h, and 48h for OD value determination.

[0045] The results showed that Priestia megaterium M23 can tolerate a maximum NaCl concentration of 13% ( Figure 4 Drought resistance experiments showed that Priestia megaterium M23 could still grow under 40% PEG concentration, indicating that Priestia megaterium M23 has a certain degree of drought resistance ( Figure 5 ).

[0046] Example 4 Priestia megaterium M23 Detection of nitrogen fixation.

[0047] Take the activated Priestia megaterium M23 The bacterial liquid was spotted onto Axubei nitrogen-free medium (KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaCO3 5.0 g, mannitol 10.0 g, CaSO4·2H2O 0.1 g, distilled water 1 L, agar 20 g, pH 7.4-7.6) for growth and cultured at 28°C for 7-10 days. The growth status and morphological characteristics of the strain were observed. If the strain grew well, it had the potential to fix nitrogen.

[0048] The experimental results show that Priestia megaterium M23 The ability to grow on the Axubei nitrogen-free medium proved that strain M23 had the ability to fix nitrogen ( Figure 1 Middle B).

[0049] Example 5 Priestia megaterium Detection of the phosphatase activity of M23.

[0050] Take 5 μL of the activated Pm M23 bacterial solution and spot-inoculate it onto an NBRIP agar medium plate. Place the plate in an incubator and invert it at 28°C for 24 hours before observation. If a transparent circle is present around the colonies, the result is positive, proving that the salt-tolerant bacteria have the ability to dissolve phosphate.

[0051] Preparation of NBRIP agar medium: Weigh 10.0 g of glucose, 25.0 g of Ca3(PO4), 25.0 g of MgCl2, 0.25 g of MgSO4·7H2O, 0.2 g of KCl, 0.1 g of (NH4)2SO4, and 15.0 g of agar, dissolve them in 1000 mL of ddH2O, and sterilize at 121°C for 15 min.

[0052] The experimental results showed that after the NBRIP agar medium was cultured at 28 ℃ for 24 h, a transparent zone appeared near the colonies of salt-tolerant growth-promoting bacteria on the plate surface, proving that the test result was positive. Priestia megaterium M23 Has a strong ability to dissolve phosphates ( Figure 1 Middle C).

[0053] Example 6 Priestia megaterium Ammonia production assay of M23 Will Priestia megateriumM23 was inoculated into 10 mL of 10 g / L peptone solution and cultured in a shaker at 28°C and 180 rpm for 48 h. Then, 0.5 mL of Nessler's reagent was added. If the color in the test tube turned from brown to yellow, the result was positive, indicating that the salt-tolerant bacteria had the ability to produce ammonia.

[0054] The results show that if Figure 1 As shown in D, after adding 0.5 mL of Nessler's reagent, the liquid in the centrifuge tube turned from brown to yellow, and the test result was positive, that is, the bacteria had obvious ammonia production ability.

[0055] Example 7 Determination of the Ability of Halophilic Bacteria to Produce Auxin Will Priestia megaterium The M23 strain was inoculated into LB medium containing 100 mg / L tryptophan (LB medium without tryptophan served as a control). The culture medium, supplemented with 100 µL of sterile water, was used as a blank control and cultured in a shaker at 37°C and 120 rpm for 2 days. An equal volume of the supernatant (4 mL) of the culture, centrifuged at 10,000 rpm for 10 minutes at 4°C, was added to a colorimetric solution (Salkowski colorimetric reagent containing 15 mL of 0.5 mol / L FeCl₃ solution, 300 mL of concentrated H₂SO₄ (specific gravity 1.84), and 500 mL of distilled water; the mixture was shaken well before use and stored in the dark). The mixture was allowed to stand in the dark for 5 hours, and the OD was immediately measured. 530nm Values ​​were calculated using the IAA standard curve Priestia megaterium In order to determine the amount of IAA produced by the M23 strain Priestia megaterium The inventors tested the IAA production of M23 under conditions of different salt concentrations in LB liquid culture containing different concentrations of NaCl (1%-5%).

[0056] It has been determined that Priestia megaterium M23 produced IAA at a concentration of 31.21 mg / L (e.g. Figure 1 Middle E, Figure 2 ) and can also produce longin under high salt conditions ( Figure 2 ).

[0057] Example 8 Priestia megaterium M23 has the function of producing extracellular polysaccharides Will Priestia megateriumAfter 48 hours of shaking culture of the M23 strain, crude polysaccharides were extracted from the fermentation broth using ethanol precipitation. 10 mL of LB broth was placed in a 50 mL centrifuge tube and centrifuged at low temperature to remove bacterial cells. The supernatant was then added with 2 volumes of pre-chilled ethanol and allowed to stand at 4°C for 12 hours before centrifugation at 10,000 rpm for 10 minutes. The resulting precipitate was dissolved in distilled water to a fixed volume, resulting in a crude polysaccharide solution. The OD490 absorbance was determined using the phenol-sulfuric acid method. A glucose standard curve was generated using glucose standard solutions of varying concentrations (0.2, 0.4, 0.6, 0.8, and 1 mg / mL). The polysaccharide content of the strain was calculated based on the standard curve.

[0058] It has been determined that Priestia megaterium M23 can produce 2.1177 mg / mL of extracellular polysaccharide. Figure 3 .

[0059] Example 9 Priestia megaterium The role of M23 in amino acid production The activated bacteria were inoculated into LB medium containing 0%, 3%, 5%, and 10% NaCl concentrations and cultured for 48 hours. 50 mL of cell suspension of bacteria treated with different NaCl levels was collected by centrifugation. The 50 mL cell suspension was pre-cooled in an ice bath and then centrifuged at 6000 rpm and 4°C for 15 minutes to collect the bacteria. The precipitated bacteria were washed twice with isotonic NaCl solution (prepared with sterile 0%, 3%, 5%, and 10% NaCl aqueous solution). After centrifugation again, the bacteria were dissolved with 5 mL of pre-cooled 0.25 mol / L perchloric acid, mixed evenly, and allowed to stand for 10 minutes. The cells were then centrifuged at room temperature for 15 minutes, and the supernatant was used to determine the content of free amino acids (LC-MS).

[0060] The results show that the present invention Priestia megateriumUnder salt-free conditions, M23 primarily produced glutamate (Glu), glutamine, proline, lysine, tyrosine, and phenylalanine. The contents of glutamate (Glu), glutamine, proline, and lysine increased significantly with increasing salinity. Proline content increased by 529.12%, 4359.38%, and 8162.14%, respectively, compared to the 0% control, under 3%, 5%, and 10% NaCl stresses. Glutamate content increased by 529.60%, 291.27%, and 543.21%, respectively, under 3%, 5%, and 10% NaCl stresses compared to the control. Glutamine content increased by 286.74%, 381.12%, and 1231.31%, respectively, under 3%, 5%, and 10% NaCl stresses compared to the control. Lysine content also increased by 56.18% under 10% NaCl stress compared to the control. The contents of Tyrosine and Phenylalanine decreased with the increase of salt stress (Table 1).

[0061] Table 1, amino acid production determination ( Priestia megaterium M23 ) (µg / ml)

[0062] Example 10 Priestia megaterium M23 plays a key role in balancing intracellular and extracellular K + / Na + Application The activated bacteria were inoculated into LB medium containing 0%, 1.5%, and 3% NaCl. The supernatant was collected at different incubation times (0 h, 24 h, 48 h, and 72 h). The supernatant was centrifuged at 12,000 g for 15 min at 4°C, and the Na+ and K+ ion contents in the supernatant were determined using a flame photometer.

[0063] The results showed that compared with the 0% treatment, the Priestia megaterium The Na content in the culture medium during the treatment of M23 + The concentration decreased significantly, while K + The content gradually increased, indicating Priestia megaterium M23 can transform and absorb Na + , and can accumulate K under salt stress conditions + To balance intracellular and extracellular K + / Na + ( Figure 6 ). Example 11 Priestia megateriumM23 is used in corn planting Corn salt tolerance test: Surface sterilized corn seeds were planted in flower pots (10*10*10cm). After the corn germinated, part of the corn was watered and part of the corn was watered with sterile water suspension. Priestia megaterium M23 Before inoculation, the bacterial solution prepared in Example 2 was diluted with sterile deionized water to 1×10 8 Using sterile deionized water as a control, dig a trench around the root (1-2 cm deep) and add the bacterial suspension into the trench at a rate of 50 mL per pot. Then grow a portion of the corn seeded with M23 bacteria under normal conditions and observe the growth of the corn. Priestia megaterium To investigate whether M23 bacteria promotes corn growth under normal growth conditions, a portion of the corn was treated with 250 mM NaCl solution, while a portion of the control corn was also treated with 250 mM NaCl. The potted corn was then cultivated in a greenhouse (temperature 25-28°C, relative humidity 30-45%, light (10,000 lux) for 16 hours, darkness for 8 hours). Priestia megaterium The effects of M23 on salt tolerance in maize were investigated. Physiological indicators related to salt tolerance (superoxide dismutase, catalase, and peroxidase activities in leaves) and plant fresh weight were measured. Superoxide dismutase (SOD) activity in maize leaves was measured using a superoxide dismutase (SOD) kit (ADS-W-KY011), catalase activity was measured using a catalase (CAT) kit (ADS-W-KY002), and peroxidase activity was measured using a peroxidase (POD) kit (ADS-W-KY003).

[0064] The results showed that under normal growth conditions, Priestia megaterium M23 can promote the growth of corn ( Figure 7 ), biomass increased, as shown by the increase in leaf fresh weight ( Figure 8 ).

[0065] Under salt stress conditions, plants will produce more ROS, which will cause varying degrees of damage after being subjected to oxidative stress. Priestia megaterium M23 can increase the activity of superoxide dismutase, catalase and peroxidase in plants, thereby clearing ROS and alleviating oxidative stress. Priestia megaterium M23 can alleviate the damage caused by salt stress by increasing the activities of three enzymes and improve the salt resistance of corn (such as Figure 9 ).

[0066] Corn drought resistance experiment: Surface sterilized corn seeds were planted in flower pots (10*10*10cm). After the corn germinated, part of the corn was watered and part of the corn was watered with sterile water suspension. Priestia megateriumBefore inoculation, the bacterial solution prepared in Example 2 was diluted with sterile deionized water to 1×10 8 Using sterile deionized water as a control, dig a trench (1-2 cm deep) around the roots and add the bacterial suspension into the trench at a rate of 50 mL per pot. Then, grow a portion of the corn treated with M23 bacteria and a portion of the control corn material (watered) under normal conditions and observe the growth of the corn. Priestia megaterium The other part was subjected to drought and water control treatment (when the corn seedlings grew to the three-leaf stage, water control treatment was started to maintain the relative moisture content of the soil at 10%) to observe whether M23 bacteria had an effect on the growth of corn under normal growth conditions; Priestia megaterium The effects of M23 on maize drought resistance were also investigated. Drought-related indicators of maize were also measured (proline and ABA content in maize leaves). ABA content was measured using a plant abscisic acid (ABA) ELISA kit (JM-01051P1). Free proline content was measured using a plant proline ELISA kit (JM-09918P1).

[0067] The results showed that applying Priestia megaterium M23 can significantly improve the drought resistance of corn ( Figure 11 ), and the inventors found that after drought stress, Priestia megaterium M23 may improve the drought resistance of maize by increasing the ABA and proline content in maize leaves ( Figure 12 ).

[0068] Example 12 Priestia megaterium M23 has the effect of increasing the soluble sugar content of plants The soluble sugar content was determined by the method described in Wang B, Li Z, Ran Q, et al. ZmNF- YB16 Overexpression Improves Drought Resistance and Yield by EnhancingPhotosynthesis and the Antioxidant Capacity of Maize Plants[J].) Take the control under normal growth conditions and Priestia megaterium The soluble sugar content of corn leaves infected with M23 bacteria was determined. The results showed that under normal conditions and drought stress, Priestia megaterium M23 can promote the accumulation of soluble sugar in maize leaves ( Figure 10 ).

[0069] Example 13 Priestia megaterium M23 is used in wheat planting Wheat salt tolerance test and its effect on tillering number: Surface sterilized wheat seeds were planted in pots (15 cm in diameter, 25 cm in height). After the wheat germinated, they were placed in a 4-degree incubator for vernalization for 35 days. Then they were grown in a greenhouse (temperature 20-23°C, relative humidity 30-45%, light 16h, dark 8h). After the seedlings resumed growth, some of the wheat was watered normally, and some was watered with sterile water suspended in water. Priestia megaterium Before inoculation, the bacterial solution prepared in Example 2 was diluted with sterile deionized water to 1×10 8 Using sterile deionized water as a control, dig a trench around the roots (1-2 cm deep) and add the bacterial suspension into the trench at a volume of 200 ml per pot. Priestia megaterium A portion of wheat grown with M23 bacteria was observed under normal conditions. Priestia megaterium Effects of M23 bacteria on wheat seed quality under normal growth conditions.

[0070] The results show: Priestia megaterium M23 can increase the VC content in wheat seeds 、 β-Glucan (β-glucan), soluble protein content (Table 2).

[0071] Table 2. Vaccination Pm M23 Effects on wheat seed quality

[0072] Note: Pm M23 is Priestia megaterium M23, **Indicates extremely significant difference, P≤0.01 Example 14 Priestia megaterium M23 for soil improvement Apply strains in normal soil and drought stress soil Priestia megaterium M23 measured the enzyme activities (soil urease activity, soil dehydrogenase activity, alkaline phosphatase activity) of corn rhizosphere soil and the auxin content in the rhizosphere soil. At the same time, it also measured the microbial diversity of the rhizosphere soil under normal conditions and drought stress conditions.

[0073] Bacterial genomic DNA from soil samples was extracted using a rapid DNA extraction kit (MP Biomedicals, Santa Ana, CA) and stored at -20°C prior to further analysis. The quantity and quality of the extracted DNA were determined by spectrophotometry (NanoDrop 1000, Thermo Scientific, USA) and gel electrophoresis, respectively. The extracted DNA was amplified using primers 338F (5'-actcctagggggcagca-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), targeting the V4 region of the bacterial 16s rRNA. High-throughput sequencing was performed on an Illumina HiSeq 2000 (Illumina Inc., San Diego, USA).

[0074] The soil tightly bound to the roots of the potted plants under normal conditions and after 20 days of drought in Example 11 (as rhizosphere soil) was collected for subsequent analysis; Urease activity in the collected soil was measured using a soil urease activity assay kit (ADS-W-TR001-96, Jiangsu Jingmei Biotechnology Co., Ltd.). Soil dehydrogenase activity was measured using a soil dehydrogenase activity assay kit (ADS-W-TR014, Jiangsu Jingmei Biotechnology Co., Ltd.). Soil alkaline phosphatase activity was measured using a soil alkaline phosphatase (S-ALP) activity assay kit (ADS-W-TR010, Jiangsu Jingmei Biotechnology Co., Ltd.). Soil auxin content was measured using a soil auxin assay kit (JM-06093401, Jiangsu Jingmei Biotechnology Co., Ltd.).

[0075] The results show that when Priestia megaterium When M23 was used for soil improvement, under normal conditions, salt stress, and drought stress, the application of Pm M23 increased the auxin content in the rhizosphere soil of plants, as well as the activities of urease, dehydrogenase, and alkaline phosphatase in the soil (Table 3).

[0076] Table 3. Inoculation under drought stress and salt stress conditions Pm Effects of M23 on soil IAA content and enzyme activity

[0077] Note: Pm M23 is Priestia megaterium M23, **Indicates extremely significant difference, P≤0.01 Moreover, under normal soil and drought stress soil growth conditions, the application of M23 can increase the diversity of rhizosphere soil microbial communities ( Figure 13 、 Figure 14 、 Figure 15 ).

[0078] For example: under normal soil growth conditions, Priestia megaterium M23 can increase Firmicutes_A, Firmicutes_D, CSP-3, Dependentiae, Gemmatimonadota, Chlamydiota, Proteobacteria, Fibrobacterota The relative abundance of bacteria.

[0079] At the phylum level, application in drought-stressed soils Priestia megaterium M23 Can increase the rhizosphere soil Actinobacteriota, Chloroflexota, Cyanobacteria, Firmicutes_D, Methylomirabilota, Nitrospirota_A, Deinococcota, Desulfobacterota_G, Desulfobacterota_I, Dependentiae The relative abundance of Nocardioides_A, GWC2-73-18, Ectobacillus, Rubellimicrobium, Sphingomicrobium, Noviherbaspirillum_ A, UBA969, Cupriavidus, Truepera, SZUA-442, Arthrobacter_D The relative abundance of Figure 16 ).

[0080] In summary, the strains involved in the present invention Priestia megaterium M23 The fungus has high salt and drought tolerance, improving the salt and drought tolerance of plants. It can produce auxins, fix nitrogen, produce exopolysaccharides, promote plant growth, increase plant biomass, increase plant soluble sugar content, increase soil microbial abundance, and increase soil urease, dehydrogenase, and alkaline phosphatase activities, among other functions, and has important application value.

Claims

1. One plant Priestia megaterium M23 bacteria were deposited in the China Center for Type Culture Collection on November 29, 2023, address: Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 20232384.

2. The strain according to claim 1 Priestia megaterium The method for culturing M23 is characterized in that: The steps include: The strain Priestia megaterium M23 was inoculated on a solid culture medium and cultured at 30-37°C for 1-2 days. Then, a colony was picked and inoculated into a liquid culture medium and cultured at 35-37°C and 150-200 rpm for 16-20 hours to obtain a fermentation broth of strain M23.

3. The culture method according to claim 2, wherein Each liter of the solid culture medium contains 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 20 g / L of agar, and the balance of water; Each liter of the liquid culture medium contains the following components: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the balance water.

4. The strain according to claim 1 Priestia megaterium Application of M23 in the production of biological agents.

5. The use according to claim 4, characterized in that The biological preparation is a liquid preparation.

6. The use according to claim 5, characterized in that The biological preparation contains bacterial species Priestia megaterium The effective viable count of M23 is 10 8 pcs / ml or more.

7. The strain according to claim 1 Priestia megaterium Application of M23 in plant cultivation.

8. The use according to claim 7, characterized in that The application includes any of the following: ① strain Priestia megaterium M23 is used to increase the soluble sugar content in plants; ② strain Priestia megaterium M23 is used to increase plant biomass; ③ strain Priestia megaterium M23 is used to increase VC in plant seeds 、 β-glucan and soluble protein content; ④ Priestia megaterium M23 strain absorbs Na in the environment under salt stress conditions + And accumulate K + To balance the K + / Na + ; ⑤ strain Priestia megaterium M23 is used to increase the salt tolerance of plants; ⑥ strain Priestia megaterium M23 can reduce Na in plant leaves under salt stress conditions. + content, increasing K in leaves + content; ⑦ strain Priestia megaterium M23 is used to increase the drought resistance of plants; Preferably, the plant is a crop; Preferably, the crops are corn and wheat.

9. The strain according to claim 1 Priestia megaterium Application of M23 in the production of one or more of glutamic acid, glutamine, proline, lysine, tyrosine and phenylalanine.

10. The strain according to claim 1 Priestia megaterium Application of M23 in soil improvement; Preferably, the application, strain Priestia megaterium The application of M23 in increasing soil microbial abundance and soil urease, dehydrogenase and alkaline phosphatase activities.

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