Xylanolytic bacillus CM263 and application thereof in phosphorus release and salt-alkali stress tolerance

Abstract

The application discloses a xylanolytic Bacillus sp CM263 and application thereof in phosphorus release and salt-alkali stress resistance, and belongs to the technical field of saline-alkali soil improvement. The xylanolytic Bacillus sp CM263 disclosed by the application has a preservation number of CGMCC No.38569. The xylanolytic Bacillus sp CM263 disclosed by the application is an advantageous strain which is extremely resistant to salt-alkali and can efficiently release phosphorus, and solves the technical problems in the prior art that microorganisms are difficult to survive in a high-salinity and high-alkali environment, have low phosphorus release activity, are not resistant to salt-alkali, have poor phosphorus release efficiency, and are not applicable to saline-alkali soil.

Description

Technical Field

[0001] This invention relates to the field of saline-alkali land improvement technology, and more specifically to a xylan-producing Bacillus CM263 and its application in phosphorus solubilization and salt-alkali stress tolerance. Background Technology

[0002] Phosphorus is one of the essential macroelements for plant growth and development, playing a vital role in plant life activities such as cell division and growth, as well as in metabolic pathways and regulation, including photosynthesis, nutrient transport, and glycolysis. In soil, phosphorus exists primarily in two forms: inorganic phosphorus (including insoluble calcium phosphate, hydroxyapatite, and ferric phosphate) and organic phosphorus (nucleic acids, phospholipids, and phosphoproteins). The content of soluble phosphorus in soil is very low, with only a small amount directly absorbed and utilized by plants. Phosphate-solubilizing bacteria are a type of growth-promoting bacteria that can convert insoluble phosphorus into phosphorus that plants can absorb and utilize, playing a crucial role in increasing the available phosphorus content in soil. Therefore, screening for dominant strains with highly efficient phosphorus-solubilizing capabilities has become a research hotspot in the field of modern agricultural microbiology.

[0003] Various types of saline-alkali soils are widely distributed in my country, covering a total area of ​​nearly 1.5 billion mu (approximately 100 million hectares), accounting for 4.5% of the country's usable soil area. Due to prominent soil constraints, strong water and heat resource limitations, large topographical limitations, and fragile ecology, saline-alkali soils have become one of the important environmental factors restricting agricultural production. Saline-alkali soils are rich in inorganic salts but lack organic matter, leading to soil compaction. Continuous application of inorganic phosphate fertilizers results in the accumulation of large amounts of insoluble phosphates in the soil, further degrading it. Furthermore, high salinity and high pH reduce the activity of various enzymes, such as alkaline phosphatase, restricting the form transformation and availability of phosphorus in the soil, thus preventing vegetation in saline-alkali areas from fully absorbing and utilizing phosphate fertilizers. Methods for improving saline-alkali land mainly include water conservancy engineering improvement, chemical improvement, and biological improvement. Among these technical measures for saline-alkali land management, biological improvement is considered an economical, effective, and environmentally friendly approach. By screening for phosphate-solubilizing bacteria adapted to saline-alkali environments, we can not only increase the phosphorus content of saline-alkali land and promote plant growth, but also improve the physical and chemical properties of the soil, which is beneficial for the bioremediation and improvement of saline-alkali soil. This will expand the saline-alkali land resources with development and utilization potential and support the improvement of national grain production capacity.

[0004] Currently reported phosphate-solubilizing bacteria mainly focus on genera such as Bacillus, Pseudomonas, Serratia, Agrobacterium, and Arthrobacter. However, reports indicate that different genera and even different species within the same genera exhibit varying phosphate-solubilizing efficiencies and mechanisms. Furthermore, their performance in saline-alkali soils varies; some phosphate-solubilizing bacteria can only tolerate high-salt environments and not high-alkali environments, while others can only tolerate high-alkali environments and not high-salt environments. Therefore, screening for phosphate-solubilizing bacteria that can tolerate both high-salt and high-alkali environments is of significant agricultural production value for developing stable and highly efficient phosphate-solubilizing microbial agents for application in saline-alkali soils.

[0005] Therefore, providing a xylan-producing Bacillus CM263 and its application in phosphorus solubilization and salt-alkali stress tolerance is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a xylan-based Bacillus CM263 and its application in phosphorus solubilization and salt-alkali stress tolerance. CM263 is an extremely salt-alkali tolerant and highly efficient phosphorus solubilizing strain, which solves the technical problems in the prior art where microorganisms have difficulty surviving in high salinity and high alkalinity environments, have low phosphorus solubilizing activity, are not salt-alkali tolerant, have poor phosphorus solubilization efficiency, and are not suitable for saline-alkali land.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A strain of xylan-based Bacillus ( Paenibacillus xylanilyticus CM263, with accession number CGMCC No. 38569, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is May 6, 2026. It is classified as *Bacillus xylanatus*. Paenibacillus xylanilyticus .

[0009] Furthermore, the application of the xylan-containing Bacillus CM263 in phosphorus solubilization.

[0010] Furthermore, the application of the xylan-containing Bacillus CM263 in phosphorus solubilization under saline-alkali conditions.

[0011] Furthermore, the application of the xylan-containing Bacillus CM263 in maize's tolerance to salt and alkali stress.

[0012] Furthermore, the application of the xylan-containing Bacillus CM263 in promoting phosphorus absorption in maize plants under saline-alkali stress.

[0013] Furthermore, the application of the xylan-containing Bacillus CM263 in promoting maize photosynthesis under saline-alkali stress.

[0014] Furthermore, the application of the xylan-producing Bacillus CM263 in promoting the activity of antioxidant enzymes in maize under saline-alkali stress.

[0015] As can be seen from the above technical solution, compared with the prior art, this invention discloses a xylan-producing Bacillus CM263 and its application in phosphorus solubilization and salt-alkali stress tolerance. The CM263 strain can tolerate both high-salt and high-alkali environments, making it more valuable for development and application than existing salt-alkali tolerant phosphorus-solubilizing bacteria. In saline-alkali or low-phosphorus soils, it can convert insoluble phosphorus into soluble phosphorus, especially in moderately to severely saline-alkali soils, still exhibiting good phosphorus solubilization effects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 This is a colony morphology diagram of strain CM263.

[0018] Figure 2 The OD600 value (A) and soluble phosphorus content (B) of strain CM263 under different pH conditions are shown.

[0019] Figure 3 The OD600 value (A) and soluble phosphorus content (B) of strain CM263 under different NaCl concentrations are shown.

[0020] Figure 4 Maize plants with CM263 added under a salt gradient; from left to right, the NaCl concentrations are 0 mM, 100 mM, 200 mM and 300 mM.

[0021] Figure 5 The effects of strain CM263 on fresh weight (A) and dry weight (B) of maize under salt-alkali stress.

[0022] Figure 6 The effects of strain CM263 on MDA content (A), POD activity (B), CAT activity (C), and SOD activity (D) in maize leaves under salt-alkali stress were investigated.

[0023] Figure 7 The effects of strain CM263 on net photosynthetic rate (A), stomatal conductance (B), intercellular CO2 concentration (C), and transpiration rate (D) of maize leaves under salt-alkali stress were investigated.

[0024] Figure 8 The effect of strain CM263 on chlorophyll content in maize leaves under salt-alkali stress.

[0025] Figure 9 The effect of strain CM263 on the total phosphorus content in maize leaves under salt-alkali stress. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Isolation and Identification of Phosphate-Solubilizing Bacteria CM263 1) Strain CM263 was isolated from extremely saline-alkali soil near Dingbian Salt Lake in Shaanxi Province using the streak plating method. The specific steps are as follows: (1) The composition of inorganic phosphorus solid medium is as follows: with slight modifications to the composition of NBRIP medium, this medium contains 10 g glucose, 5 g Ca3(PO4)2, 0.03 g FeSO4·7H2O, 0.5 g (NH4)2SO4, 0.3 g MgSO4·7H2O, 0.3 g KCl, 0.3 g NaCl, 0.03 g MnSO4 and 18 g agar per liter. Distilled water is added to 1000 ml and the pH of the medium is adjusted to 7.0-8.0.

[0028] (2) Separation and purification: Weigh 10g of saline-alkali soil and dissolve it in 90ml of sterile water. After shaking and culturing at 150rpm for 30min, dilute it to 10ml each time. -5 Inoculate 0.2 ml of diluted solution onto inorganic phosphorus solid medium and incubate upside down in a 28°C incubator for 7 days. Select colonies that produce a clear, transparent halo and streak them 2-3 times on inorganic phosphorus solid medium plates to further purify the strain until a single colony grows on the agar plate.

[0029] 2) The CM263 strain was classified and identified using pyrosequencing. The specific steps are as follows: (1) Preparation of total bacterial genomic DNA: Genomic DNA of strain CM263 was extracted using a bacterial genomic DNA extraction kit (Tiangen) and used as a template for PCR reaction.

[0030] (2) PCR amplification of genes: Following the reaction system specified in the PCR kit instructions, PCR amplification was performed using the universal full-length bacterial primers 27F and 1492R.

[0031] The primer sequences for universal primers 27F and 1492R are as follows: 27F: 5'-AGRGTTTTGATYNTGGCTCAG-3'; SEQ ID NO. 1.

[0032] 1492R: 5'-TASGGHTACCTTGTTASGACTT-3'; SEQ ID NO. 2.

[0033] The reaction conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 40 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ extension for 10 min.

[0034] (3) Purification, sequencing and analysis of PCR products The PCR amplification products were submitted to the Xi'an branch of Beijing Qingke Co., Ltd. for sequencing. The 16S rRNA gene sequence was determined as shown in SEQ ID NO.3. The sequenced product was then entered into the website of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ) and identified by BLAST comparison in the database. The results showed that strain CM263 was related to Bacillus xylanus (Bacillus). Paenibacillus xylanilyticus The 16S rRNA gene sequence of the isolated strain showed 99.36% homology, therefore the isolated strain was identified as a xylan-like Bacillus (Bacillus). Paenibacillus xylanilyticus ).

[0035]

[0036] 3) The morphological characteristics of strain CM263 are as follows: CM263 strain belongs to the phylum Firmicutes, family Bacillusaceae, genus Bacillus, and is Gram-positive. On LB agar plates, colonies appear as white, translucent colonies with irregular edges. Figure 1 It can utilize glucose, maltose, xylose, and sucrose as its sole carbon source, but cannot utilize lactose or galactose as its sole carbon source; its sole nitrogen source can be ammonium sulfate, ammonium dihydrogen phosphate, sodium nitrate, and potassium nitrate. The strain can tolerate a high-salt, high-alkalinity environment with pH=9 and 8.8% NaCl. The optimal growth conditions are: pH=5.0 or 8.0, and temperature 30℃.

[0037] Example 2: Physiological and biochemical characteristics of strain CM263 (1) Starch hydrolysis test Preparation of beef extract peptone agar medium containing 0.2% soluble starch: 3 g / L beef extract, 5 g / L sodium chloride, 10 g / L peptone, 2 g / L soluble starch, 18 g / L agar powder. Adjust pH to 7-8. Autoclave at 121℃ for 20 min. Pour plates in a clean bench for later use. Lugol's iodine solution: 1 g iodine, 2 g potassium iodide. Dissolve potassium iodide in a small amount of deionized water, then add iodine and dissolve completely. Dilute with water to 300 ml. Inoculate the bacterial strain onto the agar plates and incubate upside down at 30℃ for 3-5 days. After colony formation, add Lugol's iodine solution, spreading evenly across the plate. Observe after a short time. If the medium turns deep blue and a colorless transparent zone appears around the colony, it indicates that the strain can secrete amylase to hydrolyze starch, which is a positive reaction. If no transparent zone appears around the colony, it indicates that the strain cannot hydrolyze starch, which is a negative reaction.

[0038] (2) VP test Culture medium preparation: 5 g / L peptone, 5 g / L glucose, 5 g / L NaCl, adjust pH to 7.0-7.2, dispense 10 ml into each test tube, and sterilize at 121℃ for 20 min. Reagents: 40% KOH, 6% α-naphthol anhydrous ethanol solution. In a clean bench, inoculate the strain into the above culture medium and incubate at 30℃ with shaking for 3 days. Take 5 ml of culture medium, add 300 μl of 40% KOH solution and 300 μl of 6% α-naphthol solution to the test tube, shake vigorously to mix, and then incubate at 37℃ for 15-30 min. If the culture medium turns red, it is a positive VP reaction. Some bacteria require 4 hours to show a red color. If no red color appears after 4 hours, it can be considered a negative reaction.

[0039] (3) MR test Culture medium preparation: Same as for the VP test.

[0040] MR reagent: Weigh 0.04g of methyl red, dissolve it in 60ml of 95% ethanol solution, and then add 40ml of ultrapure water to make up to 100ml.

[0041] After culturing in the same test tube as the VP test for 3 days, use a pipette to draw 5 ml of culture medium into a sterile centrifuge tube, and add 100 μl of MR reagent (color change range pH=4.2~6.3, color changes from red to yellow). If red appears, it is an MR positive reaction; if it turns yellow, it is a negative reaction.

[0042] (4) Gelatin hydrolysis test Culture medium preparation: 0.5g peptone, 12g gelatin, 100ml deionized water, pH=7.2~7.4. Dispense 10ml into each test tube and sterilize at 121℃ for 20min.

[0043] Inside a clean bench, use a long toothpick to pick up the bacterial strain and puncture it into the gelatin to a depth of about 2 / 3. Incubate at a constant temperature of 20°C for 7 days, and observe the test results daily. If the gelatin itself liquefies due to the high incubation temperature, it should not be shaken. Let it stand in a 4°C refrigerator for 2 hours to solidify, and observe whether the gelatin is liquefied by bacteria. If it is liquefied, the gelatin hydrolysis is positive; otherwise, it is negative.

[0044] (5) Tween 80 hydrolysis test Culture medium preparation: 10 g / L peptone, 5 g / L sodium chloride, 0.2 g / L calcium chloride, 10 ml / L Tween 80, 18 g / L agar powder, pH 7-8. Autoclave at 121℃ for 20 min, then pour into plates for later use. In a clean bench, inoculate the bacterial strain onto the culture medium plates and incubate at 30℃ for 3-5 days. Observe the plates; a white halo around the colonies indicates a positive reaction, while the absence of a halo indicates a negative reaction.

[0045] (6) Catalase activity Add 600 μl of 3% hydrogen peroxide solution to a 1.5 ml centrifuge tube, then add 400 μl of bacterial culture to the solution using a pipette. Observe the generation of bubbles within 30 seconds. The presence of bubbles indicates a positive catalase reaction, while the absence of bubbles indicates a negative catalase reaction.

[0046] (7) H2S production capacity determination Culture medium preparation: 20 g / L peptone, 5 g / L NaCl, 0.5 g / L ferric ammonium citrate, 0.5 g / L Na2S2O3·5H2O, 10 g / L agar, adjust pH to 7.2. Melt the peptone and agar, cool to 60°C and add the other ingredients. Use a pipette to dispense 10 ml into test tubes, sterilize at 112.6°C for 30 min, and let stand upright to solidify before use.

[0047] Dip a sterile long toothpick into the bacterial solution, puncture and inoculate with the H2S test medium, and place in a 28℃ constant temperature incubator for 3 days. If a black precipitate line appears, it indicates that sulfide has been produced, which is a positive reaction; otherwise, it is a negative reaction.

[0048] (8) Determination of hydrolysis capacity of citrate Culture medium preparation: NH4H2PO4 1g / L, K2HPO4 1g / L, NaCl 5g / L, MgSO4·7H2O 0.2g / L, sodium citrate 2g / L, agar powder 18g / L. After heating and dissolving the above components, adjust the pH to 6.8, then add 1% bromothymol blue ethanol solution indicator, shake well, and when it turns yellow-green, use a pipette to pipette 10ml into test tubes, sterilize at 121℃ for 20min to prepare slant culture medium.

[0049] Use a pipette to inoculate 100 μl of bacterial suspension onto a citrate slant culture medium, place it in a 28°C incubator and incubate statically for 3 days. Observe whether the bacteria grow and the color change of the culture medium: yellow or blue indicates a positive reaction; green indicates a negative reaction.

[0050] The physiological and biochemical characteristics of strain CM263 are shown in Table 1.

[0051] Table 1 Physiological and biochemical characteristics of strain CM263

[0052] Note: + indicates a positive reaction; - indicates a negative reaction.

[0053] Example 3: Determination of the phosphorus-solubilizing ability of phosphate-solubilizing bacterium CM263 under different salt and alkali stress conditions. Preparation of culture for strain CM263: The test strain was inoculated into LB liquid medium and cultured at 30℃ and 180r / min with shaking until the logarithmic growth phase. The cells were collected by centrifugation, washed 2-3 times with sterile water, resuspended, and the concentration of the bacterial suspension was adjusted to OD600=0.6.

[0054] (1) The NaCl concentration was set to 0.03% (basal medium), and the pH was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. 0.5 ml of CM263 strain culture (OD600=0.6) was inoculated into 50 ml of inorganic phosphorus liquid medium with tricalcium phosphate as the sole phosphorus source. The culture was incubated at 30℃ with shaking at 180 rpm for 5 days. A plot was drawn with different culture conditions as the x-axis and phosphorus solubility concentration and strain propagation index OD600 value as the y-axis. The results are shown in […]. Figure 2 A and B.

[0055] Depend on Figure 2It is evident that pH significantly affects the stress on phosphate-solubilizing bacteria CM263. At pH=3, the OD600 value is 0.001, and the phosphate-solubilizing concentration is 0.00 mg / L, indicating that the strongly acidic environment strongly inhibits the growth of the strain. At pH=4, the OD600 value remains only 0.002, but the phosphate-solubilizing concentration rapidly increases to 284.26 mg / L. At this stage, phosphate solubilization mainly comes from the contribution of inorganic acids in the liquid culture medium. At pH=5, the OD600 value rises sharply to 2.103, significantly higher than other pH treatments, and the phosphate-solubilizing concentration simultaneously reaches a peak of 613.89 mg / L, significantly higher than other treatments, indicating that the strain proliferates vigorously and has the strongest phosphate-solubilizing ability under these conditions. At pH=6 and 7, the OD600 values ​​are 1.932 and 1.998, respectively, with no significant difference from pH=5. The strain's proliferation remains at a high level, but the phosphate-solubilizing concentrations significantly decrease to 206.21 mg / L and 303.25 mg / L, respectively. Within the alkaline pH range of 8-12, the phosphorus-solubilizing ability of the strain gradually decreased: at pH 8, the phosphorus-solubilizing concentration was 212.26 mg / L, and the OD600 value was 2.028; at pH 9, the OD600 value was 1.49, significantly lower than the pH 5-8 treatment group, while the phosphorus-solubilizing concentration was 100.48 mg / L, still higher than the national standard requirement of 70 mg / L; at pH 10, the phosphorus-solubilizing concentration decreased to 13.39 mg / L, and the OD600 value decreased to 0.008, significantly lower than the pH 5-9 treatment group; at pH 11 and 12, the phosphorus-solubilizing concentrations were only 2.95 mg / L and 0.75 mg / L, respectively, with OD600 values ​​of 0.001 and 0.000, respectively. This indicates that strain CM263 can adapt to a weakly alkaline environment to exert its phosphorus-solubilizing effect, but its growth and function are severely inhibited under strongly alkaline conditions.

[0056] (2) At pH 8.5 and NaCl concentrations of 0 M (0.03%), 0.3 M (1.75%), 0.7 M (4.10%), 1.1 M (6.43%), 1.5 M (8.77%), and 1.9 M (11.11%), 0.5 ml of CM263 strain culture (OD600 = 0.6) was inoculated into 50 ml of inorganic phosphorus liquid medium with tricalcium phosphate as the sole phosphorus source. The culture was incubated at 30°C with shaking at 180 rpm for 5 days. A plot was created with different culture conditions on the x-axis and phosphorus solubility concentration and the strain propagation index OD600 value on the y-axis. The results are shown in [Figure 1]. Figure 3 A and B.

[0057] according to Figure 3It can be seen that as the NaCl concentration increases, the solubility of tricalcium phosphate by strain CM263 first increases and then gradually decreases. Under the condition of 0.03% NaCl basal medium, the phosphorus solubility concentration is 231.90 mg / L, and the OD600 value is 2.02. When the NaCl concentration is increased to 0.3M, the phosphorus solubility concentration increases significantly to 646.15 mg / L, reaching a peak value, and the OD600 value is 1.77. Subsequently, with further increases in salt concentration, the phosphate-solubilizing ability and strain proliferation continued to decline: at 0.7M, the phosphate-solubilizing concentration was 289.89 mg / L, and the OD600 value was 1.68; at 1.1M, the phosphate-solubilizing concentration was 162.36 mg / L, and the OD600 value was 1.46, significantly lower than the basal medium conditions, but still much higher than the national standard requirement of 70 mg / L; at 1.5M, the phosphate-solubilizing concentration plummeted to 4.95 mg / L, and the OD600 value was 0.95, significantly lower than the low-salt treatment group; by 1.9M, the phosphate-solubilizing concentration dropped to 0.00 mg / L, and the OD600 value dropped to 0.17, significantly lower than all treatment groups. This indicates that strain CM263 can adapt to certain concentrations of salt and alkali stress and exert its phosphate-solubilizing effect.

[0058] In summary, the phosphate-solubilizing bacterium CM263 can adapt to both high-salt environments (1.1M NaCl) and weakly alkaline environments (pH=9) to dissolve insoluble phosphates.

[0059] Example 4: Effects of strain CM263 on the biomass of potted maize plants in saline-alkali soil Wash the river sand with running water to remove residual mineral nutrients, then sterilize at 121℃ for 1 hour to remove residual microorganisms. Weigh 1.7 kg of the treated river sand and mix it with tricalcium phosphate at 0.5% as the sole phosphorus source. Place the mixture in clean flowerpots. Disinfect the "Zhengdan 958" corn seeds with 75% anhydrous ethanol for 10 minutes, then rinse 4-5 times with sterile water to remove residual alcohol. Plant 4 seedlings in each flowerpot. One week after emergence, inoculate with 10 ml of CM263 culture (OD600=0.6) three times, 5 days apart. During the first month of the growing season, water and Hoagland nutrient solution (Hoagland nutrient solution preparation: calcium nitrate tetrahydrate 945 mg / L, potassium nitrate 607 mg / L, potassium dihydrogen phosphate 136 mg / L, magnesium sulfate heptahydrate 493 mg / L, iron salt solution) are added. 2.5 mL / L of ferrous sulfate heptahydrate and 5 mL / L of trace element solution. Ferrous salt solution: 2.78 g of ferrous sulfate heptahydrate and 3.73 g of disodium EDTA were dissolved separately in sterile deionized water, then mixed and brought to a final volume of 500 mL, adjusting the pH to 5.5. Trace element solution: 0.83 mg / L potassium iodide, 6.2 mg / L boric acid, 22.3 mg / L manganese sulfate, 8.6 mg / L zinc sulfate, 0.25 mg / L sodium molybdate, 0.025 mg / L copper sulfate, and 0.025 mg / L cobalt chloride. Seedlings were managed normally. One month later, the plants were managed without phosphorus source in the nutrient solution. Four saline-alkali conditions were set up for stress treatment: pH=8.0, and NaCl concentrations of 0 mM, 100 mM, 200 mM, and 300 mM. 100 ml was applied to each pot, with four biological replicates for each treatment. Plants were harvested one month after the stress treatment. See plant photos below. Figure 4 Results for maize biomass (dry weight and fresh weight) are shown below. Figure 5 .

[0060] Figure 5The results showed that under 0 mM NaCl treatment, the fresh weight of the CK group was 68.50 g / plant, and that of the CM263 treatment group was 65.33 g / plant, with no significant difference; the dry weight of the CK group was 37.90 g / plant, and that of the CM263 treatment group was 37.39 g / plant, with no significant difference. Under 100 mM NaCl treatment, the fresh weight of the CK group was 74.50 g / plant, and that of the CM263 treatment group was 74.73 g / plant, with no significant difference; the dry weight of the CK group was 38.90 g / plant, and that of the CM263 treatment group was 38.45 g / plant, with no significant difference. These results indicate that under low salt stress conditions, the promoting effect of strain CM263 on maize biomass is not significant. Under 200 mM NaCl treatment, the fresh weight of the CK group was 60.60 g / plant, and that of the CM263 treatment group was 69.25 g / plant, an increase of 14.3% compared to the control, but the difference was not statistically significant. The dry weight of the CK group was 37.10 g / plant, and that of the CM263 treatment group was 38.48 g / plant, an increase of 3.7% compared to the control, but the difference was not statistically significant. This indicates that under moderate salt stress, strain CM263 showed a trend of promoting maize growth. Under 300 mM NaCl treatment, the fresh weight of the CK group was 51.80 g / plant, and that of the CM263 treatment group was 71.63 g / plant, a significant increase of 38.3% compared to the control, with a statistically significant difference (P<0.05). The dry weight of the CK group was 36.40 g / plant, and that of the CM263 treatment group was 39.15 g / plant, a significant increase of 7.6% compared to the control, with a highly statistically significant difference (P<0.01). The above results indicate that, under high salt stress (300 mM NaCl), inoculation with strain CM263 can significantly promote the accumulation of maize biomass and effectively alleviate the inhibitory effect of high salt stress on maize growth.

[0061] In summary, the growth-promoting effect of strain CM263 on maize biomass is salt concentration-dependent, with the most significant growth-promoting effect under high salt stress (300 mM NaCl), indicating that this strain has good application potential in severely saline-alkali land.

[0062] Example 5: Effects of strain CM263 on antioxidant enzyme activity in saline-alkali potted maize Select maize plants, and collect 0.3g of fresh leaves from each pot for preservation. Four biological replicates are used for each treatment, for a total of 16 samples. Weigh 0.3g of fresh plant leaves (as needed for testing), chop them, and place them in a pre-chilled mortar or centrifuge tube. Add pre-chilled 50 mmol / L phosphate buffer (PBS, pH=7.8, containing 0.1 mmol / L EDTA, 0.3% Triton X-100, and 4% PVPP) at a ratio of 1:5 (w / v), and grind or homogenize thoroughly in an ice bath. Centrifuge the homogenate at 10,000–12,000 rpm for 15–20 min at 4℃, and collect the supernatant as the crude enzyme solution, which is stored at 4℃ for later use. Using the extracted crude enzyme solution, determine the activities of SOD, POD, CAT, and the content of MDA. The results are shown in [Figure number missing]. Figure 6 .

[0063] according to Figure 6 It was found that strain CM263 could alleviate the accumulation of reactive oxygen species in maize in response to salt-alkali stress. Compared with the uninoculated control group (CK), the CM263 treatment group showed slightly higher MDA content at 0 mM and 200 mM, but lower MDA content at 100 mM and 300 mM. Specifically, at 300 mM, the MDA content in the CM263 treatment group was 7.92 μmol / g FW, lower than the 10.15 μmol / g FW in the CK group, but the differences between treatments were not statistically significant. Regarding POD activity, the CM263 treatment group showed higher POD activity than the CK at all four salt-alkali gradients. Specifically, at 200 mM NaCl, the POD activity in the CK group was 50.38 U / g FW, while that in the CM263 treatment group was 66.00 U / g FW, a statistically significant difference. No significant differences were observed at 0 mM, 100 mM, and 300 mM. Regarding CAT activity, the CM263 treatment group showed higher activity than the control (CK) at 0 mM and 100 mM, but lower activity than the control (CK) at 200 mM and 300 mM, with no statistically significant differences among treatments. The CM263 strain treatment group also showed higher SOD activity than the control (CK) at 0 mM NaCl, but lower activity than the control (CK) at 100 mM, 200 mM, and 300 mM, again with no statistically significant differences among treatments.

[0064] Example 6: Effects of strain CM263 on photosynthesis of potted maize in saline-alkali soil Maize plants were selected, and three fully expanded leaves were chosen from each plant. Each leaf was counted 10 times. The response of maize photosynthesis (net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular carbon dioxide concentration) to salt-alkali stress was measured using a Li-6400 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). The results are shown in [Figure number missing]. Figure 7 .

[0065] according to Figure 7It can be seen that strain CM263 can significantly alleviate the adverse effects of salt-alkali stress on maize photosynthesis. Regarding net photosynthetic rate, the CK group under 0 mM, 100 mM, 200 mM, and 300 mM NaCl conditions showed rates of 4.74, 5.09, 5.22, and 5.09 μmol·m⁻¹, respectively. -2 ·s -1 The corresponding concentrations for the CM263 treatment groups were 12.32, 10.98, 9.21, and 9.19 μmol·m⁻¹, respectively. -2 ·s -1 The transpiration rate was significantly higher than that of the control group (CK) at all four salinity gradients. The corresponding transpiration rates for the CK group were 0.97, 0.78, 0.85, and 0.71 mmol·m⁻², respectively. -2 ·s -1 The CM263 treatment groups had concentrations of 0.90, 1.29, 1.83, and 1.81 mmol·m⁻², respectively. -2 ·s -1 Except for the 0 mM condition where no significant difference was observed, the CM263 treatment group showed significantly higher values ​​than the control (CK) at 100 mM, 200 mM, and 300 mM. Regarding stomatal conductance, the CM263 treatment group showed higher values ​​than the control (CK) at all four salinity gradients, but the differences were not statistically significant. As for intercellular CO2 concentration, the CM263 treatment group showed higher values ​​than the control (CK) at 0 mM, 100 mM, and 200 mM, but lower values ​​than the control (CK) at 300 mM, with no statistically significant differences among the treatments.

[0066] Example 7: Effect of strain CM263 on chlorophyll content of saline-alkali potted maize Corn plants were selected, and non-destructive chlorophyll content was measured using a SPAD-502 chlorophyll meter. Three fully expanded functional leaves were selected from each plant. On each leaf, the midrib was selected, and 2-3 readings were taken on both sides of the vein. The average of all readings was taken as the relative chlorophyll content (SPAD value) of that plant. The results of leaf chlorophyll content are shown below. Figure 8 .

[0067] according to Figure 8 It was found that, compared with the uninoculated CK group, the chlorophyll content of maize inoculated with CM263 strain increased under all four salinity gradients. The chlorophyll content of the CK group under 0mM, 100mM, 200mM, and 300mM NaCl conditions were 15.16, 15.84, 16.48, and 15.89, respectively; while the corresponding values ​​for the CM263 treatment group were 15.90, 16.89, 20.71, and 18.76. Under 200mM NaCl conditions, the chlorophyll content of the CM263 treatment group was significantly higher than that of the CK group; under 0mM, 100mM, and 300mM conditions, the chlorophyll content of the CM263 treatment group was higher than that of the CK group, but the differences were not statistically significant.

[0068] Example 8: Effect of strain CM263 on total phosphorus content in leaves of saline-alkali potted maize Phosphorus content in plant leaves was determined using the sulfuric acid-hydrogen peroxide digestion-molybdenum-antimony colorimetric method: Plant leaf samples were dried to constant weight at 80℃ and pulverized through a 40-mesh sieve. Approximately 0.3 g of sample was weighed and placed in a digestion tube, and 10 mL of concentrated sulfuric acid was added. Digestion was carried out on a graphite digester with progressively increasing temperatures (100℃→280℃→400℃). After cooling, 1–5 mL of 30% hydrogen peroxide was added, and digestion continued until the solution was clear and transparent. After dilution of the digest, molybdenum-antimony colorimetric reagent (ammonium molybdate-antimony-ascorbic acid mixture) was added, thoroughly mixed, and allowed to stand at 25℃ for 30 min. The absorbance was measured at a wavelength of 700 nm. A standard curve was prepared using phosphorus standard solutions, and the total phosphorus content of the sample was calculated.

[0069] Selected maize plants were analyzed, and the phytophosphate content in the leaves was measured. The results are shown below. Figure 9 .

[0070] according to Figure 9 It was found that, compared with the uninoculated CK group, the total phosphorus content of maize plants treated with strain CM263 was increased under all four salinity gradients. The total phosphorus content of the CK group under 0 mM, 100 mM, 200 mM, and 300 mM NaCl conditions was 0.85, 1.02, 1.05, and 0.84 g / kg, respectively; while the corresponding values ​​for the CM263 treatment group were 1.46, 1.40, 1.53, and 0.97 g / kg. At 0 mM, the CM263 treatment group was significantly higher than the CK group; at 100 mM and 200 mM, the CM263 treatment group was significantly higher than the CK group; at 300 mM, the CM263 treatment group was higher than the CK group, but the difference was not statistically significant.

[0071] In summary, strain CM263 can promote phosphorus absorption in maize, increase chlorophyll content, significantly enhance net photosynthetic rate and transpiration rate, and to some extent enhance antioxidant enzyme activity under pH=8.0 and various NaCl concentration stress conditions, thus alleviating the damage to maize caused by salt and alkali stress.

[0072] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of xylan-like Bacillus ( Paenibacillus xylanilyticus CM263 is characterized by, Its accession number is CGMCC No.38569, and it has been deposited at the China General Microbiological Culture Collection Center on May 6, 2026.

2. The application of the xylan-derived Bacillus CM263 as described in claim 1 in phosphorus solubilization.

3. The application of the xylan-producing Bacillus CM263 as described in claim 1 in phosphorus solubilization under saline-alkali conditions.

4. The application of the xylan-containing Bacillus CM263 as described in claim 1 in the salt-alkali stress tolerance of maize.

5. The application of the xylan-containing Bacillus CM263 as described in claim 1 in promoting phosphorus absorption in maize plants under saline-alkali stress.

6. The application of the xylan-producing Bacillus CM263 as described in claim 1 in promoting maize photosynthesis under saline-alkali stress.

7. The application of the xylan-producing Bacillus CM263 as described in claim 1 in promoting the activity of antioxidant enzymes in maize under saline-alkali stress.

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