Microorganism screening method capable of improving stress resistance of crops in saline-alkali soil and application of microorganism screening method

Abstract

The invention relates to the field of microorganism screening, in particular to a microorganism screening method capable of improving saline-alkali soil crop stress resistance and application thereof.The microorganism screening method comprises the following steps that S1, in saline-alkali soil with Na < + > larger than 0.6% and pH 8.5-10.0, suaeda glauca rhizosphere soil and root system tissue are collected and inoculated to 0.6 moL / L NaCl, and the saline-alkali soil is inoculated to a culture medium; in a liquid culture medium with the pH value of 8.0, through a gradient saline-alkaline stress enrichment-bifunctional bacterium collaborative screening technology and in combination with an embedding matrix and modified biochar compounded microbial screening agent, compared with the prior art, salt ion adsorption, flora survival rate improvement, root system nitrogen fixation and phosphate solubilizing bacterium standard-reaching rate improvement and crop biomass increase are synchronously realized, and the yield of the crops is increased. And moreover, the toxicity of salt ions is remarkably reduced, the soil micro-ecology is systematically optimized, and the method has a wide application prospect in marginal land agricultural development and saline-alkali soil ecological restoration.

Description

Technical Field

[0001] The present invention relates to the field of microbial screening, and in particular to a microbial screening method capable of improving the stress resistance of crops in saline-alkali land and its application. Background Art

[0002] Soil salinization, as a global ecological problem, seriously restricts the sustainable development of agriculture in arid and semi-arid areas. High concentrations of sodium ions and alkaline environment not only directly cause osmotic stress and ion toxicity in plants, but also systematically inhibit crop physiological functions through multiple mechanisms such as destroying soil aggregate structure and fixing nutrients (such as phosphorus precipitation and nitrogen volatilization).

[0003] In the existing technology, microbial agents have low survival rates and unstable functional expression in saline-alkali environments due to the lack of effective protection against high osmotic pressure and oxidative stress. In addition, although physically modified biochar or humic acid materials can adsorb salt in the short term, the carriers are not functionally modified and cannot continuously activate the soil nutrient cycle. At the same time, although chemical conditioners can quickly reduce alkalinity, they can easily destroy the soil microecological balance. Long-term application leads to increased compaction and a decrease in microbial diversity.

[0004] Therefore, based on the above-mentioned related technologies, there is an urgent need to develop a microbial screening method and its application that can improve the stress resistance of saline-alkali land crops. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to propose a microbial screening method and its application that can improve the stress resistance of saline-alkali land crops, so as to solve the problems in the existing technology of low survival rate of single bacterial agents, short physical improvement time and large side effects of chemical conditioning.

[0006] Based on the above objectives, the present invention provides a method for screening microorganisms that can improve the stress resistance of crops in saline-alkali land and its application.

[0007] A method for screening microorganisms capable of improving stress resistance of crops in saline-alkali land comprises the following steps: Step S1: In saline-alkali land with Na+ > 0.6% and pH 8.5-10.0, rhizosphere soil and root tissue of Suaeda salsa were collected and inoculated into a liquid culture medium containing 0.6 mol / L NaCl and pH 8.0. The NaCl concentration was increased by 0.3 mol / L NaCl and the pH of the culture medium was increased by 0.5 every 48 hours until the NaCl concentration in the liquid culture medium reached 1.5 mol / L and the pH was 9.5. The enrichment dynamics of nitrogen-fixing bacteria and phosphate-solubilizing bacteria were tracked: Step S2: The enriched bacterial solution was spread on 1.2 mol / L NaCl, pH 9.0 Ashby agar and 1.0 mol / L NaCl, pH 8.5 Montana agar, respectively, and colonies within 72 hours with a diameter greater than 1 mm were screened to obtain the primary screening strains: Step S3: Add the primary screened strains to a culture dish containing 1 mol / L NaCl and pH 8.5, retain the top 15% of functional expression, collect the bacterial sludge by centrifugation, add a microbial screening agent, and obtain a bacterial solution; Step S4: Add the plant seeds to the bacterial solution and sow them in sterilized sandy soil containing 150mmoL / LNaCl. After 7 days, the nitrogen-fixing bacteria are detected to be ≥8×10 4 CFU / g root, phosphate-solubilizing bacteria ≥1.2×10 5 CFU / g root; The mass ratio of the primary screening strain to the microbial screening agent is 0.25-0.35:1; The mass ratio of the plant seeds to the bacterial solution is 1:1.0-1.2; Through gradient saline-alkali stress enrichment and synergistic screening of bifunctional bacteria, combined with a specific ratio of microbial screening agents, the survival rate and functional expression efficiency of the strain under saline-alkali stress are significantly improved.

[0008] Preferably, the preparation steps of the microbial screening agent in step S3 are as follows: Step A1: Add sodium alginate, sodium carboxymethyl cellulose, and xanthan gum to deionized water, heat to 50-70°C, stir for 40-50 minutes until dissolution is complete, and cool to 20-40°C to obtain an embedding matrix; Step A2: adding the modified biochar to the microbial culture liquid, heating to 20-30° C., stirring for 20-40 minutes at a speed of 150-250 rpm, standing for 8-12 minutes, adding the embedding matrix, stirring for 4-6 minutes at a speed of 300-500 rpm, adding magnesium humate, chitosan, calcium citrate, glycerol and ascorbic acid, stirring for 12-16 minutes, adjusting the viscosity to 800-1000 mPa·s, placing in a vacuum degassing machine, reducing the pressure to -0.08 MPa, heating to 20-30° C., and degassing for 8-12 minutes to obtain a microbial screening agent; Hydrogel microcapsules are formed by embedding the matrix of sodium alginate-xanthan gum to maintain the intracellular osmotic balance of microorganisms. At the same time, ascorbic acid can remove reactive oxygen species and reduce the damage of oxidative stress to bacteria. Glycerol, as an osmotic protectant, can stabilize the cell membrane structure and prolong the survival period of the bacterial community under adverse conditions. In addition, magnesium humate provides Mg 2+ , promoting chlorophyll synthesis to maintain photosynthesis, while chitosan can stimulate the jasmonic acid signaling pathway of crops and upregulate stress-resistant genes. At the same time, the two synergistically enhance the activity of crop antioxidant enzymes, enhance cell membrane stability, and reduce biomass loss caused by salt stress.

[0009] Preferably, the mass ratio of sodium alginate, sodium carboxymethyl cellulose and xanthan gum in step A1 is 1:0.12-0.14:0.05-0.07; The mass ratio of the modified biochar, microbial culture medium, embedding matrix, magnesium humate, chitosan, calcium citrate, glycerol and ascorbic acid in step A2 is 0.6-0.7: 0.16-0.17: 1: 0.08-0.085: 0.04-0.06: 0.008-0.009: 0.08-0.085: 0.003-0.004.

[0010] Preferably, the steps for preparing the microbial liquid in step A2 are as follows: Step B1: Add tryptone, yeast powder, and NaCl to deionized water, stir for 10-15 minutes, adjust the pH to 7.1-7.3, heat to 110-130°C, and autoclave for 15-25 minutes to obtain LB liquid culture medium; Step B2: Under sterile conditions, add nitrogen-fixing bacteria and phosphate-solubilizing bacteria to LB liquid culture medium, raise the temperature to 25-30°C, culture for 22-26 hours, and shake at 150-200 rpm to obtain an activated bacterial solution; Step B3: Add the modified culture medium to the culture dish, heat it to 110-130℃, sterilize it for 10-30min, cool it to 25-35℃, add the activated bacterial solution, rotate it at 150-250rpm, adjust the pH to 8.3-8.7, and ferment it for 45-50h. 600 ≥2.0, extracellular polysaccharide yield ≥1.5g / L, stop fermentation, add to centrifuge tube, cool to 2-5℃, rotate at 3000-5000rpm, centrifuge for 10-20min, wash, add 0.85% NaCl, and resuspend to 10 10 CFU / mL, microbial culture liquid; Through the synergistic action of nitrogen-fixing bacteria and phosphate-solubilizing bacteria, both secrete organic acids and enzymatic substances respectively, converting the fixed nitrogen and phosphorus elements in saline-alkali land into absorbable forms, thereby significantly alleviating the problem of nutrient fixation caused by high pH in saline-alkali land and improving the efficiency of nutrient acquisition by crop roots.

[0011] Preferably, the mass ratio of tryptone, yeast powder and NaCl in step B1 is 1:0.45-0.55:0.8-1.2; The mass ratio of the nitrogen-fixing bacteria to the phosphate-solubilizing bacteria in step B2 is 1:0.65-0.67; The mass ratio of the improved culture medium to the activated bacterial solution in step B3 is 1:0.04-0.06.

[0012] Preferably, the steps for preparing the improved culture medium in step B3 are as follows: Add glucose, peptone, K2HPO4, MgSO4·7H2O and CaCl2 to deionized water, stir for 20-30 minutes, add NaCl and 0.1 mol / L Tris-HCl buffer, adjust the pH to 8.4-8.6, and obtain a modified culture medium.

[0013] Preferably, the mass ratio of the glucose, peptone, K2HPO4, MgSO4·7H2O, CaCl2, NaCl and buffer solution is 1.8-2.2:1:0.18-0.22:0.018-0.022:0.0008-0.0012:1.7-1.8:1.1-1.3.

[0014] Preferably, the modified biochar preparation steps in step A2 are as follows: Step C1: immersing the straw in a 0.1 mol / L HCl solution, heating to 70-90°C, stirring for 1-3 hours, filtering to a pH of 6.8-7.2, heating to 100-110°C, drying for 1-3 hours, placing in a grinder to a particle size of 0.5-2 mm, introducing nitrogen, placing in a tubular furnace, heating to 480-520°C, keeping warm for 1-3 hours, and cooling to 70-90°C to obtain biochar; Step C2: adding humic acid to a 0.1 mol / L sodium hydroxide solution, heating to 50-70°C, stirring for 50-70 minutes, adding biochar, reacting for 5-7 hours at a speed of 100-200 rpm, filtering, washing, and drying to obtain modified biochar; The modified biochar is acid washed and coated with humic acid to form a porous structure with high specific surface area. Combined with the cationic properties of chitosan, it can adsorb Na + 、Cl - Salt separation ions reduce the osmotic pressure of soil solution. At the same time, humic acid can enhance the soil aggregate structure, improve water retention and air permeability. Combined with chitosan, it can further fix salt through electrostatic effect and reduce the damage of ion poisoning to the root system.

[0015] Preferably, in step C1, the mass ratio of the straw to the HCl solution is 1:9-10; The mass ratio of humic acid to biochar in step C2 is 0.08-0.12:1.

[0016] The invention discloses an application of a microbial screening method capable of improving the stress resistance of crops in saline-alkali land. The microbial screening method is applied to agricultural development and ecological restoration of marginal land.

[0017] Beneficial effects of the present invention: The present invention provides a microbial screening method for improving the stress resistance of crops in saline-alkali land and its application. The present invention uses gradient salt-alkali stress enrichment-bifunctional bacteria collaborative screening technology, combined with an embedding matrix and a modified biocarbon composite microbial screening agent. Compared with the existing technology, the present invention can simultaneously achieve salt ion adsorption, improve the survival rate of the bacterial community, increase the root nitrogen fixation and phosphate solubilizing bacteria compliance rate, increase crop biomass, significantly reduce salt ion toxicity and systematically optimize the soil microecology, and has broad application prospects in marginal land agricultural development and saline-alkali land ecological restoration. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0019] The sources and properties of some of the raw materials used in the present invention are as follows: The nitrogen-fixing bacteria used in the present invention is Kosakonia radicincitans GXGL-4A, which has a public announcement number of "CN106086042A" and an invention patent titled "Azotobacterium ammonium carrier gene, mutant, azotobacterium ammonium carrier mutant and application thereof" and a deposit number of CGMCC No. 12588; The phosphate-solubilizing bacteria used in the present invention is Enterobacter cloacae, which is disclosed in the invention patent with the authorization announcement number of “CN106085896B” and the name of “Enterobacter cloacae and its use”, and the preservation number is CGMCC No.12206.

[0020] Example 1: The steps for preparing the improved culture medium are as follows: Add 180 g of glucose, 100 g of peptone, 18 g of K2HPO4, 1.8 g of MgSO4·7H2O and 0.08 g of CaCl2 to 1000 mL of deionized water, stir for 20 min, add 170 g of NaCl and 110 g of 0.1 mol / L Tris-HCl buffer, adjust the pH to 8.4-8.6, and obtain a modified culture medium.

[0021] Example 2: The steps for preparing the improved culture medium are as follows: Add 200 g of glucose, 100 g of peptone, 20 g of K2HPO4, 2 g of MgSO4·7H2O and 0.1 g of CaCl2 to 1000 mL of deionized water, stir for 25 min, add 175 g of NaCl and 120 g of 0.1 mol / L Tris-HCl buffer, adjust the pH to 8.4-8.6, and obtain an improved culture medium.

[0022] Example 3: The steps for preparing the improved culture medium are as follows: Add 220 g of glucose, 100 g of peptone, 22 g of K2HPO4, 2.2 g of MgSO4·7H2O and 0.12 g of CaCl2 to 1000 mL of deionized water, stir for 30 min, add 180 g of NaCl and 130 g of 0.1 mol / L Tris-HCl buffer, adjust the pH to 8.4-8.6, and obtain a modified culture medium.

[0023] Example 4: The steps for preparing microbial culture liquid are as follows: S1: Add 100 g of tryptone, 45 g of yeast powder, and 80 g of NaCl to 1000 mL of deionized water, stir for 10 min, adjust the pH to 7.1-7.3, heat to 130°C, and autoclave for 15 min to obtain LB liquid medium; S2: Under sterile conditions, add 100 g of nitrogen-fixing bacteria and 65 g of phosphate-solubilizing bacteria to 1000 mL of LB liquid medium, raise the temperature to 25°C, culture for 26 h, and shake at 150 rpm to obtain an activated bacterial solution; S3: Add 100g of the modified culture medium to the culture dish, heat it to 110℃, sterilize it for 30min, cool it to 25℃, add 4g of activated bacterial solution, rotate at 150rpm, adjust the pH to 8.3-8.7, ferment it for 50h, and OD 600 ≥2.0, extracellular polysaccharide production ≥1.5g / L, stop fermentation, add to centrifuge tube, cool to 2℃, rotate at 5000rpm, centrifuge for 10min, wash, add 0.85% NaCl, and resuspend to 10 10 CFU / mL, and obtain the microbial culture liquid.

[0024] Example 5: The steps for preparing microbial culture liquid are as follows: S1: Add 100 g of tryptone, 50 g of yeast powder, and 100 g of NaCl to 1000 mL of deionized water, stir for 13 min, adjust the pH to 7.1-7.3, heat to 120°C, and autoclave for 20 min to obtain LB liquid medium; S2: Under sterile conditions, add 100 g of nitrogen-fixing bacteria and 66 g of phosphate-solubilizing bacteria to 1000 mL of LB liquid medium, raise the temperature to 28°C, culture for 24 h, and shake at 180 rpm to obtain an activated bacterial solution; S3: Add 100g of the modified culture medium to the culture dish, heat it to 120℃, sterilize it for 20min, cool it to 30℃, add 5g of activated bacterial solution, rotate at 200rpm, adjust the pH to 8.3-8.7, ferment for 48h, and OD 600≥2.0, extracellular polysaccharide production ≥1.5g / L, stop fermentation, add to centrifuge tube, cool to 3℃, rotate at 4000rpm, centrifuge for 15min, wash, add 0.85% NaCl, and resuspend to 10 10 CFU / mL, and obtain the microbial culture liquid.

[0025] Example 6: The steps for preparing microbial culture liquid are as follows: S1: Add 100 g of tryptone, 55 g of yeast powder, and 120 g of NaCl to 1000 mL of deionized water, stir for 15 min, adjust the pH to 7.1-7.3, heat to 110°C, and autoclave for 25 min to obtain LB liquid medium; S2: Under sterile conditions, add 100 g of nitrogen-fixing bacteria and 67 g of phosphate-solubilizing bacteria to 1000 mL of LB liquid medium, raise the temperature to 30°C, culture for 22 h, and shake at 200 rpm to obtain an activated bacterial solution; S3: Add 100g of the modified culture medium to the culture dish, heat it to 130℃, sterilize it for 10min, cool it to 35℃, add 6g of activated bacterial solution, rotate at 150rpm, adjust the pH to 8.3-8.7, ferment it for 50h, and OD 600 ≥2.0, extracellular polysaccharide production ≥1.5g / L, stop fermentation, add to centrifuge tube, cool to 2℃, rotate at 5000rpm, centrifuge for 10min, wash, add 0.85% NaCl, and resuspend to 10 10 CFU / mL, and obtain the microbial culture liquid.

[0026] Example 7: The steps for preparing modified biochar are as follows: S1: 100g of straw was immersed in 900g of 0.1mol / L HCl solution, heated to 70℃, stirred for 3h, filtered to pH 6.8-7.2, heated to 100℃, dried for 3h, placed in a grinder to a particle size of 0.5-2mm, introduced with nitrogen, placed in a tube furnace, heated to 520℃, kept warm for 1h, and cooled to 90℃ to obtain biochar; S2: Add 8g of humic acid to 150mL of 0.1mol / L sodium hydroxide solution, heat to 50℃, stir for 70min, add 100g of biochar, react for 5h, rotate at 200rpm, filter, wash, and dry to obtain modified biochar. Example 8: The steps for preparing modified biochar are as follows: S1: 100g of straw was immersed in 1000g of 0.1mol / L HCl solution, heated to 80℃, stirred for 2h, filtered to pH 6.8-7.2, heated to 105℃, dried for 2h, placed in a grinder to a particle size of 0.5-2mm, introduced with nitrogen, placed in a tube furnace, heated to 500℃, kept warm for 2h, and cooled to 80℃ to obtain biochar; S2: Add 1g of humic acid to 150mL of 0.1mol / L sodium hydroxide solution, heat to 60℃, stir for 60min, add 100g of biochar, react for 6h, rotate at 150rpm, filter, wash, and dry to obtain modified biochar. Example 9: The steps for preparing modified biochar are as follows: S1: 100 g of straw was immersed in 1100 g of 0.1 mol / L HCl solution, heated to 90°C, stirred for 1 hour, filtered to a pH of 6.8-7.2, heated to 110°C, dried for 1 hour, placed in a grinder to a particle size of 0.5-2 mm, introduced with nitrogen, placed in a tube furnace, heated to 520°C, kept warm for 1 hour, and cooled to 90°C to obtain biochar; S2: Add 1.2g of humic acid to 150mL of 0.1mol / L sodium hydroxide solution, heat to 70℃, stir for 50min, add 100g of biochar, react for 7h, rotate at 100rpm, filter, wash, and dry to obtain modified biochar. Example 10: Preparation method of microbial screening agent S1: Add 100 g of sodium alginate, 12 g of sodium carboxymethyl cellulose, and 5 g of xanthan gum to 150 mL of deionized water, heat to 50°C, stir for 50 min, and dissolve until complete. Cool to 20°C to obtain the embedding matrix. S2: Add 60g of modified biochar to 16g of microbial culture liquid, heat to 20℃, stir for 40min, rotate at 150rpm, let stand for 12min, add 100g of embedding matrix, stir for 4min, rotate at 500rpm, add 8g of magnesium humate, 4g of chitosan, 0.8g of calcium citrate, 8g of glycerol and 0.3g of ascorbic acid, stir for 12min, adjust the viscosity to 800-1000mPa·s, place in a vacuum degassing machine, reduce the pressure to -0.08MPa, heat to 30℃, degas for 8min, and obtain the microbial screening agent.

[0027] Example 11: Preparation method of microbial screening agent S1: Add 100 g of sodium alginate, 13 g of sodium carboxymethyl cellulose, and 6 g of xanthan gum to 150 mL of deionized water, heat to 60°C, stir for 45 min, and dissolve until complete. Cool to 30°C to obtain the embedding matrix. S2: Add 65g of modified biochar to 16.5g of microbial culture liquid, heat to 25°C, stir for 20-40min, rotate at 200rpm, let stand for 10min, add 100g of embedding matrix, stir for 5min, rotate at 400rpm, add 8.3g of magnesium humate, 5g of chitosan, 0.85g of calcium citrate, 8.3g of glycerol and 0.35g of ascorbic acid, stir for 14min, adjust the viscosity to 800-1000mPa·s, place in a vacuum degassing machine, reduce the pressure to -0.08MPa, heat to 25°C, degas for 10min, and obtain a microbial screening agent.

[0028] Example 12: Preparation of microbial screening agent S1: Add 100 g of sodium alginate, 14 g of sodium carboxymethyl cellulose, and 7 g of xanthan gum to 150 mL of deionized water, heat to 70°C, stir for 40 min, and dissolve until complete. Cool to 40°C to obtain the embedding matrix. S2: Add 70g of modified biochar to 17g of microbial culture liquid, heat to 30℃, stir for 20min, rotate at 250rpm, let stand for 8min, add 100g of embedding matrix, stir for 6min, rotate at 300rpm, add 8.5g of magnesium humate, 6g of chitosan, 0.9g of calcium citrate, 8.5g of glycerol and 0.4g of ascorbic acid, stir for 16min, adjust the viscosity to 800-1000mPa·s, place in a vacuum degassing machine, reduce the pressure to -0.08MPa, heat to 20℃, degas for 12min, and obtain the microbial screening agent.

[0029] Example 13: A method for screening microorganisms that can improve the stress resistance of crops in saline-alkali soil S1: In Na + In saline-alkali land with a pH value of 8.5-10.0 and a pH value of >0.6%, rhizosphere soil and root tissue of Suaeda salsa were collected and inoculated into a liquid culture medium containing 0.6 mol / L NaCl and a pH value of 8.0. At 48-hour intervals, the NaCl concentration was increased by 0.3 mol / L NaCl and the pH value of the culture medium was increased by 0.5 until the NaCl concentration in the liquid culture medium reached 1.5 mol / L and the pH value reached 9.5. The enrichment dynamics of nitrogen-fixing bacteria and phosphate-solubilizing bacteria were tracked to obtain the enriched bacterial solution: S2: The enriched bacterial solution was spread on 1.2 mol / L NaCl, pH 9.0 Ashby agar and 1.0 mol / L NaCl, pH 8.5 Montana agar, respectively. Colonies within 72 hours with a diameter greater than 1 mm were selected to obtain the primary screening strains: S3: Add 25 g of the primary screening strain to a culture dish containing 1 mol / L NaCl and pH 8.5, retain the top 15% of functional expression, collect the bacterial sludge by centrifugation, and add 100 g of microbial screening agent to obtain bacterial liquid; S4: 100g of plant seeds were added to 100g of bacterial solution and sown in sterilized sandy soil containing 150mmoL / LNaCl. After 7 days, the number of nitrogen-fixing bacteria was detected to be ≥8×10 4 CFU / g root, phosphate-solubilizing bacteria ≥1.2×10 5 CFU / g root.

[0030] Example 14: A method for screening microorganisms that can improve the stress resistance of crops in saline-alkali soil S1: In Na + In saline-alkali land with a pH value of 8.5-10.0 and a pH value of >0.6%, rhizosphere soil and root tissue of Suaeda salsa were collected and inoculated into a liquid culture medium containing 0.6 mol / L NaCl and a pH value of 8.0. At 48-hour intervals, the NaCl concentration was increased by 0.3 mol / L NaCl and the pH value of the culture medium was increased by 0.5 until the NaCl concentration in the liquid culture medium reached 1.5 mol / L and the pH value reached 9.5. The enrichment dynamics of nitrogen-fixing bacteria and phosphate-solubilizing bacteria were tracked to obtain the enriched bacterial solution: S2: The enriched bacterial solution was spread on 1.2 mol / L NaCl, pH 9.0 Ashby agar and 1.0 mol / L NaCl, pH 8.5 Montana agar, respectively. Colonies within 72 hours with a diameter greater than 1 mm were selected to obtain the primary screening strains: S3: Add 30 g of the primary screening strain to a culture dish containing 1 mol / L NaCl and pH 8.5, retain the top 15% of functional expression, collect the bacterial sludge by centrifugation, and add 110 g of microbial screening agent to obtain bacterial liquid; S4: 100 g of plant seeds were added to 110 g of bacterial solution and sown in sterilized sandy soil containing 150 mmoL / L NaCl. After 7 days, the number of nitrogen-fixing bacteria was detected to be ≥ 8 × 10 4 CFU / g root, phosphate-solubilizing bacteria ≥1.2×10 5 CFU / g root.

[0031] Example 15: A method for screening microorganisms that can improve the stress resistance of crops in saline-alkali soil S1: In Na + In saline-alkali land with a pH value of 8.5-10.0 and a pH value of >0.6%, rhizosphere soil and root tissue of Suaeda salsa were collected and inoculated into a liquid culture medium containing 0.6 mol / L NaCl and a pH value of 8.0. At 48-hour intervals, the NaCl concentration was increased by 0.3 mol / L NaCl and the pH value of the culture medium was increased by 0.5 until the NaCl concentration in the liquid culture medium reached 1.5 mol / L and the pH value reached 9.5. The enrichment dynamics of nitrogen-fixing bacteria and phosphate-solubilizing bacteria were tracked to obtain the enriched bacterial solution: S2: The enriched bacterial solution was spread on 1.2 mol / L NaCl, pH 9.0 Ashby agar and 1.0 mol / L NaCl, pH 8.5 Montana agar, respectively. Colonies within 72 hours with a diameter greater than 1 mm were selected to obtain the primary screening strains: S3: Add 35 g of the primary screening strain to a culture dish containing 1 mol / L NaCl and pH 8.5, retain the top 15% of functional expression, collect the bacterial sludge by centrifugation, and add 100 g of microbial screening agent to obtain bacterial liquid; S4: 100 g of plant seeds were added to 120 g of bacterial solution and sown in sterilized sandy soil containing 150 mmoL / L NaCl. After 7 days, the number of nitrogen-fixing bacteria was detected to be ≥ 8 × 10 4 CFU / g root, phosphate-solubilizing bacteria ≥1.2×10 5 CFU / g root.

[0032] Comparative Example 1: Compared with Example 13, this comparative example did not add an embedding matrix during the preparation of the microbial screening agent. The microbial culture liquid, modified biochar and other components were directly mixed. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, microorganisms that can improve the stress resistance of crops in saline-alkali land were obtained.

[0033] Comparative Example 2: Compared with Example 13, this comparative example only replaces "modified biochar" with "biochar", and the remaining steps and parameters are the same, which will not be repeated in this comparative example. Finally, microorganisms that can improve the stress resistance of crops in saline-alkali land are obtained.

[0034] Comparative Example 3: Compared with Example 13, this comparative example did not add magnesium humate and chitosan during the preparation of the microbial screening agent, and the remaining components were supplemented in proportion. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, microorganisms that can improve the stress resistance of crops in saline-alkali land were obtained.

[0035] Comparative Example 4: Compared with Example 13, this comparative example did not add a microbial screening agent during the microbial screening process. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, microorganisms that can improve the stress resistance of crops in saline-alkali land were obtained.

[0036] Performance testing: Microbial survival test: Refer to the test standard of "Agricultural Microbial Agents" GB 20287-2006 and use a constant temperature incubator; 1. Weigh 1.0 g of each bacterial solution from Examples 13-15 and Comparative Examples 1-4, add 9 mL of sterile saline, vortex and oscillate for 2 minutes to obtain 10 -1 dilution, gradient dilution to 10-8 , take three serial dilution gradients, take 100 μL of dilution solution and drop it into the center of LB plate, temperature 30℃, incubate for 48 h; 2. Calculation formula: ; 3. Add the sample to the saline-alkali matrix and put it into a 200mL breathable polyethylene bag. Take 1g of the sample and measure the viable bacterial count at time zero, record it as CFU0, place it in a constant temperature box in a dark environment, temperature 30℃±1℃, water holding capacity reduced to 40%, culture for 30 days, and record the viable bacterial count, CFU, in the same way as above. 30 ; 4. Survival rate: Table 1 Test results of microbial survival rate of examples and comparative examples Salt adsorption test Refer to NY / T 3034-2016 test standard; 1. Take 10.0 g each of the microbial screening agents in Examples 13-15 and Comparative Examples 1-3; 2. Take natural low-salt soil, Na + <0.1%, add NaCl and Na2CO3, the molar ratio is 9:1, adjust to Na + The content was 1.2%, pH 8.8 ± 0.2, microbial screening agent was added at a mass ratio of 10:1, and then 200 mL of 0.01 mol / LCaCl2 solution was added. The mixture was placed in a constant temperature oscillator, heated to 25 ° C, and shaken at 150 rpm for 72 h. After standing for 30 min, the supernatant was taken and filtered with a 0.45 μm filter membrane. Na + Concentration test; 3. Na + Calculation of adsorption rate: , C1: filtrate Na of screening agent treatment group + Concentration (mg / L), C2: blank control group (no screening agent) filtrate Na + Concentration (mg / L).

[0037] Table 2 Test results of salt adsorption capacity of examples and comparative examples Crop stress resistance testing Test crops: corn; Soil treatment: Base soil: low-salinity farmland soil (EC<1 dS / m, pH 6.8); Salt stress group: NaCl was added to 0.6% (w / w, equivalent to EC = 6.4 dS / m); Screening agent treatment: 10g / kg (i.e. 1% w / w) was incorporated into the soil at the time of sowing; Control group: CK1: no salt stress, no selection agent added, CK2: 0.6% NaCl, no selection agent added; Culture conditions: greenhouse 25 / 18℃ (day / night), 2h photoperiod, 60% humidity; Sampling time: 30 days after sowing (three leaves and one heart stage); Chlorophyll fluorescence parameters (Fv / Fm) were measured using a LI-6800 portable photosynthetic instrument. 1. At 20:00 the night before the measurement, select the third fully expanded leaf and wrap it in tin foil to dark adapt for 12 hours; 2. Fix the dark-adapted leaves in the leaf chamber, trigger the saturation flash, and the instrument automatically records F0 (minimum fluorescence), F m (maximum fluorescence), ; Malondialdehyde (MDA) content was determined using an ultraviolet spectrophotometer; 1. Prepare 0.1% thiobarbituric acid solution: add 1g thiobarbituric acid to 100mL 20% trichloroacetic acid solution and stir to dissolve; 2. Take 0.2 g of fresh leaves and add 2 mL of PBS buffer, grind in an ice bath, cool to 4°C, centrifuge at 12,000 rpm for 15 min, and collect the supernatant; 3. Add 0.1% thiobarbituric acid solution to 1 mL of supernatant, heat to 100°C, react for 15 minutes, cool to 4°C, centrifuge at 5000 rpm for 10 minutes, and measure the absorbance of the supernatant at 532 nm, 600 nm, and 450 nm. 4. Extraction volume.

[0038] The aboveground biomass was measured using an oven; Cut the aboveground stems and leaves at the ground level, remove the surface soil, put them in an oven, heat to 105℃, bake for 30 minutes, cool to 80℃, dry for 48 hours, cool to room temperature, and weigh them with an analytical balance to obtain the dry weight of the aboveground part (g / plant).

[0039] Table 3 Test results of stress resistance of crops in Examples and Comparative Examples Data Analysis: As can be seen from Tables 1-3, the present invention's method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil has a higher bacterial colony survival rate, stronger salt adsorption capacity, and significantly improved crop stress resistance; In contrast, in Comparative Example 1, since no embedding matrix was added, the microbial survival rate plummeted to 0.31%. This is because the sodium alginate-xanthan gum embedding matrix formed hydrogel microcapsules, which can maintain the intracellular osmotic balance of microorganisms. At the same time, ascorbic acid can remove reactive oxygen species and reduce the damage of oxidative stress to the bacteria. Glycerol, as an osmotic protectant, can stabilize the cell membrane structure and prolong the survival period of the flora under adverse conditions. In the comparative example, the lack of the embedding matrix caused the bacteria to be directly exposed to the high osmotic pressure environment, causing the cell membrane to rupture and inactivate, thereby causing a decrease in crop photosynthetic efficiency and a sharp drop in biomass. In Comparative Example 2, the modified biochar was replaced by biochar, resulting in a Na⁺ adsorption rate of only 52.7%. The reason was that there was a lack of acid washing and pore expansion and humic acid coating, resulting in insufficient ion exchange capacity. In addition, the modified biochar formed a porous structure with a high specific surface area through acid washing and humic acid coating, which, combined with the cationic properties of chitosan, could adsorb Na⁺. + 、Cl - The salt separation ions reduce the osmotic pressure of the soil solution. At the same time, humic acid can enhance the soil aggregate structure, improve water retention and air permeability. Combined with chitosan, it can further fix salt through electrostatic action and reduce the damage of ion toxicity to the root system. Ordinary biochar cannot absorb salt, resulting in salt retention in the soil, aggravating stress and increasing the MDA content of crops to 8.1 nmol / g. Comparative Example 3: Since magnesium humate and chitosan were not added, the crop stress resistance signal pathway was not activated. The reason was that the Mg 2+ The promotion of chlorophyll synthesis and the induction of antioxidant enzymes by chitosan resulted in a decrease in the Fv / Fm value to 0.67 and a 29% reduction in biomass. In Comparative Example 4, since no microbial screening agent was added, the functional bacteria were unable to colonize the root system. The reason was that the bacteria were not protected by the embedding matrix and supported by the modified biocarbon carrier, and died in large numbers in the saline-alkali environment. The final crop dry weight was only 8.9g / plant, which verified the irreplaceable nature of the screening agent.

[0040] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0041] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for screening microorganisms that can improve the stress resistance of crops in saline-alkali land, characterized in that: The steps include: Step S1: In Na + In saline-alkali land with a pH value of 8.5-10.0 and a pH value of >0.6%, rhizosphere soil and root tissue of Suaeda salsa were collected and inoculated into a liquid culture medium containing 0.6 mol / L NaCl and a pH value of 8.

0. At 48-hour intervals, the NaCl concentration was increased by 0.3 mol / L NaCl and the pH value of the culture medium was increased by 0.5 until the NaCl concentration in the liquid culture medium reached 1.5 mol / L and the pH value reached 9.

5. The enrichment dynamics of nitrogen-fixing bacteria and phosphate-solubilizing bacteria were tracked to obtain the enriched bacterial solution: Step S2: The enriched bacterial solution was spread on 1.2 mol / L NaCl, pH 9.0 Ashby agar and 1.0 mol / L NaCl, pH 8.5 Montana agar, respectively, and colonies within 72 hours with a diameter greater than 1 mm were screened to obtain the primary screening strains: Step S3: Add the primary screened strains to a culture dish containing 1 mol / L NaCl and pH 8.5, retain the top 15% of functional expression, collect the bacterial sludge by centrifugation, add a microbial screening agent, and obtain a bacterial solution; Step S4: Add the plant seeds to the bacterial solution and sow them in sterilized sandy soil containing 150mmoL / LNaCl. After 7 days, the nitrogen-fixing bacteria are detected to be ≥8×10 4 CFU / g root, phosphate-solubilizing bacteria ≥1.2×10 5 CFU / g root; The mass ratio of the primary screening strain to the microbial screening agent is 0.25-0.35:1; The mass ratio of the plant seeds to the bacterial liquid is 1:1.0-1.

2.

2. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to claim 1, characterized in that: The preparation steps of the microbial screening agent in step S3 are as follows: Step A1: Add sodium alginate, sodium carboxymethyl cellulose, and xanthan gum to deionized water, heat to 50-70°C, stir for 40-50 minutes until dissolution is complete, and cool to 20-40°C to obtain an embedding matrix; Step A2: Add the modified biochar to the microbial culture liquid, raise the temperature to 20-30°C, stir for 20-40 minutes, rotate at 150-250 rpm, let stand for 8-12 minutes, add the embedding matrix, stir for 4-6 minutes, rotate at 300-500 rpm, add magnesium humate, chitosan, calcium citrate, glycerol and ascorbic acid, stir for 12-16 minutes, adjust the viscosity to 800-1000 mPa·s, place in a vacuum degassing machine, reduce the pressure to -0.08 MPa, raise the temperature to 20-30°C, and degas for 8-12 minutes to obtain the microbial screening agent.

3. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali land according to claim 2, characterized in that: The mass ratio of sodium alginate, sodium carboxymethyl cellulose and xanthan gum in step A1 is 1:0.12-0.14:0.05-0.07; The mass ratio of the modified biochar, microbial culture medium, embedding matrix, magnesium humate, chitosan, calcium citrate, glycerol and ascorbic acid in step A2 is 0.6-0.7: 0.16-0.17: 1: 0.08-0.085: 0.04-0.06: 0.008-0.009: 0.08-0.085: 0.003-0.

004.

4. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to claim 2, characterized in that: The steps for preparing the microbial culture solution in step A2 are as follows: Step B1: Add tryptone, yeast powder, and NaCl to deionized water, stir for 10-15 minutes, adjust the pH to 7.1-7.3, heat to 110-130°C, and autoclave for 15-25 minutes to obtain LB liquid culture medium; Step B2: Under sterile conditions, add nitrogen-fixing bacteria and phosphate-solubilizing bacteria to LB liquid culture medium, raise the temperature to 25-30°C, culture for 22-26 hours, and shake at 150-200 rpm to obtain an activated bacterial solution; Step B3: Add the modified culture medium to the culture dish, heat it to 110-130℃, sterilize it for 10-30min, cool it to 25-35℃, add the activated bacterial solution, rotate it at 150-250rpm, adjust the pH to 8.3-8.7, and ferment it for 45-50h. 600 ≥2.0, extracellular polysaccharide yield ≥1.5g / L, stop fermentation, add to centrifuge tube, cool to 2-5℃, rotate at 3000-5000rpm, centrifuge for 10-20min, wash, add 0.85% NaCl, and resuspend to 10 10 CFU / mL, and obtain the microbial culture liquid.

5. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali land according to claim 4, characterized in that: The mass ratio of tryptone, yeast powder and NaCl in step B1 is 1:0.45-0.55:0.8-1.2; The mass ratio of the nitrogen-fixing bacteria to the phosphate-solubilizing bacteria in step B2 is 1:0.65-0.67; The mass ratio of the improved culture medium to the activated bacterial solution in step B3 is 1:0.04-0.

06.

6. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali land according to claim 4, characterized in that: The steps for preparing the improved culture medium in step B3 are as follows: Add glucose, peptone, K2HPO4, MgSO4·7H2O and CaCl2 to deionized water, stir for 20-30 minutes, add NaCl and 0.1 mol / L Tris-HCl buffer, adjust the pH to 8.4-8.6, and obtain a modified culture medium.

7. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to claim 6, characterized in that: The mass ratio of the glucose, peptone, K2HPO4, MgSO4·7H2O, CaCl2, NaCl and buffer solution is 1.8-2.2:1:0.18-0.22:0.018-0.022:0.0008-0.0012:1.7-1.8:1.1-1.

3.

8. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to claim 2, characterized in that: The modified biochar preparation steps in step A2 are as follows: Step C1: immersing the straw in a 0.1 mol / L HCl solution, heating to 70-90°C, stirring for 1-3 hours, filtering to a pH of 6.8-7.2, heating to 100-110°C, drying for 1-3 hours, placing in a grinder to a particle size of 0.5-2 mm, introducing nitrogen, placing in a tubular furnace, heating to 480-520°C, keeping warm for 1-3 hours, and cooling to 70-90°C to obtain biochar; Step C2: adding humic acid to 0.1 mol / L sodium hydroxide solution, heating to 50-70° C., stirring for 50-70 min, adding biochar, reacting for 5-7 h, rotating at 100-200 rpm, filtering, washing, and drying to obtain modified biochar.

9. The method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to claim 8, characterized in that: In step C1, the mass ratio of the straw to the HCl solution is 1:9-10; The mass ratio of humic acid to biochar in step C2 is 0.08-0.12:

1.

10. Use of the method for screening microorganisms capable of improving stress resistance of crops in saline-alkali soil according to any one of claims 1 to 9, characterized in that: The microbial screening method is applied to agricultural development and ecological restoration of marginal land.

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