Screening and application of saline-alkali soil crop growth-promoting bacteria

Abstract

The invention relates to the field of growth-promoting bacteria, in particular to screening and application of saline-alkali soil crop growth-promoting bacteria, and discloses a screening method and application of the saline-alkali soil crop growth-promoting bacteria. Through gradient domestication, multi-index function verification and molecular identification, a strain which is salt-tolerant, can efficiently solubilize phosphorus (more than 150 mu g / mL), can secrete IAA (more than 30 mg / L) and has nitrogenase activity more than 20 IU / L is screened from rhizosphere soil with salt concentration of 0.8%-1.2% and pH of 7.8-8.5. Screened strains are compounded with a carrier (the ratio of decomposed sheep manure to attapulgite to humic acid to xanthine is (70-75): (20-25): (8-10): (0.3-1)), the complex microbial inoculant is prepared through secondary fermentation, and a final field test shows that the yield of corn can be increased by 18%-32%, the pH of soil is reduced by 0.5-1.0, the salinity is reduced by 0.2%-0.5%, the saline-alkaline environment is remarkably improved, and the complex microbial inoculant has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of growth-promoting bacteria, and in particular to the screening and application of growth-promoting bacteria for crops in saline-alkali land. Background Art

[0002] Saline-alkali land is a kind of salt accumulation, which means that the salt content in the soil will affect the normal growth of crops. Soil salinization is often caused by thermodynamic effects in high temperature environments. Under the action of soil capillary attraction, the water stored in the soil evaporates due to thermodynamic effects, causing deep salts to rise to the soil surface along with the water in the capillaries. These salts continue to accumulate and cause soil salinization. The formation of soil salinization is affected by many factors, among which precipitation and soil moisture are the key factors inducing soil salinization. For example, lower precipitation and soil moisture in arid and semi-arid environments, and stronger thermodynamic effects will aggravate the occurrence of soil salinization. In addition The formation of salinization also includes the following factors: groundwater level, topography, soil parent material, and unreasonable development and utilization of the natural environment by humans. At present, salinization has become a very common environmental stress, seriously affecting the growth and development of plants and reducing the yield of food crops. Soil salinization refers to the process in which land slowly deteriorates due to salt accumulation. When the soluble salts in the soil surface exceed 0.1%, it is salinized soil. The excessive accumulation of soluble salts such as sodium chloride and magnesium chloride in salinized soil directly affects the dynamic balance of sodium ions / potassium ions in plant cells, thereby affecting the absorption of nutrients in plant cells and the activity of related enzymes, thereby affecting the normal growth and development of plants.

[0003] It can be seen that salt stress has an important impact on the growth and development of plants, and compared with other types of ecosystems, the recovery of salinized ecosystems takes longer. Plant growth-promoting bacteria mainly exist in the rhizosphere soil or around the roots of plants, which can promote the plant's intake of nutrients and improve the plant's resistance to adversity. They have the potential to improve saline-alkali soil. Plant growth-promoting bacteria increase the plant's ability to absorb nutrients by biodegrading and dissociating soil particles around the plant roots. Growth-promoting bacteria secrete extracellular polysaccharides and adhesive proteins to form adhesive substances, which allow the microorganisms to closely combine with the plant roots to form a consortium. In this symbiotic process, growth hormones, amino acids and other substances will be produced to stimulate the growth of plant roots, thereby promoting the plant's ability to absorb water and nutrients. Therefore, plant growth-promoting bacteria have a significant promoting effect on plant growth and development, enabling plants to better adapt to the environment under adverse conditions.

[0004] Therefore, based on the above-mentioned related technologies, it is urgent to develop a method for screening and applying saline-alkali land crop growth-promoting bacteria. Summary of the invention

[0005] In view of this, the purpose of the present invention is to propose a method for screening and applying saline-alkali land crop growth-promoting bacteria, so as to improve the soil environment and promote crop yield by screening and compounding saline-alkali land crop growth-promoting bacteria.

[0006] Based on the above objectives, the present invention provides a method for screening and applying saline-alkali land crop growth-promoting bacteria.

[0007] A method for screening growth-promoting bacteria for crops in saline-alkali land comprises the following steps: Take rhizosphere soil, pass it through a 2 mm sieve first, remove impurities, add sterile saline at a ratio of 1:10, shake for 30 minutes to make a soil suspension, and then dilute to obtain a diluted suspension. Spread the diluted suspension on LB solid culture medium for culture, pick single colonies, and screen the strains with the fastest growth rate, phosphorus solubilization amount >150 μg / mL, IAA secretion >30 mg / L, and nitrogenase activity >20 IU / L to obtain saline-alkali land crop growth-promoting bacteria.

[0008] Preferably, the salt concentration of the rhizosphere soil is 0.8%-1.2% and the pH is 7.8-8.5.

[0009] Preferably, the concentration of the diluted suspension is 10 -6 .

[0010] Preferably, the LB solid medium contains 3% NaCl, and the pH of the LB solid medium is 8.5; The culture conditions in the LB solid medium are 37° C. for 48 h.

[0011] Preferably, the screening process is as follows: Step A1. Inoculate a single colony into a liquid culture medium containing 0.5%, 1%, 3%, and 5% NaCl and pH 7.0, 8.0, 9.0, and 10.0, and culture at 37°C with shaking for 72 h. 600 Detection and screening of the fastest growing strains; Step A2. Using NBRIP medium, culturing for 7 days after inoculation, determining the soluble phosphorus content in the supernatant by molybdenum antimony colorimetric method, and screening strains with a phosphorus solubility of >150 μg / mL; Step A3. Using the Salkowski colorimetric method, the level of IAA secretion of the strain under tryptophan induction was detected, and the strain with a secretion amount of >30 mg / L was screened; Step A4. Determine the activity of nitrogenase by acetylene reduction method, and select strains with activity > 20 IU / L.

[0012] An application of saline-alkali land crop growth-promoting bacteria comprises the following steps: Step S1. Carrier adsorption: Mix the bacterial solution and the carrier at a ratio of 1 mL:10 g, add the embedding agent, and stir evenly; Step S2. Drying and granulation: drying in a fluidized bed at 40°C until the moisture content is ≤10%, forming granules with a particle size of 2-4 mm to obtain a composite bacterial agent.

[0013] Preferably, the preparation process of the carrier in step S1 is as follows: Mixing the decomposed sheep manure, attapulgite, humic acid and prebiotic xanthine evenly, adjusting the pH to 7.5-8.0, and obtaining a carrier; The embedding agent in step S1 is sodium alginate with a concentration of 0.1%.

[0014] Preferably, the mass ratio of the decomposed sheep manure, attapulgite, humic acid and prebiotic xanthine is 70-75:20-25:8-10:0.3-1.

[0015] Preferably, the preparation process of the decomposed sheep manure is as follows: The sheep manure is composted and fermented at 60°C for 15 days to kill pathogens and parasite eggs. At the same time, the moisture content is adjusted to 38%-45% to obtain decomposed sheep manure.

[0016] Preferably, the preparation process of the attapulgite is as follows: The attapulgite was soaked in 5% HCl solution for 24 hours, then washed with water until neutral, and then dried and activated at 120°C to obtain attapulgite.

[0017] Preferably, the preparation process of the LB solid medium is as follows: Mix tryptone, yeast extract and NaCl evenly until the solute is dissolved, then adjust the pH to 7.0 with 5 mol / L NaOH, make the volume to 1 L with deionized water, add agar strips to the LB solid medium to obtain LB solid medium.

[0018] Preferably, the mass ratio of tryptone, yeast extract and NaCl is 10g:5g:5g.

[0019] Preferably, the concentration of NaOH is 5 mol / L.

[0020] Preferably, the usage ratio of the agar strip to the components in the LB solid culture medium is 2.5 g:100 mL.

[0021] Preferably, the specific process of carrier adsorption in step S1 is as follows: a single colony is picked up in a 3 mL liquid LB test tube, and after activation by shaking culture at 35°C and 170 rpm, it is transferred to a molasses liquid fermentation medium at a volume ratio of 1%, and the fermentation liquid in the stable period is selected, and the fermentation liquid and the carrier are mixed and composted and mixed, and then secondary fermentation is performed, 30-35 L of bacterial liquid is added to each ton of solid organic material, and the compost is turned in the middle to make the fermentation temperature lower than 50°C. The fermentation ends after 7 days, and the number of active bacteria reaches 1×10 8 CFU / g, then add embedding medium and stir evenly.

[0022] Beneficial effects of the present invention: The present invention provides screening of crop growth-promoting bacteria in saline-alkali land and application thereof. The present invention screens out salt-tolerant, highly efficient phosphate solubilizing (>150 μg / mL), IAA secreting (>30 mg / L) and nitrogenase activity>20 IU / L strains from rhizosphere soil with a salt concentration of 0.8%-1.2% and a pH of 7.8-8.5 through gradient domestication and multi-index function verification. The screened strains are compounded with a carrier and prepared into a composite bacterial agent through secondary fermentation. Finally, field tests show that the bacterial agent has highly efficient growth performance, strong salt tolerance and stress resistance, and also has a synergistic enhancement mechanism to improve soil, and has broad application prospects. DETAILED DESCRIPTION

[0023] 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 in conjunction with specific embodiments.

[0024] The sources and properties of some of the raw materials used in the present invention are as follows: The rhizosphere soil used in the present invention is rhizosphere soil samples of plants normally grown in saline-alkali land from different provinces.

[0025] Example 1: Screening of a saline-alkali land crop growth-promoting bacterium, comprising the following steps: S1. 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl were mixed evenly until the solute was dissolved, and then the pH was adjusted to 7.0 with 5 mol / L NaOH, and the volume was fixed to 1 L with deionized water, and an agar strip was added to the LB solid medium, wherein the ratio of the agar strip to the components in the LB solid medium was 2.5 g:100 mL, to obtain an LB solid medium; S2. 10 g of glucose, 0.1 g of ammonium sulfate, 0.2 g of potassium chloride, 5 g of calcium phosphate, 5 g of magnesium chloride hexahydrate, and 0.25 g of magnesium sulfate heptahydrate were mixed, the volume was made up to 1000 mL with distilled water, and the pH was adjusted to 7.0 to obtain NBRIP medium; S3. Take rhizosphere soil with a salt concentration of 0.8% and a pH of 7.8, pass it through a 2 mm sieve to remove impurities, add sterile saline at a ratio of 1:10, shake for 30 minutes to make a soil suspension, and then take 100 μL of the suspension and dilute it to 10 -6 The diluted suspension was spread on LB solid medium containing 3% NaCl and pH 8.5 and cultured at 37°C for 48 h. A single colony was picked and inoculated into liquid medium containing 0.5%, 1%, 3%, and 5% NaCl and pH 7.0, 8.0, 9.0, and 10.0, and cultured at 37°C with shaking for 72 h. The OD 600 Detection and screening of the fastest growing strains were carried out using NBRIP medium and cultured for 7 days after inoculation. The soluble phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method, and strains with a phosphorus solubility of >150 μg / mL were screened. The level of IAA secretion of the strains under tryptophan induction was detected using the Salkowski colorimetric method, and strains with a secretion of >30 mg / L were screened. The activity of nitrogenase was determined by the acetylene reduction method, and strains with an activity of >20 IU / L were selected.

[0026] Example 2: Screening of a saline-alkali land crop growth-promoting bacterium, comprising the following steps: S1. 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl were mixed evenly until the solute was dissolved, and then the pH was adjusted to 7.0 with 5 mol / L NaOH, and the volume was fixed to 1 L with deionized water, and an agar strip was added to the LB solid medium, wherein the ratio of the agar strip to the components in the LB solid medium was 2.5 g:100 mL, to obtain an LB solid medium; S2. 10 g of glucose, 0.1 g of ammonium sulfate, 0.2 g of potassium chloride, 5 g of calcium phosphate, 5 g of magnesium chloride hexahydrate, and 0.25 g of magnesium sulfate heptahydrate were mixed, the volume was made up to 1000 mL with distilled water, and the pH was adjusted to 7.0 to obtain NBRIP medium; S3. Take rhizosphere soil with a salt concentration of 1.0% and a pH of 8.2, pass it through a 2 mm sieve to remove impurities, add sterile saline at a ratio of 1:10, shake for 30 minutes to make a soil suspension, and then take 100 μL of the suspension and dilute it to 10 -6 The diluted suspension was spread on LB solid medium containing 3% NaCl and pH 8.5 and cultured at 37°C for 48 h. A single colony was picked and inoculated into liquid medium containing 0.5%, 1%, 3%, and 5% NaCl and pH 7.0, 8.0, 9.0, and 10.0, and cultured at 37°C with shaking for 72 h. The OD 600Detection and screening of the fastest growing strains were carried out using NBRIP medium and cultured for 7 days after inoculation. The soluble phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method, and strains with a phosphorus solubility of >150 μg / mL were screened. The level of IAA secretion of the strains under tryptophan induction was detected using the Salkowski colorimetric method, and strains with a secretion of >30 mg / L were screened. The activity of nitrogenase was determined by the acetylene reduction method, and strains with an activity of >20 IU / L were selected.

[0027] Example 3: Screening of a saline-alkali land crop growth-promoting bacterium, comprising the following steps: S1. 10 g of tryptone, 5 g of yeast extract and 5 g of NaCl were mixed evenly until the solute was dissolved, and then the pH was adjusted to 7.0 with 5 mol / L NaOH, and the volume was fixed to 1 L with deionized water, and an agar strip was added to the LB solid medium, wherein the ratio of the agar strip to the components in the LB solid medium was 2.5 g:100 mL, to obtain an LB solid medium; S2. 10 g of glucose, 0.1 g of ammonium sulfate, 0.2 g of potassium chloride, 5 g of calcium phosphate, 5 g of magnesium chloride hexahydrate, and 0.25 g of magnesium sulfate heptahydrate were mixed, the volume was made up to 1000 mL with distilled water, and the pH was adjusted to 7.0 to obtain NBRIP medium; S3. Take rhizosphere soil with a salt concentration of 1.2% and a pH of 8.5, pass it through a 2 mm sieve to remove impurities, then add sterile saline at a ratio of 1:10, shake for 30 minutes to make a soil suspension, and then take 100 μL of the suspension and dilute it to 10 -6 The diluted suspension was spread on LB solid medium containing 3% NaCl and pH 8.5 and cultured at 37°C for 48 h. A single colony was picked and inoculated into liquid medium containing 0.5%, 1%, 3%, and 5% NaCl and pH 7.0, 8.0, 9.0, and 10.0, and cultured at 37°C with shaking for 72 h. The OD 600 Detection and screening of the fastest growing strains were carried out using NBRIP medium and cultured for 7 days after inoculation. The soluble phosphorus content in the supernatant was determined by the molybdenum antimony colorimetric method, and strains with a phosphorus solubility of >150 μg / mL were screened. The level of IAA secretion of the strains under tryptophan induction was detected using the Salkowski colorimetric method, and strains with a secretion of >30 mg / L were screened. The activity of nitrogenase was determined by the acetylene reduction method, and strains with an activity of >20 IU / L were selected.

[0028] Example 4: An application of saline-alkali land crop growth-promoting bacteria, comprising the following steps: S1. The sheep manure is composted and fermented at 60°C for 15 days to kill pathogens and parasite eggs, while adjusting the moisture content to 38%-45% to obtain decomposed sheep manure; S2. Soak the attapulgite in 5% HCl solution for 24 hours, then wash with water until neutral, and then dry and activate at 120°C to obtain attapulgite; S3. 70 g of decomposed sheep manure, 20 g of attapulgite, 8 g of humic acid and 0.3 g of prebiotic xanthine were mixed and the pH was adjusted to 7.5 to obtain a carrier; S4. Carrier adsorption: Mix the bacterial solution and the carrier at a ratio of 1 mL:10 g, add 0.1% sodium alginate, and stir evenly; S5. Drying and granulation: Dry in a fluidized bed at 40°C until the moisture content is ≤10%, and make granules with a particle size of 2-4 mm to obtain a composite bacterial agent.

[0029] Example 5: An application of saline-alkali land crop growth-promoting bacteria, comprising the following steps: S1. The sheep manure is composted and fermented at 60°C for 15 days to kill pathogens and parasite eggs, while adjusting the moisture content to 38%-45% to obtain decomposed sheep manure; S2. Soak the attapulgite in 5% HCl solution for 24 hours, then wash with water until neutral, and then dry and activate at 120°C to obtain attapulgite; S3. 72 g of decomposed sheep manure, 23 g of attapulgite, 9 g of humic acid and 0.7 g of prebiotic xanthine were mixed and the pH was adjusted to 7.8 to obtain a carrier; S4. Carrier adsorption: Mix the bacterial solution and the carrier at a ratio of 1 mL:10 g, add 0.1% sodium alginate, and stir evenly; S5. Drying and granulation: Dry in a fluidized bed at 40°C until the moisture content is ≤10%, and make granules with a particle size of 2-4 mm to obtain a composite bacterial agent.

[0030] Example 6: An application of saline-alkali land crop growth-promoting bacteria, comprising the following steps: S1. The sheep manure is composted and fermented at 60°C for 15 days to kill pathogens and parasite eggs, while adjusting the moisture content to 38%-45% to obtain decomposed sheep manure; S2. Soak the attapulgite in 5% HCl solution for 24 hours, then wash with water until neutral, and then dry and activate at 120°C to obtain attapulgite; S3. 75 g of decomposed sheep manure, 25 g of attapulgite, 10 g of humic acid and 1 g of prebiotic xanthine were mixed and the pH was adjusted to 8.0 to obtain a carrier; S4. Carrier adsorption: Mix the bacterial solution and the carrier at a ratio of 1 mL:10 g, add 0.1% sodium alginate, and stir evenly; S5. Drying and granulation: Dry in a fluidized bed at 40°C until the moisture content is ≤10%, and make granules with a particle size of 2-4 mm to obtain a composite bacterial agent.

[0031] Example 7: An application of saline-alkali land crop growth-promoting bacteria, comprising the following steps: S1. Mix molasses, soybean meal, sodium chloride, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, ferrous sulfate, zinc sulfate, and manganese sulfate in percentages of 5%, 0.72%, 0.5%, 0.1%, 0.1%, 0.1%, 0.05%, 0.05%, and 0.05%, and then adjust the pH to 7.5 to obtain a molasses liquid fermentation medium; S2. Pick a single colony into a 3mL liquid LB test tube, culture and activate it at 35℃ and 180rpm, then transfer it to molasses liquid fermentation medium at a volume ratio of 1%. Select the fermentation liquid in the stable period, mix the fermentation liquid with the carrier and compost it for secondary fermentation. Add 30-35L of bacterial liquid to each ton of solid organic material, turn the compost in the middle to keep the fermentation temperature below 50℃, and end the fermentation after 7 days. The number of active bacteria reaches 1×10 8 CFU / g, then add the embedding agent and stir evenly to obtain the composite bacterial agent.

[0032] Comparative Example 1: Compared with Example 4, this comparative example did not add decomposed sheep manure during the preparation of the composite bacterial agent, and the remaining steps and parameters were the same, which will not be repeated in this comparative example, and finally a composite bacterial agent was obtained.

[0033] Comparative Example 2: Compared with Example 4, this comparative example did not add attapulgite during the preparation of the composite bacterial agent, and the remaining steps and parameters were the same, which will not be repeated in this comparative example, and finally a composite bacterial agent was obtained.

[0034] Comparative Example 3: Compared with Example 4, the prebiotic xanthine was not added during the preparation of the composite bacterial agent in this comparative example, and the remaining steps and parameters were the same, which will not be repeated in this comparative example, and the composite bacterial agent was finally obtained.

[0035] Comparative Example 4: Compared with Example 4, this comparative example only adjusts the dosage of "prebiotic xanthine" to "2 g", and the remaining steps and parameters are the same, which will not be repeated in this comparative example, and finally obtains a composite bacterial agent.

[0036] Comparative Example 5: Compared with Example 7, in this comparative example, the single colony was not inoculated into the molasses liquid culture medium for secondary fermentation. Instead, the bacterial liquid was directly mixed with the carrier for subsequent processes. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite bacterial agent was obtained.

[0037] Performance Test: The composite bacterial agents prepared in Examples 4 to 7 and Comparative Examples 1 to 5 were subjected to the following performance tests: Determination of viable bacterial count: Use the dilution plate method to accurately weigh 1g of the composite bacterial sample and put it into a conical flask containing 99mL of sterile saline. Oscillate for 30 minutes to fully disperse the bacterial agent and make 10 -2 Then use the 10-fold gradient dilution method to dilute the dilution solution into 10 -3 , 10 -4 , 10 -5 , 10 -6 Dilutions of different concentrations were prepared. 0.1 mL of dilutions of different concentrations were taken respectively and evenly spread on LB solid culture medium plates. Three parallel plates were made for each concentration. The plates were placed in a 37°C constant temperature incubator for 24-48 hours. After the colonies grew, the plates with colony counts between 30 and 300 were selected for counting. According to the colony counts on the plates, the number of viable bacteria in the sample was calculated according to the formula: number of viable bacteria (CFU / g) = average number of colonies × dilution factor × 10.

[0038] Determination of phosphorus solubilization: Take 1g of the composite bacterial agent sample and inoculate it into a triangular flask containing 100mL of NBRIP liquid culture medium, and culture it at 37℃ and 180r / min for 7 days. After the culture is completed, centrifuge the bacterial solution at 4000r / min for 15min and take the supernatant. The molybdenum antimony colorimetric method is used to determine the content of soluble phosphorus in the supernatant. The specific operation is as follows: draw a certain amount of supernatant into a colorimetric tube, add molybdenum antimony colorimetric agent, shake it well, color it at room temperature for 30min, and then use a spectrophotometer to measure the absorbance at a wavelength of 700nm, and calculate the phosphorus solubilization amount according to the phosphorus standard curve.

[0039] For the determination of IAA secretion, the composite bacterial sample was inoculated into LB liquid culture medium containing 5 mg / mL tryptophan, and cultured at 37°C and 180 r / min for 48 hours. After the culture, the bacterial solution was centrifuged at 10,000 r / min for 10 minutes, and the supernatant was taken. The IAA content was determined by the Salkowski colorimetric method. 2 mL of supernatant was taken into a test tube, 4 mL of Salkowski reagent was added, and after shaking, it was placed at room temperature away from light for 30 minutes. The solution turned pink. The absorbance was measured at a wavelength of 530 nm using a spectrophotometer, and the IAA secretion was calculated based on the IAA standard curve.

[0040] For salt tolerance test (3% NaCl survival rate), take equal amounts of composite bacterial agent samples and inoculate them into LB liquid medium containing 3% NaCl and LB liquid medium without NaCl, respectively, and culture them at 37°C and 180r / min for 24h. After the culture, the number of viable bacteria in the two culture media was determined by the dilution plate method. Salt tolerance (survival rate) = (number of viable bacteria in the medium containing 3% NaCl / number of viable bacteria in the medium without NaCl) × 100%.

[0041] Field yield increase rate determination (taking corn as an example) Select saline-alkali land as the test field, and divide the test field into several plots, each with the same area. Different composite microbial agents are evenly applied to each plot according to a certain dosage, such as the solid microbial agent in Example 4-5 and Example 6 is applied at 1.2 kg per mu, and the liquid microbial agent in Example 7 is applied at 2.3 L per mu. At the same time, a blank control plot without the composite microbial agent is set. Plant corn according to conventional planting management methods, and count the corn yield of each plot during the corn harvest period. Yield increase rate = [(yield of the plot where the composite microbial agent is applied - yield of the blank control plot) / yield of the blank control plot] × 100%.

[0042] The soil pH drop was determined by collecting soil samples from each plot before applying the compound bacterial agent and after corn harvest. The soil samples of the 0-20 cm soil layer were collected from each plot according to the five-point sampling method. The collected soil samples were mixed evenly, air-dried and sieved through a 2 mm sieve. 10 g of soil sample was weighed and placed in a 50 mL plastic centrifuge tube, 25 mL of carbon dioxide-free distilled water was added, and the mixture was shaken for 30 minutes and allowed to stand for 30 minutes. The pH value of the supernatant was measured with a pH meter, and the difference in soil pH before and after the application of the compound bacterial agent was calculated, which was the soil pH drop.

[0043] The results are shown in Table 1 below: Table 1 project Viable bacteria count / CFU / g Phosphate solubilization amount / μg / mL IAA secretion / mg / L Salt tolerance Field yield increase rate pH drop Example 4 <![CDATA[1.2×10 8 ]]> 191.65 38.9 72% 28% 0.8 Example 5 <![CDATA[1.1×10 8 ]]> 185.20 36.7 68% 25% 0.7 Example 6 <![CDATA[0.8×10 8 ]]> 162.30 29.5 55% 18% 0.5 Example 7 <![CDATA[2.1×10 9 ]]> 233.61 60.82 85% 32% 1.0 Comparative Example 1 <![CDATA[0.3×10 8 ]]> 125.40 22.1 45% 12% 0.3 Comparative Example 2 <![CDATA[0.5×10 8 ]]> 148.70 28.3 50% 15% 0.4 Comparative Example 3 <![CDATA[1.0×10 8 ]]> 178.50 31.2 60% 17% 0.5 Comparative Example 4 <![CDATA[0.7×10 8 ]]> 145.20 25.7 72% 10% 0.6 Comparative Example 5 <![CDATA[1.5×10 8 ]]> 203.53 33.1 62% 21% 0.5 Data Analysis: It can be seen from Table 1 that the number of viable bacteria in Examples 4-7 is between 1.2×10 8 to 2.1×10 9 CFU / g, while the viable bacteria counts of Comparative Examples 1-5 were lower. For example, in Comparative Example 1, no decomposed sheep manure was added, and the viable bacteria count was only 0.3×10 8 This may be because the decomposed sheep manure provides nutrition and protection, which promotes the survival of the strain. Comparative Example 2 did not add attapulgite, and the number of viable bacteria was 0.5×10 8 , indicating that the adsorption of attapulgite helps maintain the number of viable bacteria. Comparative Example 3 did not add xanthine, the number of viable bacteria was 1.0×10 8, which is lower than that in Example 4, indicating that xanthine may promote the growth of the strain. In Comparative Example 4, xanthine was increased to 2 g, and the number of viable bacteria decreased to 0.7×10 8 This may be due to the inhibitory effect of high concentration of xanthine. Comparative Example 5 did not undergo secondary fermentation, and the number of viable bacteria was 1.5×10 8 , which is lower than 2.1×10 in Example 7. 9 , indicating that secondary fermentation helps to increase the number of viable bacteria.

[0044] In terms of the amount of phosphate solubilization, the amount of phosphate solubilization of Examples 4-7 was between 162.30 and 233.61 μg / mL, while in Comparative Example 3, no xanthine was added, and the amount of phosphate solubilization was 178.50, which was lower than 191.65 in Example 4, indicating that xanthine may promote the secretion of phospholytic enzymes. The amount of xanthine used in Comparative Example 4 was too high, and the amount of phosphate solubilization decreased to 145.20, which may be because the high concentration of xanthine inhibited the activity of phospholytic enzymes.

[0045] In terms of IAA secretion, the IAA secretion of Examples 4-7 ranged from 29.5 to 60.82 mg / L, and the secretion of Comparative Example 3 was 31 mg / L without the addition of xanthine, which was lower than 38.9 of Example 4, indicating that xanthine promoted the secretion of IAA. The high dosage of Comparative Example 4 caused the secretion to drop to 25.7, which may be due to the obstruction of the metabolic pathway.

[0046] In terms of salt tolerance, the survival rates of Examples 4-7 were between 55% and 85%, while the survival rates of Comparative Examples 1-2 were lower (45% and 50%) without the addition of carrier components, indicating that the carrier components have a protective effect on the salt tolerance of the strain. Comparative Example 3 did not add xanthine, and the survival rate was 60%, which was lower than 72% of Example 4, indicating that xanthine helps to improve salt tolerance.

[0047] In terms of field yield increase rate, the yield increase rate of Examples 4-7 is between 18% and 32%, while the yield increase rate of Comparative Examples 1-5 is lower, especially Comparative Example 1 is only 12%, indicating that the addition of carrier components and xanthine has a significant effect on yield increase. The high xanthine dosage of Comparative Example 4 causes the yield increase rate to drop to 10%, indicating that exceeding the threshold will have a negative impact.

[0048] In terms of the decrease in soil pH, the pH decrease in Examples 4-7 was between 0.5 and 1.0, while the decrease in Comparative Examples 1-5 was smaller, indicating that the addition of carriers and xanthine helps to lower soil pH and improve the saline-alkali environment.

[0049] In summary, the present invention screened out salt-tolerant, efficient phosphate solubilizing (>150 μg / mL), IAA secreting (>30 mg / L) and nitrogenase activity>20 IU / L strains from rhizosphere soil with a salt concentration of 0.8%-1.2% and a pH of 7.8-8.5 through gradient domestication and multi-index function verification, compounded the screened strains with a carrier, and prepared a composite bacterial agent through secondary fermentation. The final field test showed that the bacterial agent has efficient growth performance, strong salt resistance and stress resistance, and also has a synergistic enhancement mechanism to improve the soil, and has broad application prospects.

[0050] 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. Under the concept 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.

[0051] 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 in the scope of protection of the present invention.

Claims

1. An application of a saline-alkali land crop growth-promoting bacterium, characterized in that: The following steps are involved: Step S1. Mix the bacterial solution and the carrier at a ratio of 1 mL:10 g, add the embedding agent, and stir evenly; Step S2. Drying and granulation: drying in a fluidized bed at 40°C until the moisture content is ≤10%, forming granules with a particle size of 2-4 mm to obtain a composite bacterial agent; The carrier is prepared from decomposed sheep manure, attapulgite, humic acid and prebiotic xanthine.

2. The use of the saline-alkali land crop growth-promoting bacteria according to claim 1, characterized in that: The preparation process of the carrier in step S1 is as follows: Mixing the decomposed sheep manure, attapulgite, humic acid and prebiotic xanthine evenly, adjusting the pH to 7.5-8.0, and obtaining a carrier; The embedding agent in step S1 is sodium alginate with a concentration of 0.1%.

3. The use of the saline-alkali land crop growth-promoting bacteria according to claim 2, characterized in that: The mass ratio of the decomposed sheep manure, attapulgite, humic acid and prebiotic xanthine is 70-75:20-25:8-10:0.3-1.

4. The use of the saline-alkali land crop growth-promoting bacteria according to claim 2, characterized in that: The preparation process of the decomposed sheep manure is as follows: The sheep manure is composted and fermented at 60°C for 15 days to kill pathogens and parasite eggs. At the same time, the moisture content is adjusted to 38%-45% to obtain decomposed sheep manure.

5. The use of the saline-alkali land crop growth-promoting bacteria according to claim 2, characterized in that: The preparation process of the attapulgite is as follows: The attapulgite was soaked in 5% HCl solution for 24 hours, then washed with water until neutral, and then dried and activated at 120°C to obtain attapulgite.

6. The use of the saline-alkali land crop growth-promoting bacteria according to claim 1, characterized in that: The screening of saline-alkali land crop growth-promoting bacteria comprises the following steps: Take rhizosphere soil, pass it through a 2 mm sieve first, remove impurities, add sterile saline at a ratio of 1:10, shake for 30 minutes to make a soil suspension, and then dilute to obtain a diluted suspension. Spread the diluted suspension on LB solid culture medium for culture, pick single colonies, and screen the strains with the fastest growth rate, phosphorus solubilization amount >150 μg / mL, IAA secretion >30 mg / L, and nitrogenase activity >20 IU / L to obtain saline-alkali land crop growth-promoting bacteria.

7. The use of the saline-alkali land crop growth-promoting bacteria according to claim 6, characterized in that: The salt concentration of the rhizosphere soil is 0.8%-1.2%, and the pH value is 7.8-8.

5.

8. The use of the saline-alkali land crop growth-promoting bacteria according to claim 6, characterized in that: The concentration of the diluted suspension is 10 -6 .

9. The use of the saline-alkali land crop growth-promoting bacteria according to claim 6, characterized in that: The LB solid medium contains 3% NaCl, and the pH of the LB solid medium is 8.5; The culture conditions in the LB solid medium are 37° C. for 48 h.

10. The use of the saline-alkali land crop growth-promoting bacteria according to claim 6, characterized in that: The screening process is as follows: Step A1. Inoculate a single colony into a liquid culture medium containing 0.5%, 1%, 3%, and 5% NaCl and pH 7.0, 8.0, 9.0, and 10.0, and culture at 37°C with shaking for 72 h. 600 Detection and screening of the fastest growing strains; Step A2. Using NBRIP medium, culturing for 7 days after inoculation, determining the soluble phosphorus content in the supernatant by molybdenum antimony colorimetric method, and screening strains with a phosphorus solubility of >150 μg / mL; Step A3. Using the Salkowski colorimetric method, the level of IAA secretion of the strain under tryptophan induction was detected, and the strain with a secretion amount of >30 mg / L was screened; Step A4. Determine the activity of nitrogenase by acetylene reduction method, and select strains with activity > 20 IU / L.