Bacillus pumilus, complex microbial inoculant and application of bacillus pumilus and complex microbial inoculant in four-dimensional improvement method of saline-alkali soil

Abstract

The invention discloses bacillus pumilus, a complex microbial inoculant and application of the complex microbial inoculant in a saline-alkali soil four-dimensional improvement method, and belongs to the technical field of microorganism application. According to the invention, a strain of bacillus pumilus is screened, and the bacillus pumilus has excellent saline-alkaline tolerance, has multiple functions of producing organic acid, proline, siderophore, trehalose, IAA and betaine and the like, and has obvious advantages in saline-alkali soil improvement; the stress resistance of plants is improved through multiple mechanisms, nutrient absorption is promoted, the soil structure and chemical environment are improved, the stability and diversity of soil microbial communities are enhanced, and then effective treatment on the saline-alkali soil is achieved. According to the four-dimensional improvement method for the saline-alkali soil, the microbial technology, the physical technology, the chemical technology and the nutrition technology are combined, the saline-alkali soil is improved from the four dimensions of physics, chemistry, biology and nutrition, soil can be effectively improved, and repeated planting is not likely to happen.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, and in particular to a strain of Bacillus pumilus, a composite bacterial agent and application thereof in a four-dimensional improvement method for saline-alkali land. Background Art

[0002] Saline-alkali land is a valuable reserve arable land resource. The results of the second soil survey in my country show that the area of ​​saline-alkali land in my country is nearly 36 million hm2. 2 , accounting for about 4.88% of the available land area, and the salinized soil area in the cultivated land accounts for 6.62%, reaching 9.209 million hm2. 2 . The improvement of saline-alkali land is of great strategic significance for accelerating the transformation of scientific research results into actual productivity. According to the soil salinity, saline-alkali land is divided into light saline-alkali land, moderate saline-alkali land and heavy saline-alkali land. At present, the commonly used improvement measures for saline-alkali land mainly include engineering measures, agronomic measures, biological measures and chemical measures, among which biological measures are the most ecologically and economically beneficial measures, mainly including plant improvement and microbial improvement.

[0003] Patent CN112292959A discloses a method for improving and utilizing mild and moderate saline-alkali land. The method utilizes the biological characteristics of mulberry grass, which is salt-tolerant, drought-resistant, and salt-absorbing. Microbial agents are used in conjunction with forage planting, and soil conditioners and microbial agents are used in conjunction with sunflower planting. The method can effectively improve the physical and chemical properties and aggregate structure of saline-alkali soil and improve the productivity of saline-alkali land. However, the above method is mainly used for improving mild and moderate saline-alkali land, and has limited effect on improving severe saline-alkali land.

[0004] Therefore, the management of severe saline-alkali land still faces huge challenges, and how to effectively improve severe saline-alkali land has become an important topic in the current scientific research field. Compared with mild and moderate saline-alkali land, the soil salinity of severe saline-alkali land is higher, the structure is more damaged, the vegetation is sparse or even unable to grow, and the water resource management is more complicated. For severe saline-alkali land, patent CN118614219A discloses a severe saline-alkali land planting equipment and use method based on micro-irrigation and salt regulation. The method increases the seedling belt and drip irrigation belt to hinder the transpiration of the saline-alkali soil below while continuously leaching the saline-alkali soil, thereby improving the salinity of the surface of severe saline-alkali land. Patent CN 118452004A discloses a medium and severe saline-alkali land crop / halophyte intercropping method with micro-area salt control. The method is completed by the steps of saline-alkali land selection, plant selection, construction of furrow and ridge micro-topography, driving soil salt migration and suitable intercropping, water and fertilizer management, and bio-salt silage forage production. The above technology relies on planting foreign soil and planting salt-tolerant plants on moderately to severely saline-alkali land, but it has no improvement effect on the severely saline-alkali land itself.

[0005] Microbial agents are a type of bacterial community obtained by screening and separation from soil or plants. After a series of industrial processes such as fermentation, these microorganisms are amplified and cultured and processed into a solid bacterial preparation. Microbial agents can be used to improve saline-alkali land by lowering soil pH and improving the ability of crops to resist salt-alkali stress. However, there are few reports on microbial agents suitable for severe saline-alkali improvement. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a strain of Bacillus pumilus, a composite bacterial agent and the application thereof in a four-dimensional improvement method for saline-alkali land.

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

[0008] The first aspect of the present invention provides a strain of Bacillus pumilus BLCC1-1591, which has been deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on October 14, 2024, and its biological preservation number is CCTCC NO: M 20242180.

[0009] The Bacillus pumilus BLCC1-1591 of the present invention has the following characteristics:

[0010] The strain itself has excellent salt-alkali resistance, can tolerate 15% NaCl concentration, can tolerate normal proliferation in an environment of pH 10.0, and can also grow well in an environment of pH 8 and 15% NaCl; Bacillus pumilus BLCC1-1591 can produce acid in LB culture medium with different NaCl concentrations, among which the contents of lactic acid, acetic acid, malic acid, citric acid and succinic acid are all above 1g / L, and the acid production performance is good; in addition, it also has multiple functions such as producing proline, siderophore, trehalose, IAA and betaine. Organic acids, proline, siderophore, trehalose, IAA and betaine enhance plant resistance, promote nutrient absorption, and improve soil structure and chemical environment through multiple mechanisms, and have obvious advantages in saline-alkali land improvement.

[0011] A second aspect of the present invention provides a bacterial agent containing the above-mentioned Bacillus pumilus.

[0012] In the bacterial agent, Bacillus pumilus BLCC1-1591 can exist in the form of cultured live bacteria, fermentation liquid or bacterial suspension.

[0013] Preferably, the bacterial agent may be in the form of an emulsion, a suspension, a granule, a wettable powder or a water dispersant.

[0014] The third aspect of the present invention provides the use of the above-mentioned Bacillus pumilus or a bacterial agent containing the above-mentioned Bacillus pumilus in at least one of the following (1)-(12):

[0015] (1) Produce organic acids;

[0016] (2) preparing products producing organic acids;

[0017] (3) Proline production;

[0018] (4) preparing a proline-producing product;

[0019] (5) siderophore production;

[0020] (6) preparing a product producing siderophores;

[0021] (7) Producing trehalose;

[0022] (8) preparing trehalose-producing products;

[0023] (9)Production of IAA;

[0024] (10) preparing IAA-producing products;

[0025] (11) Producing betaine;

[0026] (12) Prepare betaine-producing products.

[0027] In a fourth aspect of the present invention, a composite bacterial agent is provided, comprising an active ingredient and a carrier, wherein the active ingredient is composed of Bacillus pumilus BLCC1-1591, Aspergillus niger BLCC6-0009, Trichoderma harzianum and Bacillus subtilis NBL-B12004 in a mass ratio of (1-7):(3-10):(3-10):(1-7);

[0028] The carrier is composed of potassium humate and glucose in a mass ratio of (30-40):(60-70).

[0029] On the one hand, the carrier plays a role in protecting the bacteria, and on the other hand, the total number of live bacteria in the composite bacterial agent can be adjusted by adjusting the dosage of the carrier.

[0030] Preferably, the Aspergillus niger BLCC6-0009 has a deposit number of CCTCC NO: M 2015722; the Bacillus subtilis NBL-B12004 has a deposit number of CCTCC NO: M 20231506.

[0031] Preferably, in the bacterial agent, the number of live bacteria of Bacillus pumilus is ≥1 billion / g; the number of live bacteria of Aspergillus niger is ≥1 billion / g, the number of live bacteria of Trichoderma harzianum is ≥1 billion / g, and the number of live bacteria of Bacillus subtilis is ≥5 billion / g.

[0032] The fifth aspect of the present invention provides the use of the above-mentioned composite bacterial agent in the four-dimensional improvement method of saline-alkali land.

[0033] A sixth aspect of the present invention provides a four-dimensional improvement method for saline-alkali land, comprising the following steps:

[0034] (1) Chemical improvement: Use acidic small molecule water to irrigate the saline-alkali soil to be improved multiple times;

[0035] (2) Physical improvement: After the first irrigation, when the soil is dry, dig the soil 40-50 cm deep, lay 1-3 cm of inert material, and backfill the soil;

[0036] (3) Comprehensive nutrition and biological improvement: Apply 2-4t / mu of acidic organic fertilizer and 25-50kg / mu of compound bacterial agent to the physically improved soil, and plough 30-40cm deep;

[0037] (4) Prepare the land and make ridges, plant salt-alkali tolerant crops, and apply 2-6 kg / mu of compound fungal agent.

[0038] Preferably, in step (1), the acidic small molecule water is irrigated into the saline-alkali soil to be improved, irrigated 2-4 times, each time at an interval of 12-18 days, and the amount of acidic small molecule water irrigated is 10-20 cm of the thickness of the infiltrated soil;

[0039] The acidic small molecule water is produced by using electrolysis water technology and citric acid as a raw material to produce acidic small molecule water with a pH range of 4-5.

[0040] Preferably, in step (2), the inert material is composed of the following raw materials in parts by weight: 40-50 parts of fly ash, 30-50 parts of zeolite powder and 5-15 parts of silicon dioxide.

[0041] Preferably, in step (3), the acidic organic fertilizer is composed of the following raw materials:

[0042] 50-60 parts of livestock and poultry manure, 20-30 parts of furfural residue, 10-20 parts of phosphate rock, and 0.1-0.2 parts of organic material fermentation agent.

[0043] Preferably, the livestock and poultry manure is chicken manure or pig manure.

[0044] Beneficial effects of the present invention:

[0045] 1. The present invention screened a strain of Bacillus pumilus, which has excellent salt-alkali resistance, and has multiple functions such as producing organic acids, proline, siderophore, trehalose, IAA and betaine, and has obvious advantages in improving saline-alkali land; organic acids, proline, siderophore, trehalose, IAA and betaine enhance plant stress resistance, promote nutrient absorption, improve soil structure and chemical environment, and enhance the stability and diversity of soil microbial communities through multiple mechanisms, thereby achieving effective management of saline-alkali land.

[0046] 2. The four-dimensional improvement method of saline-alkali land of the present invention combines microbial technology, physical technology, chemical technology and nutritional technology to improve saline-alkali land from the four dimensions of physics, chemistry, biology and nutrition, which can effectively improve the soil and make continuous planting less prone to repetition. On clay-type severe saline-alkali soil, the pH value was reduced to 7.36 after two years of improvement, the soluble total salt content of the soil was reduced by 78.73%, and the soil bulk density was reduced from 1.52g / cm 3 Reduced to 1.42g / cm 3 , the soil structure is improved, the soil is looser, the soil organic matter content is increased after adding nutrients, and it can positively feedback the soil after planting plants, increase the soil organic matter content, and choose salt-alkali tolerant food crops for planting. On sandy heavy saline-alkali soil, the pH dropped to 8.11 after two years of improvement, the soil soluble total salt content dropped by 73.90%, and the soil bulk density dropped from 1.43g / cm 3 Reduced to 1.31g / cm 3 , the soil structure was improved, the soil organic matter content was increased, and after the soil was improved and corn was planted in the second year, the yield of the treatment group was 65.20% higher than that of the control group; after the improvement of the severe saline-alkali soil in Shandong coastal area, the pH showed a downward trend. After one growth cycle of corn was improved, the pH was reduced by 7.15% compared with before improvement, and the soluble total salt content of the soil was reduced by 58.22%. The soil organic matter content was increased, and after corn was planted in the year of improvement, the yield of the treatment group was 13.69% higher than that of the control group. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Colony morphology of strain RZ2;

[0048] Figure 2 :Bacterial morphology of strain RZ2;

[0049] Figure 3 : Soil profile of sandy soil type severe saline-alkali land before improvement in Test Example 3;

[0050] Figure 4 : The soil profile of the improved sandy soil type severe saline-alkali land in Test Example 3;

[0051] Figure 5: Pictures of corn at jointing stage in the control group in Experimental Example 4;

[0052] Figure 6 : Pictures of corn at jointing stage in the treatment group of Experimental Example 4; DETAILED DESCRIPTION

[0053] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0055] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0056] The phosphate rock used in the present invention was purchased from Jinan Shenglong Chemical Co., Ltd.

[0057] The organic material fermentation agent used in the present invention is purchased from Shandong Baolaililai Bioengineering Co., Ltd. The product consists of Bacillus subtilis, Saccharomyces cerevisiae, Bacillus licheniformis and their metabolites, digestive enzymes, carriers (soluble and insoluble), etc., and the total number of viable bacteria is >10 billion CFU / g.

[0058] The Aspergillus niger BLCC6-0009 used in the present invention has a deposit number of CCTCC NO: M2015722, and the strain has been recorded in the published patent CN105385609 B.

[0059] The Trichoderma harzianum used in the present invention was purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd., with a viable count of 20 billion / g;

[0060] The deposit number of Bacillus subtilis NBL-B12004 used in the present invention is CCTCCNO: M 20231506, and the strain has been recorded in the published patent CN 117487699 B.

[0061] Example 1: Isolation and identification of strains

[0062] 1. Test materials

[0063] Separation source: saline-alkali soil in Rizhao and Dongying.

[0064] LB liquid medium: 0.2% glucose, 1.0% peptone, 0.5% sodium chloride, 0.5% yeast extract. LB solid medium needs to be supplemented with 1.5% agar powder, pH 7.0, and sterilized at 121°C for 30 min.

[0065] 2. Isolation and screening of strains:

[0066] 2.1 Isolation of strains

[0067] Accurately weigh 10g of saline-alkali soil and put it in a conical flask filled with 90mL of 0.9% saline and glass beads. Oscillate it in a constant temperature shaker at 30℃ and 180r / min for about 1h. Pipette 1mL of mixed bacterial solution and add it to the saline solution. Dilute to 10 -3 , 10 -4 , 10 -5 , 10 -6 The concentration gradient was spread on LB solid medium supplemented with 5% NaCl, and cultured at 37°C for 24 h. Colonies with different morphologies, such as flat or volcano-shaped protrusions, neat or irregular edges, rough and wrinkled surfaces, dirty white or slightly yellow, were picked for plate purification. The purified single colonies were stored on LB solid medium and stored at 4°C for later use.

[0068] 2.2 Screening of salt-tolerant strains

[0069] The Bacillus strains screened in 2.1 above that tolerated 5% NaCl concentration were inoculated on LB solid culture medium supplemented with 10% and 15% NaCl, respectively, and the proliferation of the strains was detected to screen out the strains that could tolerate 15% NaCl concentration.

[0070] More than 100 Bacillus strains tolerant to 5% NaCl concentration were isolated from saline-alkali soils in Rizhao and Dongying. They were inoculated on LB solid culture medium supplemented with 10% and 15% NaCl, respectively, and the proliferation of the strains was tested. A strain that could tolerate 15% NaCl concentration was obtained and numbered RZ2.

[0071] 3. Identification of strains

[0072] 3.1 Morphological identification

[0073] The screened pure culture species that tolerate 15% NaCl concentration were inoculated on LB solid medium and cultured at 37°C for 20 hours, and the colony morphology was observed.

[0074] Strain RZ2 was cultured at 37°C for 20 h, and the colonies were off-white with smooth and neat edges (e.g. Figure 1 ). Observed under an optical microscope, the bacteria are thin rod-shaped, Gram-positive bacteria (such as Figure 2 ).

[0075] 3.2 Molecular Biological Identification

[0076] The target strain was inoculated into fresh LB liquid medium and cultured for 20 hours. The bacterial DNA was extracted using the kit of Tiangen Company and the 16S rDNA sequence was amplified. The primers used were universal primers:

[0077] 1492R: 5'-ggttaccttgttacgactt-3';

[0078] 27F: 5'-agagtttgatcctggctcag-3'.

[0079] The PCR reaction system (50 μL) was as follows: Mixture 25 μL (including Taq DNA polymerase and dNTP, etc., Tiangen Biochemical Technology Co., Ltd.), upstream and downstream primers 1 μL each, template DNA 2 μL, ultrapure water 21 μL.

[0080] The PCR amplification program was 94°C pre-denaturation for 5 min, 94°C denaturation for 1 min, 52°C annealing for 1 min, 72°C extension for 2 min, 25 cycles, and 72°C extension for 10 min. The PCR products were sent to Beijing Boshang Biotechnology Co., Ltd. for sequence determination.

[0081] The electrophoresis results of the 16S rDNA PCR product of strain RZ2 showed that a band with good specificity was obtained at a molecular weight of about 1500 bp, which was consistent with the expected result, and sequencing was performed. The 16S rDNA gene sequence is shown in SEQ ID NO. 1. The sequencing sequence was compared with the 16S rDNA gene sequences of some strains registered on the website http: / / www.ncbi.nlm.nih.gov, and the RZ2 strain was identified as Bacillus pumilus, named Bacillus pumilus BLCC1-1591, and the strain was biologically deposited. The deposit information is as follows:

[0082] Bacterial species: Bacillus pumilus BLCC1-1591

[0083] Latin name: Bacillus pumilus

[0084] Collection institution: China Center for Type Culture Collection

[0085] Abbreviation of depository institution: CCTCC

[0086] Address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0087] Deposit date: October 14, 2024

[0088] Deposit number: CCTCC NO: M 20242180.

[0089] Example 2: Performance evaluation of Bacillus pumilus BLCC1-1591

[0090] 1.1 Seed solution preparation

[0091] The Bacillus pumilus BLCC1-1591 strain was transferred to LB solid medium and cultured at 37°C for 24 hours. The colonies were picked and added to LB liquid medium supplemented with 5% NaCl. The culture was shaken at 37°C and 180 r / min for 14 hours to obtain Bacillus pumilus BLCC1-1591 seed solution for later use.

[0092] 1.2 Salt tolerance test

[0093] The seed solution of Bacillus pumilus BLCC1-1591 prepared in 1.1 was inoculated at 2% (volume percentage) into LB liquid medium supplemented with 5%, 10% and 15% NaCl, respectively, and cultured at 37°C and 180 rpm for 24 h. The pH value and OD value of the fermentation liquid were measured. 600nm Value and viable count.

[0094] Table 1 Determination of salt tolerance of Bacillus pumilus BLCC1-1591

[0095]

[0096] As shown in Table 1, the OD value of the fermentation liquid of Bacillus pumilus BLCC1-1591 was 0.0447 W / m after being cultured in a medium with a concentration of 15% NaCl for 24 h. 600 The value is 1.542, and the number of viable bacteria is 1.9×10 8 CFU / mL, indicating that the strain can tolerate 15% NaCl concentration.

[0097] 1.3 Determination of alkali resistance

[0098] The Bacillus pumilus BLCC1-1591 seed solution prepared in 1.1 was inoculated into LB liquid medium with a pH value of 8.0 and 10.0 at a 2% inoculation rate, and cultured at 37°C and 180 rpm for 24 h. The pH value and OD value of the fermentation liquid were measured. 600nm Value and viable count.

[0099] Table 2 Alkali resistance test of Bacillus pumilus BLCC1-1591

[0100]

[0101] As shown in Table 2, after culturing for 24 h in LB liquid medium with initial pH values ​​of 8.0 and 10.0, the OD 600 The viable bacteria counts were all above 2.0, and the viable bacteria counts were all at the 8th power level. The viable bacteria counts were 3.41×10 8 CFU / mL, indicating that the strain can tolerate and proliferate normally in an environment with a pH value of 10.0.

[0102] 1.4 Determination of salt and alkali resistance

[0103] The seed solution of Bacillus pumilus BLCC1-1591 prepared in 1.1 was inoculated into LB liquid medium with pH 8-5% NaCl, pH 9-5% NaCl, pH 8-10% NaCl, pH 9-10% NaCl, and pH 8-15% NaCl at a 2% inoculation rate, and cultured at 37°C and 180 rpm for 24 h. The pH value and OD value of the fermentation liquid were measured. 600nm Value and viable count.

[0104] Table 3 Simultaneous salt-alkali tolerance test of Bacillus pumilus BLCC1-1591

[0105]

[0106] As shown in Table 3, the number of viable bacteria of Bacillus pumilus BLCC1-1591 in the tested saline-alkali medium was 10 8 CFU / mL, the highest number of viable bacteria in pH 8-5% NaCl medium was 1.1×10 9 CFU / mL, the number of viable bacteria is lowest in pH 8-15% NaCl medium, but can still reach 1.5×10 8 CFU / mL, indicating that the strain has good salt-alkali resistance.

[0107] 1.5 Determination of organic acid content in fermentation broth of Bacillus pumilus BLCC1-1591

[0108] Referring to the national standard GB5009.157-2016 "National Food Safety Standard Determination of Organic Acids in Food", the high performance liquid chromatography method was used to determine the content of organic acids in the fermentation broth of Bacillus pumilus BLCC1-1591.

[0109] Table 4 Organic acid content in fermentation broth of Bacillus pumilus BLCC1-1591 (g / L)

[0110]

[0111] As shown in Table 4, Bacillus pumilus BLCC1-1591 can produce acid in LB medium with different NaCl concentrations, among which the contents of lactic acid, acetic acid, malic acid, citric acid and succinic acid are all above 1 g / L, and the acid production performance is good.

[0112] 1.6 Determination of the production of siderophore, IAA, trehalose and betaine by Bacillus pumilus BLCC1-1591

[0113] Test strain: Bacillus pumilus BLCC1-1591 obtained by screening in Example 1

[0114] Comparative strains: Bacillus pumilus BLCC1-1706, BLCC1-1823 and BLCC1-1824 were all provided by the strain resource library of the Science and Technology Innovation Center of Shandong Baolai Lilai Biotechnology Co., Ltd., and the preparation method of their seed solution was the same as the method in Example 1.1.

[0115] 1.6.1 Determination of siderophore production levels

[0116] MKB medium: 5 g / L casamino acids, 15 mL / L glycerol, K 2 HPO 4 2.5g / L, MgSO 4 7H 2 O 2.5g / L, adjust pH to 7.2, and sterilize at 115℃ for 15min.

[0117] The seed liquid of Bacillus pumilus BLCC1-1591 and the seed liquid of the control strain were inoculated into MKB culture medium at a 2% inoculation rate, respectively, and cultured at 30°C and 180r / min for 48h. The fermentation broth was centrifuged at 4000r / min for 10min to remove the bacteria. 3mL of the supernatant was mixed with an equal volume of CAS reagent, and the absorbance of the solution at 630nm was measured after reacting at room temperature for 1h (A). 3mL of uninoculated liquid culture medium was mixed with an equal volume of CAS reagent, and its absorbance at 630nm was measured as the reference value (Ar) according to the above method.

[0118] According to the sample OD 630 Value and blank OD 630 The ratio of the values ​​A / Ar is used to judge the iron carrier production. The foreign reference standards are: A / Ar: 0-0.2+++++; 0.2-0.4++++; 0.4-0.6+++; 0.6-0.8++; 0.8-1+.

[0119] 1.6.2 Determination of IAA content

[0120] The IAA content was determined by the Salkowski colorimetric method.

[0121] Specific method: The seed liquid of Bacillus pumilus BLCC1-1591 and the seed liquid of the control strain were transferred to LB liquid medium containing 0.5g / L tryptophan at a 2% inoculation rate, respectively, and cultured at 30°C and 180rpm for 48h. The fermentation liquid was centrifuged at 4000r / min for 10min, 2mL of the fermentation supernatant was taken into a 10mL centrifuge tube, and an equal amount of Salkowski reagent was added. It was placed in a dark place for half an hour to observe its color change. 1.0, 5.0, 10.0, 15.0, and 20.0mg / L indoleacetic acid standard solutions were prepared with the standard product IAA. According to the above color development method, the absorbance was measured at 530nm using an ultraviolet spectrophotometer (zeroed with distilled water and an equal amount of colorimetric solution), with concentration as the horizontal axis and OD as the horizontal axis. 530 The standard curve is drawn with the value as the vertical axis, and the standard curve equation is calculated. 530 The value was used to calculate the amount of IAA produced by the strain.

[0122] 1.6.3 Determination of betaine content

[0123] 1.6.3.1 Preparation of Betaine Standard Curve: Take 6 test tubes, add 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mL of Betaine Standard Solution, and then add 2.0, 1.8, 1.6, 1.4, 1.2, 1.0 mL of distilled water, the total volume is 3 mL, and the concentrations are: 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / mL. After storing in the refrigerator for 15 minutes, add 5 mL of Reye's solution dropwise, and then place in the refrigerator for 1 hour. After taking out, centrifuge at 10000r / min for 15 minutes, discard the supernatant, add 5 mL of petroleum ether, centrifuge as above, and evaporate naturally to dryness. Measure the absorbance at 525nm, and draw a standard curve with absorbance and concentration.

[0124] 1.6.3.2 Preparation of samples to be tested: The seed liquid of Bacillus pumilus BLCC1-1591 and the seed liquid of the control strain were inoculated into LB liquid culture medium at a 2% inoculation rate, cultured at 30°C and 180rpm for 24h, and the fermented liquid was centrifuged at 4000r / min for 10min, and the supernatant was discarded after centrifugation and freeze-dried. Weigh 100mg of freeze-dried powder and put it into a mortar, grind it into powder under liquid nitrogen, dissolve it in distilled water, filter and discard the residue, adjust the pH to about 1.0 with concentrated hydrochloric acid, and then make up to 100mL. Take 3.0mL and transfer it to a centrifuge tube, cover it and store it in a refrigerator at 4°C for 15min, add 5mL of Reye's salt solution, and then put it in the refrigerator for 1h, take it out and centrifuge it at 10000r / min for 15min, discard the supernatant, add 1mL of petroleum ether, and place it in a fume hood to evaporate to dryness for use. Add 5.0mL of 70% acetone solution to the prepared sample and measure the absorbance at 525nm.

[0125]

[0126] Where:

[0127] c-betaine content of the sample to be tested obtained from the standard curve (μg / mL);

[0128] v-the volume of distilled water used to dissolve betaine (mL);

[0129] m-sample mass (mg).

[0130] 1.6.4 Determination of trehalose content

[0131] 1.6.4.1 Preparation of standard curve: Accurately measure 0, 12.5, 25, 50, 75, and 100 μL of 0.1 g / 100 mL standard trehalose solution, dilute to 1.0 mL and shake well, add 0.5 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid, vortex mix, cool in a fume hood for 20 min to room temperature, and measure absorbance at a wavelength of 490 nm.

[0132] 1.6.4.2 Preparation of samples to be tested: The seed liquid of Bacillus pumilus BLCC1-1591 and the seed liquid of the control strain were inoculated into LB liquid culture medium at a 2% inoculation rate, cultured at 30°C and 180rpm for 24h, and the fermented liquid was centrifuged at 4000r / min for 10min, and the supernatant was discarded after centrifugation and freeze-dried. Weigh 100mg of freeze-dried powder and put it into a mortar, grind it into powder under liquid nitrogen, and extract the bacteria with 15mL 0.5mol / L trichloroacetic acid solution in an ice water bath for eight times, each time for 30min. The resulting solution only contains trehalose. Centrifuge each extract and make the supernatant volume to 25mL. Take 1mL of supernatant respectively, add 0.5mL 6% phenol first, then add 2.5mL concentrated sulfuric acid, mix well, place in a fume hood to cool to room temperature, and measure the absorbance at a wavelength of 490nm. Find the trehalose content (μg / mL) from the standard curve.

[0133]

[0134] Where:

[0135] c- Trehalose content of the sample to be tested obtained from the standard curve (μg / mL);

[0136] v-volume of trichloroacetic acid used to extract trehalose (mL);

[0137] m-sample mass (mg).

[0138] 1.6.5 Test results

[0139] Table 5 Determination of the level of iron carrier production, IAA, trehalose and betaine performance of BLCC1-1591

[0140]

[0141] Note: “—” indicates no siderophore. Data in the same column with different lowercase letters indicate significant differences (P<0.05), and data in the same column with the same lowercase letters or no letters indicate no significant differences (P>0.05).

[0142] As shown in Table 5, the siderophore level: only BLCC1-1591 and BLCC1-1706 produced siderophores among the four tested Bacillus pumilus, and BLCC1-1591 was significantly higher than BLCC1-1706 (P < 0.05); IAA production, betaine and trehalose content: all four tested Bacillus pumilus strains can produce these three substances, among which BLCC1-1591 had the highest production, which was significantly higher than the other three strains (P < 0.05). In short, Bacillus pumilus BLCC1-1591 had the highest siderophore production level, IAA production, betaine and trehalose content.

[0143] 1.7 Determination of proline production performance of Bacillus pumilus BLCC1-1591

[0144] 1.7.1 Detection Method

[0145] The seed liquid of Bacillus pumilus BLCC1-1591 was inoculated into LB liquid culture medium at an inoculation rate of 2%, and cultured in a shaking incubator at 30°C and 180 r / min for 24 h. The fermentation liquid was centrifuged at 4000 r / min for 10 min, and the supernatant was discarded and freeze-dried after centrifugation.

[0146] Reagents: ① Acidic ninhydrin solution: accurately weigh 1.25g of ninhydrin powder and dissolve it in 30mL of glacial acetic acid and 20mL of 6M phosphoric acid, heat in a 70℃ water bath, stir to dissolve, cool in a fume hood and place in a brown bottle, and store in a refrigerator at 4℃; ② 80% ethanol; ③ Proline standard solution (100μg / mL): accurately weigh 10mg of proline, dissolve it in a small amount of 80% ethanol, make up to 100mL with distilled water, and then dilute with distilled water to make 1.0, 2.5, 5.0, 10.0, 15.0, 20.0μg / mL series solutions.

[0147] Standard curve drawing: Take 7 10mL centrifuge tubes, add 2mL of standard proline series solution of 0, 1, 2.5, 5, 10, 15, 20μg / mL, 2mL of glacial acetic acid, and 2mL of ninhydrin respectively, mix well and heat in a boiling water bath for 20min. After cooling, use a blank tube (without proline standard solution) as a control and measure the optical density colorimetrically at a wavelength of 515nm.

[0148] Sample preparation and determination: ① Weigh each sample and put it into a mortar, grind it into a homogenate with 80% ethanol (a little), transfer the homogenate into a large test tube and wash the mortar with a small amount of 80% ethanol, the total volume is 10mL, cover the test tube, and extract in the dark for 1h. ② Vortex to mix evenly, centrifuge at 4000r / min for 5min at room temperature. ③ Take 2mL of supernatant, glacial acetic acid, and ninhydrin solution in a test tube, mix thoroughly, heat in a boiling water bath for 20min, cool in a fume hood, measure the absorbance at a wavelength of 515nm, and find out the proline content (μg / mL) from the standard curve.

[0149]

[0150] Where: c-proline content of the sample to be tested obtained from the standard curve (μg / mL);

[0151] V - volume of ethanol used to extract proline (mL);

[0152] m-sample mass (mg)

[0153] 1.7.2 Results

[0154] The results of the determination according to the above method showed that Bacillus pumilus BLCC1-1591 had the ability to produce proline, and the content of proline was 6.89 μg / mg.

[0155] Example 3: Evaluation of salt and alkali resistance of Aspergillus niger

[0156] Aspergillus niger BLCC6-0009 has been recorded in patent CN 105385609 B. In addition to the reported advantage of high glucose oxidase production, the inventors found that the Aspergillus niger strain also has salt-alkali resistance.

[0157] In order to verify the salt-alkali resistance of Aspergillus niger BLCC6-0009, the inventor conducted laboratory research and experiments. The specific test results are as follows:

[0158] 1 Test method

[0159] 1.1 pH tolerance:

[0160] The composition of the fermentation medium is as follows: 10% glucose, 0.1% peptone, 0.4% potassium dihydrogen phosphate, 0.4% sodium nitrate, 0.07% magnesium sulfate, 0.05% potassium chloride, 0.07% calcium carbonate, all in mass percentage, pH natural;

[0161] The pH of the fermentation medium was adjusted to 4, 5, 6, 7, 8, 9, 10, and 11, respectively, and Aspergillus niger BLCC6-0009 was inoculated at 1% inoculation rate, and cultured in a shaking incubator at 30° C. and 180 r / min for 24 h. The number of viable bacteria in the fermentation liquid was determined using the non-inoculated control.

[0162] 1.2 Salt tolerance:

[0163] The amount of sodium chloride in the fermentation medium was adjusted to make the concentration of sodium chloride in the medium 0%, 0.5%, 1%, 3% and 5%, respectively. Aspergillus niger BLCC6-0009 was inoculated at 1% inoculation amount and cultured in a shaking incubator at 30°C and 180 r / min for 24 h. The number of viable bacteria in the fermentation broth was determined using the non-inoculated culture as a control.

[0164] 2 Test results

[0165] Table 6 The number of viable Aspergillus niger in different pH medium (CFU / mL)

[0166] Viable count (Aspergillus niger BLCC6-0009) pH4 <![CDATA[6.0×10 3 ]]> pH5 <![CDATA[6.0×10 3 ]]> pH6 <![CDATA[5.4×10 4 ]]> pH7 <![CDATA[2.6×10 5 ]]> pH8 <![CDATA[2.8×10 5 ]]> pH9 <![CDATA[3.4×10 4 ]]> pH10 <![CDATA[2.0×10 4 ]]> pH11 <![CDATA[1.3×10 4 ]]>

[0167] Table 7 The number of viable Aspergillus niger in different salt contents in the culture medium (CFU / mL)

[0168] NaCl concentration Viable count (Aspergillus niger BLCC6-0009) 0％ <![CDATA[5.8×10 4 ]]> 0.5％ <![CDATA[2.6×10 5 ]]> 1％ <![CDATA[2.9×10 5 ]]> 3％ <![CDATA[3.7×10 4 ]]> 5％ <![CDATA[1.0×10 4 ]]>

[0169] As shown in Tables 6 and 7, Aspergillus niger BLCC6-0009 can survive in the pH range of 4-11, and grows best in the pH range of 7 and 8, with the viable counts of 2.6×10 5 CFU / mL and 2.8×10 5 CFU / mL, indicating that Aspergillus niger BLCC6-0009 can adapt to a wide pH range and grow well under high pH conditions.

[0170] The viable count of Aspergillus niger BLCC6-0009 at 0% salt content was 5.8×10 4 CFU / mL, the viable bacterial count at 5% salt content is 1.0×10 4 CFU / mL, with a maximum of 2.6×10 5 CFU / mL~2.9×10 5 CFU / mL.

[0171] The above results show that Aspergillus niger BLCC6-0009 has good salt and alkali resistance.

[0172] Example 4: Preparation of composite bacterial agent

[0173] The composite bacterial agent is a powder, which is prepared by the following method:

[0174] (1) Preparation of bacterial powder:

[0175] Preparation method of Aspergillus niger powder: inoculate spores of Aspergillus niger BLCC6-0009 in a solid fermentation medium at an inoculation rate of 0.01% (mass fraction), ferment at a temperature of 30°C, and ferment aerobically for 3 days. The solid fermentation medium is composed of a substrate and water at a ratio of 1g:0.6mL; wherein the substrate is composed of 100% bran. The solid fermentation product is dried and crushed, and the number of viable bacteria of the obtained Aspergillus niger powder is 20 billion / g;

[0176] Preparation method of Bacillus subtilis powder: inoculate the seed liquid of Bacillus subtilis NBL-B12004 into a fermentation tank, shake and culture for 24 hours at 30°C and 180r / min, spray dry the fermentation liquid to obtain Bacillus subtilis powder with a viable cell count of 1.0×10 11 CFU / g;

[0177] Preparation method of Bacillus pumilus powder: inoculate the seed liquid of Bacillus pumilus BLCC1-1591 into a fermentation tank, culture under shaking conditions of 37°C and 180r / min for 24h, spray dry the fermentation liquid to obtain Bacillus pumilus powder, and the number of viable bacteria is 1.1×10 11 CFU / g;

[0178] The fermentation medium used for Bacillus pumilus and Bacillus subtilis fermentation is composed of 20 g / L glucose, 10 g / L peptone, 5 g / L sodium chloride and 5 g / L yeast extract. When used, the pH is adjusted to 6.5-7.5.

[0179] Trichoderma harzianum powder was purchased from Shandong Hezhong Kangyuan Biotechnology Co., Ltd., with a viable count of 20 billion / g;

[0180] The prepared Bacillus pumilus powder, Aspergillus niger powder, Trichoderma harzianum powder and Bacillus subtilis powder were uniformly mixed in a mass ratio of 1:5:5:5 to obtain an active ingredient;

[0181] (2) mixing potassium humate and glucose in a mass ratio of 35:65 to obtain a carrier;

[0182] (3) The active ingredient and the carrier are mixed to obtain a composite bacterial agent, wherein the total viable bacterial count of the composite bacterial agent is 8 billion / g.

[0183] Comparative Example 1:

[0184] The difference between this comparative example and Example 1 is:

[0185] The composite bacterial agent is prepared by the following method:

[0186] The Trichoderma harzianum and the Bacillus subtilis were mixed uniformly in a mass ratio of 1:1 to obtain an active ingredient;

[0187] Potassium humate and glucose were stirred and mixed uniformly in a mass ratio of 35:65 to obtain a carrier;

[0188] The active ingredient and the carrier are mixed to obtain a composite bacterial agent, wherein the total number of viable bacteria in the composite bacterial agent is 8 billion / g.

[0189] Comparative Example 2:

[0190] The difference between this comparative example and Example 1 is:

[0191] The composite bacterial agent is prepared by the following method:

[0192] Bacillus pumilus, Trichoderma harzianum and Bacillus subtilis were mixed uniformly in a mass ratio of 1:5:5 to obtain an active ingredient;

[0193] Potassium humate and glucose were stirred and mixed uniformly in a mass ratio of 35:65 to obtain a carrier;

[0194] The active ingredient and the carrier are mixed to obtain a composite bacterial agent, wherein the total number of viable bacteria in the composite bacterial agent is 8 billion / g.

[0195] Comparative Example 3:

[0196] The difference between this comparative example and Example 1 is:

[0197] Aspergillus niger, Trichoderma harzianum, and Bacillus subtilis were mixed uniformly in a mass ratio of 1:1:1 to obtain an active ingredient;

[0198] Potassium humate and glucose were stirred and mixed uniformly in a mass ratio of 35:65 to obtain a carrier;

[0199] The active ingredient and the carrier are mixed to obtain a composite bacterial agent, wherein the total number of viable bacteria in the composite bacterial agent is 8 billion / g.

[0200] Test Example 1: Verification of the effect of different microbial compound agents

[0201] 5 kg of saline-alkali soil was weighed and placed in a plastic pot (26.0 cm long, 20.8 cm wide, 13.5 cm high), and different materials were selected and added to the corresponding soil at a 2% (V / V) addition amount, specifically as follows: CK (control group, no microbial agent was used), treatment 1 (composite agent prepared in comparative example 1 was applied), treatment 2 (composite agent prepared in comparative example 2 was applied), treatment 3 (composite agent prepared in comparative example 3 was applied), treatment 4 (composite agent prepared in example 4 was applied). 2.67 g of compound fertilizer with a ratio of 15:15:15 (N:P:K) was applied to each pot. According to 18 seeds per pot, regular sowing was carried out, and the sowing depth was 1 cm. The seeds were uniform and round rapeseed seeds. The saline-alkali soil was collected from Dongying City, Shandong Province. The soluble total salt content of the soil was 7.37 g / kg, and the soil pH was 8.02. After collection, it was crushed and air-dried, and passed through a 10-mesh sieve (i.e., a 1.7 mm sieve). Three replicates were randomly placed, and thinning was performed at the three-leaf stage, and field planting was performed at the five-leaf stage, and 7 plants were retained in each pot. Each group adopted the same management method throughout the growth period, and 500 mL of water was poured every 3 days to ensure water supply. The pH and soluble total salt content of the soil were measured after 30 days.

[0202] Table 8 Soil pH and soluble total salt content in different groups after 30 days

[0203] Group pH Soluble total salt content (g / kg) CK 7.95±0.05 6.56±0.08 Process 1 7.88±0.04 6.08±0.05 Process 2 7.70±0.05 5.86±0.02 Process 3 7.82±0.01 5.92±0.03 Process 4 7.62±0.02 5.29±0.06

[0204] As shown in Table 8, compared with CK, the pH of soil in treatment 4 decreased by 4.15% and the soluble total salt content of soil decreased by 19.36%, which was the best treatment compared with other treatments. Compared with CK, the pH of soil in treatment 1, treatment 2, and treatment 3 decreased by 0.88%, 3.14%, and 1.64%, respectively, and the soluble total salt content of soil decreased by 7.32%, 10.67%, and 9.76%, respectively. Therefore, the application of the composite bacterial agent prepared in Example 4 is the best treatment, which can effectively reduce the pH and soluble total salt content of soil.

[0205] Test case 2: Application of the four-dimensional improvement method for saline-alkali land in clay-type severe saline-alkali soil

[0206] The four-dimensional improvement method for saline-alkali land was used, combined with the composite bacterial agent prepared in Example 4, and physical, chemical and nutritional technologies to form an integrated method for saline-alkali land soil improvement, covering the four dimensions of physics, chemistry, biology and nutrition. Saline-alkali soil improvement was carried out in Wujiaqu City, Xinjiang Uygur Autonomous Region. The soil type was clay-type severe saline-alkali soil; the soil texture was clay, the soluble total salt content was 27.6 g / kg, and the soil pH was 8.02.

[0207] The four-dimensional improvement method of saline-alkali land specifically includes the following steps:

[0208] 1. Chemical improvement:

[0209] Deep plowing is carried out in spring, the depth of deep plowing is 0.3m, and the land is leveled. The height difference cannot exceed ±5cm. The cofferdam is built, and the acidic small molecule water is used for watering. The salt is washed out in batches. The irrigation amount is 15cm of the soil thickness. The leaching is carried out twice, with an interval of about 15 days each time. Each watering is based on the appearance of small cracks in the soil, and the soil salt content is reduced to the tolerable range of the planted plants.

[0210] The acidic small molecule water is prepared by using Japanese electrolysis water technology and the electrolysis device in patent CN 114318371 B. Water and citric acid are mixed and then electrolyzed. The voltage of the electrolytic cell is controlled to be 6V and the current is 8A to obtain acidic small molecule water with a pH in the range of 4-5.

[0211] 2. Physical improvement:

[0212] Physical technique: After the first irrigation, when the soil is dry, dig the soil to a depth of 45 cm, lay 1.5 cm of inert material, and backfill the soil;

[0213] The inert material consists of fly ash, zeolite powder and silicon dioxide in a mass ratio of 40:50:10;

[0214] 3. Comprehensive improvement of nutrition and biology:

[0215] On the physically improved soil, 3 t / mu of acidic organic fertilizer and 50 kg / mu of the composite bacterial agent prepared in Example 4 were spread, and the soil was ploughed to a depth of 30 cm; the acidic organic fertilizer was formed by fermentation of pig manure, furfural residue, phosphate rock and Tianqing organic materials in a mass ratio of 50:30:20:0.1;

[0216] 4. Level the land and lay the drip irrigation belt:

[0217] Dig shallow trenches to prepare for planting under the ridges, and lay drip irrigation tapes on the leveled soil surface with a spacing of 20 cm;

[0218] 5. Planting of salt-alkali tolerant crops: Plant shallow-rooted salt-alkali tolerant plants such as Leymus chinensis, sow in mid-May, with a sowing rate of 2kg / mu;

[0219] 6. Topdressing of bacterial fertilizer:

[0220] The composite bacterial agent prepared in Example 4 was applied at 4 kg / mu during the jointing stage of Leymus chinensis by spraying.

[0221] The soil pH, soluble total salt content, soil bulk density, and soil organic matter content are regularly measured with survey cycles of 3 months, 6 months, one year, and two years.

[0222] Table 9 Soil changes

[0223]

[0224] As shown in Table 9, after the four-dimensional improvement method of saline-alkali land of the present invention is applied to the improvement of clay-type severe saline-alkali soil, the pH shows a downward trend. After two years of improvement, the pH is reduced by 8.23% compared with that before improvement; the soluble total salt content of the soil decreases from the initial 27.60 g / kg to 5.87 g / kg, a decrease of 78.73%; the soil bulk density decreases from 1.52 g / cm2 after two years. 3 Reduced to 1.42g / cm 3 , the soil structure is improved and the soil is more loose; the soil organic matter content is increased after adding nutrients, and after the later plant planting, it can positively feedback the soil and increase the soil organic matter content, and salt-alkali tolerant food crops can be selected for planting.

[0225] Test Example 3: Application of the four-dimensional improvement method for saline-alkali land in sandy and severely saline-alkali soil

[0226] Using the four-dimensional improvement method for saline-alkali land, saline-alkali soil improvement was carried out in Moyu County, Hotan Prefecture, Xinjiang Uygur Autonomous Region. The soil type is sandy severe saline-alkali soil; the soil texture is sandy, the soluble total salt content is 26.32g / kg, and the soil pH is 8.84. 4 mu of this type of saline-alkali land was selected, of which 2 mu was used as the control group without any treatment; the adjacent 2 mu was used as the treatment group, and the four-dimensional improvement method for saline-alkali land was used. The specific steps are as follows:

[0227] 1. Chemical improvement:

[0228] Deep plowing is carried out in spring, the depth of deep plowing is 0.3m, and the land is leveled. The height difference cannot exceed ±5cm. The cofferdam is built, and the acidic small molecule water is used for watering. The salt is washed out in batches. The irrigation amount is 15cm of the soil thickness. The irrigation is carried out 3 times, with an interval of about 15 days each time. Each watering is based on the appearance of small cracks in the soil, so as to reduce the soil salinity to the tolerable range of the planted plants.

[0229] The acidic small molecule water is prepared by using Japanese electrolysis water technology and the electrolysis device in patent CN 114318371 B. Water and citric acid are mixed and then electrolyzed. The voltage of the electrolytic cell is controlled to be 6V and the current is 8A to obtain acidic small molecule water with a pH in the range of 4-5.

[0230] 2. Physical improvement:

[0231] After irrigation, when the soil is dry, dig the soil to a depth of 45 cm, lay 2.5 cm of inert material, and backfill the soil; the inert material is composed of fly ash, zeolite powder and silica in a mass ratio of 45:40:7.5;

[0232] 3. Comprehensive improvement of nutrition and biology:

[0233] On the physically improved soil, 3 t / mu of acidic organic fertilizer and 50 kg / mu of the composite bacterial agent prepared in Example 4 were spread, and the soil was ploughed to a depth of 30 cm; the acidic organic fertilizer was fermented by chicken manure, furfural residue, phosphate rock and organic material fermentation agent in a mass ratio of 60:20:20:0.2.

[0234] 4. Land preparation, ridge making and laying of drip irrigation belts:

[0235] Dig shallow trenches to prepare for planting under ridges, and lay drip irrigation tapes on the surface of saline-alkali soil with a spacing of 80 cm;

[0236] 5. Mulching: Covering with agricultural mulch before planting plants;

[0237] 6. Plant planting: Herba Pinelliae, sow in mid-May, planting density 80cm×80cm;

[0238] 7. Topdressing of bacterial fertilizer:

[0239] The composite bacterial agent prepared in Example 4 was applied at 2 kg / mu in the jointing stage of Pinus sylvestris by spraying.

[0240] 8. Changing to corn: In the second year, corn was changed to corn. The original sedge was plowed to the bottom of the soil with a plowing depth of 20 cm. Before plowing, 4 kg / mu of the composite bacterial agent prepared in Example 4 and 50 kg / mu of compound fertilizer (15-15-15, N:P:K) were applied.

[0241] In the first year, the control group planted sedge normally with a planting density of 80cm×80cm. In the second year, corn was planted instead. The original sedge was plowed to the bottom of the soil with a plowing depth of 20cm, and 50kg / mu of compound fertilizer (15-15-15, N:P:K) was applied before plowing. The field management measures of the control group and the treatment group were carried out in accordance with local management methods and remained consistent.

[0242] The corn was Jingke 665, which was sown in mid-May, and the planting density of the control group and the treatment group was 60 cm×20 cm.

[0243] Soil pH, soluble total salt content, soil bulk density, and soil organic matter content are regularly measured with survey cycles of 3 months, 6 months, one year, and two years, and corn yield is measured in the second year.

[0244] Table 10 Soil changes

[0245]

[0246] Table 11 Corn growth

[0247] Group Yield per mu (kg) Biomass (kg) Hundred Grain Weight Control group 367.43±9.85 1709.21±8.55 35.14±1.26 Treatment Group 607.01±16.33 1739.28±6.39 38.98±1.40

[0248] As shown in Table 10, after the four-dimensional improvement method of saline-alkali land of the present invention is applied to the improvement of sandy severe saline-alkali soil, the pH shows a downward trend. After two years of improvement, the pH is reduced by 8.26% compared with that before improvement; the soluble total salt content of the soil is reduced from the initial 26.32 g / kg to 6.87 g / kg, a decrease of 73.90%; the soil bulk density is reduced from 1.43 g / cm2 to 1.67 g / kg2 after two years of improvement. 3 Reduced to 1.31g / cm 3 , the soil structure is improved (see Figure 3 and Figure 4 ); The content of soil organic matter is improved after adding nutrients, and can positively feedback the soil after the plants are planted in the later stage, increasing the content of soil organic matter.

[0249] In the second year of soil improvement, corn was planted. As shown in Table 11, the yield of the treatment group was 607.01 kg / mu, and the yield of the control group was 367.43 kg / mu. The yield of the treatment group was 65.20% higher than that of the control group; the corn biomass of the treatment group was 1.73% higher than that of the control group; and the corn 100-grain weight of the treatment group was 10.93% higher than that of the control group.

[0250] After two years of soil improvement, you can continue to plant salt-alkali tolerant corn such as Jingke 665.

[0251] Test case 4: Application of the four-dimensional saline-alkali land improvement method in severe saline-alkali soil in Shandong coastal area

[0252] Using the four-dimensional improvement method for saline-alkali land, saline-alkali soil improvement was carried out in Lijin County, Dongying City, Shandong Province. The soil type is coastal severe saline-alkali soil; the soil texture is loam, the soluble total salt content is 7.85g / kg, and the soil pH is 8.81. 2 mu of this type of saline-alkali land was selected, of which 1 mu was used as the control group without any treatment; the adjacent 1 mu was used as the treatment group, and the four-dimensional improvement method for saline-alkali land was used for improvement. The specific steps are as follows:

[0253] 1. Chemical improvement:

[0254] Deep plowing is carried out in spring, the depth of deep plowing is 0.3m, and the land is leveled. The height difference cannot exceed ±5cm. The cofferdam is built, and the acidic small molecule water is used for watering. The salt is washed out in batches. The irrigation amount is 15cm of the soil thickness. The leaching is carried out twice, with an interval of about 15 days each time. Each watering is based on the appearance of small cracks in the soil, and the soil salt content is reduced to the tolerable range of the planted plants.

[0255] The acidic small molecule water is prepared by using Japanese electrolysis water technology and the electrolysis device in patent CN 114318371 B. Water and citric acid are mixed and then electrolyzed. The voltage of the electrolytic cell is controlled to be 6V and the current is 8A to obtain acidic small molecule water with a pH in the range of 4-5.

[0256] 2. Physical improvement:

[0257] After irrigation, when the soil is dry, dig the soil to a depth of 45 cm, lay 2.5 cm of inert material, and backfill the soil; the inert material is composed of fly ash, zeolite powder and silica in a mass ratio of 40:45:15;

[0258] 3. Comprehensive improvement of nutrition and biology:

[0259] On the physically improved soil, 2 t / mu of acidic organic fertilizer and 25 kg / mu of the composite bacterial agent prepared in Example 4 were spread, and the soil was ploughed to a depth of 30 cm; the acidic organic fertilizer was fermented by sheep manure, furfural residue, phosphate rock and organic material fermentation agent in a mass ratio of 55:25:20:0.2.

[0260] 4. Land preparation, ridge making and planting of salt-alkali tolerant crops:

[0261] The salt-alkali tolerant corn Liangyu 99 is sown in late April with a planting density of 50cm×20cm;

[0262] 5. Topdressing of bacterial fertilizer:

[0263] 3 kg / mu of the composite bacterial agent prepared in Example 4 was applied by spraying during the tasseling stage of corn.

[0264] The soil pH, soluble total salt content, soil organic matter content, as well as growth indicators and yield at maturity are regularly measured during each growth period of corn.

[0265] The control group was planted with the same variety of corn (Liangyu 99) with a planting density of 50 cm × 20 cm. The field management measures for both the control and treatment groups were carried out in accordance with local management methods and remained consistent.

[0266] Table 12 Soil changes

[0267]

[0268] Table 13 Corn growth

[0269] Group Plant height(cm) Stem diameter (mm) Yield per mu (kg) 100-grain weight (g) Control group 184±6.14 25.59±1.19 564.03±15.02 33.16±1.11 Treatment Group 213±8.28 28.05±1.05 641.22±13.98 38.12±2.39

[0270] It can be seen from Table 12 that after the four-dimensional improvement method of saline-alkali land of the present invention was applied to the improvement of severe saline-alkali soil in Shandong coastal area, the pH showed a downward trend, and the pH of corn after one growth cycle was reduced by 7.15% compared with that before improvement; the soluble total salt content of the soil decreased from the initial 7.85 g / kg to 3.28 g / kg, a decrease of 58.22%; the soil organic matter content was improved after adding nutrients, and it could positively feedback the soil after the later plant planting to increase the soil organic matter content.

[0271] Depend on Figure 5 , Figure 6 It can be seen that the four-dimensional saline-alkali land improvement method of the present invention can effectively improve saline-alkali soil and promote corn growth.

[0272] As shown in Table 13, for mature corn, the plant height and stem diameter of the treatment group were 15.76% and 9.61% higher than those of the control group, respectively; the yield of the treatment group was 641.22 kg / mu, and the yield of the control group was 564.03 kg / mu, which was 13.69% higher than that of the control group; the 100-grain weight of corn in the treatment group was 14.96% higher than that of the control group.

[0273] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of Bacillus pumilus, characterized in that: The Bacillus pumilus is Bacillus pumilus BLCC1-1591, and its biological preservation number is CCTCC NO: M 20242180.

2. A bacterial agent, characterized in that The method comprises the Bacillus pumilus according to claim 1.

3. Use of the Bacillus pumilus according to claim 1 or the bacterial agent according to claim 2 in at least one of the following (1) to (12): (1) Produce organic acids; (2) preparing products producing organic acids; (3) Proline production; (4) preparing a proline-producing product; (5) siderophore production; (6) preparing a product producing siderophores; (7) Producing trehalose; (8) preparing trehalose-producing products; (9)Production of IAA; (10) preparing IAA-producing products; (11) Producing betaine; (12) Prepare betaine-producing products.

4. A composite bacterial agent, characterized in that: The invention comprises an active ingredient and a carrier, wherein the active ingredient is composed of Bacillus pumilus BLCC1-1591, Aspergillus niger BLCC6-0009, Trichoderma harzianum and Bacillus subtilis NBL-B12004 according to claim 1 in a mass ratio of (1-7):(3-10):(3-10):(1-7); The carrier is composed of potassium humate and glucose in a mass ratio of (30-40):(60-70). Preferably, the deposit number of the Aspergillus niger BLCC6-0009 is CCTCC NO: M2015722; the deposit number of the Bacillus subtilis NBL-B12004 is CCTCC NO: M20231506.

5. The composite bacterial agent according to claim 4, characterized in that: In the composite bacterial agent, the number of live bacteria of Bacillus pumilus is ≥1 billion / g; the number of live bacteria of Aspergillus niger is ≥1 billion / g, the number of live bacteria of Trichoderma harzianum is ≥1 billion / g, and the number of live bacteria of Bacillus subtilis is ≥5 billion / g.

6. Use of the composite bacterial agent described in claim 4 or 5 in the four-dimensional improvement method of saline-alkali land.

7. A four-dimensional improvement method for saline-alkali land, characterized in that: The following steps are involved: (1) Chemical improvement: use acidic small molecule water to irrigate the saline-alkali soil to be improved; (2) Physical improvement: After the first irrigation, when the soil is dry, dig the soil 40-50 cm deep, lay 1-3 cm of inert material, and backfill the soil; (3) Comprehensive nutrition and biological improvement: Apply 2-4t / mu of acidic organic fertilizer to the physically improved soil, apply compound bacterial agents, and till the soil to a depth of 30-40cm; (4) Prepare the land and make ridges, plant salt-alkali tolerant crops, and apply compound microbial agents.

8. The four-dimensional improvement method for saline-alkali land according to claim 7, characterized in that: In step (1), the acidic small molecule water is irrigated into the saline-alkali soil to be improved, irrigated 2-4 times, each time at an interval of 12-18 days, and the amount of acidic small molecule water irrigated is 10-20 cm of the thickness of the infiltrated soil; Preferably, the acidic small molecule water is produced by electrolyzing water technology with citric acid as raw material to produce acidic small molecule water with a pH of 4-5.

9. The four-dimensional improvement method for saline-alkali land according to claim 7, characterized in that: In step (2), the inert material is composed of the following raw materials in parts by weight: 40-50 parts of fly ash, 30-50 parts of zeolite powder, and 5-15 parts of silicon dioxide.

10. The four-dimensional improvement method for saline-alkali land according to claim 7, characterized in that: In step (3), the acidic organic fertilizer is composed of the following raw materials: 50-60 parts of livestock and poultry manure, 20-30 parts of furfural residue, 10-20 parts of phosphate rock, and 0.1-0.2 parts of organic material fermentation agent.

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