A compound microbial agent for saline-alkali soil improvement and a preparation method and application thereof

Abstract

This invention provides a compound microbial agent for improving saline-alkali soil, its preparation method, and its application, belonging to the field of microbial technology. Specifically, the compound microbial agent comprises the following components by weight: 20-30 parts of Bacillus subtilis, 10-15 parts of Bacillus licheniformis, 10-20 parts of Acinetobacter calcium acetate, 10-20 parts of Bacillus belyssus, 5-10 parts of Aspergillus niger, 1-3 parts of citric acid, 20-30 parts of biochemical potassium humate, 10-20 parts of water, 50-60 parts of rice husk, 2-5 parts of pig manure, 10-20 parts of fish meal, and 10-20 parts of soybean meal. The compound microbial agent provided by this invention promotes potato growth in saline-alkali soil environments, increases the water content of potato leaves, and improves the physical and chemical properties of the soil.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a compound microbial agent for improving saline-alkali land, its preparation method, and its application. Background Technology

[0002] Saline-alkali soil is a general term for both saline and alkaline soils. Statistics show that China has approximately 200,000 square kilometers of saline-alkali soil, accounting for about 2.1% of the country's total land area. To date, about 80% of my country's saline-alkali soil remains undeveloped and unutilized, mainly distributed in North China, Northeast China, Northwest China, and coastal areas north of the Yangtze River. Soil salinization significantly damages the physical and chemical properties of soil, introducing more harmful anions and cations that severely hinder crop growth, easily causing seedling loss or death, thus impeding agricultural development.

[0003] Currently, the main methods for improving saline-alkali land include physical improvement, water conservancy improvement, chemical improvement, and biological improvement. Physical improvement mainly includes methods such as leveling the ground, deep plowing and sun-drying the soil, timely loosening of the soil, and raising the ground level. Water conservancy improvement includes the "well irrigation and drainage" method and the "forced drainage and forced irrigation" method, which require huge investments in water conservancy facilities and have high maintenance costs. Chemical improvement uses some acidic salts to improve saline-alkali land. Biological improvement includes planting salt-tolerant plants and halophytes, and applying microbial fertilizers, which is a sustainable approach. Existing saline-alkali land amendments generally focus on reducing soil salinity and pH value, but are lacking in improving the ecological environment of saline-alkali land itself. As a result, these amendments only treat the symptoms and not the root cause, leading to poor improvement effects on saline-alkali land. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a compound microbial agent for improving saline-alkali land, its preparation method, and its application.

[0005] The technical solution of the present invention is as follows: A compound microbial agent, comprising the following components by weight: 20-30 parts of Bacillus subtilis, 10-15 parts of Bacillus licheniformis, 10-20 parts of Acinetobacter calcium acetate, 10-20 parts of Bacillus vesicles, 5-10 parts of Aspergillus niger, 1-3 parts of citric acid, 20-30 parts of biochemical potassium humate, 10-20 parts of water, 50-60 parts of rice husk, 2-5 parts of pig manure, 10-20 parts of fish meal, and 10-20 parts of soybean meal.

[0006] Preferably, in the compound microbial agent, Bacillus subtilis has the accession number ACCC 60429, Acinetobacter calcium acetate has the accession number ACCC 01236, Bacillus licheniformis has the accession number ACCC 05186, Bacillus belyss has the accession number ACCC 03040, and Aspergillus niger has the accession number ACCC 32413.

[0007] Preferably, the number of spores of each bacterial component: Bacillus subtilis is (1-3) × 10⁻⁶. 9 The cfu / mL concentration and the spore count of Bacillus licheniformis were (1-2) × 10⁻⁶. 8 The viable count of Acinetobacter calcium acetate at cfu / mL was (1-2) × 10⁻⁶. 8 The cfu / mL and spore count of Bacillus belysinus were (1-2) × 10⁻⁶. 8 The cfu / mL concentration and the number of Aspergillus niger spores were (1-2) × 10⁻⁶. 8 cfu / mL.

[0008] Preferably, the compound microbial agent comprises the following components by weight: 30 parts of Bacillus subtilis, 10 parts of Bacillus licheniformis, 15 parts of Acinetobacter calcium acetate, 10 parts of Bacillus belye, 5 parts of Aspergillus niger, 2 parts of citric acid, 23 parts of biochemical potassium humate, 10 parts of water, 50 parts of rice husk, 3 parts of pig manure, 10 parts of fish meal, and 10 parts of soybean flour.

[0009] The preparation method of the above-mentioned compound microbial agent includes the following steps: (1) Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate, Bacillus belye and Aspergillus niger were fermented and cultured separately to obtain Bacillus subtilis fermentation broth, Bacillus licheniformis fermentation broth, Acinetobacter calcium acetate fermentation broth, Bacillus belye fermentation broth and Aspergillus niger fermentation broth; (2) Mix rice husks, pig manure, fish meal, soybean flour and water according to the corresponding weight parts and then sterilize to obtain microbial fermentation substrate; (3) The fermentation broths of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate, Bacillus belye, and Aspergillus niger from step (1) are inoculated into the microbial fermentation substrate prepared in step (2) according to the corresponding weight parts and mixed evenly. The mixture is then allowed to ferment at 28-30℃ for 60-72 hours. Before the fermentation process begins, the corresponding weight parts of citric acid are added to the fermentation substrate. After the fermentation is completed, the corresponding weight parts of biochemical potassium humate are added to the fermentation product to obtain a compound microbial agent.

[0010] Preferably, in step (1), the method for fermenting and culturing strains of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate, Bacillus belye and Aspergillus niger includes the following steps: ① Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate, and Bacillus belye were inoculated into LB medium and cultured at 30-40℃ and 180-200 r / min for 20-36 h to obtain seed culture of each strain; Aspergillus niger was inoculated into PDA medium and cultured at 28-30℃ and 180-200 r / min for 36-48 h to obtain Aspergillus niger seed culture; ② The Bacillus subtilis seed culture, Bacillus licheniformis seed culture, Acinetobacter calcium acetate seed culture and Bacillus vesalis seed culture prepared in step ① are inoculated into LB medium at an inoculation rate of 5-10% by volume, and cultured at 30-37℃ and 125-130rpm for 40-48h to obtain Bacillus subtilis fermentation broth, Bacillus licheniformis fermentation broth, Acinetobacter calcium acetate fermentation broth and Bacillus vesalis fermentation broth; The Aspergillus niger seed liquid prepared in step ① was inoculated into PDA medium at an inoculation rate of 5-10% by volume and cultured at 25-30℃ and 125-130rpm for 70-74h to obtain Aspergillus niger fermentation broth.

[0011] Preferably, in step (2), the sterilization conditions are 121°C and high-pressure steam treatment for 15-20 minutes.

[0012] The application of the above-mentioned compound microbial agents in soil improvement.

[0013] Preferably, the above-mentioned compound microbial agent is used in the improvement of saline-alkali land.

[0014] The application of the above-mentioned compound microbial agents in crop cultivation.

[0015] Beneficial effects of the present invention The compound microbial agent provided by this invention promotes potato growth in saline-alkali soil environments and increases the water content of potato leaves; it also improves the physical and chemical properties of the soil. Attached Figure Description

[0016] Figure 1 This is a diagram showing the growth status of potatoes.

[0017] Figure 2 A line graph showing the change in potato plant height over time; In the figure: Experimental group 1 did not use compound microbial inoculant and served as a blank control group; Experimental group 2 used compound microbial inoculant and served as an experimental group; Experimental group 3 used compound microbial inoculant without added citric acid; Experimental group 4 used compound microbial inoculant without added biochemical potassium humate; Experimental group 5 used compound microbial inoculant with added sterilized fermentation broth of each strain.

[0018] Figure 3This is a comparison chart of potato leaf water content at the end of Experiment 1; Among them, experimental group 1 did not use compound microbial inoculants; experimental group 2 used compound microbial inoculants; experimental group 3 used compound microbial inoculants without added citric acid; experimental group 4 used compound microbial inoculants without added biochemical potassium humate; and experimental group 5 used compound microbial inoculants with added sterilized fermentation broth of each strain. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0020] All details not described in the embodiments are based on existing technology in the field.

[0021] Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0022] The Bacillus subtilis, Acinetobacter calcium acetate, Bacillus licheniformis, Bacillus belye, and Aspergillus niger of this invention were purchased from the China Agricultural Microbial Culture Collection Center (ACCC).

[0023] This invention does not have any particular limitation on the source of LB medium; commercially available LB medium or medium prepared by conventional methods can be used.

[0024] Example 1 Preparation of fermentation broth for various strains The Bacillus subtilis accession number is ACCC 60429, Acinetobacter calcium acetate accession number is ACCC 01236, Bacillus licheniformis accession number is ACCC 05186, Bacillus belyssus accession number is ACCC 03040, and Aspergillus niger accession number is ACCC 32413.

[0025] The preparation methods for fermentation broths of various microbial strains include the following steps: (1) Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate and Bacillus vesalis strains were inoculated into LB medium and cultured at 180 r / min and 30℃ for 36 h to obtain activated seed liquids of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate and Bacillus vesalis. Aspergillus niger was inoculated into PDA medium and cultured at 180 r / min and 30 ℃ for 36 h to obtain activated Aspergillus niger seed liquid.

[0026] (2) The activated seed cultures of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcium acetate and Bacillus belye were inoculated into LB liquid medium at a volume fraction of 5% and cultured at 30°C and 125 rpm for 48 h to obtain the fermentation broth of each strain.

[0027] (3) The activated Aspergillus niger seed liquid was inoculated into PDA liquid culture medium at a volume fraction of 5% and cultured at 30℃ and 125rpm for 70h to obtain Aspergillus niger fermentation broth.

[0028] Ultimately, the number of spores in the Bacillus licheniformis fermentation broth was 1.6 × 10⁻⁶. 8 The cfu / mL concentration of Acinetobacter calcium acetate in the fermentation broth was 1.5 × 10⁻⁶. 8 The cfu / mL concentration of Bacillus subtilis fermentation broth was 1.0 × 10⁻⁶. 9 The cfu / mL concentration of Bacillus belysinus fermentation broth was 1.0 × 10⁻⁶. 8 The cfu / mL concentration of Aspergillus niger fermentation broth was 1.0 × 10⁻⁶. 8 cfu / mL.

[0029] Example 2 A compound microbial agent for improving saline-alkali land is composed of the following components in parts by weight: 30 parts Bacillus subtilis, 10 parts Bacillus licheniformis, 15 parts Acinetobacter calcareae, 10 parts Bacillus belye, 5 parts Aspergillus niger, 2 parts citric acid, 23 parts biochemical potassium humate, 10 parts water, 50 parts rice husk, 3 parts pig manure, 10 parts fish meal, and 10 parts soybean meal.

[0030] Preparation of compound microbial agents Rice husks, dried pig manure, fish meal, soybean flour, and water were mixed in the corresponding weight proportions mentioned above and sterilized by high-pressure steam treatment at 121°C for 15 minutes to serve as the substrate for microbial fermentation.

[0031] The fermentation broths of each strain prepared in Example 1 were added to the fermentation substrate according to the corresponding weight parts mentioned above. After stirring evenly, the mixture was allowed to ferment at 28°C for 72 hours to obtain the fermentation product.

[0032] Before the fermentation process begins, 3 parts of citric acid are added to the fermentation substrate; after the fermentation is completed, 23 parts of biochemical potassium humate are added to the fermentation product according to the corresponding weight and mixed evenly; finally, the finished product of compound microbial agent is obtained.

[0033] Comparative Example 1 The difference from Example 2 is that citric acid is not added, but everything else is the same, resulting in a compound microbial inoculant.

[0034] Comparative Example 2 The difference from Example 2 is that no biochemical potassium humate was added, but everything else is the same, and a compound microbial agent was prepared.

[0035] Comparative Example 3 The difference from Example 2 is that the fermentation broth of each strain was sterilized before being added, while everything else is the same, to obtain a compound microbial agent.

[0036] Comparative Example 4 The difference from Example 2 is that after adding various bacterial fermentation liquids, instead of allowing them to stand at 28°C for 72 hours for fermentation, 3 parts of citric acid and 23 parts of biochemical potassium humate are directly added and mixed evenly to obtain a compound microbial agent.

[0037] Comparative Example 5 The difference from Example 2 is that no Aspergillus niger fermentation liquid was added, but everything else was the same, and a compound microbial agent was prepared.

[0038] Experimental Example 1 Application of compound microbial agents in potato cultivation Tested variety: Potato Lishu No. 7; Soil samples were taken from different locations in the same area of ​​Yikeshu Village, Hekou District, Dongying City, Shandong Province. The resulting soil samples were saline-alkali soils with high salt content and pH > 8.

[0039] Experimental treatment: The flowerpots used in the experiment were 30cm long, 30cm wide, and 25cm high, and contained 1.5kg of soil.

[0040] The experiment consisted of five groups, each with three replicates. The experimental procedures were as follows: Experimental Group 1: Using the test soil, the treated potato tubers with buds were buried 5cm below the soil surface and finally covered with soil.

[0041] Experimental Group 2: Using the test soil, the treated potato tubers with buds were buried 5cm below the soil surface, and only 10g of the compound microbial agent prepared in Example 2 was applied around the potatoes. Finally, the soil was used to cover the potatoes.

[0042] Experimental Group 3: Using the test soil, the treated potato tubers with buds were buried 5cm below the soil surface, and only 10g of the compound microbial agent prepared in Comparative Example 1 was applied around the potatoes. Finally, the soil was used to cover the potatoes.

[0043] Experimental Group 4: Using the test soil, the treated potato tubers with buds were buried 5cm below the soil surface, and only 10g of the compound microbial agent prepared in Comparative Example 2 was applied around the potatoes. Finally, the soil was used to cover the potatoes.

[0044] Experimental Group 5: Using the test soil, the treated potato tubers with buds were buried 5cm below the soil surface, and only 10g of the compound microbial agent prepared in Comparative Example 3 was applied around the potatoes. Finally, the soil was used to cover the potatoes.

[0045] Experiment Example 2 The impact of non-fermented compound microbial agents on potato cultivation The test soil was selected, and the treated potato tubers with buds were buried 5cm below the soil surface. Only 10g of the compound microbial agent prepared in Comparative Example 4 was applied around the potatoes, and finally the soil was covered.

[0046] Experiment Example 3: Effects of Different Compound Microbial Inoculant Components on Potato Cultivation The test soil was selected, and the treated potato tubers with buds were buried 5cm below the soil surface. Only 10g of the compound microbial agent prepared in Comparative Example 5 was applied around the potatoes, and finally the soil was covered.

[0047] Plant height detection method: Using a ruler or measuring tape, measure vertically from the base of the plant (at the soil surface) to the highest point of the plant under its natural growth state. Repeat the measurement 3 times and take the average value.

[0048] Methods for detecting leaf moisture content: Weigh the fresh weight of the leaf to be tested ( ), Oven at 105℃ for 15 min to fix the green, then dry at 70-80℃ until constant weight, and weigh the dry weight ( ), calculate: leaf water content (%) = .

[0049] pH value detection: Refer to industry standard NY / T 1121.2-2006, mix and shake with a soil-to-water ratio of 1:2.5 (or 1:5), let stand, and then measure the potential value of the supernatant with a pH meter and convert it to pH.

[0050] Available phosphorus detection: Refer to the People's Republic of China Agricultural Industry Standard for Available Phosphorus in Soil NY / T 1121.7-2014. For neutral / calcareous soils, use sodium bicarbonate extraction; for acidic soils, use ammonium fluoride-hydrochloric acid extraction. Determine the absorbance of the extract using the molybdenum-antimony colorimetric method and convert it to available phosphorus content.

[0051] Available potassium detection: The concentration of potassium ions in the extract was determined by a flame photometer in accordance with the People's Republic of China agricultural industry standard NY / T 1121.14-2016, which specifies the extraction of available potassium from soil using neutral ammonium acetate solution.

[0052] The biomass indicators of potato seedlings reflect the growth status of seedlings under a certain stress treatment, indicating that alkaline salt stress inhibits the accumulation of aboveground biomass in potatoes, thereby affecting the normal growth of seedlings.

[0053] After a period of time, the salt- and alkali-tolerant compound microbial improver was applied under the conditions of an indoor temperature of 21℃, an average daily sunshine of 11-12 hours, and soil moisture of 60%-70%.

[0054] Potato plant height comparison chart as follows: Figure 1 As shown, other test results are shown in Tables 1, 2, and 3. Figure 2 and Figure 3 .

[0055] Table 1

[0056] Table 2

[0057] Table 3 Comparison of Soil Physicochemical Properties

[0058] The compound microbial agent provided by this invention provides appropriate nutrients for potato growth in saline-alkali soil, rationally controls fertilization, ensures robust and healthy potato growth, alleviates salt stress on plants growing in saline-alkali soil, improves plant salt tolerance, promotes plant growth, and increases the water content of potato leaves. In addition, the microbial agent can activate some insoluble elements in the soil, effectively improving the soil environment. The compound microbial agent achieves greater benefits when all components are added in specific proportions.

Claims

1. A complex microbial inoculant, characterized in that, The following components are included by weight parts: Bacillus subtilis 20-30 parts, Bacillus licheniformis 10-15 parts, Acinetobacter calcoaceticus 10-20 parts, Bacillus velezensis 10-20 parts, Aspergillus niger 5-10 parts, citric acid 1-3 parts, biochemical potassium fulvic acid 20-30 parts, water 10-20 parts, rice husk 50-60 parts, pig manure 2-5 parts, fish meal 10-20 parts and soybean meal 10-20 parts.

2. The complex microbial agent according to claim 1, wherein In the complex microbial agent, the preservation number of Bacillus subtilis is ACCC 60429, the preservation number of Acinetobacter calcoaceticus is ACCC 01236, the preservation number of Bacillus licheniformis is ACCC 05186, the preservation number of Bacillus velezensis is ACCC 03040, and the preservation number of Aspergillus niger is ACCC 32413.

3. The complex microbial agent according to claim 1, wherein The bacterial species components: the number of bacillus subtilis spores is (1-3) x 10 9 cfu / mL, the number of bacillus licheniformis spores is (1-2) x 10 8 cfu / mL, the number of acinetobacter calcoaceticus living bacteria is (1-2) x 10 8 cfu / mL, the number of bacillus velezensis spores is (1-2) x 10 8 cfu / mL, the number of aspergillus niger spores is (1-2) x 10 8 cfu / mL.

4. The complex microbial agent according to claim 1, wherein The complex microbial agent includes the following components by weight parts: Bacillus subtilis 30 parts, Bacillus licheniformis 10 parts, Acinetobacter calcoaceticus 15 parts, Bacillus velezensis 10 parts, Aspergillus niger 5 parts, citric acid 2 parts, biochemical potassium fulvic acid 23 parts, water 10 parts, rice husk 50 parts, pig manure 3 parts, fish meal 10 parts and soybean meal 10 parts.

5. The method for preparing the complex microbial agent according to any one of claims 1 to 4, characterized by, The following steps are included: (1) The strains of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcoaceticus, Bacillus velezensis and Aspergillus niger are respectively fermented and cultured to obtain Bacillus subtilis fermentation broth, Bacillus licheniformis fermentation broth, Acinetobacter calcoaceticus fermentation broth, Bacillus velezensis fermentation broth and Aspergillus niger fermentation broth; (2) The rice husk, pig manure, fish meal, soybean meal and water are mixed according to the corresponding weight parts and sterilized to obtain a microbial fermentation substrate; (3) The Bacillus subtilis fermentation broth, Bacillus licheniformis fermentation broth, Acinetobacter calcoaceticus fermentation broth, Bacillus velezensis fermentation broth and Aspergillus niger fermentation broth in step (1) are inoculated into the microbial fermentation substrate prepared in step (2) according to the corresponding weight parts, mixed uniformly, and then placed for fermentation at 28-30℃ for 60-72 hours; before the start of the fermentation process, the corresponding weight parts of citric acid are added to the fermentation substrate; after the end of the fermentation, the corresponding weight parts of biochemical potassium fulvic acid are added to the fermentation product to obtain a complex microbial agent.

6. The production method according to claim 5, wherein In step (1), the method for respectively fermenting and culturing the strains of Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcoaceticus, Bacillus velezensis and Aspergillus niger includes the following steps: ① Bacillus subtilis, Bacillus licheniformis, Acinetobacter calcoaceticus and Bacillus velezensis are respectively inoculated into LB culture medium and cultured at 30-40℃ and 180-200r / min for 20-36h to obtain seed liquid of each strain; Aspergillus niger is inoculated into PDA culture medium and cultured at 28-30℃ and 180-200r / min for 36-48h to obtain Aspergillus niger seed liquid; ② The Bacillus subtilis seed culture, Bacillus licheniformis seed culture, Acinetobacter calcium acetate seed culture and Bacillus vesalis seed culture prepared in step ① are inoculated into LB medium at an inoculation rate of 5-10% by volume, and cultured at 30-37℃ and 125-130rpm for 40-48h to obtain Bacillus subtilis fermentation broth, Bacillus licheniformis fermentation broth, Acinetobacter calcium acetate fermentation broth and Bacillus vesalis fermentation broth; The Aspergillus niger seed liquid prepared in step ① was inoculated into PDA medium at an inoculation rate of 5-10% by volume and cultured at 25-30℃ and 125-130rpm for 70-74h to obtain Aspergillus niger fermentation broth.

7. The production method according to claim 5, wherein In step (2), the sterilization conditions are 121°C and high-pressure steam treatment for 15-20 minutes.

8. The application of the compound microbial agent according to any one of claims 1-4 in soil improvement.

9. Use according to claim 8, wherein the compound is ###0002### Application of the compound microbial agent in the improvement of saline-alkali land.

10. The application of the compound microbial agent according to any one of claims 1-4 in crop cultivation.

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