Microbial composition for repairing saline alkali soil, microbial agent and application of microbial agent

Abstract

The invention relates to the technical field of microbial soil remediation, in particular to a microbial composition for remediation of saline alkali soil, a microbial agent and application of the microbial agent. According to the characteristics of the bacillus subtilis KC000 and the paenibacillus mucilaginosus JDY001, different compounding proportions are set and applied to the saline-alkali soil, then the salt content of the saline-alkali soil is measured, and it is found that the soil nutrition and environmental conditions are obviously improved by applying the microbial agent, and the saline-alkali soil can be applied to the saline-alkali soil. The soil conditioner is beneficial to the growth and reproduction of paenibacillus mucilaginosus and bacillus subtilis in soil, is beneficial to improving the content of organic matters, rapidly available phosphorus and rapidly available potassium in the soil, increases the content of alkali-hydrolyzable nitrogen in the soil to a certain extent, reduces the pH value of the soil, accelerates salt leaching, inhibits salt return and reduces the salt content of the surface layer of the soil. Meanwhile, the microbial composition also has the effect of preventing and treating sheath blight.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial soil remediation, and in particular to a microbial combination for remediating saline-alkali soil, a microbial agent and applications thereof. Background Art

[0002] Saline-alkali land is a typical soil degradation phenomenon characterized by high levels of salt in the soil, which can affect crop growth. Saline-alkali land is widespread globally. In China, it is primarily found in North China, Northeast China, and Northwest China.

[0003] The formation of saline-alkali land is primarily due to rising groundwater levels, which causes salt to accumulate in the soil surface. This salt is redistributed horizontally and vertically within the soil, eventually accumulating at the surface and affecting plant growth. Furthermore, saline-alkali soils are prone to compaction, which reduces soil porosity, air permeability, and water infiltration. This not only affects the growth and metabolism of functional microorganisms in the soil but also reduces the activity of enzymes such as invertase, alkaline phosphatase, and catalase, thereby reducing the conversion rate of soil organic matter.

[0004] Traditional methods for treating saline-alkali land mainly include physical improvement, hydraulic improvement and chemical improvement. Although these methods can alleviate the salinization problem to a certain extent, they are often costly, have limited effects, and may cause secondary pollution. For example, physical improvement methods (covering with sand and soil, micro-area soil improvement, deep plowing and ground covering, etc.) can reduce the salt content by changing the soil structure, but they are difficult to implement and costly. Although chemical improvement methods (applying chemical amendments) can quickly improve soil properties, long-term use may lead to soil pollution and ecological imbalance.

[0005] In recent years, with the continuous development of microbial technology, its application in saline-alkali land remediation has gradually demonstrated unique advantages. The core of microbial technology for saline-alkali land remediation lies in leveraging the metabolic activities of naturally occurring microbial flora to improve soil structure, enhance soil fertility, and reduce salinity. Microorganisms can decompose organic matter in the soil, producing humus, which in turn improves soil aeration, water permeability, and water retention. Furthermore, certain microorganisms can react with salt in the soil, converting it into harmless or low-toxic substances, thereby reducing soil salinity.

[0006] Microbial preparations are an important tool for the microbial treatment of saline-alkali land. These preparations typically contain multiple beneficial microorganisms that work synergistically to improve the soil environment. Applying these preparations not only increases the number of microorganisms in the soil and enhances its biological activity, but also promotes nutrient cycling, improving crop resistance and yield.

[0007] The core of microbial technology for saline-alkali land remediation lies in leveraging the metabolic activities of naturally occurring microbial communities to improve soil structure, enhance soil fertility, and reduce salinity. Specifically, microorganisms can decompose organic matter in the soil, producing humus, which in turn improves soil aeration, water permeability, and water retention. Furthermore, certain microorganisms can react with soil salt, converting it into harmless or low-toxic substances, thereby reducing soil salinity.

[0008] In summary, microbial agents hold broad application prospects in saline-alkali land remediation. However, applying microbial technology to saline-alkali land remediation is not a one-off process. In practice, it is necessary to formulate scientific and rational remediation plans based on local climate, soil, crop conditions, and other factors. Furthermore, efforts should be made to strengthen the research and development and promotion of microbial technology to enhance its application in saline-alkali land remediation. Summary of the Invention

[0009] The purpose of the present invention is to provide a microbial combination, a microbial agent and their application for repairing saline-alkali soil, so as to solve the problems existing in the above-mentioned prior art.

[0010] To achieve the above object, the present invention provides the following solutions:

[0011] The present invention provides a microbial combination for repairing saline-alkali soil, wherein the microbial combination comprises Bacillus subtilis KC000 and Paenibacillus mucilaginosus JDY001.

[0012] The deposit number of the Bacillus subtilis KC000 is CGMCC No. 34047; the deposit number of the Paenibacillus jelly JDY001 is CGMCC No. 34048.

[0013] Preferably, the ratio of the number of viable bacteria of the Bacillus subtilis KC000 to the number of viable bacteria of the Paenibacillus gelatinosa JDY001 in the microbial combination is (20-80):(5-20).

[0014] Preferably, the ratio of the number of viable bacteria of the Bacillus subtilis KC000 and the Paenibacillus jelly JDY001 in the microbial combination is 1:1.

[0015] The present invention provides the use of the above-mentioned microbial combination in preparing a microbial agent for repairing saline-alkali soil.

[0016] The present invention provides a microbial agent for repairing saline-alkali soil, which comprises the above-mentioned microbial combination.

[0017] Preferably, the microbial agent further comprises auxiliary materials.

[0018] The present invention provides the use of the above-mentioned microbial combination or the above-mentioned microbial agent in repairing saline-alkali land.

[0019] The present invention provides a method for repairing saline-alkali land, comprising the step of applying the above-mentioned microbial agent to the soil to be repaired.

[0020] Preferably, the application amount of the microbial agent is 20 kg / mu; the application frequency is 3 times, with an interval of 1 month between each application.

[0021] The present invention provides the use of the above-mentioned microbial combination in the preparation of a microbial preparation for resisting sheath blight.

[0022] As an additional solution, the present invention provides a microbial agent for resisting sheath blight, which comprises the above-mentioned microbial combination.

[0023] Further preferably, the microbial agent also includes auxiliary materials.

[0024] As an additional solution, the present invention provides the use of the above-mentioned microbial combination or the above-mentioned microbial agent in preventing and treating sheath blight.

[0025] As an additional solution, the present invention provides a method for preventing and controlling sheath blight, comprising the step of applying the above-mentioned microbial agent to the soil.

[0026] Further preferably, the application amount of the microbial agent is 20 kg / mu; the application frequency is 3 times, with an interval of 1 month between each application.

[0027] The present invention discloses the following technical effects:

[0028] The present invention first uses the jelly-like Bacillus species and the Bacillus subtilis species from the Microbial Fertilizer Technology Research and Extension Center as the starting strains. Through methods such as high temperature and mutagenesis, the strains are selected based on their salt and alkali tolerance. The selected strains are then tested for fermentation ability through shake flask fermentation spore formation rate experiments. Ultimately, the Bacillus subtilis KC000 and the jelly-like Bacillus JDY001 are selected. Based on the characteristics of the two strains, the present invention configures them into microbial agents with different compounding ratios to obtain microbial agents that reduce the salt content of saline-alkali soil. The agents are then applied to saline-alkali soil, and the salt content of the repaired saline-alkali soil is then measured. The application of the microbial agent significantly improves soil nutrition and environmental conditions, promotes the growth and reproduction of jelly-like Bacillus and Bacillus subtilis in the soil, and helps increase the content of soil organic matter, available phosphorus, and available potassium. It also increases the soil alkaline nitrogen content to a certain extent, reduces soil pH, accelerates salt leaching, inhibits salt return, and reduces the salt content of the soil surface. At the same time, the microbial combination also has the effect of preventing and treating sheath blight. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1 Screening of Paenibacillus mucilaginosus JDY001

[0035] 1. Preparation of different strains of bacterial cells

[0036] The emerging strains were isolated and screened from the strain bank of the Microbial Fertilizer Technology Research and Extension Center. After being heat-treated at 60°C, 80°C, and 100°C for 20 minutes, they were inoculated into potassium-dissolving culture medium and incubated in a 32°C constant-temperature incubator for 48 hours before use.

[0037] 2. Screening of salt- and alkali-tolerant strains

[0038] (1) Place the strain prepared in step 1 into 10 mL of sterile water and shake thoroughly to disperse the cells for later use.

[0039] (2) Prepare culture media with different salinity and alkali content, pour them onto plates, and set aside for use after solidification. The basic components of the culture media with different salinity contents are: peptone 1%, sucrose 8%, Na2HPO4 1.5%, different concentrations of sodium chloride, (NH4)2SO4 0.6%, MgSO4·7H2O 1.5%, CaCO3 1.5%, yeast extract 0.6%, FeCl3 0.01%, agar 1.9%. The preparation method of the culture media is: add the above ingredients to 1L of water to obtain the culture media; the specific components and pH value of the culture media are shown in Table 1.

[0040] Table 1 Culture media with different salt contents

[0041]

[0042] (3) Strain induction treatment: The bacterial solution to be used in step (1) was placed on the plate prepared in step (2) and then cultured at 32°C for 48 hours. The strains grown on the plate were selected and induced three times to select the optimal strain of Paenibacillus jelly-like, JDY001.

[0043] The key characteristic of Paenibacillus jelly is its ability to decompose potassium-containing minerals in silicates, aluminosilicates, and other ores. It has the functions of dissolving phosphorus, releasing potassium, and fixing nitrogen. It also releases soluble trace elements such as calcium, sulfur, magnesium, iron, zinc, molybdenum, and manganese, which not only improves soil fertility but also provides crops with a comprehensive range of nutrients that can be absorbed and utilized. Simultaneously, during growth and reproduction, it produces organic acids, amino acids, polysaccharides, and other substances that are beneficial for plant absorption and utilization. After propagation in the soil, it secretes plant growth stimulants and various enzymes to enhance crop resistance to certain diseases and inhibit the growth of other pathogens. In this example, different saline and alkali components were added to the primary screening solid plate culture medium to induce salt and alkali tolerance. After repeated screening and cultivation, the strain JDY001 with good salt and alkali tolerance was selected.

[0044] In the present invention, the strain JDY001 is named Paenibacillus mucilaginosus JDY001, and the strain was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration of Microorganisms on April 1, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 34048.

[0045] Example 2 Screening of Bacillus subtilis KC000

[0046] 1. Preparation of different strains of bacterial cells

[0047] The emerging strains were isolated and screened from the strain bank of the Microbial Fertilizer Technology Research and Extension Center. After being heat-treated at 60°C, 80°C, and 100°C for 30 minutes, they were inoculated into nutrient agar medium and incubated in a 35°C constant-temperature incubator for 48 hours before use.

[0048] 2. Screening of salt- and alkali-tolerant strains

[0049] (1) Place the strain prepared in step 1 into 10 mL of sterile water and shake thoroughly to disperse the cells for later use.

[0050] (2) Prepare culture media with different salinity and alkali content and pour them onto plates. Set aside for use after solidification. The basic components of the culture media with different salinity contents are: 1% peptone, 0.3% beef extract, different concentrations of sodium chloride, and 1.9% agar. The culture media are prepared by adding the above ingredients to 1 L of water. The specific components of the culture media are shown in Table 2.

[0051] Table 2 Culture media with different salt contents

[0052]

[0053] (3) Strain induction treatment: The bacterial solution to be used in step (1) was placed on the plate prepared in step (2) and then incubated at 35°C for 48 hours. The strain grown on the plate was selected and the optimal Bacillus subtilis strain, KC000, was selected after three repeated induction treatments.

[0054] The important characteristic of Bacillus subtilis is that it can reproduce rapidly in the soil, becoming a dominant bacterium, effectively inhibiting the growth and reproduction of other pathogens in the soil and preventing the occurrence of soil-borne diseases. During the reproduction process, it can produce active substances such as subtilisin, polymyxin, nystatin, and gramicidin. These active substances cause the mycelium of pathogens to break, disintegrate, or dissolve the cytoplasm, achieving the effect of killing pathogens, effectively preventing and controlling the harm of pathogens to crops. It can utilize the humus in the soil to reproduce in large quantities and decompose the organic matter in the soil. Bacillus subtilis can not only increase the available nutrients in the soil, improve soil structure, but also prevent soil from becoming compacted. During the reproduction process, it can also produce multiple hormone substances and various vitamins, which can not only increase the nutrient content of the soil, but also promote crop growth, increase yield, and improve the quality of agricultural products. In this embodiment, different saline and alkali components were added to the primary screening solid plate culture medium to induce salt and alkali tolerance. After repeated screening and cultivation, the strain with good salt and alkali tolerance, KC000, was selected.

[0055] The present invention named the strain -KC000 as Bacillus subtilis KC000, and on April 1, 2025, the strain was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 34047.

[0056] Example 3 Screening of Fermentation Medium for Bacillus jelly JDY001 and Bacillus subtilis KC000

[0057] 1. Screening process of fermentation medium for jelly-like Paenibacillus JDY001

[0058] The jelly-like Paenibacillus JDY001 in Example 1 was subjected to seed culture and fermentation culture in a fermenter in sequence to obtain a jelly-like Paenibacillus JDY001 fermentation broth; wherein the seed culture used a jelly-like Paenibacillus seed culture medium; and the fermentation culture used different jelly-like Paenibacillus fermentation culture media.

[0059] 1.1. Seed culture medium of Paenibacillus jelly JDY001:

[0060] Sucrose 10 g, K2HPO4·3H2O 0.2 g, calcium chloride 1 g, magnesium sulfate 0.2 g, ammonium sulfate 0.005 g and water 1000 mL, pH 7.0-7.2, sterilize at 121°C for 30 min.

[0061] 1.2. Seed culture conditions:

[0062] The inoculation amount is 10%, the rotation speed is 160r / min, the temperature is 30℃, and the culture is carried out for 12-15h.

[0063] 1.3. Fermentation medium for Paenibacillus jelly JDY001

[0064] Treatment 1: 10 g sucrose, 10 g starch, 0.01 g sodium chloride, 1.0 g yeast extract or 0.8 g yeast extract powder (1.0 g yeast extract was used in this example), 5 g calcium carbonate, 0.08 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.005 g ammonium sulfate, and 1000 mL water, pH 7.2-7.4;

[0065] Treatment 2: 8 g sucrose, 10 g starch, 0.01 g sodium chloride, 1.2 g yeast extract or 0.8 g yeast extract powder (1.2 g yeast extract was used in this example), 5 g calcium carbonate, 0.08 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.005 g ammonium sulfate, and 1000 mL water, pH 7.2-7.4;

[0066] Treatment 3: 10 g sucrose, 8 g starch, 0.01 g sodium chloride, 1.0 g yeast extract or 0.8 g yeast extract powder (1.0 g yeast extract was used in this example), 5 g calcium carbonate, 0.08 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.005 g ammonium sulfate, and 1000 mL water, pH 7.2-7.4;

[0067] Treatment 4: 12 g sucrose, 10 g starch, 0.01 g sodium chloride, 1.0 g yeast extract or 0.8 g yeast extract powder (1.0 g yeast extract was used in this example), 5 g calcium carbonate, 0.08 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.005 g ammonium sulfate, and 1000 mL water, pH 7.2-7.4;

[0068] Treatment 5: 10 g sucrose, 12 g starch, 0.01 g sodium chloride, 1.2 g yeast extract or 0.8 g yeast extract powder (1.2 g yeast extract was used in this example), 5 g calcium carbonate, 0.08 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.005 g ammonium sulfate, and 1000 mL water, pH 7.2-7.4.

[0069] 1.4. Shake flask fermentation conditions

[0070] The inoculation amount was 10%, the temperature was 33°C, the rotation speed was 180 r / min, and the fermentation culture was carried out for 72 hours.

[0071] 2. Investigation of the number of effective viable cells in the fermentation broth of Paenibacillus sp. JDY001 obtained from different fermentation media

[0072] Table 3 shows the results of the investigation of the effective viable bacterial count and exopolysaccharide content in the fermentation broths of Paenibacillus jelly JDY001 obtained from different fermentation media. The results showed that the bacterial count and exopolysaccharide content were the highest in Treatment 2. Therefore, the fermentation medium provided by Treatment 2 was used in subsequent experiments.

[0073] Table 3 Survey results of the effective viable bacteria count in the fermentation broth of Paenibacillus japonicus JDY001 obtained from different fermentation media

[0074]

[0075] 3. Screening process of Bacillus subtilis KC000 fermentation medium

[0076] The Bacillus subtilis KC000 in Example 2 was subjected to seed culture and fermentation culture in a fermenter in sequence to obtain a Bacillus subtilis KC000 fermentation broth; wherein the seed culture used a Bacillus subtilis seed culture medium; and the fermentation culture used different Bacillus subtilis fermentation culture media.

[0077] Bacillus subtilis KC000 seed culture medium: 10 g corn flour, 10 g soybean meal, 5 g yeast extract, 10 g bran, and 1000 mL water, pH 7.0-7.5, sterilize at 121°C for 30 min.

[0078] 3.2. Seed culture conditions:

[0079] Seed culture conditions: inoculation amount is 10%, rotation speed is 160r / min, temperature is 30℃, and culture is 12-15h.

[0080] 1.3. Bacillus subtilis KC000 fermentation medium

[0081] Treatment 1: 8 g corn flour, 10 g soybean meal, 5 g yeast extract, 10 g bran, 1.0 g calcium chloride, 0.05 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, and 1000 mL water, pH 7.0-7.5;

[0082] Treatment 2: 10 g corn flour, 10 g soybean meal, 5 g yeast extract, 10 g bran, 1.0 g calcium chloride, 0.05 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, and 1000 mL water, pH 7.0-7.5;

[0083] Treatment 3: 12 g corn flour, 10 g soybean meal, 5 g yeast extract, 10 g bran, 1.0 g calcium chloride, 0.05 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, and 1000 mL water, pH 7.0-7.5;

[0084] Treatment 4: 10 g corn flour, 8 g soybean meal, 5 g yeast extract, 10 g bran, 1.0 g calcium chloride, 0.05 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, and 1000 mL water, pH 7.0-7.5;

[0085] Treatment 5: 10 g corn flour, 12 g soybean meal, 5 g yeast extract, 10 g bran, 1.0 g calcium chloride, 0.05 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, and 1000 mL water, pH 7.0-7.5.

[0086] 3.4. Shake flask fermentation conditions

[0087] The inoculation amount was 10%, the temperature was 33°C, the rotation speed was 180 r / min, and the fermentation culture was carried out for 72 hours.

[0088] 4. Investigation of the number of effective viable cells in Bacillus subtilis KC000 fermentation broth obtained from different fermentation media

[0089] The results of the investigation of the effective viable bacterial counts in the Bacillus subtilis KC000 fermentation broths obtained using different fermentation media are shown in Table 4. The results showed that the bacterial content in Treatment 2 was the highest. Therefore, the fermentation medium provided by Treatment 2 was used in subsequent experiments.

[0090] Table 4 Survey results of effective viable bacteria count in Bacillus subtilis KC000 fermentation broth obtained from different fermentation media

[0091]

[0092] Example 4 Preparation of jelly-like Paenibacillus JDY001 and Bacillus subtilis KC000 liquid preparations

[0093] The jelly-like Paenibacillus JDY001 in Example 1 and the Bacillus subtilis KC000 in Example 2 were sequentially subjected to seed culture and fermentation culture in a fermenter to obtain a jelly-like Paenibacillus JDY001 fermentation broth and a Bacillus subtilis KC000 fermentation broth; wherein the seed culture used a jelly-like Paenibacillus and a Bacillus subtilis seed culture medium, respectively; and the fermentation culture used a jelly-like Paenibacillus and a Bacillus subtilis fermentation medium, respectively.

[0094] 1. Seed culture medium of Paenibacillus jelly JDY001:

[0095] Sucrose 10 g, K2HPO4·3H2O 0.2 g, calcium chloride 1 g, magnesium sulfate 0.2 g, ammonium sulfate 0.005 g and water 1000 mL, pH 7.0-7.2, sterilize at 121°C for 30 min.

[0096] 2. Bacillus subtilis KC000 seed culture medium:

[0097] Corn flour 10g, soybean meal 10g, yeast extract 5g, bran 10g and water 1000mL, pH 7.0-7.5, sterilize at 121℃ for 30min.

[0098] Seed culture conditions: inoculation amount is 10%, rotation speed is 160r / min, temperature is 30℃, and culture is 12-15h.

[0099] 3. Fermentation medium of Paenibacillus jelly JDY001:

[0100] Sucrose 8 g, starch 10 g, sodium chloride 0.01 g, yeast extract 1.2 g or yeast extract powder 0.8 g (1.2 g yeast extract was used in this example), calcium carbonate 5 g, dipotassium hydrogen phosphate 0.08 g, magnesium sulfate 0.5 g, ammonium sulfate 0.005 g, and water 1000 mL, pH 7.2-7.4, sterilize at 121°C for 30 min.

[0101] 4. Bacillus subtilis KC000 fermentation medium:

[0102] Corn flour 10g, soybean meal 10g, yeast extract 5g, bran 10g, calcium chloride 1.0g, potassium dihydrogen phosphate 0.05g, magnesium sulfate 0.2g and water 1000mL, pH 7.0-7.5, sterilize at 121℃ for 30min.

[0103] The inoculation amount of the fermentation tank was 10%, the temperature was 33℃, the dissolved oxygen was controlled to be above 30%, and the fermentation culture was carried out for 72h. The spores reached above 90%, and the bacterial count of Paenibacillus jellis JDY001 in the fermentation broth reached 5×10 8 CFU / mL, the bacterial count of Bacillus subtilis KC000 fermentation broth reached 50×10 8 CFU / mL, the fermentation can be terminated.

[0104] Example 5 Preparation of microbial agent

[0105] The preparation method of the microbial agent comprises the following steps: uniformly mixing a fermentation broth of jelly-like Paenibacillus sp. JDY001 and a fermentation broth of Bacillus subtilis KC000 according to different mass ratios, wherein the effective viable bacteria count in the microbial agent is ≥2 billion / mL.

[0106] Example 6 Application of microbial preparations

[0107] (1) The following experimental microbial agents were prepared according to the preparation method of Example 4. A total of 7 composite bacterial gradients and 2 control groups were set up, and each gradient was repeated 3 times. The soil of each treatment was kept moist. If applied in the field, the average dosage of the microbial agent was 20 kg / mu.

[0108] Control 1: Bacillus subtilis KC000 fermentation broth;

[0109] Control 2: Fermentation broth of Paenibacillus jelly JDY001;

[0110] Treatment 1: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 1:1;

[0111] Treatment 2: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 1:2;

[0112] Treatment 3: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 1:3;

[0113] Treatment 4: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 1:4;

[0114] Treatment 5: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 2:1;

[0115] Treatment 6: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 3:1;

[0116] Treatment 7: Bacillus subtilis KC000 fermentation broth: Paenibacillus jelly JDY001 fermentation broth = 4:1.

[0117] (2) Each pot contains 2500 g of soil, and the application rate of microbial agent is 20 mL / pot, which is applied continuously for 3 months, once a month, for a total of 3 times.

[0118] (3) Detect soil salinity, collect statistics and analyze the experimental results as shown in Table 5.

[0119] Table 5 Repair effects of different treatments

[0120] Serial number Total nitrogen (g / kg) pH Organic matter (g / kg) Total salt content (g / kg) Pristine soil 0.63 8.3 5.5 5.512 Control 1 0.68 7.9 7.1 4.489 Control 2 0.72 7.8 7.2 4.505 Process 1 0.77 7.5 9.1 3.473 Process 2 0.81 7.1 10.2 3.450 Process 3 1.12 7.4 11.0 3.397 Process 4 1.24 6.9 14.0 3.343 Process 5 1.20 6.8 13.0 3.454 Process 6 1.18 6.8 12.9 3.468 Process 7 1.18 6.5 13.6 3.471

[0121] The experimental data in Table 5 demonstrate that microbial agents at different treatment gradients exhibited a moderate remediation effect on saline-alkali soils, increasing nitrogen content, reducing salinity, and regulating pH. The microbial agent prepared from a fermentation broth of Bacillus subtilis KC000 and a fermentation broth of Paenibacillus jelly JDY001 at a mass ratio of 4:1 demonstrated the highest efficacy, reducing the salt content of the original soil from 5.512 to 3.343. This demonstrates that the microbial combination and microbial agent provided by the present invention are effective in remediating saline-alkali soils.

[0122] Example 7 Field Experiment

[0123] 1. Test time and location

[0124] Time: Corn (Zhengdan 958) planting experiment will be conducted from June 15, 2024 to October 22, 2024.

[0125] Experimental location: Zhaozhuangzi Village, Xingji Town, Cang County, Cangzhou City, Hebei Province.

[0126] 2. Experimental materials and methods

[0127] 2.1. Test microbial agent: A microbial agent was prepared by mixing the fermentation broth of Bacillus subtilis KC000 in Example 2 and the fermentation broth of Paenibacillus jelly JDY001 in Example 1 at a mass ratio of 4:1, with an effective viable cell count of ≥ 2 billion / mL.

[0128] 2.2 Test location and basic information

[0129] The test site is located in Cangzhou City. The soil type in the area is fluvo-aquic soil with a sandy loam texture. The experimental design was a fixed-point corn experiment. Before the experiment began, soil samples were collected from 0-20 cm deep as a baseline. After natural air drying, basic soil physical and chemical properties and nutrient content were determined (see Table 6).

[0130] Table 6 Nutrient status of the tested soil

[0131] Analysis Project pH Organic matter (g / kg) Alkaline nitrogen (mg / kg) Available phosphorus (mg / kg) Fast-acting potassium (mg / kg) Analysis results 8.5 14.6 99 14.3 90

[0132] 2.3. Fertilization period and method

[0133] On June 15, 2024, the experimental field was fertilized and leveled. The test fertilizer was applied as base fertilizer at a rate of 20 L / mu.

[0134] Experimental plan and method: This example has 4 treatments and 3 replications, using a completely randomized block design. The plot area is 6.00m×5.00m=30.00m 2 .

[0135] Treatment 1: conventional fertilization;

[0136] Treatment 2: conventional fertilization + test microbial agent;

[0137] Treatment 3: conventional fertilization + equal amount of substrate (this treatment uses inactivated test microbial agents);

[0138] Treatment 4: blank control.

[0139] Other field management measures were the same.

[0140] On June 15, 2024, fertilization and land preparation were carried out, and corn was sown experimentally at a sowing rate of 2 kg per mu. After sowing, the test fertilizer and an equal amount of substrate were applied with water at a rate of 20 kg per mu. Pre-emergence closed weeding was carried out on June 17. On August 4, urea was topdressed at 15 kg per mu and 45 t per mu was watered. Pest and disease control was carried out on August 15. Harvest was carried out on October 22. All data were statistically analyzed.

[0141] 3. Analysis of test results

[0142] 3.1 Effects of soil remediation agents on soil bulk density, total porosity, and proportion of soil water-stable aggregates

[0143] Table 7 Statistics of changes in soil bulk density, total porosity and proportion of water-stable aggregates before and after the test

[0144]

[0145] As shown in Table 7, by comparing the data of soil bulk density, total porosity and proportion of water-stable aggregates before and after planting, it was found that under conventional fertilization conditions, the microbial agent provided by the present invention was applied to the corn in the experimental field. Compared with the other two treatment groups and before planting, the soil bulk density was significantly reduced (P<0.05), the total porosity was significantly increased (P<0.05), and the proportion of soil water-stable aggregates was significantly increased (P<0.05).

[0146] 3.2 Effects of microbial agents on soil pH, organic matter, and available nutrients

[0147] After the crops were harvested, soil samples from the 0-20 cm tillage layer were collected in each experimental field using the diagonal sampling method. The soil pH, organic matter, and nitrogen, phosphorus, and potassium contents were measured and the experimental data were recorded.

[0148] Table 8 Statistics of changes in soil pH, organic matter and available nutrients before and after the experiment

[0149] deal with pH Organic matter (g / kg) Alkaline nitrogen (mg / kg) Available phosphorus (mg / kg) Fast-acting potassium (mg / kg) Before planting <![CDATA[8.5±0.3 a ]]> <![CDATA[14.6±0.4 c ]]> <![CDATA[99±3 d ]]> <![CDATA[14.3±0.6 e ]]> <![CDATA[90±3 d ]]> 1 <![CDATA[8.2±0.2 b ]]> <![CDATA[16.3±0.9 b ]]> <![CDATA[111±2 b ]]> <![CDATA[21.9±1.2 c ]]> <![CDATA[138±2 b ]]> 2 <![CDATA[7.7±0.1 d ]]> <![CDATA[20.1±0.8 a ]]> <![CDATA[120±3 a ]]> <![CDATA[28.8±1.7 a ]]> <![CDATA[162±5 a ]]> 3 <![CDATA[8.0±0.1 c ]]> <![CDATA[16.6±1.0 b ]]> <![CDATA[115±3 b ]]> <![CDATA[25.1±0.8 b ]]> <![CDATA[141±5 b ]]> 4 <![CDATA[8.3±0.2 b ]]> <![CDATA[14.8±0.3 c ]]> <![CDATA[108±1 c ]]> <![CDATA[18.8±0.6 d ]]> <![CDATA[125±7 c ]]>

[0150] As shown in Table 8, under conventional fertilization conditions, the application of the microbial agent provided by the present invention to the corn in the experimental field significantly increased the content of organic matter, alkaline nitrogen, available phosphorus and available potassium in the soil compared with the other two treatment groups and before planting (P<0.05), and improved the pH value of the soil to a certain extent, which can provide a more suitable growth environment for crops.

[0151] 3.3. Inhibitory effects of microbial agents on corn sheath blight and other diseases

[0152] Table 9 Statistical survey of the rate of corn sheath blight in the experimental field

[0153]

[0154] Sheath blight mainly harms the leaf sheaths and stalks of corn. In the early stages of the disease, gray-green water-soaked lesions will appear on the leaf sheaths at the base of the corn, and then the lesions will gradually expand to form large cloud-like spots. The color of the lesions is usually gray-brown to dark brown, with darker edges. As the disease progresses, the lesions will expand upward, causing the leaves to turn yellow and die. In severe cases, it can affect the development of the cobs and even cause lodging. The cause of the disease is mainly affected by the soil environment, planting density and climatic conditions: saline-alkali soil is usually humid and has poor air permeability. This environmental condition is very suitable for the growth and reproduction of sheath blight fungi; when planting corn, if the planting density is too high and the field ventilation and light conditions are poor, it will also increase the chance of the spread of the pathogen; in high temperature and high humidity climatic conditions, especially in the rainy season or after irrigation, the disease is more likely to occur.

[0155] As shown in Table 9, the use of the microbial agent of the present invention significantly improved this symptom. The various functional substances in the microbial agent promoted the formation of soil aggregates, increased soil organic matter content, balanced soil nutrients, and inhibited the spread of some crop rhizosphere pathogens, thereby reducing the incidence of sheath blight. As can be seen from the results in the table, the average incidence of sheath blight in Treatment 2 was 35.71%, 33.33%, and 50.00% lower than that in Treatments 1, 3, and 4, respectively. This demonstrates that the microbial agent has an antagonistic effect on pathogens, significantly reducing their numbers and significantly reducing the incidence of the disease.

[0156] 3.4 Effects of microbial agents on soil microbial populations

[0157] Table 10 Effects of different fertilization treatments on the number of soil bacteria, fungi and actinomycetes

[0158] deal with <![CDATA[Bacteria (×10 7 cfu·g -1 )]]> <![CDATA[Fungus (×10 2 cfu·g -1 )]]> <![CDATA[Actinomycetes (×10 5 cfu·g -1 )]]> 1 <![CDATA[24.95±0.95 c ]]> <![CDATA[64.43±0.67 b ]]> <![CDATA[54.16±0.96 c ]]> 2 <![CDATA[29.58±0.94 a ]]> <![CDATA[71.24±0.64 a ]]> <![CDATA[60.96±0.25 a ]]> 3 <![CDATA[26.90±0.25 b ]]> <![CDATA[66.76±0.93 b ]]> <![CDATA[56.39±2.31 b ]]> 4 <![CDATA[17.99±0.48 d ]]> <![CDATA[53.73±1.61 c ]]> <![CDATA[45.93±0.78 d ]]>

[0159] Bacteria, actinomycetes, and fungi in the soil are the primary components of the microbial flora within the soil ecosystem. Changes in the composition and abundance of the soil microbial flora are significantly impacted by the transformation and absorption of soil nutrients, as well as the balance of the soil microecology. They are important biological indicators of changes in soil environmental quality. After harvest, soil samples were collected from the 0-20 cm tillage layer of each experimental plot using the diagonal sampling method, and viable bacteria, fungi, and actinomycetes were counted.

[0160] As shown in Table 10, under conventional fertilization conditions, the application of the microbial agent of the present invention to the corn in the experimental field resulted in significantly higher numbers of bacteria, fungi, and actinomycetes in the soil than in the other three treatments (P<0.05). The number of bacteria in Treatment 2 increased by 18.59%, 9.99%, and 64.44% compared to Treatments 1, 3, and 4, respectively; the number of fungi in Treatment 2 increased by 10.56%, 6.71%, and 32.59% compared to Treatments 1, 3, and 4, respectively; and the number of actinomycetes in Treatment 2 increased by 12.56%, 8.10%, and 32.73% compared to Treatments 1, 3, and 4, respectively. This increase in microbial diversity effectively improved soil compaction.

[0161] 3.5 Effects of microbial agents on corn yield

[0162] Table 11 Plot Yield Statistics (Yield Unit: kg)

[0163]

[0164] As can be seen from Table 11, conventional fertilization + test microbial agents increased yield by 54.08 kg / mu compared with conventional fertilization + equal amount of substrate, with an increase rate of 8.61%; increased yield by 68.89 kg / mu compared with conventional fertilization, with an increase rate of 11.23%; and increased yield by 125.93 kg / mu compared with the blank control, with an increase rate of 22.64%.

[0165] Statistical analysis of experimental data: The variance analysis of the plot yield results is shown in the table below.

[0166] Table 12 Analysis of variance table for randomized block design

[0167] Factors of variation sum of squares degrees of freedom mean square F-number p-value Between blocks 3.26 2.00 1.63 1.38 0.3206 Processing room 48.85 3.00 16.28 13.84 0.0042 error 7.06 6.00 1.18 Total variation 59.16 11.00

[0168] As can be seen from Table 12, the p value between treatments is 0.0042, and the p value is <0.01, indicating that the yield difference between treatments is extremely significant. The LSD method was further used for multiple comparisons, and the results are shown in the table below.

[0169] Table 13 Multiple comparison table

[0170]

[0171] It can be seen from Table 13 that the yield difference between treatment 2 and treatment 3 reached a significant level; the yield difference between treatment 2 and treatment 1 reached a significant level; the yield difference between treatment 2 and treatment 4 reached an extremely significant level.

[0172] In summary, under conventional fertilization conditions, the application of microbial agents to corn in the experimental field increased the yield by 54.08 kg / mu compared with conventional fertilization + the same amount of substrate, with an increase rate of 8.61%, and the yield increase effect was significant; the yield was increased by 68.89 kg / mu compared with conventional fertilization, with an increase rate of 11.23%, and the yield increase effect was significant; the yield was increased by 125.93 kg / mu compared with the blank control, with an increase rate of 22.64%, and the yield increase effect was extremely significant.

[0173] 4. Conclusion

[0174] The microbial agent provided by the present invention has a significant repair effect on the test field soil after being applied to the soil under certain reagent dosage and conditions. By performing data statistical analysis on the test results and comparing them with various indicators before planting, the soil bulk density is reduced, the porosity is increased, the proportion of water-stable aggregates is increased, the soil becomes loose, the diversity of microorganisms is increased, the soil compaction phenomenon is effectively improved, the organic matter and nitrogen, phosphorus and potassium contents in the soil are increased to a certain extent, the pH value of the soil is improved, and during the planting process of crops, the occurrence of diseases such as corn sheath blight is suppressed, and there is a significant yield-increasing effect. Compared with other treatment groups, the water and fertilizer absorption capacity is improved, the stress resistance is enhanced, the incidence of diseases and insect pests is reduced during the test, and it can have a significant antagonistic effect on pathogens, significantly reduce the number of pathogens, and significantly reduce the occurrence of diseases.

[0175] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A microbial combination for repairing saline-alkali soil, characterized in that: The microbial combination includes Bacillus subtilis KC000 and Paenibacillus mucilaginosus JDY001; The deposit number of the Bacillus subtilis KC000 is CGMCC No. 34047; the deposit number of the Paenibacillus jelly JDY001 is CGMCC No. 34048.

2. The microbial combination according to claim 1, characterized in that The ratio of the number of viable bacteria of the Bacillus subtilis KC000 and the number of viable bacteria of the Paenibacillus jelly JDY001 in the microbial combination is (20-80):(5-20).

3. The microbial combination according to claim 1, characterized in that The ratio of the number of live bacteria of the Bacillus subtilis KC000 and the number of live bacteria of the Paenibacillus jelly JDY001 in the microbial combination is 1:

1.

4. Use of the microbial combination according to any one of claims 1 to 3 in the preparation of a microbial agent for repairing saline-alkali soil.

5. A microbial agent for repairing saline-alkali soil, characterized in that: The microbial agent comprises the microbial combination according to any one of claims 1 to 3.

6. The microbial agent according to claim 5, characterized in that The microbial agent also includes auxiliary materials.

7. Use of the microbial combination according to any one of claims 1 to 3 or the microbial agent according to claim 5 or 6 in remediating saline-alkali land.

8. A method for repairing saline-alkali land, characterized in that: The method comprises the step of applying the microbial agent according to claim 5 or 6 to the soil to be repaired.

9. The method according to claim 8, characterized in that The application amount of the microbial agent is 20 kg / mu; the application frequency is 3 times, with an interval of 1 month between each application.

10. Use of the microbial combination according to any one of claims 1 to 3 in the preparation of a microbial preparation for resisting sheath blight.

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