Preparation method and application of a composite microecological bacterial agent

Abstract

The invention relates to a preparation method and application of a composite microecological microbial agent, which effectively solves the problem of preparing the composite microecological microbial agent and realizes the application in improving wheat planting soil. The method comprises the following steps: streak culture of strains RGZ1-4-29 and SJ3-6 on nutrient agar plates respectively to activate the strains; picking a single colony of the activated strains, accessing the strains to seed liquid, and culturing the strains on a shaking table; inoculating the seed liquid into a fermentation medium to obtain a microbial suspension, adding the microbial suspension, cellulase and protease to a carrier, adding water, mixing, stacking and fermenting the mixture in a thin film, drying after the fermentation is completed, adding Guangxi potassium humate, chitosan and polyglutamic acid, and mixing again to obtain a composite microecological microbial agent. The method is rich in raw materials, has scientific and reasonable compatibility, is easy to operate, has good product quality, is effectively used for improving wheat field soil and increasing wheat yield, is a major innovation in wheat field soil improvement, and has huge economic and social benefits.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a preparation method of a composite microecological bacterial agent and application thereof. Background Art

[0002] Wheat is one of the main food sources for humans, especially in my country, where it plays an important role as a crop and is widely planted. However, in recent years, the problem of soil acidification caused by the irrational use of fertilizers and pesticides has become increasingly serious, manifested in the decrease of soil pH, the loss of nutrients such as calcium and magnesium in the soil, and the increased activity of toxic metal ions. The pH of soil suitable for wheat growth is between 6.5 and 7.5. At present, the pH of acidified soil is below 6.0, and some seriously affected plots even reach below 4.0, resulting in yellowing of wheat seedlings, difficulty in growth, reduced yields or even no yields. In addition, soil acidification also increases the incidence of soil-borne diseases, seriously affecting wheat yields. Fusarium is an important soil-borne pathogenic fungus with a wide host range and strong infection ability. It can cause wheat root rot (Fusarium, Helicoverpa graminearum and Fusarium graminearum), wheat fusarium ergot (Fusarium graminearum) and wheat stem rot (Fusarium graminearum and Pseudoceromyces graminearum). It will not only cause serious reduction in wheat production, but also produce fungal toxins that can cause acute poisoning symptoms after consumption, and at the same time reduce immunity and fertility, endangering the health of humans and animals.

[0003] The traditional chemical amendment quicklime can quickly increase soil pH, improve soil properties, and achieve a certain yield increase effect in a short period of time, but long-term application will destroy soil structure, cause soil compaction, and soil "re-acidification". In acidic soil, the iron and aluminum systems will be transformed, causing imbalance of soil phosphorus, potassium, calcium, magnesium, iron and other elements, and aggravating heavy metal pollution in the soil. After water-soluble phosphate fertilizer is applied to acidic soil, phosphate ions react with active iron and aluminum ions in acidic soil to form amorphous aluminum ferric phosphate, which is then gradually converted into crystalline pink phosphate iron ore and phosphate aluminum stone, which significantly reduces its effectiveness. In recent years, scientific researchers have used biochar to improve soil. Field tests have shown that although it has a good improvement effect, it is difficult to promote and apply it on a large scale due to its high cost and inconvenient use. Microorganisms play a role in improving soil and regulating crop growth by improving soil physical and chemical properties, improving soil fertility, inhibiting the growth of pathogens, and improving plant resistance. Therefore, it is of great significance to develop a composite microecological agent to improve the acidified soil in wheat fields, enhance the resistance of wheat to soil-borne diseases, and promote a high yield and harvest of wheat. However, there has been no public report on the use of composite microecological agents for improving wheat field soil (acidification + soil-borne diseases). Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a composite microecological bacterial agent, which can effectively solve the problem of preparing a composite microecological bacterial agent and realizing its application in improving wheat planting soil.

[0005] The technical solution provided by the present invention is a method for preparing a composite microecological bacterial agent, comprising the following steps:

[0006] (1) Activation of strains: streak the preserved strains RGZ1-4-29 and SJ3-6 on nutrient agar plates respectively and culture them at 25-30°C for 24-48 hours to activate the strains;

[0007] The strain RGZ1-4-29 was isolated from wheat farmland soil in Rigze, Tibet, and was classified and named Paenibacillus ( Paenibacillus sp. ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on October 18, 2021, with the deposit number CGMCC No: 23622, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0008] The strain SJ3-6 was isolated from wheat roots that grew well in acidified soil in Zhumadian, Henan Province, and was classified and named as Burkholderia parasitica ( Paraburkholderia fungorum ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on October 18, 2021, with the deposit number CGMCC No: 23621, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0009] The nutrient agar plate is prepared by adding water to 1000 mL of peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g and agar 15.0 g, sterilized at 121° C. for 30 min, and pH 7.2±0.2 (known technology);

[0010] (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate it in a shaking incubator at 25-30°C and 120-180 r / min until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid;

[0011] The NB medium is composed of 10 g of peptone, 3 g of beef powder and 5 g of sodium chloride, added with water to 1000 mL, and sterilized by high pressure at 121°C for 30 min;

[0012] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 2-5% of the fermentation medium, the temperature was 25-30°C, the pH was 5-8, and the fermentation time was 18-32 hours to obtain a microbial suspension. The effective viable count of Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6 in the microbial suspension was 5.0×10 8 ~5.0×10 10 cfu / mL;

[0013] The fermentation medium is composed of the following weight percentages: 0.5% casein peptone, 1% potassium humate from Guangxi, 0.5% urea, 0.1% KH2PO4, 0.05% MgSO4 and 0.02% FeSO4, and the balance is water, which are mixed evenly;

[0014] (4) Preparation of microbial agent: add microbial suspension, 3-5 kU / kg of cellulase and 5-10 kU / kg of protease to the carrier, add water to the carrier until the moisture content is 50%, mix well, cover with film after stacking, ferment at 25-35 ° C for 72-120 hours, dry after fermentation, add 3-5% of Guangxi potassium humate, 0.03-0.05% of chitosan, and 0.02-0.03% of polyglutamic acid by weight of the dried fermented material, mix well again, and form a composite microbial agent;

[0015] The protease is one of trypsin, subtilisin or papain;

[0016] The carrier is one of shiitake mushroom residue, Pleurotus eryngii or Flammulina velutipes residue (existing technology).

[0017] The composite microecological bacterial agent prepared by the method of the present invention is applied to wheat field soil, is effectively used to improve wheat field soil, inhibit wheat soil-borne diseases, ensure high yield and harvest of wheat, and is applied in wheat planting in acidified soil.

[0018] The method of the invention has abundant raw materials, scientific and reasonable compatibility, easy operation of the preparation method, good product quality, can be effectively used for improving wheat field soil and increasing wheat yield, is a major innovation in wheat field soil improvement, and has huge economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The anti-Fusarium performance of strain RGZ1-4-29 and the phosphate solubilization function of strain SJ3-6 of the present invention are shown in FIG. 1 , wherein: A is Paenibacillus ( Paenibacillus sp.) RGZ1-4-29 against Fusarium graminearum ( Fusarium graminearum ) ; B is the antagonistic effect of Bacillus ( Paenibacillus sp.) RGZ1-4-29 against Fusarium oxysporum ( Fusarium oxysporumf. sp. niveum ) ; the left side is the control pathogen, and the right side is the inhibition zone produced by different screened strains; C is the hydrolysis zone produced by SJ3-6 on the inorganic phosphorus plate.

[0020] Figure 2 This is a diagram of the antagonism test between the strains RGZ1-4-29 and SJ3-6 of the present invention on a nutrient agar plate.

[0021] Figure 3 This is a comparison chart of the potted plant effects of the composite microecological agent of the present invention on improving acidified soil. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0023] The present invention can be specifically implemented by the following examples.

[0024] Example 1

[0025] The present invention provides a method for preparing a composite microecological bacterial agent, comprising the following steps:

[0026] (1) Activation of strains: streak the preserved strains RGZ1-4-29 and SJ3-6 on nutrient agar plates respectively and culture them at 26-28°C for 30-40 h to activate the strains;

[0027] (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate at 26-28°C and 150 rpm in a shaking incubator until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid;

[0028] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 3.5% of the volume of the fermentation medium, the temperature was 26-28°C, the pH was 7, and the fermentation time was 25 h to obtain a microbial suspension. The effective viable count of Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6 in the microbial suspension was 5.0×10 8 ~5.0×10 10 cfu / mL;

[0029] (4) Preparation of microbial agents: Add microbial suspension, 4 kU / Kg of cellulase and 7 kU / kg of Bacillus subtilisin to the carrier mushroom residue, add water to the carrier to a moisture content of 50%, mix well, pile up and cover with film, ferment at 30°C for 96 h, dry after fermentation, add 4% of Guangxi potassium humate, 0.04% of chitosan and 0.025% of polyglutamic acid by weight of the dried fermented material, mix well again to form a composite microbial agent.

[0030] Example 2

[0031] The present invention provides a method for preparing a composite microecological bacterial agent, comprising the following steps:

[0032] (1) Activation of strains: The preserved strains RGZ1-4-29 and SJ3-6 were streaked on nutrient agar plates and cultured at 27°C for 45 h to activate the strains;

[0033] (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate it in a shaking incubator at 27°C and 170 rpm until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid;

[0034] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 3% of the fermentation medium, the temperature was 26°C, the pH was 5, and the fermentation time was 30 h to obtain a microbial suspension. The effective viable count of Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6 in the microbial suspension was 5.0×10 8 ~5.0×10 10 cfu / mL;

[0035] (4) Preparation of microbial agent: Add microbial suspension, 3.5 kU / Kg of cellulase and 6 kU / kg of papain to the carrier Pleurotus eryngii residue, add water to the carrier to make the moisture content of 50%, mix well, cover with film after stacking, ferment at 26℃ for 118h, dry after fermentation, add 3.5% of Guangxi potassium humate, 0.03% of chitosan and 0.02% of polyglutamic acid by weight of the dried fermented material, mix well again to form a composite microbial agent.

[0036] Example 3

[0037] The present invention provides a method for preparing a composite microecological bacterial agent, comprising the following steps:

[0038] (1) Activation of strains: The preserved strains RGZ1-4-29 and SJ3-6 were streaked on nutrient agar plates and cultured at 29°C for 25 h to activate the strains;

[0039] (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate it in a shaking incubator at 29°C and 130 rpm until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid;

[0040] (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 5% of the volume of the fermentation medium, the temperature was 29°C, the pH was 8, and the fermentation time was 19 h to obtain a microbial suspension. The effective viable count of Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6 in the microbial suspension was 5.0×10 8 ~5.0×10 10 cfu / mL;

[0041] (4) Preparation of microbial agents: Add microbial suspension, 5 kU / Kg of cellulase and 9 kU / kg of trypsin to the carrier Flammulina velutipes residue, add water to the carrier to a moisture content of 50%, mix well, cover with film after stacking, ferment at 34°C for 73 h, dry after fermentation, add 5% of Guangxi potassium humate, 0.05% of chitosan, and 0.03% of polyglutamic acid by weight of the dried fermented material, mix well again, and form a composite microbial agent.

[0042] It should be pointed out that the above is only an embodiment of the present invention, which is used to illustrate the specific implementation of the present invention, and is not intended to limit the scope of protection of the present invention. Any equivalent or equivalent modifications and substitutions made within the spirit and principles of the present invention are essentially the same as the technical solution of the present invention and are included in the scope of protection of the present invention.

[0043] The strain SJ3-6 of the present invention has good ability to solubilize phosphorus, potassium and produce iron carriers; the strain RGZ1-4-29 has good enzyme production ability, can inhibit various wheat fungal disease pathogens such as Fusarium oxysporum, Fusarium graminearum, and Pythium, and can effectively prevent and treat wheat root rot and fusarium scaber. There is no antagonism between the above two bacteria. The above two probiotics are mixed and fermented and prebiotics are added to prepare a composite microecological bacterial agent for the improvement of acidified soil in wheat fields, which can effectively increase soil pH, improve soil microecological structure, increase soil enzyme content, promote wheat growth, improve the phenomenon of yellowing, weak and even death of seedlings, reduce the occurrence of fusarium scaber and root rot, and increase wheat yield. The relevant test data are as follows:

[0044] By selecting and applying phosphorus-solubilizing, potassium-solubilizing, and enzyme-producing microorganisms, the soil is promoted to release deposited phosphorus, potassium, iron and other elements, the crop absorption and utilization rate is increased, and the pollution and compaction of the soil caused by excessive application of chemical fertilizers are alleviated; prebiotics are applied to improve the living environment of soil microorganisms, stimulate soil vitality, increase the content of soil biological enzymes, improve soil aggregate structure, and reduce soil compaction. Plant probiotics in soil improvement microbial agents increase the content of beneficial bacteria, and prebiotics promote the growth and reproduction of beneficial bacteria, which can improve the structure of plant rhizosphere microbial flora, inhibit the growth of pathogenic bacteria, and play a role in reducing the use of chemical fertilizers and pesticides, improving soil, and increasing crop yields.

[0045] In the present invention, the strain SJ3-6 is a paraburkholderia, gram-negative, oxidase, catalase, and nitrate reduction positive, and is cultured on a nutrient agar plate at 30°C for 24 hours. The colonies are milky yellow, round and opaque, with smooth surfaces and neat edges; the strain RGZ1-4-29 is a Paenibacillus, gram-positive, casein hydrolysis, catalase, gelatin hydrolysis, and nitrate reduction positive. On the nutrient agar plate, the colonies are white, transparent, round, with neat edges, and moist, smooth, and slightly raised surfaces.

[0046] The paraburkholderia SJ3-6 of the present invention is isolated from the acidified soil of the wheat field in Zhumadian, has good environmental tolerance, is harmoniously compatible with the soil ecology, is conducive to giving full play to the advantages of the strain, has the ability to fix nitrogen, dissolve inorganic phosphates, and produce plant hormones, and can promote the absorption of nitrogen and phosphorus by plants; the bacillus RGZ1-4-29 can secrete a variety of cell wall hydrolases and antibacterial substances, has antagonistic effects on a variety of plant pathogenic fungi, performs well in plant growth promotion, and is an important plant probiotic (see Figure 1 ).

[0047] Experiment 1: Antagonistic reaction test experiment between functional strains

[0048] Use the double streak method to inoculate two strains of bacteria on the same nutrient agar plate. First, streak one strain horizontally along the surface of the plate, and then streak the other strain vertically along the surface of the plate. Incubate in a 25-30℃ incubator for 3 days. Observe the growth of the two strains at the junction of the vertical and horizontal streaks on the plate. If there is obvious inhibition at the junction, it indicates that there is antagonism between the two strains and it is not suitable for mixed culture; if there is no obvious inhibition of the growth of the two strains at the interface, it indicates that there is no antagonism between the strains and they can be mixed (see Figure 2 ).

[0049] Experiment 2: Experiment on the improvement effect of composite microecological agent on acidified wheat soil (taking Example 1 as an example)

[0050] The experiment had two treatments: (1) CK: 2 kg / pot of acidified soil; (2) 1.9 kg of acidified soil + 0.1 kg of compound microecological agent / pot. Each treatment was repeated three times, and samples were taken after one month to compare the treatment effects.

[0051] The observation and measurement of biological characteristics of wheat before and after treatment and the determination of soil physical and chemical properties showed that the composite microecological agent had a significant improvement effect on acidified soil. In the control acidified soil, wheat seedlings were sparse and weak, with yellow leaves and slow growth, while in the treated soil, wheat seedlings were strong and grew well ( Figure 3), wheat plant height, root length, aboveground and underground fresh weight and dry weight increased by 2-4 times compared with the control, and chlorophyll content increased by 1.7 times. Soil pH increased significantly from pH3.8 to pH5.3. The physical and chemical properties of the soil also improved significantly, and the content of soil organic matter increased significantly. Soil phosphatase is a type of enzyme that catalyzes the mineralization of soil organic phosphorus compounds. Its activity directly affects the decomposition and transformation of organic phosphorus in the soil and its biological effectiveness. It is an indicator for evaluating the direction and intensity of soil phosphorus biological transformation. Soil catalase mainly comes from the secretions of soil microorganisms and plant roots. It is an important enzyme in soil biological metabolism. Its main function is to eliminate the toxic effects of hydrogen peroxide in the soil and plays an important role in the active oxygen scavenging system. It can be seen from Table 1 that the activity of phosphatase in the treated soil increased by 1.6 times; catalase increased by more than 2 times, indicating that the soil's ability to decompose and transform organic phosphorus and remove harmful substances has been greatly improved.

[0052] Table 1 Effect of acidified soil improving microecological agents on soil improvement in potted plants

[0053] deal with CK Compound microbial agent Plant height 10.925±0.763 22.443±0.273 Root length 5.867±0.506 12.756±0.931 Fresh weight of underground part 0.037±0.003 0.156±0.033 Dry weight of underground part 0.008±0.002 0.016±0.003 Fresh weight of aboveground part 0.133±0.020 0.348±0.005 Above ground dry weight 0.014±0.002 0.034±0.001 Chlorophyll content 21.050±4.579 35.725±1.209 Soil pH 3.828±0.112 5.330±0.087 Soil urease activity 23.532±3.544 31.305±2.359 Soil phosphatase activity 2956.239±890.9 7725.505±863.510 Soil catalase activity 3.763±0.392 12.486±3.945 Soil alkaline nitrogen content 220.500±16.663 159.250±20.903 Soil available phosphorus content 199.834±6.957 206.922±22.428 Available potassium content in soil 109.304±5.378 121.403±15.906 Soil organic matter content 5.150±0.252 8.400±1.461

[0054] Experiment 3: Effect of compound microecological agents on wheat disease resistance and yield

[0055] The wheat variety for the test was Fengdecunmai No. 21, and the test site was Yicheng District, Zhumadian City, Henan Province. The blank control was a control without any pesticides or fertilizers, the conventional fertilization treatment was a base fertilizer (N+P2O5+K2O≥45%), and the base fertilizer + (50-100 kg / mu) compound microecological microbial agent was a microbial agent treatment. Each treatment had 4 replicate plots, and each plot had an area of ​​30m 2 Before planting, the compound microecological agent and base fertilizer were evenly spread on the soil surface of the wheat field. After application, the wheat field was rotary tilled to a depth of about 15cm to 30cm, mixed evenly with the soil, and wheat was sown mechanically. The agronomic measures were consistent among the treatments.

[0056] Observation of seedling emergence: 7 days after wheat seedlings emerged, observe the differences in seedling emergence between treatments. After all seedlings have grown, survey 5 points in each treatment, and randomly select 1 m 2 , calculate the germination rate.

[0057] From the late grain filling stage to the milky stage of wheat, the fusarium head blight disease was investigated for each treatment using a 5-point sampling method. 40 ears were investigated at each point and 200 ears were investigated in each plot. The number of diseased ears and diseased spikelets of wheat was recorded, and the diseased ear rate, disease index and control effect were calculated.

[0058] Grading Standard:

[0059] Level 0: No disease;

[0060] Level 1: diseased spikelets account for less than 1 / 4 of the whole spike;

[0061] Level 3: diseased spikelets account for 1 / 4 to 1 / 2 of the whole spike;

[0062] Level 5: diseased spikelets account for 1 / 2 to 3 / 4 of the whole spike;

[0063] Level 7: diseased spikelets account for more than 3 / 4 of the entire spike.

[0064] Among them, diseased ear rate = number of diseased ears / total number of investigated ears × 100%;

[0065] Disease index = ∑ (number of diseased ears at each level × corresponding level value) / (total number of ears surveyed × 7) × 100

[0066] Control effect (%) = (average disease index of control area - average disease index of treatment area) / average disease index of control area × 100%

[0067] Before harvest, samples were collected from 5 points in each treatment, with a depth of 1 m 2 Sample plots were taken to investigate the number of effective ears, the number of grains per ear, and the thousand-grain weight, and to calculate the theoretical yield and yield increase rate. Theoretical yield per unit area = number of grains per ear × thousand-grain weight × number of ears per mu × 0.85.

[0068] Calculate the number of ears per mu, the number of grains per ear and the yield.

[0069] Table 2 Effects of compound microecological agents on disease resistance and yield of wheat

[0070] deal with Soil pH Seedling rate (%) Disease index (%) Control effect (%) Number of ears per mu (10,000) Number of grains per ear Thousand-grain weight (g) Yield (kg / mu) Blank control 4.26 58.98 0.23 0 27.85 28.28 35.98 240.87 Conventional fertilization 4.22 59.75 0.21 6.40 30.89 30.22 38.76 307.55 Compound microecological agent + conventional fertilization 5.88~6.05 89.12~95.17 9~17 40.44~60.90 40.06~41.87 31.70~32.87 40.77~41.56 463.66~484.8

[0071] As can be seen from Table 2, the composite microecological agent can regulate soil pH, improve soil microecological structure, increase wheat emergence rate, effectively reduce the incidence of wheat fusarium rust, improve root growth of wheat in the seedling stage and stem development in the later stage, improve wheat stress resistance, increase wheat thousand-grain weight and per mu yield, and increase per mu yield by 50.8-57.6%. The effect is unexpectedly good.

[0072] While conducting experiments on Example 1, the same experiments were also conducted on other examples, and the same and similar results were obtained, which will not be described one by one here.

[0073] In summary, the present invention utilizes Burkholderia parabacterium ( Burkholderia fungorum ) SJ3-6 and Paenibacillus ( Paenibacillus sp.) RGZ1-4-29 mixed fermentation to prepare acidified soil improvement microecological bacterial agent, has a significant improvement effect on severely acidified soil, by balancing the microecology to the level required by crops, helps the soil return to a state suitable for wheat growth, can effectively increase the emergence rate of wheat in acidified soil, reduce the occurrence of wheat fungal diseases, improve soil fertility, reduce pollution, and is simple to apply; the strain has strong adaptability to the environment, has nitrogen fixation and phosphorus dissolving effects, can produce plant hormones, can secrete a variety of enzymes, has the functions of disease prevention, growth promotion, and soil improvement, greatly increased wheat yield, the yield can be increased by more than 50%, and is safe and pollution-free to humans and animals, friendly to the environment, in line with the direction of green and sustainable development of agriculture, and has good development and application prospects.

Claims

1. A method for preparing a composite microecological bacterial agent, characterized in that: The following steps are involved: (1) Activation of strains: streak the preserved strains RGZ1-4-29 and SJ3-6 on nutrient agar plates respectively and culture them at 25-30°C for 24-48 hours to activate the strains; The strain RGZ1-4-29 was isolated from wheat farmland soil in Rigze, Tibet, and was classified and named Paenibacillus ( Paenibacillus sp. ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on October 18, 2021, with the deposit number CGMCC No: 23622, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The strain SJ3-6 was isolated from wheat roots that grew well in acidified soil in Zhumadian, Henan Province, and was classified and named Burkholderia parasitica ( Paraburkholderia fungorum ), deposited in the General Microbiology Center of China Microorganism Culture Collection Committee on October 18, 2021, with the deposit number CGMCC No: 23621, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The nutrient agar plate is prepared by adding water to 1000 mL of peptone 10.0 g, beef extract powder 3.0 g, sodium chloride 5.0 g and agar 15.0 g, sterilized at 121° C. for 30 min, and pH 7.2±0.2; (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate it in a shaking incubator at 25-30°C and 120-180 rpm until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid; The NB medium is composed of 10 g of peptone, 3 g of beef powder and 5 g of sodium chloride, added with water to 1000 mL, and sterilized by high pressure at 121°C for 30 min; (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 2-5% of the volume of the fermentation medium, the temperature was 25-30°C, the pH was 5-8, and the fermentation time was 18-32 hours to obtain a microbial suspension containing Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6, and the effective viable count was 5.0×10 8 ~5.0×10 10 cfu / mL; The fermentation medium is composed of the following weight percentages: 0.5% casein peptone, 1% potassium humate from Guangxi, 0.5% urea, 0.1% KH2PO4, 0.05% MgSO4 and 0.02% FeSO4, and the balance is water, which are mixed evenly; (4) Preparation of microbial agent: add microbial suspension, 3-5 kU / kg of cellulase and 5-10 kU / kg of protease to a carrier, add water to the carrier until the moisture content is 50%, mix well, cover with a film after stacking, ferment at 25-35°C for 72-120 hours, dry after fermentation, add 3-5% of Guangxi potassium humate, 0.03-0.05% of chitosan and 0.02-0.03% of polyglutamic acid based on the weight of the dried fermented product, mix well again, and form a composite microbial agent; The protease is one of trypsin, subtilisin or papain; The carrier is one of shiitake mushroom residue, king oyster mushroom residue or enoki mushroom residue.

2. The method for preparing the composite microecological agent according to claim 1, characterized in that: The following steps are involved: (1) Activation of strains: The preserved strains RGZ1-4-29 and SJ3-6 were streaked on nutrient agar plates and cultured at 26-28°C for 30-40 h to activate the strains; (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate at 26-28°C and 150 rpm in a shaking incubator until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid; (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 3.5% of the volume of the fermentation medium, the temperature was 26-28°C, the pH was 7, and the fermentation time was 25 h to obtain a microbial suspension containing Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6, and the effective viable count was 5.0×10 8 ~5.0×10 10 cfu / mL; (4) Preparation of microbial agent: Add microbial suspension, 4 kU / Kg of cellulase and 7 kU / kg of subtilisin to the carrier mushroom residue, add water to the carrier to make the moisture content of 50%, mix well, cover with film after stacking, ferment at 30°C for 96 hours, dry after fermentation, add 3-5% of Guangxi potassium humate, 0.03-0.05% of chitosan and 0.02-0.03% of polyglutamic acid by weight of the dried fermented material, mix well again, and form a composite microbial agent.

3. The method for preparing the composite microecological agent according to claim 1, characterized in that: The following steps are involved: (1) Activation of strains: The preserved strains RGZ1-4-29 and SJ3-6 were streaked on nutrient agar plates and cultured at 27°C for 45 h to activate the strains; (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Cultivate in a shaking incubator at 27°C and 170 rpm until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid; (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 3% of the fermentation medium, the temperature was 26°C, the pH was 5, and the fermentation time was 30 h to obtain a microbial suspension containing Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6, with an effective viable count of 5.0×10 8 ~5.0×10 10 cfu / mL; (4) Preparation of microbial agent: Add microbial suspension, 3.5 kU / Kg of cellulase and 6 kU / kg of papain to the carrier Pleurotus eryngii residue, add water to the carrier to make the moisture content of 50%, mix well, cover with film after stacking, ferment at 26°C for 118 h, dry after fermentation, add 3-5% of Guangxi potassium humate, 0.03-0.05% of chitosan and 0.02-0.03% of polyglutamic acid by weight of the dried fermented material, mix well again, and form a composite microbial agent.

4. The method for preparing the composite microecological agent according to claim 1, characterized in that: The following steps are involved: (1) Activation of strains: The preserved strains RGZ1-4-29 and SJ3-6 were streaked on nutrient agar plates and cultured at 29°C for 25 h to activate the strains; (2) Seed culture: Pick a single colony of the activated strain and inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of NB medium. Incubate the culture in a shaking incubator at 29°C and 130 rpm until the OD value of the seed solution reaches 0. 600 If the value is above 1.2, it becomes seed liquid; (3) Fermentation culture: The seed liquid was inoculated into the fermentation medium at a volume of 5% of the volume of the fermentation medium, the temperature was 29°C, the pH was 8, and the fermentation time was 19 h to obtain a microbial suspension containing Paenibacillus RGZ1-4-29 and Burkholderia parasitica SJ3-6, with an effective viable count of 5.0×10 8 ~5.0×10 10 cfu / mL; (4) Preparation of microbial agent: Add microbial suspension, 5 kU / Kg of cellulase and 9 kU / kg of trypsin to the carrier Flammulina velutipes residue, add water to the carrier to make the moisture content of 50%, mix evenly, cover with film after stacking, ferment at 34°C for 73 hours, dry after fermentation, add 3-5% of Guangxi potassium humate, 0.03-0.05% of chitosan and 0.02-0.03% of polyglutamic acid by weight of the dried fermented material to form a composite microbial agent.

5. Use of the composite microecological agent prepared by the method according to any one of claims 1 to 4 in wheat cultivation in acidified soil.

6. Use of the composite microecological agent prepared by the method according to any one of claims 1 to 4 in the preparation of an inhibitor of wheat soil-borne fusarium wilt.

Images