CN105886428B - A Streptomyces sp. and its application in microbial fertilizers - Google Patents

Abstract

The invention discloses a strain of Streptomyces lividans W68 (strain preservation number: CGMCC No. 12210) isolated from deep-sea sludge with broad-spectrum antifungal activity, a microbial inoculum prepared from the bacterium and a microorganism using the bacterium as a material Fertilizer application technology. The microbial inoculum prepared by Streptomyces lividans W68 isolated from deep sea sludge and the application technology of the microbial fertilizer provided by the invention are not only simple and efficient, but also environmentally friendly, and the spore concentration of the solid fermented bacterial preparation is can reach tens of billions. The pot experiment confirmed that Streptomyces lividans W68 isolated from deep-sea sludge showed obvious control effect on soil-borne fungal diseases such as wheat root rot, melon wilt, pepper phytophthora and wheat take-all. The invention has great application prospects for green prevention and control of soil-borne fungal diseases.

Description

A Streptomyces sp. and its application in microbial fertilizers

technical field

The invention relates to a marine-derived Streptomyces albidoflavus strain W68 with broad-spectrum antifungal activity and biocontrol application potential, and belongs to the technical fields of microorganisms such as the application of marine microorganisms and the prevention and control of crop diseases.

Background technique

Fungal diseases are the main reason for the reduction of crop yield and quality. The frequent use of chemical pesticides has led to a sharp increase in the resistance of some pathogenic fungi, resulting in the pollution of the environment and agricultural products, as well as destroying the balance of microorganisms inside and outside the plant and exacerbating the outbreak of diseases.

The research on the prevention and control of soil-borne plant fungal diseases has always been a hot and difficult point, and the fungicides on the market generally have no or little effect on them. Over the years, some biocontrol bacteria against soil fungi have also been screened, such as Coniothyrium minitans for the control of sheath blight, Trichoderma viride for sheath blight and Pythium ultimum. The control of various diseases including seedling disease or root rot, and the control of Streptomyces griseoviridis for cotton fusarium wilt, etc. Although these soil fungal disease biocontrol strains all have strong antifungal activity, the field test results are poor, many strains have poor fermentation performance, and usually have certain geographical limitations in application.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Streptomyces albidoflavus strain W68 with broad-spectrum anti-pathogenic fungi activity and its application in preventing and treating soil-borne fungal diseases.

In fact, the present invention relates to a Streptomyces albidoflavus strain W68.

The invention relates to a fungicide whose active ingredient is Streptomyces albidoflavus strain W68.

The invention relates to an inoculum of Streptomyces albidoflavus strain W68 for preventing and treating soil-borne fungal diseases.

The invention relates to the application of Streptomyces albidoflavus strain W68 as a microbial fertilizer in preventing and controlling soil-borne fungal diseases.

What is protected is the application technology of Streptomyces lividans W68 isolated from deep-sea sludge, microbial preparations prepared from the bacterium and microbial fertilizers using the bacterium as the material.

The deep-sea Streptomyces lividans W68 described in the present invention is separated from marine activated sludge, and the quantitative activated sludge is put into sterile water containing small glass beads for pretreatment. Gao's No. 1 medium was used for isolation and culture, and then PDA medium was used for purification and culture. The 16S rDNA sequences were compared in the NCBI database, and the bacteria were identified as belonging to the Streptomyces albidoflavus group. At present, the bacteria has been deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee on March 14, 2016. The address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing (CGMCC). The preservation number is CGMCC No.12210

The biological characteristics of the Streptomyces jaundice W68 described in the present invention are as follows:

Table 1. Colony characteristics of Streptomyces luteus W68

culture medium Colony front color Colony back color ISP2 White yellow ISP3 yellowish yellowish ISP4 White White ISP5 Micro scar growth no pigment Bennete White yellowish, white Cazpek's yellowish yellowish Glu+Asp Micro scar growth no pigment Glu+Asp+M White White PDA White yellow

The colony characteristics of Streptomyces lividans W68 on various media are shown in Table 1. The average incubation time was 5-7 days. On PDA medium, aerial hyphae were initially white and powdery with white and creamy secondary clumps. After sporulation, the colonies are yellowish. Scanning electron microscopy showed that the spore filaments were curved, without spiral, and each spore filament contained about 30 to 50 spores;

The antifungal activity of Streptomyces lividans W68 isolated from deep-sea sludge described in the present invention is as follows:

Streptomyces lividans W68 isolated from deep sea sludge has broad-spectrum antifungal activity. The plate confrontation experiment showed that Streptomyces lividans W68 was resistant to Fusarium verticillioides, Fusarium oxysporum, Bipolaris sorokiniana, Rhizoctonia solani, Dahlia Verticillium dahliae, Magnaporthe grisea and other pathogenic fungi that cause plant diseases have good antagonistic effects (Figure 2).

The biocontrol application technology and method of Streptomyces japonica W68 isolated from deep-sea sludge described in the present invention are as follows:

(1) using the prepared microbial viable bacteria preparation for seed coating technology;

(2) Microbial inoculum or microbial fertilizer using Streptomyces lividans W68 as material;

(3) Using Streptomyces lividans W68 as material and common agricultural waste as solid fermentation material, a microbial inoculum or microbial fertilizer with a spore concentration of 1×10 ¹⁰ /g can be obtained;

(4) Microbial inoculum or microbial fertilizer can be used for coating, seed soaking, root dipping, hole application, furrow application, and base fertilizer use.

The biocontrol application effect of Streptomyces sp. W68 isolated from deep sea sludge described in the present invention is as follows:

Control effect of Streptomyces lividans W68 isolated from deep sea sludge on wheat root rot (Bipolaris sorokiniana). In the pot experiment on the prevention and control of wheat root rot, the method of seed coating was adopted, and the Streptomyces luteus W68 bacterial preparation improved the emergence rate of wheat seeds in the soil containing wheat root rot, and the average plant height and dry weight of the plant were It was higher than that of the control group in the diseased group, and it could basically reach the growth level of wheat seeds in the soil without pathogenic bacteria (Fig. 3).

Control effect of Streptomyces lividans W68 isolated from deep sea sludge on Fusarium oxysporum. Through seed coating and root irrigation, the use of Streptomyces lividans W68 can significantly overcome the continuous cropping obstacles caused by Fusarium wilt: continuous planting of melons in the fields where melons were planted in the previous year, the survival rate of melon seedlings in the treatment group It can reach 93%, and the seedling survival rate of blank control group is only 13% (Fig. 4).

Control effect of Streptomyces lividans W68 isolated from deep sea sludge on Phytophthora capsici. When the pepper seedlings were transplanted, the seedlings were treated with Streptomyces lividans W68 bacteria preparation, which could significantly reduce the incidence of pepper Phytophthora disease. Compared with the control group, the growth cycle of the dipped root-treated plants was prolonged by about 1-2 weeks, and the yield of dry pepper was increased by 5% (Fig. 5).

Control effect of Streptomyces lividans W68 isolated from deep sea sludge on wheat take-all disease (Gaeumannomyces.graminis). The use of the deep-sea Streptomyces lividans W68 viable bacterial preparation can significantly reduce the diseased plant rate at the heading stage of wheat and reduce the disease index. Compared with chemical agents, the control effect of Streptomyces lividans W68 live bacteria preparation is obviously better than that of chemical reagent triazolone, and is equivalent to that of total erosion. It can be seen that the viable preparation of Streptomyces lividans W68 can completely replace the use of chemical agents (Figure 6).

Description of drawings:

Fig. 1 is the growth morphology and scanning electron microscope observation diagram of Streptomyces flavus W68 on PDA medium.

Figure 2 is a graph showing the antagonism of Streptomyces lividans W68 against various phytopathogens.

Figure 3 is a graph showing the results of a pot experiment on the prevention and control of wheat root rot by Streptomyces lividans W68.

Figure 4 is a diagram showing the results of a field test of Streptomyces lividans W68 for the prevention and control of melon fusarium wilt.

Figure 5 is a diagram showing the results of a field test of Streptomyces lividans W68 for the prevention and control of Phytophthora capsicum.

Figure 6 is a diagram showing the results of a field test of Streptomyces lividans W68 to prevent and control wheat take-all disease.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

In the following implementing regulations, the following culture medium is used:

Gao's No. 1 screening medium: soluble starch 20g, KNO ₃ 1g, K ₂ HPO ₄ 0.5g, MgSO ₄ 7H ₂ O 0.5g, NaCl 0.5g, FeSO ₄ 7H ₂ O 0.01g, NaCl 0.5g, agar 20g, dilute to 1000mL with tap water, pH 7.2～7.4.

PDA medium: Potato dextrose agar medium, boil 200 g of potato for 30 minutes, filter and collect the filtrate, 20 g of glucose, 15-20 g of agar, make up to 1000 ml with tap water, natural pH; 115 ℃ high temperature sterilization.

ISP2 medium (yeast extract malt extract agar): yeast extract 10g, glucose 4g, malt extract 10g, agar 20g, tap water to make up to 1000mL, pH 7.3.

ISP3 medium (oat flour agar): 20 g of oat flour infusion, 1 ml of trace salt solution, 20 g of agar, tap water to make up to 1000 mL, pH 7.2.

ISP4 medium (starch agar): 10 g of soluble starch, 1 g of MgCO ₃ , 1 g of K ₂ HPO ₄ , 1 g of NaCl, 1 g of NaNO ₃ , 15 g of agar, tap water to make up to 1000 ml, pH 7.2-7.4.

ISP5 medium (glycerol-aspartic agar): L-aspartic 1g, K ₂ HPO ₄ 1g, trace salt solution 1ml, glycerol 10g, agar 20g, tap water to 1000ml, pH 7.0～7.4

Bennete medium: glucose 10g, beef extract 1g, agar 15g, yeast extract 1g, hydrolyzed casein 2g, tap water to 1000mL, pH 7.3

Cazpek's medium (Cazpek's medium): sucrose 30g, NaNO ₃ 2g, K ₂ HPO ₄ 1g, MgSO ₄ ·7H ₂ O 0.5g, KCl 0.5g, FeSO ₄ ·7H ₂ O 0.01g, agar 20g, tap water to make up 1000mL, pH 7.2-7.4.

Glu+Asp medium (glucose asparagine agar): 10 g of glucose, 0.5 g of asparagine, 0.5 g of K ₂ HPO ₄ , 15 g of agar, tap water to make up to 1000 mL, pH 7.2-7.4.

Glu+Asp+M medium (glucose-asparagine meat extract agar): glucose 10g, aspartin 0.5g, beef extract 2g, K ₂ HPO ₄ 0.5g, agar 15g, tap water to 1000mL, pH 6.8.

Trace salt solution: FeSO ₄ ·7H ₂ O 0.1 g, ZnSO ₄ ·7H ₂ O 0.1 g, MnCl ₂ ·4H ₂ O 0.1 g, distilled water to 100 ml.

2×YT liquid medium: peptone 16g, yeast powder 10g, NaCl 5g, tap water to 1000mL, natural pH.

Example 1

Isolation, identification and biological characteristics of strains

1. Isolation and purification of strains

In this experiment, several strains of actinomycetes were isolated from marine activated sludge. The process is as follows: 1g of the sample was added to a sterilized 300ml conical flask containing small glass beads and 99ml of sterile saline, and incubated at 28°C for 1h on a shaker. . Take 1 ml of suspension, dilute it to ^10-4 by gradient dilution method, take stock solution, ^10-2 dilution and ^10-4 dilution respectively and apply potassium dichromate to the final concentration of 50μg/ml Gao's No. 1 medium. , on the 5th day, the 7th day and the 10th day, a single colony was picked, and then purified and isolated and cultured on the PDA medium by plate streak method.

The resulting purified strain was stored at -80°C with 20% glycerol.

The identification of strains

1. Morphological identification

Morphological identification of Streptomyces lividans W68 isolated from deep-sea sludge, using the method of plate streak to observe the morphology after inoculation and culture in different media (Table 1).

2. Molecular identification

PCR amplification of 16S rDNA using universal primers 27F and 1492R (annealing temperature of 56 ° C, the target sequence is about 1400bp, the sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTACGACTT-3'.

The PCR reaction program was: 95°C for 5 min; 95°C for 30s, 56°C for 90s, 72°C for 60s (33 cycles); 72°C for 10 min; 4°C.

After the amplified sequence was purified and sequenced, the homology was compared by the Blastn program of NCBI, and the phylogenetic tree was established by the Neighbor-Joining method using MEGA5 software.

The biological characteristics of the strains

94 phenotypic tests, including 71 carbon source utilization tests and 23 chemical susceptibility tests (Table 2, Table 3), were performed on Streptomyces flavus W68 using Biolog GEN III microplates.

Table 2. Utilization of 71 different carbon sources by Streptomyces flavus W68

Remarks: + means available, - means not available

Table 3. Tolerance of Streptomyces flavus W68 to 23 different reagents

Remarks: + means tolerance, - means intolerance

4. Preservation of strains

Streptomyces sp. W68 isolated from deep sea sludge has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on March 14, 2016. , the deposit number is CGMCC No.12210.

Example 2

Antifungal activity test of Streptomyces lividans W68 isolated from deep sea sludge

Antifungal activity of Streptomyces flavus W68 was determined by the plate confrontation method:

(1), Streptomyces culture and collection: use streaking method to inoculate Streptomyces lividans Streptomyces lividans W68 on a PDA plate, cultivate at 28 ° C for 5-7 days to produce sufficient spores; scrape the spores in an appropriate amount of sterile water to make The spore suspension is ready for use.

(2), the cultivation of pathogenic bacteria: inoculate cucumber solani, cabbage wilt, wheat root rot, rice blast, cotton fusarium wilt, cotton verticillium wilt on a PDA plate, and use after culturing at 28 ° C for one week.

(3), plate confrontation experiment: respectively inoculate the pathogenic bacteria culture block in the center of the PDA plate, inoculate 5 μl of Streptomyces flavonoids W68 spore suspension at a distance of about 2.0 cm from the pathogenic bacteria, cultivate at 28 ° C for 3-5 days, and observe the growth of the pathogenic bacteria.

Example 3

Microbial inoculum with Streptomyces lividans W68 as material:

(1) Preparation of liquid seeds

Pick the aerial hyphae and spores of Streptomyces lividans W68 grown on PDA plates for 4-5 days, inoculate them into 2×YT liquid medium, and use them as seed solution after 1-2 days of shaking at 28-30°C and 180rpm. .

(2) Preparation of solid fermented seeds

The liquid seeds are inserted into a tissue culture bottle containing solid fermentation materials at a mass ratio of 10%, and cultured at 28-30° C. for 4-6 days to obtain solid fermented seeds.

(3) Microbial agents

Using agricultural waste livestock and poultry manure, edible fungus residue, bran or soybean meal as the basic raw materials, adding a certain proportion of MnSO ₄ , (NH) ₂ SO ₄ , the ratio of material to water is 1:1. The solid fermented seeds were inoculated at a ratio of 10%. Cultivate at 28-30°C for 5-7 days, air-dry after fermentation and culture, and the spore concentration can reach 1×10 ¹⁰ /g, which can be used as Streptomyces microbial inoculum.

Example 4

Preparation of microbial fertilizer with Streptomyces japonica W68 as material:

(1) Preparation of liquid seeds

Pick the aerial hyphae and spores of Streptomyces lividans W68 grown on PDA plates for 4-5 days, inoculate them into 2×YT liquid medium, and use them as seed solution after 1-2 days of shaking at 28-30°C and 180rpm. .

(2) Preparation of solid fermented seeds

The liquid seeds are inserted into a tissue culture bottle containing solid fermentation materials at a mass ratio of 10%, and cultured at 28-30° C. for 4-6 days to obtain solid fermented seeds.

(3) Preparation of microbial fertilizer

Using agricultural waste livestock and poultry manure, edible fungus residue, bran or soybean meal as the basic raw materials, adding a certain proportion of MnSO ₄ , (NH) ₂ SO ₄ , the ratio of material to water is 1:1. The solid fermented seeds were inoculated at a ratio of 10%. Cultivated at 28-30°C for 5-7 days, air-dried after fermentation and culture, and the spore concentration can reach 1×10 ¹⁰ /g, which can be used as Streptomyces microbial fertilizer.

Example 5

The application of microbial inoculants or microbial fertilizers of Streptomyces lividans W68 isolated from deep-sea sludge to plant fungal diseases:

1. Prevention and control of wheat root rot by Streptomyces sp. W68 isolated from deep sea sludge

(1) Test site and environmental conditions: Three pot experiments were conducted in the outdoor natural environment of the Institute of Microbiology, Chinese Academy of Sciences.

(2) Test chemicals: blank control, Streptomyces lividans W68 spore suspension, 70% methotrexate powder

(3) Test method:

Seed treatment: take some wheat seeds, after 10% 84 disinfectant treatment for 5 minutes, rinse twice with sterile water, and then soak the seeds in sterile water for 4 hours;

Seed coating with Streptomyces lividans W68: Take the soaked seeds in a petri dish, use 1 ml of sodium carboxymethyl cellulose solution and 1 ml of the collected W68 spore suspension for seed coating, and place them at 28°C after mixing. overnight;

Seed suffocation with methyl bromide: According to the use method of methyl bromide, the surface sterilized wheat seeds are subjected to 6h brooding treatment.

(4) Treatment arrangement: 10 seeds were planted in each flowerpot, four parallel and random arrangements were made for each treatment, and the growth was observed under natural environment conditions for 2-3 weeks. The experiment was repeated three times.

(5) Statistical data: Record the height of the plant, the wet and dry weight of the above-ground plant, and the diameter of the plant stem, and analyze the data statistically.

(6) Analysis of the results: In the pot experiment for the prevention and control of wheat root rot, the method of seed coating was adopted, and the Streptomyces lividans W68 bacterial preparation was used to improve the emergence rate of wheat seeds in the soil containing wheat root rot. The height and dry weight of the plants were higher than those of the control group in the diseased group, and they could basically reach the growth level of wheat seeds in the soil without pathogenic bacteria (Figure 3).

2. The field test of Streptomyces lividans W68 isolated from deep-sea sludge to fusarium wilt of melon.

(1) Experimental site and environmental conditions: The experiment was set up in Donglou Township, Xinzhou City, Shanxi Province. The fertility of the experimental field is medium, and the previous crop is melon. The melon is sown, cultivated, and managed uniformly, in line with local agricultural practices.

(2) Demonstration reagents: blank control, microbial inoculants or microbial fertilizers made of Streptomyces lividans W68, and similar products from other companies.

(3) Area of plot and arrangement of treatments: each treatment was 70 m ² , covered with plastic film, and 2 rows of melons were planted in each treatment.

(4) Application method: the seed soaking treatment method is adopted. The melon is soaked with bacterial fertilizer for 1 hour before sowing.

(5) Data statistics: take pictures, count dead seedlings, and count test results.

(6) Result analysis: Through the seed soaking treatment of melon seeds, the use of microbial inoculants or microbial fertilizers with Streptomyces flavus W68 as the material can significantly overcome the continuous cropping obstacles of melon caused by Fusarium wilt. Continuous planting of melon in the field, the survival rate of melon seedlings in the treatment group can reach 93%, and the survival rate of melon seedlings in the blank control group is only 13% (Figure 4).

3. The field test of Streptomyces lividans W68 isolated from deep sea sludge against Phytophthora capsicum.

(1) Experimental site and environmental conditions: The experiment was set up in Gaocheng Village, Xinzhou City, Shanxi Province. The previous crop was pepper, and the variety was Beijing Red. Pepper seedlings.

(2) Demonstration reagents: blank control, microbial inoculum or microbial fertilizer made of Streptomyces lividans W68, and similar products from other companies.

(3) Area of plot and arrangement of treatments: each treatment was 70 m ² , covered with plastic film, and 2 rows of peppers were planted in each treatment.

(4) Application method: When the pepper seedlings are transplanted, they are treated with bacterial fertilizer dipped in the roots for 2-3 hours, and the bacterial liquid is irrigated in the seed holes after transplanting.

(5) Data statistics: take pictures and make output statistics.

(6) Analysis of the results: When transplanting pepper seedlings, using microbial inoculants or microbial fertilizers with Streptomyces lividans W68 as the material to dip the roots of the seedlings can significantly reduce the incidence of pepper Phytophthora disease. Compared with the control group, the dipped root-treated plants had about 1-2 weeks longer growth cycle and a 5% increase in dry pepper yield (Figure 5).

4. The field test of Streptomyces lividans W68 isolated from deep sea sludge on wheat take-all.

(1) Test site and environmental conditions: The test was set up in Donglizhuang Village, Wuhe Town, Jingxian County, Hebei Province. The previous crop is corn, and the average annual yield of wheat is about 950 catties per mu. Wheat sowing, 30 catties/mu. Apply 100 catties of special fertilizer for wheat under Tianfeng brand per mu. In the case of irrigation, the planting is carried out to grab moisture. After sowing, the frozen water is poured once, and then it is poured back to Qingshui, and 60 catties of urea are applied per mu. Use 10% trisulfuron + 56% sodium tetrachloride to control weeds, and use 25g/L beta-cyhalothrin to control wheat midge and aphids.

(2) Demonstration reagents: blank control, microbial inoculants or microbial fertilizers with Streptomyces lividans W68 as materials, and another chemical control with 50% triazolone WP and 12.5% total erosion FS.

(3) Area of plot and arrangement of treatments: 50g of bacterial fertilizer was used per kilogram of wheat seeds.

(4) Data statistics: Investigate the emergence rate, conduct a root system investigation at the jointing stage and heading stage of wheat, adopt the diagonal 5-point sampling method, take 20 plants from each point, investigate the root infection, and classify according to wheat take-all disease Standard records, statistics of diseased plant rate and disease index. Investigate the white ears, take a 1-meter double row at each point, investigate the total number of ears and the number of white ears, and count the white ear rate. The yield measurement survey was carried out, 5 points were randomly sampled for each treatment, the total number of ears per 1-meter double row was investigated, the number of ears per mu was counted, the number of effective ears was 20 ears per point, threshed after drying, the number of grains per ear and 1000-grain weight were counted, and the yield and 1000-grain weight were calculated. Yield effect.

(5) Analysis of results: The use of microbial inoculants or microbial fertilizers with Streptomyces lividans W68 as the material can significantly reduce the rate of diseased plants at the heading stage of wheat and reduce the disease index. Compared with chemical agents, the control effect of microbial inoculants or microbial fertilizers with Streptomyces lividans W68 as the material is obviously better than that of chemical agent triazolone, and is comparable to that of total erosion. It can be seen that microbial inoculants or microbial fertilizers made of Streptomyces flavus W68 can completely replace the use of chemical agents (Figure 6).

Claims (6)

1. one plant of Streptomycesalbidoflhaving (Streptomyces albidoflavus) bacterial strain W68, deposit number CGMCC No.12210。

2. a kind of microbial bacterial agent, it is characterised in that: its active constituent is Streptomycesalbidoflhaving bacterial strain described in claim 1 W68。

3. the application of microbial bacterial agent described in claim 2, it is characterised in that: the microbial bacterial agent is for preventing and treating soil-borne fungus Disease, the soil-borne fungus disease are by following a kind of fungi or a variety of fungus-caused: fusarium verticillioides, sharp spore reaping hook Bacterium, Bipolaris sacchari, Rhizoctonia solani Kuhn, Verticillium dahliae bacterium, grey big angle base shell bacterium, every gram of dry weight of the microbial bacterial agent Contain 1 × 10 containing the Streptomycesalbidoflhaving W68 for being isolated from deep-sea sludge¹⁰A spore.

4. the application of microbial bacterial agent described in claim 2, which is characterized in that the microbial bacterial agent is used as microbial manure.

5. the application of microbial bacterial agent as claimed in claim 3 is used to prevent and treat root rotof flax, Muskmelon Fusarium wilt, capsicum epidemic disease Mildew and take-all.

6. a kind of preparation method of microbial manure, this method comprises:

(1) preparation of liquid seeds

Picking grows aerial hyphae and the spore of 4-5 days Streptomycesalbidoflhaving W68 as described in claim 1 on PDA plate, It is seeded in 2 × YT fluid nutrient medium, 28-30 DEG C, 180rpm shaken cultivation uses after 1-2 days as seed liquor；

(2) preparation of solid fermentation seed

By the access of liquid seeds in mass ratio 10% equipped in the tissue culture bottle of solid fermentation material, 28-30 DEG C, cultivates 4-6 days, obtain To solid fermentation seed；

(3) preparation of microbial manure

Using agricultural wastes feces of livestock and poultry, edible fungi residue, wheat bran or dregs of beans as raw material, a certain proportion of MnSO is added₄、(NH)₂SO₄, material-water ratio 1:1 is inoculated with solid fermentation seed according to 10% ratio, 28-30 DEG C culture 5-7 days, after fermented and cultured It air-dries, spore concentration reaches 1 × 10¹⁰A/g, uses as microbial streptomycete fertilizer.