Compound microbial agent for preventing and treating grape downy mildew and preparation method thereof

Abstract

The invention relates to a compound microorganism bacterium agent for preventing and treating grape downy mildew. The compound microorganism bacterium agent comprises streptomyces rochei HML17 of which the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.34955 and bacillus subtilis HM-1 of which the preservation number is CGMCC No.21752. The invention also relates to a preparation method of the compound microorganism bacterium agent for preventing and treating the grape downy mildew. The invention also relates to a preparation method and application of the compound microbial agent. The compound microbial agent disclosed by the invention can be used for effectively preventing and treating grape downy mildew, improving the micro-ecological environment of soil, promoting the growth of plants and increasing the yield and income of grapes by matching two modes of spraying and root application.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to a compound microbial agent for the prevention and control of grape downy mildew and its preparation method. Background Technology

[0002] Changli County in Qinhuangdao is a nationally renowned grape-producing area, but downy mildew is a frequent disease that negatively impacts grape production. Downy mildew is caused by a fungus called *Phytospora variegata* (…). Plasmopara viticola This disease, caused by infection, is the number one disease in China's grape-producing regions, widely damaging tender above-ground tissues such as leaves, new shoots, flower clusters, and young fruits. In the early stages, it forms oily, polygonal lesions, and later, a white, frosty mold layer appears on the back of the lesions. In severe cases, it leads to premature leaf drop, dieback of new shoots, and fruit rot, not only reducing the yield and quality of the current year but also weakening the tree and affecting the following year's fruit production. In rainy years, it can even cause an epidemic throughout the entire vineyard.

[0003] Current prevention and control mainly rely on agricultural measures and chemical agents. Agricultural control reduces pathogens by clearing diseased plant debris and improving drainage and ventilation, but it is difficult to completely control reinfection. Chemical agents are divided into two categories: protective agents (such as Bordeaux mixture) and systemic agents (such as dimethomorph). Although they can take effect quickly, long-term use can easily lead to increased drug resistance in pathogens, and at the same time cause environmental problems such as pesticide residues and damage to the soil microecology, which is contrary to the needs of green agricultural development.

[0004] With the rapid development of biological control technology, the use of beneficial microorganisms to control plant diseases has become a research hotspot. However, at present, there are not many types of compound microbial agents that can be used to control grape downy mildew, and their control effects are often unsatisfactory. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent that can effectively prevent and control grape downy mildew, improve the soil micro-ecological environment, promote plant growth, and increase grape yield and income, as well as its preparation method and application.

[0006] The present invention adopts the following technical solution: A compound microbial agent comprising Streptomyces louchei ( Streptomyces rochei HML17 and Bacillus subtilis ( Bacillus subtilis HM-1.

[0007] Furthermore, the Streptomyces loucherei HML17 has the accession number CGMCC No.34955 and was deposited on June 20, 2025 at the China General Microbiological Culture Collection Center, located in Beijing, China.

[0008] Furthermore, the Bacillus subtilis HM-1 has the accession number CGMCC No.21752 and was deposited on January 28, 2021, at the China General Microbiological Culture Collection Center, located in Beijing, China.

[0009] Furthermore, the total viable count in the compound microbial agent is not less than 1×10⁻⁶. 10 cfu / g.

[0010] Furthermore, in the compound microbial agent, the ratio of viable counts of Streptomyces louchei HML17 to Bacillus subtilis HM-1 is 1~10:2.

[0011] A method for preparing the above-mentioned composite microbial agent includes the following steps: (1) Prepare Streptomyces loucheri HML17 bacterial powder and Bacillus subtilis HM-1 bacterial powder respectively; (2) Mix Streptomyces loucheri HML17 bacterial powder and Bacillus subtilis HM-1 bacterial powder according to the ratio of live bacteria to obtain the product.

[0012] In the preparation method, the Streptomyces louchei HML17 mycelial powder is prepared by the following method: (i) Preparation of Gao's No. 1 culture medium; (ii) Activation of strains: Pick one loopful of Streptomyces louchei HML17 colonies and inoculate them into a 150mL Erlenmeyer flask containing 50mL of Gao's No. 1 liquid medium. Incubate at 250rpm and 28℃ for 3 days for activation. (iii) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of Gao's No. 1 liquid culture medium. Incubate at 250 rpm and 28 °C for 3 days to obtain seed culture. (iv) Preparation of fermentation broth: 180 mL of the prepared seed liquid was inoculated into a 6 L fermenter containing 3.5 L of Gao's No. 1 liquid culture medium and cultured at 250 rpm and 28 °C for 5 days to obtain the fermentation broth of Streptomyces louchei HML17. (v) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed by freeze dryer to obtain Streptomyces louchei HML17 microbial powder.

[0013] In the preparation method, the Bacillus subtilis HM-1 bacterial powder is prepared by the following method: (I) Preparation of LB liquid culture medium; (II) Activation of strain: Pick one loop of Bacillus subtilis HM-1 colony, inoculate it into a 150 mL Erlenmeyer flask containing 50 mL LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 37 °C for 24 h. (III) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Incubate at 160 rpm and 37 °C for 24 h with constant temperature shaking to obtain seed culture. (IV) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h to obtain Bacillus subtilis HM-1 fermentation broth; (V) Preparation of bacterial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus subtilis HM-1 bacterial powder.

[0014] In the preparation method, the viable count of the Streptomyces louchei HML17 powder is not less than 1×10⁻⁶. 10 cfu / g; the viable count of the Bacillus subtilis HM-1 powder is not less than 2 × 10⁻⁶. 10 cfu / g.

[0015] Application of the above-mentioned compound microbial agent in the prevention and control of grape downy mildew.

[0016] Application of the above-mentioned compound microbial agent in the preparation of a biological pesticide for controlling grape downy mildew.

[0017] The beneficial effects of this invention are as follows: the compound microbial agent of this invention, applied by both foliar spraying and root application, can effectively control grape downy mildew, improve the soil micro-ecological environment, promote plant growth, and increase grape yield and income. At the same time, it also has the advantages of being environmentally friendly and less prone to developing resistance, providing a new direction for the green control of grape downy mildew. Attached Figure Description

[0018] Figure 1 The colony morphology of Streptomyces louchei HML17 after 48 hours of culture on Gao's No. 1 medium.

[0019] Figure 2 The morphology of Streptomyces louchei HML17 under an optical microscope after peacock blue staining.

[0020] Figure 3 Phylogenetic tree of 16S rDNA of Streptomyces loucheri HML17.

[0021] Figure 4 The results of the synergistic experiment between strain HML17 and Bacillus subtilis HM-1 are shown. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the embodiments, and any modifications made by those skilled in the art within the scope defined by the claims also fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the reagents used in the following embodiments are all available from conventional biochemical reagent stores.

[0023] Example 1: Characteristics of Streptomyces louchei HML17 Streptomyces loucheri ( Streptomyces rochei HML17 was deposited on June 20, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCCNo.34955.

[0024] (1) Morphological characteristics The fungal cells are short and fluffy, white on the surface and pale orange on the reverse. They are dry, opaque, and velvety, with a dense, wrinkled texture that is easy to pick out. Under a microscope, they appear as radial filaments. Figure 1 As shown. Peacock blue staining as... Figure 2 As shown. A phylogenetic tree of *Streptomyces louchei* HML17 constructed based on 16S rDNA is shown below. Figure 3 As shown.

[0025] (2) Physiological and biochemical characteristics The physiological and biochemical characteristics of Streptomyces louchei HML17 were identified, and the results are shown in Table 1.

[0026] Table 1. Physiological and biochemical characteristics of Streptomyces louchei HML17 .

[0027] Example 2: Biocompatibility of Streptomyces loucherei HML17 and Bacillus subtilis HM-1 Bacillus subtilis ( Bacillus subtilis HM-1 was deposited on January 28, 2021, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCCNo.21752.

[0028] Streptomyces loucherii HML17 was activated on Gao's No. 1 medium, and Bacillus subtilis HM-1 was activated using LB medium.

[0029] LB medium: 5g yeast extract, 10g peptone, 10g sodium chloride, and 20g agar are placed in a 1000mL beaker, 900mL distilled water is added and heated to dissolve, the pH is adjusted to 7.2~7.4, and the volume is brought to 1L with distilled water. The mixture is then sterilized at 121℃ for 30 minutes and set aside.

[0030] Gao's No. 1 culture medium: 20g soluble starch, 1g KNO3, 0.5g NaCl, 0.5g KH2PO4·3H2O, 0.5g MgSO4·7H2O, 0.01g FeSO4·7H2O, 20g agar, 1000mL water, pH 7.2~7.4. First, dissolve the starch in a small amount of water by heating, then mix with the other ingredients.

[0031] A colony of Bacillus subtilis HM-1 was picked up using an inoculation loop and placed in 1 mL of sterile water. The suspension was prepared by repeatedly pipetting and mixing with a sterile pipette. This suspension was then added to LB medium that had not solidified but was not hot to the touch. After mixing, the suspension was immediately poured into plates. A Streptomyces rotundifolia HML17 bacterial cake was placed in the center of the cooled and solidified plate, and the plates were labeled. This process was repeated three times. The plates were incubated at 28°C for 5 days. Bacillus subtilis HM-1 and Streptomyces rotundifolia HML17 co-grown without forming an inhibition zone, indicating that the Bacillus subtilis HM-1 strain and Streptomyces rotundifolia HML17 strain are well-compatible and do not inhibit each other. The results are shown in the figure below. Figure 4 .

[0032] Example 3: In vitro efficacy determination of Streptomyces loucherei HML17, Bacillus subtilis HM-1 and Staphylococcus aureus monococcosis. (1) Preparation of sporangium suspension of grape downy mildew: Fresh diseased leaves infected with grape downy mildew and forming pale yellow oily spots were collected from the field and brought back to the laboratory. After being rinsed with running water, the petioles were wrapped with moist defatted cotton and placed in a petri dish with moist gauze. The petioles were sealed with plastic wrap and cultured in a humidified dark environment at 20℃ for 1-2 days to promote the production of new sporangia. The sporangia were then rinsed with sterile water using a pipette to prepare a sporangium suspension for later use.

[0033] (2) Preparation of fermentation broth of Streptomyces loucheri HML17: Pick one loop of Streptomyces loucheri HML17 colony and inoculate it into a 150 mL Erlenmeyer flask containing 50 mL of Gao's No. 1 liquid culture medium. Incubate at 250 rpm and 28 °C for 3 days with constant temperature shaking.

[0034] (3) Preparation of Bacillus subtilis HM-1 fermentation broth: Pick one loop of Bacillus subtilis HM-1 colony and inoculate it into a 150 mL Erlenmeyer flask containing 50 mL LB liquid medium. Incubate at 160 rpm and 37 °C for 24 h with constant temperature shaking.

[0035] (4) Indoor efficacy test: Select healthy grape leaves of uniform size, rinse them with clean water, and dry them until there are no water droplets on the front and back of the leaves. Use a 5mm diameter punch to make uniform leaves and place them in a sterile petri dish with wet gauze, with the back of the leaves facing up. Place 80 leaves in each petri dish.

[0036] (5) The experiment included 4 treatments: Treatment 1: Use a pipette to inoculate a mixture of 2 μL of Bacillus subtilis HM-1 fermentation broth and 2 μL of sporangium suspension onto the underside of the leaf.

[0037] Treatment 2: A mixture of 2 μL of Streptomyces loucheri HML17 fermentation broth and 2 μL of sporangium suspension was pipetted and inoculated onto the underside of the leaf.

[0038] Treatment 3: A mixture of 1 μL of Streptomyces loucheri HML17 fermentation broth, 1 μL of Bacillus subtilis HM-1 fermentation broth, and 2 μL of sporangium suspension was pipetted and inoculated onto the underside of the leaf.

[0039] Treatment 4: Use a pipette to draw 2 μL of sterile water + 2 μL of sporangium suspension and inoculate it onto the underside of the leaf.

[0040] After inoculation, the cells were placed in an artificial climate chamber with alternating periods of 20°C and 2 hours of darkness for cultivation. Each treatment was repeated 3 times. On day 8 after inoculation, the number of lesions was observed and recorded, and the incidence rate and control effect were calculated.

[0041] Incidence rate (%) = Total number of cases / Total number of vaccination sites × 100; Prevention and control effect (%) = (control incidence rate - treatment incidence rate) / control incidence rate × 100.

[0042] Table 2 Results of in vitro control efficacy against *Botrytis cinerea* .

[0043] The results showed that both Streptomyces loucheri HML17 and Bacillus subtilis HM-1 were effective in controlling grape downy mildew, and the combined effect of the two strains was more than 18% higher than that of a single strain, with better control effect than that of a single strain.

[0044] Example 4: Preparation of Streptomyces loucherei HML17 inoculum (1) Preparation of Gao's No. 1 culture medium: Same as in Example 2.

[0045] (2) Activation of strains: Select one loop of Streptomyces louchei HML17 colony and inoculate it into a 150mL Erlenmeyer flask containing 50mL of Gao's No. 1 liquid culture medium. Incubate at 250rpm and 28℃ for 3 days for activation.

[0046] (3) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of Gao's No. 1 liquid culture medium. Incubate at 250 rpm and 28℃ for 3 days to obtain seed culture.

[0047] (4) Preparation of fermentation broth: 180 mL of the prepared seed liquid was inoculated into a 6 L small fermenter containing 3.5 L of Gao's No. 1 liquid culture medium and cultured at 250 rpm and 28 °C for 5 days to obtain the fermentation broth of Streptomyces louchei HML17.

[0048] (5) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by freeze dryer to obtain Streptomyces louchei HML17 powder. The effective viable count was tested to be 1.51 × 10⁻⁶. 10 cfu / g.

[0049] Example 5: Preparation of Bacillus subtilis HM-1 inoculum (1) Preparation of LB liquid culture medium: Same as in Example 2.

[0050] (2) Activation of strain: Pick one loop of Bacillus subtilis HM-1 colony, inoculate it into a 150mL Erlenmeyer flask containing 50mL LB liquid medium, and activate it by constant temperature shaking at 160rpm and 37℃ for 24h.

[0051] (3) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid culture medium. Incubate at 160 rpm and 37 °C for 24 h to obtain seed culture.

[0052] (4) Preparation of fermentation broth: 180 mL of the prepared seed liquid was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h to obtain Bacillus subtilis HM-1 fermentation broth.

[0053] (5) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1, and the mixture was sprayed by a freeze dryer to obtain Bacillus subtilis HM-1 powder with a viable count of 3.03 × 10⁻⁶ cells / day. 10 cfu / g.

[0054] Preparation of compound microbial agents in Examples 6-8 The Streptomyces louchei HML17 bacterial powder prepared in Example 4 (with an effective viable count of 1.51 × 10⁻⁶) 10 The CFU / g of Bacillus subtilis HM-1 bacterial powder prepared in Example 5 (effective viable count 3.03 × 10⁻⁶) was compared with that of Bacillus subtilis HM-1 bacterial powder prepared in Example 5 (effective viable count 3.03 × 10⁻⁶). 10 Compound microbial agents 1, 2, and 3 were prepared by mixing microbial cells (cfu / g) at viable cell ratios of 1:2, 5:2, and 5:1, respectively. Testing revealed that compound microbial agent 1 had a total viable cell count of 22 billion CFU / g, compound microbial agent 2 had a total viable cell count of 18 billion CFU / g, and compound microbial agent 3 had a total viable cell count of 16 billion CFU / g.

[0055] Example 9: Field trial of the compound microbial inoculant used in Examples 6-8 (1) Test conditions Experimental variety: Cabernet Sauvignon (wine grape).

[0056] Experimental location: A grape planting base in Changli County, Hebei Province (5 consecutive years of cropping, downy mildew occurs year-round).

[0057] Experimental design: Six treatment groups were set up, sprayed with Streptomyces loucheri HML17 inoculum (T1), Bacillus subtilis HM-1 inoculum (T2), compound inoculum 1 (T3), compound inoculum 2 (T4), compound inoculum 3 (T5), and blank control (CK). Each group was replicated three times, and each replicate consisted of 100 grapevines.

[0058] (2) Application method Root application: On March 15 (budding period), mix different treatment inoculants with well-rotted organic fertilizer (inoculant: organic fertilizer = 1:50). The blank control does not use inoculants. Apply the mixture along the root distribution area (make a 20cm deep circular trench 50cm away from the trunk), cover with soil and water.

[0059] Foliar spraying: Two applications were made: First application: July 20th, the inoculant was diluted with water at a ratio of 1:300 and sprayed evenly onto the surface of the grapevines, ensuring the surface was moist but not dripping; Second application: August 20th (peak season for downy mildew), the application method was the same as above. Blank control (CK): Sprayed with water, the application method was the same as for the inoculant treatment.

[0060] (3) Survey indicators and methods Grape growth index survey: On June 20, the height of grapevines and the number of new shoots in each treatment were surveyed, with 10 vines randomly selected from each replicate.

[0061] Downy mildew disease survey: On August 30, the incidence of downy mildew on grape leaves and fruits under each treatment was investigated and recorded according to the following grading standards.

[0062] Leaf disease severity classification: Grade 0: No lesions; Grade 1: Lesions cover ≤5% of the total leaf area; Grade 2: Lesions cover 6% to 15% of the total leaf area; Grade 3: Lesions cover 16% to 30% of the total leaf area; Grade 4: Lesions cover 31% to 50% of the total leaf area; Level 5: Lesions cover more than 50% of the total leaf area or the leaves fall off.

[0063] Fruit disease severity classification: Grade 0: No lesions; Grade 1: The area of ​​lesions accounts for ≤5% of the fruit surface area; Grade 2: Lesions cover 6% to 15% of the fruit surface area; Grade 3: Lesions cover 16% to 30% of the fruit surface area; Grade 4: Lesions cover 31% to 50% of the fruit surface area; Grade 5: Lesions cover more than 50% of the fruit surface area or the fruit is rotten.

[0064] Calculation indicators: Incidence rate (%) = (Number of cases / Total number of cases surveyed) × 100; Disease index = ∑ (number of diseased leaves / fruits at each level × corresponding level value) / (total number of leaves / fruits surveyed × highest level value) × 100; Prevention and control effect (%) = (disease index of blank control group - disease index of treatment group) / disease index of blank control group × 100.

[0065] Production targets: Yield per plant: 20 plants were randomly selected for each replicate, and the total weight of the fruit per plant (kg) was measured. Yield per mu: calculated based on a plant spacing of 2m × 3m (111 plants per mu), unit: kg / mu; Yield increase rate (%) = (yield per mu of treatment group - yield per mu of blank control group) / yield per mu of blank control group × 100.

[0066] Soil microecological indicators: Survey period: March 10 (before application of pesticides) and October 15 (15 days after harvest). Investigation method: Soil samples were taken from the 0-20cm soil layer for each replicate (five points were mixed). The dilution plate method was used to determine the number of beneficial bacteria in the soil: Bacillus subtilis and Streptomyces colony counts (cfu / g soil); the number of harmful bacteria: Botrytis cinerea oospore counts (cells / g soil) and Fusarium colony counts (cfu / g soil); the ratio of beneficial bacteria to harmful bacteria was calculated.

[0067] (4) Experimental results and analysis The control effect of downy mildew is shown in Table 3.

[0068] Table 3 Results of leaf downy mildew disease control .

[0069] The analysis in Table 3 shows that the compound microbial agent treatment groups (T3~T5) are significantly better than the single microbial agent (T1~T2) and the blank control (CK) in controlling downy mildew of leaves. Among them, the T3 treatment has the highest control effect of up to 94.9%.

[0070] Table 4 Results of downy mildew control in fruits .

[0071] The analysis in Table 4 shows that microbial inoculants are effective in controlling downy mildew on grapes. Among them, the T3 treatment had the lowest disease incidence and disease index, with a good fruit rate of 97.9% and no fruit rot. The control effect of the compound inoculant treatment was better than that of the single inoculant treatment.

[0072] Table 5. Survey data on grapevine height and number of new shoots .

[0073] The analysis of Table 5 shows that the plant height and number of new shoots in the compound microbial agent treatment group were significantly higher than those in other treatments. The plant height in the T3 group increased by 36.7% compared with the CK group, and the number of new shoots increased by 5.8 per plant, indicating that Streptomyces loucheri and Bacillus subtilis can synergistically promote grape growth.

[0074] Table 6 Results of the Production Index Survey .

[0075] The analysis of Table 6 shows that the yield of the T3 treatment reached 588.3 kg per mu, which is 35.9% higher than that of the CK treatment. Moreover, there were no small or deformed fruits. The yield of the compound microbial agent treatment group was higher than that of the single microbial agent treatment group.

[0076] Table 7 Changes in soil microbiota before and after pesticide application. .

[0077] The analysis of Table 7 shows that the number of beneficial bacteria in the soil treated with T3 increased by 258% compared with that before the application of the pesticide, and the ratio of beneficial bacteria to harmful bacteria reached 5264.7, indicating that the compound microbial treatment can improve the soil micro-ecological environment.

[0078] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. A compound microbial agent, characterized in that, It includes Streptomyces loucheri ( Streptomyces rochei HML17 and Bacillus subtilis ( Bacillus subtilis HM-1.

2. The compound microbial agent according to claim 1, characterized in that, The preservation number of *Streptomyces loucherii* HML17 is CGMCC No. 34955; the preservation number of *Bacillus subtilis* HM-1 is CGMCC No. 21752.

3. The compound microbial agent according to claim 1, characterized in that, The ratio of viable counts of Streptomyces loucherii HML17 to Bacillus subtilis HM-1 is 1 to 10:

2.

4. The compound microbial agent according to claim 1, characterized in that, The total viable count in the compound microbial agent is not less than 1×10⁻⁶. 10 cfu / g.

5. A method for preparing a composite microbial agent as described in any one of claims 1 to 4, characterized in that, It includes the following steps: (1) Prepare Streptomyces loucheri HML17 bacterial powder and Bacillus subtilis HM-1 bacterial powder respectively; (2) Mix Streptomyces loucheri HML17 bacterial powder and Bacillus subtilis HM-1 bacterial powder according to the ratio of live bacteria to obtain the product.

6. The preparation method according to claim 5, characterized in that, The Streptomyces louchei HML17 mycelial powder was prepared by the following method: (i) Preparation of Gao's No. 1 culture medium; (ii) Activation of strains: Pick one loopful of Streptomyces louchei HML17 colonies and inoculate them into a 150mL Erlenmeyer flask containing 50mL of Gao's No. 1 liquid medium. Incubate at 250rpm and 28℃ for 3 days for activation. (iii) Preparation of seed culture: Take 5 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 250 mL of Gao's No. 1 liquid culture medium. Incubate at 250 rpm and 28℃ for 3 days to obtain seed culture. (iv) Preparation of fermentation broth: 180 mL of the prepared seed liquid was inoculated into a 6 L fermenter containing 3.5 L of Gao's No. 1 liquid culture medium and cultured at 250 rpm and 28 °C for 5 days to obtain the fermentation broth of Streptomyces louchei HML17. (v) Preparation of microbial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed by freeze dryer to obtain Streptomyces louchei HML17 microbial powder.

7. The preparation method according to claim 5, characterized in that, The Bacillus subtilis HM-1 bacterial powder was prepared by the following method: (I) Preparation of LB liquid culture medium; (II) Activation of strain: Pick one loop of Bacillus subtilis HM-1 colony, inoculate it into a 150 mL Erlenmeyer flask containing 50 mL LB liquid medium, and activate it by constant temperature shaking at 160 rpm and 37 °C for 24 h; (III) Preparation of seed culture: Take 4 mL of activated bacterial culture and inoculate it into a 1000 mL Erlenmeyer flask containing 200 mL of LB liquid medium. Incubate at 160 rpm and 37 °C for 24 h with constant temperature shaking to obtain seed culture. (IV) Preparation of fermentation broth: 180 mL of the prepared seed culture was inoculated into a 6 L fermenter containing 3.5 L of LB liquid medium and cultured at 160 rpm and 37 °C for 48 h to obtain Bacillus subtilis HM-1 fermentation broth; (V) Preparation of bacterial agent: The fermentation broth and soluble starch were mixed evenly at a mass ratio of 10:1 and sprayed through a freeze dryer to obtain Bacillus subtilis HM-1 bacterial powder.

8. The preparation method according to claim 5, characterized in that, The viable count of the Streptomyces louchei HML17 bacterial powder is not less than 1×10⁻⁶. 10 cfu / g; the viable count of the Bacillus subtilis HM-1 powder is not less than 2 × 10⁻⁶. 10 cfu / g.

9. The application of a compound microbial agent as described in claims 1 to 4 in the prevention and control of grape downy mildew.

10. The application of the compound microbial agent as described in claims 1-4 in the preparation of a biological pesticide for controlling grape downy mildew.

Images