Microbial agent for preventing and treating strawberry black spot and preparation method thereof

Abstract

The invention relates to a microbial agent for preventing and treating strawberry black spot and a preparation method thereof, and belongs to the technical field of microbial agents. The microbial agent is prepared from the following components: 50-60 parts by weight of a composite microbial liquid, 15-18 parts by weight of a stabilizer and 11-13 parts by weight of a protective agent, the stabilizer contains glycine and taurine, and the protective agent contains sodium alginate and ergothioneine. The microbial agent provided by the invention can be used for effectively preventing and treating strawberry black spot and improving the yield and quality of strawberries.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial agents, and relates to a microbial agent for preventing and controlling strawberry black spot disease and a preparation method thereof. Background Art

[0002] Strawberries belong to the genus Fragaria of the Rosaceae family and are perennial herbaceous plants. Strawberries are rich in nutrients such as vitamin C, vitamin A, and vitamin E, which can relieve night blindness, maintain the health of epithelial tissues, improve eyesight and nourish the liver, and promote growth and development. The rich dietary fiber can promote gastrointestinal motility, food digestion, improve constipation, and prevent the occurrence of acne and intestinal cancer. It is known as the "queen of fruits" because of its bright fruit color and unique taste, and is therefore deeply loved by consumers.

[0003] During the process of strawberry cultivation, various diseases and pests are likely to occur. Strawberry black spot disease is one of the common diseases of strawberries, which is widely distributed and occurs in all strawberry production areas in China. The pathogen of strawberry black spot disease is Alternaria of the Deuteromycotina. Under the microscope, the hyphae are colorless and without septa, the conidia are dark brown, pear-shaped, oval or obclavate, brownish, with 3-8 transverse septa and 1-4 longitudinal septa. The septa are slightly constricted and have short beaks. The main transmission methods of the pathogen are through seedlings, etc. The mycelium overwinters on diseased plants or diseased residues and is transmitted through seedlings. Among them, the pathogen spores can also directly infect and cause disease; Alternaria in the air can also infect; it is easy to get sick in high-temperature and high-humidity weather, and the disease is more serious when there are more rainy days or the field humidity is high; the disease is more serious in continuous cropping fields.

[0004] Strawberry black spot disease mainly damages leaves, petioles, stolons and berries. When the leaves are infected, black-brown irregular lesions are produced, with a diameter of 5-8 mm, slightly showing a concentric ring pattern. The center of the lesion is grayish-brown, and a yellow halo often appears around the lesion. Brown small sunken spots appear on the petioles and stolons. When the lesion encircles the petiole or stem for one week, the petiole and stem break, and the diseased part constricts. When the berries are infected, black spots are produced, with a black sooty mold layer on them, and the lesions are relatively shallow.

[0005] Currently, the commonly used control measures are mainly as follows: (1) Select strawberry varieties resistant to black spot disease; promptly remove and burn the diseased leaves when found. (2) At the initial stage of the disease, spray 600 times the liquid of 10% polyoxin wettable powder, or 1500 times the liquid of 25% azoxystrobin suspension, or 1200 times the liquid of 25% pyraclostrobin ester emulsifiable concentrate, or 1000-1500 times the liquid of 10% difenoconazole water dispersible granules, or 800 times the liquid of 75% chlorothalonil wettable powder, once every 10 days, and control for 2-3 times. However, the prevention and control of strawberry diseases rely too much on chemical pesticides. Especially, the number of pesticide applications is large and the interval period is short. The unreasonable use of pesticides has enhanced the drug resistance of the pathogen. The strawberry fruit harvesting period is long, and chemical pesticides are often used to control diseases and pests during the fruit harvesting period, and the fruits are not picked and sold according to the requirements of the pesticide safety interval. Therefore, the problem of pesticide residues in fresh strawberry fruits is relatively prominent, and the food safety management risk is large. Summary of the Invention

[0006] The main object of the present invention is to provide a microbial inoculant, which has a good control effect on strawberry black spot disease, can achieve a good control effect without using any chemical agents, has less pollution, and can ensure the food safety of strawberries.

[0007] The present invention adopts the following technical solutions to achieve the above object:

[0008] A microbial inoculant is mainly prepared from the following components:

[0009] 50 - 60 parts by weight of compound microbial bacterial liquid, 15 - 18 parts by weight of stabilizer, and 11 - 13 parts by weight of protective agent.

[0010] Further, the compound microbial bacterial liquid contains Streptomyces crystallinus, Bacillus amyloliquefaciens, and Streptomyces catenulisporus;

[0011] Further, the total number of effective viable bacteria in the compound microbial bacterial liquid is not less than 10×10 9 cfu / mL.

[0012] Further, the stabilizer contains glycine and taurine in a weight ratio of 1:(0.5 - 0.8).

[0013] Further, the protective agent contains sodium alginate and ergothioneine in a weight ratio of 1:(0.1 - 0.3).

[0014] The present invention provides a preparation method of the above microbial inoculant, which mainly includes the following steps:

[0015] Step A, preparing a fermentation substrate: inoculating a seed solution of Streptococcus equi subsp. zooepidemicus and a seed solution of Vishniacozyma victoriae into a culture medium containing 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate, culturing at 37°C for 36 h, and after culturing, centrifuging and reserving the supernatant;

[0016] Step B, adding gelatinized starch, L-theanine, and peony seed oil to the above-obtained supernatant, mixing evenly, and then inoculating a seed solution of Bacillus amyloliquefaciens, a seed solution of Streptomyces catenulisporus, and a seed solution of Streptomyces crystallinus, and culturing at 30°C to obtain a compound microbial bacterial liquid;

[0017] Step C, adding a stabilizer to the compound microbial bacterial liquid, mixing evenly, concentrating, and drying to obtain a mixture;

[0018] Step D, adding the protective agent after mixing it with water to the above mixture, mixing evenly, and drying to obtain the microbial inoculant.

[0019] Further, the inoculation amount of the Streptococcus equi subsp. zooepidemicus seed liquid in step A is 10% of the volume of the culture medium, and the inoculation amount of the Vishniacozyma victoriae seed liquid is 8% of the volume of the culture medium.

[0020] Further, the effective viable count of the Streptococcus equi subsp. zooepidemicus seed liquid in step A is not less than 1×10 8 cfu / mL, and the effective viable count of the Vishniacozyma victoriae seed liquid is not less than 1×10 8 cfu / mL.

[0021] Further, the addition amount of the gelatinized starch in step B is 30 - 40 g / L, the addition amount of L-theanine is 5 - 8 g / L, and the addition amount of peony seed oil is 1 - 1.5 g / L.

[0022] Furthermore, the preparation method of the gelatinized starch is as follows: make a suspension by adding water to corn starch, heat and stir until it becomes viscous and transparent, cool, dry, and pulverize to obtain the gelatinized starch.

[0023] Further, the effective viable count of the Bacillus amyloliquefaciens seed liquid in step B is not less than 1×10 9 cfu / mL, the effective spore amount of the Streptomyces catenulisporus seed liquid is not less than 1×10 8 per mL, and the effective spore amount of the Streptomyces crystallinus seed liquid is not less than 2×10 8 per mL.

[0024] Further, the inoculation amount of the Bacillus amyloliquefaciens seed liquid in step B is 10%, the inoculation amount of the Streptomyces catenulisporus seed liquid is 10%, and the inoculation amount of the Streptomyces crystallinus seed liquid is 10%.

[0025] Further, the total effective viable count of the composite microbial liquid in step B is not less than 10×10 9 cfu / mL.

[0026] The present invention also provides a specific use of the above microbial inoculant, that is, the microbial inoculant can be used to prevent and control strawberry black spot disease.

[0027] Further, the usage method of the microbial inoculant is to dilute the microbial inoculant 100 - 200 times and spray it on the leaf surface before the onset or at the initial stage of strawberry black spot disease.

[0028] The present invention has the following beneficial effects:

[0029] 1. The microbial inoculant provided by the present invention has a good prevention and control effect on strawberry black spot disease, can effectively reduce the incidence rate, reduce the disease index, and the prevention and control effect is above 80%, with a significant effect.

[0030] 2. In the microbial inoculant of the present invention, Streptomyces catenulae and Streptomyces crystallinus are the main acting flora, and Bacillus amyloliquefaciens is the auxiliary flora. When used before the onset of strawberry black spot disease, a protective layer can be formed on the plant surface to protect it from pathogen infection. When used in the initial stage of the disease, the flora in the present invention can eliminate the invaded pathogens, inhibit the growth of the pathogens, and prevent their continuous amplification. During the preparation of the microbial inoculant, the fermentation broth of Streptococcus equi subsp. zooepidemicus and Vishniacozyma victoriae is used as the fermentation basis of the inoculant. The fermentation broth contains hyaluronic acid and microbial metabolites. Hyaluronic acid has certain film-forming properties. When sprayed on the leaf surface of the plant, it will form a protective film to isolate the pathogens in the air. For the invaded pathogens, they will be isolated in the environment containing the microbial inoculant, and the invaded pathogens will be eliminated by using the microbial antagonistic effect. The metabolites in the fermentation broth have certain bactericidal effects and can assist in eliminating the invaded pathogens. To avoid the death of the bacteria caused by the exposure of the microorganisms to the air and affect their control effect, glycine and taurine are added as stabilizers, and sodium alginate and ergothioneine are added as protectants during the preparation of the inoculant, which can effectively ensure the number of effective live bacteria and achieve better control effects. Detailed implementation mode

[0031] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope protected by the claims of this application. The bacterial strains used in the present invention are all commercial strains and can be purchased; the raw materials used are all commercially available products and can be purchased.

[0032] Example 1

[0033] Raw material preparation:

[0034] After activating the purchased Streptococcus equi subsp. zooepidemicus (Guangdong Microbial Culture Collection Center, strain number GDMCC NO.1.437) strain according to the instructions, it was inoculated into the seed medium and cultured until the number of effective live bacteria was not less than 1×10 8 cfu / mL of Streptococcus equi subsp. zooepidemicus seed liquid.

[0035] After activating the purchased Vishniacozyma victoriae (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) strain according to the instructions, it was inoculated into the seed medium and cultured until the number of effective live bacteria was not less than 1×10 8 cfu / mL of Vishniacozyma victoriae seed liquid.

[0036] After activating the purchased Streptomyces catenulae strain (China General Microbiological Culture Collection Center, strain number CGMCC 4.6283) according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 1×10 8 cfu / mL of Streptomyces catenulae seed liquid.

[0037] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC 1.10901) according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0038] After activating the purchased Streptomyces crystallinus strain (China General Microbiological Culture Collection Center, strain number CGMCC 4.1600) according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 2×10 8 cfu / mL of Streptomyces crystallinus.

[0039] Prepare the medium: Weigh each component according to the composition ratio of 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. Add water and mix well, then adjust the pH to 7.0 and sterilize to obtain the medium.

[0040] Prepare the gelatinized starch: Make a suspension of corn starch with water, heat and stir until it becomes viscous and transparent, cool and dry, then pulverize to obtain the gelatinized starch.

[0041] Prepare the microbial inoculum:

[0042] Step A: Inoculate the seed liquid of Streptococcus equi subsp. zooepidemicus into the above-prepared medium at an inoculation amount of 10% by volume, and inoculate the seed liquid of Vishniacozyma victoriae at an inoculation amount of 8% by volume. Then place it at 37°C and culture according to the conventional fermentation production process for 36 h. After the culture is completed, centrifuge and reserve the supernatant.

[0043] Step B: Add 40 g / L of gelatinized starch, 8 g / L of L-theanine, and 1 g / L of peony seed oil to the above-obtained supernatant, mix well, and then inoculate Bacillus amyloliquefaciens at an inoculation amount of 10% by volume, inoculate the seed liquid of Streptomyces catenulae at an inoculation amount of 10% by volume, and inoculate the seed liquid of Streptomyces crystallinus at an inoculation amount of 10%. Carry out the conventional fermentation process at 30°C for culture to obtain a composite microbial liquid with a total effective viable count of not less than 10×10 9 cfu / mL;

[0044] Step C: Take 60 kg of the composite microbial liquid prepared by the above method, add 10 kg of glycine and 5 kg of taurine thereto, mix well, concentrate and dry to obtain a mixture.

[0045] Step D: Mix 10 kg of sodium alginate and 1 kg of ergothioneine, add an appropriate amount of water and stir until viscous, add the above mixture, mix well, dry and crush to obtain a microbial inoculant.

[0046] Example 2

[0047] Raw material preparation:

[0048] After activating the purchased Streptococcus equi subsp. zooepidemicus (Guangdong Microbial Culture Collection Center, strain number GDMCC NO.1.437) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 8 cfu / mL of Streptococcus equi subsp. zooepidemicus seed liquid.

[0049] After activating the purchased Vishniacozyma victoriae (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 8 cfu / mL of Vishniacozyma victoriae seed liquid.

[0050] After activating the purchased Streptomyces catenulisporus (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) strain according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 1×10 8 spores / mL of Streptomyces catenulisporus seed liquid.

[0051] After activating the purchased Bacillus amyloliquefaciens (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0052] After activating the purchased Streptomyces crystallinus (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) strain according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 2×10 8 spores / mL of Streptomyces crystallinus.

[0053] Prepare the medium: Weigh each component according to the component ratio of 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. After adding water and mixing well, adjust the pH to 7.0 and sterilize to obtain the medium.

[0054] Preparation of gelatinized starch: Corn starch is added with water to form a suspension, then heated and stirred until it becomes viscous and transparent. After cooling, it is dried and pulverized to obtain gelatinized starch.

[0055] Preparation of microbial inoculum:

[0056] Step A: Inoculate the prepared culture medium with the seed liquid of Streptococcus equi subsp. zooepidemicus at an inoculation amount of 10% by volume and the seed liquid of Vishniacozyma victoriae at an inoculation amount of 8% by volume, and then place it under the condition of 37°C and culture for 36 h according to the conventional fermentation production process. After the culture is completed, centrifuge and reserve the supernatant.

[0057] Step B: Add 30 g / L of gelatinized starch, 5 g / L of L-theanine, and 1.5 g / L of peony seed oil to the above-obtained supernatant, mix well, and then inoculate Bacillus amyloliquefaciens at an inoculation amount of 10% by volume, the seed liquid of Streptomyces catenulisporus at an inoculation amount of 10% by volume, and the seed liquid of Streptomyces crystallinus at an inoculation amount of 10% into it. Carry out the conventional fermentation process culture at 30°C to obtain a composite microbial liquid with a total effective viable count of not less than 10×10 9 cfu / mL;

[0058] Step C: Take 50 kg of the composite microbial liquid prepared by the above method, add 10 kg of glycine and 8 kg of taurine to it, mix well, concentrate and dry to obtain a mixture.

[0059] Step D: Mix 10 kg of sodium alginate and 2 kg of ergothioneine, add an appropriate amount of water and stir until it becomes viscous, add the above mixture, mix well, dry and pulverize to obtain the microbial inoculum.

[0060] Example 3

[0061] Raw material preparation:

[0062] After activating the purchased strain of Streptococcus equi subsp. zooepidemicus (Guangdong Microbial Culture Collection Center, strain number GDMCC NO.1.437) according to the instructions, inoculate it into the seed culture medium and culture until the effective viable count is not less than 1×10 8 cfu / mL of the seed liquid of Streptococcus equi subsp. zooepidemicus.

[0063] After activating the purchased strain of Vishniacozyma victoriae (China General Microbiological Culture Collection Center, strain number CGMCC 2.5592) according to the instructions, inoculate it into the seed culture medium and culture until the effective viable count is not less than 1×10 8 cfu / mL of the seed liquid of Vishniacozyma victoriae.

[0064] After activating the purchased Streptomyces catenulae strain (China General Microbiological Culture Collection Center, strain number CGMCC 4.6283) according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 1×10 8 cfu / mL of Streptomyces catenulae seed liquid.

[0065] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC 1.10901) according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0066] After activating the purchased Streptomyces crystallinus strain (China General Microbiological Culture Collection Center, strain number CGMCC 4.1600) according to the instructions, inoculate it into the seed medium and culture until the effective spore count is not less than 2×10 8 cfu / mL of Streptomyces crystallinus.

[0067] Prepare the medium: Weigh each component according to the component ratio of 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. Add water and mix well, then adjust the pH to 7.0 and sterilize to obtain the medium.

[0068] Prepare the gelatinized starch: Make a suspension of corn starch with water, heat and stir until it becomes viscous and transparent, cool and dry, then crush to obtain the gelatinized starch.

[0069] Prepare the microbial inoculant:

[0070] Step A, inoculate the seed liquid of Streptococcus equi subsp. zooepidemicus into the above-prepared medium at an inoculation amount of 10% by volume, and inoculate the seed liquid of Vishniacozyma victoriae at an inoculation amount of 8% by volume. Then place it at 37°C and culture for 36 h according to the conventional fermentation production process. After the culture is completed, centrifuge and reserve the supernatant.

[0071] Step B, add 35 g / L of gelatinized starch, 6 g / L of L-theanine, and 1.3 g / L of peony seed oil to the above-obtained supernatant and mix well. Then inoculate Bacillus amyloliquefaciens into it at an inoculation amount of 10% by volume, inoculate the seed liquid of Streptomyces catenulae at an inoculation amount of 10% by volume, and inoculate the seed liquid of Streptomyces crystallinus at an inoculation amount of 10%. Carry out the conventional fermentation process at 30°C for culture to obtain a composite microbial liquid with a total effective viable count of not less than 10×10 9 cfu / mL;

[0072] Step C: Take 55 kg of the composite microbial inoculum prepared by the above method, add 10 kg of glycine and 7 kg of taurine thereto, mix well, concentrate and dry to obtain a mixture.

[0073] Step D: Mix 10 kg of sodium alginate and 1 kg of ergothioneine, add an appropriate amount of water and stir until viscous, add the above mixture, mix well, dry and pulverize to obtain a microbial inoculant.

[0074] Comparative Example 1

[0075] Raw material preparation:

[0076] After activating the purchased Streptomyces catenulae (China General Microbiological Culture Collection Center, strain number CGMCC 4.6283) strain according to the instructions, inoculate it into the seed medium and culture until the effective spore amount is not less than 1×10 8 cfu / mL of Streptomyces catenulae seed liquid.

[0077] After activating the purchased Bacillus amyloliquefaciens (China General Microbiological Culture Collection Center, strain number CGMCC 1.10901) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable bacteria number is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0078] After activating the purchased Streptomyces crystallinus (China General Microbiological Culture Collection Center, strain number CGMCC 4.1600) strain according to the instructions, inoculate it into the seed medium and culture until the effective spore amount is not less than 2×10 8 cfu / mL of Streptomyces crystallinus.

[0079] Prepare gelatinized starch: Make a suspension of corn starch with water, heat and stir until viscous and transparent, cool, dry and pulverize to obtain gelatinized starch.

[0080] Prepare the medium: Weigh each component according to the component ratio of 35 g / L of gelatinized starch, 6 g / L of L-theanine, 1.3 g / L of peony seed oil, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate, add water and mix well, adjust the pH to 7.0, and sterilize to obtain the medium.

[0081] Prepare the microbial inoculant:

[0082] Step A: Inoculate the medium prepared by the above method with Bacillus amyloliquefaciens at an inoculation amount of 10% by volume, inoculate the Streptomyces catenulae seed liquid at an inoculation amount of 10% by volume, and inoculate the Streptomyces crystallinus seed liquid at an inoculation amount of 10%, and carry out conventional fermentation process culture at 30°C to obtain a composite microbial inoculum with a total effective viable bacteria number not less than 10×10 9 cfu / mL.

[0083] Step B: Take 55 kg of the compound microbial bacteria liquid prepared by the above method, add 10 kg of glycine and 7 kg of taurine thereto, mix well, concentrate and dry to obtain a mixture.

[0084] Step C: Mix 10 kg of sodium alginate and 1 kg of ergothioneine, add an appropriate amount of water and stir until viscous, add the above mixture, mix well, dry and crush to obtain a microbial inoculant.

[0085] Comparative Example 2

[0086] Raw material preparation:

[0087] After activating the purchased Streptococcus equi subsp. zooepidemicus (Guangdong Microbial Culture Collection Center, strain number GDMCC NO.1.437) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable bacteria count is not less than 1×10 8 cfu / mL of Streptococcus equi subsp. zooepidemicus seed liquid.

[0088] After activating the purchased Streptomyces catenulisporus (China General Microbiological Culture Collection Center, strain number CGMCC 4.6283) strain according to the instructions, inoculate it into the seed medium and culture until the effective spore amount is not less than 1×10 8 spores / mL of Streptomyces catenulisporus seed liquid.

[0089] After activating the purchased Bacillus amyloliquefaciens (China General Microbiological Culture Collection Center, strain number CGMCC 1.10901) strain according to the instructions, inoculate it into the seed medium and culture until the effective viable bacteria count is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0090] Prepare the culture medium: Weigh each component according to the component ratio of 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. After adding water and mixing well, adjust the pH to 7.0 and sterilize to obtain the culture medium.

[0091] Prepare the gelatinized starch: Make a suspension of corn starch with water, heat and stir until it becomes viscous and transparent, cool and dry, and crush to obtain the gelatinized starch.

[0092] Prepare the microbial inoculant:

[0093] Step A: Inoculate the Streptococcus equi subsp. zooepidemicus seed liquid into the above-prepared culture medium at an inoculation amount of 10% by volume, then place it at 37°C and culture according to the conventional fermentation production process for 36 h. After the culture is completed, centrifuge and reserve the supernatant.

[0094] Step B: Add 35 g / L of gelatinized starch, 6 g / L of L-theanine, and 1.3 g / L of peony seed oil to the supernatant obtained above. After mixing evenly, inoculate Bacillus amyloliquefaciens at an inoculation amount of 10% by volume and inoculate the seed liquid of Streptomyces catenulisporus at an inoculation amount of 10% by volume, and carry out conventional fermentation process cultivation at 30 °C to obtain a composite microbial inoculum with a total effective viable count of not less than 10×10 9 cfu / mL;

[0095] Step C: Take 55 kg of the composite microbial inoculum prepared by the above method, add 10 kg of glycine and 7 kg of taurine to it. After mixing evenly, concentrate and dry to obtain a mixture;

[0096] Step D: Mix 10 kg of sodium alginate and 1 kg of ergothioneine evenly, add an appropriate amount of water and stir until viscous, add the above mixture, mix evenly, dry, and pulverize to obtain a microbial inoculant.

[0097] Comparative Example 3

[0098] Raw material preparation:

[0099] After activating the purchased strain of Vishniacozyma victoriae (China General Microbiological Culture Collection Center, strain number CGMCC 2.5592) according to the instructions, inoculate it into the seed medium and culture until the total effective viable count is not less than 1×10 8 cfu / mL of Vishniacozyma victoriae seed liquid.

[0100] After activating the purchased strain of Streptomyces catenulisporus (China General Microbiological Culture Collection Center, strain number CGMCC 4.6283) according to the instructions, inoculate it into the seed medium and culture until the total effective spore count is not less than 1×10 8 spores / mL of Streptomyces catenulisporus seed liquid.

[0101] After activating the purchased strain of Streptomyces crystallinus (China General Microbiological Culture Collection Center, strain number CGMCC 4.1600) according to the instructions, inoculate it into the seed medium and culture until the total effective spore count is not less than 2×10 8 spores / mL of Streptomyces crystallinus.

[0102] Prepare the medium: Weigh each component according to the component ratio of 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. After adding water and mixing evenly, adjust the pH to 7.0 and sterilize to obtain the medium.

[0103] Prepare the microbial inoculant:

[0104] Step A: Inoculate the prepared culture medium with the seed liquid of Vishniacozyma victoriae at an inoculation amount of 8% by volume, and then place it at 37 °C and culture for 36 h according to the conventional fermentation production process. After the culture is completed, centrifuge, and reserve the supernatant.

[0105] Step B: Add 35 g / L of corn starch, 6 g / L of L-theanine, and 1.3 g / L of peony seed oil to the above-mentioned obtained supernatant. After mixing, inoculate the seed liquid of Streptomyces catenulis at an inoculation amount of 10% by volume and inoculate the seed liquid of Streptomyces crystallinus at an inoculation amount of 10% into it, and carry out conventional fermentation process culture at 30 °C to obtain a composite microbial bacterial liquid with a total effective viable count of not less than 10×10 9 cfu / mL.

[0106] Step C: Take 55 kg of the composite microbial bacterial liquid prepared by the above method, add 10 kg of glycine and 7 kg of taurine to it, mix well, concentrate and dry to obtain a mixture.

[0107] Step D: Mix 10 kg of sodium alginate and 1 kg of ergothioneine, add an appropriate amount of water and stir until viscous, add the above mixture, mix well, dry and pulverize to obtain a microbial inoculant.

[0108] Comparative Example 4

[0109] Raw material preparation:

[0110] After activating the purchased strain of Streptomyces catenulis (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, inoculate it into the seed medium and culture until the effective spore amount is not less than 1×10 8 cfu / mL of Streptomyces catenulis seed liquid.

[0111] After activating the purchased strain of Bacillus amyloliquefaciens (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, inoculate it into the seed medium and culture until the effective viable count is not less than 1×10 9 cfu / mL of Bacillus amyloliquefaciens seed liquid.

[0112] After activating the purchased strain of Streptomyces crystallinus (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, inoculate it into the seed medium and culture until the effective spore amount is not less than 2×10 8 cfu / mL of Streptomyces crystallinus.

[0113] Prepare gelatinized starch: Make a suspension of corn starch with water, heat and stir until viscous and transparent, cool and dry, and pulverize to obtain gelatinized starch.

[0114] Preparation of culture medium: Weigh each component according to the component ratio of 50 g / L of gelatinized starch, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate. After adding water and mixing evenly, adjust the pH to 7.0 and sterilize to obtain the culture medium.

[0115] Preparation of microbial inoculum:

[0116] Step A: Inoculate Bacillus amyloliquefaciens into the above-prepared culture medium at an inoculation amount of 10% by volume, inoculate the seed liquid of Streptomyces catenulisporus at an inoculation amount of 10% by volume, and inoculate the seed liquid of Streptomyces crystallinus at an inoculation amount of 10%. Carry out conventional fermentation process cultivation at 30 °C to obtain a composite microbial liquid with a total effective viable count of not less than 10×10 9 cfu / mL;

[0117] Step B: Take 55 kg of the composite microbial liquid prepared by the above method, add 1 kg of hyaluronic acid, mix evenly, concentrate and dry to obtain a mixture;

[0118] Step C: Add 10 kg of sodium alginate to an appropriate amount of water and stir until viscous. Add the above mixture, mix evenly, dry and pulverize to obtain the microbial inoculum.

[0119] Performance test

[0120] Pot experiment 1: Preventive effect of microbial inoculum on strawberry black spot

[0121] 1. Materials and methods

[0122] This experiment was carried out in the greenhouse of the cooperative agricultural base of our hospital. The strawberry black spot pathogen was taken from diseased strawberry fruits, and the strain was obtained after isolation, purification and identification, and stored for later use.

[0123] A total of 10 treatments were set in this experiment, including 1 blank treatment (water treatment), 1 chemical agent control treatment (600-fold solution of 10% polyoxin wettable powder), and 8 microbial inoculum treatments (the microbial inoculum was diluted with 10 g of water and 5 kg of water before use).

[0124] Strawberry plants (Miaoxiang) with basically the same growth and no pests and diseases were transplanted into pots, 1 plant per pot. 10 days after transplantation, select strawberry plants with good growth, randomly divide them into 9 groups, 20 pots per group, namely the blank group, the control group, and experimental groups 1-7. Set up barriers between groups. Each group was sprayed with the corresponding agent according to the corresponding treatment method. 2 days after spraying the agent, each group was sprayed with a suspension of strawberry black spot pathogen. After 10 days, observe the disease occurrence of strawberries, calculate the incidence rate, and randomly pick 10 diseased leaves from each group to calculate the lesion area.

[0125]

[0126] 2. Results and Analysis

[0127] As can be seen from the results in Table 1, the microbial inoculant prepared by the present invention has a good preventive effect on strawberry black spot disease. After spraying the microbial inoculant of the present invention, a protective layer can be formed on the strawberry plants, effectively avoiding the infection of the plants by the pathogenic bacteria, and it can be maintained for a long time with remarkable effects.

[0128] Table 1 Preventive effects of different microbial inoculants on strawberry black spot disease

[0129] Group Treatment method Incidence rate (%) <![CDATA[Lesion area (mm 2 )]]> Blank group Clear water 75 736 Control group 10% polyoxin 10 188 Experimental group 1 Microbial inoculant of Example 1 15 192 Experimental group 2 Microbial inoculant of Example 2 10 176 Experimental group 3 Microbial inoculant of Example 3 20 201 Experimental group 4 Microbial inoculant of Comparative Example 1 65 369 Experimental group 5 Microbial inoculant of Comparative Example 2 50 313 Experimental group 6 Microbial inoculant of Comparative Example 3 55 328 Experimental group 7 Microbial inoculant of Comparative Example 4 35 297

[0130] Pot experiment 2: Inhibitory effect of microbial inoculant on strawberry black spot disease

[0131] 1. Materials and Methods

[0132] This experiment was carried out in the greenhouse of the cooperative agricultural base of our hospital. The pathogenic bacteria of strawberry black spot disease were taken from diseased strawberry fruits, and the strains were obtained after isolation, purification and identification, and stored for later use.

[0133] A total of 10 treatments were set in this experiment, including 1 blank treatment (treated with water), 1 chemical agent control treatment (800-fold solution of 75% chlorothalonil wettable powder), and 8 microbial inoculant treatments (the microbial inoculant was diluted with 10 g of water and 5 kg of water before use).

[0134] The "Miaoxiang" strawberry plants with basically the same growth and no pests and diseases were transplanted into pots, 1 plant per pot. 10 days after transplantation, the strawberry plants with good growth were selected and randomly divided into 9 groups, with 20 pots in each group, namely the blank group, the control group, and experimental groups 1-7. Barriers were set between groups. Each group was sprayed with a suspension of pathogenic bacteria of black spot disease. After the strawberries showed symptoms of the disease, each group was sprayed with the corresponding agent according to the corresponding treatment method. After 10 days, the incidence of strawberry black spot disease was observed and calculated, and the disease was graded according to the following criteria, and the disease index and control effect were calculated:

[0135] Grade 0: No disease spots;

[0136] Grade 1: 0 < proportion of disease spot area ≤ 10%;

[0137] Grade 2: 10% < proportion of disease spot area ≤ 30%;

[0138] Grade 3: 30% < proportion of disease spot area ≤ 50%;

[0139] Grade 4: 50% < proportion of disease spot area ≤ 70%;

[0140] Grade 5: 70% < proportion of disease spot area ≤ 100%.

[0141]

[0142] 2. Results and analysis

[0143] As can be seen from the results in Table 2 below, the microbial inoculant of the present invention has a good control effect on strawberry plants already infected by pathogenic bacteria, with its incidence rate being able to drop to below 40%, and the disease index being relatively low. The control effect is above 80%, and the control effect is significant, significantly better than the microbial inoculants obtained in Comparative Example 1 - Comparative Example 4.

[0144] Table 2 Inhibitory effects of different microbial inoculants on strawberry black spot

[0145] Group Treatment method Incidence rate (%) Disease index Control effect (%) Blank group Clear water 100 3.15 — Control group 75% chlorothalonil 40 0.7 77.78 Experimental group 1 Microbial inoculant of Example 1 30 0.5 84.13 Experimental group 2 Microbial inoculant of Example 2 35 0.6 80.95 Experimental group 3 Microbial inoculant of Example 3 40 0.55 82.54 Experimental group 4 Microbial inoculant of Comparative Example 1 75 1.65 47.62 Experimental group 5 Microbial inoculant of Comparative Example 2 65 1.25 60.32 Experimental group 6 Microbial inoculant of Comparative Example 3 60 1.3 58.73 Experimental group 7 Microbial inoculant of Comparative Example 4 55 1.2 61.90

Claims

1. A microbial agent, characterized in that: Prepared from the following ingredients: 50-60 parts by weight of composite microbial liquid, 15-18 parts by weight of stabilizer, and 11-13 parts by weight of protective agent.

2. The microbial agent according to claim 1, characterized in that The composite microbial liquid contains Streptomyces crystalline, Bacillus amyloliquefaciens and Streptomyces spp.; the total effective viable count of the composite microbial liquid is not less than 10×10 9 cfu / mL.

3. The microbial agent according to claim 1, characterized in that The stabilizer contains glycine and taurine in a weight ratio of 1:(0.5-0.8).

4. The microbial agent according to claim 1, characterized in that The protective agent contains sodium alginate and ergothioneine in a weight ratio of 1:(0.1-0.3).

5. The method for preparing the microbial agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: Step A, preparing a fermentation matrix: inoculating a culture medium containing 50 g / L of glucose, 30 g / L of egg yolk powder, 5 g / L of yeast powder, 2 g / L of dipotassium hydrogen phosphate, and 1 g / L of magnesium sulfate heptahydrate with a seed solution of Streptococcus equi subsp. zooepidemicus and a seed solution of Vichysoma victoriae, culturing at 37° C. for 36 hours, and after the culturing is completed, centrifuging and using the supernatant for later use; Step B: add gelatinized starch, L-theanine and peony seed oil to the supernatant obtained above, mix well, inoculate Bacillus amyloliquefaciens seed liquid, Streptomyces spp. seed liquid and Streptomyces crystalline seed liquid into the supernatant, and culture at 30°C until the total effective viable count is not less than 10×10 9 cfu / mL of composite microbial culture; Step C, adding a stabilizer to the composite microbial liquid, mixing, concentrating, and drying to obtain a mixture; Step D, adding water to the protective agent and mixing evenly, then adding the above mixture, mixing evenly, and drying to obtain the microbial agent.

6. The preparation method according to claim 5, characterized in that: In the step A, the inoculation amount of the Streptococcus equi subspecies zooepidemicus seed solution is 10% of the volume of the culture medium, and the inoculation amount of the Victoria yeast seed solution is 8% of the volume of the culture medium; in the step B, the inoculation amount of the Bacillus amyloliquefaciens seed solution is 10%, the inoculation amount of the Streptomyces microfasciculus seed solution is 10%, and the inoculation amount of the Streptomyces crystallis seed solution is 10%.

7. The preparation method according to claim 5, characterized in that: In the step B, the amount of gelatinized starch added is 30-40 g / L, the amount of L-theanine added is 5-8 g / L, and the amount of peony seed oil added is 1-1.5 g / L.

8. The preparation method according to claim 5, characterized in that: The preparation method of the gelatinized starch comprises the following steps: adding water to corn starch to prepare a suspension, heating and stirring the suspension until the suspension becomes viscous and transparent, cooling the suspension, drying the suspension, and crushing the suspension to obtain the gelatinized starch.

9. Use of the microbial agent obtained by the preparation method according to any one of claims 5 to 8 in preventing and controlling strawberry black spot disease.

10. The use according to claim 9, characterized in that The method for using the microbial agent is to dilute the microbial agent 100-200 times and then spray it on the leaf surface before the onset of strawberry black spot disease or in the early stage of the disease.

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