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

Abstract

This invention relates to a microbial inoculant for controlling strawberry black spot disease and its preparation method, belonging to the field of microbial inoculant technology. The microbial inoculant of this invention is prepared from the following components: 50-60 parts by weight of a compound microbial inoculant solution, 15-18 parts by weight of a stabilizer, and 11-13 parts by weight of a protectant. The stabilizer contains glycine and taurine, and the protectant contains sodium alginate and ergothioneine. The microbial inoculant provided by this invention can effectively control strawberry black spot disease and improve strawberry yield and quality.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, and relates to a microbial inoculant for the prevention and control of strawberry black spot disease and its preparation method. Background Technology

[0002] Strawberries belong to the genus *Fragaria* of the Rosaceae family and are perennial herbaceous plants. They are rich in nutrients such as vitamin C, vitamin A, and vitamin E, which can alleviate night blindness, maintain the health of epithelial tissues, improve eyesight and liver function, and promote growth and development. Their abundant dietary fiber promotes gastrointestinal motility and digestion, improves constipation, and helps prevent acne and colon cancer. Known as the "Queen of Fruits," strawberries are loved by consumers for their vibrant color and unique taste.

[0003] Strawberry cultivation is susceptible to various diseases and pests. Strawberry black spot is one of the most common diseases, widely distributed and occurring in all strawberry-producing areas of my country. The pathogen of strawberry black spot is *Alternaria*, a fungus belonging to the Deuteromycetes. Under a microscope, the mycelium is colorless and septate; the conidia are dark brown, obpipiceal, ovate, or obclature, brownish-red, with 3-8 transverse septa and 1-4 longitudinal septa, slightly constricted at the septa, and have a short beak. The main mode of transmission is through seedlings, etc. The mycelium overwinters on diseased plants or plant debris and spreads via seedlings. Pathogen spores can also directly infect plants. *Alternaria* in the air can also cause infection. High temperature and humidity promote disease development; frequent rain or high field humidity exacerbates the disease; continuous cropping also leads to more severe outbreaks.

[0004] Strawberry black spot disease primarily affects leaves, petioles, runners, and berries. Infected leaves develop irregular, dark brown spots, 5-8 mm in diameter, with slightly concentric rings. The center of the spot is grayish-brown, often surrounded by a yellow halo. Small, sunken brown spots appear on petioles and runners. When the spot encircles the petiole or stem, the petiole or stem breaks, and the affected area constricts. Infected berries develop black spots covered with a dark, smoky mold; the spots are relatively light in color.

[0005] The commonly used prevention and control measures are mainly: (1) Select strawberry varieties resistant to black spot disease; promptly remove and burn diseased leaves. (2) Spray 10% polyoxin wettable powder at 600 times dilution, or 25% azoxystrobin suspension at 1500 times dilution, or 25% pyraclostrobin emulsifiable concentrate at 1200 times dilution, or 10% difenoconazole water-dispersible granules at 1000-1500 times dilution, or 75% chlorothalonil wettable powder at 800 times dilution, once every 10 days, for 2-3 treatments. However, strawberry diseases rely excessively on chemical pesticides for control, especially with frequent and short-interval applications, and unreasonable use of pesticides leads to increased resistance of pathogens. The strawberry fruit harvest period is long, and chemical pesticides are frequently used to control pests and diseases during the harvest period. Moreover, the strawberries are not harvested and marketed in accordance with the pesticide safety interval requirements, so the problem of pesticide residues in fresh strawberries is relatively prominent, and the risk of food safety management is high. Summary of the Invention

[0006] The main objective of this invention is to provide a microbial agent that has a good control effect on strawberry black spot disease. It can achieve good control without the use of any chemical agents, with less pollution, and can ensure the food safety of strawberries.

[0007] The present invention employs the following technical solutions to achieve the above objectives:

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

[0009] The mixture consists of 50-60 parts by weight of compound microbial inoculum, 15-18 parts by weight of stabilizer, and 11-13 parts by weight of protectant.

[0010] Furthermore, the composite microbial solution contains *Streptomyces crystallineis*, *Bacillus amyloliquefaciens*, and *Streptomyces simonii*.

[0011] Furthermore, the total number of viable bacteria in the composite microbial solution is not less than 10 × 10⁻⁶. 9 cfu / mL.

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

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

[0014] This invention provides a method for preparing the above-mentioned microbial inoculant, which mainly includes the following steps:

[0015] Step A, preparation of fermentation substrate: Inoculate Streptococcus equi seed liquid and Saccharomyces victoriae seed liquid into a culture medium containing 50 g / L glucose, 30 g / L egg yolk powder, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate and 1 g / L magnesium sulfate heptahydrate. Incubate at 37°C for 36 h. After the culture is completed, centrifuge and keep the supernatant for later use.

[0016] Step B: Add gelatinized starch, L-theanine, and peony seed oil to the supernatant obtained above, mix well, and then inoculate it with Bacillus amyloliquefaciens seed liquid, Streptomyces simulans seed liquid, and Streptomyces crystallineis seed liquid. Cultivate at 30°C to obtain a compound microbial culture.

[0017] Step C: Add a stabilizer to the compound microbial inoculum, mix well, concentrate and dry to obtain a mixture;

[0018] Step D: Add the preservative to water and mix well, then add to the above mixture, mix well, and dry to obtain the microbial inoculant.

[0019] Furthermore, in step A, the inoculation amount of Streptococcus equi subsp. veterinaria seed liquid is 10% of the culture medium volume, and the inoculation amount of Vichynik's yeast seed liquid is 8% of the culture medium volume.

[0020] Furthermore, in step A, the effective viable count of *Streptococcus equi* subsp. *epidemicus* seed solution is not less than 1 × 10⁻⁶. 8 The effective viable count of *Saccharomyces vincristinae* seed culture should be no less than 1 × 10⁻⁶ cfu / mL. 8 cfu / mL.

[0021] Furthermore, in 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.

[0022] Furthermore, the method for preparing the gelatinized starch is as follows: corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

[0023] Furthermore, in step B, the number of viable bacteria in the Bacillus amyloliquefaciens seed solution is not less than 1 × 10⁻⁶. 9 CFU / mL, and the effective spore count in *Streptomyces simulans* seed culture is not less than 1×10⁻⁶. 8 The effective spore count in the *Streptomyces crystallineis* seed culture is not less than 2 × 10⁶ spores / mL. 8 per mL.

[0024] Furthermore, in step B, the inoculation amount of Bacillus amyloliquefaciens seed liquid is 10%, the inoculation amount of Streptomyces simulans seed liquid is 10%, and the inoculation amount of Streptomyces crystallineis seed liquid is 10%.

[0025] Furthermore, in step B, the total number of effective viable bacteria in the composite microbial solution is not less than 10 × 10⁻⁶. 9 cfu / mL.

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

[0027] Furthermore, the method of using the microbial agent is to dilute the microbial agent 100-200 times and spray it on the leaves before or at the early stage of strawberry black spot disease.

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

[0029] 1. The microbial agent provided by this invention has a good control effect on strawberry black spot disease, which can effectively reduce the incidence rate and the disease index, with a control effect of over 80%, and the effect is significant.

[0030] 2. In the microbial agent of this invention, *Streptomyces simonii* and *Streptomyces crystallinus* are the main active bacteria, and *Bacillus amyloliquefaciens* is the auxiliary bacteria. When used before the onset of strawberry black spot disease, it can form a protective layer on the plant surface, protecting it from pathogen infection. When used in the early stages of disease, the bacteria in this invention can eliminate already invading pathogens, inhibit their growth, and prevent their continued proliferation. In the preparation of the microbial agent, the fermentation broth of *Streptococcus equi* subsp. *porphyria* and *Vichyva victoriae* is used as the fermentation base. This fermentation broth contains hyaluronic acid and microbial metabolites. Hyaluronic acid has certain film-forming properties; when sprayed onto the plant leaves, it forms a protective film, isolating airborne pathogens. For already infected pathogens, it isolates them in an environment containing the microbial agent, utilizing microbial antagonism to eliminate the infected pathogens. The metabolites in the fermentation broth have a certain bactericidal effect, which can further help eliminate already infected pathogens. To avoid microbial death caused by exposure to air, which would affect the control effect, glycine and taurine are added as stabilizers, and sodium alginate and ergothioneine are added as protectants during the preparation of the microbial agent. This can effectively ensure the number of viable bacteria and achieve better control effect. Detailed Implementation

[0031] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. After reading this invention, any modifications of the invention in various equivalent forms by those skilled in the art fall within the scope of protection of the claims of this application. All bacterial strains used in this invention are commercially available strains; all raw materials used are commercially available products.

[0032] Example 1

[0033] Raw material preparation:

[0034] After activating the purchased Streptococcus equi subsp. porphyria (Guangdong Provincial Microbial Culture Collection Center, strain number GDMCC NO. 1.437) strain according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Streptococcus equi subsp. veterinary seed solution.

[0035] After activating the purchased *Veinia vincristinae* strain (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Victoria vischnik yeast seed culture.

[0036] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0037] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0038] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

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

[0040] Preparation of gelatinized starch: Corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

[0041] Preparation of microbial inoculants:

[0042] Step A: Inoculate the above-prepared culture medium with Streptococcus equi seed liquid at a volume of 10% and Saccharomyces victoriae seed liquid at a volume of 8%. Then, incubate at 37°C for 36 hours according to the conventional fermentation process. After the culture is completed, centrifuge and keep the supernatant for later use.

[0043] Step B: Add 40 g / L gelatinized starch, 8 g / L L-theanine, and 1 g / L peony seed oil to the obtained supernatant. After mixing, inoculate with *Bacillus amyloliquefaciens*, *Streptomyces simonii* seed culture (10% by volume), and *Streptomyces crystallineis* seed culture (10% by volume). Culture using conventional fermentation processes at 30°C to obtain a total viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0044] Step C: Take 60 kg of the compound microbial culture prepared by the above method, add 10 kg of glycine and 5 kg of taurine to it, 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 pulverize to obtain the microbial inoculant.

[0046] Example 2

[0047] Raw material preparation:

[0048] After activating the purchased Streptococcus equi subsp. porphyria (Guangdong Provincial Microbial Culture Collection Center, strain number GDMCC NO. 1.437) strain according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Streptococcus equi subsp. veterinary seed solution.

[0049] After activating the purchased *Veinia vincristinae* strain (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Victoria vischnik yeast seed culture.

[0050] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0051] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0052] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

[0053] Preparation of culture medium: Weigh out each component according to the following ratio: glucose 50g / L, egg yolk powder 30g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 1g / L. Add water and mix well. Adjust the pH to 7.0 and sterilize to obtain the culture medium.

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

[0055] Preparation of microbial inoculants:

[0056] Step A: Inoculate the above-prepared culture medium with Streptococcus equi seed liquid at a volume of 10% and Saccharomyces victoriae seed liquid at a volume of 8%. Then, incubate at 37°C for 36 hours according to the conventional fermentation process. After the culture is completed, centrifuge and keep the supernatant for later use.

[0057] Step B: Add 30 g / L gelatinized starch, 5 g / L L-theanine, and 1.5 g / L peony seed oil to the obtained supernatant, mix well, and then inoculate with *Bacillus amyloliquefaciens*, *Streptomyces simonii* seed culture, and *Streptomyces crystallineis* seed culture at 10% of their respective volumes. Culture using conventional fermentation processes at 30°C to obtain a total viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0058] Step C: Take 50 kg of the compound microbial culture 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 viscous, add the above mixture, mix well, dry, and pulverize to obtain the microbial agent.

[0060] Example 3

[0061] Raw material preparation:

[0062] After activating the purchased Streptococcus equi subsp. porphyria (Guangdong Provincial Microbial Culture Collection Center, strain number GDMCC NO. 1.437) strain according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Streptococcus equi subsp. veterinary seed solution.

[0063] After activating the purchased *Veinia vincristinae* strain (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Victoria vischnik yeast seed culture.

[0064] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0065] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0066] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

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

[0068] Preparation of gelatinized starch: Corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

[0069] Preparation of microbial inoculants:

[0070] Step A: Inoculate the above-prepared culture medium with Streptococcus equi seed liquid at a volume of 10% and Saccharomyces victoriae seed liquid at a volume of 8%. Then, incubate at 37°C for 36 hours according to the conventional fermentation process. After the culture is completed, centrifuge and keep the supernatant for later use.

[0071] Step B: Add 35 g / L gelatinized starch, 6 g / L L-theanine, and 1.3 g / L peony seed oil to the obtained supernatant, mix well, and then inoculate with *Bacillus amyloliquefaciens*, *Streptomyces simonii* seed culture (10% by volume), and *Streptomyces crystallineis* seed culture (10% by volume). Culture using conventional fermentation processes at 30°C to obtain a total viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0072] Step C: Take 55 kg of the compound microbial culture 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;

[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 the microbial inoculant.

[0074] Comparative Example 1

[0075] Raw material preparation:

[0076] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0077] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0078] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

[0079] Preparation of gelatinized starch: Corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

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

[0081] Preparation of microbial inoculants:

[0082] Step A: Inoculate the culture medium prepared by the above method with Bacillus amyloliquefaciens at a volume of 10%, with Streptomyces simonii seed culture at a volume of 10%, and with Streptomyces crystallineis seed culture at a volume of 10%. Culture using conventional fermentation processes at 30°C to obtain a total viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0083] Step B: Take 55 kg of the compound microbial culture 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;

[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 pulverize to obtain the microbial agent.

[0085] Comparative Example 2

[0086] Raw material preparation:

[0087] After activating the purchased Streptococcus equi subsp. porphyria (Guangdong Provincial Microbial Culture Collection Center, strain number GDMCC NO. 1.437) strain according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Streptococcus equi subsp. veterinary seed solution.

[0088] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0089] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0090] Preparation of culture medium: Weigh out each component according to the following ratio: glucose 50g / L, egg yolk powder 30g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 1g / L. Add water and mix well. Adjust the pH to 7.0 and sterilize to obtain the culture medium.

[0091] Preparation of gelatinized starch: Corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

[0092] Preparation of microbial inoculants:

[0093] Step A: Inoculate the above-prepared culture medium with Streptococcus equi subsp. veterinary seed liquid at an inoculation rate of 10% by volume, then incubate at 37°C for 36 hours using conventional fermentation production process. After incubation, centrifuge and reserve the supernatant.

[0094] Step B: Add 35 g / L gelatinized starch, 6 g / L L-theanine, and 1.3 g / L peony seed oil to the obtained supernatant, mix well, and then inoculate with Bacillus amyloliquefaciens and Streptomyces simonii seed liquid at 10% by volume. Culture using conventional fermentation processes at 30°C to obtain a total effective viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0095] Step C: Take 55 kg of the compound microbial culture 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;

[0096] 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 the microbial inoculant.

[0097] Comparative Example 3

[0098] Raw material preparation:

[0099] After activating the purchased *Veinia vincristinae* strain (China General Microbiological Culture Collection Center, strain number CGMCC2.5592) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 8 cfu / mL Victoria vischnik yeast seed culture.

[0100] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0101] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

[0102] Preparation of culture medium: Weigh out each component according to the following ratio: glucose 50g / L, egg yolk powder 30g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 1g / L. Add water and mix well. Adjust the pH to 7.0 and sterilize to obtain the culture medium.

[0103] Preparation of microbial inoculants:

[0104] Step A: Inoculate the above-prepared culture medium with 8% of the volume of *V. victorienicum* seed culture, and then culture it at 37°C for 36 hours according to the conventional fermentation process. After the culture is completed, centrifuge and keep the supernatant for later use.

[0105] Step B: Add 35 g / L corn starch, 6 g / L L-theanine, and 1.3 g / L peony seed oil to the obtained supernatant. After mixing, inoculate with *Streptomyces simonii* seed culture at 10% inoculation rate and *Streptomyces crystallineis* seed culture at 10% inoculation rate. Culture using conventional fermentation process at 30°C to obtain a total effective viable count of not less than 10 × 10⁻⁶ cells / day. 9 CFU / mL composite microbial culture;

[0106] Step C: Take 55 kg of the compound microbial culture 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 the microbial inoculant.

[0108] Comparative Example 4

[0109] Raw material preparation:

[0110] After activating the purchased *Streptomyces simonii* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.6283) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 1×10⁻⁶. 8 Streptomyces simulans seed culture per mL.

[0111] After activating the purchased Bacillus amyloliquefaciens strain (China General Microbiological Culture Collection Center, strain number CGMCC1.10901) according to the instructions, it was inoculated into seed culture medium and cultured until the effective viable count was not less than 1×10⁻⁶. 9 CFU / mL Bacillus amyloliquefaciens seed solution.

[0112] After activating the purchased *Streptomyces crystallineis* strain (China General Microbiological Culture Collection Center, strain number CGMCC4.1600) according to the instructions, it was inoculated into seed culture medium and cultured until the effective spore count was not less than 2 × 10⁻⁶. 8 crystalline Streptomyces per mL.

[0113] Preparation of gelatinized starch: Corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

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

[0115] Preparation of microbial inoculants:

[0116] Step A: Inoculate the prepared culture medium with *Bacillus amyloliquefaciens*, *Streptomyces simonii* seed culture (10% by volume), and *Streptomyces crystallineis* seed culture (10% by volume). Culture using conventional fermentation processes at 30°C to obtain a total viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture;

[0117] Step B: Take 55 kg of the composite microbial culture prepared by the above method, add 1 kg of hyaluronic acid to it, mix well, 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 it becomes viscous. Add the above mixture, mix well, dry, and pulverize to obtain the microbial inoculant.

[0119] Performance testing

[0120] Potted Experiment 1: The Preventive Effect of Microbial Inoculants on Strawberry Black Spot Disease

[0121] 1. Materials and Methods

[0122] This experiment was conducted in the greenhouse of our cooperative agricultural base. The pathogen of strawberry black spot disease was obtained from strawberry fruits infected with black spot disease. After isolation, purification, and identification, the strain was obtained and stored for later use.

[0123] Ten treatments were set up in this experiment: one blank treatment (water treatment), one chemical reagent control treatment (600 times dilution of 10% polyoxin wettable powder), and eight microbial agent treatments (microbial agents were diluted at a ratio of 10g to 5kg of water before use).

[0124] Strawberry plants of similar growth and free from pests and diseases (Miaoxiang) were transplanted into pots, one plant per pot. Ten days after transplanting, healthy strawberry plants were selected and randomly divided into 9 groups of 20 pots each: a blank group, a control group, and experimental groups 1-7. Barriers were set up between the groups. Each group was sprayed with the corresponding pesticide according to the treatment method. Two days after the pesticide was sprayed, each group was sprayed with a suspension of black spot pathogen. Ten days later, the disease incidence of strawberries was observed and the incidence rate was calculated. Ten diseased leaves were randomly picked from each group and the area of ​​the lesions was calculated.

[0125]

[0126] 2. Results and Analysis

[0127] As shown in Table 1, the microbial agent prepared by this invention has a good preventive effect against strawberry black spot disease. After spraying with the microbial agent of this invention, a protective layer can be formed on the strawberry plant, effectively preventing the infection of the plant by pathogens, and the effect can be maintained for a long time, with significant effect.

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

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

[0130] Pot Experiment 2: Inhibitory Effect of Microbial Inoculants on Strawberry Black Spot Disease

[0131] 1. Materials and Methods

[0132] This experiment was conducted in the greenhouse of our cooperative agricultural base. The pathogen of strawberry black spot disease was obtained from strawberry fruits infected with black spot disease. After isolation, purification, and identification, the strain was obtained and stored for later use.

[0133] Ten treatments were set up in this experiment: one blank treatment (water treatment), one chemical reagent control treatment (800 times dilution of 75% chlorothalonil wettable powder), and eight microbial agent treatments (microbial agents were diluted at a ratio of 10g to 5kg of water before use).

[0134] 'Miaoxiang' strawberry plants with similar growth and no pests or diseases were transplanted into pots, one plant per pot. Ten days after transplanting, healthy strawberry plants were selected and randomly divided into 9 groups of 20 pots each: a control group, a control group, and experimental groups 1-7. Barriers were set up between the groups. Each group was sprayed with a suspension of black spot pathogens. After the strawberries developed symptoms, each group was sprayed with the appropriate pesticide according to the corresponding treatment method. Ten days later, the incidence of black spot disease was observed, and the disease was graded according to the following standards. The disease index and control effect were calculated:

[0135] Grade 0, no lesions;

[0136] Grade 1, 0 < lesion area percentage ≤ 10%;

[0137] Grade 2, 10% < lesion area ≤ 30%;

[0138] Grade 3, 30% < lesion area ≤ 50%;

[0139] Grade 4, 50% < lesion area ≤ 70%;

[0140] Grade 5, 70% < lesion area ≤ 100%.

[0141]

[0142] 2. Results and Analysis

[0143] As shown in Table 2 below, the microbial agent of the present invention has a good control effect on strawberry plants that have been infected by pathogens. The incidence rate can be reduced to below 40%, and the disease index is low. The control effect is above 80%, and the control effect is significant, which is significantly better than the microbial agents obtained by comparative examples 1-4.

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

[0145] Group Handling method Incidence rate (%) Disease index Preventive efficacy (%) Blank group Clear water 100 3.15 — control group 75% Chlorothalonil 40 0.7 77.78 Experiment 1 Group Example 1 Microbial inoculants 30 0.5 84.13 Experiment 2 group Example 2 Microbial inoculants 35 0.6 80.95 Experimental Group 3 Example 3 Microbial inoculants 40 0.55 82.54 Experiment 4 group Comparative Example 1: Microbial Inoculant 75 1.65 47.62 Experiment 5 group Comparative Example 2: Microbial Inoculants 65 1.25 60.32 Experiment 6 group Comparative Example 3: Microbial Inoculants 60 1.3 58.73 Experiment 7 group Comparative Example 4: Microbial Inoculants 55 1.2 61.90

Claims

1. A method for preparing a microbial inoculant, characterized by, Includes the following steps: Step A, preparation of fermentation substrate: Inoculate Streptococcus equi seed liquid and Saccharomyces victoriae seed liquid into a culture medium containing 50 g / L glucose, 30 g / L egg yolk powder, 5 g / L yeast powder, 2 g / L dipotassium hydrogen phosphate and 1 g / L magnesium sulfate heptahydrate. Incubate at 37°C for 36 h. After the culture is completed, centrifuge and keep the supernatant for later use. Step B: Add 30-40 g / L of gelatinized starch, 5-8 g / L of L-theanine, and 1-1.5 g / L of peony seed oil to the obtained supernatant, mix well, and then inoculate with Bacillus amyloliquefaciens seed solution, Streptomyces simulans seed solution, and Streptomyces crystallineis seed solution. Incubate at 30°C to obtain a total effective viable count of not less than 10 × 10⁻⁶. 9 CFU / mL composite microbial culture; Step C: Add 15-18 parts by weight of stabilizer to 50-60 parts by weight of compound microbial inoculum, mix well, concentrate and dry to obtain a mixture; the stabilizer contains glycine and taurine in a weight ratio of 1:(0.5-0.8); Step D: Add 11-13 parts by weight of the protectant to water and mix well, then add to the above mixture, mix well, and dry to obtain the microbial agent; the protectant contains sodium alginate and ergothioneine in a weight ratio of 1:(0.1-0.3).

2. The production method according to claim 1, wherein In step A, the inoculation amount of *Streptococcus equi* subsp. *pneumoniae* seed solution is 10% of the culture medium volume, and the inoculation amount of *Vichynica victoriae* seed solution is 8% of the culture medium volume; in step B, the inoculation amount of *Bacillus amyloliquefaciens* seed solution is 10%, the inoculation amount of *Streptomyces simulans* seed solution is 10%, and the inoculation amount of *Streptomyces crystallineis* seed solution is 10%.

3. The preparation method according to claim 1, characterized in that, The method for preparing the gelatinized starch is as follows: corn starch is mixed with water to form a suspension, heated and stirred until it becomes viscous and transparent, cooled, dried, and pulverized to obtain gelatinized starch.

4. The application of a microbial agent prepared by any one of claims 1-3 in the prevention and control of strawberry black spot disease.

5. The application as described in claim 4, characterized in that, The method of using the microbial agent is to dilute it 100-200 times and spray it on the leaves before or at the early stage of strawberry black spot disease.

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