Preparation method of microbial solid fumigation preparation and application thereof in botrytis cinerea prevention and treatment

Abstract

The application discloses a preparation method of a microbial solid fumigation preparation and application of the microbial solid fumigation preparation in grey mould prevention and treatment. Fresh wheat kernels are washed, boiled until the kernels are opened, dried, and then weighed and put into a conical flask, and the bottom of the flask is shaken and flattened, and the wheat kernels are subjected to high-temperature sterilization treatment twice to obtain a wheat kernel culture medium; a streptomyces xiyangyangensis WY228 fermentation liquor; the streptomyces xiyangyangensis WY228 fermentation liquor is added into the wheat kernel culture medium, mixed thoroughly, and then cultured, and the flask is shaken twice a day during the culture, and after the culture is completed, the microbial solid fumigation preparation is obtained by drying and storing. The microbial solid fumigation preparation can significantly inhibit the growth of botrytis cinerea by producing bacteriostatic gas, does not introduce live bacteria and excess moisture on the surface of the treated agricultural products, has safe and efficient prevention and treatment capacity on the grey mould, and has a good market application prospect.

Description

A method for preparing a microbial solid fumigation agent and its application in the control of gray mold. Technical Field

[0001] This invention relates to the field of gray mold control technology, specifically to a method for preparing a microbial solid fumigation agent and its application in gray mold control. Background Technology

[0002] Botrytis cinerea, belonging to the family Sclerotiniaceae and the genus Botrytis, is a typical necrotrophic pathogen with a wide host range. It can cause surface rot and the formation of a gray mold layer on 1400 plant species, including tomatoes, strawberries, grapes, and cherries. Furthermore, Botrytis cinerea can infect multiple parts of plants, including fruits, flowers, leaves, and stems, causing severe disease both before and after harvest. The non-specificity of its host and the diversity of infected organ types lead to enormous agricultural and postharvest losses (Jiao et al., Organic acid, a virulence factor for pathogenic fungi, causing postharvest decay in fruits. Mol Plant Pathol. 2022). It is estimated that the global economic losses due to various crop gray mold diseases reach as high as US$100 billion annually, posing a significant threat to global agricultural development.

[0003] For many years, the application of chemical gases and chemical fungicides has been an effective way to control gray mold. However, the long-term use of chemical pesticides has led to environmental pollution, food safety issues, and adverse effects on human health. Furthermore, due to the rapid spread of Botrytis cinerea, its wide host range, and high genetic variation, severe resistance has developed over the past few decades due to the extensive use of chemical agents. Currently, resistant strains to various fungicides such as benzimidazoles, dicarboximides, and N-phenylcarbamates have emerged (Han Zhiqi et al., Research Progress on Fungicide Resistance of Botrytis cinerea, China Vegetables, 2014).

[0004] Microbial volatile organic compounds (mVOCs) are lipophilic compounds with low boiling points, low molecular weight (<300 Da), and high vapor pressure. Some VOCs produced by microorganisms have inhibitory effects on pathogens, effectively controlling various plant and crop diseases. Compared to traditional liquid pesticides, mVOCs have the advantage of being perceived at low concentrations and easily diffuse over long distances in soil, air, and other environments. Microorganisms can inhibit pathogen growth through their antimicrobial VOCs without direct contact with pathogens and plants, demonstrating significant application potential in plant disease control (Sharifi et al., Belowground plant-microbe communications via volatile compounds. J Exp Bot. 2022). Currently, reports on the application of mVOCs in the control of gray mold are relatively rare. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing a microbial solid fumigation agent and its application in the control of gray mold. This microbial solid fumigation agent does not introduce live bacteria into the treated agricultural products and is a safe and efficient biocontrol agent.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] A method for preparing a microbial solid fumigation agent includes the following steps:

[0008] Step 1: Take fresh wheat grains, rinse them clean, add water and boil until the grains open, filter and air dry, weigh the air-dried wheat grains into an Erlenmeyer flask, shake well and spread evenly at the bottom of the flask, sterilize at high temperature twice to obtain wheat grain culture medium; sterilize at 121℃ for 15 min, repeat sterilization twice to obtain the culture medium.

[0009] Step 2: Take Streptomyces xitangense WY228, inoculate it into ISP-2 medium, and culture it at 28℃ and 180 rpm for 2 days to obtain the fermentation broth of Streptomyces xitangense WY228.

[0010] Step 3: Inoculate the fermentation broth of Streptomyces xitangensis WY228 into wheat grain culture medium, mix thoroughly, and let it stand for culture. Shake the flask twice a day during the culture period. After the culture is completed, dry and store to obtain the microbial solid fumigation preparation.

[0011] As an improvement, in step 1, the amount of air-dried wheat grains weighed and added to the conical flask is 10-40g / 50g, the moisture content of the air-dried wheat grains is 33%-50%, and the high-temperature sterilization conditions are 121℃ for 15 min.

[0012] As an improvement, in step 3, the inoculation amount of the Xitang Streptomyces WY228 fermentation broth is 5-10%, the static culture temperature is 20-28℃, and the culture period is 2 days.

[0013] As an improvement, in step 3, drying is carried out in an oven at 35°C for 24 hours, and storage is carried out in a low-temperature vacuum environment at 4°C.

[0014] The microbial solid fumigation preparation obtained by the above preparation method.

[0015] The application of the microbial solid fumigation preparation obtained by the above preparation method in inhibiting Botrytis cinerea.

[0016] As an improvement, after rehydration treatment with the microbial solid fumigation agent, it is placed together with the agricultural products to be treated in a sealed container. The concentration of the microbial solid fumigation agent used is 26.67 g / L. The prevention or treatment of gray mold is achieved through the inhibitory effect of the generated VOCs on Botrytis cinerea.

[0017] A further improvement is that the rehydration treatment uses sterile water or nutrient solution.

[0018] A further improvement is that the nutrient solution consists of 6 g / L tryptone, 4 g / L glucose, and 1% CaCl2.

[0019] A biological agent for controlling gray mold in strawberries, wherein the biological agent contains a microbial solid fumigation preparation obtained by the above-described preparation method. Beneficial effects.

[0020] Compared with existing technologies, the present invention provides a method for preparing a microbial solid fumigation agent and its application in the control of gray mold, which has the following specific advantages:

[0021] 1. The microbial solid fumigation preparation provided by the present invention can generate antibacterial gas, which can easily diffuse over long distances in environments such as soil and air. The preparation does not come into direct contact with the treated agricultural products and does not introduce live bacteria on their surface.

[0022] 2. The microbial solid fumigation preparation provided by this invention does not require spraying water-based solvents on the surface of the treated agricultural products, and is especially suitable for crops whose surfaces are easily damaged or rotten and are not suitable for treatment with liquid agents, such as strawberries, blueberries, and lettuce;

[0023] 3. The microbial solid fumigation preparation provided by this invention is safe, efficient, low-cost, and easy to use, and has good market application prospects in the prevention and control of gray mold. Attached Figure Description

[0024] Figure 1 shows the optimized fermentation process of the Xitang Streptomyces WY228 fumigation preparation, where A represents different inoculum amounts, B represents bottling amounts, C represents the material-to-water ratio, D represents the culture temperature, E represents different dosages, and F represents different culture times.

[0025] Figure 2 is a flowchart of the optimized preparation process of the Xitang Streptomyces WY228 fumigation preparation;

[0026] Figure 3 shows the in vitro antibacterial activity of the dominant compounds in the Xitang Streptomyces WY228 fumigation preparation;

[0027] Figure 4 shows a photograph of an example of the control effect of Xitang Streptomyces WY228 fumigation preparation on gray mold in strawberries;

[0028] Figure 5 shows the effect of Xitang Streptomyces WY228 fumigation preparation on the control of gray mold in strawberries. The physicochemical indicators of strawberries were measured after the application. Among them, (A) is the disease index, (B) is the weight loss rate, (C) is ascorbic acid, (D) is titratable acid, (E) is firmness, (F) is soluble solids, (G) is total phenols, (H) is flavonoids, (I) is malondialdehyde, and (J) is superoxide dismutase (SOD) activity. Detailed Implementation

[0029] Potato glucose agar medium: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, pH at rest, sterilize at 115℃ for 30 min.

[0030] ISP-2: 4 g yeast extract, 4 g glucose, 10 g malt extract, 15 g agar, 1000 mL distilled water, pH 7.2, sterilized at 115℃ for 30 min.

[0031] ISP-3: Add 20 g of oat flakes to 1000 mL of distilled water to make a homogenate, filter through four layers of gauze, add distilled water to make up to 1000 mL, add 15 g of agar, pH 6.0, sterilize at 121℃ for 20 min.

[0032] ISP-5: 1.0 g L-asparagine, 10 g glycerol, 1.0 g dipotassium hydrogen phosphate, 15 g agar, 1000 mL distilled water, pH 7.2, sterilized at 121℃ for 20 min.

[0033] Tryptone-Soybean Agar Medium (TSA Medium): 15 g tryptone, 5 g soybean peptone, 5 g NaCl, 1000 mL distilled water, 15 g agar, pH 7.2, sterilized at 121℃ for 20 min.

[0034] Wheat grain culture medium: Wash wheat grains and boil them until they open, filter them through gauze at the bottom, dry them, weigh an appropriate amount of wheat grains and put them into an Erlenmeyer flask, sterilize at 121℃ for 30 min.

[0035] Streptomyces xitangense WY228 is an endophytic fungus of burdock, isolated from healthy burdock plants in the field in Hekou Town, Peixian County, Xuzhou City, Jiangsu Province, by our laboratory. It was deposited at the Guangdong Provincial Culture Collection Center for Microbial Cultures on November 8, 2017, at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 60274 and taxonomic name: Streptomycessp.

[0036] Fermentation broth of Streptomyces xitangensis WY228: Activated Streptomyces xitangensis WY228 was inoculated into ISP-2 medium and cultured at 28℃ and 180rpm for 2 days.

[0037] Botrytis cinerea was purchased from the Guangdong Provincial Center for Microbial Culture Collection, with the culture collection number GDMCC 3.47.

[0038] Rehydration method for wheat grain preparations: Add sterile water or nutrient solution to make the final water content of the wheat grain preparations 33%-50%, and incubate overnight in an incubator at 28℃.

[0039] Method for testing viable cell count: Take a certain amount of rehydrated wheat grain preparation and place it in a sterile centrifuge tube. Add an appropriate amount of sterile water and place it on a vortex shaker for shaking. Take 1 mL of the vortexed bacterial solution and dilute it on ISP-2 medium. Place it in a 28℃ incubator for static culture for 2 days and count the viable cells.

[0040] Method for detecting antibacterial activity: A certain amount of rehydrated wheat grain preparation was placed on one side of a separated Petri dish, and simultaneously, a 5 mm Botrytis cinerea cake was inoculated onto the other side of the PDA medium. The dish was then sealed and incubated at 28°C for 3 days. The colony diameter was measured, and the inhibition rate was calculated.

[0041] Example 1: Detection of the inhibitory effect of *Streptomyces xitangensis* WY228 on *Botrytis cinerea* on different culture media.

[0042] Add an appropriate amount of potato dextrose agar medium to the left compartment of the partitioned plate, and inoculate with *Botrytis cinerea* cakes with a diameter of 5 mm. Add appropriate amounts of ISP-2, ISP-3, ISP-5, tryptone soybean agar medium, or wheat grain medium to the right compartment of the partitioned plate, and evenly inoculate with 500 μL of *Streptomyces setonii* WY228 fermentation broth. After sealing with sealing film, incubate the plates at 28°C for 3 days, measure the colony diameter, and calculate the inhibition rate.

[0043] The results showed that VOCs produced by *Streptomyces xitangensis* WY228 on ISP-2, ISP-3, and ISP-5 media had almost no inhibitory effect on *Botrytis cinerea*, while VOCs produced on TSA and wheat grain media exhibited a strong inhibitory effect on *Botrytis cinerea*. Wheat grains are a nutrient-rich natural medium, containing abundant starch and small-molecule proteins, and their loose structure facilitates good ventilation. Therefore, wheat grains were chosen for the preparation of a specific microbial agent for gray mold.

[0044] Example 2: Preparation method of a microbial fumigation agent specifically for gray mold caused by Streptomyces xitangense WY228.

[0045] Rinse fresh wheat grains three times, boil them in water until all the grains open, filter and drain the water, then air dry at room temperature. Weigh 10 g of wheat grains and dispense them into 50 mL Erlenmeyer flasks, shake well and spread evenly at the bottom of the flask, sterilize at 121℃ for 30 min to obtain wheat grain culture medium.

[0046] 2.1 Optimization of the fermentation process for WY228 wheat grain formulation

[0047] (1) Effect of different inoculum amounts on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0048] 10 g of wheat grain culture medium was placed in a 50 mL Erlenmeyer flask. Inoculation amounts of *Streptomyces xitangensis* WY228 fermentation broth were added at 1% (100 μL), 3% (300 μL), 5% (500 μL), and 10% (1000 μL), respectively. After thorough mixing with the wheat grains, the flask was incubated statically at 28℃ for 2 days, shaking it twice daily during the incubation period. The flask was then transferred to one side of a partition plate, and simultaneously, *Botrytis cinerea* cakes (5 mm) were inoculated onto the other side of the partition plate on a PDA plate. The plate was sealed and incubated at 28℃ for 3 days. The colony diameter was measured to calculate the inhibition rate.

[0049] (2) Effect of different culture temperatures on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0050] 10 g of wheat grain culture medium was placed in a 50 mL Erlenmeyer flask. Fermentation broth of strain WY228 was added at a 5% inoculum rate and thoroughly mixed with the wheat grains. The flasks were then incubated at 20, 25, and 28°C for 2 days, shaking twice daily during this period. After incubation, the wheat grains were transferred to one side of a partitioned plate, and simultaneously, *Botrytis cinerea* (5 mm) was inoculated onto the other side of the plate. The plates were sealed and then incubated at 20, 25, and 28°C for another 3 days. The colony diameter was measured to calculate the inhibition rate.

[0051] (3) Effect of different bottling amounts on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0052] 10, 20, 30, and 40 g of wheat grain culture medium were placed in 50 mL Erlenmeyer flasks, respectively. Fermentation broth of strain WY228 was added at a 5% inoculum rate, and the mixture was thoroughly mixed with the wheat grains. The flasks were then incubated at 28°C for 2 days, shaking twice daily. For each batch size, 10 g of the wheat grain preparation was weighed and placed on one side of a partitioned plate. Simultaneously, a 5 mm *Botrytis cinerea* cake was inoculated onto the other side of the plate. The plates were sealed and incubated at 28°C for another 3 days. The colony diameter was measured to calculate the inhibition rate.

[0053] (4) Effects of different moisture contents on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0054] Wheat grains were weighed and directly bottled for sterilization without boiling, with a material-to-water ratio of 1:0 (0% moisture content). A certain amount of wheat grains was weighed, boiled until cracked, and then dried to 1.5 times its original weight, with a material-to-water ratio of 2:1 (33% moisture content). A certain amount of wheat grains was weighed, boiled until cracked, and then dried to twice its original weight, with a material-to-water ratio of 1:1 (50% moisture content). A certain amount of wheat grains was weighed, boiled until cracked, and then dried to three times its original weight, with a material-to-water ratio of 1:2 (67% moisture content). The *Streptomyces xitangense* WY228 fermentation broth was inoculated at a 5% inoculum rate. After thoroughly mixing the bacteria with the wheat grain culture medium, the culture was incubated at 28℃ for 2 days, shaking the flask twice daily. 10 g of wheat grain preparations with different moisture contents were weighed and placed on one side of a separate plate, while simultaneously inoculating *Botrytis cinerea* cakes (5 mm) onto the other side of the plate on PDA medium. The plates were sealed and incubated at 28℃ for another 3 days. The colony diameter was measured to calculate the inhibition rate.

[0055] (5) Effects of different dosages on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0056] The fermentation broth of *Streptomyces xitangensis* WY228 was inoculated at a 5% inoculum rate into a 50 mL Erlenmeyer flask containing 10 g of wheat grain culture medium. After thorough mixing, the flask was incubated at 28℃ for 2 days, shaking it twice daily. After incubation, 2, 4, and 8 g of the wheat grain preparation were weighed onto one side of a separate Petri dish, and simultaneously, *Botrytis cinerea* cakes (5 mm) were inoculated onto the other side of a PDA plate. The dishes were then sealed and incubated at 28℃ for another 3 days. The colony diameter was measured to calculate the inhibition rate.

[0057] (6) Effect of different culture days on the inhibition of Botrytis cinerea by WY228 wheat grain formulation:

[0058] The fermentation broth of *Streptomyces xitangensis* WY228 was inoculated at a 5% inoculum rate into a 50 mL Erlenmeyer flask containing 10 g of wheat grain culture medium. After thorough mixing, the flasks were incubated at 28℃ for 2, 3, and 4 days, with the flasks shaken twice daily. 4 g of the wheat grain preparation from each incubation period was weighed onto one side of a separate Petri dish, and simultaneously, *Botrytis cinerea* cakes (5 mm) were inoculated onto the other side of a PDA plate. The dishes were then sealed and incubated at 28℃ for another 3 days. The colony diameter was measured to calculate the inhibition rate.

[0059] Through optimization of a series of fermentation processes, the antibacterial activity of WY228 wheat grain preparation was gradually improved. As shown in Figure 1, within the range of 20-28℃, with an inoculum amount of 5-10% of *Streptomyces xitangensis* WY228, a bottling amount of 10-40 g, a material-to-water ratio of 2:1 (moisture content 33%) to 1:1 (moisture content 50%), a wheat grain culture medium dosage of 4-8 g, and a culture period of 2 days, the WY228 wheat grain preparation achieved a 100% inhibition rate against *Botrytis cinerea*, which is the optimal fermentation process.

[0060] 2.2 Optimization of the preparation process of WY228 wheat grain formulation

[0061] (1) Two schemes for optimizing the best drying method:

[0062] Option 1, to ensure that the final moisture content is 15% after different drying treatments, is operated as follows: Natural drying: Dispense the WY228 wheat grain preparation into disposable sterile petri dishes, place them in an open laminar flow hood, and let them air dry at room temperature; Oven drying: Adjust the oven temperature to 35℃, dispense the WY228 wheat grain preparation into disposable sterile petri dishes, place them in a UV-sterilized oven, and dry them with the oven open; Vacuum freeze-drying: Dispense the WY228 wheat grain preparation into 50 mL sterile centrifuge tubes, freeze them overnight in an ultra-low temperature freezer, and then vacuum freeze-dry them.

[0063] Option 2 ensures consistent drying times for different drying processes. The WY228 wheat grain formulation was dried using the three methods described above: natural drying, oven drying, and vacuum freeze drying, with a drying time of 24 hours each.

[0064] None of the three drying methods in Drying Scheme 1 completely dried the moisture, which may cause the wheat grain preparation to become moldy during storage.

[0065] The oven drying in Option 2 can completely evaporate the moisture, ensuring the dryness of the wheat grains and preventing mold growth. The low moisture content keeps the bacteria in a dormant state, which helps to ensure the antibacterial activity of the bacteria.

[0066] Therefore, drying in a 35℃ oven for 24 hours is the optimal drying method.

[0067] (2) Optimization of the optimal storage method: WY228 wheat grain preparations dried in a 35℃ oven for 24 h were stored under vacuum at room temperature, 4℃, and 4℃. The antibacterial activity and viable cell count of the wheat grain preparations were tested, and 4℃ vacuum storage was found to be the optimal storage method. Under this storage condition, after a shelf life of 90 days, the antibacterial activity of the wheat grain preparations remained above 70%, and the viable cell count did not decrease significantly, which was superior to storage at 4℃ and room temperature vacuum storage.

[0068] (3) Optimization of optimal rehydration conditions: The rehydration solutions used were sterile water, a self-made nutrient solution, and a TSA nutrient solution. The self-made nutrient solution (g / L) consisted of 6 g of tryptone (nitrogen source), 4 g of glucose (carbon source), and 1% CaCl2. The TSA nutrient solution was prepared by diluting TSA medium 5 times and adding 1% CaCl2. Different rehydration solutions were added to achieve a final water content of 33% and 50% for the wheat grain preparations, respectively. The preparations were then incubated overnight at 28°C. The activity of the wheat grain preparations was tested the next day by measuring antibacterial activity and the number of viable bacteria.

[0069] Shelf life evaluation of wheat grain formulations: Using the optimal fermentation process, drying method and storage method obtained in the previous stage, samples were taken at 15 days, 30 days and 90 days and rehydrated under different rehydration conditions to evaluate the antibacterial activity and viable bacteria count of the wheat grain formulations.

[0070] Regarding the choice of rehydration solution, both sterile water and homemade nutrient solution showed good results. However, from an economic perspective, sterile water can be chosen as the rehydration solution. After rehydration, there was no significant difference in the antibacterial effect of 33% and 50% water content on the wheat grain preparation. As shown in Figure 2, the optimal preparation process for WY228 wheat grain preparation is drying in an oven at 35℃ for 24 hours, and it can be stored for about 90 days under vacuum at 4℃. Homemade nutrient solution or sterile water is the best rehydration solution.

[0071] Example 3: Application Test of Xitang Streptomyces WY228 Inoculant

[0072] 3.1 Identification of VOCs in WY228 wheat grain formulation

[0073] Headspace solid-phase microextraction-gas chromatography-mass spectrometry analysis revealed that the WY228 wheat grain formulation produced a total of 128 VOCs. Among them, the compounds with a content exceeding 1% were: terbinafine (36.12%), dimethyl 2,4-furandicarboxylate (11.80%), 2-methylbutyric acid (9.32%), 4-methylhexanoic acid (3.57%), 2-methylpropionic acid (1.59%), 1,4-dimethyladamantane (1.38%), 4-methylpropionic acid (1.21%), methyl furoate (1.13%), and cyclopentanone (1.12%).

[0074] Four commercially available monomeric compounds showed strong inhibitory effects against *Botrytis cinerea* at 28℃ and 22℃. As shown in Figure 3, all four compounds exhibited inhibitory effects against *Botrytis cinerea* at 28℃. 4-Methylvalerate achieved an inhibition rate of approximately 80% at a concentration of 50 μL / plate; methyl furoate and 2-methylbutyric acid achieved nearly 100% inhibition rates at a concentration of 30 μL / plate; and cyclopentanone completely inhibited *Botrytis cinerea* growth at a concentration of 50 μL / plate. At 22℃, methyl furoate completely inhibited *Botrytis cinerea* mycelial growth at a concentration of 50 μL / plate. 2-Methylbutyric acid achieved an inhibition rate of nearly 90% at a concentration of 50 μL / plate. 4-Methylvalerate achieved an inhibition rate of approximately 70% at a concentration of 50 μL / plate. Cyclopentanone achieved an inhibition rate of 80% at a concentration of 50 μL / plate.

[0075] 3.2 The control effect of WY228 grain preparation on gray mold in strawberries

[0076] Select strawberries with no surface damage, uniform ripeness, and similar size. Wash them with clean water, then soak them in 75% ethanol for 30 seconds. Rinse twice with sterile water and air dry in a laminar flow hood. Make wounds on the sides of the strawberries with sterile bamboo skewers and inoculate them with 10 μL of Botrytis cinerea spore suspension (1×10⁻⁶). 6 Place 100 spores / mL into a sterile container (volume 0.75 L). Place a petri dish in the container away from the strawberries and add 20 g or 40 g of WY228 wheat grain preparation to test the control effect of this fumigation preparation on strawberry gray mold. Direct spraying of carbendazim (1:1000 dilution) was used as a positive control.

[0077] After fumigation with WY228 grain preparation, strawberries inoculated with Botrytis cinerea did not develop gray mold, and strawberries not inoculated with Botrytis cinerea did not show any changes in fruit texture or color. As shown in Figure 4, Control represents the blank control group, BC represents the Botrytis cinerea inoculation group, Carbendazim represents the carbendazim group, 20 g wheat seeds represent the 20 g grain preparation fumigation group, 20 g wheat seeds + BC represents the Botrytis cinerea inoculation group combined with 20 g grain preparation fumigation, 40 g wheat seeds represent the 40 g grain preparation fumigation group, and 40 g wheat seeds + BC represents the Botrytis cinerea inoculation group combined with 40 g grain preparation fumigation. As shown in the figure, in the BC group inoculated only with Botrytis cinerea, gray mold lesions were obvious in strawberries. After fumigation with WY228 grain preparation, both 20g and 40g of WY228 grain preparation in the 20g Wheat seeds + BC and 40g Wheat seeds + BC groups completely inhibited Botrytis cinerea infection, and no gray mold symptoms appeared in the strawberries, achieving the same control effect as direct spraying of carbendazim in the Carbendazim group. In the 20g Wheat seeds and 40g Wheat seeds groups not inoculated with Botrytis cinerea, the 20g and 40g WY228 grain preparations did not change the color and texture of the strawberries, and were no different from the strawberries in the control group.

[0078] As shown in Figure 5, fumigation treatment with 26.67 g / L WY228 wheat grain preparation not only completely inhibited the occurrence of strawberry gray mold (Figure 5(A)), but also controlled the reduction of fruit weight loss (Figure 5(B)), maintained the content of vitamin C (Figure 5(C)) and titratable acid (Figure 5(D)) in the fruit, did not reduce fruit firmness (Figure 5(E)), delayed the increase of soluble solids in the fruit, controlled the rapid ripening and aging of the fruit (Figure 5(F)), increased total phenols (Figure 5(G)) and flavonoids (Figure 5(H)) related to stress resistance, decreased the content of malondialdehyde, a marker of membrane lipid peroxidation (Figure 5(I)), and increased superoxide dismutase activity (Figure 5(J)), significantly improved the resistance of strawberry fruit, and helped improve the quality of strawberries during post-harvest storage.

[0079] In summary, the microbial solid fumigation preparation of the present invention significantly inhibits the growth of Botrytis cinerea by generating antibacterial gases, and does not introduce live bacteria or excess moisture into the surface of the treated agricultural products. It has a safe and efficient control capability against gray mold and has good market application prospects.

Claims

1. The application of a microbial solid fumigation preparation in inhibiting Botrytis cinerea, characterized in that, After rehydration treatment, the microbial solid fumigation agent is placed together with the agricultural products to be treated in a sealed container. The concentration of the microbial solid fumigation agent used is 26.67 g / L. The prevention or treatment of gray mold is achieved through the inhibitory effect of the generated VOCs on Botrytis cinerea. The application is for the control of gray mold in strawberries. The preparation method of the microbial solid fumigation agent includes the following steps: Step 1, take fresh wheat grains, wash them clean, add water and boil until the wheat grains open, filter and air dry until the moisture content is 33%-50%. Weigh the air-dried wheat grains into an Erlenmeyer flask at a rate of 10-40 g / 50 mL, shake well and spread evenly at the bottom of the flask, and sterilize at 121℃ for 30 min. Step 1: Obtain wheat grain culture medium; Step 2: Take *Streptomyces xitangensis* WY228, inoculate it into ISP-2 medium, and culture at 28℃ and 180 rpm for 2 days to obtain *Streptomyces xitangensis* WY228 fermentation broth; Step 3: Inoculate the *Streptomyces xitangensis* WY228 fermentation broth into wheat grain culture medium, mix thoroughly, and then incubate statically. Shake the flask twice a day during the incubation period. After the incubation is completed, dry and store to obtain the microbial solid fumigation preparation; In Step 3, the inoculation amount of *Streptomyces xitangensis* WY228 fermentation broth is 5-10%, the static incubation temperature is 20-28℃, and the incubation period is 2 days; In Step 3, drying is carried out in an oven at 35℃ for 24 hours, and storage is carried out at 4℃ under low temperature and vacuum.

2. The application according to claim 1, characterized in that, The rehydration treatment uses sterile water or nutrient solution.

3. The application according to claim 2, characterized in that, The nutrient solution consists of 6 g / L tryptone, 4 g / L glucose, and 1% CaCl2.

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