Mixed biocontrol microbial inoculant for preventing and treating blueberry gray mold and application of mixed biocontrol microbial inoculant

Abstract

The invention relates to the technical field of biopesticides, in particular to a mixed biocontrol microbial inoculum for preventing and treating blueberry gray mold and application of the mixed biocontrol microbial inoculum. The invention relates to a mixed biocontrol microbial agent for preventing and treating blueberry gray mold. The mixed biocontrol microbial agent is prepared from the following components: pseudomonas chlororaphis A4, bacillus altitudinis LY-3, bacillus altitudinis Y-14 and bacillus altitudinis An-2. The four bacteria are mixed and prepared, the four bacteria have an inhibiting effect on the blueberry botrytis cinerea when being independently used, and the four bacteria are mixed and prepared to cooperate with one another to inhibit growth of botrytis cinerea hyphae; the four components are used together, so that gray mold of blueberry fruits in the field can be effectively prevented and treated, and the yield of blueberries is increased.

Description

Technical Field

[0001] This application relates to the field of biopesticide technology, specifically to a mixed biocontrol agent for controlling gray mold in blueberries and its application. Background Technology

[0002] Blueberries, a deciduous shrub belonging to the genus Vaccinium in the family Ericaceae, are also known as bilberries. Native to eastern Canada and the United States, they are now cultivated in China, Japan, New Zealand, and various European countries. Blueberries are loved by consumers for their unique flavor, rich nutritional value, and antioxidant properties. In particular, blueberries are rich in anthocyanins, vitamin C, vitamin K, dietary fiber, and various trace elements, offering health benefits such as preventing cardiovascular disease, anti-oxidation, anti-vision degeneration, anti-arteriosclerosis and thrombosis, anti-cancer properties, and delaying aging. They are listed by the Food and Agriculture Organization of the United Nations as one of the five healthiest foods for humans and are hailed as "golden berries."

[0003] Blueberry gray mold is a fungal disease caused by *Botrytis cinerea*, affecting blueberries. Also known as botrytis, it is a major disease of blueberries, primarily damaging flowers, fruits, leaves, pedicels, and branches. The disease spreads rapidly and widely, causing significant losses to blueberry production under low temperature and high humidity conditions. The damage from gray mold manifests in two main ways: yield loss and quality degradation. Once infected, grayish-white mold appears on the surface of blueberry fruits, leading to fruit rot and, in severe cases, the death of the entire plant. During the fruiting stage, gray mold infection renders blueberries unmarketable, significantly reducing yield and affecting fruit color, flavor, and nutritional quality. Furthermore, gray mold reduces the blueberry's resistance to other pests and diseases, increasing the chances of infection. Control of blueberry gray mold primarily relies on chemical control, supplemented by biological and ecological methods. Due to the resistance of pathogens to chemical pesticides, it is difficult to completely eliminate pathogens in the soil, resulting in a sustained decrease in the effectiveness of chemical pesticides. Summary of the Invention

[0004] To address the increasing resistance of blueberry gray mold to pesticides, which makes it difficult for existing chemical pesticides to effectively control the disease, this application provides a mixed biocontrol agent for controlling blueberry gray mold and its application. This application involves mixing Pseudomonas aeruginosa A4, Bacillus lysinus LY-3, and Bacillus lysinus Y-14. Each of these three fungi has an inhibitory effect on blueberry gray mold when used individually. When mixed, they work synergistically to accelerate the dissolution of gray mold cells, thereby effectively controlling blueberry gray mold.

[0005] In a first aspect, this application provides a mixed biocontrol agent for preventing and controlling gray mold in blueberries, employing the following technical solution: A mixed biocontrol agent for controlling gray mold in blueberries comprises the following components: Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14.

[0006] By adopting the above technical solution, this application formulates a mixture of Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14. Each of these three fungi has an inhibitory effect on blueberry gray mold when used alone, and the mixture can work synergistically to accelerate the dissolution of gray mold cells. This mixed biocontrol agent is not only beneficial for the prevention and control of blueberry gray mold, but also has a certain growth-promoting effect on blueberry growth, which can improve the blueberry planting industry and increase its economic value.

[0007] Preferably, the volume ratio of the three bacterial solutions—*Pseudomonas aeruginosa* A4, *Bacillus lysaefolia* LY-3, and *Bacillus lysaefolia* Y-14—is 1:1:1.

[0008] Preferably, the mixed biocontrol agent for preventing and controlling blueberry gray mold also includes Bacillus hygroscopicus An-2.

[0009] By adopting the above technical solution, the addition of Bacillus hygroscopicus An-2 to the mixture of the three bacterial solutions has a further promoting effect, thereby improving the control effect of the biocontrol agent.

[0010] Preferably, the volume ratio of the four bacterial solutions—*Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* An-2, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14—is 1:1:1:1.

[0011] Secondly, this application provides an application of a mixed biocontrol agent for controlling gray mold in blueberries, employing the following technical solution: The application of a mixed biocontrol agent for controlling gray mold in blueberries includes the following steps: Preparation: Mix the components according to the formula to obtain a mixed biocontrol agent for controlling gray mold in blueberries; Spraying: At the beginning of the blueberry flowering period, spray the blueberry plants with a mixed biocontrol agent to prevent blueberry gray mold. Spray once every 8-12 days, for a total of three sprays.

[0012] By employing the above-mentioned technical solutions, gray mold pathogens primarily infect blueberry flowers, young fruits, and tender shoots. Its conidia are spread by air currents, preferentially attaching to the surface of flowers, young fruits, and residual petals to germinate, forming a gray mold layer and causing fruit rot. When the mixed biocontrol agent is sprayed onto blueberry plants, the agent can directly cover high-risk areas such as flowers and fruits, forming a biofilm barrier and preventing spores from contacting the host.

[0013] Applying botrytis cinerea every 8-12 days ensures that protective barriers are rebuilt before the next peak infection period. Continuous, high-frequency spraying leads to thickened biofilms, hindering gas exchange on the fruit surface, causing abnormal respiration and increased fruit drop. Decreased light transmittance affects color change and reduces soluble solids content. Longer intervals make it difficult to rebuild protective barriers before the next peak infection period, reducing the effectiveness of spraying.

[0014] Preferably, the peak flowering period of blueberries is marked by the simultaneous opening of more than 70% of the flower buds on the blueberry plant.

[0015] Preferably, each blueberry plant is sprayed with 350-450 mL of a mixed biocontrol agent to prevent blueberry gray mold.

[0016] By employing the above technical solutions, when the spraying amount is low, the mixed biocontrol agent is prone to incomplete coverage, creating infection gaps and resulting in an effective bacterial density below the inhibition threshold. When the spraying amount is high, competition for carbon sources occurs between the mixed biocontrol agent and the fruit, reducing the soluble solids content. Furthermore, the prolonged canopy humidity negates the antibacterial effect of the mixed biocontrol agent.

[0017] In summary, this application has the following beneficial effects: 1. This application uses a mixture of Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14. Each of these three fungi has an inhibitory effect on blueberry gray mold when used individually, and the mixture works synergistically to accelerate the dissolution of gray mold cells. This mixed biocontrol agent is not only beneficial for the control of blueberry gray mold, but also has a certain growth-promoting effect on blueberry growth, which can improve the blueberry planting industry and increase its economic value. 2. This application, by adding Bacillus hygroscopicus An-2 to the above three bacterial solutions, has a further promoting effect and improves the control effect of the biocontrol agent; 3. This application involves spraying a mixed biocontrol agent every 8-12 days starting from the peak blueberry flowering period to ensure that protection is rebuilt before the next peak of infection. Attached Figure Description

[0018] Figure 1 This is a graph showing the results of a plate confrontation experiment between *Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* An-2, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14 and *Botrytis cinerea*. Detailed Implementation

[0019] The raw materials in this application include the following: Pseudomonas aeruginosa A4: A commercially available product from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., product number ZKCC25942; Bacillus altitudinis LY-3: This product is deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 23, 2023, and is classified as Bacillus altitudinis with accession number CGMCC No. 26884. Bacillus hygroscopicus An-2: A commercially available product from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., with product number ZKCC25231; Bacillus highlandii Y-14: A commercially available product from Beijing Zhongke Quality Inspection Biotechnology Co., Ltd., with product number ZKCC25900; The above four bacteria were subjected to a plate confrontation experiment to detect their inhibitory effect on Botrytis cinerea. The results are as follows: Figure 1 As shown, all four bacteria have a certain inhibitory effect, with Pseudomonas aeruginosa A4 showing the best effect.

[0020] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0021] Example 1 The application of a mixed biocontrol agent for controlling gray mold in blueberries includes the following steps: Preparation: *Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14 were cultured in liquid LB medium. The concentrations of all three bacteria were kept within 3.5 × 10⁻⁶. 9 CPU / mL -4.5*10 9 When the concentration of CPU / mL is 200 mL, 200 mL of each of Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14 are mixed to obtain a mixed biocontrol agent for the prevention and control of gray mold in blueberries. Spraying: Dilute the mixed biocontrol agent for controlling blueberry gray mold 100 times and spray the blueberry plants with the mixed biocontrol agent at the beginning of the blueberry flowering period, spraying once every 10 days, for a total of three sprays; spray 400mL of the diluted mixed biocontrol agent for controlling blueberry gray mold on each blueberry plant; the blueberry plants should be healthy and free from pests and diseases of the Emerald variety.

[0022] The peak flowering period for blueberries is marked by the simultaneous opening of more than 70% of the flower buds on the blueberry plant. After each application of the mixed biocontrol agent for controlling blueberry gray mold, the agent should remain moist on the leaves and fruit surfaces of the blueberry plant without dripping.

[0023] Examples 2-4 Examples 2-4 are based on the application of Example 1, with adjustments made to the amount of the three bacterial solutions added when mixed. The specific adjustments are shown in Table 1.

[0024] Comparative Examples 1-2 Comparative Example 1, based on the application in Example 1, replaced the spraying of diluted mixed biocontrol agent for controlling blueberry gray mold with spraying water.

[0025] Comparative Example 2, based on the application in Example 1, involved spraying a diluted pesticide for controlling blueberry gray mold. Any effective chemical pesticide was used; this application only uses iprodione diluted 1000 times for illustration.

[0026] The blueberry fruits from Examples 1-4 and Comparative Examples 1-2 were subjected to the following performance tests.

[0027] Performance testing 1. Yield per plant At the early stage of fruit ripening (when about 70% of the fruit has changed color), five blueberry plants were randomly selected. The number of fruits on each plant was counted (including ripe and immature fruits to avoid missing the later ripening fruits). Ten ripe blueberries were randomly selected from each plant and their fresh weight was measured using an electronic balance. The average number of fruits per plant and the average fresh weight of a single fruit were then calculated. The yield per plant was calculated based on the average fresh weight of a single fruit and the average number of fruits per plant.

[0028] 2. Soluble solids content and titratable acid content The soluble solids content of blueberries was determined according to GB / T 12295-1990, "Determination of Soluble Solids Content in Fruits and Vegetables - Refractometer Method".

[0029] The titratable acid content of blueberry fruit was determined according to GB / T 12456-2021 "Determination of total acid in food".

[0030] Table 1. Dosage and performance test results of the three bacterial cultures in Examples 1-4 and Comparative Examples 1-2.

[0031] Referring to Table 1, a comparison of Examples 1-4 and Comparative Examples 1-2 shows that, during the blueberry growth period, spraying the mixed biocontrol agent is significantly superior to spraying water and carbendazim. The mixture of *Pseudomonas aeruginosa* A4, *Bacillus lysus* LY-3, and *Bacillus lysus* Y-14 can synergistically accelerate the dissolution of *Botrytis cinerea* cells. This mixed biocontrol agent is not only beneficial for the control of blueberry gray mold but also has a certain growth-promoting effect on blueberry growth, which can improve the blueberry planting industry and increase its economic value. A comparison of Examples 1-4 shows that the mixed biocontrol agent is more effective when the volume ratio of the three bacterial solutions is 1:1:1.

[0032] Example 5 Example 5, based on the application of Example 1, adjusts the application steps as follows: Preparation: Incubate *Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* An-2, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14 in liquid LB medium until the concentrations of all three bacteria are within 3.5 × 10⁻⁶. 9 CPU / mL -4.5*10 9 At a concentration of CPU / mL, 100 mL of each of *Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* An-2, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14 were mixed to obtain a mixed biocontrol agent for controlling blueberry gray mold.

[0033] The blueberry fruits from Example 5 were subjected to the performance tests described above, and the test results are shown in Table 2.

[0034] Table 2. Data on the dosage and performance of Bacillus hygroscopicus An-2 in Examples 1 and 5.

[0035] Referring to Table 2, a comparison of Example 1 and Example 5 shows that adding Bacillus hygroscopicus An-2 to the mixed biocontrol agent yields better results.

[0036] Examples 6-9 Examples 6-9 are based on the preparation method of Example 5, but the amount of the four bacterial solutions added when they are mixed is adjusted. The specific adjustments are shown in Table 3.

[0037] The blueberry fruits from Examples 6-9 were subjected to the performance tests described above, and the test results are shown in Table 3.

[0038] Table 3. Dosage and performance test data of the four bacterial solutions mixed in Examples 1 and 5-9.

[0039] Referring to Table 3, a comparison of Examples 1 and 5-9 shows that the mixed biocontrol agent is more effective when the volume ratio of the four bacterial solutions is 1:1:1.

[0040] Examples 10-11 Examples 10-11 are based on the preparation method of Example 1, but the interval of "every 10 days" in the spraying step is adjusted. The specific adjustments are shown in Table 4.

[0041] Comparative Examples 3-4 Comparative Examples 3-4 are based on the preparation method of Example 1, but the interval of "every 10 days" in the spraying step is adjusted. The specific adjustments are shown in Table 4.

[0042] The blueberry fruits from Examples 10-11 and Comparative Examples 3-4 were subjected to the performance tests described above, and the test results are shown in Table 4.

[0043] Table 4. Interval days and performance test data for Examples 1, 10-11 and Comparative Examples 3-4

[0044] Referring to Table 4, comparing Examples 1, 10-11, and 3-4, it is evident that spraying the mixed biocontrol agent at 8-12 day intervals is more effective. This is because *Botrytis cinerea* requires an 8-12 day interval to ensure protective regeneration before the next infection peak. Continuous, high-frequency spraying in a short period leads to biofilm thickening, hindering gas exchange on the fruit surface, resulting in abnormal respiration and increased fruit drop. Decreased light transmittance affects color change, and soluble solids content decreases. Longer intervals make it difficult to rebuild protective regeneration before the next infection peak, reducing the spraying effect.

[0045] Examples 12-15 Examples 12-15 are based on the preparation method of Example 1, but the dosage of the mixed biocontrol agent for controlling blueberry gray mold per blueberry plant is adjusted. The specific adjustments are shown in Table 5.

[0046] The blueberry fruits from Examples 12-15 were subjected to the performance tests described above, and the test results are shown in Table 5.

[0047] Table 5. Dosage and performance test data of the mixed biocontrol agent per blueberry plant in Examples 1 and 12-15.

[0048] Referring to Table 5, a comparison of Examples 1 and 12-15 shows that the effectiveness of the mixed biocontrol agent initially increases and then decreases with increasing spraying dosage. This is because as the spraying dosage increases, the density of effective bacteria increases, gradually covering the infection gaps and exerting an antibacterial effect. However, beyond a certain range, competition for carbon sources arises between the mixed biocontrol agent and the fruit, reducing the soluble solids content. Furthermore, the prolonged duration of canopy humidity negates the antibacterial effect of the mixed biocontrol agent.

[0049] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A mixed biocontrol agent for controlling gray mold in blueberries, characterized in that, It includes the following components: Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14.

2. The mixed biocontrol agent for controlling blueberry gray mold according to claim 1, characterized in that: The volume ratio of the three bacterial solutions—Pseudomonas aeruginosa A4, Bacillus lysaefolia LY-3, and Bacillus lysaefolia Y-14—was 1:1:

1.

3. The mixed biocontrol agent for controlling blueberry gray mold according to claim 1, characterized in that: It also includes Bacillus angustifolius An-2.

4. The mixed biocontrol agent for controlling blueberry gray mold according to claim 3, characterized in that: The volume ratio of the four bacterial solutions—*Pseudomonas aeruginosa* A4, *Bacillus hygroscopicus* An-2, *Bacillus hygroscopicus* LY-3, and *Bacillus hygroscopicus* Y-14—was 1:1:1:

1.

5. The application of a mixed biocontrol agent for controlling gray mold in blueberries, characterized in that: The mixed biocontrol agent for controlling blueberry gray mold is any one of the mixed biocontrol agents for controlling blueberry gray mold as described in claims 1-4.

6. The application of the mixed biocontrol agent for controlling blueberry gray mold according to claim 5, characterized in that, Includes the following steps: Preparation: Mix the components according to the formula to obtain a mixed biocontrol agent for controlling gray mold in blueberries; Spraying: At the beginning of the blueberry flowering period, spray the blueberry plants with a mixed biocontrol agent to prevent blueberry gray mold. Spray once every 8-12 days, for a total of three sprays.

7. The application of the mixed biocontrol agent for controlling blueberry gray mold according to claim 6, characterized in that: The sign of peak blueberry bloom is when more than 70% of the flower buds on the blueberry plant open simultaneously.

8. The application of the mixed biocontrol agent for controlling blueberry gray mold according to claim 6, characterized in that: Spray each blueberry plant with 350-450 mL of a mixed biocontrol agent to prevent blueberry gray mold.

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