A Green Control Method for Blueberry Gray Mold Based on Compound Microbial Agents

Abstract

This application relates to the field of plant disease biological control technology, and discloses a green control method for blueberry gray mold based on a compound microbial agent. The method involves using methyltrophic Bacillus, Bacillus amyloliquefaciens, and Bacillus buddingus to prepare a highly active surface tension regulating liquid and a hydrophobic modified polysaccharide matrix liquid through induced fermentation. Subsequently, calcium glycinate is added, and a self-emulsifying system is constructed through a high-shear process. This invention solves the problems of conventional microbial agents being difficult to wet and adhere to the high-waxy surface of blueberries and being susceptible to rain erosion. The hydrophobic modified polysaccharide and biosurfactant in the system work synergistically to significantly reduce the contact angle and enhance colonization ability. Simultaneously, the chemical precipitation-biodegradation coupling mechanism constructed by calcium glycinate and Bacillus amyloliquefaciens can specifically remove oxalic acid, the pathogenic factor of gray mold. This composition exhibits good stability and significant control efficacy, making it suitable for the green control of blueberry gray mold.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, specifically a green control method for blueberry gray mold based on compound microbial agents. Background Technology

[0002] Blueberry gray mold is a significant fungal disease caused by *Botrytis cinerea*, severely restricting the yield and quality of the blueberry industry. Currently, production mainly relies on chemical fungicides for control. However, with the emergence of pathogen resistance and increasing food safety requirements, the use of chemical pesticides is strictly limited, making biological control using microbial agents an important alternative.

[0003] However, existing microbial agents have significant limitations in controlling blueberry gray mold. Firstly, the thick waxy layer covering blueberry fruit skin exhibits significant hydrophobic properties. Conventional aqueous microbial agents have high surface tension, making it difficult to effectively wet and spread on the fruit surface. The solution easily rolls off and is lost, resulting in low adhesion of functional strains to the target site and poor resistance to rain washout. Secondly, the pathogenic mechanism of *Botrytis cinerea* mainly involves secreting oxalic acid into the host tissue, using oxalic acid to lower the tissue pH and chelate calcium ions in the cell wall to disrupt the host's defenses. Most existing biocontrol agents rely solely on producing antimicrobial substances or spatial competition to exert their effects, lacking a specific clearance or blocking mechanism against oxalic acid, the core pathogenic factor. This makes it impossible to effectively interrupt the pathogen's pathogenesis in the early stages of infection, leading to unstable control effects in practical applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a green control method for blueberry gray mold based on compound microbial agents. This method solves the technical problems of poor adhesion of existing biological agents to the high-waxy surface of blueberries and the lack of an effective blocking mechanism against oxalic acid, the pathogenic factor of gray mold, which leads to poor control effects.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a green control method for blueberry gray mold based on a compound microbial agent. This method constructs a self-emulsifying biological composition by regulating the metabolic pathways of different microorganisms and combining it with a physical shearing process to control blueberry gray mold. The method includes the following steps: S1. Activate methylotrophic Bacillus, Bacillus amyloliquefaciens and budding short-stem mold respectively to provide seed liquid for subsequent fermentation preparation; S2. The methyltrophic Bacillus and the amyloliquefaciens are inoculated into a liquid culture medium for co-fermentation.

[0006] In this process, specific induction conditions are controlled to regulate metabolites: Inoculation and environmental control: The inoculation volume ratio of the methyltrophic Bacillus to the amyloliquefaciens Bacillus was 1:2. The co-fermentation temperature was controlled at 28-32℃, and the rotation speed was controlled at 200-240 rpm.

[0007] Culture medium basis: The basic components of the liquid culture medium include 10-15 g / L peptone, 5-8 g / L yeast extract and 5-10 g / L sodium chloride, which provide nitrogen source and mineral elements for strain growth and secondary metabolite synthesis.

[0008] Metabolic induction: When fermentation reaches an OD600 of 0.6-0.8, L-leucine is added to maintain a final concentration of 0.5-1.0 g / L, serving as a precursor for the synthesis of lipopeptide biosurfactants. After 12-14 hours of fermentation, methanol is added to maintain a final concentration of 0.8%-1.2% v / v, and sterile air containing 50-100 ppm oxalic acid is continuously introduced for 2 hours to induce the expression of methanol-utilizing enzymes and oxalic acid-degrading enzymes, respectively. Through this process, a highly active surface tension regulating solution rich in biosurfactants and zymogens is obtained. S3. The budding short-stem mold is inoculated into a specific culture medium containing methyl oleate for fermentation. Through substrate guidance and shear force regulation, an extracellular polysaccharide with hydrophobic side chain modification is synthesized.

[0009] The specific synthesis conditions for this step are as follows: The culture medium containing methyl oleate comprises: sucrose 60-80 g / L as a carbon source for the polysaccharide skeleton; ammonium sulfate 1.5-2.5 g / L and yeast extract 1.0-2.0 g / L as nitrogen sources; dipotassium hydrogen phosphate 3.0-5.0 g / L, magnesium sulfate 0.2-0.4 g / L, and sodium chloride 0.5-1.0 g / L as cofactors; and methyl oleate 3.0-5.0 g / L as a hydrophobic modification group donor. Fermentation temperature was controlled at 26-28℃. A segmented rotation speed control strategy was adopted: the rotation speed was 180 rpm for the first 24 hours of fermentation to promote cell growth; after 24 hours of fermentation, the rotation speed was reduced to 120-150 rpm to prevent the breakage of long-chain polysaccharides and to promote the transesterification or physical entanglement of hydrophobic groups. The total fermentation cycle was 96-120 hours. The hydrophobic modified polysaccharide matrix liquid prepared by the above process has a viscosity of 500-800 mPa·s at 25℃ and 100 rpm, and has rheological properties as an emulsion stabilizing framework. S4. The two-phase fermentation broth is assembled using fluid shear force.

[0010] By weight, measure 30-40 parts of the highly active surface tension regulating liquid, 60-70 parts of the hydrophobic modified polysaccharide matrix liquid, 0.02-0.05 parts of riboflavin, and 0.8-1.2 parts of calcium glycine; The riboflavin and the calcium glycine are dissolved and dispersed in the highly active surface tension regulating solution; The highly active surface tension regulating liquid containing additives is injected into the hydrophobic modified polysaccharide matrix liquid. The mixture is sheared at 1200-1500 rpm for 20-30 minutes at 20-25°C, followed by standing to defoam for 1-2 hours. During this process, the biosurfactants in the highly active surface tension regulating liquid adsorb onto the hydrophobic modified polysaccharide interface in the hydrophobic modified polysaccharide matrix liquid, forming a stable self-emulsifying composition. S5. Dilute the self-emulsifying composition with water 50-100 times and spray it onto the surface of the blueberry plant under a pressure of 0.3-0.5 MPa. Utilizing the self-emulsifying properties of the composition, the solution wets, spreads, and forms a film on the surface of the hydrophobic waxy layer.

[0011] The second aspect of this invention provides the technical essence of the reaction mechanism and process parameter control of the method. The method described in this invention achieves the following technical effects and reaction processes through the combination of specific fermentation process parameters and physical shear conditions: Regarding the construction of the co-fermentation system, in step S2, the inoculation volume ratio of the methyltrophic Bacillus to the amyloliquefaciens was set to 1:2, and the temperature was controlled at 28-32℃ and the rotation speed at 200-240 rpm. Under these conditions, the added L-leucine was utilized by the amyloliquefaciens to synthesize lipopeptide metabolites such as surfactants and oxytocin; the added methanol and oxalic acid gases induced the expression of methanol dehydrogenase in the methyltrophic Bacillus and the expression of oxalate decarboxylase in the amyloliquefaciens, respectively. The resulting highly active surface tension regulating solution possesses the physical property of reducing liquid-solid interfacial tension and exhibits enzyme catalytic activity targeting specific substrates.

[0012] Regarding the hydrophobic modification of the polysaccharide matrix, in step S3, the culture medium containing methyl oleate comprises 60-80 g / L sucrose and 3.0-5.0 g / L methyl oleate. Under conditions of 26-28℃ and segmented rotation speed control (180 rpm for the first 24 hours, then reduced to 120-150 rpm), the *Brugia buddingis* integrates the long-chain fatty acid groups from methyl oleate into the pullulan polysaccharide structure. The resulting hydrophobically modified polysaccharide matrix solution has a viscosity of 500-800 mPa·s at 25℃ and 100 rpm, exhibiting amphiphilic rheological characteristics.

[0013] Regarding phase separation and self-emulsifying assembly, in step S4, when the highly active surface tension regulating liquid is mixed with the hydrophobic modified polysaccharide matrix liquid, surface-active lipopeptides and hydrophobic modified polysaccharide chains exist in the system. Under a shear field of 1200-1500 rpm, the surface-active lipopeptides adsorb onto the interface of the hydrophobic modified polysaccharide chains, undergoing microscopic phase separation and reassembly to form a pseudoplastic fluid with a small dispersed phase particle size. When the self-emulsifying composition is sprayed onto the blueberry surface, the lipophilic-hydrophilic balance of the fluid matches the surface energy of the waxy layer on the blueberry peel, achieving wetting and spreading of the solution.

[0014] Regarding the operation of the controlled reaction system, the composition forms a solid membrane containing microorganisms and additives after drying on the blueberry surface. When oxalic acid is present in the environment, the calcium glycinate dispersed within the membrane undergoes a displacement reaction with oxalic acid to generate calcium oxalate precipitate. The calcium oxalate precipitate acts as a signaling molecule, inducing the Bacillus amyloliquefaciens in a stable state within the membrane to secrete antifungal secondary metabolites. Simultaneously, riboflavin acts as a photosensitizer in response to ambient light signals, regulating the metabolic activity of microorganisms within the membrane. The methyltrophic Bacillus utilizes methanol released from damaged plant tissue as a carbon source to maintain survival and carry out metabolic activities.

[0015] This invention provides a green control method for blueberry gray mold based on a compound microbial agent. It has the following beneficial effects: 1. This invention utilizes hydrophobically modified polysaccharides prepared by methyl oleate-induced fermentation and lipopeptide surfactants for shearing and assembly, solving the problem of water-based preparations being difficult to adhere to the high-waxy surface of blueberries. This composition can reduce the liquid-solid interfacial tension, allowing the liquid to quickly wet and spread on the fruit surface. After drying and forming a film, the hydrophobic groups on the polysaccharide chains undergo physical adsorption with the waxy layer of the fruit peel, improving the residue rate of the preparation under rain washout conditions and prolonging the colonization time of functional strains at the target site.

[0016] 2. This invention constructs a dual blocking system against oxalic acid, the pathogenic factor of gray mold, by adding calcium glycinate and combining it with Bacillus amyloliquefaciens that has been specifically induced. Calcium glycinate preferentially reacts with oxalic acid secreted by the pathogen to form an insoluble precipitate, inhibiting its acidification and damage to plant tissues. The precipitate then acts as a substrate signal to activate the expression of oxalic acid-degrading enzymes in the strain, continuously decomposing oxalic acid. This combination of chemical precipitation and biodegradation effectively blocks the infection pathway of the pathogen.

[0017] 3. The co-fermentation and segmented regulation process of this invention improves the synthesis efficiency of active metabolites and the physical stability of the system. By adding precursor substances, it promotes the synthesis of surfactants and uses fluid shear force to drive the interfacial assembly of hydrophobic polysaccharides and surfactants, so that the formulation remains uniform and stable without the addition of chemically synthesized emulsifiers. This avoids stratification or water separation during long-term storage and meets the requirements of green agriculture for the environmental safety of formulations. Detailed Implementation

[0018] Example: Example 1 This embodiment provides a green control method for blueberry gray mold based on compound microbial agents. The specific steps are as follows: S1, strain activation Glycerol preservation tubes of methyltrophic Bacillus, Bacillus amyloliquefaciens, and budding short-stem mold were streaked onto solid plates for activation. Then, single colonies were picked and inoculated into LB liquid medium (Bacillus) and YPD liquid medium (budding short-stem mold) to prepare seed culture with an OD600 of 1.0.

[0019] S2. Preparation of highly active surface tension regulating liquid Prepare a liquid culture medium with the following components: 12.5 g / L peptone, 6.5 g / L yeast extract, 7.5 g / L sodium chloride, and pH 7.0. Take the methyltrophic Bacillus seed solution from step S1 and the Bacillus amyloliquefaciens seed solution, mix them at a volume ratio of 1:2, and inoculate them into the above liquid culture medium at a 3% inoculation rate. The fermentation temperature was controlled at 30℃, and the shaking speed was set to 220 rpm; When fermentation reached the 7th hour (at which point OD600 was approximately 0.7), a sterile L-leucine solution was added to the fermentation broth to bring the final concentration to 0.75 g / L. When fermentation reached the 13th hour, sterile methanol was added to the fermentation broth to make its final concentration reach 1.0% (v / v), and at the same time, sterile air containing 75 ppm oxalic acid gas was continuously introduced into the fermentation system for 2 hours. Continue culturing until the total fermentation time reaches 42 hours, then stop fermentation. The resulting fermentation broth is the highly active surface tension regulating solution.

[0020] S3. Preparation of hydrophobically modified polysaccharide matrix solution Prepare an induction medium with the following components: 70 g / L sucrose, 2.0 g / L ammonium sulfate, 1.5 g / L yeast extract, 4.0 g / L dipotassium hydrogen phosphate, 0.3 g / L magnesium sulfate, 0.75 g / L sodium chloride, and 4.0 g / L methyl oleate, pH 6.2. Take the budding *Brachystomata* seed culture from step S1 and inoculate it into this medium at a 6% inoculum. Fermentation temperature was controlled at 27℃; From the start of fermentation to the 24th hour, the rotation speed was set to 180 rpm; after the 24th hour, the rotation speed was reduced to 135 rpm. The total fermentation cycle was 108 hours. After fermentation, the viscosity of the fermentation broth was measured at 25°C and 100 rpm. The viscosity obtained was within the expected range, and the fermentation broth was the hydrophobic modified polysaccharide matrix solution.

[0021] S4. Preparation of self-emulsifying compositions By weight, measure 35 parts of high-activity surface tension regulating solution, 65 parts of hydrophobic modified polysaccharide matrix solution, 0.035 parts of riboflavin, and 1.0 part of calcium glycine. First, riboflavin and calcium glycine were added to a highly active surface tension regulating solution and ultrasonically dispersed for 5 minutes to dissolve them. Then, the mixture was injected into a shearing vessel containing a hydrophobically modified polysaccharide matrix solution. The system temperature was controlled at 22℃. The shearing machine was turned on, and the speed was set to 1350 rpm. Shearing was continued for 25 minutes. After shearing, the mixture was allowed to stand for 1.5 hours to defoam, and the finished product composition was obtained.

[0022] Example 2 This embodiment provides a green control method for blueberry gray mold based on compound microbial agents. The specific steps are as follows: S1, strain activation Same as Example 1.

[0023] S2. Preparation of highly active surface tension regulating liquid The liquid culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, and 5 g / L sodium chloride, pH 7.0. The inoculum volume ratio of methyltrophic Bacillus to Bacillus amyloliquefaciens was 1:2, with an inoculum size of 2%. Fermentation temperature: 28℃; rotation speed: 200 rpm; When fermentation reaches an OD600 of 0.6, L-leucine is added to a final concentration of 0.5 g / L. After 12 hours of fermentation, methanol was added to a final concentration of 0.8% (v / v), and sterile air containing 50 ppm oxalic acid gas was introduced for 2 hours. The total fermentation time is 36 hours.

[0024] S3. Preparation of hydrophobically modified polysaccharide matrix solution The induction culture medium consisted of: 60 g / L sucrose, 1.5 g / L ammonium sulfate, 1.0 g / L yeast extract, 3.0 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.5 g / L sodium chloride, and 3.0 g / L methyl oleate, pH 6.0. The inoculum size was 5%. Fermentation temperature 26℃; For the first 24 hours, the engine speed is 180 rpm; after 24 hours, the engine speed drops to 120 rpm. The total fermentation cycle is 96 hours.

[0025] S4. Preparation of self-emulsifying compositions By weight, measure 30 parts of high-activity surface tension regulating solution, 70 parts of hydrophobic modified polysaccharide matrix solution, 0.02 parts of riboflavin, and 0.8 parts of calcium glycine; The mixing method is the same as in Example 1; The system temperature was 20℃, the shearing speed was 1200 rpm, and the shearing was continued for 20 minutes. The system was then allowed to stand for 1 hour to defoam.

[0026] Example 3 This embodiment provides a green control method for blueberry gray mold based on compound microbial agents, and the specific steps are as follows: S1, strain activation Same as Example 1.

[0027] S2. Preparation of highly active surface tension regulating liquid The liquid culture medium consisted of 15 g / L peptone, 8 g / L yeast extract, and 10 g / L sodium chloride, with a pH of 7.2. The inoculum volume ratio of methyltrophic Bacillus to Bacillus amyloliquefaciens was 1:2, and the inoculum size was 4%. Fermentation temperature 32℃, rotation speed 240rpm; When fermentation reaches an OD600 of 0.8, L-leucine is added to a final concentration of 1.0 g / L. After 14 hours of fermentation, methanol was added to a final concentration of 1.2% (v / v), and sterile air containing 100 ppm oxalic acid gas was introduced for 2 hours. The total fermentation time is 48 hours.

[0028] S3. Preparation of hydrophobically modified polysaccharide matrix solution The induction culture medium consisted of: 80 g / L sucrose, 2.5 g / L ammonium sulfate, 2.0 g / L yeast extract, 5.0 g / L dipotassium hydrogen phosphate, 0.4 g / L magnesium sulfate, 1.0 g / L sodium chloride, and 5.0 g / L methyl oleate, pH 6.5. The inoculum size was 8%. Fermentation temperature 28℃; For the first 24 hours, the engine speed is 180 rpm; after 24 hours, the engine speed is reduced to 150 rpm. The total fermentation cycle is 120 hours.

[0029] S4. Preparation of self-emulsifying compositions By weight, measure 40 parts of highly active surface tension regulating solution, 60 parts of hydrophobic modified polysaccharide matrix solution, 0.05 parts of riboflavin, and 1.2 parts of calcium glycine. The mixing method is the same as in Example 1; The system temperature was 25℃, the shearing speed was 1500 rpm, and the shearing was continued for 30 minutes. The system was then allowed to stand for 2 hours to defoam.

[0030] Comparative example: Comparative Example 1 Compared with Example 1, the difference is that methyl oleate was not added to the induction medium in step S3, while the other steps and parameters are the same as in Example 1.

[0031] By removing the hydrophobic modified substrate, *Bacillus buddingus* produced only ordinary hydrophilic pullulan. The aim was to demonstrate that without hydrophobic side chains, the system could not form a self-emulsifying structure and could not effectively wet and colonize on hydrophobic blueberry surfaces.

[0032] Comparative Example 2 Compared with Example 1, the difference is that calcium glycine was not added to the ingredients in step S4, while the other steps and parameters are the same as in Example 1.

[0033] The key components of the mineralization reaction were eliminated. The aim was to demonstrate that without calcium ion-mediated in-situ crystallization of oxalate, lesions could not be physically sealed, and the synthesis of secondary metabolites of StrainB could not be triggered, leading to a decrease in efficacy.

[0034] Comparative Example 3 Compared with Example 1, the difference is that L-leucine was not added during the fermentation process in step S2, while the other steps and parameters are the same as in Example 1.

[0035] This study aims to block the efficient synthetic pathway of lipopeptide biosurfactants. The goal is to demonstrate that without high concentrations of biosurfactants, interfacial rearrangement in a two-phase system cannot be facilitated, leading to excessively high surface tension and poor spreadability of the drug solution.

[0036] Comparative Example 4 Compared with Example 1, the difference is that in the mixing and emulsification operation of step S4, continuous shearing at 1350 rpm for 25 minutes is replaced by ordinary mechanical stirring at 100 rpm for 25 minutes. The remaining steps and parameters are the same as in Example 1.

[0037] The key energy input was eliminated. The aim was to demonstrate that the two-phase fermentation broth is not a simple physical mixture, but rather requires high shear force to drive microscopic phase separation and self-assembly between surfactants and hydrophobic polysaccharides to form a stable emulsion system.

[0038] Test example: Test Example 1: Rheological Properties and Physical Stability Testing of the Composition System 1. Experiment Description This test case aims to determine the dynamic viscosity and centrifugal stability of each composition to evaluate the effects of different preparation processes and components on the physical structure of the system.

[0039] S1. The self-emulsifying compositions prepared in Examples 1, 2, and 3, and the control samples prepared in Comparative Examples 1 and 4 were selected. All samples were allowed to stand for 24 hours to defoam after preparation before testing. S2. Place 500 mL of each sample in a constant temperature beaker and maintain the sample temperature at 25 ± 0.5℃. Use an NDJ-8S rotational viscometer for measurement. Select rotor #2 and set the rotation speed to 60 rpm. Record the viscosity value (mPa·s) after the reading stabilizes for 30 seconds. Perform three parallel measurements for each sample, record the raw data, and calculate the average value. S3. Take 10 mL of each sample and inject it into a graduated centrifuge tube, recording the initial liquid level height H0. Place the centrifuge tube in a benchtop high-speed centrifuge, set the speed to 3000 rpm, and the centrifugation time to 20 minutes. After centrifugation, remove the centrifuge tube, observe the layering, and measure the height H1 of the upper aqueous phase or clear liquid layer. Calculate the water separation rate of each sample group according to the formula: Water separation rate = (H1 / H0) × 100%.

[0040] 2. Test Results The dynamic viscosity and water separation rate test results of each group of samples are shown in Table 1.

[0041] Table 1. Viscosity and stability test data of each group of compositions.

[0042] 3. Results Analysis Data from Comparative Example 1 showed that its viscosity was significantly lower than that of Examples 1-3, and the water separation rate reached 28.5%. This is because Comparative Example 1 lacked methyl oleate induction during fermentation, and the budding short-stem mold only synthesized hydrophilic pullulan polysaccharide, lacking hydrophobic side chain modification. During mixing, the hydrophilic polysaccharide could not form an effective hydrophobic anchoring effect with the lipopeptide biosurfactants in the highly active surface tension regulating liquid. The lack of interfacial affinity between the two phase components prevented the formation of a stable polymer-surfactant complex framework, leading to phase separation under centrifugal force.

[0043] Comparative Example 4 showed the highest water separation rate, reaching 84.3%, and also exhibited lower viscosity. This indicates that the energy provided by simple low-speed mechanical stirring is insufficient to overcome the energy barrier at the two-phase interface. The highly active surface tension regulating liquid and the hydrophobic modified polysaccharide matrix liquid only formed a macroscopic physical mixture, failing to drive the directional alignment and assembly of lipopeptide molecules at the hydrophobic modified polysaccharide interface through shear force. The lack of a high-shear input self-emulsification process prevented the system from forming a microscopic emulsion structure, resulting in each component maintaining its own physical state and exhibiting extremely poor stability.

[0044] Data from Examples 1-3 show that, by controlling the biphasic fermentation parameters and the high-shear emulsification process, the prepared compositions exhibit high dynamic viscosity (512.3-745.8 mPa·s) and extremely low water separation rate (0.4%-1.2%). This is because the hydrophobic groups on the hydrophobically modified polysaccharide chains physically entangle and hydrophobically associate with the hydrophobic tail chains of the lipopeptide biosurfactants, self-assembling under high shear force to form a stable pseudoplastic fluid structure. This structure increases the viscosity of the continuous phase and creates a steric hindrance effect on the dispersed phase droplets, thereby effectively hindering droplet aggregation and phase separation.

[0045] Test Example 2: Blueberry Surface Wetting and Rain Washback Resistance Test 1. Experiment Description This test case aims to determine the contact angle of each composition on the waxy surface of blueberry fruit and the residual rate of microbial community after simulated rainfall, in order to evaluate the colonization and adhesion performance of the formulation on the target surface.

[0046] S1. Select the self-emulsifying compositions prepared in Examples 1, 2, and 3, and the control samples prepared in Comparative Examples 1 and 3. Dilute all samples 100 times with sterile water before testing; S2. Select fresh, ripe blueberries with intact bloom and uniform size as the test matrix. Fix the berries on the stage of the optical contact angle measuring instrument. Use a micro-syringe to draw up the diluted solution to be tested and drop it vertically at the equator of the berry, with the droplet volume precisely controlled at 2 μL. Five seconds after the droplet contacts the berry surface, the instrument's built-in image analysis software automatically captures a side projection image of the droplet and calculates the contact angle (°). Repeat the test on 10 berries for each sample group, record all raw data, and take the average value. S3. Using a handheld sprayer, evenly spray each group of diluted solutions onto the surface of blueberry fruits until the fruit surface is completely wet but no liquid drips. Place the treated fruits in a 25℃ environment to air dry for 24 hours to form a solidified film. Then, place the fruits under an artificial rainfall device with a rainfall intensity of 20 mm / h for 30 minutes. After rinsing, place the fruits in an Erlenmeyer flask containing sterile buffer solution and shake to elute. Take the eluent, perform serial dilutions, and spread it on LB agar plates. After incubating overnight at 37℃, count the colony count of Bacillus amyloliquefaciens (CFU / cm³). 2 Simultaneously, the surface bacterial count of the fruit was measured before rinsing. The rain rinsing resistance of each group of samples was calculated according to the formula: Residual rate = (Bacterial count after rinsing / Bacterial count before rinsing) × 100%.

[0047] 2. Test Results The contact angle and scouring residue rate of each group of samples on the blueberry surface are shown in Table 2.

[0048] Table 2. Contact angle and rainwater erosion resistance data for each group of compositions.

[0049] 3. Results Analysis Comparative Example 3 showed a contact angle as high as 95.4°, with a rinsing residue rate of only 12.3%. This is because this group lacked L-leucine precursors during co-fermentation, limiting the metabolic synthesis of lipopeptide biosurfactants. The lack of surfactants resulted in a failure to significantly reduce the surface tension of the solution, making it unable to overcome the hydrophobic potential energy of the thick waxy layer on the blueberry skin. The droplets exhibited a spherical shape with a high contact angle on the fruit surface, resulting in a very small contact area, making effective wetting and spreading difficult. Consequently, most of the solution rolled off and was lost before drying and forming a film, failing to establish initial adhesion.

[0050] Comparative Example 1 showed a contact angle of 45.3°, similar to the Example group, but with a rinsing residue rate of only 15.6%. Although this group contained sufficient biosurfactants to achieve wetting and spreading at a low contact angle, the polysaccharide matrix synthesized by *Brugia buddingis* was a purely hydrophilic structure due to the lack of methyl oleate induction. After drying and film formation, the hydrophilic polysaccharide backbone could not form hydrophobic interactions or anchoring effects with the waxy layer of the blueberry skin. When subjected to simulated rainfall, the hydrophilic film layer rapidly absorbed water, swelled, and dissociated, causing the bacteria encapsulated within the film to be completely washed away by the water flow, thus failing to achieve rain-resistant colonization.

[0051] Data from Examples 1-3 show that the compositions prepared via a dual induction and assembly process exhibit both low contact angles (40.1°-48.2°) and high residue rates (81.2%-88.7%). The biosurfactants in the system first reduce the liquid-solid interfacial tension, allowing the drug solution to spread rapidly on the waxy fruit surface. Subsequently, as water evaporates, the long-chain fatty acid side chains in the hydrophobically modified polysaccharide matrix undergo physical adsorption and intermolecular intercalation with the waxy fruit peel layer through hydrophobic interactions, forming a dense film with a hydrophobic surface. This film not only locks in the functional bacterial strains but also exhibits resistance to aqueous erosion due to its hydrophobically modified structure, thus maintaining a high bacterial population retention rate even in humid environments.

[0052] Test Example 3: In vitro oxalate scavenging capacity test Experimental instructions This test case aims to quantitatively determine the scavenging efficiency of the cured film formed by each composition on oxalic acid in the environment, in order to verify the inhibitory effect of calcium glycinate and the microbial metabolic system on pathogenic factors in the system.

[0053] S1. Select the self-emulsifying composition stock solution prepared in Examples 1, 2 and 3, and the control sample prepared in Comparative Example 2 (without added calcium glycinate); S2. Take a clean glass slide with dimensions of 25.4mm × 76.2mm. Use a micropipette to draw 1.0mL of the sample to be tested and spread it evenly on one side of the slide, controlling the spreading area to be within 20cm². 2 The coated glass slides were placed in a sterile laminar flow hood and air-dried at 25°C for 24 hours until a dry, solid film was formed. S3. Prepare a 15.0 mmol / L oxalic acid aqueous solution as a simulated reaction substrate. Pour 50 mL of the oxalic acid solution into a 100 mL stoppered conical flask. Completely immerse a glass slide with the cured film in the oxalic acid solution in the conical flask. Place the conical flask in a constant temperature shaking incubator, set the temperature to 30℃, the rotation speed to 100 rpm, and react in the dark for 4 hours. S4. After the reaction is complete, transfer 10.0 mL of supernatant from each conical flask to a titration flask. Acidify with 5 mL of 2 mol / L sulfuric acid solution. Titrate with 0.02 mol / L potassium permanganate standard solution until the solution turns slightly pink and does not fade within 30 seconds, recording the volume of potassium permanganate consumed. Calculate the residual oxalic acid concentration in the solution after the reaction based on stoichiometry. Calculate the oxalic acid removal capacity of each group of samples using the formula: Removal rate = [(Initial concentration - Residual concentration) / Initial concentration] × 100%.

[0054] Test Results The results of the oxalic acid removal rate test for each group of samples are shown in Table 3.

[0055] Table 3. Test data on oxalic acid scavenging ability of each group of compositions.

[0056] 3. Results Analysis Comparative Example 2 showed an oxalic acid clearance rate of only 25.3%. No calcium glycine was added to this group of samples, resulting in a lack of a calcium source for rapid chemical reactions with oxalic acid. Therefore, oxalic acid consumption relied entirely on the basal metabolic activities of the *Bacillus amyloliquefaciens* and *Bacillus methyltrophicus* embedded in the membrane. Since substrate uptake and intracellular enzymatic digestion require a certain amount of time, and in the absence of specific signaling molecules, the metabolic activity of the strains was not fully activated, resulting in only a small amount of oxalic acid being degraded within the 4-hour test period.

[0057] Data from Examples 1-3 show that the oxalic acid removal rate ranged from 79.2% to 87.8%, significantly higher than that of Comparative Example 2. This result is attributed to the synergistic effect of calcium glycinate and the microbial metabolic system in the system. When the membrane comes into contact with a solution containing oxalic acid, the calcium glycinate dispersed in the membrane releases calcium ions. These calcium ions rapidly undergo a chemical displacement reaction with oxalate ions in the solution, forming a poorly soluble calcium oxalate precipitate. This chemical process rapidly reduces the concentration of free oxalic acid in the solution in the initial stage of the reaction, achieving physical solidification and blockade of pathogenic factors.

[0058] During the chemical precipitation process, the in-situ generated calcium oxalate microcrystals did not simply exist as inert products. Combined with the oxalate gas induction step used in the preparation process, the *Bacillus amyloliquefaciens* in the example system pre-expressed an oxalate decarboxylase system. Calcium oxalate precipitation, acting as a specific substrate signal, further induced and activated the oxalate degradation metabolic pathway of the strain, prompting the microorganisms to secrete more extracellular enzymes or accelerate intracellular transport, decomposing oxalate into formic acid and carbon dioxide. This coupled mineralization-capture-biodegradation mechanism ensures that the composition can efficiently and continuously remove oxalate from the environment, thereby blocking the pathogenic pathway by which pathogens utilize oxalate to acidify plant tissues and damage cell walls.

[0059] Test Example 4: Efficacy Test of Blueberry Gray Mold Control in Potted Plants Experimental instructions This test case aims to determine the inhibitory effect of each composition on gray mold on actual plants through pot inoculation experiments, so as to comprehensively evaluate the colonization performance and pathological blocking ability of the formulation.

[0060] S1. Select three-year-old potted blueberry plants that are growing uniformly and are in the young fruit stage. 15 plants were selected for each treatment group; A highly pathogenic strain of *Botrytis cinerea*. After culturing on PDA plates for 7 days, the spores were washed away with sterile water, and the spore suspension concentration was adjusted to 1×10⁻⁶. 6spores / mL; S2. Dilute the stock solutions of the compositions prepared in Examples 1-3 and Comparative Examples 1-4 with water by 100 times. Set up a water control group. Use a handheld pressure sprayer to spray the fruit clusters of blueberry plants in a directional manner until the fruit surface is completely wet and a small amount of liquid drips down; After treatment, the plants are left to stand for 24 hours under natural ventilation to allow the solution to solidify into a film layer after the moisture on the fruit surface evaporates. After the membrane has solidified, the ears of fruit in all treatment groups are evenly sprayed with spore suspension for inoculation. After inoculation, the plants were placed in an artificial climate chamber with the temperature controlled at 25±1℃ and the relative humidity maintained at 90%-95% (the first 24 hours were dark and moist, followed by 12 hours of light / 12 hours of darkness) to induce disease. S3. Investigate fruit disease incidence 7 days after inoculation. Grade the fruit based on the proportion of lesions to the total fruit surface area: Grade 0: No lesions; Grade 1: Lesion area ≤ 5%; Grade 3: 5% < Lesion area ≤ 25%; Grade 5: <25%< Lesion area ≤50%; Level 7: Lesion area > 50% or fruit rot.

[0061] The calculation formula is as follows: Disease index = [∑(number of diseased fruits at each level × relative level value) / (total number of fruits surveyed × 7)] × 100; Relative efficacy (%) = [(CK disease index − treatment group disease index) / CK disease index] × 100%.

[0062] 2. Test Results The disease index and relative prevention efficacy of each group are shown in Table 4.

[0063] Table 4. Test data on the control efficacy against blueberry gray mold in potted plants.

[0064] 3. Results Analysis The relative efficacy of Comparative Examples 1, 3, and 4 was all below 35%, significantly lower than that of the Example Group. Comparative Example 3 lacked metabolically induced biosurfactants, preventing the pesticide from spreading on the hydrophobic, highly waxy blueberry surface, resulting in insufficient coverage. Comparative Example 1 lacked a hydrophobically modified polysaccharide matrix; although it could spread, it could not withstand environmental humidity or liquid erosion during inoculation, leading to dissociation and loss of the film layer at the inoculation stage. Comparative Example 4, lacking high-shear emulsification, suffered from severe system stratification, resulting in uneven spraying and some fruits failing to receive effective protective components. These results indicate that constructing a self-emulsifying system with low surface tension and strong hydrophobic anchoring ability is the physical basis for achieving stable colonization and efficacy of the pesticide on the blueberry surface.

[0065] The relative efficacy of Comparative Example 2 was 58.2%, which, while better than the other comparative examples, was still significantly lower than that of Examples 1-3 (83.2%-88.5%). The system of Comparative Example 2 contained a complete film-forming matrix and surfactant, enabling it to form a physical barrier and thus exhibiting some efficacy. However, due to the lack of calcium glycinate, this group was unable to cope with the oxalic acid attack secreted by the pathogen. The pathogen penetrated the physical defense by secreting oxalic acid to acidify the fruit tissue and chelate calcium in the cell wall. In contrast, the Example groups, through the in-situ reaction of calcium ions with oxalic acid, not only eliminated the chemical toxicity of oxalic acid, but the resulting precipitate also acted as a signaling molecule to activate the secondary metabolism of *Bacillus amyloliquefaciens*. This chemical-biological linkage mechanism significantly improved the blocking efficiency against gray mold infection.

[0066] Examples 1-3 maintained a relative efficacy of over 83% under different process parameters, and Example 3 showed similar results to Example 1. This confirms that the technical solution of the present invention has good robustness within the set parameter range. The carrier system assembled under shear force using a highly active surface tension regulating liquid prepared by co-fermentation and a hydrophobically modified polysaccharide matrix liquid successfully solved the problems of poor adhesion of aqueous preparations to the blueberry wax layer and poor retention of active ingredients. Furthermore, the calcium-bacterial coupling mechanism enabled precise targeted removal of pathogenic factors, thus achieving a highly efficient level of prevention and control.

Claims

1. A green control method for blueberry gray mold based on a composite microbial inoculant, characterized by, Includes the following steps: S1, respectively activate methyltrophic Bacillus, Bacillus amyloliquefaciens and budding short-stem mold; S2. The methyltrophic Bacillus and the amyloliquefaciens Bacillus are inoculated into a liquid culture medium for co-fermentation, and a highly active surface tension regulating solution is prepared by induced culture. S3. The budding short-stem mold is inoculated into a culture medium containing methyl oleate for fermentation to prepare a hydrophobic modified polysaccharide matrix solution. S4. Measure 30-40 parts by weight of the highly active surface tension regulating liquid, 60-70 parts by weight of the hydrophobic modified polysaccharide matrix liquid, 0.02-0.05 parts by weight of riboflavin, and 0.8-1.2 parts by weight of calcium glycine; mix the highly active surface tension regulating liquid with the hydrophobic modified polysaccharide matrix liquid, and perform shear emulsification at a speed of 1200-1500 rpm to obtain a self-emulsifying composition; S5. Dilute the self-emulsifying composition with water and spray it onto the surface of the blueberry plant.

2. The blueberry gray mold green prevention and control method based on the composite microbial agent according to claim 1, characterized in that, In step S2, the inoculation volume ratio of the methyltrophic Bacillus to the amyloliquefaciens is 1:2; the co-fermentation temperature is controlled at 28-32℃ and the rotation speed is controlled at 200-240rpm.

3. The blueberry gray mold green prevention and control method based on the composite microbial agent according to claim 1, characterized in that, In step S2, the basic components of the liquid culture medium include: 10-15 g / L peptone, 5-8 g / L yeast extract, and 5-10 g / L sodium chloride.

4. The green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S2, the induction culture specifically includes: when fermentation reaches OD600 of 0.6-0.8, adding L-leucine to maintain the final concentration at 0.5-1.0 g / L; After fermentation for 12-14 hours, methanol is added to maintain the final concentration at 0.8%-1.2% v / v, and sterile air containing 50-100 ppm oxalic acid gas is continuously introduced for 2 hours.

5. A green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S3, the culture medium containing methyl oleate comprises the following components: sucrose 60-80 g / L, ammonium sulfate 1.5-2.5 g / L, yeast extract 1.0-2.0 g / L, dipotassium hydrogen phosphate 3.0-5.0 g / L, magnesium sulfate 0.2-0.4 g / L, sodium chloride 0.5-1.0 g / L, and methyl oleate 3.0-5.0 g / L.

6. The green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S3, the fermentation temperature is controlled at 26-28℃; the rotation speed is 180 rpm for the first 24 hours of fermentation, and the rotation speed is reduced to 120-150 rpm after 24 hours of fermentation; the total fermentation cycle is 96-120 hours.

7. The green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S3, the viscosity of the prepared hydrophobic modified polysaccharide matrix solution is 500-800 mPa·s at 25°C and 100 rpm.

8. A green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S4, the specific operations of mixing and shear emulsification are as follows: First, the riboflavin and the calcium glycine are dissolved and dispersed in the highly active surface tension regulating solution. Then, the highly active surface tension regulating solution containing additives is injected into the hydrophobic modified polysaccharide matrix solution for shearing.

9. A green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S4, the shearing emulsification time is 20-30 minutes, the system temperature during the shearing process is controlled at 20-25℃, and after the shearing is completed, the system is allowed to stand for 1-2 hours to defoam.

10. A green control method for blueberry gray mold based on compound microbial agents according to claim 1, characterized in that, In step S5, the self-emulsifying composition is diluted with water by a factor of 50-100; the spraying pressure is controlled at 0.3-0.5 MPa.

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