Green compound bactericide for preventing and treating apple mold core and preparation method thereof

Abstract

The invention discloses a green compound bactericide for preventing and treating apple moldy core and a preparation method thereof, and relates to the technical field of apple disease prevention and control, and the preparation method specifically comprises the following steps: S1, preparing epsilon-polylysine microspheres; s2, preparing a perillaldehyde nano-liposome; s3, preparing a rhodovulum sulfidophilum fermentation solution; s4, compounding a finished product; and S5, enzyme sensitive coating. According to the compound bactericide disclosed by the invention, 1 + 1gt can be realized through a multi-mechanism synergistic effect (targeted delivery, direct bacteriostasis, induced resistance and ecological occupation); and 2, the composition has the characteristics of green, safety, high efficiency and stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of apple disease prevention and control, and particularly relates to a green compound fungicide for preventing and treating apple moldy core and a preparation method thereof. BACKGROUND

[0002] Apple moldy core is one of the main diseases in apple production, which is mainly caused by Alternaria alternata (Fr.) Keissler, Stemphylium vesicarium Buckley, Botryosphaeria dothidea (Moug. ex Fr.) Ces. et De Not., etc., and affects the internal quality of fruits, and even causes a large number of rot during postharvest storage, resulting in significant economic losses. At present, the main prevention and control methods for apple moldy core include chemical fungicides, single biological agents and chemical + biological compound fungicides. Alternaria Trichothecium roseum The chemical fungicides (such as tebuconazole, carbendazim, etc.) have a quick effect, but long-term use may lead to problems such as increased resistance of pathogenic bacteria, excessive pesticide residues and environmental pollution. The single biological agents (such as Bacillus subtilis and Trichoderma) are environmentally friendly and do not easily produce resistance, but have unstable prevention and control effects, are greatly affected by environmental temperature and humidity, have a short effective period, and need to be frequently applied, thereby increasing the cost. The chemical + biological compound fungicides can delay resistance, but still rely on chemical pesticides, which does not meet the requirements of green agriculture.

[0003] Therefore, it is urgent to develop a green, safe, efficient and stable compound fungicide which takes into account the prevention and control effect and environmental safety.

[0004] The present application provides a green compound fungicide for preventing and treating apple moldy core and a preparation method thereof. SUMMARY

[0005] The present application provides a green compound fungicide for preventing and treating apple moldy core and a preparation method thereof.

[0006] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows: The present application provides a preparation method of a green compound fungicide for preventing and treating apple moldy core, which specifically comprises the following steps: S1: preparing ε-polylysine (ε-PL) microspheres: S11: dissolving ε-polylysine in 0.1M phosphate buffer (pH 6.5) to obtain a 5% ε-polylysine solution; S12: dissolving sodium alginate in deionized water, and stirring at 60℃ and 500rpm for 30min until completely dissolved, and then cooling to 25℃ to obtain a 1.2% sodium alginate solution; S13: uniformly mixing chitosan, acetic acid and water to obtain a 0.5% chitosan solution, and adjusting the pH to 5; S14: mixing the ε-polylysine solution and the sodium alginate solution, and stirring at 300rpm for 30min to obtain a mixed solution;​ S15: Use a syringe (22G needle, 0.41mm inner diameter) to dropwise add 1.5% CaCl2 solution at a rate of 5mL / min, let it stand and solidify for 30min to form calcium cross-linked microspheres; suspend the microspheres in the chitosan solution, stir at 150rpm for 1h, centrifuge and wash three times, prefreeze at -40℃ for 24h, and vacuum freeze-dry for 48h to obtain white powder, namely ε-polylysine microspheres; S2: Preparation of perillaldehyde nanoliposomes: Soybean lecithin, cholesterol, and perillaldehyde were dissolved in a 10-fold volume of a chloroform-methanol mixed solvent and rotary evaporated at 50 rpm at 40°C to remove the solvent to form a uniform lipid film. PBS (pH 7.0) with the same volume as the mixed solvent was added, vortexed for 5 minutes, sonicated at 50W on ice for 10 minutes, and filtered through a 0.22 μm filter to obtain perillaldehyde nanoliposomes. The mixture was stored at 4°C. S3: Preparation of sulfur-loving red oomycetes ( Rhodovulum sulfidophilum ) Fermentation broth: Take 1 freeze-dried powder, add 0.3 mL of sterile saline to the culture tube, gently flick until mixed, inoculate 5% of the inoculum into the liquid culture medium, and ferment at 30°C and 2000 lux for 2-3 days until the OD 600 =0.5-0.8; transfer 10% of the inoculum into fresh culture medium and expand the culture at 30℃ and 2000lux for 3-5 days until OD 600 =1.5-2.0, 4°C, 8000 rpm centrifugation for 15 min, take the supernatant, filter through 0.22 μm, and obtain the fermentation broth of R. sulfidophilus; S4: Finished product compounding: S41: dissolving xanthan gum and water at 60° C., cooling, and adding vitamin E to obtain a premixed auxiliary; S42: Add the fermentation broth of R. thiophilum to the premixed additives and stir at 200 rpm for 5 min. Then add the perillaldehyde nanoliposomes and stir at 300 rpm for 10 min. Then add the ε-polylysine microspheres and stir at a low speed of 150 rpm for 15 min. Finally, high-pressure homogenization (50 MPa, 3 cycles) is performed to ensure uniform particle size to obtain composite microspheres. The pH is adjusted to 6.0 ± 0.2 with 0.1 M NaOH or HCl. S5: Enzyme-sensitive coating: S51: Preparation of pectin-cellulase substrate polymer: Dissolve pectin: Dissolve high methoxyl pectin in 0.1 M pH 5.0 acetic acid buffer at 60°C and 500 rpm with magnetic stirring for 2 h until completely dissolved. Disperse cellulose: Add microcrystalline cellulose (MCC) to the above solution and sonicate at 300W for 10 minutes (pulse mode 5s on / 5s off) until uniformly suspended. Cross-linking reaction: Genipin (cross-linking agent) was added, and the reaction was carried out at 50°C for 3h (pH 5.0); Purification and drying: the reaction solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was washed with ethanol for 3 times, and was freeze-dried at -50°C for 24h to obtain a complex powder; S52: Preparation of spraying liquid: the above complex powder was dispersed in 60% ethanol aqueous solution, and was treated with ultrasonic at 200W for 5min to obtain a uniform suspension; S53: Spraying: the spraying liquid was uniformly sprayed on the surface of the composite microspheres by using an electrostatic spraying instrument, and was vacuum dried at 40°C for 2h after spraying to form an enzyme-sensitive coating (about 10-20μm thick).

[0007] Further, in the step S13, the mass ratio of chitosan to acetic acid is 1:1; In the step S14, the mass ratio of ε-polylysine solution to sodium alginate solution is 1:1; In the step S15, the volume ratio of the mixed solution to CaCl2 solution is 1:15; the mass ratio of the microspheres to chitosan solution is 1:5.

[0008] Further, in the step S2, the mass ratio of soybean lecithin, cholesterol and perilla aldehyde is 7:2:1; the volume ratio of chloroform to methanol in the mixed solvent is 2:1.

[0009] Further, in the step S3, the formula of the culture medium is as follows: NaCl 10g / L, MgCl2·6H2O 0.2g / L, CaCl2·2H2O 0.1g / L, NH4Cl 0.5g / L, KCl 0.5g / L, KH2PO4 0.5g / L, Na2S·9H2O 0.48g / L, sodium acetate 2g / L, vitamin 1mg / L, biotin 0.1mg / L, trace elements 1mL / L, pH 7.0-7.5, sterilized at 121°C for 20min.

[0010] Further, in the step S41, the mass ratio of xanthan gum, vitamin E and water is 0.1:0.05:50; In the step S42, the mass ratio of premixing auxiliary agent, Rhodovulum sulfidophilum fermentation broth, perilla aldehyde nanoliposome and ε-polylysine microsphere is 0.3:6:1.6:2.4; the particle size of the composite microspheres is 150-250nm.

[0011] Further, in the step S51, the solid-liquid ratio of high-methoxyl pectin to acetic acid buffer is 1g:100mL; the mass ratio of microcrystalline cellulose, genipin and high-methoxyl pectin is 0.5:0.05:1; In the step S52, the solid-liquid ratio of the compound powder and 60% ethanol aqueous solution is 0.2g:10mL; In the step S53, the electrostatic spraying voltage is 15kV, the nozzle diameter is 0.3mm, and the spraying flow rate is 0.5mL / min.

[0012] The application further provides a green compound fungicide for preventing and treating apple mildew heart disease, which is prepared by the method.

[0013] Further, the application method of the compound fungicide is as follows: the compound fungicide is mixed with 500 times of water by mass, stirred for 5 minutes, and used immediately; the use period and the amount of fungicide are as follows: Apple initial flowering period and full flowering period: whole tree spraying is performed once, the flower column and calyx are mainly sprayed, and the spraying amount is 50-70L / acre each time; Apple young fruit period: the fruit surface, fruit stem and residual calyx are sprayed twice, the interval between each spraying is 10 days, and the spraying amount is 100-150L / acre each time.

[0014] Further, the application of the compound fungicide further includes the following synergistic management measures: the residual flowers and diseased fruits in the orchard are removed 3 days before spraying to reduce the source of bacteria; the spraying is avoided during the peak period of honeybee visiting flowers to protect pollinating insects; and the bagging is completed within 24 hours after the second spraying in the young fruit period.

[0015] Compared with the prior art, the application has the following beneficial effects: In the present application, the epsilon-polylysine is a cationic antibacterial peptide, which destroys the fungal cell membrane and leads to the leakage of intracellular substances; the perillaldehyde is a plant-derived aldehyde, which inhibits the synthesis of the mycotoxin and interferes with the growth of the hyphal tip; the Rhodovulum sulfidophilum fermentation broth contains various active substances (such as pigments, polysaccharides, coenzyme Q10, IAA, 5-amino levulinic acid, etc.), which have the effects of directly inhibiting the pathogenic bacteria and inducing systemic resistance. After the compounding by the method of the present application, on the one hand, the cellulase secreted by the pathogenic bacteria specifically degrades the coating of the microspheres, triggers the release of the microspheres, reduces the non-target exposure, realizes the localized release of the disease spot and the targeted delivery of the drug; on the other hand, after the destruction of the cell membrane by the epsilon-polylysine, the perillaldehyde is more easily entered into the bacteria and interferes with the synthesis of the mycotoxin; the combination of the liposome and the microbial fermentation broth solves the problems of the easy volatilization of the plant essential oil and the low survival rate of the microorganisms. By loading the epsilon-PL on the sodium alginate-chitosan microspheres, the antibacterial peptide can be released slowly, and the effective period is prolonged; by wrapping the perillaldehyde with the nano-liposome, the stability of the perillaldehyde can be improved, and the perillaldehyde can be released to the infection site of the pathogenic bacteria; by compounding the photosynthetic bacterial metabolites and the plant essential oil, the antibacterial effect is improved, the ecological niche is competed, and the colonization of the pathogenic bacteria is reduced. In addition, all the components of the compounding fungicide of the present application are derived from nature or microbial fermentation, and the nano-carrier is biodegradable and has no effect on bees and soil microorganisms, which has the green safety. In summary, the compounding fungicide of the present application can realize the prevention and treatment effect of 1+1>2 through the synergistic effect of multiple mechanisms (targeted delivery + direct inhibition + induced resistance + ecological occupation), and has the characteristics of green safety, high efficiency and stability. DETAILED DESCRIPTION

[0016] In order to make the purpose and advantages of the present application more clear and explicit, the present application is further described in detail below in combination with examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0017] The instruments, reagents, materials and the like involved in the following examples, if not specifically stated, are all conventional instruments, reagents, materials and the like in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods and the like involved in the following examples, if not specifically stated, are all conventional experimental methods, detection methods and the like in the prior art.

[0018] The formula of the Rhodovulum sulfidophilum culture medium in the following examples is as follows: NaCl 10 g / L, MgCl2·6H2O 0.2 g / L, CaCl2·2H2O 0.1 g / L, NH4Cl 0.5 g / L, KCl 0.5 g / L, KH2PO4 0.5 g / L, Na2S·9H2O 0.48 g / L, sodium acetate 2 g / L, vitamin 1 mg / L, biotin 0.1 mg / L, trace elements 1 mL / L, pH 7.0-7.5, sterilized at 121 ℃ for 20 min. When preparing, first dissolve the inorganic salts in deionized water, adjust the pH to 7.0-7.5, and then add deionized water to 1 L; sterilize at 121 ℃ for 20 min; Na2S·9H2O (sulfur source), vitamin (B1), biotin, trace elements (SL-10 formula) need to be added to the culture medium after separate filtration sterilization, and gently shake; pass high-purity nitrogen for 20 min to remove dissolved oxygen, and then seal immediately.

[0019] Example 1 The present embodiment provides a preparation method of a green compound fungicide for preventing and treating apple mycotic heart disease, which specifically comprises the following steps: S1: preparing ε-polylysine (ε-PL) microspheres: S11: dissolving 50 g of ε-polylysine in 950 g of 0.1 M phosphate buffer (pH 6.5) to obtain a 5% ε-polylysine solution; S12: dissolving 120 g of sodium alginate in 9880 g of deionized water, and stirring at 60 ℃ and 500 rpm for 30 min until completely dissolved, and then cooling to 25 ℃ to obtain a 1.2% sodium alginate solution; S13: uniformly mixing 50 g of chitosan, 50 g of acetic acid and 9900 g of water to obtain a 0.5% chitosan solution, and adjusting the pH to 5; S14: mixing the ε-polylysine solution and the sodium alginate solution in a mass ratio of 1:1, and stirring at 300 rpm for 30 min to obtain a mixed solution; S15: using a syringe (needle 22G, inner diameter 0.41 mm) to drop the mixed solution into a 1.5% CaCl2 solution (mixed solution:CaCl2 solution = 1:15, v / v) at a speed of 5 mL / min, and standing for 30 min to form calcium cross-linked microspheres; suspending the microspheres in 5 times the mass of the chitosan solution, stirring at 150 rpm for 1 h, centrifugal washing 3 times, pre-freezing at -40 ℃ for 24 h, and vacuum freeze-drying for 48 h to obtain a white powder, which is ε-polylysine microspheres (≈200 nm); S2: Preparation of perillyl alcohol nanoliposomes: 70 g of soybean lecithin, 20 g of cholesterol, and 10 g of perillyl alcohol were dissolved in 1000 mL of a chloroform-methanol (2:1, v / v) mixed solvent, and a uniform lipid film was formed by rotary evaporation (removal of solvent) at 40°C and 50 rpm. 1000 mL of PBS (pH 7.0) was added, vortexed for 5 min, and ultrasonicated for 10 min at 50 W in an ice bath. The perillyl alcohol nanoliposomes (≈150 nm) were obtained by filtration through a 0.22 μm filter membrane, and stored at 4°C; S3: Preparation of Rhodovulum sulfidophilum fermentation broth: Take one vial of freeze-dried powder, add 0.3 mL of sterile normal saline to the strain tube, and mix gently until uniform. Inoculate the liquid medium with a 5% inoculum, and ferment at 30°C under 2000 lux light for 2-3 days until the OD 600 =0.5-0.8. Transfer the culture into fresh medium with a 10% inoculum, and expand the culture at 30°C under 2000 lux light for 3-5 days until the OD 600 =1.5-2.0. Centrifuge at 4°C and 8000 rpm for 15 min, take the supernatant, filter through a 0.22 μm filter, and obtain the Rhodovulum sulfidophilum fermentation broth; S4: Preparation of finished product: S41: Dissolve 10 g of xanthan gum in 5000 g of water at 60°C, and after cooling, add 5 g of vitamin E to obtain a premixing aid; S42: Add the Rhodovulum sulfidophilum fermentation broth to the premixing aid, stir at 200 rpm for 5 min, then add the perillyl alcohol nanoliposomes, stir at 300 rpm for 10 min, and then add the ε-polylysine microspheres (mass ratio of 0.3:6:1.6:2.4), stir at 150 rpm for 15 min at low speed. Finally, high-pressure homogenization (50 MPa, 3 cycles) is used to ensure uniform particle size, and the composite microspheres (particle size of 150-250 nm) are obtained. Adjust the pH to 6.0±0.2 with 0.1 M NaOH or HCl; S5: Enzyme-sensitive coating: S51: Preparation of pectin-cellulase substrate polymer: Dissolve pectin: Dissolve high-methoxyl pectin in 0.1 M pH 5.0 acetic acid buffer (solid-liquid ratio of 1 g:100 mL), and completely dissolve at 60°C and 500 rpm magnetic stirring for 2 h; Disperse cellulose: Add microcrystalline cellulose (pectin:MCC=1:0.5) to the above solution, and uniformly suspend by ultrasonic treatment at 300 W for 10 min (pulse mode of 5 s on / 5 s off); Crosslinking reaction: Add genipin (pectin:genipin=1:0.05), and react at 50°C for 3 h (pH 5.0); Purification and drying: centrifugation of the reaction solution at 8000 rpm for 10 min, discarding the supernatant, washing the precipitate with ethanol for 3 times, freezing at -50℃ and drying for 24 h to obtain the complex powder; S52: Preparation of the spraying solution: dispersing the above complex powder in 60% ethanol aqueous solution (solid-liquid ratio 0.2 g: 10 mL), ultrasonic treatment at 200 W for 5 min to obtain a uniform suspension; S53: Spraying: uniformly spraying the spraying solution on the surface of the composite microspheres by using an electrostatic spraying instrument (voltage 15 kV, nozzle diameter 0.3 mm, flow rate 0.5 mL / min, nozzle-substrate distance 10 cm, temperature 25℃, humidity ≤40%), vacuum drying at 40℃ for 2 h after spraying to form an enzyme-sensitive coating.

[0020] The key parameters of the green complex bactericide prepared were detected, the particle size distribution (dynamic light scattering particle size analyzer) was 150-250 nm; the encapsulation efficiency (ultrafiltration-HPLC method) was ε-PL≥85%, perillaldehyde≥90%; the stability (54℃, 14 days) was active loss≤10%.

[0021] Application method: dilute the complex bactericide with 500 times mass of clean water, stir for 5 min, and use immediately; the use period and the amount of drug are: Apple initial flowering period and full flowering period: spray the whole tree once, focus on spraying the flower column and calyx, and the spraying amount is 50-70 L / acre each time; Apple young fruit period: spray the fruit surface, fruit stem and residual calyx twice, with an interval of 10 days each time, and the spraying amount is 100-150 L / acre each time.

[0022] Synergistic management measures: remove the residual flowers and diseased fruits in the orchard 3 days before spraying to reduce the source of bacteria; avoid the peak period of honeybee visiting flowers when spraying during the flowering period to protect pollinating insects; complete the bagging within 24 hours after the second spraying during the young fruit period.

[0023] Comparative Example 1 Conventional chemical control.

[0024] Comparative Example 2 No control (blank control).

[0025] Experimental Example 1 Evaluation of control effect Apple mildew heart disease was prevented according to the methods of the above Example 1 and Comparative Examples 1-2, respectively. At the harvest period, 500 fruits were randomly taken for each method, and the occurrence of mildew heart disease was checked (judgment standard: normal appearance of fruit, no obvious disease spots, and mildew, browning or rotting in the heart chamber after the fruit was cut vertically, which may be accompanied by white, gray, pink or black mold), the disease rate was counted, and the control effect was calculated: control effect (%) = 1- (treatment group incidence rate / control group incidence rate) x 100%, and the results are shown in Table 1.

[0026] Table 1 Rate of apple fruit infected with core rot

[0027] As can be seen from Table 1, the green compound fungicide prepared in Example 1 has a significant control effect on apple core rot, which is significantly higher than chemical control and the blank control. It is also safe for apple tree growth and no adverse reactions were found in the field.

[0028] Comparative Example 3 ε-PL microspheres were used alone.

[0029] Comparative Example 4 Perillaldehyde nanoliposomes were used alone.

[0030] Comparative Example 5 The fermentation broth of R. sulfidophilus was used alone.

[0031] Comparative Example 6 ε-PL microspheres, perillaldehyde nanoliposomes and fermentation broth of Rhodopseudomonas thiophilus are simply physically mixed for use.

[0032] Experimental Example 2 Antibacterial Activity Test 1. Pathogen culture Alternaria alternata ( Alternaria alternata ), Pink Trichothecene ( Trichothecium roseum ) were cultured on PDA plates at 25°C for 5 days.

[0033] 2. Antibacterial activity test Each plate was inoculated with a suspension of pathogenic bacteria (1 × 10 6 spores / mL), 200 μL of the treatment solutions of Example 1 (compound group), Comparative Examples 3-5 (single use groups), and Comparative Example 6 (physical mixing group) were added respectively (the concentrations of the corresponding components in each treatment group were consistent), and the cells were cultured at 25°C for 48 h. The diameters of the inhibition zones (mm) were measured. The results are shown in Table 2.

[0034] Table 2 Comparison of antibacterial activity

[0035] Note: * Indicates that the difference between the combination group and the single use group is significant ( P <0.05), # Indicates that the difference between the compound group and the physical mixing group is significant ( P <0.05). As can be seen from Table 2, the inhibition zone of the compound group against Alternaria alternata and Trichothecene rosea was significantly greater than that of each single group and the physical mixing group ( P <0.05), indicating that the active ingredients have a synergistic effect after compounding, and the inhibitory effect on the two pathogens is not a simple addition of the active ingredients.

[0036] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a green compound fungicide for preventing and treating apple core rot, characterized in that: The specific steps include: S1: Preparation of ε-polylysine microspheres: S11: dissolving ε-polylysine in 0.1 M phosphate buffer to obtain a 5% ε-polylysine solution; S12: Sodium alginate was dissolved in deionized water at 60°C and 500 rpm with magnetic stirring for 30 min until completely dissolved, and then cooled to 25°C to obtain a 1.2% sodium alginate solution; S13: mixing chitosan, acetic acid and water to obtain a chitosan solution with a concentration of 0.5%, and adjusting the pH to 5; S14: mixing the ε-polylysine solution and the sodium alginate solution, and magnetically stirring at 300 rpm for 30 min to obtain a mixed solution; S15: Use a syringe to drop the mixed solution into a 1.5% CaCl2 solution at a rate of 5 mL / min, let it stand and solidify for 30 minutes to form calcium cross-linked microspheres; suspend the microspheres in the chitosan solution, stir at 150 rpm for 1 hour, centrifuge and wash three times, pre-freeze at -40°C for 24 hours, and vacuum freeze-dry for 48 hours to obtain a white powder, which is ε-polylysine microspheres; S2: Preparation of perillaldehyde nanoliposomes: Soybean lecithin, cholesterol, and perillaldehyde were dissolved in 10 volumes of a chloroform-methanol mixed solvent and rotary evaporated at 50 rpm at 40°C to form a uniform lipid film. PBS was added, vortexed for 5 minutes, and sonicated in an ice bath at 50W for 10 minutes. The perillaldehyde nanoliposomes were filtered through a 0.22 μm filter and stored at 4°C. S3: Prepare the fermentation broth of R. thiophilus: Take 1 vial of freeze-dried powder, add 0.3 mL of sterile saline to the culture tube, gently flick until mixed, inoculate 5% of the inoculum into the liquid culture medium, and ferment at 30°C and 2000 lux for 2-3 days until the OD 600 =0.5-0.8; transfer 10% of the inoculum into fresh culture medium and expand the culture at 30℃ and 2000lux for 3-5 days until OD 600 =1.5-2.0, 4°C, 8000 rpm centrifugation for 15 min, take the supernatant, filter through 0.22 μm, and obtain the fermentation broth of R. sulfidophilus; S4: Finished product compounding: S41: dissolving xanthan gum and water at 60° C., cooling, and adding vitamin E to obtain a premixed auxiliary; S42: Add the fermentation broth of R. thiophilum to the premixed additive, stir at 200 rpm for 5 minutes, then add perillaldehyde nanoliposomes, stir at 300 rpm for 10 minutes, then add ε-polylysine microspheres, stir at a low speed of 150 rpm for 15 minutes; finally, high-pressure homogenization is performed to ensure uniform particle size to obtain composite microspheres, and the pH is adjusted to 6.0±0.2; S5: Enzyme-sensitive coating: S51: Preparation of pectin-cellulase substrate polymer: Dissolve pectin: Dissolve high methoxyl pectin in 0.1 M pH 5.0 acetic acid buffer at 60°C and 500 rpm with magnetic stirring for 2 h until completely dissolved. Disperse cellulose: add microcrystalline cellulose and sonicate at 300W for 10 minutes until uniformly suspended; Cross-linking reaction: add genipin and react at 50℃ for 3h; Purification and drying: The reaction solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, the precipitate was washed three times with ethanol, and freeze-dried at -50°C for 24 h to obtain the complex powder; S52: Preparation of spray solution: The above composite powder was dispersed in 60% ethanol aqueous solution and ultrasonicated at 200W for 5 minutes to obtain a uniform suspension; S53: Spraying: Use an electrostatic sprayer to evenly spray the spray liquid on the surface of the composite microspheres. After spraying, vacuum dry at 40° C. for 2 hours to form an enzyme-sensitive coating.

2. The method for preparing a green compound fungicide for preventing and treating apple core rot according to claim 1, characterized in that: In step S13, the mass ratio of chitosan to acetic acid is 1:1; In step S14, the mass ratio of the ε-polylysine solution to the sodium alginate solution is 1:1; In step S15, the volume ratio of the mixed solution to the CaCl2 solution is 1:15; the mass ratio of the microspheres to the chitosan solution is 1:

5.

3. The method for preparing a green compound fungicide for preventing and treating apple core rot according to claim 1, characterized in that: In step S2, the mass ratio of soybean lecithin, cholesterol, and perillaldehyde is 7:2:1; and the volume ratio of chloroform to methanol in the mixed solvent is 2:

1.

4. The method for preparing a green compound fungicide for preventing and treating apple core rot according to claim 1, characterized in that: In step S3, the culture medium has the following formula: NaCl 10 g / L, MgCl2·6H2O 0.2 g / L, CaCl2·2H2O 0.1 g / L, NH4Cl 0.5 g / L, KCl 0.5 g / L, KH2PO4 0.5 g / L, Na2S·9H2O 0.48 g / L, sodium acetate 2 g / L, vitamins 1 mg / L, biotin 0.1 mg / L, trace elements 1 mL / L, pH 7.0-7.5, and sterilization at 121°C for 20 min.

5. The method for preparing a green compound fungicide for preventing and treating apple core rot according to claim 1, characterized in that: In step S41, the mass ratio of xanthan gum, vitamin E and water is 0.1:0.05:50; In step S42, the mass ratio of the premixed auxiliary agent, the fermentation broth of R. thiophilus, the perillaldehyde nanoliposomes, and the ε-polylysine microspheres is 0.3:6:1.6:2.4; and the particle size of the composite microspheres is 150-250 nm.

6. The method for preparing a green compound fungicide for preventing and treating apple core rot according to claim 1, characterized in that: In step S51, the solid-liquid ratio of high methoxy pectin to acetic acid buffer is 1 g:100 mL; the mass ratio of microcrystalline cellulose, genipin and high methoxy pectin is 0.5:0.05:1; In step S52, the solid-to-liquid ratio of the composite powder to the 60% ethanol aqueous solution is 0.2 g:10 mL; In step S53, the electrostatic spraying voltage is 15 kV, the nozzle diameter is 0.3 mm, and the spraying flow rate is 0.5 mL / min.

7. A green compound fungicide prepared by the method for preparing a green compound fungicide for preventing and treating apple core rot according to any one of claims 1 to 6.

8. The green compound fungicide for preventing and treating apple core rot according to claim 7, characterized in that: The application method of the compound fungicide is as follows: add the compound fungicide to 500 times the mass of clean water, stir for 5 minutes, and use immediately after preparation; the application period and dosage are as follows: During the early and full flowering period of apples: spray the whole tree once, focusing on the style and calyx, with a spraying rate of 50-70L / mu each time; Young apple fruit stage: spray the fruit surface, fruit stalk and residual calyx twice, with an interval of 10 days between each spraying, and the amount of each spraying is 100~150L / mu.

9. The green compound fungicide for preventing and treating apple core rot according to claim 8, characterized in that: The application of the compound fungicide also includes coordinated management measures: removing dead flowers and diseased fruits in the orchard 3 days before spraying to reduce the base number of fungal sources; avoiding the peak period of bee visits when spraying during the flowering period to protect pollinating insects; and completing bagging within 24 hours after the second spraying during the young fruit stage.

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