Application of bacillus velezensis P15 fungicide in preservation of picked strawberries

Abstract

The invention relates to the technical field of biological prevention and control, and discloses application of a bacillus velezensis P15 fungicide in preservation of harvested strawberries, and the application comprises the step of applying the bacillus velezensis P15 fungicide to the harvested strawberries so as to prevent and control strawberry postharvest diseases caused by pathogenic fungi. The preservation number of the bacillus velezensis P15 is CGMCC (China General Microbiological Culture Collection Center) NO.31995. The bacillus velezensis P15 microbial inoculum is used for preservation of picked strawberries, can efficiently inhibit more than 90% of picked pathogenic bacteria such as botrytis cinerea and the like, has a control effect superior to that of prochloraz, and does not enable the pathogenic bacteria to resist drugs after being used for a long time; the strawberry preservative is harmless to human, animals, plants and the environment, has no microbial agent residue, can be used for fresh keeping of green and organic strawberries, solves the food safety problem of chemical preservative residue, and improves the acceptability of consumers.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically the application of Bacillus vesiculosus P15 in the postharvest preservation of strawberries. Background Technology

[0002] Modern intensive agricultural production methods and the long-term, large-scale application of chemical pesticides have led to increasingly serious crop diseases. This phenomenon is particularly prominent in the post-harvest storage stage of fruits and vegetables. Strawberries, as a fruit with high nutritional value but delicate skin and short shelf life, are highly susceptible to infection by pathogenic fungi during post-harvest storage and transportation, leading to rot and spoilage, and causing huge economic losses. The pathogens that cause post-harvest rot in strawberries mainly include Botrytis cinerea (causing gray mold), Colletotrichum spp. (causing anthracnose), Fusarium spp., and Rhizopus spp., among others. Among them, gray mold caused by Botrytis cinerea is one of the most important post-harvest diseases of strawberries.

[0003] Currently, the main method for controlling postharvest diseases in strawberries is the use of chemical fungicides, such as imazalil and pyrimethanil. While this method is effective for a certain period, long-term reliance on chemical control has led to a series of serious problems. First, pathogens easily develop resistance under the continuous selective pressure of chemical agents, resulting in a continuous decline in the efficacy of the agents. Second, the residue of chemical pesticides on the fruit surface is increasingly raising consumer concerns about food safety, and it also does not meet the requirements of green agriculture and sustainable development. In addition, international standards for pesticide residue limits in agricultural products are becoming increasingly stringent, posing a technical barrier to my country's strawberry export trade. Therefore, we propose the application of Bacillus vesiculosus P15 inoculant in postharvest preservation of strawberries to address the aforementioned problems. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of Bacillus vesiculosus P15 inoculant in postharvest preservation of strawberries. This solves the problem that controlling postharvest diseases in strawberries mainly relies on chemical fungicides such as prochloraz and pyrimethanil, but this method has significant drawbacks: long-term use leads to increased drug resistance in pathogens, resulting in a continuous decline in control efficacy; at the same time, pesticide residues not only raise food safety concerns and violate the concept of green agriculture, but also become a problem restricting my country's strawberry export trade due to increasingly stringent international residue limits.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: the application of Bacillus vesiculosus P15 inoculant in postharvest preservation of strawberries, the application comprising applying the Bacillus vesiculosus P15 inoculant to postharvest strawberries to prevent postharvest diseases of strawberries caused by pathogenic fungi; the preservation number of Bacillus vesiculosus P15 is CGMCCNO.31995.

[0006] Preferably, the pathogenic fungus includes one or more of Botrytis cinerea, Colletotrichum spp, Fusarium spp, and Rhizopus spp.

[0007] Preferably, the Bacillus vesiculus P15 inoculant is the fermentation broth of Bacillus vesiculus P15.

[0008] Preferably, the Bacillus belyss P15 inoculum further includes an adjuvant, which includes one of a stabilizer and a dispersant.

[0009] Preferably, the stabilizer is glycerol or sucrose, and the amount added is 2%-5% of the total mass of the bacterial agent; the dispersant is Tween 80 or gum arabic, and the amount added is 0.1%-1% of the total mass of the bacterial agent, so as to improve the adhesion of the bacterial agent to the surface of strawberry fruit and the shelf life of live bacteria.

[0010] This invention provides a method for post-harvest preservation of strawberries, including the aforementioned Bacillus leuciscin P15 inoculant, comprising the following steps:

[0011] S1. Select undamaged, disease-free, and uniformly sized strawberry fruits. Rinse them gently with sterile water 2-3 times to remove surface impurities, and then place them on sterile filter paper to air dry naturally until there is no obvious moisture on the surface.

[0012] S2. Prepare a bacterial treatment solution using Bacillus vesiculosus P15 inoculum, wherein the viable bacterial concentration of Bacillus vesiculosus P15 is 1×10⁻⁶. 7 CFU / mL to 1×10 10 CFU / mL was used to treat strawberry fruits;

[0013] S3. Store the treated strawberry fruits in an environment with a temperature of 4-8℃ and a relative humidity of 85%-90%.

[0014] Preferably, the treatment includes: spraying method: spraying the fungicide treatment solution evenly onto the surface of strawberry fruit, with a spraying amount of fungicide treatment solution of 10mL-20mL per 100g of strawberry fruit;

[0015] Soaking method: Immerse the strawberry fruit completely in the bacterial treatment solution for 10-30 minutes;

[0016] Atomization method: The bacterial agent treatment liquid is atomized by atomization equipment, so that the atomized bacterial agent is evenly attached to the surface of the strawberry fruit.

[0017] Beneficial effects

[0018] This invention provides an application of Bacillus vesiculosus P15 inoculant in postharvest preservation of strawberries. Compared with existing technologies, it has the following advantages:

[0019] The application of Bacillus vesiculosus P15 in postharvest preservation of strawberries demonstrates its highly effective inhibitory effect on major postharvest pathogenic fungi of strawberries, including Botrytis cinerea, Anthracnose, Fusarium, and Rhizopus. Its antibacterial spectrum covers more than 90% of postharvest pathogens in strawberries, outperforming the traditional chemical preservative imazalil. Furthermore, long-term use does not lead to drug resistance in pathogens, and it poses no harm to humans, animals, plants, or the environment. The product has been tested and found to have no residues, making it suitable for postharvest preservation of green and organic strawberries. This addresses the food safety concerns associated with chemical preservative residues and increases consumer acceptance. Attached Figure Description

[0020] Figure 1 The images show the colony, cell morphology, and Gram staining of Bacillus belyssus P15 of this invention; A shows the colony of Bacillus belyssus P15, and B shows the cell morphology and Gram staining.

[0021] Figure 2 The table shows the experimental results of this invention.

[0022] Figure 3 This is a comparison table of strawberry fruit quality indicators for this invention;

[0023] Figure 4 This is a flowchart of a method for post-harvest preservation of strawberries according to the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] like Figure 1 As shown:

[0026] The application of Bacillus vesiculosus P15 in the postharvest preservation of strawberries includes applying the Bacillus vesiculosus P15 inoculo to postharvest strawberries to prevent postharvest diseases of strawberries caused by pathogenic fungi; the preservation number of Bacillus vesiculosus P15 is CGMCC NO.31995;

[0027] Morphological characteristics: When cultured on LB solid medium at 25℃ for 24-48 hours, the colonies are initially white and smooth, gradually developing wrinkles in the later stages; the cells are rod-shaped, 1.5-5.2 μm in length and 0.4-0.6 μm in diameter, arranged singly or in pairs, and Gram-positive.

[0028] It can form spores, tolerate a temperature range of 4-45℃, and a pH tolerance range of 4.0-9.0. It can reproduce normally in the low temperature and high humidity environment of strawberry post-harvest storage.

[0029] Disease control is achieved through two pathways: first, by secreting lipopeptide antibacterial substances such as ituronidin and surfactants, which directly inhibit the growth of pathogenic mycelia and spore germination; second, by producing volatile organic compounds, such as 2,3-butanediol, which inhibit the spread of pathogens through airborne transmission and simultaneously induce disease resistance in strawberry fruits.

[0030] Bacillus belyssus P15 inoculant includes two core types, which can be selected according to the actual application scenario:

[0031] Liquid bacterial agent, the active ingredient of which is the fermentation broth of Bacillus vesiculosus P15, with a viable cell concentration of not less than 1×10⁻⁶. 7 CFU / mL;

[0032] Solid microbial agents or wettable powders: Based on liquid microbial agents, with the addition of carriers such as diatomaceous earth and maltodextrin, and adjuvants, they are prepared by spray drying, with a viable bacteria concentration of not less than 1×10⁻⁶. 10 CFU / mL, which facilitates long-term storage and transportation;

[0033] To improve the adhesion, water resistance, and shelf life of the inoculant on the surface of strawberry fruit, the following adjuvants are added to the inoculant, with each component calculated as a percentage by mass:

[0034] The stabilizer is glycerol or sucrose, and its addition amount is 2%-5%. It can maintain the osmotic pressure of the bacterial cells, slow down the inactivation of the bacterial cells during low-temperature storage, and extend the shelf life of the bacterial agent.

[0035] The dispersant used is Tween 80 or gum arabic, with an addition amount of 0.1%-1%. It can reduce the surface tension of the inoculant, so that the inoculant is evenly dispersed on the surface of the strawberry, avoiding damage to the fruit caused by excessive local concentration. At the same time, it can improve water resistance and reduce the loss of inoculant caused by washing or condensation.

[0036] The synergist uses 0.5% trehalose, with an addition amount of 0.5%-1%, which can further improve the spore germination rate, form a dominant microbial community on the surface of strawberries, and enhance the antibacterial effect;

[0037] Taking liquid bacterial agent as an example, the preparation steps are as follows:

[0038] Bacillus belye P15 strain was inoculated from slant culture medium into LB liquid medium (10 g / L tryptone, 10 g / L NaCl, 5 g / L yeast extract, pH 7.0) and cultured at 25°C and 150 rpm for 12 hours to prepare a seed culture (viable cell concentration approximately 1 × 10⁻⁶). 8 CFU / mL);

[0039] Inoculate the seed culture into the fermenter (LB liquid medium) at a 1% (v / v) inoculation rate, control the temperature at 25-28℃, the stirring speed at 200 rpm, and the aeration rate at 1:1.5 (v / v·min), and incubate for 24 hours. After fermentation, the viable cell concentration can reach 1×10⁻⁶. 9 -1×10 10 CFU / mL;

[0040] The fermentation broth was filtered through a 300-mesh filter to remove impurities. Stabilizers and dispersants were added according to the formula, and after thorough mixing, the viable cell concentration was tested and adjusted to the target concentration, such as 1×10⁻⁶. 7 -1×10 9 CFU / mL yields the liquid bacterial agent;

[0041] This fungicide is effective in controlling postharvest diseases of strawberries caused by the following pathogenic fungi, and its antibacterial spectrum covers the main postharvest pathogens of strawberries:

[0042] Botrytiscinerea: Inhibition rate ≥75%;

[0043] Anthrax spp.: including Colletotrichum spp., Colletotrichum candida, etc., with an inhibition rate of ≥70%;

[0044] Fusarium spp.: Inhibition rate ≥65%;

[0045] Rhizopus spp.: Inhibition rate ≥60%.

[0046] This method involves mixing liquid bacterial agent with diatomaceous earth at a mass ratio of 1:2, spray drying the mixture to produce a wettable powder, pulverizing it, and then passing it through an 80-mesh sieve to determine the viable bacterial concentration.

[0047] like Figure 4 As shown:

[0048] This invention provides a method for post-harvest preservation of strawberries, comprising the following steps:

[0049] S1. Select strawberry fruits that are free from mechanical damage, pests and diseases, and are of uniform size, and remove overripe and unripe fruits;

[0050] Rinse the fruit gently 2-3 times with sterile water or clean tap water to remove dirt, pesticide residues and other impurities from the surface. Avoid rubbing the fruit vigorously to prevent damage to the skin.

[0051] Place the washed strawberries on sterile filter paper and air dry naturally until there is no obvious moisture on the surface, about 30-60 minutes at room temperature of 25℃, or use cool air at 15-20℃ and wind speed of 0.5m / s to dry them, to prevent moisture residue from promoting the reproduction of pathogens.

[0052] S2. If using liquid bacterial agent, the viable bacteria concentration is 1×10⁻⁶. 9 CFU / mL, diluted with sterile water or clean tap water to a viable bacteria concentration of 1×10⁻⁶. 7 CFU / mL to 1×10 10 CFU / mL, recommended concentration is 1×10⁻⁶ 8 CFU / mL;

[0053] If using a wettable powder, the viable bacteria concentration is 1×10⁻⁶. 9 For CFU / g, first add a small amount of sterile water and stir until completely dissolved, then dilute to the target concentration. During the dilution process, you can stir gently at 50 rpm to avoid vigorous stirring that could cause cell inactivation.

[0054] S3. Select the following treatment method according to the production scale to ensure that the fungicide evenly covers the fruit surface;

[0055] When spraying, use a manual or electric sprayer. Fill the sprayer with the fungicide solution and spray it evenly on the surface of the strawberry fruit. Keep the nozzle 15-20cm away from the fruit. Spray 10-20mL of fungicide solution per 100g of strawberries. After spraying, gently turn the fruit over to ensure that the stem, skin folds and other parts are covered with fungicide to avoid missing any areas.

[0056] When using the soaking method, use a stainless steel or food-grade plastic soaking tank with a stirring device. Completely immerse the strawberries in the bacterial treatment solution for 10-30 minutes, with 20 minutes recommended. Keep stirring during the soaking process to ensure uniform bacterial concentration. Change the bacterial treatment solution once for every 100kg of strawberries treated to avoid cross-contamination by pathogens.

[0057] When using the atomization method, an ultrasonic atomizer is used in conjunction with a sealed treatment chamber. The strawberries are laid flat on a tray and placed in the sealed treatment chamber. The atomizer is then activated to atomize the bacterial treatment solution for 15-30 minutes. After atomization, the chamber is kept sealed for 10 minutes to ensure that the atomized bacterial agent fully adheres to the surface of the fruit. The atomized particles are fine and can evenly cover the surface of the fruit and the gaps between the stems, with no risk of mechanical damage, resulting in the best preservation effect.

[0058] S4. Store the treated strawberries in an environment with a temperature of 4-8℃ and a relative humidity of 85%-90%. It is recommended to store them in an environment with a temperature of 6℃ and a relative humidity of 88%.

[0059] It should be noted that for detailed methods of isolation and screening of Bacillus belyss P15, please refer to the patent literature, publication number: CN119162042A.

[0060] Example 1

[0061] The postharvest preservation effect of the Bacillus vesiliflorus P15 inoculant of this invention on strawberries was investigated through the following comparative experiments, as detailed below:

[0062] Strawberry variety: Hongyan; Inoculum agent: Bacillus berberis P15 liquid inoculum agent of this invention; Control agent: chemical preservative imazalil; Pathogen: Botrytiscinerea strain;

[0063] Three treatment groups were set up, with 100 strawberries (each weighing 20-25g) treated in each group, and the treatment was repeated 3 times. For the blank control group, strawberries were pretreated and then sprayed with sterile water and stored in an environment of 6℃ and 88% relative humidity. For the chemical preservative group (imazalil): strawberries were pretreated and then sprayed with a diluted imazalil solution, and the storage conditions were the same as the control group. For the P15 inoculant group: strawberries were pretreated and then sprayed with a P15 inoculant solution, and the storage conditions were the same as the control group.

[0064] The indicators are determined by storage period, decay rate, disease index, and fruit quality indicators; the specific methods are as follows:

[0065] Storage period: Record the time from the start of storage of strawberries until the rate of decay reaches 20%;

[0066] Rot rate: The number of rotten fruits is counted every 3 days during storage. Rot rate = (number of rotten fruits / total number of fruits) × 100%;

[0067] Disease index: Refer to strawberry gray mold;

[0068] Fruit quality indicators: After 12 days of storage, the firmness, soluble solids content, and vitamin C content of strawberries were measured.

[0069] Test results table, such as Figure 2 As shown:

[0070] Comparison of strawberry fruit quality indicators, such as Figure 3 As shown:

[0071] Results analysis: The storage period of strawberries in the P15 inoculant group reached 16 days, which was 100% longer than the control group and 6.7% longer than the prochloraz group; the rot rate after 12 days of storage was 18.7%, which was 71.5% lower than the control group and 16.1% lower than the prochloraz group; the control effect reached 76.8%, which was significantly better than the prochloraz group, indicating that the P15 inoculant was more effective in controlling postharvest diseases in strawberries. The fruit firmness, soluble solids content, and vitamin C content of strawberries in the P15 inoculant group were significantly higher than those in the control group. The P15 inoculant was slightly higher than that in the control group and the prochloraz group, indicating that while inhibiting pathogens, it can effectively slow down the deterioration of strawberry fruit quality and maintain the taste and nutritional value of the fruit. The strawberries in the P15 inoculant group were tested and found to have no bacterial residue (compliant with GB4789.2 "National Food Safety Standard for Microbiological Examination of Food - Determination of Total Colony Count"), while the strawberries in the prochloraz group were found to have trace amounts of the agent residue (0.02 mg / kg, which, although compliant with national standards, poses a potential health risk), indicating that the P15 inoculant is safer.

[0072] This formulation utilizes Bacillus vesiculosus P15, which exhibits highly effective inhibition against major postharvest pathogens of strawberries, including Botrytis cinerea, Anthracnose, Fusarium, and Rhizopus. Its antibacterial spectrum covers over 90% of postharvest pathogens in strawberries, outperforming the traditional chemical preservative imazalil. Furthermore, long-term use does not lead to antibiotic resistance in pathogens, and it poses no harm to humans, animals, plants, or the environment. The product has been tested and found to have no residues, making it suitable for postharvest preservation of green and organic strawberries. This solution addresses the food safety concerns associated with chemical preservative residues and enhances consumer acceptance.

[0073] It should be noted that the strain B. velezensis P15 provided in this invention was deposited on September 18, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code: 100101), with accession number CGMCC No. 31995, and classified as Bacillus velezensis.

[0074] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of Bacillus velezensis P15 inoculum in postharvest preservation of strawberries, characterized in that: The application comprises applying the Bacillus velezensis P15 agent to postharvest strawberries to prevent and treat postharvest diseases of strawberries caused by pathogenic fungi; the Bacillus velezensis P15 has a preservation number of CGMCC NO.31995.

2. The application of Bacillus velezensis P15 inoculant in the postharvest preservation of strawberries according to claim 1, characterized in that: The pathogenic fungi comprise one or more of Botrytis cinerea, Colletotrichum spp, Fusarium spp and Rhizopus spp.

3. The application of Bacillus velezensis P15 inoculant in the postharvest preservation of strawberries according to claim 1, characterized in that: The Bacillus velezensis P15 agent is a fermentation liquor of the Bacillus velezensis P15.

4. The application of Bacillus velezensis P15 inoculant in the postharvest preservation of strawberries according to claim 1, characterized in that: The Bacillus velezensis P15 agent further comprises an additive, and the additive comprises one of a stabilizer and a dispersant.

5. The use of Bacillus velezensis P15 inoculant in the postharvest preservation of strawberries according to claim 4, characterized in that: The stabilizer is glycerol or sucrose, and the addition amount is 2%-5% of the total mass of the agent; the dispersant is Tween 80 or gum arabic, and the addition amount is 0.1%-1% of the total mass of the agent, so as to improve the adhesion of the agent to the surface of the strawberry fruit and the survival period of the live bacteria.

6. A method for postharvest preservation of strawberry, comprising using the Bacillus velezensis P15 inoculant according to any one of claims 1-5, characterized in that: The method comprises the following steps: S1, selecting undamaged, pest-free and uniform strawberry fruits, washing the strawberry fruits with sterile water for 2-3 times to remove surface impurities, and then placing the strawberry fruits on sterile filter paper to dry naturally until no obvious water is present on the surface; S2, the bacillus velezensis P15 fungicide is prepared into a fungicide treatment liquid, wherein the viable bacterial concentration of the bacillus velezensis P15 is 1×10 7 CFU / mL to 1×10 10 CFU / mL, to treat the strawberry fruits; S3, placing the treated strawberry fruits in an environment with a temperature of 4-8 ℃ and a relative humidity of 85%-90% for storage.

7. The method for postharvest preservation of strawberry according to claim 6, characterized in that: The treatment comprises: a spraying mode: uniformly spraying the agent treatment liquid on the surface of the strawberry fruits, and the spraying amount of the agent treatment liquid per 100 g of strawberry fruits is 10 mL-20 mL; an immersion mode: completely immersing the strawberry fruits in the agent treatment liquid, and the immersion time is 10 min-30 min; an atomization mode: atomizing the agent treatment liquid through atomization equipment, so that the atomized agent is uniformly attached to the surface of the strawberry fruits.

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