A novel preparation for inhibiting botrytis cinerea and its application

Abstract

The application discloses a novel preparation for inhibiting botrytis cinerea and application thereof, and relates to the technical field of bacteriostatic drugs, and has the technical scheme as follows: the active ingredient of the preparation is composed of isoeugenol and bacillus velezensis fermentation liquor.The half bacteriostatic concentration EC50 value of the novel preparation reaches 59.7 ug / mL, and the bacteriostatic capacity is higher than that of the existing technology of bacto in the same concentration.Based on the influence on the membrane permeability of filamentous fungi, the application can synergistically inhibit the pathogenic activity of postharvest fungi of fruits, and is expected to be used for preparing a novel preparation for preventing and treating high-sugar fruits after harvesting.The application is simple to use, environment-friendly, can be directly smeared on the surface of fruits in the planting stage, can not be directly contacted with the fruits in the storage period, and can provide the bacteriostatic capacity for the storage period by using the fumigation mode.The application provides a new choice for the biological prevention of fruits, and has good economic benefits and social benefits.

Description

Technical Field

[0001] This invention relates to the field of antibacterial drug technology, and more specifically, to a novel formulation for inhibiting Botrytis cinerea and its application. Background Technology

[0002] Botrytis cinerea is a very common and highly damaging plant pathogenic fungus. The disease it causes is commonly known as gray mold. Due to its extremely wide host range, strong adaptability, and high destructive power, Botrytis cinerea is one of the most important and troublesome pathogens in global agricultural production, especially in the Vitaceae family and the Vitis genus.

[0003] Shine Muscat (Vitis labrusca × vinifera 'Shine Muscat') is a hybrid grape variety originating in Japan and developed in Europe and America. It possesses a unique rose aroma and a soluble solids content of around 20%, sometimes reaching as high as 26%, resulting in a sweet taste, thin skin without astringency, and firm fruit. However, the high sugar content provides a high-quality carbon source for microbial growth, which presents a challenge during storage. Gray mold is the most damaging and troublesome fungal disease affecting Shine Muscat, especially during flowering and fruit ripening. Infected Shine Muscat flowers exhibit a scalded appearance, soft rot, and a dense layer of gray mold. The fruit surface develops brown, sunken lesions, later forming a grayish-brown mold layer, leading to extensive fruit rot.

[0004] Currently, traditional methods for suppressing Botrytis cinerea (gray mold) mainly rely on chemical pesticides. However, because Botrytis cinerea readily develops resistance, these methods are ineffective and unsustainable. Commonly used chemical fungicides such as carbendazim, iprodione, boscalid, and propiconazole mostly have single sites of action. Botrytis cinerea reproduces rapidly and undergoes frequent genetic mutations, easily developing resistance through gene mutations, leading to reduced or even complete ineffectiveness of the pesticides. Compared to traditional chemical methods, using plant essential oils to suppress Botrytis cinerea offers significant advantages, primarily in terms of environmental friendliness, resistance to pesticides, and multiple antibacterial properties.

[0005] Isoeugenol, belonging to the phenylpropanoid class of compounds, can be considered a derivative of o-methoxyphenol. It is a naturally occurring aromatic compound with applications in fragrances, food, and pharmaceuticals due to its unique aroma and diverse biological activities. It can insert itself into the cell membrane of fungi, interacting with lipid and protein components. This interaction disrupts the orderly structure of the cell membrane, increasing its permeability and leading to the leakage of intracellular ions and essential substances, ultimately causing cell death. Furthermore, isoeugenol can also affect protein synthesis, and its non-specific antibacterial mechanism makes it difficult for drug-resistant bacteria to develop.

[0006] Bacillus velezensis exhibits outstanding advantages as a biocontrol bacterium in inhibiting filamentous fungi. Its comprehensive capabilities far surpass those of traditional chemical fungicides and are even superior to many other biological control strains. Its multi-target mechanism makes it difficult for drug resistance to develop, and it is easily fermented on a large scale. It possesses excellent colonization capabilities and combines preventative, curative, and disease-inducing functions. It is safe for humans, animals, and the environment, meeting the requirements of green agriculture. Summary of the Invention

[0007] The purpose of this invention is to provide a novel formulation for inhibiting Botrytis cinerea and its application, which organically combines isoeugenol and Bacillus belye to achieve a synergistic antibacterial effect through complementary advantages, thus providing technical support for new preservative formulations.

[0008] The above-mentioned technical objective of this invention is achieved through the following technical solution: a novel formulation for inhibiting Botrytis cinerea, the formulation comprising an oil phase and an aqueous phase, wherein the active ingredient in the oil phase is isoeugenol, and the active ingredient in the aqueous phase is Bacillus belysium BV-J11 fermentation broth. The Bacillus belysium BV-J11 is derived from Hubei Blue Valley Microbial Technology Co., Ltd., and is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan City, Hubei Province, on August 17, 2023, with accession number CCTCC NO: M20231502.

[0009] The present invention is further configured such that the volume ratio of the oil phase to the water phase in the formulation is 1:1.

[0010] The present invention is further configured such that the preparation method of the formulation is as follows:

[0011] (1) Add rhamnolipin to isoeugenol and shake thoroughly to prepare the oil phase;

[0012] (2) While stirring, add gellan gum to an equal volume of Bacillus vesiculosus BV-J11 fermentation broth of oil phase to prepare an aqueous phase;

[0013] (3) After the oil phase and the water phase are preheated at 50°C for 5 min, the oil phase is slowly added to the water phase and stirred at 50°C and 80 r / min for 6 h to obtain an emulsion, which is the preparation.

[0014] The present invention is further configured such that the rhamnolipid in the formulation accounts for 5% (v / v) in the oil phase.

[0015] The present invention is further configured such that the gellan gum in the formulation accounts for 0.3% (v / v) in the aqueous phase.

[0016] The present invention further provides the application of this novel formulation in inhibiting gray mold in Sunshine Rose.

[0017] The present invention further provides the application of this formulation in inhibiting gray mold during the cultivation of high-sugar fruits.

[0018] The present invention further provides the application of this formulation in inhibiting gray mold during the storage of high-sugar fruits.

[0019] In summary, the present invention has the following beneficial effects:

[0020] The novel formulation of this invention utilizes multiple natural antibacterial agents to synergistically inhibit bacteria, with isoeugenol having a half-maximal inhibitory concentration (EC50) of 76 ug / mL against Botrytis cinerea.

[0021] This invention provides a potential antifungal agent and application method for gray mold. Compared with traditional plant essential oil preparations, this invention has good water solubility and strong dilution stability, which lays a good foundation for the development and application of natural pesticides.

[0022] Bacillus belye typically has an unpleasant odor, caused by the decomposition of proteins in the culture medium, producing substances such as hydrogen sulfide and methanethiol. Isoeugenol, as a natural fragrance, can effectively mask this odor, thus eliminating negative sensory impacts in practical applications.

[0023] The novel formulation of this invention is primarily applied via direct contact, meaning it is directly applied to the surface of plants or fruits infected with Botrytis cinerea. In addition, this invention also possesses certain volatile antibacterial activity, providing a new strategy for preserving fruit in warehouses. Attached Figure Description

[0024] Figure 1 This is the antibacterial effect curve of isoeugenol against Botrytis cinerea in this invention;

[0025] Figure 2 This invention demonstrates the inhibitory effect of Bacillus belye fermentation broth treated with different methods on Botrytis cinerea.

[0026] Figure 3 The curves show the antibacterial effects of different concentrations of Bacillus belye fermentation broth on Botrytis cinerea in this invention.

[0027] Figure 4 This is a stability evaluation of the formulation in this invention;

[0028] Figure 5 This shows the encapsulation effect of the formulation in this invention (unit: μm).

[0029] Figure 6 This is the antibacterial effect curve of Botrytis cinerea after different concentration dilutions in this invention;

[0030] Figure 7 This invention demonstrates the inhibitory effect of different concentrations of the product on Botrytis cinerea;

[0031] Figure 8 This is the inhibitory effect curve of the positive control compound, Beco-3, on Botrytis cinerea in this invention;

[0032] Figure 9 This invention describes the in vivo antibacterial effects of different treatment methods.

[0033] Figure 10 These are images of the product appearance of this invention. Detailed Implementation

[0034] The following is in conjunction with the appendix Figure 1-9 The present invention will be described in further detail below.

[0035] The culture medium formulation used in this embodiment of the invention is as follows:

[0036] PDA medium: Take 200g of diced potatoes, add an appropriate amount of distilled water, boil for 30 minutes, filter through 8 layers of gauze to remove residue, take the clear liquid, add distilled water to make up to 1L, add 20g of glucose, add 20g of agar powder to the solid medium, sterilize at 121℃ for 20 minutes.

[0037] Optimize YPD medium: Take 28.5g of glucose, 15.2g of yeast extract, and 8.3g of peptone, and add distilled water to a final volume of 1L.

[0038] Example 1: Determination of the half-maximal inhibitory concentration (EC50) of isoeugenol

[0039] Isoeugenol was diluted to concentrations of 20, 40, 60, 80, and 100 μg / mL, respectively, and the concentration of Botrytis cinerea spore suspension was 10. 7 CFU / mL. Use a 10mm punch to collect mycelial cakes from the edge of Botrytis cinerea colonies cultured for 3-5 days. Use the mycelial cake method to conduct antibacterial experiments. Each treatment is repeated three times. Incubate upside down at 28℃ for 3-5 days, measure the colony diameter, and calculate the antibacterial rate.

[0040] Antibacterial rate (%) =

[0041] The results are shown in Table 1:

[0042]

[0043] Table 1. Antibacterial rate of different concentrations of isoeugenol against Botrytis cinerea

[0044] Note: The inhibition rate is the average of three replicates.

[0045] Example 2: Tracing the active substances of Bacillus belyssus

[0046] Bacillus belye was streaked onto YPD agar plates. After incubation at 37°C for 24 hours, single colonies from the YPD agar plates were inoculated onto YPD liquid agar and incubated for another 24 hours to obtain the fermentation broth. The fermentation broth was then subjected to centrifugation, acid precipitation, and no treatment. The centrifuged fermentation broth contained no Bacillus belye, and the acid-precipitated fermentation broth contained no Bacillus belye or lipopeptides. The spore concentration was set at 10... 7 100 μL of CFU / mL *Botrytis cinerea* was evenly spread onto PDA medium. A 15 mm diameter filter paper disc was placed in the center of the medium. 50 μL of the fermentation broth from the above treatment was added to each filter paper disc. The mixture was incubated at 28°C in the dark for 3-5 days. The antibacterial effect was observed, and the results are shown in Table 2.

[0047]

[0048] Table 2. Inhibitory effects of different fermentation broth treatments on Botrytis cinerea

[0049] Note: The inhibition rate is the average of three replicates.

[0050] This example shows that the fermentation broth with acid precipitation to remove protein has no antibacterial effect, while the fermentation broth after removing the bacteria still has an antibacterial effect. This suggests that the active substances in the Bacillus brevis fermentation broth are mainly in its fermentation broth.

[0051] Example 3: Evaluation of the antibacterial efficacy of Bacillus belyss BV-J11 fermentation broth

[0052] The Bacillus belye fermentation broth was diluted to concentrations of 20, 40, 60, 80, and 100 mg / mL, respectively, and the concentration of Botrytis cinerea spore suspension was 10. 7 CFU / mL. Antibacterial experiments were conducted using the double-layer plate method, with three replicates for each treatment. The plates were incubated upside down at 28°C for 3-5 days, and the colony diameter was measured to calculate the inhibition rate.

[0053] Antibacterial rate (%) =

[0054] The results are shown in Table 3:

[0055]

[0056] Table 3. Inhibition rate of Botrytis cinerea fermentation broth at different concentrations against Botrytis cinerea

[0057] Note: The antibacterial rate in the table is the average of three experiments.

[0058] The above two examples demonstrate that both isoeugenol, a plant essential oil, and Bacillus vesiculosus have antibacterial effects against Botrytis cinerea. Therefore, it is considered to combine the two to test whether they have a synergistic effect. However, fermentation broth using water as a solvent is difficult to mix with plant essential oils. This invention uses an encapsulation method to mix isoeugenol with Bacillus vesiculosus fermentation broth.

[0059] Example 4: Evaluation of the water solubility and stability of the emulsion

[0060] Isoeugenol belongs to the phenylpropanoid class of compounds, which are insoluble in water, making their performance unstable in practical applications. By replicating existing technology, Tween 80 was used as an emulsifier to dissolve isoeugenol, achieving a solubility of over 80%. Its stability was determined by heating in a water bath at 40°C for 24 hours, using the following formula:

[0061]

[0062] The results are shown in Table 4:

[0063]

[0064] Table 4. Stability evaluation of existing technology emulsions

[0065] As can be seen from Table 4, the stability of emulsification using Tween 80 as an emulsifier decreases significantly over time. To solve this problem, the present invention encapsulates the active material, mainly non-polar material, on the basis of using an emulsifier.

[0066] 5% (v / v) rhamnolipid was added to isoeugenol and shaken thoroughly to prepare the oil phase. 0.3% (m / m) gellan gum was added to an equal volume of Bacillus bereaves BV-J11 fermentation broth while stirring to prepare the aqueous phase. The oil and aqueous phases were preheated at 50°C for 5 min, then the oil phase was slowly added to the aqueous phase, and the mixture was stirred at 50°C and 80 rpm for 6 h to obtain an emulsion.

[0067] The prepared emulsion was heated in a water bath at 40°C for 24 hours, and its stability was determined according to the following formula:

[0068]

[0069] The stability of the emulsion obtained by this method is shown in Table 5.

[0070]

[0071] Table 5. Stability evaluation of the emulsion of the present invention

[0072] Example 5: Evaluation of the product's antibacterial ability

[0073] The finished emulsion was serially diluted to 15, 30, 45, 60, and 75 μg / mL, and antibacterial experiments were conducted using the double-layer plate method. Each experiment was repeated three times, with 50 μL of drug per experimental group. The antibacterial rate was calculated using the same method as in Example 1, and the results are shown in Table 6.

[0074]

[0075] Table 6. Antibacterial rates of products at different concentrations

[0076] Note: The antibacterial rate in the table is the average of three experiments.

[0077] The antibacterial rate of the positive control, Beclomethasone, is shown in Table 7.

[0078]

[0079] Table 7. Antibacterial rates of different concentrations of Becochloridium

[0080] Note: The antibacterial rate in the table is the average of three experiments.

[0081] Example 6: Evaluation of Usage Method

[0082] The concentration of Botrytis cinerea spore suspension was 10. 7 CFU / mL. Using a 10mm punch, collect mycelial cakes from the edge of *Botrytis cinerea* colonies cultured for 3-5 days. Inoculate these cakes onto PDA medium. Separately, apply 70ug / mL of isoeugenol to a 15mm diameter filter paper disc, ensuring the filter paper does not directly contact the medium. Seal the disc with plastic wrap and incubate at 28°C in the dark for 3 days. Perform three replicates to confirm reproducibility. The direct contact method also involves three groups, using the same treatment method as in Example 1.

[0083] The results are shown in Table 8:

[0084]

[0085] Table 8. Inhibitory effects of direct contact and fumigation methods on Botrytis cinerea

[0086] Note: The inhibition rate is the average of three replicates.

[0087] Example 7: In vivo antifungal activity assay

[0088] The effects of this study on the development of fungi on the surface of Shine Muscat grapes (Vitis labrusca × vinifera 'Shine Muscat') and common grapes were observed. Each fruit was placed in a moist, 1-liter sealed container at a temperature of 4–8°C. Ten fruits were used for each treatment. Filter paper discs (15 mm in diameter) were attached to the container lids, with 100 μL of the present invention applied to the filter paper. In the blank control, after the pathogen completely covered the fruit surface, the area of ​​lesions on the treated fruit was measured (in millimeters) using calipers. The experiment was conducted three times. The proportion of rotten lesions was calculated using the following formula:

[0089] Proportion of rotten lesions (mm²) = π × a × b

[0090] Relative inhibition rate (%) = [(C - T) / C] × 100

[0091] Where a and b represent the vertical and horizontal lesion diameters (mm) of the same source point, respectively, and C and T represent the average proportion of rotten lesions (mm²) of the control group and the treatment group, respectively.

[0092] The invention was also applied to the surface of Shine Muscat (Vitis Shine Muscat) fruits, with each fruit placed in a moistened 1-liter sealed container. Measurements were taken after the pathogen had completely covered the fruit surface (as in the blank control). Ten fruits were used for each treatment. The relative inhibition rate was calculated as described above. The results are shown in Table 9.

[0093]

[0094] Table 9. Antibacterial rate of emulsions at different concentrations

[0095] Example 8: Application Security Evaluation

[0096] Ten volunteers were recruited to evaluate the Shine Muscat grapes in the control and experimental groups on a nine-point scale after washing. The evaluation criteria are as follows:

[0097] Color: The naturalness and freshness of the fruit's surface color (1-9 points, the higher the score, the closer the color is to the ideal state).

[0098] Odor: The acceptability of odor in the treatment group and the control group (1-9 points, with high scores indicating no irritating odor).

[0099] Overall acceptability: Based on a comprehensive evaluation of appearance, smell, and texture, 10 volunteers independently rated the overall quality. The results are shown in Table 10:

[0100]

[0101] Table 10. Nine-point scale evaluation of control group and experimental group

[0102] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A novel formulation for inhibiting Botrytis cinerea, characterized by: The formulation is divided into an oil phase and an aqueous phase. The active ingredient in the oil phase is isoeugenol, and the active ingredient in the aqueous phase is Bacillus belysium BV-J11 fermentation broth. The preservation number of Bacillus belysium BV-J11 is CCTCC NO:M20231502.

2. A novel formulation for inhibiting Botrytis cinerea according to claim 1, characterized by: The volume ratio of the oil phase to the aqueous phase in the formulation is 1:

1.

3. The novel formulation for inhibiting Botrytis cinerea according to claim 1, characterized in that: The preparation method of the formulation is as follows: (1) Add rhamnolipin to isoeugenol and shake thoroughly to prepare the oil phase; (2) While stirring, add gellan gum to an equal volume of Bacillus vesiculosus BV-J11 fermentation broth of oil phase to prepare an aqueous phase; (3) After the oil phase and the water phase are preheated at 50°C for 5 min, the oil phase is slowly added to the water phase and stirred at 50°C and 80 r / min for 6 h to obtain an emulsion, which is the preparation.

4. The novel formulation for inhibiting Botrytis cinerea according to claim 3, characterized in that: In the formulation, rhamnolipids account for 5% (v / v) in the oil phase.

5. A novel formulation for inhibiting Botrytis cinerea according to claim 3, characterized in that: In the formulation, gellan gum accounts for 0.3% (v / v) in the aqueous phase.

6. The application of the novel formulation for inhibiting Botrytis cinerea according to claim 1, characterized in that: The application of the formulation in inhibiting gray mold in Sunshine Rose.

7. The application of the novel formulation for inhibiting Botrytis cinerea according to claim 1, characterized in that: The application of the formulation in inhibiting gray mold during the cultivation of high-sugar fruits.

8. The application of the novel formulation for inhibiting Botrytis cinerea according to claim 1, characterized in that: The application of the formulation in inhibiting gray mold during the storage of high-sugar fruits.

Images