Application of benzocaine in the prevention of postharvest apple blue mould disease

Abstract

The application discloses application of benzocaine in prevention and treatment of postharvest apple black spot disease. 50 The application finds that the fruit extract of Schisandra chinensis has an antibacterial effect on Alternaria alternata, and the phenylpropanoid compound benzocaine in the fruit extract has a strong inhibitory effect on the Alternaria alternata, with an EC 50 of 42.99 mg / L. The benzocaine accounts for 14.19% in the fruit extract of Schisandra chinensis, and is a main antibacterial component in the fruit of Schisandra chinensis. The benzocaine can effectively prevent the occurrence of apple black spot disease, has a certain treatment effect on the apple black spot disease, and mainly has a protection effect.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, and in particular relates to the application of benzocaine in the prevention and control of postharvest apple black spot disease. Background Technology

[0002] apple( Malus domestica Apple is a fruit widely cultivated in northern my country. Apple black spot disease is caused by *Alternaria alternata*, also known as fine *Alternaria* or *Alternaria*. Alternaria alternata Apple black spot disease, caused by fungal infection (Fr.) Keissl., can infect fruit both before and after harvest for commercial processing. During the later stages of storage, black spots appear on the fruit surface, leading to rot. The affected areas are densely covered with black or dark green mold, topped with white flocculent material, impacting storage, transportation, and sales, causing significant economic losses. Currently, synthetic fungicides are widely used to control the disease. While synthetic fungicides are effective in controlling post-harvest diseases, they pose risks to human health and the environment and can lead to drug resistance in pathogens. Therefore, researching and utilizing alternatives to existing chemical fungicides for the prevention and control of fruit and vegetable diseases is imperative. Benzocaine, also known as ethyl para-aminobenzoate or ethyl 4-aminobenzoate, has the chemical formula C9H. 11 NO2, belonging to the phenylpropanoid class of compounds, is poorly soluble in water but readily soluble in organic solvents such as alcohols, ethers, and chloroform. Benzocaine is commonly used as a local anesthetic and clinically for analgesia and antipruritic effects on wounds, ulcers, burns, skin fissures, and hemorrhoids. Due to its antibacterial, anti-inflammatory, and anticancer properties, benzocaine is widely used in medicine. Compound benzocaine gel is effective in treating oral ulcers and toothaches and exhibits antibacterial activity against Staphylococcus aureus. Currently, there are no reports of benzocaine being used for the control of plant pathogens. Summary of the Invention

[0003] The purpose of this invention is to provide the application of benzocaine in the control of postharvest apple black spot disease. Benzocaine has a strong inhibitory effect on Alternaria alternifolia (Alternaria alternifolia). Alternaria alternata The effects of (Fr.) Keissl. can effectively prevent the occurrence of apple black spot disease and have a certain therapeutic effect on apple black spot disease, with the main function being protection.

[0004] In a first aspect, the present invention protects the use of benzocaine in any of the following: A1) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.); A2) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.) Mycelial growth; A3) promotes the growth of Alternaria alternata ( Alternaria alternata(Fr.) Keissl.) The surface of the hyphae is ruptured.

[0005] Secondly, this invention protects the use of benzocaine in the prevention and / or treatment of plant fungal diseases, wherein the fungus is Alternaria alternata (…). Alternaria alternata (Fr.) Keissl.).

[0006] Thirdly, the present invention protects the use of benzocaine in the prevention and / or treatment of postharvest apple black spot disease.

[0007] In some embodiments of the present invention, the application method is spraying in the form of a benzocaine solution with a concentration of 42.99 mg / L. To improve the solubility of benzocaine in water, preferably, the benzocaine solution is composed of benzocaine, dimethylformamide (DMF), emulsifier OP-10, and water. In some embodiments of the present invention, the mass concentration of dimethylformamide in the benzocaine solution is 5%; the mass concentration of the emulsifier in the benzocaine solution is 10%.

[0008] Fourthly, the present invention protects the use of compositions containing benzocaine in any of the following: A1) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.) A2) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.) Mycelial growth; A3) promotes the growth of Alternaria alternata ( Alternaria alternata (Fr.) Keissl.) The surface of the hyphae is ruptured.

[0009] Fifthly, the present invention protects the use of compositions containing benzocaine in the prevention and / or treatment of fungal diseases of plants, wherein the fungus is Alternaria alternata (…). Alternaria alternata (Fr.) Keissl.).

[0010] In a sixth aspect, the present invention protects the use of compositions containing benzocaine in the prevention and / or treatment of postharvest apple black spot disease.

[0011] Seventhly, the present invention protects the use of the Schisandra chinensis fruit extract containing benzocaine in any of the following: A1) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.); A2) Inhibits Alternaria alternifolia ( Alternaria alternata (Fr.) Keissl.) Mycelial growth; A3) promotes the growth of Alternaria alternata ( Alternaria alternata (Fr.) Keissl.) The surface of the hyphae is ruptured.

[0012] Eighthly, the present invention protects the use of a Schisandra chinensis fruit extract containing benzocaine in the prevention and / or treatment of plant fungal diseases, wherein the fungus is Alternaria alternata (…). Alternaria alternata (Fr.) Keissl.).

[0013] Ninthly, the present invention protects the use of Schisandra chinensis fruit extract containing benzocaine in the prevention and / or treatment of postharvest apple black spot disease.

[0014] In the above applications, the Schisandra chinensis fruit extract is the Schisandra chinensis plant of the genus Schisandra in the family Magnoliaceae. S chisandra chinensis (Turcz.) Baill . The fruit extract of Schisandra chinensis can be obtained directly from commercial sources or according to preparation methods disclosed in the literature. It is understood that the Schisandra chinensis fruit extract can be any commercially available product or obtained through any extraction and separation method, as long as the extract contains benzocaine, and the benzocaine content is sufficient to ensure the effectiveness of the Schisandra chinensis fruit extract against Alternaria alternata (Alternaria alternata). Alternaria alternata (Fr.) Keissl.) has ideal antibacterial effect or ideal control effect on plant fungal diseases including apple black spot. For example, the extract of Schisandra chinensis fruit is an ethanol extract of Schisandra chinensis fruit. In some embodiments of the present invention, the preparation method of the extract of Schisandra chinensis fruit includes: 1) extracting Schisandra chinensis fruit powder with 70% ethanol three times at a material-to-liquid ratio of 1:20, 60 min / time, combining the filtrates, and filtering to obtain crude extract of Schisandra chinensis fruit; 2) extracting with petroleum ether at a material-to-liquid ratio of 1:20 twice at a material-to-liquid ratio of 4 h / time, defatting, extracting and separating, retaining the ethanol layer, and rotary evaporating at 60°C water bath until no ethanol odor is found to obtain the extract of Schisandra chinensis fruit.

[0015] In the above applications, the interspaced algae ( Alternaria alternata (Fr.) Keissl.) refers to pathogens that can cause black spot disease, including but not limited to the pathogens of apple black spot disease, such as the Alternaria alternata strain YLPGl7092603-1 or YLPGl7092603-2 isolated from diseased apple fruits.

[0016] The present invention has the following beneficial effects: This invention discovers that the extract of Schisandra chinensis fruit has the effect of... A. alternata It has antibacterial effects; at a concentration of 8 mg / mL, the extract of Schisandra chinensis fruit has antibacterial properties. A. alternataThe inhibition rate was 91.9%. Compound detection was performed using liquid chromatography-mass spectrometry (LC-MS). The extract of Schisandra chinensis fruit contained a large number of organic acid compounds and phenylpropanoid compounds, among which benzocaine, a phenylpropanoid compound, exhibited strong inhibitory activity. A. alternata The effect of EC 50 The concentration was 42.99 mg / L; and benzocaine accounted for 14.19% of the extract of Schisandra chinensis fruit, which is the main antibacterial component in Schisandra chinensis fruit; the results showed that benzocaine can effectively prevent the occurrence of apple black spot disease and has a certain therapeutic effect on apple black spot disease, mainly with a protective effect. Attached Figure Description

[0017] Figure 1 The types and relative contents of compounds in the Schisandra chinensis fruit extract in Example 1 of this invention are shown.

[0018] Figure 2 The benzocaine in Example 2 of this invention A. alternata The mycelial growth inhibition effect is shown in the figure. (a) Control (benzocaine concentration 0 mg / L); (b) Benzocaine concentration 25 mg / L; (c) Benzocaine concentration 50 mg / L; (d) Benzocaine concentration 100 mg / L; (e) Benzocaine concentration 200 mg / L; (f) Benzocaine concentration 400 mg / L.

[0019] Figure 3 Different processing methods in Embodiment 2 of the present invention A. alternata Scanning electron micrographs of hyphae morphology, A - control; B - treatment with 42.99 mg / L benzocaine.

[0020] Figure 4 The diagram shows the effect of benzocaine on postharvest apple black spot disease in Example 3 of the present invention. a-control group, b-treatment group, c-protective group. Detailed Implementation

[0021] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0023] The following examples contain the extract of Schisandra chinensis fruit: Schisandra chinensis, annual fruits of Schisandra chinensis collected from a Schisandra chinensis plantation (124.8°E; 40.8°N) (http: / / www.hywwz.cn; Kuandian County, Liaoning Province, China).

[0024] The washed Schisandra chinensis fruits were dried in an electric constant-temperature drying oven at 60℃ until constant weight. They were then pulverized into powder using a multi-functional pulverizer. The active substances in the Schisandra chinensis fruits were extracted using ethanol extraction. 100g of the Schisandra chinensis fruit powder was weighed and placed in an Erlenmeyer flask. At a material-to-liquid ratio of 1:20, it was extracted three times with 70% ethanol, 60 min each time, and the filtrates were combined. After filtration, a crude extract of Schisandra chinensis fruits was obtained. This crude extract was then extracted twice with petroleum ether at a material-to-liquid ratio of 1:20, 4 h each time, and defatted. The extraction and separation were completed, retaining the ethanol layer. The extract was then rotary evaporated at 60℃ in a water bath until no ethanol odor remained, yielding the Schisandra chinensis fruit extract, which was weighed.

[0025] The benzocaine (analytical grade, 99% purity) used in the following examples was produced by Shanghai Maclean Biochemical Technology Co., Ltd., with product code Lot#:C12579078.

[0026] The following examples of interspaced spores ( Alternaria alternata (Fr.) Keissl.) YLPGl7092603-1 is recorded in the literature "Wu Haiyan, Li Changshui, Ma Qingzhou, Yin Xinming, Geng Yuehua, Zhang Meng. Alternaria ( Alternaria alternata Study on pathogenicity differentiation in jujube, pear and apple fruits [J]. Tianjin Agricultural Sciences Tianjin Agricultural Sciences In 2023, 29(5): 47-50, the public can obtain it from Henan Agricultural University. This biological material is only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0027] The apples used in the following examples are all Fuji apples, purchased from Beinong Supermarket and stored at 4°C until use.

[0028] Example 1: Evaluation of the effects of Schisandra chinensis fruit extract on... A. alternata Antibacterial effect and component analysis (1) Antibacterial test Experimental methods: Will A. alternata Activated, the fungi were inoculated onto PDA medium and incubated at 28°C for 72 h. The extract of Schisandra chinensis fruit was diluted with sterile water to PDA medium containing concentrations of 0, 0.5, 1, 2, 4, and 8 mg / mL, with 0 serving as the control group. After pouring into petri dishes and allowing to cool and solidify, activated plant pathogenic fungi with a diameter of 6 mm were inoculated and incubated at 28°C for 72 h. The diameter of the colonies was measured using the cross-crossing method, and the mycelial growth inhibition rate was calculated using the formula.

[0029]

[0030] Experimental results: The inhibition rates of Schisandra chinensis fruit extract at concentrations of 0, 0.5, 1, 2, 4 and 8 mg / mL were 0, 11.5, 31.1, 51.4, 70.3 and 91.9%, respectively.

[0031] The results showed that the extract of Schisandra chinensis fruit had an effect on... A. alternata It has antibacterial effects; at a concentration of 8 mg / mL, the extract of Schisandra chinensis fruit has antibacterial properties. A. alternata The inhibition rate was 91.9%.

[0032] (2) Compound extraction Accurately weigh 200 μg of Schisandra chinensis fruit extract into a 2 mL centrifuge tube, add 600 µL of methanol (1:3 w / v) containing 2-chloro-L-phenylalanine (4 ppm), vortex for 30 s; add 100 mg of glass beads, place in a tissue homogenizer, and homogenize at 60 Hz for 90 s; sonicate at room temperature for 15 min; centrifuge at 12000 rpm at 4 ℃ for 10 min, collect the supernatant and filter through a 0.22 μm membrane, add the filtrate to the detection bottle for LC-MS detection.

[0033] (3) On-machine testing Chromatographic conditions were as follows: ultra-high performance liquid chromatography (UHPLC) system, (2.1 × 150 mm, 1.8 µm) column, flow rate of 0.25 mL / min, column temperature of 40 °C, and injection volume of 2 μL. Positive ion mode was used. The mobile phase consisted of 0.1% formic acid acetonitrile (C) and 0.1% formic acid water (D). The gradient elution program was: 0–1 min, 2% °C; 1–9 min, 2%–50% °C; 9–12 min, 50%–98% °C; 12–13.5 min, 98% °C; 13.5–14 min, 98%–2% °C; 14–20 min, 2% °C. In negative ion mode, the mobile phase consisted of acetonitrile (A) and 5 mM ammonium formate water (B), with the gradient elution program as follows: 0–1 min, 2% A; 1–9 min, 2%–50% A; 9–12 min, 50%–98% A; 12–13.5 min, 98% A; 13.5–14 min, 98%–2% A; 14–17 min, 2% A. Mass spectrometry conditions were as follows: mass spectrometer detector, electrospray ionization (ESI) source, and data acquisition in both positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 30 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325 °C. A first-stage full scan was performed at a resolution of 60,000 m / z, with a first-stage ion scan range of 100–1000 m / z. Second-stage fragmentation was performed using HCD at a collision voltage of 30% and a second-stage resolution of 15,000 m / z. The first four ions acquired were fragmented, and unnecessary MS / MS information was removed using dynamic exclusion.

[0034] (4) Compound identification Compound identification was first confirmed based on precise molecular weight (molecular weight error <= 30 ppm). Subsequently, the compounds were confirmed and annotated using MS / MS fragmentation mode in the Human Metabolome Database (http: / / www.hmdb.ca), METLIN (http: / / metlin.scripps.edu), Massbank (http: / / www.massbank.jp / ), LipidMaps (http: / / www.lipidmaps.org), mzClond (https: / / www.mzcloud.org), and Panomick's self-built standard database.

[0035] A total of 157 compounds were detected in the extract of Schisandra chinensis fruit using liquid chromatography-mass spectrometry (LC-MS). These compounds were broadly classified into 11 categories, among which (… Figure 1 Organic acids were the most abundant, with 56 types, accounting for 43.09% of the total detected amount; followed by phenylpropanoids, with 6 types, accounting for 21.77%; sugars were the third most abundant, with 12 types, accounting for 9.55%; and phenols ranked fourth, accounting for 8.49%. In addition to the above compounds, the extract of Schisandra chinensis fruit also contained polyols (3.81%), flavonoids (1.10%), alkaloids (1.16%), glycerides (1.96%), amines (0.66%), terpenes (1.23%), and inorganic salts (4.41%).

[0036] Based on the relative content of compounds, there are 22 compounds with a content of more than 1% (as shown in Table 1). Among them, two compounds have a content of more than 10%, namely nonanoic acid and benzocaine, accounting for 18.93% and 14.19% of the total detected, respectively.

[0037] Table 1. Summary of compounds with a content of 1% or higher in Schisandra chinensis extract.

[0038] Example 2: Evaluation of benzocaine's effect on... A. alternata Antibacterial effect (1) Antibacterial effect Experimental Methods: Antibacterial activity was determined using the growth rate method. Benzocaine is poorly soluble in water; therefore, 5 wt% dimethylformamide (DMF) and 10 wt% OP-10 emulsifier were used as adjuvants, and distilled water was added to prepare the required concentration. A PDA culture medium with a specific concentration gradient was then prepared. After pouring the medium into petri dishes and allowing it to cool and solidify, activated plant pathogenic fungi with a mycelial growth diameter of 6 mm were inoculated. The mixture was incubated at 28℃ for 72 h. The colony diameter was measured using the cross-crossing method, and the antibacterial effect was calculated based on the mycelial growth inhibition rate.

[0039]

[0040] Experimental results: The inhibition rate results are shown in Table 2; the virulence regression equation is 1.2652x + 6.7291 (R²). 2 =0.9847), EC 50 The concentration was 42.99 mg / L. The inhibitory effect was as follows: Figure 2 As shown, by Figure 2 It can be seen that as the concentration of benzocaine increases, the effect on... A. alternate The inhibition rate of benzocaine also increased with increasing inhibition rate, showing a clear linear relationship. A. alternata The inhibitory effect was significantly higher than that of Schisandra chinensis fruit extract, and 43.77 times that of Schisandra chinensis fruit extract. Table 2. Benzocaine's effect on... A. alternata Mycelial growth inhibitory activity

[0041] (2) Benzocaine to A. alternata Electron microscopic observation of the influence of hyphal morphology A. alternata After 3 days of solid culture, in A. alternata A 0.6 cm diameter bacterial disc was taken from the edge of the colony and placed in the center of PDA solid medium in a 90 mm Petri dish. The concentration was determined based on the half-maximal effect (EC50). 50The mycelium was treated with 42.99 mg / L benzocaine, and the control group received the same concentration. The mycelium was fixed with 2.5% glutaraldehyde (4℃ for 24 h). The mycelium was then washed three times with 0.1 mol / L pH 7.4 phosphate buffer. It was then dehydrated sequentially with 30%, 50%, 70%, 80%, 90%, and 95% ethanol solutions, followed by three dehydration cycles with anhydrous ethanol for 8 min each. It was then treated twice with tert-butanol for approximately 8 min each time. The mycelium was then frozen at -20℃ for 20 min and dried using a critical dryer. Finally, gold was sputtered: one side of a carbon conductive tape was attached to a petri dish, and the other side was attached to the dried mycelium. After sputtering, the samples were observed using SEM, and images were acquired for analysis.

[0042] Scanning electron microscopy results showed that, via EC 50 There was a significant difference between the treatment group (B) and the control group (A) obtained from treatment with a concentration of (42.99 mg / L). Figure 3 The hyphae in the control group were long, plump, and relatively regular in shape. The hyphae in the treatment group were significantly different from those in the control group. The hyphae in the treatment group had broken surfaces, irregular shapes, and were fragmented.

[0043] Example 3: Analysis of the control effect of benzocaine on postharvest apple black spot disease (1) Preventive effect Experimental Method: Select uniformly sized apples. Wipe the apple surface three times with cotton soaked in 75% ethanol in a clean bench. Spray 50 μL of benzocaine (42.99 mg / L) evenly onto the surface of the Fuji apples, forming a 3 cm circle with the inoculation point as the diameter. The control was sprayed with an equal volume of sterile water. Let stand for 12 hours. A. alternata Activate, inoculate into PDA medium, and incubate at 28℃ for 72 hours for later use. Take a 0.6cm diameter sample... A. alternata Inoculate the fungal patch onto the spraying center; wrap the apple with plastic wrap and incubate at 28°C, measure the diameter of the lesion on the 7th day and calculate the control effect.

[0044]

[0045] Experimental results: Inoculation A. alternata Seven days later, the black spot disease on the apples treated with benzocaine spread. Figure 4 a) The lesion diameter reaches 3.55 cm; after inoculation A. alternata Previously, 50 μL (42.99 mg / L) of benzocaine was sprayed. Figure 4 c) Apple black spot disease was effectively prevented, with an average lesion diameter of 0.6 cm, indicating that benzocaine has a protective effect against apple black spot disease, with an efficacy of 83.1%.

[0046] (2) Therapeutic effect Experimental method: Inoculate the surface of Fuji apples with a seed at a depth of 0.6 cm. A. alternata Wrap the lesions in plastic wrap and let them stand at 28°C for 12 hours. Spray 50 μL of benzocaine (42.99 mg / L) onto the inoculation site, forming a 3 cm circle with the inoculation site as the diameter. For the control, spray an equal amount of sterile water. Wrap the lesions in plastic wrap again. Measure the diameter of the lesions on the 7th day and calculate the treatment effect.

[0047]

[0048] A. alternata After invasion, spray benzocaine before the apples show symptoms. Figure 4 b) The average lesion diameter was 2.8 cm, indicating that benzocaine has a therapeutic effect on apple black spot disease, with an efficacy of 21.13%.

[0049] Therefore, benzocaine can effectively prevent the occurrence of apple black spot disease and has a certain therapeutic effect on apple black spot disease, with the main effect being protection.

[0050] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of benzocaine in inhibiting Alternaria alternata, characterized in that, The applications include, Inhibit the growth of Alternaria alternata hyphae; or, It causes the surface of the hyphae of Alternaria alternata to rupture.

2. The use of benzocaine in the prevention and / or treatment of fungal diseases of plants, wherein the fungus is Alternaria alternifolia.

3. The use of benzocaine in the prevention and / or treatment of postharvest apple black spot disease, characterized in that, The apple black spot disease is caused by Alternaria alternata.

4. The application according to claim 3, characterized in that: The application method is to spray in the form of benzocaine solution with a concentration of 42.99 mg / L.

5. The use of a composition containing benzocaine in inhibiting Alternaria alternata, characterized in that, The applications include, Inhibit the growth of Alternaria alternata hyphae; or, It causes the surface of the hyphae of Alternaria alternata to rupture.

6. The use of a composition containing benzocaine in the prevention and / or treatment of fungal diseases of plants, wherein the fungus is Alternaria alternata.

7. The use of a composition containing benzocaine in the prevention and / or treatment of postharvest apple black spot disease, characterized in that, The apple black spot disease is caused by Alternaria alternata.

8. The application of Schisandra chinensis fruit extract containing benzocaine in the inhibition of Alternaria alternata, characterized in that, The applications include, Inhibit the growth of Alternaria alternata hyphae; or, It causes the surface of the hyphae of Alternaria alternata to rupture.

9. The use of Schisandra chinensis fruit extract containing benzocaine in the prevention and / or treatment of plant fungal diseases, wherein the fungus is Alternaria alternata.

10. The use of Schisandra chinensis fruit extract containing benzocaine in the prevention and / or treatment of postharvest apple black spot disease, characterized in that, The apple black spot disease is caused by Alternaria alternata.

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