Application of cinnamic acid derivatives in prevention and treatment of bacterial perforation disease of peach

Abstract

The application discloses application of cinnamic acid derivatives in prevention and treatment of peach bacterial perforation disease, and utilizes broad-target metabolome to screen differential metabolites of peach leaf blades responding to pathogenic bacteria infection, and finds that p-coumaric acid, methyl caffeate and methyl ferulate have better bacteriostatic activities on the pathogenic bacteria (Xanthomonas arboricola pv. pruni, Xap) of the peach bacterial perforation disease through determination of the bacteriostatic activities of the differential metabolites, and the minimum inhibitory concentrations are 0.0625 mg / mL, 0.0625 mg / mL and 0.125 mg / mL respectively, and the minimum inhibitory concentration of oxytetracycline, a positive control drug, is 0.125 mg / mL. The bacteriostatic activities of the p-coumaric acid and the methyl ferulate are better than that of the oxytetracycline, and the bacteriostatic activity of the methyl caffeate is equivalent to that of the oxytetracycline, so that the p-coumaric acid, the methyl caffeate and the methyl ferulate can be used as the agents for preventing and treating the peach bacterial perforation disease.

Description

[Technical Field]

[0001] This invention belongs to the field of plant-derived fungicides for the prevention and control of bacterial leaf spot in peaches, specifically involving the application of cinnamic acid derivatives in the prevention and control of bacterial leaf spot in peaches. [Background Technology]

[0002] Bacterial leaf spot of peach is a common global disease affecting peach trees and is one of the most important diseases in peach-producing areas of my country. The pathogen is *Xanthomonas arboricola* pv. pruni, Xap, a pathogen that causes leaf blight. This fungus primarily infects leaves, branches, and fruits, entering tissues through stomata and bud scars on leaves and lenticels on fruits. On leaves, it manifests as water-soaked lesions, which later dry and fall off, leaving perforations and causing premature leaf drop. Fruit infection leads to decreased fruit quality, and in some areas, severe infection results in significant yield reduction or even crop failure, causing substantial economic losses. Besides peaches, this pathogen also affects stone fruits such as plums, apricots, cherries, and almonds, and is listed as a quarantine pathogen by the European Union.

[0003] In 2022, my country's peach cultivation area reached 15 million mu (approximately 1 million hectares), accounting for 52.0% of the world's total, and its output reached 15.295 million tons, accounting for 57.96% of the world's total (FAO statistics). China ranked first in both area and output. With the prevalence of peach bacterial spot disease in most peach-producing areas of my country, the demand for effective control agents is increasing. In production, the types of chemical agents available for controlling peach bacterial spot disease are limited. Copper-based agents and antibiotics are widely used and effective agents for controlling peach bacterial spot disease, but their dependence and long-term use have led to increasingly prominent problems such as drug resistance, tree toxicity, environmental pollution, and pesticide residues.

[0004] According to data from the China Pesticide Information Network, as of July 2023, 11,859 fungicides were registered in my country, with approximately 280 agents used to control bacterial diseases, accounting for 2.4% of the total registered fungicide products in the country. These agents contained approximately 32 active ingredients, mainly copper-based fungicides, antibiotics, microbial inoculants, and other types of bactericides. Compared to copper-based fungicides and antibiotics, plant-derived fungicides are more environmentally friendly, safer, and less likely to induce resistance. Currently, several plant-derived fungicides have achieved significant effects in controlling crop diseases, such as eugenol, berberine, osthol, and ethoxyquin.

[0005] Bacterial diseases affect a wide range of crops and cause serious damage. There are few registered control agents and limited registered active ingredients. Furthermore, the routine use of copper-based fungicides and antibiotics has a huge negative impact on the industry. Therefore, the development of plant-derived fungicides is urgent and necessary. [Summary of the Invention]

[0006] In view of the problems that existing technologies for controlling peach bacterial spot disease mainly use copper-based preparations and antibiotics, leading to drug resistance, tree toxicity, environmental pollution, and pesticide residues, this invention provides the application of cinnamic acid derivatives in the control of peach bacterial spot disease. By using broad-target metabolomics analysis to identify differential metabolites in susceptible and resistant peach bacterial spot disease materials in response to Xap infection, and by measuring the antibacterial activity of differential metabolites, antibacterial substances superior to or equivalent to positive control drugs were screened. A total of three cinnamic acid derivatives that inhibit the pathogen of peach bacterial spot disease were found: methyl ferulic acid, p-coumaric acid, and methyl caffeate.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of cinnamic acid derivatives in the prevention and control of bacterial leaf spot of peach, wherein the cinnamic acid derivative is selected from one of methyl ferulic acid (CAS: 2309-07-1), p-coumaric acid (CAS No.: 501-98-4), and methyl caffeate (CAS No.: 3843-74-1);

[0009] The aforementioned peach bacterial spot disease refers to a disease caused by infection of peach trees with the pathogenic fungus Xanthomonas sarboricola pv. Pruni (Xap).

[0010] Furthermore, the application of the cinnamic acid derivatives in the prevention and control of peach bacterial spot disease includes the following: methyl ferulic acid has a minimum inhibitory concentration (MIC) of 0.0625 mg / mL against the pathogen of peach bacterial spot disease; p-coumaric acid has a MIC of 0.0625 mg / mL against the pathogen of peach bacterial spot disease; and methyl caffeate has a MIC of 0.125 mg / mL against the pathogen of peach bacterial spot disease.

[0011] Furthermore, the aforementioned application involves combining an effective amount of cinnamic acid derivatives with pesticide-acceptable excipients or additives to formulate different formulations of drugs suitable for controlling peach bacterial leaf spot.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] This invention relates to the application of cinnamic acid derivatives in the prevention and control of peach bacterial leaf spot. It provides three cinnamic acid derivatives (methyl ferulic acid, p-coumaric acid, and methyl caffeate) with good antibacterial effects. While there are numerous studies on the pharmacological effects of methyl caffeate, a natural product, there are no reports on its application in the prevention and control of peach bacterial leaf spot. These derivatives show significant effects in inhibiting the pathogen of peach bacterial leaf spot, with minimum effective inhibitory concentrations (MICs) of 0.0625, 0.0625, and 0.0125 mg / mL, respectively. The MIC of the positive control oxytetracycline is 0.125 mg / mL. Methyl ferulic acid and p-coumaric acid exhibit superior antibacterial activity compared to the positive control oxytetracycline, while methyl caffeate shows comparable antibacterial activity. These three cinnamic acid derivatives are expected to become effective antibacterial agents. [Attached Image Description]

[0014] Figure 1 This is a K-Means diagram of the differential metabolites of cinnamic acid derivatives in the prevention and control of bacterial leaf spot in peaches, according to an embodiment of the present invention.

[0015] Figure 2 This is an example of the application of cinnamic acid derivatives in the prevention and control of bacterial leaf spot in peaches. The antibacterial effect of cinnamic acid derivatives at a concentration of 2.0 mg / mL compared with the positive control oxytetracycline is shown in the figure (A: methyl ferulic acid; B: p-coumaric acid; C: methyl caffeate; D: positive control drug oxytetracycline; E: 10% DMSO).

Detailed Implementation Methods

[0016] The specific embodiments of the present invention will be further described below with reference to examples.

[0017] Example:

[0018] The application of cinnamic acid derivatives in the prevention and control of bacterial leaf spot in peaches includes the following steps:

[0019] Discovery and antibacterial activity testing of coumaric acid, methyl caffeate, and methyl ferulic acid.

[0020] 1. Materials:

[0021] 1.1 Test materials:

[0022] The peach variety "Yanbao," which is resistant to bacterial spot disease, and the susceptible variety "Frederic" were introduced from the National Peach Germplasm Resource Nursery of the Beijing Academy of Agricultural and Forestry Sciences and planted in Guilin City, Guangxi Province.

[0023] 1.2 Medicines and Reagents:

[0024] DMSO, purchased from Xilong Scientific Co., Ltd.

[0025] p-Coumaric acid, methyl caffeate and methyl ferulic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0026] LB broth powder and nutrient agar (NA) were purchased from Hybo Biotechnology Co., Ltd.

[0027] Culture medium:

[0028] LB liquid culture medium was prepared by weighing 33.0 g of LB broth powder, adding it to 1000 mL of distilled water, boiling to dissolve it, adjusting the pH to 7.0 ± 0.2, dispensing it into Erlenmeyer flasks, and sterilizing it at 120 °C for 20 min.

[0029] The solid culture medium was prepared by weighing 33.0 g of LB broth powder, adding it to 1000 mL of distilled water, boiling to dissolve it, adjusting the pH to 7.0 ± 0.2, dispensing it into 250 mL Erlenmeyer flasks, and sterilizing it at 120 °C for 20 min.

[0030] LB solid plates are prepared by sterilizing solid culture medium at 120°C, cooling it to about 55°C, taking 15mL of the medium into a sterile petri dish, and allowing it to solidify.

[0031] 1.3 Experimental bacterial suspension:

[0032] 1.3.1 Strain activation: The strain was inoculated into a test tube containing 2 mL of LB liquid medium and incubated at 37℃±1℃ for 12h~18h; the bacterial suspension was picked up with an inoculation loop and streaked onto LB solid plates and incubated at 37℃±1℃ for 18h~24h; then a single colony was picked from the plate and inoculated into the slant of LB solid medium in a test tube and incubated at 37℃±1℃ for 18h~24h; the slant was then stored in a refrigerator at 1℃~4℃ as a preservation strain.

[0033] 1.3.2 Preparation of test bacterial suspension: Take the preserved bacteria with an inoculation loop and inoculate them onto LB solid plates using the streak method. Incubate at 37℃±1℃ for 24h. Add 20mL of LB liquid medium to a 100mL sterile Erlenmeyer flask. Inoculate a single colony from the LB solid plate into the LB liquid medium with an inoculation loop. Incubate at 37℃±1℃ for 12h~18h. Adjust the bacterial concentration after incubation to an OD value of 0.65 with LB liquid medium. Use this as the test bacterial suspension.

[0034] 2. Experimental Methods:

[0035] 2.1 Material preparation: Select leaves that have just turned green and inoculate them with bacteria by needle pricking. Eight holes are punctured on each leaf for inoculation. Leaves at 0 and 1 day after inoculation are collected, frozen in liquid nitrogen, dried in a freeze dryer, pulverized thoroughly, and stored at 4℃. The samples are entrusted to Wuhan Metawell Biotechnology Co., Ltd. for extensive targeted metabolomics analysis.

[0036] 2.2 Data Processing: Mass spectrometry data were processed using Analyst 1.6.3 software; based on a self-built standard database, qualitative and quantitative mass spectrometry analysis of metabolites in the samples was performed; characteristic ions of each substance were screened using a triple quadrupole detector, and the signal intensity (CPS) of the characteristic ions was obtained in the detector. The sample mass spectrometry file was opened using MultiaQuant software, and chromatographic peak integration and correction were performed. The peak area (Area) of each chromatographic peak represents the relative content of the corresponding substance. Finally, all chromatographic peak area integration data were exported and saved.

[0037] 2.3 Screening of Differential Metabolites: Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed on the three-dimensional data matrix using R software (ropls package). Based on the OPLS-DA results, the variable importance in projection (VIP) of the obtained multivariate analysis OPLS-DA model was used to initially screen out differential metabolites among leaf samples of susceptible and resistant varieties. At the same time, differential metabolites can be further screened by combining the p-value of univariate analysis with the fold change.

[0038] Screening criteria: Metabolites with Fold Change ≥ 2 and Fold Change ≤ 0.5 were selected; a difference of more than 2 or less than 0.5 between the control and experimental groups was considered significant; volcano plots were generated using R software to characterize the accumulation patterns of metabolites between the infected and resistant groups; the relative contents of all differentially expressed metabolites identified according to the screening criteria in all group comparisons were normalized by z-score, followed by K-means cluster analysis;

[0039] Finally, potential resistant substances were screened based on the K-means clustering analysis results of differential metabolites. The screening criteria were: compounds whose content increased significantly after inoculation of resistant varieties and decreased significantly in susceptible varieties.

[0040] 2.4 Antibacterial activity test of resistance substances:

[0041] 2.4.1 Preparation of test samples: Using 10% DMSO solution as solvent, prepare a 2.0 mg / mL solution of differential metabolites and oxytetracycline, filter to remove contaminants, and set aside.

[0042] 2.4.2 Preparation of test plates: The solid culture medium is sterilized at 120℃ and cooled to about 55℃. The bacterial suspension is diluted with the solid culture medium at a ratio of 1:10. After thorough mixing, 15mL is taken into a sterile petri dish and allowed to solidify before use.

[0043] 2.4.3 Determination of antibacterial activity of differential metabolites: Place three sterilized Oxford cups on the test plate, and add 200 μL of the prepared test sample solution to each Oxford cup using a sterile pipette. Each treatment is repeated three times. Place the plate upright in a 37℃ constant temperature incubator and incubate for 72 h. Remove the plate and measure the diameter of the inhibition zone formed by the test sample and the blank control group. Select the sample with an inhibition zone larger than or close to that of the positive control oxytetracycline as the resistance substance for minimum inhibitory concentration testing.

[0044] 2.4.4 Determination of minimum inhibitory concentration of resistance substances: Prepare solutions of differential metabolites and oxytetracycline at concentrations of 2.0, 1.0, 0.5, 0.25, 0.125, and 0.065 mg / mL, filter to remove contaminating bacteria, and operate according to the methods in 2.4.2 and 2.4.3. The minimum inhibitory concentration is the sample whose inhibition zone is just larger than that of the blank control group.

[0045] 3. Experimental Results:

[0046] 3.1 K-means cluster analysis of differentially metabolized metabolites:

[0047] See attached diagram for differential metabolite K-Means. Figure 1 The relative content trends of different metabolites in the leaves of different peach varieties in response to Xap infection were divided into six categories (Sub Class 1-6). Compounds whose relative content increased significantly after inoculation of "Yanbao" and decreased significantly in "Frederic" were selected as potential antibacterial compounds. Among them, 22 compounds in Sub Class 5 met the criteria, and a total of 14 standards were obtained. Detailed compound information is shown in Appendix 1.

[0048] Table 1 Information on potentially antibacterial compounds

[0049]

[0050] Based on the results of the differential metabolite antibacterial activity test, the inhibition zone diameter of oxytetracycline at 2.0 mg / mL was 14.5 mm, while that of the blank control was 8.0 mm. At equivalent concentrations, three of the 14 differential metabolites showed antibacterial activity superior to or close to that of oxytetracycline: methyl ferulic acid, p-coumaric acid, and methyl caffeate, with inhibition zone diameters of 17.2, 15.2, and 14.3 mm, and minimum inhibitory concentrations (MICs) of 0.0625, 0.0625, and 0.125 mg / mL, respectively. The MIC of oxytetracycline was 0.125 mg / mL. The inhibition zone sizes and MICs of the antibacterial compounds are shown in Table 2, and the inhibition zone experimental diagram is shown in [Figure number missing]. Figure 2 ;

[0051] Table 2. Size of the inhibition zone and minimum inhibitory concentration of antibacterial compounds

[0052]

[0053]

[0054] Experimental conclusion:

[0055] The three cinnamic acid derivatives proposed in this invention—methyl ferulic acid, p-coumaric acid, and methyl caffeate—have minimum inhibitory concentrations (MICs) of 0.0625, 0.0625, and 0.125 mg / mL against the pathogen of peach bacterial leaf spot, respectively. The MIC of oxytetracycline is 0.125 mg / mL. The antibacterial activity of methyl ferulic acid and p-coumaric acid is superior to that of oxytetracycline, while the antibacterial activity of methyl caffeate is comparable to that of oxytetracycline.

[0056] Figure 2 This example shows the antibacterial effect of a 2.0 mg / mL concentration of cinnamic acid derivative compared to the positive control oxytetracycline in the prevention and control of bacterial leaf spot of peaches (A: methyl ferulic acid; B: p-coumaric acid; C: methyl caffeate; D: positive control drug oxytetracycline; E: 10% DMSO).

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, any improvements and changes made without departing from the inventive concept of the present invention are within the protection scope of the present invention.

Claims

1. The application of cinnamic acid derivatives in the prevention and control of bacterial leaf spot in peaches, characterized by: The cinnamic acid derivative is methyl ferulic acid. The aforementioned peach bacterial leaf spot refers to the infection of peach trees by the pathogenic fungus *Xanthomonas auricula-judae*. Xanthomonas arboricola pv. Pruni, Xap Diseases caused by; The minimum inhibitory concentration of methyl ferulic acid against the pathogen of peach bacterial perforation is 0.0625 mg / mL.

2. The application of the cinnamic acid derivative according to claim 1 in the prevention and control of bacterial leaf spot of peach, characterized in that: The application described involves combining an effective amount of cinnamic acid derivatives with pesticide-acceptable additives to create different formulations of drugs suitable for controlling bacterial leaf spot disease in peaches.

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