Application of Prunus cerasifera leaf extract in the prevention and control of bacterial leaf spot of peach

Abstract

This invention discloses the application of *Prunus cerasifera* leaf extract in the prevention and control of bacterial leaf spot disease in peaches. The invention utilizes ultrasound-assisted extraction to extract the active ingredients from *Prunus cerasifera* leaves. Antibacterial tests demonstrate that the 80% ethanol extract of *Prunus cerasifera* leaves exhibits good antibacterial activity against the pathogen of peach bacterial leaf spot disease (*Xanthomonas arboricola* pv. pruni, Xap). The minimum inhibitory concentration (MIC) is the same as that of the positive control drug oxytetracycline, both being 0.125 mg / mL. Therefore, it can be used as a raw material for the prevention and control of peach bacterial leaf spot disease.

Description

[Technical Field]

[0001] This invention belongs to the technical field of plant-derived fungicides for the prevention and control of bacterial leaf spot disease in peaches, specifically involving the application of extracts from purple-leaf plum leaves in the prevention and control of bacterial leaf spot disease 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 of them used to control bacterial diseases, accounting for 2.4% of the total registered fungicide products in the country. These fungicides contain approximately 32 active ingredients, mainly copper-based agents, antibiotics, microbial inoculants, and other types of bactericides. Compared to copper-based agents 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 response to the problems of the wide range of crops affected by existing bacterial diseases, the severity of damage, the scarcity of registered control agents, the limited number of registered effective ingredients, and the huge negative impact of the routine use of copper preparations and antibiotics on the industry, this invention provides the application of Prunus cerasifera leaf extract in the control of peach bacterial spot disease, and uses Prunus cerasifera leaves to prepare plant-derived control agents for peach bacterial spot disease.

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

[0008] The active ingredients in the leaves of Prunus cerasifera were extracted using methanol and ethanol solutions of different concentrations to obtain Prunus cerasifera leaf extracts. The inhibitory zone method was used to determine the inhibitory activity of different Prunus cerasifera leaf extracts against the pathogen of Prunus perforatum bacterial spot disease of peach, so as to confirm that Prunus cerasifera leaf extracts can be used for the prevention and control of Prunus perforatum bacterial spot disease of peach.

[0009] The application of purple-leaf plum leaf extract in the prevention and control of bacterial leaf spot disease in peach includes the following steps:

[0010] 1) Raw material preparation: Collect purple-leaf plum leaves, dry them in the shade, crush them, and set aside for later use;

[0011] 2) Preparation of crude extract: The antibacterial substances of Prunus cerasifera leaves were extracted by ultrasonic-assisted extraction, filtered to obtain the extract, concentrated and vacuum dried to obtain Prunus cerasifera leaf extract.

[0012] The extraction conditions for the ultrasonic-assisted extraction method in the preparation of the crude extract are as follows: material-to-liquid ratio of 1:40, room temperature, ultrasonic extraction for 4 hours, and extraction frequency of 40KHz.

[0013] The extraction solvent for the preparation of the crude extract is selected from one of the following: 100%, 90%, 80%, 70%, or 60% (v / v) ethanol aqueous solution or 100%, 90%, 80%, 70%, or 60% (v / v) methanol solution.

[0014] 3) Antibacterial activity test of Prunus cerasifera leaf extract: Using 10% DMSO solution as solvent, Prunus cerasifera leaf extract solutions with concentrations of 2.0, 1.0, 0.5, 0.25, 0.125 and 0.0625 mg / mL were prepared. Oxytetracycline was used as a positive control and 10% DMSO was used as a blank control. The inhibition zone size and minimum inhibitory concentration of different Prunus cerasifera leaf extracts against the strains were determined by the inhibition zone method, verifying that the antibacterial activity of Prunus cerasifera leaf extract was comparable to that of oxytetracycline.

[0015] The strain used in the antibacterial activity test refers to the pathogenic species of *Xanthomonas sarboricola* (pv. pruni, Xap).

[0016] In this invention:

[0017] The raw material mentioned in step 1) is the leaf of a shrub or small tree (Prunus cerasifera 'Atropurpurea') belonging to the genus Prunus in the family Rosaceae.

[0018] Furthermore, in step 2), the extraction solvent for the preparation of the crude extract is selected from an 80% ethanol aqueous solution by volume.

[0019] Furthermore, the aforementioned application involves combining an effective amount of Prunus cerasifera leaf extract with pesticide-acceptable excipients or additives to formulate different formulations of drugs suitable for controlling bacterial leaf spot of peach.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] In the prior art, there are no reports of using purple-leaf plum leaves for the control of peach bacterial leaf spot. This invention describes the application of purple-leaf plum leaf extract in the control of peach bacterial leaf spot, providing a purple-leaf plum leaf extract with good antibacterial effect. The plant source is abundant, and the preparation method is simple. The purple-leaf plum leaf extract shows good inhibitory effect on the pathogenic bacteria of peach bacterial leaf spot, with a minimum inhibitory concentration (MIC) of 0.125 mg / mL, the same as the MIC of the positive control drug oxytetracycline. The antibacterial activity of the purple-leaf plum leaf extract is comparable to that of oxytetracycline, and it is expected to become an effective agent for the control of peach bacterial leaf spot. [Attached Image Description]

[0022] Figure 1 This is an example of the application of Prunus cerasifera leaf extract in the prevention and control of bacterial leaf spot of peach. The antibacterial effect of 80% ethanol extract of Prunus cerasifera leaf at a concentration of 2.0 mg / mL and the positive control oxytetracycline is shown in the figure (left, middle and right are 80% ethanol extract of Prunus cerasifera leaf, oxytetracycline and blank control, respectively).

Detailed Implementation Methods

[0023] The specific embodiments of the present invention will be further described below with reference to examples.

[0024] Example:

[0025] The application of purple-leaf plum leaf extract in the prevention and control of bacterial leaf spot disease in peach includes the following steps:

[0026] 1.1 Raw material preparation: Collect the above-ground parts of purple-leaf plum leaves in summer, dry them in the shade, crush them, and set aside for later use;

[0027] 1.2 Preparation of crude extract: 10g of Prunus cerasifera leaf powder was weighed and extracted with 400mL of 100%, 90%, 80%, 70%, and 60% (v / v) ethanol aqueous solution or 100%, 90%, 80%, 70%, and 60% (v / v) methanol solution, respectively. Extraction conditions: room temperature extraction, ultrasonic extraction for 4h, extraction frequency 40kHz. The residue was removed by filtration to obtain the extract. The extract was concentrated and vacuum dried to obtain Prunus cerasifera leaf extracts with different extracts.

[0028] 2. Antibacterial activity test of Prunus cerasifera leaf extract:

[0029] 2.1 Activation of the strain: 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.

[0030] The LB liquid culture medium is prepared by weighing 10.0g of tryptone, 5.0g of yeast extract, and 10.0g of sodium chloride, then adding the above components to 1000mL of distilled water, boiling to dissolve, adjusting the pH to 7.0±0.2, dispensing into Erlenmeyer flasks, and sterilizing at 120℃ for 20min.

[0031] The LB solid culture medium is prepared by weighing 10.0 g of tryptone, 5.0 g of yeast extract, 10.0 g of sodium chloride, and 20.0 g of agar, then adding the above components to 1000 mL of distilled water, boiling to dissolve, adjusting the pH to 7.0 ± 0.2, dispensing into 250 mL Erlenmeyer flasks, and sterilizing at 120 °C for 20 min.

[0032] The LB solid plate is prepared by sterilizing LB solid culture medium at 120°C, cooling it to about 55°C, taking 15mL into a sterile petri dish, and allowing it to solidify.

[0033] 2.2 Preparation of bacterial suspension: Using an inoculation loop, take the preserved bacteria and inoculate them onto LB solid plates using the streak method. Incubate at 37℃±1℃ for 24 h. Add 20 mL of LB liquid medium to a 100 mL sterile Erlenmeyer flask. Use an inoculation loop to take a single colony from the LB solid plate and inoculate it into the LB liquid medium. Incubate at 37℃±1℃ for 12 h–18 h. Adjust the bacterial concentration after incubation to an OD value of 0.65 using LB liquid medium, and use this as the test bacterial suspension.

[0034] 2.3 Preparation of test plates: The solid culture medium was sterilized at 120℃ and cooled to about 55℃. The bacterial suspension was diluted with the solid culture medium at a ratio of 1:10. After thorough mixing, 10 mL was taken into a sterile petri dish and allowed to solidify before use.

[0035] 2.4 Preparation of test samples: Using 10% DMSO solution as solvent, extracts of different purple-leaf plum leaves at concentrations of 2.0 mg / mL were prepared.

[0036] 2.5 Antibacterial activity determination: Three sterilized Oxford cups were placed on the test plate. 200 μL of the prepared test sample solution at different concentrations was added to each Oxford cup using a sterile pipette. 200 μL of 10% DMSO was added to the blank control group. Each treatment was repeated three times. The plates were incubated upright in a 37℃ incubator for 72 h. After incubation, the diameter of the inhibition zone of different extracts was measured using an inhibition zone measuring instrument. The optimal extraction solution was determined based on the size of the inhibition zone diameter.

[0037] Determination of the minimum inhibitory concentration (MIC) of Prunus cerasifera leaf extract: Extracts obtained from the optimal extract solution were used to prepare Prunus cerasifera leaf extract solutions at concentrations of 2.0, 1.0, 0.5, 0.25, 0.125, and 0.065 mg / mL with a positive control oxytetracycline solution using 10% DMSO. The solutions were filtered to remove contaminants and set aside. Antibacterial activity experiments were conducted according to methods 2.1–2.5. The MIC was defined as the minimum inhibitory concentration (MIC) where the inhibition zone was just larger than that of the blank control group.

[0038] Experimental results:

[0039] 1. Antibacterial activity of extracts from purple-leaf plum leaves obtained with different extracts:

[0040] As shown in Table 1, the inhibition zones of the extracts from methanol solutions of different concentrations are all smaller than those from ethanol solutions of different concentrations. The inhibition zones of the extracts from ethanol solutions of different concentrations are of different sizes, with the largest inhibition zone observed in the 80% ethanol solution.

[0041] Table 1. Size of the inhibition zone at 2.0 mg / mL for different Prunus cerasifera leaf extracts.

[0042]

[0043]

[0044] Minimum inhibitory concentration of 2.80% ethanol extract:

[0045] According to Table 2, at a concentration of 2.0 mg / mL, the inhibition zones of the Prunus cerasifera leaf extract extracted with 80% ethanol solution (v / v) and the positive control drug oxytetracycline were 14.3 and 14.5 mm, respectively, with a minimum inhibitory concentration of 0.125 mg / mL for both.

[0046] Table 2. Inhibition zone size and minimum inhibitory concentration of Prunus cerasifera leaf extract and oxytetracycline.

[0047]

[0048] in conclusion:

[0049] This invention prepares an extract of *Prunus cerasifera* leaves obtained from an 80% ethanol solution. The size of its inhibition zone at an equivalent concentration (2.0 mg / mL) is similar to that of the positive control drug oxytetracycline, and its minimum inhibitory concentration is the same as oxytetracycline, both being 0.125 mg / mL. The antibacterial activity of the *Prunus cerasifera* leaf extract obtained from an 80% ethanol solution is comparable to that of oxytetracycline against the pathogen of peach bacterial leaf spot, making it a potential plant-derived agent for the control of peach bacterial leaf spot.

[0050] Figure 1 This embodiment shows the antibacterial effect of 2.0 mg / mL concentration of 80% ethanol extract of Prunus cerasifera leaf in the prevention and control of bacterial leaf spot of peach, compared with the positive control oxytetracycline (left, middle, and right are respectively 80% ethanol extract of Prunus cerasifera leaf, oxytetracycline, and blank control).

[0051] 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. Use of a leaf extract of Prunus cerasifera in the control of bacterial spot of peach, characterized in that: The preparation of the purple-leaf plum leaf extract includes the following steps: 1) Preparation of raw material: collect purple leaf plum leaves, dry in the shade, crush, and reserve; the raw material is the leaves of the plant Prunus cerasifera Atropurpurea' ​​ 2) Preparation of crude extract: The antibacterial substances of Prunus cerasifera leaves were extracted by ultrasonic-assisted extraction, filtered to obtain the extract, concentrated and vacuum dried to obtain Prunus cerasifera leaf extract; The extraction conditions for the ultrasonic-assisted extraction method in the preparation of the crude extract are as follows: material-to-liquid ratio of 1:40, room temperature, ultrasonic extraction for 4 h, and extraction frequency of 40 kHz. The extraction solvent for the preparation of the crude extract is selected from one of the following: 100%, 90%, 80%, 70%, 60% ethanol aqueous solution or 100%, 90%, 80%, 70%, 60% methanol solution (volume concentration). The pathogen causing the bacterial leaf spot of peach is the variegated species of *Xanthomonas aurea*. Xanthomonas arboricola pv. pruni.

2. The application of the purple-leaf plum leaf extract according to claim 1 in the prevention and control of bacterial leaf spot disease in peaches, characterized in that: In step 2), the extraction solvent for the preparation of the crude extract is selected from an 80% (v / v) aqueous ethanol solution.

3. The application of the purple-leaf plum leaf extract according to claim 1 in the prevention and control of bacterial leaf spot disease in peaches, characterized in that: The application described involves combining an effective amount of Prunus cerasifera leaf extract with pesticide-acceptable additives to create different formulations of drugs suitable for controlling bacterial leaf spot disease in peaches.

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