Application of Polygonum aviculare extract in the prevention and treatment of bacterial leaf spot in peach

Abstract

This invention discloses the application of *Polygonum aviculare* extract in the prevention and control of bacterial leaf spot in peach. The invention utilizes ultrasound-assisted extraction to extract the active components of *Polygonum aviculare*. Antibacterial experiments demonstrated that the extract exhibits good antibacterial activity against the pathogen of peach bacterial leaf spot (*Xanthomonas arboricola* pv. pruni, Xap), with a minimum inhibitory concentration (MIC) identical to that of the positive control drug oxytetracycline, both at 0.125 mg / mL. The antibacterial activity of *Polygonum aviculare* extract is comparable to that of oxytetracycline, making it a potential agent for the prevention and control of peach bacterial leaf spot.

Description

[Technical Field]

[0001] This invention belongs to the technical field of plant-derived fungicides for the prevention and control of bacterial leaf spot in peaches, specifically involving the application of extracts from *Polygonum cuspidatum* 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, but their dependence and long-term use have led to increasingly prominent problems such as drug resistance, tree toxicity, environmental pollution, and pesticide residues. According to data from the China Pesticide Information Network, as of July 2023, there were 11,859 registered fungicides in my country, of which approximately 280 were used to control bacterial diseases, accounting for 2.4% of the total registered fungicide products in China. The active ingredients were approximately 32, mainly copper-based agents, antibiotics, microbial agents, and other types of bactericides. Compared with copper-based fungicides and antibiotics, plant-derived fungicides are green, safe, and less likely to cause drug resistance. Currently, many plant-derived fungicides have achieved significant results in the prevention and control of crop diseases, such as eugenol, berberine, and osthol.

[0004] Polygonum multiflorum contains flavonoids, phenylpropionates, terpenes, and phenolic acids, and has anti-inflammatory, antibacterial, antiviral, and antitumor effects. The minimum inhibitory concentrations (MICs) of the 75% ethanol extract against Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Proteus mirabilis were 0.05, 0.01, 0.01, and 0.05 mg / mL, respectively. The ethyl acetate fraction showed MICs against Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa, respectively, at 0.07, 0.12, 0.07, and 0.12 mg / mL. However, no studies have been reported on the use of Polygonum multiflorum in controlling bacterial leaf spot of peaches. [Summary of the Invention]

[0005] 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 *Polygonum cuspidatum* extract in the control of peach bacterial spot disease, and uses *Polygonum cuspidatum* to prepare plant-derived control agents for peach bacterial spot disease.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The active ingredients in *Polygonum cuspidatum* were extracted using an 80% methanol solution to obtain *Polygonum cuspidatum* extract. The inhibitory activity of the extract against the pathogen of peach bacterial leaf spot was determined by the inhibition zone method to confirm that the extract can be used for the prevention and control of peach bacterial leaf spot.

[0008] The application of Polygonum aviculare extract in the prevention and control of bacterial leaf spot in peaches includes the following steps:

[0009] 1) Raw material preparation; collect the above-ground parts of the stalks, air-dry them, crush them, and set them aside for later use;

[0010] 2) Extract preparation: The bactericidal substance of Polygonum aviculare was extracted by ultrasonic-assisted extraction with 80 (v / v)% methanol solution at a material-to-liquid ratio of 1:(5-50). The extract was obtained by filtration, concentration and vacuum drying to obtain Polygonum aviculare extract.

[0011] 3) Antibacterial activity test of Polygonum aviculare extract: Polygonum aviculare extract solutions with concentrations of 2.0, 1.0, 0.5, 0.25, 0.125 and 0.0625 mg / mL were prepared using 10% DMSO solution as solvent. Oxytetracycline was used as a positive control and 10% (v / v) DMSO solution was used as a blank control. The size of the inhibition zone and the minimum inhibitory concentration of Polygonum aviculare extract were determined by the inhibition zone method to verify that the antibacterial activity of Polygonum aviculare extract was comparable to that of oxytetracycline.

[0012] The strain used in the antibacterial activity test refers to the pathogenic species of *Xanthomonas sarboricola* (pv. pruni, Xap).

[0013] In this invention:

[0014] The raw material mentioned in step 1) is Polygonum perfoliatum L., which is a plant of the Polygonaceae family and the Polygonum genus. It is an annual herbaceous plant, including the whole plant, flowers, leaves, or the above-ground and underground parts with flowers and leaves.

[0015] The extract preparation described in step 2) preferably uses an ultrasonic-assisted method, using an 80 (v / v)% methanol solution to extract the bactericidal substance of *Polygonum aviculare*. The extraction conditions are: a material-to-liquid ratio of 1:40, room temperature, ultrasonic extraction for 4 hours, and an extraction frequency of 40 kHz.

[0016] Furthermore, the 80 (v / v)% methanol solution mentioned in step 2) can be replaced with an aqueous ethanol solution or a methanol solution with a volume concentration of 100%, 90%, 80%, 70%, or 60%.

[0017] Furthermore, the aforementioned application involves combining an effective amount of *Polygonum aviculare* or its extract with pesticide-acceptable excipients or additives to formulate different formulations of drugs suitable for controlling bacterial leaf spot of peaches.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] In the prior art, there are no reports of using *Ligustrum lucidum* extract for the control of peach bacterial leaf spot. This invention provides an *Ligustrum lucidum* extract with good antibacterial effect in the control of peach bacterial leaf spot. It has abundant plant sources, a simple preparation method, and exhibits good inhibitory effects against the pathogen of peach bacterial leaf spot. The minimum inhibitory concentration is the same as that of the positive control drug oxytetracycline, both being 0.125 mg / mL. At the same concentration (2.0 mg / mL), the inhibition zones of *Ligustrum lucidum* extract and oxytetracycline are 14.4 and 14.5 mm, respectively. The antibacterial activity of *Ligustrum lucidum* extract is comparable to that of oxytetracycline, and it shows promise as a highly effective agent for the control of peach bacterial leaf spot. [Attached Image Description]

[0020] Figure 1 This is an example of the application of *Codonopsis pilosula* extract in the prevention and control of bacterial leaf spot in peaches according to an embodiment of the present invention. The antibacterial effect of 80% methanol extract of *Codonopsis pilosula* at a concentration of 2.0 mg / mL compared with the positive control oxytetracycline (left, middle, and right are 80% methanol extract of *Codonopsis pilosula*, oxytetracycline, and blank control, respectively).

Detailed Implementation Methods

[0021] The specific embodiments of the present invention will be further described below with reference to examples.

[0022] Example:

[0023] The application of Polygonum aviculare extract in the prevention and control of bacterial leaf spot in peaches includes the following steps:

[0024] 1. Raw material preparation: In summer, collect the above-ground parts of the *Ligustrum lucidum*, air-dry them, crush them, and set them aside for later use;

[0025] 2. Extraction preparation: Weigh 10g of *Polygonum aviculare* powder and add 400mL of 100%, 90%, 80%, 70%, and 60% ethanol aqueous solution and 100%, 90%, 80%, 70%, and 60% methanol solution respectively for extraction. Extraction conditions: room temperature extraction, ultrasonic extraction for 4h, extraction frequency 40KHz; filter to remove residue to obtain extracts, concentrate the extracts, and vacuum dry to obtain *Polygonum aviculare* extracts with different extracts.

[0026] 3. Antibacterial activity test of Polygonum multiflorum extract:

[0027] 1) Activation of the strain: Inoculate the strain into a test tube containing 2 mL of LB liquid medium and incubate at 37℃±1℃ for 12h~18h; use an inoculation loop to pick up the bacterial suspension and inoculate it onto LB solid plates by streak method, and incubate at 37℃±1℃ for 18h~24h. Then pick a single colony from the plate and inoculate it into the slant of LB solid medium in a test tube, and incubate at 37℃±1℃ for 18h~24h. After that, store the test tube slant in a refrigerator at 1℃~4℃ as the preserved strain.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 2) Preparation of 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 onto 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 and use this as the test bacterial suspension.

[0032] 3) Preparation of test samples: Using 10% (v / v) DMSO solution as solvent, prepare extract solutions of 2.0, 1.0, 0.5, 0.25, 0.125, and 0.065 mg / mL of Polygonum aviculare extract, filter to remove impurities, and set aside for use.

[0033] 4) 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, 10mL is taken into a sterile petri dish and allowed to solidify before use.

[0034] 5) Antibacterial activity assay: Place three sterilized Oxford cups on the test plate, and add 200 μL of the prepared test sample solution of different concentrations to the Oxford cups using a sterile pipette. Add 200 μL of 10% DMSO to the blank control group. Repeat each treatment three times. Place the plate upright in a constant temperature incubator at 37℃ for 72 h. Remove the plate and measure the diameter of the inhibition zone of different extracts using an inhibition zone measuring instrument. Determine the optimal extraction solution based on the size of the inhibition zone diameter.

[0035] 6) Determination of the minimum inhibitory concentration of Polygonum aviculare extract: Select the extract extracted with the best extract solution, and prepare Polygonum aviculare extract and positive control oxytetracycline solutions with 10% DMSO solution at concentrations of 2.0, 1.0, 0.5, 0.25, 0.125, and 0.065 mg / mL. Filter to remove impurities and set aside.

[0036] The antibacterial activity experiment was conducted according to methods 1) to 5). The minimum inhibitory concentration was defined as the inhibition zone that was just larger than that of the blank control group.

[0037] Experimental results:

[0038] 1) Antibacterial activity of extracts of *Polygonum aviculare* extracted with different extract solutions

[0039] As shown in Table 1, the optimal extraction solution is an 80% methanol solution.

[0040] Table 1. Inhibition zone size of different extracts of *Polygonum aviculare* extract at 2.0 mg / mL

[0041]

[0042]

[0043] 2) Minimum inhibitory concentration of 80% methanol extract

[0044] At the same concentration (2.0 mg / mL), the inhibition zone size of the 80% methanol extract of Polygonum aviculare was 14.4 mm, while that of oxytetracycline was 14.5 mm. The minimum inhibitory concentration of the 80% methanol extract of Polygonum aviculare was the same as that of oxytetracycline, both being 0.125 mg / mL, indicating that the antibacterial activity of Polygonum aviculare extract against Xap was comparable to that of oxytetracycline.

[0045] Table 1. Inhibition zone size and minimum inhibitory concentration of *Polygonum cuspidatum* extract and oxytetracycline.

[0046]

[0047] in conclusion:

[0048] The 80% methanol extract of *Polygonum cuspidatum* prepared in this invention exhibits good inhibitory effects against the pathogen of peach bacterial leaf spot. At a concentration of 2.0 mg / mL, the inhibition zone size of *Polygonum cuspidatum* extract is 14.4 mm, while that of oxytetracycline is 14.5 mm. The minimum inhibitory concentration (MIC) is the same as that of oxytetracycline, both being 0.125 mg / mL. The antibacterial activity of *Polygonum cuspidatum* extract against the pathogen of peach bacterial leaf spot is comparable to that of oxytetracycline, making it a potential plant-derived agent for the control of peach bacterial leaf spot.

[0049] Figure 1 This example illustrates the antibacterial effect of 2.0 mg / mL *Codonopsis pilosula* extract in preventing and treating bacterial leaf spot of peaches, comparing it with the positive control oxytetracycline. (Left, middle, and right represent 80% methanol extract of *Codonopsis pilosula*, oxytetracycline, and blank control, respectively.)

[0050] 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 *Polygonum aviculare* extract in the prevention and control of bacterial leaf spot in peaches, characterized by: The preparation of the extract of *Polygonum aviculare* includes the following steps: 1) Raw material preparation; collect the above-ground parts of *Ligustrum lucidum*, air-dry, crush, and set aside; the raw material is *Ligustrum lucidum*. Polygonum perfoliatum The flowers, leaves, or above-ground parts with both flowers and leaves of L.; 2) Extract preparation: The bactericidal substances of Polygonum aviculare were extracted using an ultrasonic-assisted method. The extraction solvent was selected from 100%, 90%, 80%, 70%, and 60% ethanol aqueous solution or 100%, 90%, 80%, 70%, and 60% methanol solution by volume. The material-to-liquid ratio was 1:(5-50). The extract was obtained by filtration, concentration, and vacuum drying to obtain Polygonum aviculare extract. 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 extract of *Polygonum aviculare* according to claim 1 in the prevention and control of bacterial leaf spot of peach, characterized in that: The extract preparation described in step 2) is carried out using an ultrasonic-assisted method. The extraction conditions are: a material-to-liquid ratio of 1:40, room temperature, ultrasonic extraction for 4 hours, and an extraction frequency of 40 kHz.

3. The application of the extract of *Polygonum aviculare* according to claim 1 in the prevention and control of bacterial leaf spot of peach, characterized in that: The extraction solvent mentioned in step 2) is selected from an 80% methanol solution by volume.

4. The application of the extract of *Polygonum aviculare* 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 *Polygonum aviculare* extract with pesticide-acceptable additives to create different formulations of drugs suitable for controlling bacterial leaf spot disease in peaches.

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