Application of Bromus japonicus extract in prevention and treatment of bacterial puncture disease of peach

Abstract

This invention discloses the application of awnless bromegranate extract in the prevention and control of bacterial leaf spot disease in peaches. The active ingredients in awnless bromegranate are extracted using an alkaline extraction and acid precipitation method, and the antibacterial component is obtained by column chromatography purification. The antibacterial component mainly contains p-coumaric acid and ferulic acid, with contents of 694.2 and 259.6 mg / g, respectively. The antibacterial component and its main components exhibit good antibacterial activity against the pathogen of peach bacterial leaf spot disease (Xanthomonas arboricola pv. pruni, Xap). The minimum inhibitory concentrations (MICs) of the antibacterial component, p-coumaric acid, ferulic acid, and the positive control drug oxytetracycline are 0.25, 0.0625, 0.50, and 0.125 mg / mL, respectively. The advantages compared to existing technologies are: awnless bromegranate is abundant, the preparation method of the antibacterial component is simple, and the inhibitory activity of its main component against coumaric acid is superior to that of oxytetracycline.

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 awnless brome extract 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 green, safe, and less likely to cause drug resistance. Currently, several plant-derived fungicides have achieved significant effects in controlling crop diseases, such as eugenol, berberine, osthol, and allicin. Bacterial diseases affect a wide range of crops and are extremely harmful. The scarcity of registered fungicides and the limited number of registered active ingredients, coupled with the widespread use of copper-based fungicides and antibiotics which have a significant negative impact on the industry, make the development of plant-derived fungicides both urgent and necessary. [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 awnless brome extract in the control of peach bacterial spot disease, and uses awnless brome 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 antibacterial components of awnless bromegrass were extracted by alkaline extraction and acid precipitation and purified by silica gel column chromatography. The inhibitory effects of the components and their main parts on the pathogen of peach bacterial leaf spot were determined by the inhibition zone method, confirming that the antibacterial components of awnless bromegrass can be used to prevent and control peach bacterial leaf spot.

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

[0009] 1) Raw material preparation: Collect awnless bromegranates, dry them, grind them, and set them aside for later use;

[0010] 2) Preparation of crude extract: The antibacterial substance of awnless brome was extracted by alkaline extraction and acid precipitation to obtain an extract. The extract was then extracted with 3 times the volume of ethyl acetate, and the extraction was repeated 3 times. The extracts were combined and the ethyl acetate was recovered to obtain the crude extract.

[0011] The alkaline extraction and acid precipitation method refers to extraction with 0.5 mol / L NaOH solution at a solid-liquid ratio of 1:50, extraction conditions: 50℃, ultrasonic extraction for 3 hours, after extraction, adding 2.0 mol / L hydrochloric acid solution to adjust the pH to neutral, and filtering to obtain the extract;

[0012] 3) Preparation of antibacterial components from awnless brome; The obtained extract was purified by silica gel column chromatography to obtain the antibacterial components from awnless brome;

[0013] The silica gel column chromatography described refers to the process where the active substance is dissolved in methanol, and the active substance is adsorbed onto silica gel (300-400 mesh) at a mass ratio of 20 to obtain sample-loaded silica gel. A suitable sized chromatography column is selected for column chromatography of the sample-loaded silica gel, ensuring that the height of the sample-loaded silica gel in the chromatography column is 2-3 cm, and the height of the chromatography silica gel is twice that of the sample-loaded silica gel. Elution is performed using a dichloromethane-ethyl acetate system, with dichloromethane eluting for 2 column volumes, and dichloromethane is recovered. Then, dichloromethane:ethyl acetate = 3:1 is used to elute for 4 column volumes, the fraction is collected, the solvent is evaporated, and then dried to obtain the awnless bromelain antibacterial component.

[0014] 4) Determination of the content of the main components of the antibacterial component; The content of p-coumaric acid and ferulic acid in the antibacterial component was determined by high performance liquid chromatography.

[0015] 5) Antibacterial activity test: Six different concentrations of awnless brome antibacterial components, p-coumaric acid, and ferulic acid solutions were prepared using 10% DMSO solution as solvent. Oxytetracycline was used as a positive control, and 10% DMSO solution was used as a blank control. The size of the inhibition zone and the minimum inhibitory concentration were determined by the inhibition zone method.

[0016] The strain used in the antibacterial activity test refers to the lesion-causing species of *Xanthomonas sarboricola* (pv. pruni, Xap).

[0017] In this invention:

[0018] Step 1) describes collecting awnless bromeliads, which involves collecting the above-ground parts of Bromusinermis Leyss., a perennial herbaceous plant belonging to the genus Bromusinermis in the Poaceae family.

[0019] Furthermore, in step 2), the active material is adsorbed onto silica gel at 20 times its mass to obtain sample-carrying silica gel, wherein the silica gel has a pore size of 300-400 mesh.

[0020] Furthermore, the aforementioned application involves combining an effective amount of awnless brome extract with pesticide-acceptable excipients or additives to formulate different formulations of drugs suitable for controlling bacterial leaf spot disease in peaches.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] In the prior art, there are no reports of using awnless bromegrass for the control of peach bacterial leaf spot. The application of awnless bromegrass extract in the control of peach bacterial leaf spot of this invention provides an awnless bromegrass extract with good antibacterial effect. It has abundant plant sources, a simple preparation method, and good inhibitory effect on the pathogen of peach bacterial leaf spot, with a minimum effective inhibitory concentration (MIC) of 0.25 mg / mL, compared to 0.125 mg / mL for the positive control drug oxytetracycline. The main components of the awnless bromegrass antibacterial component are coumaric acid and ferulic acid, with contents of 694.2 and 259.6 mg / g, respectively, and MICs of 0.0625 and 0.50 mg / mL, respectively. The MIC of coumaric acid, at 0.0625 mg / mL, is superior to that of oxytetracycline. Therefore, the awnless bromegrass antibacterial component is a potential agent for the control of peach bacterial leaf spot. [Attached Image Description]

[0023] Figure 1 This is an example of the application of awnless brome extract in the prevention and treatment of bacterial leaf spot in peaches. The antibacterial effect of the awnless brome extract at a concentration of 2.0 mg / mL compared with the positive control oxytetracycline is shown in the figure (A: awnless brome antibacterial component; B: p-coumaric acid; C: ferulic acid; D: positive control drug oxytetracycline; E: blank control).

Detailed Implementation Methods

[0024] The specific embodiments of the present invention will be further described below with reference to examples.

[0025] Example:

[0026] The application of awnless brome extract in the prevention and control of bacterial leaf spot in peaches includes the following steps:

[0027] 1.1. Raw material preparation: Collect the above-ground parts of awnless bromegrass, dry at 50℃, pulverize, and set aside;

[0028] 1.2. Preparation of crude extract: Weigh 100.0 g of pretreated awnless bromegrass, add 5.0 L of 0.5 mol / L NaOH, and extract ultrasonically for 3 h at an extraction temperature of 50℃ and an extraction frequency of 40 kHz. Adjust the pH to neutral with 2.0 mol / L hydrochloric acid, filter, add 3 times the volume of ethyl acetate to the filtrate for extraction, extract 3 times, combine the extracts, and evaporate the ethyl acetate to obtain the crude extract;

[0029] 1.3. Preparation of antibacterial component of awnless bromelain: The crude extract was dissolved in methanol, and the active substance was adsorbed onto 20 times the mass of silica gel (300-400 mesh) to obtain sample-loaded silica gel. A suitable chromatographic column was selected for column chromatography to ensure that the height of the sample-loaded silica gel in the chromatographic column was 2-3 cm, and the height of the chromatographic silica gel was twice that of the sample-loaded silica gel. After eluting with dichloromethane for 2 column volumes, it was eluted with dichloromethane:ethyl acetate = 3:1 for 4 column volumes. The fraction was collected, the solvent was evaporated, and then dried to obtain the antibacterial component of awnless bromelain.

[0030] 2. Determination of p-coumaric acid and ferulic acid content in crude extract and antibacterial components of awnless bromelain:

[0031] 2.1. Construction of standard curves for coumaric acid and ferulic acid: Coumaric acid and ferulic acid standards at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL were prepared and analyzed by HPLC. Standard curves were plotted with the concentrations of coumaric acid and ferulic acid as the abscissa (x) and the peak areas as the ordinate (y). The regression equation for coumaric acid was: y = 718.48x - 1.4166 (R² = 0.9999); the regression equation for ferulic acid was: y = 1983.8x + 0.6693 (R² = 0.9996).

[0032] 2.2. Content of p-coumaric acid and ferulic acid in crude extract and antibacterial component; the crude extract and antibacterial component were partitioned into 1.0 mg / mL fractions using 80% methanol solution, and the contents of p-coumaric acid and ferulic acid were determined by high-performance liquid chromatography (HPLC); chromatographic column: YMC-PackODS-AM (150×4.6 mm, 3 μm); mobile phase A: 0.1% formic acid aqueous solution, mobile phase B: methanol; column temperature: 35.0℃; flow rate: 0.5 mL / min; injection volume: 5.0 μL; detection wavelength 245nm; time gradient: (0-33.00min: 25%B), (33.01-34.00min: 100%B), (34.01-46.00min: 100%B), (46.01-47.00min: 40%B), (47.01-60.00min: 40%B); the contents of p-coumaric acid and ferulic acid in the alkaline hydrolysate and alkaline purified hydrolysate were calculated based on the peak area and regression equation;

[0033] 3. Antibacterial activity test of awnless bromelain antibacterial components and their main components

[0034] 3.1. 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 streak it onto LB solid plates, 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, incubate at 37℃±1℃ for 18h~24h; finally, store the slant in a refrigerator at 1℃~4℃ as the preserved strain.

[0035] 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.

[0036] 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.

[0037] The LB solid plate is prepared by sterilizing LB solid culture medium at 120°C, cooling it to about 55°C, taking 15mL of it into a sterile petri dish, and allowing it to solidify.

[0038] 3.2. 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 24h. Add 20mL of LB liquid medium to a 100mL 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 12h~18h. 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.

[0039] 3.3. Preparation of test samples: Using 10% DMSO solution as solvent, prepare solutions of 2.0, 1.0, 0.5, 0.25, 0.125, and 0.065 mg / mL of the antibacterial component of *Acer buergerianum*, p-coumaric acid, ferulic acid, and the positive control oxytetracycline. Filter to remove contaminants and set aside.

[0040] 3.4. 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.

[0041] 3.5. Antibacterial activity determination: Place three sterilized Oxford cups on the test plate. Use a sterile pipette to add 200 μL of the prepared test sample solution of different concentrations to the Oxford cups respectively. Add 200 μL of 10% DMSO to the blank control group. Each treatment is repeated 3 times. Place the plate upright in a 37℃ constant temperature incubator and incubate for 72 h. Remove the plate and use an inhibition zone measuring instrument to measure the diameter of the inhibition zone formed by the test sample and the blank control group using the cross method. Take the average value. The minimum inhibitory concentration is when the inhibition zone is just larger than that of the blank control group.

[0042] Experimental results:

[0043] 1. Content of p-coumaric acid and ferulic acid in crude extract and antibacterial component of awnless bromelain:

[0044] The crude extract of awnless bromelain contained 155.6 mg / g of p-coumaric acid and 58.2 mg / g of ferulic acid. After purification, the antibacterial components contained 694.2 mg / g of p-coumaric acid and 259.6 mg / g of ferulic acid, with p-coumaric acid accounting for 69.4% and ferulic acid accounting for 26.0%.

[0045] 2. Size of the inhibition zone and minimum inhibitory concentration of the antibacterial component of awnless bromelain and oxytetracycline:

[0046] At a concentration of 2.0 mg / mL, the inhibition zones of the awnless bromelain antibacterial component, coumaric acid, ferulic acid, and oxytetracycline were 14.0, 15.1, 11.0, and 14.5 mm, respectively, with minimum inhibitory concentrations of 0.25, 0.0625, 0.50, and 0.125 mg / mL.

[0047] Table 1. Inhibition zone size and minimum inhibitory concentration of awnless bromelain and oxytetracycline.

[0048]

[0049] result:

[0050] The main components of the awnless brome antibacterial component prepared in this invention are coumaric acid and ferulic acid, with contents of 694.2 and 259.6 mg / g, respectively. At a concentration of 2.0 mg / mL, the inhibition zones of the awnless brome antibacterial component, coumaric acid, ferulic acid, and oxytetracycline are 14.0, 15.1, 11.0, and 14.5 mm, respectively, and the minimum inhibitory concentrations are 0.25, 0.0625, 0.50, and 0.125 mg / mL, respectively. The minimum inhibitory concentration of coumaric acid is superior to that of oxytetracycline.

[0051] Figure 1 This embodiment shows the antibacterial effect of awnless brome extract at a concentration of 2.0 mg / mL on the antibacterial component of awnless brome and the positive control oxytetracycline in the prevention and treatment of bacterial leaf spot of peach (A: antibacterial component of awnless brome; B: p-coumaric acid; C: ferulic acid; D: positive control drug oxytetracycline; E: blank control).

[0052] 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 awnless brome extract in the prevention and control of bacterial leaf spot pathogens in peaches, characterized in that: The preparation method of the awnless brome extract includes the following steps: 1) Raw material preparation: Collect awnless bromegranates, dry them, crush them, and set them aside; The aforementioned collection of awnless bromegrass refers to the collection of perennial herbaceous plants belonging to the genus Bromus of the Poaceae family. Bromus inermis Leyss. The above-ground portion; 2) Preparation of crude extract: The antibacterial substance of awnless brome was extracted by alkaline extraction and acid precipitation to obtain an extract. The extract was then extracted with 3 times the volume of ethyl acetate, and the extraction was repeated 3 times. The extracts were combined and the ethyl acetate was recovered to obtain the crude extract. The alkaline extraction and acid precipitation method refers to extraction with 0.5 mol / L NaOH solution at a solid-liquid ratio of 1:

50. The extraction conditions are: 50℃, ultrasonic extraction for 3 h, extraction frequency of 40 kHz. After extraction, 2.0 mol / L hydrochloric acid solution is added to adjust the pH to neutral, and the extract is obtained by filtration. 3) Preparation of antibacterial component of awnless bromelain: The crude extract was dissolved in methanol, and the active substance was adsorbed onto silica gel at 20 times its mass to obtain sample-loaded silica gel. A suitable chromatographic column was selected for column chromatography to ensure that the height of the sample-loaded silica gel in the chromatographic column was 2-3 cm, and the height of the chromatographic silica gel was twice that of the sample-loaded silica gel. The sample was eluted with a dichloromethane-ethyl acetate system, with 2 column volumes of dichloromethane eluted and the dichloromethane recovered. Then, 4 column volumes of dichloromethane:ethyl acetate = 3:1 were eluted. The fraction was collected, the solvent was evaporated, and the sample was dried to obtain the antibacterial component of awnless bromelain. The pathogen causing bacterial leaf spot of peach is a diseased species of *Xanthomonas aurea*. Xanthomonas arboricola pv. pruni, Xap .

2. The application of the awnless brome extract according to claim 1 in the prevention and control of bacterial leaf spot pathogens in peaches, characterized in that: The application described involves combining an effective amount of awnless bromegranate extract with pesticide-acceptable excipients or additives to formulate different formulations of drugs suitable for controlling bacterial leaf spot disease in peaches.

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