Plant extract for preventing and treating walnut black spot disease and preparation method and application thereof

Abstract

The invention discloses a plant extract for preventing and treating walnut black spot disease, a preparation method and application thereof, and belongs to the technical field of plant protection and biological control; the plant extract for preventing and treating walnut black spot disease is an Artemisia macrophylla extract, and its antibacterial components include cypermethrin. The plant extract or cypermethrin is used for preventing and treating walnut black spot disease, which can effectively inhibit the growth of pathogenic bacteria, reduce the incidence of walnut black spot disease, and reduce the size of walnut black spot disease spots.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection and biological control, and particularly relates to a plant extract for preventing and treating walnut black spot disease, and a preparation method and application thereof. Background Art

[0002] Walnut (Juglans regia L.), also known as walnut and Qiang peach, is a deciduous tree of the genus Juglans in the family Juglandaceae. It has extremely high economic and nutritional value.

[0003] Since the rain and heat coincide during the leafing period of walnuts, it provides good prerequisites for the infection of black spot pathogens, making black spot disease common during the fruit ripening period. The main pathogen of walnut bacterial black spot is Xanthomonas. At present, the prevention and control measures for walnut black spot are mainly chemical control, using inorganic copper preparations, organic copper preparations and antibiotic chemical agents (such as kasugamycin). However, the long-term application of chemical agents can easily cause problems such as pesticide residues and food safety, and lead to a series of ecological environmental damage. Therefore, the research on biological control methods is of great significance to the sustainable and healthy development of the walnut industry in the future. Summary of the invention

[0004] The invention discloses a plant extract for preventing and treating walnut black spot disease and a preparation method thereof. The plant extract or its effective antibacterial components are used to prevent and treat the walnut black spot disease, and the effect is remarkable.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] A plant extract for preventing and treating walnut black spot disease, which is an Artemisia macrophylla extract.

[0007] Artemisia grandis is a plant of the genus Artemisia in the Asteraceae family. It is mainly used to prepare hay and serve as winter reserve feed for livestock. There are currently no reports on its use in the prevention and control of walnut diseases.

[0008] Furthermore, the above-mentioned plant extract includes Artemisia annua water extract, Artemisia annua ethanol extract or Artemisia annua petroleum ether extract.

[0009] Furthermore, the antibacterial component in the above plant extract includes Absinthin, the molecular formula of which is: 30 H 40 O6.

[0010] Artemisia selengensis has been confirmed to be a component of Artemisia grandis, and the effect of artemisia selengensis in preventing and treating walnut black spot disease is verified in the present invention.

[0011] The method for preparing a plant extract for preventing and treating walnut black spot disease is characterized by comprising the following steps:

[0012] Take Artemisia grandis plant material, add extraction reagent, perform ultrasonic extraction or heating reflux extraction, centrifuge to obtain supernatant after extraction, and concentrate by reduced pressure distillation.

[0013] Furthermore, the extraction reagent includes water, ethanol solution or petroleum ether.

[0014] The extracts obtained by extracting Artemisia sphaerocephala with the above different extraction reagents all have the efficacy of preventing and treating walnut black spot disease, among which the ethanol extract has the best effect. The selection of the extraction reagent for Artemisia sphaerocephala extract is not limited to the several ones described in the present invention.

[0015] Further, the Artemisia argyi plant material includes fresh powder of the aerial part of Artemisia argyi;

[0016] The volume fraction of ethanol solution is 70%;

[0017] The preparation method further comprises soaking before extraction;

[0018] The above preparation method further comprises filtering using a filter membrane after concentration.

[0019] Using the above plant extract or the plant extract prepared by the above method or citronella for the prevention and treatment of walnut black spot disease can effectively inhibit the growth of pathogens, reduce the incidence of walnut black spot disease, and reduce the size of walnut black spot disease spots.

[0020] The medicament for preventing and treating walnut black spot disease comprises the above-mentioned plant extract.

[0021] Agents for preventing and treating walnut black spot disease include acetaminophen.

[0022] The method for preventing and controlling walnut black spot disease is to spray the above-mentioned agent on walnut branches, leaves and fruits. The pathogen of walnut black spot disease often parasitizes on walnut branches and leaves. Spraying the extract can effectively inhibit the growth and proliferation of the pathogen of walnut black spot disease, and play a certain degree of prevention and control effect.

[0023] In summary, the Artemisia grandiflora extract of the present invention has a strong inhibitory effect on Xanthomonas, the pathogen of walnut black spot disease, and has a significant effect on inhibiting the occurrence of black spot disease on walnut leaves and fruits; its component acylindole has a strong inhibitory effect on Xanthomonas, the pathogen of walnut black spot disease, and has a significant preventive effect on walnut leaf black spot disease, which is beneficial to reducing the incidence of walnut black spot disease and plays a good biological control role. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure shows the antibacterial rate of the Artemisia annua water extract in Example 1.

[0025] Figure 2 The figure shows the antibacterial rate of the Artemisia annua ethanol extract in Example 1.

[0026] Figure 3The figure shows the antibacterial rate of the Artemisia annua petroleum ether extract in Example 1.

[0027] Figure 4 Shown are the antibacterial rates of different plant extracts in Example 2.

[0028] Figure 5 (A) shows the state of leaves at day 0 of treatment in Example 3 of the present invention; Figure 5 (B) shows the state of leaves after 7 days of treatment in Example 3 of the present invention; Figure 5 (C) is an enlarged view of the leaf state after 7 days of treatment in Example 3 of the present invention;

[0029] Among them, the experimental group: a single walnut leaf inoculated with the black spot pathogen after being treated with Artemisia annua extract of different concentrations (concentration 1, concentration 1 / 2, concentration 1 / 4); the negative control group: a single walnut leaf inoculated with the black spot pathogen after being treated with sterile water (inoculation); the blank control group: a single walnut leaf inoculated with sterile water after being treated with sterile water (control).

[0030] Figure 6 Shown is the morbidity of different treatment groups after 7 days of treatment in Example 3 of the present invention.

[0031] Figure 7 Shown is the size of lesions in different treatment groups after 7 days of treatment in Example 3 of the present invention.

[0032] Figure 8 The fruit state after 30 days of treatment in Example 4 of the present invention is shown;

[0033] Among them, the experimental group: the walnut fruits were treated with Artemisia annua extract and then inoculated with the black spot pathogen; the control group: the walnut fruits were treated with sterile water and then inoculated with the black spot pathogen.

[0034] Fig. 9 Shown is a clustering heat map of differential metabolites by AS and MS in Example 5 of the present invention.

[0035] The horizontal direction is the sample name, the vertical direction is the differential metabolite information, Group is the sample grouping, and Class is the primary classification of the substance; the clustering line on the left side of the figure is the metabolite clustering line, and the clustering line on the top of the figure is the sample clustering line.

[0036] Fig.10 Shown is the antibacterial rate of oxaliplatin in Example 6 of the present invention.

[0037] Fig.11 The figure shows the state of leaves after 5 days of treatment in Example 7 of the present invention;

[0038] Among them, the experimental group: leaves treated with icarin, kasugamycin, and oleanolic acid; the negative control group: a single walnut leaf inoculated with black spot pathogens after being treated with sterile water (inoculation); the blank control group: a single walnut leaf inoculated with sterile water after being treated with sterile water (control).

[0039] Fig.12 The incidence rates of different treatment groups after 5 days of treatment in Example 7 of the present invention.

[0040] Fig.13 The sizes of lesions in different treatment groups after 5 days of treatment in Example 7 of the present invention. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The plant materials used in the examples were taken from Wuling Mountain, Hebei Province, located in the northern part of Xinglong County, Hebei Province.

[0043] Example 1 Co-culture test of different Artemisia grandis extracts and pathogenic bacteria

[0044] (1) Preparation of Artemisia grandis aqueous extract by reflux method

[0045] Add liquid nitrogen to the fresh sample of the aerial part of Artemisia grandis and grind it into powder. Take 2.0g of the powder, place it in a 100mL conical flask, and soak it in 30mL ultrapure water for 24h. After soaking, put it in a flask, heat and reflux for 1h, and use two layers of gauze for coarse filtration to collect the first filtrate; add 30mL ultrapure water to the residue, heat and reflux for 1h, and use two layers of gauze for coarse filtration to collect the second filtrate; combine the two filtrates, centrifuge for 20min (4000r / min), take the supernatant, concentrate the sample to 15mL by vacuum distillation (about 40min), and filter it with a filter membrane for later use.

[0046] (2) Preparation of Artemisia argyi ethanol extract by ultrasonic vibration method

[0047] The fresh sample of the aerial part of Artemisia grandis was ground into powder by adding liquid nitrogen, 2.0 g of the powder was taken and placed in a 100 mL conical flask, and a 70% volume fraction ethanol solution was added at a solid-liquid ratio of 1:30 (g / mL), and stirred evenly. Under room temperature, the sample was treated with 250 W ultrasonic oscillation for 30 minutes, and the sample was immediately centrifuged for 20 minutes (4000 r / min) after being taken out, and the supernatant was taken, and the sample was concentrated by vacuum distillation at 50 ° C (40min), and filtered with a filter membrane for later use.

[0048] (3) Preparation of Artemisia grandiflora petroleum ether extract by reflux method

[0049] Add liquid nitrogen to the fresh sample of the aerial part of Artemisia grandis and grind it into powder. Take 2.0g of the powder, wrap it with filter paper, put it into a flask, add 30mL of petroleum ether, heat and reflux for 2h (60℃), collect the first extract; add 30mL of petroleum ether to the residue, heat and reflux for 2h, collect the second extract; combine the two extracts, distill under reduced pressure at 60℃, evaporate the organic solvent, and concentrate it into oil to eliminate the antibacterial effect of the solvent itself on the strain, then dissolve it with 0.2% DMSO and make up to 15mL. Store at 4℃ for later use.

[0050] (3) Co-culture

[0051] The antibacterial rates of three Artemisia grandis extracts against black spot pathogens were determined by double isogradient dilution 96-well plate method:

[0052] The pathogen of black spot disease (Xanthomonas sp., isolated from walnut black spot disease plants) was inoculated into YEP liquid medium and cultured at 28°C and 180 r / min until the logarithmic phase (OD 600 =0.6), each Artemisia selengensis extract was diluted to different concentrations with sterile water (1-6: initial concentration, 1 / 2 initial concentration, 1 / 4 initial concentration, 1 / 8 initial concentration, 1 / 16 initial concentration, 1 / 32 initial concentration), 50 μL of bacterial solution and 50 μL of Artemisia selengensis extracts of different concentrations were mixed evenly in a 96-well plate as the experimental group (test), and sterile water and bacterial solution were mixed as the control (control), and each group was cultured at 28°C for 24 hours. After the culture was completed, the absorbance of the bacterial solution at 600nm was measured by an ELISA instrument to calculate the inhibition rate.

[0053] Inhibition rate = 100-(OD test *100 / OD control ).

[0054] The results are as follows Figure 1-3 As shown in the results, all three Artemisia annua extracts had inhibitory effects on the pathogen of black spot disease, among which the Artemisia annua ethanol extract had the best antibacterial effect.

[0055] Example 2 Antibacterial test of co-culture of different plant extracts with pathogenic bacteria

[0056] The inhibition rate of different plant extracts against black spot pathogens was determined by double equal gradient dilution 96-well plate method:

[0057] The pathogen of black spot disease (Xanthomonas sp., isolated from walnut black spot disease plants) was inoculated into YEP liquid medium and cultured at 28°C and 180 r / min until the logarithmic phase (OD 600=0.6), 50 μL of bacterial solution and 50 μL of plant extract were mixed evenly in a 96-well plate as the experimental group (test), and sterile water and bacterial solution were mixed as the control (control). They were cultured at 28°C for 24 h, and the absorbance of the bacterial solution at 600 nm was measured by an enzyme marker to calculate the inhibition rate.

[0058] Inhibition rate = 100-(OD test *100 / OD control )

[0059] The experimental group was composed of the ethanol extract of Artemisia annua (prepared by the same method as in Example 1), and the control groups were composed of the ethanol extracts of Potentilla, Elsholtzia, Scutellaria baicalensis, Epilobium angustifolium, Astragalus membranaceus, Rock fennel, Clematis macrophylla, and Vitex truncatula (each extract was prepared by the same method as the ethanol extract of Artemisia annua) and the conventional agent kasugamycin (2% kasugamycin diluted 1000 times).

[0060] The results are as follows Figure 4 As shown, except for the ethanol extract of Artemisia annua and the ethanol extract of Aster chrysanthemum, the plant extracts had no inhibitory effect on pathogens, and the antibacterial effect of the ethanol extract of Aster chrysanthemum was significantly lower than that of the ethanol extract of Artemisia annua.

[0061] Example 3 Disease status of leaves treated with Artemisia argyi extract at different concentrations

[0062] Mature walnut leaves were collected, the base of the leaves were wrapped with wet cotton balls, placed on a tray covered with wet gauze, covered with plastic wrap, and cultured in a 25°C culture room. Different concentrations of Artemisia selengensis ethanol extract (preparation method is the same as in Example 1) were applied to the surface of the detached leaves, 150 μL was applied to the ventral surface, 50 μL was applied to the dorsal surface, and 50 μL was applied to the ventral surface again after 20 minutes. Sterile water was used as a negative control. The leaves coated with the extract and sterile water were punctured with 10 inoculation holes, and 20 μL OD was dripped into each hole. 600 The concentration of the bacterial solution of walnut black spot pathogen (Xanthomonas sp., isolated from walnut black spot disease plants) was 0.6, and each treatment was repeated 5 times. In addition, a blank control was set up with leaves smeared with sterile water. Ten holes were punctured with a needle and 20 μL of sterile water was added to each hole.

[0063] The results are as follows Figure 5-7 As shown, after 7 days of treatment, there was no change in the leaves of the blank control (control), and obvious black spots appeared at the inoculation holes of the negative control (inoculation). The incidence rate and black spot size of leaves treated with different concentrations of Artemisia annua extract were significantly reduced compared with the negative control.

[0064] Example 4 Disease status of fruits treated with Artemisia argyi extract

[0065] Walnut fruits were collected. The experimental group was treated with the extract of Artemisia grandis (prepared by the same method as in Example 1) and then inoculated with the pathogen of walnut black spot disease (Xanthomonas sp., isolated from walnut black spot disease plants) by acupuncture. The control group was treated with sterile water and then inoculated with the pathogen of walnut black spot disease by acupuncture. The disease condition of the fruits was observed and recorded.

[0066] The results are as follows Figure 8 As shown, after 30 days of treatment, large black spots appeared on the walnut fruits in the control group, while there were no black spots at the puncture sites in the experimental group or the black spots only remained the size of the puncture sites.

[0067] Example 5 Determination of plant extract components

[0068] Mixed samples (MS) of Artemisia sieversiana ethanol extract with antibacterial effect and 8 plant extracts with basically no antibacterial effect (Elsholtzia ciliata, Epilobium angustifolium, Astragalus membranaceus, Rock fennel, Clematis macrophylla, Dioscorea zingiberensis, Potentilla officinalis, Vitex negundo) were sent to Myvi Metabolism for secondary metabolomics detection.

[0069] The results are as follows Fig. 9 As shown in the figure, among the differential metabolites between AS and MS, the content of acetaminophen in AS was higher.

[0070] Example 6 Antibacterial effect of co-culture of yam and pathogenic bacteria

[0071] Since cypermethrin is a component of Artemisia grandis extract, the inhibition rate of cypermethrin against black spot pathogens was determined by double equal gradient dilution 96-well plate method:

[0072] Take 2 mg of yamidine and dissolve it in 4 mL of anhydrous ethanol to obtain a mother solution with a concentration of 0.5 mg / mL. Take 1 mL of the mother solution and dilute it to 10 mL with sterile water. The initial concentration is 0.05 mg / mL (50 μg / mL), and then dilute it twice in a gradient (50 μg / mL, 25 μg / mL, 12.5 μg / mL). Add sterile water to 10 mL with 1 mL of anhydrous ethanol, and then dilute it twice in a gradient as a gradient control of ethanol concentration content such as yamidine ethanol solution. The walnut black spot pathogen (Xanthomonas, obtained by self-isolation from walnut black spot plants) was inoculated into YEP liquid culture medium and cultured to the logarithmic phase (OD600=0.6) at 28°C and 180 r / min. Each group of reagents was mixed with an equal volume of bacterial solution for co-cultivation test.

[0073] The results are as follows Fig.10 As shown, acylatin has a significant inhibitory effect on the pathogen of walnut black spot disease.

[0074] Example 7 Disease status of detached leaves treated with cypermethrin

[0075] Experimental group: 50 μg / mL of ciprofloxacin was applied to the leaf surface, 250 μL was applied to the ventral surface, and 50 μL was applied to the dorsal surface. Ten inoculation holes were punctured on the leaves, and 20 μL of OD was dripped into each hole. 600 The control group: Sterile water was applied to the leaf surface, 250 μL on the ventral side and 50 μL on the back side. Ten inoculation holes were punctured on the leaves, and 20 μL OD was added to each hole. 600 The pathogen solution was 0.6 as a negative control; 4 mg / mL oleanolic acid was prepared and applied to the leaf surface, 250 μL was applied to the ventral side and 50 μL was applied to the back side. Ten inoculation holes were punctured on the leaves, and 20 μL OD was added to each hole. 600 The pathogenic bacteria liquid with a concentration of 0.6 was used as a control for drugs without antibacterial effect; 2% kasugamycin diluted 1000 times was used as a control for conventional drugs; and sterile water was dripped at the acupuncture site as a blank control.

[0076] The results are as follows Figure 11-13 As shown, kasugamycin can effectively reduce the incidence and size of walnut black spot disease, and its effect is better than kasugamycin.

[0077] The above are only preferred embodiments of the present invention. The protection scope of the present invention is not limited to the embodiments shown herein. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some modifications and embellishments without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. The application of Artemisia grandiflora extract in the prevention and treatment of walnut black spot disease is characterized in that: The pathogen of walnut black spot disease is Xanthomonas; The preparation method of the Artemisia grandiflora extract comprises the following steps: Take Artemisia grandis plant material, add extraction reagent, perform ultrasonic extraction or heating reflux extraction, centrifuge to obtain supernatant after extraction, and concentrate by vacuum distillation; The extraction reagent includes water, ethanol solution or petroleum ether.

2. The use according to claim 1, characterized in that: The antibacterial component in the Artemisia grandiflora extract includes cypermethrin; the molecular formula of cypermethrin is: 30 H 40 O6.

3. The use according to claim 1, characterized in that: The Artemisia argyi plant material includes fresh powder of the aerial part of Artemisia argyi; The volume fraction of the ethanol solution is 70%; It also involves soaking before extraction; It also includes filtration using a membrane filter after concentration.

4. Application of cypermethrin in the prevention and treatment of walnut black spot disease, wherein the molecular formula of cypermethrin is: C 30 H 40 O6, the pathogen of walnut black spot disease is Xanthomonas.

5. A method for preventing and controlling walnut black spot disease, characterized in that: Spraying Artemisia grandis extract or Artemisia selengensis extract on walnut branches, leaves and fruits; The pathogen of walnut black spot disease is Xanthomonas; The Artemisia selengensis extract includes Artemisia selengensis water extract, Artemisia selengensis ethanol extract or Artemisia selengensis petroleum ether extract; the antibacterial component in the Artemisia selengensis extract includes cypermethrin; The molecular formula of the oxaliplatin is: 30 H 40 O6.

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