A plant-derived extract that effectively inhibits the spider web pathogen of Odetteria oosporei and its application

Abstract

The present invention discloses a plant-derived extract that effectively inhibits the spiderweb disease of Oudemansiella raphanipes and its use. The present invention provides the use of a star anise methanol extract for any of the following purposes: preventing and controlling the spiderweb disease of Oudemansiella raphanipes and / or promoting the growth of mycelium; inhibiting Bacillus heterocladus; and simultaneously inhibiting Bacillus heterocladus and other common crop pathogens. The star anise methanol extract provided by the present invention can effectively prevent and control the spiderweb disease of Oudemansiella raphanipes. Compared with traditional chemical control methods, this method is environmentally friendly, pollution-free, and has a low potential risk of pesticide residues.

Description

Technical Field

[0001] The present invention relates to the technical field of crop disease prevention and control, and in particular to a plant-derived extract capable of effectively inhibiting the pathogenic bacteria of Odetteria oosporea and an application thereof. Background Art

[0002] Oudemansiella raphanipes, also known as "black-skinned chicken mushroom," is a rare and valuable high-end health-promoting mushroom with both edible and medicinal value. It is now cultivated in many provinces and cities across China and is highly sought after by mushroom farmers and consumers. Spiderweb disease has been discovered for the first time as a pest of edible mushrooms. Its primary host is Agaricus bisporus, affecting 25% of fruiting bodies. With the gradual development of the edible mushroom industry, the host range of spiderweb disease has also gradually expanded. Spiderweb disease of Oudemansiella raphanipes is an emerging disease discovered by our research team in Tongzhou, Beijing in 2019. The main symptom in the field is the appearance of grayish-white, spider-web-like hyphae on the soil, which gradually spread to the base of the stipe, then upwards to the gills and even the cap, ultimately completely covering and enveloping the fruiting bodies, causing necrosis and rot. The pathogen was isolated and identified as Cladobotryum varium. This disease severely impacts the yield and quality of Oudemansiella raphanipes, causing significant distress and economic losses for mushroom farmers.

[0003] Currently, physical and chemical control are commonly used to control fungal diseases of edible fungi. However, with the use of chemical agents in production, there are also varying degrees of pesticide residues on the fruiting bodies of edible fungi, posing a safety hazard to agricultural products. Biological control is a green control method with no residues. It mainly uses the interaction between pathogens, biological metabolites with antibacterial effects, or extracts of plants and Chinese herbal medicines for control, thereby reducing the application of chemical agents for comprehensive disease prevention and control. Star anise is a common spice in life and an economic crop that is both a medicine and a food. As a green and healthy food, edible fungi have higher requirements for disease control inputs. As a natural plant, star anise extracts have the prospect of developing botanical pesticides. However, no relevant research reports have been reported in the field of edible fungi diseases. Summary of the Invention

[0004] The present invention aims to provide a plant-derived extract for effectively inhibiting the pathogenic bacteria of Odetteria oosporea and application thereof.

[0005] In the first aspect, the present invention claims the use of anise methanol extract in any of the following:

[0006] (A1) preventing and controlling spider web disease of Oudemansiella raphanipes and / or promoting hyphae growth of Oudemansiella raphanipes;

[0007] (A2) preparing a product for inhibiting spider web disease of Oudemansiella raphanipes and / or promoting mycelial growth of Oudemansiella raphanipes;

[0008] (A3) inhibition of Cladobotryum varium;

[0009] (A4) Preparation of products for inhibiting Cladobotryum varium;

[0010] (A5) simultaneously inhibiting Cladobotryum varium and all or part of the following pathogens: Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae, Alternaria alternata, Colletotrichum capsici, Corynespora acassiicola, Diploospora longispora, and Hypomyces sp.;

[0011] (A6) Preparing a product for simultaneously inhibiting Cladobotryum varium and all or part of the following pathogens: watermelon wilt pathogen (Fusarium oxysporum), strawberry gray mold pathogen (Botrytis cinerea), cotinus wilt pathogen (Verticillium dahliae), potato leaf spot pathogen (Alternaria alternata), pepper anthracnose pathogen (Colletotrichum capsici), hydrangea leaf spot pathogen (Corynespora cassiicola), morel cap rot pathogen (Diploospora longispora) and wood ear spider web pathogen (Hypomyces sp.).

[0012] The star anise methanol extract can be prepared according to a method comprising the following steps: extracting star anise with methanol, collecting the extract and removing the methanol to obtain the star anise methanol extract.

[0013] Furthermore, the star anise methanol extract can be prepared according to a method comprising the following steps: adding pure methanol (38 ml) to star anise powder (2 g) in a ratio of 2 g:38 ml, mixing and placing at room temperature (25° C.) for 10 hours, then ultrasonically crushing the mixture (power 220 W, ultrasound 6 seconds, intermittent 9 seconds) for 60 minutes, filtering (0.015 MPa), removing the filter residue, and then rotary evaporating at 60° C. to obtain an extract.

[0014] Furthermore, after obtaining the extract, the method may further include drying the extract at 60° C. for 12 hours.

[0015] Furthermore, after drying, the method may further include a reconstitution step with sterile water. The ratio of sterile water used during reconstitution to the initially weighed star anise powder is 4.5 mL sterile water: 2 g star anise powder. Alternatively, the concentration of the star anise methanol extract in the resulting solution after reconstitution is 0.02 g / mL.

[0016] The star anise powder is prepared according to a method comprising the following steps: drying the star anise in an oven at 55° C. to a constant weight (e.g., 10 hours), crushing the star anise with a wall breaker, and then passing the crushed star anise through a 100-mesh sieve to obtain the star anise powder.

[0017] In a second aspect, the present invention claims a method for preparing anise methanol extract.

[0018] The method for preparing the methanol extract of star anise claimed in the present invention may include the steps described in the first aspect above.

[0019] In a third aspect, the present invention claims protection for the star anise methanol extract prepared by the method described in the second aspect above.

[0020] In a fourth aspect, the present invention claims the use of star anise in preparing any of the following products (B1) to (B3):

[0021] (B1) preparing a product for preventing and treating spider web disease of Oudemansiella raphanipes and / or promoting mycelial growth of Oudemansiella raphanipes;

[0022] (B2) preparing a product for inhibiting Cladobotryum varium;

[0023] (B3) preparing a product for simultaneously inhibiting Cladobotryum varium and all or part of the following pathogens: watermelon wilt pathogen (Fusarium oxysporum), strawberry gray mold pathogen (Botrytis cinerea), cotinus wilt pathogen (Verticillium dahliae), potato leaf spot pathogen (Alternaria alternata), pepper anthracnose pathogen (Colletotrichum capsici), hydrangea leaf spot pathogen (Corynespora cassiicola), morel cap rot pathogen (Diploospora longispora), and wood ear spider web pathogen (Hypomyces sp.);

[0024] The active ingredient in the product comprises or is the star anise methanol extract described in the third aspect above.

[0025] In a fifth aspect, the present invention claims protection for a product having any of the functions shown below (C1)-(C3), wherein the active ingredient comprises or is the star anise methanol extract described in the third aspect above.

[0026] (C1) preventing and controlling spider web disease of Oudemansiella raphanipes and / or promoting hyphae growth of Oudemansiella raphanipes;

[0027] (C2) inhibits Cladobotryum varium;

[0028] (C3) Simultaneously inhibits Cladobotryumvarium and all or part of the following pathogens: Fusarium oxysporum, Botrytis cinerea, Verticillium dahliae, Alternaria alternata, Colletotrichum capsici, Corynespora cassicola, Diploospora longispora, and Hypomyces sp.

[0029] In a sixth aspect, the present invention claims a method for preventing and controlling spider web disease of Oudemansiella raphanipes and / or promoting hyphae growth of Oudemansiella raphanipes.

[0030] The method for preventing and controlling spider web disease of Oudemansiella raphanipes and / or promoting mycelial growth of Oudemansiella raphanipes claimed in the present invention may include the following steps: applying the star anise methanol extract described in the third aspect above to Oudemansiella raphanipes or its culture medium.

[0031] In a seventh aspect, the present invention claims a method for inhibiting Cladobotryum varium.

[0032] The method for inhibiting Cladobotryum varium claimed in the present invention may comprise the following step: applying the star anise methanol extract described in the third aspect above to Cladobotryum varium or its culture medium.

[0033] In an eighth aspect, the present invention claims a method for simultaneously inhibiting Cladobotryum varium and all or part of the following pathogens: watermelon wilt pathogen (Fusarium oxysporum), strawberry gray mold pathogen (Botrytis cinerea), cotinus wilt pathogen (Verticillium dahliae), potato leaf spot pathogen (Alternaria alternata), pepper anthracnose pathogen (Colletotrichum capsici), hydrangea leaf spot pathogen (Corynespora cassiicola), morel cap rot pathogen (Diploospora longispora) and wood ear spider web disease pathogen (Hypomyces sp.).

[0034] The method claimed in the present invention for simultaneously inhibiting Cladobotryumvarium and all or part of the above pathogens may include the following steps: applying the star anise methanol extract described in the third aspect above to the target bacteria or its culture medium.

[0035] In the sixth to eighth aspects above, the star anise methanol extract may be used at a concentration of 0.001-0.002 g / mL.

[0036] In the present invention, the raw material for preparing the star anise methanol extract is dried star anise fruit.

[0037] Experiments have shown that the star anise methanol extract provided by the present invention can effectively prevent and control the spider web pathogen of Oudemansiella raphanipes and promote the growth of Oudemansiella raphanipes mycelium. Compared with traditional chemical control methods, this method has the advantages of being environmentally friendly, pollution-free, and having a low potential risk of pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The methanol extraction process for star anise. A: Star anise after drying for 10 hours; B: Star anise powder after crushing and sieving; C: Star anise after 10 hours of methanol soaking; D: Ultrasonic treatment; E and F: Extracts from star anise extracted with methanol.

[0039] Figure 2 Figure 3. Inhibitory effects of star anise extracts and carbendazim in different solvents on Staphylococcus heterocladus (PDA, 4 days). A: ethanol star anise extract; B: acetone star anise extract; C: ethyl acetate star anise extract; D: dichloromethane star anise extract; E: chloroform star anise extract; F: 2% sodium chloride star anise extract; G: 2% sodium chloride solution; H: methanol star anise extract; I: carbendazim; J: sterile water.

[0040] Figure 3 Figure 3. Inhibitory effects of star anise extracts and carbendazim in different solvents on Odette oosporidis (PDA, 4 days). A: ethanol star anise extract; B: acetone star anise extract; C: ethyl acetate star anise extract; D: dichloromethane star anise extract; E: chloroform star anise extract; F: 2% sodium chloride star anise extract; G: 2% sodium chloride solution; H: methanol star anise extract; I: carbendazim; J: sterile water.

[0041] Figure 4 The mycelial growth of different pathogens under sterile water control (PDA, 4 days): A: Potato leaf spot pathogen; B: Auricularia auriculariae cochinchinensis; C: Morchella oleracea cap rot pathogen; D: Hydrangea leaf spot pathogen; E: Pepper anthracnose pathogen; F: Strawberry gray mold pathogen; G: Fusarium wilt pathogen; H: Cotinus coggygria wilt pathogen.

[0042] Figure 5 The effect of star anise methanol extract on the mycelial growth of different pathogens (PDA, 4 days). A: potato leaf spot pathogen; B: wood ear spider web pathogen; C: morel cap rot pathogen; D: hydrangea leaf spot pathogen; E: pepper anthracnose pathogen; F: strawberry gray mold pathogen; G: yellow watermelon wilt pathogen; H: cotinus coggygria wilt pathogen. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0044] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0045] Oudemansiella raphanipes and Cladobotryum varium: Both are recorded in the article "First Report of Cobweb Disease in Oudemansiella raphanipes Caused Cladobotryum varium in Beijing, China. Plant Disease, Volume 105, Number 12, 18 November 2021". With the applicant's consent, the public may obtain them from the applicant and may only be used to repeat the experiments of the present invention and may not be used for other purposes.

[0046] Example 1. Preparation of Star Anise Extract

[0047] Weigh 10 g of star anise fruit (purchased from the dry material market), dry it in a 55°C oven for 10 h until constant weight (the weight difference after two consecutive drying and weighing is less than 0.5 g), crush it with a wall breaker, and then pass it through a 100-mesh sieve.

[0048] Weigh 2 g of ground star anise powder, add 38 mL of different solvents (methanol, ethanol, acetone, ethyl acetate, dichloromethane, chloroform, 2% (i.e., 20 g / L) sodium chloride solution), stir rapidly until completely mixed, and place at room temperature (25°C) for 10 hours; the mixture that has been placed for 10 hours is ultrasonically broken (power 220 W, ultrasonic 6s, intermittent 9s) for 60 minutes, and the resulting solution is filtered (0.015 MPa) until no liquid drips from the Buchner funnel, and then the filter residue is removed and rotary evaporated using a rotary evaporator (60°C, 35 rpm) to concentrate it into an extract; the extract is placed in a 50 mL centrifuge tube and dried in a 60°C oven for 12 hours to constant weight (about 0.09 g), and 4.5 mL of sterile water is added for reconstitution. The solution is the star anise plant extract (concentration is 0.02 g / mL), which is sealed and stored at 4°C for later use.

[0049] Weigh 2 g of ground star anise powder, add 18 mL of sterile water, stir rapidly until completely mixed, place at room temperature (25 ° C) for 10 hours, and then ultrasonically crush for 60 min (power 220, ultrasound 6s, intermittent 9s). The obtained solution is filtered (0.015 MPa), and after removing the filter residue, centrifuge at 6000 r / min for 15 min. The supernatant is filtered and sterilized (0.22 μm) to obtain the star anise water extract, which is sealed and stored at 4 ° C for later use.

[0050] A solution with a concentration of 0.97 g / L was prepared using sterile water according to the recommended dosage (175 g / mu) of carbendazim (50% carbendazim wettable powder; Jiangsu Sanshan Co., Ltd.) and filtered sterilized (0.22 μm) as a positive control stock solution; the solution was stored at 4°C until use.

[0051] Figure 1 The process of extracting star anise with methanol solvent.

[0052] Example 2: Effects of carbendazim and anise extract on the mycelial growth of Staphylococcus heterocladus and Odetteria oosporeensis

[0053] After sterilization, 9 mL of the carbendazim solution (concentration of 0.97 g / L) prepared in Example 1 or different amounts of anise extracts (concentration of 0.02 g / mL) of different solvents were added to the flask (the final concentration of each plate after addition is shown in Table 1), and the PDA medium cooled to 55°C after sterilization was added and the volume was adjusted to 90 mL. The medium was shaken and evenly poured into the culture dish (15 mL per culture dish). After cooling for 24 hours, 8 mm in diameter and the same growth state of Cladonia ovata were respectively inoculated. obotryumvarium) and mycelial blocks of Agaricus ootryum, with the culture medium diluted to 15 mL after adding sterile water as control. Each treatment was repeated 3 times and cultured at 25°C. After 48 h, the colony diameters of Agaricus ootryum and S. heterocladus were determined by the cross-cross method, and the inhibition rates of different treatments on S. heterocladus mycelium were calculated [inhibition rate = (mycelial diameter of control group - mycelial diameter of experimental group) / (mycelial diameter of control group - diameter of inoculated cake) × 100%].

[0054] The results are shown in Table 1. The positive control chemical agent carbendazim had the highest inhibition rate of 90.1%. The most effective solvent, ethanol, could directly stop the growth of Staphylococcus heterocladus, but at the same time had a strong inhibitory effect on the host Agaricus oosporus mycelia. When methanol was used as the extractant, the inhibition rate against the pathogen Staphylococcus heterocladus could reach 88.57%. By changing the amount of extract added to the plate, a concentration gradient inhibition experiment was conducted to calculate the inhibition rate. Among them, the methanol extract had no negative impact on the host Agaricus oosporus mycelia (and had a certain promoting effect on the growth of Agaricus oosporus mycelia) and had a strong inhibitory effect on the growth of Staphylococcus heterocladus. The amount added to the extracts of different solvents was the smallest, and the inhibitory effect was closest to that of the chemical agent carbendazim.

[0055] Figure 2 The inhibitory effects of star anise extracts in different solvents and carbendazim on Staphylococcus axylodis (PDA, 4d). Figure 3 The inhibitory effects of star anise extracts in different solvents and carbendazim on Odette mushroom (PDA, 4d).

[0056] Table 1 Effects of different solvents of star anise extract and carbendazim on the mycelial growth of Staphylococcus heterocladus and Odetteria oosporeensis

[0057]

[0058]

[0059] Note: x: logarithmic value of concentration; y: bioprobability value of inhibition percentage. Different lowercase letters in the same column indicate significant differences (P<0.05).

[0060] Example 3: Effects of different star anise extracts on the mycelial growth of common pathogens of other crops

[0061] 9 mL of the carbendazim solution (concentration of 0.97 g / L) or the star anise extract (concentration of 0.02 g / mL) prepared in Example 1 was added to a sterilized triangular flask, and the PDA medium cooled to 55° C. after sterilization was added and the volume was adjusted to 90 mL. The medium was shaken and evenly poured into a culture dish. After cooling for 24 h, 8 different pathogen mycelial blocks (PDA, 5 d) with a diameter of 8 mm were respectively inoculated (see Table 2). 15 mL of sterile water was added to the culture medium as a control. Each treatment was repeated 3 times and cultured at 25° C. After 48 h, the colony diameter was determined by the cross-cross method. The average inhibition rate of different treatments on the mycelial growth of other common crop pathogens was calculated [inhibition rate = (mycelial diameter of the control group - mycelial diameter of the experimental group) / (mycelial diameter of the control group - inoculated cake diameter) × 100%].

[0062] The results are shown in Table 2. The methanol extract has a certain degree of antibacterial effect on different pathogens on different crops and has a certain broad spectrum.

[0063] Figure 4 The mycelial growth of different pathogens under sterile water control (PDA, 4 days). Figure 5 The effect of star anise methanol extract on the mycelial growth of different pathogens (PDA, 4d).

[0064] Table 2. Inhibition rate of star anise extracts from different solvents on the mycelial growth of different crop pathogens

[0065]

[0066]

[0067] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0068] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application of star anise methanol extract in any of the following: (A1) Preventing and controlling spider web disease of Odetteria oosporei and / or promoting the growth of Odetteria oosporei hyphae; (A2) preparing products for inhibiting spider web disease of Odetteria oosporida and / or promoting the growth of Odetteria oosporida hyphae; (A3) inhibiting Staphylococcus heterocladus; (A4) preparing a product for inhibiting Staphylococcus heterocladus; (A5) Simultaneously inhibiting all or part of the following pathogens: watermelon wilt pathogen, strawberry gray mold pathogen, cotinus wilt pathogen, potato leaf spot pathogen, pepper anthracnose pathogen, hydrangea leaf spot pathogen, morel cap rot pathogen and wood ear spider web disease pathogen; (A6) Preparing a product for simultaneously inhibiting Bacillus heteroclada and all or part of the following pathogens: watermelon wilt pathogen, strawberry gray mold pathogen, cotinus wilt pathogen, potato leaf spot pathogen, pepper anthracnose pathogen, hydrangea leaf spot pathogen, morel cap rot pathogen and wood ear spider web pathogen.

2. The use according to claim 1, characterized in that: The star anise methanol extract is prepared according to a method comprising the following steps: extracting the star anise with methanol, collecting the extract and removing the methanol to obtain the star anise methanol extract.

3. The use according to claim 1, characterized in that: The star anise methanol extract is prepared according to a method comprising the following steps: adding methanol to star anise powder in a ratio of 2g:38ml, mixing and placing at room temperature for 10 hours, then ultrasonically crushing the mixture, filtering, removing the filter residue and rotary evaporating to obtain an extract.

4. The use according to claim 3, characterized in that: After obtaining the extract, the method further comprises the step of drying the extract.

5. Use of star anise in the preparation of any of the following products (B1) to (B3): (B1) Products for controlling spider web disease of Odetteria oosporei and / or promoting the mycelial growth of Odetteria oosporei; (B2) Products for inhibiting Staphylococcus heterocladus; (B3) Products for simultaneously inhibiting Staphylococcus axylodis and all or part of the following pathogens: the pathogen of watermelon wilt, the pathogen of strawberry gray mold, the pathogen of cotinus wilt, the pathogen of potato leaf spot, the pathogen of pepper anthracnose, the pathogen of hydrangea leaf spot, the pathogen of morel cap rot and the pathogen of wood ear spider web disease; The active ingredient in the product comprises or is an star anise methanol extract; the star anise methanol extract is prepared according to the steps described in any one of claims 2-4.

6. A method for preventing and treating spider web disease of Odetteria oosporosa and / or promoting mycelial growth of Odetteria oosporosa, comprising the following steps: applying aniseed methanol extract to Odetteria oosporosa or its culture medium; the aniseed methanol extract is prepared according to the steps described in any one of claims 2 to 4.

7. A method for inhibiting Staphylococcus heterocladus, comprising the following steps: applying an anise methanol extract to Staphylococcus heterocladus or its culture medium; the anise methanol extract is prepared according to the steps described in any one of claims 2 to 4.

8. A method for simultaneously inhibiting Staphylococcus heterocladus and all or part of the following pathogens, comprising the steps of: applying an anise methanol extract to the target bacteria or a culture medium thereof; the anise methanol extract is prepared according to the steps of any one of claims 2 to 4; The pathogens are: watermelon wilt pathogen, strawberry gray mold pathogen, cotinus wilt pathogen, potato leaf spot pathogen, pepper anthracnose pathogen, hydrangea leaf spot pathogen, morel cap rot pathogen and wood ear spider web disease pathogen.

9. The method according to any one of claims 6 to 8, characterized in that: The use concentration of the star anise methanol extract is 0.001-0.002 g / mL.

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