Osthole compound pesticide composition for preventing and treating plant pathogenic fungi and application thereof

Abstract

The invention discloses an osthole compound pesticide composition for preventing and treating plant pathogenic fungi and application of the osthole compound pesticide composition. The composition comprises an effective component A and an effective component B, the effective component A is cnidium lactone, and the effective component B is eugenol, azoxystrobin or hydroxychloroquine. The composition is compounded by using the cnidium lactone and eugenol, the cnidium lactone and azoxystrobin as well as the cnidium lactone and hydroxychloroquine as effective components, so that the composition has an obvious synergistic effect on preventing and treating tea leaf spot and tomato gray mold, and the generation of drug resistance and drug resistance of plants can be delayed by compounding the effective components; more choices are provided for prevention and treatment of pathogenic fungi, and the bacillus subtilis has a wide application prospect in prevention and treatment of plant pathogenic fungi diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pesticides, and particularly relates to a cuminol compound pesticide composition for preventing and treating plant pathogenic fungi and application thereof. BACKGROUND

[0002] Fungi are eukaryotic organisms with a nucleus, filamentous vegetative bodies, spore production, and no chlorophyll, which absorb nutrients in a heterotrophic manner, and some of which can live parasitically on plants. Fungi are a large group of organisms that are widely distributed in nature. Currently, there are 87,861 species of fungi, of which more than 10,000 species of fungi can cause plant diseases, and are the most important plant pathogenic group. Plant fungal diseases caused by fungal infection account for about 70% to 80% of the total number of plant diseases. Fungi can cause important threats and disasters to ecological safety and biodiversity.

[0003] Osthole (Osthole, structural formula as shown in Figure 1 Osthole has excellent antibacterial activity on crop pathogenic fungi, and can be used for preventing and treating fungal diseases of important crops such as tea trees, tomatoes, cowpeas, rice, and grapes, and has a significant antibacterial effect on diseases caused by Botrytis cinerea. The osthole of the present application can delay the development of drug resistance of pathogenic bacteria, expand the fungicidal spectrum of pesticides, reduce the pollution of pesticides to the environment, and meet the policy of reducing the amount of pesticides and increasing efficiency. The osthole has a broad application prospect in the field of crop disease prevention and treatment.

[0004] Eugenol, also known as 4-allyl-2-methoxyphenol, has a chemical formula of C 10 H 12 O2 (structural formula as shown in Figure 2Eugenol (shown in Figure 2) is a colorless to pale yellow essential oil that is slightly soluble in water and soluble in organic solvents such as ethanol, acetone, ethyl acetate, ether, and chloroform. It is primarily used as a primary flavoring agent in flavors such as cloves, carnations, and dianthus. It can also be used as a food flavoring, pesticide, and preservative (Anna, M., Ramona, B., et al. Antimicrobial activity of eugenol and essential oils containing eugenol: a mechanistic viewpoint. Critical Reviews in Microbiology, 2017, 43:668-689). Eugenol has antibacterial activity, therapeutic effects on diabetes, and inhibits cholinesterase activity (Hayriye, GB, Ali, K., et al. Novel eugenol bearing oxypropanolamines: synthesis, characterization, antibacterial, antidiabetic, and anticholinergic potentials. Bioorganic Chemistry, 2019, 88: 102931-102931).

[0005] Azoxystrobin (structural formula: Figure 3 As shown), the chemical name is (E)-2-{2-[6(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate, the chemical formula is C 22 H 17N3O5, is a methoxy acrylate fungicide. Azoxystrobin belongs to QoI class of fungicides, which inhibits the mitochondrial respiration by blocking the cytochrome c reductase. It has high and broad spectrum fungicidal activity against almost all fungal (Ascomycotina, Basidiomycotina, Chytridiomycotina and Deuteromycotina) pathogens, such as powdery mildew, rust, leaf blight, net blotch, downy mildew, rice blast and so on. It can be used for foliar spray, seed treatment and soil treatment, mainly for the control of plant diseases in cereals, rice, peanuts, grapes, potatoes, fruit trees, vegetables, coffee, lawns and so on. The QoI class of fungicides is characterized by a very high resistance risk, which interferes with its biological activity (Balandzic, M., Stojsin, V., et al. Sensitivity of Cercospora Beticola isolates to azoxystrobin. Contemporary Agriculture, 2020, 69: 1-4.). Azoxystrobin has short-term developmental effects on larval and adult zebrafish, which can cause mitochondrial dysfunction in larval and adult zebrafish, induce oxidative stress in larval and adult zebrafish, and induce apoptosis in larval zebrafish, but has no effect on adult zebrafish. It can activate the immune response of larval and adult zebrafish (Fang, J.C., Pei, Z.W., et al. Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio). Aquatic Toxicology, 2018, 198: 129-140).

[0006] Hydroxychloroquine (HCC) (structure formula as Figure 4Hydroxychloroquine (HCQ, Plaquenil®, Quenamox®, Quenchil®, Aclora®, Solquin®, Sanum®) is a 4-aminoquinoline derivative antimalarial drug with similar action and mechanism to chloroquine, but with only half the toxicity. Hydroxychloroquine has anti-inflammatory, immunomodulatory, and anti-infective effects. Bioinformatics and drug chemistry studies on 15 selected potential targets of SARS-CoV-2 and 6 FDA drugs (chloroquine, hydroxychloroquine, favipiravir, lopinavir, remdesivir, and ritonavir) showed that hydroxychloroquine also has antiviral potential (Sinosh, S., Dharshini, G., et al. Structural and molecular basis of the interaction mechanism of selected drugs towards multiple targets of SARS-CoV-2 by molecular docking and dynamic simulation studies-deciphering the scope of repurposed drugs. Computers in Biology and Medicine, 2020, 55: 105960). Hydroxychloroquine is also commonly used to prevent and treat skin infections, often in combination with other antibiotics.

[0007] It has been found that long-term, continuous, and single application of one fungicide active ingredient can easily lead to the development of drug resistance in pathogens. The reasonable mixing of pesticides has many advantages, such as improving the control effect, delaying the development of drug resistance, reducing the cost of use, and expanding the fungicidal spectrum. In view of the fact that osthole has the effect of treating cancer in medicine, there is no osthole used in the agricultural field at present. In the present study, osthole, eugenol, and azoxystrobin or hydroxychloroquine were mixed, and the activity of the mixture on plant pathogenic fungi was determined, in order to lay a foundation for the development of crop fungicides. SUMMARY

[0008] In view of this, one of the purposes of the present application is to provide an osthole compound pesticide composition for preventing and treating plant pathogenic fungi, which comprises effective component A and effective component B, the effective component A is osthole, and the effective component B is eugenol, azoxystrobin, or hydroxychloroquine.

[0009] Preferably, the mass ratio of the effective component A and the effective component B is 1:1, 1:2, or 2:1.

[0010] Preferably, the above-mentioned composition is prepared as a dosage form of a suspension or a water dispersible granule. The above-mentioned effective components can be prepared into different dosage forms with adjuvants known to those skilled in the art.

[0011] The second purpose of the present application is to provide a preparation method of the above-mentioned composition, comprising the following steps:

[0012] 1) Take 0.012g of osthole and dissolve it in DMSO (dimethyl sulfoxide) to prepare mother liquor I with the concentration of osthole being 100mg / mL; take Eugenol, Azoxystrobin or hydroxychloroquine and dissolve them in DMSO respectively to prepare mother liquor II with the concentration being 100mg / mL;

[0013] 2) Mix mother liquor I and mother liquor II according to the proportion to prepare the compound.

[0014] The third object of the present application is to provide the application of the above-mentioned composition in inhibiting the disease caused by plant pathogenic fungi.

[0015] Preferably, the plant pathogenic fungi include Botrytis cinerea and Lasiodiplodia theobromae.

[0016] Preferably, the use concentration of the composition is greater than or equal to 10μg / mL.

[0017] The bactericidal composition of osthole, Eugenol, Azoxystrobin or hydroxychloroquine prepared by the present application has synergistic effect and is safe and environmentally friendly, which is used for preventing and treating common plant diseases such as tea leaf spot and tomato gray mold; compared with the existing prevention and treatment agents, the present application realizes the purposes of improving the efficacy, reducing the pollution and lowering the cost. The compound pesticide composition of the present application has strong synergistic effect; the action mode of the effective components is unique and has no cross resistance with the existing fungicides; the dosage form is advanced and environmentally friendly, which has little environmental pollution and does not add organic components in the preparation; the preparation has fast speed and long effective period, and has broad application prospect in the prevention and treatment of plant pathogenic fungal diseases. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the structural formula of osthole;

[0019] Figure 2 is the structural formula of Eugenol;

[0020] Figure 3 is the structural formula of Azoxystrobin;

[0021] Figure 4 is the structural formula of hydroxychloroquine;

[0022] Figure 5 is the antibacterial activity of osthole and Eugenol with the compound ratio of 1:1, 1:2 and 2:1 on Botrytis cinerea mycelium after 48h of treatment, and the scale is 1cm;

[0023] Figure 6Antifungal activity of osthole and azoxystrobin at 1:1, 1:2 and 2:1 ratio against Botrytis cinerea after 48h treatment, scale = 1cm.

[0024] Figure 7 Antifungal activity of osthole and hydroxychloroquine at 1:1, 1:2 and 2:1 ratio against Botrytis cinerea after 48h treatment, scale = 1cm.

[0025] Figure 8 Antifungal activity of osthole and eugenol at 1:1, 1:2 and 2:1 ratio against Lasiodiplodia theobromae after 48h treatment, scale = 1cm.

[0026] Figure 9 Antifungal activity of osthole and azoxystrobin at 1:1, 1:2 and 2:1 ratio against Lasiodiplodia theobromae after 48h treatment, scale = 1cm.

[0027] Figure 10 Antifungal activity of osthole and hydroxychloroquine at 1:1, 1:2 and 2:1 ratio against Lasiodiplodia theobromae after 48h treatment, scale = 1cm.

[0028] Figure 11 Antifungal activity of osthole and eugenol at 1:1, 1:2 and 2:1 ratio against Botrytis cinerea on living tomato fruits after 48h treatment, scale = 1cm.

[0029] Figure 12 Antifungal activity of osthole and eugenol at 1:1, 1:2 and 2:1 ratio against Botrytis cinerea on living tomato fruits after 48h treatment, scale = 1cm. DETAILED DESCRIPTION

[0030] The application will be described in greater detail below with reference to the embodiments. These embodiments are merely exemplary and are not intended to limit the scope of the application. The application is not limited to the embodiments or examples described below, and modifications and variations of the application that do not depart from the spirit and scope of the application will be considered within the scope of the application. The experimental materials used in the following examples are commercially available unless otherwise specified. The percentages mentioned in the following examples are mass percentages unless otherwise specified.

[0031] To determine the synergistic effect of osthole with eugenol, azoxystrobin or hydroxychloroquine, first, the two components were used for indoor bioassay test. After analyzing the characteristics of the two active ingredients, the objects of bioassay were determined as B. cinerea (strain number: GZFQ-1, accession number: CGMCC3.20932) and L. theobromae (strain number: GZHS-2017-010, accession number: CGMCC3.20151) for the following specific experiments. Among them, B. cinerea belongs to the genus Botrytis, which can cause gray mold disease of vegetables, grapes and the like. L. theobromae belongs to the genus Colletotrichum, which can cause tea leaf spot disease. In tropical and subtropical regions, it can cause diseases such as top bud death, wilt, stem wilt, root rot, stem rot, fruit rot and the like on various different hosts. Host plants such as guavas, coconuts, papayas and grapes and the like.

[0032] Experimental Example 1 Toxicity Experiment

[0033] The indoor bioassay experiment method was used: referring to the standard NY / T1156.7-2006, the growth rate method was used to determine the inhibition effect of the fungicides on the mycelial growth of tea leaf spot and gray mold pathogenic fungi.

[0034] (1) Preparation of the fungicides

[0035] 1) 0.102 g of osthole technical material (98%) (purchased from Chongqing Platinum Titanium Technology Co., Ltd.) was weighed and dissolved in 1 mL of DMSO to prepare a 100 mg / mL stock solution.

[0036] 2) 0.102 g of eugenol technical material (98%) (purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.) was weighed and dissolved in 1 mL of DMSO to prepare a 100 mg / mL stock solution.

[0037] 3) 0.102 g of azoxystrobin technical material (98%) (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) was weighed and dissolved in 1 mL of DMSO to prepare a 100 mg / mL stock solution.

[0038] 4) 0.102 g of hydroxychloroquine technical material (98%) (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) was weighed and dissolved in 1 mL of DMSO to prepare a 100 mg / mL stock solution.

[0039] 5) Compounding: osthole was compounded with eugenol, azoxystrobin or hydroxychloroquine, and the compounding ratio was set as 1:1, 1:2 and 2:1. The above stock solutions were configured according to the ratio to obtain the compounding agents of each ratio.

[0040] (2) Indoor test of osthole, eugenol, azoxystrobin or hydroxychloroquine single agent and compounding agent

[0041] On the basis of preliminary experiments, four concentration gradients were designed in the range of 10% to 90% of the mycelial growth inhibition rate of each fungicide, i.e. each fungicide (single agent and complex agent) was added to the PDA medium which had been melted and cooled to room temperature, so that the final concentration was 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL (single agent or part of complex agent) or 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL (part of complex agent), and then poured into sterile culture dishes (100 mm) to make drug-containing plates, each treatment was repeated 5 times, and the treatment with sterile water was used as a blank control. L. theobromae or B. cinerea was inoculated aseptically on the central part of the drug-containing plate with mycelium facing down (wherein the strain and the concentration gradient of the complex agent are shown in Table 1-7), and then cultured in a constant temperature incubator at 25°C. After 3-9 days of culture, the colony diameter was measured by the cross method, the inhibition rate was calculated according to formula (1), and then the regression equation, r Figures 5-10 , and EC 2 were calculated by DPS v 9.01 software (http: / / www.dpsw.cn / dps_eng / ). 50 The regression equation represents the relationship between the mycelial growth rate and the amount of fungicide used; EC 50 represents the median effective concentration.

[0042]

[0043] The biological activity of the mixture of A and B was evaluated by Wadley method, and SR (synergistic ratio) was calculated according to formulas (2) and (3).

[0044]

[0045] In the formulas, a and b are the proportions of A and B single agents in the complex agent; EC(A) 50 and EC(B) 50 are the EC 50 values of A and B, respectively; and EC 50(th) is the EC 50 value of the complex agent.

[0046]

[0047] SR represents the interaction of A and B after mixing: EC 50(ob) represents the measured EC 50 value after mixing. SR > 1.5 indicates synergistic or synergistic effect, 0.5 ≤ SR ≤ 1.5 indicates additive effect, and SR < 0.5 indicates antagonistic effect.

[0048] The results are shown in Tables 1-7.

[0049] Table 1 Toxicity test results of osthol on tomato gray mold and tea leaf spot fungus

[0050]

[0051] Table 2 Toxicity test results of eugenol on tomato gray mold and tea leaf spot fungus

[0052]

[0053] Table 3 Toxicity test results of azoxystrobin on tomato gray mold and tea leaf spot fungus

[0054]

[0055] Table 4 Toxicity test results of hydroxychloroquine on tomato gray mold and tea leaf spot fungus

[0056]

[0057] Table 5 Toxicity test results of different proportions of osthol and eugenol on tomato gray mold and tea leaf spot fungus

[0058]

[0059] Table 6 Toxicity test results of different proportions of osthol and azoxystrobin on tomato gray mold and tea leaf spot fungus

[0060]

[0061]

[0062] Table 7 Toxicity test results of different proportions of osthol and hydroxychloroquine on tomato gray mold and tea leaf spot fungus

[0063]

[0064] From the above data, it can be concluded that when osthol is compounded with eugenol or azoxystrobin in different proportions, the control ability on tomato gray mold and tea leaf spot fungus has different proportions of synergistic effect; when osthol is compounded with hydroxychloroquine in different proportions, the control ability on tomato gray mold and tea leaf spot fungus has different proportions of additive effect. It can be seen that the compound preparation of osthol, eugenol, azoxystrobin or hydroxychloroquine can be used as an ideal pesticide for controlling L. theobromae and B. cinerea.

[0065] Experimental Example 2 In-vivo test

[0066] According to the above indoor SR value of the osthole and eugenol complex composition, the indoor living body test of preventing and treating tomato gray mold B. cinerea is carried out, and the prevention and treatment effect of the osthole and eugenol complex on tomato gray mold B. cinerea is determined.

[0067] (1) The bacteriostatic activity of the osthole and eugenol complex composition on B. cinerea living body

[0068] Tomato fruits are selected from a plantation in Guizhou, and after being transported back to the laboratory, fruits with uniform size and full color are selected, wiped clean with moist sterile cotton, and naturally dried to remove surface moisture. Each treatment group is set up with 5 replicates. Osthole and eugenol are prepared in a ratio of 1:1, 1:2, and 2:1, respectively, and complex solutions with concentrations of 10, 100, 500, 1000, and 2000 μg / mL are prepared (preparation method refers to experimental example 1). The fruits are sprayed with the treatment, and the spraying concentrations are 10, 100, 500, 1000, and 2000 μg / mL, respectively, with sterile water as the positive control group. After the fruit surface is naturally dried, a syringe is used to punch four small holes on the fruit, with a depth of 2 mm. Then, a sterilized punch with a diameter of (4 mm) is used to punch a fungus cake on the colony of fresh B. cinerea cultured for 3 days, and the punched fungus cake is inoculated onto the front of the fruit wound, with sterile PDA medium inoculation as the negative control group and sterile water spraying as the positive control group. After 48 h, the pathogenicity is observed, photographed, and counted.

[0069] Disease index = lesion area

[0070] Prevention and treatment effect % = [(control disease index - treatment disease index) / control disease index] * 100

[0071] Table 8 Results of indoor living body efficacy test for preventing and treating pathogenic fungi

[0072]

[0073] The results are shown in Table 8 and Figure 11 (From left to right: CK (sprayed with water, blank PDA), CK (not sprayed with medicine, fungus disc pathogenicity), 10 μg / mL complex solution spraying, 100 μg / mL complex solution spraying, 500 μg / mL complex solution spraying, 1000 μg / mL complex solution spraying, 2000 μg / mL complex solution spraying, fungus disc pathogenicity for 48 h) As shown, after spraying the osthole and eugenol complex combination 1:1, 1:2, and 2:1, the inhibition rate of the pathogenic fungus B. cinerea lesion showed an upward trend with increasing drug concentration. As shown in Table 8 and Figure 12As shown in the figure (the abscissa represents the concentration of osthole and eugenol after compounding, and the ordinate represents the disease spot area and inhibition rate of tomato fruit after inoculation with B. cinerea after spraying osthole and eugenol after compounding), after spraying the combination of osthole and eugenol, with the increase of the concentration of the added drug, the disease spot area of the pathogenic bacteria B. cinerea showed a downward trend, and the inhibition rate of the disease spot showed an upward trend; compared with the control, the plaque area and inhibition rate of the low-concentration drug of 10 μg / mL had a significant difference.

[0074] In summary, the combination of osthole and eugenol has obvious prevention and control effect on the spread of pathogenic fungi, which is conducive to delaying the drug resistance of plants. The formula found in the present study has low cost and good environmental compatibility.

[0075] The conventional techniques and schemes not described in detail in the above examples are well known in the art, and therefore will not be described in detail here. The above examples and / or experimental examples describe the preferred embodiments of the present application in detail, however, the present application is not limited to the specific details in the above embodiments, and within the scope of the technical concept of the present application, various simple modifications can be made to the technical scheme of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. An osthole compound pesticide composition for controlling plant pathogenic fungi, characterized in that: The composition comprises an active ingredient A and an active ingredient B, wherein the active ingredient A is osthole, and the active ingredient B is eugenol, azoxystrobin or hydroxychloroquine.

2. The composition according to claim 1, wherein The mass ratio of active ingredient A to active ingredient B is 1:1, 1:2 or 2:

1.

3. The composition according to any one of claims 1 to 2, characterized in that The composition is prepared in the form of a suspension or water-dispersible granules.

4. The method for preparing the composition according to any one of claims 1 to 3, characterized in that: The steps include: 1) Weighing osthole and dissolving it in DMSO to prepare a stock solution I with an osthole concentration of 100 mg / mL; weighing eugenol, azoxystrobin, or hydroxychloroquine and dissolving them in DMSO to prepare a stock solution II with a concentration of 100 mg / mL; 2) Mix the mother liquor I and the mother liquor II in proportion for compounding.

5. Use of the composition according to any one of claims 1 to 3 in preventing and controlling plant pathogenic fungi.

6. The use according to claim 5, characterized in that The plant pathogenic fungi include two types: Botrytis cinerea and Lasiodiplodia theobromae.

7. The use according to claim 6, characterized in that The composition is used at a concentration of greater than or equal to 10 μg / mL.

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