Application of pinnatifone A in preparation of plant fungicide and plant fungicide

Abstract

The invention discloses an application of pinnatifone A in preparation of a plant fungicide and the plant fungicide. The mass percentage content of the pinnatifone A in the plant fungicide is 1%-10%. The effective component of the prepared bactericide is pinnatifone A, and the pinnatifone A can be prepared into a soluble concentrate, a microemulsion and an aqueous solution with the assistance of an auxiliary agent. The bactericide has the advantages of high efficiency, low toxicity, safety to non-target organisms, good environmental compatibility, no residual toxicity after use and simple preparation method, not only has a good control effect on gray mold on crops such as tomatoes and strawberries, but also can be used for controlling various plant diseases caused by phytopathogens such as apple tree canker, and has wide application prospects. The application and popularization values are realized.

Description

Technical Field

[0001] The invention belongs to the field of plants and their applications, and particularly relates to application of pinnatifone A in preparing a plant fungicide and the plant fungicide. Background Art

[0002] Plant diseases are one of the important reasons for crop yield reduction. They not only reduce crop yield and quality, but also often cause serious agricultural economic losses. Among plant diseases, fungal diseases and oomycete diseases are particularly difficult to control. Plant diseases caused by pathogenic fungi account for 70% to 80%. Their infection, occurrence and spread will lead to serious outbreaks of diseases in forests and crops. Botanical pesticides have good environmental compatibility, target organisms are not likely to develop resistance, low toxicity and low residue, etc., which have attracted much attention in the industry. The development of botanical pesticides has also become an important trend and development direction of the future pesticide market. In particular, new products such as botanical fungicides, antiviral agents, immune inducers and botanical stimulants have gradually become a hot spot in the research and development of biological pesticides.

[0003] Mountain agarwood is a commonly used medicine in Inner Mongolian medicine. It comes from the peeled and dried stems, roots and thick branches of Syringa pinnatifolia, a species endemic to the genus Syringa in the family Oleaceae. This medicine is also known as Helan Mountain Clove or its Mongolian name Alashan-Agaru. Syringa pinnatifolia is endemic to China. It is mainly produced in western China, including the Helan Mountain area at the junction of Inner Mongolia and Ningxia, as well as in the hillside shrubs in southern Shaanxi, Gansu, eastern Qinghai and western Sichuan. It is scattered in local areas such as the central Qinling Mountains in Shaanxi, Sichuan, Qinghai and Helan Mountain. Mountain agarwood has the effects of suppressing Hei, clearing away heat and relieving pain. It is mainly used to treat symptoms such as heart and chest pain, bloody pain, Hei heart pain, shortness of breath, and Hei disease of the main pulse. It is often used in the treatment of coronary heart disease, angina pectoris, chest tightness and shortness of breath, and myocardial ischemia and other cardiopulmonary diseases.

[0004] In addition to the two main components, lignans and sesquiterpenes, mountain agarwood also contains cyclopentane, triterpenes, phenylpropanoids, coumarins, phenylethanol, small molecule phenols and alkaloids. Nearly 100 compounds have been reported to be isolated from mountain agarwood. Modern pharmacological studies have shown that mountain agarwood extracts and their active ingredients have multiple effects such as myocardial protection, analgesia, anti-tumor, antibacterial, anti-inflammatory and antioxidant. Summary of the invention

[0005] The object of the present invention is to provide an application of pinnatifone A for preparing a botanical fungicide and the botanical fungicide, which not only has a good control effect on gray mold on crops such as tomatoes and strawberries, but can also be used for the control of various plant diseases such as apple tree rot.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of pinnatifone A in the preparation of plant fungicides.

[0008] Optionally, the mass percentage of the pinnatifone A in the plant fungicide is 1% to 10%.

[0009] Optionally, the plant fungicide is used to control plant diseases caused by tomato gray mold B. cinerea, apple rot V. mali, pepper phytophthora P. capsici, watermelon wilt F. oxysporum, mango anthracnose C. asianum, wheat take-all G. graminis, tobacco brown spot A. alternata, corn leaf blight S. turcica, apple anthracnose C. gloeosporioides and / or cabbage black spot A. brassicicola.

[0010] A botanical fungicide, wherein the botanical fungicide is prepared by using pinnatifone A as an active ingredient and adding an auxiliary agent, and the preparation form is a soluble solution;

[0011] The soluble solution is prepared from the following raw materials by weight percentage:

[0012] pinnatifone A1% to 10%, surfactant 5% to 30%, the balance is organic solvent, the sum of the weight percentages of the raw materials is 100%;

[0013] The organic solvent is at least one of ethyl acetate, sec-butyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, solvent oil 120#, solvent oil 150# and solvent oil 200#;

[0014] The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates.

[0015] Optionally, the preparation of the soluble liquid comprises, by weight:

[0016] Weigh 1 to 10 parts of pinnatifone A, dissolve in 30 parts of sec-butyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate and 5 parts of polyoxyethylene styrene acid ether, and make up to 100 parts with ethanol.

[0017] A botanical fungicide, wherein the botanical fungicide is prepared by using pinnatifone A as an active ingredient and adding an auxiliary agent, and the preparation form is a microemulsion;

[0018] The microemulsion is prepared from the following raw materials by weight percentage:

[0019] 1% to 10% pinnatifone A, 20% to 30% organic solvent, 5% to 20% surfactant, 5% to 10% antifreeze agent, and the balance is water, the sum of the weight percentages of the raw materials is 100%;

[0020] The organic solvent is at least one of ethyl acetate, sec-butyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, solvent oil 120#, solvent oil 150# and solvent oil 200#;

[0021] The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates;

[0022] The antifreeze agent is ethylene glycol or propylene glycol.

[0023] Optionally, the microemulsion is prepared by weight:

[0024] Weigh 1 to 10 parts of pinnatifone A, dissolve in 30 parts of sec-butyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate and 5 parts of polyoxyethylene styrene acid ether, mix under high-speed stirring, and heat to 40°C; add water dropwise to make up to 100 parts under high-speed stirring, and keep the temperature at 40°C during the process of adding water; after the water is added dropwise, heat to 50°C and stir for 1 hour.

[0025] A botanical fungicide, wherein the botanical fungicide is prepared by using pinnatifone A as an active ingredient and adding an auxiliary agent, and the preparation form is an aqueous solution;

[0026] The aqueous solution is prepared from the following raw materials by weight percentage:

[0027] pinnatifone A 1% to 10%, organic solvent 1% to 10%, surfactant 5% to 30%, antifreeze 5% to 10%, the balance is water, the sum of the weight percentages of the raw materials is 100%;

[0028] The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates;

[0029] The antifreeze agent is ethylene glycol or propylene glycol.

[0030] Optionally, the preparation of the aqueous solution includes, by weight:

[0031] Weigh 1 to 10 parts of pinnatifone A, add 10 parts of ethyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate, 5 parts of polyoxyethylene styrene acid ether, and make up to 100 parts with water.

[0032] The technical solution of the present invention has the following advantages:

[0033] 1. It not only has a good control effect on gray mold on crops such as tomatoes and strawberries, but can also be used to prevent and control various plant diseases such as apple tree rot;

[0034] 2. Safe for humans, animals and other non-target organisms, and has good environmental compatibility;

[0035] 3. Green and environmentally friendly, low toxicity and low residue, not easy to produce drug resistance;

[0036] 4. The preparation method is simple and suitable for popularization and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:

[0038] Figure 1 The separation process of SCX-1 and SCX-5 of the present invention;

[0039] Figure 2 The separation process of SCX-2 and SCX-9 of the present invention;

[0040] Figure 3 The separation process of SCX-3 of the present invention;

[0041] Figure 4 The separation process of SCX-4, SCX-6 and SCX-8 of the present invention;

[0042] Figure 5 This is the separation process of SCX-7 of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation cases. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0044] During the research on botanical pesticides, the Biopesticide Engineering Technology Research Center of Northwest A&F University found that agarwood extract has good inhibitory activity against plant pathogens, and applied for a patent for "A kind of agarwood extract fungicide and its application" (CN 115918685 A). On this basis, the applicant conducted a systematic study on the antibacterial active ingredients of agarwood and obtained the antibacterial active compound pinnatifone A.

[0045] pinnatifone A, (3S,4aR)-6-hydroxy-4a,5-dimethyl-3-(prop-1-en-2-yl)-1,2,3,4,4a,7,8,8a-octahydronaphthalene-1,7-dione, molecular formula C 15 H 20 O3, molecular weight 248.32, light yellow flaky crystals, easily soluble in dichloromethane. Pinnatifone A is an elimofenane-type sesquiterpenoid compound isolated from Aquilaria sinensis. Currently, its research is mostly focused on the field of chemistry, mostly involving the structure and separation of compounds, and its activity is less studied, mainly in the medical field, such as antioxidant activity, and its related applications in the prevention and control of plant diseases caused by pathogens have not yet been found.

[0046] The botanical fungicide of the present invention uses pinnatifone A as an active substance, and a certain proportion of auxiliary agents is added for dosage form processing to prepare an environmentally friendly preparation, namely a microemulsion, an aqueous solution or a soluble solution.

[0047] Among them, the applicant separated the chemical components of the 95% ethanol extract of Syringa pinnatifolia and obtained 9 compounds, and clarified the structures of 8 compounds, namely, zingiberenone (SCX-1), 3-methoxy-4-hydroxybenzaldehyde (SCX-2), oleanolic acid (SCX-3), salidroside (SCX-4), α-caryophyllene (SCX-5), 3,4-dihydroxyphenylethanol (SCX-6), (8E)-ligustrum lucidum glycoside (SCX-7) and Pinnatifone A (SCX-9). The active compound was confirmed to be SCX-9 through activity assay, and pinnatifone A was identified. The separation process is as follows: Figure 1-5 :

[0048] The petroleum ether extract of the Aquilaria sinensis extract was separated. The eluent was a mixed system of petroleum ether, ethyl acetate and methanol. The specific ratio of gradient elution was petroleum ether: ethyl acetate: methanol (V / V / V) 100:1:0, 50:1:0, 30:1:0, 10:1:0, 5:1:0, 3:1:0, 1:1:0, 1:3:0, 0:1:0, 0:0:1, a total of 10 sections, each ratio eluted about 5 column volumes, each column volume was about 3.5L, after silica gel column chromatography elution, thin layer chromatography detection and merging, the primary column product was combined into 10 components. Activity tracking was carried out by indicating the mycelial growth rate of pathogens. Components 2, 3, 4, 5 and 6 showed good inhibitory activity, and a total of 5 components were further separated for active components. The separation process of each component is as follows:

[0049] Separation of component 2: Component 2 was subjected to silica gel column chromatography. The eluent was a mixed system of petroleum ether and ethyl acetate. The specific elution ratio was petroleum ether:ethyl acetate (V / V) of 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1 and other system gradient elutions. The separation was detected by thin layer chromatography. The separation conditions were combined or separated according to the separation conditions of each point to obtain 6 fractions, which were named 2-1 to 2-6 respectively. Fraction 2-3 and fraction 2-5 were further subjected to silica gel column chromatography. Fraction 2-3 was subjected to silica gel column chromatography, the eluent was a mixed system of petroleum ether and ethyl acetate, the specific elution ratio was petroleum ether: ethyl acetate (V / V) 70:1, 60:1, 50:1 and other system gradient elution, and three fractions were obtained, named 2-3-1 to 2-3-3, and fraction 2-3-3 was eluted with petroleum ether: ethyl acetate (V / V) 60:1 to obtain compound SCX-1. Fraction 2-5 was subjected to silica gel column chromatography, the eluent was a mixed system of petroleum ether and ethyl acetate, the specific elution ratio was petroleum ether: ethyl acetate (V / V) 50:1, 40:1, 30:1 and other system gradient elution, and three fractions were obtained, named 2-5-1 to 2-5-3, and fraction 2-5-2 was purified by Sephadex LH-20 gel column, eluted with dichloromethane: methanol 1:1 to obtain compound SCX-5.

[0050] Separation of component 3: Component 3 was subjected to silica gel column chromatography, and the eluent was a mixed system of petroleum ether and ethyl acetate, and the specific elution ratio was petroleum ether: ethyl acetate (V / V) of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 0:1, pure methanol and other system gradient elution, and the separation was detected by thin layer chromatography. According to the separation of each point, it was combined or separated to obtain 6 fractions named 3-1 to 3-6, and fractions 3-2 and 3-5 were further subjected to silica gel column chromatography. Fraction 3-2 was subjected to silica gel column chromatography, and the eluent was a mixed system of dichloromethane and methanol, and the specific elution ratio was dichloromethane: methanol (V / V) of 50:1, and 3 fractions were obtained, named 3-2-1 to 3-2-3, and fraction 3-2-3 was eluted with petroleum ether: ethyl acetate (V / V) 20:1 to obtain compound SCX-2. Fraction 3-5 was subjected to silica gel column chromatography. The eluent was a mixed system of petroleum ether and ethyl acetate. The specific elution ratios were petroleum ether: ethyl acetate (V / V) of 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:3, 0:1, etc., and four fractions were obtained, respectively named 3-5-1 to 3-5-4. Fraction 3-5-3 was purified by Sephadex LH-20 gel column and eluted with dichloromethane: methanol in a ratio of 1:1 to obtain compound SCX-9.

[0051] Separation of component 4: Component 4 was subjected to silica gel column chromatography. The eluent was a mixed system of petroleum ether and ethyl acetate. The specific elution ratios were petroleum ether:ethyl acetate (V / V) of 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1. Pure methanol was used for system gradient elution. The separation was detected by thin layer chromatography. The six fractions were combined or separated according to the separation conditions of each point, and were named 4-1 to 4-6 respectively. Fraction 4-3 and fraction 3-5 were further subjected to silica gel column chromatography. Fraction 4-3 was subjected to silica gel column chromatography. The eluent was a mixed system of dichloromethane and methanol. The specific elution ratios were dichloromethane: methanol (V / V) 100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, and 1:1. Five fractions were obtained and named 4-3-1 to 4-3-5. Fraction 4-3-4 was purified by Sephadex LH-20 gel column elution with dichloromethane: methanol 1:1 to obtain compound SCX-3.

[0052] Separation of component 5: Component 5 was subjected to silica gel column chromatography. The eluent was a mixed system of dichloromethane and methanol. The specific elution ratio was dichloromethane: methanol (V / V) of 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 0:1 and other system gradient elutions. The separation was detected by thin layer chromatography. The separation conditions were combined or separated according to the separation conditions of each point to obtain 9 fractions, which were named 5-1 to 5-7 respectively. Fraction 5-3 and fraction 5-6 were further subjected to silica gel column chromatography. Fraction 5-3 was subjected to silica gel column chromatography, the eluent was a mixed system of dichloromethane and methanol, the specific elution ratios were dichloromethane: methanol (V / V) 3:1, 2:1, 1:1, 0:1, and 4 fractions were obtained and named 5-3-1 to 5-3-4. Fraction 5-3-3 was purified by Sephadex LH-20 gel column, eluted with dichloromethane: methanol 1:1, to obtain compound SCX-6. Fraction 5-6 was subjected to silica gel column chromatography, the eluent was a mixed system of ethyl acetate and methanol, the specific elution ratio was ethyl acetate: methanol (V / V) 10:1, 8:1, 6:1, 4:1, 3:1, 2:1, 1:1, 1:3, 0:1 and other system gradient elutions, and 3 fractions were obtained, respectively named 5-6-1 to 5-6-3. Fraction 5-6-1 was subjected to silica gel column chromatography, the eluent was a mixed system of petroleum ether and ethyl acetate, the specific elution ratio was petroleum ether: ethyl acetate (V / V) 6:1, and compound SCX-4 was obtained. Fraction 5-6-3 was purified by Sephadex LH-20 gel column, eluted with dichloromethane: methanol 1:1, and compound SCX-8 was obtained.

[0053] Separation of component 6: Component 6 was subjected to silica gel column chromatography, and the eluent was a mixed system of dichloromethane and methanol. The specific elution ratios were dichloromethane: methanol (V / V) of 10:1, 8:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 0:1, etc., and the separation was detected by thin layer chromatography. The separation was combined or separated according to the separation conditions of each point, and 5 fractions were obtained, respectively named 6-1 to 6-5. Fraction 6-3 was further subjected to silica gel column chromatography, and the eluent was a mixed system of petroleum ether and acetone. The specific elution ratios were petroleum ether: acetone (V / V) of 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 4 fractions were obtained, respectively named 6-3-1 to 6-3-4. Fraction 6-3-2 was purified by Sephadex chromatography. The compound SCX-7 was obtained by purification on an LH-20 gel column with elution using dichloromethane:methanol 1:1.

[0054] The specific spectral data of each compound are as follows:

[0055] Spectral data of SCX-1: colorless flaky crystals, easily soluble in dichloromethane, 1H NMR (400MHz, Chloroform-d) δ6.00(d,J=13.1Hz,1H),5.95(s,1H),5.85(d,J=16.4Hz,1H),5.24(d,J=15.5Hz,1H),2.34(t,J= 12.7Hz,2H),2.23(d,J=13.7Hz,2H),1.89(d,J=13.1Hz,1H),1.79(s,3H),1.71(s,1H),1.53(s,3H),1.19(s,3H),1.06(s,3H). 13 C NMR (101MHz, Chloroform-d) δ204.59, 160.96, 149.04, 138.05, 136.41, 127.26, 125.10, 42.50, 39.55, 37.99, 29.54, 24.32, 15.34, 11.92. The above spectral data are basically consistent with the zerumbone reported in the literature (Zhang Guobin et al. 1994), so the compound was identified as a zerumbone-type sesquiterpene compound.

[0056] Spectral data of SCX-2: white crystalline powder compound, easily soluble in dichloromethane, ESI-HR-MS (-) m / z: 151.03 [MH]-, 1 H NMR (400MHz, Chloroform-d) δ9.66 (s, 1H), 7.30-7.23 (m, 2H), 6.88 (d, J = 8.6Hz, 1H), 6.31 (s, 1H), 3.80 (s, 3H). 13 C NMR (101MHz, Chloroform-d) δ190.81, 151.57, 146.99, 129.60, 127.36, 114.23, 108.62, 55.89. The above spectral data are basically consistent with the 3-methoxy-4-hydroxybenzaldehyde reported in the literature (Li et al. 2018), so the compound was identified as a small molecule phenol compound, 3-methoxy-4-hydroxybenzaldehyde (vanillin).

[0057] Spectral data of SCX-3: white powdery compound, easily soluble in dichloromethane, ESI-HR-MS (-) m / z: 455.37 [MH]-, 1H NMR (400 MHz, Chloroform-d). The compound contains one olefin proton signal δH5.27 (1H, s) and one oxymethyl signal δH3.21 (1H, m). 13C NMR (101MHz,CDCl3)δ183.69,143.48,122.75,79.18,55.33,53.45,47.74,46.67,45.99,44.02,41.70,41.06,39.38,38.89,38.52,37.22,33.92,33.21,32.71,30.82,28.23,27.80,27.29,26.08,23.70,23.02,18.41,17.27,15.69,15.45. The above spectral data are basically consistent with the oleanolic acid reported in the literature (Liu Yu et al. 2019), so the compound was identified as a pentacyclic triterpenoid compound, oleanolic acid.

[0058] Spectral data of SCX-4: brown powder compound, easily soluble in methanol, ESI-HR-MS (-) m / z: 299.17 [MH]-, 1 H NMR (400MHz, Methanol-d4) δ7.06(d,J=8.4Hz,2H),6.69(d,J=8.5Hz,2H),4.28(s,1H),4.09-3.97(m,1H),3.86(d,J=12.1Hz ,1H),3.75-3.61(m,2H),3.34(dd,J=17.6,8.7Hz,2H),3.27(d,J=3.6Hz,2H),3.18(d,J=16.8Hz,1H),2.83(t,J=8.4Hz,2H). 13 C NMR (101MHz, Methanol-d4) δ155.38, 129.55, 129.33, 114.73, 102.95, 76.66, 76.53, 73.70, 70.72, 70.22, 61.33, 34.97. The above spectral data are basically consistent with the salidroside reported in the literature (Nishimura et al. 1990), so the compound was identified as a phenylethanol compound, salidroside.

[0059] Spectral data of SCX-5: light yellow oily liquid, easily soluble in dichloromethane, ESI-HR-MS (+) m / z: 204.20 [M] +, 1H NMR(400MHz,Chloroform-d)δ5.65-5.54(m,1H),5.16(d,J=15.9Hz,1H),4.95(s,1H),4. 87(s,1H),2.52(s,2H),2.11(s,4H),1.92(s,2H),1.64(s,3H),1.43(s,3H),1.06(s,6H). 13 C NMR (101MHz, Chloroform-d) δ141.03, 139.27, 133.18, 127.74, 125.83, 124.98, 41.97, 40.43, 39.75, 37.39, 23.35, 17.96, 15.10. The above spectral data are basically consistent with the α-humulene reported in the literature (Bao et al. 2015), so the compound was identified as a humulane-type sesquiterpene compound, α-humulene.

[0060] Spectral data of SCX-6: brown powder compound, easily soluble in dichloromethane, ESI-HR-MS (-) m / z: 153.10 [MH]-, 1 H NMR(400MHz,Chloroform-d)δ6.81(d,J=8.0Hz,1H),6.74(s,1H),6.66(d,J=9.4Hz,1H ),5.48(s,1H),5.25(s,1H),3.82(t,J=6.4Hz,2H),3.50(s,1H),2.76(t,J=6.4Hz,2H). 13 C NMR (101MHz, Chloroform-d) δ142.31, 131.47, 121.55, 116.18, 115.65, 63.89, 38.52. The above spectral data are basically consistent with the 3,4-dihydroxyphenylethanol reported in the literature (Bai Ruifeng et al. 2017), so the compound was identified as a humulane-type sesquiterpene compound, 3,4-dihydroxyphenylethanol (hydroxytyrosol).

[0061] Spectral data of SCX-7: brown powder compound, easily soluble in methanol, ESI-HR-MS (-) m / z: 709.01 [M + Na] +, 1H NMR (400MHz, Methanol-d4) δ = 7.51 (s, 1H), 7.08-7.00 (m, 2H), 6.72-6.64 (m, 2H), 6.14-6.04 (m, 1H), 5.90 (t, J = 1. 7,1H),4.80(d,J=7.7,1H),4.59(s,0H),4.33(dd,J=11.9,2.1,1H),4.29(d,J=7.8,1H),4.20(dd,J=11.9,5.7,1H ),4.04-3.91(m,2H),3.88(dd,J=12.0,1.7,1H),3.74-3.62(m,1H),3.68(s,4H),3.49-3.25(m,6H),3.20(t,J=8. 2,1H),2.82(tt,J=9.5,4.8,2H),2.75(dd,J=14.3,4.8,1H),2.48(dd,J=14.3,8.8,1H),1.72(dd,J=7.1,1.5,3H). 13 C-NMR(125MHz,Methanol-d4)δ:95.0,155.12,109.26,31.65,41.21,172.95,124.91,130.27,13.75,168.61,51.94,100.69,74.59,77 .82,71.34,78.26,62.60,72.16,36.32,130.53,130.85,116.08,156.63,116.08,130.85,104.30,74.83,77.82,71.41,75.02,64.90. The above spectral data are basically consistent with the (8E)-nüzhenide reported in the literature (Machida et al. 2002), so the compound was identified as an iridoid ether terpenoid compound, (8E)-nüzhenide.

[0062] Spectral data of SCX-9: light yellow flaky crystalline compound, easily soluble in dichloromethane, ESI-HR-MS m / z: 247.13 [MH] -,1H NMR(400MHz,Chloroform-d)δ6.19(s,1H),4.77(d,J=22.3Hz,2H),3.08-3.01(m,1H),2.69(d,J=5.4Hz,1H),2.60(dd,J =17.2,4.5Hz,1H),2.49-2.44(m,1H),2.33-2.18(m,3H),1.79(s,3H),1.75(s,3H),1.64(d,J=13.6Hz,1H),1.39(s,3H). 13 C NMR (101MHz, Chloroform-d) δ209.02, 191.64, 146.67, 144.60, 129.62, 110.40, 54.35, 46.04, 42.90, 42.13, 41.19, 32.01, 27.13, 20.47, 10.83. The above spectral data are basically consistent with the pinnatifone A reported in the literature (Zhang et al. 2014), so the compound was identified as the erimofenane-type sesquiterpenoid compound pinnatifone A.

[0063]

[0064] SCX-1, 3-methoxy-4-hydroxybenzaldehyde (SCX-2), oleanolic acid (SCX-3), salidroside (SCX-4), α-caryophyllene (SCX-5), 3,4-dihydroxyphenylethanol (SCX-6), (8E)-ligustrin (SCX-7) and Pinnatifone A

[0065] (SCX-9) compound structure;

[0066] After isolating pinnatifone A and other compounds, the applicant first screened the inhibitory activity of pinnatifone A and other compounds against plant pathogenic fungi and oomycetes at a concentration of 100 μg / mL. The results are shown in Table 1.

[0067] Table 1. Inhibitory activity of 9 compounds against apple rot pathogen and tomato gray mold pathogen

[0068]

[0069] Note: The final concentration of the 9 compounds in the table is 100 μg / mL. The data are the mean ± standard error of 3 replicates. “--” indicates no inhibitory activity.

[0070] The results in Table 1 show that pinnatifone A has the highest activity against indicator bacteria. The antibacterial spectrum of pinnatifone A and its toxicity against several plant pathogens were then determined, and the results are shown in Tables 2 and 3.

[0071] The pathogenic fungi and oomycetes tested included apple rot pathogen (Valsa mali), tomato gray mold pathogen (Botrytis cinerea), watermelon wilt pathogen (Fusarium oxysporum), pepper blight pathogen (Phytophthora capsici), mango anthracnose pathogen (Colletotrichum asianum), wheat take-all pathogen (Gaeumannomyces graminis), rice sheath blight pathogen (Rhizoctonia solani), tobacco brown spot pathogen (Alternaria alternata), potato dry rot pathogen (Fusarium solanum), corn leaf blight pathogen (Setosphaeriaturcica), apple anthracnose pathogen (Colletotrichum gloeosporioides), rapeseed sclerotinia pathogen (Sclerotinia sclerotiorum), cabbage black spot pathogen (Alternaria brassicicola), and wheat stem rot pathogen (Fusarium pseudograminearum).

[0072] The determination method is the mycelium growth rate method. Pinnatifone A is prepared into a 20 mg / mL stock solution for standby use. Plant pathogenic fungi and oomycetes are cultured with PDA solid culture medium. During the experiment, a certain amount of compound stock solution is added to the melted solid culture medium, shaken and poured into a sterilized culture dish to prepare a drug-containing culture medium with a final concentration of 100 μg / mL, and the activated pathogen cake is inoculated and inverted in a 25°C constant temperature incubator for culture. The experiment sets a solvent control, and each treatment is repeated three times. After the control is basically full of the culture medium, the colony diameter is measured and the inhibition rate is calculated. The colony diameter and inhibition rate are calculated according to the following formula:

[0073] Colony growth diameter = colony outer diameter - diameter of bacterial cake;

[0074] Inhibition rate (%) = (control group diameter - treatment group diameter) / (control group diameter - bacterial cake diameter) × 100;

[0075] Table 2. Inhibitory activity of pinnatifone A against 14 pathogenic fungi and oomycetes

[0076]

[0077]

[0078] Note: The final concentration of pinnatifone A in the dish is 100 μg / mL. The data are the mean ± standard error of 3 replicates. “--” indicates no inhibitory activity.

[0079] Table 3. Inhibitory effects of pinnatifone A on the mycelial growth of 10 pathogenic fungi and oomycetes

[0080]

[0081] The experimental results showed that pinnatifone A had a good inhibitory effect on the mycelial growth of 10 of the 14 plant pathogenic fungi and oomycetes tested, and indicated that the compound had a broad-spectrum antibacterial activity.

[0082] In order to better understand the essence of the invention, the technical content of the invention is described in detail with examples below, but the invention is not limited to these examples.

[0083] Example 1 Preparation of 1% pinnatifone A soluble liquid

[0084] Weigh 1 kg of pinnatifone A, dissolve it in 30 kg of sec-butyl acetate, then add 15 kg of calcium dodecylbenzenesulfonate, 5 kg of polyoxyethylene styrene acid ether, and ethanol to make up to 100 kg, mix well, and stir at a stirring speed of 800 to 1000 rpm for 10 to 30 minutes to obtain 100 kg of 1% pinnatifone A soluble liquid. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0085] Example 2 Preparation of 10% pinnatifone A soluble liquid

[0086] Weigh 10 kg of pinnatifone A, dissolve it in 30 kg of sec-butyl acetate, then add 15 kg of calcium dodecylbenzenesulfonate, 5 kg of polyoxyethylene styrene acid ether, and ethanol to make up to 100 kg, mix well, and stir at a stirring speed of 800 to 1000 rpm for 10 to 30 minutes to obtain 100 kg of 10% pinnatifone A soluble liquid. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0087] Example 3 Preparation of 1% pinnatifone A microemulsion

[0088] Weigh 1 kg of pinnatifone A, dissolve it in 30 kg of sec-butyl acetate, then add 15 kg of calcium dodecylbenzene sulfonate and 5 kg of polyoxyethylene styrene acid ether, mix under high-speed stirring, and heat to about 40°C; add deionized water dropwise under high-speed stirring to make up to 100 kg, and keep the temperature at about 40°C during the process of adding deionized water; after the water is added dropwise, heat to 50°C, stir for 1 hour, and prepare 100 kg of 1% pinnatifone A microemulsion. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0089] Example 4 Preparation of 10% pinnatifone A microemulsion

[0090] Weigh 10 kg of pinnatifone A, dissolve it in 30 kg of sec-butyl acetate, then add 15 kg of calcium dodecylbenzene sulfonate and 5 kg of polyoxyethylene styrene acid ether, mix under high-speed stirring, and heat to about 40°C; add deionized water dropwise under high-speed stirring to make up to 100 kg, and keep the temperature at about 40°C during the process of adding deionized water; after the water is added dropwise, heat to 50°C, stir for 1 hour, and prepare 100 kg of 10% pinnatifone A microemulsion. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0091] Example 5 Preparation of 1% pinnatifone A aqueous solution

[0092] Weigh 1 kg of pinnatifone A, dissolve it in 10 kg of ethyl acetate, add 15 kg of calcium dodecylbenzene sulfonate, 5 kg of polyoxyethylene styrene acid ether, and make up to 100 kg with water. After mixing evenly, stir at a stirring speed of 800 to 1000 rpm for 10 to 30 minutes to prepare 100 kg of 1% pinnatifone A aqueous solution. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0093] Example 6 Preparation of 10% pinnatifone A aqueous solution

[0094] Weigh 10 kg of pinnatifone A, dissolve it in 10 kg of ethyl acetate, add 15 kg of calcium dodecylbenzene sulfonate and 5 kg of polyoxyethylene styrene acid ether, make up to 100 kg with water, mix well, stir at a stirring speed of 800 to 1000 rpm for 10 to 30 minutes, and 100 kg of 10% pinnatifone A aqueous solution can be prepared. The stability and appearance of the preparation meet the requirements of commercial preparations.

[0095] Example 7 Control effects of six pinnatifone A preparations on apple rot on detached branches

[0096] The scald inoculation method was adopted. Apple twigs of uniform thickness were selected and cut into small sections of about 15 cm. They were completely disinfected with 1% NaClO solution, then rinsed with clean water, and the ends of the branches were sealed with melted wax to keep them moisturized. They were dried for later use. The bark was scalded with a red-hot iron nail. After the burn, a small amount of sterile water was dripped into the area to facilitate the adhesion of the fungus cake. For the protective activity determination, the scalded area was first sprayed with the solution of the test agent, and apple rot bacteria were inoculated thereon one day later. For the therapeutic activity determination, the activated apple rot bacteria were first inoculated at the scalded area, and then cultured in a dark and moisturizing environment for one day and sprayed with the test agent. After the treatment, each experimental treatment included 5 apple branches, which were placed in a tray covered with moist filter paper and covered with a sealing film. The lesion size was measured after constant temperature and moisturizing culture for 5 to 7 days under dark conditions. The lesion area was calculated according to the ellipse, and the control effect was calculated using the following formula. The control results are shown in Table 3.

[0097] Lesion area = 1 / 4 × π × major diameter × minor diameter;

[0098] Control effect (%) = (control group area - treatment group area) / control area × 100;

[0099] Table 4. Control effects of six pinnatifone A preparations on apple rot on detached branches

[0100]

[0101] Example 8 Control effects of six pinnatifone A preparations on tomato gray mold on detached fruits

[0102] Select green tomatoes of the same size, completely disinfect them with 1% NaClO solution, then rinse with clean water, dry and set aside. Use a sterilized inoculation needle to pierce the tomato skin, scratch a circular wound with a diameter of about 1 cm, and drip a small amount of sterile water there to facilitate the adhesion of the bacterial cake; for protective activity determination, first spray the wound with the test agent, and then inoculate tomato gray mold on it one day later; for therapeutic activity determination, first inoculate the activated tomato gray mold on the scald, then culture it in a dark and moisturizing environment for one day, and spray the wound with the test agent. After treatment, each experimental treatment includes 3 tomato fruits, which are placed in a tray covered with moist filter paper and covered with sealing film, and cultured in a constant temperature and moisturizing environment for 3 to 5 days under dark conditions before measuring the size of the lesions. The lesion area is calculated according to the ellipse, and the control effect is calculated using the following formula. The control results are shown in Table 4.

[0103] Lesion area = 1 / 4 × π × major diameter × minor diameter;

[0104] Control effect (%) = (control group area - treatment group area) / control area × 100;

[0105] Table 5. Control effects of six pinnatifone A preparations on tomato gray mold on detached fruits

[0106]

[0107] The experiment showed that the six preparations had good control effects on apple rot on detached branches and tomato gray mold on detached fruits.

[0108] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims

1. Application of pinnatifone A in the preparation of plant fungicides.

2. The use according to claim 1, characterized in that: The mass percentage of the pinnatifone A in the plant fungicide is 1% to 10%.

3. The use according to claim 1 or 2, characterized in that: The plant fungicide is used for preventing and controlling plant diseases caused by tomato gray mold B. cinerea, apple rot pathogen V. mali, pepper phytophthora P. capsici, watermelon wilt pathogen F. oxysporum, mango anthracnose pathogen C. asianum, wheat take-all pathogen G. graminis, tobacco brown spot pathogen A. alternata, corn leaf spot pathogen S. turcica, apple anthracnose pathogen C. gloeosporioides and / or cabbage black spot pathogen A. brassicicola.

4. A botanical fungicide, characterized in that: The plant fungicide is made of pinnatifone A as an active ingredient and an auxiliary agent, and the preparation form is a soluble solution; The soluble solution is prepared from the following raw materials by weight percentage: pinnatifone A1% to 10%, surfactant 5% to 30%, the balance is organic solvent, the sum of the weight percentages of the raw materials is 100%; The organic solvent is at least one of ethyl acetate, sec-butyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, solvent oil 120#, solvent oil 150# and solvent oil 200#; The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates.

5. The botanical fungicide according to claim 4, characterized in that The preparation of the soluble liquid comprises, by weight: Weigh 1 to 10 parts of pinnatifone A, dissolve in 30 parts of sec-butyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate and 5 parts of polyoxyethylene styrene acid ether, and make up to 100 parts with ethanol.

6. A botanical fungicide, characterized in that: The plant fungicide is made of pinnatifone A as an active ingredient and an auxiliary agent, and the preparation form is a microemulsion; The microemulsion is prepared from the following raw materials by weight percentage: 1% to 10% pinnatifone A, 20% to 30% organic solvent, 5% to 20% surfactant, 5% to 10% antifreeze agent, and the balance is water, the sum of the weight percentages of the raw materials is 100%; The organic solvent is at least one of ethyl acetate, sec-butyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, solvent oil 120#, solvent oil 150# and solvent oil 200#; The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates; The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates. The antifreeze agent is at least one of glycerol and propylene glycol.

7. The botanical fungicide according to claim 6, characterized in that The microemulsion is prepared by weight: Weigh 1 to 10 parts of pinnatifone A, dissolve in 30 parts of sec-butyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate and 5 parts of polyoxyethylene styrene acid ether, mix under high-speed stirring, and heat to 40°C; add water dropwise to make up to 100 parts under high-speed stirring, and keep the temperature at 40°C during the process of adding water; after the water is added dropwise, heat to 50°C and stir for 1 hour.

8. A botanical fungicide, characterized in that: The plant fungicide is made of pinnatifone A as an active ingredient and an auxiliary agent, and the preparation form is an aqueous solution; The aqueous solution is prepared from the following raw materials by weight percentage: pinnatifone A 1% to 10%, organic solvent 1% to 10%, surfactant 5% to 30%, antifreeze 5% to 10%, the balance is water, the sum of the weight percentages of the raw materials is 100%; The surfactant is at least one of alkylbenzene sulfonates, alkylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers, phenethylphenol polyoxyethylene ethers and similar substances, adducts of castor oil and ethylene oxide, and arylalkylphenol polyoxyethylene ether formaldehyde condensates; The antifreeze agent is ethylene glycol or propylene glycol.

9. The botanical fungicide according to claim 8, characterized in that The preparation of the aqueous solution comprises, by weight: Weigh 1 to 10 parts of pinnatifone A, add 10 parts of ethyl acetate, then add 15 parts of calcium dodecylbenzenesulfonate, 5 parts of polyoxyethylene styrene acid ether, and make up to 100 parts with water.

Images