Alkylated citrate compound and application thereof in preventing and treating plant diseases

Abstract

The invention relates to the technical field of extraction of green pesticides from natural products of trichoderma asperellum, relates to an alkylated citrate compound, relates to application of the alkylated citrate compound in preparation of drugs for preventing and treating plant diseases, and further relates to application of the alkylated citrate compound in preparation of drugs for preventing and treating plant diseases by using a computer deep learning model and combining with pathogenic proteins of phytopathogens. And predicting the antibacterial activity of the alkylated citrate compound.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, the field of separation and application of strain metabolites, the separation of alkylated citrate compounds from the solid fermentation product of Trichoderma asperellum TCS007, the application of alkylated citrate compounds in plant disease activity, and the activity simulation of alkylated citrate compounds binding to the pathogenic protein of Sclerotinia sclerotiorum. Background Art

[0002] Trichoderma asperellum is a new type of biocontrol fungus, belonging to the Hyphomycetes class, Hyphomycetales order, Moniliaceae family of Deuteromycetes. Some Trichoderma strains can produce aerial hyphae, mostly in the form of floccose or arachnoid, and their colonies expand rapidly, showing radial concentric circles. They usually start as white and then produce pigments over time, mostly green, and there are also gray and brown, etc. Trichoderma has strong adaptability and can reproduce quickly. It is usually the dominant population and has obvious advantages in biological control and growth promotion. It is one of the most widely used biocontrol bacteria.

[0003] The new Trichoderma asperellum TCS007 (T. asperellum TCS007) was isolated from Antarctic marine sediments and identified and named Trichoderma asperellum TCS007 after mutagenic stability. At present, this strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 15677. This strain was first published in patents ZL201810935616.6 and ZL201810936028.4. Trichoderma asperellum TCS007 grows rapidly on PDA medium. Under the condition of 28 °C, the mycelium can cover a 9-cm petri dish in about 3 days. Its mycelium has a moderate thickness, showing a felt-like shape, white and flat in the early stage, and later forming dark green concentric conidia. On CMD medium, the strain forms clustered conidial masses, the main branches of the conidiophores are tree-like, the phialides are short, gradually tapering at the base, and swollen in the middle, showing an ampoule shape. The length of the conidia of Trichoderma asperellum TCS007 is 3.4 - 5.0 μm, the width is 2.8 - 4.0 μm, and the shape is mostly spherical, sub-spherical or oval, with a rough surface covered with small spines.

[0004] In previous studies by the research group where the inventors of this application belong, it was found that the specific rice medium (80 g of rice + 120 mL of distilled water) for Trichoderma asperellum TCS007 to produce secondary metabolites, and the solid crude extract obtained by ethyl acetate extraction after fermentation had an inhibition rate of 95.8% against Sclerotinia sclerotiorum (see Zheng Kebin, Antibacterial, Growth Promotion and Stress Resistance of Trichoderma asperellum TCS007 from Marine Habitats, Master's Thesis of Zhejiang A&F University, Online publication time: January 16, 2021 - February 15, 2021).

[0005] It is known in the art that Trichoderma asperellum can secrete a variety of secondary metabolites with antibacterial activity. However, the metabolites in the solid fermentation product of Trichoderma asperellum TCS007 are not yet clear. In the present invention, Trichoderma asperellum TCS007 is fermented by a solid medium, the metabolites are extracted and separated, and the biological activities of the metabolites are determined to analyze the antibacterial mechanism of Trichoderma asperellum TCS007.

[0006] Upon retrieval, patents EP1795206A1, EP2886530A1, CN1819990A, CN101489580A, TW201010692A1 disclose the use of alkylated citrate compounds for the treatment or prevention of HCV infections. Patent WO9418157A1 discloses that the compound shown in formula (I) is used as an effective antifungal agent for the treatment of fungal infections, mainly for the treatment of infections caused by fungi such as Candida albicans, Cryptococcus, Ustilago maydis, Aspergillus, etc. It also discloses the use for the treatment of cancer. However, there is no literature in the prior art that discloses the application of alkylated citrate compounds for the prevention and control of plant diseases. Summary of the Invention

[0007] One object of the present invention is to isolate an alkylated citrate compound from the solid fermentation product of Trichoderma asperellum TCS007 and identify the chemical structure of the alkylated citrate compound;

[0008] The present invention provides a preparation method of an alkylated citrate compound, wherein the alkylated citrate compound is shown in formula (I) or formula (II), and is characterized in that the alkylated citrate compound is isolated from the solid fermentation product of Trichoderma asperellum TCS007; the separation methods include but are not limited to acid precipitation method, salting-out method, organic solvent precipitation method, organic solvent extraction method, etc.;

[0009]

[0010] Wherein,

[0011] R1 and R3 each independently selected from H, OH, halogen, C 1-12 alkyl, C 1-12 haloalkyl, C 1-12 alkoxy, C 1-12 haloalkoxy, C 3-7 cycloalkyl;

[0012] R2, R4, and R5 each independently selected from H, OH, C 1-12 alkyl, C 1-12 haloalkyl, C 1-12 alkoxy, C 1-12 haloalkoxy, C 3-7 cycloalkyl.

[0013] The Trichoderma asperellum TCS007 solid fermentate is obtained by inoculating a Trichoderma asperellum TCS007 spore suspension into a solid medium for fermentation culture;

[0014] The Trichoderma asperellum TCS007 seed liquid is obtained by culturing an activated Trichoderma asperellum TCS007 strain in a PDB culture solution;

[0015] The solid fermentation medium comprises 72 g of rice, 3.5% soybean powder (based on the weight of the rice, the weight percentage of the soybean powder is 3.5% of the weight of the rice), and 47 mL of distilled water;

[0016] The fermentation process is as follows: the bottling amount is 72 g / 300 mL, the initial pH is 7.0, the fermentation temperature is 28 °C, the inoculation amount is 1 mL (spore concentration 1×10 7 cells / mL), the light cycle time is 12 hours, and the culture time is 29 days.

[0017] The purpose of the present invention is to determine the biological activities of the whole and parts of the main metabolites in the Trichoderma asperellum TCS007 fermentate.

[0018] The present invention provides an alkylated citrate compound, its derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, and pharmaceutically acceptable salts represented by formula (I) or formula (II),

[0019]

[0020] wherein,

[0021] R1 and R3 are each independently selected from H, OH, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl;

[0022] R2, R4, and R5 are each independently selected from H, OH, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, C 3-7 cycloalkyl.

[0023] Preferably, R1 and R3 are each independently selected from H, OH; R2 is selected from H, CH 3 ; R4 and R5 are each independently selected from H, OH, CH 3 , OCH 3 .

[0024] Preferably, the alkylated citrate compound represented by formula (I) and / or formula (II) is selected from

[0025]

[0026]

[0027] Another object of the present invention is to conduct a bioactivity screening on the application of the alkylated citrate compound in the prevention and control of plant diseases;

[0028] Use of the alkylated citrate compound represented by formula (I) and / or formula (II) in the preparation of a drug for preventing and controlling plant diseases;

[0029] Use of the alkylated citrate compound represented by formula (I) and / or formula (II) in the prevention and control of plant diseases;

[0030] A method for preventing and controlling plant diseases, the method comprising applying an effective amount of the alkylated citrate compound represented by formula (I) and / or formula (II) to a plant;

[0031] Use of the alkylated citrate compound represented by formula (I) and / or formula (II) in the preparation of an agriculturally acceptable formulation for preventing and controlling plant diseases;

[0032] The plant diseases are selected from diseases caused by plant pathogenic fungi, and the plant pathogenic fungi are selected from diseases caused by Magnaporthe oryzae, Alternaria spp., Fusarium spp., Botrytis cinerea, Erysiphe spp., Phytophthora spp., Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum spp., Mycosphaerella spp., etc.

[0033] The plant diseases are Sclerotinia sclerotiorum of rapeseed, brown rot of peach, Colletotrichum gloeosporioides of camellia oleifera, rice blast, apple brown spot, cucumber gray mold, rice sheath blight, wheat scab, etc.

[0034] The drug or formulation further comprises one or more surfactants and / or carriers; based on the weight of the drug or formulation, the weight percentage content of the alkylated citrate compound represented by formula (I) and / or formula (II) is 1% to 99%; the concentration of the surfactant and / or carrier is 1% to 90%.

[0035] The drug or formulation is selected from conventional formulation types in the field of pesticide formulations such as emulsifiable concentrates, dispersible oil suspensions, soluble solutions, seed treatment suspensions, wettable powders, microemulsions, emulsifiable concentrates, suspensions or water dispersible granules, etc.

[0036] Advantageous technical effects of the present invention

[0037] The present invention for the first time isolates alkylated citrate compounds from the solid fermentation product of Trichoderma asperellum TCS007, and determines the chemical structures of six alkylated citrate compounds and their biological activities against eight plant diseases; according to the single-channel theory of compound biological activities, the inventors can conclude that the alkylated citrate compounds represented by formula (I) or formula (II) summarized in the present invention have the same or similar or better control effects against these eight plant diseases.

[0038] The present invention for the first time clarifies the types of alkylated citrate secondary metabolites produced by the fermentation of Trichoderma asperellum TCS007, clarifies the chemical structures and biological activities of the main alkylated citrate compounds. Since the physical and chemical properties of the alkylated citrate active substances are more stable than those of Trichoderma asperellum TCS007 itself, they have broad application values in the control of field diseases.

[0039] The alkylated citrate secondary metabolites extracted and separated in the present invention are secreted by Trichoderma asperellum TCS007 and have been proven to have significant antibacterial activities. The main active components and structures of the metabolites of Trichoderma asperellum TCS007 are clarified, which can be effectively applied to the control of plant diseases and provide an effective control means for agricultural production.

[0040] The present invention uses the methods of reverse docking and molecular docking, and uses a computer deep learning prediction model to predict the biological activities of alkylated citrate compounds in the field of plant disease control. The prediction results show that the alkylated citrate compounds represented by formula (I) or formula (II) summarized in the present invention have the uses of controlling various plant diseases.

[0041] The alkylated citrate compounds represented by formula (I) or formula (II) provided by the present invention have the effects or functions of binding to the pathogenic proteins of multiple Sclerotinia sclerotiorum, Colletotrichum spp., and Magnaporthe oryzae, resulting in the inability of Sclerotinia sclerotiorum, Colletotrichum spp., and Magnaporthe oryzae to carry out normal physiological activities, and can inhibit Sclerotinia sclerotiorum, Colletotrichum spp., and Magnaporthe oryzae. Description of the Drawings

[0042] Figure 1 Schematic diagram for the preparation of the crude extract of Trichoderma asperellum TCS007;

[0043] Figure 2 Flow chart for the separation of the crude extract of Trichoderma asperellum TCS007;

[0044] Figure 3 Negative ion high-resolution mass spectrum of Compound 1;

[0045] Figure 4 For Compound 1 1 1H-NMR spectrum;

[0046] Figure 5 For Compound 113 C-NMR;

[0047] Figure 6 Structure of Compound 1;

[0048] Figure 7 Negative ion high resolution mass spectrum of Compound 2;

[0049] Figure 8 of Compound 2 1 H-NMR spectrum;

[0050] Figure 9 of Compound 2 13 C-NMR;

[0051] Figure 10 Structure of Compound 2;

[0052] Figure 11 Negative ion high resolution mass spectrum of Compound 3;

[0053] Figure 12 of Compound 3 1 H-NMR spectrum;

[0054] Figure 13 of Compound 3 13 C-NMR spectrum;

[0055] Figure 14 Structure of Compound 3;

[0056] Figure 15 Negative ion high resolution mass spectrum of Compound 4;

[0057] Figure 16 of Compound 4 1 H-NMR spectrum;

[0058] Figure 17 of Compound 4 13 C-NMR spectrum;

[0059] Figure 18 Structure of Compound 4

[0060] Figure 19 Negative ion high resolution mass spectrum of Compound 5;

[0061] Figure 20 of Compound 5 1 H-NMR spectrum;

[0062] Figure 21 of Compound 5 13 C-NMR spectrum;

[0063] Figure 22 Structure of Compound 5

[0064] Figure 23 Negative high-resolution mass spectrum of Compound 6;

[0065] Figure 24 of Compound 6 1 H-NMR spectrum;

[0066] Figure 25 of Compound 6 13 C-NMR spectrum;

[0067] Figure 26 Structure of Compound 6

[0068] Figure 27 Fermentation diagram of Trichoderma asperellum TCS007;

[0069] Figure 28 Determination results of antibacterial activities of Compounds 1-3;

[0070] Figure 29 Preparation process flow of alkylated citrate compounds;

[0071] Figure 30 Random forest RF prediction results of alkylated citrate compounds;

[0072] Figure 31 XGBoost prediction results of alkylated citrate compounds. Specific embodiments

[0073] Example 1

[0074] Isolate a mixture containing alkylated citrate compounds from the fermentation broth of Trichoderma asperellum TCS007, and separate, purify and identify the alkylated citrate compounds in the mixture.

[0075] Test materials

[0076] Test strains

[0077] Biocontrol fungus: Trichoderma asperellum TCS007 (T. asperellum TCS007) was isolated from Antarctic marine sediments, identified and named as Trichoderma asperellum TCS007 after mutagenesis stability, and has been deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 15677.

[0078] Culture media

[0079] PDA solid medium: anhydrous glucose 20.0 g / L, potato 200.0 g / L, agar powder 18.0 g / L, distilled water 1 L, pH natural.

[0080] CM-C solid medium: 50 mL of 20× nitrate, 1 mL of trace elements, 10 g of D-glucose, 2 g of peptone, 1 g of yeast extract, 1 g of casein amino acid, 1 mL of vitamin solution, pH 6.5.

[0081] PDB seed culture medium: 20.0 g / L of anhydrous glucose, 200.0 g / L of potato, 1 L of distilled water, pH natural.

[0082] Solid fermentation medium for Trichoderma asperellum TCS007: 72 g of rice, 3.5% soybean powder (based on the weight of rice, the weight percentage of soybean powder is 3.5% of the weight of rice), and 47 mL of distilled water.

[0083] Main instruments

[0084] MGC-250BP artificial climate chamber (Shanghai Yiheng Scientific Instrument Co., Ltd.);

[0085] BSA233S electronic precision balance (Sartorius Scientific Instrument Co., Ltd.);

[0086] HH-4 digital display constant temperature water bath (Changzhou Guohua Electric Appliance Co., Ltd.);

[0087] FE28 pH meter (Mettler-Toledo Instruments Co., Ltd.);

[0088] SW-CJ-2FD type clean bench (Suzhou Antai Air Technology Co., Ltd.);

[0089] ZQZY-CF type shaking incubator (Shanghai Zhichu Instruments Co., Ltd.);

[0090] YB102 electronic balance (Hangzhou Micronpie Technology Co., Ltd.);

[0091] Thin layer chromatography silica gel plate (Hangzhou Kaiying Instrument Business Department);

[0092] Shaker (NewBrunswickScientific);

[0093] Silica gel (Qingdao Ocean Chemical Factory);

[0094] Ultrasonic oscillator (Hangzhou Farant Ultrasonic Technology Co., Ltd.);

[0095] Hot air constant temperature drying oven (Shanghai Shibei Instrument Equipment Factory);

[0096] Rotary evaporator (DigitalwaterbathSB-1000) (EYELA Co., Ltd., Japan);

[0097] High-performance liquid preparative chromatography (Shimadzu LC-8A, Shimadzu-C18, 5 μm, 250×20 mm i.d) (Shimadzu Corporation, Japan);

[0098] Superconducting nuclear magnetic resonance spectrometer (Bruber AVANCE-400) (Bruker, Rheinstetten, Germany);

[0099] Ultraviolet spectrometer (Varian Cary 300 Biospectrophotometer) (Varian Technologies China Co., Ltd., USA);

[0100] (VARIAN) Infrared spectrometer (Nicolet Magna FT-IR 750 spectrometer) (Varian, Palo Alto, CA, USA);

[0101] Test method

[0102] Activation culture of test strains

[0103] Activation of phytopathogenic fungi: Inoculate Magnaporthe oryzae on a CM-C plate and incubate it in the dark in an inverted position in an incubator at 25 °C. When it grows to 3 / 4 of the culture dish, set it aside. Inoculate Sclerotinia sclerotiorum, Monilinia fructicola, Colletotrichum gloeosporioides, Diplocarpon mali, Botrytis cinerea, Rhizoctonia solani, and Fusarium graminearum on a PDA plate and incubate it in the dark in an inverted position in an incubator at 26 °C. When it grows to 3 / 4 of the culture dish, set it aside.

[0104] Activation of Trichoderma asperellum strain TCS007: Take 10 μL of TCS007 bacterial liquid from the thawed glycerol tube, spread it on a PDA plate, and incubate it in the dark in an inverted position at 28 °C for 5 days. Then, cut a mycelial cake with a sterile punch with a diameter of 8 mm under a sterile environment, and place the mycelial cake in the center of the PDA plate with a sterile inoculation needle, and incubate it in the dark in an inverted position at 28 °C for 5 days, set it aside.

[0105] Extraction of crude extract from solid fermentation of Trichoderma asperellum TCS007

[0106] As Figure 1 shown, under a sterile environment, when TCS007 grows to 3 / 4 of the diameter of the culture dish on the PDA medium, rinse it with sterile water, filter the mycelium with sterile gauze to obtain a suspension of TCS007 conidia, then use a hemocytometer to measure the spore content under an optical microscope, and adjust the spore concentration to 1×10 7Use 1×10⁶ cfu / mL as the seed solution for standby. Weigh 72 g of rice, pour it into a 300 mL Erlenmeyer flask, add 47 mL of distilled water, stir well, seal it, sterilize at 121 °C for 20 minutes, then inoculate 1 mL of TCS007 seed solution in a sterile environment, and finally place it in an illumination incubator for cultivation. The cultivation temperature is 28 °C, the light cycle is 12 hours, and the cultivation time is 29 days. The fermented TCS007 solid fermentation product is extracted by equi - volume miscible extraction with ethyl acetate solvent three times, then filtered by suction. First, anhydrous sodium sulfate is added for drying, and then it is concentrated and evaporated to dryness under reduced pressure using a rotary evaporator. After re - dissolving with ethyl acetate, it is collected in a centrifuge tube, and the TCS007 organic - phase crude extract is obtained after the solvent volatilizes.

[0107] Separation and Purification of Trichoderma asperellum TCS007 Solid - Fermentation Crude Extract

[0108] As Figure 2 shown, the TCS007 crude extract is evenly mixed with silica gel of 100 - 200 mesh, and placed in a fume hood to completely dry the sample. After the solvent volatilizes completely, it is loaded into a column, and the volume ratio of the sample to the column - loading silica gel is 1:3. Silica - gel column chromatography is used, and gradient elution is carried out with dichloromethane / methanol as the elution phase (CH 2 Cl 2 / CH 3 OH = 98:2 / 95:5 / 90:10 / 85:15 / 80:20 / 70:30), the elution volume of each gradient is 2000 mL, and fractions are collected with a 500 mL conical flask, with a filling volume of 200 mL per flask. TLC detection is used, and the collected fractions are concentrated and combined according to the ultraviolet and color - development conditions. Finally, 7 concentrated fractions are obtained (Ⅰ(4 - 13), Ⅱ(14 - 18), Ⅲ(19 - 23), Ⅳ(24 - 31), Ⅴ(32 - 36), Ⅵ(37 - 44), Ⅶ(45 - 53)). Preliminary activity tracking shows that fraction Ⅲ(19 - 23) has antibacterial activity.

[0109] Fraction Ⅲ(19 - 23) is subjected to gel Sephadex LH - 20 column chromatography, and elution is carried out using the CH 2 Cl 2 / CH 3 OH(1:1, v / v) system, the elution volume is 2000 mL, and fractions are collected with 20 mL test tubes. Each of the collected fractions is concentrated and combined after TLC detection, and finally 5 concentrated fractions are obtained (1 - 10, 11 - 21, 22 - 34, 35 - 60, 61 - 70). Activity tracking is carried out again, and fraction (22 - 34) has antibacterial activity.

[0110] Fraction (22 - 34) is subjected to secondary silica - gel column chromatography, and gradient elution is carried out with dichloromethane / methanol as the elution phase (CH 2 Cl2 / CH 3 OH = 95:5 / 90:10 / 85:15), with each gradient elution volume being 500 mL. The fractions were collected using a conical flask with a specification of 250 mL, and each flask was filled with 100 mL. TLC detection was used, and the collected fractions were rotary evaporated and concentrated and combined according to the ultraviolet and color development conditions. Finally, 3 concentrated fractions (2 - 5, 6 - 9, 10 - 19) were obtained. Then, activity tracking was carried out, and fraction (2 - 5) had antibacterial activity.

[0111] Six compounds were further purified from fraction (2 - 5) using semi - preparative HPLC. For semi - preparative HPLC, an acetonitrile:0.1% TFA (70:30, v / v) system was used, with a flow rate of 1.5 mL / min and a detection wavelength of λ = 220 nm, obtaining compound 1, compound 2, compound 3, compound 4, compound 5, and compound 6.

[0112] Structural identification of alkylated citrate monomer compounds

[0113] After the monomer compound was prepared by HPLC and concentrated, it was placed in a vacuum drying oven. After drying was completed, the weight of the compound was weighed. The sample was dissolved in a suitable deuterated solvent, and nuclear magnetic resonance (NMR) spectra ( 1 H spectrum, 13 C spectrum, DEPT spectrum, 1 H - 1 H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum) were collected, and the structure of the compound was identified by combining high - resolution mass spectrometry determination.

[0114] Compound 1 (TCS007 - 1), this compound is a pale yellow oil and was identified as Viridiofungin A, with the molecular formula C 31 H 45 NO 10 . The negative ion high - resolution mass spectrometry diagram of compound 1, 1 H - NMR spectrum diagram, 13 C - NMR, the structure is as Figures 3 - 6 shown;

[0115] Compound 2 (TCS007 - 2), this compound is a pale yellow oil and was identified as Viridiofungin A′, with the molecular formula C 32 H 47 NO 10 . The negative ion high - resolution mass spectrometry diagram of compound 2, 1 H - NMR spectrum diagram, 13 C - NMR, the structure is as Figures 7 - 10 shown;

[0116] Compound 3 (TCS007-3), which is a pale yellow oil and is identified as Viridiofungin B, with the molecular formula C 31 H 45 NO 9 。The negative ion high-resolution mass spectrum of Compound 3, 1 H-NMR spectrum, 13 C-NMR, and structure are as Figures 11 - 14 shown;

[0117] Compound 4 (TCS007-4), which is a pale yellow oil and is identified as Viridiofungin, with the molecular formula C 31 H 43 NO 9 。The negative ion high-resolution mass spectrum of Compound 4, 1 H-NMR spectrum, 13 C-NMR, and structure are as Figures 15 - 18 shown;

[0118] Compound 5 (TCS007-5), which is a pale yellow oil and is identified as Viridiofungin, with the molecular formula C 33 H 49 NO 10 。The negative ion high-resolution mass spectrum of Compound 5, 1 H-NMR spectrum, 13 C-NMR, and structure are as Figures 19 - 22 shown;

[0119] Compound 6 (TCS007-6), which is a pale yellow oil and is identified as Viridiofungin, with the molecular formula C 32 H 45 NO 9 。The negative ion high-resolution mass spectrum of Compound 6, 1 H-NMR spectrum, 13 C-NMR, and structure are as Figures 23 - 26 shown;

[0120] Determination of the antibacterial activities of Compounds 1 to 6

[0121] The antibacterial activities of the isolated pure compounds were determined by the growth rate method to measure the inhibitory effects of the compounds on plant pathogenic fungi.

[0122] The compounds were prepared into a stock solution with a concentration of 1%, and then serially diluted to 10 mg / L. Boscalid with the same concentration was used as the control agent. The liquid medicine was pipetted into the sterilized medium to prepare the medicated medium with the corresponding concentration, and the medium without the agent was used as the blank control.

[0123] Inoculate a vigorously growing pathogen disc with a diameter of 8 mm in the center of the flat plate, with the mycelium side facing down, and incubate it upside down at 26 °C for 24 - 96 hours. When the diameter of the pathogen in the control group reaches 3 / 4 of the culture dish, use the cross method to measure the diameter of the pathogen colony and calculate the inhibition rate. The inhibition rate is calculated according to formula (1):

[0124] Inhibition rate (%) = (Colony diameter of the control group - Colony diameter of the treatment group) / (Colony diameter of the control group - 0.8 cm) × 100 (1)

[0125] Test results

[0126] The antibacterial activities of Compounds 1 to 6 were determined against 8 kinds of pathogens, including Sclerotinia sclerotiorum, Monilinia fructicola, Colletotrichum gloeosporioides, Magnaporthe oryzae, Marssonina coronaria, Botrytis cinerea, Rhizoctonia solani, and Fusarium graminearum. The results are shown in Table 1 and Table 2. At a concentration of 10 mg / L, the 6 compounds all had certain antibacterial activities against 8 kinds of pathogenic fungi. Among them, the antibacterial activity against Sclerotinia sclerotiorum was the best, and the inhibition rate reached over 98%, which was equivalent to the activity of the commercial fungicide boscalid. The antibacterial activities against Colletotrichum gloeosporioides and Monilinia fructicola were significantly higher than that of boscalid, and the inhibition rate reached over 90%. The 6 compounds showed certain antibacterial activities against Rhizoctonia solani, Magnaporthe oryzae, Fusarium graminearum, Marssonina coronaria, and Botrytis cinerea, and the inhibition rate was between 32.50% and 55.65%.

[0127] Table 1 Inhibitory effects of Compounds 1 to 3 of TCS007 at 10 mg / L on the tested plant pathogenic fungi

[0128]

[0129] Table 2 Inhibitory effects of Compounds 4 to 6 of TCS007 at 10 mg / L on the tested plant pathogenic fungi

[0130]

[0131]

[0132] Example 2

[0133] The chemical synthesis preparation method of the alkylated citrate compound provided by the present invention is as Figure 29 shown.

[0134] Step 1:

[0135]

[0136] At 0 °C, add BH 3 ·SMe 2And THF, (+)-A-pinene was added dropwise, and after stirring the reaction, Compound 1 was added, followed by the addition of acetaldehyde and H 2 O, and the alkynyl group was borylated. After the reaction was completed, column chromatography was performed to obtain Compound 2.

[0137] Step 2:

[0138]

[0139] N 2 Under N-protection conditions, in a solution of Compound 2 in DMF, K 3 PO 4 and a catalytic amount of Pd(dppf)Cl 2 and pyruvic acid were added, and the reaction was stirred at 80 °C. After the reaction was complete, it was quenched with H 2 O, extracted with AcOEt, and the organic layer was dried over MgSO 4 and column chromatography was performed to obtain Compound 3.

[0140] Step 3:

[0141]

[0142] At room temperature, Mg shavings were added to anhydrous THF, and a catalytic amount of EDB and Compound 3 were added to obtain a Grignard reagent solution. At 0 °C, under N 2 -protection conditions, the Grignard reagent was added to a solution of LaCl 3 ·2LiCl; at -50 °C, the Grignard reagent and a mixed solution of LaCl 3 ·2LiCl were added to a THF solution of N-methoxy-N-methyl octanamide, and the reaction was continued with stirring. It was quenched with a saturated NH 4 Cl aqueous solution, extracted with CHCl 3 and the organic layer was dried over Na 2 SO 4 filtered, concentrated in vacuo, and column chromatography was performed to obtain Compound 4.

[0143] Step 4:

[0144]

[0145] At room temperature, Compound 4 and CCl 4 were added to a round-bottom flask, and an excess of PPh 3 was added in two batches. The reaction was heated to 75 °C and the reaction was continued. After the reaction was completed, it was filtered and column chromatography was performed to obtain Compound 5.

[0146] Step 5:

[0147]

[0148] Under room temperature conditions, the Grignard reagent was prepared from compound 5 and Mg shavings in Et 2 O. The 2-oxo-1,4-dicarboxylate in Et 2 O solution was added dropwise to the Grignard reagent. After the reaction was completed, filtration was carried out, and saturated NH 2 Cl aqueous solution was added to the suspension of the filter cake in Et 4 O. The organic phase was separated, the aqueous layer was extracted with Et 2 O, and the combined organic extracts were dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure, and column chromatography was carried out to obtain compound 6.

[0149] Step 6:

[0150]

[0151] The DMF solution of compound 6 and L-tyrosine methyl ester / L-tyrosine was cooled to -10 °C, the catalyst DIEA and HATU were added successively, and the temperature was slowly raised to room temperature, followed by stirring overnight. After the reaction was completed, it was quenched with NH 4 Cl aqueous solution, extracted with EA, washed successively with H 2 O and saturated NaCl, dried over anhydrous Na 2 SO 4 , and column chromatography was carried out to obtain the alkylated citrate compound 7.

[0152] In addition, the preparation method of the alkylated citrate compound provided by the present invention can also be prepared by referring to the preparation methods of the alkylated citrate compounds disclosed in Patent EP1795206A1, EP2886530A1, CN1819990A, CN101489580A, and TW201010692A1.

[0153] Example 3

[0154] Prediction of the biological activity of the alkylated citrate compound provided by the present invention

[0155] Reverse docking and target modulation

[0156] Reverse docking and target adjustment is a structure-based compound discovery method that identifies potential protein targets by predicting the binding mode of small molecule compounds to target proteins. First, perform the reverse docking operation on each molecule, sort out the possible binding targets of each molecule (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6), select the targets with a frequency of occurrence ≥ 4 (the first column of Table 3), and then perform molecular docking with each compound (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6). The detailed information of the binding targets can be viewed from the PDB database (https: / / www.rcsb.org / ).

[0157] Molecular docking

[0158] Molecular docking is a commonly used method in the development process of small molecule compounds. It can predict the interaction force (score) between small molecule compounds and proteins, providing a theoretical basis for pesticide design and screening. The inventors of this application performed molecular docking on each molecule (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6) with possible targets. The lower the score, the stronger the binding force between the small molecule compound and the target, and the higher the possible activity. The molecular docking results are shown in Table 3.

[0159] Table 3 Molecular docking results of Compounds 1 - 6 of TCS007 with target molecules

[0160]

[0161]

[0162] Antibiotic-like prediction

[0163] An antibiotic-like activity prediction model was constructed using the AI autonomous modeling module of PDAI, and compounds 1 - 6 were predicted for antibiotic-like activity. The results are shown in Table 4.

[0164] Table 4 Evaluation results of Compounds 1 - 6 as antibiotic-like activities

[0165]

[0166]

[0167] In addition, the inventors of this application also used a prediction model based on deep learning. They input the SMILES of the compound structure and used a neural network to predict the antibacterial activity of the compound (https: / / www.nature.com / articles / s41586-023-06887-8#code-availability). The results are as Figure 30 、Figure 31 As shown, the output of the deep learning model is a probability value (in the range of 0 to 1), indicating the likelihood that compounds 1-6 have antibacterial activity.

[0168] Binding to the pathogenic proteins of plant pathogens Sclerotinia sclerotiorum, Colletotrichum spp., and Magnaporthe oryzae

[0169] Use Chem3D to convert the format of small ligands and save them in mol2 format. Then use AutoDockTools to continue converting the format of small molecule ligands and save them as "pdbqt" format. Use PyMOL and AutoDockTools to remove the original ligands, water molecules, and add hydrogen atoms to the target protein receptor, and find the active pocket, and save it as "pdbqt" format; finally, use AutoDockvina for molecular docking to obtain the experimental results in this table. The value is the scoring value, and the smaller the value, the stronger the affinity between the protein and the molecule. The results are shown in Tables 5, 6, and 7.

[0170] Table 5 Evaluation results of the binding of compounds 1-6 to the pathogenic protein of Sclerotinia sclerotiorum

[0171]

[0172]

[0173]

[0174] As can be seen from Table 5, compounds 1-6 bind reasonably well to the protein structures numbered 6, 9, 11, 13, 14, 17, 19, 30, 32, 38-43, resulting in changes in the functions of these proteins and the inability to complete the normal physiological activities of Sclerotinia sclerotiorum; among them, AF-A7E6B5-F1-model_v4.pdb numbered 9 and AF-A7E9A5-F1-model_v4.pdb numbered 13 showed the best performance. It can be seen from this that the alkylated citrate compounds provided by the present invention have the effect or function of binding to multiple pathogenic proteins of Sclerotinia sclerotiorum, resulting in the inability of Sclerotinia sclerotiorum to carry out normal physiological activities and being able to strongly inhibit Sclerotinia sclerotiorum.

[0175] Table 6 Evaluation results of the binding of compounds 1-6 to the pathogenic protein of Colletotrichum spp.

[0176]

[0177] As can be seen from Table 6, compounds 1-6 bind reasonably well to the protein structures numbered 3, 4, 5, and 6, resulting in changes in the functions of these proteins and preventing the normal physiological activities of Bacillus anthracis. Among them, CgNLP1_2025_02_20_18_36_model_0 numbered 3 showed the best performance. It can be seen from this that the alkylated citrate compounds provided by the present invention have the effect or function of binding to multiple pathogenic proteins of Bacillus anthracis, resulting in the inability of Bacillus anthracis to carry out normal physiological activities and can effectively inhibit Bacillus anthracis.

[0178] Table 7 Evaluation results of the binding of compounds 1-6 to pathogenic proteins of Magnaporthe oryzae

[0179]

[0180] As can be seen from Table 7, compounds 1-6 bind reasonably well to the protein structures numbered 6, 7, and 8, resulting in changes in the functions of these proteins and preventing the normal physiological activities of Magnaporthe oryzae. Ati1 numbered 7 showed the best performance. It can be seen from this that the alkylated citrate compounds provided by the present invention have the effect or function of binding to multiple pathogenic proteins of Magnaporthe oryzae, resulting in the inability of Magnaporthe oryzae to carry out normal physiological activities and can inhibit Magnaporthe oryzae.

Claims

1. Use of the alkylated citrate compound represented by formula (I) and / or formula (II) in the preparation of a medicament for preventing and controlling plant diseases, in, R1 and R3 are each independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl.

2. Use of the alkylated citrate compounds represented by formula (I) and / or formula (II) for preventing and controlling plant diseases, in, R1 and R3 are each independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl.

3. A method for controlling plant diseases, comprising applying an effective amount of an alkylated citrate compound represented by formula (I) and / or formula (II) to the plant, in, R1 and R3 are each independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl.

4. Use of an alkylated citrate compound of formula (I) and / or formula (II) in the preparation of an agriculturally acceptable formulation for controlling plant diseases, in, R1 and R3 are each independently selected from H, OH, halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Haloalkoxy, C 3-7 Cycloalkyl.

5. The use or purpose or method according to any one of claims 1 to 4, characterized in that: Said R1 and R3 are each independently selected from H, OH; R2 is selected from H, CH3; R4 and R5 are each independently selected from H, OH, CH3, and OCH3.

6. The use or purpose or method according to claim 5, characterized in that: The alkylated citrate compound represented by formula (I) and / or formula (II) is selected from 7. The use or purpose or method according to claims 1-4, characterized in that: The plant diseases are selected from diseases caused by plant pathogenic fungi; preferably, the plant pathogenic fungi are selected from diseases caused by rice blast, alternaria, fusarium, botrytis cinerea, powdery mildew, phytophthora, Rhizoctonia, sclerotinia, anthracnose, and mycosphaeria.

8. The use or purpose or method according to claim 7, characterized in that: The plant diseases are rapeseed sclerotinia, peach brown rot, tea oil anthracnose, rice blast, apple brown spot, cucumber gray mold, rice sheath blight, wheat fusarium head blight and the like.

9. An alkylated citrate compound represented by formula (1) or formula (II), its derivatives, analogs, tautomeric forms, stereoisomers, polymorphs and pharmaceutically acceptable salts, in, R1 and R3 are each independently selected from H, OH, halogen, C 1-12 Alkyl, C 1-12 Haloalkyl, C 1-12 Alkoxy, C 1-12 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-12 Alkyl, C 1-12 Haloalkyl, C 1-12 Alkoxy, C 1-12 Haloalkoxy, C 3-7 Cycloalkyl.

10. A method for preparing an alkylated citrate compound, wherein the alkylated citrate compound is represented by formula (1) or formula (II), characterized in that: The alkylated citrate compound is separated from the solid fermentation product of Trichoderma aspergillus TCS007; preferably, the separation method includes but is not limited to acid precipitation method, salting-out method, organic solvent precipitation method, organic solvent extraction method, etc.; in, R1 and R3 are each independently selected from H, OH, halogen, C 1-12 Alkyl, C 1-12 Haloalkyl, C 1-12 Alkoxy, C 1-12 Haloalkoxy, C 3-7 Cycloalkyl; R2, R4, R5 are each independently selected from H, OH, C 1-12 Alkyl, C 1-12 Haloalkyl, C 1-12 Alkoxy, C 112 Haloalkoxy, C 3-7 Cycloalkyl.

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