A black tiger extract, a preparation method thereof and a use thereof for preventing and treating plant diseases

Abstract

The application discloses use of an extract of black tiger in preparation of a fungicide for preventing and treating plant diseases caused by pathogenic fungi and oomycetes. The black tiger extract has the effects of inhibiting spore germination, germ tube growth or zoospore formation, and vegetative hypha growth of the fungi and oomycetes, can effectively prevent growth of the pathogenic fungi and infection on host plants, and has certain prevention and treatment effects on rice rice blast, rice rice smut, potato late blight, cucumber anthracnose, grape anthracnose, corn ear rot, pepper blight and camphor blight, and provides a possibility for development of a plant source fungicide with diversified effective components, high prevention and treatment efficiency and a wide spectrum of prevention and treatment objects.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control, specifically relating to a black tiger extract, its preparation method, and its use in controlling plant diseases. Background Technology

[0002] Plant fungal and oomycete diseases are numerous, accounting for approximately 70% of all plant diseases. Several, even dozens, of different fungi and oomycetes can cause diseases on a single plant. For example, rice blast, rice false smut, sheath blight, sesame spot, and bakanae disease; wheat scab, powdery mildew, stripe rust, stem rust, leaf rust, root rot, and sheath blight; and corn large leaf spot, small leaf spot, stem base rot, rust, sheath blight, Curvularia leaf spot, head smut, and powdery mildew are all caused by fungi. These fungal diseases account for over 90% of the area affected and the losses caused by crop diseases. Besides fungal diseases, oomycete diseases are also serious in many crops, such as potato late blight, soybean Phytophthora blight, pepper Phytophthora blight, grape downy mildew, and cucumber downy mildew. Statistics show that fungal and oomycete diseases cause 10%-20% of crop yield losses annually.

[0003] Chemical control remains the primary method for controlling plant diseases, with widely used synthetic fungicides including tricyclazole, azoxystrobin, pyraclostrobin, boscalid, cyazofamid, chlorothalonil, thiophanate-methyl, carbendazim, prothiophanate-methyl, and imazalil. Reports indicate that many plant pathogenic fungi in agricultural production have developed significant resistance and cross-resistance to fungicides. For example, rice blast fungus, wheat scab fungus, and gray mold fungus have shown marked resistance to fungicides such as carbendazim, prothiophanate-methyl, and boscalid, respectively. Furthermore, the long-term use of synthetic fungicides has led to a series of social problems, including environmental pollution, pesticide residues, and toxicity to humans and animals.

[0004] Plant-derived fungicides, as effective alternatives to chemically synthesized fungicides, are an important branch of biological pesticides. They possess both antibacterial and bactericidal activities, while also offering advantages such as safety for humans and animals, environmental friendliness, low likelihood of developing resistance, and easy degradation. Currently used plant-derived fungicides mainly include extracts from Sophora flavescens, bulb extracts from garlic and other Allium family plants, Cnidium monnieri, Neem seed extract, and Oregano extract. These plant-derived fungicides have been widely used for the control of various diseases in crops and fruits and vegetables. For example, matrine is used to control downy mildew, powdery mildew, gray mold, and blight; allicin is used to control anthracnose and blight in peppers.

[0005] Black Tiger ( Scarlet Kadsura Black Tiger (Hemiberlesia lataniae) is a traditional medicinal plant. Studies have shown that it contains abundant lignans, terpenes, and other secondary metabolites, exhibiting good activity in anti-inflammatory and antioxidant properties. However, there are currently no reports on the application of Black Tiger extract as a plant-derived fungicide for the control of plant diseases. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a black tiger extract that inhibits the germination of conidia, germ tube growth or appressorium formation and hyphal growth of fungi and oomycetes, effectively preventing the growth of pathogens and their infection of host plants, and has a certain preventive and control effect on plant diseases.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] The first aspect of the present invention is to provide the use of black tiger extract in the preparation of fungicides for the prevention and control of plant diseases caused by pathogenic fungi and oomycetes;

[0009] The plant disease is caused by rice blast fungus. Magnaporthe rice Rice blast disease caused by Aspergillus oryzae; or the plant disease mentioned above is caused by Aspergillus oryzae. Virens stilaginoidea Rice blast disease caused by pathogenic fungus; or the plant disease described is caused by pathogenic fungus Phytophthora. Phytophthora infestans Potato late blight caused by [the disease]; or the plant disease described is caused by [a specific type of fungus, possibly *Colletotrichum spp.*] of the Cucurbitaceae family. Colletotrichum orbiculare Arx. Anthracnose of cucumber caused by *Colletotrichum candida*; or the plant disease is caused by *Colletotrichum candida*. Colletotrichum gloeosporioides Caused by anthracnose in grapes; or the plant disease is caused by the anthracnose fungus of the pepper plant. Colletotrichum capsici Anthracnose of peppers caused by *Cyclocarya paliurus*; or the plant disease described herein is caused by *Cyclocarya paliurus*. Cladosporium herbarium Corn ear rot caused by; or the plant disease mentioned is caused by Phytophthora capsici. Phytophthora capsicum Caused by Phytophthora blight in peppers; or the plant disease is caused by Phytophthora camphorata. Phytophthora cinnamon Camphor tree blight caused by this disease;

[0010] The extraction steps for the black tiger extract are as follows:

[0011] (1) After grinding the black tiger root into coarse powder, it was soaked in a solvent for the first extraction, and the first filtrate was obtained after filtration;

[0012] (2) Add solvent to the filter residue for a second extraction. After filtration, the second filtrate is obtained. Combine the two filtrates and concentrate the filtrate into an extract to obtain black tiger extract.

[0013] The solvent is methanol, ethanol, cyclohexanol, petroleum ether, n-hexane, n-pentane, dodecane, dichloromethane, 1,2-dichloroethane, diethyl malonate, 1,4-dioxane, acetone, or acetonitrile.

[0014] The first and second extractions include one or two of mechanical stirring extraction, ultrasound-assisted extraction, and Soxhlet extraction.

[0015] In one optional embodiment, the mechanical stirring extraction time is 1 to 5 days; the ultrasonic-assisted extraction time is 0.5 to 2 hours; and the Soxhlet extraction time is 0.5 to 2 hours.

[0016] In one optional embodiment, the fungicide is formulated as an emulsifiable concentrate, wettable powder, suspension concentrate, powder, soluble powder, aqueous solution, water-dispersible granules, fumigant, granules, or seed coating agent.

[0017] A second aspect of the present invention is to provide the application of the lignin compounds in the black tiger extract described above in the preparation of a fungicide for controlling plant diseases caused by pathogenic fungi and oomycetes, wherein the amount of the lignin compounds is 100~400 mg / L, and the chemical structural formula of the lignin compounds is shown in Formula I below:

[0018] .

[0019] The plant disease is caused by rice blast fungus. Magnaporthe rice Rice blast disease caused by Aspergillus oryzae; or the plant disease mentioned above is caused by Aspergillus oryzae. Virens stilaginoidea Rice blast disease caused by pathogenic fungus; or the plant disease described is caused by pathogenic fungus Phytophthora. Phytophthora infestans Potato late blight caused by [the disease]; or the plant disease described is caused by [a specific type of fungus, possibly *Colletotrichum spp.*] of the Cucurbitaceae family. Colletotrichum orbiculare Arx. Anthracnose of cucumber caused by *Colletotrichum candida*; or the plant disease is caused by *Colletotrichum candida*. Colletotrichum gloeosporioides Grape anthracnose caused by; or the plant disease is caused by Lychee anthracnose fungus. Colletotrichum litchii Anthracnose of litchi caused by *Cyclocarya paliurus*; or the plant disease described is caused by *Cyclocarya paliurus*. Cladosporium herbarium Corn ear rot caused by; or the plant disease mentioned is caused by Phytophthora capsici. Phytophthora capsicum Caused by blight of peppers; or the plant disease is caused by strawberry anthracnose fungus. Colletotrichum fragariae Brooks Strawberry anthracnose caused by Botrytis cinerea; or the plant disease mentioned above is caused by Botrytis cinerea. Botrytis cinerea Pers. Fr. Tomato gray mold caused by Botrytis cinerea; or the plant disease mentioned above is caused by Botrytis cinerea. Botrytis cinerea Pers. This causes gray mold disease in strawberries.

[0020] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0021] The black tiger extract of this invention inhibits the germination of fungal and oomycete conidia, germ tube growth or appressorium formation, and growth of vegetative hyphae. It can effectively prevent the growth of pathogens and their infection of host plants. It has certain control effects on rice blast, rice false smut, potato late blight, cucumber anthracnose, grape anthracnose, corn ear rot, pepper blight, and camphor blight. It provides a possibility for the development of plant-derived fungicides with diverse active ingredients, high control efficacy, and broad-spectrum control targets. Attached Figure Description

[0022] Figure 1 These are the results of the safety evaluation of the extract HL1-B1 in rice at different concentrations.

[0023] Figure 2 The results show the safety evaluation of the monomer compound Anwuzhixin of this invention in rice at different concentrations. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0025] Example 1: Preparation of Black Tiger Extract HL1-A1

[0026] Black tiger root was crushed into coarse powder, soaked in water, and ultrasonically extracted for 1 hour. Then it was mechanically stirred and soaked at room temperature for 1 day, followed by steam distillation for 4 hours. The oil layer was recovered, and water was removed by anhydrous sodium sulfate to obtain black tiger extract HL1-A1.

[0027] Example 2 Preparation of Black Tiger Extract HL1-A2

[0028] Black tiger root was crushed into coarse powder and soaked in 95% ethanol (ethanol to black tiger coarse powder ratio of 1g:10mL). It was then extracted by ultrasonication (80Hz) for 1 hour, followed by mechanical stirring extraction at 20℃ for 2 days.

[0029] After filtration, the first filtrate was obtained; the residue was added to 95% ethanol and ultrasonically extracted for 1 h, followed by mechanical stirring and extraction at 20℃ for 2 days, and then filtered to obtain the second filtrate; the two filtrates were combined and concentrated into an extract to obtain black tiger extract HL1-A2.

[0030] Example 3 Preparation of Black Tiger Extract HL1-A3

[0031] Black tiger root was crushed into coarse powder and soaked in 95% ethanol (the ratio of ethanol to black tiger coarse powder was 1g:10mL). Soxhlet extraction was performed for 1 h, and the first filtrate was obtained after filtration. The residue was added to 95% ethanol and extracted by Soxhlet extraction for 1 h. The second filtrate was obtained after filtration. The two filtrates were combined and concentrated into an extract to obtain black tiger extract HL1-A3.

[0032] Example 4 Preparation of Black Tiger Extract HL1-A4

[0033] The extraction process was the same as in Example 2, except that ethanol was replaced with methanol to obtain black tiger extract HL1-A4.

[0034] Example 5 Preparation of Black Tiger Extract HL1-A5

[0035] The extraction process was the same as in Example 2, except that ethanol was replaced with cyclohexanol to obtain black tiger extract HL1-A5.

[0036] Example 6 Preparation of Black Tiger Extract HL1-B1

[0037] The extraction process was the same as in Example 2, except that ethanol was replaced with petroleum ether (60-90℃) to obtain black tiger extract HL1-B1.

[0038] Example 7 Preparation of Black Tiger Extract HL1-B2

[0039] The extraction process was the same as in Example 3, except that ethanol was replaced with petroleum ether (60-90℃) to obtain black tiger extract HL1-B2.

[0040] Example 8 Preparation of Black Tiger Extract HL1-B3

[0041] The extraction process was the same as in Example 2, except that ethanol was replaced with n-hexane to obtain black tiger extract HL1-B3.

[0042] Example 9 Preparation of Black Tiger Extract HL1-B4

[0043] The extraction process was the same as in Example 2, except that ethanol was replaced with n-pentane to obtain black tiger extract HL1-B4.

[0044] Example 10 Preparation of Black Tiger Extract HL1-B5

[0045] The extraction process was the same as in Example 2, except that ethanol was replaced with dodecane to obtain black tiger extract HL1-B5.

[0046] Example 11 Preparation of Black Tiger Extract HL1-B6

[0047] The extraction process was the same as in Example 2, except that ethanol was replaced with toluene to obtain black tiger extract HL1-B6.

[0048] Example 12 Preparation of Black Tiger Extract HL1-B7

[0049] The extraction process was the same as in Example 2, except that ethanol was replaced with dichloromethane to obtain black tiger extract HL1-B7.

[0050] Example 13 Preparation of Black Tiger Extract HL1-B8

[0051] The extraction process was the same as in Example 3, except that ethanol was replaced with dichloromethane to obtain black tiger extract HL1-B8.

[0052] Example 14 Preparation of Black Tiger Extract HL1-B9

[0053] The extraction process was the same as in Example 2, except that ethanol was replaced with carbon tetrachloride to obtain black tiger extract HL1-B9.

[0054] Example 15 Preparation of Black Tiger Extract HL1-B10

[0055] The extraction process was the same as in Example 2, except that ethanol was replaced with 1,2-dichloroethane to obtain black tiger extract HL1-B10.

[0056] Example 16 Preparation of Black Tiger Extract HL1-C1

[0057] The extraction process was the same as in Example 2, except that ethanol was replaced with chloroform to obtain black tiger extract HL1-C1.

[0058] Example 17 Preparation of Black Tiger Extract HL1-C2

[0059] The extraction process was the same as in Example 2, except that ethanol was replaced with ethyl acetate to obtain black tiger extract HL1-C2.

[0060] Example 18 Preparation of Black Tiger Extract HL1-C3

[0061] The extraction process was the same as in Example 3, except that ethanol was replaced with ethyl acetate to obtain black tiger extract HL1-C3.

[0062] Example 19 Preparation of Black Tiger Extract HL1-C4

[0063] The extraction process was the same as in Example 2, except that ethanol was replaced with diethyl malonate to obtain black tiger extract HL1-C4.

[0064] Example 20 Preparation of Black Tiger Extract HL1-C5

[0065] The extraction process was the same as in Example 2, except that ethanol was replaced with diethyl ether to obtain black tiger extract HL1-C5.

[0066] Example 21 Preparation of Black Tiger Extract HL1-C6

[0067] The extraction process was the same as in Example 3, except that ethanol was replaced with diethyl ether to obtain black tiger extract HL1-C6.

[0068] Example 22 Preparation of Black Tiger Extract HL1-C7

[0069] The extraction process was the same as in Example 2, except that ethanol was replaced with tetrahydrofuran to obtain black tiger extract HL1-C7.

[0070] Example 23 Preparation of Black Tiger Extract HL1-C8

[0071] The extraction process was the same as in Example 2, except that ethanol was replaced with 1,4-dioxane to obtain the black tiger extract HL1-C8.

[0072] Example 24 Preparation of Black Tiger Extract HL1-C9

[0073] The extraction process was the same as in Example 2, except that ethanol was replaced with acetone to obtain black tiger extract HL1-C9.

[0074] Example 25 Preparation of Black Tiger Extract HL1-C10

[0075] The extraction process was the same as in Example 2, except that ethanol was replaced with acetonitrile to obtain black tiger extract HL1-C10.

[0076] Example 25 Preparation of Black Tiger Extract HL1-D1

[0077] Black tiger root was crushed into coarse powder and added to the material bottle of a supercritical carbon dioxide equipment. The pressure was set to 20 MPa and the extraction temperature to 45 °C. Extraction was carried out for 1.5 h to obtain black tiger extract HL1-D1.

[0078] Example 26 Preparation of Black Tiger Extract HL1-E1

[0079] The black tiger extract HL1-A2 obtained in Example 2 was further extracted three times with petroleum ether (60-90℃) and water. The organic phases were combined, and a large amount of solvent was removed by rotary evaporation. The extract was further separated and purified by column chromatography (the column packing material was 200-300 mesh silica gel, the sample amount to silica gel mass ratio was 1:20, and isocratic elution was performed using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 20:1) to obtain the purified extract HL1-E1.

[0080] Effect verification example

[0081] I. Inhibitory effect of black tiger extract on rice blast fungus

[0082] (I) Inhibitory effect of black tiger extract on conidial germination and appressorium formation of rice blast fungus

[0083] 1. Experimental Methods

[0084] (1) Production of conidia of rice blast fungus: The rice blast fungus to be tested ( Magnaporthe rice Strain P131 (provided by the College of Plant Protection, China Agricultural University) was inoculated onto tomato oat agar (OTA) plates and incubated in a 28℃ constant temperature and light incubator. After 3-5 days, rice blast fungus mycelia were scraped from the OTA plates, broken up, and evenly spread onto new OTA plates, which were then incubated in a 28℃ constant temperature and light incubator. When new mycelia emerged from the surface of the culture medium, they were broken up with cotton swabs and incubated at 28℃ under light for 48 h. A large number of conidia were produced on the surface of the OTA plates.

[0085] (2) Preparation of *Blastomyces oryzae* conidial suspension: After scraping OTA plates with sterile water, the suspension was filtered through three layers of lens paper. The filtrate was the conidial suspension. The conidia were counted using a hemocytometer, and the conidial concentration in the suspension was adjusted to 4 × 10⁻⁶. 5 per mL.

[0086] (3) Treatment of rice blast fungus conidia with the test extracts: Equal volumes of the stock solutions of the test extracts at different concentrations were added to the conidia suspension, and each test extract was prepared to a working concentration of 200 mg / L and a conidia concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ cells / mL and a working concentration of 50 mg / L conidia. 5 A mixed inoculum of 1 conidia / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. Twelve hours after inoculation, the conidia germination rate and appressorium formation rate were observed and tallied under a microscope.

[0087] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germinations and appressorium formations were investigated, and the conidia germination rate and appressorium formation rate were calculated. The conidia germination rate and appressorium formation rate of solvent treatment (1% dimethyl sulfoxide) were used as controls to calculate the inhibitory effect of the test sample on the germination of conidia and the formation of appressorium of *Blastomyces oryzae*.

[0088] 2. Experimental Results

[0089] The results are shown in Table 1 below. In Table 1, "+" indicates that the inhibition effect at 200 mg / L is less than 40%, "++" indicates that the inhibition effect at 50 mg / L is 40-80%, and "+++" indicates that the inhibition effect at 50 mg / L is greater than 80%.

[0090] Table 1

[0091]

[0092] The above results indicate that only extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 showed inhibitory effects of more than 80% on both conidial germination and appressorium formation at low concentrations (50 mg / L), demonstrating good inhibitory activity. Therefore, extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 were selected for in vivo efficacy testing.

[0093] The inhibitory effects of extracts with 40-80% concentration on conidial germination and appressorium formation at a concentration of 200 mg / L are shown in Table 2 below.

[0094] Table 2

[0095]

[0096] The above results indicate that when the concentration of the extract, which had an inhibitory effect on conidial germination and appressorium formation of 40-80%, was increased to 200 mg / L, its inhibitory effect on conidial germination and appressorium formation was greater than 80%.

[0097] (II) The preventive effect of black tiger extract on barley leaves infected with rice blast fungus.

[0098] 1. Experimental Methods

[0099] (1) Preparation of barley leaves: Take barley leaves (Emai 9) at the one-leaf-one-heart stage and place them in the inoculation box for later use.

[0100] (2) Production of rice blast fungus conidia: Same as step (1) in the experimental method described in “(I)” above.

[0101] (3) Preparation of rice blast fungus conidial suspension: Same as step (2) in the experimental method of “(I)” above.

[0102] (4) Barley leaf inoculation: To evaluate the preventive effect, 4 μL of black tiger extract (extracts HL1-B1, HL1-B2, HL1-B3, HL1-E1) (working concentration: 200 mg / L) was spot-inoculated onto barley leaves, 3 spots per leaf, 3 leaves per treatment; 12 h later, 4 μL (working concentration: 4 × 10⁻⁶) was spot-inoculated at the droplet site. 5 Rice blast fungus conidia (number of spores / mL) were cultured in the dark at 28°C for 24 h, then transferred to light at 28°C. The results were collected after 4 days.

[0103] (5) Statistics and analysis: The area of ​​barley leaf lesions was investigated, and the germination rate of conidia treated with solvent (1% dimethyl sulfoxide) was used as a control to evaluate the control effect of the test extract on barley leaves infected with rice blast fungus.

[0104] 2. Experimental Results

[0105] The results are shown in Table 3 below. In Table 3, "+++" indicates that the inhibition effect of 200 mg / L is greater than 80%.

[0106] Table 3

[0107]

[0108] The above results indicate that extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 all showed greater than 80% control efficacy against diseases, and had a good control effect against barley and rice blast.

[0109] (III) The preventive effect of black tiger extract on rice leaves infected with rice blast fungus.

[0110] 1. Experimental Methods

[0111] (1) Preparation of rice leaves: Take rice leaves at the four-leaf stage (susceptible varieties: CO39 and Xiangwanxian 11) and place them in an inoculation box for later use.

[0112] (2) Production of rice blast fungus conidia: Same as step (1) in the experimental method described in “(I)” above.

[0113] (3) Preparation of rice blast fungus conidial suspension: Same as step (2) in the experimental method of “(I)” above.

[0114] (4) Rice leaf inoculation: To evaluate the preventive effect, 4 μL of black tiger extract (working concentration: 200 mg / L) was spot-inoculated onto rice leaves, 3 spots per leaf, 3 leaves per treatment; 12 h later, 4 μL (working concentration: 4 × 10⁻⁶) was spot-inoculated at the droplet site. 5 Rice blast fungus conidia (number of spores / mL) were cultured in the dark at 28°C for 24 h, then transferred to light at 28°C. The results were collected after 6 days.

[0115] (5) Statistics and analysis: The area of ​​lesions on rice leaves was investigated. The germination rate of conidia treated with solvent (1% dimethyl sulfoxide) was used as a control to evaluate the control effect of the extract on rice leaves infected with rice blast fungus.

[0116] 2. Experimental Results

[0117] The results are shown in Table 4 below. In Table 4, "+++" indicates that the inhibition effect of 200 mg / L is greater than 80%.

[0118] Table 4

[0119]

[0120] The above results indicate that extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 all showed greater than 80% control efficacy against the disease, and had a good control effect against rice blast.

[0121] (iv) The preventive effect of black tiger extract (HL1-B1, HL1-B2, HL1-B3, HL1-E1) on potted rice infected with rice blast fungus.

[0122] 1. Experimental Methods

[0123] (1) Preparation of rice plants: Take rice plants at the four-leaf stage (susceptible variety: CO39) and place them in an inoculation box for later use.

[0124] (2) Production of rice blast fungus conidia: Same as step (1) in the experimental method described in “(I)” above.

[0125] (3) Preparation of rice blast fungus conidial suspension: Same as step (2) in the experimental method of “(I)” above.

[0126] (4) Rice plant inoculation: Spray 10 mL of black tiger extract (200 mg / L) evenly onto rice plants, and 24 h later spray 10 mL of rice blast fungus conidial solution (working concentration: 1×10⁻⁶). 5 (number / mL), cultured in the dark and moist for 24 h, then in the dark and moist for 24 h at 28℃, and then cultured under light at 28℃. The results were collected after 6 days.

[0127] (5) Statistics and Analysis: Rice blast leaf blast disease survey was conducted in accordance with the agricultural industry standard "Technical Specification for Field Monitoring of Rice Blast Resistance" (NYT3685-2020). The specific standards are as follows: Grade 0: No disease on the whole leaf; Grade 1: Brown necrotic spots the size of pinheads on the leaves; Grade 2: Larger brown necrotic spots (1-2 mm in diameter) on the leaves, but no typical lesions; Grade 3: Typical rice blast lesions, lesion area <2%; Grade 4: Typical rice blast lesions, 2% ≤ lesion area <5%; Grade 5: Typical rice blast lesions, 5% ≤ lesion area <10%; Grade 6: Typical rice blast lesions, 10% ≤ lesion area <25%; Grade 7: Typical rice blast lesions, 25% ≤ lesion area <50%; Grade 8: Typical rice blast lesions, 50% ≤ lesion area <75%; Grade 9: Typical rice blast lesions, lesion area ≥75%.

[0128] 2. Experimental Results

[0129] The results are shown in Table 5 below. In Table 5, "1-9" represent the occurrence level of rice blast disease.

[0130] Table 5

[0131]

[0132] The above results indicate that rice leaves treated with extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 showed no typical lesions and exhibited good control effects against rice blast leaf blast. Among these, HL1-E1, being an extract obtained after further separation and purification of HL1-B1, had a higher content of active ingredients than HL1-B1, and its antibacterial activity was superior to that of HL1-B1, HL1-B2, and HL1-B3 (which were not further purified).

[0133] (V) The control effect of black tiger extract (HL1-B1, HL1-B2, HL1-B3, HL1-E1) on rice blast disease in the field.

[0134] 1. Experimental Methods

[0135] (1) Rice planting: Rice (variety: Yanfeng 47) is planted in plots, with each plot being 20 square meters.

[0136] (2) Application plan: spray once each at the heading stage and the full heading stage of rice, with the working solution concentration of the extract being 1200 mg / L and the amount of pesticide applied per plot being 2.7 L.

[0137] (3) Statistics and Analysis: Rice blast leaf blast disease survey was conducted in accordance with the agricultural industry standard "Technical Regulations for Field Monitoring of Rice Blast Resistance" (NYT3685-2020). The specific standards are as follows: Grade 0: No disease on the whole leaf; Grade 1: Brown necrotic spots the size of pinheads on the leaves; Grade 2: Larger brown necrotic spots (1-2 mm in diameter) on the leaves, but no typical lesions; Grade 3: Typical rice blast lesions, lesion area <2%; Grade 4: Typical rice blast lesions, 2% ≤ lesion area <5%; Grade 5: Typical rice blast lesions, 5% ≤ lesion area <10%; Grade 6: Typical rice blast lesions, 10% ≤ lesion area <25%; Grade 7: Typical rice blast lesions, 25% ≤ lesion area <50%; Grade 8: Typical rice blast lesions, 50% ≤ lesion area <75%; Grade 9: Typical rice blast lesions, lesion area ≥75%.

[0138] 2. Experimental Results

[0139] The results are shown in Table 6 below. In Table 6, "1-7" represent the occurrence level of rice blast disease.

[0140] Table 6

[0141]

[0142] The results indicate that although rice leaves treated with extracts HL1-B1, HL1-B2, and HL1-B3 showed typical rice blast lesions, the lesion area was less than 2%, indicating that the treatments generally had good control effects against rice blast leaf blast. Among these, HL1-E1 showed the best control effect.

[0143] To further demonstrate that the concentration "1200 mg / L" applied in this invention is a safe concentration (harmless to plants), the extract HL1-B1 was further used to conduct a safety evaluation of rice at this concentration. The specific results are as follows:

[0144] (1) Seed germination. Healthy japonica rice (Nipponbare) seeds were selected and germinated with pesticides at concentrations of 300, 600, and 1200 μg·mL⁻¹. -1 Rice seeds were soaked in black tiger extract (HL1-B1) and cultured in the dark at 30℃ for 48 h. Germination was then observed and the germination rate was recorded. DMSO (1%) was used as a solvent control. Each treatment consisted of 100 seeds and was repeated three times.

[0145] (2) Physiological indicators. Japonica rice (Nipponbare) was cultured in seedling boxes until 2 weeks of age, and then the plant surface was evenly sprayed with a concentration of 300, 600 and 1200 μg·mL. -1Black tiger extract was cultured at 30℃ under alternating light and dark conditions for 7 days, and the plant height and root length of rice plants in each treatment were measured. DMSO (1%) treatment was used as a solvent control. Forty rice plants were investigated for each treatment, and each treatment was repeated three times.

[0146] like Figure 1 As shown in Figure A, the result is 1200 μg·mL -1 600 μg·mL -1 and 300 μg·mL -1 The germination rate of rice seeds treated with high concentrations of black tiger extract was over 98%, which was not different from that treated with 1% DMSO.

[0147] like Figure 1 As shown in Figures B and C, the result indicates 1200 μg·mL -1 600 μg·mL -1 and 300 μg·mL -1 Rice plants treated with the concentration of black tiger extract showed no difference in morphological parameters (plant height, root length) compared to those treated with 1% DMSO.

[0148] The results showed that the black tiger extract at 1200 μg·mL⁻¹ was effective. -1 At concentrations of [specific concentration], it has no effect on the germination process of rice seeds or the physiological development of the plant, and is non-toxic to rice.

[0149] II. Inhibitory effect of black tiger extract on Aspergillus oryzae

[0150] (I) Inhibitory effect of black tiger extract on the germination of Aspergillus oryzae conidia

[0151] 1. Experimental Methods

[0152] (1) Production of Aspergillus oryzae conidia: The Aspergillus oryzae to be tested ( Virens stilaginoidea The PJ52 strain (provided by the College of Plant Protection, China Agricultural University) was inoculated onto potato dextrose agar (PDA) plates and cultured in a constant temperature and light incubator at 28℃. After 7 days, the mycelial cake was inoculated onto potato sucrose agar (PSB) and cultured at 28℃ and 180 rpm for 7 days, after which a large number of conidia were produced.

[0153] (2) Preparation of Aspergillus oryzae conidia suspension: The culture medium was filtered through three layers of lens paper, and the filtrate was the conidia solution. Conidia were counted using a hemocytometer, and the conidia concentration in the conidia solution was adjusted to 4 × 10⁻⁶. 5 per mL.

[0154] (3) Treatment of Aspergillus oryzae conidia with the test extracts: Stock solutions of test extracts of different concentrations were added to an equal volume of conidial suspension. Each test extract was prepared to a working concentration of 200 mg / L and a conidial concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ cells / mL and a working concentration of 50 mg / L conidia. 5 A mixed inoculum of 1 conidia / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. Twelve hours after inoculation, the conidia germination rate and appressorium formation rate were observed and tallied under a microscope.

[0155] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germination and appressorium formation were investigated, and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1% dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of Aspergillus oryzae conidia.

[0156] 2. Experimental Results

[0157] The results are shown in Table 7 below. Table 7 shows some biological data of black tiger extract: "+" indicates that the inhibition effect at 200 mg / L is less than 40%, "++" indicates that the inhibition effect at 50 mg / L is 40-80%, and "+++" indicates that the inhibition effect at 50 mg / L is greater than 80%.

[0158] Table 7

[0159]

[0160] The above results indicate that only extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 showed a greater than 80% inhibitory effect on both conidial germination and appressorium formation of *Aspergillus oryzae* at low concentrations (50 mg / L), demonstrating good inhibitory activity. Therefore, extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 were selected for in vivo efficacy testing.

[0161] The inhibitory effects of extracts with 40-80% concentration on conidial germination and appressorium formation at a concentration of 200 mg / L are shown in Table 8 below.

[0162] Table 8

[0163]

[0164] The above results indicate that when the concentration of the extract, which had an inhibitory effect on conidial germination and appressorium formation of 40-80%, was increased to 200 mg / L, its inhibitory effect on conidial germination and appressorium formation was greater than 80%.

[0165] (II) The control effect of black tiger extract (HL1-B1, HL1-B2, HL1-B3, HL1-E1) on rice false smut in the field.

[0166] 1. Experimental Methods

[0167] (1) Rice planting: Rice (variety: Liaoxing No. 1) is planted in plots, with each plot being 60 square meters.

[0168] (2) Application plan: spray once each at the heading stage and the full heading stage of rice, with the working solution concentration of the extract being 1200 mg / L and the amount of pesticide applied per plot being 2.7 L.

[0169] (3) Statistics and Analysis: A survey of rice panicle blast disease was conducted 15 days after the second application of the extract. The specific standards are as follows: Grade 0: Healthy rice panicle with no blasted grains; Grade 1: 1 blasted grain on the panicle; Grade 3: 2 blasted grains on the panicle; Grade 5: 3-5 blasted grains on the panicle; Grade 7: 6-9 blasted grains on the panicle; Grade 9: More than 10 blasted grains on the panicle. The disease index was calculated based on the disease severity level.

[0170] 2. Experimental Results

[0171] The results are shown in Table 9 below. In Table 9, "1-9" represent the occurrence level of rice false smut disease.

[0172] Table 9

[0173]

[0174] The above results indicate that rice treated with extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 had fewer rice panicles with false smut, demonstrating good field control effects against rice false smut. Among them, HL1-E1 showed the best control effect.

[0175] III. Inhibitory effect of black tiger extract on late blight pathogens

[0176] (a) Inhibitory effect of black tiger extract on spore germination of potato late blight pathogen.

[0177] 1. Experimental Methods

[0178] (1) Preparation of zoosporangia of potato late blight pathogen: The zoosporangia of the potato late blight pathogen to be tested ( Phytophthora infesting The MZ strain was cultured on 10% V8 medium. After the zoosporangia were produced, the zoosporangia were washed off with sterile water, filtered through gauze, and a zoosporangia suspension was prepared. The suspension was then treated in the dark at 4°C for 3 h.

[0179] (2) Preparation of zoospore suspension: The prepared zoospore solution, which had been treated with sterile water at 4°C, was counted using a hemocytometer, and the zoospore concentration was adjusted to 4 × 10⁻⁶. 4 per mL.

[0180] (3) Treatment of zoospores with the test extract: Equal volumes of the stock solutions of the test extracts of different concentrations were added to the spore suspension. Each test extract was prepared to a working concentration of 500 mg / L and a conidial concentration of 2×10⁻⁶. 5 A mixed inoculum of 250 mg / L conidia at a concentration of 2 × 10⁶ / mL and a working concentration of 250 mg / L conidia at a concentration of 2 × 10⁶ / mL 5 A mixed inoculum of spores / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. The spore germination rate was observed and tallied under a microscope 12 h after inoculation.

[0181] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germinations was investigated and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1% dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of the conidia.

[0182] 2. Experimental Results

[0183] The results are shown in Table 10. In Table 10, "+" means that the inhibition effect of 500 mg / L is less than 40%, "++" means that the inhibition effect of 250 mg / L is 40-80%, and "+++" means that the inhibition effect of 250 mg / L is greater than 80%.

[0184] Table 10

[0185]

[0186] The results showed that only extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 had an inhibitory effect of more than 80% on the conidia of Phytophthora blight, while the control effects of other extracts were all below 80%.

[0187] The inhibitory effects of extracts with 40-80% concentration on conidial germination and appressorium formation at a concentration of 500 mg / L are shown in Table 11 below.

[0188] Table 11

[0189]

[0190] The above results indicate that when the concentration of the extract, which had an inhibitory effect on conidial germination and appressorium formation of 40-80%, was increased to 500 mg / L, its inhibitory effect on conidial germination and appressorium formation was greater than 80%.

[0191] (II) The preventive effect of black tiger extract (HL1-B1, HL1-B2, HL1-B3, HL1-E1) on late blight infection of potted potatoes.

[0192] 1 Experimental Methods

[0193] (1) Potato preparation: Take potato seedlings (susceptible variety: Desiree) and place them in an inoculation box for later use.

[0194] (2) Preparation of zoosporangia of Potato Late Blight: The MZ strain of Potato Late Blight to be tested was cultured on 10% V8 medium. After the zoosporangia were produced, the zoosporangia were washed off with sterile water, filtered with gauze, and a zoosporangia suspension was prepared. The suspension was then treated in the dark at 4°C for 3 h.

[0195] (3) Preparation of zoospore suspension: The prepared zoospore solution, which had been treated with sterile water at 4°C, was counted using a hemocytometer, and the zoospore concentration was adjusted to 4 × 10⁻⁶. 4 per mL.

[0196] (4) Spray inoculation: Before inoculation, the potato plants to be treated were placed in an artificial climate greenhouse at 20℃ for acclimatization 4 hours in advance. After acclimatization, the upper and lower surfaces of the leaves of the treated plants were evenly sprayed with black tiger extract (HL1-B1, HL1-B2, HL1-B3, HL1-E1, concentration of 500 mg / L). 24 hours later, the spore suspension (spore concentration of 2×10⁻⁶) was evenly sprayed. 5 The black tiger extract was treated with dark humidity for 24 h and then cultured under normal light (20℃, 18 h light / 6 h dark). The preventive effect of the black tiger extract on potato late blight was evaluated after 7 days.

[0197] (5) Statistics and analysis: The disease severity of potato late blight was investigated according to the grading standards. The specific standards are as follows: Grade 0: No lesions; Grade 1: Lesion area accounts for less than 5% of the total leaf area; Grade 3: Lesion area accounts for 5-10% of the total leaf area; Grade 5: Lesion area accounts for 10.1-20% of the total leaf area; Grade 7: Lesion area accounts for 20.1-50% of the total leaf area; Grade 9: Lesion area accounts for more than 50% of the total leaf area.

[0198] 2. Experimental Results

[0199] The results are shown in Table 12 below. "1-9" represent the incidence levels of potato late blight at a concentration of 500 mg / L.

[0200] Table 12

[0201]

[0202] The above results indicate that the lesion area on potato leaves treated with extracts HL1-B1, HL1-B2, HL1-B3, and HL1-E1 accounted for less than 5% of the total leaf area, demonstrating a good control effect against potato late blight.

[0203] (III) The control effect of black tiger extracts (HL1-B1, HL1-B2, HL1-B3, HL1-E1) on late blight of potato in the field.

[0204] 1. Experimental Methods

[0205] (1) Potato planting: Potatoes are planted in plots of 60 square meters each.

[0206] (2) Application plan: During the potato growth period, apply the agent (HL1-B1, HL1-B2, HL1-B3, HL1-E1) once a week for a total of 3 treatments. The concentration of the extract working solution is 1200 mg / L, and the amount of agent applied to each plot is 5 L.

[0207] (3) Statistics and analysis: The disease severity of potato late blight was investigated according to the grading standards. The specific standards are as follows: Grade 0: No lesions; Grade 1: Lesion area accounts for less than 5% of the total leaf area; Grade 3: Lesion area accounts for 5-10% of the total leaf area; Grade 5: Lesion area accounts for 10.1-20% of the total leaf area; Grade 7: Lesion area accounts for 20.1-50% of the total leaf area; Grade 9: Lesion area accounts for more than 50% of the total leaf area.

[0208] 2. Experimental Results

[0209] The results are shown in Table 13, where "1-9" represent the incidence levels of potato late blight.

[0210] Table 13

[0211]

[0212] The above results indicate that potato leaves treated with extracts HL1-B1, HL1-B2, and HL1-B3 showed grade 5 disease, while potato leaves treated with HL1-E1 showed grade 3 disease, demonstrating good control over potato late blight.

[0213] IV. Inhibitory effect of black tiger extract on other pathogens

[0214] To further investigate the inhibitory effects of the extract of this invention on other pathogens, *Anthracnose spp.*, *Anthracnose spp.*, *Anthracnose spp.*, *Anthracnose spp.*, *Anthracnose spp.*, *Anthracnose spp.*, *Anthracnose spp.*, *Phytophthora capsici*, *Phytophthora spp.*, *Phytophthora camphorata*, *Gray mold* of strawberry, and *Gray mold* of tomato were selected for testing. All pathogens were provided by the College of Plant Protection, China Agricultural University.

[0215] (I) Inhibitory effect of black tiger extract on spore germination of grape anthracnose fungus

[0216] 1. Experimental Methods

[0217] (1) Production of conidia of grape anthracnose fungus: The grape anthracnose fungus to be tested ( Colletotrichum gloeosporioides The strain was inoculated onto potato dextrose agar (PDA) plates and incubated in a constant temperature and light incubator at 28°C. After 7 days, the mycelial cake was inoculated onto potato dextrose agar (PDA) medium and incubated at 28°C and 180 rpm for 7 days, after which a large number of conidia were produced.

[0218] (2) Preparation of conidial suspension: The culture medium was filtered through three layers of lens paper, and the filtrate was the conidial solution. Conidia were counted using a hemocytometer, and the conidial concentration in the conidial solution was adjusted to 4 × 10⁻⁶. 5 per mL.

[0219] (3) Treatment of Grape anthracnose conidia with the test extracts: Equal volumes of stock solutions of different concentrations of the test extracts were added to the conidial suspensions, and each test extract was prepared to a working concentration of 600 mg / L and a conidial concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ spores per mL and a working concentration of 300 mg / L. 5 A mixed inoculum of 1 conidia / mL was prepared and inoculated sequentially onto hydrophobic glass slides, then incubated at 28°C in the dark. The conidia germination rate was observed and tallied under a microscope 12 h after inoculation.

[0220] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germination and appressorium formation were investigated, and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1‰ dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of conidia of Grape anthracnose fungus.

[0221] (II) Black Tiger Extract's Effects on Cucumber Anthracnose ( Colletotrichum orbiculare Arx. Inhibition of spore germination

[0222] The experimental procedure is the same as the above section on "(I) Inhibitory effect of black tiger extract on spore germination of grape anthracnose".

[0223] (III) Black Tiger Extract's Effects on Lychee Anthracnose ( Colletotrichum litchii Inhibition of spore germination

[0224] The experimental procedure is the same as the above section on "(I) Inhibitory effect of black tiger extract on spore germination of grape anthracnose".

[0225] (iv) Black Tiger Extract's Effects on Strawberry Anthracnose ( Colletotrichum fragariae Brooks. Inhibition of spore germination

[0226] The experimental procedure is the same as the above section on "(I) Inhibitory effect of black tiger extract on spore germination of grape anthracnose".

[0227] (v) Inhibitory effect of black tiger extract on spore germination of maize ear rot fungus

[0228] 1. Experimental Methods

[0229] (1) Production of conidia of the corn ear rot pathogen: The corn ear rot pathogen to be tested ( Cladosporium herbs The strain was inoculated onto potato dextrose agar (PDA) plates and incubated in a constant temperature and light incubator at 28°C. After 7 days, the mycelial cake was inoculated onto potato dextrose agar (PDA) medium and incubated at 28°C and 180 rpm for 7 days, after which a large number of conidia were produced.

[0230] (2) Preparation of conidial suspension: The culture medium was filtered through three layers of lens paper, and the filtrate was the conidial solution. Conidia were counted using a hemocytometer, and the conidial concentration in the conidial solution was adjusted to 4 × 10⁻⁶. 5 per mL.

[0231] (3) Treatment of maize ear rot fungus conidia with the test extracts: Equal volumes of different concentrations of the test extract stock solution were added to the conidia suspension, and each test extract was prepared to a working concentration of 600 mg / L and a conidia concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ spores per mL and a working concentration of 300 mg / L. 5 A mixed inoculum of 1 conidia / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. The conidia germination rate was observed and tallied under a microscope 24 h after inoculation.

[0232] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germination and appressorium formation were investigated, and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1‰ dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of conidia of *Hymenococcus faecium*.

[0233] (vi) Inhibitory effect of black tiger extract on spore germination of strawberry wilt fungus

[0234] 1. Experimental Methods

[0235] (1) Production of conidia of Fusarium wilt pathogen: The Fusarium wilt pathogen to be tested (… Fusarium oxysporum The strain was inoculated onto potato dextrose agar (PDA) plates and incubated in a constant temperature and light incubator at 28°C. After 7 days, the mycelial cake was inoculated onto potato dextrose agar (PDA) medium and incubated at 28°C and 180 rpm for 7 days, after which a large number of conidia were produced.

[0236] (2) Preparation of conidial suspension: The culture medium was filtered through three layers of lens paper, and the filtrate was the conidial solution. Conidia were counted using a hemocytometer, and the conidial concentration in the conidial solution was adjusted to 4 × 10⁻⁶. 5 per mL.

[0237] (3) Treatment of strawberry wilt fungus conidia with the test extracts: Equal volumes of the stock solutions of the test extracts at different concentrations were added to the conidia suspensions, and each test extract was prepared to a working concentration of 600 mg / L and a conidia concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ spores per mL and a working concentration of 300 mg / L. 5 A mixed inoculum of 1 conidia / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. The conidia germination rate was observed and tallied under a microscope 24 h after inoculation.

[0238] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germination and appressorium formation were investigated, and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1‰ dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of strawberry wilt fungus conidia.

[0239] (vii) Black Tiger Extract's Effects on Cucumber Wilt Bacterium ( Fusarium oxysporum f. sp. cucumerinum Inhibition of spore germination

[0240] The experimental procedure is the same as the above section on "(VI) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0241] (viii) Black tiger extract against pepper anthracnose bacteria ( Colletotrichum capsici Inhibition of Cs25 spore germination

[0242] The experimental procedure is the same as the above section on "(VI) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0243] (ix) Black Tiger Extract's Effects on Anthracnose Bacterium tumefaciens ( Colletotrichum capsici Inhibitory effect of Cgg50 spore germination

[0244] The experimental procedure is the same as the above section on "(VI) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0245] (x) Inhibitory effect of black tiger extract on spore germination of Phytophthora capsici.

[0246] 1. Experimental Methods

[0247] (1) Preparation of zoosporangia of Phytophthora capsici: The zoosporangia of Phytophthora capsici to be tested ( Phytophthora capsicum The BYA5 strain was cultured on 10% V8 medium. After the zoosporangia were produced, the zoosporangia were washed off with sterile water, filtered through gauze, and a zoosporangia suspension was prepared. The suspension was then treated in the dark at 4°C for 3 h.

[0248] (2) Preparation of zoospore suspension: The prepared zoospore solution, which had been treated with sterile water at 4°C, was counted using a hemocytometer, and the zoospore concentration was adjusted to 4 × 10⁻⁶. 4 per mL.

[0249] (3) Treatment of zoospores with the test extract: Equal volumes of the stock solutions of the test extracts of different concentrations were added to the spore suspension. Each test extract was prepared to a working concentration of 600 mg / L and a conidial concentration of 2×10⁻⁶. 5 A mixed inoculum of 2 × 10⁶ spores per mL and a working concentration of 300 mg / L. 5 A mixed inoculum of spores / mL was prepared and sequentially spotted onto hydrophobic glass slides, then incubated at 28°C in the dark. The spore germination rate was observed and tallied under a microscope 12 h after inoculation.

[0250] (4) Statistics and analysis: Three fields of view were counted for each treatment, and 100 conidia were counted for each field of view. The number of germinations was investigated and the conidia germination rate was calculated. The conidia germination rate of solvent treatment (1% dimethyl sulfoxide) was used as a control to calculate the inhibitory effect of the test sample on the germination of the conidia.

[0251] (xi) Black Tiger Extract's Effects on Soybean Phytophthora ( Phytophthora sojaeThe experimental procedure for the inhibitory effect of fungal spore germination is the same as the above-mentioned "(X) Inhibitory effect of black tiger extract on spore germination of strawberry wilt fungus".

[0252] (xii) Black Tiger Extract's Effect on Phytophthora camphorata ( Phytophthora cinnamon Inhibition of fungal spore germination

[0253] The experimental procedure is the same as the above section on "(X) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0254] (xiii) Black Tiger Extract on Strawberry Gray Mold ( Botrytis cinerea Pers. Inhibition of fungal spore germination

[0255] The experimental procedure is the same as the above section on "(X) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0256] (xiv) Black Tiger Extract on Tomato Gray Mold ( Botrytis cinerea Pers. Fr. Inhibition of fungal spore germination

[0257] The experimental procedure is the same as the above section on "(X) Inhibitory effect of black tiger extract on the germination of strawberry wilt fungus spores".

[0258] The above-mentioned inhibitory effects on the germination of different pathogen spores are shown in Table 14 below. "+" indicates that the inhibition effect at 600 mg / L is less than 40%, "++" indicates that the inhibition effect at 300 mg / L is 40-80%, and "+++" indicates that the inhibition effect at 300 mg / L is greater than 80%.

[0259] Table 14

[0260]

[0261] The above results indicate that extract HL1-B1, at a low concentration (300 mg / L), exhibits an inhibitory effect of over 80% against cucumber anthracnose, demonstrating good control efficacy. Its inhibitory effects on grape anthracnose, corn stem and ear rot pathogens, *Phytophthora capsici* BYA5, and *Phytophthora camphorata* are less pronounced, ranging from 40% to 80%. At high concentrations (600 mg / L), the inhibitory effects on strawberry anthracnose, strawberry wilt pathogens, cucumber wilt pathogens, as well as *Phytophthora capsici* Cs25 and Cgg50, *Phytophthora soybeanata*, and *Botrytis cinerea* are less than 40%, showing some inhibitory activity, but the effect is relatively poor. These results demonstrate that the black tiger extract obtained in this invention has a good inhibitory effect on some pathogens.

[0262] Anthracnose fungi with spore germination inhibition effects below 80% were selected, including *A. sarcodactylis* (grape anthracnose), *A. sarcodactylis* (strawberry anthracnose), *A. sarcodactylis* (lychee anthracnose), *A. sarcodactylis* (corn stalk and ear rot), *A. sarcodactylis* (strawberry wilt), *A. sarcodactylis* (cucumber wilt), *A. sarcodactylis* (pepper anthracnose Cs25), *A. sarcodactylis* (pepper anthracnose Cgg50), *A. sarcodactylis* (pepper anthracnose BYA5), *A. sarcodactylis* (soybean anthracnose), *A. sarcodactylis* (camphor tree anthracnose), *A. sarcodactylis* (tomato gray mold), and *A. sarcodactylis* (strawberry gray mold). Further inhibition tests were conducted at higher extract concentrations (900 mg / L), and the results are shown in Table 15 below. "+" indicates an inhibition effect below 40% at 900 mg / L; "++" indicates an inhibition effect of 40-80% at 900 mg / L; and "+++" indicates an inhibition effect greater than 80% at 900 mg / L.

[0263] Table 15

[0264]

[0265] The above results indicate that increasing the concentration of the extract to 900 mg / L further enhances its inhibitory effect on pathogens, achieving an inhibition rate of over 80% against *Phytophthora indicum*, *Phytophthora indicum*, *Phytophthora indicum* Cs25, *Phytophthora indicum* BYA5, and *Phytophthora indicum*.

[0266] V. Inhibitory effects of the monomeric compound styrosinol isolated from black tiger extract on different pathogens.

[0267] (I) Isolation of the monomeric compound Antenylene

[0268] A suitable amount of distilled water was added to the black tiger extract HL1-B1 to disperse and mix it. The extract was then extracted three times sequentially with equal volumes of petroleum ether, ethyl acetate, and n-butanol. The extracts were combined, and the solvents were removed by rotary evaporation under reduced pressure, and the samples were recovered, yielding petroleum ether extract, ethyl acetate extract, n-butanol extract, and aqueous extract of black tiger extract. Bioassays of the extracts for controlling rice blast were conducted, identifying the petroleum ether extract as the active component of the black tiger extract. The active components were then separated and purified using chromatographic techniques such as normal silica gel column chromatography.

[0269] Stage I column chromatography: The petroleum ether extract of the black tiger was crudely separated by normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase (PE / EA, from 200 / 1 to 1 / 10, v / v), yielding 19 fractions (Fr. 1-Fr. 19). Bioassays identified Fr. 8 as the major active fraction.

[0270] Stage II column chromatography: Fr. 8 was separated into six subfractions (Fr. 8.1-Fr. 8.6) by normal-phase silica gel column chromatography with petroleum ether / ethyl acetate as the mobile phase (PE / EA, from 20 / 1 to 1 / 5, v / v). Bioassays confirmed Fr. 8.5 as the major active fraction.

[0271] Stage III column chromatography: Fr. 8.5 was purified by Sephadex LH-20 column chromatography with dichloromethane / methanol as the mobile phase (CH2Cl2 / CH3OH=1 / 1, v / v) to finally obtain monomeric compound A. After identification, compound A was identified as styrosinase, and its structural formula and NMR data are shown below:

[0272]

[0273]

[0274] (II) Inhibitory effect of monomeric compound styrosin on spore germination of different pathogens

[0275] To further investigate the inhibitory effect of the monomeric compound acetoxin isolated from the extract HL1-B1 of this invention on pathogens, all pathogens used in the above-mentioned experiments were selected for testing (blast fungus, aspergillus oryzae, late blight fungus, grape anthracnose fungus, cucumber anthracnose fungus, strawberry anthracnose fungus, litchi anthracnose fungus, corn ear rot fungus, strawberry wilt fungus, cucumber wilt fungus, pepper anthracnose fungus, pepper phytophthora, soybean phytophthora, camphor phytophthora, strawberry gray mold, tomato gray mold). Therefore, all pathogens were provided by the College of Plant Protection, China Agricultural University.

[0276] 1. Experimental Methods

[0277] The method for inhibiting the germination of spores of various pathogens is the same as the method for inhibiting bacteria using the extracts described above, and the specific process will not be repeated here. The concentrations of the monomeric compound anisodamine were prepared at 200 and 100 mg / L.

[0278] 2. Experimental Results

[0279] The results are shown in Table 16. "+" indicates that the inhibition effect at 200 mg / L is less than 40%, "++" indicates that the inhibition effect at 100 mg / L is 40-80%, and "+++" indicates that the inhibition effect at 100 mg / L is greater than 80%.

[0280] Table 16

[0281]

[0282] The above results indicate that the monomeric compound Anwuzhixin, at low concentrations (100 mg / L), exhibits an inhibitory effect of over 80% against *Anthracnose of cucumber*, *Anthracnose of grape*, *Anthracnose of corn*, *Blastomyces oryzae*, *Aspergillus oryzae*, and *Late Blight of potato*, demonstrating good control efficacy. However, its inhibitory effects on *Anthracnose of litchi*, *Phytophthora capsici* BYA5, *Gyromitra esculenta*, and *Gyromitra esculenta* are less pronounced, ranging from 40% to 80%. At high concentrations (200 mg / L), the inhibitory effect on *Anthracnose of strawberry*, *Fusarium wilt of strawberry*, *Fusarium wilt of cucumber*, *Anthracnose of pepper* Cs25, *Anthracnose of pepper* Cgg50, *Phytophthora soyensis*, and *Phytophthora camphorata* is less than 40%, showing some inhibitory effect, but the effect is poor. These results demonstrate that the monomeric compound Anwuzhixin obtained in this invention exhibits good inhibitory effects against some pathogens at low concentrations.

[0283] Anthracnose fungi of strawberry, lychee, strawberry wilt, cucumber wilt, pepper anthracnose Cs25, pepper anthracnose Cgg50, pepper Phytophthora BYA5, soybean Phytophthora, camphor tree Phytophthora, tomato gray mold, and strawberry gray mold, with spore germination inhibition effects below 80%, were further tested at higher concentrations (400 mg / L) of anthraquinone. The results are shown in Table 17 below. "+" indicates an inhibition effect below 40% at 400 mg / L; "++" indicates an inhibition effect of 40-80% at 400 mg / L; "+++" indicates an inhibition effect greater than 80% at 400 mg / L.

[0284] Table 17

[0285]

[0286] The above results indicate that, with increasing concentration, except for the cucumber wilt pathogen, pepper anthracnose pathogen Cs25, and pepper anthracnose pathogen Cgg50, whose inhibition effects remained below 40%, the strawberry anthracnose pathogen, tomato gray mold pathogen, and strawberry gray mold pathogen all showed inhibition effects above 80%. The litchi anthracnose pathogen, strawberry wilt pathogen, soybean phytophthora, and camphor phytophthora all increased to between 40% and 80%, indicating that the inhibition effect of these pathogens became better and better with increasing working solution concentration.

[0287] VI. Safety evaluation of the monomeric compound anthocyanin isolated from black tiger extract on rice.

[0288] (1) Seed germination. Healthy japonica rice (Nipponbare) seeds were selected and germinated with pesticides at concentrations of 300, 600, and 1200 μg·mL⁻¹. -1Rice seeds were soaked in acetone and cultured in the dark at 30℃ for 48 h. Germination was then observed and the germination rate was recorded. DMSO (1%) was used as a solvent control. Each treatment consisted of 100 seeds and was repeated three times.

[0289] Physiological indicators. Japonica rice (Nipponbare) was cultured in seedling trays for 2 weeks, and then the plant surface was uniformly sprayed with pesticides at concentrations of 300, 600, and 1200 μg·mL⁻¹. -1 After culturing rice plants in alternating light and dark conditions at 30℃ for 7 days, the plant height, root length, and fresh weight of each treatment were measured. DMSO (1%) was used as a solvent control. Forty rice plants were investigated for each treatment, and each treatment was replicated three times.

[0290] like Figure 2 As shown in Figure A, the result is 1200 μg·mL -1 600 μg·mL -1 and 300 μg·mL -1 The germination rate of rice seeds treated with acetoquinone was over 98%, which was not different from that of the 1% DMSO treatment.

[0291] like Figure 2 As shown in Figure B, the result is 1200 μg·mL -1 600 μg·mL -1 and 300 μg·mL -1 Rice plants treated with 1% DMSO showed no difference in morphological parameters (root length) compared to those treated with 1% DMSO.

[0292] The above experimental results show that acetaminophen at 1200 μg·mL -1 At concentrations of [specific concentration], it has no effect on the germination process of rice seeds or the physiological development of the plant, and is non-toxic to rice.

[0293] Although the present invention has been described using the above preferred embodiments, it is not intended to limit the scope of protection of the present invention. Any changes and modifications made by those skilled in the art to the above embodiments without departing from the spirit and scope of the present invention shall still fall within the scope of protection of the present invention.

Claims

1. The application of black tiger extract in the preparation of fungicides for controlling plant diseases caused by pathogenic fungi, wherein the plant diseases are caused by Aspergillus oryzae. Ustilaginoidea virens Rice blast disease caused by [the disease / infection]; The extraction steps for the black tiger extract are as follows: (1) After grinding the black tiger root into coarse powder, it was soaked in a solvent for the first extraction, and the first filtrate was obtained after filtration; (2) Add solvent to the filter residue for a second extraction. After filtration, the second filtrate is obtained. Combine the two filtrates and concentrate the filtrate into an extract to obtain black tiger extract. in, The solvent is methanol, ethanol, cyclohexanol, petroleum ether, n-hexane, n-pentane, dodecane, dichloromethane, 1,2-dichloroethane, diethyl malonate, 1,4-dioxane, acetone, or acetonitrile. The first and second extractions include one or two of mechanical stirring extraction, ultrasound-assisted extraction, and Soxhlet extraction.

2. The application according to claim 1, characterized in that, The mechanical stirring extraction time is 1 to 5 days; the ultrasonic-assisted extraction time is 0.5 to 2 hours; and the Soxhlet extraction time is 0.5 to 2 hours.

3. The application according to claim 1, characterized in that, The formulation of the bactericide is emulsifiable concentrate, wettable powder, suspension concentrate, powder, soluble powder, aqueous solution, water-dispersible granules, fumigant, granules, or seed coating agent.

4. The use of lignan compounds in the black tiger extract of claim 1 in the preparation of a fungicide for controlling plant diseases caused by pathogenic fungi, wherein the plant disease is caused by Aspergillus oryzae. Ustilaginoidea virens The lignan compound is used in rice blast disease caused by the disease; the dosage of the lignan compound is 100~400 mg / L, and the chemical structural formula of the lignan compound is shown in Formula I below: 。

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