DHE fluorescence intensity-based high-flux targeted phytophthora elicitin antibacterial compound screening method and application

Abstract

The invention discloses a DHE fluorescence intensity-based high-flux targeted phytophthora elicitin antibacterial compound screening method and application, and belongs to the field of biotechnology and agricultural disease control. According to the method, a to-be-detected compound, an elicitin protein and fluorescent sterol DHE are jointly incubated in a PBS buffer system, the fluorescence value after the DHE and the elicitin are combined is used as a contrast, and the fluorescence intensity is reduced after the to-be-detected compound is competitively combined with the elicitin, and the fluorescence intensity of the to-be-detected compound is detected by detecting the change of the fluorescence intensity of the reaction system. And rapid and high-throughput screening of the active compound capable of specifically targeting the elicitin-sterol binding site is realized. The screened compound has a wide antibacterial spectrum and good inhibitory activity on various important plant pathogenic fungi, and shows huge potential as a broad-spectrum botanical fungicide lead compound.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and agricultural disease control, specifically relating to a method and application of high-throughput targeted screening of antibacterial compounds for Phytophthora elicitin based on DHE fluorescence intensity. Background Technology

[0002] Phytophthora is a highly destructive plant pathogen with nearly 200 species. It can infect many important food and economic crops such as potatoes, soybeans, tomatoes, peppers, and cucumbers, causing devastating diseases.

[0003] Although Phytophthora resembling fungi in morphology, it belongs to the phylum Oomycetes in the kingdom Trichophyton, and its cell walls contain cellulose instead of chitin. Its zoospores also possess biflagellates, significantly distinguishing it from fungi. This taxonomic position renders traditional fungicides largely ineffective against Phytophthora, posing a significant challenge to disease control. Even more serious is the fact that Phytophthora exhibits complex genetic diversity, strong genome plasticity, and rapid mutation under natural conditions, making it highly susceptible to the emergence of drug-resistant strains and strains that overcome host resistance. The pathogen overwinters as oospores through sexual reproduction, increasing the primary source of infection. Simultaneously, it spreads via zoospores using rainwater or wind, reaching distances exceeding 10 kilometers, leading to rapid disease spread. Phytophthora's rapid adaptability allows it to quickly overcome chemical fungicides and host genetic resistance, limiting the effectiveness of existing control measures.

[0004] In plant-pathogen interactions, sterols, as essential lipids in eukaryotes, are not only structural components of cell membranes but also act as signaling molecules regulating the growth, development, and reproduction of organisms. Oomycetes such as *Phytophthora* are typical sterol auxotrophs, unable to synthesize sterols themselves and must obtain them from their host plants to maintain their vegetative growth, asexual reproduction, and sexual reproduction. *Phytophthora* acquires host sterols by secreting a class of effector proteins called elicitins. Elicitins are small-molecule, cysteine-rich secretory proteins whose hydrophobic cavities can bind sterol molecules with high affinity, thus helping *Phytophthora* "plunder" sterols from the host cell membrane. As a key effector protein for sterol acquisition in *Phytophthora*, elicitins play an irreplaceable role in the pathogenic process and are an ideal target for developing novel anti-oomycete drugs.

[0005] However, while elicitin is known to bind to sterols, there is a lack of a direct, rapid, and high-throughput method for screening compounds targeting the elicitin-sterol binding site. Traditional screening methods typically rely on complex bioactivity assays, such as pathogen growth inhibition assays or spore germination inhibition assays. These methods are cumbersome, time-consuming, have low throughput, and struggle to distinguish whether compounds act directly on the elicitin target or exert their antibacterial effect through other non-specific pathways.

[0006] Therefore, there is an urgent need in this field to develop a compound screening method based on elicitin targets that is simple to operate, highly sensitive, and suitable for high-throughput screening, in order to provide an effective technical platform for the development of novel anti-oomycete drugs. Summary of the Invention

[0007] The purpose of this invention is to provide a method and application for high-throughput screening of antibacterial compounds targeting Phytophthora elicitin based on DHE fluorescence intensity.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks protection for the use of an active compound that specifically targets the elicitin-sterol binding site in competitively inhibiting the binding of elicitin to sterols, wherein the active compound is at least one selected from glycyrrhizin, carvacrol, aloin, scutellarin A, glycyrrhizin isoflavone A, emodin anthrone, baicalin methyl ester, aloe-emodin, 6-feruloylspinosin, punice, aloe-emodin-8-O-β-D-glucoside, xanthohumol, scutellarin B, (E / Z)-demethoxycurcumin, theaflavin-3-gallate, sophoranone G, and 3'-demethylhesperidin.

[0010] Preferably, the active compound is glycyrrhizin, glycyrrhizin A, or glycyrrhizin A.

[0011] Secondly, the present invention seeks protection for the use of the active compounds described above, glycyrrhizin, glycyrrhizin A, or glycyrrhizin isoflavone A, in any of the following:

[0012] (1) Application in inhibiting the growth of oomycetes or fungi, which is achieved by competitively inhibiting elicitin-sterol binding;

[0013] (2) Application in the preparation of pesticide products that inhibit the growth of oomycetes or fungi;

[0014] The oomycetes are selected from at least one of Phytophthora capsici, Phytophthora hygroscopica, Phytophthora lychee, Phytophthora tobacco, Pythium cerevisiae, Phytophthora soybean, and Phytophthora pathogenica; the fungi are selected from at least one of Sclerotinia sclerotiorum, Fusarium graminearum, and Botrytis cinerea.

[0015] Thirdly, the present invention claims protection for the use of the active compound glycyrrhizin as described above in any of the following:

[0016] (1) Application in the prevention and control of soybean root rot;

[0017] (2) Application in the preparation of pesticide products for the prevention and control of soybean root rot;

[0018] (3) Application in promoting soybean growth;

[0019] (4) Application in the preparation of pesticide products for promoting soybean growth.

[0020] This invention develops a high-throughput method for screening antibacterial compounds by targeting Phytophthora elicitin based on DHE fluorescence intensity. The method includes the following steps:

[0021] (1) Mix the test compound, elicitin protein and DHE (dehydroergosterol) in a buffer solution to form a reaction system;

[0022] (2) Incubate the reaction system;

[0023] (3) Detect the fluorescence intensity of the reaction system;

[0024] (4) Based on the changes in fluorescence intensity, compounds that can inhibit the binding of DHE to elicitin protein are screened.

[0025] Further, in step (1), the concentration of DHE in the reaction system is 0.1-50 μM, the concentration of elicitin protein is 0.05-25 μM, and the concentration of the test compound is 0.1-100 μM. Preferably, the concentration of DHE in the reaction system is 1-30 μM, the concentration of elicitin protein is 0.1-15 μM, and the concentration of the test compound is 1-50 μM. More preferably, the concentration of DHE in the reaction system is 1-10 μM, the concentration of elicitin protein is 0.5-5 μM, and the concentration of the test compound is 1-20 μM. Most preferably, the concentration of DHE in the reaction system is 5 μM, the concentration of elicitin protein is 2.5 μM, and the concentration of the test compound is 5 μM.

[0026] Furthermore, in step (1), the buffer solution is a PBS buffer with a pH of 7.0-8.0 (preferably 7.2-7.6).

[0027] Furthermore, the elicitin protein is the SOJ1B protein of *Phytophthora sojae* (the amino acid sequence of the SOJ1B protein is shown in SEQ ID NO: 2; the nucleotide sequence of the gene encoding the SOJ1B protein is shown in SEQ ID NO: 1). However, this method is not limited to the specific elicitin protein disclosed (such as *Phytophthora sojae* SOJ1B), and homologous proteins from other species of the *Phytophthora* genus, as well as effector proteins with similar sterol-binding functions, can also be used.

[0028] Furthermore, in step (2), the incubation temperature is 20-25℃ and the incubation time is 0.5-2 h (preferably 30 minutes).

[0029] Furthermore, in step (3), the fluorescence intensity is detected at an excitation wavelength of 315-335 nm (preferably 324 nm) and an emission wavelength of 345-360 nm (preferably 350 nm). The fluorescence intensity is detected using a high-throughput fluorescence detection device (e.g., an ELISA reader).

[0030] Furthermore, in step (4), if the fluorescence intensity of the reaction system is lower than that of the control group, it is determined that the test compound can inhibit the binding of DHE to elicitin protein.

[0031] The above method was used to screen compounds with anti-oomycete and / or fungal activity. Seventeen compounds with anti-oomycete and / or fungal activity were screened using this method, specifically at least one of the following: glycyrrhizin, carvacrol, aloin, scutellarin A, glycyrrhizin isoflavone A, emodin anthrone, baicalin methyl ester, aloe-emodin, 6-feruloylspinosin, punice, aloe-emodin-8-O-β-D-glucoside, xanthohumol, scutellarin B, (E / Z)-demethoxycurcumin, theaflavin-3-gallate, sophoranone G, and 3'-demethylhesperidin. Preferably, the compound is glycyrrhizin, scutellarin A, or glycyrrhizin isoflavone A.

[0032] The aforementioned compounds exert their anti-oomycete and / or fungal effects by inhibiting the binding of elicitin protein to sterols. The oomycete is *Phytophthora*, selected from at least one of *Phytophthora capsici*, *Phytophthora malathi*, *Phytophthora lychee*, *Phytophthora tobacco*, *Pythium oxysporum*, *Phytophthora sojae*, and *Phytophthora pathogenica*. The fungus is selected from at least one of *Sclerotinia sclerotiorum*, *Fusarium graminearum*, and *Botrytis cinerea*.

[0033] Dehydroergosterol (DHE) is a naturally fluorescent sterol analog with a structure highly similar to sterols, making it a functional fluorescent probe for sterol-binding proteins. When DHE binds to elicitin proteins, its fluorescence emission intensity undergoes a detectable change. This property allows for the establishment of a fluorescence-based elicitin ligand binding detection system, providing a direct and quantitative detection method for screening compounds targeting elicitin. This invention involves co-incubating the test compound, elicitin protein, and the fluorescent sterol DHE in a PBS buffer system. Using the fluorescence intensity after DHE binds to elicitin as a control, and observing the decrease in fluorescence intensity after the test compound competitively binds to elicitin, the change in fluorescence intensity in the reaction system enables rapid and high-throughput screening of active compounds that specifically target the elicitin-sterol binding site. Using this method, this invention successfully screened 17 natural products with significant elicitin-sterol binding inhibitory activity from a compound library. Further biological validation revealed that, at specific concentrations, three compounds inhibited the growth of Phytophthora in soybean, demonstrating promising application potential in controlling plant diseases such as soybean root rot caused by Phytophthora in soybean. This invention not only provides a highly efficient drug screening platform targeting elicitin but also offers important candidate compounds for developing novel, environmentally friendly fungicides targeting new Phytophthora in soybean.

[0034] The beneficial effects of this invention are:

[0035] The inventors of this invention have established a high-throughput method for screening antibacterial compounds targeting elicitin in *Phytophthora indicum* based on the fluorescence intensity of DHE. This method utilizes the principle that the fluorescence intensity increases after DHE binds to elicitin, and that the competitive binding of the test compound to elicitin reverses this fluorescence intensity increase. By detecting changes in the fluorescence intensity of the reaction system, rapid and high-throughput screening of active compounds that specifically target the elicitin-sterol binding site is achieved. Using this method, this invention successfully screened 17 natural products from a compound library, including glycyrrhizin, glycyrrhizin A, and glycyrrhizin isoflavone A, which significantly inhibit the binding of elicitin to sterols. These compounds can effectively block the sterol uptake pathway of *Phytophthora indicum* by competitively inhibiting elicitin-sterol binding, thereby inhibiting the growth of *Phytophthora indicum*, and show good application potential in the prevention and control of plant diseases such as soybean root rot caused by *Phytophthora indicum*. Attached Figure Description

[0036] Figure 1 The purified recombinant SOJ1B-His protein was analyzed by SDS-PAGE.

[0037] Figure 2To establish and validate the DHE fluorescence detection system.

[0038] Figure 3 This is the result of high-throughput screening based on the DHE fluorescence detection system.

[0039] Figure 4 The antibacterial effect of the screened compounds was detected using a multi-well plate assay; where A is the multi-well plate antibacterial assay procedure; and B is the multi-well plate compound antibacterial phenotype.

[0040] Figure 5 The broad-spectrum antibacterial phenotypes of glycyrrhizin, glycyrrhizin A, and glycyrrhizin isoflavone against different pathogens are shown. Among them, A represents the plate phenotype of colony growth after treatment of different pathogens with glycyrrhizin, glycyrrhizin A, and glycyrrhizin isoflavone A; B represents the inhibition rate of different pathogens after treatment with glycyrrhizin, glycyrrhizin A, and glycyrrhizin isoflavone A.

[0041] Figure 6 This study aims to investigate the inhibitory effect of glycyrrhizin on the pathogenicity of Phytophthora in soybean and its pot-plant protective effect on soybean seedlings. Specifically, A represents the pot-plant effect of glycyrrhizin on soybean growth; B represents the root growth of soybean under different concentrations of glycyrrhizin; C represents the survival rate of soybeans under different treatments; D represents the fresh weight of aboveground parts under different treatments; E represents the dry weight of underground parts under different treatments; and F represents the fresh weight of underground parts under different treatments. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the specific embodiments shown in the accompanying drawings. However, these embodiments do not limit the present invention, and any structural, methodological, or functional modifications made by those skilled in the art based on these embodiments are included within the scope of protection of the present invention.

[0043] Example 1: Cloning of SOJ1B protein from Phytophthora soybeanis

[0044] Step 1) Total RNA extraction

[0045] Using Phytophthora soybean mycelia cultured in liquid V8 as material, total RNA was extracted using the Wingfly RNA Extraction Kit. The specific operation was performed according to the instructions, and the content and quality of the extracted total RNA were detected by spectrophotometer.

[0046] Step 2) Reverse transcription to generate the first strand

[0047] Take 1 μg of the total RNA obtained in step 1) above as a template and use the Novozymes HiScript II 1st Strand cDNA Synthesis Kit to synthesize cDNA (refer to the reagent instructions for specific operation, and adjust the volume to 20 μl for the reaction). The obtained cDNA will be used as a template for subsequent gene cloning.

[0048] Step 3) PCR amplification of the SOJ1B sequence

[0049] Based on the sequence of the SOJ1B encoding gene shown in SEQ ID NO: 1, the inventors designed the following PCR amplification primer sequence:

[0050] Upstream primer (as shown in SEQ ID NO: 3):

[0051] 5'- gggtatctctcgagaaaagagaattcATGACGGCCTGCACGGCG-3'

[0052] Downstream primer (as shown in SEQ ID NO: 4):

[0053] 5'-ctcaatgatgatgatgatggtcgacGAGCGACGCGCACTTGGT -3'

[0054] Table 1 PCR amplification reaction system

[0055] Components volume <![CDATA[ddH2O]]> 18 μl 2 × Phanta Max Buffer 25 μl dNTP Mix (10 mM each) 1 μl Upstream primer (10 μM) 2 μl Downstream primer (10 μM) 2 μl Phanta Max Super-Fidelity DNA Polymerase 1 μl Step 2) Obtaining the template cDNA 1 μl

[0056] Add the corresponding components to the 50 μl reaction system according to Table 1; the PCR amplification system is 95 °C pre-denaturation for 3 min, 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 90 s, for 35 cycles, and finally 72 °C extension for 5 min.

[0057] The bands were separated by agarose gel electrophoresis, stained with ethidium bromide (EB) for 15 min, photographed, and the results were recorded. The PCR product encoding the SOJ1B gene sequence was then excised from the gel and recovered (the electrophoretic bands were recovered using the Novozymes FastPure Gel DNA Extraction Mini Kit).

[0058] The PCR products recovered from the gel were ligated into the restriction endonuclease-linearized pPIC9K vector (a commercially available vector from Invitrogen) using the Vazyme ClonExpress® II One Step Cloning Kit, following the manufacturer's instructions, to obtain the pPIC9K-SOJ1B-His recombinant plasmid. The ligation product was transformed into competent E. coli JM109 cells, plated on LB agar plates (containing 50 μg / mL bleomycin), and incubated at 37 °C for 16 h. Two positive clones were screened by colony PCR. The plasmid was then extracted using the Vazyme FastPure® Plasmid Mini Kit plasmid extraction kit according to the manufacturer's instructions and sent to Nanjing Qingke Company for sequencing. The sequence obtained was identical to SEQ ID NO: 1 (the nucleotide sequence encoding the SOJ1B gene), confirming that the pPIC9K::SOJ1B-His plasmid was correctly ligated.

[0059] Example 2: Eukaryotic expression and purification of SOJ1B protein

[0060] Step 1) Eukaryotic expression of pPIC9K::SOJ1B-His protein

[0061] The correctly sequenced pPIC9K-SOJ1B-His recombinant plasmid was linearized using the restriction endonuclease Pme1 (selected according to the vector multiple cloning site), and the linearized product was purified and recovered. The linearized product was transformed into Pichia pastoris strain KM71 by electroporation and evenly spread on His-deficient plates. After incubation in the dark at 30°C for approximately 48 h, positive clones were screened by colony PCR. The positive clones were initially cultured in glycerol medium for approximately 24 h, and the cells were collected by centrifugation. Expression was then induced in methanol medium for 72 h.

[0062] Step 2) Protein purification

[0063] Using the AKTA automated purification system TM The Avant 25 (GE Healthcare) protein purification system requires passing the protein through a desalting column first. After desalting, the protein solution is passed through a nickel column, eluted with low-concentration imidazole to remove impurities, then eluted with high-concentration imidazole to remove the target protein, and finally passed through a desalting column again. The size of the target product is then determined by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. (See...) Figure 1 . Figure 1 The left side shows the standard marker band, and the right side shows the size of the PpIC9K::SOJ1B-His protein.

[0064] like Figure 1 It can be seen that a purified eukaryotic expression protein (SOJ1B-His) with a size of approximately 12 kD was obtained, and its concentration was then determined for subsequent experiments.

[0065] Example 3 Construction of DHE fluorescence detection system

[0066] In a standard black 96-well microplate, set up the following four treatment groups, with 3 replicates in each group:

[0067] 1) Blank control: Add only PBS buffer (pH 7.4);

[0068] 2) DHE control: Add PBS buffer and dehydroergosterol (DHE) to a final concentration of 5 μM;

[0069] 3) Protein binding control: PBS buffer, DHE (5 μM) and SOJ1B protein (2.5 μM) prepared in Example 2 were added.

[0070] 4) Sample group to be tested: Add PBS buffer, DHE (5 μM), SOJ1B protein prepared in Example 2 (2.5 μM) and β-sitosterol (5 μM).

[0071] Centrifuge the microplate at 1000 rpm for 1 minute to thoroughly mix all components, and incubate at room temperature (25℃) in the dark for 30 minutes. Detect the fluorescence intensity of each treatment group using a Tecan multi-sensor microplate reader. The excitation wavelength was set to 324 nm, and the emission wavelength to 350 nm. Fluorescence intensity is expressed in arbitrary units (au). Process the obtained data by subtracting the blank control value from all wavelength values, and plot the results using Graphpad Prism 9.5. See [Figure showing results]. Figure 2 . Figure 2 The horizontal axis represents the excitation wavelength (nm), and the vertical axis represents the fluorescence intensity (au). The results of three different treatments (DHE treatment alone, DHE and SOJ1B co-treatment, and DHE, SOJ1B, and β-sitosterol co-treatment) are indicated by different color curves.

[0072] like Figure 2 It was found that, compared with the DHE-treated group alone, the fluorescence intensity of the system was significantly increased after co-treatment with DHE and SOJ1B protein. After adding β-sitosterol, the fluorescence intensity of the system was significantly decreased compared with the DHE+SOJ1B treated group, but still higher than that of the DHE-treated group alone. These results indicate that SOJ1B protein can bind to DHE and significantly enhance its fluorescence intensity, while β-sitosterol can effectively inhibit the binding of SOJ1B protein to DHE, thereby reducing the fluorescence signal of the system, thus confirming the validity of the system.

[0073] Example 4: High-throughput screening of compound libraries

[0074] The experimental system and operating procedures were as described in Example 3. This system was used to perform high-throughput screening of the candidate compound library PhenolsLibrary (MedChemExpress, catalog number: HY-L057), which contains 1220 compounds.

[0075] The fluorescence detection data for each compound group were processed. First, outliers were removed. Then, a wavelength of 350 nm, near the highest point of all data, was selected as the data processing target. The fluorescence intensity values ​​of different compound treatment groups at this wavelength were calculated and compared with the fluorescence intensity values ​​of the DHE+SOJ1B protein treatment group. The inhibition rate of each compound was calculated, and the results are shown below. Figure 3 .

[0076] Figure 3 The horizontal axis represents the corresponding compound code, and the vertical axis represents the inhibition rate.

[0077] like Figure 3 It can be seen that among all the compounds, there are 17 compounds with an inhibition rate greater than 40%, namely glycyrrhizin, carvacrol, aloin, scutellarin A, glycyrrhizin isoflavone A, emodin anthrone, baicalin methyl ester, aloe bitter, 6-feruloylspinosin, punice, aloe-emodin-8-O-β-D-glucoside, xanthohumol, scutellarin B, (E / Z)-demethoxycurcumin, theaflavin-3-gallate, sophoranone G and 3'-demethylhesperidin.

[0078] Example 5: Antibacterial activity assay of 17 compounds at 50 μM concentration using multi-well plates.

[0079] Phytophthora soybeanae was cultured in the dark at 25°C on 70 cm V8 medium. When the mycelium in the petri dish nearly covered the entire plate, zoospore production was induced. During induction, the mycelium was washed four times with sterile tap water every 30 minutes until most of the mycelium produced sporangia. Then, 5 mL of sterile tap water was added at 25°C and the mixture was incubated for about 4-6 hours to allow the release of zoospores.

[0080] All materials used in the experiment, including 24-well plates, pipettes, pipette tips, sealing film, markers, sterile tape, V8 liquid culture medium, and rifampicin antibiotic, were sterilized under UV light for 30 min in a laminar flow hood. Rifampicin antibiotic was added to the culture medium and mixed thoroughly. Then, x μL of zoospores, y μL of the compound (final concentration 50 μM), and 1000-xy μL of V8 culture medium were added sequentially to the wells of the 24-well plate. The 24-well plates were sealed and incubated in a shaker at 25°C and 120 rpm in the dark for 72 h. Phenotypic observation and image acquisition were then performed. The flowchart and screening results are shown below. Figure 4 .

[0081] Figure 4 Figure A in the diagram is a flowchart of the antibacterial process in a multi-well plate, and Figure B shows the inhibitory effect of 17 compounds on zoospores of Phytophthora soybean at a concentration of 50 μM.

[0082] like Figure 4 It was found that when the compound was screened at a final concentration of 50 μM, three compounds, namely glycyrrhizin, glycyrrhizin A, and glycyrrhizin A, significantly inhibited the growth of Phytophthora spp. in soybean.

[0083] Example 6: Broad-spectrum antibacterial effects of glycyrrhizin, glycyrrhizin A and glycyrrhizin A

[0084] An in vitro antibacterial assay was conducted using the plate growth rate method, targeting eight important plant pathogens from different taxa. Five 50 μM solutions of glycyrrhizin, 100 μM solutions of glycyrrhizin A, and glycyrrhizin isoflavone A were added to the respective culture media, with plates containing an equal volume of DMSO serving as blank controls. After inoculation with mycelial cakes, the plates were cultured under suitable conditions until the control colonies essentially filled the culture dishes. The results are shown below. Figure 5 .

[0085] like Figure 5 It was found that, compared with the blank control, treatments with glycyrrhizin, cypermethrin A, and glycyrrhizin isoflavone A significantly inhibited the mycelial growth of all tested pathogenic fungi. Specifically, colony expansion was significantly inhibited in the treated groups, and the inhibition zones were clear. This indicates that glycyrrhizin, cypermethrin A, and glycyrrhizin isoflavone A are not only effective against Phytophthora soybeanis, but also have a broad spectrum of inhibition and good inhibitory activity against a variety of important plant pathogenic fungi, demonstrating their great potential as lead compounds for broad-spectrum plant-derived fungicides.

[0086] Example 7: Inhibition of Phytophthora virulence in soybean by glycyrrhizin

[0087] Different concentrations (3 μM, 10 μM) of glycyrrhizin solution were mixed with Phytophthora soybean spore suspension using a root-dipping method. Sterile water was used as a blank control, and an equal volume of DMSO was used as a solvent control. Soybean seedling roots were immersed in the mixture for 30 min before transplanting to suitable conditions. The control effect of glycyrrhizin on soybean root rot was evaluated by observing and statistically analyzing the survival rate, weighing the fresh and dry weight of the aboveground parts, and measuring the fresh weight of the underground parts. Results are shown below. Figure 6 .

[0088] Figure 6 Figure A shows the aboveground phenotypes of soybean seedlings in different treatment groups. From left to right: Mock (simulated treatment), DMSO (solvent control), 3 μM Glabridin, 10 μM Glabridin; Figure B shows the root phenotypes of soybean seedlings in the corresponding treatment groups; Figure C shows the survival rate statistics; Figure D shows the fresh weight of the aboveground parts; Figure E shows the dry weight of the aboveground parts; and Figure F shows the fresh weight of the underground parts.

[0089] like Figure 6 The results showed that, compared with the control group, the plants treated with glycyrrhizin were more robust, especially the 10 μM treatment group, which exhibited the best performance in leaf color, stem morphology, and root development. Furthermore, the plant survival rate increased significantly with increasing glycyrrhizin concentration, and root length and plant height were also significantly better than the control group. This indicates that glycyrrhizin can effectively inhibit the pathogenicity of *Phytophthora indica*, reduce the severity of plant disease, significantly improve the survival rate of soybean seedlings, and promote their growth and development, demonstrating its potential as a plant immune inducer.

[0090] The applicant has developed a high-throughput method for screening antibacterial compounds targeting elicitin in *Phytophthora infestans* based on DHE fluorescence intensity, and has successfully screened 17 natural products with significant elicitin binding inhibitory activity, including glycyrrhizin, glycyrrhizin A, and glycyrrhizin isoflavone A. Those skilled in the art, upon reviewing this application, can reasonably expand the scope of application of this invention.

[0091] First, the screening method of the present invention is not limited to the specific elicitin proteins disclosed (such as Soybean Phytophthora SOJ1B), but can also use homologous proteins from other species of Phytophthora, as well as effector proteins with similar sterol binding functions.

[0092] Secondly, the active compounds such as glycyrrhizin, cymoxanil A, and glycyrrhizin isoflavone A obtained through this method can be rationally combined with conventional plant disease control ingredients to form pesticide compositions with synergistic effects. For example, glycyrrhizin can be combined with metalaxyl, cymoxanil A, or glycyrrhizin isoflavone A with fludioxonil. In this combination, glycyrrhizin, cymoxanil A, and glycyrrhizin isoflavone A block the uptake of Phytophthora sterols by targeting and inhibiting elicitin function, while metalaxyl and fludioxonil control oomycete and fungal diseases through other mechanisms of action, respectively, achieving multi-target synergistic control.

[0093] Third, the active compounds of the present invention can be formulated into pesticide formulations of different forms with suitable pesticide excipients (such as emulsifiers, dispersants, stabilizers, etc.) according to actual application needs, including but not limited to soluble concentrates, emulsifiable concentrates, water-dispersible granules, etc., to adapt to various application methods such as foliar spraying, root irrigation, and seed treatment.

[0094] The above application extensions are all based on the technical solutions and experimental results disclosed in this invention, and fall within the technical scope that can be reasonably implemented by those skilled in the art after mastering the content of this invention.

[0095] It should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for the sake of clarity. Those skilled in the art should regard the specification as an organization, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0096] The detailed descriptions listed above are merely specific descriptions of feasible implementations of the present invention, and are not intended to limit the scope of protection of the present invention. All equivalent implementations or modifications made without departing from the technical spirit of the present invention should be included within the scope of protection of the present invention.

[0097] sequence list

[0098] SOJ1B protein-coding gene (SEQ ID NO: 1)

[0099] ACGGCCTGCACGGCGACCCAGCAAACCGCCGCGTACAAGACGCTCGTCAGCATCCTGTCCGAGTCGTCCTTCTCGCAGTGTTCCAAGGACTCGGGCTACTCCATGCTCACGGCCAAGGCCCTGCCCACCAACGCGCAGTACAAGCTCATGTGCGCGTCCACGGCCTGCAACACCATGATCAAGAAGATCGTGGCGCTCAACCCGCCCGACTGCGACCTGACCGTTCCCACCAGCGGCCTCGTGCTGGACGTGTACACGTACGCCAACGGCTTCTCGACCAAGTGCGCGTCGCTCTAA

[0100] SOJ1B protein (SEQ ID NO: 2)

[0101] TACTATQQTAAYKTLVSILSESSFSQCSKDSGYSMLTAKALPTNAQYKLMCASTACNTMIKKIVALNPPDCDLTVPTSGLVLDVYTYANGFSTKCASL.

Claims

1. The application of active compounds that specifically target the elicitin-sterol binding site in competitively inhibiting the binding of elicitin to sterols, characterized in that, The active compound is at least one of the following: glycyrrhizin, carvacrol, aloin, scutellarin A, glycyrrhizin isoflavone A, emodin anthrone, baicalin methyl ester, aloe-emodin, 6-feruloylspinosin, punice, aloe-emodin-8-O-β-D-glucoside, xanthohumol, scutellarin B, (E / Z)-demethoxycurcumin, theaflavin-3-gallate, sophoranone G, and 3'-demethylhesperidin.

2. The application as described in claim 1, characterized in that, The active compounds are glycyrrhizin, glycyrrhizin A, or glycyrrhizin A.

3. The use of the active compound as described in claim 1 or 2, glycyrrhizin, glycyrrhizin A, or glycyrrhizin isoflavone A, in any of the following: (1) Application in inhibiting the growth of oomycetes or fungi, which is achieved by competitively inhibiting elicitin-sterol binding; (2) Application in the preparation of pesticide products that inhibit the growth of oomycetes or fungi; The oomycetes are selected from at least one of Phytophthora capsici, Phytophthora hygroscopica, Phytophthora lychee, Phytophthora tobacco, Pythium cerevisiae, Phytophthora soybean, and Phytophthora pathogenica; the fungi are selected from at least one of Sclerotinia sclerotiorum, Fusarium graminearum, and Botrytis cinerea.

4. The use of the active compound glycyrrhizin as described in claim 1 or 2 in any of the following: (1) Application in the prevention and control of soybean root rot; (2) Application in the preparation of pesticide products for the prevention and control of soybean root rot; (3) Application in promoting soybean growth; (4) Application in the preparation of pesticide products for promoting soybean growth.

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