Use of evodigen in preparing anti-plant fungus preparation

Abstract

This invention provides the application of Evodia rutaecarpa root extract in the preparation of antifungal agents, belonging to the field of agricultural fungal control technology. The active ingredient of the Evodia rutaecarpa root extract is naturally derived and easily obtained; its unique chemical structure and novel mechanism of action effectively avoid the problem of pathogen resistance caused by the concentrated mechanisms of action of existing fungicides, filling the market gap for quinolone-based plant-derived pesticides. The Evodia rutaecarpa root extract exhibits a precise antifungal spectrum and significant inhibitory effect, showing obvious inhibitory activity against soil-borne and interplant-borne plant fungi that are serious threats in agricultural production, such as Phytophthora tobaccoii, Fusarium equisetifolium, Colletotrichum gloeosporioides, and Fusarium moniliforme.

Description

Technical Field

[0001] This invention belongs to the field of agricultural fungal control technology, specifically relating to the application of evodiamine rhizome extract in the preparation of antifungal agents. Background Technology

[0002] Plant fungal diseases are a significant factor restricting the high-quality and efficient development of agricultural production. Among them, soil-borne and interplant-borne plant fungal diseases, due to their wide transmission routes, strong concealment of damage, and difficulty in control, cause serious losses to crop yield and quality. Soil-borne plant fungi are widely present in the soil and can infect crops through the root system, causing diseases such as root rot, stem base rot, and wilt. The pathogens can survive in the soil for a long time, and under continuous cropping conditions, they are prone to outbreaks and epidemics, leading to stunted crop growth, reduced yield, or even crop failure. Interplant-borne plant fungi mainly spread through airflow, wind and rain splash, and plant contact, infecting the leaves, fruits, and tender stems of crops, causing epidemic diseases such as anthracnose, which seriously affect the commercial value of crops.

[0003] With increasing environmental awareness and the demand for green agriculture, the use of chemical fungicides is strictly limited. Plant-derived fungicides, due to their natural origin, good environmental compatibility, low susceptibility to pathogen resistance, and safety for non-target organisms, have become the core direction of fungicide research and development. Currently available plant-derived fungicides mainly include berberine, azadirachtin, emodin methyl ether, eugenol, and matrine. These products have played a certain role in the control of some plant fungal diseases, but significant limitations remain. On the one hand, the chemical structures of these plant-derived fungicides are relatively simple, mainly concentrated in alkaloids, terpenes, and phenolic acids, resulting in a relatively concentrated mechanism of action. Long-term single use can easily lead to pathogen resistance. On the other hand, existing plant-derived fungicides lack specificity against certain types of plant fungi, making it difficult to meet the needs of precise control in production. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide the application of evodiamine rhizome in the preparation of antifungal agents; the evodiamine rhizome has a highly efficient inhibitory effect on plant fungi and can be used to prevent and control plant fungal diseases.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides the application of Evodia rutaecarpa root extract in the preparation of antifungal agents; the fungi include any one or more of Fusarium moniliforme, Phytophthora nicotineum, Fusarium equisetifolium, and Colletotrichum gloeosporioides.

[0006] Preferably, the concentration of the evodiamine root extract is ≥0.01 μg / mL.

[0007] Preferably, the concentration of the evodiamine root extract is 0.2~1 μg / mL.

[0008] Preferably, the antifungal agent further includes a pesticide-acceptable carrier or excipient.

[0009] Preferably, the pesticide-acceptable carrier includes any two or more of the following: adjuvants, wetting agents, emulsifiers, suspending agents, preservatives, salts that affect osmotic pressure, buffers, sweeteners, flavoring agents, and coloring agents.

[0010] This invention provides the application of evodiamine rhizome extract in the prevention and control of plant fungal diseases.

[0011] Preferably, the plant fungal diseases include soil-borne plant fungal diseases and / or interplant-borne fungal diseases.

[0012] Preferably, the plant fungal diseases include those caused by Phytophthora tobaccoii, Fusarium equisetifolium, Fusarium moniliforme, and / or Colletotrichum gloeosporioides.

[0013] This invention provides the application of evodiamine rhizome extract in the preparation of plant-derived pesticides.

[0014] The beneficial effects of this invention are: This invention provides the application of Evodia rutaecarpa root extract in the preparation of antifungal agents; the fungi include any one or more of Fusarium moniliforme, Phytophthora tobaccoii, Fusarium equisetifolium, and Colletotrichum gloeosporioides. The Evodia rutaecarpa root extract is a characteristic secondary metabolite of the root of Evodia rutaecarpa, a plant in the Rutaceae family. It can be obtained by isolation and purification from Evodia rutaecarpa root extracts. The raw material sources are widely available, and the extraction and preparation processes are mature, conforming to the green and environmentally friendly development trend of plant-derived pesticides. The application cost is controllable, and it has good environmental compatibility. The Evodia rutaecarpa root extract has an alkaloid structure containing a quinolone core, specifically 2,4-dihydroxyquinoline as the core, modified by oxygen methylation, nitrogen methylation, and 3-position isopentenylation. Its chemical structure is relatively simple and unrelated to the structural types of existing mainstream plant-derived fungicides on the market. This unique structure gives it a novel mechanism of action, effectively avoiding the problem of pathogen resistance caused by the concentrated mechanisms of action of existing fungicides, filling the market gap for quinolone-based plant-derived pesticides. The described Evodia rutaecarpa root extract exhibits significant inhibitory activity against both soil-borne and interplant-borne fungi that pose serious threats to agricultural production. It specifically addresses the issues of poor efficacy or excessively high applicable concentrations of existing plant-derived fungicides against these key pathogenic fungi. It can serve as an effective ingredient or one of the effective ingredients in antifungal agents for plant diseases, providing an efficient and specific technical solution for the precise control of plant fungal diseases. Based on its natural origin, unique structure, and excellent antifungal activity, the described Evodia rutaecarpa root extract can be widely applied to the control of fungal diseases in various crops. It is suitable for soil treatment or root application for soil-borne diseases, as well as foliar spraying for interplant-borne diseases. It meets the demand for efficient, safe, and low-toxicity fungicides in green agricultural production and has significant industrial application potential and economic value. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 The chemical structural diagram of evodiamine; Figure 2 The graph shows the antibacterial activity of 0.2 and 1.0 μg / mL evodiamine against Phytophthora nicotine on day 5. Figure 3 The antibacterial activity of 0.2 and 1.0 μg / mL evodiamine against Fusarium equisetifolium on day 5 is shown in the figure. Figure 4 The antibacterial activity of 0.2 and 1.0 μg / mL evodiamine against Colletotrichum gloeosporioides on day 5 is shown in the figure. Figure 5 The antibacterial activity of 0.2 and 1.0 μg / mL evodiamine against Fusarium moniliforme on day 5 is shown in the figure. Figure 6 This figure shows the results of the antimicrobial activity of Evodia rutaecarpa root extract and other plant-derived antimicrobial molecules against Phytophthora intoxin. Detailed Implementation

[0017] This invention provides the application of evodiamine rhizome extract in the preparation of antifungal agents; the fungi include any one or more of Fusarium moniliforme, Phytophthora tobaccotae, Fusarium equisetifolium, and Colletotrichum gloeosporioides. As an optional embodiment of this invention, the fungi include soil-borne pathogenic fungi and / or plant-borne pathogenic fungi. As an optional embodiment of this invention, the concentration of evodiamine rhizome extract used is ≥0.01 μg / mL, which can be 0.2~1.0 μg / mL, specifically 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μg / mL.

[0018] As an optional embodiment of the present invention, the antifungal preparation further includes a pesticide-acceptable carrier or excipient. In the present invention, the pesticide-acceptable carrier may be any one or more of the following: adjuvants, wetting agents, emulsifiers, suspending agents, preservatives, salts that affect osmotic pressure, buffers, sweeteners, flavoring agents, and coloring agents.

[0019] The rutin provided by this invention is one of the characteristic secondary metabolites of the root of *Evodia rutaecarpa*, a plant of the Rutaceae family, and its chemical structural formula is as follows: Figure 1 As shown in the figure, phytochemical analysis revealed that evodiamine is mainly concentrated in the root bark of *Evodia rutaecarpa*, suggesting that it exerts an inhibitory effect on pathogenic fungi in the root bark. The evodiamine is an alkaloid structure containing a quinolone core, with a relatively simple chemical structure, specifically involving oxygen methylation, nitrogen methylation, and isopentenylation at the 3-position of the 2,4-dihydroxyquinoline core. The simple chemical structure of evodiamine facilitates its preparation through a combination of biosynthesis and chemical synthesis, ensuring a reliable drug supply for its use as an antifungal agent to control fungal diseases.

[0020] The results of the embodiments of this invention show that the evodia root extract exhibits significant inhibitory activity against soil-borne and interplant-borne plant fungi that cause serious damage in agricultural production, such as Phytophthora tobaccoii, Fusarium equisetifolium, Colletotrichum gloeosporioides, and Fusarium moniliforme. It can specifically solve the problems of poor control effect and high applicable concentration of existing plant-derived fungicides against these key pathogenic fungi. It can be used as an effective ingredient or one of the effective ingredients in antifungal agents for plant diseases, providing an efficient and specific technical solution for the precise control of plant fungal diseases.

[0021] This invention provides the application of evodiamine root extract in the control of plant fungal diseases. As an optional embodiment of this invention, the plant fungal diseases include soil-borne plant fungal diseases and / or inter-plant-borne fungal diseases. In this invention, the plant fungal diseases include those caused by *Phytophthora nicotineae*, *Fusarium equisetifolium*, *Fusarium moniliforme*, and / or *Colletotrichum gloeosporioides*.

[0022] This invention also provides the application of evodiamine rhizome extract in the preparation of plant-derived pesticides.

[0023] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] The sources of the strains involved in the following technical solutions: Fusarium equisetifolium ( Fusarium equiseti ): National Microbial Culture Resource Center, number CFCC 50599.

[0025] Colloidal anthrax ( Colletotrichum gloeosporioides ): National Microbial Resource Bank, number ACCC37608.

[0026] Phytophthora indicum ( Phytophthora nicotiana ): National Microbial Resource Bank, number ACCC 36285.

[0027] Fusarium moniliforme ( Fusarium fujikuroi ): National Microbial Culture Resource Center, number CFCC 85556.

[0028] Saccharomyces cerevisiae ( Saccharomyces cerevisiae ): National Microbial Resource Bank, number CCTCC CY20081301.

[0029] Example 1 The preparation steps of evodiamine are as follows: Take 460g of fresh Evodia rutaecarpa root, slice it, and then crush it to obtain Evodia rutaecarpa powder with a particle size ≤60 mesh. Mix the Evodia rutaecarpa powder with acetone at a mass-to-volume ratio of 460g:1L, and perform a first extraction by soaking in acetone for 24 hours at a temperature of 30℃. After soaking, perform solid-liquid separation to obtain the first extract and Evodia rutaecarpa powder residue. Add 1L of acetone to the Evodia rutaecarpa powder residue and perform a second extraction by soaking in acetone for 24 hours at a temperature of 30℃. After soaking, perform solid-liquid separation to obtain the second extract and Evodia rutaecarpa powder residue. Add 1L of acetone to the Evodia rutaecarpa powder residue and perform a third extraction by soaking in acetone for 24 hours at a temperature of 30℃. After soaking, perform solid-liquid separation to obtain the third extract.

[0030] The first, second, and third extracts of Evodia rutaecarpa were combined to obtain an Evodia rutaecarpa extract. The solvent in the Evodia rutaecarpa extract was recovered by vacuum extraction using a rotary evaporator at 37°C in a water bath to obtain 15g of crude extract.

[0031] 15g of crude extract was separated by normal-phase silica gel column chromatography with dichloromethane-methanol gradient elution to obtain 10 fractions Fr. A~J. Fr. B (1% MeOH) was found to contain the target fraction, evodiamine, by HPLC and was concentrated to 0.869g. Fr. B was separated by ODS reverse-phase column chromatography to obtain Fr. B1~B55 and Fr. B40~B44, which were concentrated to 0.423g. This was purified by semi-preparative HPLC (mobile phase: CH3CN-H2O=55%) to obtain 270mg of pure evodiamine. The structural formula of evodiamine is shown below. Figure 1 As shown.

[0032] Example 2 1. Experimental materials: Evodia rutaecarpa root extract (refrigerated at -20℃ for later use), PDA culture medium, mycelial cake of the test pathogen (5mm in diameter), sterile petri dish (9cm), calipers, constant temperature water bath.

[0033] 2. Operating steps: Preparation of drug-containing plates: Cool the melted PDA medium to 50°C, add Evodia rutaecarpa DMSO solution (1 mg / mL), determine the final concentration of Evodia rutaecarpa by controlling the added volume, shake quickly and gently, pour into sterile petri dishes (about 20 mL per dish), and wait for solidification before use.

[0034] Control treatment: PDA medium that has been melted and cooled to 50°C was mixed with an equal volume of sterile PDB medium (equal volume means equal volume to the DMSO solution containing the drug in the experimental group) to prepare blank control plates.

[0035] Inoculation: Using a sterile inoculation needle, inoculate the pathogenic fungal discs (mycelial side down) into the center of the drug-containing plate and the control plate. Incubation: All plates were incubated in a 25℃ incubator, and colony growth was observed during the incubation process. The selected pathogens were: *Phytophthora tobaccoii*, *Fusarium equisetifolium*, *Colletotrichum gloeosporioides*, and *Fusarium moniliforme*.

[0036] Each experiment was conducted in triplicate.

[0037] 3. The inhibitory effects of different concentrations of evodiamine on four types of fungi, as follows: Figures 2-5 As shown in Table 1.

[0038] Table 1. Inhibition rates of different concentrations of evodiamine against four fungi after 5 days of culture (mean ± standard deviation)

[0039] From Table 1 and Figure 2 It was found that within the test concentration range of 0.01–0.1 μg / mL, the inhibition rate of evodiamine against the test target showed a significant dose-dependent positive correlation with increasing concentration. In the low concentration range (0.01–0.04 μg / mL), the inhibitory effect was weak, with a relatively low inhibition rate; doubling the concentration resulted in a small increase in the inhibition rate, indicating unremarkable activity. In the medium to high concentration range (0.08–0.1 μg / mL), the inhibitory activity rapidly increased. At high concentrations (0.2–1.0 μg / mL), the antibacterial activity was significantly enhanced, reaching over 80%. In summary, evodiamine exhibits clear inhibitory activity within this test concentration range, with its inhibitory effect characterized by weak efficacy at low concentrations and strong efficacy at medium to high concentrations.

[0040] The results above indicate that, in order to further evaluate the antifungal effect of evodiamine, its EC50 was determined. 50 Values ​​were determined. Different concentration gradients of the target compound, evodiamine, were established at 0.01, 0.02, 0.04, 0.08, and 0.1 μg / mL. Its inhibitory activity was monitored from day 3 post-inoculation (when growth was low) until the control group reached full colony size. The results showed that the inhibition rate of evodiamine against *Colletotrichum gloeosporioides* fluctuated at different time points, reaching its highest level on day 5.

[0041] Comparative Example 1 Effects of different concentrations of evodiamine on single-celled yeast 1. Experimental materials: PDA culture medium, test pathogen mycelial cake (5mm in diameter), and Saccharomyces cerevisiae ( Saccharomyces cerevisiae AS2.399) Mycelium cake, sterile petri dish (9cm), calipers, constant temperature water bath.

[0042] 2. Operating steps: Preparation of drug-containing plates: Cool the melted PDA medium to 50°C, add Evodia rutaecarpa DMSO solution, and determine the final concentration of Evodia rutaecarpa DMSO by controlling the added volume to 1 mg / mL. Shake gently and quickly, and pour into sterile petri dishes (about 20 mL per dish). Let it solidify before use.

[0043] Control treatment: PDA medium that has been melted and cooled to 50°C is mixed with an equal volume of sterile PDB medium to prepare blank control plates.

[0044] Inoculation: Using a sterile inoculation needle, inoculate the *Saccharomyces cerevisiae* bacterial cake (mycelial side down) into the center of the drug-containing plate and the control plate. Incubation: All plates are placed in a 25°C incubator and the colony growth is observed during the incubation process.

[0045] Each experiment was conducted in triplicate.

[0046] 3. The results showed that evodiamine at a concentration of 1 mg / mL had no inhibitory effect on Saccharomyces cerevisiae in PDA medium, as shown in Table 2.

[0047] Table 2. Inhibition rate of evodiamine rhizome on brewer's yeast

[0048] Example 3 1. Experimental materials: Evodia rutaecarpa root extract, resveratrol, allicin, carvacrol, and osthol (Evodia rutaecarpa root extract, resveratrol, allicin, carvacrol, and osthol were refrigerated at -20℃ for later use) as test samples, PDA culture medium, bacterial cakes of the test pathogen (5 mm in diameter), sterile petri dishes (9 cm), calipers, and a constant temperature water bath.

[0049] 2. Operating steps: Preparation of drug-containing plates: Cool the melted PDA medium to 50°C, add DMSO solutions of evodiamine, resveratrol, allicin, carvacrol, and osthol respectively, and determine the final concentration of the corresponding test sample by controlling the added volume. Shake quickly and gently, pour into sterile petri dishes (about 20 mL per dish), and let solidify before use.

[0050] Control treatment: PDA medium that has been melted and cooled to 50°C is mixed with an equal volume of sterile PDB medium to prepare blank control plates.

[0051] Inoculation: Using a sterile inoculation needle, inoculate the pathogenic fungal disc (mycelial side down) into the center of the drug-containing plate and the control plate. Incubation: All plates were incubated in a 25℃ incubator, and colony growth was observed during the incubation process. The selected pathogen was *Phytophthora tobaccoii*.

[0052] Each experiment was conducted in triplicate.

[0053] 3. The inhibitory effects of different samples on Phytophthora indicum, such as Figure 6 As shown in Table 3.

[0054] Table 3. Inhibitory effects of different samples on Phytophthora indicum (mean ± standard deviation)

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of evodiamine rhizome extract in the preparation of antifungal agents; wherein the fungi include: One or more of Fusarium moniliforme, Phytophthora tobaccoii, Fusarium equisetifolium, and Colletotrichum gloeosporioides.

2. The application according to claim 1, characterized in that, The concentration of the evodiamine root extract used is ≥0.01 μg / mL.

3. The application according to claim 1 or 2, characterized in that, The concentration of the evodiamine root extract used is 0.2~1.0 μg / mL.

4. The application according to claim 1, characterized in that, The antifungal agents also include pesticide-acceptable carriers and / or excipients.

5. The application according to claim 4, characterized in that, The pesticide-acceptable carriers include any one or more of the following: adjuvants, wetting agents, emulsifiers, suspending agents, preservatives, salts that affect osmotic pressure, buffers, sweeteners, flavoring agents, and coloring agents.

6. Application of Evodia rutaecarpa root extract in the prevention and control of plant fungal diseases.

7. The application according to claim 6, characterized in that, The plant fungal diseases mentioned include soil-borne plant fungal diseases and / or interplant-borne fungal diseases.

8. The application according to claim 7, characterized in that, The plant fungal diseases mentioned include those caused by Phytophthora tobaccoii, Fusarium equisetifolium, Fusarium moniliforme, and / or Colletotrichum candida.

9. Application of Evodia rutaecarpa root extract in the preparation of plant-derived pesticides.

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