Etoxazole as a chitin synthase inhibitor for fungi and oomycetes and its new application in fungicides

Abstract

The present invention relates to etoxazole as a fungal and oomycete chitin synthase inhibitor and its novel application in sterilization. In vitro enzyme activity inhibition tests and sterilization tests demonstrate that etoxazole can significantly inhibit the activity of fungal and oomycete chitin synthases, and can also inhibit the growth of fungal hyphae, the production of oomycete sporangia, and the toxicity of infection. Therefore, etoxazole can be used as a bactericide for the prevention and treatment of fungal and oomycete diseases.

Description

Technical Field

[0001] The present invention relates to the field of pesticides, specifically to the field of application technology, and in particular to etoxazole as a chitin synthase inhibitor for fungi and oomycetes and its new application in sterilization. Background Art

[0002] Chitin, a linear polymer composed of β-N-acetylglucosamine, is the most abundant amino polysaccharide in nature. It is primarily found in tissues such as fungal cell walls, oomycete spore walls, and the exoskeleton of insects. Therefore, chitin biosynthesis is essential for the growth, development, and reproduction of these organisms. Chitin synthase (CHS) (EC 2.4.1.16) is the key core protein responsible for chitin biosynthesis (Zhu, KY, Merzendorfer, H., Zhang, W., Zhang, J. & Muthukrishnan, S. Biosynthesis, turnover, and functions of chitin in insects. Annu. Rev. Entomol. 2016, 61, 177-196). Since chitin does not exist in plants and mammals, chitin synthase may be one of the safest targets among the more than 30 pesticide molecular targets currently used to control plant pathogens and pests (Li, X., Yang, X., Zheng, X., Bai, M. & Hu, D. Review on structures of pesticide targets. Int. J. Mol. Sci. 2020, 21). Small molecule compounds that can inhibit chitin synthase have important application prospects as insecticides and fungicides.

[0003] Etoxazole, chemically known as 4-(4-tert-butyl-2-ethoxyphenyl)-2-(2,6-difluorophenyl)-4,5-dihydro-1,3-oxazole, was developed by Sumitomo Chemical Co., Ltd. in Japan in the mid-1980s and commercialized in 1998 as an insecticide and acaricide. Etoxazole has excellent control effects against mites such as spider mites, E. punctatus, Panonychus mites, Tetranychus urticae, and Tetranychus cinnabarinus in crops like citrus. It is highly rain-resistant, with a long-lasting effect of up to 50 days. It is environmentally safe and poses minimal or no risk to beneficial insects and mites. Currently, no research has demonstrated the use of etoxazole as a chitin synthase inhibitor for fungi and oomycetes, nor has there been any research reports, patent applications, or pesticide registrations regarding its fungicidal properties. Summary of the Invention

[0004] To address this gap in the aforementioned field, the present invention provides etoxazole as a fungal and oomycete chitin synthase inhibitor and its novel application in sterilization. At a concentration of 20 μg / mL, etoxazole exhibits significant bactericidal effects against fungi and oomycetes and can be used as a pesticide. Specifically, etoxazole significantly inhibits the activity of fungal and oomycete chitin synthases, inhibiting fungal hyphae growth, suppressing the production of oomycete sporangia, and inhibiting the virulence of oomycetes.

[0005] Use of etoxazole as a chitin synthase inhibitor of fungi or oomycetes.

[0006] Application of etoxazole in the preparation of fungicides and oomycicide.

[0007] The application of etoxazole in preventing and controlling crop diseases caused by fungi or oomycetes.

[0008] Use of etoxazole in preparing a medicament for preventing and treating diseases caused by fungi or oomycetes or inhibiting chitin synthase of fungi or oomycetes.

[0009] A method for preventing and controlling crop diseases caused by fungi or oomycetes, characterized in that a fungicide and an oomyceticide containing etoxazole as an active ingredient are applied to the crops.

[0010] The fungus is Fusarium or Aspergillus; the oomycete is Phytophthora; further preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus; the oomycete is Phytophthora sojae or Phytophthora capsici.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention provides, for the first time, a novel application of etoxazole as a chitin synthase inhibitor for fungi and oomycetes. In vitro inhibitory activity assays demonstrate that at concentrations of 10 μg / mL and 5 μg / mL, etoxazole significantly inhibits the chitin synthase activities of Fusarium graminearum, Aspergillus fumigatus, Phytophthora sojae, and Phytophthora capsici. The inhibition rates for Fusarium graminearum chitin synthase were 48% and 29%, respectively; for Aspergillus fumigatus chitin synthase, 53% and 31%, respectively; for Phytophthora sojae chitin synthase, 63% and 37%, respectively; and for Phytophthora capsici chitin synthase, 70% and 39%, respectively. The catalytic domains of fungal and oomycete chitin synthases are very conserved, and nine motifs related to catalytic activity (TMYNE, DGR, KASKL, DVGT, QHFEY, VLPG, EDR, QRKRW, SWG) are all present (for details, see the literature: Chen W, Cao P, Liu Y, YuA, Wang D, Chen L, Sundarraj R, Yuchi Z, Gong Y, Merzendorfer H, Yang Q. Structural basis for directional chitin biosynthesis. Nature. 2022, 610: 402-408). Therefore, etoxazole can also inhibit the chitin synthases of other fungi and oomycetes.

[0013] Furthermore, the present invention provides a new application of etoxazole as a fungicide for the first time. The present invention has been shown to have a fungicidal activity assay test result. At a concentration of 20 μg / mL, etoxazole can significantly inhibit the mycelial growth and spore production of Fusarium graminearum, inhibit the growth and infection toxicity of soybean phytophthora sporangium, and achieve the effect of killing fungi and killing oomycetes. The present invention is achieved by inhibiting the activity of chitinase, thereby inhibiting the activity of fungi or oomycetes, and therefore can also kill other fungi or kill oomycetes. The experiment further proves that etoxazole can be used as a fungus and oomycete chitin synthase inhibitor, and can also be used as a pesticide fungicide. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The inhibitory effect of etoxazole on the mycelial growth of Fusarium graminearum was determined by the disc diffusion method in Example 2;

[0015] Figure 2 The inhibitory effect of etoxazole on the virulence of Phytophthora sojae was determined in Example 4. (A) Photograph of etiolated soybean hypocotyls infected with Phytophthora sojae spores; (B) Statistical results of lesion length; (C) Relative biomass of Phytophthora sojae. Different lowercase letters indicate statistically significant differences between treatments (t-test, P < 0.01). DETAILED DESCRIPTION

[0016] The following experiments and operations are provided to further illustrate the present invention and should not be considered as limitations of the present invention.

[0017] The etoxazole and other reagents used in the present invention are all commercially available products.

[0018] Example 1: Determination of the inhibitory activity of etoxazole on chitin synthase of Phytophthora sojae and Fusarium graminearum

[0019] (1) Chitin synthase activity assay

[0020] Add coating solution (50mM Tris-HCl, 0.1mg / mL WGA, pH 7.5) to a 96-well microtiter plate (100μL / well) and incubate at room temperature overnight. Empty the plate and wash once using a microplate washer. Add 300μL of blocking solution (50mM Tris-HCl, 20mg / mL BSA, pH 7.5) to each well and block for at least 3 hours. After blocking, the plate can be stored at -20°C until needed.

[0021] Remove the blocking buffer and wash the plate once. Add the appropriate amount of PsChs1, 1mM UDP-GlcNAc, and 5mM MnCl2 to the wells and make up to 100μL with Buffer W (20mM Tris, 150mM NaCl, pH 8.0). Perform three replicates for each experiment. Incubate at 30°C, 350r / min for 1 hour. Remove the plate and rinse three times with distilled water. Because HRP activity gradually decreases after thawing, the binding buffer should be prepared freshly for use. The binding buffer composition is as follows: 2μg / mL WGA-HRP, 20mg / mL BSA, and make up to volume with Buffer W. Add 100μL of binding buffer to each well and incubate at 30°C, 350r / min for 30 minutes. Remove the plate and rinse three times with distilled water. Add 100 μL of one-component TMB colorimetric solution to each well and measure absorbance at 652 nm every 20 seconds for a total of 5 minutes. Shake the plate for 3 seconds before each measurement to thoroughly mix the solution. Plot a dot plot with time on the horizontal axis and absorbance on the vertical axis. Calculate the slope of the fitted line as the relative chitin synthase activity.

[0022] (2) Chitin synthase inhibitory activity assay

[0023] Before the reaction began, different concentrations of etoxazole or an equal volume of DMSO were mixed with soybean chitin synthase (PsChs), pepper chitin synthase (PcChs), Fusarium graminearum chitin synthase (FgChs), or Aspergillus fumigatus chitin synthase (AfChs) as the experimental group and incubated at room temperature for 10 minutes. After the incubation was completed, ions and substrates were added to the system. All compounds were dissolved in 100% DMSO, and the volume of compound added to all reactions was equal (2 μL). In the control group, an equal volume of compound or DMSO was added without enzyme to ensure that the final DMSO concentration in all reactions was the same. Chitin synthase inhibitory activity was calculated according to the following formula:

[0024] Inhibitory activity (%) = 1-(activity of positive control group - activity of negative control group) / (activity of test group - activity of negative control group) Inhibitory activity (%) = 1-(K3-K2) / (K1-K0) × 100

[0025] K3: Slope of the experimental group with added compound

[0026] K2: Slope of the control group with added compound

[0027] K1: Slope of the experimental group with DMSO added

[0028] K0: Slope of the control group with DMSO added

[0029] The results showed that at 10 μg / mL, the inhibitory activities of etoxazole against the chitin synthase of Fusarium graminearum, chitin synthase of Aspergillus fumigatus, chitin synthase of Phytophthora sojae and chitin synthase of Phytophthora capsici were 48%, 53%, 63% and 70%, respectively; at 5 μg / mL, the inhibitory activities of etoxazole against the chitin synthase of Fusarium graminearum, chitin synthase of Aspergillus fumigatus, chitin synthase of Phytophthora sojae and chitin synthase of Phytophthora capsici were 29%, 31%, 37% and 39%, respectively (see Table 1 for details).

[0030] Table 1 Inhibitory activity of etoxazole against chitin synthases of oomycetes and fungi

[0031]

[0032] Note: 1) The data format in the table is: Represents the mean of three replicates, SE is the standard error;

[0033] Example 2: Determination of the inhibitory activity of etoxazole on the mycelial growth of Fusarium graminearum

[0034] (1) Paper diffusion method: Inoculate Fusarium graminearum in YPD medium containing agar, spread the bacterial solution evenly so that it covers every corner of the entire plate, and leave it at room temperature for a while (3-5 minutes) to allow the agar to absorb excess surface moisture before placing the paper containing the drug, but the placement time should not exceed 15 minutes. Stick the special drug-sensitive paper containing etoxazole on the agar surface inoculated with the test bacteria. Each paper must be pressed down to ensure complete contact with the agar surface. Whether it is a single paper or a paper distribution device, the paper must be evenly distributed. After the paper is placed, the agar culture medium is inverted and incubated in an incubator for 48 hours, and the size of the inhibition ring is observed. The concentration of etoxazole is 20μg / mL, with the solvent DMSO as the negative control and carbendazim as the positive control.

[0035] (2) Liquid culture method: Take the same initial amount of Fusarium graminearum mycelium and add it to 1 mL of YPD medium. Add filter-sterilized etoxazole solution to a final concentration of 20 μg / mL. Incubate on a shaker for 48 hours. After that, remove the mycelium, dry it, and weigh the dry weight. Use the solvent DMSO as a negative control, and carbendazim as a positive control.

[0036] The results showed that, whether by paper diffusion method or liquid culture method, etoxazole at a concentration of 20 μg / mL could significantly inhibit the growth of mycelium of Fusarium graminearum (see Figure 1 and Table 2)

[0037] Table 2 Inhibitory effect of etoxazole on mycelial growth of Fusarium graminearum determined by liquid culture method

[0038]

[0039] Note: 1) The data format in the table is: Represents the mean of three replicates, SE is the standard error;

[0040] 2) The data in the table are the average of three replicates. Different letters after the data in the same column indicate significant differences at 5% (DMRT).

[0041] Example 3: Determination of the inhibitory activity of etoxazole on the sporangium production of Phytophthora sojae

[0042] Inoculate soybean phytophthora hyphae onto a V8 solid plate and grow for 3-4 days. Pour in 25 mL of liquid culture medium, use a 2.5 mm diameter puncher to punch out an appropriate amount of bacterial cake into a culture dish, and culture at 26°C in the dark for 1.5-2 days. Gently shake once every half a day to prevent the bacterial cakes from touching each other or adhering to the wall. Wash with sterile water 6 times, pour in 5 mL of sterile water (containing etoxazole at a final concentration of 10 μg / mL and 20 μg / mL), keep it in the dark, and observe the growth and germination of sporangia with an inverted microscope after 5-10 hours. Pour the mycelium and liquid in the culture dish into a centrifuge tube, make up to 5 mL, and homogenize the mycelium by ultrasonication. Take 100 μL onto a glass slide, cover it with a coverslip, and count under a microscope. Use the solvent DMSO as a negative control and metalaxyl as a positive control.

[0043] The results showed that etoxazole at concentrations of 10 μg / mL and 20 μg / mL could significantly inhibit the production of sporangia in soybean Phytophthora (see Figure 2 )

[0044] Table 3 The inhibitory effect of etoxazole on the growth of soybean phytophthora sporangium

[0045]

[0046] Note: 1) The data format in the table is: Represents the mean of three replicates, SE is the standard error;

[0047] 2) The data in the table are the average of three replicates. Different letters after the data in the same column indicate significant differences at 5% (DMRT).

[0048] Example 4: Determination of the inhibitory activity of etoxazole against the infection toxicity of soybean Phytophthora

[0049] The seedlings of soybean variety Hefeng 47 were dark cultured at 25 ° C for 4 days. Sporangia were induced according to the method of Example 4 and cultured at 25 ° C until zoospores were produced. Zoospores were counted using a hemocytometer and the concentration was adjusted to about 100 spores / 5 μL. Ethoxydazole was mixed with the spores at a final concentration of 20 μg / mL, and then about 100 spores were inoculated onto the hypocotyls of Hefeng 47 seedlings. After inoculation, the plants were cultured at 25 ° C under high humidity and dark conditions for 2 days and then evaluated. The lesions were quantified and the relative pathogen biomass (the ratio of pathogen DNA to host DNA in infected tissue) was evaluated using qRT-PCR. Infected plant tissues were collected and genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen). The actin gene for soybean phytophthora and the soybean GmCYP2 gene were used as internal reference genes, the solvent DMSO was used as a negative control, and metalaxyl was used as a positive control.

[0050] The results showed that after treating soybean phytophthora with etoxazole at a concentration of 20 μg / mL, the length of soybean lesions and the relative biomass of soybean phytophthora were significantly reduced compared with the control, which was reduced by more than 90%, and the effect was comparable to that of the control agent metalaxyl (see Figure 2 ), indicating that etoxazole can significantly inhibit the toxicity of soybean phytophthora to soybean.

Claims

1. Use of etoxazole in the preparation of a fungicide, wherein the fungus is Fusarium graminearum.

2. Use of etoxazole in preventing and controlling crop diseases caused by fungi, wherein the fungus is Fusarium graminearum.

3. Use of etoxazole in the preparation of a medicament for preventing and treating diseases caused by a fungus, wherein the fungus is Fusarium graminearum.

4. A method for preventing and controlling crop diseases caused by fungi, characterized in that: A fungicide containing etoxazole as an active ingredient is applied to crops, and the fungus is Fusarium graminearum.

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