New application of etoxazole as fungus and oomycete chitin synthase inhibitor and in sterilization aspect

Abstract

The invention relates to etoxazole as a fungus and oomycete chitin synthase inhibitor and a novel application of etoxazole in sterilization. In-vitro enzyme activity inhibition test and sterilization test results prove that etoxazole can significantly inhibit the activity of fungal chitin synthase and oomycete chitin synthase, can inhibit the growth of fungal hyphae and the generation and infection toxicity of oomycete sporangium, and can be used as a bactericide for preventing and treating 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 is a linear polymer composed of β-N-acetylglucosamine. It is the most abundant amino polysaccharide in nature and is mainly found in fungal cell walls, oomycete spore walls, and insect exoskeletons. Therefore, the biosynthesis of chitin 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 the biosynthesis of chitin (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 the mid-1980s and commercialized as an insecticide and acaricide in 1998. Etoxazole has excellent control effects on mites such as spider mites, Tetranychus urticae, Panonychus mites, Tetranychus urticae, and Tetranychus cinnabarinus in crops such as citrus. It is highly rain-resistant, with a long-lasting effect of up to 50 days, is environmentally safe, and has no or minimal harm to beneficial insects and mites. Currently, no research has shown that etoxazole can be used as a chitin synthase inhibitor for fungi and oomycetes, nor has there been any research report, patent application, or pesticide registration for its fungicidal effects. Summary of the invention

[0004] In view of the blank in the above-mentioned field, the present invention provides a new application of etoxazole as a chitin synthase inhibitor for fungi and oomycetes and in sterilization, which has a significant bactericidal effect on fungi and oomycetes at a concentration of 20 μg / mL and can be used as a pesticide. Specifically, etoxazole can significantly inhibit the activity of chitin synthase in fungi and oomycetes, inhibit the growth of fungal hyphae, inhibit the production of oomycete sporangia and the toxicity 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 oomycicides.

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

[0008] The invention discloses an application of etoxazole in the preparation of a medicament for preventing and controlling diseases caused by fungi or oomycetes or inhibiting chitin synthase of fungi or oomycetes.

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

[0010] The fungus is Fusarium or Aspergillus; the oomycete is Phytophthora; more 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 a new application of etoxazole as a chitin synthase inhibitor for fungi and oomycetes for the first time. The results of in vitro inhibition activity assay test of the present invention prove that at concentrations of 10 μg / mL and 5 μg / mL, etoxazole can significantly inhibit the chitin synthase activity of Fusarium graminearum, Aspergillus fumigatus, Phytophthora sojae and Phytophthora capsici, and the inhibition rates of chitin synthase of Fusarium graminearum are 48% and 29%, respectively, the inhibition rates of chitin synthase of Aspergillus fumigatus are 53% and 31%, respectively, the inhibition rates of chitin synthase of Phytophthora sojae are 63% and 37%, respectively, and the inhibition rates of chitin synthase of Phytophthora capsici are 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 have an inhibitory effect on the chitin synthases of other fungi and oomycetes.

[0013] Further, the present invention provides a new application of etoxazole as a bactericide for the first time. The present invention has been proved by the bactericidal activity determination test results that 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 can achieve the effect of killing fungi and killing oomycetes. The present invention is achieved by inhibiting the activity of chitinase, so as to inhibit the activity of fungi or oomycetes, and therefore other fungi agents or oomycetes can also be killed. The experiment further proves that etoxazole can be used as a fungal and oomycete chitin synthase inhibitor, and can also be used as a pesticide bactericide. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 The inhibitory effect of etoxazole on the infection toxicity of soybean Phytophthora in Example 4 was determined. (A) Photograph of yellowed soybean hypocotyls infected with soybean Phytophthora spores; (B) Statistical results of lesion length; (C) Relative biomass of soybean Phytophthora. Different lowercase letters on the bar indicate statistically significant differences among 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 regarded as limiting 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 the coating solution (50mM Tris-HCl, 0.1mg / mL WGA, pH 7.5) to a 96-well ELISA plate (100μL / well) and let stand overnight at room temperature. Empty the ELISA plate and wash it once using a plate washer. Add 300μL of blocking solution (50mM Tris-HCl, 20mg / mL BSA, pH 7.5) to each well and block for at least 3h. After blocking, the ELISA plate can be stored in a -20℃ refrigerator for later use.

[0021] Empty the blocking solution and wash the ELISA plate once. Add appropriate amount of PsChs1, 1mM UDP-GlcNAc, 5mM MnCl2 to the ELISA wells, and make up to 100μL with Buffer W (20mM Tris, 150mM NaCl, pH 8.0). Set up three replicates for each experiment. Incubate at 30℃, 350r / min for 1h, empty the ELISA plate and wash the ELISA plate three times with single distilled water. Since the activity of HRP will gradually decay after thawing, the binding solution must be prepared and used immediately. The components of the binding solution are as follows: 2μg / mL WGA-HRP, 20mg / mL BSA, and make up to the volume with Buffer W. Add 100μL of binding solution to each well, incubate at 30℃, 350r / min for 30min, empty the ELISA plate, and wash the ELISA plate three times with single distilled water. Add 100 μL of single-component TMB colorimetric solution to each well, measure the absorbance at 652 nm, measure once every 20 seconds, and measure for a total of 5 minutes. Shake the ELISA plate for 3 seconds before each test to allow the solution to mix thoroughly. Draw a dot graph with time as the horizontal axis and absorbance as the vertical axis, and calculate the slope of the fitted line as the relative activity of chitin synthase.

[0022] (2) Chitin synthase inhibitory activity assay

[0023] Before the reaction, different concentrations of etoxazole or an equal volume of DMSO were mixed with soybean phytophthora chitin synthase (PsChs), pepper phytophthora chitin synthase (PcChs), graminearum fusarium chitin synthase (FgChs) or Aspergillus fumigatus chitin synthase (AfChs) as the experimental group, incubated at room temperature for 10 minutes, and then ions and substrates were added to the system after the incubation. All compounds were dissolved in 100% DMSO, and the volume of compounds added to all reactions was equal (2μL). In the control group, an equal volume of compound or DMSO was added, and no enzyme was added to ensure that the final concentration of DMSO in all reactions was the same. The 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 on 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 on 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 oomycete and fungal chitin synthase

[0031]

[0032] Note: 1) The data format in the table is: Represents the mean of 3 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 to 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 placing the paper, the agar medium is inverted in an incubator and incubated 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 hyphae and add it to 1 mL YPD medium, add the filtered and sterilized etoxazole solution to a final concentration of 20 μg / mL, place it on a shaker for 48 hours, then take out the hyphae and dry them, and weigh the dry weight. The solvent DMSO was used as a negative control, and carbendazim was used as a positive control.

[0036] The results showed that, whether by paper diffusion or liquid culture, 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 Liquid culture method to determine the inhibitory effect of etoxazole on the mycelial growth of Fusarium graminearum

[0038]

[0039] Note: 1) The data format in the table is: Represents the mean of 3 replicates, SE is the standard error;

[0040] 2) The data in the table are the averages 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 the mycelium of soybean phytophthora into V8 solid plates and grow for 3-4 days. Pour in 25mL of liquid culture medium, use a 2.5mm diameter puncher to punch 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 sticking to the wall. Wash with sterile water 6 times, pour in 5mL of sterile water (containing etoxazole with a final concentration of 10μg / mL and 20μg / 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 5mL, and homogenize the mycelium by ultrasound. Take 100μL on a slide, cover it with a coverslip, and count under a microscope. The solvent DMSO was used as a negative control, and metalaxyl was used as a positive control.

[0043] The results showed that etoxazole at 10 μg / mL and 20 μg / mL concentrations 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 3 replicates, SE is the standard error;

[0047] 2) The data in the table are the averages 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. Ethoxazole was mixed with spores at a final concentration of 20 μg / mL, and then about 100 spores were inoculated into the hypocotyls of Hefeng 47 seedlings. After inoculation, the plants were cultured at 25 ° C for 2 days under high humidity and dark conditions and then evaluated. The lesions were quantified and the relative pathogen biomass (the ratio of pathogen DNA to host DNA in infected tissues) was evaluated using qRT-PCR. Infected plant tissues were collected and genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen). Actin genes for Phytophthora sojae and soybean GmCYP2 genes were used as internal reference genes, with solvent DMSO as a negative control and metalaxyl as a positive control.

[0050] The results showed that after the treatment of 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 could be reduced by more than 90%, which was equivalent to the effect of the control agent metalaxyl (see Figure 2 ), indicating that etoxazole can significantly inhibit the toxicity of soybean phytophthora to soybean.

Claims

1. The use of etoxazole as a chitin synthase inhibitor of fungi or oomycetes.

2. Application of etoxazole in the preparation of fungicides and oomycicides.

3. The use according to claim 2, characterized in that: The fungus is Fusarium or Aspergillus; preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus.

4. The use according to claim 2, characterized in that: The oomycete is Phytophthora; preferably, the oomycete is Phytophthora sojae or Phytophthora capsici.

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

6. The use according to claim 5, characterized in that The fungus is Fusarium or Aspergillus; the oomycete is Phytophthora; more preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus; the oomycete is Phytophthora sojae or Phytophthora capsici.

7. Use of etoxazole in the preparation of an agent for preventing and controlling diseases caused by fungi or oomycetes or inhibiting chitin synthase of fungi or oomycetes.

8. The use according to claim 7, characterized in that The fungus is Fusarium or Aspergillus; the oomycete is Phytophthora; more preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus; the oomycete is Phytophthora sojae or Phytophthora capsici.

9. A method for preventing and controlling crop diseases caused by fungi or oomycetes, characterized in that: Apply fungicides and oomycicide containing etoxazole as an active ingredient to crops.

10. The method according to claim 9, characterized in that: The fungus is Fusarium or Aspergillus; the oomycete is Phytophthora; more preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus; the oomycete is Phytophthora sojae or Phytophthora capsici.

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