Buprofezin as fungus and oomycete chitin synthase inhibitor and application of buprofezin in sterilization

Abstract

The invention relates to buprofezin as a fungus and oomycete chitin synthase inhibitor and novel application of buprofezin in sterilization. In-vitro enzyme activity inhibition test and sterilization test results prove that buprofezin can significantly inhibit the activity of fungal chitin synthase and oomycete chitin synthase, can inhibit fungal spore production and hypha growth, can inhibit oomycete sporangium production and infection toxicity, 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 thiazolinone as a chitin synthase inhibitor for fungi and oomycetes and its 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] Buprofezin, also known as buprofezin, has a chemical name of (Z)-2-tert-butylimino-5-phenyl-3-propan-2-yl-1,3,5-thiadiazin-4-one, and is an insect growth regulator insecticide. The mechanism of action of buprofezin is not very clear, but it is speculated that it inhibits the chitin biosynthesis of insects (including whiteflies) and interferes with the normal growth and development of insects. Buprofezin is highly effective against Hemiptera pests, but ineffective against Lepidoptera. It has persistent larvicidal activity against Coleoptera, some Homoptera, and Acarina. At present, most of the research on buprofezin is focused on its application as an insecticide in pest control in plant culture. No research has shown that buprofezin can be used as a chitin synthase inhibitor for fungi and oomycetes, nor has there been any research report, patent application, or pesticide registration in terms of fungicides. Summary of the invention

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

[0005] Therefore, the present invention provides the use of thiazolinone in preparing a fungal or oomycete chitin synthase inhibitor for sterilization.

[0006] The present invention further provides the use of thiazolinone in the preparation of fungicides and oomycetes

[0007] Preferably, the fungus is Fusarium or Aspergillus. More preferably, the fungus is Fusarium graminearum or Aspergillus fumigatus.

[0008] In addition, preferably, the oomycete is Phytophthora. More preferably, the oomycete is Phytophthora of soybean or Phytophthora of pepper.

[0009] The present invention also provides the use of thiamethoxam in preventing and controlling diseases caused by fungi or oomycetes.

[0010] Specifically, 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] The present invention also provides the use of thiamethoxam in preparing a medicament for preventing and controlling diseases caused by fungi or oomycetes or inhibiting chitin synthase of fungi or oomycetes.

[0012] Specifically, 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.

[0013] The present invention further provides a method for preventing and controlling diseases caused by fungi or oomycetes, which comprises applying a fungicide and an oomycete containing buprofezin as an active ingredient to crops. Specifically, 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;

[0014] The diseases are plant diseases caused by Fusarium, Aspergillus, and Phytophthora, such as crop diseases caused by Fusarium graminearum, Aspergillus fumigatus, Phytophthora sojae, or Phytophthora sojae. For example, Fusarium graminearum can infect the ears, stems, stem bases, and roots of cereal crops such as wheat (Triticum aestivum), barley (Hordeum vulgare), rice (Oryza sativa), and oats (Arena sativa), causing ear rot, stem rot, stem base rot, and root rot. It can also infect other plants. Aspergillus fumigatus infects cotton bolls (cotton boll aspergillosis) and apples, causing fruit rot, or is stored on seeds, and can cause pulmonary aspergillosis and other diseases in humans, livestock, and poultry. Phytophthora sojae mainly infects soybeans, causing soybean root rot. Phytophthora sojae causes pepper phytophthora, etc.

[0015] The administration method is spraying, and the administration concentration is 1-100 ug / ml, such as 10-50 ug / ml, more specifically 20 ug / ml.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a new application of thiamethoxam 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 a concentration of 10 μg / mL and 5 μg / mL, thiamethoxam 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 69% and 24% respectively, the inhibition rates of chitin synthase of Aspergillus fumigatus are 72% and 32% respectively, the inhibition rates of chitin synthase of Phytophthora sojae are 80% and 37% respectively, and the inhibition rates of chitin synthase of Phytophthora capsici are 85% and 40% respectively.

[0018] Furthermore, the present invention provides for the first time the use of thiamethoxam as a fungicide. The fungicidal activity assay results of the present invention show that at a concentration of 20 μg / mL, thiamethoxam can significantly inhibit the mycelial growth and spore production of Fusarium graminearum, inhibit the growth and toxicity of the sporangium of Phytophthora sojae, and achieve the effect of killing fungi and oomycetes.

[0019] Among them, the catalytic domains of chitin synthases of fungi and oomycetes 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, Yu A, 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, the present invention adopts the inhibitory activity of the inhibitor to determine two fungi and two oomycetes, and determines the related bactericidal activity. Therefore, the present invention also has an inhibitory effect on other fungal and oomycete chitin synthases, and can therefore be used for fungal and oomycete antibacterial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The inhibitory effect of thiamethoxam on the spore production of Fusarium graminearum was determined in Example 2.

[0021] Figure 2 The inhibitory effect of thiamethoxam on the hyphae growth of Fusarium graminearum was determined by the paper diffusion method in Example 3.

[0022] Figure 3 The inhibitory effect of buprofezin on the infection toxicity of soybean Phytophthora in Example 5. (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 bars indicate statistically significant differences among treatments (t test, P<0.01). DETAILED DESCRIPTION

[0023] The following experiments and operations are provided to further illustrate the present invention and should not be regarded as limiting the present invention.

[0024] The thiamethoxam used in the present invention is a commercially available product.

[0025] Example 1: Determination of inhibitory activity of thiamethoxam against chitin synthase of oomycetes and fungi

[0026] (1) Chitin synthase activity assay

[0027] Add the coating solution (50mMTris-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 (50mMTris-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.

[0028] 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 (20mMTris, 150mMNaCl, pH 8.0). Set up three replicates for each group of experiments. 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.

[0029] (2) Chitin synthase inhibitory activity assay

[0030] Before the reaction, different concentrations of buprofezin or an equal volume of DMSO were mixed with chitin synthase from Phytophthora sojae (PsChs), Phytophthora capsici (PcChs), Fusarium graminearum (FgChs) or Aspergillus fumigatus (AfChs) as the experimental group and incubated at room temperature for 10 minutes. After the incubation, ions and substrates were added to the system. All compounds were dissolved in 100% DMSO, and the volume of compounds added to all reactions was equal (2 μL). 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:

[0031] 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

[0032] K3: Slope of the experimental group with added compound

[0033] K2: Slope of the control group with added compound

[0034] K1: Slope of the experimental group with DMSO added

[0035] K0: Slope of the control group with DMSO added

[0036] The results showed that at 10 μg / mL, the inhibitory activity of thiamethoxam against chitin synthase of Fusarium graminearum and chitin synthase of Phytophthora sojae was 48% and 63%, respectively; at 5 μg / mL, the inhibitory activity of thiamethoxam against chitin synthase of Fusarium graminearum and chitin synthase of Phytophthora sojae was 29% and 37%, respectively (see Table 1 for details).

[0037] Table 1 Inhibitory activity of thiamethoxam against chitin synthases of oomycetes and fungi

[0038]

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

[0040] Example 2: Determination of the inhibitory activity of buprofezin on the spore production of Fusarium graminearum

[0041] Take the same initial amount of Fusarium graminearum hyphae and add them to 4 mL CMC medium to induce spore expression, add filtered sterilized thiamethoxam solution to a final concentration of 20 μg / mL, filter the hyphae after 4 days of culture, and count the produced spores. The solvent DMSO was used as a negative control, and carbendazim was used as a positive control.

[0042] The results showed that thiamethoxam at a concentration of 20 μg / mL could significantly inhibit the production of spores in Fusarium graminearum (see Figure 1 and Table 2)

[0043] Table 2 The inhibitory effect of thiamethoxam on the spore production of Fusarium graminearum

[0044]

[0045] Note: 1) The data format in the table is: Represents the mean of 3 replicates, SE is the standard error;

[0046] 2) The data in the table are the average values ​​of 3 replicates. Different letters after the data in the same column indicate significant differences at 5% (DMRT). Example 3: Determination of inhibitory activity of buprofezin on hyphae growth of Fusarium graminearum

[0047] (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 thiamethoxam 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 to observe the size of the inhibition ring. The concentration of thiamethoxam is 20μg / mL, with the solvent DMSO as the negative control and carbendazim as the positive control.

[0048] (2) Liquid culture method: Take the same initial amount of Fusarium graminearum hyphae and add it to 1 mL YPD medium, add filtered sterilized thiamethoxam 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. Solvent DMSO was used as a negative control, and carbendazim was used as a positive control.

[0049] The results showed that, whether by paper diffusion or liquid culture, thiamethoxam at a concentration of 20 μg / mL could significantly inhibit the growth of mycelium of Fusarium graminearum (see Figure 2 and Table 3)

[0050] Table 3 Inhibitory effect of thiamethoxam on mycelial growth of Fusarium graminearum determined by liquid culture method

[0051]

[0052] Note: 1) The data format in the table is: Represents the mean of 3 replicates, SE is the standard error;

[0053] 2) The data in the table are the average values ​​of 3 replicates. Different letters after the data in the same column indicate significant differences at 5% (DMRT). Example 4: Determination of inhibitory activity of buprofezin on the production of conidia of Phytophthora sojae

[0054] 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 thiamethoxam with a final concentration of 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. Use the solvent DMSO as a negative control and metalaxyl as a positive control.

[0055] The results showed that 20 μg / mL thiamethoxam could significantly inhibit the production of sporangia after treating soybean phytophthora (see Table 4 for details). Table 4 Inhibitory effect of thiamethoxam on the growth of soybean phytophthora sporangia

[0056]

[0057] Note: 1) The data format in the table is: Represents the mean of 3 replicates, SE is the standard error;

[0058] 2) The data in the table are the average values ​​of 3 replicates. Different letters after the data in the same column indicate significant differences at 5% (DMRT). Example 5: Determination of inhibitory activity of buprofezin on the infection toxicity of soybean phytopathogenes

[0059] Seedlings of soybean variety Hefeng 47 were cultured in the dark 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. Thiazide was mixed with 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 tissues) 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.

[0060] The results showed that after the soybean lesion length and the relative biomass of soybean lesion were significantly reduced after the soybean lesion was treated with 20 μg / mL thiamethoxam compared with the control (see Figure 3), indicating that thiamethoxam can significantly inhibit the toxicity of soybean phytophthora to soybean.

Claims

1. Application of thiazolinone in the preparation of fungal or oomycete chitin synthase inhibitors for sterilization.

2. Application of thiazolinone in the preparation of fungicides and oomycetes.

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 thiamethoxam in the prevention and treatment of 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 thiamethoxam 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 diseases caused by fungi or oomycetes, characterized in that: Application of fungicides and oomycicide containing buprofezin as 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; The diseases are ear rot, stem rot, stem base rot and root rot of cereal crops, cotton boll Aspergillus disease, soybean root rot, pepper phytophthora disease, etc.; The administration method is spraying, and the administration concentration is 1-100 ug / ml, such as 10-50 ug / ml, more specifically 20 ug / ml.

Images