Buprofezin as a chitin synthase inhibitor of fungi and oomycetes and its application in bactericidal treatment

Abstract

The present invention relates to novel applications of thiazolinone as a fungal and oomycete chitin synthase inhibitor and its sterilization application. In vitro enzyme activity inhibition tests and sterilization tests demonstrate that thiazolinone can significantly inhibit the activity of fungal and oomycete chitin synthases, inhibit fungal spore production and hyphal growth, and inhibit the production and infection toxicity of oomycete sporangia. The invention can be used as a sterilant 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 thiazolinone as a chitin synthase inhibitor for fungi and oomycetes and its 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] Buprofezin, also known as buprofezin, is a pesticide belonging to the insect growth regulator class. Its chemical name is (Z)-2-tert-butylimino-5-phenyl-3-propan-2-yl-1,3,5-thiadiazin-4-one. While its mechanism of action is not entirely clear, it is speculated to inhibit chitin biosynthesis in insects (including whiteflies), interfering with their normal growth and development. Buprofezin is highly effective against Hemipteran pests but ineffective against Lepidoptera. It exhibits persistent larvicidal activity against Coleoptera, some Homoptera, and Acarina. Currently, research on buprofezin primarily focuses on its use as an insecticide for pest control in plant culture. No studies have demonstrated its potential as a chitin synthase inhibitor for fungi and oomycetes, nor have there been any reports, patent applications, or pesticide registrations for its fungicidal properties. Summary of the Invention

[0004] To address the gaps in the aforementioned field, the present invention provides a novel application of thiazolinone as a chitin synthase inhibitor for fungi and oomycetes, and its novel application in sterilization. At a concentration of 20 μg / mL, thiazolinone exhibits significant sterilization against fungi and oomycetes, and can be used as a fungicide and oomycete. Specifically, thiazolinone can significantly inhibit the activity of chitin synthase in fungi and oomycetes, inhibiting fungal hyphae growth and spore production, and inhibiting the production of oomycete sporangia and the virulence of oomycetes.

[0005] Therefore, the present invention provides the use of thiazolinone in preparing fungal or oomycete chitin synthase inhibitors 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 sojae or Phytophthora capsici.

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

[0010] Specifically, 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] The present invention also provides the use of thiazolinone in preparing a medicament for preventing and treating 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; further 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, comprising applying a fungicide and an oomyceticide 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; and the oomycete is Phytophthora sojae or Phytophthora capsici.

[0014] The diseases are plant diseases caused by Fusarium, Aspergillus, and Phytophthora, specifically 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 estivum), 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 it can be stored on seeds and 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 blight, 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, for the first time, a novel application of thiazolinone as a chitin synthase inhibitor for fungi and oomycetes. In vitro inhibitory activity assays demonstrate that thiazolinone significantly inhibits the chitin synthase activities of Fusarium graminearum, Aspergillus fumigatus, Phytophthora sojae, and Phytophthora capsici at concentrations of 10 μg / mL and 5 μg / mL, with inhibition rates of 69% and 24% for Fusarium graminearum chitin synthase, 72% and 32% for Aspergillus fumigatus chitin synthase, 80% and 37% for Phytophthora sojae, and 85% and 40% for Phytophthora capsici, respectively.

[0018] Furthermore, the present invention provides for the first time the use of thiazolinone as a fungicide. Fungicidal activity assays demonstrate that at a concentration of 20 μg / mL, thiazolinone significantly inhibits mycelial growth and spore production of Fusarium graminearum, and inhibits the growth and virulence of sporangia of Phytophthora sojae, achieving both fungicidal and oomycete-killing effects.

[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 uses 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 the chitin synthase of other fungi and oomycetes, and can therefore be used for antibacterial applications of fungi and oomycetes. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 2 The disc diffusion method in Example 3 was used to determine the inhibitory effect of thiazolinone on the mycelial growth of Fusarium graminearum.

[0022] Figure 3 The inhibitory effect of buprofezin on the virulence of Phytophthora sojae was determined in Example 5. (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 above the bars indicate statistically significant differences between 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 considered as limitations of the present invention.

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

[0025] Example 1: Determination of inhibitory activity of buprofezin on chitin synthase of oomycetes and fungi

[0026] (1) Chitin synthase activity assay

[0027] Add coating solution (50 mM Tris-HCl, 0.1 mg / mL WGA, pH 7.5) to a 96-well microtiter plate (100 μL / well) and incubate at room temperature overnight. Empty the plate and wash once using a microplate washer. Add 300 μL of blocking solution (50 mM Tris-HCl, 20 mg / 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.

[0028] 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 (20mMTris, 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 must 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.

[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 synthases from Phytophthora sojae (PsChs), Phytophthora capsici (PcChs), Fusarium graminearum (FgChs), or Aspergillus fumigatus (AfChs) as experimental groups 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 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 the same final DMSO concentration in all reactions. 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 the chitin synthase of Fusarium graminearum and the chitin synthase of Phytophthora sojae was 48% and 63%, respectively; at 5 μg / mL, the inhibitory activity of thiamethoxam against the chitin synthase of Fusarium graminearum and the chitin synthase of Phytophthora sojae was 29% and 37%, respectively (see Table 1 for details).

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

[0038]

[0039] Note: 1) The data format in the table is: The data represent the mean of three 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] An equal initial amount of Fusarium graminearum mycelium was added to 4 mL of CMC medium to induce spore expression. A filter-sterilized thiazolinone solution was added to a final concentration of 20 μg / mL. After 4 days of incubation, the mycelium was filtered and the spores were counted. DMSO solvent was used as a negative control, and carbendazim was used as a positive control.

[0042] The results showed that thiazolinone 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 Inhibitory effect of thiazolinone on spore production of Fusarium graminearum

[0044]

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

[0046] 2) The data in the table are the average of 3 replicates. Data in the same column with different letters after them are significantly different at 5% (DMRT). Example 3: Determination of the inhibitory activity of buprofezin on the mycelial 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 thiazolinone on the agar surface inoculated with the test bacteria. Each paper must be pressed down to ensure complete contact with the agar surface. Whether the paper is placed alone or with a paper distribution device, the paper must be evenly distributed. After the paper is placed, the agar medium is inverted and incubated in an incubator for 48 hours, and the size of the inhibition ring is observed. The concentration of thiazolinone is 20μg / mL, the solvent DMSO is used as the negative control, and carbendazim is used as the positive control.

[0048] (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 buprofezin solution to a final concentration of 20 μg / mL. Incubate on a shaker for 48 h. 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.

[0049] The results showed that, whether by paper diffusion method or liquid culture method, thiazolinone 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 thiazolinone 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 three replicates, SE is the standard error;

[0053] 2) The data in the table are the average of 3 replicates. Data in the same column with different letters after them are significantly different at 5% (DMRT). Example 4: Determination of the inhibitory activity of buprofezin on the sporangium production of Phytophthora sojae

[0054] Inoculate soybean phytophthora hyphae onto a V8 solid plate and grow for 3-4 days. Pour in 25 mL of liquid culture medium and use a 2.5 mm diameter puncher to punch out an appropriate amount of bacterial cake into a culture dish. Incubate at 26°C in the dark for 1.5-2 days. Gently shake 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 thiamethoxam at a final concentration of 20 μg / mL), incubate 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 slide, cover 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 of thiamethoxam could significantly inhibit the production of sporangia after treating Phytophthora sojae (see Table 4 for details). Table 4 Inhibitory effect of thiamethoxam on the growth of sporangia of Phytophthora sojae

[0056]

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

[0058] 2) The data in the table are the average of 3 replicates. The data in the same column are marked with different letters, which indicate significant difference at 5% (DMRT). Example 5: Determination of inhibitory activity of buprofezin on the infection toxicity of soybean phytophthora

[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 their concentration was adjusted to about 100 spores / 5 μL. Thiamethoxam 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 the DNeasy Plant Mini Kit (Qiagen). The actin gene for Phytophthora sojae 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 the lesion length and relative biomass of soybean P. sojae were significantly reduced after treatment with 20 μg / mL thiamethoxam compared with the control (see Figure 3), indicating that thiazolinone can significantly inhibit the toxicity of soybean phytophthora to soybean.

Claims

1. The use of thiazolinone in the preparation of a fungicide, characterized in that: The fungus is Fusarium graminearum.

2. The use of thiazolinone in preventing and treating diseases caused by fungi, characterized in that: The fungus is Fusarium graminearum.

3. The use according to claim 2, characterized in that The diseases are ear rot, stem rot, stem base rot and root rot of cereal crops caused by Fusarium graminearum.

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

5. The method according to claim 4, wherein The diseases are ear rot, stem rot, stem base rot and root rot of cereal crops caused by Fusarium graminearum.

6. The method according to claim 4, characterized in that: The method of application is spray.

7. The method according to any one of claims 4 to 6, characterized in that: The fungicide is applied at a concentration of 1-100 μg / ml.

8. The method according to claim 7, characterized in that: The fungicide was applied at a concentration of 10-50 μg / ml.

9. The method according to claim 8, characterized in that The fungicide was applied at a concentration of 20 μg / ml.

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