Application of Chymostatin in the Prevention and Control of Rice Blast Disease

Abstract

The application relates to the technical field of pesticides for rice, and aims to solve the problem of ecological environment damage caused by existing chemical pesticides, and provides application of Chymostatin in prevention and control of rice blast, including: S1, inhibiting rice Magnaporthe oryzae; S2, preventing and controlling rice blast; the CAS No of the Chymostatin is 9076-44-2. Chymostatin has a strong inhibiting effect on rice Magnaporthe oryzae, has low harm degree on animals such as human and livestock, is easy to degrade, is friendly to the environment, is beneficial to ecological balance and stability. Chymostatin can significantly inhibit the formation of Magnaporthe oryzae appressorium, and then inhibit the infection. Chymostatin has good inhibiting activity on various rice Magnaporthe oryzae and has broad spectrum.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology for rice, specifically to the application of chymostatin in the control of rice blast. Background Technology

[0002] Rice blast fungus ( Magnaporthe oryzae This disease, caused by rice blast, can occur throughout the entire growth period of rice. Based on the timing and location of its infection, it is classified into seedling blast, leaf blast, node blast, panicle blast, and grain blast. The rice blast pathogen primarily overwinters as conidia and mycelium on diseased plant debris. The following year, when temperature and humidity are suitable, the pathogen rapidly grows and produces a large number of conidia, which are spread by wind and rain to healthy rice plants. The conidia germinate to form appressoriums that infect the rice. Symptoms appear after approximately 5-7 days, followed by further mycelial differentiation to produce conidiophores, which generate a large number of conidia that are then spread again by wind and rain, leading to further infection.

[0003] Chemical control is currently the most efficient, rapid, and convenient method for controlling rice blast, especially in years with high incidence or in susceptible planting areas. Conventional blast fungicides, such as isoprothiolane from the last century, have strong penetrability, but long-term use can cause a foul odor in the rice, affecting its quality. Isoprothiolane is a major control agent widely used in production, but resistant strains were discovered in 1976. Currently, the commonly used systemic fungicide tricyclazole is the benchmark agent for rice blast control, possessing extremely strong systemic properties (absorbed and translocated by plants) and protective effects, effectively inhibiting the germination and invasion of pathogen conidia. However, reports of resistant strains have emerged.

[0004] Currently, microbial pesticides are defined as pesticides synthesized through artificial modification using microorganisms themselves, their secondary metabolites, or compounds whose active components are their metabolites. Among these, proteins or polypeptides produced by plants, animals, or microorganisms exert their control effect by interfering with the life activities of pathogens or pests; these belong to the protein or polypeptide class of biological pesticides.

[0005] Jinggangmycin, an agricultural antibiotic developed in the 1970s, has a helical polycyclic peptide chemical structure. Its main mechanism of action is to inhibit the trehalase enzyme in pathogens, preventing the conversion of trehalose into two glucose molecules, thereby inhibiting mycelial elongation. Its main active ingredient is Jinggangmycin A. In my country, it is widely used to control rice sheath blight, covering nearly 10 million hectares annually, and remains a major effective agent for controlling this disease. Currently, chitosan, a commonly used agent, is mainly derived from the exoskeleton of crustaceans (its main component is chitin). It stimulates the plant's immune response by interfering with the chitin modification of the pathogen's cell wall, inhibiting fungal growth and reproduction to control fungal diseases such as gray mold. It is derived from extreme thermophilic fungi. Thermus thermophilusThermolides, a natural product, possess a unique polyketide-nonribosomal peptide hybrid macrolide structure and significant anti-plant parasitic nematode activity, demonstrating good potential for green pesticide development.

[0006] Chymostatin is derived from actinomycetes ( Streptomyces Chymostatin, a short peptide composed of several amino acids, was isolated from [a specific organism / organism]. Current research has found that chymostatin is a competitive inhibitor. Its molecular structure is very similar to that of the natural substrate of chymotrypsin, allowing it to preemptively bind to the enzyme's active site, thereby preventing the actual substrate from binding to the enzyme and inhibiting its catalytic activity. Studies have shown that this substance can reduce high-mobility group box 1 (HMGB1) and pro-inflammatory cytokines (IL-1β and IL-6), has a protective effect against acute lung injury induced by paraquat, and exhibits anti-cancer activity against lung cancer. Currently, there are no reports in China regarding the inhibitory effect of chymostatin on rice blast fungus. Summary of the Invention

[0007] To comprehensively address the aforementioned problems, the purpose of this invention is to provide the application of chymostatin in the control of rice blast. This invention is the first to discover that chymostatin has a strong inhibitory effect on rice blast fungus, providing a new scientific approach for the further development and creation of novel bio-based fungicides and their application in the prevention and control of rice blast.

[0008] To achieve the above objectives and technical ideas, the first aspect of the present invention provides an application of Chymostatin, as S1) or S2):

[0009] S1) Inhibits rice blast fungus;

[0010] S2) Control of rice blast;

[0011] The CAS No. of the chymostatin is 9076-44-2.

[0012] Preferably, inhibition of rice blast fungus manifests as inhibition of rice blast fungus conidial germination and / or inhibition of rice blast fungus appressorium formation.

[0013] The second aspect of the present invention provides a method for preventing and controlling rice blast disease, wherein the method directly or indirectly treats rice blast fungus with the aforementioned chymostatin to achieve prevention and control of rice blast disease.

[0014] Preferably, the inhibition of rice blast fungus is manifested in the inhibition of rice blast fungus conidial germination and / or the inhibition of rice blast fungus appressorium formation.

[0015] A third aspect of the present invention provides a bio-based fungicide for the control of rice blast, the bio-based fungicide comprising Chymostatin.

[0016] Preferred, Chymostatin, CAS No: 9076-44-2.

[0017] Preferably, the bio-derived bactericide can be formulated into powder, granule, dispersible powder, dispersible granule, dispersible tablet, soluble solid, soluble liquid, oil, ultra-low volume, dispersible liquid, emulsion, suspension, suspension emulsion, or seed coating agent.

[0018] Preferably, the powdered formulation is selected from powders, contact powders, or floating powders;

[0019] Granular formulations are selected from granules, large granules, fine granules, microparticles, or microcapsules.

[0020] Dispersible powder formulations are selected from wettable powders or oil-dispersible powders;

[0021] Dispersible granular formulations are selected from water-dispersible granules, emulsion granules, or effervescent granules;

[0022] Dispersible tablet formulations are selected from dispersible tablets or effervescent tablets;

[0023] Soluble solid dosage forms are selected from soluble powders, soluble granules, or soluble tablets;

[0024] Soluble formulations are selected from soluble agents, aqueous solutions, or soluble gels;

[0025] Oil-based preparations are selected from oil-based or film-spreading oil-based preparations;

[0026] Ultra-low volume formulations are selected from ultra-low volume liquids or ultra-low volume microcapsule suspensions;

[0027] Dispersible liquid formulations are selected from emulsifiable concentrates or dispersible liquids;

[0028] Emulsion formulations are selected from water-based emulsions, oil-based emulsions, or microemulsions;

[0029] The suspension formulation is selected from suspension concentrates, microcapsule suspension concentrates, or oil suspension concentrates;

[0030] The seed coating agent is selected from water suspension seed coating agents, dispersible powder seed coating agents, or dispersible granule seed coating agents.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. Currently, rice blast fungus has developed resistance to some fungicides. Chymostatin, as a novel short peptide antibiotic, has a strong inhibitory effect on rice blast fungus.

[0033] 2. Chymostatin can inhibit the infection of rice blast fungus by significantly suppressing the formation of appressorium.

[0034] 3. It exhibits good inhibitory activity against multiple races of rice blast fungus, demonstrating broad-spectrum activity. Attached Figure Description

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0036] In the attached diagram:

[0037] Figure 1 The inhibitory effect of hymostatin on appressorium formation of different rice blast fungi races;

[0038] Figure 2 The effect of chymostatin on the germination of conidia from different races of rice blast fungus;

[0039] Figure 3 To investigate the inhibitory effect of different concentrations of chymostatin on the pathogenicity of different races of rice blast fungus through in vitro rice inoculation;

[0040] Figure 4 To investigate the effect of adding chymostatin on the control of rice blast through direct spray inoculation. Detailed Implementation

[0041] The following combination Figures 1-4 The preferred embodiments of the present invention are described herein. It should be understood that the preferred embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available. The following embodiments are provided to better understand the present invention, but do not limit the present invention.

[0042] Example 1:

[0043] The application of chymostatin is S1) or S2):

[0044] S1) Inhibits rice blast fungus;

[0045] S2) Application in the prevention and control of rice blast;

[0046] Chymostatin is found in actinomycetes ( StreptomycesThe short peptide tetrapeptide aldehyde, composed of several amino acids, was isolated from MCE. Its CAS No.: 9076-44-2 was purchased from MCE under the product number HY-P3042.

[0047] The inhibition of rice blast fungus is manifested in inhibiting the germination of rice blast fungus conidia and / or inhibiting the formation of rice blast fungus appressorium.

[0048] Example 2:

[0049] A method for inhibiting rice blast fungus involves directly or indirectly treating the fungus with chymostatin to control rice blast disease. The inhibition of rice blast fungus is manifested in inhibiting the germination of conidia and / or inhibiting the formation of appressoriums.

[0050] Example 3:

[0051] A bio-based fungicide for the control of rice blast includes Chymostatin as described in Example 1, Chymostatin's CAS No: 9076-44-2.

[0052] Biological bactericides can be formulated into powder formulations, granular formulations, dispersible powder formulations, dispersible granular formulations, dispersible tablet formulations, soluble solid formulations, soluble liquid formulations, oil formulations, ultra-low volume formulations, dispersible liquid formulations, emulsion formulations, suspension formulations, suspension emulsions, or seed coating agents.

[0053] Powdered formulations are selected from powders, contact powders, or floating powders;

[0054] Granular formulations are selected from granules, large granules, fine granules, microparticles, or microcapsules.

[0055] Dispersible powder formulations are selected from wettable powders or oil-dispersible powders;

[0056] Dispersible granular formulations are selected from water-dispersible granules, emulsion granules, or effervescent granules;

[0057] Dispersible tablet formulations are selected from dispersible tablets or effervescent tablets;

[0058] Soluble solid dosage forms are selected from soluble powders, soluble granules, or soluble tablets;

[0059] Soluble formulations are selected from soluble agents, aqueous solutions, or soluble gels;

[0060] Oil-based preparations are selected from oil-based or film-spreading oil-based preparations;

[0061] Ultra-low volume formulations are selected from ultra-low volume liquids or ultra-low volume microcapsule suspensions;

[0062] Dispersible liquid formulations are selected from emulsifiable concentrates or dispersible liquids;

[0063] Emulsion formulations are selected from water-based emulsions, oil-based emulsions, or microemulsions;

[0064] The suspension formulation is selected from suspension concentrates, microcapsule suspension concentrates, or oil suspension concentrates;

[0065] The seed coating agent is selected from water suspension seed coating agents, dispersible powder seed coating agents, or dispersible granule seed coating agents.

[0066] Experimental verification:

[0067] 1. Inhibitory effect of chymostatin on conidial germination and appressorium formation of rice blast fungus:

[0068] The pathogenicity of *Strombus oryzae* was determined by measuring appressorium formation on hydrophobic slides and by inoculating treated rice. Three physiological races of *Strombus oryzae* (B157, Guy11, and Zhong1) were inoculated onto rice bran medium (20 g rice bran, 2 g yeast extract, 20 g agar, 1000 mL water) plates and cultured under alternating light and dark for 10 days to induce conidia. A certain amount of sterile water was added to the plates, which were then scraped with a spreader and filtered to obtain a conidial suspension. Conidia were counted using a hemocytometer, and the conidia count was adjusted to 10-1. 5 Units / mL are available for use.

[0069] B157 is directly derived from 70-15, and its genome sequence is almost identical to that of 70-15. Therefore, the genome information of 70-15 is usually directly queried in databases. In databases such as NCBI, the genome assembly accession number of 70-15 is GCA_000002495.2. 70-15 itself was obtained from strain CHNO.115 (belonging to physiological race IA-1) collected from the southern United States through three consecutive single-spore isolations.

[0070] Guy11 is a strain isolated from rice in French Romanoia in 1986. It is a mating type. MAT1-2 This is a representative strain widely used in genetic and pathological research, and is another very important reference strain besides 70-15. Genome Assembly Accession Number: GCA_002368045.1.

[0071] Zhong1 is a strain isolated from rice in Zhejiang, China in 1993. It is a representative strain of the mating type MAT1-1 and exhibits strong pathogenicity in Chinese rice varieties. Therefore, it is an important model for studying the interaction between rice blast fungus and rice, particularly its pathogenic mechanism in Chinese varieties. Genome Assembly Accession Number: GCA_025077495.1.

[0072] Using DMSO as solvent, 6 mg / mL of chomostatin stock solution was prepared by adding chomostatin. Then, it was diluted to obtain four different concentration gradients of chomostatin working solution: 3 μg / mL, 6 μg / mL, 30 μg / mL and 60 μg / mL. A control group with a chomostatin concentration of 0 μg / mL was also set up.

[0073] The prepared working solutions of different concentrations were added to the above conidial suspension, and 20 μL of each solution was dropped onto a hydrophobic glass slide. After incubation in the dark for 6-8 h, the germination rate and appressorium formation rate were recorded under an optical microscope.

[0074] The inhibitory effects of chymostatin on the appressorium of rice blast fungus are shown in Table 1 and... Figure 1 As shown in Table 1 and Figure 1 The results showed that chymostatin had a significant inhibitory effect on the formation of appressorium of rice blast fungus. In particular, at a dosage of 6 μg / mL, the appressorium formation rates of rice blast fungus races B157, Guy11 and Zhong1 were reduced to 34.4%, 35.3% and 38.3%, respectively.

[0075] Table 1. Inhibition of appressorium formation by Chymostatin against rice blast fungus:

[0076] ;

[0077] Chymostatin also showed a significant inhibitory effect on the germination of rice blast fungus, as shown in Table 2 and... Figure 2 As shown in Table 2 and Figure 2 The results showed that chymostatin also has inhibitory activity against the germination of rice blast fungus. In particular, at a dosage of 60 μg / mL, the germination rates of rice blast fungus races B157, Guy11 and Zhong1 were reduced to 40.8%, 45.4% and 32.6%, respectively.

[0078] Table 2. Inhibition of rice blast fungus germination by Chymostatin:

[0079] ;

[0080] 2. Chymostatin can significantly reduce the pathogenicity of rice blast fungus:

[0081] (1) Effects of chymostatin on inoculation symptoms in detached rice leaves:

[0082] Healthy rice leaves were cut and fixed flat in a square petri dish with the upper surface facing up. The dish was kept moist with moist filter paper and absorbent cotton. 20 μL of conidial suspension was inoculated onto the detached rice leaves, with approximately 7-8 drops of the suspension (with or without the agent) evenly added to each leaf. The leaves were placed in a dark, humidified incubator at 25°C for 24-48 hours, then transferred to a light-dark incubator. During this period, the condition of lesions was observed, and water was replenished as needed to prevent the leaves from drying out. After 6-7 days, the disease development on the detached leaves was observed and photographed. The results are as follows: Figure 3 As shown.

[0083] like Figure 3 As shown, gradient inoculation revealed that with increasing chymostatin concentration, the area of ​​lesions formed by rice blast fungus decreased, and it had a significant inhibitory effect on different rice blast fungal races, indicating that chymostatin can significantly reduce the pathogenicity of rice blast fungus, and exhibits a concentration-dependent reduction in pathogenicity.

[0084] (2) Effects of chymostatin on inoculation diseases in live rice seedlings:

[0085] Rice seeds are disinfected and germinated, then transferred to seedling trays. After approximately three weeks of growth, rice blast conidia are collected and the concentration is adjusted to 10. 5 The disease incidence rate was determined by spraying rice with or without chymostatin at a concentration of [number] cells / mL, followed by a moisturizing treatment. After 6-7 days, the disease incidence on the leaves was observed and photographed. Results are as follows: Figure 4 As shown.

[0086] like Figure 4 As shown, spray inoculation revealed that the number of lesions formed by rice blast fungus was significantly reduced after the addition of chymostatin, indicating that chymostatin can significantly reduce the pathogenicity of rice blast fungus.

[0087] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of Chymostatin, for S1) or S2): S1) Application in inhibiting rice blast fungus; S2) Application in the prevention and control of rice blast; in, CAS No:9076-44-2 for Chymostatin.

2. The application according to claim 1, characterized in that, Inhibition of rice blast fungus manifests as inhibition of conidial germination or inhibition of appressorium formation.

3. A method for inhibiting rice blast fungus, characterized in that, This method achieves the prevention and control of rice blast disease by directly or indirectly treating rice blast fungus with chymostatin.

4. The method according to claim 3, characterized in that, The inhibition of rice blast fungus is manifested in the inhibition of rice blast fungus conidia germination or the inhibition of rice blast fungus appressorium formation.

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