A fungus inhibitor and its application

Abstract

The present invention belongs to the field of pesticides, and in particular relates to a fungal inhibitor and its use. The present invention provides a fungal inhibitor comprising at least carvacrol, cuminaldehyde, and isoniazid. Compared to carvacrol, cuminaldehyde, or isoniazid alone, the fungal inhibitor exhibits significantly improved inhibitory effects against Neurospora crassa. This is demonstrated by the combination of carvacrol, cuminaldehyde, and isoniazid exhibiting a synergistic inhibitory effect, resulting in a reduction in the required minimum inhibitory concentration (MIC), yielding a technical benefit unexpected to those skilled in the art.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticides, and in particular relates to a fungus inhibitor and application thereof. Background Art

[0002] In nature, there are many types of fungi and they are widely distributed. Many of them are pathogenic or opportunistic. These fungi not only disrupt agricultural planting and animal husbandry, but may also pose a threat to human health. The prior art has disclosed some pesticides for preventing and treating fungal diseases, such as carbendazim, chlorpyrifos and myclobutanil. However, the aforementioned pesticides for preventing and treating fungal diseases have the following technical defects: some toxic and harmful substances, such as lime nitrogen and calcium cyanamide, are used in their production process; they are highly toxic and harmful to mammals, and can even cause teratogenicity, and their residues pollute the environment. Therefore, the development of green, environmentally friendly, low-cost, and less toxic fungal inhibitors or pesticides has become an urgent problem to be solved. Biological secondary metabolites provide a rich resource for the selection of natural fungicides.

[0003] Carvacrol is a volatile monoterpene phenol. A green and safe natural plant extract, it is abundant in Lamiaceae plants such as oregano, thyme, and winter savory. It has a thymol aroma and is used as a flavoring, additive, and pharmaceutical intermediate. Existing research has shown that carvacrol can inhibit the proliferation of cervical cancer spores, reduce oxidative stress damage in rats with diabetic nephropathy, inhibit the antibacterial activity of Zygosaccharomyces rouxii, and regulate the intestinal microbiome of lambs.

[0004] Cumaldehyde, with its cinnamon-like aroma, is commonly used as a component of plant essential oils. Naturally occurring in over 50 essential oils, including cumin oil, cassia oil, and cassia bark oil, cuminaldehyde has been shown to modulate the intestinal flora of rats, inhibit the white spot syndrome virus, and is not prone to drug resistance.

[0005] Isoniazid is an important anti-tuberculosis drug with high selectivity.

[0006] Hyphae are the basic structural units of fungi, providing nutrition and forming conidia. Conidia are the primary cells for fungal reproduction and spread. Furthermore, conidia possess a strong ability to withstand adverse environmental conditions. Inhibiting the formation of mycelium and conidia is crucial for inhibiting fungal spread and fungal diseases. Summary of the Invention

[0007] Technical problem: Provide a highly effective, green, environmentally friendly and low-cost fungal inhibitor for Neurospora crassa.

[0008] Technical solution:

[0009] In one aspect, a fungal inhibitor is provided, comprising at least carvacrol, cumaldehyde and isoniazid.

[0010] In some embodiments, the weight ratio of carvacrol, cumaldehyde, and isoniazid is 1:(1-10):(40-50).

[0011] In some embodiments, the weight ratio of carvacrol, cumaldehyde, and isoniazid is 1:10:50.

[0012] In some embodiments, the fungal inhibitor further comprises a solvent; the solvent is capable of dissolving carvacrol, cumaldehyde and isoniazid simultaneously.

[0013] In some embodiments, the solvent is Tween 80 solution.

[0014] In some embodiments, the solvent is a 0.1-2.0% Tween 80 solution.

[0015] In another aspect, the present invention provides use of the aforementioned fungal inhibitor for inhibiting Neurospora crassa and closely related ascomycetes.

[0016] In some embodiments, the closely related Ascomycetes include Aspergillus nidulans, Aspergillus fumigatus, and Stemphylium eturmiunum.

[0017] In some embodiments, 10 μL of a 2×10 6 A spore suspension of Neurospora crassa at a concentration of 100 μg / mL is dropped into a solid culture medium containing the fungal inhibitor for static culture, wherein the total concentration of the fungal inhibitor is not less than 100 μg / mL, and the weight ratio of carvacrol, cumaldehyde and isoniazid in the fungal inhibitor is 1:10:50.

[0018] In some embodiments, 100 μL of a 2×10 6 A spore suspension of Neurospora crassa at a concentration of spores / mL is dropped into 1.5 mL of a liquid culture medium containing the fungal inhibitor and cultured statically, wherein the total concentration of the antibacterial agent of the fungal inhibitor is not less than 9 μg / mL, and the weight ratio of carvacrol, cumaldehyde and isoniazid in the fungal inhibitor is 1:10:50.

[0019] Beneficial effects:

[0020] The present invention discovers and verifies that carvacrol, cuminic aldehyde, isoniazid, and their combination significantly inhibit the growth of Neurospora crassa. The antibacterial effect increases with increasing antibacterial concentration. The composition containing carvacrol, cuminic aldehyde, and isoniazid significantly improves the inhibitory effect on Neurospora crassa compared to single carvacrol, cuminic aldehyde, or isoniazid. This is demonstrated by the combination of carvacrol, cuminic aldehyde, and isoniazid exerting a synergistic inhibitory effect, producing unexpected technical effects for those skilled in the art in terms of the "amount" of the minimum antibacterial concentration required to delay the germination of Neurospora crassa, cause mycelial bleaching, and inhibit conidia formation.

[0021] The antibacterial composition of the present invention comprises carvacrol, isoniazid, and cuminaldehyde. The antibacterial composition of the present invention has a simple composition, low cost, and high antibacterial efficiency against Neurospora crassa and closely related ascomycetes (such as Aspergillus nidulans, Aspergillus fumigatus, and Stemphylium eturmiunum). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The inhibitory effects of different antibacterial agents in Example 1 on Neurospora crassa in solid culture medium;

[0023] Figure 2 The inhibitory effects of different antibacterial agents in Example 2 on Neurospora crassa in liquid culture medium;

[0024] Figure 3 The inhibitory effect of the volatilization of different antibacterial agents in Example 3 on Neurospora crassa;

[0025] Figure 4 This is the inhibitory effect of spraying different antibacterial agents on Neurospora crassa in Example 4. DETAILED DESCRIPTION

[0026] Example 1 - Synergistic inhibition of Neurospora crassa by carvacrol, cumaldehyde and isoniazid - solid culture medium

[0027] (1) Preparation of different antibacterial agent mother solutions

[0028] Preparation of 1 mg / mL carvacrol stock solution: Dissolve 10 mg of carvacrol in 10 mL of 0.1% Tween 80 solution and mix well to obtain 1 mg / mL carvacrol stock solution.

[0029] Preparation of 50 mg / mL cuminaldehyde stock solution: Add 500 mg of cuminaldehyde to 10 mL of 0.1% Tween 80 solution and mix well to obtain 50 mg / mL cuminaldehyde stock solution;

[0030] Preparation of 50 mg / mL isoniazid stock solution: Add 500 mg of isoniazid to 10 mL of 0.1% Tween 80 solution and mix thoroughly to obtain a 50 mg / mL isoniazid stock solution.

[0031] Preparation of a 1 mg / mL stock solution of the three-component composition: Carvacrol, cumaldehyde, and isoniazid were mixed in a weight ratio of 1:10:50 to obtain a three-component composition; 10 mg of the three-component composition was added to 10 mL of 0.1% Tween 80 solution and mixed to obtain a 1 mg / mL stock solution of the three-component composition.

[0032] (2) Preparation of Neurospora crassa spore suspension

[0033] Conidia of Neurospora crassa in solid culture medium were collected using 0.1% Tween 20 solution, counted using a hemocytometer, and diluted to a spore suspension concentration of 2 × 10 6 pieces / mL.

[0034] (3) Inhibitory effects of different antibacterial agents on Neurospora crassa in solid culture medium

[0035] Test method: 10 μL of a 2×10 6 After dropping a spore suspension of 10000 spores / mL onto the center of a Minimal Medium plate containing different concentrations of different antibacterial agents and a Minimal Medium plate without antibacterial agents (recorded as the control group), the plates were placed at 30°C for 8 days. The growth of the colonies on each plate was observed. The results are as follows: Figure 1 shown.

[0036] The composition and concentration of each solid culture medium antibacterial agent are as follows:

[0037] Carvacrol alone: ​​the concentrations of carvacrol in the culture medium were 100, 300, 400, and 500 μg / mL;

[0038] Cuminaldehyde alone: ​​the concentrations of cuminaldehyde in the culture medium were 100, 300, 400, and 500 μg / mL;

[0039] Isoniazid alone: ​​the concentrations of isoniazid in the culture medium were 100, 300, 400, and 500 μg / mL;

[0040] Three-component composition: The total antibacterial concentrations of the three-component composition in the culture medium (carvacrol, cumaldehyde and isoniazid weight ratio Car:Cum:Iso=1:10:50) were 10, 50, 100 and 200 μg / mL, respectively.

[0041] The study found that:

[0042] The inhibitory effect of carvacrol alone on Neurospora crassa in solid culture medium: When the concentration of carvacrol in the culture medium was not less than 100 μg / mL, the germination time of Neurospora crassa was prolonged, the mycelium turned white and grew slowly, brown spots appeared on the surface of the culture medium, and conidia were not formed ( Figure 1 ); when the concentration of carvacrol in the culture medium is not less than 300 μg / mL, the growth of Neurospora crassa can be completely inhibited.

[0043] The inhibitory effect of cumaldehyde alone on Neurospora crassa in solid culture medium: When the cumaldehyde concentration in the culture medium was not less than 300 μg / mL, the hyphal growth of Neurospora crassa was inhibited and the number of conidia was significantly reduced ( Figure 1 ); when the concentration of cuminaldehyde in the culture medium is not less than 400 μg / mL, the growth of Neurospora crassa is completely inhibited.

[0044] The inhibitory effect of isoniazid alone on Neurospora crassa in solid culture medium: When the isoniazid concentration in the culture medium was not less than 300 μg / mL, the mycelial morphology of Neurospora crassa was abnormal and the number of conidia was significantly reduced ( Figure 1 ); when the isoniazid concentration in the culture medium is not less than 400 μg / mL, the growth of Neurospora crassa is completely inhibited.

[0045] The inhibitory effect of carvacrol, cumaldehyde and isoniazid on Neurospora crassa in solid culture medium: when the total concentration of the three-component composition (carvacrol, cumaldehyde and isoniazid weight ratio Car:Cum:Iso=1:10:50) in the culture medium is not less than 50μg / mL, the germination time of Neurospora crassa is prolonged, the mycelium turns white and grows slowly, and the number of conidia is reduced ( Figure 1 ); when the total concentration of the three-component composition in the culture medium (carvacrol, cuminaldehyde and isoniazid weight ratio Car:Cum:Iso=1:10:50) is not less than 100 μg / mL, the growth of Neurospora crassa is completely inhibited.

[0046] By comparison, it can be seen that the combined use of carvacrol, cumaldehyde and isoniazid significantly enhances the inhibitory effect on Neurospora crassa in solid culture medium compared to the use of carvacrol, cumaldehyde and isoniazid alone, and this enhanced effect reduces the "amount" of the minimum inhibitory concentration required to delay the germination of Neurospora crassa, cause mycelium whitening and inhibit the formation of conidia, as well as the minimum inhibitory concentration required to completely inhibit the growth of Neurospora crassa, to an unexpected degree for those skilled in the art.

[0047] Example 2 - Carvacrol, cuminaldehyde and isoniazid synergistically inhibit Neurospora crassa - liquid culture medium

[0048] (1) Preparation of different antibacterial agent mother solutions: same as in Example 1;

[0049] (2) Preparation of Neurospora crassa spore suspension: same as in Example 1;

[0050] (3) Inhibitory effects of different antibacterial agents on Neurospora crassa in liquid culture medium

[0051] Test method: 100 μL of a 2×10 6 The spore suspension of 1000 / mL was dropped into 1.5mL Minimal Medium liquid culture medium containing different concentrations of different antibacterial agents and incubated at 30℃ for 7 days. The growth of colonies on each liquid culture medium was observed. The results are as follows: Figure 2 shown.

[0052] The composition and concentration of antibacterial agents in each liquid culture medium are as follows:

[0053] Carvacrol alone: ​​the final concentrations of carvacrol in the liquid culture medium were 0, 100, 200, 400, 600, and 800 μg / mL;

[0054] Cuminaldehyde alone: ​​the final concentrations of cuminaldehyde in the liquid culture medium were 0, 1000, 2000, 3000, 4000, 5000, and 6000 μg / mL;

[0055] Isoniazid alone: ​​the final concentrations of isoniazid in the liquid culture medium were 0, 1000, 2000, 3000, 4000, and 5000 μg / mL;

[0056] Three-component composition: The total final concentration of the antibacterial agent of the three-component composition (carvacrol, cumaldehyde and isoniazid weight ratio Car:Cum:Iso=1:10:50) in the liquid culture medium was 0, 1, 3, 5, 7, and 9 μg / mL, respectively.

[0057] The study found that:

[0058] The inhibitory effect of carvacrol alone on Neurospora crassa in liquid culture medium: when the concentration of carvacrol in the liquid culture medium is not less than 400 μg / mL, the amount of mycelium of Neurospora crassa is reduced and no conidia are formed; when the concentration of carvacrol in the liquid culture medium is not less than 600 μg / mL, the growth of Neurospora crassa is completely inhibited ( Figure 2 ).

[0059] The inhibitory effect of cumaldehyde alone on Neurospora crassa in liquid culture medium: when the cumaldehyde concentration in the liquid culture medium is not less than 4000 μg / mL, the amount of Neurospora crassa hyphae is reduced and no conidia are formed; when the cumaldehyde concentration in the liquid culture medium is not less than 6000 μg / mL, the growth of Neurospora crassa is completely inhibited ( Figure 2 ).

[0060] The inhibitory effect of isoniazid alone on Neurospora crassa in liquid culture medium: when the isoniazid concentration in the liquid culture medium is not less than 2000 μg / mL, the amount of mycelium of Neurospora crassa is reduced and no conidia are formed; when the isoniazid concentration in the liquid culture medium is not less than 3000 μg / mL, the growth of Neurospora crassa is completely inhibited ( Figure 2 ).

[0061] The inhibitory effect of the combined use of carvacrol, cumaldehyde and isoniazid on Neurospora crassa in liquid culture medium: when the concentration of the three-component composition in the liquid culture medium (the weight ratio of carvacrol, cumaldehyde and isoniazid Car:Cum:Iso=1:10:50) is not less than 7 μg / mL, the amount of Neurospora crassa hyphae is reduced and no conidia are formed; when the concentration of the three-component composition in the liquid culture medium (the weight ratio of carvacrol, cumaldehyde and isoniazid Car:Cum:Iso=1:10:50) is not less than 9 μg / mL, the growth of Neurospora crassa is completely inhibited ( Figure 2 ).

[0062] By comparison, it can be seen that the combined use of carvacrol, cumaldehyde and isoniazid significantly enhances the inhibitory effect on Neurospora crassa in liquid culture medium compared to the use of carvacrol, cumaldehyde and isoniazid alone, and this enhancement is a "quantitative" reduction in the minimum inhibitory concentration required to inhibit the growth of Neurospora crassa and the formation of conidia, as well as the minimum inhibitory concentration required to completely inhibit the growth of Neurospora crassa, which has reached an unexpected level for those skilled in the art.

[0063] Example 3 - Volatile Synergistic Inhibition of Neurospora crassa by Carvacrol, Cumaldehyde, and Isoniazid

[0064] (1) Preparation of Neurospora crassa spore suspension: same as in Example 1;

[0065] (2) The volatility of different antibacterial agents on the inhibitory effect of Neurospora crassa in solid culture medium

[0066] Take 10 μL of the solution with a concentration of 2×10 6 A spore suspension of 100 μL / mL was added to Minimal Medium solid culture medium. Then, 100 μL of a 0.1% Tween 80 solution (control), 100 μg of carvacrol, 100 μg of cumaldehyde, 100 μg of isoniazid, and a three-component combination (composition: 1 μg carvacrol, 10 μg cumaldehyde, 50 μg isoniazid) were added dropwise to filter paper in a separate plate and cultured for another 14 days.

[0067] The study found that:

[0068] The volatiles of carvacrol alone, cuminaldehyde alone, and the three-component combination (composition: carvacrol 1μg, cuminaldehyde 10μg, and isoniazid 50μg) could completely inhibit the growth of Neurospora crassa within 14 days. Among them, the three-component combination used the least amount of antibacterial agent and had the best antibacterial effect. However, the volatiles of isoniazid had no obvious inhibitory effect on Neurospora crassa ( Figure 3 ).

[0069] Example 4 - Spraying of carvacrol, cumaldehyde and isoniazid synergistically inhibits Neurospora crassa

[0070] (1) Preparation of different antibacterial agent mother solutions: same as in Example 1;

[0071] (2) Preparation of Neurospora crassa spore suspension: same as in Example 1;

[0072] (3) Inhibitory effect of spraying different antibacterial agents on Neurospora crassa

[0073] Test method: 10 μL of a 2×10 6 The spore suspension of 10000 / mL was dropped into Minimal Medium solid culture medium (plate) and placed in static culture at 30℃ for 5 days. Then different antibacterial agents with different concentrations were sprayed onto the plate, with the amount of antibacterial agent sprayed being 1mL. After spraying, the plate was placed in static culture at 30℃ for 14 days and the growth of colonies on each solid culture medium was observed. Figure 4 shown.

[0074] The composition of each solid culture medium and the concentration of antibacterial agent are as follows:

[0075] Control group: the culture medium did not contain antibacterial agent, and the concentration of Tween 80 solution was 0.1%;

[0076] Carvacrol alone: ​​the concentration of carvacrol in the culture medium was 100 mg / mL;

[0077] Cuminaldehyde alone: ​​the concentration of cuminaldehyde in the culture medium was 100 mg / mL;

[0078] Isoniazid alone: ​​the concentration of isoniazid in the culture medium was 100 mg / mL;

[0079] Three-component composition: The final concentrations of carvacrol, cuminaldehyde, and isoniazid in the three-component composition in the culture medium are 2 mg / mL, 20 mg / mL, and 100 mg / mL, respectively.

[0080] The study found that:

[0081] Compared with the control group, which began to germinate and grow hyphae and spores after 3 days of culture, the growth of Neurospora crassa was significantly inhibited after spraying carvacrol alone, cumaldehyde alone, isoniazid alone and the three-component combination (carvacrol, cumaldehyde and isoniazid weight ratio Car:Cum:Iso=1:10:50); within 14 days of culture after spraying the antibacterial agent, no hyphae and spores germinated on the plates sprayed with carvacrol, cumaldehyde, isoniazid and the three-component combination ( Figure 4 ).

Claims

1. A fungal inhibitor, characterized in that At least: Carvacrol, cumaldehyde and isoniazid, with the weight ratio of carvacrol, cumaldehyde and isoniazid being 1:(1-10):(40-50); and a solvent capable of simultaneously dissolving carvacrol, cumaldehyde and isoniazid.

2. The fungus inhibitor according to claim 1, characterized in that The weight ratio of carvacrol, cumaldehyde and isoniazid is 1:10:

50.

3. The fungus inhibitor according to claim 1, characterized in that The solvent is Tween 80 solution or dimethyl sulfoxide.

4. The fungus inhibitor according to claim 3, characterized in that The solvent is 0.1-2.0% Tween 80 solution.

5. Use of the fungal inhibitor according to any one of claims 1 to 4 for inhibiting Neurospora crassa and closely related ascomycetes.

6. The use according to claim 5, characterized in that The closely related ascomycetes include Aspergillus nidulans, Aspergillus fumigatus, and Stemphylium eturmiunum.

7. The use according to claim 5, characterized in that 10 μL of the solution was diluted to a concentration of 2 × 10 6 A spore suspension of Neurospora crassa at a concentration of 100 μg / mL is dropped into a solid culture medium containing the fungal inhibitor for static culture, wherein the total concentration of the fungal inhibitor is not less than 100 μg / mL, and the weight ratio of carvacrol, cumaldehyde and isoniazid in the fungal inhibitor is 1:10:

50.

8. The use according to claim 5, characterized in that 100 μL of the solution was added to a concentration of 2 × 10 6 A spore suspension of Neurospora crassa at a concentration of 1 μg / mL is dropped into 1.5 mL of a liquid culture medium containing the fungal inhibitor and cultured statically, wherein the total concentration of the antibacterial agent of the fungal inhibitor is not less than 9 μg / mL, and the weight ratio of carvacrol, cumaldehyde and isoniazid in the fungal inhibitor is 1:10:50.

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