Pharmaceutical composition for inhibiting fusarium graminearum and application thereof
By using compositions of serpentin, isopsoralen and serpentin, the drug resistance and environmental pollution problems of existing chemical drugs to prevent and treat wheat gibberellia were solved, and the effective and low-toxic inhibitory effect of Fusarium granium was achieved.
Patent Information
- Application Number
- CN202411906887.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-28
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing chemical drugs to prevent and treat wheat gibberellia have problems with drug resistance and environmental pollution, and there is a lack of low-toxic and low-pollution plant-source pesticides.
A combination of serpentin, isopsoralen and serpentin is used to prepare a drug or preparation with inhibitory activity of Fusarium granulis through synergistic action and spray it on plants to prevent and treat gibberellosis.
The inhibition rate of Fusarium granulis is improved. Compared with monomeric compounds, the composition has a higher antibacterial rate and has a synergistic effect, which slows down the generation of bacterial resistance and has important application value.
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Figure CN119699352A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pharmaceutical compositions, and in particular relates to a pharmaceutical composition for inhibiting Fusarium graminearum and an application thereof. Background Art
[0002] Wheat scab is mainly caused by Fusarium graminearum and is a common fungal disease that affects wheat yield. China is one of the most serious wheat scab-affected areas in the world's wheat-growing regions. The high incidence of scab has a serious impact on wheat yield, with a maximum reduction of 45%. Currently, the use of chemical drugs to prevent and control wheat scab is the most economical and effective method, but chemical control will cause the bacteria to develop drug resistance on the one hand, and will also cause certain environmental pollution on the other hand. Therefore, finding low-toxic and low-pollution botanical pesticides has become an important issue. At the same time, seeking to prevent and control wheat scab by combining drugs is also conducive to slowing down the development of drug resistance in bacteria. Summary of the invention
[0003] The present invention provides a composition with Fusarium graminearum inhibitory activity, wherein the composition comprises monomers of two or three compounds selected from osthole, isopsoralen and Zanthoxylum bungeanum toxin, and synergistic effects exist between the compounds.
[0004] In a specific embodiment, the composition consists of osthole and isopsoralen, and the mass ratio of osthole to isopsoralen is 1:1.
[0005] In a specific embodiment, the composition consists of osthole and xanthophyll toxin, and the mass ratio of osthole to xanthophyll toxin is 1:1.
[0006] In a specific embodiment, the composition consists of isopsoralen and xanthophyllotoxin, and the mass ratio of isopsoralen to xanthophyllotoxin is 1:1.
[0007] In a specific embodiment, the composition consists of osthole, isopsoralen and xanthoxylum bungeanum, and the mass ratio of osthole, isopsoralen and xanthoxylum bungeanum is 1:1:1.
[0008] The present invention provides the use of the above compound monomer or composition in the preparation of a medicine or preparation having Fusarium graminearum inhibitory activity.
[0009] The invention provides a preparation having the activity of inhibiting Fusarium graminearum, wherein the preparation contains the above compound monomer or combination.
[0010] In the above preparation, the concentration of the compound monomer or the composition is selected from 50 to 1000 μg / mL; preferably 250 μg / mL or 500 μg / mL.
[0011] The present invention provides a method for preventing and controlling Fusarium graminearum in plants, which comprises spraying a preparation containing the above compound monomer or combination on the plant body to achieve the prevention and control effect.
[0012] The beneficial effects of the present invention are:
[0013] The present invention provides a composition having the activity of inhibiting Fusarium graminearum, wherein the composition comprises two or three compound monomers of osthole, isopsoralen and zanthoxylum toxin. The inhibition rate of each composition on Fusarium graminearum is higher than that of each monomer, and there is a synergistic effect between the components in the composition, so the composition has important application value in the field of wheat head blight prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is the inhibition rate of mycelium of Fusarium graminearum by compounds and compositions at different concentrations;
[0015] Figure 2 The preventive and therapeutic effects of each compound monomer on Fusarium graminearum at 250 μg / mL; A is a blank control (no inoculation), B-E are preventive experiments, F is a negative control (inoculation), and G-J are therapeutic experiments; B, G: carbendazim, C, H: isopsoralen, D, I: osthole, E, J: Zanthoxytoxin;
[0016] Figure 3 The preventive and therapeutic effects of each compound monomer on Fusarium graminearum at 500 μg / mL; A is a blank control (no inoculation), B-E are preventive experiments, F is a negative control (inoculation), and G-J are therapeutic experiments; B, G: carbendazim, C, H: isopsoralen, D, I: osthole, E, J: Zanthoxytoxin;
[0017] Figure 4 The preventive effect of each composition on Fusarium graminearum at 250 μg / mL and 500 μg / mL; wherein A is a blank treatment; B-F are preventive experiments of different drug combinations at 250 μg / mL, B is carbendazim treatment, C is osthole + isopsoralen, D is osthole + zanthoxylum toxin, E is zanthoxylum toxin + isopsoralen, and F is osthole + zanthoxylum toxin + isopsoralen; G is a blank inoculation; H-L are preventive experiments of different drug combinations at 500 μg / mL, H is carbendazim treatment, I is osthole + isopsoralen, J is osthole + zanthoxylum toxin, K is zanthoxylum toxin + isopsoralen, and L is osthole + zanthoxylum toxin + isopsoralen;
[0018] Figure 5 The therapeutic effects of each composition on Fusarium graminearum at 250 μg / mL and 500 μg / mL; wherein A is a blank treatment; B-F are treatment experiments with different drug combinations at 250 μg / mL; B is carbendazim treatment, C is osthole + zanthoxylum toxin + isopsoralen, D is osthole + zanthoxylum toxin, E is osthole + isopsoralen, and F is zanthoxylum toxin + isopsoralen; G is a blank inoculation; H-L are treatment experiments with different drug combinations at 500 μg / mL; H is carbendazim treatment, I is osthole + zanthoxylum toxin + isopsoralen, J is osthole + zanthoxylum toxin, K is osthole + isopsoralen, and L is zanthoxylum toxin + isopsoralen. DETAILED DESCRIPTION
[0019] In the present invention, wheat seeds (Jimai 22) were provided by the College of Agriculture of Qingdao Agricultural University; osthole, isopsoralen and zanthoxylum toxin standards were purchased from Solebao Beijing Technology Co., Ltd., with a purity of HPLC ≥ 98%, and carbendazim was purchased from Solebao Beijing Technology Co., Ltd., with an active ingredient content of 50%. Fusarium graminearum was provided by the College of Plant Protection of Qingdao Agricultural University.
[0020] Other materials used in the present invention, if not otherwise stated, can be obtained through commercial channels. Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.
[0021] Example 1 In vitro antibacterial experiment of Fusarium graminearum
[0022] Prepare spore suspension:
[0023] The activated Fusarium graminearum was inoculated into sodium carboxymethyl cellulose (CMC) liquid culture medium, cultured in a shaking incubator at 28°C and 200 rpm for 6 h, and dispensed into 50 mL sterile centrifuge tubes. The supernatant was removed by centrifugation, and the spore concentration was adjusted with sterile ddH2O to a final concentration of 1×10 6 pcs / mL, for future use.
[0024] Weigh 2 mg of osthole, isopsoralen, zanthoxylum toxin, and carbendazim respectively and dissolve them in 1 mL of DMSO to prepare a 2000 μg / mL stock solution.
[0025] Using the doubling dilution method, add 50 μL of MH broth medium and 50 μL of spore suspension to each well of a 96-well plate. Add 100 μL of the mother solution to the first well, aspirate 100 μL and add it to the second well, blow it evenly, then aspirate 100 μL and add it to the next well, and remove 100 μL of the solution from the last well. Make the concentrations of columns 1 to 12 range from 1000 μg / mL to 0.488 μg / mL, and use 200 μL of sterile dd H2O as a blank control. Add 200 μL of spore suspension to column 12 as a positive control. Each well is paralleled with 3 copies, and cultured at 25°C for 7 days. Observe with the naked eye, and take the lowest concentration in the well without turbidity as the MIC value. Use the MIC value to set the concentration of subsequent experiments.
[0026] The experimental results showed that the minimum inhibitory concentration of each compound was 62.5 μg / mL, so 62.5 μg / mL was set as 1MIC value, and the concentrations were screened with MIC, 2MIC, 4MIC, and 8MIC. The mycelial growth rate inhibition method was used to prepare drug-containing plates with concentrations of 62.5 μg / mL, 125 μg / mL, 250 μg / mL, and 500 μg / mL for osthole, isopsoralen, cyperus rotten, osthole + isopsoralen (1:1), osthole + cyperus rotten (1:1), isopsoralen + cyperus rotten (1:1), and osthole + isopsoralen + cyperus rotten (1:1:1), respectively. An equal amount of DMSO was used as a blank control, and 2 mg / mL carbendazim was used as a positive control. After culturing at 27°C for 5 days in a constant temperature incubator, the mycelial growth diameter of each group against Fusarium graminearum was compared, and the inhibition rate was calculated. The inhibition rate formula is:
[0027] Inhibition rate = [colony diameter (CK) - colony diameter (treatment)] / colony diameter (CK) × 100%;
[0028] The test results are as follows Figure 1 As shown:
[0029] Depend on Figure 1 It can be seen that with the increase of different liquid concentrations, the antibacterial rate of each monomer compound and its combined compound liquid increases, and the antibacterial rate of the composition at 250μg / mL and 500μg / mL concentrations can basically match that of carbendazim. When the concentration reaches 500μg / mL, the antibacterial rates of osthole, isopsoralen, and zanthoxylum toxin reach 74.65%, 73.02%, and 65.68%, respectively, while the antibacterial rates of osthole + zanthoxylum toxin, osthole + isopsoralen, zanthoxylum toxin + isopsoralen, osthole + zanthoxylum toxin + isopsoralen can be as high as 95.45%, 98.77%, 100%, 100%, which is close to the 100% antibacterial rate of carbendazim, and can basically achieve the antibacterial effect of carbendazim.
[0030] In summary, osthole, zanthoxylum toxin and isopsoralen all have high inhibition rates against Fusarium graminearum. When they are combined in pairs or in combination, the inhibition rate is further improved when the usage amount of each monomer is the same, indicating that there is a synergistic effect among the three.
[0031] Example 2 In vivo resistance experiment of Fusarium graminearum
[0032] From the above experiments, it can be seen that the antibacterial effect of each compound monomer and the combination is more obvious at 250 μg / mL and 500 μg / mL. Therefore, these two concentrations were selected for the in vivo antibacterial test on wheat.
[0033] Spray test solution: Accurately weigh 10 mg each of osthole, isopsoralen, zanthoxylum toxin, and carbendazim, dissolve the above original drugs in 1 mL of DMSO as solvent, prepare a 10 mg / mL mother solution, dilute the mother solution to 250 μg / mL and 500 μg / mL, and prepare 2 mg / mL of carbendazim solution. Prepare 250 μg / mL and 500 μg / mL of osthole + isopsoralen (1:1), osthole + zanthoxylum toxin (1:1), isopsoralen + zanthoxylum toxin (1:1), and osthole + isopsoralen + zanthoxylum toxin (1:1:1) according to the volume ratio.
[0034] In vitro leaf method: Wheat (Jimai 22) was planted in disposable seedling pots, with 10 wheat plants planted in each pot, and cultured in a 22°C light incubator with a light / dark ratio of 12 / 12h. The test was carried out when the wheat plants grew to the 2-leaf stage. Using the in vitro leaf method, flat wheat leaves with uniform width were selected, divided into 3-3.5cm leaf segments, inserted into the green culture medium, and cultured in a 25°C constant temperature light incubator.
[0035] Prevention group: Spray 250μg / mL and 500μg / mL of osthole, isopsoralen, Zanthoxylum toxin, osthole + isopsoralen, osthole + Zanthoxylum toxin, isopsoralen + Zanthoxylum toxin, osthole + isopsoralen + Zanthoxylum toxin in a constant temperature and light incubator for 1 day. Use carbendazim solution as a positive contrast, make a circular wound on the upper surface of the leaf segment, and drip 10μL of spore suspension. Continue to culture for 2 days. Observe and record the spread of the lesions, and the experiment is repeated three times.
[0036] Treatment group: a circular wound was made in the middle of the leaf, 10 μL of spore suspension was dripped, and the leaves were cultured in a constant temperature incubator at 25°C for 2 days. After the growth of bacterial plaques, 250 μg / mL and 500 μg / mL of osthole, isopsoralen, zanthoxylum toxin, osthole + isopsoralen, osthole + zanthoxylum toxin, isopsoralen + zanthoxylum toxin, and osthole + isopsoralen + zanthoxylum toxin were sprayed respectively, and carbendazim solution was used as a positive contrast. After 2 days of culture in a constant temperature and light incubator, the control of lesions was observed, and the experiment was repeated three times.
[0037] The test results are as follows Figure 2 , Figure 3 , Figure 4 and Figure 5 As shown:
[0038] Depend on Figure 2 It can be seen that the uninfected wheat leaves (A) are healthy, with no signs of mycelium growth, the leaves are flat and bright green; the untreated infected wheat leaves (F) have obvious plaques, vigorous mycelium growth, and the leaves are yellow-green and wrinkled. When treated with a concentration of 250μg / mL, the results of the prevention experiment show that the wheat leaves treated with isopsoralen (C), osthole (D), and prickly ash toxin (E) all have a preventive effect on disease susceptibility, with small plaques, mainly distributed around the circular wounds, showing a white disease state, without a large number of mycelium, the leaves remain intact, there is yellowing, and the shrinkage phenomenon is not obvious. The results of the treatment experiment show that isopsoralen (H), osthole (I), and prickly ash toxin (J) all have good treatment effects. Spraying drugs after mycelium growth can remove most of the mycelium, and the leaves remain intact, but the yellowing phenomenon of the leaves still exists.
[0039] Depend on Figure 3 It can be seen that at a concentration of 500 μg / mL, the results of the prevention experiment show that wheat leaves treated with isopsoralen (C), osthole (D), and prickly ash toxin (E) all have a good effect in preventing disease, no large amount of hyphae are produced, and the leaves remain intact. The results of the treatment experiment show that isopsoralen (H), osthole (I), and prickly ash toxin (J) all have a good treatment effect. Spraying the drugs after the hyphae grows can remove the hyphae, but the leaves turn yellow and have a slight shrinkage phenomenon.
[0040] Depend on Figure 4 It can be seen that under the treatment of 250μg / mL and 500μg / mL concentrations, the wheat leaves without inoculation can remain bright green on the culture medium (A), while the mycelium of the wheat without spraying the solution grows obviously, showing reddish brown, and spreads to the culture medium to continue growing (G); the prevention effect of carbendazim is relatively good, the leaves remain intact, and only show yellowing due to fungal infection, without shrinkage (B);
[0041] When treated with 250 μg / mL concentration, the leaves of osthole + isopsoralen turned yellow due to fungal infection (C); no hyphae appeared under the treatment of osthole + zanthoxylum toxin, and although the leaves turned yellow, there was no shrinkage (D); under the treatment of zanthoxylum toxin + isopsoralen, the wheat leaves remained bright green, the surface hyphae were completely removed, and no shrinkage or yellowing occurred (E); under the treatment of osthole + zanthoxylum toxin + isopsoralen, no obvious hyphae were produced on the leaves, and the yellowing phenomenon existed, but compared with the treatment of carbendazim, the phenomenon was mild (F);
[0042] When treated with 500 μg / mL concentration, the leaves of osthole + isopsoralen turned yellow due to fungal infection (I); no hyphae appeared under the treatment of osthole + zanthoxylum toxin, and although the leaves turned yellow, there was no shrinkage (J); under the treatment of zanthoxylum toxin + isopsoralen, the wheat leaves remained bright green, the surface hyphae were completely removed, and no shrinkage or yellowing occurred (K); under the treatment of osthole + zanthoxylum toxin + isopsoralen, no obvious hyphae were produced on the leaves, and the leaves yellowed, but compared with the treatment of carbendazim, the phenomenon was mild (L).
[0043] Depend on Figure 5 It can be seen that under the treatment of 250μg / mL and 500μg / mL concentrations, the wheat leaves without inoculation can remain bright green on the culture medium (A), while the mycelium of the wheat inoculated without spraying the solution grows obviously, showing reddish brown, and spreads to the culture medium to continue growing (G); the treatment effect of carbendazim is better, the leaves remain intact, the wounds show brown spots, and they turn yellow due to fungal infection, with very small amounts of mycelium produced and no shrinkage (B);
[0044] When treated with 250 μg / mL concentration, hyphae were produced at the wound and leaves turned yellow under the treatment of osthole + prickly ash + isopsoralen, which was similar to the effect under the treatment of carbendazim (C); no large amount of hyphae appeared under the treatment of osthole + prickly ash, and although the leaves turned yellow, there was no shrinkage (D); the leaves of osthole + isopsoralen turned yellow due to fungal infection, and hyphae appeared, and the effect was average (E); under the treatment of prickly ash + isopsoralen, the wheat leaves remained bright green, the surface hyphae were completely removed, and no shrinkage or yellowing occurred (E);
[0045] When treated with 500 μg / mL concentration of osthole + zanthoxylum toxin + isopsoralen, almost no hyphae were produced at the wound, and yellowing of leaves occurred, which was better than the treatment with low concentration (I); no large amount of hyphae appeared under the treatment of osthole + zanthoxylum toxin, and although the leaves turned yellow, there was no shrinkage (J); due to fungal infection, the leaves of osthole + isopsoralen turned yellow, hyphae appeared, and the dark brown phenomenon at the wound was more obvious (K); under the treatment of zanthoxylum toxin + isopsoralen, the wheat leaves remained bright green, the surface hyphae were completely removed, and no shrinkage or yellowing occurred (L).
[0046] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.
Claims
1. A composition having Fusarium graminearum inhibitory activity, characterized in that: The components of the composition include two or three of osthole, isopsoralen and zanthoxylum bungeanum toxin.
2. The composition according to claim 1, characterized in that The composition consists of osthole and isopsoralen, and the mass ratio of osthole to isopsoralen is 1:
1.
3. The composition according to claim 1, characterized in that The composition consists of osthole and xanthoxylum bungeanum toxin, and the mass ratio of osthole to xanthoxylum bungeanum toxin is 1:
1.
4. The composition according to claim 1, characterized in that The composition consists of isopsoralen and zanthoxylum bungeanum toxin, and the mass ratio of isopsoralen to zanthoxylum bungeanum toxin is 1:
1.
5. The composition according to claim 1, characterized in that The composition consists of osthole, isopsoralen and xanthoxylum bungeanum toxin, and the mass ratio of osthole, isopsoralen and xanthoxylum bungeanum toxin is 1:1:
1.
6. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament or preparation having Fusarium graminearum inhibitory activity.
7. A preparation having Fusarium graminearum inhibitory activity, characterized in that: The preparation contains the composition according to any one of claims 1 to 5.
8. The preparation according to claim 7, characterized in that The concentration of the composition in the preparation is selected from 50 to 1000 μg / mL.
9. The preparation according to claim 8, characterized in that The concentration of the composition in the formulation is 250 μg / mL or 500 μg / mL.
10. A method for controlling Fusarium graminearum in plants, characterized in that: The method comprises spraying the preparation described in any one of claims 7 to 9 on plants to achieve the control effect of Fusarium graminearum.