Use of resveratrol in inhibiting deoxynivalenol production by fusarium graminearum
By using resveratrol to disrupt the cell membrane integrity of Fusarium graminearum, the growth and toxin production of Fusarium graminearum were significantly inhibited, solving the problem of poor control of fungal toxins in existing technologies and achieving a significant reduction in the content of fungal toxins.
Patent Information
- Application Number
- CN202411151424.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-21
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2044-08-21
AI Technical Summary
Existing technologies are not very effective in controlling fungal toxin contamination caused by Fusarium graminearum, especially the preparation methods for Fusarium deoxynivalensis, which suffer from incomplete adsorption, low adsorption of nutrients, and low in vitro degradation efficiency of microorganisms.
Using resveratrol as the main component, it significantly inhibits mycelial growth and the expression of the toxin-producing gene DON by disrupting the cell membrane integrity of Fusarium graminearum, causing cell death, and significantly inhibiting the expression of the key gene for DON synthesis. This demonstrates the application of resveratrol.
It has achieved an efficient and economical solution to the problem of mycotoxin contamination that is difficult to solve with existing technologies, significantly inhibiting the growth and toxin production of Fusarium graminearum and reducing the content of mycotoxins.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of fungi and mycotoxin prevention and control in agriculture, and particularly relates to an inhibitor containing resveratrol for fusarium producing deoxynivalenol (DON). BACKGROUND
[0002] Fusarium graminearum is the main pathogenic fungus causing wheat scab, which has caused serious threat to the yield and quality of wheat. Fusarium graminearum can produce mycotoxins such as deoxynivalenol after contaminating wheat and other cereals, thereby causing serious food safety and feed safety problems. Eating food contaminated by mycotoxins can cause acute and chronic poisoning in animals and humans, among which DON can cause vomiting and diarrhea in animals, induce gastrointestinal inflammation, inhibit the synthesis of proteins and nucleic acids, and induce cell apoptosis. At present, the methods for detoxifying mycotoxin-contaminated cereals and feed raw materials mainly include physical adsorption such as montmorillonite and microbial / enzyme degradation, but the actual application effect is poor, including incomplete adsorption, adsorption of nutrients, and low efficiency of microbial in vitro degradation.
[0003] The present application mainly aims at the problem of poor effect of existing mycotoxin prevention and control technology, takes the main toxin-producing fungus fusarium graminearum as the research object, and takes inhibiting fusarium producing DON as the main research idea, screens natural active substances that can significantly inhibit the growth and toxin production of fusarium from common Chinese herbal medicine natural products, and provides a technical approach for fusarium graminearum and its main mycotoxin prevention and control. SUMMARY
[0004] The first object of the present application is to provide a substance for preventing and controlling fusarium graminearum.
[0005] The second object of the present application is to provide the application of resveratrol in inhibiting fusarium graminearum producing DON.
[0006] The present application is realized by the following technical scheme:
[0007] The present application screens a variety of natural products such as resveratrol and lycium barbarum polysaccharide, and finds that resveratrol has the best performance in inhibiting the growth and toxin production of fusarium graminearum.
[0008] Specifically, resveratrol can significantly inhibit the mycelial growth of fusarium graminearum at a concentration of 80-320 μg / ml, and the growth inhibition rate of fusarium graminearum is about 50% and the DON production is reduced by about half under the treatment of 320 μg / mL resveratrol.
[0009] More specifically, resveratrol can significantly destroy the cell membrane integrity of fusarium graminearum, cause cell death, and significantly inhibit the expression of key genes Tri6 and Tri10 for producing DON. BRIEF DESCRIPTION OF DRAWINGS
[0010] Figure 1 is the mycelium diameter of F. graminearum at different culture time under different concentrations of resveratrol treatment.
[0011] Figure 2 is the growth inhibition rate of F. graminearum under different concentrations of resveratrol.
[0012] Figure 3 is the cell membrane damage effect of F. graminearum under different concentrations of resveratrol. A is the normal growth of F. graminearum mycelium, and B to D are F. graminearum mycelium treated with 80 μg / mL, 160 μg / mL and 320 μg / mL resveratrol respectively.
[0013] Figure 4 is the content of mycotoxin DON in F. graminearum culture under different concentrations of resveratrol treatment.
[0014] Figure 5 is the relative expression amount of mRNA of key genes Tri6 and Tri10 of DON produced by F. graminearum under different concentrations of resveratrol treatment. DETAILED DESCRIPTION
[0015] The present application will be further described below in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.
[0016] In the embodiment of the present application, the F. graminearum is F. graminearum isolated from moldy wheat.
[0017] Related terms are explained as follows: PDA: potato agar medium; PDB: potato medium; GYEP: glucose medium; PI: propidium iodide; FDA: fluorescein diacetate.
[0018] Example 1 Effect of resveratrol on inhibition of F. graminearum growth
[0019] 1. Experimental method
[0020] (1) Preparation of resveratrol stock solution In the clean bench, take 1.6 g of resveratrol sample and dissolve it in 100 mL of DMSO to obtain a 16 mg / mL resveratrol stock solution, which is stored in a -20℃ refrigerator.
[0021] (2) Preparation of PDA medium Weigh 2.4 g of PDB medium powder and 1.5 g of agar, pour them into a 200 mL conical flask, add deionized water to make up to 100 mL, use a high-pressure sterilization pot to sterilize at 121℃ for 25 min, and cool at room temperature for standby.
[0022] (3) Preparation of resveratrol PDA medium In the clean bench, resveratrol stock solution was added to PDA medium to a final concentration of 80, 160 and 320 μg / mL. The control group was added with an equal amount of DMSO solution.
[0023] (4) Solid culture of Fusarium graminearum and determination of the growth inhibition rate of resveratrol on Fusarium graminearum
[0024] ① Fusarium graminearum suspension was inoculated into PDA medium and evenly coated.
[0025] ② The inoculated petri dishes were placed in a 35°C constant temperature incubator.
[0026] ③ After the Fusarium graminearum covered the PDA plate, a sterile punch was used to make a 6mm diameter fungus cake at a distance of 1 / 3 from the center of the colony, which was inoculated in the center of the PDA petri dish containing resveratrol.
[0027] ④ The petri dish inoculated with the fungus cake was placed in a 35°C constant temperature incubator for 5 days.
[0028] ⑤ The diameter of the colony in each petri dish was measured every 24 hours.
[0029] ⑥ According to the growth inhibition rate (MGIR) formula, the percentage of the growth inhibition of resveratrol on Fusarium graminearum mycelium on the eighth day was calculated.
[0030] MGIR (%) = [(C-N) / (C-0.6)]x100 In the formula, C is the average diameter of the radial growth of the mycelium of the control group, unit: cm; N is the average diameter of the radial growth of the mycelium of the test group, 0.6 is the diameter of the fungus cake at the time of inoculation; unit: cm.
[0031] 2. Experimental results
[0032] ① The effect of resveratrol on the mycelial growth of Fusarium graminearum is shown in Table 1. Compared with the control group, the resveratrol treatment group significantly reduced the mycelial growth rate of Fusarium graminearum. Figure 1 ).
[0033] ② With the increase of the concentration of resveratrol, the inhibition rate of resveratrol on Fusarium graminearum gradually increased, and at a concentration of 320 μg / mL, the inhibition rate was about 50% ( Figure 2 ).
[0034] Example 2 Effect of resveratrol on the cell membrane of Fusarium graminearum mycelium
[0035] The effect of resveratrol on the cell membrane integrity of Fusarium graminearum hyphae was explored using the FDA-PI dual fluorescence counterstaining method. FDA (green) stained the cytoplasm of live cells, while PI (red) stained the nuclei of dead cells with damaged cell membranes (FDA excitation wavelength was 535 nm, and PI excitation wavelength was 617 nm).
[0036] 1. Experimental Methods
[0037] (1) Preparation of PDB medium containing resveratrol: Weigh 2.4g of PDB medium powder, pour it into a 200mL Erlenmeyer flask, add deionized water to make up to 100mL, autoclave at 121℃ for 25min, and cool at room temperature for later use.
[0038] (2) Liquid culture of Fusarium graminearum and determination of the effect of resveratrol on the cell membrane integrity of Fusarium graminearum.
[0039] ① Inoculate the Fusarium graminearum suspension into PDB medium and incubate at 180 rpm and 35°C for 7 days.
[0040] ② Take the Fusarium graminearum mycelium cultured for 7 days, centrifuge at 8000 rpm for 5 min at 4℃, remove the supernatant, and resuspend and wash with PBS (pH 7.4).
[0041] ③ Soak the mycelium in 1 mL of resveratrol at concentrations of 80, 160 and 320 μg / mL for 12 hours.
[0042] ④ Centrifuge at 8000 rpm for 5 min to remove liquid, add FDA and PI staining solution to a final concentration of 50 μg / mL and stain at room temperature in the dark for 15-20 min.
[0043] ⑤ Take a small amount of mycelium from the sample and place it on a glass slide. Press it gently with a coverslip to ensure that no air bubbles are generated.
[0044] ⑥ Use a fluorescence microscope to observe the staining of Fusarium hyphae at excitation wavelengths of 535 nm and 617 nm, and take pictures to record the results.
[0045] 2. Experimental Results
[0046] like Figure 3 As shown, compared with the control group ( Figure 3 Compared to group A, the resveratrol treatment group ( Figure 3 (BD) exhibits good killing effects on Fusarium graminearum mycelium, and with increasing resveratrol concentration, cell membrane damage worsens, and the number of dead cells increases. Note: Figure 3 The BD values were 80, 160, and 320 μg / mL of resveratrol, respectively.
[0047] Example 3: Determination of the inhibitory effect of resveratrol on the fungal toxin DON produced by Fusarium graminearum
[0048] 1. Experimental Methods
[0049] ① In 4 mL of Fusarium graminearum spore suspension (CFU = 1 × 10⁻⁶), 6 Add resveratrol to the solution at final concentrations of 0, 80, 160, and 320 μg / mL and incubate for 24 h.
[0050] ② Inoculate the incubated bacterial solution onto 100g of sterilized rice and culture it in an Erlenmeyer flask for 21 days, shaking and mixing once a day.
[0051] ③After fermentation is complete, dry and grind the rice.
[0052] ④ Extract 1g of powder sample with three times its volume of water, then add 600μL of ethyl acetate and shake to extract.
[0053] ⑤ Centrifuge at 3000 rpm for 1 min, collect the supernatant solution, and dry it with nitrogen gas.
[0054] ⑥ The sample was resuspended in 200 μL of 25% methanol, filtered through a 0.22 μm organic phase filter membrane, and the DON content in the sample was determined by HPLC.
[0055] ⑦ The mobile phase of HPLC is water and acetonitrile. The elution program is as follows: 0-12 min, the acetonitrile concentration increases from 12% to 29%; 12-14 min, the acetonitrile concentration increases from 29% to 90%; 14-15 min, the acetonitrile concentration remains unchanged; 15-16 min, the acetonitrile concentration decreases from 90% back to 12%; finally, equilibrate with 12% acetonitrile for 4 min.
[0056] 2. Experimental Results
[0057] like Figure 4 As shown, in rice inoculated with Fusarium graminearum, the DON content in the control group without resveratrol was approximately 7000 μM, the DON content in the 80 μg / mL resveratrol treatment group was approximately 7000 μM, and the DON content in the 320 μg / mL resveratrol treatment group was approximately 3000 μM, suggesting that resveratrol can significantly inhibit the production of DON by Fusarium graminearum.
[0058] Example 4: Effect of resveratrol on the expression of toxin-producing genes in Fusarium graminearum
[0059] 1. Experimental Methods
[0060] (1) Extraction and reverse transcription of resveratrol-treated Fusarium graminearum RNA
[0061] ①Collect the mycelium of Fusarium graminearum treated with different concentrations of resveratrol for 7 days, and add it to a 50 mL centrifuge tube. Centrifuge at 7000 rpm for 30 min at 4°C.
[0062] ②Discard the supernatant, resuspend with PBS, and repeat the previous steps 2-3 times to remove the culture medium impurities.
[0063] ③Weigh 50 mg of Fusarium graminearum mycelium and add it to a 1 mL RNase-free centrifuge tube. In a fume hood, add 1 mL Trizol reagent to each tube.
[0064] ④Grind the mycelium with a tissue homogenizer until there are no obvious solids.
[0065] ⑤Add the solution obtained in the previous step to a 1 mL RNase-free centrifuge tube and let it stand at room temperature for 5 min to allow complete lysis.
[0066] ⑥Add chloroform at a ratio of 1:5 with Trizol and mix well. Let it stand at room temperature for 2-3 min.
[0067] ⑦Centrifuge at 12000 rpm for 15 min at 4°C.
[0068] ⑧Transfer the upper aqueous phase to a new centrifuge tube and add an equal volume of isopropanol. Mix well by inverting and let it stand at 4°C for 5-10 min.
[0069] ⑨Centrifuge at 12000 rpm for 10 min at 4°C and discard the supernatant.
[0070] ⑩Add 1 mL of 75% ethanol and mix gently by inverting 15 times to suspend the precipitate.
[0071] ⑾Centrifuge at 12000 rpm for 5 min at 4°C and discard the supernatant.
[0072] ⑿Air dry at room temperature for 5 min, then dissolve the RNA with 30 μL of RNase-free deionized water. Store at -80°C for future use.
[0073] ⒀Use IIIAll-in one RT SuperMix kit to perform reverse transcription and synthesis of cDNA according to the manufacturer's instructions.
[0074] (2) RT-qPCR detection of Fusarium graminearum toxin gene Tri6 and Tri10 expression
[0075] Refer to Yeason qPCR SYBR Green quantitative PCR detection kit according to the manufacturer's instructions. Use Yeason qRT-PCR was performed by qPCR SYBR Green, and the relevant primers are shown in Table 1.
[0076] Table 1 Design of primers related to Fusarium graminearum toxinogenic genes
[0077]
[0078] 2. Experimental results
[0079] As shown in Figure 5 compared with the blank control group (without resveratrol), the expression of key genes Tri6 and Tri10 in the synthesis of DON in the resveratrol treatment group was significantly reduced, indicating that resveratrol can inhibit the ability of Fusarium graminearum to produce DON by affecting the expression of genes related to the synthesis of DON.
[0080] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.
Claims
1. Use of resveratrol in inhibiting the production of deoxynivalenol (DON) by Fusarium graminearum.
2. Use according to claim 1, characterized in that, Resveratrol inhibits the expression of genes Tri5 and Tri10 of Fusarium graminearum for producing DON.
3. Use according to claim 1, characterized in that, Use of resveratrol in preventing and controlling the contamination of DON in feed.