Application of kaempferide and 4, 7-dimethyl naringenin in prevention and treatment of wheat scab

Abstract

The invention discloses an application of kaempferide and 4, 7-dimethyl naringenin in inhibition of synthesis of deoxynivalenol, in particular to an application of kaempferide and 4, 7-dimethyl naringenin in prevention and treatment of wheat scab, and a pathogenic bacterium of the wheat scab is fusarium graminearum. The kaempferide and the 4, 7-dimethyl naringenin are used for preventing and treating the wheat scab by inhibiting the biosynthesis of deoxynivalenol and / or inhibiting the expression of a key gene for synthesizing deoxynivalenol. The kaempferide and the 4, 7-dimethyl naringenin involved in the invention are wheat endogenous secondary metabolites, are derived from plants, have low toxicity to crops, are easy to degrade in the environment, and do not have the problem of residual pollution of chemical bactericides. Meanwhile, as the action mechanism is different from that of the existing chemical bactericide, the bactericide has no cross resistance with the common bactericide, and is suitable for resistance treatment and a green agricultural production system.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of kaempferol and 4,7-dimethylnaringenin in the prevention and control of wheat scab. Background Technology

[0002] Wheat head blight (FHB) is caused by Fusarium graminearum (… Fusarium graminearum Fusarium head blight is a global wheat disease caused by Fusarium head blight. Due to the generally low resistance of wheat to this disease and the scarcity of highly resistant varieties, it typically results in yield losses of over 10% annually, and in severe cases, can cause yield reductions of up to 50%. More seriously, during its infection of wheat ears, Fusarium graminearum produces various mycotoxins, including deoxynivalenol (DON). DON not only accelerates pathogen infection and promotes disease spread but also poses a serious threat to human and livestock health. Ingestion of DON-contaminated grains can cause nausea, vomiting, and diarrhea in humans and animals; long-term exposure may damage the immune system and pose a carcinogenic risk. Furthermore, DON exhibits extremely high thermal and chemical stability, making it difficult to degrade under weakly acidic, high-temperature, and high-pressure conditions. Once contaminated with grain products, it is almost impossible to remove through conventional processing methods. Therefore, effectively controlling Fusarium head blight and reducing DON contamination levels in grains has become a major issue for ensuring food safety and promoting sustainable agricultural development.

[0003] Currently, production mainly relies on chemical fungicides to control Fusarium head blight, thereby indirectly reducing DON accumulation. However, numerous studies have found significant limitations in the application of chemical fungicides. When fungicide concentrations drop to sub-lethal levels, they not only fail to effectively inhibit DON synthesis but may even exacerbate its accumulation. For example, methoxyacrylate fungicides (such as azoxystrobin) can upregulate key genes in DON biosynthesis by increasing acetyl-CoA levels. Tri5 , Tri6 The expression of triazole fungicides (such as tebuconazole) increases toxin production by inhibiting sterol synthesis, leading to the diversion of precursors into the DON synthesis pathway. Furthermore, the residue problem of chemical fungicides is becoming increasingly prominent, and their bioaccumulation effects in the environment and organisms pose potential ecological risks. Therefore, there is an urgent need to find novel active substances with lower toxicity, better environmental compatibility, and the ability to simultaneously inhibit the growth of Fusarium graminearum or its toxin-producing capacity.

[0004] In recent years, plant- or microbial secondary metabolites have attracted much attention due to their low toxicity and environmental friendliness. Existing studies have shown that some natural products, such as thymol, can inhibit the growth of *Fusarium graminearum*, while benzoxazines and flavonoids can inhibit DON accumulation. However, these natural products are numerous, and currently only a very small number have been proven to possess bactericidal or antiviral activity against *Fusarium graminearum*, far from meeting the needs of green pesticide development. In particular, research on the inhibitory effects of wheat endogenous flavonoids such as kaempferide and 4,7-di-O-methylnaringenin on toxin production by *Fusarium graminearum* has not been reported. Therefore, screening more substances with antibacterial or antiviral activity from plant endogenous metabolites, especially exploring new uses for natural flavonoids such as kaempferide and 4,7-di-O-methylnaringenin, is of significant practical importance for developing new green pesticides and ensuring food security. Summary of the Invention

[0005] This invention aims to solve the above-mentioned problems. In a first aspect, this invention provides the application of kaempferide in inhibiting the synthesis of deoxynivalenols by Fusarium oxysporum. Kaempferide belongs to the flavonoid class of substances and has the molecular formula C2. 16 H 12 O6 has a relative molecular mass of 300.06.

[0006] Secondly, this invention provides the application of 4,7-di-O-methylnaringenin in inhibiting the synthesis of deoxynivalenol. 4,7-Di-O-methylnaringenin belongs to the flavonoid family and has the molecular formula C2. 17 H 16 O5 has a relative molecular mass of 300.10.

[0007] Thirdly, this invention provides the application of kaempferol and 4,7-dimethylnaringenin in the control of wheat scab, wherein the pathogen of wheat scab is Fusarium graminearum (…). Fusarium graminearum ).

[0008] Furthermore, the kaempferol and 4,7-dimethylnaringenin control wheat scab by inhibiting the biosynthesis of deoxynivalenol and / or inhibiting the expression of key genes for deoxynivalenol synthesis.

[0009] Furthermore, the key gene for deoxynivalenol synthesis is... TRI5 Genes and / or TRI6 Gene.

[0010] Furthermore, the concentration of kaempferol used is not less than 30 μg / mL, and the concentration of 4,7-dimethylnaringenin used is not less than 30 μg / mL.

[0011] Fourthly, the present invention provides a biological inhibitor for wheat scab, wherein the effective active ingredient of the biological inhibitor comprises kaempferol and / or 4,7-dimethylnaringenin.

[0012] Furthermore, the concentration of the kaempferol and / or 4,7-dimethylnaringenin used is not less than 30 μg / mL.

[0013] Fifthly, the present invention provides a composition for preventing and controlling wheat scab, comprising kaempferol and / or 4,7-dimethylnaringenin as active ingredients.

[0014] The present invention has the following beneficial effects: 1. This invention is the first to discover that the endogenous wheat flavonoids kaempferol and 4,7-dimethylnaringenin can significantly inhibit the toxin production capacity of Fusarium graminearum. In vitro experiments showed that treatment with 30 μg / mL kaempferol and 4,7-dimethylnaringenin reduced the DON content in the culture medium by 30.98% and 20.10%, respectively, and reduced the toxin production per unit dry weight of mycelium by 27.28% and 22.17%, respectively. Comparative experiments also confirmed that eight other endogenous wheat metabolites (such as 4-hydroxyacetophenone, taurine, and farnesin) did not show significant DON inhibition under the same conditions, indicating that kaempferol and 4,7-dimethylnaringenin are specific for inhibiting DON synthesis.

[0015] 2. This invention further elucidates the mechanism by which kaempferol and 4,7-dimethylnaringenin inhibit DON synthesis. Experiments have demonstrated that kaempferol can significantly inhibit key genes in DON biosynthesis. Tri5 The expression of [a specific gene] was inhibited at a rate of 52.41%; 4,7-dimethylnaringenin significantly inhibited the expression of another key gene. Tri6 The expression of [a specific gene] was inhibited at a rate of 16.89%. Both [the methods] block the DON synthesis pathway at the transcriptional level by regulating the expression of different toxin-producing genes, providing a theoretical basis for the development of targeted antitoxin agents.

[0016] 3. This invention verifies the efficacy of kaempferol and 4,7-dimethylnaringenin under in vivo conditions. After wheat was infected with Fusarium graminearum, spraying with 30 μg / mL of kaempferol and 4,7-dimethylnaringenin significantly reduced the DON content in wheat leaves, with inhibition rates reaching 34.27% and 20.82%, respectively. This indicates that these two compounds can still effectively inhibit toxin accumulation and reduce the harm of DON to wheat quality in actual agricultural production scenarios.

[0017] 4. This invention discovered that the combined use of kaempferol and 4,7-dimethylnaringenin exhibits a significant synergistic effect. After treatment with the mixture, the DON synthesis inhibition rate reached 36.47% and the DON content reduction rate reached 38.46% under in vitro conditions; under in vivo conditions, the DON content reduction rate in wheat leaves reached 41.53%, both significantly higher than the effects of either single agent treatment. This synergistic effect allows for better antitoxic effects at lower concentrations, which is beneficial for reducing usage costs.

[0018] 5. This invention confirms that kaempferol and 4,7-dimethylnaringenin are safe and non-toxic to wheat growth. At a concentration of 30 μg / mL, wheat leaves did not exhibit symptoms of phytotoxicity such as yellowing, stunting, or wrinkling. Compared with the control group, the fresh weight of wheat in the kaempferol-treated group, the 4,7-dimethylnaringenin-treated group, and the mixed treatment group increased by 16.00%, 16.89%, and 19.56%, respectively, demonstrating a certain growth-promoting effect. This indicates that the compounds of this invention can effectively inhibit toxins while also promoting healthy crop growth.

[0019] 6. The kaempferol and 4,7-dimethylnaringenin involved in this invention are both endogenous secondary metabolites of wheat, derived from plants, with low toxicity to crops, and are easily degraded in the environment, thus avoiding the residual pollution problems of chemical fungicides. Furthermore, because their mechanisms of action differ from existing chemical fungicides, they do not exhibit cross-resistance with commonly used fungicides, making them suitable for resistance management and green agricultural production systems. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0021] Figure 1 Effects of kaempferol and 4,7-dimethylnaringenin on DON synthesis (A) and DON content (B) in vitro and in vitro. Figure 2 Kaempferol and 4,7-dimethylnaringenin are key genes in DON synthesis. TRI5 (A) and TRI6 (B) The impact of expression; Figure 3 Effects of kaempferol and 4,7-dimethylnaringenin on DON content in wheat; Figure 4 Effects of kaempferol and 4,7-dimethylnaringenin on wheat growth. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0023] This invention screened endogenous wheat metabolites whose levels were significantly upregulated compared to the control group by measuring the broad-target metabolome of wheat ears infected with Fusarium graminearum. A total of 652 endogenous metabolites with significant changes in content were detected, of which 396 showed significant upregulation. Subsequently, based on factors such as the upregulation factor and cost, 15 endogenous metabolites were selected for further experiments, of which 5 showed inhibitory effects on Fusarium graminearum mycelial growth or DON synthesis (Tables 1 and 2).

[0024] Table 1. Effects of 15 endogenous wheat metabolites on the mycelial growth of Fusarium graminearum.

[0025] Table 2. Effects of 15 wheat endogenous metabolites on DON biosynthesis in Fusarium graminearum

[0026] This invention investigated the applications of wheat endogenous metabolites kaempferol and 4,7-dimethylnaringenin in preventing wheat scab and the fungal toxin deoxynivalenol (DON). The study demonstrated that kaempferol and 4,7-dimethylnaringenin inhibited DON biosynthesis and the relative expression levels of key DON synthesis genes under in vitro and indoor conditions.

[0027] Example 1: Indoor and in vitro effects of kaempferol and 4,7-dimethylnaringenin on DON yield 1.1 Experimental Materials and Methods The metabolites used in this invention are kaempferide (Shanghai Maclean Biochemical Technology Co., Ltd.) and 4,7-di-O-methylnaringenin (Shanghai Yuanye Biotechnology Co., Ltd.).

[0028] PDA medium (200 g potato, 20 g glucose, 15 g agar powder, 1 L deionized water) was used to activate the strain, and CMC medium (15 g carboxymethyl cellulose, 2 g NaNO3, 0.5 g MgSO4·7H2O, 1 g KH2PO4, 1 g Yeast Extract, 1 L deionized water) was used to culture the spores. TBI medium (30 g sucrose, 1 g KH2PO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 g FeSO4·7H2O, 0.8 g putrescine, 200 μL trace element B, 1 L deionized water) was used to induce Fusarium graminearum to produce DON. Trace element B (100 mL) contains 5 g Citric acid, 5 g ZnSO4·7H2O, 0.25 g CuSO4·5H2O, 0.05 g MnSO4·H2O, 0.05 g H3BO4, and 0.05 g Na2MoO4·2H2O.

[0029] The strain is the standard wild-type strain of Fusarium graminearum. Fusarium graminearum PH-1 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.)

[0030] Using a punch, fungal discs were extracted from the edges of activated colonies in PDA medium and transferred to CMC medium. The cultures were incubated at 25°C in the dark with shaking at 200 rpm for 5 days. The resulting fungal culture was filtered to obtain a spore suspension, which was then adjusted to a final concentration of 1×10⁻⁶. 6 / mL. Take 1 mL of this spore suspension and inoculate it into a 50 mL Erlenmeyer flask containing TBI medium. All flasks are placed in a 28°C incubator in the dark. After 2 days of culture, certain concentrations of kaempferol, 4,7-dimethylnaringenin, and a mixture of both are added to the medium to achieve a concentration of 30 μg / mL for each of the three substances (EC50 cannot be calculated because the two metabolites do not inhibit the growth of *Fusarium graminearum*). 50 Considering solubility, 30 μg / mL was chosen as the uniform experimental concentration. An equal volume of DMSO was added to the blank control group. After 4 days of incubation, 1 mL of culture medium was extracted from 50 mL of TBI medium and filtered through a 0.22 μm aqueous filter membrane into a brown vial for subsequent HPLC analysis. The remaining culture medium was vacuum filtered, dried, and weighed. All treatments were performed in triplicate.

[0031] DON content reduction rate (%) = (DON content in control group - DON content in treatment group) / DON content in control group × 100% Toxin production per unit dry weight mycelium (μg / g) = Total DON content in TBI medium / Mycelium per unit dry weight DON synthesis inhibition rate (%) = (Toxin production per unit dry weight of mycelium in control group - Toxin production per unit dry weight of mycelium in treatment group) / Toxin production per unit dry weight of mycelium in control group × 100% 1.2 Experimental Results Experimental results are as follows Figure 1 As shown, the significance analysis between the control and treatment groups using an independent samples t-test revealed that treatment with 30 μg / mL kaempferol and 4,7-dimethylnaringenin significantly reduced the DON content in the culture medium (B), with reduction rates of 30.98% and 20.10%, respectively. Furthermore, calculation of mycelial toxin production per unit dry weight showed that treatment with 30 μg / mL kaempferol and 4,7-dimethylnaringenin also significantly inhibited mycelial toxin production per unit dry weight (A), with DON synthesis inhibition rates of 27.28% and 22.17%, respectively. This indicates that kaempferol and 4,7-dimethylnaringenin can significantly inhibit DON synthesis and reduce the toxin-producing capacity of *Fusarium graminearum*.

[0032] Furthermore, the combined treatment with kaempferol and 4,7-dimethylnaringenin also significantly reduced the DON content and the toxin production per unit dry weight of mycelium in TBI medium. The DON content reduction rate and synthesis inhibition rate were 38.46% and 36.47%, respectively, which were greater than the inhibition rates of the two metabolites alone. This indicates that the combination of kaempferol and 4,7-dimethylnaringenin can more effectively inhibit DON synthesis.

[0033] Example 2: Kaempferol and 4,7-Dimethylnaringenin on key toxin-producing genes in Fusarium graminearum TRI5 , TRI6 The effect of expression on measurement 2.1 Experimental Materials and Methods Using a punch, collect fungal discs from the edge of the growing colony and transfer them to CMC medium. Incubate at 25°C in the dark with shaking at 200 rpm for 5 days. Filter the resulting fungal culture to obtain a spore suspension, then adjust to a final concentration of 1×10⁻⁶. 6 / mL. 1 mL of the spore suspension was inoculated into a 50 mL Erlenmeyer flask containing TBI medium. All flasks were incubated at 28°C in the dark. After 2 days of incubation, kaempferol and 4,7-dimethylnaringenin were added to the medium to achieve a concentration of 30 μg / mL for each, respectively. An equal volume of DMSO was added to the blank control group. After 4 days of incubation, 50 mL of TBI medium was vacuum filtered under reduced pressure, flash-frozen in liquid nitrogen, and stored at -80°C. All treatments were performed in triplicate.

[0034] Gene expression inhibition rate (%) = (relative gene expression level in control group - relative gene expression level in treatment group) / relative gene expression level in control group × 100%.

[0035] The culture medium, reagents, and test strains used in Example 2 were the same as those in Example 1.

[0036] 2.2 Experimental Results Kaempferol and 4,7-dimethylnaringenin target key toxin-producing genes in Fusarium graminearum TRI5 , TRI6 The experimental results of the effect of relative expression level are as follows Figure 2 As shown. Under in vitro conditions, kaempferol significantly inhibited... TRI5 The relative expression level was inhibited by 52.41%, while 4,7-dimethylnaringenin significantly inhibited [the expression level]. TRI6 The relative expression level was inhibited by 16.89%. Combined with the analysis in Example 1, it can be seen that kaempferol and 4,7-dimethylnaringenin can inhibit... TRI5 or TRI6 Gene expression inhibits DON synthesis, thereby reducing the toxin-producing capacity of Fusarium graminearum.

[0037] Example 3: Determination of the effects of kaempferol and 4,7-dimethylnaringenin on DON content in wheat 3.1 Experimental Materials and Methods The wheat variety used in this invention is Jimai 22, a semi-winter wheat widely planted in the Huang-Huai-Hai region.

[0038] Using a punch, collect fungal discs from the edge of the growing colony and transfer them to CMC medium. Incubate at 25°C in the dark with shaking at 200 rpm for 5 days. Filter the resulting fungal culture to obtain a spore suspension, then adjust to a final concentration of 1×10⁻⁶. 5 / mL. 10 μL was inoculated onto wheat leaves, followed by spraying with water and covering with a bag to maintain humidity. Three days later, 30 μg / mL of kaempferol, 4,7-dimethylnaringenin, and a mixture thereof were sprayed onto the wheat leaves. The control group was treated with water. Five days after the first spraying, the same concentration of kaempferol, 4,7-dimethylnaringenin, and a mixture thereof was sprayed again. Four days after the second spraying, the wheat leaves were removed with scissors, flash-frozen in liquid nitrogen, and stored at -80℃. DON was extracted from the wheat leaves using the QuEChERS method, and the DON content was determined by HPLC.

[0039] DON content inhibition rate (%) = (DON content per unit weight of wheat in the control group - DON content per unit weight of wheat in the treatment group) / DON content per unit weight of wheat in the control group × 100% The culture medium, reagents, and test strains used in Example 3 were the same as those in Example 1.

[0040] 3.2 Experimental Results The effects of kaempferol, 4,7-dimethylnaringenin, and mixtures thereof on the DON content in wheat are as follows: Figure 3 As shown, treatment with 30 μg / mL kaempferol and 4,7-dimethylnaringenin after Fusarium graminearum infection of wheat significantly inhibited the DON content in wheat leaves, with inhibition rates of 34.27% and 20.82%, respectively. These results indicate that kaempferol and 4,7-dimethylnaringenin can effectively inhibit the DON content in wheat in outdoor in vivo experiments, reducing DON contamination and mitigating its harm to host crops such as wheat.

[0041] Furthermore, treating wheat infected with Fusarium graminearum with a mixture of 30 μg / mL kaempferol and 4,7-dimethylnaringenin reduced the DON content in wheat by 41.53%, and the inhibitory effect of the mixture on the DON content in wheat was stronger than that of either metabolite alone.

[0042] Example 4: Evaluation of the effects of kaempferol and 4,7-dimethylnaringenin on wheat growth 4.1 Experimental Materials and Methods A mixture of 30 μg / mL kaempferol, 4,7-dimethylnaringenin, and kaempferol was sprayed onto healthy, uniformly growing wheat leaves. The control group was treated with water. The spraying was repeated after 5 days. Four days after the second spraying, wheat growth was observed and the fresh weight of the wheat was measured to calculate the fresh weight inhibition rate.

[0043] Fresh weight inhibition rate (%) = (fresh weight of wheat leaves in control group - fresh weight of wheat leaves in treatment group) / fresh weight of wheat leaves in control group × 100%.

[0044] The reagents used in Example 4 are the same as those in Example 1.

[0045] 4.2 Experimental Results The effects of treatments with 30 μg / mL concentrations of kaempferol, 4,7-dimethylnaringenin, and a mixture thereof on wheat growth are as follows: Figure 4 As shown in Table 3, the results indicate that treatment with 30 μg / mL kaempferol, 4,7-dimethylnaringenin, and their mixture did not result in significant stunting, lodging, yellowing, or wrinkling of wheat leaves. Furthermore, the fresh weight of wheat in all three treatment groups increased compared to the control group. These results suggest that treatment with 30 μg / mL kaempferol, 4,7-dimethylnaringenin, and their mixture not only does not adversely affect wheat growth but also promotes it to some extent.

[0046] Table 3. Effects of kaempferol, 4,7-dimethylnaringenin, and mixtures thereof on wheat fresh weight.

[0047] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. Application of kaempferol in inhibiting deoxynivalenol synthesis by Fusarium oxysporum. Application of 2,4,7-Dimethylnaringenin in inhibiting the synthesis of deoxynivalenol by Fusarium oxysporum.

3. The application of kaempferol and 4,7-dimethylnaringenin in the control of wheat scab, characterized in that, The pathogen causing wheat scab is Fusarium graminearum (Fusarium graminearum). Fusarium graminearum ).

4. The application of kaempferol and 4,7-dimethylnaringenin according to claim 3 in the control of wheat scab, characterized in that, Kaempferol and 4,7-dimethylnaringenin control wheat scab by inhibiting the biosynthesis of deoxynivalenol and / or inhibiting the expression of key genes involved in deoxynivalenol synthesis.

5. The application of kaempferol and 4,7-dimethylnaringenin according to claim 4 in the control of wheat scab, characterized in that, The key gene for deoxynivalenol synthesis is... TRI5 Genes and / or TRI6 Gene.

6. The application of kaempferol and 4,7-dimethylnaringenin according to claim 3 in the control of wheat scab, characterized in that, The concentration of kaempferol used is not less than 30 μg / mL, and the concentration of 4,7-dimethylnaringenin used is not less than 30 μg / mL.

7. A biological inhibitor for wheat scab, characterized in that, The active ingredients of the bioinhibitor include kaempferol and / or 4,7-dimethylnaringenin.

8. A biological inhibitor for wheat scab according to claim 7, characterized in that, The concentration of kaempferol and / or 4,7-dimethylnaringenin used shall not be less than 30 μg / mL.

9. A composition for controlling wheat scab, characterized in that, It contains kaempferol and / or 4,7-dimethylnaringenin as active ingredients.

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