Application of hararine in prevention and treatment of wheat scab

Abstract

The invention discloses an application of hararine as a bacteriostatic agent of a plant pathogen fusarium graminearum, and an application of the hararine in prevention and treatment of wheat scab. The hararine is used for preventing and treating wheat scab by inhibiting mycelial growth of fusarium graminearum, inhibiting biosynthesis of deoxynivalenol and / or inhibiting expression of deoxynivalenol synthesis key genes. It is confirmed for the first time that the hararine has significant bacteriostatic activity on the main pathogenic bacterium fusarium graminearum of wheat scab, and the half inhibitory concentration (EC50) of the hararine is 56.71 [mu] g / mL. Meanwhile, the hararine can obviously inhibit the biosynthesis of DON toxin, the inhibition rate of the hararine to DON synthesis under the EC50 concentration can reach 25.04%, the inhibition rate of the hararine to the DON content in wheat leaves reaches up to 46.30%, and the hararine shows good dual effects of bacteriostasis and toxicity inhibition. The hararine is expanded to the field of agricultural disease prevention and control for the first time, and a new active substance source and a technical path are provided for green prevention and control of wheat scab.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of halogen alkaloids in the prevention and control of wheat scab. Background Technology

[0002] Wheat head blight (FHB) is caused by Fusarium graminearum (… Fusarium graminearum Fusarium graminearum is a global wheat disease that severely impacts wheat yield and quality. This disease not only reduces wheat production, but more seriously, Fusarium graminearum produces various mycotoxins during infection, among which deoxynivalenol (DON) is the most common and harmful. DON is highly toxic, highly stable, and difficult to remove, allowing it to persist in grains for extended periods. Ingestion by humans and animals can cause health problems such as vomiting, immunosuppression, and intestinal damage. Therefore, DON has been listed as a key monitoring target among natural food contaminants by the Food and Agriculture Organization of the United Nations and the World Health Organization.

[0003] Currently, the control of wheat scab mainly relies on chemical fungicides, such as triazoles and methoxyacrylates. However, the long-term and extensive use of chemical agents has brought about many problems: firstly, some agents, at sub-lethal concentrations, actually stimulate the synthesis of key genes for DON (such as...). TRI5 , TRI6 The expression of DON leads to increased accumulation; secondly, pesticide residues pose a potential risk to the environment and food safety; and thirdly, pathogens are prone to developing drug resistance, affecting control efficacy. Therefore, developing novel green pesticides that can effectively inhibit the growth of Fusarium graminearum and reduce DON accumulation has become an important direction in current research on the control of wheat scab.

[0004] Plant-derived natural metabolites have become an important resource for the development of novel fungicides due to their wide availability, structural diversity, good environmental compatibility, and novel mechanisms of action. Existing studies have shown that some plant secondary metabolites, such as thymol, flavonoids, and alkaloids, have a certain inhibitory effect on Fusarium graminearum. However, the currently reported active substances are still limited, and there is an urgent need to discover more natural products with both antibacterial and antitoxic functions.

[0005] Harmine is a natural β-carboline alkaloid widely found in *Phyllostachys edulis* (camel thorn). Peganum harmala Haraldine possesses various biological activities, including antioxidant, anti-inflammatory, and antitumor effects, in many plants. However, a literature search has revealed no research reports on the inhibitory effect of haraldine on Fusarium graminearum, nor any studies on its use in controlling wheat scab or inhibiting DON toxin synthesis.

[0006] This invention, starting from plant endogenous metabolites, has for the first time discovered and verified the inhibitory effect of harine on mycelial growth, DON synthesis and key gene expression of Fusarium graminearum, providing a new technical solution for the green control of wheat scab. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned problems. In a first aspect, the present invention provides harine as a plant pathogen of Fusarium graminearum (…). Fusarium graminearum Uses of antibacterial agents.

[0008] Secondly, the present invention provides the application of halogen alkaloids in the control of wheat scab, wherein the pathogen of wheat scab is *Fusarium graminearum* as described in the first aspect. Fusarium graminearum ).

[0009] Furthermore, the haline controls wheat scab by inhibiting the mycelial growth of Fusarium graminearum, inhibiting the biosynthesis of deoxynivalenol, and / or inhibiting the expression of key genes for deoxynivalenol (DON) synthesis.

[0010] Furthermore, the key gene for deoxynivalenol (DON) synthesis is... TRI5 Genes and / or TRI6 Gene.

[0011] Furthermore, the concentration of the halogen alkali used is not less than 56.71 μg / mL.

[0012] Thirdly, based on the same invention, this invention also provides a biological inhibitor for wheat scab, wherein the effective active ingredient of the biological inhibitor includes halogen alkaloid.

[0013] Harmine is an alkaloid with the molecular formula C2. 13 H 12 N2O has a relative molecular mass of 212.10.

[0014] Furthermore, the content of halogen in the biological inhibitor is not less than 56.71 μg / mL.

[0015] Furthermore, the biological inhibitor is applied by foliar spraying.

[0016] The present invention has the following beneficial effects: 1. This invention is the first to demonstrate that halogen alkaloids have significant antibacterial activity against Fusarium graminearum, the main pathogen of wheat scab, with a half-maximal inhibitory concentration (EC50) of [missing value]. 50 The concentration was 56.71 μg / mL. Simultaneously, halamine significantly inhibited the biosynthesis of DON toxin, at EC... 50At the specified concentration, the inhibition rate of DON synthesis reached 25.04%, and the inhibition rate of DON content in wheat leaves reached as high as 46.30%, showing good dual effects of antibacterial and antitoxic.

[0017] 2. This invention further reveals that harine inhibits key genes involved in DON synthesis in Fusarium graminearum. TRI5 and TRI6 The expression of [something] reduces the accumulation of DON toxin. Among them, [something] TRI5 The gene expression inhibition rate reached 32.64%. TRI6 The gene expression inhibition rate reached 12.22%, providing a theoretical basis for the application of halogen.

[0018] 3. Halalkaloid is a natural plant-derived alkaloid, derived from endogenous plant metabolites, exhibiting good biocompatibility and environmental compatibility. Experimental data show that at effective antibacterial concentrations, halalkaloid has no significant adverse effects on wheat growth, with a wheat fresh weight inhibition rate of only 2.22%. No symptoms of phytotoxicity such as dwarfing, yellowing, or wilting were observed, demonstrating its potential for development as a green biological pesticide.

[0019] 4. Currently, there are no research reports on the use of halalkaloids for the control of wheat scab or the inhibition of Fusarium graminearum and DON toxin. This invention is the first to extend halalkaloids to the field of agricultural disease control, providing a new source of active substances and technical pathways for the green control of wheat scab. 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 halogen alkaloids on the mycelial growth of Fusarium graminearum; Figure 2 The effect of halogen on the in vitro synthesis of DON, where A represents DON synthesis and B represents DON content; Figure 3 Haraldine's role in key genes of DON synthesis TRI5 (A) and TRI6 (B) The impact of expression; Figure 4 The effect of halogenated alkaloids on the DON content in wheat; Figure 5 The effects of halogenated alkaloids 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 utilizes the plant broad-target metabolome of wheat ears infected with *Fusarium graminearum* to screen for endogenous wheat metabolites whose levels were significantly upregulated compared to the control group. 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 fold increase in content and cost, 15 endogenous metabolites were selected, including 5 metabolites such as halogen alkaloid that inhibited *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 application of the wheat endogenous metabolite harzine in preventing wheat scab and the fungal toxin deoxynivalenol (DON). The study showed that harzine inhibited Fusarium graminearum mycelial growth, DON biosynthesis, and the relative expression levels of key DON synthesis genes under in vitro conditions.

[0027] Example 1: Indoor toxicity determination of halalkaloids against Fusarium graminearum 1.1 Experimental Materials and Methods The metabolite used in this invention is harmine (purchased from Shanghai Maclean's Biochemical Technology Co., Ltd.); the culture medium is PDA medium (200 g potato, 20 g glucose, 15 g agar powder, 1 L deionized water); and the bacterial strain is the standard wild-type strain of Fusarium graminearum. Fusarium graminearum PH-1 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.)

[0028] This invention uses the mycelial growth rate method to determine the antibacterial activity of halogen alkaloids against Fusarium graminearum.

[0029] Harmonic acid was dissolved in DMSO (dimethyl sulfoxide) and diluted to different concentrations, then added to different PDA media to form treatment groups with different concentration gradients (0, 5, 10, 20, 40, 60, 80 μg / mL). An equal volume of DMSO was added to the control group. Each treatment was repeated three times. The solutions were then poured into disposable petri dishes with a diameter of 9 cm. After cooling and solidification, Fusarium graminearum activated for 4 days was used to create mycelial discs at the colony edge using a 0.6 cm diameter punch and inoculated into these media containing different concentrations of harmonic acid. The culture dishes were incubated upside down in a 25 ℃ incubator in the dark for 4 days. The colony diameter of each treatment and the control group was measured using the cross-cross method, and the inhibition rate of harmonic acid on mycelial growth was calculated.

[0030] Mycelial growth inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / (Coronary diameter of control group) 0.6) × 100%.

[0031] 1.2 Experimental Results Using the logarithm of the concentration of different treatment groups as x and the probability value of mycelial growth inhibition rate as y, a linear regression equation was established, and the results are shown in Table 3.

[0032] Table 3. Results of indoor toxicity assay of harzine against Fusarium graminearum (PH-1)

[0033] Experimental results are as follows Figure 1 The concentrations added from left to right are 0, 5, 10, 20, 40, 60, and 80 μg / mL. As the concentration of harzine increases, the colony diameter of *Fusarium graminearum* decreases, indicating that the inhibitory effect of harzine on mycelial growth gradually increases with increasing concentration. Table 3 shows a correlation coefficient of R0. 2 =0.9512. The closer the correlation coefficient is to 1, the stronger the linear relationship between the concentration logarithm and the inhibition rate probability value established in this invention. This indicates a very strong causal relationship between concentration change and antibacterial effect. Based on this, the EC50 of harzine against Fusarium graminearum (PH-1) was calculated. 50 The concentration was 56.71 μg / mL. The above experimental results indicate that halogen alkaloids have a good inhibitory effect on the mycelial growth of Fusarium graminearum.

[0034] Example 2: Indoor and in vitro effects of halogenated alkaloids on DON production by Fusarium graminearum 2.1 Experimental Materials and Methods The culture medium used in this invention: 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 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.

[0035] 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. Take 1 mL of this spore suspension and inoculate it into a 50 mL Erlenmeyer flask containing TBI medium. All flasks are incubated at 28°C in the dark. After 2 days of incubation, add a certain concentration of harzine to the medium to achieve a concentration of 56.71 μg / mL (EC). 50 The blank control group received an equal volume of DMSO. 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.

[0036] DON reduction rate (%) = (DON content in control group - DON content in treatment group) / DON content in control group × 100%; Toxin production per unit dry weight of mycelium (μg / g) = Total DON content in TBI medium / Mycelium per unit dry weight; DON 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% The test reagents and test strains used in Example 2 were from the same sources as those in Example 1.

[0037] 2.2 Experimental Results Experimental results are as follows Figure 2As shown, the significance of EC between the control group and the treatment group was determined through one-way ANOVA. 50 Treatment with halogenated sodium hydroxide significantly reduced the DON content in the culture medium, with a reduction rate of up to 30.01%. Furthermore, calculations of mycelial toxin production per unit dry weight showed that EC... 50 Treatment with halaline at a certain concentration can also significantly inhibit the toxin production per unit dry weight of mycelium, with a DON inhibition rate of up to 25.04%, indicating that halaline can significantly inhibit DON synthesis and reduce the toxin production capacity of Fusarium graminearum.

[0038] As shown in Example 1, under in vitro indoor conditions, haline can not only effectively inhibit the mycelial growth of Fusarium graminearum, the pathogen of wheat scab, but also significantly inhibit the synthesis of DON, further reducing the damage caused by Fusarium graminearum.

[0039] Example 3: Haraldine's effect on key toxin-producing genes in Fusarium graminearum TRI5 , TRI6 The effect of expression on measurement 3.1 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. Take 1 mL of this spore suspension and inoculate it into a 50 mL Erlenmeyer flask containing TBI medium. All flasks are incubated at 28°C in the dark. After 2 days of incubation, add a certain concentration of harzine to the medium to achieve a concentration of 56.71 μg / mL (EC). 50 The blank control group received an equal volume of DMSO. After 4 days of incubation, 50 mL of TBI medium was vacuum filtered, flash-frozen in liquid nitrogen, and stored at -80 °C. All treatments were performed in triplicate.

[0040] 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%.

[0041] The culture medium, reagents, and test strains used in Example 3 were the same as those in Example 2.

[0042] 3.2 Experimental Results Haraldine affects key toxin-producing genes in Fusarium graminearum TRI5 , TRI6 The experimental results of the effect of relative expression level are as follows Figure 3 As shown, under indoor in vitro conditions, halogen treatment can simultaneously inhibit TRI5 and TRI6The relative expression levels and inhibition rates were 32.64% and 12.22%, respectively. Combined with the analysis of Example 2, it can be seen that halamine can inhibit... TRI5 and TRI6 Gene expression inhibits DON synthesis, thereby reducing the toxin-producing capacity of Fusarium graminearum.

[0043] Example 4: Determination of the effect of halogenated alkaloids on the DON content in wheat 4.1 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.

[0044] 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. Inoculate 10 μL onto wheat leaves, then spray with water and cover with a bag to maintain moisture. After 3 days, EC... 50 A concentration of halogenated sodium hydroxide was sprayed onto wheat leaves, while the control group was treated with water. Three days after the first spraying, the same concentration of halogenated sodium hydroxide 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°C. DON was extracted from the wheat leaves using the QuEChERS method, and its content was determined by HPLC.

[0045] 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 4 were the same as those in Example 2.

[0046] (2) Experimental results The effect of halogenated alkaloids on the DON content in wheat is as follows: Figure 4 As shown, when Fusarium graminearum infects wheat and then EC is used... 50 Treatment with halogenated alkaloids significantly inhibited the DON content in wheat leaves, with an inhibition rate of up to 46.30%. This result indicates that halogenated alkaloids can effectively inhibit DON content in outdoor in vivo experiments, reducing DON pollution and mitigating its harm to host crops such as wheat.

[0047] Example 5: Assessment of the effects of halogenated alkaloids on wheat growth 5.1 Experimental Materials and Methods EC 50A concentration of halogenated alkali was sprayed onto the leaves of healthy, uniformly growing wheat, while the control group was treated with water. The spraying was repeated three days later. Four days after the second spraying, the wheat growth was observed and the fresh weight of the wheat was weighed to calculate the fresh weight inhibition rate.

[0048] 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%.

[0049] The reagents used in Example 5 are the same as those in Example 1.

[0050] 5.2 Experimental Results EC 50 The effects of halogenated alkali treatment on wheat growth, such as Figure 5 And as shown in Table 4. From Figure 5 As can be seen from EC 50 Under treatment with EC, wheat leaves did not show obvious symptoms such as stunting, lodging, yellowing, chlorosis, or wrinkling, and the fresh weight of wheat did not decrease significantly compared with the control group. This result indicates that EC... 50 Treatment of wheat with halogenated alkali at a certain concentration will not have an adverse effect on wheat growth.

[0051] Table 4 Effects of halogenated alkaloids on wheat fresh weight

[0052] 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. Haraldine is used as a plant pathogen by Fusarium graminearum (… Fusarium graminearum Uses of antibacterial agents.

2. The application of halogen alkaloid in the control of wheat scab, characterized in that, The pathogen causing wheat scab is Fusarium graminearum as described in claim 1. Fusarium graminearum ).

3. The application of halogen alkaloids according to claim 2 in the control of wheat scab, characterized in that, The haline controls wheat scab by inhibiting the mycelial growth of Fusarium graminearum, inhibiting the biosynthesis of deoxynivalenol, and / or inhibiting the expression of key genes for deoxynivalenol synthesis.

4. The application of halogen alkaloids according to claim 2 in the control of wheat scab, characterized in that, The key gene for deoxynivalenol synthesis is... TRI5 Genes and / or TRI6 Gene.

5. The application of halogen alkaloids according to claim 2 in the control of wheat scab, characterized in that, The concentration of the halogenated base used is not less than 56.71 μg / mL.

6. A biological inhibitor for wheat scab, characterized in that, The active ingredient of the biological inhibitor includes halogen.

7. A biological inhibitor for wheat scab according to claim 6, characterized in that, The content of halogen in the bio-inhibitor is not less than 56.71 μg / mL.

8. A biological inhibitor for wheat scab according to claim 6, characterized in that, The biological inhibitor is applied by foliar spraying.

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