Volatile substance and method for inhibiting growth of fusarium oxysporum

Abstract

The invention belongs to the technical field of using L-menthyl acetate as a novel plant source HDAC activator for inhibiting fungal growth, and discloses a volatile substance and a method for inhibiting growth of fusarium oxysporum by reducing H3K14ac and H3K27ac levels. According to the present invention, the monomer component L-menthyl acetate of the mint volatile oil can inhibit the activity of Fusarium oxysporum histone deacetylase (FoArginase HDAC), and inhibit the expression of the ribosome pathway key gene of Fusarium oxysporum; molecular docking shows that the L-menthyl acetate can interact with amino acid residues such as His240, His203, Phe242 and the like in Fusarium oxysporum histone deacetylase FoArginaseHDAC, so that the activity of the FoArginaseHDAC is improved, and the H3K14ac level and the H3K27ac level of the Fusarium oxysporum are reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of utilizing L-menthyl acetate as a novel plant-derived HDAC activator for inhibiting fungal growth, and particularly relates to a volatile substance and a method for inhibiting the growth of Fusarium oxysporum by reducing the levels of H3K14ac and H3K27ac. Background Art

[0002] Fusarium oxysporum is the key pathogenic factor of Panax notoginseng root rot. During the cultivation process of Panax notoginseng, it invades the roots through soil media, causing root tissue rot, posing a serious threat to the growth and development of Panax notoginseng and its final quality. In view of this, Panax notoginseng growers widely use chemical fungicides such as methyl thiophanate, carbendazim and genkang, in order to effectively curb the spread of pathogens and prevent and control diseases. At present, foreign researchers have made significant progress, and pesticide technology and products are undergoing profound changes. Despite this, my country still faces the challenge of scarce original targets, which limits the independent research and development capabilities of high-efficiency and low-risk small molecule pesticides based on molecular design technology. Therefore, strengthening the discovery and utilization of original targets, combined with advanced molecular design strategies, has become an urgent need to promote green pesticide innovation and promote green agricultural development.

[0003] Histone deacetylation is one of the most critical epigenetic regulatory mechanisms in eukaryotes. It precisely regulates fungal growth, development cycle, and adaptive response to environmental stress, and affects the transcriptional activity of a series of key genes. Excessive histone deacetylase (HDAC) activators can induce cell apoptosis. This property has been successfully applied in the field of human cancer treatment and has important medical value. In recent years, with the in-depth study of biological control strategies, the method of using volatile organic compounds (VOCs) as environmentally friendly biological control agents has gradually emerged and has received widespread attention from the scientific research and agricultural communities. However, although volatile small molecules have shown great potential in biological control, there is still a lack of research on whether they can act as HDAC activators, directly participate in regulating the histone deacetylation process in fungi, and further affect the growth and development mechanism of fungi. Therefore, exploring the possibility of volatile small molecules as HDAC activators and revealing their specific mechanisms of how they regulate growth and development and respond to environmental stress in fungi by affecting histone deacetylation will not only provide new strategies and targets for the biological control of fungal diseases, but also promote a deeper understanding of the epigenetic regulation mechanisms of eukaryotes, opening up new avenues for sustainable agricultural development and the development of new drugs.

[0004] The problems and defects of the prior art are:

[0005] There is no research on how volatile small molecules regulate histone deacetylation in Fusarium oxysporum and affect the growth and development of the fungus. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a volatile substance that inhibits the growth of Fusarium oxysporum by reducing the levels of H3K14ac and H3K27ac.

[0007] The present invention is achieved in that a method for inhibiting the growth of Fusarium oxysporum by reducing the levels of H3K14ac and H3K27ac comprises:

[0008] Step 1, sample processing:

[0009] 2 g of Fusarium oxysporum mycelium was added to a conical flask containing 40 mL of L-menthyl acetate solution of different concentrations, and the conical flask was placed in a constant temperature incubator at 28°C for constant temperature shaking culture for 24 h, and then the sample was collected and ground into powder using a cryogenic grinder for later use;

[0010] Step 2: Prepare histones of the treated samples according to the instructions of the histone extraction kit.

[0011] Furthermore, the L-menthyl acetate can be used as a FoArginase_HDAC activator.

[0012] Furthermore, the L-menthyl acetate is 1 mmol / L and 2 mmol / L.

[0013] Further, the histone extraction kit is

[0014] Further, the method for preparing histones of the treated sample according to the instructions of the histone extraction kit is as follows:

[0015] Protein separation was performed using 15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), followed by immunoblotting using anti-H3K14ac, anti-H3K27ac, anti-H3K9ac, and anti-H3 antibodies to analyze the acetylation status of H3 lysine. Anti-H3 antibody immunoblotting was used as a control. Finally, ImageJ software was used to quantify the intensity of the immunoblot bands.

[0016] Furthermore, the antiH3K14ac is: Jingjie, PTM157.

[0017] Furthermore, the antiH3K27ac is: Abcan, Cat#: ab24404.

[0018] Furthermore, the antiH3K9ac is: Abcam, Cat#: ab16635.

[0019] Furthermore, the antiH3 is: Jingjie, PTM1002.

[0020] The invention provides a volatile substance for inhibiting the growth of Fusarium oxysporum. The substance effectively inhibits the growth of Fusarium oxysporum by inhibiting the levels of H3K14ac and H3K27ac. The substance mainly comprises L-menthyl acetate as an effective ingredient and can significantly reduce the levels of H3K14ac and H3K27ac at a specific concentration, thereby achieving the effect of inhibiting the growth of Fusarium oxysporum.

[0021] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0022] First, the present invention relates to the use of L-menthyl acetate, a monomer component of peppermint volatile oil, as a new plant-derived HDAC activator for inhibiting the growth of Fusarium oxysporum in inhibiting the growth and reproduction of pathogenic fungi, and belongs to the technical field of pesticide bactericides. L-menthyl acetate, a monomer component of peppermint volatile oil, can inhibit the activity of Fusarium oxysporum histone deacetylase (FoArginase_HDAC) and inhibit the expression of key genes of the ribosomal pathway of Fusarium oxysporum. Molecular docking shows that L-menthyl acetate can interact with amino acid residues His240, His203, Phe242, etc. in Fusarium oxysporum histone deacetylase FoArginase_HDAC, thereby increasing the activity of FoArginase_HDAC and inhibiting the levels of H3K14ac and H3K27ac of Fusarium oxysporum. It was found that L-menthyl acetate, a monomer component of mint volatile oil, as a volatile small molecule, can inhibit the growth of Fusarium oxysporum; it was found that L-menthyl acetate, a monomer component of mint volatile oil, inhibits the histone deacetylation level of Fusarium oxysporum; L-menthyl acetate, a monomer component of mint volatile oil, can inhibit the histone deacetylase activity of Fusarium oxysporum and inhibit the expression of key genes in the ribosome pathway.

[0023] Second, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0024] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0025] Expected Returns

[0026] Market expansion and demand satisfaction: With the increasing demand for sustainable agricultural development and green pesticides, L-menthyl acetate, as a new plant-derived HDAC activator pesticide, is expected to fill the gap in the market for high-efficiency, low-toxic, and environmentally friendly pesticides. Especially in the field of significant control of fungal diseases, its market demand will further expand. The development and application of this new pesticide will provide more options for agricultural production, and also provide new solutions to meet the needs of sustainable agricultural development.

[0027] Improved economic benefits: By improving the yield and quality of crops and reducing losses caused by diseases, farmers' income can be directly increased. Pesticide manufacturers can achieve significant improvements in economic benefits through large-scale production and sales.

[0028] Technical barriers and competitive advantages: As a new type of pesticide, L-menthyl acetate may have certain technical barriers in technology research and development and production processes, which will help companies establish competitive advantages in the market. With the continuous maturity of technology and the promotion of application, companies are expected to obtain continuous benefits from technological innovation.

[0029] Business Value

[0030] Environmental protection and sustainable development: As a plant-based pesticide, L-menthyl acetate is natural and low-toxic, which is in line with the current concept of environmental protection and sustainable development, and helps to enhance the market competitiveness of pesticide products. The addition of the environmental label will help attract more consumers and partners who pay attention to green agriculture.

[0031] Brand building and market recognition: Companies that successfully launch L-menthyl acetate pesticides can enhance their visibility and influence in the pesticide industry through active marketing and brand building. A good brand image will help companies gain more opportunities and resources in the market.

[0032] Industrial chain integration and synergistic effects: The promotion and application of L-menthyl acetate pesticides is expected to drive the development of related industrial chains, including raw material supply, production and processing, sales and services, etc. Through industrial chain integration, companies can achieve resource sharing and complementary advantages, further enhancing the overall commercial value.

[0033] International cooperation and market expansion: With the continuous development of global agricultural trade, L-menthyl acetate pesticide is expected to go abroad and participate in international market competition. Through international cooperation and market expansion, enterprises can further enhance their brand influence and market share.

[0034] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0035] L-Menthyl acetate, a plant-derived compound, has been developed as an HDAC activator, which is an innovation in the field of pesticides. Traditional HDAC inhibitors have been widely studied and applied in fields such as cancer treatment, but its application in the field of pesticides, especially as an activator to inhibit fungal growth, is novel.

[0036] As people pay more attention to environmental protection and sustainable development, botanical pesticides have attracted attention due to their low toxicity, low residue, and environmental friendliness. L-Menthyl acetate, as a botanical HDAC activator, conforms to this development trend.

[0037] At present, the prevention and control of fungal diseases mainly relies on chemical pesticides, but these pesticides often have problems such as environmental pollution and drug resistance. L-Menthyl acetate, as an HDAC activator, inhibits the growth of fungi by regulating the epigenetic mechanism of fungi, providing a new strategy for fungal control. In the field of pesticides, the application of HDAC activators is still blank. The research and development of L-Menthyl acetate fills the gap in this field and provides a new direction for the innovation and development of pesticides.

[0038] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0039] In the Chinese medicinal materials planting industry, some Chinese medicinal materials such as Paris polyphylla and Panax notoginseng are very susceptible to root rot pathogens such as Fusarium oxysporum, resulting in a serious decline in yield and quality. If it can be applied to Chinese medicinal materials planting, the occurrence of root rot can be prevented and controlled, and the quality and yield of Chinese medicinal materials can be improved, which will promote the innovative development of the Chinese medicinal materials industry. In crop planting, the use of chemical pesticides can be reduced or avoided, pesticide residues can be reduced, and crop quality can be improved.

[0040] (4) The technical solution of the present invention overcomes technical prejudice:

[0041] Technological bias: Traditionally, HDAC inhibitors have been widely studied for their ability to inhibit the growth of tumor cells, while the application of HDAC activators in the field of pesticides has been relatively less explored.

[0042] Overcoming: The application of L-menthyl acetate as an HDAC activator in pesticides may affect the resistance mechanism of plants to fungal diseases by activating HDAC in plants. This novel idea breaks the traditional application limitations of HDAC inhibitors in the field of pesticides and opens up a new direction for pesticide innovation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a flow chart of a method for inhibiting the growth of Fusarium oxysporum by inhibiting the levels of H3K14ac and H3K27ac provided by an embodiment of the present invention.

[0044] Figure 2 1 is a graph showing the effect of different concentrations of L-menthyl acetate on the growth of Fusarium oxysporum provided in an embodiment of the present invention.

[0045] Figure 3 3 is a graph showing the effect of different concentrations of L-menthyl acetate on H3K27ac provided in an embodiment of the present invention.

[0046] Figure 4 Graph showing the effect of L-menthyl acetate on enzyme activity provided by an embodiment of the present invention.

[0047] Figure 5 It is a molecular docking result diagram of L-menthyl acetate and FoArginase_HDAC provided in an embodiment of the present invention.

[0048] Figure 6 This is a verification diagram of RTqPCR and ChIPqPCR in the L-menthyl acetate treatment group provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0050] like Figure 1 As shown, a method for inhibiting the growth of Fusarium oxysporum by reducing the levels of H3K14ac and H3K27ac provided by an embodiment of the present invention comprises the following steps:

[0051] S101, sample processing:

[0052] 2 g of Fusarium oxysporum mycelium was added to a conical flask containing 40 mL of L-menthyl acetate solution of different concentrations, and the conical flask was placed in a constant temperature incubator at 28°C for constant temperature shaking culture for 24 h, and then the sample was collected and ground into powder using a cryogenic grinder for later use;

[0053] S102, preparing histones of the treated sample according to the instructions of the histone extraction kit.

[0054] The L-menthyl acetate provided in the embodiment of the present invention can be used as a FoArginase_HDAC activator.

[0055] The L-menthyl acetate provided in the embodiment of the present invention is 1 mmol / L and 2 mmol / L.

[0056] The histone extraction kit provided in the embodiment of the present invention is

[0057] The method for preparing histones from treated samples according to the instructions of the histone extraction kit provided in the embodiment of the present invention is as follows:

[0058] Protein separation was performed using 15% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), followed by immunoblotting using anti-H3K14ac, anti-H3K27ac, anti-H3K9ac, and anti-H3 antibodies to analyze the acetylation status of H3 lysine. Anti-H3 antibody immunoblotting was used as a control. Finally, ImageJ software was used to quantify the intensity of the immunoblot bands.

[0059] The antiH3K14ac provided in the embodiment of the present invention is: Jingjie, PTM157.

[0060] The antiH3K27ac provided in the embodiment of the present invention is: Abcan, Cat#: ab24404.

[0061] The antiH3K9ac provided in the embodiment of the present invention is: Abcam, Cat#: ab16635.

[0062] The volatile substance that inhibits the growth of Fusarium oxysporum at the levels of H3K14ac and H3K27ac provided in an embodiment of the present invention is characterized in that the antiH3 is: Jingjie, PTM1002. Specific implementation method:

[0064] Example 1: Treatment of Fusarium oxysporum with L-menthyl acetate

[0065] L-Menthyl acetate was diluted by two-fold dilution method and added to 20 mL of potato dextrose agar (PDA) medium (final monomer concentration gradient was 8, 4, 2, 1, 0.5, 0.25 mmol / L). The colony diameter was measured once every 24 hours by the "cross" method until the 7th day, and the obtained values ​​were plotted into a line graph to calculate the half inhibition concentration (EC50).

[0066] The minimum inhibitory concentration (MIC) value was determined by the 96-well plate method, and a spore suspension was prepared. The spore concentration was adjusted to 2×105 / mL. The initial concentration of L-menthyl acetate was 4 mg / mL, and L-menthyl acetate was diluted into eight concentration gradients by two-fold dilution. The 96-well plate was incubated at 28°C for 20 h, and the absorbance of each well was measured at a wavelength of 595 nm to calculate the MIC value of L-menthyl acetate.

[0067] Example 2: Effect of L-menthyl acetate on histone H3K14ac and H3K27ac of Fusarium oxysporum

[0068] Sample treatment: Take 2 g of Fusarium oxysporum mycelium and add it into a conical flask containing 40 mL of L-menthyl acetate solution (1 mmol / L and 2 mmol / L) of different concentrations. Place the conical flask in a constant temperature incubator at 28°C for constant temperature shaking incubation for 24 hours, then collect the sample and grind it into powder using a low-temperature grinder for later use.

[0069] Histone extraction kit The histones of the treated samples were prepared according to the instructions. 15% SDS PAGE was used for separation, followed by immunoblotting using antiH3K14ac (PTM157), antiH3K27ac (Abcan, Cat#: ab24404), antiH3K9ac (Abcam, Cat#: ab16635) and antiH3 (PTM1002) antibodies to detect H3 lysine acetylation / status. AntiH3 immunoblotting was used as a control. The band intensity was quantitatively analyzed using ImageJ software.

[0070] Example 3: Effect of L-menthyl acetate on FoArginase_HDAC enzyme activity

[0071] In order to determine the activity of FoArginase_HDAC, we used gene recombination technology to transfer the target gene into an expression vector (pET28a, kan+), and expressed the protein through Escherichia coli (BL21). The concentration of the expressed protein was determined using the BCA method (BCA protein concentration determination kit (SK1070)). FluorimetricHDACActivityAssayKit*GreenFluorescence* measures the activity of histone HDAC deacetylase.

[0072] Example 4: Molecular docking of L-menthyl acetate and FoArginase_HDAC

[0073] AutodockVina was used for molecular docking simulation. The 3D structure of FoArginase_HDAC protein was downloaded from the Unipot official website (https: / / www.uniprot.org / uniprotkb / Q9FE17 / entry#names_and_taxonomy), and the 3D structure of L-menthyl acetate was downloaded from the Pubchem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. The protein receptor was dehydrated, hydrogenated, and energy minimized using AutoDockTool software, and molecular docking and calculation results were performed using AutodockVina, and the results were visualized using PyMOL and Ligplot software.

[0074] Example 5: RTqPCR and ChIPqPCR analysis of Fusarium oxysporum treated with L-menthyl acetate

[0075] Sample treatment: Take 2 g of Fusarium oxysporum mycelium and add it into a conical flask containing 40 mL of L-menthyl acetate solution (1 mmol / L and 2 mmol / L) of different concentrations. Place the conical flask in a constant temperature incubator at 28°C for constant temperature shaking incubation for 24 hours, then collect the sample and grind it into powder using a low-temperature grinder for later use.

[0076] Take 2g of Fusarium oxysporum powder treated with monomers of different concentrations, extract the total RNA of the sample with Trizol reagent, divide the above fungal sample powder into 1.5mL EP tubes, and immediately add Trizol reagent to fully lyse. Then add chloroform, isopropanol and ethanol to remove proteins and DNA. Finally, elute with DEPC water. Use a reverse transcription kit to synthesize the first-chain cDNA from the total RNA. QTUB was used as a reference gene, and RTqPCR was performed using the SYBR Premix Ex Taq kit (TaKaRa Biomedicals) in the CFX Manager 3.1 system (BioRad). Use 2△△Cq Methods The relative expression of genes was analyzed.

[0077] Take 2g of Fusarium oxysporum powder after monomer treatment with different concentrations, add 30mL 1% formaldehyde solution and shake for cross-linking for 5min, then add 2.5M glycine and shake for 5min to terminate cross-linking. Add 30mL extraction buffer I and mix well, then centrifuge at low temperature and discard the supernatant, repeat the washing twice, then use 300μL extraction buffer III for density gradient centrifugation, discard the supernatant, and obtain the cell nucleus. Resuspend the cell nucleus with cell nucleus lysis buffer and perform high-power ultrasound at 4℃ for 50min (30son, 30soff). Take 10μL of the ultrasound product to decrosslink and detect whether the DNA fragments after ultrasound are distributed around 500bp. If the DNA detection quality meets the standard, the ChIP experiment can be carried out. The ultrasonic sample was added to the ProteinA magnetic beads (with bound antibodies H3K14ac and H3K27ac), incubated overnight in a 4°C refrigerator, and the complex magnetic beads were washed with low salt, high salt, LiCl and TE buffer in sequence. Finally, the binding product was eluted with 250 μL elution buffer in a constant temperature metal bath at 65°C for 25 min (mixed every 2 min), and the supernatant was aspirated into a clean EP tube, repeated twice for a total of 500 μL, and 20 μL 5M NaCl was added to the eluted collection solution to decrosslink in a metal bath at 65°C overnight. Finally, the decrosslinked product was removed and purified to obtain DNA, and ChIPqPCR was performed in the CFXManager3.1 system (BioRad) using the SYBR Premix Ex Taq kit (TaKaRa Biomedicals). The relative expression of genes was analyzed using the Percent Input method.

[0078] Figure 2 This is the antibacterial effect diagram of L-menthyl acetate on Fusarium oxysporum. Figure 1 It can be seen that after treatment with monomer L-menthyl acetate, the growth of Fusarium oxysporum was significantly inhibited. Its EC50 value is 1.872mmol / L. In comparison, the EC50 value of the commercial fungicide, oxadipamide, is 0.428mmol / L. Although its EC50 value is lower, the antibacterial activity at higher concentrations shows similar effects. In addition, the minimum inhibitory concentration of L-menthyl acetate against Fusarium oxysporum is 2.60mmol / L, which has a good inhibitory effect on Fusarium oxysporum.

[0079] Figure 3The figure shows the effect of L-menthyl acetate on H3K14ac and H3K27ac of Fusarium oxysporum. Western blot was performed on samples treated with L-menthyl acetate to detect changes in the levels of H3K9ac, H3K14ac and H3K27ac. As shown in the figure, after treatment with different concentrations of L-menthyl acetate, the abundance of H3K27ac showed a downward trend, while 1mmol / L L-menthyl acetate could increase the H3K14ac modification level of Fusarium oxysporum, and the abundance decreased after 2mmol / L treatment. The antibacterial activity results show that compared with the 1mmol / L treatment group, the inhibitory effect of 2mmol / L L-menthyl acetate on Fusarium oxysporum was significantly increased, indicating that L-menthyl acetate can indeed act as an external factor to affect the modification of histone acetylase, thereby inhibiting the growth of Fusarium oxysporum.

[0080] Figure 4 This is the effect of L-menthyl acetate on the enzyme activity of FoArginase_HDAC. The HDAC protein activity test kit was used to determine the effect of L-menthyl acetate on the activity of FoArginase_HDAC. First, the target protein FoArginase_HDAC was expressed through prokaryotic protein expression, and the activity was good. After adding the commercial inhibitor guqustatin A, its activity was significantly reduced to 11910.9RFU / min / g. After treatment with L-menthyl acetate, the protein activity was significantly activated, and the protein activity increased to 19144.4RFU / min / g, which shows that citronellol can activate the activity of FoArginase_HDAC, thereby inhibiting the level of histone acetylation.

[0081] Figure 5 Molecular docking results of L-menthyl acetate and FoArginase_HDAC. L-menthyl acetate can bind to FoArginase_HDAC through hydrophobic forces and hydrogen bonds, with a binding energy of 6.3 kcal / mol, indicating that the binding between them has good stability. L-menthyl acetate forms a hydrogen bond with the amino acid residue His240 of the protein FoArginase_HDAC, and forms hydrophobic forces with five amino acids including His203, Phe242, and Gly211, as shown in the figure.

[0082] Figure 6The RTqPCR and ChIPqPCR validation diagrams of gene expression in the L-menthyl acetate treatment group. ChIPqPCR further verified that after 2mmol / L L-menthyl acetate treatment, the H3K14ac enrichment multiples of the key genes FOBCDRAFT_13939, FOBCDRAFT_160800, FOBCDRAFT_171928, and FOBCDRAFT_215340 in the ribosomal pathway of Fusarium oxysporum were significantly reduced, which was consistent with the results of western blot detection, which was a decrease. The RTqPCR experiment showed that L-menthyl acetate can significantly inhibit the expression of these genes.

[0083] The H3K27ac enrichment multiples of the key ribosomal pathway genes FOBCDRAFT_221167, FOBCDRAFT_162204, and FOBCDRAFT_225593 of Fusarium oxysporum significantly decreased, which was consistent with the results of western blot detection. Further experiments through RTqPCR showed that L-menthyl acetate could significantly inhibit the expression of these genes.

[0084] It further shows that L-menthyl acetate can inhibit the activity of histone deacetylase FoArginase_HDAC in Fusarium oxysporum, thereby reducing the modification levels of H3K14ac and H3K27ac, and finally leading to the inhibition of gene expression in the ribosomal pathway of Fusarium oxysporum.

[0085] Application Areas

[0086] Control of fungal diseases of crops: L-Menthyl acetate has a strong bactericidal effect and can prevent and control fungal diseases on crops, such as root rot, leaf spot, powdery mildew, etc., by inhibiting the growth of fungi.

[0087] Crop pest control: In addition to its fungicidal effect, L-menthyl acetate can also be used to control agricultural pests such as aphids and caterpillars.

[0088] Forestry and horticultural disease control: L-Menthyl acetate can also be used to control diseases and insect pests on trees and flowers to protect the healthy growth of plants.

[0089] Eco-friendly pesticides: Compared with traditional chemical pesticides, L-menthyl acetate, as a plant-based pesticide, does not pollute the ecological environment and meets the requirements of green agriculture and sustainable development.

[0090] Related Products

[0091] L-Menthyl acetate pesticide preparation: L-Menthyl acetate is mixed with other auxiliary ingredients (such as surfactants, stabilizers, etc.) to make an easy-to-spray pesticide preparation for controlling diseases and insect pests on crops.

[0092] Compound pesticides: L-Menthyl acetate is compounded with other active pesticide ingredients (such as other botanical pesticides, biological pesticides or low-toxic chemical pesticides) to form a compound pesticide product with a broader fungicide and insecticide spectrum.

[0093] Plant growth regulators: Utilize the regulatory effect of L-menthyl acetate on plant growth to develop plant growth regulators that can promote crop growth and improve stress resistance.

[0094] Through antibacterial tests, L-menthyl acetate and the commercial fungicide benomyl showed similar antibacterial activity at higher concentrations, indicating that L-menthyl acetate has a good antibacterial effect on Fusarium oxysporum. Further verification of its mechanism of action showed that L-menthyl acetate has a significant activation effect on FoArginase_HDAC enzyme activity, thereby reducing the modification levels of H3K14ac and H3K27ac in Fusarium oxysporum. Molecular docking simulation verification showed that L-menthyl acetate can bind to FoArginase_HDAC through hydrophobic forces and hydrogen bonds, with a binding energy of 6.3kcal / mol. Five amino acid residues, including His203, Phe242, and Gly211, are the main binding sites, with good stability. RTqPCR and ChIPqPCR results further proved that after treatment with L-menthyl acetate, the key genes of the ribosomal pathway of Fusarium oxysporum, such as FOBCDRAFT_221167 and FOBCDRAFT_162204, were at low H3K14ac and H3K27ac acetylation levels, and their expression was inhibited. In summary, L-menthyl acetate can activate the activity of histone deacetylase in pathogenic fungi, resulting in low acetylation of histones, thereby inhibiting gene expression.

[0095] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A volatile substance for inhibiting the growth of Fusarium oxysporum, characterized in that: The inhibitory effect is achieved by reducing the levels of histone H3K14ac and H3K27ac. The substance includes L-menthyl acetate, which acts as a FoArginase_HDAC activator and is able to inhibit the growth of Fusarium oxysporum at different concentrations.

2. The volatile substance according to claim 1, characterized in that The concentration of the L-menthyl acetate is 1 mmol / L or 2 mmol / L.

3. The volatile substance according to claim 1, characterized in that The method for treating Fusarium oxysporum with the volatile substance comprises the following steps: Fusarium oxysporum hyphae were added into solutions containing different concentrations of L-menthyl acetate; Incubate in a 28°C incubator with constant temperature shaking for 24 hours; The incubated samples were ground into powder using a cryogenic grinder for further histone extraction and analysis.

4. A method for inhibiting the growth of Fusarium oxysporum by reducing the levels of H3K14ac and H3K27ac, characterized in that: The following steps are involved: 2 g of Fusarium oxysporum mycelium was added into a conical flask containing 40 mL of L-menthyl acetate solution of different concentrations; The conical flask was placed in a constant temperature incubator at 28°C and incubated with constant temperature shaking for 24 hours, and the sample was collected; Use a cryogenic grinder to grind the cultured sample into powder for later use; The histones of the treated samples were prepared according to the instructions of the histone extraction kit.

5. The method according to claim 4, characterized in that The concentration of L-menthyl acetate is 1 mmol / L and 2 mmol / L, which is used to effectively reduce the levels of H3K14ac and H3K27ac in Fusarium oxysporum.

6. The method according to claim 4, characterized in that The histone extraction kit is Used to extract histones from processed samples and perform further analysis.

7. The method according to claim 4, characterized in that The extracted histones were separated by 15% SDS PAGE gel, and the H3 lysine acetylation status was detected by immunoblotting using antiH3K14ac, antiH3K27ac, antiH3K9ac and antiH3 antibodies. The band intensity was quantitatively analyzed by ImageJ software.

8. The method according to claim 4, characterized in that The L-menthyl acetate can be used as a FoArginase_HDAC activator to inhibit the growth of Fusarium oxysporum by regulating the histone acetylation level.

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