Application of alantolactone in preparation of fusarium oxysporum inhibitor

Abstract

The invention relates to the technical field of plant disease control, in particular to application of alantolactone in preparation of a fusarium oxysporum inhibitor. Specifically, on the basis that alantolactone has a fusarium oxysporum inhibition effect, the invention develops an environment-friendly bactericide containing alantolactone. In the bactericide, the use concentration of alantolactone is 1mM, the use concentration of N-aminoethyl piperazine is 5mM, and the bactericide disclosed by the invention can also inhibit fusarium oxysporum chlamydospore.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease control, and more specifically to the application of costunolide in the preparation of an inhibitor against Fusarium oxysporum. Background Art

[0002] Fusarium oxysporum is one of the major threats in global agricultural production, capable of causing severe diseases such as wilt and root rot in many economically important crops (such as wheat, banana, tomato, etc.). These diseases usually manifest as plant wilting, yellowing, and root rot, ultimately leading to a decline in crop yields or even crop failures, posing a major challenge to global food security. According to relevant statistics, plant diseases caused by Fusarium oxysporum result in billions of dollars in economic losses every year, seriously affecting the sustainability of agricultural production and the quality of agricultural products.

[0003] To control plant pathogenic fungi, the currently widely used method is to apply synthetic chemical pesticides. These traditional chemical pesticides do have significant antibacterial effects in the initial stage, but their long-term and extensive use also brings the following problems: enhanced pathogen resistance, the mechanism of action of chemical pesticides is single, and after long-term use, pathogens are prone to develop resistance to pesticides through gene mutation, enzyme activity regulation, etc., resulting in a gradual decline in the control effect; environmental pollution, chemical pesticides are likely to remain in the soil and water during application, causing pollution to the ecological environment and potentially affecting the survival of non-target organisms; food safety hazards, the problem of excessive pesticide residues may pose a threat to human health, especially for people with weaker immune systems (such as children and the elderly).

[0004] Based on the above problems, the development of highly efficient, low-toxic, and environmentally friendly natural plant-derived bacteriostatic agents has gradually become an important research direction in the field of plant disease control. In recent years, natural plant extracts have attracted much attention from the scientific and industrial communities due to their multi-target effects, low environmental residues, and good biodegradability.

[0005] Inula helenium is a traditional medicinal plant, and its roots are rich in sesquiterpene compounds, especially costunolide. Existing studies have shown that costunolide has biological activities such as anti-microbial, anti-inflammatory, antioxidant, and anti-tumor. For example, costunolide exhibits significant inhibitory effects on certain bacteria and fungi by inhibiting protein synthesis and disrupting cell membrane structure. However, current research on costunolide mainly focuses on its antibacterial effects against human pathogens (such as Staphylococcus aureus, Candida albicans), and its research on plant pathogenic fungi (especially Fusarium oxysporum) is relatively blank.

[0006] The inhibitory mechanism of plant pathogenic fungi is complex and may involve multiple aspects such as cell membrane, cell wall, leakage of contents, and metabolic regulation. Therefore, it is particularly necessary to study a natural plant bacteriostatic agent that can efficiently inhibit plant pathogenic fungi and is harmless to the environment and crops. Based on this, the present invention takes costunolide as the research object and develops an inhibitor with high efficiency in inhibiting Fusarium oxysporum. Summary of the Invention

[0007] The present invention provides a plant pathogenic Fusarium oxysporum inhibitor based on costunolide, aiming to solve the serious harm caused by plant pathogenic fungi to crops, as well as problems such as drug resistance and environmental pollution caused by traditional chemical pesticides. This method is based on natural plant extracts, and through optimizing the extraction process, separating and purifying active ingredients, studying the bacteriostatic effect and analyzing the mechanism of action, a highly efficient, low-toxic, and environmentally friendly plant disease control agent is developed. The specific content is as follows:

[0008] In the first aspect of the present invention, the application of costunolide in the preparation of an inhibitor of Fusarium oxysporum is provided. In some embodiments, the use concentration of costunolide is 0.25 mM - 1 mM.

[0009] On the other hand, based on the research that costunolide has the ability to inhibit Fusarium oxysporum, the present application further develops an agricultural fungicide composition, in which the use concentration of costunolide is 1 mM and the use concentration of N - aminoethylpiperazine is 5 mM.

[0010] Combining all the above technical solutions, the advantages and positive effects of the present invention are: the fungicide developed in the present application can effectively kill the chlamydospores of Fusarium oxysporum. The fungicide is highly efficient, low-toxic, environmentally friendly, and has no impact on the germination and growth of tobacco. Brief Description of the Drawings

[0011] Figure 1 is the structural formula of costunolide;

[0012] Figure 2 is of the extract in Example 1 1 HNMR, 13 C NMR, HSQC spectrum and HMBC spectrum, (A) is 1 H NMR (400 MHz, CDCl3), (B) is 13 C NMR (100 MHz, CDCl3), (C) is the HSQC spectrum, (D) is the HBMC spectrum;

[0013] Figure 3It is the dose - response curve under different culture times in Example 2. (A) is the dose - response curve during the 2 - day culture period, (B) is the dose - response curve during the 4 - day culture period, and (C) is the dose - response curve during the 6 - day culture period;

[0014] Figure 4 It is the effect of costunolide bacteriostatic solution at different concentrations on the growth of Fusarium oxysporum in Example 3. (A) is the OD600 value of Fusarium oxysporum at different concentrations, and (B) is the change in the OD600 value of Fusarium oxysporum at different concentrations;

[0015] Figure 5 It is the statistical histogram of the chemotaxis index of costunolide on Fusarium oxysporum in Example 4. The right figure is a typical microscopic image of spore germination. The blue mid - line is the spore inoculation line, the brown - red solid line is the solvent control area, and the dotted line is the test compound area;

[0016] Figure 6 It is the effect of different fungicides on the cell membrane permeability of chlamydospores of Fusarium oxysporum in Example 5. (A) is the result of fungicide A, (B) is the result of fungicide B, (C) is the result of fungicide C, and (D) is the result of fungicide D;

[0017] Figure 7 It is the measurement result of the content leakage of different fungicides on Fusarium oxysporum in Example 5;

[0018] Figure 8 It is the differential expression volcano plot in Example 7;

[0019] Figure 9 It is the gene function enrichment and pathway analysis diagram in Example 7. (A) is the GO enrichment analysis diagram, and (B) is the KEGG pathway analysis diagram. Detailed implementation manners

[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained from commercial channels. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] Based on the information included in this application, those skilled in the art can easily make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for schematically illustrating specific aspects of the present invention. In fact, all various changes that those skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0022] For a better understanding of the present invention rather than limiting the scope thereof, all numbers representing amounts, percentages, and other numerical values used in this application should be understood to be modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties to be obtained. Each numerical parameter should be considered at least as obtained by the reported significant figures and by conventional rounding methods. In the present invention, "about" means within 10% of a given value or range, preferably within 5%.

[0023] Unless otherwise specified, the temperature in the following examples of the present invention is at normal temperature conditions. Normal temperature refers to the natural room temperature conditions in the four seasons without additional cooling or heating treatment. Generally, the normal temperature is controlled at 10 - 30 °C, preferably 15 - 25 °C.

[0024] Unless otherwise specified, the reagents and materials used in the examples of the present invention can be obtained by commercially available means.

[0025] The detailed implementation process and content are as follows:

[0026] Example 1 Isolation and Purification of Alantolactone

[0027] S1: Select fresh roots of Inula helenium (purchased from the Chinese medicinal materials planting base in Enshi City, Hubei Province), place them in low-temperature drying (below 60 °C) for drying. The dried roots are ground into fine powder with a high-speed grinder and screened through a 100-mesh sieve to ensure that the powder particle size is less than 150 μm. Accurately weigh 5 g of the powder, add 50 mL of anhydrous methanol, transfer it to a 100-mL flask, and use an ultrasonic-assisted extractor for extraction. The extraction conditions are set at 50 °C, a power of 200 W, and an extraction time of 60 minutes. After the extract is cooled to room temperature, it is centrifuged at 12,000 rpm / min for 10 minutes using a high-speed refrigerated centrifuge. The supernatant is filtered through a 0.45-μm microporous membrane to finally obtain a clear methanol extract of Inula helenium, which is stored in a refrigerator at 4 °C for standby.

[0028] S2: Separate the methanol extract of Inula helenium using a high-performance liquid chromatograph (HPLC, model: Agilent1260, Agilent Technologies). The chromatographic column is DiamonsilPlus C18 (4.6 mm × 250 mm, 5 μm, Dima Technologies). The mobile phase is a mixture of acetonitrile and 0.10% phosphoric acid solution (50:50, volume ratio). The chromatographic conditions are a flow rate of 1.0 mL / min, a detection wavelength of 254 nm, a column temperature of 40 °C, an injection volume of 20 μL, and an isocratic elution mode. The samples are collected by peak splitting according to the peak shape and retention time of the chromatogram.

[0029] Structure Identification

[0030] The structure of the compound was identified by testing the results of 1H NMR, 13C NMR, HSQC, and HMBC two-dimensional spectra using a nuclear magnetic resonance spectrometer, and the connection mode of the molecular skeleton was verified by matching the chemical shift (δ value) and coupling constant (J value) and combining with the long-range correlation signals of HMBC.

[0031] of the extract 1 The results of the 1H NMR spectrum are as follows: 1 1H NMR (400 MHz, CDC l3 ) δ 6.20 (s, 1H, H13A), 5.62 (s, 1H, H13B), 5.15 (d, J = 3.9 Hz, 1H, H6), 4.86–4.77 (m, 1H, H8), 3.64–3.49 (m, 1H, H7), 2.54–2.36 (m, 1H, H4), 2.11 (dd, J = 14.8, 2.8 Hz, 1H, H9A), 1.89–1.72 (m, 1H, H9B), 1.66–1.49 (m, 4H, H2,3), 1.49–1.37 (m, 1H, H1A), 1.19 (s, 3H, H15), 1.14 (dd, J = 13.5, 3.7 Hz, 1H, H1B), 1.09 (d, J = 7.4 Hz, 3H, H14).

[0032] of the extract 13 The results of the 13C NMR spectrum are as follows: 13 13C NMR (100 MHz, CDCl3) δ 170.47 (C12), 149.07 (C5), 139.80 (C11), 121.69 (C13), 118.74 (C6), 76.44 (C8), 42.64 (C9), 41.71 (C1), 39.48 (C7), 37.59 (C4), 32.71 (C3), 32.65 (C10), 28.57 (C15), 22.55 (C14), 16.74 (C2).

[0033] of the extract 1 In the 1H NMR spectrum, it shows that ( Figure 2 -A) there are three proton signals of sp2 hybridization, 6.20 (s, 1H, H13A), 5.62 (s, 1H, H13B), 5.15 (d, J = 3.9 Hz, 1H, H6). Through the HSOC spectrum ( Figure 2-C) It was determined that 6.20 (s, 1H, H13A) and 5.62 (s, 1H, H13B) belong to the protons on the same carbon, and their corresponding carbon signals are δc 121.69 (C13), while the carbon signal corresponding to 5.15 (d, J = 3.9 Hz, 1H, H6) is 118.74 (C6); there are two methyl proton signals 1.19 (s, 3H, H15) and 1.09 (d, J = 7.4 Hz, 3H, H14), and their corresponding carbon signals are δc 28.57 (C15) and 22.55 (C14); there is one methine proton signal 4.86–4.77 (m, 1H, H8) connected to an oxygen atom, and the corresponding carbon signal is δc 76.44 (C8); there are two allylic methine proton signals 3.64–3.49 (m, 1H, H7) and 2.54–2.36 (m, 1H, H4), and the corresponding carbon signals are δc 39.48 (C7) and 37.59 (C4) respectively; there are four pairs of enantiomeric methylene proton signals 2.11 (dd, J = 14.8, 2.8 Hz, 1H, H9A), 1.89–1.72 (m, 1H, H9B), 1.66–1.49 (m, 4H, H2,3), 1.49–1.37 (m, 1H, H1A), 1.14 (dd, J = 13.5, 3.7 Hz, 1H, H1B), and the corresponding carbon signals are 42.64 (C9), 16.74 (C2), 32.71 (C3) and 41.71 (C1) respectively;

[0034] The 13 13C NMR spectrum shows that ( Figure 2 -B) There is also one ester carbonyl signal 170.47 (C12). Combining with the HSQC spectrum, it can also be seen that there are three quaternary carbon signals 149.07 (C5), 32.65 (C10) and 139.80 (C11) in the compound. Combining with the HMBC spectrum ( Figure 2 -A), the correlation signals of H13 with C12 and C7, the correlation signals of H6 with C10 and C8, the correlation signals of H11 with C9 and C10, the correlation signals of H14 with C5 and C4, and the correlation signals of H15 with C1 and C9, it is inferred that the extract is costunolide. Performing liquid phase analysis on the costunolide standard product, it is found that under the conditions of the same program, the peak emergence time of the costunolide standard product is consistent with that of the compound.

[0035] Example 2 Determination of IC50 antibacterial activity

[0036] The dose - effect relationship of costunolide was evaluated by the CLSI M27 - A3 micro - dilution method. Prepare a spore suspension of Fusarium oxysporum (1×10 6CFU / mL, calibrated by the McFarland turbidity standard); A two-fold concentration gradient (0.031 - 1 mM) was established in a 96-well cell culture plate, and 100 μL of PDB medium and 100 μL of Fusarium oxysporum spore suspension were added to each well; A blank control (sterile medium) and an equal-volume solvent control (0.1% DMSO) were set; After shaking culture at 28 °C for 48 hours, the absorbance (OD 600 ) was measured at 600 nm using a BioTek Synergy H1 microplate reader. The formula for calculating the inhibition rate is:

[0037]

[0038] A four-parameter logistic model was constructed using GraphPad Prism 9.5: y = Bottom + 1 + 10(LogIC 50 -x)HillSlopeTop - Bottom to calculate the IC 50 value and the 95% confidence interval, and each experiment was independently repeated three times.

[0039] By measuring the OD600 values of the target strain under treatment with different concentrations of the antibacterial solution (culture time: 2 days, 4 days, 6 days), a four-parameter logistic regression model (4PL) was used to fit the dose-response curve, and the half-inhibitory concentration (IC50) of Fusarium oxysporum spores was calculated. The results showed that the IC50 value increased significantly with the extension of the culture time ( Figure 3 ). The antibacterial activity decreased significantly with the extension of the action time. The IC50 was 0.1719 mL / L at 2 days of culture (R 2 = 0.9550), increased to 0.2217 mL / L at 4 days (R 2 = 0.9689), and further increased to 0.2803 mL / L at 6 days (R 2 = 0.9521), indicating that a 63.1% increase in the drug concentration was required to achieve the same antibacterial effect (2 days vs 6 days).

[0040] The 4PL model had a good fit for the dose-response curves at all time points (R 2 > 0.95), conforming to the typical S-shaped pharmacodynamic characteristics. The above results indicate that costunolide shows significant concentration-time dual dependence on the growth inhibition of Fusarium oxysporum within the tested concentration range

[0041] (0.031 - 1 mM) (p < 0.05). Its short-term (2 days) high-efficiency antibacterial property (IC50 < 0.2 mM) and long-term (6 days) effect maintenance ability (IC50 < 0.3 mM) highlight the development potential of this natural compound as a substitute for chemical pesticides, especially in terms of delaying the occurrence of drug resistance and environmental compatibility.

[0042] Example 3 Determination of Minimum Inhibitory Concentration (MIC)

[0043] According to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guideline, the antibacterial activity of costunolide against Fusarium oxysporum was determined by the microbroth dilution method. A two-fold concentration gradient (0.031 - 1 mM) of costunolide solution was prepared, with the solvent being 0.1% DMSO (v / v); 100 μL of standardized bacterial suspension (1×10 6 CFU / mL, calibrated by the McFarland turbidity standard) and 100 μL of PDB were added to a 96-well cell culture plate, and different concentrations of costunolide solution were added, with a negative control (0.1% DMSO) set; the culture plate was placed in a thermostatic shaking incubator (Thermo Scientific TM ) and incubated at 28 °C and 150 rpm for 0 days, 2 days, 4 days, and 6 days; the absorbance (OD 600 ) at 600 nm was measured using a multi-functional microplate reader (BioTek Synergy H1), and the data collection time points were 0 h, 48 h, 96 h, and 144 h.

[0044] Calculate ΔOD = OD t - OD0 (t is the culture time), and the MIC is defined as the lowest drug concentration that inhibits the growth of bacteria by ≥ 99% (statistically significant difference from the negative control, p < 0.05, Student's t-test).

[0045] The results are shown in Table 1 and Figure 4 show that there was no significant difference in the initial OD600 values among all concentration groups, excluding the interference of immediate drug toxicity. The ΔOD600 in the 0.500 mM treatment group decreased significantly starting from the 4th day (ΔOD600 = 0.001 ± 0.001) and continued until the 6th day (ΔOD600 = -0.047 ± 0.005), meeting the MIC standard; the ΔOD600 in the low-concentration groups (≤ 0.250 mM) did not reach the threshold (0.018 - 0.583), while the high-concentration groups (≥ 1.000 mM) showed a rapid antibacterial effect on the 1st day (ΔOD600 = -0.036 ± 0.011). In summary, the MIC of costunolide against Fusarium oxysporum is 0.500 mM, and its antibacterial effect shows a significant time-concentration dependence (p < 0.01).

[0046] Table 1: Statistics of OD600 values of each well after treatment with different concentrations of costunolide for 6 days

[0047]

[0048]

[0049] With the prolongation of the culture time, we observed the inhibitory effects of costunolide at different concentrations on the mycelial growth, especially in the concentration group of (0.25 mM - 1 mM). Under the treatment of costunolide at a concentration of 0.5 mM, the results of four parallel experiments showed that its inhibitory effect on fungal growth was different at different time points. On the 1st day and the 2nd day, the change range of the OD600 values of the four groups was within 0.05, which were 0.002 to 0.003 (from Day 0 to Day 1) and 0.001 to -0.001 (from Day 0 to Day 2) respectively, and no significant change was achieved, indicating that the inhibitory effect of costunolide on the growth of Fusarium oxysporum was weak at these two time points and there was almost no obvious change. However, on the 3rd day, the OD600 value decreased significantly, and the change ranges of groups 1 to 4 were -0.046, -0.047, -0.042, and -0.054 respectively, indicating that costunolide had an obvious inhibitory effect on fungal growth at this concentration. Thus, it can be seen that the inhibitory effect of costunolide at a concentration of 0.5 mM on Fusarium oxysporum began to appear on the 3rd day and remained continuously below the MIC level, showing a strong antibacterial effect. In the concentration groups of 0.25 mM and below (such as 0.125 mM, 0.063 mM, 0.031 mM, etc.), the change of the OD600 value was relatively weak, the antibacterial effect was not obvious, and the OD600 values of each group on the 2nd day, the 4th day, and the 6th day did not reach obvious changes (less than 0.05), indicating that the inhibitory effect of costunolide at these concentrations on the growth of Fusarium oxysporum was not obvious.

[0050] According to the definition of MIC: when the change of the OD600 value is less than 0.05, this concentration can be confirmed as the MIC. Therefore, the costunolide at a concentration of 0.5 mM was confirmed as the MIC concentration in this experiment because the change range of OD600 (0.04725 < 0.05) from the 1st day to the 6th day met the MIC standard, indicating that costunolide significantly inhibited the growth of Fusarium oxysporum at this concentration.

[0051] Example 4 Chemotaxis analysis of costunolide on Fusarium oxysporum

[0052] Test the chemotactic response of Fusarium oxysporum spores to DMSO, costunolide (0.25 mM, 0.5 mM, 1 mM). Using DMSO as the negative control, the dotted line separates different concentration compound-solvent combinations. The proportion of hyphal directional growth of about 900 spores was statistically analyzed in each group of experiments, and the data were the average values of three replicates (the error bars represent the standard error). The experiment showed ( Figure 5),The chemotactic response of costunolide to Fusarium oxysporum showed a concentration-dependent shift. The chemotactic index of the 1 mM experimental group (35.6 ± 0.7%) was significantly lower than that of the control group (64.4 ± 7.2%, P < 0.001), and it was significantly away from the costunolide source point; at a medium concentration (0.5 mM), the chemotactic index (42.0 ± 1.6%) was significantly lower than that of the control group (58.0 ± 1.7%, P < 0.001), and the hyphal growth direction was inhibited to a certain extent, and chemotaxis began to appear, and the hyphae would gradually deviate from the costunolide source point; at a lower concentration (0.25 mM), the inhibitory effect of costunolide on hyphal growth was weak, and chemotaxis was not obvious; the above results indicate that the increase in the concentration of costunolide will enhance its inhibitory effect and may affect its growth direction by inducing an avoidance response of the hyphae.

[0053] Example 5 Preparation and Testing of Fungicides

[0054] In the later stage of Example 2, it was observed that the inhibitory effect of costunolide on the chlamydospores of Fusarium oxysporum was poor. In order to further develop an agricultural fungicide, this example was formulated based on costunolide, specifically as follows:

[0055] Grouping Composition A 1 mM DMSO B 5 mM N-(2-Aminoethyl)piperazine C 1 mM DMSO + 1 mM Costunolide D 1 mM Costunolide + 5 mM N-(2-Aminoethyl)piperazine

[0056] Effect of Fungicides on the Cell Membrane Permeability of Chlamydospores of Fusarium oxysporum

[0057] Transfer 15 mL of the chlamydospore suspension of Fusarium oxysporum to a 50 mL sterile centrifuge tube, and add 0.2 mL of the fungicides in groups A - D above. Incubate at 28 °C and 180 rpm for 6 hours, and set three replicates for each group of experiments.

[0058] After the incubation, centrifuge at 5000 × g and 4 °C for 5 minutes using a refrigerated centrifuge, discard the supernatant, collect the spore precipitate, and wash the spores 3 times with PBS buffer (50 mM, pH 7.4). Add 7.5 mg / mL of PI dye to the spores, stain them at 37 °C in the dark for 20 minutes, wash the spores 3 times with PBS buffer again, and then evenly spread the spores on a glass slide. Observe the fluorescence signal under a fluorescence microscope (Zeiss LSM710, Carl Zeiss, Germany) at an excitation wavelength of 535 nm and an emission wavelength of 590 nm, and analyze the changes in cell membrane permeability after treatment with different fungicides based on the signal of the negative control group.

[0059] In the PI dye staining experiment, the treatment effects of different fungicides were observed using a fluorescence microscope, and the results are as Figure 6It was shown that fungicide D had the most significant fluorescence intensity, indicating a substantial increase in cell membrane permeability, suggesting that the cell membrane was most severely damaged at this concentration, the leakage of cell contents was most obvious, and it could effectively damage the cell membrane of the chlamydospores of Fusarium oxysporum, ultimately leading to a large amount of leakage of intracellular substances.

[0060] Determination of the leakage of cell contents of chlamydospores of Fusarium oxysporum by fungicides

[0061] Take 15 mL of the chlamydospore suspension of Fusarium oxysporum into a 50 mL centrifuge tube, and then add 0.2 mL of fungicides A - D respectively. Set three replicates for each treatment group. The bacterial suspension was sampled at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Each time, 10 μL of the supernatant was taken and mixed with 200 μL of Coomassie Brilliant Blue dye, and the OD value was measured at a wavelength of 600 nm. According to the change of the OD value after dye binding, the leakage amount of cell contents was estimated. The experiment referred to the analysis method of Zhou et al. (2023) and was statistically analyzed by SPSS 26.0 software.

[0062] The leakage of cell contents was determined using the Coomassie Brilliant Blue dye method. The least squares method was used to fit a linear model in Python for linear regression fitting. The standard curve of bovine serum albumin (BSA) (0.025 - 2 mg / mL) was used to quantify the protein leakage amount. The fitted standard curve equation was:

[0063] y = 0.3593x + 0.2291 (R2 = 0.9553)

[0064] where y is the OD595 value and x is the protein concentration (mg / mL). The experimental results showed ( Figure 7 ) that after 4 hours of treatment in the fungicide D group, the increase in the OD595 value was the most obvious, indicating that this group of fungicides could strongly damage the cell membrane, resulting in a large amount of release of cell contents ( Figure 9 B).

[0065] Example 6 Effect of fungicides on the germination of tobacco seeds

[0066] Select large-flowered tobacco seeds with neat and uniform grains. Surface disinfect the seeds with 75% ethanol for 30 s, wash them three times with sterile water, and dry them with sterile filter paper. Then immerse the seeds in compounds with different concentrations for 2 h. Take out the seeds and place them evenly and equidistantly in a petri dish lined with 4 layers of moist filter paper, with 50 seeds per dish. Set 3 replicates for each concentration treatment. Incubate them in the dark in a light incubator for 14 d (humidity 80%, temperature 28 ± 2 °C). Uniformly supplement a fixed amount of sterile water every 2 d to keep the filter paper moist. Start counting the seed germination potential on the 5th day and the seed germination rate on the 15th day. Measure the main root length of each germinated seedling at 15 d. Germination potential (%) = the number of germinated seeds in the first 5 days / the total number of tested seeds × 100%; Germination rate (%) = the number of germinated seeds on the 15th day / the total number of tested seeds × 100%; Germination index (GI) = Σ(Gt / Dt).

[0067] Compared with the blank control, there were no significant changes in the seed germination rate and seedling growth in the fungicide A-D treatment groups, indicating that the prepared fungicides would not have an adverse effect on the germination of tobacco seeds.

[0068] Example 7 Transcriptome analysis

[0069] Add 15 mL of chlamydospore solution of Fusarium oxysporum to a 50 mL sterile centrifuge tube, and then add 1 mL of fungicide D. Use DMSO as the control group, and set three replicates for each treatment group. After the treatment, centrifuge at 12,000 rpm / min at 4 °C for 10 minutes with a refrigerated centrifuge, discard the supernatant, and collect the bacterial cell precipitate. Store the precipitate in a refrigerator at 80 °C and then send it to Novogene for transcriptome sequencing analysis.

[0070] Use DESeq2 for differential expression analysis, and set the threshold as p value < 0.05,

[0071] |log2FoldChange| > 1. The volcano plot ( Figure 8 ) shows the significantly differentially expressed genes between the treatment group and the control group. Specifically, there were 746 genes significantly up-regulated and 540 genes significantly down-regulated in the fungicide D treatment group. The up-regulated genes were mainly involved in processes such as cell membrane repair, antioxidant stress response, and energy metabolism, while the down-regulated genes were concentrated in mechanisms such as cell cycle and DNA repair. These differentially expressed genes indicate that the fungicide D treatment group inhibits the growth of pathogenic microorganisms by regulating the stability of the cell membrane, enhancing the antioxidant ability of cells, and regulating metabolic pathways.

[0072] The bacteriostatic effect of the bactericide D treatment group on the chlamydospores of Fusarium oxysporum showed significant biological function changes at the transcriptional level. Through gene ontology (GO) functional enrichment analysis, we found that the differentially expressed genes were mainly enriched in multiple important biological processes and molecular functions. In particular, the enrichment in cofactor binding, RNA polymerase II transcription factor activity, sequence-specific DNA binding, DNA-binding transcription factor activity, and transcription regulator activity indicated that the bactericide D treatment group might affect the growth and metabolism of Fusarium oxysporum chlamydospores by regulating gene activities related to transcription( Figure 9 ). These functional categories reflect the transcriptional regulatory effect of the bactericide on chlamydospores, which may inhibit the basic life processes within cells by altering the activity of transcription factors.

[0073] Further KEGG pathway analysis showed that the metabolic pathways mainly affected by the bactericide included Biosynthesis of amino acids, Carbon metabolism, and Biosynthesis of secondary metabolites, etc. These pathways are closely related to the basic growth and metabolic processes of chlamydospores, especially the biosynthesis of amino acids, carbon metabolism, and the synthesis of secondary metabolites, which may be the key pathways for exerting the bacteriostatic effect. By regulating these metabolic pathways, the bactericide may limit the energy source and synthesis ability of Fusarium oxysporum chlamydospores, thereby inhibiting their growth and reproduction.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of scutellaria lactone in the preparation of inhibitors of Fusarium oxysporum.

2. The use according to claim 1, wherein the concentration of the inulin lactone is 0.25 mM-1 mM.

3. An agricultural fungicide composition, characterized in that It contains at least inulinolactone.

4. The bactericidal composition according to claim 3, characterized in that Also included is N-aminoethylpiperazine.

5. The bactericidal composition according to claim 3, characterized in that The concentration of the inulin lactone was 1 mM, and the concentration of N-aminoethylpiperazine was 5 mM.

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