Application of plant secondary metabolite in inhibiting pathogenicity of fusarium oxysporum

Abstract

The invention relates to an application of a plant secondary metabolite in inhibiting pathogenicity of fusarium oxysporum. The application comprises the step of realizing an antibacterial effect through at least one mechanism of inhibiting hypha growth, spore germination, cell membrane permeability change, defensive enzyme activity reduction, cell wall degrading enzyme activity inhibition or toxin synthesis reduction of pathogenic bacteria. The antibacterial action mode of the 2, 4-di-tert-butylphenol is that the 2, 4-di-tert-butylphenol can destroy the completeness of cell membranes of fusarium oxysporum of cucumbers and disturb the functions of the cell membranes to cause leakage of cell contents and oxidative damage, so that normal physiological activities of cells are inhibited. Meanwhile, 2, 4-di-tert-butylphenol can inhibit defensive enzyme activity and cell wall degrading enzyme activity of fusarium oxysporum and has a remarkable inhibition effect on toxin synthesis, so that the purpose of inhibiting fusarium oxysporum hyphae from infecting host plants is achieved, and a basis is provided for subsequent deeper research on a fusarium oxysporum inhibition mechanism.

Description

Technical Field

[0001] The invention belongs to the technical field of plant antibacterial agents, and particularly relates to application of a plant secondary metabolite, 2,4-di-tert-butylphenol, in inhibiting the pathogenicity of cucumber wilt pathogen. Background Art

[0002] Cucumber (Cucumis sativus L.) is an important economic crop in my country, and its large-scale production has long been severely restricted by Fusarium wilt. Under the continuous cropping cultivation model, the incidence of the disease can be as high as 70% or more, and severely affected areas may even lead to crop failure, causing significant economic losses. Pathogenic studies have shown that cucumber wilt is caused by Fusarium oxysporum f.sp.cucumerinum, and its pathogenic mechanism mainly involves two synergistic mechanisms: one is the vascular blockage hypothesis, in which the pathogen destroys the host vascular tissue by secreting cell wall degrading enzymes (CWDEs) such as cellulase and pectinase, resulting in xylem blockage and water transport obstruction; the other is the toxin pathogenic hypothesis, in which the fusaric acid produced by the pathogen can destroy the permeability of plant cell membranes, interfere with water metabolism and induce systemic wilting. The synergistic effect of the two types of pathogenic factors significantly accelerates the development of the disease.

[0003] The current prevention and control strategy is mainly agricultural prevention and control such as crop rotation, supplemented by chemical treatment. However, the enrichment of soil pathogens caused by continuous cropping has limited the effectiveness of traditional agricultural measures, and chemical prevention and control faces the following key bottlenecks: 1) Conventional fungicides (such as benzimidazoles) easily induce pathogens to develop resistance; 2) There is a lack of specific inhibitors for key pathogenic targets (such as toxin synthesis or cell wall degradation enzyme secretion); 3) The ecological and environmental risks and pesticide residues of chemical agents are becoming increasingly prominent. Therefore, it is urgent to develop new plant-based agents that have both high-efficiency antibacterial activity and environmental compatibility.

[0004] 2,4-di-tert-butylphenol (2,4-DTBP) is a phenolic compound widely found in plant secondary metabolites. Recent studies have revealed that it has multiple biological activities: in terms of antibacterial mechanism, this substance can destroy the normal development of pathogens by inhibiting fungal spore germination and inducing abnormal hyphae branching; in terms of plant interaction, low-concentration treatment can enhance the colonization ability of beneficial microorganisms in crop roots and improve plant systemic disease resistance. However, existing studies have not yet clarified the regulatory relationship between 2,4-DTBP and key pathogenic factors of Fusarium oxysporum (such as fusaric acid biosynthesis and cell wall degradation enzyme activity), and there is also a lack of systematic research on its role in the prevention and control of cucumber wilt.

[0005] Based on this, the present invention proposes an innovative application of 2,4-DTBP in the control of cucumber Fusarium wilt: by analyzing the dose-effect relationship of its inhibition on the growth of pathogenic bacteria, and combining the dynamic changes of pathogenicity-related indicators such as toxin accumulation and cell wall degrading enzyme activity, its multi-target antibacterial mechanism is systematically analyzed. This method not only provides a scientific basis for the development of new plant-derived fungicides, but also is expected to break through the limitation of the lack of targeted regulation of the synergistic effect of pathogenic factors in the existing technology, and has important application value for realizing the green production of cucumbers. Summary of the Invention

[0006] Technical problem to be solved: In order to solve the above problems, the present invention provides an application of a plant secondary metabolite in inhibiting the pathogenicity of cucumber Fusarium wilt bacteria, using 2,4-di-tert-butylphenol as an antibacterial agent to achieve the effect of inhibiting the pathogenicity of cucumber Fusarium wilt bacteria, and providing a new and sustainable effective prevention and control agent for inhibiting cucumber Fusarium wilt bacteria.

[0007] Technical solution: An application of 2,4-di-tert-butylphenol (2,4-DTBP) in inhibiting the pathogenicity of cucumber Fusarium wilt bacteria (Fusarium oxysporum f. sp. cucumerinum), wherein the application realizes the antibacterial effect through at least one of the mechanisms of inhibiting the mycelial growth, spore germination, cell membrane permeability change, defense enzyme activity reduction, cell wall degrading enzyme activity inhibition or toxin synthesis reduction of the pathogenic bacteria.

[0008] The application concentration of the above 2,4-DTBP is 0.05 mg / mL to 1.6 mg / mL.

[0009] The half-maximal effective inhibitory concentration of the above 2,4-DTBP on the mycelial growth of cucumber Fusarium wilt bacteria is 0.104 mg / mL, and the half-maximal effective inhibitory concentration on spore germination is 1.024 mg / mL.

[0010] The above application is achieved by inhibiting the mycelial growth of the pathogenic bacteria. Specifically, it includes: in the drug-containing medium, when the concentration of 2,4-DTBP is 0.1 mg / mL, the colony diameter is reduced by more than 50% compared with the control group, and the mycelial growth inhibition rate shows a significant positive correlation with the concentration increase.

[0011] The above application is achieved by inhibiting the spore germination of the pathogenic bacteria. Specifically, it includes: when the concentration of 2,4-DTBP is 0.1 mg / mL, the spore germination rate is reduced by 13.8% compared with the control group, and the inhibitory effect is enhanced with the increase of the concentration.

[0012] The above application is achieved by destroying the integrity of the cell membrane of the pathogenic bacteria. Specifically, it is manifested as: after treatment with 2,4-DTBP, the relative electrical conductivity of the mycelium increases by 48.71% to 135.20% within 48 hours.

[0013] The above application is achieved by inhibiting the activity of defense enzymes of pathogenic bacteria, specifically including: after treatment with 2,4-DTBP, the inhibition rate of superoxide dismutase (SOD) activity is 36.23% to 64.78%, and the inhibition rate of peroxidase (POD) activity is 53.82% to 77.08%.

[0014] The above application is achieved by inhibiting the activity of cell wall degrading enzymes of pathogenic bacteria, specifically including: when the concentration of 2,4-DTBP is 1.0 mg / mL, the inhibition rates of the activities of carboxymethyl cellulase, β-glucanase, polygalacturonase, and polymethylgalacturonase are 51.85%, 78.13%, 33.90%, and 57.41% respectively.

[0015] The above application is achieved by inhibiting the synthesis of fusaric acid by pathogenic bacteria, specifically including: when the concentration of 2,4-DTBP is 1.0 mg / mL, the content of fusaric acid is reduced by 64.94% compared with the control group.

[0016] A plant-derived antibacterial composition contains the above-mentioned 2,4-DTBP as an active ingredient, and an agriculturally acceptable carrier or adjuvant. The composition is used for the prevention and control of cucumber fusarium wilt.

[0017] Beneficial effects: The present invention shows that 2,4-ditert-butylphenol has a good effect on inhibiting cucumber fusarium wilt bacteria and a long effective time. The EC 50 value for mycelial growth is 0.104 mg / mL. In terms of inhibiting the spore germination of cucumber fusarium wilt bacteria, significant inhibitory effects start to appear from the treatment with 2,4-ditert-butylphenol at a concentration of 0.05 mg / mL. The research shows that the EC 50 value of 2,4-ditert-butylphenol for spore germination is 1.024 mg / mL, and the relative conductivity of cucumber fusarium wilt bacteria is significantly increased after treatment. The above research shows that the treatment with 2,4-ditert-butylphenol has the effect of reducing the growth activity of cucumber fusarium wilt bacteria. Using the defense enzyme activity, cell wall degrading enzyme activity, and toxin production of cucumber fusarium wilt bacteria as indicators for detecting the pathogenicity of cucumber fusarium wilt bacteria, the results show that after treatment with 2,4-ditert-butylphenol, it has significant inhibitory effects on the defense enzyme activity, secreted cell wall degrading enzyme activity, and toxin of cucumber fusarium wilt bacteria. Therefore, 2,4-ditert-butylphenol has a strong inhibitory effect on the pathogenicity of cucumber fusarium wilt bacteria.

[0018] The antibacterial mode of action of 2,4 - di - tert - butylphenol lies in its ability to disrupt the integrity of the cell membrane of Fusarium oxysporum f. sp. cucumerinum, disrupt the cell membrane function, resulting in the leakage of cell contents, cause oxidative damage, and thus inhibit the normal physiological activities of cells. At the same time, 2,4 - di - tert - butylphenol can inhibit the activities of defense enzymes and cell wall - degrading enzymes of Fusarium oxysporum f. sp. cucumerinum and significantly inhibit the toxin synthesis, thereby achieving the purpose of inhibiting the hyphae of Fusarium oxysporum f. sp. cucumerinum from infecting host plants, providing a basis for further research on the inhibition mechanism of cucumber fusarium wilt disease. Description of the Drawings

[0019] Table 1 shows the effect of 2,4 - di - tert - butylphenol on the hyphal diameter of Fusarium oxysporum f. sp. cucumerinum.

[0020] Table 2 shows the results of the virulence determination of 2,4 - di - tert - butylphenol on the hyphae of Fusarium oxysporum f. sp. cucumerinum.

[0021] Table 3 shows the results of the virulence determination of 2,4 - di - tert - butylphenol on the spore germination rate of Fusarium oxysporum f. sp. cucumerinum.

[0022] Figure 1 shows the effect of 2,4 - di - tert - butylphenol on the sporulation amount of Fusarium oxysporum f. sp. cucumerinum.

[0023] Figure 2 shows the effect of 2,4 - di - tert - butylphenol on the spore germination rate of Fusarium oxysporum f. sp. cucumerinum.

[0024] Figure 3 shows the effect of 2,4 - di - tert - butylphenol on the relative electrical conductivity of the hyphae of Fusarium oxysporum f. sp. cucumerinum.

[0025] Figure 4 shows the effect of 2,4 - di - tert - butylphenol on the activity of defense enzymes of the hyphae of Fusarium oxysporum f. sp. cucumerinum.

[0026] Figure 5 shows the effect of 2,4 - di - tert - butylphenol on the activity of cell wall - degrading enzymes produced by Fusarium oxysporum f. sp. cucumerinum.

[0027] Figure 6 shows the effect of 2,4 - di - tert - butylphenol on the content of fusaric acid produced by Fusarium oxysporum f. sp. cucumerinum. Detailed Embodiments

[0028] The present invention will be further described below in conjunction with the drawings and the description of the drawings. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

[0029] The strain of Fusarium oxysporum f. sp. cucumerinum is preserved in the laboratory of Professor Chen Xuehao of the College of Horticulture and Landscape Architecture, Yangzhou University.

[0030] Example 1

[0031] The mycelial growth inhibition rate was determined by the mycelial growth rate method. 2,4-Di-tert-butylphenol was dissolved in DMSO and then mixed evenly with PDA medium to prepare medicated media with concentrations of CK(0), 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL. One mycelial disc was taken with a mycelial disc puncher and inoculated in the center of the medicated medium for cultivation. It was cultured in the dark in a constant temperature incubator at 28 °C for 6 days. The colony diameter of each petri dish was measured by the cross method every 24 h, and 3 experimental replicates were set for each group.

[0032] The results are shown in Tables 1 and 2. Compared with the control, the inhibition rate of 2,4-di-tert-butylphenol during the cultivation process was 27.53%-91.31%. The virulence regression equation for mycelial growth was y = 2.306x + 2.267 (R 2 = 0.968), and the EC50 value was 0.104 mg / mL.

[0033] Table 1 Effects of 2,4-Di-tert-butylphenol on the Colony Diameter of Fusarium oxysporum f. sp. cucumerinum

[0034]

[0035] Table 2 Results of the Virulence Determination of 2,4-Di-tert-butylphenol on the Mycelia of Fusarium oxysporum f. sp. cucumerinum

[0036]

[0037] Example 2

[0038] 2,4-Di-tert-butylphenol was dissolved in DMSO and then mixed evenly with PDB medium to prepare medicated media with concentrations of CK(0), 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL. Five mycelial discs were taken and inoculated into the medicated PDB medium. After cultivation at 28 °C and 180 r / min, the mycelia were filtered with four layers of sterilized lens paper to collect conidia, and the conidia were counted by the hemocytometer method. 6 experimental replicates were set for each group.

[0039] The results are as Figure 1 shown. Compared with the control, 2,4-di-tert-butylphenol had a significant inhibitory effect. A significant inhibitory effect began to be shown at the concentration of 0.05 mg / mL, and the inhibitory effect became stronger with the increase in concentration.

[0040] Example 3

[0041] A fresh mycelial disc was taken with a puncher and inoculated in the drug-free PDB medium for 3 days to collect conidia. The conidia were counted using a hemocytometer and the spore concentration was adjusted to 10 6The spore suspension was drawn and mixed with the agent to form CK (0), 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 mg / mL concentrations. 0.2 mL of the mixture was drawn into a 96-well plate and placed in a 28°C constant temperature incubator for 12 h. The spore germination was observed and 6 experimental replicates were set for each group.

[0042] The results are as follows Figure 2 As shown in Table 3, the spore germination rate of 2,4-di-tert-butylphenol was significantly reduced at a concentration of 0.1 mg / mL, and there was a concentration effect. According to the calculation of the inhibition rate of spore germination, the toxicity regression equation of 2,4-di-tert-butylphenol on spore germination was y=1.839x-0.019(R 2 =0.989), and the EC50 value was 1.024 mg / mL.

[0043] Table 3 Toxicity test results of 2,4-di-tert-butylphenol on spore germination rate of cucumber wilt pathogen

[0044]

[0045] Example 4

[0046] Pipette 2mL 1×10 6 The spore suspension with a concentration of 0.5 mg / mL was shaken in PDB medium for 3 days. After filtering and washing, 0.3 g of fresh mycelium was weighed and placed in 30 mL of CK(0), 0.1, and 1.0 mg / mL solution formed by sterile 0.9% NaCI solution and 2,4-di-tert-butylphenol. The culture was shaken at 28°C and 180 r / min. The conductivity values C at 2, 6, 12, 24, and 48 hours and the conductivity value F of the mycelium after boiling were measured using a conductivity meter. The relative conductivity = C / F×100% (where: C-conductivity value during treatment; F-conductivity value after boiling). Three experimental replicates were set for each group.

[0047] The results are as follows Figure 3 As shown in the figure, at 48h, the relative conductivity of the 0.1mg / mL and 1.0mg / mL 2,4-di-tert-butylphenol treatments increased by 48.71% and 135.20% respectively compared with the CK group. Overall, the relative conductivity increased fastest between 6h and 12h, and then tended to be flat.

[0048] Example 5

[0049] Pipette 2mL 1×10 6The spore suspension at [X] spores / mL was added to the PDB medium and cultured with shaking at 28 °C and 180 r / min for 3 days. Then, after filtration and washing, fresh mycelium was weighed and added to the medicated PDB medium with the concentration of 2,4-di-tert-butylphenol being CK(0), 0.1, and 1.0 mg / mL. Samples were taken at 12, 24, and 48 h, ground in liquid nitrogen, and stored in a -80 °C refrigerator. The SOD enzyme activity was determined according to the instruction manual of the Superoxide Dismutase (SOD) Activity Content Kit from Suzhou Keming Biotechnology Co., Ltd. The POD enzyme activity was determined according to the instruction manual of the Superoxide Dismutase (POD) Activity Content Kit from Suzhou Keming Biotechnology Co., Ltd.

[0050] The results are as Figure 4 shown. With the increase of the treatment time, the SOD activity under the treatment of 0.1 mg / mL concentration showed a trend of first increasing and then decreasing, and under the treatment of 1.0 mg / mL concentration, it showed a decreasing trend. At 48 h, the inhibition rates of 2,4-di-tert-butylphenol with concentrations of 0.1 mg / mL and 1.0 mg / mL on SOD were 36.23% and 64.78% respectively. After the treatment of 2,4-di-tert-butylphenol, the POD activity of the mycelium decreased significantly. The inhibition rates of 2,4-di-tert-butylphenol with concentrations of 0.1 mg / mL and 1.0 mg / mL on SOD were 53.82% and 77.08% respectively.

[0051] Example 6

[0052] 2,4-Di-tert-butylphenol was dissolved in an appropriate amount of DMSO to prepare a medicated Czapek medium with concentrations of CK(0), 0.1, and 1.0 mg / mL. A fungal disc was inoculated into the medicated medium and cultured in the dark at 28 °C and 200 rpm on a shaker for 7 days. The supernatant was filtered through a microporous membrane to obtain a sterile fermentation broth, which was the cell wall degrading enzyme solution sample. Three experimental replicates were set for each group.

[0053] Determination of carboxymethyl cellulase activity: Take 1 mL of the 1% sodium carboxymethyl cellulose (CMC) buffer prepared in advance, add 1 mL of the enzyme solution sample and 1 mL of 0.05 M citric acid - sodium citrate buffer, place it in a 50 °C constant temperature water bath for 30 min, then add 2 mL of DNS solvent to terminate the reaction, keep it in a boiling water bath for 8 min, and cool it immediately after taking it out. When at room temperature, make up the volume to 10 mL and then measure the OD 540 value. The control was the same as the above operations except that the substrate was added after terminating the reaction. Determination of β-glucosidase activity: The reaction substrate was replaced with 1% salicin buffer, and the other operations were the same as above. Determination of polygalacturonase activity: The substrate was 1% polygalacturonic acid buffer, and the constant temperature water bath time was 60 min, and the other operations were the same as above. Determination of polymethylgalacturonase activity: Replace the reaction substrate in the above steps with 1% pectin buffer, and the constant temperature water bath time is 60 min, and the other operations are the same as above.

[0054] The results are as follows Figure 5 shown. 2,4-Di-tert-butylphenol at 0.1 mg / mL and 1.0 mg / mL significantly inhibited the activities of all four cell wall-lysing enzymes, and the inhibitory effect was stronger at higher concentrations.

[0055] Example 7

[0056] Dissolve 2,4-di-tert-butylphenol in an appropriate amount of DMSO to prepare Czapek-Dox media containing the drug at concentrations of CK(0), 0.1, and 1.0 mg / mL. Punch out fungal discs and inoculate them into 100 mL of the drug-containing Czapek-Dox media. Incubate in the dark on a shaker at 28 °C and 200 rpm for 15 days. Take the supernatant and filter it through a microporous membrane to obtain a crude toxin solution sample, which is stored in a refrigerator at 4 °C for later use. Weigh 4 g of the sample, add sodium chloride, then add 15 mL of acetonitrile. After extraction and centrifugation, take 8 mL of the supernatant, purify it with n-hexane saturated with acetonitrile, then rotary evaporate to dryness, dissolve it in water, purify it through an HLB column, rotary evaporate the eluate to dryness, dissolve it in 1 mL of methanol aqueous solution, filter it, and perform quantitative detection on the machine.

[0057] The results are as follows Figure 6 shown. The regression equation of the fusaric acid standard curve is y = 0.9856x + 2.014 (R 2 = 0.999), proving that this standard curve can be used. Through quantitative detection, it was found that 2,4-di-tert-butylphenol at 0.1 mg / mL and 1.0 mg / mL both inhibited the content of fusaric acid in FOC, and the inhibition rates reached 40.18% and 64.94% respectively, showing a significant difference from the content in the CK group.

[0058] The above embodiments describe the preferred embodiments of the present invention and do not limit the present invention. Without departing from the spirit or scope of the present invention, technical improvements and equivalent substitutions made by relevant technical personnel to the present invention are all within the protection scope of the present invention.

Claims

1. Application of 2,4 - di - tert - butylphenol (2,4 - DTBP) in inhibiting the pathogenicity of Fusarium oxysporum f.sp. cucumerinum ), characterized in that The application includes achieving antibacterial effects through at least one of the mechanisms of inhibiting the hyphal growth, spore germination, change in cell membrane permeability, decrease in defense enzyme activity, inhibition of cell wall degrading enzyme activity, or reduction in toxin synthesis of the pathogenic bacterium.

2. The application according to claim 1, characterized in that, The application concentration of the 2,4-DTBP is 0.05 mg / mL to 1.6 mg / mL.

3. The application according to claim 2, wherein The median effective inhibitory concentration of the 2,4-DTBP against the hyphal growth of Fusarium oxysporum f. sp. cucumerinum is 0.104 mg / mL, and the median effective inhibitory concentration against spore germination is 1.024 mg / mL.

4. The application according to claim 1, characterized in that, The application is achieved by inhibiting the hyphal growth of the pathogenic bacterium. Specifically, in a drug-containing culture medium, when the concentration of 2,4-DTBP is 0.1 mg / mL, the colony diameter is reduced by more than 50% compared with the control group, and the hyphal growth inhibition rate shows a significant positive correlation with the increase in concentration.

5. The application according to claim 1, characterized in that The application is achieved by inhibiting the spore germination of the pathogenic bacterium. Specifically, when the concentration of 2,4-DTBP is 0.1 mg / mL, the spore germination rate is reduced by 13.8% compared with the control group, and the inhibitory effect is enhanced with the increase in concentration.

6. The application according to claim 1, characterized in that, The application is achieved by destroying the integrity of the cell membrane of the pathogenic bacterium. Specifically, after treatment with 2,4-DTBP, the relative electrical conductivity of the hyphae increases by 48.71% to 135.20% within 48 hours.

7. The application according to claim 1, wherein The application is achieved by inhibiting the defense enzyme activity of the pathogenic bacterium. Specifically, after treatment with 2,4-DTBP, the inhibition rate of superoxide dismutase (SOD) activity is 36.23% to 64.78%, and the inhibition rate of peroxidase (POD) activity is 53.82% to 77.08%.

8. The application according to claim 1, wherein The application is achieved by inhibiting the cell wall degrading enzyme activity of the pathogenic bacterium. Specifically, when the concentration of 2,4-DTBP is 1.0 mg / mL, the inhibition rates of the activities of carboxymethyl cellulase, β-glucanase, polygalacturonase, and polymethylgalacturonase exceed 51.85%, 78.13%, 33.90%, and 57.41% respectively.

9. The application according to claim 1, characterized in that, The application is achieved by inhibiting the synthesis of fusaric acid by the pathogenic bacterium. Specifically, when the concentration of 2,4-DTBP is 1.0 mg / mL, the content of fusaric acid is reduced by 64.94% compared with the control group.

10. A plant-derived antibacterial composition, characterized in that, Comprising the 2,4-DTBP according to any one of claims 1-9 as an active ingredient, and an agriculturally acceptable carrier or adjuvant, the composition is used for the prevention and control of cucumber Fusarium wilt.

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