Method for preventing and treating fusarium oxysporum by using trans-cinnamic acid and palmitic acid

Abstract

The invention discloses a method for preventing and treating fusarium oxysporum by using trans-cinnamic acid and palmitic acid, and belongs to the technical field of biological prevention and treatment of plant diseases. The bacillus subtilis B579 and the fusarium oxysporum (FOC) are co-cultured to induce bacteriostatic metabolites, key bacteriostatic metabolites including trans-cinnamic acid (CA) and palmitic acid (PA) are screened out through metabonomics analysis, the optimal bacteriostatic concentration of the key bacteriostatic metabolites is determined to be 50, and the bacteriostatic effect is achieved by destroying the completeness of cell membranes of pathogenic bacteria and initiating membrane lipid peroxidation. The invention further provides a biological agent containing CA and PA and an application method, greenhouse pot experiment verifies that the prevention and control effects on cucumber fusarium wilt respectively reach 67.58% (CA) and 47.29% (PA), the environmental compatibility is excellent, and an efficient and safe prevention and control scheme is provided for sustainable development of the facility cucumber industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease biological control, and in particular to a method for controlling Fusarium oxysporum by using trans-cinnamic acid and palmitic acid. BACKGROUND

[0002] Cucumber fusarium wilt, also known as wilt disease, vine cutting disease and dead seedling disease, is a devastating disease caused by Fusarium oxysporum f. sp. cucumeris (FOC). The disease is particularly harmful in continuous cropping plots, with a 70% incidence rate after 3 years of continuous cropping, resulting in a 10%-50% yield loss, and even absolute yield loss in severe cases, which has become a key factor restricting the sustainable development of the greenhouse cucumber industry.

[0003] Current common methods for controlling cucumber fusarium wilt mainly rely on chemical control, such as using thiophanate-methyl, chlorothalonil and pyraclostrobin to disinfect seeds and seedbeds, which can achieve significant control effect. However, excessive and frequent use of these chemicals can cause soil environmental pollution, increase pathogen resistance and reduce field efficacy. The control effect of biocontrol microbial agents is highly dependent on soil microenvironment (such as pH, humidity and indigenous bacterial competition), and has poor stability in the field. Single plant-derived antibacterial substances have narrow activity spectrum and limited action strength, which cannot meet the high-efficiency control requirements of commercial production. The core of the above technical defects is the lack of bio-derived antibacterial active ingredients with clear action targets, high biological activity and excellent environmental compatibility, as well as standardized application technology systems. SUMMARY

[0004] The present application aims to provide a method for controlling Fusarium oxysporum by using trans-cinnamic acid and palmitic acid, to solve the problem of lack of bio-derived antibacterial active ingredients with clear action targets, high biological activity and excellent environmental compatibility, as well as standardized application technology systems in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides a method for controlling Fusarium oxysporum by using trans-cinnamic acid and palmitic acid, which comprises soil treatment or root irrigation, and the concentration of trans-cinnamic acid is 50 , and the concentration of palmitic acid is 50 .

[0006] In another aspect, the present application provides a bio-agent for controlling Fusarium oxysporum, and the active ingredients are trans-cinnamic acid and palmitic acid, the concentration of trans-cinnamic acid is 50 , and the concentration of palmitic acid is 50 .

[0007] In another aspect, the present application provides the use of trans-cinnamic acid and palmitic acid in the preparation of a bio-agent for controlling Fusarium oxysporum.

[0008] Therefore, the method for controlling Fusarium oxysporum using trans-cinnamic acid and palmitic acid of the present invention has the following beneficial effects: (1) When Bacillus subtilis B579 is cultured alone, it may not produce trans-cinnamic acid (CA) and palmitic acid (PA) in small amounts. However, when co-cultured with the pathogen Fusarium oxysporum, the interaction between the two activates the specific metabolic pathways of Bacillus subtilis, thereby inducing it to produce large amounts of these antibacterial substances. This invention uses the technical route of "co-culture induction - non-targeted metabolomics screening - targeted function verification" to accurately identify CA and PA from 15 candidate metabolites, forming a complete chain of evidence, and solving the problems of low screening efficiency and poor targeting of biological antibacterial components.

[0009] (2) This invention not only confirmed the antibacterial activity of CA and PA, but also revealed their mechanism of action at the cellular and molecular levels. By detecting multiple indicators such as conductivity, protein leakage, MDA content, and CDA content, MDA was increased by 4.04 times and 3.27 times compared with the control, and CDA content was increased by 1.99 times and 1.55 times compared with the control, respectively. This strongly proves that these two metabolites exert their antibacterial effect by destroying the integrity of the pathogen cell membrane and inducing membrane lipid peroxidation, providing theoretical support for their standardized application.

[0010] (3) Greenhouse pot experiment showed that the disease index of CA and PA treatment groups was 14.81 and 24.08, respectively, which were 67.58% and 47.29% more effective than FOC treatment alone (disease index 45.68). Compared with chemical agents, there is no risk of environmental pollution, the pathogens are not likely to develop drug resistance, and compared with traditional biocontrol agents, the field stability is stronger and there is no need to rely on specific soil microenvironment.

[0011] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The antagonistic effect of different concentrations of CFs (B579) on the mycelial growth of pathogenic fungi; data are average values. Standard deviation; different lowercase letters on the bar chart represent significant differences between different treatments ( P 0.05); Figure 2Metabolomic differences of CFs in different culture groups; where A represents metabolomic differences under positive ion mode and B represents metabolomic differences under negative ion mode. Figure 3 The images show differential metabolite volcano plots of CFs in different culture groups under different modes. Specifically, A represents the differential metabolic volcano plots of the Bacillus subtilis B579 monoculture group and the Bacillus subtilis B579 co-culture group under positive ion mode; B represents the differential metabolic volcano plots of the Bacillus subtilis B579 monoculture group and the Bacillus subtilis B579 co-culture group under negative ion mode; and C represents the differential metabolic volcano plots of the Bacillus subtilis B579 monoculture group and the Fusarium oxysporum monoculture group under negative ion mode. Figure 4 The heatmap analysis of metabolic components in CFs from different culture groups is shown; where A is the heatmap analysis of metabolic components under positive ion mode and B is the heatmap analysis of metabolic components under negative ion mode. Figure 5 Preliminary in vitro plate assay of the antagonistic activity of metabolites against spore germination of Fusarium wilt pathogens; Figure 6 The antagonistic effects of CA and PA at different concentrations on the germination of pathogenic spores are shown. Here, A represents the antagonistic effect of CA at different concentrations on the germination of pathogenic spores, and B represents the antagonistic effect of PA at different concentrations on the germination of pathogenic spores. Figure 7 For CA and PA (50) The cell membrane permeability and intracellular protein content of pathogenic mycelia under treatment, where A is the relative conductivity of pathogenic mycelia; and B is the intracellular protein content. Figure 8 For CA and PA (50) The MDA content and CAT activity of pathogenic mycelia under treatment are given by A, where A is the MDA content of pathogenic mycelia and B is the CAT activity of pathogenic mycelia. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0016] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0017] Bacillus subtilis ( Bacillus subtilis B579 (accession number: CGMCC2207) was stored at -80°C on LB agar plates.

[0018] Fusarium wilt pathogen of cucumber: Fusarium oxysporum ( Fusarium oxysporum f.sp. cucumerinum (FOC for short) is stored at 4°C in PDA medium.

[0019] The cucumber variety used in this example is "Prickly Eggplant".

[0020] Example 1 Under aseptic conditions, frozen Bacillus subtilis B579 was plated on LB agar plates and incubated at 37°C for 1 day. The activated bacterial suspension was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was discarded, and the cells were resuspended in sterile water and adjusted to a pH of 10. 4 CFU / mL.

[0021] Fusarium oxysporum was inoculated onto PDA plates and incubated at 28°C for 6 days. The spore suspension was then diluted to a concentration of 10 with sterile water. 4 Conidia / mL.

[0022] Bacillus subtilis B579 culture (10) grown in PDB medium for 12 hours 4 (CFU / mL) filtered through a membrane (0.22) The membrane was used to dilute the CFs (2:1 and 1:2, filtrate: PDB) at different volume ratios (high concentration and low concentration).

[0023] 300 Pathogenic spore suspension (10 4 Conidia ( / mL) were inoculated into Erlenmeyer flasks containing 30 mL of two concentrations of CFs dilution and incubated on a shaker at 180 rpm and 28°C for 3 days. The culture contents were then poured into Petri dishes and dried at 60°C to constant weight. The dry weight of the mycelium was measured. 30 mL of PDB medium was taken and 300 mL of CFs dilution solution was added. The pathogen spore suspension served as a control, and each treatment was repeated three times.

[0024] The results are as follows Figure 1 As shown, the culture filtrate (CFs) of Bacillus subtilis B579 can significantly inhibit the growth of the pathogen of cucumber wilt in a concentration-dependent manner.

[0025] Example 2 To study the microbial metabolome under single-culture and co-culture conditions, Bacillus subtilis B579 was co-cultured with Fusarium wilt pathogens. First, 300... Pathogenic spore suspension (10 4 (1 conidia / mL) were inoculated into an Erlenmeyer flask containing 30 mL of PDB medium and incubated on a shaker at 180 rpm and 28°C for 48 h. Then 300 (10) 4 A pre-culture medium of Bacillus subtilis (CFU / mL) was added to an Erlenmeyer flask containing Fusarium wilt pathogens, and the flask was incubated for 12 hours. Single cultures of Bacillus subtilis B579 and FOC were used as controls. The mixture was filtered through a 0.22 μL filter membrane. The culture was filtered to obtain CFs. 1 mL of CFs was lyophilized in a freeze dryer and then resuspended at 100 °C. The sample was placed in a methanol-water solution (4:1, v / v) and incubated on ice for 6 min. The mixture was then centrifuged at 15000 rpm and 4°C for 15 min. The supernatant was transferred to a new tube, and the methanol content was diluted to 53% with mass spectrometry-grade water. The tube was then centrifuged under the same conditions as the previous step, and the supernatant was retained. 50 μL of each sample was used for quality control analysis, and the remaining samples were used for metabolomics analysis.

[0026] Metabolomic differences of CFs in different culture groups as follows Figure 2 As shown, A represents the metabolomic differences under positive ion mode, and B represents the metabolomic differences under negative ion mode. Principal component analysis (PCA) shows that, regardless of whether it is under positive or negative ion mode, QC sample points and sample points within each treatment group cluster well together, indicating good stability and high data quality of the entire method. However, the sample points between different treatments are more dispersed in the PCA plot, proving that there are significant differences in the metabolome among treatments in CFs.

[0027] Differential metabolite volcano diagrams of CFs under different culture modes in different culture groups are shown below. Figure 3 As shown, A represents the differential metabolic volcano plots of the Bacillus subtilis B579 single-culture group and the Bacillus subtilis B579 co-culture group under positive ion mode; B represents the differential metabolic volcano plots of the Bacillus subtilis B579 single-culture group and the Bacillus subtilis B579 co-culture group under negative ion mode; and C represents the differential metabolic volcano plots of the Bacillus subtilis B579 single-culture group and the Bacillus subtilis B579 co-culture group under negative ion mode. In the figures, green indicates down-regulation, red indicates up-regulation, and gray indicates no significant change.

[0028] The following three criteria were used for screening: 1. Metabolites not secreted in the filtrate of single-culture FOC cultures; 2. Metabolites secreted by Bacillus subtilis B579; 3. Metabolites highly secreted by Bacillus subtilis B579 under co-culture conditions, subject to the constraints of the first two criteria.

[0029] Thermographic analysis of metabolic components in CFs from different culture groups is as follows: Figure 4As shown, A represents the heatmap analysis of metabolic components under positive ion mode, and B represents the heatmap analysis of metabolic components under negative ion mode; Cluster 1 and Cluster 2 represent the metabolites highly secreted by Bacillus subtilis B579 in the co-culture filtrate after condition screening.

[0030] After screening, a total of 49 target metabolites (cluster 1 and cluster 2) were identified. Table 1. B579 hypersecreting metabolites (positive ionic compounds) identified in the co-culture filtrate.

[0031] Table 2. B579 hypersecreting metabolites (anionic compounds) identified in the co-culture filtrate

[0032] Example 3 To screen for metabolites in CFs that resist the germination of Fusarium wilt conidia, commercial standards of 15 relevant metabolites were purchased based on metabolomics information. 100 μL of the standard was prepared according to the manufacturer's provided method. A stock solution with a pH of 7. Take 100... Pathogenic spore suspension (10 4 Add conidia ( / mL) to a PDA plate, spread evenly, and allow to air dry at room temperature. Place sterile filter paper discs (5 mm in diameter) at equal intervals (2.5 cm) in the plate, and add 10 mL of conidia to each disc. Metabolites, to be added to 10 The solvent used to dissolve the metabolites served as a control. Plates were placed in a constant temperature incubator at 28°C, and the diameter of the halo around the filter paper was calculated on day 4 (halo diameter = total diameter of the halo - diameter of the filter paper). Each metabolite was tested three times.

[0033] Preliminary results of in vitro plate assays of the antagonistic activity of metabolites against spore germination of Fusarium wilt pathogens are as follows: Figure 5 As shown, four metabolites significantly inhibited the germination of Fusarium wilt spores. Trans-cinnamic acid (CA) exhibited the strongest inhibitory effect, followed by palmitic acid (PA).

[0034] Example 4 Further investigation was conducted on CA and PA, which showed the best results. Different concentration gradients (0, 25, 50, 100) were set up. The antibacterial effects of these two metabolites were tested using the same method as in Example 3.

[0035] The results are as follows Figure 6As shown, A represents the antagonistic effect of CA at different concentrations on the germination of pathogenic spores, and B represents the antagonistic effect of PA at different concentrations on the germination of pathogenic spores. The results indicate that the solvent has no antagonistic activity against the germination of pathogenic spores. PA at a concentration of 25% showed antagonistic activity against the germination of pathogenic spores. L showed no antagonistic activity, while CA (25) showed no antagonistic activity. At this time, they exhibit antagonistic activity. The antagonistic ability of both CA and PA against pathogen spore germination is at least 50%. The strongest antagonistic effect was observed at [specific concentration], producing halo diameters of 0.63 cm and 1.27 cm, respectively. At the same concentration, CA exhibited stronger antagonistic ability against pathogenic spore germination.

[0036] Example 5 Take 3 mL of pathogenic spores (10 4 A suspension of 50 spores / mL was inoculated into an Erlenmeyer flask containing 100 mL of PDB medium and incubated at 28°C for 3 days. Then, 3 mL of the suspension (50 spores / mL) was taken. Metabolites were added to mycelial cultures grown for 3 days, with an equal volume of the corresponding solvent added as a control. The cultures were incubated for 12 hours at 28°C and 180 rpm in a shaker. The mycelia were collected, washed, and blotted dry with absorbent paper. The mycelia (0.2 g / mL) were then suspended in distilled water. Conductivity was measured every half hour. After 4 hours, the samples were boiled in a water bath for 10 minutes, and the final conductivity was measured. Each treatment was repeated three times. The relative conductivity was calculated using the following formula: ; Transfer 15 mL of the culture medium co-cultured with metabolites for 12 h to a new tube and centrifuge at 5000 rpm for 8 min at 4 °C. Resuspend the precipitate in 1 mL of sterile distilled water and disrupt the resuspended material using an ultrasonic homogenizer, repeating this process twice with a 30 s interval. Centrifuge the disrupted mixture at 20000 rpm for 15 min at 4 °C, discard the supernatant, and measure the remaining intracellular soluble protein using the Bradford assay. Each treatment is repeated three times.

[0037] The results are as follows Figure 7 As shown, where A represents CA and PA (50 The relative conductivity of pathogenic mycelia treated with CA and PA was measured, where B represents the soluble protein content within the pathogenic mycelia exposed to the two metabolites. The results showed that, compared to the control group, the conductivity of mycelia treated with the metabolites significantly increased over time, indicating that CA and PA can affect the permeability of the pathogenic plasma membrane. Compared to PA (50... In CA treatment, the plasma membrane of pathogens showed higher permeability within 90-240 minutes, indicating that CA caused more severe plasma membrane damage than PA at this time. Figure 7 As can be seen from B, after 12 hours of treatment, use 50 The soluble protein content of bacteria treated with CA or PA was significantly reduced compared to the control, decreasing by 36.58% and 24.71%, respectively. P <0.05).

[0038] Example 6 Take 15 mL of the culture medium after co-culturing with metabolites for 12 h, centrifuge at 8000 rpm for 5 min at 4℃, discard the supernatant to obtain mycelium, and determine the MDA content and CAT activity in the mycelium according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd.).

[0039] The results are as follows Figure 8 As shown, Figure 8 In the middle, A represents CA and PA (50 The MDA content in the mycelia of the pathogen treated with CA and PA was 50%. Under the specified conditions, after 12 hours of treatment, the MDA content of the mycelium was 9.04%. and 7.15 The levels were 4.04 times and 3.27 times higher than the control, respectively. These results indicate that CA and PA can disrupt the cell membrane of pathogenic bacteria, thereby triggering membrane lipid peroxidation.

[0040] Figure 8 In the middle, B represents CA and PA (50) CAT activity in pathogenic mycelia treated with CA and PA (50) When co-cultured for 12 hours, the CAT activities in the mycelia were 266.73 U / g and 218.91 U / g, respectively, which were 1.99 times and 1.55 times that of the control, respectively. This indicates that CA and PA can affect CAT activity, and stressed mycelia may reduce the degree of stress by increasing the activity of this enzyme.

[0041] Example 7 To investigate the effects of CA and PA on plants facing pathogen invasion, the experimental group used a sterile syringe to administer 30 mL of CA and PA (50 mL of each). Slowly inject the solution into the rhizosphere soil of cucumbers with two true leaves grown in a greenhouse. The control and FOC treatment groups (inoculated only with the pathogen) received 30 mL of sterile water. On day 1 of treatment, the control group received 30 mL of sterile water, while the other treatments received 30 mL of pathogen spore suspension (10...). 4 Inject conidia (1 / mL) into the cucumber rhizosphere soil. Culture conditions: natural light, day / night temperature 25℃ / 18℃, relative humidity 60%-70%. Each treatment was replicated 3 times, for a total of 108 plants, three plants per pot. Thirteen days after inoculation, the disease incidence in cucumbers was assessed, and the disease index and relative control effect were calculated using the formula: ; .

[0042] The following methods were used to investigate the disease incidence: Grade 0, healthy and asymptomatic; Grade 1, slight wilting of cotyledons; Grade 2, wilting symptoms in seedlings (yellowing of cotyledons); Grade 3, withered, yellowed, and twisted plants; Grade 4, withered and dead plants.

[0043] The results of the pot experiment are shown in Table 3: Table 3. Control effects of target metabolites on cucumber wilt disease.

[0044] Note: Data are averages. Standard deviation; different lowercase letters in the same column represent significant differences between different treatment groups ( ).

[0045] The results showed that cucumber seedlings in the control group grew normally, while cucumbers treated with FOC alone showed disease symptoms, with a disease index of 45.68. The disease indices of CA and PA treatments were 14.81 and 24.08, respectively, and their control effects on cucumber wilt were 67.58% and 47.29%, respectively.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for controlling Fusarium oxysporum using trans-cinnamic acid and palmitic acid, characterized by: including soil treatment or drench application, at a concentration of 50 mg / L of trans-cinnamic acid mg / L of palmitic acid.

2. A biological agent for controlling Fusarium oxysporum, characterized in that, The active ingredients are trans-cinnamic acid and palmitic acid, the concentration of trans-cinnamic acid being 50 , and the concentration of palmitic acid being 50 .

3. Use of trans-cinnamic acid and palmitic acid for the preparation of a biological preparation for the control of Fusarium oxysporum.

Images