Application of bromocamphor in inhibiting pseudomonas syringae and preventing and treating mulberry epidemic diseases

Abstract

The invention discloses application of bromocamphor in inhibition of pseudomonas syringae and prevention and treatment of mulberry epidemic diseases, the invention provides the application of bromocamphor in inhibition of pseudomonas syringae for the first time, a bacteriostatic rate determination result shows that the bacteriostatic effect of bromocamphor on pseudomonas syringae is in a concentration dependent form, and when the concentration is 1.5 mg.mL <-1 >, the bacteriostatic effect of bromocamphor on pseudomonas syringae is not in a concentration dependent form, and when the concentration is not in a concentration dependent form, the bacteriostatic effect of bromocamphor on pseudomonas syringae is not in a concentration dependent form. The bacteriostasis rate of bromocamphor reaches 90% or above. At the same time, the pseudomonas syringae treated by bromocamphor is seriously deformed and shrunk, and the morphological structure is destroyed, which shows that bromocamphor has good bacteriostatic activity. Besides, by measuring the influence of bromocamphor on biofilm formation, motility, active oxygen generation and exopolysaccharide generation of pseudomonas syringae, the bacteriostatic mechanism of active substances of root exudates is further studied, and a new thought is provided for development of novel plant source biopesticides. The composition of the mulberry root exudates under the neutral pH condition is determined, and a certain theoretical basis is provided for green prevention and control of mulberry epidemic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microbial control, and particularly relates to application of bromocamphor in inhibiting Pseudomonas syringae and preventing and controlling mulberry blight. Background Art

[0002] The various compounds released by plant roots into the surrounding environment in an active or passive manner are collectively referred to as root exudates. In recent years, root exudates have become a research hotspot for plant-soil-microorganism interactions and material cycles. The composition of root exudates is complex and diverse, and they play a vital role in signal transduction, nutrient transport and stress resistance. There are more than 220 types of root exudates, which can be divided into high molecular weight exudates and low molecular weight exudates according to their molecular weight. The former mainly includes mucilage and exoenzymes, among which mucilage contains polysaccharides and polyuronic acid; the latter mainly contains low molecular weight organic acids, sugars, phenols and various amino acids (including non-protein amino acids, such as plant iron carriers). In addition, root exudates also contain some physiologically active substances, such as hormones, vitamins and various self-injurious and other-injurious compounds. Plants release these compounds to change the physical, chemical or biological properties of the rhizosphere, thereby improving the nutrient absorption of the roots and regulating the microecological functions of the rhizosphere.

[0003] Mulberry blight caused by Pseudomonas syringae causes huge economic losses to the sericulture industry. At present, physical and chemical measures are mainly used to prevent and control mulberry blight, but due to the complex pathogenic mechanism of Pseudomonas syringae and the pollution of traditional chemical agents to the soil environment, there is still a lack of efficient and environmentally friendly prevention and control strategies. Therefore, it is of great significance to explore new biocontrol agents for the prevention and control of mulberry blight caused by Pseudomonas syringae. Using plant secondary metabolites as biocontrol agents is a good strategy to protect plants from disease infection. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention proposes for the first time the antibacterial effect of bromocamphor on Pseudomonas syringae, providing a theoretical basis for the development of new plant-derived agents for preventing and treating mulberry blight.

[0005] Technical solution: In order to achieve the above purpose, the bromocamphor of the present invention is used in inhibiting Pseudomonas syringae.

[0006] Among them, the bromocamphor is used in the preparation of an antibacterial agent for Pseudomonas syringae.

[0007] Wherein, the bromocamphor is artificially synthesized or extracted from mulberry root secretions.

[0008] The bromocamphor is obtained from neutral components of mulberry root secretions.

[0009] Wherein, the content of bromocamphor in the neutral components of the mulberry root secretions exceeds 10%.

[0010] The invention discloses an application of bromocamphor in preventing and treating mulberry blight caused by Pseudomonas syringae.

[0011] Among them, the bromocamphor is used in the preparation of a medicament for preventing and treating mulberry blight caused by Pseudomonas syringae.

[0012] The concentration of bromocamphor in the preparation of the agent for preventing and treating mulberry blight caused by Pseudomonas syringae is 0.5-1.5 mg·mL -1 .

[0013] Preferably, the concentration of bromocamphor in the preparation of the agent for preventing and treating mulberry blight caused by Pseudomonas syringae is 1.5 mg·mL -1 .

[0014] The pharmaceutical composition for preventing and treating mulberry blight of the present invention comprises bromocamphor as a main active ingredient, and pharmaceutically acceptable carriers and auxiliary materials. The mulberry blight is caused by Pseudomonas syringae.

[0015] Wherein, the dosage form of the pharmaceutical composition includes emulsion, suspension and granule.

[0016] The present invention purifies the collected mulberry root secretions under hydroponic conditions, separates and screens bromocamphor, a substance with good antibacterial effect on Pseudomonas syringae, and analyzes its antibacterial mechanism, and uses the substance with high content and good antibacterial effect to prevent and control mulberry blight. The present invention provides a basis for the application of root secretions in the field of disease prevention and control.

[0017] Specifically, the present invention purifies the collected mulberry root exudates under hydroponic conditions according to the following method:

[0018] 1. 14 days after hydroponically cultivating mulberry seedlings, the variety is Fengchi. Collect the culture solution and separate and purify the root secretions.

[0019] 2. The bromocamphor mother solution was co-cultured with the Pseudomonas syringae suspension, and the biofilm was stained with 1% crystal violet to clarify the effect of bromocamphor treatment on the formation of Pseudomonas syringae biofilm.

[0020] Furthermore, GC-MS (Thermo Trace-1300 ISQ) was used to analyze and identify the composition of root exudates and calculate the relative content.

[0021] Furthermore, the absorbance value was used to detect the inhibitory ability of root exudate bromocamphor on Pseudomonas syringae.

[0022] Furthermore, the morphological and structural changes of Pseudomonas syringae after treatment with bromocamphor were observed under a scanning electron microscope.

[0023] Furthermore, 1.5 mg mL -1 The concentrations of bromocamphor were co-cultured with Pseudomonas syringae, and the changes in the swimming ability of Pseudomonas syringae were measured on NB semi-solid medium.

[0024] Furthermore, the water-soluble alcohol precipitation phenol-sulfuric acid method was used to determine the changes in the content of extracellular polysaccharides of Pseudomonas syringae after treatment with bromocamphor.

[0025] Furthermore, an active oxygen species kit was used to detect changes in the content of active oxygen (ROS) in Pseudomonas syringae after treatment with bromocamphor.

[0026] The present invention proposes for the first time the application of bromocamphor (a component of root secretions) to prevent and control the blight caused by Pseudomonas syringae. The composition of mulberry root secretions under neutral pH conditions was analyzed by GC-MS technology, which mainly included hydrocarbons, alcohols, esters, organic acids, aldehydes and other substances. It was found that the relative content of bromocamphor was relatively high, accounting for 18.33%. The results of the inhibition rate determination showed that the antibacterial effect of bromocamphor on Pseudomonas syringae was concentration-dependent. As the concentration increased, the inhibition rate also increased, and the concentration was 1.5 mg·mL -1 When the bacteriostatic rate of bromocamphor reaches more than 90%. At the same time, scanning electron microscopy observation found that the Pseudomonas syringae treated with bromocamphor was severely deformed and wrinkled, and the morphological structure was destroyed, indicating that bromocamphor has good antibacterial activity. In addition, the present invention further studies the antibacterial mechanism of active substances in root secretions by measuring the effects of bromocamphor on biofilm formation, motility, reactive oxygen generation and extracellular polysaccharide generation of Pseudomonas syringae, providing new ideas for the development of new plant-based biological pesticides. The present invention clarifies the composition of mulberry root secretions under neutral pH conditions, providing a certain theoretical basis for green prevention and control of mulberry blight.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] The invention firstly proposes the application of bromocamphor in inhibiting Pseudomonas syringae and preventing and treating mulberry blight.

[0029] The present invention successfully determined the composition of neutral components in the root secretions of healthy mulberry trees through GC-MS technology, and detected bromocamphor in the neutral components with a relatively high content of 18.33%. The results of the antibacterial rate showed that bromocamphor at different concentrations had an inhibitory effect on the growth of Pseudomonas syringae. When the concentration of bromocamphor reached 0.5-1.5 mg·mL -1When the antibacterial rate of bromocamphor was 70%-90%, the plate antibacterial test results were consistent with the antibacterial rate results. Bromocamphor showed a good antibacterial effect. The EC50 of bromocamphor was calculated based on the linear equation to be 0.35 mg·mL -1 , indicating that when the antibacterial rate reaches 50%, the required concentration of bromocamphor is 0.35 mg·mL -1 . In addition, scanning electron microscopy observations revealed that the Pseudomonas syringae treated with bromocamphor underwent severe deformation and shrinkage, and its morphological structure was destroyed, confirming that bromocamphor had good antibacterial activity against Pseudomonas syringae. Through research on the antibacterial mechanism, it was found that the treatment with bromocamphor significantly inhibited the biofilm formation and motility of Pseudomonas syringae, induced the release of reactive oxygen species in Pseudomonas syringae, caused oxidative damage to the bacteria, and thus affected the growth activity of Pseudomonas syringae. The present invention provides a reference for the regulatory pathway of the virulence mechanism of Pseudomonas syringae, and provides an environmentally friendly and effective strategy for the research and development of mulberry blight control agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the total ion current chromatogram of the neutral components of the root exudates of mulberry seedlings (Note: the numbers in the figure are the retention times of the root exudates, and the red boxes emphasize the substances with relatively high contents at each retention time);

[0031] Figure 2 The antibacterial effect of bromocamphor, a secretion from the roots of mulberry trees, on Pseudomonas syringae;

[0032] Figure 3 The plate-type antibacterial effect of bromocamphor, a mulberry root secretion, on Pseudomonas syringae (Note: the yellow rectangular box in the figure is located at the same position on each plate, the enlarged effect diagram below the plate shows the growth of the colony in the corresponding area, CK is the blank control, and ethanol is the solvent control);

[0033] Figure 4 1.5 mg mL -1 Scanning electron microscopy analysis of Pseudomonas syringae after treatment with active substances (Note: A: Cell morphology of Pseudomonas syringae treated with solvent control; B: Morphological changes of Pseudomonas syringae after treatment with bromocamphor, red arrows indicate damaged parts of the bacteria);

[0034] Figure 5 1.5 mg mL -1 Effect of bromocamphor on biofilm formation of Pseudomonas syringae (Note: A: Microscope observation of biofilm formation of Pseudomonas syringae under bromocamphor treatment; B: The effect of bromocamphor on the amount of biofilm formation of Pseudomonas syringae at absorbance of 595nm, CK is blank control, ethanol is solvent control, different letters indicate significant differences, p<0.05);

[0035] Figure 6 1.5 mg mL -1 Effect of bromocamphor on the motility of Pseudomonas syringae (Note: the formation of bacterial circles of Pseudomonas syringae on semi-solid culture medium after 2 days of treatment, 1: CK; 2: bromocamphor, red line segment: swimming bacterial circle = outer circle diameter - inner circle diameter, A: the original bacterial circle size of Pseudomonas syringae after bromocamphor treatment; B: the colony diameter of Pseudomonas syringae after 2 days of treatment, CK is the blank control, ethanol is the solvent control. Different letters indicate significant differences, p<0.05);

[0036] Figure 7 1.5 mg mL -1 Effects of bromocamphor on the extracellular polysaccharides of Pseudomonas syringae (Note: CK is the solvent control; different letters indicate significant differences, p<0.05);

[0037] Figure 8 1.5 mg mL -1 Effect of bromocamphor on the reactive oxygen burst in Pseudomonas syringae (Note: A: bromocamphor, B: solvent control). DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with embodiments and drawings.

[0039] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0040] Bacteria strain: Pseudomonas syringae was isolated and preserved by Jiangsu University of Science and Technology, and is a wild-type Pseudomonas syringae strain MYC18. The 16s rDNA sequence accession number of MYC18 on NCBI is PQ130153.1; the gyrB sequence accession number is PQ300031.1; or other wild-type Pseudomonas syringae or commercially available model bacteria can be used.

[0041] Plant: Fengchi mulberry is preserved by Jiangsu University of Science and Technology / Sericulture Research Institute of Chinese Academy of Agricultural Sciences.

[0042] Sources of experimental reagents and equipment:

[0043] Experimental reagents: Bromocamphor used in the examples was purchased from Aladdin Reagent Company ((+)-3-bromocamphor, CAS No.: 10293-06-8, Article No.: B153198), triphenyltetrazolium chloride (TTC) was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., hydrolyzed casein was purchased from Sigma-Aldrich, glucose, peptone, agar powder, ethyl acetate, hydrochloric acid, sodium hydroxide, anhydrous ethanol, pentanediol, PBS buffer, crystal violet, 5% phenol, concentrated sulfuric acid, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd., and a reactive oxygen species (ROS) detection kit was purchased from Beyotime Biotechnology Co., Ltd.

[0044] Experimental equipment: -80℃ refrigerator (WiseCryo, Korea), high-speed refrigerated centrifuge (Thermo FRESCO17, Thermo Fisher, America), small centrifuge (BE-6100), SpectraMaxi3 microplate reader (BUCHI (Switzerland)), clean bench (Zhejiang Fuxia Medical Technology Co., Ltd.), digital display gas bath constant temperature oscillator THZ-82A (Nanjing Lisigao Instrument Equipment Co., Ltd.), circulating water vacuum pump, GC-MS gas spectrometer (Thermo Trace-1300 ISQ, Thermofishe), high pressure sterilizer (Zhiwei Instrument), constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.), light incubator (Nanjing Sanshi Biotechnology Co., Ltd.).

[0045] Solution and test medium configuration:

[0046] Bromocamphor mother solution: weigh bromocamphor solid powder and add anhydrous ethanol to prepare concentrations of 0.5, 1, 3, 5, 10, and 15 mg mL -1 The mother liquor is then put aside for later use after it is fully dissolved.

[0047] NA medium: 0.3g beef extract, 1g peptone, 0.5g sodium chloride, add ddH2O to make up to 100mL, stir evenly, put into a 121℃ high pressure sterilizer for 30min, if it is a solid culture medium, add 1.5g agar powder.

[0048] NB medium: Do not add agar powder to the above NA medium.

[0049] NA semisolid medium: add 0.75g agar powder, and the remaining ingredients and contents are the same as the above NA medium.

[0050] Example 1

[0051] Identification and acquisition of the main components of mulberry root exudates

[0052] After culturing the mulberry seedlings of the Healthy Fengchi variety for 14 days, the culture fluid was taken out and collected. 600 mL of the collected fluid was measured and filtered using a circulating water vacuum pump. The pore size of the organic filter membrane was 0.22 μM. After filtration, ethyl acetate was used for extraction. The filtered collected fluid was extracted with ethyl acetate in a ratio of 2:1 to obtain the ethyl acetate phase as a neutral component. The neutral component extract was concentrated to 5 mL using a rotary evaporation concentrator (45°C, 50rpm) and analyzed and identified using GC-MS technology. The chromatographic column used a DB-5MS column (30m×0.25mm×0.25μm), the ion source was EI+, the temperature was 250°C, and the inlet temperature was 250°C; the column temperature used a temperature increase program, 80°C at 15°C·min -1 Raise the temperature to 200℃ and then increase the temperature at 1.5℃·min -1 The temperature was raised to 250°C; the carrier gas was He, and the flow rate was 1 mL min -1 The injection volume was 1 μL. Each chromatographic peak was automatically searched in the NIST2014 and other mass spectrometry databases by computer and combined with manual analysis, and the relative content of each component in the GC-MS spectrum was calculated by peak area normalization method to determine its composition.

[0053] After GC-MS analysis, the total ion current chromatogram of mulberry seedling root secretions under the extraction condition of pH = 7.0 was obtained. Figure 1 The results showed that a total of 28 main components were detected in the root exudates of mulberry seedlings extracted under neutral conditions, including 1 organic acid, ketorolac; 8 esters, namely butyl acetate, ethyl acetate, labdanum, dibutyl phthalate, geranyl isovalerate, diisooctyl phthalate, imiprolin, and monoglyceride; 2 amides, erucamide and oleamide; 10 alkanes, namely decane, 4-methyl-decane, dodecane, undecane, nonadecane, tridecane, hexadecane, p-xylene, polystyrene, and squalene; 2 alcohols, geraniol and docosanol, with relatively low contents; acetophenone, isophorone, dodecyl nonyl ether, 2,6-di-tert-butyl-p-cresol, bromocamphor and other main components were also detected in the root exudates. Among them, bromocamphor had the highest relative content in the exudates under neutral extraction conditions, which was 18.33%.

[0054] Example 2

[0055] Analysis on the antibacterial effect of bromocamphor, an active substance in root exudates, on Pseudomonas syringae

[0056] Bromocamphor, a component with a relatively high content in the root secretions of healthy mulberry trees under neutral conditions, was selected to explore its effect on the growth of Pseudomonas syringae. The inhibitory ability of bromocamphor on Pseudomonas syringae was detected by absorbance value.

[0057] Commercially purchased bromocamphor was prepared into a stock solution, and 20 μL of bromocamphor of different concentrations was added to a 96-well plate (the concentration gradient was 0.05, 0.1, 0.3, 0.5, 1 and 1.5 mg mL -1 ), add 140 μL of NB liquid medium and OD 600 40 μL of 0.6 Pseudomonas syringae suspension (NB culture medium) was prepared into a mixed solution with a total system of 200 μL. 20 μL of anhydrous ethanol was taken as a solvent control, and 20 μL of NB culture medium was used as a blank control. After the addition was completed, the absorbance of the mixed solution was measured using a SpectraMaxi3 microplate reader, the detection wavelength was 600 nm, and the read value was recorded as T0. The 96-well plate was then placed in a bacterial constant temperature incubator and cultured at 30°C for 24 hours. After the culture was completed, the solution was gently shaken to make it evenly suspended and the absorbance of the solution was detected again. The resulting value was recorded as TF. The antibacterial efficiency of drugs of different concentrations on Pseudomonas syringae was calculated, and the calculation formula is as follows:

[0058]

[0059] In the formula, T0 sample Represents the absorbance value of bacterial culture before adding drugs; TF sample Represents the absorbance value of bacterial culture after adding drugs; T0 blank Represents the absorbance value of the bacterial solution before culture in the control group; TF blank Represents the absorbance value of the bacterial solution after culture in the control group.

[0060] Absorb OD 600 = 0.6 of the Pseudomonas syringae suspension was treated with bromocamphor at different concentrations. At the same time, anhydrous ethanol was used as a solvent control and NB liquid culture medium was used as a blank control to treat Pseudomonas syringae. After overnight culture, the bacterial suspension was diluted to 1×10 8 CFU·mL -1 , aspirate 200 μL of Pseudomonas syringae suspension and spread it evenly on the plate, place it in a 30℃ constant temperature incubator, and observe the growth of colonies on the plate after 3 days.

[0061] Figure 2 The results showed that different concentrations of bromocamphor had an inhibitory effect on the growth of Pseudomonas syringae. The antibacterial effect of bromocamphor gradually increased with the increase of concentration. When the concentration was 1.5 mg·mL -1 When bromocamphor was added, the antibacterial rate of Pseudomonas syringae reached more than 90%. The results of the plate antibacterial experiment were consistent with the results of the antibacterial rate. Figure 3 As shown in Table 1, bromocamphor exhibited a good antibacterial effect. Table 1 shows the EC values ​​of bromocamphor against Pseudomonas syringae 50 The EC of bromocamphor was calculated based on the linear equation. 500.35 mg·mL -1 , indicating that when the antibacterial rate reaches 50%, the required concentration of bromocamphor is 0.35 mg·mL -1 .

[0062] Table 1 Effect of bromocamphor from mulberry root exudates on Pseudomonas syringae EC 50 Determination of

[0063]

[0064] Example 3

[0065] Effects of Bromocamphor from Root Exudates of Mulberry Tree on Colony Morphology and Growth of Pseudomonas syringae

[0066] The cell slides were placed in a 12-well plate and NB culture medium and Pseudomonas syringae bacterial solution (OD 600 =0.5), cultured at 30°C for 12 h, and treated with bromocamphor (1.5 mg mL -1 , anhydrous ethanol as solvent control), and continue to culture for 8 hours. The cultured slides were pre-fixed with pentanediol, dehydrated and dried with different concentrations of alcohol, and then placed in an SCD 500 ion sputtering instrument to spray gold on the sample surface. The morphological changes of Pseudomonas syringae were observed under a scanning electron microscope and the images were recorded.

[0067] The growth morphology of Pseudomonas syringae after treatment with bromocamphor, an active substance in root exudates, was observed by scanning electron microscopy ( Figure 4 ). The surface structure of the bacteria in the solvent control group was complete and the morphology was full. Compared with the solvent control group, 1.5 mg·mL -1 The morphology of Pseudomonas syringae strains treated with bromocamphor was severely deformed, and the bacteria were flat and shrunken, as indicated by the red arrows in the figure, which further shows that bromocamphor has a significant inhibitory effect on Pseudomonas syringae.

[0068] Example 4

[0069] Effects of bromocamphor from mulberry root exudates on biofilm formation of Pseudomonas syringae

[0070] This example measured the concentration of 1.5 mg mL -1 The biofilm formation ability of Pseudomonas syringae under the treatment of bromocamphor. 150 μL bromocamphor stock solution (1.5 mg mL -1 ) and 300 μL of Pseudomonas syringae suspension (OD 6000.5) was added to a 24-well plate, and NB culture medium was added to a total system of 1.5mL. The inoculated 24-well plate was placed in a 30°C constant temperature incubator for 24 hours, and anhydrous ethanol was used as a solvent control. After the culture is completed, the supernatant culture medium was carefully removed and washed three times with PBS buffer. 200μL of 1% crystal violet was added to stain the biofilm, and after standing for 15 minutes, it was washed twice with PBS and dried at room temperature for 30 minutes. In order to quantify the biomass of the biofilm, 200μL of 95% ethanol was added to each well to dissolve the crystal violet adsorbed on the biofilm. After 30 minutes, the absorbance value at 595nm was detected using an enzyme reader to reflect the formation of the Pseudomonas syringae biofilm. Each treatment was repeated 3 times.

[0071] Under the microscope, it can be clearly observed that the biofilm in the treatment group is relatively sparse, in sharp contrast to the continuous large-area biofilm in the control group ( Figure 5 A). The absorbance value test showed that the amount of biofilm formation of Pseudomonas syringae after treatment with the active substance bromocamphor was significantly reduced compared with the solvent control ( Figure 5 B).

[0072] Example 5

[0073] Effects of Bromocamphor from Mulberry Root Exudates on the Motility of Pseudomonas syringae

[0074] This example investigates the effect of bromocamphor on the motility of Pseudomonas syringae.

[0075] Use 1.5 mg mL -1 The concentration of bromocamphor treated OD 600 = 0.6, and cultured overnight in a shaker at 30°C, 220 rpm, with anhydrous ethanol as the solvent control. 8 μL of bacterial solution was added to the semi-solid plate, and then cultured in a 30°C constant temperature incubator for 2 days. The growth of the plate was observed, and the size of the bacterial circle was measured by the cross method. Each treatment was repeated 3 times.

[0076] 1.5 mg·mL -1 After treatment with the active substances, the motility of Pseudomonas syringae was significantly reduced, and the bacterial rings formed on the semi-solid medium were smaller than those in the control group ( Figure 6 ). The diameter is measured by the cross method. The diameter of the colony after 2 days minus the original colony diameter is the growth of the colony. Figure 6 The red line in the lower left corner of A shows the colony diameter of Pseudomonas syringae after treatment was significantly reduced compared with the control group ( Figure 6 B).

[0077] Example 6

[0078] Effects of Bromocamphor from Mulberry Root Exudates on the Exopolysaccharide Production of Pseudomonas syringae

[0079] The content of extracellular polysaccharide of Pseudomonas syringae was determined by the water-soluble alcohol precipitation phenol-sulfuric acid method. 5 mL of Pseudomonas syringae was centrifuged at 10000 rpm for 10 min and the OD 600 = 0.5 bacterial suspension, placed in 5mL1.5mg·mL -1 The samples were treated overnight in bromocamphor, and 4 times the volume of anhydrous ethanol was added to the supernatant, and then the extracellular polysaccharides were precipitated in a refrigerator at 4°C overnight. After centrifugation for 10 minutes, the supernatant was removed and dissolved in water to obtain a crude polysaccharide solution. The specific operation of the polysaccharide content determination is as follows: glucose was used as the standard control, and a glucose standard curve was prepared. 200 μL of crude polysaccharide solution with different treatments, 100 μL of 4% phenol solution and 500 μL of concentrated sulfuric acid were mixed in turn, and the absorbance was obtained at 490 nm after 15 minutes. Finally, the extracellular polysaccharide content in the treatment group was calculated according to the standard curve. Ethanol was used as a solvent control, and each treatment was repeated 3 times.

[0080] Extracellular polysaccharides (EPS) are one of the important pathogenic factors that can help bacteria attach to the surface of host tissues or non-biological materials, increasing their survival and transmission capabilities.

[0081] This leads to vascular bundle blockage, obstruction and impact on water transport, and ultimately leads to plant wilting. -1 After treatment with bromocamphor, the EPS content of Pseudomonas syringae increased significantly ( Figure 7 ), which was speculated to be due to the increased cell membrane permeability of Pseudomonas syringae strains after bromocamphor treatment.

[0082] Example 7

[0083] Bromocamphor from mulberry root exudates induces reactive oxygen burst in Pseudomonas syringae

[0084] The reactive oxygen species (ROS) content in Pseudomonas syringae treated with erucamide, oleamide and bromocamphor was detected using an active oxygen species kit. The reactive oxygen species in the bacteria can oxidize non-fluorescent DCFH-DA to generate fluorescent DCF, thereby observing red fluorescence under a microscope. The specific operation is as follows: Pseudomonas syringae is cultured overnight, and the OD of the bacterial solution is 2.34. 600 =0.6, the samples were divided into 4 groups and 1 mg·mL -1Pseudomonas syringae was treated with bromocamphor, and an equal amount of anhydrous ethanol was added as a solvent control, and the culture was continued at 30°C for 8 hours. Subsequently, the treated bacterial solution was centrifuged at 8000rpm in a centrifuge for 2 minutes, the precipitate was retained to remove the bacterial culture solution, and it was gently washed 3 times with sterile water, and 10mM DCFH-DA diluted 1:1000 with serum-free culture medium was added, and it was protected from light at 30°C for 30 minutes (the added volume should be sufficient to cover the bacteria). After the culture is completed, DCFH-DA is removed and the sample is thoroughly washed with sterile water. Finally, the sample fluorescence is measured using a scanning confocal laser microscope. Each treatment is repeated 3 times.

[0085] Excessive reactive oxygen species (ROS) in bacteria can cause oxidative stress to the bacteria themselves, leading to oxidative loss and even affecting their activity. By observing the amount of ROS generated under a scanning confocal laser microscope, the treatment intensity of bromocamphor at a specific concentration on Pseudomonas syringae was reflected, and its antibacterial strength was evaluated. The results showed that at a concentration of 1.5 mg mL -1 When bromocamphor is used, a large amount of ROS is generated in Pseudomonas syringae, as shown by the red dots in the figure ( Figure 8 A). Compared with the solvent control group ( Figure 8 B), the ROS of Pseudomonas syringae treated with bromocamphor increased significantly under the same area, indicating that bromocamphor caused oxidative damage to Pseudomonas syringae, thereby affecting bacterial growth.

[0086] Like many plant pathogens, Pseudomonas syringae forms biofilm aggregates at the roots of host plants, leading to bacterial invasion and infection. Pseudomonas syringae can stay in the soil outside the host plant for a long time, which causes great difficulty in the development of prevention and control work, and the use of traditional chemical pesticides can cause damage to the environment and human body. Therefore, it is urgent to develop new ecological agents to effectively prevent and control the occurrence of mulberry blight. Plants contain abundant antibacterial substances, which are easy to decompose, environmentally friendly, and low in cost. However, the specific mechanism of action of bromocamphor in plant-derived root secretions has not been reported. The present invention has determined that bromocamphor has antibacterial activity against Pseudomonas syringae, and confirmed that it can deform, shrink and destroy the cell membrane of Pseudomonas syringae. In summary, the bromocamphor screened by the present invention can be used as a potential plant antibacterial agent and become a key drug resource for the development of future field applications of mulberry blight.

Claims

1. Application of bromocamphor in inhibiting Pseudomonas syringae.

2. The use according to claim 1, characterized in that: The invention discloses an application of the bromocamphor in preparing an antibacterial agent for Pseudomonas syringae.

3. The use according to claim 1 or 2, characterized in that: The bromocamphor is preferably obtained by artificial synthesis or extraction from mulberry root secretions.

4. The use according to claim 3, characterized in that: The bromocamphor is obtained from the neutral components of mulberry root exudates.

5. The use according to claim 3, characterized in that: The content of bromocamphor in the neutral components extracted from the mulberry root secretions exceeds 10%.

6. Application of bromocamphor in preventing and treating mulberry blight caused by Pseudomonas syringae.

7. The use according to claim 6, characterized in that: The bromocamphor is used in preparing a medicament for preventing and treating mulberry blight caused by Pseudomonas syringae.

8. The use according to claim 6, characterized in that: The concentration of the bromocamphor in the preparation of the agent for preventing and treating mulberry blight caused by Pseudomonas syringae is 0.5-1.5 mg·mL -1 .

9. A pharmaceutical composition for preventing and treating mulberry blight, characterized in that: The pharmaceutical composition comprises bromocamphor as a main active ingredient, as well as pharmaceutically acceptable carriers and auxiliary materials. The mulberry blight is caused by Pseudomonas syringae.

10. The pharmaceutical composition for preventing and treating mulberry blight according to claim 8, characterized in that: The dosage forms of the pharmaceutical composition include emulsion, suspension and granules.

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