Application of p-allylphenol in preparation of inhibitor for preventing and treating pear fire blight and preparation of p-allylphenol

Abstract

The invention discloses application of p-allylphenol in preparation of an inhibitor for preventing and treating pear fire blight and preparation of p-allylphenol, and relates to the technical field of biology. The preparation of p-allyl phenol comprises the following steps: obtaining spores of Streptomyces sp.D67, inoculating the spores to a culture medium for cultivation, and carrying out purification treatment on the obtained crude extract to obtain p-allyl phenol. The effective concentration of p-allylphenol is 0.125 mg / mL-0. 5mg / mL, and it is found that p-allylphenol has quorum sensing inhibition activity on pear fire disease pathogenic bacteria. The p-allylphenol is applied to the preparation of the inhibitor for preventing and treating pear fire blight, the inhibition rate of the p-allylphenol on the generation of a pear fire blight pathogenic bacteria biofilm reaches 69.3%, and the inhibition rate of the p-allylphenol on the formation of exopolysaccharides reaches 39.9%; the p-allylphenol has an outstanding technical effect on solving the pear fire blight, and provides a new choice for biological prevention and control application of the pear fire blight.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to the application of p-allylphenol in the preparation of an inhibitor for controlling pear fire blight and the preparation of p-allylphenol. Background Technology

[0002] my country ranks first in the world in both pear cultivation area and total output. However, in recent years, the occurrence of pear fire blight has severely restricted the development of the pear industry. Therefore, the control of this disease has become a focus of attention within the industry. Pear fire blight is a devastating disease, and its pathogen is *Erwinia*, a member of the Enterobacteriaceae family. Currently, the main methods for controlling pear fire blight include plant detection and quarantine, breeding of disease-resistant varieties, biological control, and chemical control. Combining multiple control methods can improve the effectiveness of pear fire blight control.

[0003] Against this backdrop, traditional control methods rely on antibiotics (such as streptomycin) or biocontrol agents prepared using antagonistic microorganisms to control pear fire blight. However, the effectiveness of traditional antibiotics and fungicides is significantly limited by biofilm barriers and drug resistance. Quorum sensing inhibitors, on the other hand, can interfere with the quorum sensing system, typically by targeting quorum sensing signaling molecules or their receptors or downstream regulators to control biofilm formation and effectively inhibit the expression of pathogen virulence factors. They neither kill nor inhibit bacterial growth, thus placing less pressure on bacterial survival. This reduces the occurrence and spread of plant diseases, a mechanism entirely different from that of antibiotics, which kill bacteria. However, the preparation of quorum inhibitors is still under investigation.

[0004] Furthermore, due to the numerous metabolites produced by microorganisms during fermentation, quorum sensing inhibitors have been discovered from various metabolites from extreme environments both domestically and internationally. Current technologies have not reported any inhibitory effects of allylphenol on the quorum sensing phenotype of pear fire blight pathogens, and existing quorum sensing inhibitors have shown limited efficacy in controlling pear fire blight. Summary of the Invention

[0005] This application provides an application of allylphenol in the preparation of inhibitors for controlling pear fire blight and a technical solution for the preparation of allylphenol, solving the problems of poor inhibitory effects of existing quorum sensing inhibitors against pear fire blight pathogens and unstable ecological safety caused by biological control. The activity of crude extract of *Streptomyces* sp. D67 against *Vibrio vulnificus* and pear fire blight pathogens was verified by preparing the extract, and the active substances of the metabolites were also verified, showing that the metabolites have an inhibitory effect on the pathogens. The technical solution for the application of allylphenol in the preparation of inhibitors for controlling pear fire blight can effectively inhibit the expression of virulence factors of pear fire blight pathogens, thereby reducing the occurrence of plant diseases and their infection and spread.

[0006] To solve the above technical problems, the technical solution provided in this application is: In the first aspect, this application provides the application of p-allylphenol in the preparation of an inhibitor for controlling pear fire blight. By using p-allylphenol as an inhibitor for controlling pear fire blight, significant technical effects are achieved.

[0007] In this application, the preferred effective concentration of allylphenol is 0.125 mg / mL to 0.5 mg / mL.

[0008] Secondly, this application provides a method for preparing p-allylphenol, which involves obtaining spores of Streptomyces p. D67 and inoculating them into a culture medium, namely Gao's No. 1 medium, to obtain a bacterial suspension; centrifuging the bacterial suspension at 10,000 r / min for 10 min at 4 °C, collecting the supernatant, adding ethyl acetate to the supernatant and extracting at 100 r / min for 3 h, collecting the organic phase, evaporating to dryness at 40 °C to obtain a crude extract; purifying the crude extract to obtain p-allylphenol.

[0009] This application further provides the application of the screening method for quorum sensing inhibitors in the control of pear fire blight pathogen, wherein the pear fire blight pathogen is Erwinia amylovora.

[0010] In this application, a strain with strong quorum sensing inhibitory activity, *Streptomyces sp. D67, was isolated from soil samples grown over many years in the arid region surrounding the Kumtag Desert. This strain was deposited on October 7, 2022, at the China Center for Type Culture Collection (CCTCC), a Budapest Treaty International Collection Unit for Microorganisms, located at Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M20221542. The gene sequence of *Streptomyces sp. D67* is shown in SEQ ID NO: 1. In-depth research was conducted on the quorum sensing inhibitory activity of the crude extract of *Streptomyces sp. D67* against allylphenol, revealing a significant technical effect of allylphenol on the inhibition of pear fire blight, providing a new option for the biological control of pear fire blight.

[0011] Through the above technical solutions, this application achieves the following technical effects: This application utilizes allylphenol in the preparation of an inhibitor for controlling pear fire blight and describes the preparation of allylphenol. Quorum sensing inhibitor experiments show that different sub-inhibitory concentrations (0.06, 0.13, 0.25, 0.5, and 1 mg / mL) of allylphenol inhibit the motility of pear fire blight pathogens. During the critical stage of biofilm formation (1-3 days), different concentrations of allylphenol significantly inhibited the formation of extracellular polysaccharides and biofilms in pear fire blight pathogens. On the first day of cultivation, at a concentration of 0.1 mg / mL, the inhibition rate of biofilm formation reached 69.3%, and the inhibition rate of extracellular polysaccharide formation reached 39.9%. Therefore, the application of allylphenol in the preparation of an inhibitor for controlling pear fire blight achieves significant technical effects. This invention provides a method for preparing allylphenol, preferentially using *Streptomyces* strains obtained through prior screening. Streptomyces sp. D67 was fermented and metabolized. The bacterial broth was centrifuged at 10,000 r / min for 10 min at 4 °C. The supernatant was collected and extracted with ethyl acetate at 100 r / min for 3 h. The organic phase was collected and evaporated to dryness at 40 °C to obtain the crude extract. The purified p-allylphenol has shown stable and significant technical effects in the preparation of inhibitors for controlling pear fire blight. Attached Figure Description

[0012] Figure 1 The figure shows the effect of metabolites on the production of violacein by *Vibrio violaceus* CV026; among which, Figure 1-1 Aseptic water control Figure 1-2 p-Allylphenol, Figure 1-3 Eugenol, Figure 1-4 2-pyrrolecarboxylic acid, Figure 1-5 Phenylic acid, Figure 1 -6 represents 3,4-dihydroxyphenylpropionic acid.

[0013] Figure 2 The figure shows the inhibition rate of metabolite components on the biofilm of pear blight pathogen.

[0014] Figure 3 The figure shows the inhibition rate of metabolite components on the extracellular polysaccharide of pear blight pathogen.

[0015] Figure 4 The figure shows the effect of metabolite components on the motility of pear fire blight pathogen; among them, Figure 4-1 For DMSO control, Figure 4-2 p-Allylphenol, Figure 4-3 Eugenol, Figure 4-4 2-pyrrole hydroxy acid, Figure 4-5 Phenylic acid, Figure 4-6 is 3,4-dihydroxyphenylpropionic acid.

[0016] Figure 5 The figure shows the effect of metabolite components on the clustering of pear fire blight pathogens; among them, Figure 5-1 For DMSO control, Figure 5-2 p-Allylphenol, Figure 5-3 Eugenol, Figure 5-4 2-pyrrole hydroxy acid, Figure 5-5 Phenylic acid, Figure 5 -6 is 3,4-dihydroxyphenylpropionic acid. Detailed Implementation

[0017] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0018] The *Porphyromonas violaceus* CV026 used in this application was kindly provided by Professor Wang Yan of the College of Marine Life Sciences, Ocean University of China; *Erwinia amylovora*, isolated from *Pyrus pyrifolia* plants infected with fire rot in Korla, Xinjiang, was kindly provided by Researcher Bao Huifang of the Institute of Microbiology, Xinjiang Academy of Agricultural Sciences. Both *Porphyromonas violaceus* CV026 and *Erwinia amylovora* can be obtained by those skilled in the art through commonly known technical means, and the use of different *Porphyromonas violaceus* and *Erwinia amylovora* does not affect the implementation of the technical solution of this application.

[0019] A strain with strong quorum sensing inhibition activity, Streptomyces sp. D67, was isolated from soil samples grown over many years in the arid region surrounding the Kumtag Desert. This strain was deposited on October 7, 2022, at the China Center for Type Culture Collection (CCTCC), a Budapest Treaty International Collection Unit for Microorganisms, located at Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M20221542. The gene sequence of Streptomyces sp. D67 is shown in SEQ ID NO: 1.

[0020] Culture media and other materials: The culture media selected are cluster culture medium, migratory culture medium, LB broth medium and LB agar medium.

[0021] The clustering medium ( / L) consisted of: peptone 10.0 g / L, D(+)-glucose 5.0 g / L, NaCl 5.0 g / L, agar powder 5.0 g / L, and pH = 7.

[0022] Migratory medium ( / L): tryptone 10.0 g / L, NaCl 3.0 g / L, agar powder 3.0 g / L, pH=7.

[0023] LB broth medium, LB agar medium, and Gao's No. 1 medium were all purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.

[0024] The reagents used in this application were: dimethyl sulfoxide (DMSO), anthrone, crystal violet, peptone, sulfuric acid, skim milk powder, ethanol, tryptone, agar powder, ethyl acetate, D(+)-glucose, NaCl, and N-hexanoyl-L-homoserine lactone standard (C6-HSL) (all analytical grade or biochemical reagents), purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. The instruments and equipment used in this application were: Clean bench, SW-CJ-1F, Suzhou Antai Air Technology Co., Ltd.; Biochemical incubator, SPX-250BF, Shanghai Fuma Experimental Equipment Co., Ltd.; Shaking incubator, BSD-250, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory; Automatic autoclave, HVE-50, HIRAYAMA Corporation, Japan; Microplate reader, Biotek Epoch-2, Biotek Instruments, Inc., USA; High-speed refrigerated centrifuge, PLCD, Sigma-Aldrich, Germany; Electric thermostatic water bath, DK-8D, Shanghai Qixin Scientific Instruments Co., Ltd.

[0025] All strains and raw materials used in this application, as well as the culture conditions and methods for the strains used, are well known in the art. All percentages mentioned in this application are weight percentages unless otherwise specified.

[0026] Example 1: Preparation of p-Allylphenol This application provides a method for preparing p-allylphenol, which involves obtaining spores of Streptomyces sp. D67 and inoculating them into a culture medium (Gao's No. 1 medium) to obtain a bacterial suspension. The bacterial suspension is centrifuged at 10,000 r / min for 10 min at 4 °C, and the supernatant is collected. The supernatant is then extracted with ethyl acetate at 100 r / min for 3 h. The organic phase is collected and evaporated to dryness at 40 °C to obtain a crude extract. The crude extract is then purified to obtain p-allylphenol.

[0027] In this application, Streptomyces sp. D67 was isolated from soil samples grown over many years in the arid region surrounding the Kumtag Desert. This strain exhibits strong quorum sensing inhibition activity and is known as Streptomyces sp. D67. Streptomyces sp. D67 was deposited on October 7, 2022, at the Budapest Treaty International Depository for Microorganisms: China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China, 430072, with accession number CCTCC NO: M20221542. The gene sequence of Streptomyces sp. D67 is shown in SEQ ID NO: 1.

[0028] Although the preparation of crude extracts is simple, it can be used to better analyze the composition of metabolites in biological samples, and serves as a physical carrier and source of evidence for realizing and demonstrating the activity of metabolites.

[0029] Example 2: Verification of the inhibitory activity of allylphenol on pear blight pathogen Based on the above embodiments, this embodiment uses a control experiment to test the quorum sensing inhibition activity of the crude extract of Streptomyces sp. D67 against the pathogen of pear fire blight.

[0030] Use an inoculation loop to scrape a loopful of fresh Erwinia mycelium and inoculate it into LB liquid medium. Incubate at 30°C and 150 r / min for 12 h. Add 2% of the bacterial culture to LB solid medium cooled to about 45°C, mix well, pour into a plate, and punch holes after solidification.

[0031] Different concentrations of crude extract were added to the wells and incubated at 30°C for 24 hours. The presence of a clear zone was observed, with DMSO used as a negative control. The MIC of the crude extract against *Pyctomyces boulardii* was 2 mg / mL, and the appearance of a clear zone on the plate indicated that the growth of *Pyctomyces boulardii* was significantly inhibited at this concentration. However, at a crude extract concentration of 1 mg / mL, no clear zone was produced, and there was no significant difference compared to the sterile water negative control, indicating that this concentration did not inhibit the growth of *Pyctomyces boulardii*.

[0032] Example 3: Determination of Minimum Inhibitory Concentration (MIC) Based on the experiments provided in Example 1 above, Erwinia and CV026 were inoculated into LB liquid medium and cultured at 30°C and 150 rpm for 12 h to prepare seed culture. Using the 96-well plate method, the test strains were inoculated at a 2% inoculum into LB liquid medium containing different concentrations (8, 4, 2, 1, 0.5, 0.25, 0.13 mg / mL) of crude extract and eugenol. The medium was then placed in a microplate reader and cultured at 30°C with shaking for 24 h. Measurements were taken every 2 h. The value was used to determine the antibacterial properties of the crude extract.

[0033] Table 1-1 MIC (mg / mL) of metabolites

[0034] Table 1-1 shows the minimum inhibitory concentrations (MICs) of the metabolites against *Pyrrosia lingua*, the pathogen of pear fire blight. As can be seen from the table, allylphenol exhibits strong antibacterial activity against *Pyrrosia lingua*, with a MIC of 0.25 mg / mL. Eugenol, 3,4-dihydroxyphenylpropionic acid, and DL-4-hydroxyphenyllactic acid show weaker antibacterial activity against *Pyrrosia lingua*, with MICs of 0.5 mg / mL for each. In all subsequent experiments, a concentration of the metabolites that does not inhibit the growth of *Pyrrosia lingua* was used. Based on their MICs against *Pyrrosia lingua*, a uniform concentration of 0.1 mg / mL was selected for the experiments. This experiment served as a further verification experiment for Example 2.

[0035] Example 4: Validation of QSI active substances As a control experiment for Example 1, a loopful of fresh *Cyclocarya violacea* CV026 bacterial culture was scraped from the culture medium and inoculated into LB liquid medium. The culture was incubated at 30°C and 150 r / min for 16 h. 150 μL of C6-HSL solution (0.1 mmol / mL) and 7.5 mL of CV026 bacterial culture were then added to 300 mL of LB solid medium cooled to about 45°C. After mixing, the medium was poured into a plate and allowed to solidify before punching holes for later use.

[0036] Add crude extracts of different concentrations to the wells, incubate at 30°C for 24 hours, and observe whether a clear zone is formed. Use DMSO as a negative control.

[0037] Since the concentration of the metabolites selected in the experiment was 10 mg / mL, inhibition zones would first form on the plates. The somewhat blurred transparent zones indicated quorum sensing inhibitory activity. Figure 1-1 As shown, compared with the sterile water negative control, all metabolites inhibited the production of violacein by CV026, indicating that all metabolites have quorum sensing inhibitory activity; different metabolites produced different sizes of clear zones, and their quorum sensing inhibitory abilities also differed. Figure 1-2The strong inhibition zone of p-allylphenol indicates that it has strong antibacterial activity against CV026. This experiment serves as a further verification experiment for Example 2.

[0038] Example 5: Determination of the effect of metabolites on biofilm formation by pear blight pathogen As a control experiment for Example 1, a 96-well plate method was used. First, 2% Erwinia bacteria suspension was inoculated into 20 mL of LB liquid medium. Then, 120 μL of the inoculation solution was added to each well, and a certain amount of crude extract was added until the concentration of the crude extract in the medium was 0.06, 0.13, 0.25, 0.5, and 1 mg / mL. An equal amount of dimethyl sulfoxide was added as a control. The plates were incubated at 30°C with shaking for 24 h using a microplate reader. The biofilm content was determined by crystal violet staining. The bacterial suspension was discarded, rinsed three times with distilled water, air-dried, stained with 1% crystal violet for 15 min, rinsed thoroughly, dried, and then eluted with 95% ethanol. The values ​​are calculated, and the results are statistically analyzed.

[0039] Biofilms enable pathogens to adhere tightly to plant surfaces, facilitating their colonization in the rhizosphere, foliage, and other parts of the plant. Extracellular polymers within biofilms can bind nutrients from the environment, providing pathogens with more abundant nutrients and giving them an advantage in nutrient competition with plants and other microorganisms. For example... Figure 2 As shown, the vertical axis represents the biomembrane inhibition rate, and the horizontal axis represents the metabolite components. The numbers 1-6 represent eugenol, p-allylphenol, phenylacrylic acid, 3,4-dihydroxyphenylpropionic acid, DL-4-hydroxyphenyllactic acid, and 2-pyrrolic acid, respectively. p-Allylphenol showed the best inhibitory effect, with an inhibition rate of 69.3%, followed by 3,4-dihydroxyphenylpropionic acid. 2-pyrrolic acid and eugenol showed the worst inhibitory effects, with inhibition rates of only 37% and 34%, respectively. Figure 4 It can be seen that metabolites reduce the initial attachment ability of pathogens by inhibiting their motility and reduce biofilm formation by inhibiting the production of extracellular polysaccharides.

[0040] Example 6: Determination of the effect of metabolites on the formation of extracellular polysaccharides by pear blight pathogen The bacterial culture was cultured using the method described in Example 3, and the polysaccharide content was determined according to the anthrone-sulfuric acid method described by Yu Fuhao et al. The bacterial culture was centrifuged at 10,000 rpm for 8 min at room temperature. The supernatant was collected, extracted twice with ethyl acetate, and the remaining organic phase was evaporated to dryness at 40°C. The solution was dissolved in 100 μL of ultrapure water, and 300 μL of 2% anthrone-sulfuric acid solution was added. The reaction was carried out under boiling water conditions for 15 min, then cooled to room temperature for determination. The value of is calculated, and the results are statistically analyzed.

[0041] Once a biofilm begins to form, the sessile cells secrete an extracellular polysaccharide matrix. Therefore, the biofilm structure is highly viscoelastic and exhibits a gel-like state, such as... Figure 3 As shown, the vertical axis represents the extracellular polysaccharide inhibition rate, and the horizontal axis represents the metabolite components. 1-6 represent eugenol, p-allylphenol, phenylacrylic acid, 3,4-dihydroxyphenylpropionic acid, DL-4-hydroxyphenyllactic acid, and 2-pyrrolic acid, respectively. p-Allylphenol showed the best inhibitory effect on the extracellular polysaccharide production ability of pear blight pathogen, with an inhibition rate of 39.9%. Phenylic acid and 3,4-dihydroxyphenylpropionic acid showed the next best inhibitory effects, while eugenol and DL-4-hydroxyphenyllactic acid showed the worst inhibitory effects, with inhibition rates of only 14.8% and 19.2%, respectively. This experiment served as a further verification experiment for Example 2.

[0042] Example 7: Determination of the motility of pear blight pathogens by metabolites Following the method of Liu Jiayi et al., cluster detection agar plates and migration detection agar plates were prepared respectively, and a certain amount of crude extract was added to them to make the final concentration of crude extract in the medium 0.06, 0.13, 0.25, 0.5 and 1 mg / mL. An equal volume of dimethyl sulfoxide was added as a control. Pathogens were inoculated and cultured at 30℃ for 24 h. The clustering and migration of the strains were observed, the results were statistically analyzed and photographed.

[0043] All cells within a biofilm structure depend on the interaction between the surface and bacterial cells for survival. The ability of bacteria to attach to the surface is crucial for the initial attachment of bacterial biofilms, while motility influences the early attachment process. Figure 4 and Figure 5 It can be seen that, compared with the DMSO control group, the metabolites all had varying degrees of inhibitory effects on the motility of pear fire blight pathogens. Among them, allylphenol had a better inhibitory effect, eugenol had a poorer inhibitory effect, and 3,4-dihydroxyphenylpropionic acid and 2-pyrrolecarboxylic acid had moderate inhibitory effects. Although the effect of swarming motility on pear fire blight pathogens was not obvious, it can be seen that the metabolites still had an inhibitory effect. Except for 3,4-dihydroxyphenylpropionic acid, which had a poor inhibitory effect, the inhibitory effects of the other metabolites were comparable. This experiment served as a further verification experiment for Example 2.

[0044] The above embodiments are only for illustrating the technical concept and features of this application in a specific scenario. Their purpose is to enable those who need this technology to understand the content of this application and implement it. They do not limit the scope of protection of this application. All equivalent changes or modifications made in accordance with the spirit and essence of this application should be covered within the scope of protection of this application.

Claims

1. The application of p-allylphenol in the preparation of an inhibitor for controlling pear fire blight.

2. The application of p-allylphenol as described in claim 1 in the preparation of an inhibitor for controlling pear fire blight, characterized in that, The effective concentration of p-allylphenol is 0.125 mg / mL to 0.5 mg / mL.

3. A method for preparing p-allylphenol, characterized in that, Spores of Streptomyces sp. D67 were obtained and inoculated into a culture medium, namely Gao's No. 1 liquid medium, to obtain a bacterial suspension. The bacterial suspension was centrifuged at 10,000 r / min for 10 min at 4 °C, and the supernatant was collected. The supernatant was added to ethyl acetate and extracted at 100 r / min for 3 h. The organic phase was collected and evaporated to dryness at 40 °C to obtain a crude extract. The crude extract was purified to obtain p-allylphenol.

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