Application of disulfide in prevention and treatment of pear fire blight

Abstract

The invention belongs to the technical field of prevention and treatment of bacterial diseases, and particularly relates to application of disulfide in prevention and treatment of pear fire blight. According to the present invention, the disulfide can strongly inhibit the T3SS of the pear fire blight bacteria while the growth of the pear fire blight bacteria is not affected, such that the pathogenicity of the pathogenic bacteria is significantly reduced, and the effect of treating the pear fire blight caused by the pear fire blight bacteria is achieved while the growth of the pear fire blight bacteria is not affected. Therefore, the disulfide or the pharmaceutically acceptable salt thereof can be used as a pathogenic bacterium T3SS inhibitor, can be prepared into a medicament for preventing and / or treating the pear fire blight, and also has the effect of reducing and delaying the drug resistance of the pear fire blight to the compound, so that the disulfide or the pharmaceutically acceptable salt thereof can be used for preparing the medicament for preventing and / or treating the pear fire blight. The effective service life is relatively long in the aspect of preventing and / or treating pear fire blight, and the application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and control of bacterial diseases. More specifically, it relates to the application of disulfide in the prevention and control of fire blight of pears. Background Art

[0002] Fire blight of pears caused by Erwinia amylovora is one of the most devastating diseases on Rosaceae plants such as pear trees and apple trees, and is an important object of entry quarantine in China. Once Erwinia amylovora invades a plant, it will colonize comprehensively and penetrate all above-ground tissue parts, including flowers, tender branches, tree trunks, main trunks, fruits, and rootstocks, and at the same time spread rapidly, making it difficult to control and eradicate. For fruit trees, the invasion of Erwinia amylovora will lead to extremely low fruit yield, causing serious economic losses. Fire blight of pears was first reported in 1780 and has now spread to various parts of the world one after another. In order to prevent the further spread of fire blight of pears, it is crucial to adopt effective prevention and control measures.

[0003] In addition, the pathogenicity of Erwinia amylovora is mainly caused by the type III secretion system (T3SS) and exopolysaccharides (fire blight toxin and fructan). T3SS is a very conservative and key virulence factor in Gram-negative pathogenic bacteria, but it is not the most critical factor for bacterial growth. This characteristic makes T3SS an ideal target for developing new antibacterial drugs. Screening inhibitors against T3SS, clarifying their inhibitory effects, and elucidating their inhibitory mechanisms will provide directions for formulating new fire blight prevention and control measures oriented towards interfering with T3SS in the future. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies in the prior art of lacking relatively effective drugs for fire blight of pears, and to provide the application of disulfide or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating fire blight of pears.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] The present invention protects the application of disulfide or its pharmaceutically acceptable salt in the preparation of drugs for preventing and / or treating fire blight of pears, and the structure of the disulfide is shown in formula (I):

[0007]

[0008] In formula (I), the R 1 , R 2 each independently selected from hydrogen, C 1～6 alkyl, C 3～6 cycloalkyl, C 2～6 alkenyl, R 3 (n) substituted benzene ring or R 4(m) Substituted heteroaryl;

[0009] Said R 3 is mono-substituted, multi-substituted or unsubstituted; n represents the number of Rs on the benzene ring 3 and n is any integer from 0 to 5; said R 3 are each independently selected from hydrogen, hydroxyl, C 1～6 alkyl, -NH2, -NO2 or halogen;

[0010] Said R 4 is mono-substituted, multi-substituted or unsubstituted; m represents the number of Rs on the heteroaryl 4 and m is any integer from 0 to 4; said R 4 are each independently selected from hydrogen, hydroxyl, C 1～6 alkyl, -NH2, -NO2 or halogen;

[0011] Said heteroaryl is a 5- to 6-membered aromatic monocyclic ring containing 1, 2, 3 or 4 heteroatoms each independently selected from N, O or S, and the remaining ring atoms are carbon.

[0012] While not affecting the growth of pathogenic bacteria, the disulfide of the present invention can strongly inhibit the T3SS of pathogenic bacteria, thereby significantly reducing the pathogenicity of pathogenic bacteria. That is, while not affecting the growth of pathogenic bacteria, the effect of treating diseases caused by pathogenic bacteria is achieved. Therefore, the disulfide or a pharmaceutically acceptable salt thereof can be used as a pathogenic bacterial T3SS inhibitor or prepared into a drug for Erwinia amylovora disease, for preventing and / or treating Erwinia amylovora disease, and at the same time, it also has the effect of reducing and delaying the generation of drug resistance of Erwinia amylovora to the compound, and has a long effective use period in the prevention and / or treatment of Erwinia amylovora disease, and has broad application prospects.

[0013] Preferably, said R 1 , R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 3～6 cycloalkyl, C 2～6 alkenyl, R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; at least one of said R 1 , R 2 is selected from R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl;

[0014] Said R 3 is mono-substituted, multi-substituted or unsubstituted; n represents the number of Rs on the benzene ring 3The number of, n is any integer from 0 to 5; the R 3 Each independently selected from hydrogen, hydroxyl, C 1～6 alkyl, -NH2, -NO2 or halogen;

[0015] The R 4 is mono-substituted, multi-substituted or unsubstituted; m represents the number of R 4 on the heteroaryl, m is any integer from 0 to 4; the R 4 Each independently selected from hydrogen, hydroxyl, C 1～6 alkyl, -NH2, -NO2 or halogen;

[0016] The heteroaryl is a 5- to 6-membered aromatic monocyclic ring, which contains 1, 2, 3 or 4 heteroatoms each independently selected from N, O or S, and the remaining ring atoms are carbon.

[0017] More preferably, the R 1 , R 2 Each independently selected from C 3～6 alkyl, C 5～6 cycloalkyl, C 3～6 alkenyl, R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; at least one of the R 1 , R 2 is selected from R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl;

[0018] The R 3 is mono-substituted or unsubstituted; n represents the number of R 3 on the benzene ring, n is 0 or 1; the R 3 Each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine;

[0019] The R 4 is mono-substituted or unsubstituted; m represents the number of R 4 on the heteroaryl, m is 0 or 1; the R 4 Each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine;

[0020] The heteroaryl is a 5- to 6-membered aromatic monocyclic ring, which contains 1, 2, 3 heteroatoms each independently selected from N or S, and the remaining ring atoms are carbon.

[0021] More preferably, the R1 , R 2 are each independently selected from propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, vinyl, allyl, R 3 (n) -substituted benzene ring or R 4 (m) -substituted heteroaryl; the R 1 , R 2 at least one of which is selected from R 3 (n) -substituted benzene ring or R 4 (m) -substituted heteroaryl;

[0022] The R 3 is monosubstituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is 0 or 1; the R 3 are each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine;

[0023] The R 4 is monosubstituted or unsubstituted; m represents the number of R 4 on the heteroaryl, and m is 0 or 1; the R 4 are each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine;

[0024] The heteroaryl is selected from a thiazole ring, a thiadiazole ring or a pyridine ring.

[0025] More preferably, the R 3 is monosubstituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is 0 or 1; the R 3 are each independently selected from hydrogen, 2-hydroxy, 3-hydroxy, 4-hydroxy, 2-methyl, 3-methyl, 4-methyl, 2-amino, 3-amino, 4-amino, 2-nitro, 3-nitro, 4-nitro, 2-chloro, 3-chloro, 4-chloro, 2-fluorine, 3-fluorine, 4-fluorine, 2-bromine, 3-bromine or 4-bromine;

[0026] The R 4 is monosubstituted or unsubstituted; m represents the number of R 4 on the heteroaryl, and m is 0 or 1; the R 4 are each independently selected from hydrogen, 2-hydroxy, 3-hydroxy, 4-hydroxy, 2-methyl, 3-methyl, 4-methyl, 2-amino, 3-amino, 4-amino, 2-nitro, 3-nitro, 4-nitro, 2-chloro, 3-chloro, 4-chloro, 2-fluorine, 3-fluorine, 4-fluorine, 2-bromine, 3-bromine or 4-bromine.

[0027] Specifically, the structure of the disulfide ether is shown as any of the following structures:

[0028]

[0029] Preferably, the structure of the disulfide ether is shown as any of the following structures:

[0030]

[0031] More preferably, the structure of the disulfide ether is shown as any of the following structures:

[0032]

[0033] Furthermore, the disulfide ether or its pharmaceutically acceptable salt is used as an inhibitor of the virulence factor T3SS in Erwinia amylovora.

[0034] Even further, the disulfide ether or its pharmaceutically acceptable salt is used as an inhibitor of the virulence factor T3SS in Erwinia amylovora and does not affect the growth of Erwinia amylovora.

[0035] Furthermore, the disulfide ether or its pharmaceutically acceptable salt inhibits the expression of related genes in the T3SS of Erwinia amylovora.

[0036] Even further, the related genes in the T3SS include one or more of hrpY, hrpS, hrpA, hrpN, hrpX, hrpL, dspE, rpoN.

[0037] Even further, the disulfide ether or its pharmaceutically acceptable salt inhibits the secretion of related pathogenic proteins in the T3SS of Erwinia amylovora.

[0038] Furthermore, the dosage form of the drug for Erwinia amylovora disease is powder, granule, water dispersible granule, suspension, emulsifiable concentrate, microemulsion or aqueous solution.

[0039] Furthermore, the disulfide ether or its pharmaceutically acceptable salt of the present invention can also be used in combination with other various drugs.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] While not affecting the growth of Erwinia amylovora, the disulfide can strongly inhibit the T3SS of Erwinia amylovora, thus significantly reducing the pathogenicity of the pathogen. That is, while not affecting the growth of Erwinia amylovora, the effect of treating the fire blight disease caused by Erwinia amylovora is achieved. Therefore, the disulfide or a pharmaceutically acceptable salt thereof can be used as a T3SS inhibitor of pathogenic bacteria, prepared into a drug for fire blight disease, for preventing and / or treating fire blight disease, and at the same time, it also has the effect of reducing and delaying the generation of drug resistance of Erwinia amylovora to the compound, and has a long effective use period in the prevention and / or treatment of fire blight disease, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a statistical chart of the structures of disulfide compounds and their corresponding numbers.

[0043] Figure 2 It is a statistical chart of the data on the effect of disulfide compounds on the activity of the hrpA promoter in the T3SS of Erwinia amylovora.

[0044] Figure 3 It is a statistical chart of the data on the effect of disulfide compounds on the activity of the hrpL promoter in the T3SS of Erwinia amylovora.

[0045] Figure 4 It is a statistical chart of the data on the effect of the disulfide compound to be tested on the growth of Erwinia amylovora in LB(A) and HMM(B).

[0046] Figure 5 Statistical chart of the data on the effect of compounds 1g(A), 2g(B), 3h(C), 3i(D), 5f(E), 6h(F), and 6i(G) on the HR of Erwinia amylovora on tobacco.

[0047] Figure 6 It is an external view of the infection degree of Erwinia amylovora on the flowers (A), leaves (D), branches (E), and immature fruits (B) of Cuiguan pears after treatment with the disulfide compound 3i. Among them, Figure (C) is a statistical chart of the infection area of Erwinia amylovora on the immature fruits of Cuiguan pears after treatment with the disulfide compound 3i, and Figure (F) is a statistical chart of the infection length of Erwinia amylovora on the branches of Cuiguan pears after treatment with the disulfide compound 3i.

[0048] Figure 7 It is a statistical chart of the data on the RT-qPCR detection of T3SS-related genes of Erwinia amylovora by the disulfide compound 3i.

[0049] Figure 8 It is a statistical chart of the data on the effect of the disulfide compound 3i on the exudation of the pathogenicity HrpN protein of Erwinia amylovora. DETAILED DESCRIPTION OF THE INVENTION

[0050] The present invention will be further described below in conjunction with the specification drawings and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0051] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0052] Example 1 Screening of T3SS inhibitors of Erwinia amylovora

[0053] The reporter strains phrpA-lux and phrpL-lux were incubated overnight in LB medium (supplemented with 50 μg / mL kanamycin) until the logarithmic growth phase. Centrifuge at 4000 r / min for 10 min, pour off the supernatant, and resuspend the cells with an equal volume of poor medium (hrp-inducing minimal medium, HMM) containing 20 mM fructose, and adjust the OD 600 to 0.3. Aliquot 500 μL of the mother liquor for each sample, add the test compound to a final concentration of 20 μg / L, DMSO as the solvent control, and benzoic acid (BA) as the positive control. After adding the test compound, aliquot 150 μL of each sample in triplicate into a 96-well plate. Shake and culture at 220 r / min and 28 °C for 18 h, then measure the luminescence and absorbance of the samples with a microplate reader, record the data and process it. This experiment was independently repeated more than three times.

[0054] To detect and quantify the inhibitory effects of the test compounds (as Figure 1 shown), this study used a lux reporter system to screen for T3SS inhibitors. The specific operations are as follows: First, use PCR to fuse the promoters of the hrpA and hrpL genes with the plasmid carrying the luxCDABE reporter gene to construct the target vector. Then, integrate this vector into Erwinia amylovora to obtain the reporter strains phrpA-lux and phrpL-lux, so as to evaluate the expression levels of the hrpA and hrpL genes by detecting the fluorescence level.

[0055] Next, grow the phrpA-lux and phrpL-lux reporter strains in a medium that induces the expression of the hrpA and hrpL genes, and measure the fluorescence intensity after incubating with each compound for 18 hours. To standardize the data, the fluorescence intensity will be adjusted according to the cell density (OD 600 ). This can effectively evaluate the effects of the compounds on the T3SS activity of Erwinia amylovora and further screen out the compounds with significant inhibitory effects. The specific algorithm is:

[0056]

[0057] The results are as Figure 2 and Figure 3 shown. Disulfides 1g, 2a, 2e, 2f, 2g, 3f, 3h, 3i, 5f, 6h, and 6i all showed good inhibitory activities against hrpA and hrpL of Erwinia amylovora, indicating that the above disulfides can strongly inhibit the T3SS of the pathogen, thereby significantly reducing the pathogenicity of the pathogen.

[0058] Example 2 Effect of Highly Active Disulfide Compounds on the Growth of Erwinia amylovora

[0059] Select some compounds in Example 1 as representative compounds for further exploration.

[0060] The growth curves of Erwinia amylovora were measured separately in the rich medium LB and the T3SS induction medium HMM in the presence of the test compounds. The wild-type Erwinia amylovora was cultured in LB medium at 220 r / min and 28 °C on a shaker until the OD 600 reached 1.0. The cells were collected by centrifugation, resuspended in HMM, and the OD 600 was adjusted to approximately 0.3. Small molecule compounds with a final concentration of 20 μg / L were added separately, and an equal volume of DMSO was used as a solvent control, and the same concentration of BA was used as a positive control. After pipetting and mixing, the mixture was added to a growth curve plate, with 300 μL added to each well. Each treatment was set with 5 parallel replicates, and this experiment was independently repeated more than three times. The growth curve of Erwinia amylovora was measured using a growth curve analyzer, and data was read every 3 h for a total of 48 h. This experiment was independently repeated 3 times. The results of the effects of the test disulfide compounds (1g, 2g, 3h, 3i, 5f, 6h, and 6i) on the growth of Erwinia amylovora in LB (Figure A) and HMM (Figure B) are shown in Figure 4 .

[0061] As can be seen from the figure, compared with DMSO and BA, 1g, 2g, 3h, 3i, 5f, 6h, and 6i did not show obvious antibacterial effects during each growth period of Erwinia amylovora. Combining the results of Example 1, it shows that these disulfides can strongly inhibit the T3SS of the pathogen while not affecting the growth of the pathogen, thereby significantly reducing the pathogenicity of the pathogen. Among them, WT is the wild-type Erwinia amylovora, WT+DMSO is the solvent control, and WT+BA is the positive control.

[0062] Example 3 Effect of Disulfide Compounds on the Hypersensitive Response (HR) of Erwinia amylovora on Tobacco

[0063] In this example, further studies were carried out on the above 7 compounds (1g, 2g, 3h, 3i, 5f, 6h, and 6i).

[0064] The wild-type Erwinia amylovora was cultured overnight in LB medium, and the cells were collected by centrifugation, resuspended in sterile water, and the OD was adjusted 600 to 0.3. The test compounds were added to a final concentration of 20 μg / mL, and an equal volume of DMSO was used as a control. The samples were treated at 28 °C for 0.5 h. Then, the treated Erwinia amylovora was inoculated onto Nicotiana benthamiana using a needleless syringe. After inoculation, the tobacco plants were continued to be cultured in the greenhouse, and the phenomena were observed and photographed 24 h later. The results of the effects of compounds 1g, 2g, 3h, 3i, 5f, 6h, and 6i on the HR of Erwinia amylovora on tobacco are shown in Figure 5 .

[0065] As Figure 5 shown, compared with DMSO and BA, at a concentration of 20 μg / mL, 3i had the most obvious inhibitory effect on HR. Therefore, disulfide 3i was further studied. Among them, H2O represents the blank control, DMSO represents the solvent control, and BA is the positive control.

[0066] Example 4 Pathogenicity test of disulfide 3i on Erwinia amylovora on the host Cuiguan pear

[0067] The Erwinia amylovora grown to the logarithmic phase was centrifuged and washed twice with sterile water, and then resuspended to an OD 600 of approximately 0.3. Then, disulfide 3i (20 μg / mL) was added, and the samples were incubated at 28 °C for 0.5 h. An equal volume of DMSO was used as the solvent control, and the same concentration of BA was used as the positive control. The bacterial suspension was inoculated onto the flowers (leaf clipping method), leaves (injection, 10 μL), branches (creating wounds, wrapped with 20 μL), and immature fruits (injection, 5 μL) of Cuiguan pears. Inoculation with sterile water was used as the blank control. The disease conditions of the leaves were observed 1 day later, and the disease conditions of the flowers and branches were observed 3 days later.

[0068] The experimental results are as Figure 6 shown. After treatment with disulfide 3i, the infection degree of Erwinia amylovora on the flowers, leaves, branches, and immature fruits of Cuiguan pears was significantly reduced. Compared with WT, the disease severity of the leaves, young branches, and fruits of Cuiguan pears was reduced by 100%, 65%, and 48% respectively, all better than the positive control BA. Among them, WT is the wild-type Erwinia amylovora, WT+DMSO is the solvent control, and WT+BA is the positive control.

[0069] Example 5 RT-qPCR detection of T3SS-related genes of Erwinia amylovora by disulfide 3i

[0070] Cultured overnight in LB medium until the OD 600 reached 1.0. Then, the bacterial cells were resuspended in HMM medium, and the OD 600Adjust to 0.3. Add DMSO and 3i to the resuspended cells and incubate at 28 °C for 12 h. After incubation, harvest the cells, extract the total bacterial RNA using the RNAprep Pure Bacteria Kit, synthesize cDNA using the HiScript II Q RT SuperMix Kit, and perform real-time fluorescence quantitative PCR using the SYBR Green Master Mix. 2 -ΔΔCt Analyze the expression of related genes by this method. Use the expression of the 16S rRNA gene to normalize the gene expression levels among samples.

[0071] The experimental results are as Figure 7 shown. As can be seen from the figure, the treatment with disulfide 3i caused a significant decrease in gene expression, indicating that disulfide 3i inhibits the expression of T3SS-related genes in Erwinia amylovora.

[0072] Example 6 Effect of disulfide 3i on the exocytosis of the pathogenic HrpN protein synthesized by the T3SS of Erwinia amylovora

[0073] To detect and quantify the inhibitory effect of disulfide 3i, the hrpN gene was fused with a plasmid carrying an HA tag by PCR to construct the target vector pBB-hrpN-HA. Then, this vector was integrated into Erwinia amylovora to obtain an Erwinia amylovora strain with an HA tag for detecting the level of HrpN protein exocytosis.

[0074] Overnight culture the Erwinia amylovora strain with an HA tag in LB medium (supplemented with 50 μg / mL gentamicin) until the logarithmic growth phase, centrifuge to collect the cells, and resuspend them in HMM medium to OD 600It was 0.3. Disulfide 3i with a final concentration of 20 μg / mL or an equal volume of DMSO was added, and the cells were induced to culture for 12 h. Then, at 4 °C, the cells were precipitated with 10% trichloroacetic acid (TCA) overnight, and the supernatant was collected to obtain protein particles. The particles were resuspended in 50 μL of PBS and mixed with 5× sodium dodecyl sulfate (SDS) loading buffer, and then boiled for 10 minutes. 20 μL of the sample was taken for polyacrylamide gel electrophoresis separation, and transferred to the membrane at 250 mA for 1 h under ice bath conditions. The membrane was rinsed 3 times with 0.05% PBST (PBS containing 0.05% Tween 20). The membrane was blocked with PBST containing 5% non-fat milk at room temperature for 2 h. The primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated at 4 °C for 1 h. The membrane was rinsed 3 times with 0.05% PBST, and finally, chemiluminescent reagent was added for imaging analysis, and Image J was used for image processing. Western blot was used to detect HrpN protein. The primary antibody was a mouse monoclonal antibody against HA tag (D191044, Sangon Biotech), diluted 1:2500. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (D110087, Sangon Biotech), diluted 1:5000.

[0075] The results were as Figure 8 shown. Compared with the DMSO control group, compound 3i significantly inhibited the exocytosis of HrpN protein, indicating that compound 3i not only affected the expression of T3SS-related genes in Erwinia amylovora but also reduced the secretion of virulence proteins.

[0076] In summary, the above examples all illustrate that the disulfide compound of the present invention can prevent and / or treat fire blight disease caused by Erwinia amylovora, indicating that disulfide has inhibitory activity against the type III secretion system of Erwinia amylovora. Disulfide can be used to inhibit the expression of genes related to the type III secretion system of Erwinia amylovora and reduce the exocytosis of related virulence proteins, thereby reducing pathogenicity, while not affecting the normal growth of Erwinia amylovora, and further reducing the generation of drug resistance of Erwinia amylovora, thus extending the service life of the drug.

[0077] The above examples are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a disulfide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating fire blight disease, characterized in that, The structure of the disulfide is shown by formula (I): In formula (I), said R 1 , R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 3～6 cycloalkyl, C 2～6 alkenyl, R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; The R 3 is mono-substituted, multi-substituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is any integer from 0 to 5; the R 3 are each independently selected from hydrogen, hydroxyl group, C 1～6 alkyl, -NH2, -NO2 or halogen; The R 4 is mono-substituted, multi-substituted or unsubstituted; m represents the number of R 4 on the heteroaryl group, and m is any integer from 0 to 4; the R 4 are each independently selected from hydrogen, hydroxy, C 1～6 alkyl, -NH2, -NO2 or halogen; The heteroaryl is a 5- to 6-membered aromatic monocyclic ring, which contains 1, 2, 3 or 4 heteroatoms each independently selected from N, O or S, and the remaining ring atoms are carbon.

2. The application according to claim 1, wherein Said R 1 and R 2 are each independently selected from hydrogen, C 1～6 alkyl, C 3～6 cycloalkyl, C 2～6 alkenyl, R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; at least one of said R 1 and R 2 is selected from R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; The R 3 is mono-substituted, multi-substituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is any integer from 0 to 5; the R 3 are each independently selected from hydrogen, hydroxyl group, C 1～6 alkyl, -NH2, -NO2 or halogen; The R 4 is mono-substituted, multi-substituted or unsubstituted; m represents the number of R 4 on the heteroaryl group, and m is any integer from 0 to 4; the R 4 are each independently selected from hydrogen, hydroxy, C 1～6 alkyl, -NH2, -NO2 or halogen; The heteroaryl is a 5- to 6-membered aromatic monocyclic ring, which contains 1, 2, 3 or 4 heteroatoms each independently selected from N, O or S, and the remaining ring atoms are carbon.

3. The application according to claim 1, wherein The said R 1 and R 2 are each independently selected from C 3～6 alkyl, C 5～6 cycloalkyl, C 3～6 alkenyl, R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; at least one of the said R 1 and R 2 is selected from R 3 (n) substituted benzene ring or R 4 (m) substituted heteroaryl; The R 3 is monosubstituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is 0 or 1; the R 3 are each independently selected from hydrogen, hydroxyl group, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine; The R 4 is monosubstituted or unsubstituted; m represents the number of R 4 on the heteroaryl, and m is 0 or 1; the R 4 are each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine; The heteroaryl is a 5- to 6-membered aromatic monocyclic ring, which contains 1, 2, 3 heteroatoms each independently selected from N or S, and the remaining ring atoms are carbon.

4. The application according to claim 1, wherein The R 1 and R 2 are each independently selected from propyl, isopropyl, butyl, isobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, vinyl, allyl, R 3 (n) -substituted benzene ring or R 4 (m) -substituted heteroaryl; at least one of the R 1 and R 2 is selected from R 3 (n) -substituted benzene ring or R 4 (m) -substituted heteroaryl; The R 3 is monosubstituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is 0 or 1; the R 3 are each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine; The R 4 is monosubstituted or unsubstituted; m represents the number of R 4 on the heteroaryl, and m is 0 or 1; the R 4 are each independently selected from hydrogen, hydroxyl, C 1～3 alkyl, -NH2, -NO2, chlorine, fluorine, bromine or iodine; The heteroaryl is selected from a thiazole ring, a thiadiazole ring or a pyridine ring.

5. The application according to claim 1, characterized in that The R 3 is monosubstituted or unsubstituted; n represents the number of R 3 on the benzene ring, and n is 0 or 1; the R 3 are each independently selected from hydrogen, 2-hydroxy, 3-hydroxy, 4-hydroxy, 2-methyl, 3-methyl, 4-methyl, 2-amino, 3-amino, 4-amino, 2-nitro, 3-nitro, 4-nitro, 2-chloro, 3-chloro, 4-chloro, 2-fluoro, 3-fluoro, 4-fluoro, 2-bromo, 3-bromo or 4-bromo; The R 4 is monosubstituted or unsubstituted; m represents the number of R 4 on the heteroaryl, and m is 0 or 1; the R 4 are each independently selected from hydrogen, 2-hydroxy, 3-hydroxy, 4-hydroxy, 2-methyl, 3-methyl, 4-methyl, 2-amino, 3-amino, 4-amino, 2-nitro, 3-nitro, 4-nitro, 2-chloro, 3-chloro, 4-chloro, 2-fluoro, 3-fluoro, 4-fluoro, 2-bromo, 3-bromo or 4-bromo.

6. The application according to claim 1, wherein The structure of the disulfide is shown by any of the following structures:

7. The application according to claim 5, characterized in that, The structure of the disulfide is shown by any of the following structures:

8. The application according to claim 7, wherein The structure of the disulfide is shown by any of the following structures:

9. The use according to any one of claims 1 to 8, characterized in that: The disulfide or a pharmaceutically acceptable salt thereof is used as an inhibitor of the virulence factor T3SS in Erwinia amylovora.

10. The application according to claim 8, wherein The disulfide or a pharmaceutically acceptable salt thereof is used as an inhibitor of the virulence factor T3SS in Erwinia amylovora and does not affect the growth of Erwinia amylovora.

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