Application of 1-methyl-5-mercapto-1H-tetrazole in preventing and controlling citrus canker

Abstract

The present invention belongs to the technical field of plant disease control, and particularly relates to the application of 1-methyl-5-mercapto-1H-tetrazole in controlling citrus canker. This compound has a strong inhibitory effect on the promoter of the hpa1 gene in the type III secretion system of the pathogen, and at the same time does not affect the normal growth of the pathogen. While not affecting the growth of plant pathogens, it can strongly inhibit the virulence factors of plant pathogens, significantly reduce the pathogenicity of plant pathogens, and achieve the effect of controlling citrus canker; moreover, it also has the effect of reducing the generation of resistance of pathogens to the compound, has a long effective service life in the control of plant diseases, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant disease control. More specifically, it relates to the application of 1-methyl-5-mercapto-1H-tetrazole in controlling citrus canker disease. Background Art

[0002] Citrus is the world's largest category of fruits and is also the fruit tree with the widest cultivation area and the most important economic status in southern China. Citrus canker disease is one of the most serious bacterial diseases on citrus, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xcc). Citrus canker disease is easy to prevent but difficult to treat. Once infected, the plant will carry the virus for life. In China, the average annual economic loss caused by citrus canker disease is as much as 500 million yuan, which greatly threatens the healthy development of the citrus industry. Therefore, developing new and effective disease control drugs is one of the key issues that urgently need to be solved.

[0003] Traditional antibiotics usually target the key factors for bacterial survival. However, the long-term and irregular use of antibiotics has made bacterial resistance more and more common and serious. To solve the problem of drug resistance, Chinese Patent Application CN111713514A discloses a medicament for citrus canker disease, which combines an organic copper bactericide, nano-silver sol, and plant volatile oil. Through the synergistic effect of the three, the bactericidal ability is improved, and the generation of pathogen drug resistance is delayed. Although this method delays the generation of drug resistance, with long-term use, according to the law of natural selection in nature, citrus canker pathogens will still gradually cause the generation of pathogen drug resistance, which is not conducive to long-term use.

[0004] Therefore, there is an urgent need to provide a method for controlling citrus canker disease that has a good effect on controlling citrus canker disease and does not cause drug resistance in citrus canker pathogens even after long-term use. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing methods for controlling citrus canker disease, which are prone to cause pathogen drug resistance, and to provide the application of 1-methyl-5-mercapto-1H-tetrazole in controlling citrus canker disease, which can achieve a good effect on controlling citrus canker disease and does not cause drug resistance in citrus canker pathogens even after long-term use.

[0006] The purpose of the present invention is to provide the application of 1-methyl-5-mercapto-1H-tetrazole as an inhibitor of the type III secretion system of plant pathogens.

[0007] Another purpose of the present invention is to provide a composition for controlling plant diseases.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] Natural products are a treasure trove bestowed upon humanity by nature. From them, people can obtain drugs for preventing and treating diseases that seriously endanger human health, medical and agricultural antibiotics, develop highly effective and low-toxic pesticides, as well as plant growth hormones and other substances with economic value, and natural products are environmentally friendly. The inventor has discovered that the natural active product 1-methyl-5-mercapto-1H-tetrazole has a significant preventive and control effect on citrus canker by inhibiting the expression of the hpa1 gene promoter in the type III secretion system of plant pathogens.

[0010] Therefore, the present invention claims the application of 1-methyl-5-mercapto-1H-tetrazole in the prevention and control of citrus canker. The 1-methyl-5-mercapto-1H-tetrazole is abbreviated as compound Z-10 and has the following structure:

[0011]

[0012] The prevention and control is prevention and / or treatment, that is, preventing citrus canker and / or treating citrus canker to avoid plants from being invaded by citrus canker.

[0013] Furthermore, the pathogen of citrus canker is Xanthomonas citrisubsp. citri.

[0014] Even further, the compound Z-10 inhibits the type III secretion system of plant pathogens. The type III secretion system (T3SS) is a key virulence factor in Gram-negative pathogenic bacteria, mainly regulating the expression of the main regulatory factors HrpG and HrpX, the assembly of needle-like structures, and the expression of effector proteins, which is the main cause of the pathogenicity of such bacteria. The compound Z-10 of the present invention acts on the virulence factor of bacteria, using it as a target. While reducing the pathogenicity of bacteria, it does not affect the normal growth of bacteria, can achieve the effect of reducing the generation of bacterial drug resistance, and realize the long-term and effective prevention and control of citrus canker.

[0015] Furthermore, the compound Z-10 inhibits the expression of the hpa1 gene promoter in the type III secretion system of plant pathogens.

[0016] Even further, the 1-methyl-5-mercapto-1H-tetrazole can also be its pharmaceutically acceptable salt or hydrate. The pharmaceutically acceptable salt or hydrate of 1-methyl-5-mercapto-1H-tetrazole can be used as a prodrug to achieve an activity effect similar to that of 1-methyl-5-mercapto-1H-tetrazole.

[0017] Based on this, the present invention also claims the use of 1-methyl-5-mercapto-1H-tetrazole as an inhibitor of the type III secretion system of plant pathogens. The 1-methyl-5-mercapto-1H-tetrazole is abbreviated as compound Z-10 and has the following structure:

[0018]

[0019] Furthermore, the 1-methyl-5-mercapto-1H-tetrazole may also be a pharmaceutically acceptable salt or hydrate thereof. The pharmaceutically acceptable salt or hydrate of 1-methyl-5-mercapto-1H-tetrazole can be used as a prodrug to achieve an activity effect similar to that of 1-methyl-5-mercapto-1H-tetrazole.

[0020] Even further, the dosage form of the inhibitor is a solid preparation or a liquid preparation. Preferably, the solid preparation includes a directly used solid preparation, a dispersible solid preparation, and a soluble solid preparation; the liquid preparation includes a liquid preparation, a dispersed liquid preparation, an emulsion preparation, a suspension preparation, and a multiphase preparation.

[0021] In addition, the present invention also provides a composition for preventing and controlling plant diseases. The composition contains 1-methyl-5-mercapto-1H-tetrazole or a pharmaceutically acceptable salt or hydrate thereof, and one or more of a bactericide, a biocontrol agent, a plant growth regulator, and an insecticide.

[0022] Furthermore, the plant disease is citrus canker. The pathogen of citrus canker is Xanthomonas citri subsp. citri.

[0023] Preferably, the bactericide includes: thiodiazole copper, quinoline copper, thiazole zinc, kasugamycin, mancozeb, copper hydroxide, Bordeaux mixture, etc.

[0024] Preferably, the biocontrol agent includes Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methylotrophicus, etc.

[0025] Preferably, the plant growth regulator includes: gibberellin, 6-benzylaminopurine, sodium nitrophenolate, brassinolide, etc.

[0026] Preferably, the insecticide includes: etoxazole, spirodiclofen, avermectin, etoxafenprox, etc.

[0027] The present invention has the following beneficial effects:

[0028] The present invention provides the application of 1-methyl-5-mercapto-1H-tetrazole in preventing and controlling citrus canker. This compound has a strong inhibitory effect on the promoter of the hpa1 gene in the type III secretion system of the pathogen, and at the same time does not affect the normal growth of the pathogen. While not affecting the growth of plant pathogens, it can strongly inhibit the virulence factors of plant pathogens, significantly reduce the pathogenicity of plant pathogens, and achieve the effect of preventing and controlling citrus canker; moreover, it also has the effect of reducing the generation of resistance of pathogens to the compound, has a long effective use period in the prevention and control of plant diseases, and has broad application prospects. Brief Description of the Drawings

[0029] Figure 1 It is the growth curve of Xcc jx-6 in NB medium or XVM2 medium after adding compound Z-10 in Example 2 of the present invention.

[0030] Figure 2 It is the diagram showing the effect of compound Z-10 on tobacco HR after treating Xcc jx-6 in Example 4 of the present invention.

[0031] Figure 3 It is the diagram showing the prevention and control of citrus canker by different concentrations of compound Z-10 in Example 5 of the present invention.

[0032] Figure 4 It is the spreading result diagram of the colony number of Xcc jx-6 in citrus leaves after inoculating with different concentrations of compound Z-10 in Example 5 of the present invention.

[0033] Figure 5 It is the statistical result diagram of the colony number of Xcc jx-6 in citrus leaves after inoculating with different concentrations of compound Z-10 in Example 5 of the present invention.

[0034] Figure 6 It is the spreading result diagram of the colony number of Xcc jx-6 in citrus leaves after compound Z-10 is compounded with biocontrol bacterium HN-8 and inoculated in Example 6 of the present invention.

[0035] Figure 7 It is the statistical result diagram of the colony number of Xcc jx-6 in citrus leaves after compound Z-10 is compounded with biocontrol bacterium HN-8 and inoculated in Example 6 of the present invention. Detailed Embodiments

[0036] The following further illustrates the present invention in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0037] Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is an important bacterial disease of citrus. Developing new and effective drugs for disease control is one of the key issues that urgently need to be solved. The type III secretion system (T3SS) is a key virulence factor of Xcc, which is highly homologous and conserved in the genus Xanthomonas. When targeting T3SS, it can reduce the pathogenicity of Xcc without affecting its growth, reduce the occurrence of the disease, and delay the development of drug resistance in the pathogen, which is beneficial for the long-term and effective use of drugs.

[0038] The following examples are for testing the inhibitory effect of compounds on the T3SS of Xanthomonas citri subsp. citri. The tested pathogen strains are: the wild-type strain Xcc jx-6 of Xcc, and the strain Xcc jx-6 carrying a reporter plasmid (lux reporter system containing the hpa1 promoter sequence, regulated by the T33SS regulator), which mainly causes citrus canker. Compound Z-10 was dissolved in dimethyl sulfoxide (DMSO) to prepare a test solution with a concentration of 200 mM.

[0039] Unless otherwise specified, all reagents and materials used in the following examples were purchased commercially.

[0040] Example 1 Inhibitory effect of compound Z-10 on the promoter of the hpa1 gene in the T3SS system

[0041] 1. Experimental method:

[0042] Streak and activate Xcc jx-6 with the reporter plasmid on an NA plate. After 2 - 3 days, pick a single colony and place it in NB medium, add kanamycin (final concentration 50 μg / mL), and culture at 28 °C and 200 rpm until the OD 600 is approximately 1.0. Centrifuge to collect the bacterial cells, wash the cells once with XVM2 medium (20 mM NaCl, 10 mM (NH4)2SO4, 5 mM MgSO4, 1 mM CaCl2, 0.16 mM KH2PO4, 0.32 mM K2HPO4, 0.01 mM FeSO4, 10 mM fructose, 10 mM sucrose, 0.03% Casamino Acids, pH 6.7), and then resuspend in XVM2 medium, add kanamycin, and adjust the OD 600To 0.1, add compound Z-10 to its final concentration of 200 μM, mix well, use an equal volume of DMSO as the solvent control, aliquot the bacterial suspension containing the compound into a black 96-well plate with a transparent bottom, 150 μL per well, use the same volume of XVM2 culture as the background for subsequent calculation of the inhibition rate, set 3 parallels for each treatment; place the 96-well plate at 28 °C and 200 rpm for 16 h. Use a multi-functional microplate reader to measure the bioluminescence value (LUX) and OD respectively 600 , after subtracting the background value of the XVM2 medium, calculate the inhibition of the compound using the formula. The calculation formula is as follows:

[0043]

[0044] 2. Experimental results:

[0045] The results are shown in Table 1

[0046] Table 1 Inhibitory effect of compound Z-10 on the promoter of hpa1 gene in the T3SS system

[0047] Compound <![CDATA[LUX / OD 600 > Inhibition rate % DMSO 1811.70±94.29 - Z-10 203.61±28.13* 88.76

[0048] Note: "*" represents a significant difference compared with the DMSO treatment group, indicating that the compound has a strong inhibitory effect on the hpa1 promoter

[0049] As can be seen from the table, compound Z-10 has a strong inhibitory effect on the hpa1 promoter

[0050] Example 2 Effect of compound Z-10 on the growth of wild-type strain Xcc jx-6

[0051] 1. Experimental method:

[0052] Measure the growth curves of Xcc jx-6 in the presence of compound Z-10 in the rich medium NB and the T3SS induction medium XVM2 respectively. Streak and activate the wild-type strain Xcc jx-6 on the NA plate, pick a single colony after 2 - 3 days and place it in the NB medium, grow until the OD 600 is about 1.0, centrifuge to collect the bacterial cells, resuspend them in the NB medium or the XVM2 medium, adjust the OD 600 to 0.1, add compound Z-10 respectively, mix well to make its final concentration 200 μM, place in a 96-well plate, use an equal volume of DMSO as the solvent control, set 3 parallels for each treatment. Incubate at 28 °C and 200 rpm, measure the data (with the plate lid) every 4 h using a multi-functional microplate reader until 48 h

[0053] 2. Experimental results:

[0054] The experimental results are shown in Figure 1 , where the left figure is the growth curve of Xcc jx-6 in NB medium supplemented with compound Z-10, and the right figure is the growth curve of Xcc jx-6 in XVM2 medium supplemented with compound Z-10. The results show that, compared with the control group DMSO, compound Z-10 did not show an obvious inhibitory effect on the growth of Xcc jx-6 in NB medium or XVM2 medium during the lag phase, logarithmic phase, and stationary phase of Xcc jx-6 growth.

[0055] Example 3 Determination of the minimum inhibitory concentration (MIC) of compound Z-10 against Xcc jx-6

[0056] 1. Experimental method:

[0057] The Xcc jx-6 strain was cultured in NB medium until the OD 600 was approximately 0.6 - 0.8. The cells were collected by centrifugation, resuspended in NB medium, and the OD 600 was adjusted to 0.2. At this time, the cell concentration of the bacterial suspension was approximately 2×10 8 CFU / mL. According to a ratio of 1:500, the bacterial suspension was diluted to a concentration of 4×10 5 CFU / mL; a certain volume of the mother liquor of Z-10 was added to the diluted bacterial suspension and mixed thoroughly to make the initial compound concentration 12800 μM; kasugamycin with an initial concentration of 12800 μg / mL was used as a positive drug control; the same volume of DMSO was added to the bacterial suspension as a solvent control; the same volume of NB medium without bacteria was used as a blank control. In a 96-well plate, the compound concentration was diluted to 6.25 μM or 6.25 μg / mL (a total of 12 gradients) using a 2-fold serial dilution method and incubated at 28 °C for 1 - 2 days. Taking the turbidity of the solvent control as a reference standard, in the treatment group of compound Z-10, the compound concentration corresponding to the clear bacterial suspension in the microplate was the minimum inhibitory concentration (MIC).

[0058] 2. Experimental results:

[0059] The results are shown in Table 2.

[0060] Table 2 Determination results of the minimum inhibitory concentration (MIC) of compound Z-10 against Xcc jx-�

[0061]

[0062]

[0063] As can be seen from the table, the minimum inhibitory concentration of the compound Z-10 of the present invention against Xcc jx-6 is 743 μg / mL.

[0064] Effect of Compound Z-10 on Hypersensitive Response (HR) of Xcc jx-6 on Tobacco

[0065] 1. Experimental method:

[0066] The Xcc jx-6 strain was cultured in NB medium until the OD 600 was approximately 0.6 - 0.8. The cells were collected by centrifugation, resuspended in sterile water, and the OD 600 was adjusted to 0.5. Compound Z-10 was added and mixed well to a final concentration of 200 μM. DMSO of equal volume was used as the solvent control, and sterile water was used as the blank control (CK). The mixture was treated at 28 °C for 2 h. Then, the treated Xcc jx-6 bacterial suspension was inoculated onto Nicotiana benthamiana plants cultured in the greenhouse for two months using a needleless syringe. After inoculation, the plants were continued to be cultured in the greenhouse and photographed and observed after 4 - 6 days.

[0067] 2. Experimental results:

[0068] For the experimental results, see Figure 2 . As can be seen from the figure, Compound Z-10 has an obvious inhibitory effect on the HR of Xcc jx-6 on the non-host plant tobacco.

[0069] Effect of Compound Z-10 on the Pathogenicity of Xcc Strains on Citrus

[0070] 1. Experimental method:

[0071] The citrus variety was Wogan and was cultured in the greenhouse. The Xcc jx-6 strain was cultured in NB medium until the OD 600 was approximately 0.6 - 0.8. The cells were collected by centrifugation, resuspended in sterile water, and the OD 600 was adjusted to 0.2 (1.0×10 8 CFU / mL). Compounds Z-10 with final concentrations of 100 μM, 150 μM, and 200 μM were added respectively. Kasugamycin with a final concentration of 200 μM was used as the positive agent control, DMSO of equal volume was used as the solvent control, and sterile water and sterile water containing 200 μM Compound Z-10 were used as the blank controls. The compounds were mixed well with the bacterial suspension and treated at 28 °C for 2 h. 5 - 10 μL of the treated bacterial suspension or sterile water was taken and injected into the back of citrus leaves using a 1 mL needleless syringe. After inoculation, the plants were continued to be cultured in the greenhouse for 3 - 7 days to observe the disease incidence.

[0072] 2. Experimental results:

[0073] The inoculation results are shown in Figure 3As shown in the figure. From top to bottom on the right side of the leaf vein are: sterile water blank control, sterile water blank control of compound Z-10, DMSO+WT (wild-type Xcc jx-6 strain) solvent control, kasugamycin+WT positive agent control; among them, there are no signs of disease in the two blank controls, the DMSO+WT solvent control shows normal disease development, and obvious lesions also appear in the kasugamycin+WT positive agent control. From top to bottom on the left side of the leaf vein are: experimental groups of compound Z-10+WT at concentrations of 100 μM, 150 μM, and 200 μM; it can be seen that obvious lesions appear at a concentration of 100 μM of compound Z-10, the lesions are significantly smaller at a concentration of 150 μM, and there is no disease development at a concentration of 200 μM.

[0074] To more accurately count the control effect of the compound, the inventor used a punch with a diameter of 5 mm, centered on the inoculation point, intercepted a leaf with a diameter of 5 mm, ground the leaf into a homogenate with sterile water in a sterilized mortar, diluted it stepwise to an appropriate concentration, and then took 100 μL and spread it on an NA plate. The plate was cultured at 28 °C for 2-3 days for colony counting. The spreading results are as Figure 4 shown, and the counting results are as Figure 5 shown. The results show that, compared with the control, compound Z-10 has a very significant control effect on citrus canker at a concentration of 200 μM, and has a better effect than the kasugamycin positive agent at the same concentration.

[0075] Example 6 Control Effect of Compound Z-10 in Combination with Biocontrol Strain HN-8 on Citrus Canker

[0076] 1. Experimental method:

[0077] Citrus variety: Wogan. The Xcc jx-6 strain and the biocontrol strain HN-8 (Burkholderia anthina HN-8, patent number: 201711405448.1) were cultured in NB medium until the OD 600 was about 0.6-0.8, the cells were collected by centrifugation, resuspended with sterile water, and the OD 600 of Xcc jx-6 was adjusted to 0.2 (1.0×10 8 CFU / mL). The concentration of compound Z-10 was selected as 100 μM with basically no control effect, and the concentration of the biocontrol bacterium HN-8 was selected as twice the OD 600 value of the pathogen Xcc jx-6, that is, adjusted to OD 600= 0.4. The following experimental groups were set up: Z-10+WT, HN-8+WT, Z-10+HN-8+WT, DMSO+WT as solvent control, and sterile water as blank control. They were treated for 2 h at 28 °C. Take 5 - 10 μL of the treated bacterial suspension and sterile water, and squeeze them into the back of citrus leaves with a 1 mL syringe without a needle. After inoculation, continue to culture in the greenhouse for 3 - 7 days, and observe the disease incidence. And refer to the coating and counting method in Example 4 to count the colonies at the inoculation site.

[0078] 2. Experimental results:

[0079] The coating results are as Figure 6 shown, and the counting statistics results are as Figure 7 shown. It can be seen that after the non-control-effective 100 μM compound Z-10 is compounded with the biocontrol strain HN-8, the effect is better than the individual use of the two, with an obvious synergistic effect, indicating that compound Z-10 has good compounding potential.

[0080] In summary, the above examples all illustrate that the compound Z-10 of the present invention, that is, compound 1-methyl-5-mercapto-1H-tetrazole, while not affecting the growth of the pathogen Xcc jx-6, can strongly inhibit the expression of the promoter of the hpa1 gene of its T3SS system, thus significantly reducing the pathogenicity of the pathogen. It can achieve the effect of preventing and / or treating plant diseases caused by the pathogen without affecting the growth of the pathogen, thereby avoiding the generation of pathogen drug resistance and prolonging the effective use period of the compound.

[0081] 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. Application of 1-methyl-5-mercapto-1H-tetrazole in preventing and controlling citrus canker, characterized in that, The 1-methyl-5-mercapto-1H-tetrazole is abbreviated as compound Z-10 and has the following structure: The pathogen of citrus canker is Xanthomonas citri subsp. citri; The compound Z-10 inhibits the type III secretion system of plant pathogens.

2. The application according to claim 1, wherein The compound Z-10 inhibits the expression of the hpa1 gene promoter in the type III secretion system of plant pathogens.

3. The application according to claim 1 or 2, characterized in that, The 1-methyl-5-mercapto-1H-tetrazole also includes its pharmaceutically acceptable salts.

4. Application of 1-methyl-5-mercapto-1H-tetrazole as an inhibitor of the type III secretion system of plant pathogens, characterized in that, The 1-methyl-5-mercapto-1H-tetrazole is abbreviated as compound Z-10 and has the following structure:

5. The application according to claim 4, wherein The 1-methyl-5-mercapto-1H-tetrazole also includes its pharmaceutically acceptable salts.

6. The application according to claim 4 or 5, characterized in that, The dosage form of the inhibitor is a solid preparation or a liquid preparation.

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