Application of salinomycin in prevention and treatment of pathogenic microorganisms

Abstract

The invention discloses application of salinomycin to prevention and treatment of pathogenic microorganisms. According to the present invention, the compound provides significant inhibition activity for agricultural fungi such as Sclerotinia sclerotiorum, Botrytis cinerea, Pyricaria oryzae, Moniliniafacticola ACCC 36263, food-borne bacteria such as Escherichia coli O157: H7ATCC 35150, Bacillus cereus BN CC 103930, Listeria monocytogenes ATCC 19115 and Salmonella enteritidis ATCC 14028, and further provides a certain inhibition effect on other agricultural pathogenic bacteria, such that the compound is expected to be developed into the one type of the compound,

Description

Technical Field

[0001] The invention belongs to the field of natural drug chemistry and discloses a new use of salinomycin in preventing and controlling plant pathogens and food-borne pathogens. Specifically, salinomycin can be used to prevent and control agricultural fungal diseases such as damping-off disease caused by agricultural fungi Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, Pyricularia oryzae, Fusariumpseudograminearum, Aspergillus flavus ACCC32601, Penicillium expansum ACCC 30898, and Monilinia fructicola ACCC 36263, sclerotinia rot of rapeseed, gray mold of tomato, fusarium head blight of wheat, rice blast, wheat stem rot, aspergillosis, penicillium, and brown rot of drupe; and agricultural fungal diseases such as Xanthomonas oryzae PXO99A and Xanthomonas citrus canker caused by plant bacteria. axonopodis pv.citri jx-6, Pseudomonas solanacearum (Smith) Smith BNCC335855 and other plant bacterial diseases; and foodborne bacterial diseases caused by foodborne bacteria Escherichia coli O157:H7 ATCC35150, Bacillus cereus BNCC 103930, Listeria monocytogenes ATCC 19115, and Salmonella enterica subsp.enterica ATCC14028. Background Art

[0002] Agricultural development is vital to my country. It is the foundation of the national economy and is related to food security and social stability. However, agricultural pathogens and diseases such as damping-off and rapeseed sclerotinia, as well as foodborne diseases caused by foodborne pathogens and diseases such as Escherichia coli and Bacillus cereus, seriously threaten the quality of agricultural products and food safety. These diseases not only reduce crop yields and quality, but may also lead to a decline in the competitiveness of agricultural products in the market, affecting farmers' income and sustainable agricultural development. At the same time, foodborne pathogens and diseases may also cause public health problems and increase medical burdens.

[0003] Therefore, effective prevention and control of microbial diseases is of great significance for ensuring the output and quality of agricultural products, protecting human health, maintaining national food security and economic development. my country has taken a variety of measures to prevent and control agricultural pathogens and foodborne pathogens. At present, fungicides are still one of the main means of preventing and controlling microbial pathogens. However, long-term use and excessive use lead to increased drug resistance of pathogens, which reduces the treatment effect and ultimately leads to a continuous increase in the infection rate of crops and related products by pathogenic microorganisms. Therefore, research and development of new and environmentally friendly fungicides is the future development direction.

[0004] Salinomycin is a polyether antibiotic extracted from the culture medium of Streptomyces albus. It is currently widely used in animal husbandry as anticoccidial treatment and growth promoter. In addition, salinomycin has a selective targeting effect on a variety of tumor stem cells, can kill mouse breast cancer stem cells, inhibit the generation of new tumor cells, and slow the growth of existing tumors. The research team tested the antibacterial activity of salinomycin against plant pathogens and foodborne pathogens. The results showed that the compound has excellent antibacterial activity against plant fungi and foodborne pathogens. This discovery has expanded the new use of salinomycin, provided a new prevention and control strategy for agricultural production and food safety, and ensured the quality of agricultural products and the health of consumers. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of salinomycin in resisting agricultural pathogens and food-borne pathogens, and to prevent and treat various diseases caused by plant pathogens and food-borne pathogens.

[0006] The compound salinomycin of the present invention has the following structural formula:

[0007]

[0008] The antibacterial agent provided by the present invention has the following advantages:

[0009] 1) The present invention found that salinomycin has significant antibacterial activity against agricultural fungi, especially against Sclerotinia sclerotiorum, Botrytis cinerea, Magnaporthe oryzae, and stone fruit brown rot, and the antibacterial activity can still reach more than 90% at 50 μg / mL. 50 The value range can reach 0.77~3.36μg / mL, and it can be developed and utilized as a lead compound of biological pesticides.

[0010] 2) The salinomycin in the present invention has significant antibacterial activity against foodborne pathogenic bacteria, and its MIC value is 0.39-0.78 μg / mL. The development and utilization of the compound is helpful to ensure food safety and reduce the occurrence of foodborne diseases. DETAILED DESCRIPTION

[0011] In order to better understand the present invention, the above content of the present invention is further described in detail by the following specific examples. However, this should not be construed as limiting the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified.

[0012] Example 1: Indoor antibacterial activity determination of compound salinomycin against agricultural pathogenic fungi

[0013] The agricultural pathogenic fungi used in this experiment were strains stored at 4°C in the laboratory, and the culture medium used for all plant pathogenic fungi in the laboratory was potato agar glucose medium (PDA for short). PDA medium formula: 200g potatoes (peeled), 20g glucose, 15g agar, 1000mL deionized water, sterilized at 121°C with high pressure steam for 20min and then taken out for use.

[0014] The mycelium growth rate method was used for the indoor antibacterial activity determination. Activation of strains: The agricultural pathogenic fungi were cultured on PDA plates at 25°C for 3 to 6 days. Preparation of drug plates: Pour the PDA culture medium sterilized at 121°C into a culture dish, cool it to 45-50°C, and add different concentrations of the test compounds to make drug-containing plates. Inoculation and culture: In a clean bench, use a puncher to punch a bacterial cake (5 mm in diameter) at the edge of the mycelium that has been cultured for 3 to 6 days (the growth conditions should be as consistent as possible), and then use an inoculation needle to pick it to the center of the drug plate, and then culture it upside down in an incubator (25°C). Result determination: After the mycelium of the blank control group is fully grown, the growth diameter of the mycelium of the drug group is measured by the cross method, and the growth inhibition rate of the mycelium by this type of compound is calculated.

[0015]

[0016] Three parallel experiments were conducted for each concentration to determine the inhibition rate of the compound. For pathogens with an inhibition rate of more than 80%, the concentration was reduced and the median effective concentration (EC50) was calculated using the statistical analysis software IBM statistics SPSS 27.0. 50 ), and the results are shown in Table 1.

[0017] Table 1 In vitro antibacterial effect of salinomycin on agricultural pathogenic fungi

[0018]

[0019] Note: “-” means the inhibition rate of this concentration was not tested.

[0020] The above results show that the salinomycin disclosed in the present invention has a good inhibitory effect on most agricultural fungi at 50 μg / mL, with an inhibition rate of more than 50%, and can reach an inhibition rate of more than 90% for Sclerotinia sclerotiorum, Botrytis cinerea, Rice blast fungus, and stone fruit brown rot fungus. The inhibitory activity of salinomycin on these four strains at a series of low concentrations was further evaluated and the EC 50 The compound was found to have significant antibacterial activity against Sclerotinia sclerotiorum, Botrytis cinerea, Magnaporthe oryzae, and stone fruit brown rot. 50 The value was 0.77-3.36 μg / mL, and at a low concentration of 5 μg / mL, it still showed an inhibition rate of more than 64%, which was close to or equivalent to the positive control drug boscalid in the prevention and control of rapeseed sclerotinia and tomato gray mold, and was slightly lower than the positive drug prochloraz in the prevention and control of stone fruit brown rot. Overall, the in vitro evaluation of the inhibitory activity of salinomycin against agricultural pathogens showed that salinomycin has potential development value in the prevention and control of crop diseases.

[0021] Example 2: Indoor antibacterial activity determination of salinomycin against plant pathogenic bacteria

[0022] The indoor antibacterial activity was determined by the micro-broth dilution method. The strain used in this experiment was a strain frozen at -80°C in the laboratory containing 30% glycerol. The frozen strains were taken out and streaked on NB solid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH 7.0; sterilized at 121°C for 20min), and cultured at a constant temperature of 28°C until a single colony grew. Single colonies on the solid culture medium were picked and transferred to NB liquid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, distilled water: 1L; sterilized at 121°C for 20min), and cultured on a constant temperature shaker at 28°C and 180rpm until the logarithmic growth phase. The strains in the logarithmic growth phase were diluted to about 10 with NB liquid culture medium. 6 Dissolve the compounds in DMSO, add them to the liquid culture medium, mix them evenly, and prepare a drug-containing liquid culture medium with a concentration of 200 μg / mL. Take 50 μL of the drug-containing culture medium and the same volume of the drug-containing medium containing about 10 6 CFU / mL bacterial culture was added to the wells of a 96-well plate, and the final drug concentration was 100μg / mL. 100μL of bacterial solution of the same concentration containing an equal amount of DMSO was used as a control. The 96-well plate was cultured in a 28°C constant temperature incubator for 24-48h until the bacterial solution in the control group grew (OD value was 0.25-0.35), and the OD value of the bacterial solution in the well was measured on an ELISA instrument (OD 600 ). In addition, the OD values ​​of 100 μL of liquid culture medium and the drug with a concentration of 100 μg / mL were measured to correct the OD values ​​caused by the culture medium and the drug itself. The calculation formula for the corrected OD value and inhibition rate is as follows:

[0023] Corrected OD 600 =OD of culture medium containing bacteria 600 - Axenic culture OD 600 ;

[0024]

[0025] All experiments were set up with three replicates to determine the inhibitory activity of salinomycin against three plant pathogenic bacteria. The compound-containing liquid culture medium was diluted in a 96-well plate by a two-fold dilution method to obtain 50 μL of a series of concentrations of the drug-containing culture medium. Then, the inhibition rate corresponding to the series of concentrations was determined according to the above-mentioned test method. The lowest concentration with an inhibition rate greater than 90% was defined as MIC (μg / mL). The MIC activity data obtained by the measurement are shown in Table 2.

[0026] Table 2 In vitro antibacterial effect of compound salinomycin on plant pathogenic bacteria at 100 ppm

[0027]

[0028] From the results in Table 2, it can be seen that salinomycin has good inhibitory activity against Xanthomonas oryzae and Xanthomonas citrifolia at 100 μg / mL, with an inhibition rate of more than 85%, while the inhibitory effect on Pseudomonas solanacearum is weak. By further determining the MIC of salinomycin, it was found that the MIC values ​​of salinomycin and the positive drug thiophanate-methyl against Xanthomonas oryzae were both 100 μg / mL, while the MIC values ​​against Xanthomonas citrifolia and Pseudomonas solanacearum were both >100 μg / mL.

[0029] Example 3: Indoor antibacterial activity determination of salinomycin against foodborne pathogenic bacteria

[0030] The microbroth dilution method was used, and the culture medium used for foodborne pathogenic bacteria was Müller-Hinton culture medium (MH culture medium). The specific experimental operation method and calculation formula are the same as those in Example 2. All experiments were set up with three replicates to determine the inhibition rate of salinomycin on four foodborne pathogenic bacteria. The drug-containing liquid culture medium of the compound was diluted in a 96-well plate by a two-fold dilution method to obtain 50 μL of drug-containing culture medium with a series of concentrations, and then the inhibition rate corresponding to the series of concentrations was determined according to the above test method. The lowest concentration with an inhibition rate greater than 90% was defined as MIC (μg / mL), and the results are shown in Table 3.

[0031] Table 3 In vitro antibacterial effect (MIC) of salinomycin on foodborne bacteria

[0032]

[0033] In summary, the compound salinomycin described in the present invention exhibits certain antibacterial activity against 12 agricultural pathogens and 4 foodborne pathogens, especially against plant fungi Sclerotinia sclerotiorum, Botrytis cinerea, Rice blast fungus, stone fruit brown rot fungus and foodborne pathogens, and has the value of further research and development in preventing and controlling agricultural diseases and ensuring food safety.

[0034] Inventors: Liu Yingqian, Zhang Lijing, Sun Yuchen, Zhang Baoqi, Lei Yaya, Zhang Zhijun, Wang Yanze

[0035] Inventing unit: Lanzhou University.

Claims

1. The present invention relates to the use of salinomycin in preventing or controlling agricultural pathogenic fungi.

2. The present invention relates to the use of salinomycin in preventing or controlling agricultural pathogenic bacteria.

3. The present invention relates to the use of salinomycin in preventing or controlling foodborne bacteria.

4. The salinomycin according to claims 1 to 3 has the following molecular structure:

5. Use of salinomycin according to claim 1 or 4 in preventing or controlling Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, Magnaporthe oryzae, Pseudofusarium graminearum, Aspergillus flavus, Penicillium expansum, and stone fruit brown rot.

6. Use of salinomycin according to claim 2 or 4 in preventing or controlling Xanthomonas oryzae, Xanthomonas citri odoratus and Pseudomonas solanacearum.

7. Use of salinomycin according to claim 3 or 4 in preventing or controlling foodborne pathogenic bacteria such as Escherichia coli, Bacillus cereus, Listeria monocytogenes and Salmonella enteritidis.

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