Application of polypeptide compound in prevention and treatment of pathogenic microorganisms

Abstract

The invention discloses application of polypeptide compounds in prevention and treatment of pathogenic microorganisms. The compound has a synergistic effect on agricultural fungi such as rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, Pyricaria oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae, pyricularia oryzae and pyricularia oryzae. The method comprises the following steps: carrying out sterilization on plant bacteria such as Xanthomonas oryzae PXO99A, Xanthomonas axonophora dispsp.citrijx-6, Pseudomonas solanacearum (Smith) BNCC335855, Pseudomonas sp. The method is characterized by comprising the following steps of: killing Aspergillus flavus ACCC32601 (Aspergillus flavus), Penicillium expansum ACCC 30898 (Penicillium expansum. EXPANSum ACCC 30898), Monilinia coerulea ACCC 36263 (Aspergillus oryzae, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger and Aspergillus niger); the compound disclosed by the invention has a certain inhibition activity on various diseases caused by streptococcus agalactiae ATCC 27956, streptococcus dysgalactiae ATCC27957, Escherichia coli ATCC 25922 and Staphylococcus aureus, particularly, the compound Thiostreptin has a particularly remarkable inhibition effect on agricultural pathogenic bacteria, food-borne germs and cow mastitis germs, and is expected to be developed into a novel antibacterial drug, so that the compound disclosed by the invention can be used for preparing the antibacterial agent for preventing and treating various diseases caused by the streptococcus agalactiae ATCC 27956, the streptococcus dysgalactiae ATCC27957, the Escherichia coli ATCC 25922 and the Staphylococcus aureus.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, discloses the use of polypeptide compounds in preventing and controlling pathogenic microorganisms, and specifically relates to preventing and controlling agricultural fungal diseases such as damping-off disease caused by agricultural fungi such as Rhizoctonia solani, Sclerotiniasclerotiorum, Botrytis cinerea, Fusarium graminearum and Pyricularia oryzae, sclerotinia rot of rapeseed, gray mold of tomato, fusarium head blight of wheat and rice blast; and preventing and controlling agricultural fungal diseases such as Xanthomonas oryzae PXO99A, Xanthomonas axonopodis pv.citri jx-6 and Pseudomonas solanacearum (Smith) Smith caused by plant bacteria such as Xanthomonas oryzae PXO99A, Xanthomonas axonopodis pv.citri jx-6 and Pseudomonas solanacearum (Smith) Smith caused by plant bacteria such as Xanthomonas oryzae PXO99A, Xanthomonas oxypodis pv.citri jx-6 and Pseudomonas solanacearum (Smith) Smith caused by plant bacteria such as Xanthomonas oryzae PXO99A, Xanthomonas oxypodis pv.citri jx-6 and Pseudomonas solanacearum (Smith) Smith caused by Plant bacterial diseases such as rice white leaf blight, citrus canker, and bacterial wilt caused by BNCC335855; foodborne fungal diseases caused by foodborne fungi Aspergillus flavus ACCC32601, Penicillium.expansumACCC 30898, and Monilinia fructicola ACCC 36263; cow mastitis caused by Streptococcus agalactiae ATCC 27956, Streptococcus dysgalactiae ATCC27957, Escherichia coli ATCC 25922, and Staphylococcus aureus Newman. Background Art

[0002] As a major agricultural producer, my country has a wide variety of crops. It is also a country where biological disasters such as pests and diseases are frequent, recurring, and widespread. Pests and diseases occur in many types, with severe degrees, high frequencies, and wide regions, posing a great threat to the safe growth of my country's crops, especially grain crops. Throughout history, plant diseases have caused a large amount of food losses every year, and also affected product quality, posing a major threat to agricultural output. Some pathogens can also produce toxic substances, causing poisoning in humans and animals. The corruption of fruits and cereals caused by foodborne fungi such as stone fruit brown rot, Aspergillus flavus, and Penicillium expansum also poses a great threat to agriculture and human health. On the other hand, as one of the most important economic industries in animal husbandry, the common disease of dairy cows, mastitis, has a serious impact on the quality and output of dairy products, resulting in reduced milk production, reduced quality of dairy products, increased costs, and even the elimination of dairy cows, causing significant economic losses. Therefore, effective prevention and control of microbial diseases is of great significance for ensuring the output and quality of crops and dairy products, protecting human health, and maintaining national food security and economic development.

[0003] At present, fungicides are still one of the main means of preventing and controlling microbial pathogens. Fungicides play a vital role in agriculture, but there are also problems such as drug resistance and pesticide residues. Therefore, research and development of new and environmentally friendly fungicides is the future development direction.

[0004] Peptide compounds are a class of small molecule polypeptides that are widely present in organisms and have antimicrobial and immunomodulatory effects. They can effectively resist external microbial invasion, participate in physiological processes such as immune response and wound healing, and are not easy to develop drug resistance. Antimicrobial peptides have shown good efficacy in combating bacterial infections by destroying bacterial cell membranes and hindering their survival and reproduction. At present, dozens of antimicrobial peptides have entered the drug development stage and some compounds have shown good effects in clinical applications with no obvious side effects. Our research group tested the activity of a series of peptide compounds against plant pathogens, foodborne pathogenic fungi, and bovine mastitis pathogens. The results showed that peptide compounds have certain inhibitory activity against these pathogens, especially the peptide compound Thiostreptin has better antibacterial activity against plant bacteria Rice Xanthomonas oryzae and Citrus canker. Summary of the invention

[0005] The purpose of the present invention is to study the new use of polypeptide compounds in resisting agricultural and animal husbandry pathogens, and to prevent and treat various plant and animal diseases caused by plant pathogens, food-borne pathogenic fungi, and bovine mastitis pathogens.

[0006] The names and chemical structural formulas or amino acid sequences of the polypeptide compounds involved in the present invention are as follows:

[0007]

[0008]

[0009] The antibacterial agent provided by the present invention has the following advantages:

[0010] 1) The present invention found that most of the polypeptide compounds showed excellent antibacterial activity against the pathogenic bacteria of bovine mastitis, providing a new direction for the development of safe, efficient and green prevention and control technology of bovine mastitis.

[0011] 2) The polypeptide compound Thiostreptin of the present invention has significant antibacterial activity against Xanthomonas oryzae and Xanthomonas citri odoratus, with an MIC value of 1.56-6.25 μg / mL, and can be developed and utilized as a lead compound for biopesticides. DETAILED DESCRIPTION

[0012] 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.

[0013] Example 1: Indoor antibacterial activity determination of polypeptide compounds against agricultural pathogenic fungi

[0014] 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.

[0015] 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.

[0016]

[0017] Three parallel experiments were performed for each concentration to determine the inhibition rate of this type of compound. The results are shown in Table 1.

[0018] Table 1 In vitro antibacterial effect of polypeptide compounds on agricultural pathogenic fungi at 50 ppm

[0019]

[0020] Note: “-” indicates that the inhibition rate of the compound was not tested.

[0021] The above results show that the polypeptide compounds of the present invention exhibit weak antibacterial effects on most agricultural pathogenic fungi, while the inhibition rate of the compounds Nosiheptide and Thiostreptin on rice blast fungus at 50 μg / mL can reach more than 60%, which is better than the positive control drug boscalid (26.95%), indicating that Nosiheptide and Thiostreptin have certain potential effects in the prevention and control of rice blast.

[0022] Example 2: Indoor antibacterial activity determination of polypeptide compounds against plant pathogenic bacteria

[0023] 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:

[0024] Corrected OD 600=OD of culture medium containing bacteria 600 - Axenic culture OD 600 ;

[0025]

[0026] All experiments were repeated three times to determine the inhibitory activity of the peptide compounds against three plant pathogenic bacteria. The results are shown in Table 2.

[0027] Table 2 In vitro antibacterial effect of polypeptide compounds on plant pathogenic bacteria at 50 ppm

[0028]

[0029]

[0030] The drug-containing liquid culture medium of the compound was diluted by the two-fold dilution method in a 96-well plate to obtain 50 μL of drug-containing culture medium of a series of concentrations. The inhibition rate corresponding to the series of concentrations was then 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 3.

[0031] Table 3 MIC values ​​of highly active peptide compounds against plant pathogenic bacteria

[0032]

[0033] From the results of Table 2 and Table 3, it can be seen that Gramicidin, Thiostreptin and Bacitracin Zinc have the most significant inhibitory activity against Xanthomonas oryzae, and the inhibition rate is above 95% at 50 μg / mL. In addition, the inhibition rate of Thiostreptin against Xanthomonas citrifolia is as high as 97%, which is significantly better than the positive control Thiostreptin. By further determining the MIC of Gramicidin, Thiostreptin and Bacitracin Zinc, it was found that the MIC values ​​of Gramicidin and Bacitracin Zinc against Xanthomonas oryzae were 25 and 50 μg / mL, respectively, while the inhibitory activity of the compound Thiostreptin against Xanthomonas oryzae and Xanthomonas citrifolia was more significant, and its MIC value was 1.56-6.25 μg / mL. This shows that the compound Thiostreptin is worthy of further development as a fungicide.

[0034] Example 3: Indoor antibacterial activity determination of polypeptide compounds against foodborne pathogenic fungi

[0035] The mycelium growth rate method was used, and the culture medium used for foodborne pathogenic fungi was potato dextrose agar medium (PDA medium). The specific experimental operation method and calculation formula were the same as in Example 1. All experiments were set up with three replicates to determine the inhibition rate of the polypeptide compounds on the three foodborne pathogenic fungi. The results are shown in Table 4.

[0036] Table 4 In vitro antibacterial effect of polypeptide compounds on foodborne pathogenic fungi at 50 ppm

[0037]

[0038] The above results indicate that the polypeptide compounds disclosed in the present invention only exhibit weak antibacterial activity against foodborne pathogenic fungi.

[0039] Example 4: Indoor antibacterial activity determination of polypeptide compounds against pathogenic bacteria of bovine mastitis

[0040] The strain used in this experiment was a strain frozen at -80℃ in the laboratory with 30% glycerol. The frozen strain was taken out and streaked on the sheep blood agar plate, and cultured at 37℃ until a single colony grew. Single colonies were picked and added to brain heart infusion broth (BHI) liquid culture medium (tryptone: 10.0g, beef heart extract powder: 17.5g, sodium chloride: 5.0g, glucose: 2.0g, disodium hydrogen phosphate (12H2O): 2.5g, distilled water: 1L; sterilized at 121℃ for 20min), and cultured at 37℃, 140rpm constant temperature shaker until the logarithmic growth phase. Similarly, the frozen strains of Escherichia coli ATCC 25922 and Staphylococcus aureus were taken out and streaked on MH solid medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, agar: 20g, distilled water: 1L; sterilized at 121°C for 20min), and cultured at 37°C until a single colony grew. Single colonies were picked to MH broth medium (beef powder: 2.0g, soluble starch: 1.5g, acid hydrolyzed casein: 17.5g, distilled water: 1L; sterilized at 121°C for 20min) liquid medium. The remaining experimental operation methods and calculation formulas are the same as in Example 2. All experiments were set up with three replicates to determine the inhibition rate of polypeptide compounds on 4 types of bovine mastitis pathogenic bacteria, and the results are shown in Table 5.

[0041] Table 5 In vitro antibacterial effect of polypeptide compounds on bovine mastitis pathogens at 50 ppm

[0042]

[0043] The drug-containing liquid culture medium of the compound was diluted by a two-fold dilution method in a 96-well plate to obtain 50 μL of drug-containing culture medium of a series of concentrations. Then, the inhibition rate corresponding to the series of concentrations was determined according to the same test method in Example 2. The lowest concentration with an inhibition rate greater than 90% was defined as MIC (μg / mL). The activity data obtained by the measurement are shown in Table 6.

[0044] Table 6 MIC values ​​of highly active peptide compounds against pathogenic bacteria of bovine mastitis

[0045]

[0046]

[0047] From the results of Table 5 and Table 6, it can be seen that the gramicidin, enramycin, nosiheptide, Thiostreptin, Bacitracin Zinc and bacitracin involved in the present invention have significant antibacterial activity against Streptococcus agalactiae ATCC 27956 and Streptococcus agalactiae ATCC27957, which is much better than the positive drug norfloxacin. Some compounds have good inhibitory activity against Staphylococcus aureus, which is close to or equivalent to the inhibitory activity of positive drugs. In addition, this type of compound has only weak inhibitory activity against Escherichia coli.

[0048] In summary, the polypeptide compounds described in the present invention show certain antibacterial activity against 8 agricultural pathogens, 3 foodborne pathogenic fungi, and 4 bovine mastitis pathogens, especially Thiostreptin shows excellent antibacterial effect against plant bacteria oryzae, citrus canker, plant fungi, rice blast, and bovine mastitis pathogens, and may be worth further research and development in agriculture and animal husbandry.

Claims

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

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

3. The present invention relates to the use of polypeptide compounds in preventing or controlling foodborne pathogenic fungi.

4. The present invention relates to the use of polypeptide compounds in preventing or controlling pathogenic bacteria of bovine mastitis.

5. The polypeptide compound according to claims 1 to 5 has the following molecular structure or amino acid sequence:

6. Use of the polypeptide compounds according to claims 1 and 5 in preventing or controlling Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum and Magnaporthe oryzae.

7. Use of the polypeptide compounds according to claims 2 and 5 in preventing or controlling Xanthomonas oryzae, Xanthomonas citri odoratus and Pseudomonas solanacearum.

8. Use of the polypeptide compounds according to claims 3 and 5 in preventing or controlling postharvest fruit decay fungi: Penicillium expansum, drupe brown rot fungus, and Aspergillus flavus.

9. Use of the polypeptide compounds according to claims 4 and 5 in preventing or controlling Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Staphylococcus aureus.

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