Application of South American tree frog antibacterial peptide DMS-PS1 in preparation of bacteriostatic agent and pesticide for preventing and treating rice bacterial leaf blight

Abstract

This invention discloses the application of the South American tree frog antimicrobial peptide DMS-PS1 in the preparation of antimicrobial agents and pesticides for the control of rice bacterial blight. This antimicrobial peptide is an α-helical amphiphilic cationic peptide, which exhibits significantly better antimicrobial and bactericidal activity against rice bacterial blight pathogens than streptomycin. It employs a multi-target synergistic bactericidal mechanism, making it less likely to induce drug resistance in pathogens. It also features low hemolysis, high cell safety, no phytotoxicity to rice, easy degradation, and no residue. It has excellent pot-plant control efficacy, effectively inhibiting lesion expansion and pathogen proliferation. It fills the technological gap of traditional chemical pesticides, which have high residues, are prone to drug resistance, and lack sufficient biosafety. It provides a new green and efficient control solution for bacterial diseases of rice, meets the requirements for the development of green biological pesticides, and has significant agricultural transformation and application value.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to the application of an antimicrobial peptide DMS-PS1 derived from the South American tree frog in the preparation of a fungicide and pesticide for controlling bacterial blight in rice. Background Technology

[0002] Rice bacterial blight is a bacterial disease caused by the fungus *Bacillus oryzae*. Along with rice blast and sheath blight, it is considered one of the "three major diseases" of rice. It can occur throughout the entire rice growth cycle, but is particularly severe in the rice-growing areas of East, South, and Central China due to suitable water and heat conditions. After infection, rice leaves wither rapidly, leading to yield reductions of 10%-30%, and in severe cases, over 90% or even total crop failure. It also affects rice quality, reducing the commercial value of rice and seriously threatening my country's food security.

[0003] Currently, the control of rice bacterial blight mainly relies on chemical pesticides, such as copper-based pesticides, thiazoles, and antibiotics. However, these traditional chemical pesticides have significant drawbacks: copper-based pesticides and thiazoles easily remain in the soil, leading to pesticide resistance in pathogens; while antibiotics are relatively easy to degrade, they are easily absorbed by crops and remain, posing not only occupational exposure risks but also the possibility of entering the human body through the food chain, causing chronic toxicity to organs such as the liver and kidneys over long-term accumulation. The effectiveness of agricultural control is affected by various factors such as climate, soil, and management level, and cannot directly act on pathogens; biological control suffers from unstable efficacy and slow onset of action, making it difficult to cope with sudden outbreaks of disease. Cassugamycin is currently the mainstream antibiotic agent for controlling rice bacterial blight, but long-term use has led to varying degrees of pesticide resistance in pathogens in some areas. Therefore, developing new green control agents that are highly effective, low in toxicity, residue-free, and unlikely to induce resistance has become an urgent need to ensure the safe production of rice.

[0004] Antimicrobial peptides, as core components of the natural immune system, possess advantages such as broad-spectrum antimicrobial activity, unique mechanisms of action, low susceptibility to inducing drug resistance, and biodegradability. Among them, the antimicrobial peptide DMS-PS1, isolated from the skin secretions of the South American tree frog (Phyllomedusa sauvagii), has been reported to have antitumor activity (publication number: CN114835791B), but no studies have yet disclosed its inhibitory activity against plant pathogens, especially rice bacterial blight pathogen, or its application in disease control. Summary of the Invention

[0005] The technical problem to be solved by this invention is that existing pesticides for controlling bacterial blight of rice have the defects of high residue, easy development of drug resistance, and insufficient biosafety. The known DMS-PS1 has only been used in the field of anti-tumor treatment, and its application in the control of plant diseases has not yet been developed.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] Using the secretions from the skin of the South American tree frog as the research object, the specific method was based on patent CN114835791 to obtain the amino acid sequence SEQ ID NO. 1: GLWKSLFKNVGKAAGKAALNAVTDMVNQ-NH2, with a molecular weight of 2931.43 Daltons, an amidation modification at the C-terminus, and α-helical amphiphilic structure characteristics. Its net charge is +3, hydrophobicity value is 0.323, hydrophobic moment is 0.267, and the α-helix content can reach 44.0% in a membrane simulation environment, which is an antimicrobial peptide.

[0008] Through further experiments, the core conclusions of this invention are as follows:

[0009] The MIC (microinhibitory concentration) of DMS-PS1 against *Xanthomonas oryzae* pv. oryzae, Xoo, the causal agent of rice bacterial blight, was 2 μg / mL, which is 14.65 times that of kasugamycin (29.3 μg / mL) under the same conditions. The MBC (microinhibitory concentration) was 16 μg / mL, which is 7.325 times that of kasugamycin (117.2 μg / mL) under the same conditions. In pot experiments, the control efficacy was 66.24% higher than that of kasugamycin 14 days after inoculation. The antibacterial effect is significantly better than that of commonly used pesticides like kasugamycin, demonstrating its potential as a highly effective control agent. Furthermore, the MIC against rice bacterial leaf streak was 8 μg / mL, and the MBC was 16 μg / mL.

[0010] DMS-PS1 achieves its bactericidal effect through a multi-target synergistic mechanism. This antimicrobial peptide works synergistically through multiple pathways, including targeting and binding to pathogen outer membrane LPS, disrupting cell membranes to form large-pore channels, disrupting proton kinetic potential, inducing ROS oxidative damage, and binding to and interfering with pathogen genomic DNA. Its unique mode of action makes it less likely to induce drug resistance in pathogens.

[0011] DMS-PS1 has high biocompatibility and is suitable for agricultural development. It exhibits extremely low hemolytic activity, no significant toxicity to human skin cells, and no adverse effects on rice plant growth, meeting the safety requirements of green biological pesticides and carrying low application risk.

[0012] DMS-PS1 degrades rapidly in soil, completely degrading within 60 minutes.

[0013] DMS-PS1 exhibits excellent and sustained efficacy in live rice. In rice inoculation experiments, DMS-PS1 significantly inhibited lesion expansion and pathogen proliferation, maintaining high efficacy even with delayed application, demonstrating significant practical application value.

[0014] Furthermore, the present invention also includes an agricultural method for preventing and controlling rice bacterial blight. During the susceptible period of rice bacterial blight pathogen or the early stage of disease, including but not limited to when leaf damage occurs due to typhoons, hail, agricultural operations, etc., an effective amount of antimicrobial peptide DMS-PS1 bacteriostatic agent or a pesticide composition containing said antimicrobial peptide DMS-PS1 is sprayed onto rice plants. The application concentration range of said antimicrobial peptide DMS-PS1 is 2~32μg / mL.

[0015] In summary, this invention has the following beneficial effects: it is the first time that DMS-PS1 has been applied to the control of rice bacterial blight, and its antibacterial and bactericidal activity is far superior to that of streptomycin. Moreover, through synergistic action of multiple mechanisms, it is not easy to induce drug resistance in pathogens. It is safe for humans, non-target organisms, and rice, and can be rapidly degraded in the soil without residue. It can be prepared as a soluble agent, which is convenient for application. It provides a new solution for the green control of rice bacterial blight and also provides a reference for the control of bacterial diseases of other crops such as rice bacterial leaf streak. Attached Figure Description

[0016] Figure 1 The graph shows the MIC determination results of DMS-PS1 and streptomycin against Xoo.

[0017] Figure 2 This is a schematic diagram illustrating the mechanism of action of DMS-PS1 on the Xoo membrane.

[0018] Figure 3 This is a schematic diagram illustrating the mechanism of DMS-PS1 in the metabolism and oxidative damage of Xoo.

[0019] Figure 4 This is a schematic diagram illustrating the genetic mechanism of DMS-PS1 on Xoo.

[0020] Figure 5 This is a diagram showing the results of a human-derived biosafety assessment.

[0021] Figure 6 The residual results of DMS-PS1 after incubation in soil for 0, 30, and 60 min are shown in the figure.

[0022] Figure 7 A schematic diagram of the control effect test of DMS-PS1 rice inoculation experiment. Detailed Implementation

[0023] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, where there is no conflict, the embodiments and features described in these embodiments can be combined with each other.

[0024] Figure 1The graph shows the MIC determination results of DMS-PS1 and streptomycin against Xoo, where:

[0025] (A) MIC measurement results of Xoo by DMS-PS1;

[0026] (B) MIC determination results of streptomycin against Xoo;

[0027] (C) MIC determination results of Xoc using DMS-PS1;

[0028] (D) Time-sterilization curve of co-culturing DMS-PS1 with Xoo.

[0029] Figure 2 The study focused on the mechanism of action of DMS-PS1 on the Xoo membrane, including:

[0030] (A) Changes in dual fluorescence staining signals of live and dead Xoo strains after treatment with different concentrations of DMS-PS1;

[0031] (B) Real-time kinetic curves of Xoo calcein leakage under different concentrations of DMS-PS1 treatment;

[0032] (C) Changes in membrane potential of Xoo bacteria were detected using the DiSC3(5) fluorescent probe under different concentrations of DMS-PS1 treatment;

[0033] (D) Curves showing the relationship between time and OD280 (protein leakage) after treatment with different concentrations of antimicrobial peptide DMS-PS1;

[0034] (E) Curves showing the relationship between time and OD260 (nucleic acid leakage) after treatment with different concentrations of antimicrobial peptide DMS-PS1;

[0035] (F) BC fluorescent probe was used to detect the binding of different concentrations of DMS-PS1 to LPS.

[0036] Figure 3 The study focused on the mechanisms of DMS-PS1 on Xoo metabolism and oxidative damage, including:

[0037] (A) Intracellular NADH fluorescence intensity of Xoo cells treated with different concentrations of DMS-PS1;

[0038] (B) Antibacterial activity of different concentrations of DMS-PS1 after the addition of NAC;

[0039] (C) Reactive oxygen species accumulation in Xoo after treatment with different concentrations of DMS-PS1;

[0040] (D) Intracellular ATP content of Xoo strain after treatment with different concentrations of DMS-PS1 peptide.

[0041] Figure 4 To study the genetic mechanism of DMS-PS1 on Xoo.

[0042] Figure 5 The results of the human-derived biosafety assessment are shown in the figure, in which:

[0043] (A) Survival rate of eukaryotic cells (HaCaT) after treatment with MTT and different concentrations of DMS-PS1;

[0044] (B) Hemolysis rate of horse erythrocytes after treatment with different concentrations of DMS-PS1.

[0045] Figure 6 The residual results of DMS-PS1 after incubation in soil for 0, 30, and 60 min are shown in the figure.

[0046] Figure 7 The control effect of DMS-PS1 inoculation experiment on rice was tested, including:

[0047] (A) The length of lesions (cm) in the DMS-PS1 treatment group 0 h after Xoo inoculation, simulating prophylactic application;

[0048] (B) The length of lesions (cm) in the DMS-PS1 treatment group 6 h after Xoo inoculation was simulated to simulate therapeutic drug administration;

[0049] (C) Colony count in leaves of the DMS-PS1 treatment group 0 h after Xoo inoculation;

[0050] (D) Count of bacterial colonies in leaves of the group treated with DMS-PS1 6 h after Xoo inoculation.

[0051] Example

[0052] The antimicrobial peptide DMS-PS1 of the present invention is prepared by solid-phase synthesis, and the specific method is based on the implementation of the prior patent CN114835791B. The obtained peptide has a purity of ≥95%.

[0053] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0054] Antimicrobial activity assay of antimicrobial peptide DMS-PS1:

[0055] MIC determination: DMS-PS1 and streptomycin were diluted twofold and co-cultured with Xoo bacterial culture to determine the MIC of DMS-PS1 as 2 μg / mL. Figure 1 As shown in A, the concentration of kasugamycin is 29.3 μg / mL. Figure 1 As shown in B.

[0056] Similarly, the MIC of DMS-PS1 for Xoc was obtained as 8 μg / mL. Figure 1 C.

[0057] MBC determination: Bacterial suspensions treated with different concentrations of DMS-PS1 and streptomycin were serially diluted and spread on NA plates, incubated at 37℃ for 48 h, and colony counts were recorded. The MBC of DMS-PS1 against Xoo was determined to be 16 μg / mL, that of streptomycin was 117.2 μg / mL, and that of DMS-PS1 against Xoc was 16 μg / mL.

[0058] Time-based sterilization dynamic detection: DMS-PS1 at concentrations of MIC, MBC, and 2MBC were mixed with Xoo bacterial suspension, and samples were taken and plated at 0h, 1h, 2h, 3h, 6h, and 8h, respectively, and the colony count was counted. Results are as follows: Figure 1 As shown in Figure D, no viable bacteria were found in the MBC and 2MBC groups at 8 h, while the colony count in the MIC group was significantly lower than that in the control group.

[0059] Study on the mechanism of action of DMS-PS1 on Xoo membrane:

[0060] SYTO9 / PI double staining of Xoo, fluorescence confocal microscopy observation: the control group showed predominantly green fluorescence, while the DMS-PS1 treatment group showed a significantly increased proportion of red fluorescence. Figure 2 As shown in Figure A; calcein leakage detection showed that the extracellular calcium ion concentration in the treatment group was significantly higher than that in the control group, as shown in Figure A. Figure 2 As shown in B; In the experiment of detecting Xoo bacterial membrane depolarization using the DiSC3(5) fluorescent probe, the fluorescence intensity of the DMS-PS1 group was higher than that of the streptomycin group. The fluorescence intensity of the MBC concentration was 243.17% of that of the streptomycin group, and the fluorescence intensity of the 2MBC concentration was 311.87% of that of the streptomycin group. Figure 2 As shown in C; monitoring OD260 and OD280 values, the leakage of nucleic acids and proteins in the DMS-PS1 treatment group increased with time and concentration, as shown in Figure C. Figure 2 As shown in D and E; LPS binding experiments show that DMS-PS1 can bind to LPS in a dose-dependent manner, such as Figure 2 As shown in F.

[0061] Study on the mechanisms of DMS-PS1 in Xoo metabolism and oxidative damage:

[0062] NADH assays showed that the higher the DMS-PS1 concentration, the lower the NADH fluorescence value. Figure 3 As shown in Figure A; ROS accumulation detection showed that the fluorescence intensity of the DMS-PS1 treatment group was significantly higher than that of the control group, and the ROS accumulation level was not concentration-dependent, reaching its peak at the MIC concentration. Figure 3As shown in B; ROS scavenging experiments showed that the MIC of antimicrobial peptides doubled under the action of ROS scavengers. ROS mainly plays an important antibacterial role at low concentrations, but at high concentrations of DMS-PS1, other antibacterial mechanisms dominate the antibacterial effect, such as... Figure 3 As shown in C; after DMS-PS1 treatment, the ATP levels in all concentration groups decreased and were lower than those in the control group, such as... Figure 3 As shown in D.

[0063] DMS-PS1 influences Xoo genetic mechanisms:

[0064] Different concentrations of DMS-PS1 were incubated with Xoo genomic DNA for 1.5 h followed by electrophoresis. Concentrations of 32 μg / mL and above inhibited DNA migration. Results are as follows... Figure 4 As shown.

[0065] Human-based safety assessment of DMS-PS1:

[0066] HaCaT cell toxicity assay: After treating HaCaT cells with serial concentrations of DMS-PS1 for 24 h, cell viability was detected by MTT assay. Viability was above 90% at concentrations ranging from 1 to 128 μg / mL. Results are as follows: Figure 5 As shown in Figure A.

[0067] Hemolysis test: A series of concentrations of DMS-PS1 were incubated with 4% horse blood erythrocyte suspension at 37℃ for 2 h. The absorbance was measured at 550 nm, and the hemolysis rate was calculated. All results were below 3%. The results are as follows: Figure 5 As shown in B.

[0068] Degradation of DMS-PS1 in soil: A 10-fold concentration of 2 MBC (320 μg / mL) was mixed with 1 gram of soil. RP-HPLC experiments were performed at 0, 30, and 60 min. The results showed that 51.324% degradation occurred at 30 min, and complete degradation occurred at 60 min. Figure 6 As shown.

[0069] DMS-PS1 rice inoculation experiment control effect test:

[0070] The rice variety was G Liangyou 727, and the DMS-PS1 inoculum concentration was 1×10⁻⁶. 8 CFU / mL, inoculate 3 leaves per plant, and apply the pesticide by spraying.

[0071] After Xoo 0h or 6h of inoculation, treatment with DMS-PS1 resulted in lesion lengths in all groups that were shorter than those in the control group and the streptomycin group. Figure 7 As shown in A and B;

[0072] After Xoo inoculation for 0h or 6h, treatment with DMS-PS1 resulted in lower colony counts in all groups compared to the control group and the streptomycin group. Figure 7 As shown in C and D.

[0073] As can be seen from the comparison of the above embodiments, the antibacterial and bactericidal activity of DMS-PS1 of the present invention is far superior to that of streptomycin, and it is less likely to induce drug resistance in pathogens through synergistic effects of multiple mechanisms: Specifically, the MIC of DMS-PS1 against *Xanthomonas oryzae* pv. oryzae, Xoo, the causal agent of rice bacterial blight, is 2 μg / mL, which is 14.65 times that of streptomycin (29.3 μg / mL) under the same conditions; the MBC is 16 μg / mL, which is 7.325 times that of streptomycin (117.2 μg / mL) under the same conditions; in addition, in pot experiments, the control efficacy of DMS-PS1 was 66.24% higher than that of streptomycin 14 days after inoculation. With a significantly higher antibacterial effect than sclerotin, a commonly used pesticide, DMS-PS1 has the potential to become a highly effective control agent. This indicates that DMS-PS1, as a fungicide and pesticide raw material for controlling rice bacterial blight, is safe for humans, non-target organisms, and rice. It can be rapidly degraded in the soil without residue and can be prepared as a soluble agent, making application convenient. This provides a new solution for the green control of rice bacterial blight and also serves as a reference for the control of other bacterial diseases in crops, such as rice bacterial leaf streak.

Claims

1. The application of a South American tree frog antimicrobial peptide DMS-PS1 in the preparation of an antimicrobial agent for controlling rice bacterial blight, wherein the amino acid sequence of the antimicrobial peptide DMS-PS1 is GLWKSLFKNVGKAAGKAALNAVTDMVNQ-NH2, and includes homologs and extensions of DMS-PS1.

2. The application according to claim 1, characterized in that, The minimum inhibitory concentration (MIC) against rice bacterial blight pathogens was 2 μg / mL, and the minimum fungicide concentration (MFFC) was 16 μg / mL. The MIC against rice bacterial leaf streak was 8 μg / mL, and the MFFC was 16 μg / mL.

3. The application according to claim 1, characterized in that, The formulation is a soluble concentrate, suspension concentrate, water-dispersible granule, or microemulsion.

4. The application according to claim 1, characterized in that, The antimicrobial peptide DMS-PS1 exhibits a hemolysis rate of <3% within a concentration range of 1–512 μg / mL, a HaCaT cell survival rate of >90%, and complete degradation in soil within 60 min.

5. The application according to claim 1, characterized in that, The antimicrobial peptide DMS-PS1 has no adverse effects on rice plant growth and its control effect is superior to that of streptomycin.

6. A pesticide composition for controlling bacterial leaf blight in rice, characterized in that, It contains an effective amount of the antimicrobial peptide DMS-PS1 and agriculturally acceptable adjuvants.

7. The pesticide composition according to claim 6, characterized in that, It also contains one or more antibacterial active ingredients from the following: kasugamycin, zinc thiazolidin, and kasugamycin.

8. A method for controlling bacterial leaf blight in rice, characterized in that, During the susceptible period of rice bacterial blight or at the early stage of disease, spray rice plants with an effective amount of antimicrobial peptide DMS-PS1 bacteriostatic agent or a pesticide composition containing said antimicrobial peptide DMS-PS1; the application concentration range of said antimicrobial peptide DMS-PS1 is 2~32μg / mL.

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