Salt-tolerant antibacterial peptide Cla-H and application thereof in prevention and control of fusarium graminearum and botrytis cinerea

Abstract

The application discloses a salt-tolerant antibacterial peptide Cla-H and application thereof in prevention and control of fusarium graminearum and botrytis cinerea. In the application, it is found for the first time that Clavanin-AK from a marine animal sea squirt has antibacterial activity on fusarium graminearum and botrytis cinerea. The application discloses an antibacterial peptide Cla-H, the amino acid sequence of the antibacterial peptide is a Clavanin-AK variant with six histidines fused at a C terminal, and the antibacterial peptide sequence is shown as SEQ ID NO. 2. In a 100 mM KCl high-salt environment, the Cla-H can still maintain good inhibitory activity, and the Cla-H has good thermal stability, thereby solving the technical bottleneck that existing antibacterial peptides have poor salt tolerance and lose antibacterial activity in a high-salt environment. The Cla-H antibacterial peptide can be applied to agricultural fungal disease prevention and control, as an effective component of a new green pesticide, has the advantages of low drug resistance inducibility and environmental friendliness, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide preparation technology, specifically a salt-tolerant antimicrobial peptide Cla-H and its application in the control of Fusarium graminearum and Botrytis cinerea. Background Technology

[0002] Fusarium graminearum and Botrytis cinerea are common fungal pathogens in agricultural production, infecting various food crops and cash crops such as wheat and tomatoes, leading to reduced yields and decreased quality, causing significant economic losses. Currently, chemical pesticides are mainly used to control these fungal diseases in agricultural production. However, long-term use of chemical pesticides easily leads to fungal resistance, and pesticide residues pollute the environment and harm human health, which does not meet the needs of green agriculture development. Antimicrobial peptides are a class of small molecule polypeptides with antimicrobial activity, widely present in organisms. They have advantages such as broad antimicrobial spectrum, rapid bactericidal speed, low likelihood of inducing resistance, and environmental friendliness, making them ideal candidate drugs to replace traditional chemical pesticides.

[0003] However, most natural antimicrobial peptides have poor salt tolerance and easily lose their antimicrobial activity in high-salt environments (such as farmland soil and fermentation systems), limiting their practical applications. Clavanin-AK is a natural antimicrobial peptide derived from the marine animal sea squirt. Existing data have reported its antimicrobial effects against bacteria such as Escherichia coli and Listeria. The antimicrobial activity of Clavanin-AK against agricultural fungi such as Fusarium graminearum and Botrytis cinerea is a first discovery in this patent.

[0004] To address the technical challenges of poor salt tolerance and easy inactivation under high-salt conditions in existing antimicrobial peptides, this study modified Clavanin-AK by fusing six histidine residues at its C-terminus to obtain the antimicrobial peptide variant Cla-H, which was then synthesized in pure form using chemical methods. Under normal low-salt conditions, Cla-H exhibited comparable antimicrobial activity to Clavanin-AK against Fusarium graminearum and Botrytis cinerea. However, under high-salt conditions (100 mM KCl), Clavanin-AK almost completely lost its activity, while Cla-H significantly improved its salt tolerance and effectively inhibited the growth of the target fungi. The invention of the antimicrobial peptide Cla-H provides a new technical solution for the green control of agricultural fungal diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a salt-tolerant antimicrobial peptide Cla-H and its application in the control of Fusarium graminearum and Botrytis cinerea in order to solve the problems mentioned above.

[0006] The technical solution adopted in this invention is as follows: a salt-tolerant antimicrobial peptide Cla-H, characterized in that: the antimicrobial peptide is a Clavanin-A variant with 6 histidine residues fused to its C-terminus, and its amino acid sequence is as follows:

[0007] VFQFLGKIIKKVGNFVKGFSKVFHHHHHH

[0008] The amino acid sequence of the Clavanin-A variant is as follows:

[0009] VFQFLGKIIKKVGNFVKGFSKVF.

[0010] In a preferred embodiment, the application of a salt-tolerant antimicrobial peptide Cla-H includes its use for the control of Fusarium graminearum and / or Botrytis cinerea.

[0011] In a preferred embodiment, the application of a salt-tolerant antimicrobial peptide Cla-H includes its use in the preparation of agricultural antifungal agents.

[0012] In a preferred embodiment, the concentration of Cla-H antimicrobial peptide in the agricultural antifungal agent is 3.125 μM-50 μM, and it can be used for the control of fungal diseases in crops such as wheat and tomatoes.

[0013] In a preferred embodiment, the Cla-H antimicrobial peptide exhibits a minimum inhibitory concentration (MIC) of 25 μM against both Fusarium graminearum and Botrytis cinerea in a high-salt environment of 100 mM KCl.

[0014] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0015] In this invention, Cla-H maintains good inhibitory activity even under high-salt conditions, effectively inhibiting the growth of Fusarium graminearum and Botrytis cinerea, thus overcoming the technical bottleneck of poor salt tolerance in existing antimicrobial peptides. The prepared antimicrobial peptide exhibits good thermal stability, with no significant loss of antimicrobial activity after treatment at 90℃, facilitating storage and transportation. Simultaneously, this antimicrobial peptide demonstrates rapid bactericidal action, initiating antimicrobial activity within 1 hour and achieving highly efficient killing of target fungi within 4 hours. Furthermore, it is unlikely to induce fungal resistance, is environmentally friendly, and is suitable as an effective ingredient in novel green pesticides, showing broad application prospects. Attached Figure Description

[0016] Figure 1 The HPLC and mass spectra of Clavanin-AK purified by chemical synthesis in this invention are shown.

[0017] Figure 2 The image shows the HPLC and mass spectra of Cla-H purified by chemical synthesis in this invention.

[0018] Figure 3 The inhibitory effects of 3.125 μM Cla-H peptide on Fusarium graminearum and Botrytis cinerea at different temperatures in this invention are shown.

[0019] Figure 4 This is a microscopic observation of the inhibition of Fusarium graminearum spore germination after treatment with 3.125 μM Cla-H peptide for different times in this invention;

[0020] Figure 5 This is a microscopic observation of the inhibition of Botrytis cinerea spore germination after treatment with 3.125 μM Cla-H peptide for different times in this invention;

[0021] Figure 6 This diagram illustrates the minimum inhibitory concentration (MIC) of Clavanin-AK and Cla-H against Fusarium graminearum (GZ) and Botrytis cinerea (BC) under low-salt and high-salt conditions in this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] Reference Figures 1-6 ,

[0024] Example 1:

[0025] An antimicrobial peptide, Clavanin-AK, has the amino acid sequence shown in SEQ ID NO.1.

[0026] An antimicrobial peptide, Cla-H, is a Clavanin-A variant with six histidine residues fused to its C-terminus, and its amino acid sequence is shown in SEQ ID NO. 2. Based on the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2, the corresponding antimicrobial peptide was synthesized by Suzhou Bio-Tech Co., Ltd., with a purity >95%. The results are shown in [see attached table]. Figure 1 and Figure 2 .

[0027] SEQ ID NO.1 is:

[0028] VFQFLGKIIKKVGNFVKGFSKVF;

[0029] SEQ ID NO.2: is:

[0030] VFQFLGKIIKKVGNFVKGFSKVFHHHHHH.

[0031] Example 2: Determination of the antimicrobial activity of antimicrobial peptides:

[0032] The antimicrobial activity of the purified antimicrobial peptide prepared in Example 1 against Fusarium graminearum and Botrytis cinerea was detected using a combination of micro-broth dilution and resazu staining. Low-salt (KCl-free) and high-salt (100 mM KCl) environments were established, and the specific steps are as follows:

[0033] (1) Prepare a 2×SFM medium suspension containing 2×10⁴ CFU / mL spores for later use;

[0034] (2) Dilute the pure Cla-H and Clavanin-AK antimicrobial peptides with sterile water to prepare a series of antimicrobial peptide solutions with a final concentration of 2× (Cla-H: 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, 1.563μM, 0.782μM; Clavanin-AK: same as Cla-H);

[0035] (3) In a 96-well microplate, add 50 μL of antimicrobial peptide solution to each well and 50 μL of sterile water to the negative control; then add 50 μL of spore suspension to each well to make the final volume of the system 100 μL and the final spore concentration 1×104 CFU / mL.

[0036] (4) Incubate at 25 °C for 48 h, add 10 μL of 0.01% resazurin detection solution to each well, and continue incubation for 4 h. After the blue color of the negative control well has completely faded, observe the color change of each well. The concentration of the replicate wells that show no color change compared to the blank control is the minimum inhibitory concentration (MIC).

[0037] Test results are shown Figure 6 Under low-salt conditions, the MIC of Cla-H against Fg was 3.125 μM and the MIC against Bc was 12.5 μM, consistent with the MIC of Clavanin-AK. Under high-salt conditions, Clavanin-AK lost its complete inhibitory activity in the range of ≤50 μM, while the MIC of Cla-H against both Fg and Bc was 25 μM, indicating that the salt tolerance of Cla-H is significantly better than that of Clavanin-AK.

[0038] Example 3: Thermal stability test of Cla-H antimicrobial peptide:

[0039] The Cla-H antimicrobial peptide pure product prepared in Example 1 was prepared into a solution with a concentration of 2×MIC and incubated at 25 ℃, 50 ℃, 75 ℃, and 90 ℃ for 30 min, respectively. After incubation, the antimicrobial activity against Fusarium graminearum was detected using the method in Example 2, and the changes in its antimicrobial effect were observed. Two controls were set up: CK1 was 1×SFM medium + 0.01% resazurin detection solution, and CK2 was 1×104 CFU / mL Fusarium graminearum spores + 0.01% resazurin detection solution.

[0040] Test results are shown Figure 3 The antibacterial effect of Cla-H antimicrobial peptide did not differ significantly after treatment at different temperatures, indicating that the antimicrobial peptide has good thermal stability, can withstand high temperature treatment, and is convenient for practical storage and application.

[0041] Example 4: Microscopic observation of the inhibition of target fungal spore germination by Cla-H antimicrobial peptide:

[0042] This embodiment uses bright-field microscopy to observe and clarify the inhibitory effect of the modified antimicrobial peptide variant Cla-H on the germination of target plant fungal pathogens (Fusarium graminearum and Botrytis cinerea) and the changes in spore morphology, providing intuitive experimental evidence for the application of Cla-H as a novel antifungal agent.

[0043] (1) Preparation of antimicrobial peptide solution:

[0044] In a clean bench, Cla-H stock solution was diluted with sterile water to prepare 2×MIC concentration solutions of the corresponding target bacteria (final Cla-H concentration was 3.125 μM). After preparation, if not used immediately, the solutions were sealed and stored in a refrigerator at 4 ℃ and used within 12 h.

[0045] (2) Preparation of fungal spore suspension:

[0046] At each time point, under sterile conditions, prepare a 2×SFM culture medium suspension with a spore concentration of 2×10⁶ CFU / mL, shake thoroughly, and aliquot into sterile 1.5 mL centrifuge tubes, 50 μL per tube. The aliquot volume must ensure that each incubation group reaches the preset time endpoint synchronously.

[0047] (3) Treatment and incubation of antimicrobial peptides:

[0048] The prepared 2×MIC concentration antimicrobial peptide solution was added to a centrifuge tube containing twice the final concentration of spore suspension, mixed thoroughly, and incubated at room temperature. A negative control group (nc, with only an equal volume of sterile water and no antimicrobial peptide) was set up, with 3 biological replicates in each group, and incubation time points of 1 h, 2 h, and 6 h.

[0049] (4) Washing and recovery culture:

[0050] After incubation, add 1 mL of 1×SFM medium to each centrifuge tube, centrifuge at 1800 g for 7 min at room temperature, discard 1 mL of supernatant; add another 1 mL of 1×SFM medium and repeat the washing operation to remove residual antimicrobial peptides.

[0051] After washing, add 100 μL of 2×PDB medium to the tube and incubate overnight at 25 ℃ (extend the culture time for Botrytis cinerea to 20 h and for Fusarium graminearum to 16 h).

[0052] (5) Microscopic observation:

[0053] After the culture was completed, each culture tube was centrifuged at 2000 g for 7 min, and 160 μL of supernatant was collected. The remaining precipitate was resuspended by pipetting, and an appropriate amount of bacterial solution was added to the concave part of a single concave glass slide. A coverslip was then placed on the slide, and the germination of fungal spores and the growth of hyphae were observed and recorded under an optical microscope (bright field mode, scale bar 20 μm).

[0054] The fungal growth state of Fusarium graminearum is as follows Figure 5 As shown. Wherein:

[0055] Negative control group (nc): After 1 h of incubation, spores began to germinate and grow germ tubes; after 2 h of incubation, germ tubes elongated significantly and formed primary hyphae; after 6 h of incubation, hyphae extended extensively and branched, forming a dense hyphal network, and the fungus completed the normal growth process from spore to mature hyphae.

[0056] Cla-H treatment group (3.125 μM): The effect was consistent with the positive control group. During the incubation period of 1 to 6 h, the fungal spores did not germinate at all, and there was no germ tube elongation or hyphal growth. The original spore morphology was maintained throughout.

[0057] Negative control group (nc): Botrytis cinerea germinated and formed hyphae within 1 hour, and completed the growth process from spores to developed mycelia within 6 hours, indicating that the growth activity of fungi in the experimental system was normal and the experimental conditions were reliable.

[0058] Cla-H treatment group (12.5 μM): In the Cla-H treatment group, fungal spores did not germinate within 1-6 h, and there were no signs of hyphal growth.

[0059] The minimum inhibitory concentrations (MICs) of Clavanin-AK and Cla-H against Fusarium graminearum and Botrytis cinerea are shown in [reference needed]. Figure 6In summary, this invention demonstrates that Cla-H maintains good inhibitory activity even under high-salt conditions, effectively inhibiting the growth of Fusarium graminearum and Botrytis cinerea, thus overcoming the technical bottleneck of poor salt tolerance in existing antimicrobial peptides. The prepared antimicrobial peptide exhibits good thermal stability, showing no significant loss of antimicrobial activity after treatment at 90℃, facilitating storage and transportation. Furthermore, this antimicrobial peptide demonstrates rapid bactericidal action, initiating antimicrobial activity within 1 hour and achieving highly efficient killing of target fungi within 4 hours. It is also less likely to induce fungal resistance, is environmentally friendly, and is suitable as an effective ingredient in novel green pesticides, showing broad application prospects.

[0060] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the term "comprising" or any other variations thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A salt-tolerant antimicrobial peptide Cla-H, characterized in that: The antimicrobial peptide is a Clavanin-A variant with 6 histidine residues fused to its C-terminus.

2. The application of the salt-tolerant antimicrobial peptide Cla-H as described in claim 1, characterized in that: The applications include the control of Fusarium graminearum and / or Botrytis cinerea.

3. The application of the salt-tolerant antimicrobial peptide Cla-H as described in claim 1, characterized in that: The applications include the preparation of agricultural antifungal agents.

4. The salt-tolerant antimicrobial peptide Cla-H as described in claim 3 and its application in the control of Fusarium graminearum and Botrytis cinerea, characterized in that: The concentration of Cla-H antimicrobial peptide in the agricultural antifungal preparation is 3.125μM-50μM, and it is used for the prevention and control of fungal diseases in crops such as wheat and tomatoes.

5. The salt-tolerant antimicrobial peptide Cla-H as described in claim 1 and its application in the control of Fusarium graminearum and Botrytis cinerea, characterized in that: The Cla-H antimicrobial peptide exhibits a minimum inhibitory concentration (MIC) of 25 μM against both Fusarium graminearum and Botrytis cinerea in a high-salt environment of 100 mM KCl.

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