Application of trichoderma hamatum antimicrobial peptide in prevention and treatment of potato late blight and insecticide

Abstract

The application belongs to the technical field of plant disease prevention and treatment, and particularly relates to application of a Trichoderma hamatum antibacterial peptide in prevention and treatment of potato late blight. The amino acid sequence of the Trichoderma hamatum antibacterial peptide is Ac-Aib-Gly-Phe-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Leuol. The application finds that a metabolic product of the Trichoderma hamatum has an inhibiting effect on the potato late blight, and thus proposes the application of the Trichoderma hamatum antibacterial peptide in prevention and treatment of the potato late blight.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to the application of Trichoderma hookeri antimicrobial peptides in the control of potato late blight and its insecticide. Background Technology

[0002] Potato late blight, caused by the pathogenic fungus *Phytophthora infestans*, is one of the most devastating diseases, characterized by its wide range of occurrence, rapid spread, severe damage, and extreme difficulty in control. The disease primarily overwinters as mycelium in tubers, while oospores survive for extended periods in the soil, serving as the primary source of infection. Under suitable conditions, infected seed potatoes, diseased plant debris in the soil, and oospores infect plants, developing into central diseased plants that produce sporangia and release zoospores. These zoospores spread under external conditions, producing germ tubes that penetrate the host, leading to disease outbreaks and posing a serious threat to agricultural production.

[0003] Currently, the main control measures for potato late blight include agricultural control, physical control, chemical control, biological control, and integrated management. However, these measures all have limitations. Long crop rotation cycles make them difficult to implement within limited arable land resources. Biological control is unstable in the field and easily affected by the environment. Commonly used chemical agents are costly and pollute the environment, making them unacceptable to farmers. Therefore, for the sustainable development of green agriculture, the development of green, efficient, and low-toxicity microbial-derived agents is urgently needed. Microbial-derived agents refer to agents with antagonistic activity processed from the secondary metabolites of microorganisms. These agents possess characteristics such as low toxicity, non-toxicity, environmental friendliness, and safety for non-target organisms, aligning with the current green concept of sustainable agricultural development. Therefore, the development of green, efficient, and safe microbial-derived agents is of great significance for crop disease control and provides a basis for promoting the healthy development of green agriculture. Most Trichoderma fungi can colonize plant roots and produce secondary metabolites with contact-killing activity, making them a hot topic in microbial-derived agent research.

[0004] Trichoderma hamatum is a filamentous biocontrol fungus belonging to the genus Trichoderma in the subphylum Ascomycota, order Hypocreales, and family Hypocreaceae. It is widely distributed in plant root ecosystems, maintaining the dynamic balance of microorganisms in the soil environment. It is easy to cultivate artificially and can colonize plant roots and leaves. Existing technologies have shown that Trichoderma hamatum can control anthracnose in Epimedium sagittatum and root-knot nematodes. The metabolites of Trichoderma hamatum have the effect of controlling black rot of Trichoderma hamatum and cabbage. However, the effect of Trichoderma hamatum and its metabolites on potato late blight is unknown. Summary of the Invention

[0005] To address the above problems, this invention provides the application of Trichoderma hookeri antimicrobial peptides in the prevention and control of potato late blight and an insecticide.

[0006] The application of Trichoderma hookeri antimicrobial peptides in the control of potato late blight, wherein the amino acid sequence of the Trichoderma hookeri antimicrobial peptides is Ac-Aib-Gly-Phe-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Leuol, wherein Aib is α-aminoisobutyric acid and Ac-Aib is acetylated α-aminoisobutyric acid.

[0007] Preferably, the pathogen causing potato late blight is Phytophthora infestans.

[0008] Preferably, the preparation method of the Trichoderma hookeri antimicrobial peptide includes the following steps:

[0009] Trichoderma hookeri T21 with accession number CGMCC NO.10923 was first fermented using seed culture medium and then second fermented using potato dextrose medium. The duration of the second fermentation was longer than that of the first fermentation.

[0010] After fermentation, the crude extract was obtained by extraction with ethyl acetate.

[0011] The crude extract was first separated using a gel column elution system to obtain the first separated product. The first separated product was then separated a second time using a reversed-phase semi-preparative chromatography column to obtain the Trichoderma hookeri antimicrobial peptide.

[0012] Preferably, the conditions for the first fermentation are 26℃~30℃, 180rpm~220rpm, and 2 days~4 days.

[0013] Preferably, the conditions for the second fermentation are 26℃~30℃ for 13 days~18 days.

[0014] Preferably, the seed culture medium is formulated as follows: 8g-12g peptone, 35g-45g maltose, 8g-12g yeast extract, 3g-5g agar powder, and ultrapure water to a final volume of 1L.

[0015] Preferably, the formula for the potato glucose culture medium is 190g-210g potato, 18g-22g glucose, 16g-20g agar powder, and ultrapure water to a final volume of 1L.

[0016] Preferably, the elution system of the gel column is a mixture of methanol and dichloromethane.

[0017] Preferably, the gradient elution process for the second separation is: 0 min to 2 min, B phase volume fraction 50%, C phase volume fraction 50%;

[0018] From 2 min to 32 min, the volume fraction of phase B was 100%, and the volume fraction of phase C was 0%.

[0019] 42 min–45 min, B phase volume fraction 50%, C phase volume fraction 50%;

[0020] Phase B is acetonitrile, and phase C is water.

[0021] Preferably, the concentration of the Trichoderma hookeri antimicrobial peptide is greater than 102.4 μg / mL.

[0022] An insecticide whose active ingredient is the aforementioned Trichoderma hookeri antimicrobial peptide.

[0023] Preferably, the insecticide is a solution of the Trichoderma hookeri antimicrobial peptide with a concentration greater than 102.4 μg / mL.

[0024] Preferably, the solvent in the solution is dimethyl sulfoxide or water.

[0025] Compared with the prior art, the advantages of the present invention are:

[0026] This invention discovers that the metabolites of Trichoderma hookeri have an inhibitory effect on potato late blight, and thus proposes the application of Trichoderma hookeri antimicrobial peptides in the prevention and control of potato late blight.

[0027] The antimicrobial peptides of Trichoderma hookeri provided by this invention have a minimum inhibitory concentration of 102.4 μg / mL and significant antimicrobial activity in the prevention and control of potato late blight. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of a monomeric compound antibacterial test.

[0029] Figure 2 Liquid phase results for the ethyl acetate extract fraction of T. hamatum T21.

[0030] Figure 3 For the structural determination of Tricholongins BⅠ, (A) LC-MS analysis, (B) HRESI-MS spectrum, (C) ESI-MS-MS spectrum, (D) ESI-MS-MS ion fragments.

[0031] Figure 4 The inhibitory effect of Tricholongins BⅠ monomers on pathogenic Phytophthora indicum is shown in Figure 1. a is the monomer compound, b is DMSO, c is H2O, and d is tricholongins.

[0032] Figure 5 This study determined the minimum inhibitory concentration (MIC) of Tricholongins BⅠ. Detailed Implementation

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0034] The culture medium formula used in this invention is as follows:

[0035] Potato glucose medium (PDA, 1L): 200g potato, 20g glucose, 17g agar powder, and ultrapure water to a final volume of 1L.

[0036] Potato Glucose Broth (PDB, 1L): 200g potatoes, 20g glucose, and ultrapure water to a final volume of 1L.

[0037] Seed culture medium (SMYA, 1L): 10g peptone, 40g maltose, 10g yeast extract, 4g agar powder, and ultrapure water to 1L.

[0038] Rye medium (1L): Weigh 100g rye grains, 20g sucrose, and 15g agar powder. Soak the rye grains in water for 24-36 hours, crush them into powder, filter through 4 layers of gauze, bring the volume to 1000mL, and sterilize by high-temperature and high-pressure steam at 121℃ for 20 minutes. This medium is used to culture potato late blight pathogen.

[0039] 1. Fermentation by bacterial strain

[0040] Trichoderma hamatum was isolated from tobacco leaves. The full name of Trichoderma hamatum is Tham21. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China. The classification name is Trichoderma hamatum, the strain accession number is CGMCC NO.10923, and the accession date is May 27, 2015.

[0041] Trichoderma harzianum strain T21 grows rapidly with well-developed aerial hyphae. When cultured on PDA plates, the hyphae are initially white, later turning green. Colonies are dense and cottony, with hyphal branching typically opposite. Conidia are oval or spherical with a smooth surface, measuring 2.1–3.2 μm in length and 2.3–4.0 μm in width. Amplification and sequencing of strain T21 were performed using ITS, TEF, and RPB primers. The sequencing results were compared with the NCBI database. Through morphological and molecular biological identification, the strain was ultimately confirmed as Trichoderma harzianum.

[0042] The preserved strain *Trichoderma hookeri* T21 was removed from the -80℃ freezer, thawed, and transferred to a PDA plate to activate the strain. It was then incubated at 28℃ for 2 days. After further transfer to a PDA plate and incubation for 3 days, purification was performed. Two to three mycelial discs were punched into the plate and inoculated into seed culture medium. The culture was then placed on a constant-temperature shaker and incubated for 3 days at 28℃ and 200 rpm. Finally, the inoculum was transferred to PDA medium and incubated at 28℃ for 15 days.

[0043] 2. Crude extract extraction

[0044] After fermentation, the culture from the PDA plate was collected into a 1000 mL Erlenmeyer flask, 500 mL of ethyl acetate was added, the PDA culture medium was crushed with a glass rod, and the mixture was placed in an ultrasonic vibrator for 30 min to accelerate the extraction of ethyl acetate. The ethyl acetate was collected by filtration through filter paper, concentrated using a rotary evaporator, and dried to obtain the crude extract.

[0045] 3. Compound separation and preparation

[0046] The crude extract was separated using a gel column chromatography system with equal volumes of methanol and dichloromethane (1:1 ratio) as the elution rate, controlled at 1 s / drop. 5 mL of each fraction was collected, for a total of 45 fractions. HPLC chromatogram comparison revealed chromatograms corresponding to the wild type in fractions Fr5, Fr6, Fr7, and Fr8. The metabolites in these four fractions exhibited consistent retention times; therefore, the four fractions were combined for semi-preparative HPLC purification.

[0047] HPLC preparation method and conditions: Phase B was acetonitrile, and Phase C was water. Separation and preparation were performed using a Kromasil 100-5-C18 reversed-phase semi-preparative column (10 μm, 10 × 250 mm) at a flow rate of 2.0 mL / min. Gradient elution process: 0 min–2 min, 50% B phase and 50% C phase; 2 min–32 min, 100% B phase and 0% C phase; 42 min–45 min, 50% B phase and 50% C phase, finally yielding 13.8 mg of the compound Tricholongins BⅠ.

[0048] Structural identification

[0049] The monomeric compound was completely dissolved in methanol and then transferred to a 1 mL sample vial for mass spectrometry analysis.

[0050] Compound 1 is readily soluble in methanol and acetone. Based on comparative analysis of the secondary metabolites from the knockout mutant and wild-type, liquid chromatography-mass spectrometry data shows that compound 1 has [M+H]... + The molecular ion peak is at 1911.0928, [M+2Na] 2+The molecular ion peak is at 978.5954. m / z 1911.1154 [M+H] + Fragmentation of ions produces a series of observable b-series and y-series fragments. Figure 3 C (Table 1), including characteristic ions m / z 740.4673 (b9) and m / z 1170.6533 (y5). Through comparison with the peptide fragmentation pattern and references, the structure of compound 1 reported in the literature is consistent, named Tricholongins BⅠ, abbreviated as LBⅠ. The molecular formula of compound LBⅠ is determined to be C 89 H 148 N 22 O2. Figure 1 Compound 1 in the text is LBⅠ, and its structure is as follows: Figure 2 As shown, the amino acid sequence of LBⅠ is Ac-Aib-Gly-Phe-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Leuol. Detailed data on secondary fragments are shown in Table 1 and [Table data would be inserted here]. Figure 3 Furthermore, this compound is the first isolation from Trichoderma hookeri.

[0051] Table 1. Amino acid composition and fragment ions in the mass spectra of Tricholongins BⅠ.

[0052]

[0053]

[0054] 4. Antibacterial activity assay

[0055] (1) Activation of pathogens. Take a piece of fungal cake from the cryopreservation tube of Phytophthora infectans stored at -80℃ and inoculate it onto PDA medium. Incubate at 28℃ for 24h.

[0056] (2) Prepare fungal plates. When the pathogenic fungi are cultured on PDA medium to a diameter of about 2 cm, a preliminary screening test for antibacterial activity is performed.

[0057] (3) Add the test drug and control. Draw a cross with the center of the petri dish as the intersection point. Take two points on each straight line so that the distance from the four points to the intersection point is 2 mm. Make holes at the four points with a disposable syringe (without the needle part), and then add the sample into the hole. Add the test drug, negative control, blank control and positive control in a clockwise direction.

[0058] (4) Culture observation and recording. Place in a 28℃ incubator and observe every 12 hours. Take photos and record when a clear inhibition zone appears.

[0059] Schematic diagram of antibacterial test of monomeric compound as shown in Figure Figure 1 As shown, the inhibitory effect of LBⅠ monomer compound on the growth of pathogenic *Phytophthora infestans* was tested by observing the cell growth. After 2 days of culture, as... Figure 4 As shown, the growth of pathogenic bacteria near the pores where the monomeric compound was added was inhibited, and a clear inhibition zone appeared. The results indicate that the LBⅠ monomeric compound has a significant inhibitory effect on pathogenic *Phytophthora*.

[0060] 5. Determination of minimum inhibitory concentration (MIC)

[0061] (1) After culturing the pathogenic fungus *Phytophthora infestans* for approximately 7 days, the fungus was washed with PDB medium, and conidia were collected. The concentration of the original conidia suspension was determined using a hemocytometer, ensuring the conidia suspension concentration was 10. 4 per mL.

[0062] (2) Activity testing using the microdilution method. A 96-well microdilution plate (12 columns) was used to obtain 10 drug concentrations using the microdilution method with DMSO as the solvent: 204.8 μg / mL, 102.4 μg / mL, 51.2 μg / mL, 25.6 μg / mL, 12.8 μg / mL, 6.4 μg / mL, 3.2 μg / mL, 1.6 μg / mL, 0.8 μg / mL, and 0.4 μg / mL, located in columns 1-10. Columns 1-10 were designated as sample wells, and columns 11 and 12 as control wells. 180 μL of pathogenic bacterial suspension was added to the sample well in column 1 using a sterile pipette, followed by 100 μL of pathogenic bacterial suspension in each of columns 2-11. Column 11 contained 100 μL of pathogenic bacterial suspension, and column 12 contained 100 μL of PDB medium as a blank control. Rows 1-3 are triple replicates, and row 4 is the positive control. The stock solution concentration of the test drug and the positive control drug was prepared to be 2.048 mg / mL. 20 μL was added to column 1, and then the solution was diluted half-fold from the sample wells in column 1 to the other columns: the pipette was adjusted to 100 μL, and after mixing thoroughly in the sample wells of column 1, 100 μL of the mixture was transferred to the next column, and so on until column 10. The 100 μL of mixture drawn from column 10 was discarded.

[0063] (3) Observation and Recording. Place the 96-well plate with the samples added in a 28℃ incubator and observe every 6 hours. After 24 hours of incubation, add 10 μL of PrestoBlue resazurin dye to each well for staining. After staining for 30 minutes, find the well at the boundary between blue and red to determine the minimum inhibitory concentration. Take photos and record the results.

[0064] Ten drug concentrations were obtained using the microdilution method: 204.8 μg / mL, 102.4 μg / mL, 51.2 μg / mL, 25.6 μg / mL, 12.8 μg / mL, 6.4 μg / mL, 3.2 μg / mL, 1.6 μg / mL, 0.8 μg / mL, and 0.4 μg / mL, listed in columns 1-10. Column 11 is the positive control (CK+, pathogenic bacterial culture), and column 12 is the blank control (CK-, LB medium). LBⅠ monomeric compound solutions and their replicates are listed in rows 1-3, and the positive control drug hygromycin is listed in row 4. After treatment with LBⅠ, cell viability chromogenic solution was added as follows... Figure 5 As shown, columns 1-2 in rows 1-3 are blue, column 3 is purple, and columns 4-10 are red. Row 4 is the positive control hygromycin B, with columns 1-5 in blue, column 6 in purple, and columns 7-10 in red, indicating that viable conidia of *Tricholongins BⅠ* are present starting from column 3. The minimum inhibitory concentration (MIC) of the monomeric compound *Tricholongins BⅠ* against *Tricholongins BⅠ* is the concentration in column 2, i.e., MIC = 102.4 μg / mL.

[0065] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0066] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0067] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of Trichoderma hookeri antimicrobial peptides in the control of potato late blight, characterized in that, The amino acid sequence of the *Trichoderma hookeri* antimicrobial peptide is Ac-Aib-Gly-Phe-Aib-Aib-Gln-Aib-Aib-Aib-Ser-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Leuol, where Aib is α-aminoisobutyric acid and Ac-Aib is acetylated α-aminoisobutyric acid.

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