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

Abstract

The invention belongs to the technical field of plant disease prevention and treatment, and particularly relates to application of 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-Ser-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Leuol, and the amino acid sequence of the trichoderma hamatum antibacterial peptide is shown in the description. The invention finds that the metabolite of the trichoderma hamatum has an inhibiting effect on the potato late blight, so that the application of the trichoderma hamatum antibacterial peptide in preventing and treating the potato late blight is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease prevention and control, and particularly relates to application of hooked Trichoderma antimicrobial peptide in preventing and controlling potato late blight and an insecticide. Background Art

[0002] Potato late blight is one of the most devastating diseases caused by Phytophthora infestans, which has the characteristics of wide occurrence, fast spread, heavy damage and loss, and extremely difficult to control. The disease mainly overwinters in tubers as mycelium, and the oospores survive in the soil for a long time, becoming the primary source of infection. When conditions are suitable, the infected seed potatoes, the diseased residues and oospores remaining in the soil infect the plants, develop into central diseased plants and produce sporangia, emitting zoospores. The zoospores spread under external conditions, produce germ tubes that penetrate the host, and cause the disease to break out, posing a serious threat to agricultural production safety.

[0003] At present, the main prevention and control measures for potato late blight include agricultural prevention, physical prevention, chemical prevention, biological prevention and integrated prevention. However, these measures have limitations, long rotation cycles, and are not easy to implement within limited arable land resources. Biological control is unstable in the field and is easily affected by the environment. Commonly used chemical agents are expensive and pollute the environment, which is difficult for farmers to accept. Therefore, for the sustainable development of green agriculture, it is urgent to develop green, efficient and low-toxic microbial source agents. Microbial source agents refer to agents with antagonistic activity processed from secondary metabolites of microorganisms. These agents have the characteristics of low toxicity, non-toxicity, environmental friendliness and safety to non-target organisms, which are in line with the green concept of sustainable agricultural development at this stage. Therefore, the development of green, efficient and safe microbial source agents is of great significance to crop diseases and provides a basis for promoting the healthy development of green agriculture. Most Trichoderma can colonize plant roots and produce secondary metabolites with contact activity, which is a hot spot in the research of microbial source agents.

[0004] Trichoderma hamatum is a filamentous biocontrol fungus belonging to the genus Trichoderma of the subphylum Ascomycota, the orders Hypocreales, and the family Hypocreaceae. It is widely distributed in the plant root ecosystem, maintains the dynamic balance of microorganisms in the soil ecological environment, is easy to be artificially cultivated, and can colonize in the plant roots and leaves. The prior art has the function of preventing and controlling anthracnose of Epimedium sagittatum, root-knot nematodes, etc. The metabolites of Trichoderma hamatum have the functions of preventing Trichoderma hamatum and cabbage black rot, but the functions of Trichoderma hamatum and its metabolites on potato late blight are unknown. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of hooked Trichoderma antimicrobial peptide in preventing and controlling potato late blight and insecticide.

[0006] Application of a hooked Trichoderma antimicrobial peptide in preventing and controlling potato late blight, wherein the amino acid sequence of the hooked Trichoderma antimicrobial peptide 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 of potato late blight is Phytophthora infestans.

[0008] Preferably, the preparation method of the hook-shaped Trichoderma antimicrobial peptide comprises the following steps:

[0009] The first fermentation of Trichoderma hookii T21 with the deposit number of CGMCC NO.10923 was carried out using seed culture medium, and the second fermentation was carried out using potato glucose culture medium, and the duration of the second fermentation was longer than the duration of the first fermentation;

[0010] After fermentation, extraction was performed using ethyl acetate to obtain a crude extract;

[0011] The crude extract is subjected to a first separation using an elution system of a gel column to obtain a first separation product, and the first separation product is subjected to a second separation using a reverse phase semi-preparative chromatography column to obtain the hook-shaped Trichoderma antimicrobial peptide.

[0012] Preferably, the conditions of the first fermentation are 26°C to 30°C, 180rpm to 220rpm, and 2 to 4 days.

[0013] Preferably, the second fermentation is carried out at 26°C to 30°C for 13 to 18 days.

[0014] Preferably, the formula of the seed culture medium is 8g-12g of peptone, 35g-45g of maltose, 8g-12g of yeast extract, 3g-5g of agar powder, and ultrapure water is supplemented to 1L.

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

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

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

[0018] 2min~32min, volume fraction of phase B is 100%, volume fraction of phase C is 0%;

[0019] 42min~45min, volume fraction of phase B is 50%, volume fraction of phase C is 50%;

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

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

[0022] An insecticide, the active ingredient of which is the hooked Trichoderma antimicrobial peptide.

[0023] Preferably, the pesticide is a solution of the hooked Trichoderma antimicrobial peptide having 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 present invention is beneficial in that:

[0026] The invention finds that the metabolites of hooked Trichoderma have an inhibitory effect on potato late blight, thereby proposing the application of hooked Trichoderma antimicrobial peptides in preventing and controlling potato late blight.

[0027] The hooked Trichoderma antimicrobial peptide provided by the present invention is used in preventing and treating potato late blight, and the minimum inhibitory concentration is 102.4 μg / mL, and the antibacterial activity is significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the antibacterial test of monomer compounds.

[0029] Figure 2 These are the liquid phase results of the ethyl acetate extract of T. hamatum T21.

[0030] Figure 3 Structural elucidation of Tricholongins BⅠ, (A) LC-MS analysis, (B) HRESI-MS spectrum, (C) ESI-MS-MS spectrum, (D) ESI-MS-MS ion fragmentation.

[0031] Figure 4 The inhibitory effect of Tricholongins BⅠ monomer compound on Phytophthora infestans, a is the monomer compound, b is DMSO, C is H2O, and d is tricholongins.

[0032] Figure 5 The minimum inhibitory concentration of Tricholongins BⅠ was determined. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

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

[0035] Potato dextrose medium (PDA, 1L): 200g potatoes, 20g glucose, 17g agar powder, and make up to 1L with ultrapure water.

[0036] Potato dextrose broth (PDB, 1 L): 200 g potatoes, 20 g glucose, and make up to 1 L with ultrapure water.

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

[0038] Rye culture medium (1L): weigh 100g rye grains, 20g sucrose, and 15g agar powder. Soak the rye grains in water for 24-36h, crush them into powder, filter them with 4 layers of gauze, make the volume to 1000mL, and sterilize them with high temperature and high pressure steam at 121℃ for 20min for culturing potato late blight pathogen.

[0039] 1. Strain fermentation

[0040] The hooked Trichoderma T21 was isolated from tobacco leaves. The full name of the hooked Trichoderma T21 is Tham21. The strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection (CGMCC), the deposit address is Beijing, China, the classification name is Trichoderma hamatum, the strain deposit number is CGMCC NO.10923, and the deposit date is May 27, 2015.

[0041] The hooked Trichoderma T21 strain grows rapidly and has well-developed aerial hyphae. When cultured on PDA plates, the color of the hyphae is white in the early stage and turns green in the later stage. The colonies are dense and cotton-like, and the hyphae branches are often opposite. The conidia are oval or spherical and have a smooth surface. They are 2.1 to 3.2 μm long and 2.3 to 4.0 μm wide. The T21 strain was amplified and sequenced using ITS, TEF and RPB primers. The sequencing results were compared in the NCBI database. Through morphological and molecular biological identification, it was finally determined that the strain was a hooked Trichoderma.

[0042] The strain Trichoderma hooked T21 was taken out from the -80℃ refrigerator, thawed and transferred to the PDA plate to activate the strain, and placed in a 28℃ incubator for 2 days. It was transferred to the PDA plate again for 3 days for purification, and 2-3 bacterial cakes were punched on the plate with a hole puncher and inoculated into the seed culture medium, which was placed in a constant temperature shaker for 3 days, and the culture conditions were 28℃, 200rpm, and finally the seed solution was transferred to the PDA culture medium and placed in a 28℃ incubator for 15 days.

[0043] 2. Extraction of crude extract

[0044] After the fermentation, the culture in 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 placed in an ultrasonic oscillator for 30 minutes to accelerate the extraction of ethyl acetate. The ethyl acetate was collected by filtering with filter paper, concentrated with a rotary evaporator, and dried to obtain a crude extract.

[0045] 3. Compound separation and preparation

[0046] The crude extract was separated using a gel column, with an elution system of equal volumes of methanol and dichloromethane (ratio 1:1), and a rate of 1s / drop, 5mL of each component was collected, and a total of 45 components were collected. By comparing the HPLC spectra, components Fr5, Fr6, Fr7, and Fr8 had spectra corresponding to the wild type, and the retention times of the metabolites in the four components were consistent, so the four components were combined for HPLC semi-preparative liquid phase purification.

[0047] HPLC preparation method and conditions: Phase B is acetonitrile and phase C is water. A Kromasil 100-5-C18 reverse phase semi-preparative column (10 μm, 10×250 mm) was used for separation and preparation, with a flow rate of 2.0 mL / min. Gradient elution process: 0 min to 2 min, 50% volume fraction of phase B, 50% volume fraction of phase C; 2 min to 32 min, 100% volume fraction of phase B, 0% volume fraction of phase C; 42 min to 45 min, 50% volume fraction of phase B, 50% volume fraction of phase C, and finally 13.8 mg of compound Tricholongins BⅠ was obtained.

[0048] Structural identification

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

[0050] Compound 1 is easily soluble in methanol and acetone. According to the analysis and comparison of the liquid chromatography mass spectrometry data of the secondary metabolites of the knockout mutant and the wild type, the [M+H] + The molecular ion peak is 1911.0928, [M+2Na] 2+The molecular ion peak is 978.5954. m / z 1911.1154[M+H] + The fragmentation of the ion 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 the comparison of the fragmentation rules of the peptide and the reference literature, the structure of compound 1 reported in the literature is consistent, named Tricholongins BⅠ, abbreviated as LBⅠ. The molecular formula of compound LBⅠ was determined to be C 89 H 148 N 22 O2. Figure 1 Compound 1 in the formula is LBⅠ, and its structure is as follows Figure 2 As shown in Table 1 , 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. The detailed data of the secondary fragments are shown in Tables 1 and Figure 3 In addition, this compound was isolated for the first time from Trichoderma hamata.

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

[0052]

[0053]

[0054] 4. Determination of antibacterial activity

[0055] (1) Activation of pathogens. From the frozen tube of pathogenic fungus Phytophthora infestans stored at -80°C, a bacterial cake was picked and inoculated onto PDA medium and placed in a 28°C incubator for 24 h.

[0056] (2) Prepare bacterial plates. When the pathogenic fungi grow to a diameter of about 2 cm on PDA medium, perform preliminary screening by antibacterial experiments.

[0057] (3) Add the drug to be tested and the control. Draw a "cross" with the center of the culture dish as the intersection point, select two points on each straight line, and make the distance from the four points to the intersection 2 mm. Use a disposable syringe (without the needle part) to make holes at the four points, then drop the sample into the hole, and add the test drug, negative control, blank control and positive control in a clockwise direction.

[0058] (4) Culture observation and record. Place the culture in a 28°C incubator and observe every 12 hours. Take photos and record when obvious inhibition zones appear.

[0059] Schematic diagram of the antibacterial test of monomer compounds Figure 1 As shown in the figure, the inhibitory effect of LBⅠ monomer compound on the growth of Phytophthora infestans was tested by observing the growth of bacteria. Figure 4 As shown in the figure, the growth of pathogenic bacteria near the orifice where the monomer compound was added was inhibited, and an obvious inhibition zone appeared. The results showed that the LBⅠ monomer compound had a significant inhibitory effect on Phytophthora infestans.

[0060] 5. Minimum inhibitory concentration (MIC) determination

[0061] (1) The pathogenic fungus Phytophthora infestans was cultured for about 7 days, washed with PDB medium, and conidia were collected. The concentration of the spore suspension stock solution was counted using a hemocytometer to make the conidia suspension concentration 10 4 Pieces / mL.

[0062] (2) Activity test by microdilution method. Take a 96-well microdilution plate with 12 columns. Use the microdilution method to obtain 10 drug concentrations, using DMSO as the solvent: 204.8μg / mL, 102.4μg / mL, 51.2μg / mL, 25.6μg / mL, 12.8μg / mL, 6.4μg / mL, 3.2μg / mL, 1.6μg / mL, 0.8μg / mL, and 0.4μg / mL, respectively in columns 1 to 10. Columns 1 to 10 are set as sample wells, and columns 11 and 12 are set as control wells. Use a sterile pipette to add 180μL of pathogen solution to the sample wells in column 1, and then add 100μL of pathogen solution to the sample wells in columns 2 to 11 respectively. Column 11 is 100μL of pathogen solution, and column 12 is added with 100μL of PDB culture medium as a blank control. Rows 1 to 3 are repeated three times, and row 4 is a positive control. The concentration of the stock solution of the drug to be tested and the positive control drug is 2.048 mg / mL. Pipette 20 μL and add it to column 1, then dilute half of the sample wells in column 1 to other columns: adjust the pipette to 100 μL, pipette evenly in the sample wells in column 1, take 100 μL of the mixed solution and move it to the next column, and then proceed to column 10. The 100 μL mixed solution aspirated from column 10 is discarded.

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

[0064] The microdilution method was used to obtain 10 drug concentrations: 204.8μg / mL, 102.4μg / mL, 51.2μg / mL, 25.6μg / mL, 12.8μg / mL, 6.4μg / mL, 3.2μg / mL, 1.6μg / mL, 0.8μg / mL, and 0.4μg / mL, which are respectively in columns 1 to 10. The 11th column is the positive control (CK+, pathogenic bacteria solution), and the 12th column is the blank control (CK-, LB culture medium). The LBⅠ monomer compound solution and its replicates are in rows 1-3, and the positive control drug hygromycin is in row 4. After treatment with LBⅠ, the cell activity colorimetric solution was added as shown in Figure 5 As shown, the 1-2 columns of the 1-3 rows are blue, the 3rd column is purple, and the 4th to 10th columns are red. The 4th row is the positive control hygromycin B, the 1-5th columns are blue, the 6th column is purple, and the 7th to 10th columns are red, indicating that there are surviving conidia of potato late blight from the 3rd column, and the minimum inhibitory concentration of the Tricholongins BⅠ monomer compound against potato late blight is the concentration in the 2nd column, that is, MIC = 102.4μg / mL.

[0065] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The application of hooked Trichoderma antimicrobial peptide in preventing and controlling potato late blight, characterized in that: The amino acid sequence of the hooked Trichoderma antimicrobial peptide 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.

2. The use according to claim 1, characterized in that: The preparation method of the hook-shaped Trichoderma antimicrobial peptide comprises the following steps: The first fermentation of Trichoderma hookii T21 with the deposit number of CGMCC NO.10923 was carried out using seed culture medium, and the second fermentation was carried out using potato glucose culture medium, and the duration of the second fermentation was longer than the duration of the first fermentation; After fermentation, extraction was performed using ethyl acetate to obtain a crude extract; The crude extract is subjected to a first separation using an elution system of a gel column to obtain a first separation product, and the first separation product is subjected to a second separation using a reverse phase semi-preparative chromatography column to obtain the hook-shaped Trichoderma antimicrobial peptide.

3. The use according to claim 2, characterized in that: The conditions of the first fermentation are 26°C to 30°C, 180rpm to 220rpm, and 2 to 4 days.

4. The use according to claim 3, characterized in that: The second fermentation is carried out under conditions of 26°C to 30°C for 13 to 18 days.

5. The use according to claim 2, characterized in that: The formula of the seed culture medium is 8g-12g of peptone, 35g-45g of maltose, 8g-12g of yeast extract, 3g-5g of agar powder, and ultrapure water is added to 1L.

6. The use according to claim 2, characterized in that: The formula of the potato glucose culture medium is 190g-210g of potato, 18g-22g of glucose, 16g-20g of agar powder, and ultrapure water is added to 1L.

7. The use according to claim 2, characterized in that: The elution system of the gel column is a mixed solution of methanol and dichloromethane.

8. The use according to claim 2, characterized in that: The gradient elution process of the second separation is: 0min-2min, the volume fraction of phase B is 50%, and the volume fraction of phase C is 50%; 2min~32min, volume fraction of phase B is 100%, volume fraction of phase C is 0%; 42min~45min, volume fraction of phase B is 50%, volume fraction of phase C is 50%; Phase B is acetonitrile and phase C is water.

9. The use according to claim 1, characterized in that: The concentration of the hooked Trichoderma antimicrobial peptide is greater than 102.4 μg / mL.

10. An insecticide, characterized in that: The effective ingredient is the hook-shaped Trichoderma antimicrobial peptide described in claim 1.

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