Application of small peptide PEP914 or PEP890 in preparation of biopesticide

Abstract

The invention discloses an application of a small peptide PEP914 or PEP890 in preparation of a biopesticide, and belongs to the technical field of biology. The biopesticide comprises a small peptide PEP914 or PEP890 and is a biopesticide for resisting bacterial leaf spot, rust disease or phytophthora root rot, the amino acid sequence of the small peptide PEP914 is shown as SEQ ID No.1, and the amino acid sequence of the small peptide PEP890 is shown as SEQ ID No.2. The small peptide PEP914 or PEP890 is applied to preparation of the biopesticide, a novel biopesticide thought is provided, and the problems that in the prior art, chemical pesticides are prone to remaining, environment problems are prone to being caused, and pathogens generate drug resistance are solved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of small peptide PEP914 or PEP890 in the preparation of biological pesticides. Background Art

[0002] As a major food crop and cash crop, soybean faces a variety of diseases during production. The large-scale use of chemical pesticides poses a serious threat to the human living environment and food security. It is particularly important to develop environmentally friendly small peptide biopesticides to improve plant resistance to soybean pathogens. However, excessive immune stimulation will have negative effects on plant growth and food yield. Selecting suitable small peptide molecules with immune activation activity, using them to prepare biopesticides to improve plant disease resistance, and analyzing the balance between immune activation and growth are important scientific issues that need to be solved. Summary of the invention

[0003] In view of the above-mentioned prior art, the present invention provides the use of small peptides PEP914 or PEP890 in the preparation of biological pesticides, provides a new idea for biological pesticides, and solves the problems in the prior art that chemical pesticides are prone to residues, environmental problems, and drug resistance of pathogens.

[0004] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide the use of small peptide PEP914 or PEP890 in the preparation of biological pesticides, the amino acid sequence of small peptide PEP914 is shown in SEQ ID No.1, and the amino acid sequence of small peptide PEP890 is shown in SEQ ID No.2.

[0005] Based on the above technical solution, the present invention can also be improved as follows.

[0006] Furthermore, the biopesticide is a biopesticide resistant to bacterial leaf spot, rust or Phytophthora root rot.

[0007] Furthermore, the biopesticide comprises a small peptide PEP914 or PEP890, the amino acid sequence of the small peptide PEP914 is shown as SEQ ID No.1, and the amino acid sequence of the small peptide PEP890 is shown as SEQ ID No.2.

[0008] Further, the biopesticide is applied to leguminous plants or cucurbitaceous plants.

[0009] Furthermore, the legume plant is soybean, mung bean, kidney bean, adzuki bean, pea, peanut or alfalfa; the cucurbitaceae plant is cucumber, pumpkin or bitter gourd.

[0010] Furthermore, the effective concentration of the small peptide PEP914 or PEP890 in the biopesticide is 1 nM to 1 μM.

[0011] Furthermore, the effective concentration of small peptides PEP914 or PEP890 in the biopesticide was 1 μM.

[0012] The beneficial effects of the present invention are as follows: the present invention discovers the anti-disease effect of small peptide PEP914 or small peptide PEP890 in bacterial leaf spot, rust or Phytophthora root rot of leguminous plants and cucurbitaceous plants, and provides the application of small peptide PEP914 or PEP890 in the preparation of biological pesticides. After the application of the small peptide, the disease condition of the plant is significantly improved, and effective plant pathogen immune defense can be achieved; small peptide PEP914 or PEP890 as a biological pesticide has little impact on the environment; small peptide PEP914 or PEP890 itself has no direct killing effect on pathogenic bacteria, but stimulates the plant's own disease resistance signal through receptor proteins P98R1 and P98R2 to enhance the plant's immune ability. The present invention provides a new idea of ​​biological pesticides to solve the problems of chemical pesticides in the prior art that are easy to remain, easy to cause environmental problems, and make pathogens resistant. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 To enhance soybean resistance to Pseudomonas leaf spot using small peptide PEP914;

[0014] Figure 2 To enhance soybean resistance to rust using small peptide PEP914;

[0015] Figure 3 To use small peptide PEP890 or small peptide PEP914 to enhance soybean resistance to Phytophthora root rot; wherein a is the result of observation under a fluorescence microscope, and b is the result of observation with the naked eye and taking a photo;

[0016] Figure 4 The small peptide PEP914 enhanced soybean disease resistance and was dependent on receptor proteins P98R1 and P98R2;

[0017] Figure 5 is the ROS burst level in leguminous plants; a is soybean cultivar Wm82, b is soybean cultivar k116, c is soybean cultivar Dongnong 50, d is mung bean, e is kidney bean, and f is adzuki bean;

[0018] Figure 6 is the ROS burst level in Leguminosae and Cucurbitaceae plants; a is pea, b is peanut, c is alfalfa, d is cucumber, e is pumpkin, and f is bitter gourd;

[0019] Figure 7 The ROS burst levels of soybean cultivar Wm82 under treatment with different concentrations of small peptide PEP914. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0021] Biological materials used in the examples:

[0022] 1. The pathogen of soybean bacterial leaf spot, Pseudomonas syringae pv.glycinea (Psg), was preserved in the laboratory. The soybean root rot fungus strains, Phytophthora sojae (P.sojae) P6497-RFP and Phytophthorasojae (P.sojae) P7076, were commonly used research strains and were obtained from public channels. The soybean rust fungus was obtained from an expert compound in Zhujia Town, Renshou County, Meishan City, Sichuan Province.

[0023] 2. The soybean cultivar Glycine max Williams 82 (Wm82) was preserved in the laboratory; the gene silencing vector and the silencing sites of the two receptor genes were completed by Weimi Biotechnology (Jiangsu) Co., Ltd., and the transgenic plants p98r1p98r2 with receptor protein P98R1 and P98R2 gene silencing were provided.

[0024] 3. Primer synthesis and sequencing were completed by Beijing Qingke Biotechnology Co., Ltd.

[0025] 4. Cultivation conditions of soybean seedlings: Soybean seeds were cultured in a 25°C incubator (16h light / 8h dark, humidity of about 60%) for about two weeks. The cultivation soil used was 100% vermiculite and watered every three days.

[0026] 5. Cultivation conditions of yellow soybean seedlings: Soybean seeds are cultured in a 25°C incubator (humidity is about 60%) in complete darkness for about a week. The cultivation soil used is 100% vermiculite and water is applied every three days.

[0027] 6. The small peptide PEP914, the small peptide PEP890, the mutant PEP914m (Y8A) of the small peptide PEP914, and the control small peptide flg22 were all completed by Sangon Biotechnology (Shanghai) Co., Ltd. The synthesis purity is above 95%, the powder is packaged at 1 mg per tube, and it is frozen in a -80°C ultra-low temperature refrigerator for long-term storage and use. After the small peptide powder is obtained, take out a tube, dissolve it with ddH2O to a concentration of 1mM according to the molecular weight and purity of the small peptide, and freeze it in a -20°C refrigerator, which can be used in a short period of time (half a year); each time the small peptide is used, it is prepared into a working solution with a lower concentration. The amino acid sequence of the small peptide PEP914 is shown in SEQ ID No. 1, the amino acid sequence of the small peptide PEP890 is shown in SEQ ID No. 2, and the amino acid sequence of the mutant PEP914m (Y8A) is shown in SEQ ID No. 3.

[0028] Experimental reagents: Fluorescence quantitative kit was purchased from Novozyme Biotechnology Co., Ltd.; Geneticin G418 was purchased from Beijing Rambolide Trading Co., Ltd.; V8 vegetable juice was purchased from Beijing Xile Trading Co., Ltd.; calcium carbonate (analytical grade) was purchased from Xilong Science Co., Ltd.; agar powder was purchased from Beijing Solebow Technology Co., Ltd.; BD Proteose Peptone was purchased from Gibco; Tween-20 was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; rifampicin antibiotics were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; Silwet L-77 was purchased from GE; potassium dihydrogen phosphate, glycerol and magnesium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd.; peroxidase (V900503-100MG) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; luminol (A8511) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; trypsin was purchased from Oxoid; sucrose was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; L-glutamic acid sodium salt monohydrate was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; tap water and sterile double distilled water (ddH2O) were both obtained from our unit.

[0029] Experimental consumables: Vermiculite was purchased from Beijing Jixiang Feiyun Garden Engineering Co., Ltd.; 150 mm disposable round dishes were purchased from Beijing Yiyan Shengke Biotechnology Co., Ltd.; 96-well white ELISA plates (3922) were purchased from CORNING; Eppendorf pipettes were purchased from Beijing Shengang Technology Co., Ltd.; culture boxes and culture covers were all taken from the greenhouse of this unit; sterile gauze was purchased from JD.com; Erlenmeyer flasks, markers, scissors, small blades, tweezers, centrifuge tubes, pipette tips and hole punchers were all purchased from Kashma Mall.

[0030] Experimental equipment: PCR instrument was purchased from Bio-Rad; quantitative PCR instrument was purchased from Bio-Rad; high temperature and high pressure sterilizer MLS-3750 was purchased from Sanyo Company of Japan; light incubator was purchased from Fujian Jiupu Biotechnology Co., Ltd.; microplate reader (Envision) was purchased from Perkin Elmer; small low temperature incubator (Asone / Azovang) was purchased from Beijing Jianqiang Weiye Technology Co., Ltd.; Olympus optical microscope (OLYMPUSBX41) was purchased from Beijing Zhongshi Tiangong Instrument Equipment Co., Ltd.; Eppendorf centrifuge was purchased from Eppendorf (Shanghai) International Trading Co., Ltd.; spectrophotometer was purchased from Beckman Coulter; shaker was purchased from Shanghai Zhichu Instrument Co., Ltd.; Bosch (BOSCH) GBM 350RE electric drill was purchased from Beijing Castmer Technology Development Co., Ltd.; -20℃ refrigerator and ultra-low temperature refrigerator were both purchased from Haier Company.

[0031] Example 1

[0032] Small peptide PEP914 as a biopesticide to improve plant resistance to bacterial diseases

[0033] The soybean cultivar Wm82 was planted in 100% vermiculite and placed in a 25°C light incubator (humidity 60%) and allowed to grow for about 2 weeks. When the first trifoliate compound leaf of the soybean was fully expanded, the soybean seedlings were taken out of the incubator.

[0034] (1) KB medium and TSA medium were prepared. After the medium was prepared according to the formula shown in Table 1 and Table 2, it was divided into small triangular bottles, sterilized at 121° C. for 15 min, and then stored in a cold room at 4° C. for later use;

[0035] Table 1 Formula of KB medium (1L)

[0036]

[0037] Table 2 Formulation of TSA medium (1L)

[0038]

[0039] (2) Preparation of bacterial leaf spot pathogen Psg: Take Psg frozen in a -80°C ultra-low temperature freezer and streak it onto a new KB solid medium containing rifampicin antibiotics (50 mg / mL), culture it at 28°C for two days, then take a single colony and add it to KB liquid medium (about 10 mL) containing rifampicin antibiotics (50 mg / mL), and culture it overnight at 28°C in a shaker at 200 rpm;

[0040] (3) Marking soybean leaves: Use a marker to lightly draw a line on the petiole of the first pair of true leaves of soybeans to mark them. Four plants were selected for each treatment, and two leaves were marked on each plant.

[0041] (4) Preparing pre-treated peptide solution and control solution: Prepare the treatment solution according to the usage amount. For each treatment of 8 soybean leaves, prepare 8 mL of peptide solution containing 0.015% (v / v) Silwet L-77 and 1 μM peptide PEP914, 8 mL of control solution containing 0.015% (v / v) Silwet L-77, and 8 mL of control solution containing 0.015% (v / v) Silwet L-77 and 1 μM peptide flg22;

[0042] (5) Applying leaves: Use tweezers to hold sterile gauze and dip it into the solution prepared in step (4), and gently apply it to the front and back of the marked soybean leaves, ensuring that the action of applying to each leaf is consistent, and do not apply too much to avoid damaging the leaves. After covering the plants with a lid, return them to the incubator and culture them overnight;

[0043] (6) Inoculation: The bacteria Psg cultured overnight in step (2) were collected by centrifugation at 4000 rpm for 4 min, then washed three times with ddH2O, and the OD of the bacterial suspension was measured by a spectrophotometer. 600 Value; Prepare OD 600 = 0.5, containing 100 mL of Silwet L-77 at a concentration of 0.015% (v / v), sprayed on the front and back sides of the marked soybean leaves, and finally covered with the lid and returned to the incubator for 4 days;

[0044] (7) Collect bacteria from leaves: Take 8 replicates of soybean leaves from three groups of different treatments (Mock, small peptide flg22, small peptide PEP914); punch the leaves with a 7 mm diameter puncher, put them into a 1.5 mL centrifuge tube, add 100 μL ddH2O, grind them thoroughly until there is no large residue, then add 900 μL ddH2O to dilute each sample 10 times, apply it to TSA medium containing rifampicin antibiotics, and culture in a 28°C incubator for two days;

[0045] (8) Counting the number of bacteria: The statistical formula for each replicate is log 10 (cfu / cm 2 )=log 10 (Number of bacteria × dilution factor / (2×πr 2 ))(where π(pi) is the ratio of a circle to a circle, approximately equal to 3.14159, and r represents the radius of the leaf blade), the average of the eight replicates of each treatment was taken as the final statistical bacterial count;

[0046] (9) Microsoft Excel and Prism 9 software were used to analyze the number of Psg growth on soybean leaves.

[0047] The results of Psg growth number are as follows Figure 1 As shown, compared with the control group Mock and the leaves treated with the small peptide flg22, the use of the small peptide PEP914 enhanced the resistance of soybean to Pseudomonas leaf spot Psg, indicating that the small peptide PEP914 can be used as a biological pesticide to improve the ability of plants to resist bacterial diseases.

[0048] The soybean cultivar Wm82 was planted in a natural environment (in the field) and the soybean plants were treated using the same method. The treated group also had a certain resistance to bacterial leaf spot disease compared with the control group (not photographed).

[0049] Example 2

[0050] Small peptide PEP914 as a biopesticide to improve plant resistance to rust

[0051] (1) Cryopreservation of rust spores: Scrape off the rust spores on soybean leaves where rust broke out in an expert compound in Zhujia Town, Renshou County, Meishan City, Sichuan Province with a small blade and collect them in a 1.5 mL centrifuge tube; open the cap of the centrifuge tube containing the spores, put it in a color-changing silica gel bottle, seal and dry it overnight, and the remaining dried spores are quickly frozen with liquid nitrogen and placed in a -80°C ultra-low temperature refrigerator, which can be stored for half a year;

[0052] (2) Planting soybean seedlings: Soybean cultivar Wm82 was planted in 100% vermiculite and placed in a 25°C light incubator (humidity 60%). It was allowed to grow for about 2 weeks, and the first pair of true leaves or trifoliate leaves were marked as experimental materials.

[0053] (3) Activation of rust fungus spores: A small amount of spores were taken out and suspended in a solution containing 0.04% (v / v) Tween, and then inoculated onto detached soybean leaves (the soybean leaves were soaked in a solution containing 0.04% (v / v) Tween before inoculation), placed in a 25°C light incubator, and cultured for about 14 days;

[0054] (4) Prepare the pre-treated peptide solution and control solution and treat the leaves: prepare the treatment solution according to the usage amount. For each treatment, 8 soybean leaves are used. Prepare 10 mL of the peptide solution containing 0.04% (v / v) Tween and 1 μM PEP914, and 10 mL of the control solution containing 0.04% (v / v) Tween. Gently and evenly apply them to the front and back of the soybean leaves marked in step (2) and treat for 24 hours.

[0055] (5) Collecting spores: Collect all rust spores cultured for about 14 days. The specific method is to scrape the spores into a 50 mL centrifuge tube, add a solution containing 0.04% (v / v) Tween to suspend the spores; centrifuge at 4000 rpm for 10 min, remove the supernatant, and then wash twice with a solution containing 0.04% (v / v) Tween;

[0056] (6) Spore inoculation: The rust spores collected in step (5) were prepared to a concentration of about 8×10 4 spores / mL solution containing 0.04% (v / v) Tween, and then spraying the solution onto the soybean leaves pretreated in step (4), spraying the spores on both the front and back sides, and spraying each leaf three times to ensure uniform infection;

[0057] (7) Observe the phenotype and take photos: After inoculation, incubate the cells at room temperature in the dark for 24 h, then place them in a 25°C light incubator for about 3 weeks, observe the phenotype and take photos.

[0058] After about 3 weeks of cultivation, the results are as follows Figure 2As shown, compared with the leaves treated with the control group Mock, the treatment with the small peptide PEP914 enhanced the resistance of soybean leaves to rust spores, indicating that the small peptide PEP914 can be used as a biopesticide to improve the ability of plants to resist soybean rust.

[0059] Example 3

[0060] Small peptides PEP914 or PEP890 as biopesticides to improve plant resistance to Phytophthora root rot

[0061] 1. Detection of small peptide pretreatment to improve soybean resistance to root rot fungus strain Phytophthora sojae (P.sojae)

[0062] The specific experimental steps for the resistance of P6497-RFP are as follows:

[0063] (1) The soybean cultivar Wm82 was planted in 100% vermiculite, placed in a 25°C light incubator (humidity 60%), and grown under normal light for about 4 days;

[0064] (2) Preparation of 10% V8 medium: Prepare V8 medium according to the formula shown in Table 3 and dispense into small triangular bottles.

[0065] Sterilize at 121℃ for 15min, and store in a 4℃ cold room for later use;

[0066] Table 3 Formula of 10% V8 medium (1L)

[0067]

[0068] (3) Activation of the Phytophthora sojae (P. sojae) P6497-RFP: While waiting for the soybean seedlings to grow, the strain P6497-RFP was activated; the specific method was as follows: take a culture plate of the Phytophthora sojae P6497-RFP stored in a 10°C incubator, gently poke a little hyphae with a pipette tip, smear it on a new 10% V8 medium containing the antibiotic G418 (50 mg / mL), and culture it at room temperature for about 4 days;

[0069] (4) Cleaning the soybean roots: After the soybeans have grown for 4 days, gently wash the vermiculite from the soybean roots with water;

[0070] (5) Prepare pre-treated peptide solution and control solution and treat soybean roots: prepare 10 mL of 1 μM PEP914 peptide solution and ddH2O as the control solution, and soak soybean roots in the peptide solution and control solution for 24 h, respectively. Each treatment involves about 20 roots.

[0071] (6) Infection of soybean roots with Phytophthora sojae strain P6497-RFP: The main and lateral roots of the treated soybeans were cut off and treated in vitro. The roots were spread flat in a round dish with the roots in contact with sterile filter paper soaked in ddH2O. The Phytophthora sojae strain P6497-RFP, which had been cultured for about 4 days, was made into small discs containing mycelial agar blocks using a 4 mm punch. The mycelial surface of each small disc was contacted with each soybean root and cultured at room temperature in the dark for 48 h. The infection of spores in the soybean roots was then observed under an optical microscope and photographed. About 12 photos were taken for each replicate. The images were then processed using Fiji (ImageJ enhanced version) and the number of P. sojae P6497-RFP spores growing was counted. Finally, the average number of spores under each treatment was calculated.

[0072] The results are as follows Figure 3 As shown in a, the white straight line is a ruler, indicating a length of 200 μm; compared with the control group, the resistance of soybeans to Phytophthora root rot was enhanced after pretreatment with small peptides PEP914 or PEP890, indicating that small peptides PEP914 or PEP890 can be used as biological pesticides to improve the ability of soybeans to resist Phytophthora root rot.

[0073] 2. Detect the effect of small peptide pretreatment on improving soybean resistance to Phytophthora sojae (P.sojae) P7076, the root rot fungus strain. The specific experimental steps are as follows:

[0074] (1) Planting yellow soybean seedlings: Plant soybean cultivation seed Wm82 in 100% vermiculite and culture in the dark for about 3 days;

[0075] (2) Preparation of 10% V8 medium: The preparation method of 10% V8 medium (1 L) is the same as above;

[0076] (3) Activation of Phytophthora sojae (P. sojae) P7076: While waiting for the soybean seedlings to grow, the P7076 strain should be activated; the specific method is: take a culture plate of the P. sojae strain stored in a 10°C incubator, gently poke a little hyphae with a pipette tip, smear it on a new 10% V8 medium without antibiotics, and culture it at room temperature for about 5 days;

[0077] (4) Cleaning the soybean roots: After the soybeans have grown for 3 days, gently wash the vermiculite from the soybean roots with water;

[0078] (5) Prepare pre-treated small peptide solution and control solution and treat yellowing soybean seedlings: prepare 10 mL of small peptide solution with a concentration of 1 μM PEP914, 10 mL of small peptide solution with a concentration of 1 μM PEP914m, and ddH2O as the control solution. Soak the yellowing soybean seedlings with the small peptide solution and the control solution for 24 h, respectively. Each treatment is for about 20 yellowing seedlings;

[0079] (6) Infection of the hypocotyls of etiolated soybean seedlings by the root rot fungus strain P7076: Spread the etiolated soybean seedlings one by one in a round dish, with the whole seedling in contact with sterile filter paper soaked in ddH2O; Use a 4 mm hole puncher to make small discs containing mycelial agar blocks of the root rot fungus strain P7076 that has been cultured for about 5 days, and contact the mycelial surface of each small disc to the hypocotyl of each etiolated seedling, and culture at room temperature in the dark for 48 h; Then observe the infection of spores in the soybean hypocotyls with the naked eye and take pictures. Take about 12 photos for each repeat, and process the pictures with Adobe Photoshop 2021 software.

[0080] like Figure 3 b The results show that the black straight line is a ruler, indicating a length of 1 cm; compared with the control group (Mock and small peptide PEP914m), the resistance of soybeans to Phytophthora root rot was enhanced after pretreatment with small peptide PEP914, indicating that small peptide PEP914 can be used as a biological pesticide to improve resistance to soybean Phytophthora root rot.

[0081] Example 4

[0082] Small peptide PEP914 enhances soybean disease resistance in a manner dependent on receptor proteins P98R1 and P98R2

[0083] The CRISPR / CAS9 technology was used to construct the soybean gene editing mutant genetic material p98r1p98r2 with silenced receptor proteins P98R1 and P98R2, which was completed by Weimi Biotechnology (Jiangsu) Co., Ltd. The amino acid sequence of receptor protein P98R1 is shown in SEQ ID No.4, the gene sequence of receptor protein P98R1 is shown in SEQ ID No.6, the amino acid sequence of receptor protein P98R2 is shown in SEQ ID No.5, and the gene sequence of receptor protein P98R2 is shown in SEQ ID No.7.

[0084] After pretreatment with the small peptide PEP914, the soybean gene-edited mutant genetic material (plant p98r1p98r2) in which the receptor proteins P98R1 and P98R2 were silenced constructed using CRISPR / CAS9 technology was detected to see whether the resistance was reduced. The treatment method was the same as in Example 1.

[0085] The results are as follows Figure 4As shown, p98r1r2-L1 and p98r1r2-L2 are two independent p98r1p98r2 homozygous mutants. Compared with the control cultivated variety Wm82, both the control treatment and the small peptide PEP914 treatment are more susceptible to the bacterial strain Psg. The control group Wm82, compared with the ddH2O treatment, has stronger disease resistance under the small peptide PEP914 treatment. This result shows that the small peptide PEP914 mediates the resistance of the small peptide PEP914 to plant diseases by interacting with the receptor proteins soybean P98R1 and P98R2.

[0086] Example 5

[0087] Small peptides PEP914 or PEP890 can activate the immune response of legumes or cucurbitaceae plants

[0088] The outbreak of reactive oxygen species (ROS) is one of the earliest observable responses of plants in defense against pathogens. The specific experimental steps are as follows (taking soybean cultivar Wm82 as an example):

[0089] (1) The soybean cultivar Wm82 was planted in 100% vermiculite, placed in a 25°C light incubator (humidity 60%), and grown under normal light for about 14 days;

[0090] (2) Add 200 μL ddH2O to each well of a 96-well white ELISA plate and use a hole punch to take leaves. Take 8 replicates for each treatment (i.e., take 8 4 mm small circular leaves);

[0091] (3) Gently clamp the small round leaf with tweezers and place it in a 96-well white ELISA plate. Cover the plate with a piece of white paper and place it under fluorescent light for overnight incubation.

[0092] (4) Add 200 μL of detection solution to each well of the ELISA plate. For 8 replicates, 1600 μL of control solution and small peptide solution need to be prepared respectively. The formula of the detection solution for ROS reaction is to add 1600 μL of ddH2O, 1.6 μL of peroxidase and 1.6 μL of substrate luminol in a 2 mL centrifuge tube, and mix well to obtain the control solution. Add small peptide (final concentration is 1 μM, small peptide is small peptide PEP914, small peptide PEP890 or small peptide flg22) to the control solution, and mix well to obtain the small peptide solution.

[0093] (5) After adding the test solution and treating for different time periods (0 to 160 min), the ROS burst level was detected using an ELISA instrument;

[0094] (6) Draw graphs using Excel and Prism 9.

[0095] Using the method of Example 5, ROS bursts in legumes were detected, including soybean cultivars Wm82, Dongnong 50 and k116, as well as mung beans, kidney beans, red beans, peas, peanuts and alfalfa; at the same time, ROS bursts were also found in Cucurbitaceae plants such as cucumber, pumpkin and bitter melon; the detection results of legumes and cucurbitaceae plants after different treatment times are as follows Figure 5 , Figure 6 As shown, it was shown that treatment with small peptides PEP914 or PEP890 could activate the immune response of Leguminosae and Cucurbitaceae plants.

[0096] Example 6

[0097] Different concentrations of small peptide PEP914 activate soybean immune response

[0098] (1) The soybean cultivar Wm82 was planted in 100% vermiculite, placed in a 25°C light incubator (humidity 60%), and grown under normal light for about 14 days;

[0099] (2) Add 200 μL ddH2O to each well of a 96-well white ELISA plate and use a hole punch to take leaves. Take 8 replicates for each treatment (i.e., take 8 4 mm small circular leaves);

[0100] (3) Gently clamp the small round leaf with tweezers and place it in a 96-well white ELISA plate. Cover the plate with a piece of white paper and place it under fluorescent light for overnight incubation.

[0101] (4) Add 200 μL of detection solution to each well of the ELISA plate. For 8 replicates, 1600 μL of control solution and small peptide solution are required. The formula of the detection solution for ROS reaction is to add 1600 μL of ddH2O, 1.6 μL of peroxidase and 1.6 μL of substrate luminol in a 2 mL centrifuge tube, and mix well to obtain the control solution. Add PEP914 small peptide (final concentrations are 1 nM, 10 nM, 100 nM and 1 μM, respectively) to the control solution, and mix well to obtain the small peptide solution.

[0102] (5) After adding the test solution and treating for different time periods (0 to 140 min), the ROS burst level was detected using an ELISA instrument;

[0103] (6) Draw graphs using Excel and Prism 9.

[0104] The results of the detection of ROS burst levels in soybean cultivar Wm82 at different concentrations of small peptide PEP914 are as follows Figure 7 As shown, it was shown that 1 nM of the small peptide PEP914 was able to activate the immune response of soybean Wm82, and 1 μM of the small peptide PEP914 activated the highest immune response.

[0105] Amino acid sequence of small peptide PEP914: DHPRGGNY (SEQ ID No. 1);

[0106] Amino acid sequence of small peptide PEP890: DLPRGGNY (SEQ ID No. 2);

[0107] The amino acid sequence of the mutant PEP914m (Y8A) of the small peptide PEP914: DHPRGGNA (SEQ ID No. 3); the amino acid sequence of the receptor protein P98R1:

[0108] MNNHTCYACFAIPATLLLVLMVLFQGTVAQTQAQTLLRWKQSLPHQSILDSWIINSTATTLSPCSWRGITCDSKGTVTIINLAYTGLAGTLLNLNLSVFPNLLRLDLKENNLTGHIPQNIGVLSKLQFLDLSTNFLNGTLPLSIANLTQVFELDLSRNNITGTLDPRLFPDGSDRPQSGLIGIRNLLFQDTLLGGRIPNEIGNIRNLTLLALDGNNFFGPIPSSLGNCTHLSILRMSENQLSGPIPPSIAKLTNLTDVRLFKNYLNGTVPQEFGNFSSLIVLHLAENNFVGELPPQVCKSGKLVNFSAAYNSFTGPIPISLRNCPALYRVRLEYNQLTGYADQDFGVYPNLTYMDLSYNRVEGDLSTNWGACKNLQVLNMAGNEISGYIPGEIFQLDQLHKLDLSSNQISGDIPSQIGNSFNLYELNLSDNKLSGIIPAEIGNLSNLHSLDLSMNKLLGPIPNQIGDISDLQNLNLSNNDLNGTIPYQIGNLRDLQYFLDLSYNSLSGEIPTDLGKLSNLISLNMSHNNLSGSIPHSLSEMFSLSTINLSYNNLEGMVPKSGIFNSSYPLDLSNNKDLCGQIRGLKPCNLTNPNGGSSERNKVVIPIVASLGGALFISLGLLGIVFFCFKRKSRAPRQISSFKSPNPFSIWYFNGKVVYRDIIEATKNFDNKYCIGEGALGIVYKAEMSGGQVFAVKKLKCDSNNLNIESIKSFENEIEAMTKTRHRNIIKLYGFCCEGMHTFLIYEYMNRGNLADMLRDDKDALELDWHKRIHIIKGVTSALSYMHHDCAPPLIHRDVSSKNILLSSNLQAHVSDFGTARFLKPDSAIWTSFAGTYGYAAPELAYTMEVTEKCDVFSFGVLALEVLTGKHPGDLVSSIQTCTEQKVNLKEILDPRLSPPAKNHILKEVDLIANVALSCLKTNPQSRPTMQSIAQLLEMEIADII(SEQ ID No.4);

[0109] Amino acid sequence of receptor protein P98R2:

[0110] (SEQ ID No. 5);

[0111] Gene sequence encoding receptor protein P98R1:

[0112]

[0113] Gene sequence encoding receptor protein P98R2:

[0114]

[0115] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. Application of small peptide PEP914 or PEP890 in the preparation of biological pesticides, characterized in that: The amino acid sequence of the small peptide PEP914 is shown in SEQ ID No.1, and the amino acid sequence of the small peptide PEP890 is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that: The biological pesticide is a biological pesticide resistant to bacterial leaf spot, rust or phytophthora root rot.

3. The use according to claim 2, characterized in that: The biopesticide comprises the small peptide PEP914 or PEP890, the amino acid sequence of the small peptide PEP914 is shown as SEQ ID No.1, and the amino acid sequence of the small peptide PEP890 is shown as SEQ ID No.

2.

4. The use according to claim 3, characterized in that: The biological pesticide is applied to leguminous plants or cucurbitaceous plants.

5. The use according to claim 4, characterized in that: The legume plant is soybean, mung bean, kidney bean, adzuki bean, pea, peanut or alfalfa; the cucurbitaceous plant is cucumber, pumpkin or bitter gourd.

6. The use according to claim 3, characterized in that: The effective concentration of the small peptide PEP914 or PEP890 in the biological pesticide is 1 nM to 1 μM.

7. The use according to claim 6, characterized in that: The effective concentration of the small peptide PEP914 or PEP890 in the biopesticide is 1 μM.

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