Preparation of pyrrole peptide and application of pyrrole peptide in agriculture

Abstract

The invention discloses preparation of pyrrole peptide and application of the pyrrole peptide in agriculture, and relates to the field of application of the pyrrole peptide in agriculture, the preparation of the pyrrole peptide and the application of the pyrrole peptide in agriculture are obviously different from traditional chemical pesticides through the application of the pyrrole peptide in agriculture, and the problem of environmental pollution is effectively reduced. Pyrrole peptide is used as a bioactive compound, shows high efficiency in plant disease control, is good in biodegradability, and reduces pollution risks of soil, water sources and air. Through comparison of embodiments 2 to 4, the control effect of the pyrrolopeptide on tobacco mosaic virus, tomato gray mold and rice bacterial leaf blight under different concentrations is superior to that of traditional chemical pesticides, such as amino-oligosaccharin, carbendazim and kasugamycin. The pyrrole peptide is not only environment-friendly, but also remarkable in effect in practical application, and provides powerful support for development of green agriculture.

Description

TECHNICAL FIELD

[0001] The application relates to a pyrrole peptide preparation method and application thereof in agriculture. BACKGROUND

[0002] Plant diseases are a big problem in agricultural production, which seriously affects the yield and quality of crops. Although traditional chemical pesticides can effectively control diseases, long-term use will lead to environmental pollution and enhanced pathogen resistance. Therefore, developing new, environmentally friendly and efficient plant disease control methods has become a hot spot in agricultural research. As a kind of biologically active compound, pyrrole peptide has shown great potential in plant disease resistance in recent years. The application provides a pyrrole peptide preparation method and application thereof in agricultural disease control, aiming to solve the problems caused by traditional pesticides and promote the development of green agriculture. SUMMARY

[0003] The application aims to provide a pyrrole peptide preparation method and application thereof in agriculture, so as to solve the problems of environmental pollution and enhanced pathogen resistance caused by long-term use of traditional chemical pesticides in the prior art.

[0004] In order to achieve the above-mentioned purpose, the application provides the following technical scheme: a pyrrole peptide preparation method, comprising the following steps:

[0005] Step one, selecting L-proline with a purity of greater than or equal to 99% as a reaction raw material;

[0006] Step two, adding L-proline into a dry 250mL single-necked round-bottom flask, and then adding an appropriate amount of analytical pure ethylene glycol as a reaction solvent and dehydration promoter, so that the L-proline is completely dissolved to form a uniform and transparent reaction system;

[0007] Step three, fixing the single-necked flask containing the reaction mixture on an electric heating jacket, installing a spherical condenser, connecting the condenser water, and ensuring that the condensing system is unobstructed;

[0008] Step four, turning on the electric heating jacket to heat, slowly warming up to the reflux state of the reaction system, and controlling the reflux temperature at 190-200 DEG C, and maintaining the temperature for 8h;

[0009] Step five, after the reaction is completed, naturally cooling to room temperature, transferring the reaction liquid together with the solid product to a Buchner funnel, and performing suction filtration, and washing the filter cake with a small amount of cold ethylene glycol for 2-3 times;

[0010] Step six, transferring the filter cake to a vacuum drying oven, drying at 60 DEG C and-0.08MPa for 4h until the constant weight, and obtaining a white powdery product pyrrole peptide.

[0011] Further, the feeding amount of L-proline is 10-20 g, and the amount of ethylene glycol is 3-5 times of the mass of the raw material.

[0012] Further, the reflux temperature is strictly controlled at 190-200℃ to avoid decomposition of the raw material or slow down the reaction rate.

[0013] The application of a pyrrole peptide in agriculture, the pyrrole peptide is formulated into a solution with different concentrations, sprayed on the surface of tobacco leaves, the concentration is 200-10000 ng / mL, used for preventing and treating tobacco mosaic virus.

[0014] Further, the optimal use concentration of the pyrrole peptide is 500 ng / mL, at which the prevention and treatment effect is best.

[0015] Further, the pyrrole peptide is formulated into a solution with different concentrations, sprayed on the surface of tomato leaves, the concentration is 200-10000 ng / mL, used for preventing and treating tomato gray mold.

[0016] Further, the optimal use concentration of the pyrrole peptide is 1000 ng / mL, at which the prevention and treatment effect is most prominent.

[0017] Further, the pyrrole peptide is formulated into a solution with different concentrations, sprayed on the surface of rice leaves, the concentration is 200-10000 ng / mL, used for preventing and treating rice white leaf blight.

[0018] Further, the optimal use concentration of the pyrrole peptide is 500 ng / mL, at which the indoor prevention and treatment effect is best.

[0019] A preparation method of a pyrrole peptide, characterized in that it comprises the following steps:

[0020] Step one, inoculate the acidophilic actinomyces into PDA solid medium, incubate at 25℃ for 6 days, after the strain is activated, transfer to a triangular flask containing 250 ml of PDA liquid seed culture medium by agar block method, and shake culture for 2-4 days to obtain a first seed liquid;

[0021] Step two, sterilize and cool a 5L fermenter, then inoculate 7% of the first seed liquid, and culture for 6 days to obtain a fermentation liquid;

[0022] Step three, centrifuge the fermentation liquid into mycelium and filtrate, dry the mycelium at 60℃ after washing, weigh and crush, extract with equal volume of ethanol for 3 times, mix each extraction liquid uniformly with a magnetic stirrer, ultrasonic oscillation for 1h, vacuum filtration, and collect the filtrate, which is the extraction liquid;

[0023] Step four, after the extraction liquid is concentrated by rotary evaporation, extract with equal volume of ethyl acetate containing 5% acetone for 3 times, collect the supernatant, rotary evaporate and nitrogen blow to dryness with a rotary evaporator, and obtain the crude extract;

[0024] Step five, the crude extract is subjected to 200-300 mesh silica gel column chromatography and ODS pressure chromatography for preliminary separation, combined with TLC, and then subjected to HPLC purification and NMR analysis to obtain p-met;

[0025] The acidophilic columnia is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 42393.

[0026] Compared with the prior art, the preparation of the pyrrole peptide and its application in agriculture provided by the present application have the following beneficial effects:

[0027] The pyrrole peptide preparation method and its application in agriculture provided by the present application are significantly different from traditional chemical pesticides, effectively reducing environmental pollution problems. As a kind of bioactive compound, pyrrole peptide shows high efficiency in plant disease control, and its biodegradability is good, reducing the pollution risk of soil, water and air. As can be seen from the comparison of Examples 2 to 4, the control effect of pyrrole peptide on tobacco mosaic virus, tomato gray mold and rice bacterial leaf blight is better than that of traditional chemical pesticides such as amino oligosaccharide, carbendazim and kasugamycin. This shows that pyrrole peptide is not only environmentally friendly, but also has remarkable effect in practical application, providing strong support for the development of green agriculture.

[0028] Another great benefit of the present application is that the use of pyrrole peptide with other biological pesticides significantly improves the disease control effect. Examples 5 and 6 respectively show the control effect of pyrrole peptide combined with lentinan and zinc thiazole on tobacco virus disease and rice bacterial leaf blight, and the results show that the control effect of the combination is better than that of any single agent. This synergistic strategy not only improves the control efficiency, but also widens the application range of pyrrole peptide, enabling it to deal with more types of plant diseases. At the same time, the use of combination reduces the amount of single agent, further reducing the risk of environmental pollution and pathogen resistance, providing a new solution for sustainable agricultural development. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments or prior art of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art based on these drawings.

[0030] Figure 1 A preparation flow chart of the pyrrole peptide of the present application;

[0031] Figure 2 A preparation principle schematic diagram of the pyrrole peptide of the present application. DETAILED DESCRIPTION

[0032] In order for those skilled in the art to better understand the technical solutions of the present application, the present application will be further described in detail below with reference to the drawings.

[0033] Example 1

[0034] I. Experimental Preparation

[0035] Reagents and instruments: L-proline with purity ≥ 99% was selected as the reaction raw material to ensure no impurity interference with the cyclization reaction; ethylene glycol was selected as the reaction solvent and dehydration promoter. The instruments included a 250 mL standard single-neck round-bottom flask, a spherical condenser, an electric heating jacket, a thermometer (accuracy ± 0.1 °C), a Buchner funnel, a vacuum drying oven, etc. All the instruments were washed with distilled water and dried before use to avoid water affecting the reaction efficiency.

[0036] Feeding ratio: L-proline was accurately weighed according to the reaction ratio (the feeding amount was adjusted according to the target product yield, usually 10-20 g), added to the dry 250 mL single-neck flask, and then an appropriate amount of ethylene glycol was added to dissolve the L-proline completely to form a uniform transparent reaction system. The solvent amount was usually 3-5 times the mass of the raw material.

[0037] II. Reaction Operation

[0038] The single-neck flask containing the reaction mixture was fixed on the electric heating jacket, the spherical condenser was installed, and the condensing water was connected (the lower end was in, and the upper end was out) to ensure that the condensing system was unobstructed. The electric heating jacket was turned on and heated slowly to the reflux state of the reaction system (the boiling point of ethylene glycol was about 197 °C, and the reflux temperature was controlled at 190-200 °C). The temperature was maintained for 8 h. During the reaction, the system state was observed regularly. The initial state was a transparent solution, and white solids gradually precipitated as the cyclization reaction proceeded. The system was kept slightly boiling in the later stage to ensure that the dehydration cyclization reaction proceeded fully.

[0039] III. Post-treatment Steps

[0040] After the reaction was completed, the electric heating jacket was turned off, and the heating was stopped. The reaction system was allowed to cool naturally to room temperature (about 25 °C), at which time a large amount of white solid product precipitated. The reaction liquid together with the solid product was transferred to the Buchner funnel, and the vacuum pump was turned on for filtration. The filter cake was washed with a small amount of cold ethylene glycol for 2-3 times to remove the residual raw materials and impurities. The filter cake was transferred to the vacuum drying oven and dried at 60 °C and -0.08 MPa for 4 h until the constant weight, to obtain the white powdery product pyrrole peptide.

[0041] The structure of the pyrrole peptide is:

[0042]

[0043] IV. Key points

[0044] The reflux temperature needs to be strictly controlled, too high is easy to lead to raw material decomposition, too low is to slow down the reaction rate, reduce the conversion rate; The condensate water needs to be continuously imported during the reaction process, avoid solvent volatilization loss, ensure the stability of the reaction system; The ethylene glycol used for washing needs to be pre-cooled during the post-processing, reduce the loss of product dissolution, improve the yield; The drying process needs to ensure the vacuum degree, completely remove the residual solvent, ensure the product purity.

[0045] Example 2 Effect of different concentrations of pyrrole peptide on tobacco virus disease

[0046] (1) Test design:

[0047] This test is an indoor potting test, Denmark product peat soil is selected as the seedling substrate, after sterilization treatment, add water and mix thoroughly, put into the seedling tray and flatten the surface of the substrate. Then sow seeds, put the seedling tray in the climate room, the environmental conditions are set as temperature 23℃, humidity 50-60%, light 6000-7000Lux. Select healthy seedlings without pests and diseases and uniform growth, transplant them into flowerpots. The test sets 7 treatments, as shown in Table 1, among which CK is used as a control, each treatment is set 3 times. The test agent is evenly sprayed on the leaf surface, 4h after application, select tobacco seedlings with uniform growth, evenly sprinkle a little quartz powder on the front surface of the 3 unfolded leaves below the tobacco, use rubbing inoculation method to inoculate virus on the 3 unfolded leaves, each leaf inoculates 50μL of tobacco mosaic virus (TMV) containing virus juice, put it in the artificial climate room for further cultivation for 5-7d, observe the tobacco disease, count the number of disease spots in the later stage of the test, and determine the virus expression amount by ELISA.

[0048] Table 1 Test design

[0049]

[0050] (2) Test results:

[0051] From the data in Table 2, it can be seen that different treatments have different degrees of antiviral effect. Treatment 1 did not implement any control means, and the incidence was the most serious, serving as a reference standard for judging the control effect of the remaining treatments. The number of tobacco spots in treatment 4 was 4.00, the virus expression was 26.03 pg / mL, and the control effect reached 73.33%, with a significant control effect. Treatment 2 was the positive control, with a control effect of 55.53%; the control effect of treatment 3 was 35.53%; the control effect of treatment 5 was 51.13%; the control effect of treatment 6 was 42.20%; and the control effect of treatment 7 was 33.33%. The spot number and virus expression of these five treatments were less than those of the control, and they had a certain control effect, but the overall control level was lower than that of treatment 4.

[0052] Under the conditions of this test, pyrrole peptide has a certain control effect on tobacco mosaic virus, and the control effect on tobacco mosaic virus is best at a concentration of 500 ng / mL.

[0053] Table 2 Control effect of different treatments on tobacco virus disease

[0054]

[0055] Note: Treatment 1: CK; Treatment 2: Amino oligosaccharide 60 mL / acre; Treatment 3: Pyrrole peptide 200 ng / mL; Treatment 4: Pyrrole peptide 500 ng / mL; Treatment 5: Pyrrole peptide 1000 ng / mL; Treatment 6: Pyrrole peptide 5000 ng / mL; Treatment 7: Pyrrole peptide 10000 ng / mL.

[0056] Example 3 Effect of different concentrations of pyrrole peptide on tomato gray mold

[0057] (1) Test design:

[0058] This test is an indoor potting test, and Danish peat soil is used as the seedling substrate. After sterilization, the substrate is thoroughly mixed with water, placed in a seedling tray, and the surface of the substrate is leveled. Then, the tomato seeds are sown, with a sowing depth of 0.5 cm. The seedling tray is placed in a climate room, and the environmental conditions are set to a temperature of 23°C, a humidity of 50%-60%, and a light intensity of 6000-7000 Lux. Healthy seedlings with no pests or diseases and uniform growth are selected and transplanted into pots. The test has 7 treatments, as shown in Table 3, with CK as the control. The test agent is evenly sprayed on the surface of the tomato leaves, and 4 hours after application, tomato seedlings with uniform growth are selected, and detached leaves are inoculated with gray mold. Each plant has 3 leaves, with 4 inoculation points on each leaf. A small hole is made in the midrib of the leaf, and a small piece of the fungus is picked up with a needle and placed in the wound of the leaf. The test is conducted in a 25°C, light:dark = 16:8h, 6000 Lux incubation room to induce disease. The tomato lesion area is counted and photographed at the end of the test, and the disease index and disease control effect are calculated.

[0059] Table 3 Test design

[0060]

[0061] (2) Test results:

[0062] According to the data in Table 4, different treatments have different degrees of disease resistance effect on tomato gray mold. The incidence of tomato in treatment 1 is 60.67%; the incidence of tomato in treatment 2 is 36.33%, and the control effect is 40.12%; the incidence of tomato in treatment 3 is 38.33%, and the control effect is 36.82%; the incidence of tomato in treatment 4 is 34.67%, and the control effect is 42.85%; the incidence of tomato in treatment 5 is 25.67%, and the control effect is 57.69%; the incidence of tomato in treatment 6 is 40.33%, and the control effect is 33.53%; the incidence of tomato in treatment 7 is 37.67%, and the control effect is 37.91%.

[0063] In summary, pyrrole peptide can prevent and control tomato gray mold to some extent, and the control effect on tomato gray mold is most prominent when the concentration is 1000 ng / mL, which provides an efficient and feasible solution for the green prevention and control of tomato gray mold.

[0064] Table 4 Control effect of different treatments on tomato gray mold

[0065]

[0066] Note: Treatment 1: CK; Treatment 2: recommended dosage of carbendazim; Treatment 3: pyrrole peptide 200 ng / mL; Treatment 4: pyrrole peptide 500 ng / mL; Treatment 5: pyrrole peptide 1000 ng / mL; Treatment 6: pyrrole peptide 5000 ng / mL; Treatment 7: pyrrole peptide 10000 ng / mL.

[0067] Example 4 Effect of different concentrations of pyrrole peptide on rice bacterial leaf blight

[0068] (1) Test design:

[0069] Xoo culture and inoculation: Xoo was inoculated into liquid NB medium and cultured at 28°C overnight for 2-3 days; the overnight bacterial solution was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded; the bacterial body was resuspended and washed once with 10 mM MgCl2, and then the bacterial solution was resuspended again with MgCl2 solution; adjust OD600 value: adjust the OD600 value of the bacterial body in the plate to 0.5 with sterile water; select leaves: 14-day-old rice seedlings with the same growth and the same leaf position; cut and inoculate leaves: sterilize the scissors, dip the bacterial solution, and cut the leaves at 4-5 cm from the leaf tip, inoculate 15-20 replicates per treatment, and ensure uniform inoculation position; spray the leaves with an appropriate amount of sterile water. This experiment has 7 treatments, as shown in Table 5, and the pesticides are configured according to the required concentration. The method of spraying pesticides first and cutting and inoculating leaves after 4 hours is used, and the length of the inoculated leaf lesion is counted and compared after 14 days, and the control effect of each treatment group is calculated.

[0070] Table 5 Test design

[0071]

[0072] (2) Test results:

[0073] According to the data in Table 6, under the conditions of this test, compared with the CK control group, after treatment with different concentrations of pyrrole peptide, the incidence of rice bacterial leaf blight was reduced, and the best indoor control effect was pyrrole peptide 500 ng / mL, with an indoor control effect of 68.96%.

[0074] Table 6 Length of lesion and indoor control effect of rice inoculation Day 14

[0075]

[0076] Note: Treatment 1: CK; Treatment 2: Springomycin 160 µg / mL; Treatment 3: Pyrrole peptide 200 ng / mL; Treatment 4: Pyrrole peptide 500 ng / mL; Treatment 5: Pyrrole peptide 1000 ng / mL; Treatment 6: Pyrrole peptide 5000 ng / mL; Treatment 7: Pyrrole peptide 10000 ng / mL.

[0077] Example 5 Effect of Pyrrole Peptide and Lentinan Compound on Tobacco Virus Disease

[0078] (1) Test design:

[0079] The experiment is an indoor pot experiment. Danish product peat soil is selected as the seedling substrate, which is sterilized and mixed with water, then filled into seedling trays and the surface of the substrate is leveled. Then the seeds are sown, and the seedling trays are placed in the climate room with the environmental conditions set at 23°C, 50-60% humidity, and 6000-7000 Lux light. Select seedlings without pests and diseases and uniform growth, and transplant them into flowerpots. The experiment has 3 treatments, as shown in Table 7, with CK as the control, and 3 replicates for each treatment. The test agent is uniformly sprayed on the leaf surface, and 4 hours after application, select tobacco seedlings with uniform growth, and evenly sprinkle a little quartz powder on the front of the 3 unfolded leaves below the fully unfolded young leaves, and use rubbing inoculation to inoculate the virus on the 3 unfolded leaves, each leaf inoculated with 50 μL of virus juice containing tobacco mosaic virus (TMV), and placed in an artificial climate room for further cultivation for 5-7 days. Observe the disease incidence of tobacco, count the number of disease spots in the late stage of the experiment, and analyze the data.

[0080] Table 7 Experimental design

[0081]

[0082] (2) Test results:

[0083] According to the data in Table 8, treatment 2 and treatment 3 both produced varying degrees of antiviral effect. The number of tobacco dry spots in treatment 2 was 8.67, with a control effect of 33.31%; the number of tobacco dry spots in treatment 3 was 5.00, with a control effect of 61.54%. Under the conditions of this experiment, the complex of pyrrole peptide and lentinan had a significant control effect on tobacco mosaic virus, and the control effect was significantly higher than that of lentinan alone.

[0084] Table 8 Control effect of different treatments on tobacco virus disease

[0085]

[0086] Note: Treatment 1: CK; Treatment 2: Lentinan; Treatment 3: Pyrrole peptide + Lentinan.

[0087] Example 6 Effect of Pyrrole Peptide and Zinc Thiazole Complex on Rice Bacterial Leaf Blight

[0088] (1) Experimental design:

[0089] Xoo culture and inoculation: Xoo was inoculated into liquid NB medium and cultured at 28°C for 2-3 days overnight; the overnight bacterial solution was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded; the bacterial body was resuspended and washed once with 10 mM MgCl2, and then the bacterial solution was resuspended again with MgCl2 solution; adjust OD600 value: adjust the OD600 value of the bacterial body in the plate to 0.5 with sterile water; select leaves: 14-day-old rice seedlings with the same growth and the same leaf position; cut and inoculate leaves: sterilize the scissors, dip the bacterial solution, and cut the leaves at 4-5 cm from the leaf tip, inoculate 15-20 replicates per treatment, and ensure uniform inoculation position; and keep the leaves moist: spray an appropriate amount of sterile water. This test has 3 treatments, as shown in Table 9, and the pesticides are configured according to the required concentration. The method of spraying pesticides first and cutting and inoculating leaves after 4 hours is used, and the length of the inoculated leaf lesion is counted and compared after 14 days, and the control effect of each treatment group is calculated.

[0090] Table 9 Test design

[0091]

[0092] (3) Test results:

[0093] According to the data in Table 10, under the conditions of this test, compared with the CK control group, treatment 2 and treatment 3 can reduce the incidence of rice bacterial leaf blight, among which the indoor control effect after spraying thiazolyl zinc is 46.96%, and the indoor control effect after spraying pyrrole peptide + thiazolyl zinc is 62.37%, that is, the pyrrole peptide and thiazolyl zinc complex have a significant control effect on rice bacterial leaf blight, and the control effect is significantly higher than that of thiazolyl zinc alone.

[0094] Table 10 Length of lesion and indoor control effect of rice inoculation Day 14

[0095]

[0096] Note: Treatment 1: CK; Treatment 2: thiazolyl zinc; Treatment 3: pyrrole peptide + thiazolyl zinc.

[0097] Comparative example

[0098] Prior art solution content

[0099] In the existing plant disease control technology, it mainly relies on the use of chemical pesticides.

[0100] These chemical pesticides are sprayed on the surface of crop leaves to form a protective film or directly kill pathogenic bacteria, thereby achieving the purpose of controlling plant diseases.

[0101] The specific operation steps are as follows:

[0102] Pesticide Selection: Choose appropriate chemical pesticides based on the type of crop disease, such as amino oligosaccharide for tobacco virus disease, carbendazim for tomato gray mold, and streptomycin or zinc thiazole for rice bacterial leaf blight.

[0103] Prepare pesticide solution: Mix the pesticide with water uniformly according to the recommended concentration on the pesticide instruction, and prepare the solution with the required concentration.

[0104] Spraying operation: Use a sprayer to uniformly spray the prepared pesticide solution on the surface of the crop leaves, ensuring that the front and back of the leaves are covered with the pesticide solution.

[0105] Subsequent management: After spraying, keep the field well ventilated and avoid rainwater washing to ensure the effective adhesion and action time of the pesticide on the leaf surface.

[0106] Problems

[0107] Although chemical pesticides show certain effects in plant disease control, long-term use brings many problems, including:

[0108] Environmental pollution: The extensive use of chemical pesticides can cause soil, water and air pollution, destroy ecological balance and harm non-target organisms.

[0109] Increased resistance of pathogenic bacteria: Long-term use of the same or similar chemical pesticides can promote the development of drug resistance in pathogenic bacteria, reducing the effectiveness of pesticides and even rendering them ineffective.

[0110] This leads farmers to increase pesticide use or switch to more effective pesticides, further exacerbating environmental pollution and ecological damage.

[0111] Pesticide residues: Residues of chemical pesticides on crops can enter the human body through the food chain, posing potential threats to human health.

[0112] Long-term intake of food containing pesticide residues can cause various diseases.

[0113] Single action: Traditional chemical pesticides can only target specific diseases for control, and for the simultaneous occurrence of multiple diseases, multiple pesticides need to be used for compounding, increasing the cost and complexity of control.

[0114] Non-persistent: Chemical pesticides have poor stability in the environment and are easily affected by factors such as light, temperature and precipitation, leading to decomposition and failure, requiring frequent application to maintain control effect.

[0115] In view of the above problems, the present application provides a preparation method of pyrrole peptide and its application in agricultural disease control, aiming to develop a new, environmentally friendly and efficient plant disease control method to solve the problems of environmental pollution and enhanced pathogen resistance caused by traditional chemical pesticides, and promote the development of green agriculture.

[0116] The preparation of one pyrrole peptide of example 1 to example 6 and its application in agriculture are compared with the control example, and the following table is obtained:

[0117]

[0118] Examples 1 to 6 show significant beneficial effects by preparing pyrrole peptides and applying them to agricultural disease control compared with traditional chemical pesticides.

[0119] Specifically, examples 2 to 4 respectively aim at tobacco mosaic virus, tomato gray mold and rice bacterial leaf blight, and by treating with different concentrations of pyrrole peptides, all achieve better control effect than traditional chemical pesticides (amino oligosaccharide, carbendazim, kasugamycin).

[0120] In addition, examples 5 and 6 further significantly improve the control effect on tobacco virus disease and rice bacterial leaf blight by compounding pyrrole peptide with biological pesticides (lentinan, thiazole zinc), which proves the synergistic effect of pyrrole peptide in compounding use.

[0121] These examples not only verify the feasibility of pyrrole peptide as a new, environmentally friendly and efficient plant disease control method, but also effectively solve the problems of environmental pollution and enhanced pathogen resistance caused by traditional chemical pesticides, and promote the development of green agriculture.

[0122] The above only describes some exemplary embodiments of the present application by way of illustration, and it is needless to say that those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present application. Therefore, the above figures and description are illustrative in nature and should not be understood as limiting the scope of protection of the claims of the present application.

Claims

1. A method for preparing pyrrole peptides, characterized in that, Includes the following steps: Step 1: Select L-proline with a purity of ≥99% as the reaction raw material; Step 2: Add L-proline to a dry 250mL single-necked round-bottom flask, and then add an appropriate amount of analytical grade ethylene glycol as a reaction solvent and dehydration promoter to completely dissolve L-proline and form a homogeneous and transparent reaction system. Step 3: Fix the single-necked bottle containing the reaction mixture onto the heating mantle, install the spherical condenser tube, connect the cooling water, and ensure that the condensation system is unobstructed; Step 4: Turn on the electric heating mantle and slowly raise the temperature to the reflux state of the reaction system. The reflux temperature is controlled at 190-200℃, and the reaction is maintained at this temperature for 8 hours. Step 5: After the reaction is complete, allow it to cool naturally to room temperature. Transfer the reaction solution along with the solid product to a Buchner funnel for filtration. Wash the filter cake 2-3 times with a small amount of cold ethylene glycol. Step 6: Transfer the filter cake to a vacuum drying oven and dry it at 60℃ and -0.08MPa for 4 hours until constant weight is obtained, yielding a white powdery product, pyrrole peptide.

2. The method for preparing pyrrole peptide according to claim 1, characterized in that, The amount of L-proline added is 10-20g, and the amount of ethylene glycol used is 3-5 times the mass of the raw materials.

3. The method for preparing pyrrole peptide according to claim 1, characterized in that, The reflux temperature is strictly controlled at 190-200℃ to avoid decomposition of raw materials or slowing down of the reaction rate.

4. A method for preparing pyrrole peptide according to any one of claims 1-3, for the application of pyrrole peptide in agriculture, characterized in that, Pyrrole peptides were prepared into solutions of different concentrations and sprayed onto the surface of tobacco leaves at concentrations of 200-10000 ng / mL to control tobacco mosaic virus.

5. A method for preparing pyrrole peptide according to any one of claims 1-3, for the application of pyrrole peptide in agriculture, characterized in that, The optimal concentration of the pyrrole peptide is 500 ng / mL, at which point the prevention and control effect is best.

6. A method for preparing pyrrole peptide according to any one of claims 1-3, for the application of pyrrole peptide in agriculture, characterized in that, Pyrrole peptides were prepared into solutions of different concentrations and sprayed onto the surface of tomato leaves at concentrations of 200-10000 ng / mL to control tomato gray mold.

7. The preparation of a pyrrole peptide according to claim 6 and its application in agriculture, characterized in that, The optimal concentration of the pyrrole peptide is 1000 ng / mL, at which the prevention and treatment effect is most prominent.

8. A method for preparing pyrrole peptide according to any one of claims 1-3, for the application of pyrrole peptide in agriculture, characterized in that, Pyrrole peptides were prepared into solutions of different concentrations and sprayed onto the surface of rice leaves at concentrations of 200-10000 ng / mL to control rice bacterial blight.

9. The preparation of a pyrrole peptide according to claim 8, characterized in that, The optimal concentration of the pyrrole peptide is 500 ng / mL, at which point the indoor control effect is the best.

10. A method for preparing pyrrole peptides, characterized in that, Includes the following steps: Step 1: Inoculate *Pyrrosia acidophilus* onto PDA solid medium and incubate at 25°C for 6 days. After the strain is activated, transfer it to an Erlenmeyer flask containing 250 ml of PDA liquid seed medium using the agar block method. Shake and incubate for 2-4 days to obtain the first-stage seed culture. Step 2: After sterilizing and cooling the 5L fermenter, inoculate it with 7% primary seed liquid and culture for 6 days to obtain the fermentation broth; Step 3: Centrifuge the fermentation broth to separate mycelium and filtrate. After washing the mycelium, dry it at 60°C, weigh it and crush it. Extract it three times with an equal volume of ethanol. Mix the extracts three times with a magnetic stirrer, sonicate for 1 hour, vacuum filter, and collect the filtrate, which is the extract. Step 4: After the extract is concentrated by rotary evaporation, it is extracted three times with an equal volume of ethyl acetate containing 5% acetone. The supernatant is collected, and the extract is evaporated by rotary evaporation and dried under nitrogen to obtain the crude extract. Step 5: The crude extract was initially separated by 200-300 mesh silica gel column chromatography and ODS pressure chromatography, and then combined by TLC, purified by HPLC and analyzed by NMR to obtain para-methyl peptide; The acidophilic spp. is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 42393.

Images