CN114097780B

CN114097780B - Method for preparing bactericide for inhibiting botrytis cinerea and application

Info

Publication number: CN114097780B
Application number: CN202111448978.0A
Authority: CN
Inventors: 高岩; 尤丽茹
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-11-08
Anticipated expiration: 2041-12-01
Also published as: CN114097780A

Abstract

The invention provides a method for preparing a bactericide for inhibiting botrytis cinerea and application, and belongs to the technical field of plant disease control. The method of the invention is that the wastes of agriculture and/or animal husbandry are liquefied at high temperature and high pressure in a high-temperature high-pressure reaction kettle, solid-liquid separation is carried out after cooling and decomposing, and liquid components are collected to obtain the bactericide. The method disclosed by the invention is simple to operate, does not need any chemical reagent except water, can quickly treat a large amount of agricultural and/or animal husbandry wastes, realizes the recycling of resources, accords with the environmental protection concept, solves the pollution problem of the existing agricultural and/or animal husbandry waste resources, and also solves the problems of high toxicity and more residues of the bactericide. The bactericide for inhibiting the botrytis cinerea prepared by the method can effectively inhibit the botrytis cinerea.

Description

Method for preparing bactericide for inhibiting botrytis cinerea and application

Technical Field

The invention belongs to the technical field of plant disease control, and particularly relates to a method for preparing a bactericide for inhibiting botrytis cinerea and application thereof.

Background

Conidiophores of Botrytis cinerea (Botrytis cinerea) are slender, grey black, and have irregular tree-shaped branches, many small stems grow on the top of the spherical branches, conidia grow on the small stems, the spores are colorless when single growing, oval or oval, gray when aggregated, and have the appearance like spike grape berries, so that the Botrytis cinerea is also called as Botrytis cinerea. The host range of the botrytis cinerea is very wide, and the botrytis cinerea can damage solanaceous vegetables such as tomatoes, berry fruits such as grapes and strawberries, and plant varieties such as flowers and crops. The gray mold control in China is mainly realized by a chemical control method, the chemical control is the simplest and most effective way, and the key point of the chemical control is to prevent plants from being infected by mold and inhibit the generation of mold spores on infected parts. The bactericide which is chemically synthesized is a specific implementation form for chemical prevention and treatment, and the chemical bactericides for preventing and treating gray mold at home and abroad at present mainly comprise the following 9 types: benzimidazoles, dicarboximides, N-phenyl carbamates, anilinopyrimidines, methoxyacrylates, pyridinamines, phenylpyrroles, succinate dehydrogenase inhibitors and sterol biosynthesis inhibitors. The chemical control has quick effect and obvious control effect, can efficiently control the disease spread in a short period, but has high propagation speed, large genetic variation and strong adaptability, and under the targeting action of the medicament, the botrytis cinerea can easily generate drug resistance or even multiple drug resistance to common bactericides along with the long-term dependence use of the bactericides. At present, a botrytis cinerea bactericide which is green and environment-friendly and has a good sterilizing effect is lacked.

Disclosure of Invention

In view of the above, the invention aims to provide a method for preparing a bactericide for inhibiting botrytis cinerea and application thereof.

The invention provides a method for preparing a bactericide for inhibiting botrytis cinerea, which comprises the following steps:

adding livestock and poultry excrement and/or straws and water into a high-temperature high-pressure reaction kettle, heating, stopping heating when the temperature rises to 190-270 ℃, cooling, taking out materials in the reaction kettle, and carrying out solid-liquid separation to obtain a liquid phase component which is a bactericide for inhibiting botrytis cinerea.

Preferably, the heating time is 0.01 to 24 hours.

Preferably, the time for cooling is 0.5-8 h.

Preferably, the pressure in the high-temperature high-pressure reaction kettle is less than or equal to 9Mpa.

Preferably, the solid-liquid separation mode comprises suction filtration; the pressure of the suction filtration is 0.01-0.1 Mpa.

Preferably, the time for stopping heating is when the temperature is increased to 230 ℃.

Preferably, the mass ratio of the livestock and poultry excrement and/or straw to water is 1: (1-12.5).

Preferably, the mass ratio of the straws to the livestock and poultry excrement is (1-10): (1-20).

Preferably, the ratio of the total volume of the livestock and poultry excrement and/or straws and water to the volume of the high-temperature high-pressure reaction kettle is (1-3): (3-4).

The invention also provides application of the bactericide prepared by the method in the scheme in prevention and treatment of botrytis cinerea.

The invention provides a method for preparing a bactericide for inhibiting botrytis cinerea, which takes livestock and poultry excrement and/or straws as raw materials, the raw materials are simple and easy to obtain, no chemical reagent except water is needed, the straws are rich in cellulose, hemicellulose and lignin, and the livestock and poultry excrement is rich in crude fiber and lignin. The presence of crude fiber and lignin can promote the decomposition of cellulose into small molecule products, while the presence of cellulose favors the formation of phenolic compounds by lignin, which supplement each other and interact with each other. The livestock and poultry excrement and/or the straws are liquefied under the conditions of high temperature and high pressure, and then cooled and decomposed, so that various phenolic substances can be generated, and the phenolic substances have the effect of inhibiting botrytis cinerea. Through carrying out high temperature high pressure liquefaction with beasts and birds excrement and/or straw, not only realize the recycle of resource, accord with the environmental protection theory, still solved the pollution problem of the discarded object resource of current agriculture and/or animal husbandry, also provided a new biological bactericide, solved the chemical synthesis bactericide and used singly, toxicity is big, the problem that the residue is many. The bactericide prepared by the invention is a biological agent, the raw materials are renewable resources, the bactericide can be degraded after being used, and is beneficial to soil, pollution-free, and the invention conforms to the research direction of new pollution-free bactericides. In conclusion, the bactericide of the invention has the advantages of simple production process, short production time, simple and easily obtained raw materials, low production cost, easy realization of industrialization and high economic benefit.

Drawings

FIG. 1 is the inhibition ratios for different concentrations for the fungicides of examples 1 to 5, wherein a is the fungicide corresponding to example 1, b is the fungicide corresponding to example 2, c is the fungicide corresponding to example 3, d is the fungicide corresponding to example 4, and e is the fungicide corresponding to example 5;

FIG. 2 is a hypha growth diagram of examples 5, 4, 3, 2 and 1 from top to bottom, corresponding to the fungicides of examples 1 to 5;

FIG. 3 shows the tomato rot status of different treatments, wherein a is the presence of wound and no fungicide spraying; b, spraying an antibacterial agent on wounds; c, spraying and sterilizing without wound.

Detailed Description

In the present invention, the bactericide is preferably an agricultural bactericide.

In the present invention, the heating time is preferably 0.01 to 24 hours, more preferably 0.5 to 12 hours, and most preferably 2 to 5 hours. In the invention, the mixture of livestock and poultry excrement and/or straw and water is liquefied by heating, so that macromolecules in the straw and the livestock and poultry excrement are destroyed, and phenolic substances are generated.

In the present invention, the time for stopping heating is preferably when the temperature is increased to 230 ℃. In the present invention, the time for the temperature reduction is preferably 0.5 to 8 hours, and more preferably 2 to 4 hours. In the present invention, the temperature after the temperature reduction is preferably not more than 30 ℃, more preferably 10 to 30 ℃, and most preferably 20 to 25 ℃. In the invention, in the cooling stage, the liquefied material is decomposed to further generate phenolic substances.

The livestock and poultry excrement and/or the straw are liquefied under the conditions of high temperature and high pressure, and then are cooled and decomposed, so that various phenolic substances can be generated, and the phenolic substances have the effect of inhibiting botrytis cinerea.

In the present invention, the phenolic substance in the bactericide preferably includes one or more of 2, 6-dimethoxy-4-methoxymethylphenol, 2, 6-dimethoxyphenol, p-ethylphenol, o-methoxyphenol, phenol, 2-methoxy-3-ethylphenol, 3, 4-dioxo-6-hydroxy-2H-1-benzopyran-2-one, 1, 2-benzenediol, 1- (2, 6-dihydroxy-4-methoxyphenyl) -1-butanone, 6-methoxy-1, 2-benzenediol, 4-methoxy-3- (methoxymethyl) phenol, 1- (2, 4, 6-trihydroxyphenyl) -2-pentanone, 3- (hydroxymethyl) -5-methoxyphenol, 2-methoxy-4-methylphenol, and 3-ethylphenol.

In the present invention, the pressure in the high-temperature high-pressure reaction kettle is preferably not more than 9MPa, more preferably 1 to 9MPa, and most preferably 3.5 to 5.2MPa.

In the invention, the solid-liquid separation mode comprises suction filtration; the pressure of the suction filtration is preferably 0.01-0.1 Mpa; the suction filtration is preferably performed by using a vacuum pump.

In the invention, the mass ratio of the livestock and poultry excrement and/or straw to water is 1: (1 to 12.5), more preferably 1: (2-5). In the invention, the mass ratio of the straws to the livestock and poultry excrement is preferably (1-10): (1-20).

In the invention, the ratio of the total volume of the livestock and poultry excrement and/or straw and water to the volume of the high-temperature high-pressure reaction kettle is preferably (1-3): (3-4).

In the present invention, the livestock and poultry excreta preferably includes: one or more of feces of sheep, cattle, pig, horse, deer, chicken, duck and goose which are bred by livestock and poultry, or unseparated water-soaked feces, stable manure, human excrement and urine; the straws are one or more of crop straws, cellulose and plant wastes with hemicellulose structures; the invention has no special requirements on the size of the straw; the invention has no special requirement on the solid content of the livestock and poultry excrement.

In the invention, the crop straws preferably comprise one or more of corn straws, corncobs, rice straws, wheat straws, sunflower straws, cotton straws, sorghum straws, tomato straws, pepper straws or castor straws.

In the invention, the bactericide is preferably diluted for use, and the volume concentration of the bactericide in the diluted bactericide solution is preferably 6-8%; the reagent used for the dilution is preferably water, more preferably sterile water. In the invention, the bactericide is used by spraying on leaf surfaces, and the application amount of a single plant is preferably 3-5 mL; the type of crop to which the fungicide is applied includes flowers, vegetables or fruits.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

Example 1

Putting 200g of livestock and poultry excrement (water soaking manure) and 200mL of water into a high-temperature high-pressure reaction kettle according to the proportion of 1g/1mL, heating to 230 ℃, the pressure of 5.2Mpa, the heating time of 2 hours, stopping heating, naturally cooling, cooling to room temperature, taking out a solid-liquid mixture, carrying out suction filtration by using a vacuum pump, the pressure of 0.1Mpa, collecting filtrate to obtain a bactericide, discharging air, and storing at 4 ℃ for later use.

Example 2

100g of corn straw, 100g of livestock and poultry excrement and 250mL of water are placed into a high-temperature high-pressure reaction kettle according to a ratio of 1.

Example 3

20g of corn straw, 80g of livestock and poultry excrement and 100mL of water are placed into a 1L high-temperature high-pressure reaction kettle according to the proportion of 1.

Example 4

Putting 80g of corn straws, 20g of livestock and poultry excrement and 200mL of water into a high-temperature high-pressure reaction kettle according to the proportion of 4.

Example 5

Putting 200g of corn straw and 500mL of water into a 1L high-temperature high-pressure reaction kettle according to the proportion of 2g/5mL, heating to 270 ℃, the pressure is 9Mpa, the heating time is 24 hours, naturally cooling after stopping heating, taking out a solid-liquid mixture after cooling to room temperature, carrying out suction filtration by using a vacuum pump, the pressure is 0.1Mpa, collecting filtrate, obtaining a bactericide, exhausting air, and storing at 4 ℃ for later use.

Example 6 measurement of bacteriostatic activity of crop straw liquefacients at different temperatures and different concentrations

Determination of the bacteriostatic Rate

A certain amount of PDA medium was prepared, sterilized and cooled to a certain temperature, and then sterile water and the bactericide prepared in examples 1 to 5 were added to prepare 2% of the preparation (92 lpda medium, 6mL of sterile water and 2mL of bactericide), 4% (92 lpda medium, 6mL of sterile water and 2mL of bactericide), 6% (92 lpda medium, 6mL of sterile water and 2mL of bactericide), 8% (92 lpda medium, 6mL of sterile water and 2mL of bactericide) of media of different concentrations. Pouring into a sterile culture dish with the diameter of 90mm, inoculating a botrytis cinerea (from Shanghai preservation Biotechnology center) block with the diameter of 5mm into a flat plate after a culture medium is solidified, repeating each experiment for 3 times, performing inverted culture in a constant-temperature incubator with the set condition temperature of 25 ℃ and the humidity of 60%, measuring the diameter of a bacterial colony by using a cross method, and taking the average value. The bacteriostatic rate (%) = [ (control colony diameter-diameter of colonies)/(control colony diameter-5 mm) ] × 100% was calculated by the following formula. See table 2, fig. 1 and fig. 2 for results.

Table 2 different embodiments with different concentrations of the bacteriostatic ratio

As can be seen from Table 2, FIG. 1 and FIG. 2, in example 1, at a temperature of 230 ℃ and a pressure of 5.2MPa, the ratio of livestock and poultry breeding excrements to water is 1:1, the bactericide obtained under the condition has the best bacteriostasis effect, and the concentration of the bactericide can reach 84.30 percent when the concentration is 8 percent.

Example 7

The bactericide prepared in example 1 and ethyl acetate were mixed in a volume ratio of 1:2, placing the mixture into a separating funnel for extraction, shaking for 5min, standing for 15min, removing the aqueous solution, then extracting the aqueous solution for 3 times, and combining the solutions after extraction. Then, an appropriate amount of anhydrous sodium sulfate was added to carry out dehydration. Then, the mixture was filtered through a 0.22 μm filter and placed in a chromatographic flask for GC-MS analysis. The analysis result shows that the phenolic substances in the bactericide are 2, 6-dimethoxy-4-methoxymethylphenol, 2, 6-dimethoxyphenol, p-ethylphenol, o-methoxyphenol, phenol, 2-methoxy-3-ethylphenol, 3, 4-dioxo-6-hydroxy-2H-1-benzopyran-2-one, 1, 2-benzenediol, 1- (2, 6-dihydroxy-4-methoxyphenyl) -1-butanone, 6-methoxy-1, 2-benzenediol, 4-methoxy-3- (methoxymethyl) phenol, 1- (2, 4, 6-trihydroxyphenyl) -2-pentanone, 3- (hydroxymethyl) -5-methoxyphenol, 2-methoxy-4-methylphenol and 3-ethylphenol, and the chemical structural formula is shown as follows:

the results of measuring the total content of phenolic compounds in the fungicides of examples 1 to 5 are shown in table 1.

Table 1 total content of phenolic substances in fungicides of examples 1 to 5

Examples	Total phenolic content (%)
		1	57.656
2	54.219
		3	51.846
4	43.803
		5	41.128

As can be seen from Table 1, the main components of the bactericide for agricultural and/or animal husbandry waste and water, which is treated at high temperature and high pressure, are phenols, and the phenols have antibacterial and anti-inflammatory effects and have the effect of inhibiting Botrytis cinerea.

Example 8

10 fresh tomatoes are taken, 5 tomatoes are taken to treat one wound, hyphae are inoculated respectively, 2 treated wounds are taken as controls, no bactericide is sprayed, and the rest 3 tomatoes are sprayed with 3-5 mL of the bactericide prepared in the example 1 for about 1-2 h. The remaining tomatoes which are not treated with the wound are sprayed with the bactericide prepared in example 1, and 3-5 mL of the bactericide is sprayed on each tomato.

The results are shown in FIG. 3.a is that no bactericide is sprayed on the surface of the wound; b, spraying an antibacterial agent on wounds; c, spraying and sterilizing without wound. As is apparent from FIG. 3, the tomatoes with wounds without spraying the bactericide are quickly rotten, and the tomatoes with the sprayed bactericide are not easy to rot and have a certain fresh-keeping effect.

Although the present invention has been described in detail with reference to the above embodiments, it is to be understood that the present invention is not limited to the details of the embodiments, and that other embodiments may be devised without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing a fungicide for inhibiting botrytis cinerea comprises the following steps:

adding livestock and poultry excrement and water into a high-temperature high-pressure reaction kettle, heating, stopping heating when the temperature rises to 230 ℃, cooling, taking out materials in the reaction kettle, and carrying out solid-liquid separation to obtain a liquid phase component which is a bactericide for inhibiting botrytis cinerea;

the mass ratio of the livestock and poultry excrement to the water is 1:1;

the pressure in the high-temperature high-pressure reaction kettle is 5.2Mpa;

the heating time is 2h.

2. The method according to claim 1, wherein the time for cooling is 0.5 to 8 hours.

3. The method of claim 1, wherein the solid-liquid separation comprises suction filtration; the pressure of the suction filtration is 0.01-0.1 Mpa.

4. The method as claimed in claim 1, wherein the ratio of the total volume of the livestock and poultry excrement and water to the volume of the high-temperature high-pressure reaction kettle is (1-3): (3-4).

5. Use of a fungicide prepared by the method according to any one of claims 1 to 4 for the control of botrytis cinerea.