CN113142213A

CN113142213A - Application of surfactin family lipopeptide in pest control

Info

Publication number: CN113142213A
Application number: CN202110445481.7A
Authority: CN
Inventors: 刘洪伟; 张丽萍; 李文雅; 王应洁; 杨雪苗; 王雅娜; 董尧坤
Original assignee: Institute of Biology of Hebei Academy of Sciences
Current assignee: Institute of Biology of Hebei Academy of Sciences
Priority date: 2021-04-25
Filing date: 2021-04-25
Publication date: 2021-07-23
Anticipated expiration: 2041-04-25
Also published as: CN113142213B

Abstract

The invention discloses an application of surfactin family lipopeptide in pest control, relating to the technical field of pest control; experiments prove that the surfactin family compound has very obvious insecticidal activity on homoptera insects and coleoptera insects, the 24-hour insecticidal effect can reach 100%, and the surfactin family compound can be applied to the prevention and control of agricultural insects.

Description

Application of surfactin family lipopeptide in pest control

Technical Field

The invention relates to the technical field of pest control, in particular to application of surfactin family lipopeptide in pest control.

Background

Surfactin (Surfactin) was a compound isolated from the extracellular secondary metabolite of Bacillus subtilis by Arima et al in 1968 and identified as a lipopeptide structure. The fatty acids of the Surfactin group have a carbon chain length of 13-16, the chiral heptapeptide with LLDLLDL is connected with a beta-hydroxyl group of a fatty acid chain carbon atom through a lactone bond, the chiral heptapeptide is molecularly in a saddle-shaped configuration in aqueous solution, and members of the family comprise surfactant produced by bacillus subtilis (Surfactin), bacillus licheniformis (lichenyiishin), surfactant produced by bacillus pumilus (pulimacidin) and Esperin (Esperin), wherein the lichenyiishin, the pulimacidin and the Esperin are mainly applied to industrial and environmental treatment. The Surfactin is a lipopeptide antibacterial substance and consists of beta-hydroxy fatty acid and small peptides of 7 amino acid residues, and carboxyl on the 7 th amino acid of a peptide chain and the beta-hydroxy of the fatty acid are condensed to form a ring structure. There are two main types of Surfactin, namely Leu and Val as the amino acids at position 7. The typical sequence of amino acids in the peptide chain is (L-) Glu- (L-) Leu- (D-) Leu- (L-) Val- (L-) Asp- (D-) Leu- (L-) Leu. However, many analogs thereof are available due to the difference in amino acids at positions 2 (Leu/Ile/Val), 4 (Val/Leu/Ile/Ala) and 7 (Leu/Val/Ile) and the difference in fatty acid chain length (C13-C15). In 1991, Baumgart proves that Surfactin extracted from culture solutions of Bacillus subtilis ATCC21332 and Bacillus subtilis OKB105 has three structural analogs which are named as Surfactin A, Surfactin B and Surfactin C respectively, wherein the Surfactin A is a main component, the seventh amino acid on a cyclic peptide chain of the Surfactin A is Leu, the seven amino acid on the Surfactin B is Val, and the seven amino acid on the Surfactin C is Ile by two-dimensional H-NMR. Oka et al used optimized High Performance Liquid Chromatography (HPLC) in 1992 to successfully isolate six surfactin-derived substances from Bacillus subtilis for the first time, and discovered two new surfactin-derived substances with different fatty acid substituents. Russian scientists in 1995 studied 5 Surfactin structural analogs from fermentation broths, whose structures were determined by mass spectrometry, chemical modification and two-dimensional nuclear magnetic resonance. Subsequent studies have more than ten homologs of Surfactin been determined more precisely. Surfactin exhibits antiviral, antitumor and mycoplasma as well as a degree of antibacterial activity, which itself does not possess antifungal activity, but enhances the antifungal activity of other lipopeptides, particularly iturin.

The pest control is an important component in agricultural production, and with the change of agricultural production modes and the progress of scientific technology, pest control strategies go through the stages of agricultural control, chemical control, comprehensive control and green control in sequence. With the continuous development of various pest control technologies such as monitoring and early warning technology, accurate pesticide application technology, biological control, physical control, transgenic technology and the like, a comprehensive control technology system taking a single crop production process and major disaster-causing pests as objects is established in sequence, and the method plays a key role in guaranteeing the grain yield and food safety in China and promoting the green control of agricultural pests. In recent years, under the influence of various factors such as global climate change, farming mode change, biological invasion which is brought by industrial structure adjustment and international trade communication is more and more frequent, the occurrence types and the damage rules of agricultural pests in China are obviously changed, and the agricultural pest control faces new challenges.

Chemical prevention and control is a measure widely applied at present, but the problems of pesticide residue, biological drug resistance and the like caused by long-term use of chemical pesticides are increasingly prominent. In recent years, with the growing concern of people on environmental ecology and food safety, development of new pesticides for the purpose of ecological friendliness has become an inevitable trend. The Surfactin family lipopeptide is suitable for being developed into a novel pest control medicament in view of the advantages of wide fungus inhibiting spectrum, low toxicity, easiness in biodegradation, no anaphylactic reaction and the like.

Disclosure of Invention

The invention aims to provide application of a surfactin family lipopeptide as an active ingredient in pest control so as to solve the problems in the prior art.

To achieve the above object, the present invention provides the use of a surfactin family lipopeptide as an active ingredient for controlling pests.

Further, the surfactin family lipopeptide is one or more of surfactin A, surfactin B, surfactin C, lichenicin, surface active agent of Bacillus pumilus and Espressosin mixture.

Further, the pests are homoptera insects or coleoptera insects.

Further, the homoptera insect is one or more of bean aphid, cotton aphid, cabbage aphid and sitophilus avenae.

Further, the coleoptera insect is one or more of a platyphylla chelonian, a Holotrichia parallela, a Holotrichia diomphalia Bates, and a Holotrichia parallela.

Furthermore, the application is to prepare the surfactant family lipopeptide into liquid medicine to post-treat the plant or soil to prevent and control pests.

The invention also provides a method of protecting a seed and plant organs formed thereafter from a pest, said method comprising treating the seed with a surfactin-family lipopeptide as described herein.

Further, the mass concentration of the surfactin family lipopeptide is 0.01-100 mu g/mL.

Further, the seeds are coated with a coating agent comprising the surfactin family lipopeptide.

Furthermore, the coating agent also comprises a film forming agent and a binding agent.

The invention discloses the following technical effects: the invention discloses a new application of surfactant, namely an application in pest control. Experiments prove that the surface active agent has very obvious insecticidal activity on homoptera insects or coleoptera insects, the 24-hour insecticidal effect can reach 100 percent, and the surface active agent can be applied to the prevention and control of agricultural pests.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The present invention will be described in further detail with reference to specific embodiments in order to make the above objects, features and advantages more apparent and understandable.

The materials used in the present invention are commercially available unless otherwise specified; the experimental methods used are all routine experimental methods in the field unless otherwise specified.

EXAMPLE 1 preparation of surfactin

1.1 preparation of surfactin family Compounds derived from Bacillus subtilis

(1) Carrying out expanded culture on a bacillus subtilis BSD-2 (with the preservation number of CGMCC No.8440) strain to obtain a fermentation liquid;

(2) centrifuging the fermentation liquor to remove thallus to obtain sterile supernatant;

(3) adding Amberlite XAD-7HP macroporous resin into the sterile supernatant for adsorption, and shaking up for 12h at 4 ℃;

(4) filtering and collecting the resin, washing with distilled water for 3 times, washing with 60% ethanol (volume/volume), and concentrating the eluate to obtain crude extract;

(5) further purification using high performance liquid chromatography: mobile phase a was acetonitrile containing 0.1% (v/v) trifluoroacetic acid, and mobile phase B was ultrapure water containing 0.1% (v/v) trifluoroacetic acid. The sample is loaded on a chromatographic column with the particle size of 5 mu m and the Wondasil C18(4.6mm multiplied by 150mm), and the elution is carried out by taking 10 to 90 percent acetonitrile as a linear gradient, wherein the flow rate is 1mL/min, the detection wavelength is 214nm, and the column temperature is 30 ℃. Fractions corresponding to 15-20min were collected and detected by mass spectrometry (Table 1) to confirm the production of surfactant.

TABLE 1 Mass spectrometric detection of the purified products

EXAMPLE 2 insecticidal Effect of surfactin on Homoptera insects

The surfactant prepared in example 1 was dissolved in methanol to prepare a mother liquor of 200. mu.g/mL, and the mother liquor was diluted with water to give solutions of 5. mu.g/mL, 10. mu.g/mL, and 20. mu.g/mL, and treated with clear water as a control. Broad bean young stem used in the disinsection experiment of bean aphid (Aphis craccivora Koch), cotton young stem used in the disinsection experiment of cotton aphid (Aphis gossypii), cabbage young leaf used in the disinsection experiment of cabbage aphid (Brevicoryne brassicae), and wheat young stem used in the disinsection experiment of wheat pipe aphid (Macrosiphe avenae) are soaked in the liquid medicine for 10min, then the water is sucked by filter paper, the stem is taken out and placed in a culture dish filled with filter paper, 2mL of deionized water is added for moisture preservation, the flat dish is covered by a preservative film, holes are pricked, and 1mL of water is added every 12 h. The tested aphids are respectively 50 heads at each concentration, the test aphids are repeatedly carried out for 3 times, each aphid with the age of 50 years and the similar size is picked by a brush head and placed on a prepared culture dish to be starved for 4 hours, broad bean caulicles, cotton caulicles, cabbage tender leaves and wheat caulicles which are treated by the liquid medicine are respectively added, the components are replaced once every 12 hours, the components are cultured in an incubator at 25 ℃ for overnight, the number of dead heads of the aphids is recorded, and the death rate of the aphids under different dilution times is calculated.

Determining the death rate of aphids: after the broad bean aphids are treated overnight, the broad bean aphids are touched by using the writing brush tips, if the aphids do not move, the aphids are regarded as dead, the number of the dead aphids is counted and recorded, and the data result implemented in the table 2 shows that the surfactant has a good control effect on homoptera insects.

TABLE 2 control Effect of surfactin on Homoptera insects

EXAMPLE 3 insecticidal Effect of surfactant on Coleoptera insects

Dissolving surfactant in methanol to obtain 200 μ g/mL mother liquor, diluting the mother liquor with water to obtain 5 μ g/mL, 10 μ g/mL, and 20 μ g/mL medicinal liquid, and making into control with clear water and medicinal soil. Broken peanuts are used as food, a plastic bucket with the capacity of 4L is used as a container, 2L of uniformly mixed medicine soil is placed in each bucket, and a proper amount of food with the same quantity is added and uniformly stirred. Selecting white star-shaped golden cuora (Protaetia brevitarsis), Holotrichia parallela, Aerugo chrysotrichia corpulenta and Holotrichia parviflora (Polyphylla gradulifera) larva grubs with the same size of the larva, repeating the treatment for 3 times, and keeping the temperature for 24 hours in an environment with the relative humidity of 50-70% and the temperature of 25-28 ℃. The number of each test insect treated is 15, and the test insects are repeated for 3 times. After inoculation, the cells are placed in a room for normal-temperature culture, and the death rate is counted after 56 hours, as shown in table 3.

TABLE 3 killing of different Coleoptera larvae by surfactin

Example 4

Dissolving surfactant in methanol to obtain 200 μ g/mL mother liquor, diluting the mother liquor with water to obtain 0.1 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL, and 100 μ g/mL medicinal liquid, soaking peanut in clear water as control. A plastic bucket with the capacity of 4L is used as a container, 2L of uniformly mixed soil is placed in each bucket, and 50 treated peanuts are added and uniformly mixed. Selecting Holotrichia parallela (Holotrichia parallela) grubs with consistent larva sizes, repeating the treatment for 3 times, and keeping the grubs for 24 hours in an environment with the relative humidity of 50-70% and the temperature of 25-28 ℃. The number of the test insects per treatment is 15 respectively, and the test insects are repeated for 3 times. After inoculation, the plants are placed in a room for normal temperature culture, and the mortality and the peanut integrity are counted after 56 hours, as shown in Table 4.

TABLE 4 insecticidal and protective action of surfactant treated seeds

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. The application of surfactin lipopeptide as active component in preventing and controlling pests.

2. Use according to claim 1, characterized in that: the surfactant family lipopeptide is one or more of surfactant A, surfactant B, surfactant C, lichenicin, surfactant of Bacillus pumilus and elsinorin.

3. The use according to claim 1, wherein the pest is a homoptera or a coleoptera insect.

4. Use according to claim 3, wherein the insects of the order Homoptera are one or more of Aphis fabae, Aphis gossypii, Aphis brassicae and Aphis gramineus.

5. The use of claim 3, wherein the coleopteran insect is one or more of a platyphylla, a Holotrichia parallela, a Holotrichia diomphalia Bates, and a Holotrichia parallela.

6. The use of a surfactin family lipopeptide for pest control according to any one of claims 1 to 5, wherein the use is for post-treating a plant or soil to control pests by formulating the surfactin family lipopeptide as a medicinal solution.

7. A method for protecting a seed and plant organs formed thereafter from a pest, the method comprising treating the seed with a surfactin-family lipopeptide according to any one of claims 1 to 2.

8. The method of claim 7, wherein the surfactant family lipopeptide is present at a concentration of 0.01 to 100 μ g/mL by mass.

9. The method of protecting a seed and subsequently formed plant organs from a pest according to any one of claims 7 to 8, wherein said seed is coated with a coating agent comprising said surfactin-family lipopeptide.

10. The method of claim 9, wherein said coating further comprises a film forming agent and a binding agent.