Preparation method and application of Ageratum conyzoides extract for preventing and controlling Spodoptera exigua

Abstract

The present invention provides a preparation method and application of an Ageratum conyzoides extract for controlling Spodoptera exigua, relating to the technical field of pest control. The Ageratum conyzoides extract is obtained by subjecting fresh Ageratum conyzoides stems and leaves to lactic acid bacteria fermentation and then performing ice-water bath ultrasonic alcohol extraction and heating alcohol extraction with ethanol of different concentrations; and both the Ageratum conyzoides extract and the petroleum ether extraction of the extract can effectively contact and kill Spodoptera exigua, and the corresponding components are plant-derived components, which can effectively reduce environmental pollution during use and comprehensively reduce the harm of pesticide residues, providing a new direction for the control of Spodoptera exigua.

Description

Technical Field

[0001] The present invention relates to the technical field of pest control, and particularly relates to a preparation method and application of an extract of Ageratum conyzoides for controlling Spodoptera exigua. Background Art

[0002] Spodoptera exigua Hubner belongs to the Lepidoptera Noctuidae insects and is a world-wide agricultural pest. In addition to damaging cruciferous vegetables, it also seriously damages crops such as corn, cotton, tobacco, flowers and fruit trees.

[0003] At present, chemical pesticides are mainly used to control Spodoptera exigua. Common chemical agents include beta-cypermethrin, flufenoxuron, cis-cypermethrin, cartap, spinosad, tebufenozide, spinosyns and chlorpyrifos, etc. Among them, insecticides such as beta-cypermethrin, spinosyns and chlorpyrifos have developed varying degrees of resistance, and some drugs such as cis-cypermethrin and cartap have basically become ineffective. Although pesticides such as spinosad and tebufenozide are still sensitive, long-term repeated use will also lead to the generation of pest resistance, causing problems such as pesticide residues, ecological environmental pollution, decline in biodiversity, and food safety.

[0004] Botanical pesticides, one of the biological pesticides, have the characteristics of less residue, low toxicity, high selectivity and difficult to generate resistance. They are a new type of environmentally friendly pesticide with good environmental compatibility. Based on the current control of Spodoptera exigua, it is crucial to develop a new botanical pesticide. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of an extract of Ageratum conyzoides for controlling Spodoptera exigua. Using the extract of Ageratum conyzoides to control Spodoptera exigua can effectively carry out contact killing on Spodoptera exigua, while reducing pollution and ensuring the control effect.

[0006] To achieve the above objectives, the technical solution of the present invention is realized through the following technical solutions:

[0007] A preparation method of an extract of Ageratum conyzoides for controlling Spodoptera exigua, the preparation method of the extract of Ageratum conyzoides includes the following steps:

[0008] S1. Select fresh stems and leaves of Ageratum conyzoides, grind them with water into a slurry, and then inoculate lactic acid bacteria for preliminary fermentation for 6-10h to obtain a preliminary fermentation material for standby;

[0009] S2. Dry and crush the above-mentioned preliminary fermentation material to obtain a pretreated powder for standby;

[0010] S3. Add an ethanol solution with a concentration of 70%-75% to the above-mentioned pretreated powder under ice bath conditions, perform ultrasonic extraction in an ice water bath, and then filter to obtain a first filtrate and a first filter residue for standby;

[0011] S4. Add the above first filter residue to an ethanol solution with a concentration of 95%-100%, perform ultrasonic extraction in an ice-water bath, and then filter to obtain a second filtrate and a second filter residue for standby;

[0012] S5. Add the above second filter residue to an ethanol solution with a concentration of 80%-85%, raise the temperature to 50-60 °C, keep warm and extract for 10-20 min, then restore to room temperature and filter to obtain a third filtrate;

[0013] S6. Combine the first filtrate, the second filtrate, and the third filtrate, and then dry to obtain the Ageratum conyzoides extract.

[0014] Preferably, in step S1, the mass ratio of the Ageratum conyzoides stems and leaves to water for grinding is 1:2-3, and the inoculation amount of lactic acid bacteria in the slurry is 0.2%-0.3% of the total mass of the slurry.

[0015] Preferably, in step S2, the drying temperature is 30-35 °C, and the pretreated powder after grinding passes through a 40-mesh sieve.

[0016] Preferably, in steps S3 and S4, the ultrasonic extraction method is to treat at an ultrasonic power of 400-600 W for 2-3 min and then let it stand for 2-3 min, and repeat this operation 5-8 times to complete the ultrasonic extraction.

[0017] Preferably, the drying method in step S6 is freeze-drying.

[0018] The Ageratum conyzoides extract is applied to control Spodoptera exigua.

[0019] Preferably, the application method is to directly use the Ageratum conyzoides extract to control Spodoptera exigua, or use the petroleum ether extract of the Ageratum conyzoides extract to control Spodoptera exigua.

[0020] Preferably, when directly using the Ageratum conyzoides extract to control Spodoptera exigua, the LC 50 values for Spodoptera exigua at 24 h, 72 h, 120 h, and 168 h are 15.67 mg / mL, 1.54 mg / mL, 0.61 mg / mL, and 0.61 mg / mL, respectively.

[0021] Preferably, the petroleum ether extract of the Ageratum conyzoides extract is obtained by adding water to the Ageratum conyzoides extract to form a suspension, then extracting with petroleum ether, and then removing the solvent.

[0022] Preferably, when using the petroleum ether extract of the Ageratum conyzoides extract to control Spodoptera exigua, the LC 50They are 5.69 mg / mL, 3.86 mg / mL, 3.42 mg / mL, and 3.13 mg / mL respectively.

[0023] The present invention provides a preparation method and application of an Ageratum conyzoides extract for controlling Spodoptera exigua. Compared with the prior art, the advantages are as follows:

[0024] The Ageratum conyzoides extract prepared by the present invention and the petroleum ether extract of the Ageratum conyzoides extract have good contact toxicity against Spodoptera exigua, can effectively control Spodoptera exigua, and the Ageratum conyzoides extract is a plant-derived component, which can effectively reduce environmental pollution during use, comprehensively reduce the harm of pesticide residues, and provide a new direction for the control of Spodoptera exigua. Specific embodiments

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Example 1:

[0026] Preparation of Ageratum conyzoides extract:

[0027] (1) Select fresh Ageratum conyzoides stems and leaves, grind them into a slurry with a material-liquid mass ratio of 1:2.5, then inoculate lactic acid bacteria accounting for 0.2% of the total mass of the slurry and ferment preliminarily for 8 h to obtain a preliminarily fermented material for standby;

[0028] (2) Dry the above-mentioned preliminarily fermented material at 35 °C and then crush it through a 40-mesh sieve to obtain a pretreated powder for standby;

[0029] (3) Add an ethanol solution with a concentration of 75% to the above-mentioned pretreated powder under ice bath conditions, perform ultrasonic treatment at 400 W for 2 min in an ice-water bath and then let it stand for 2 min, perform the cycle of ultrasonic treatment and standing for 5 times, and then filter to obtain a first filtrate and a first filter residue for standby;

[0030] (4) Add an ethanol solution with a concentration of 98% to the above-mentioned first filter residue, perform ultrasonic treatment at 600 W for 3 min in an ice-water bath and then let it stand for 3 min, perform the cycle of ultrasonic treatment and standing for 8 times, and then filter to obtain a second filtrate and a second filter residue for standby;

[0031] (5) Add an ethanol solution with a concentration of 85% to the above-mentioned second filter residue, raise the temperature to 55 °C, keep it warm for extraction for 15 min, then return to room temperature and filter to obtain a third filtrate;

[0032] (6) Combine the first filtrate, the second filtrate, and the third filtrate, and then lyophilize to obtain the Ageratum conyzoides extract. Example 2:

[0033] Petroleum ether extraction of the Ageratum conyzoides extract:

[0034] Add the Ageratum conyzoides extract prepared in Example 1 above to 5 times the volume of pure water to make a suspension, and then add petroleum ether to the suspension according to a volume ratio of 1:3 for extraction, and then remove the solvent to obtain the petroleum ether extract.

[0035] Comparative Example 1:

[0036] Preparation of the Ageratum conyzoides extract:

[0037] (1) Select fresh Ageratum conyzoides stems and leaves, grind them into a slurry with water according to a material-liquid mass ratio of 1:2.5, dry them at 35 °C and then crush them through a 40-mesh sieve to obtain a pretreated powder for standby;

[0038] (2) Add the above pretreated powder to an ethanol solution with a concentration of 75% under ice bath conditions, and perform ultrasonic treatment at 400 W for 2 min under ice-water bath conditions and then let it stand for 2 min. Repeat the ultrasonic treatment and standing process 5 times, and then filter to obtain the first filtrate and the first filter residue for standby;

[0039] (3) Add the above first filter residue to an ethanol solution with a concentration of 98% under ice bath conditions, and perform ultrasonic treatment at 600 W for 3 min under ice-water bath conditions and then let it stand for 3 min. Repeat the ultrasonic treatment and standing process 8 times, and then filter to obtain the second filtrate and the second filter residue for standby;

[0040] (4) Add the above second filter residue to an ethanol solution with a concentration of 85%, raise the temperature to 55 °C, keep it warm for extraction for 15 min, and then restore to room temperature and filter to obtain the third filtrate;

[0041] (5) Combine the first filtrate, the second filtrate, and the third filtrate, and then lyophilize to obtain the Ageratum conyzoides extract.

[0042] Comparative Example 2:

[0043] Preparation of the Ageratum conyzoides extract:

[0044] (1) Select fresh Ageratum conyzoides stems and leaves, grind them into a slurry with water according to a material-liquid mass ratio of 1:2.5, and then inoculate 0.2% of the total mass of the slurry with lactic acid bacteria for preliminary fermentation for 8 h to obtain a preliminarily fermented material for standby;

[0045] (2) Dry the above preliminarily fermented material at 35 °C and then crush it through a 40-mesh sieve to obtain a pretreated powder for standby;

[0046] (3) Add the above pretreated powder to an ethanol solution with a concentration of 98% under ice bath conditions. Under ice-water bath conditions, perform ultrasonic treatment at 400 W for 2 min and then let it stand for 2 min. Repeat the ultrasonic treatment and standing process 18 times, and then filter to obtain the first filtrate and the first filter residue for later use;

[0047] (4) Add the above first filter residue to an ethanol solution with a concentration of 85%. Heat it to 55 °C and keep it warm for extraction for 15 min. Then, after returning to room temperature, filter to obtain the second filtrate;

[0048] (5) Combine the first filtrate and the second filtrate, and then freeze-dry to obtain the Ageratum conyzoides extract.

[0049] Comparative Example 3:

[0050] Preparation of Ageratum conyzoides extract:

[0051] (1) Select fresh Ageratum conyzoides stems and leaves, grind them into a slurry with a material-liquid mass ratio of 1:2.5, and then inoculate 0.2% of the total mass of the slurry with lactic acid bacteria and perform preliminary fermentation for 8 h to obtain the preliminary fermentation material for later use;

[0052] (2) Dry the above preliminary fermentation material at 35 °C and then crush it through a 40-mesh sieve to obtain the pretreated powder for later use;

[0053] (3) Add the above pretreated powder to an ethanol solution with a concentration of 75% under ice bath conditions. At room temperature, perform ultrasonic treatment at 400 W for 2 min and then let it stand for 2 min. Repeat the ultrasonic treatment and standing process 5 times, and then filter to obtain the first filtrate and the first filter residue for later use;

[0054] (4) Add the above first filter residue to an ethanol solution with a concentration of 98%. At room temperature, perform ultrasonic treatment at 600 W for 3 min and then let it stand for 3 min. Repeat the ultrasonic treatment and standing process 8 times, and then filter to obtain the second filtrate and the second filter residue for later use;

[0055] (5) Add the above second filter residue to an ethanol solution with a concentration of 85%. Extract at room temperature for 15 min, and then filter to obtain the third filtrate;

[0056] (6) Combine the first filtrate, the second filtrate, and the third filtrate, and then freeze-dry to obtain the Ageratum conyzoides extract.

[0057] Detection:

[0058] Use the insect immersion method to determine the insecticidal activity of the extracts / extracts prepared in the above Examples 1-2 and Comparative Examples 1-3 against the 3rd instar larvae of Spodoptera exigua;

[0059] The extracts / extractives of five concentration gradients of 1.25 mg / mL, 2.5 mg / mL, 5.0 mg / mL, 10.0 mg / mL, and 20.0 mg / mL were used for contact toxicity tests (set as treatment groups); 10 larvae were treated with each concentration of each group of substances, and the experiment was repeated 3 times; an aqueous solution of 0.5% Tween 80 was used as the control group, and pyrethrin was used as the verification group. The survival status of the larvae was checked at 1, 3, 5, and 7 days after treatment, and the corrected mortality rate of each group was calculated. And the LC 50 .

[0060] Among them: ;

[0061] The calculation methods of the mortality rates of the control group and the treatment group are as follows:

[0062] .

[0063] Among them, the corrected mortality rates of the extracts prepared in Example 1 against Spodoptera exigua at different concentrations are shown in Table 1 below:

[0064] Table 1

[0065]

[0066] And the single-factor Anova test analysis was performed using SPSS 23.0 statistical analysis software, and then the regression probability test analysis was carried out to obtain the 95% confidence interval of the median lethal concentration LC 50 , LC 50 value, slope ± standard error, chi-square value ( X 2 ), and P value, as shown in Table 2 below:

[0067] Table 2

[0068]

[0069] It can be seen from the above detections that the extracts prepared in Example 1 and Example 2 have good contact toxicity against Spodoptera exigua at lower concentrations.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an ageratum leaf extract for controlling beet armyworm, characterized in that: The preparation method of the ageratum leaf extract comprises the following steps: S1. Select fresh stems and leaves of Ageratum cochinchinensis, add water and grind into slurry, then inoculate with lactic acid bacteria for preliminary fermentation for 6-10 hours to obtain preliminary fermentation material for later use, and the inoculation amount of lactic acid bacteria is 0.2%-0.3% of the total mass of the slurry; S2, drying and crushing the above-mentioned preliminary fermentation material to obtain pre-treated powder for standby use; S3, adding 70%-75% ethanol solution to the pretreated powder under ice bath conditions, ultrasonic extraction in an ice water bath, and then filtering to obtain a first filtrate and a first filter residue for later use; S4, adding the first filter residue to an ethanol solution with a concentration of 95%-100%, performing ultrasonic extraction in an ice-water bath, and then filtering to obtain a second filtrate and a second filter residue for standby use; S5, adding the second filter residue to an ethanol solution with a concentration of 80%-85%, heating to 50-60°C, extracting at this temperature for 10-20 minutes, returning to room temperature and filtering to obtain a third filtrate; S6, combining the first filtrate, the second filtrate, and the third filtrate, and then drying to obtain an ageratum leaf extract; The ultrasonic extraction in the above steps S3 and S4 is performed by treating at an ultrasonic power of 400-600 W for 2-3 minutes and then standing for 2-3 minutes, and this operation is repeated 5-8 times to complete the ultrasonic extraction.

2. The preparation method according to claim 1, characterized in that: In step S1, the stems and leaves of Ageratum are ground with water to obtain a material-liquid mass ratio of 1:2-3.

3. The preparation method according to claim 1, characterized in that: The drying temperature in step S2 is 30-35° C., and the crushed pre-treated powder is sieved through a 40-mesh sieve.

4. The preparation method according to claim 1, characterized in that: The drying method in step S6 is freeze-drying.

5. Use of the Ageratum cochinchinensis extract obtained by the preparation method according to any one of claims 1 to 4 in controlling beet armyworm.

6. The use according to claim 5, characterized in that: The application method is to directly use the ageratum extract to control the beet armyworm, or to use the petroleum ether extract of the ageratum extract to control the beet armyworm.

7. The use according to claim 6, characterized in that: The LC values ​​of the ageratum extract on beet armyworm at 24 h, 72 h, 120 h and 168 h were 50 They are 15.67 mg / mL, 1.54 mg / mL, 0.61 mg / mL and 0.61 mg / mL respectively.

8. The use according to claim 6, characterized in that: The petroleum ether extract of the ageratum extract is obtained by adding water to the ageratum extract to prepare a suspension, then extracting with petroleum ether, and then removing the solvent.

9. The use according to claim 8, characterized in that: The LC values ​​of the petroleum ether extract of the sweet thistle extract against the beet armyworm at 24 h, 72 h, 120 h and 168 h 50 They are 5.69 mg / mL, 3.86 mg / mL, 3.42 mg / mL and 3.13 mg / mL respectively.

Images