Method for extracting chemical substance with obvious activity on root-knot nematode

Abstract

The invention belongs to the technical field of biological control, and particularly relates to a method for extracting a chemical substance with obvious activity on root-knot nematode. According to the method for extracting the chemical substance with the obvious activity on the root-knot nematode, the hedyotis diffusa sample is pretreated, impurities on the surface of the sample can be removed easily, and the purity of the subsequent active substance is improved; a sample is soaked in an ethanol solution with the volume concentration of 95%, effective active substances in the sample can be separated out easily, and effective chemical substances in the sample are promoted to be completely released through two times of ultrasonic extraction. Therefore, the chemical substance with obvious activity on the root-knot nematodes can be used for preventing and treating the root-knot nematodes or preparing biological products for preventing and treating the root-knot nematodes, not only is the death rate of the root-knot nematodes improved, but also green and health of plants are guaranteed, and the economic value of the plants is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and in particular relates to a method for extracting chemical substances with obvious activity against root-knot nematodes. Background Art

[0002] Root-knot nematodes are a type of plant parasitic nematodes that seriously harm agricultural production. They mainly parasitize on vegetables, grains, cash crops, fruit trees and other hosts, causing serious reductions in plant yields, with the maximum reduction reaching more than 75%. At present, the methods for preventing and controlling root-knot nematodes are mainly divided into chemical control and biological control. Although chemical control is more effective, it is highly toxic and seriously threatens food health and human safety; while biological control is more environmentally friendly, the control effect is often not ideal.

[0003] Hedyotis diffusa is an annual loose herb, 15-50cm tall; its roots are slender, branched, and white; its stems are slightly square or oblate, smooth and hairless, with multiple branches from the base; its finished medicine tastes bitter and light, and is cold in nature. Its main functions are to clear away heat and detoxify, relieve pain and resolve knots, and promote diuresis and remove dampness. However, there is no research or report on the use of Hedyotis diffusa extracts in the prevention and treatment of root-knot nematodes. Summary of the invention

[0004] The object of the present invention is to provide a method for extracting a chemical substance having obvious activity against root-knot nematodes. The chemical substance obtained by extracting Hedyotis diffusa has obvious activity against root-knot nematodes, and can thus be used for efficiently preventing and controlling root-knot nematodes.

[0005] The present invention provides a method for extracting a chemical substance having obvious activity against root-knot nematodes, comprising the following steps:

[0006] The Hedyotis diffusa sample is sterilized, dried and crushed in sequence to obtain a crushed sample;

[0007] Soaking the crushed sample in an ethanol solution with a volume concentration of 95% to obtain a soaked sample;

[0008] Performing a first ultrasonic extraction on the soaked sample to obtain a first filtrate and a first filter residue;

[0009] The first filtrate is mixed with an ethanol solution having a volume concentration of 95%, and then subjected to a second ultrasonic extraction to obtain a second filtrate;

[0010] The first filtrate and the second filtrate are mixed and concentrated into a paste-like substance to obtain a chemical substance having obvious activity against root-knot nematodes.

[0011] Preferably, the Hedyotis diffusa sample is derived from one or more of the roots, stems and leaves of Hedyotis diffusa.

[0012] Preferably, during the soaking, the mass volume ratio of the crushed sample to the ethanol solution is 1 g: 20-30 mL.

[0013] Preferably, the duration of the first ultrasound and the second ultrasound is 35-48 minutes respectively;

[0014] The temperatures of the first ultrasound and the second ultrasound are 25-27° C. respectively;

[0015] The power of the first ultrasound and the second ultrasound are 310-350W respectively.

[0016] Preferably, when performing the second ultrasonic extraction, the amount of ethanol solution added is based on the mass of the crushed sample, and the mass-to-volume ratio of the crushed sample to the ethanol solution is 1 g: 20-30 mL.

[0017] Preferably, the soaking time is 15-18 hours.

[0018] The present invention provides a chemical substance extracted by the chemical substance extraction method described in the above technical solution.

[0019] The present invention provides the use of chemical substances extracted by the chemical substance extraction method described in the above technical solution in controlling root-knot nematodes and / or preparing biological products for controlling root-knot nematodes.

[0020] Preferably, the root-knot nematodes include: cucumber root-knot nematodes, tomato root-knot nematodes or tobacco root-knot nematodes.

[0021] Beneficial effects:

[0022] The present invention provides a method for extracting chemical substances with obvious activity against root-knot nematodes. By sequentially disinfecting, drying and crushing the Hedyotis diffusa sample, it is beneficial to remove impurities on the sample surface and improve the purity of subsequent active substances; by soaking the sample in an ethanol solution with a volume concentration of 95%, it is beneficial to precipitate the effective active substances in the sample, and by adding two ultrasonic extractions, it is more beneficial to completely release the effective chemical substances in the sample, and then the chemical substances with obvious activity against root-knot nematodes can be obtained.

[0023] Based on the above technical advantages, the present invention provides the use of the chemical substance extracted by the chemical substance extraction method described in the above technical solution in the prevention and control of root-knot nematodes and / or the preparation of biological products for preventing and controlling root-knot nematodes. By using the chemical substance to prevent and control biological pests, the mortality rate of root-knot nematodes is increased, the green health of plants is guaranteed, and the economic value of plants is improved. DETAILED DESCRIPTION

[0024] The invention sequentially disinfects, dries and crushes the oldenlandia diffusa sample to obtain a crushed sample; the disinfection comprises: soaking the oldenlandia diffusa sample in alcohol with a volume concentration of 75%; the soaking time is preferably 1-2 minutes, more preferably 1 minute; the drying temperature is preferably 50-60°C, more preferably 55°C; there is no special requirement for the drying time, and the sample is completely dried; there is no special requirement for the crushing method, and the crushed sample with a length of 0.6-1.0 mm is preferably obtained.

[0025] The Hedyotis diffusa sample of the present invention is preferably derived from: one or more of the roots, stems and leaves of Hedyotis diffusa, more preferably the roots, stems and leaves of Hedyotis diffusa; when the roots, stems and leaves are contained, there is no special requirement for the addition amount of the three, and the total amount can meet the sample requirements.

[0026] In the present invention, unless otherwise specified, the reagents and equipment used are conventional reagents and equipment.

[0027] Example 1

[0028] A method for extracting a chemical substance having significant activity against root-knot nematodes, comprising the steps of:

[0029] (1) Soak the roots of Hedyotis diffusa in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0030] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0031] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0032] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste is a chemical substance that has obvious activity against root-knot nematodes.

[0033] Example 2

[0034] A method for extracting a chemical substance having significant activity against root-knot nematodes, comprising the steps of:

[0035] (1) Soak the stems of Hedyotis diffusa in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0036] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0037] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0038] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste is a chemical substance that has obvious activity against root-knot nematodes.

[0039] Example 3

[0040] A method for extracting a chemical substance having significant activity against root-knot nematodes, comprising the steps of:

[0041] (1) Soak the leaves of Hedyotis diffusa in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0042] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0043] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0044] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste is a chemical substance that has obvious activity against root-knot nematodes.

[0045] Example 4

[0046] A method for extracting a chemical substance having significant activity against root-knot nematodes, comprising the steps of:

[0047] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0048] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0049] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0050] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste is a chemical substance that has obvious activity against root-knot nematodes.

[0051] Example 5

[0052] A method for extracting a chemical substance having significant activity against root-knot nematodes, comprising the steps of:

[0053] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0054] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0055] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 48 min, the temperature is 27° C., and the power is 350 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0056] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste is a chemical substance that has obvious activity against root-knot nematodes.

[0057] Comparative Example 1

[0058] A method for extracting a chemical substance, comprising the following steps:

[0059] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0060] (2) soaking 100 g of the sample crushed in step (1) in 1000 mL of 95% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0061] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0062] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste-like substance is the chemical substance.

[0063] Comparative Example 2

[0064] A method for extracting a chemical substance, comprising the following steps:

[0065] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0066] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 80% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0067] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0068] (4) Mixing the first filtrate in step (2) and the second filtrate in step (3) to obtain a mixed solution, and concentrating the mixed solution into an extract using a rotary evaporator. The paste-like substance is the chemical substance.

[0069] Comparative Example 3

[0070] A method for extracting a chemical substance, comprising the following steps:

[0071] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0072] (2) soaking 100 g of the sample crushed in step (1) in 1000 mL of 80% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0073] (3) using 1000 mL of ethanol solution with a volume concentration of 80%, performing a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, performing suction filtration to obtain a second filtrate;

[0074] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste-like substance is the chemical substance.

[0075] Comparative Example 4

[0076] A method for extracting a chemical substance, comprising the following steps:

[0077] (1) Soak the Hedyotis diffusa sample (including roots, stems and leaves) in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0078] (2) soaking 100 g of the sample crushed in step (1) in 2000 mL of 95% ethanol solution for 20 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0079] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0080] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste-like substance is the chemical substance.

[0081] Comparative Example 5

[0082] A method for extracting a chemical substance, comprising the following steps:

[0083] (1) Soak the roots of Hedyotis diffusa in 75% alcohol by volume for 1 min for disinfection, then rinse the disinfected sample twice with clean water, dry it in an oven at 55° C., and grind it after drying to obtain a sample with a length of 0.8 mm for later use;

[0084] (2) soaking 100 g of the sample crushed in step (1) in 1000 mL of 80% ethanol solution for 15 h; then performing a first ultrasonic extraction on the soaked sample (the first ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and filtering after the first ultrasonic extraction to obtain a first filtrate and a first filter residue;

[0085] (3) using 2000 mL of 95% ethanol solution to perform a second ultrasonic extraction on the first filter residue in step (2) (the second ultrasonic extraction time is 35 min, the temperature is 26° C., and the power is 310 W), and after the second ultrasonic extraction, filtering is performed to obtain a second filtrate;

[0086] (4) The first filtrate in step (2) and the second filtrate in step (3) are mixed to obtain a mixed solution, and the mixed solution is concentrated into an extract using a rotary evaporator. The paste-like substance is the chemical substance.

[0087] Application Example 1 Determination of Nematode Killing Effect

[0088] The chemical substances prepared in Examples 1-5 and Comparative Examples 1-5 were respectively dissolved with sterile water to obtain solutions of the chemical substances to be tested with final concentrations of 0.5 mg / mL, 1 mg / mL and 1.5 mg / mL, respectively;

[0089] The corresponding verification experiment was carried out using a 96-well plate. Nematode suspension (about 100-150 nematodes), 65 μL of sterile water, and 5 μL of the chemical solution to be tested were added to each well; 5 μL of sterile water was added to the blank control instead of the chemical solution to be tested; 5 μL of 1 mg / mL avermectin aqueous solution was used as the positive control instead of the chemical solution to be tested; each well was a treatment, and each treatment was repeated 3 times in parallel. The 96-well plate was incubated at room temperature for 24 hours, and the number of nematode deaths was counted under a microscope to calculate the nematode mortality rate. The results are shown in Table 1.

[0090] Table 1 Nematode mortality in different treatments

[0091]

[0092]

[0093] From the data in Table 1, it can be seen that by adopting the technical solution of the present invention, after extracting chemical substances from the roots, stems and leaves of Hedyotis diffusa as samples, the killing effect on other nematodes is the strongest, up to 98.3%; and in Examples 1-5, when the concentration of the solution containing the chemical substances is 1 mg / mL, the killing effect on nematodes is the best.

[0094] Application Example 2 Field Experiment

[0095] The plots with uniform and severe root-knot nematode disease and uniform fertility were selected, and the field experiment was arranged according to the randomized block design. The flue-cured tobacco variety was Yunyan 87 (the tobacco seedlings with uniform growth were selected for the following treatments, and each treatment group was repeated three times, and 10 tobacco plants were planted in each repeat and the conventional management method was the same);

[0096] Treatment group: The chemical substance extracted in Example 4 was dissolved in sterile water to obtain a solution with a concentration of 1 mg / mL (referred to as solution A);

[0097] Control group: Solution A in the treatment group was replaced with sterile water;

[0098] Experimental process:

[0099] (1) Root soaking treatment: The tobacco seedlings were soaked in solution A and sterile water for 10 min.

[0100] (2) When transplanting, the tobacco seedlings were irrigated with solution A and sterile water once, with 40 mL of the corresponding solution used for each tobacco seedling;

[0101] (3) After that, the tobacco seedlings were irrigated with solution A and sterile water once every 10 days, with 40 mL of the corresponding solution used for each tobacco seedling;

[0102] (4) 60 days after transplanting, the plant height and the number of diseased tobacco plants in different treatments were observed. The specific indicators are shown in Table 2.

[0103] Table 2 Average plant height and number of diseased plants in tobacco under different treatments

[0104] deal with Average plant height (cm) Number of diseased plants (plants) Treatment Group 60.27 3 Control group 43.30 10

[0105] From the data in Table 2, it can be seen that the chemical substance extracted by the present invention with obvious activity against root-knot nematodes can significantly inhibit the outbreak of tobacco root-knot nematodes. After 60 days of transplanting, the number of diseased plants was only 3, and the incidence rate was 10%, while the incidence rate in the control group was as high as 33.3%; in addition, the chemical substance with obvious activity against root-knot nematodes provided by the present invention is also beneficial to the improvement of tobacco plant height, thereby indirectly improving the economic value of tobacco.

[0106] In summary, the chemical substance provided by the present invention that has obvious activity against root-knot nematodes can not only significantly inhibit the occurrence of tobacco root-knot nematodes, but also help promote the growth of plant height, thereby obtaining healthy and green plants.

[0107] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting a chemical substance having significant activity against root-knot nematodes, characterized in that: The steps include: The Hedyotis diffusa sample is sterilized, dried and crushed in sequence to obtain a crushed sample; Soaking the crushed sample in an ethanol solution with a volume concentration of 95% to obtain a soaked sample; Performing a first ultrasonic extraction on the soaked sample to obtain a first filtrate and a first filter residue; The first filtrate is mixed with an ethanol solution having a volume concentration of 95%, and then subjected to a second ultrasonic extraction to obtain a second filtrate; The first filtrate and the second filtrate are mixed and concentrated into a paste-like substance to obtain a chemical substance having obvious activity against root-knot nematodes.

2. The method for extracting a chemical substance according to claim 1, characterized in that: The Hedyotis diffusa sample is derived from one or more of the roots, stems and leaves of Hedyotis diffusa.

3. The method for extracting a chemical substance according to claim 1, characterized in that: During the soaking, the mass volume ratio of the crushed sample to the ethanol solution is 1 g: 20-30 mL.

4. The method for extracting a chemical substance according to claim 1, characterized in that: The duration of the first ultrasound and the second ultrasound is 35-48 minutes respectively; The temperatures of the first ultrasound and the second ultrasound are 25-27° C. respectively; The power of the first ultrasound and the second ultrasound are 310-350W respectively.

5. The method for extracting a chemical substance according to claim 1, characterized in that: When performing the second ultrasonic extraction, the amount of ethanol solution added is based on the mass of the crushed sample, and the mass-to-volume ratio of the crushed sample to the ethanol solution is 1 g: 20-30 mL.

6. The method for extracting a chemical substance according to claim 1, characterized in that: The soaking time is 15-18 hours.

7. The chemical substance extracted by the method for extracting a chemical substance according to any one of claims 1 to 6.

8. Use of the chemical substance extracted by the method for extracting chemical substances according to any one of claims 1 to 6 in controlling root-knot nematodes and / or preparing biological products for controlling root-knot nematodes.

9. The use according to claim 8, characterized in that: The root-knot nematodes include: cucumber root-knot nematodes, tomato root-knot nematodes or tobacco root-knot nematodes.

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