A photophilous flower extract nematicide, its preparation method and application

Abstract

The application provides a nematocide of a light-loving flower extract and a preparation method and application thereof, and particularly relates to application of the light-loving flower extract in preparation of a nematocide. Through determination of a mortality rate and a hatching rate of southern root-knot nematodes, it is found that ethyl naphthalene in the light-loving flower has good insecticidal activity on nematodes and has obvious inhibition on hatching of eggs of the southern root-knot nematodes. The nematocidal activity of ethyl naphthalene increases with increase of a concentration of the medicine, and the inhibition rate on hatching of the nematode eggs increases with increase of a treatment time and the concentration of the medicine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nematicides, in particular to a light-loving flower extract nematicide and a preparation method and application thereof. BACKGROUND

[0002] Root-knot nematode is considered to be one of the most harmful nematodes, which is a worldwide disease, and the loss caused by it accounts for more than 90% of the loss of the whole cultivated crops, causing more than 170 billion US dollars of agricultural loss in the world every year. Chemical nematicides such as abamectin, thiazolidine phosphorus and fluopyram have been recognized as the most effective method for controlling root-knot nematodes. However, in the rapid development of global agriculture, the application amount and frequency of traditional chemical nematicides are increasing, which leads to the generation of root-knot nematode resistance, and accordingly increases the cost of root-knot nematode control. In addition, while killing harmful nematodes, chemical nematicides also kill a large number of natural enemies, destroy the ecological balance and pollute the human living environment. The application of chemical nematicides is increasingly inconsistent with the concept of modern human green consumption, so it is becoming more and more important to develop green nematicides with new nematicidal mechanisms.

[0003] Plant source pesticides are the results of evolution and co-evolution of plant defense functions adapting to harmful organisms and environmental stress, are secondary metabolites produced by plants extracted from plants, have the characteristics of high selectivity, environmental friendliness and non-resistance, and meet the requirements of modern pesticides and environment compatibility. In recent years, the research on extracting natural nematicidal active substances from plants has gradually attracted the interest of scholars at home and abroad, but the application of plant source compounds in the control of root-knot nematodes has been slow, so it is urgent to explore and apply efficient nematicidal plants to control root-knot nematodes.

[0004] Light-loving flower is a shrub, a plant of the genus Jatropha, and a Hainan endemic plant. At present, scholars at home and abroad have less research on light-loving flower. Song Xiaoping et al. determined 19 kinds of chemical components such as alkane, alkene and alcohol in the leaf volatile oil of light-loving flower by GC-MS. Kan Sujun et al. determined 32 components in the fruit volatile oil of light-loving flower by GC-MS, and the main components are esters, ketones, alkenes and alcohols. Tang Bei et al. found that the leaves of light-loving flower contain antitumor components, and 36 chemical components were separated from the leaves of light-loving flower, including acids, ketones, esters, alcohols and alkaloids. The existing research results show that light-loving flower may have antitumor activity, but no one has reported the agricultural activity of light-loving flower, especially the nematicidal activity, so it is extremely meaningful to study the nematicidal activity of light-loving flower. SUMMARY

[0005] Therefore, the present application provides a light-loving flower extract nematicide and a preparation method and application thereof.

[0006] The technical scheme of the present application is implemented as follows:

[0007] The application of the extract of Helipticum or ethynyl naphthalene in preparing nematocide.

[0008] Further, the application concentration of ethynyl naphthalene is 100 μg·mL-1 or above.

[0009] Further, the application of the extract of Helipticum or ethynyl naphthalene in preparing plant nematocide.

[0010] Further, the application of the extract of Helipticum or ethynyl naphthalene in preparing nematocide for pepper seedlings and tomato seedlings.

[0011] Further, the nematode is Meloidogyne incognita.

[0012] A preparation method of a nematocide of the extract of Helipticum, wherein the extract of Helipticum is prepared according to the following method:

[0013] S1. Drying Helipticum leaves at 45-55 ℃ for 3-5 days, crushing, and adding a solvent to prepare a crude extract; the mass ratio of Helipticum leaves to the solvent is 10-15:1;

[0014] S2. Dissolving the crude extract prepared in step S1 in water to prepare a crude extract aqueous solution, and extracting with an organic solvent for 3-5 times to prepare an organic phase, and concentrating the organic phase under vacuum at 40-50 ℃ to prepare an organic solvent extract;

[0015] S3. Column chromatography separation of the organic solvent extract prepared in step S2 with an organic solvent system to prepare a separation product;

[0016] S4. Adding the separation product prepared in step S3 to a gel column, and isocratic elution with an eluent to prepare a fraction;

[0017] S5. Adding the fraction prepared in step S4 to a semi-preparative liquid chromatograph for further purification to prepare the extract of Helipticum; the extract of Helipticum contains the active ingredient ethynyl naphthalene.

[0018] Further, the solvent in step S1 is at least one of methanol, ethyl acetate, petroleum ether, acetone, dichloromethane or water; and the organic solvent in step S2 is one or more of petroleum ether, chloroform, ethyl acetate and n-butanol.

[0019] Further, the organic solvent system in step S3 is at least one of petroleum ether / ethyl acetate, petroleum ether / chloroform or petroleum ether / n-butanol; and the volume ratio of the two components in the organic solvent system is 1:4-6.

[0020] Further, the gel column in step S4 is a hydroxypropyl dextran gel column; the eluent is at least one of methanol, ethanol or dichloromethane; and the volume concentration of the eluent is 90% to 100%.

[0021] Further, the mobile phase in step S5 is methanol and water, the volume ratio of methanol to water in the mobile phase is 55 to 65:33 to 45, and the flow rate of the mobile phase is 4 to 6 mL / min.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] (1) The present application first reports the nematicidal activity of ethyl naphthol extract of Heteropanax fragilis, providing a new resource for developing green nematicides with a new nematicidal mechanism.

[0024] (2) The extract of Heteropanax fragilis of the present application not only has excellent in-vitro nematicidal activity, but also has obvious inhibitory effect on nematode egg hatching, has the advantages of high efficiency, low toxicity and good persistence, and effectively prevents and controls southern root-knot nematode disease in pepper seedlings and tomato seedlings.

[0025] (3) The present application provides an extraction method of leaf crude extract of Heteropanax fragilis and a separation method of active ingredients, effectively improving the purity of active compounds and the nematicidal effect of Heteropanax fragilis extract. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0027] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0028] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0029] Example 1 - Preparation method of Heteropanax fragilis extract nematicide

[0030] S1. 5 kg of Heteropanax fragilis leaves were dried at 45℃ for 5 days, crushed, and added into 500 g of methanol solution to prepare 400 g of methanol crude extract;

[0031] S2. The 400 g of methanol crude extract prepared in step S1 was dissolved in 2 L of water to prepare a methanol crude extract aqueous solution, which was then extracted with 2 L of petroleum ether for 5 times to prepare an organic phase, and the organic phase was concentrated under vacuum at 40℃ to prepare 20 g of petroleum ether extract;

[0032] S3. The 20 g of petroleum ether extract prepared in step S2 was subjected to column chromatography separation with a petroleum ether / ethyl acetate solvent system in a volume ratio of 1:5 to prepare 2.1 g of separation product;

[0033] S4. The 2.1 g isolate prepared in step S3 was added to a Sephadex LH-20 gel column and eluted isocratically with 100% methanol eluent at a flow rate of 3 mL / min for 150 min to produce 400 mg fractions;

[0034] S5. The 400 mg fractions prepared in step S4 were added to a semi-preparative liquid chromatograph with a mobile phase of 60:40 methanol and water by volume at a flow rate of 5 mL / min for 9 min to further purify to produce 20 mg of the H. liukiuensis extract; the H. liukiuensis extract contains the active ingredient ethynyl naphthalene.

[0035] Example 2 - Preparation of a nematocide from H. liukiuensis extract

[0036] S1. 5 kg of H. liukiuensis leaves were dried at 50°C for 4 days, ground, and added to 500 g of methanol solution to produce 400 g of methanol crude extract;

[0037] S2. The 400 g of methanol crude extract prepared in step S1 was dissolved in 2 L of water to produce a methanol crude extract aqueous solution, which was extracted with 2 L of petroleum ether four times to produce an organic phase, which was concentrated under vacuum at 45°C to produce 20 g of petroleum ether extract;

[0038] S3. The 20 g of petroleum ether extract prepared in step S2 was column chromatographed with a petroleum ether / ethyl acetate solvent system at a volume ratio of 1:5 to produce 2.3 g of isolate;

[0039] S4. The 2.3 g of isolate prepared in step S3 was added to a Sephadex LH-20 gel column and eluted isocratically with 100% methanol eluent at a flow rate of 3 mL / min for 130 min to produce 400 mg fractions;

[0040] S5. The 400 mg fractions prepared in step S4 were added to a semi-preparative liquid chromatograph with a mobile phase of 60:40 methanol and water by volume at a flow rate of 5 mL / min for 9 min to further purify to produce 20 mg of the H. liukiuensis extract; the H. liukiuensis extract contains the active ingredient ethynyl naphthalene.

[0041] Example 3 - Preparation of a nematocide from H. liukiuensis extract

[0042] S1. 5 kg of H. liukiuensis leaves were dried at 55°C for 3 days, ground, and added to 500 g of methanol solution to produce 400 g of methanol crude extract;

[0043] S2. Dissolve 400 g of the methanol crude extract prepared in step S1 in 3 L of water to prepare a methanol crude extract aqueous solution, and then extract the aqueous solution with 3 L of petroleum ether three times to prepare an organic phase. Concentrate the organic phase under vacuum at 50°C to prepare 20 g of a petroleum ether extract;

[0044] S3. Perform column chromatography on the 20 g of the petroleum ether extract prepared in step S2 using a petroleum ether / ethyl acetate solvent system in a volume ratio of 1:5 to prepare 2.6 g of a fraction;

[0045] S4. Load the 2.6 g of the fraction prepared in step S3 into a Sephadex LH-20 gel column, and perform isocratic elution using a methanol eluent with a volume fraction of 100%, at a flow rate of 3 mL / min, for 120 min to prepare 400 mg of a fraction;

[0046] S5. Load the 400 mg of the fraction prepared in step S4 into a semi-preparative liquid chromatograph, and perform further purification using a mobile phase of methanol and water in a volume ratio of 60:40, at a flow rate of 5 mL / min, for 9 min to prepare 20 mg of a Hoya curranii extract. The Hoya curranii extract contains the active ingredient ethynyl naphthalene.

[0047] Test Example 1 - Study on the nematicidal activity of ethynyl naphthalene

[0048] The nematicidal activity of ethynyl naphthalene on the second instar larvae of Meloidogyne incognita was determined by the immersion method. The second instar larvae of Meloidogyne incognita were prepared into a 100 larvae / mL aqueous solution of nematodes using sterile water. 10 mg of the Hoya curranii extract of Test Examples 1 to 3 were weighed, respectively, dissolved in 1 mL of DMF (N,N-dimethylformamide), and then made up to 5 mL with a 0.5% aqueous solution of polysorbate-80 to prepare a 2000 μg / mL aqueous solution of the Hoya curranii extract. The solution was diluted with 45 mL of water to prepare a 100 μg / mL aqueous solution of the Hoya curranii extract. 1 mL of the aqueous solution of nematodes was added to a 12-well plate, and 1 mL of the prepared 100 μg / mL aqueous solution of the Hoya curranii extract was added to the sample well at the same time to prepare a mixed test solution. The solution was mixed by shaking, and incubated at 28°C in the dark. Abamectin was used as a positive control, and sterile water containing the same amount of DMF and polysorbate-80 was used as a blank control. The death of the nematodes was observed and recorded, and the death of the nematodes was identified by the body posture. The live nematodes could move, and the dead nematodes were stiff and immobile. The death of the nematodes was also identified by the needle prick method. The mortality and corrected mortality of the nematodes were calculated.

[0049] Mortality (%) = (number of dead nematodes / total number of nematodes) x 100%

[0050] Corrected mortality (%) = (treatment group mortality - control group mortality) / (1 - control group mortality) x 100%

[0051] Table 1. Nematicidal activity of ethynyl naphthol and abamectin in Examples 1-3

[0052]

[0053] The results show that the active ingredient ethynyl naphthol in Examples 1-3 has extremely excellent nematicidal activity, and when the concentration is 100 μg·mL-1, the lethal activity on southern root-knot nematodes is 100%, which is equivalent to the commercial drug abamectin. The EC50 of ethynyl naphthol is 38.0 μg·mL-1.

[0054] Test Example 2 - Effect of ethynyl naphthol on egg hatching of southern root-knot nematodes

[0055] The samples of Examples 1-3 were diluted with water to a concentration of 200 μg·mL-1, 400 μg·mL-1 of ethynyl naphthol solution, 1 mL of southern root-knot nematode egg solution was added to 1 mL of 200 μg·mL-1, 400 μg·mL-1 of ethynyl naphthol solution, respectively, to make a concentration of 100 μg·mL-1, 200 μg·mL-1 of the solution, sterile water as control, under the condition of 25℃, after 2, 4, 8, 16, 24h treatment, keep the egg grains deposited on the bottom of 12-hole plate, aspirate the drug solution with a pipette and replace it with sterilized water, incubate in a (25±1)℃ incubator, each experiment is repeated 3 times, check the hatching of the eggs under the stereomicroscope after 7 days, and calculate the hatching inhibition rate.

[0056] Hatching inhibition rate (%) = (control group hatching rate - treatment group hatching rate) / control group hatching rate x 100%

[0057] Table 2. Effect of ethynyl naphthol on hatching of nematode eggs in Test Examples 1-3

[0058]

[0059] a “-” means not tested

[0060] The results of Test Example 2 show that ethynyl naphthol also has obvious inhibition effect on the hatching of southern root-knot nematode eggs, and with the increase of treatment time and drug concentration, the inhibition rate of ethynyl naphthol on the hatching of nematode eggs will increase.

[0061] Test Example 3 - Tomato seedling potting control effect experiment of ethynyl naphthol on southern root-knot nematodes

[0062] In the plot where the root-knot nematode is more serious, 50-60 cm tomato seedlings are selected, 50 seedlings are transplanted into a prepared 210 mm x 170 mm flowerpot, one seedling per pot, and the sample of Example 2 is configured into 200 μg·mL-1and 500 μg·mL-1concentrations of ethyl naphthalene solution. After 7 days of tomato seedling planting, the prepared solution is irrigated into the rhizosphere of the plant, and 10 μg·mL-1of abamectin is used as a positive control, and water is used as a control solution, with 5 replicates per treatment. After 60 days, the number of root knots on the tomato roots is counted, and the control effect is calculated, and the test results are shown in Table 3:

[0063] Inhibition effect (%) = (number of nematodes in the control group - number of nematodes in the treatment group) / number of nematodes in the control group x 100%

[0064] Table 3. Ethyl naphthalene on tomato seedling potting of southern root-knot nematode control effect experiment

[0065]

[0066] a “-” means no data

[0067] The results of Test Example 3 show that the nematicidal activity of ethyl naphthalene increases with the increase of the concentration of the drug, and the protection activity data shows that when the concentration of ethyl naphthalene is 500 μg·mL-1, 200 μg·mL-1, 100 μg·mL-1, the inhibition effect on southern root-knot nematode is 51.65%, 34.64%, 23.50% respectively, and when the concentration of ethyl naphthalene is 500 μg·mL-1, the protection activity is slightly lower than that of the commercial drug abamectin. The treatment activity data shows that when the concentration of ethyl naphthalene is 500 μg·mL-1, 200 μg·mL-1, 100 μg·mL-1, the inhibition effect on southern root-knot nematode is 51.53%, 33.29%, 16.03% respectively, and when the concentration of ethyl naphthalene is 500 μg·mL-1, the treatment activity is equivalent to that of the commercial drug abamectin. Under the same concentration, the protection activity and treatment activity of ethyl naphthalene do not change, while the protection activity of abamectin decreases compared with the treatment activity.

[0068] Test Example 4 - Ethyl naphthalene on southern root-knot nematode pepper seedling field efficacy experiment

[0069] Select 15-20 cm of 360 pepper seedlings, configure the sample of example 2 into 200 μg·mL-1, 500 μg·mL-1 of ethyl naphthalene solution, and after transplanting the pepper seedlings, irrigate the planting hole with 200 μg·mL-1, 500 μg·mL-1 of ethyl naphthalene solution, and the liquid amount of each hole is 100 mL, and the control is water. Divide the plot into four small areas, 30 pepper seedlings in each small area, use the drug once every 10 days, a total of three times, and harvest the peppers after 60 days of transplanting. After washing the pepper roots, count and record the number of root nodules of each pepper and the yield of each group of peppers, calculate the field control effect and the influence on yield.

[0070] Field control efficiency (%) = 1-(treated group produces root nodule number / control group produces root nodule number) x 100%

[0071] Table 4. Ethyl naphthalene field experiment on pepper seedlings of southern root-knot nematode

[0072] Treatment Root nodulation number Inhibition rate Control group 18.9±6.2 a- 500 pg / mL -1 ]] 6.4±2.4 66.14％ 200 μg / mL -1 ]] 7.3±3.2 61.38％

[0073] a “-” means no data

[0074] The results of test example 4 show that in the field experiment, when the concentration of ethyl naphthalene is 200 μg·mL-1, 500 μg·mL-1, the inhibition rate of the pepper plant invaded by southern root-knot nematode to produce root nodules is 61.38%, 66.14% respectively, and it has good inhibition effect on southern root-knot nematode.

[0075] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of an extract of Solanum nigrum Linn or ethyl naphthol in the preparation of a plant nematocide, characterized in that, the plant is pepper seedlings and tomato seedlings; The nematode is Meloidogyne arenaria (Southern root-knot nematode) Meloidogyne incognita ) the extract of Solanum nigrum Linn is prepared according to the following method: S1. Dry Solanum nigrum Linn leaves at 45-55°C for 3-5 days, crush them, add a solvent, and prepare a crude extract; the mass ratio of Solanum nigrum Linn leaves to solvent is 10-15:1; S2. Dissolve the crude extract prepared in step S1 in water to prepare a crude extract aqueous solution, and extract the crude extract aqueous solution with an organic solvent 3-5 times to prepare an organic phase; concentrate the organic phase under vacuum at 40-50°C to prepare an organic solvent extract; S3. Perform column chromatography separation on the organic solvent extract prepared in step S2 using an organic solvent system to prepare a separation product; S4. Add the separation product prepared in step S3 to a gel column, add an eluent, and perform isocratic elution to prepare a fraction; S5. Add the fraction prepared in step S4 to a semi-preparative liquid chromatograph, further purify the fraction, and prepare an extract of Solanum nigrum Linn; the extract of Solanum nigrum Linn contains the active ingredient ethyl naphthol.

2. Use as claimed in claim 1, characterised in that, The administration concentration of the ethynyl naphthol is 100 μg·mL -1 The above.

3. The use according to claim 1, wherein The solvent in step S1 is at least one of methanol, ethyl acetate, petroleum ether, acetone, dichloromethane, or water; the organic solvent in step S2 is one or more of petroleum ether, chloroform, ethyl acetate, and n-butanol.

4. The use according to claim 1, wherein The organic solvent system in step S3 is at least one of petroleum ether / ethyl acetate, petroleum ether / chloroform, or petroleum ether / n-butanol; the volume ratio of the two components in the organic solvent system is 1:4-6.

5. The use according to claim 1, wherein The gel column in step S4 is a hydroxypropyl dextran gel column; the eluent is at least one of methanol, ethanol, or dichloromethane; the volume concentration of the eluent is 90%-100%.

6. The use according to claim 1, wherein The mobile phase in step S5 is methanol and water; the volume ratio of methanol to water in the mobile phase is 55-65:33-45; the flow rate of the mobile phase is 4-6 mL / min.

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