Application of rice leaf extract in insecticidal activity

Abstract

The present invention discloses the application of rice leaf extract in insecticidal use. The present invention provides the application of rice leaf extract in any one of the following: insecticidal use; preparation of insecticides. The rice leaf extract provided by the present invention has a certain control effect on aphids, diamondback moths and spider mites. The present invention has important significance for controlling these three pests on crops.

Description

Technical Field

[0001] The present invention relates to the field of agriculture, and particularly to the application of rice leaf extract in insecticidal treatment. Background Art

[0002] Flavonoids are natural metabolites widely distributed in plants, containing the basic skeleton C6-C3-C6. According to the degree of oxidation of the central three-carbon chain, whether it forms a ring, and the position of the B-ring connection (2- or 3-position), etc., the main natural flavonoids can be classified into the following categories: flavones, flavonols, dihydroflavones, dihydroflavonols, anthocyanins, isoflavones, etc. Most flavonoids in plants are combined with sugars to form glycosides, and some exist in the free state (aglycone form). Flavonoids have strong anti-inflammatory and anti-cancer effects and have great application prospects in the treatment of diseases such as coronary heart disease and other cardiovascular diseases and tumors.

[0003] The leaves of rice contain a large number of secondary metabolites, including flavonoids, which play an important role in rice's resistance to ultraviolet rays and diseases (Peng et al., 2017; Zhan et al., 2020). However, currently, it is not very clear whether rice leaf metabolites can kill insects and other issues. Summary of the Invention

[0004] The purpose of the present invention is to provide a rice leaf extract and its application in insecticidal treatment.

[0005] In the first aspect, the present invention claims the application of the rice leaf extract in any one of the following:

[0006] P1. Insecticidal;

[0007] P2. Preparing an insecticide.

[0008] In the second aspect, the present invention claims an insecticide.

[0009] The insecticide claimed by the present invention is the rice leaf extract.

[0010] In the first aspect and the second aspect, the rice leaf extract is the ethanol extract of rice leaves.

[0011] Furthermore, the rice leaf extract is the extract obtained by extracting rice leaves with 75% (volume percentage) ethanol.

[0012] Among them, the rice leaves can be fresh rice leaves.

[0013] Further, the rice leaf extract can be prepared by a method including the following steps: adding rice leaves to 75% (volume percentage) ethanol at a ratio of 1 Kg (fresh weight) to 1 L, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid.

[0014] Further, after soaking, there is also a step of rotary evaporation and concentration.

[0015] Further, the mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract can be (1 - 1.5):1, such as 1.35:1.

[0016] In a specific embodiment of the present invention, the total concentration of the target substances (tricin-5-O-glucoside and isoorientin) in the stock solution of the rice leaf extract obtained after rotary evaporation and concentration reaches about 2000 mg / L, such as 2040 mg / L (the concentration of tricin-5-O-glucoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L).

[0017] In the third aspect, the present invention claims to protect a method for killing insects on crops.

[0018] The method for killing insects on crops claimed by the present invention may include the following steps: applying the rice leaf extract described in the second aspect above to the surface of the crops.

[0019] Specifically, it can be: spraying the solution of the rice leaf extract described in the second aspect above (the total working concentration of the target substances tricin-5-O-glucoside and isoorientin is 1440 mg / L) on the surface of the crops. It is only necessary to moisten the surface of the plants.

[0020] In the first aspect to the third aspect, the rice is indica rice.

[0021] Further, the indica rice can be any variety of indica rice such as Montakcl, J.P.5, PD 46, PATNAI 6, Shui Ya Jien, and / or YOU-I B, etc.

[0022] In a specific embodiment of the present invention, the rice leaves are a quality mixture of Montakcl, J.P.5, PD 46, PATNAI6, Shui Ya Jien, and YOU-I B leaves.

[0023] In the first aspect to the third aspect, the insects are agricultural pests.

[0024] Further, the pests may be aphids, Tetranychus cinnabarinus Boisduval, or Plutella xylostella (Linnaeus).

[0025] In a specific embodiment of the present invention, the aphids are Aphis laburni Kaltenbach.

[0026] In the present invention, the crops may be selected from broad beans, rape, radishes, corn, and kidney beans.

[0027] Fourthly, the present invention claims the use of a mixture composed of tricin-5-O-glucoside and isoorientin in any of the following:

[0028] P1. Insecticidal;

[0029] P2. Preparation of pesticides.

[0030] Among them, in the mixture, the mass ratio of tricin-5-O-glucoside to isoorientin may be (1 - 1.5):1. Such as 1.35:1.

[0031] In the said use, the mixture composed of tricin-5-O-glucoside and isoorientin is dissolved in ethanol (such as 75% ethanol), so that the total working concentration of tricin-5-O-glucoside and isoorientin is 1440 μg / mL.

[0032] Fifthly, the present invention claims a pesticide, the active ingredients of which are tricin-5-O-glucoside and isoorientin.

[0033] Among them, the mass ratio of tricin-5-O-glucoside to isoorientin may be (1 - 1.5):1. Such as 1.35:1.

[0034] In the fourth and fifth aspects, the pests are agricultural pests. Further, the pests may be aphids, Tetranychus cinnabarinus Boisduval, or Plutella xylostella (Linnaeus). Still further, the aphids may be Aphis laburni Kaltenbach.

[0035] In the present invention, the insecticidal can also be understood as controlling the corresponding agricultural pests.

[0036] Experiments have proved that the rice leaf extract provided by the present invention has insecticidal effects, especially strong lethality against three agricultural pests, namely aphids, Tetranychus cinnabarinus Boisduval, and Plutella xylostella (Linnaeus). The present invention is of great significance for controlling these three insect pests of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the result of the insecticidal effect on Aphis fabae.

[0038] Figure 2 It is the insecticidal effect on Tetranychus cinnabarinus.

[0039] Figure 3 It is the insecticidal effect on Plutella xylostella.

[0040] Figure 4 It is the insecticidal effect on Mythimna separata.

[0041] Figure 5 It is the insecticidal effect on Culex pipiens larvae. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will be further described in detail below in conjunction with the specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0043] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0044] Example 1: Preparation of Rice Leaf Extract

[0045] (1) Rice (indica) about 60 days after sowing, collect fresh leaves (equal masses of fresh leaves of indica rice varieties Montakcl, J.P.5, PD46, PATNAI 6, Shui Ya Jien, and YOU-I B are mixed. These indica rice varieties are all recorded in "Supplemental Table 1" of "Wang et al., The power of inbreeding: NGS based GWAS of rice reveals convergent evolution during rice domestication. 2016 Molecular Plant 9:975", which is available to the public from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes), and then soak the rice leaves in 75% ethanol (volume percentage, with 25% being water) for 30 min at a ratio of 1 Kg of fresh leaves to 1 L of 75% ethanol; then collect the soaking solution to obtain a rice leaf extract, and add an appropriate amount of 75% ethanol to the leaves again and repeat the extraction once;

[0046] (2) Collect all the leaf extracts (the liquid after soaking) obtained and concentrate them using a rotary evaporator. During this process, continuously detect the concentration of the concentrate until the total concentration of the target substances (tricin-5-O-glucopyranoside and isoorientin) in the rice leaf extract reaches approximately 2000 mg / L. The method for detecting the content of the target substances is as follows: Pipette 200 μL of the concentrate of the rice leaf extract into a 1.5 mL Eppendorf centrifuge tube, centrifuge at 4 °C and 12,000 rpm for 15 min. Pipette 100 μL of the supernatant into an Agilent injection vial containing a liner with a volume of 200 μL. Then detect it using UPLC-MS. Mobile phase A: 0.1% formic acid aqueous solution (% represents volume percentage); Mobile phase B: acetonitrile. Elution gradient: 0 - 2 min: 5% B - 10% B, 2 - 12 min: 10% B - 25% B, 12 - 18 min: 25% B - 70% B, 18 - 23 min: 70% B - 90% B, 23 - 25 min: 90% B - 100% B, 25 - 30 min: 100% B, post-run for 5 min, and % all represent volume percentages. Flow rate: 0.3 mL / min, column temperature: 40 °C, injection volume: 5 μL. Use an electrospray ionization source (ESI), detect in positive ion mode, the carrier gas is high-purity nitrogen, pressure 40 psi, temperature 325 °C. Finally, using the standard curve of the target substances (tricin-5-O-glucopyranoside and isoorientin) drawn, convert the peak area of the target substances in the obtained mass spectrum into the absolute content of the target substances in the concentrate. After detection, the total concentration of the target substances tricin-5-O-glucopyranoside and isoorientin in the concentrate is 2040 mg / L (the concentration of tricin-5-O-glucopyranoside is 1173 mg / L, and the concentration of isoorientin is 867 mg / L). Dilute the obtained rice leaf extract with water to make the total amount of the active substances (the total amount of the target substances tricin-5-O-glucopyranoside and isoorientin) 1440 mg / L for subsequent experiments.

[0047] The structural formula of tricin-5-O-glucopyranoside is as follows:

[0048]

[0049] The structural formula of isoorientin is as follows:

[0050]

[0051] Example 2. Verification of the insecticidal effect of biological pesticide WSE

[0052] I. Experimental materials

[0053] Test targets: Spodoptera frugiperda, Helicoverpa armigera, Strinia nubilalis (Hubner), Mythimna separata (Walker), Culex pipiens pallens, Aphis laburni Kaltenbach, Tetranychus cinnabarinus (Boisduval), Plutella xylostella (Linnaeus).

[0054] Test crops: Maize, broad bean, kidney bean, rape, etc.

[0055] Test agents: The original solution of the biological pesticide WSE prepared in Example 1 (the final concentration of the solid extract is 1440 mg / L) and its 2-, 4-, and 8-fold dilution solutions (the dilution solution is water).

[0056] II. Pesticide application method

[0057] 1. Activity against Helicoverpa armigera

[0058] Helicoverpa armigera, a normal population reared indoors. Test method: Leaf dipping method. For the leaf dipping method, maize leaves are immersed in liquid medicines of different concentrations. After the liquid medicine dries, they are ground and placed in 24-well plates. One 3rd instar larva is introduced into each well, 10 larvae are used each time, with three replicates, for a total of 30 test insects. It mainly has stomach toxicity and contact toxicity effects, and at the same time, the feeding phenomenon of the larvae is observed. The mortality rate is checked after 72 hours. Maize leaves soaked with water for feeding are used as the blank control.

[0059] 2. Activity against Strinia nubilalis

[0060] Strinia nubilalis (Hubner), a normal population reared indoors. Test method: Leaf dipping method. For the leaf dipping method, maize leaves are immersed in liquid medicines of different concentrations. After the liquid medicine dries, 10 3rd instar larvae are introduced into a petri dish, with three replicates, for a total of 30 test insects used. It mainly has stomach toxicity and contact toxicity effects, and at the same time, the feeding phenomenon of the larvae is observed. The mortality rate is checked after 72 hours. Maize leaves soaked with water for feeding are used as the blank control.

[0061] 3. Activity against Mythimna separata

[0062] Mythimna separata (Walker), a normal population reared indoors. By the leaf-dipping method, corn leaves were dipped in the medicated solutions prepared at different concentrations. After the medicated solutions dried, 10 fourth-instar larvae were introduced into a petri dish, with three replicates, and a total of 30 test insects were used. The main effects were stomach poisoning and contact killing, and the feeding behavior of the larvae was observed simultaneously. The mortality rate was checked after 24 hours. Corn leaves soaked in water used for feeding served as the blank control.

[0063] 4. Activity against Culex mosquito larvae

[0064] Culex pipiens pallens, a normal population reared indoors. The prepared medicated solutions at different concentrations were added to a 100 mL beaker. Ten fourth-instar larvae were selected and poured into the beaker together with 10 mL of rearing solution (tap water standing for more than 20 days), with three replicates, and a total of 30 test insects were used. The treatment was placed in a standard treatment room, and the mortality rate was checked after 24 hours. The group with an equal amount of aqueous solution (replacing the medicated solution) and rearing solution served as the blank control.

[0065] 5. Activity against Aphis fabae

[0066] Test method for Aphis fabae Kaltenbach: dipping method. Broad bean seedlings with nymphs were dipped in medicated solutions at different concentrations for 3 - 5 seconds, and the excess solution was shaken off. The plants were inserted into a foam board and covered with a kerosene lamp shade (the upper opening of the shade was sealed with gauze and rubber bands). The test was placed in a treatment room. Each medicament had 3 replicates. Broad bean plants with insects dipped in aqueous solution served as the blank control. The results were checked after 24 hours. The number of dead and alive insects was checked with a magnifying glass, and the standard for death was that the test insect could not crawl its own body length.

[0067] 6. Activity against mites

[0068] Test method for Tetranychus cinnabarinus (Boisduval): dipping method. Kidney bean seedlings with spider mites were dipped in medicated solutions at different concentrations for 5 seconds, gently shaken to remove the excess solution, and the plants were inserted into a hydroponic tank with a square glass cover. The periphery of the glass cover was coated with lanolin to prevent the mites from escaping. The test was placed in a treatment room. Each medicament had 3 replicates. Kidney bean plants with spider mites dipped in aqueous solution served as the blank control. The number of dead spider mites in the medicament-treated samples was checked after 24 hours.

[0069] 7. Activity against Plutella xylostella

[0070] Diamondback moth (Plutella xylostella (Linnaeus)), a normal population reared indoors. The leaf-dipping method proposed by the Insecticide Resistance Action Committee (IRAC) was adopted. Using the prepared test liquid medicine, dip rape leaves with straight-tip ophthalmic forceps for 3 - 5 seconds, then shake off the excess liquid. One leaf each time, with a total of 3 leaves for each sample. Place them on the treatment paper in the order of sample marking. After the liquid medicine dries, put them into a 10-cm long straight tube with a label, introduce 3rd instar diamondback moth larvae, and cover the tube mouth with gauze. Place the test treatments in a standard treatment room, with 3 replicates for each pesticide. Check the results after 72h. Gently touch the insect body with a needle, and those that do not move are considered dead. Calculate the mortality rate. Use rape leaves dipped in water as the blank control group.

[0071] 8. Activity against Spodoptera frugiperda

[0072] Fall armyworm (Spodoptera frugiperda), a normal population reared indoors. The leaf-dipping method proposed by the Insecticide Resistance Action Committee (IRAC) was adopted. Dip corn leaves in liquid medicines of different concentrations, gently shake to remove the excess liquid medicine. After the liquid medicine dries, introduce 10 3rd instar larvae into a petri dish, with three replicates, using a total of 30 test insects. It mainly has stomach toxicity and contact killing effects, and at the same time observe the feeding phenomenon of the larvae. Check the mortality rate after 72 hours. Gently touch the insect body with a needle, and those that do not move are considered dead. Use corn leaves soaked in water for feeding as the blank control.

[0073]

[0074]

[0075] Note: If the mortality rate of the blank control is <5%, no correction is required; if the mortality rate of the blank control is between 5% - 20%, it should be corrected according to the corrected mortality rate formula; if the mortality rate of the blank control >20%, the data is not reliable and the test needs to be redone.

[0076] III. Results and Analysis

[0077] Investigation of the insecticidal activity results shows that the high-concentration biological pesticide WSE has a certain control effect on Aphis craccivora, Plutella xylostella and Tetranychus cinnabarinus, but the effect on other pests is not ideal. Among them, at the original liquid concentration of 1440 mg / L, the lethality rate to Aphis craccivora is 90%, the lethality rate to Tetranychus cinnabarinus is 95%, and the lethality rate to Plutella xylostella is 70%. However, when the concentration decreases, the lethality rate is not ideal. See Table 1 for details.

[0078] Table 1. Control effects of WSE at different concentrations on different pests

[0079]

[0080]

[0081] In addition, the insecticidal effect on Aphis fabae is as Figure 1 shown. The insecticidal effect on Tetranychus cinnabarinus is as Figure 2 shown. The insecticidal effect on Plutella xylostella is as Figure 3 shown (the left side is 1440 mg / L WSE; the right side is the blank control). The insecticidal effect on Mythimna separata is as Figure 4 shown (the left side is 1440 mg / L WSE; the right side is the blank control). The insecticidal effect on Culex mosquito larvae is as Figure 5 shown (the upper side is 720 mg / L WSE; the lower side is 1440 mg / L WSE).

[0082] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of a rice leaf extract in any of the following: P1. Insecticidal; P2. Preparation of an insecticide; The rice leaf extract is prepared by a method comprising the following steps: adding rice leaves in a ratio of 1 kg fresh weight to 1 L of 75% (v / v) ethanol, soaking for 30 min, extracting twice continuously, and obtaining the rice leaf extract from the soaked liquid; after soaking, there is also a step of rotary evaporation and concentration; The rice is indica rice; the indica rice is a mass mixture of Montakcl, J.P. 5, PD 46, PATNAI 6, Shui Ya Jien, and YOU-I B, etc.; The insect is an agricultural pest; The agricultural pest is aphid, Tetranychus cinnabarinus, or Plutella xylostella.

2. The use according to claim 1, characterized in that: The mass ratio of tricin-5-O-glucoside to isoorientin in the rice leaf extract is (1 - 1.5):

1.

3. A method for insecticidal treatment of crops, comprising the following steps: applying the rice leaf extract described in claim 1 or 2 to the surface of the crops; wherein, The total working concentration of the target substances tricin-5-O-glucoside and isoorientin in the rice leaf extract is 1440 mg / L.

4. The method according to claim 3, characterized in that: The crops are selected from broad beans, rapeseed, radishes, corn, and kidney beans.

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