A method for preventing and controlling Nilaparvata lugens in rice based on slow-release nano matrine

Abstract

The present invention discloses a method for controlling rice brown planthopper based on sustained-release nano-matrine, belonging to the field of nano-pesticides. In the present invention, matrine is loaded into the mesoporous structure of mesoporous silica, and then encapsulated with chitosan to obtain sustained-release nano-matrine. Then, the sustained-release nano-matrine is formulated into a sustained-release nano-matrine solution, which is then applied as a pesticide to the leaves of rice seedlings to control rice brown planthopper. The sustained-release nano-matrine adopted in the present invention can induce rice seedlings to produce the plant defense hormone jasmonic acid precursor 12-oxophytodienoic acid and the brown planthopper-resistant substances luteolin and cis-aconitine. These substances have a targeted toxic effect and can inhibit the reproduction of offspring, thereby better controlling rice brown planthopper.

Description

Technical Field

[0001] The present invention relates to a method for controlling rice brown planthopper based on sustained-release nano matrine, belonging to the field of nano pesticides. Background Art

[0002] In modern agriculture, traditional chemical pesticides play a leading role in controlling weeds, pests and diseases, and ensuring the safety of agricultural food production such as grains, and have played a substantial role in ensuring global food production. However, more than 90% of pesticides are lost during application due to environmental factors (temperature, light, hydrolysis, microorganisms, etc.); traditional pesticide formulations have problems of low utilization rate and large dosage, which will cause problems of agricultural product safety, and then cause a series of adverse effects on human health and the ecological environment. Therefore, the development of safe, environmentally friendly and green nano pesticide formulations is of great significance for ensuring agricultural product safety, reducing environmental pollution, etc., and shows good application prospects in promoting pesticide reduction and efficiency increase, and promoting the sustainable development of the pesticide industry.

[0003] Nowadays, nano pesticides have become an effective and safe control tool for crop pest management. It can improve the transport and distribution of pesticides in plants, improve the utilization efficiency, reduce the direct contact between pesticides and environmental media, reduce environmental pollution, effectively prolong the persistence of pesticides, and reduce non-target toxicity. Mesoporous silica nanoparticles (MSNs) have been widely used in several fields such as molecular loading (nucleic acids, proteins, pesticides, etc.) due to their surface modification, large surface area, high drug loading rate and stability. However, MSNs still need to be modified to improve their control efficacy and safety. For example, the premature release of pesticides in MSNs reduces their drug efficacy.

[0004] Therefore, a nano delivery system based on an encapsulation strategy can prevent the premature release of drugs in MSNs; however, there is no literature mentioning how to encapsulate matrine. Summary of the Invention

[0005] [Technical Problem]

[0006] There is no literature publicly disclosing the preparation and application of nano matrine.

[0007] [Technical Solution]

[0008] To solve the above problems, the present invention uses sustained-release nano matrine to control rice brown planthopper. The method of the present invention is simple to operate, has strong versatility and does not require special equipment; and the prepared sustained-release nano matrine has good pH, temperature, light and long-term storage stability, and has broad application value in the field of controlling rice brown planthopper.

[0009] The first object of the present invention is to provide a method for preventing and controlling rice brown planthoppers based on sustained-release nano-matrine, comprising the following steps:

[0010] Prepare the sustained-release nano-matrine into a sustained-release nano-matrine solution, and then apply it as a pesticide on the leaves of rice seedlings to prevent and control rice brown planthoppers.

[0011] In an embodiment of the present invention, the sustained-release nano-matrine solution is an aqueous solution of sustained-release nano-matrine with a concentration of 10 - 30 mg / L.

[0012] In an embodiment of the present invention, the dosage of the sustained-release nano-matrine is 0.05 - 0.2 mg / plant, and further preferably 0.1 mg / plant.

[0013] In an embodiment of the present invention, the application period is the tillering stage.

[0014] In an embodiment of the present invention, the application is carried out in 2 times with an interval of 9 days between the two applications.

[0015] In an embodiment of the present invention, the density of adult rice brown planthoppers in the rice seedlings is 0 - 20 individuals / plant.

[0016] In an embodiment of the present invention, the sustained-release nano-matrine can induce rice to produce the plant defense hormone jasmonic acid precursor 12-oxo-phytodienoic acid and the insect-resistant substances luteolin and cisaconitine.

[0017] In an embodiment of the present invention, the preparation method of the sustained-release nano-matrine is as follows:

[0018] Disperse the mesoporous silica nanocarrier in the matrine solution, mix evenly to obtain a mixture; then add the chitosan solution to the mixture, stir evenly, concentrate and freeze-dry to obtain the sustained-release nano-matrine;

[0019] Among them, the ratio of the mesoporous silica nanocarrier, the matrine solution and the chitosan solution is 100 mg: 5 mL: 20 mL;

[0020] The concentration of the matrine solution is 20 mg / L;

[0021] The concentration of the chitosan solution is 0.6% (w / v), the pH is 6, and the solvent is an aqueous acetic acid solution with a volume fraction of 0.5%.

[0022] In one embodiment of the present invention, the mesoporous silica nanocarrier uses cetyltrimethylammonium bromide (CTAB) as a template to prepare a mesoporous silica nanocarrier with a two-dimensional tubular pore structure containing the template; then the template is removed by calcination to obtain the mesoporous silica nanocarrier.

[0023] In one embodiment of the present invention, the mixing evenly is first by vortexing and then by ultrasonic treatment.

[0024] In one embodiment of the present invention, the stirring evenly is first by vortexing and then by ultrasonic treatment.

[0025] The second object of the present invention is the application of the method of the present invention in the field of agricultural pest control.

[0026] [Beneficial effects]

[0027] (1) Matrine used in the present invention is a substance extracted from the roots of Sophora flavescens, belonging to a plant-derived pesticide, which has the characteristics of easy degradation and low toxicity, and also has broad-spectrum insecticidal properties.

[0028] (2) The mesoporous silica nanocarrier used in the present invention is composed of mesoporous silicon and chitosan. Specifically, matrine is loaded into the mesoporous structure of mesoporous silicon, and then encapsulated with chitosan. On the one hand, it increases its drug loading rate, and on the other hand, it can protect the matrine component from being easily lost, thereby improving the stability of matrine and avoiding the inactivation of the active ingredient due to interference by external environmental factors.

[0029] (3) Due to the property of chitosan used in the present invention to dissolve in acid itself, the nano-matrine can be slowly released under weak acid conditions in the environment, thus achieving a better nano-pesticide delivery effect and having great application potential in agricultural pest control.

[0030] (4) The sustained-release nano-matrine used in the present invention can induce rice seedlings to produce the plant defense hormone jasmonic acid precursor 12-oxophytodienoic acid and the anti-brown planthopper substances luteolin and cis-aconitine. These substances have targeted toxic effects and can inhibit the reproduction of pests, thereby better controlling the brown planthopper in rice.

[0031] (5) The sustained-release nano-matrine used in the present invention has basically no significant difference in the release rate at room temperature and low temperature, has good optical stability and pH stability, and has high biosafety. Description of the drawings

[0032] Figure 1 Transmission electron microscopy (A), Zeta potential and hydrodynamic diameter (B) of the sustained-release nano-matrine prepared in Example 1.

[0033] Figure 2 Specific surface area and pore size (A) and thermogravimetric analysis graph (B) of the sustained-release nano matrine prepared in Example 1.

[0034] Figure 3 Stability test of the sustained-release nano matrine prepared in Example 1; wherein, (A) pH stability; (B) temperature stability; (C) light stability.

[0035] Figure 4 Bio-safety assessment of the sustained-release nano matrine prepared in Example 1, the matrine raw drug of Comparative Example 1, and the commercial matrine of Comparative Example 2; wherein (A) seed germination toxicity experiment; (B) non-target organism tadpole safety experiment.

[0036] Figure 5 Control effect assessment of the sustained-release nano matrine prepared in Example 1 and the commercial matrine of Comparative Example 2 against the brown planthopper of rice; wherein, (A) number of brown planthopper larvae; (B) rice insect-resistant substances; MNPs (nano matrine); CM (commercial matrine). Detailed implementation manners

[0037] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0038] Testing methods:

[0039] 1. TEM test:

[0040] Dissolve 10 mg of the nanomaterial in deionized water to prepare a suspension of 100 mg / L. After vortexing, ultrasonicate for 2 hours. Then, use a pipette to aspirate an appropriate amount onto a copper grid and let it dry naturally. After that, take a transmission electron microscope (TEM) image of the nanoparticles on a transmission electron microscope at an acceleration voltage of 100 kV.

[0041] 2. Zeta potential and hydrodynamic diameter test:

[0042] Dissolve 10 mg of the nanomaterial in deionized water to obtain a suspension of 100 mg / L. Then, vortex the suspension evenly and ultrasonicate for 2 hours. Next, aspirate 1 mL of the suspension into a potential cup and a colorimetric cuvette respectively, and use a Malvern nanoparticle size analyzer (Nano-ZS90, Malvern Instruments, Malvern, UK) to test the nano Zeta potential and hydrodynamic diameter of the material.

[0043] 3. Specific surface area and pore size test:

[0044] The porosity of the samples was studied by measuring nitrogen adsorption at -196 °C using a surface area and pore size analyzer (Autosorb-iQ, Quantachrome Instruments, USA). Before analysis, the MSNs were degassed at 40 °C for 12 hours.

[0045] 4. Thermogravimetric analysis test:

[0046] Thermogravimetric analysis (TGA) was used to record the weight loss data of the material as a function of temperature. 5 mg of the material was weighed into a small crucible, and then the small crucible was placed in a heating furnace. Subsequently, the temperature was increased from 40 °C to 600 °C at a heating rate of 10 °C / min and a nitrogen flow rate of 20 mL / min.

[0047] 5. Stability test:

[0048] (1) pH stability test:

[0049] 5 mg of MNPs was uniformly dispersed in 5 mL of deionized water to obtain a suspension; then the suspension was transferred into a dialysis bag and dialyzed in a brown wide-mouth bottle. 50 wt% methanol aqueous solutions with different pH values (5.0, 6.0, 7.0, 8.0, 9.0) were used as the release medium. At certain time intervals (0, 1, 2, 4, 12, 18, 24, 36, 48 hours), 1 mL of the dialysis solution was collected and the same volume of fresh release medium was added. Subsequently, the concentration of matrine was determined at 212 nm using a UV-visible spectrometer. Each sample was repeated 3 times.

[0050] (2) Temperature stability test:

[0051] The MNPs aqueous solution (0.2 mg / mL) was added to a brown glass bottle and stored at 4 °C, 25 °C, and 37 °C for 48 hours to evaluate the storage stability at low or high temperatures. Then, the concentration of matrine was determined at 212 nm using a UV-visible spectrometer. Each sample was repeated 3 times.

[0052] (3) Photo-stability test:

[0053] 10 mg of MNPs was dispersed in 20 mL of deionized water to obtain a dispersion; the dispersion was poured into a 50 mL quartz tube, and the samples were irradiated at room temperature in the dark and under ultraviolet light (253 nm) for 0, 1, 2, 4, 12, 18, 24, 36, 48 hours, and the concentration of matrine was determined at 212 nm using a UV-visible spectrometer. Each sample was repeated 3 times.

[0054] 6. Biosafety:

[0055] A seed germination toxicity experiment and a non-target organism tadpole safety experiment were set up;

[0056] Among them, a total of 9 treatments were set up for the seed germination toxicity experiment, namely CK, 25 mg / L MNPs, 100 mg / L MNPs, 200 mg / L MNPs, 500 mg / L MNPs, 25 mg / L CM, 100 mg / L CM, 200 mg / L CM, and 500 mg / L CM. The seeds were placed in a petri dish with a diameter of about 10 cm lined with filter paper. Then, the medicaments of each concentration were prepared respectively, and each preparation was dropped onto the filter paper once a day in a certain amount to moisten the seeds. After 72 hours, their growth status was observed and the leaf tip length, root length, and germination rate were recorded.

[0057] For the safety of non-target organism tadpoles, MSNs, CS, MNPs, matrine raw drug (Matrine), and commercial matrine (CM) solutions were prepared according to concentration gradients (5, 25, 50, 100 mg / L); with pure water as the control, the safety of the materials was verified by observing the survival status of tadpoles at 0, 12, 24, and 48 hours.

[0058] 7. Test for anti-insect substances in rice: Take 100 mg of rice leaf samples, grind them in liquid nitrogen, add 1.5 mL of 80% methanol (0.1% formic acid, L-phenylalanine), vortex for 15 min, and then ultrasonicate in an ice bath for 15 min; centrifuge at 4°C and 13000 g for 10 min, filter the supernatant through a 0.22 μm filter membrane, concentrate, and re-dissolve in 200 μL of 70% methanol. After vortexing and centrifuging, aspirate the supernatant into a sample vial and detect it on the machine.

[0059] Among them, the mass spectrometry scanning mode is the Full MS / dd-MS2 mode. For analysis, ESI- and ESI+ need to be tested once each, and the single duration is 14 min.

[0060] Liquid phase conditions:

[0061] ((ID=16))(1) Chromatographic column: Waters HSS T3; column temperature: 35°C

[0062] ((ID=19))(2) Mobile phase: A = 0.1% formic acid / water; B = 0.1% formic acid / acetonitrile; flow rate 0.35 mL / min

[0063] ((ID=22))(3) Elution gradient is shown in Table 1:

[0064] Table 1

[0065] Time / min ％B 0 5 1.5 5 10 100 11 100 11.5 5 14 5

[0066] ((ID=31))(4) Injection volume: 5 μL.

[0067] The matrine technical drug used in the examples was purchased from Beijing Innochem Science & Technology Co., Ltd., with a purity of 98%; the commercial 1.3% matrine used in the comparative examples was purchased from Tianjin Hengyuan Weiye Biotechnology Development Co., Ltd.

[0068] Example 1

[0069] A method for preparing sustained-release nano matrine, comprising the following steps:

[0070] (1) Preparation of mesoporous silica nanocarrier MSNs:

[0071] Dissolve 2.96 g of cetyltrimethylammonium bromide in 100 mL of Milli-Q water, adjust its pH value to 10.0 with ammonium hydroxide, then heat to 80 °C and keep stirring for 30 minutes to obtain a mixture;

[0072] While stirring at room temperature, cool the temperature of the mixture to 30 °C, dropwise add 1.86 mL of tetraethyl orthosilicate (TEOS), react at a stirring speed of 550 rpm for 24 hours, and then age at 80 °C for 24 hours to obtain a reaction product;

[0073] Filter the reaction solution product through a cellulose nitrate membrane (Whatman TM ) with a pore size of 100 nm, wash it 3 times by centrifugation with absolute ethanol (8000 rpm, 15 min) to obtain white MSNs containing the template (CTAB@MSNs);

[0074] Calcine CTAB@MSNs at 600 °C for 5 hours to prepare mesoporous silica nanocarrier MSNs without the template, cool to room temperature, and set aside.

[0075] (2) Preparation of chitosan CS solution:

[0076] Add 600 mg of CS to 0.5% (v / v) of 100 mL of acetic acid aqueous solution, then adjust the pH of the solution to 6.0 with 2M sodium hydroxide, ultrasonically treat for 30 minutes, and stir overnight at 500 rpm to obtain a 0.6% (w / v) chitosan CS solution;

[0077] (3) Preparation of sustained-release nano matrine:

[0078] Disperse 100 mg of MSNs in 5 mL of 20 mg / L matrine aqueous solution, vortex for several minutes to mix well; then ultrasonically treat for 30 minutes to obtain a mixture;

[0079] Add 20 mL of chitosan CS solution to the mixture, vortex and stir well, then sonicate for 1 hour; finally, stir at 600 rpm for 48 hours at room temperature to obtain a sustained-release nano matrine solution; concentrate and lyophilize the sustained-release nano matrine solution to obtain sustained-release nano matrine (MNPs).

[0080] Perform performance tests on the obtained sustained-release nano matrine (MNPs), and the test results are as follows:

[0081] Figure 1 Transmission electron microscopy (A), Zeta potential and hydrodynamic diameter (B) of the sustained-release nano matrine prepared in Example 1. From Figure 1 it can be seen that: the average size of the sustained-release nano matrine (MNPs) is 85.6 nm ( Figure 1 in A), and the Zeta potential is reversed from -21.3 ± 0.5 mV to 19.5 ± 0.6 mV, further confirming that silica is successfully encapsulated by chitosan ( Figure 1 in B).

[0082] Figure 2 Specific surface area and pore size (A) and thermogravimetric analysis chart (B) of the sustained-release nano matrine prepared in Example 1. Figure 2 The isotherm in Figure 2 shows typical type IV adsorption-desorption curves and H3 hysteresis loop characteristics, indicating that mesoporous silica has a mesoporous structure ( 2 in A), its surface area is 437.1 m 2 / g, while the surface area of nano matrine is 137.1 m Figure 2 / g, which indicates that the mesoporous silica nanocarrier has been successfully encapsulated by chitosan and its mesoporous structure has also changed. The three-stage thermal decomposition of MNPs corresponds to the decomposition stages of chitosan and matrine respectively, indicating that the sustained-release nano matrine has been successfully prepared, and the drug loading rate of MNPs can be obtained to be approximately 19.7% through the decomposition temperatures of each substance (

[0083] Figure 3 Stability test of the sustained-release nano matrine prepared in Example 1; among them, (A) pH stability; (B) temperature stability; (C) light stability. From Figure 3It can be seen from Figure A that: MNPs under different pH conditions basically reached the release equilibrium after 48 hours. This can be explained that chitosan was decomposed by water or acid to release matrine on it, and matrine in the mesoporous silica nanocarrier was also released to a certain extent due to the loss of chitosan protection, and the release amount basically maintained at about 80%. Under acidic conditions, the release rate of matrine was faster. At pH 5, the release of MNPs reached equilibrium within 4 hours. It should be noted that under alkaline conditions, the release of MNPs decreased slightly, which may be due to the chemical reaction between matrine and alkali. From Figure 3 It can be seen from Figure B that: MNPs released matrine faster at 37°C, while there was basically no significant difference in the release rate of matrine at room temperature and low temperature, but the overall cumulative release amount was about 80%. From Figure 3 It can be seen from Figure C that: the cumulative release amount of sustained-release nano-matrine under different light conditions also basically remained at about 80%, that is, although the release rate under ultraviolet light irradiation was slightly faster, the change was not significant, indicating that MNPs had good optical stability.

[0084] Comparative Example 1

[0085] Matrine raw drug (Matrine) was used.

[0086] Comparative Example 2

[0087] Commercial matrine (CM) was used.

[0088] Figure 4 For the biosafety evaluation of the sustained-release nano-matrine prepared in Example 1, the matrine raw drug in Comparative Example 1, and the commercial matrine in Comparative Example 2. It can be seen from the seed germination toxicity experiment that: when the seeds were exposed to the sustained-release nano-matrine (MNPs), no obvious toxic effects were observed. Even when the MNPs exposure concentrations were 25mg / L and 100mg / L, there was a certain promoting effect on the germination and growth of rice seeds, while at a higher concentration (500mg / L), there was a slight inhibitory effect on rice growth, indicating that the MNPs had no toxic effects on seeds at appropriate concentrations. When the seeds were exposed to commercial matrine (CM), the toxic effects were more obvious, and the toxicity was positively correlated with the concentration. In particular, when the concentration of CM was 200mg / L and 500mg / L, the seed germination rates decreased by 49.7% and 59.5% respectively, the leaf tip lengths decreased by 66.2% and 82.2% respectively, and the root lengths decreased by 67.6% and 80.1% respectively. Although the concentrations of 25mg / L and 100mg / L had no significant effect on seed germination, the above-ground part and root length were significantly inhibited, and there were signs of withering and poisoning at the leaf tips ( Figure 4In A), from the safety results of non-target organism tadpoles, it can be seen that: tadpoles exposed to MNPs did not show obvious toxicity at higher concentrations (50 mg / L, 100 mg / L), and the activity of tadpoles was not significantly affected under MNPs exposure. When tadpoles were exposed to the original matrine drug (Matrine), no significant mortality was observed within 12 hours; however, after 24 hours of exposure, toxic effects began to appear with the increase in concentration. Compared with the control group (CK), when exposed to 50 mg / L and 100 mg / L of the original matrine drug (Matrine), the mortality rates of tadpoles increased by approximately 20% and 40% respectively. When tadpoles were exposed to commercial matrine, it was obvious that this compound was highly toxic to tadpoles, and the mortality rate of tadpoles was 100% after 12 hours, indicating that commercial matrine is highly toxic to non-target organisms. In summary, compared with the toxicity test results of the other two matrines (commercial matrine - CM and original matrine drug - Martine), the sustained-release nano matrine (MNPs) obviously has higher biosafety. Figure 4 In B).

[0089] Example 2

[0090] A method for controlling brown planthopper of rice based on sustained-release nano matrine includes the following steps:

[0091] Transplant rice seedlings into flower pots, with 2 plants per pot. When they reach the tillering stage after 35 days of cultivation, spray 4 mL of 25 mg / L aqueous solution of sustained-release nano matrine on the leaf surface of each plant according to the dosage of 0.1 mg matrine per plant. Then, after an interval of 9 days, spray 4 mL again; inoculate 14 adult brown planthoppers per pot; continue cultivation; cover the soil with tin foil during the whole spraying process, and evenly spray along the leaves to the stems with a sprayer; subsequently, count the population number of the offspring of brown planthoppers on rice plants within 18 days to evaluate the control effect of nano matrine on brown planthoppers.

[0092] At the same time, use pure water to replace the aqueous solution of sustained-release nano matrine for cultivation as the control group (CK).

[0093] The results are as Figure 5 .

[0094] Comparative Example 3

[0095] Prepare the commercial matrine in Comparative Example 2 into an aqueous solution of commercial matrine (CM) and replace the aqueous solution of sustained-release nano matrine; then cultivate according to Example 2.

[0096] The results are as Figure 5 .

[0097] From Figure 5It can be seen that the average number of the offspring of Nilaparvata lugens in the control group (CK) was 82, the average number of the offspring of Nilaparvata lugens in the slow-release nano-matrine (MNPs) treatment group was 40, and the inhibition rate of MNPs on the offspring of Nilaparvata lugens was 51.2%; the average number of the final offspring in the commercial matrine treatment group was 69, and its inhibition rate on the offspring of Nilaparvata lugens was 15.8% ( Figure 5 in A); meanwhile, in the slow-release nano-matrine (MNPs) treatment group, MNPs increased the contents of jasmonic acid precursor 12-oxophytodienoic acid, luteolin and cis-aconitine by 82.3%, 48.4% and 71.6% respectively; commercial matrine increased the contents of jasmonic acid precursor 12-oxophytodienoic acid, luteolin and cis-aconitine by 43.5%, 10.1% and 18.5% respectively ( Figure 5 in B).

[0098] In summary, by comparing the results of Example 2 and Comparative Example 3, compared with commercial matrine, slow-release nano-matrine has a better prevention and control effect on the reproduction of Nilaparvata lugens in rice, and a stronger induction effect on rice insect-resistant substances.

[0099] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for controlling Nilaparvata lugens in rice based on sustained-release nano-matrine, characterized in that, It includes the following steps: Prepare the sustained-release nano matrine into a sustained-release nano matrine solution, and then apply it as a pesticide on the leaves of rice seedlings to control the brown planthopper of rice; the application period is the tillering stage; The sustained-release nano matrine solution is an aqueous solution of sustained-release nano matrine, and the concentration is 10-30 mg / L; The dosage of the sustained-release nano matrine is 0.05-0.2 mg / plant; The sustained-release nano matrine can induce rice to produce the plant defense hormone jasmonic acid precursor 12-oxophytodienoic acid and the insect-resistant substances luteolin and cisaconitine; The preparation method of the sustained-release nano matrine is as follows: Disperse the mesoporous silica nanocarrier in the matrine solution, mix evenly to obtain a mixture; then add the chitosan solution to the mixture, stir evenly, concentrate and freeze-dry to obtain the sustained-release nano matrine; The sustained-release nano matrine is that matrine is loaded in the mesoporous structure of nano silica, and then encapsulated with chitosan; The ratio of the mesoporous silica nanocarrier, the matrine solution and the chitosan solution is 100 mg: 5 mL: 20 mL.

2. The method according to claim 1, wherein The application is carried out in two times, and the interval between the two applications is 9 days.

3. The method according to claim 1, wherein The concentration of the matrine solution is 20 mg / L; the concentration of the chitosan solution is 0.6% (w / v), the pH is 6, and the solvent is an aqueous acetic acid solution with a volume fraction of 0.5%.

4. Application of the method according to any one of claims 1-3 in the field of agricultural pest control.

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