Application of small molecule substance TH287 in prevention and treatment of rice blast

Abstract

The application belongs to the field of plant protection, and particularly relates to application of a small molecule substance TH287 in preventing and treating rice blast. The small molecule inhibitor TH287 is obtained based on screening of a Magnaporthe oryzae NUDIX hydrolase structure, and has strong specificity. The small molecule inhibitor TH287 can be applied to inhibiting Magnaporthe oryzae NUDIX hydrolase activity, inhibiting pathogenicity of Magnaporthe oryzae, enhancing resistance of rice to Magnaporthe oryzae, preventing and treating rice blast caused by Magnaporthe oryzae, preparing a bactericide for preventing and treating rice blast caused by Magnaporthe oryzae, and improving rice yield. The application has great application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant protection, and particularly relates to application of a small molecule TH287 in preventing and treating rice blast. BACKGROUND

[0002] Rice (Oryza sativa) is an important global food crop, and its high yield and safe and stable yield are directly related to the stability and development of the national economy. Rice blast caused by Magnaporthe oryzae is the most devastating rice disease, ranking first among fungal diseases. Globally, rice blast causes an average annual yield reduction of rice of about 10-30%, and in severe cases, the yield reduction is up to 40-50%, or even no harvest. Therefore, controlling rice blast is of great significance for improving rice yield and ensuring food production safety. At present, the prevention and control of the disease is mainly based on breeding of disease-resistant varieties and chemical control. Due to the complex pathogenic types of M. oryzae in the field and the rapid change of population composition, the disease resistance of disease-resistant varieties is lost after 3-5 years of popularization. Chemical control is the fastest and most direct control method, but it is also the most harmful to the environment, and it is easy to cause resistance of the pathogen.

[0003] At present, the fungicides used for the prevention and control of M. oryzae mainly include sterol demethylation inhibitors (DMIs), mitochondrial respiration inhibitors (QoIs), and melanin biosynthesis inhibitors (MBIs), but the long-term use of the above fungicides has gradually caused resistance of M. oryzae. Therefore, it is urgent to develop new targets to develop new types of high-efficiency and low-toxicity fungicides.

[0004] In the interaction between plant pathogens and hosts, a complex "arms race" is staged. When attacked by a pathogen, the receptors on the cell membrane of the plant recognize the conserved pattern molecules PAMPs (Pathogen-associated Molecular Patterns) secreted by the pathogen, triggering PTI (PAMP-triggered immunity) and accompanied by accumulation of a large amount of active oxygen. In order to inhibit the PTI of the host, the pathogen secretes a large amount of effectors into the plant cells or extracellular space via vesicle transport, thereby inhibiting the defense response of the host. Vesicle transport is a key process of secretion of effectors of M. oryzae, and abnormality of this process will cause abnormal secretion of effectors, accumulation in the cell and cause stress. Therefore, analyzing the molecular mechanism of secretion and transport of effectors not only helps to understand the pathogenic mechanism of the pathogen, but also has important reference value for designing new types of low-toxicity and high-efficiency fungicides. SUMMARY

[0005] In order to overcome at least one problem existing in the prior art, the present application identifies a small molecule substance TH287 (small molecule inhibitor TH287) by protein structure-mediated small molecule substance screening, which can inhibit the NUDIX hydrolase activity of Magnaporthe oryzae and is of great significance to inhibit the pathogenicity of the pathogen and increase the yield of rice.

[0006] To achieve the above-mentioned object, the present application adopts the following technical solutions:

[0007] The present application first provides the application of NUDIX12 protein in screening drugs for preventing and treating rice blast caused by Magnaporthe oryzae.

[0008] Specifically, based on the structure model of NUDIX12 protein and the structure model of nucleic acid oxidative damage repair enzyme MTH1 protein, the structure model of the compound to be tested is docked with them, the docking results are analyzed using Auto Dock Tools, and the docking model is visualized using PyMOL software; if the compound to be tested occupies the pocket position of the NUDIX12 enzyme active site, so that MTH1 cannot directly contact the substrate NADH, then the candidate compound for preventing and treating rice blast is determined.

[0009] Optionally, the substrate NADH structure model is also used as a control to determine whether the candidate compound can hinder the correct combination of NADH and the NUDIX12 enzyme active site.

[0010] Specifically, the PDB ID of the NUDIX12 protein structure model is G4NFV7, and the PDB ID of the structure model of the nucleic acid oxidative damage repair enzyme MTH1 protein is P36639.

[0011] Through the above-mentioned method, the present application screens a small molecule substance TH287 (6-(2,3-dichlorophenyl)-N4-methylpyrimidine-2,4-diamine), the molecular formula of which is shown in the following formula (I):

[0012]

[0013] The compound is a known compound, which has been used as an inhibitor of MTH1 in mammals and has been reported in the treatment of cancer. However, there is no report on the prevention and treatment of rice blast.

[0014] Therefore, the present application further provides a method for controlling or preventing rice blast in rice, which comprises applying a compound of formula (I) or an agrochemically active salt thereof, such as base addition salts of inorganic bases and organic bases, more specifically potassium salt, sodium salt, ammonium salt, dimethylamine salt, and isopropylamine salt, to rice, its planting site, or its propagation material.

[0015] Specifically, the compound of formula (I) is prepared into a medicine in the form of one of emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, bait, stock solution and stock powder; the content of the medicine is 1-99% by weight, preferably 10-80% by weight, preferably 15-50% by weight; the effective dose concentration when used in the medicine is 0.1 ug / ml-100 ug / ml, and the application amount is 10-500 g / ha, preferably 50-200 g / ha.

[0016] The application also provides the use of the compound of formula (I) or an agrochemically active salt thereof for enhancing the resistance of rice to Magnaporthe grisea, preferably the agrochemically active salt such as the base addition salt of inorganic base and organic base, more particularly the potassium salt, sodium salt, ammonium salt, dimethylamine salt, isopropylamine salt thereof.

[0017] The application further provides the use of the compound of formula (I) or an agrochemically active salt thereof in the preparation of a pesticide for controlling or preventing the infection of rice by Magnaporthe grisea or causing diseases.

[0018] Specifically, the pesticide is a medicine in the form of one of emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, bait, stock solution and stock powder, which further comprises a pharmaceutically acceptable auxiliary ingredient.

[0019] Preferably, the pesticide is prepared into a form suitable for spray application.

[0020] Compared with the prior art, the application has the following beneficial effects:

[0021] (1) The application provides an effective method for screening a medicine for preventing and treating rice blast caused by Magnaporthe grisea, wherein the small molecule inhibitor TH287 identified by screening based on the structure of NUDIX hydrolase of Magnaporthe grisea has strong specificity; (2) The small molecule inhibitor TH287 is obtained by screening based on the structure of NUDIX hydrolase of Magnaporthe grisea, and the homologous proteins in other species are quite different, so the small molecule inhibitor TH287 has the characteristics of low toxicity, high safety and the like as a fungicide; (3) The small molecule inhibitor TH287 has strong inhibitory activity on the pathogenicity of Magnaporthe grisea, and can improve the yield of rice to a certain extent; (4) The small molecule inhibitor TH287 can also play an important biological function in regulating the pathways of conidial production and appressorium formation of the pathogen. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a schematic diagram of the TH287 and NUDIX binding region in an embodiment of the application;

[0023] Figure 2A schematic diagram of the results of NUDIX12 enzyme activity verification in an embodiment of the present application; wherein the NUDIX12 hydrolytic enzyme can degrade a variety of dinucleotide substances;

[0024] Figure 3 A schematic diagram of the results of the inhibition of NUDIX12 and its rice homolog proteinase activity by the compound TH287 in an embodiment of the present application; wherein the substrate uses NADH;

[0025] Figure 4 A schematic diagram of the results of the verification of the interaction between NUDIX12 and its rice homolog protein and the compound TH287 in an embodiment of the present application; wherein the micro thermal swing (MST) technology is used to verify the binding ability of NUDIX12 and its rice homolog protein and TH287 in vitro, and the Kd dissociation constant is smaller, the stronger the binding is;

[0026] Figure 5 A schematic diagram of the results of the inhibition of the pathogenicity activity of Magnaporthe oryzae by the compound TH287 in an embodiment of the present application. DETAILED DESCRIPTION

[0027] The present application will be further described below through specific embodiments, so as to better understand the present application, the schematic embodiments of the present application and the description thereof are only used to explain the present application, and do not constitute a limitation on the present application.

[0028] Example 1: Screening of the compound TH287

[0029] TH287 was identified based on the structure screening of Magnaporthe oryzae NUDIX hydrolase. The steps include: the structure model (PDB ID: G4NFV7) of MGG_08699 protein (NUDIX12, see: Chen, Long, et al. "Bioinformatics and expression characteristics of NUDIX hydrolase family in Magnaporthe oryzae." Molecular Plant Breeding 14.5 (2016): 7.) was derived from Alpha Fold protein structure database (https: / / alphafold.ebi.ac.uk / ), and the 3D structure model of ligand molecule was downloaded from PubChem website. Auto Dock Tools (version 1.5.6) software was used to add polar hydrogen to the protein structure and calculate Gasteiger charge. Auto Dock Tools ligand preparation program was called for batch conversion, and Auto Dock Vina was run for batch docking. The top 10 docking models from low to high affinity value were output for each molecule, and a python program was used to extract the top 10 docking models from low to high affinity value and the corresponding molecular structure in all docking results. AutoDock Tools was used to analyze the docking results, and PyMOL (version 2.6.0a0) software was used to visualize the docking models. From which the small molecule TH287 was screened.

[0030] The screening and identification results are shown in Figure 1 Figure 1 Part A and part B of the above figure respectively represent the binding state of nucleic acid oxidative damage repair enzyme MTH1 protein and substrate NADH and small molecule TH187, both of which are combined at the same position, that is, TH287 occupies the position of NADH, causing MTH1 to be unable to directly contact the substrate NADH. And Figure 1 Part C of the above figure identifies the binding region of NUDIX12, TH287 and substrate NADH, TH287 is combined in the enzyme active bag of NUDIX12, which hinders the combination of NUDIX12 and NADH. This shows that there is a significant difference between the binding sites of TH287 and MTH1 and NUDIX12.

[0031] Example 2: Determination of the function of compound TH287 inhibiting Magnaporthe oryzae target protein MoNud5

[0032] ​1. The coding sequence of NUDIX12-GFP was constructed in pYF11 vector, and transformed into Magnaporthe oryzae by protoplast transformation. After 48 hours of shaking culture in CM medium, the mycelial pellets were collected and dried with absorbent paper. After grinding in liquid nitrogen, 1 mL of protein lysis solution (Bi Yun Tian P0013B) was added. The protein was extracted and then purified by GFP beads (ChromoTek GFP-Trap Agrose, GTA-20) to obtain the corresponding protein.

[0033] NUDIX12-HIS protein and HIS protein were constructed in pET32a vector and transformed into E. coli BL21 to obtain prokaryotic expression strains. The prokaryotic expression induction conditions were as follows: 200 mL of bacterial solution was added with 100 μM of isopropyl-β-D-thiogalactoside (IPTG), the induction temperature was 25°C, and the induction time was 4 hours. The bacterial body was obtained by centrifugation and added with 1 mL of PBS buffer, and then broken by ultrasonic wave (power 20%, breaking time 5 s, interval time 5 s) until clear. The protein was obtained by centrifugation at 3600 rpm for 15 minutes. Then, the NUDIX12-HIS protein and HIS protein were purified by HIS beads.

[0034] 2. The function of TH287 in inhibiting NUDIX12 was verified by the following method, which includes the following steps:

[0035] The activity determination method of Nudix hydrolase: the determination reaction was performed in a 50 μL reaction system, and the system components included 50 mM Tris-HCl (pH 8.5), 2 μg NUDIX12-GFP, NUDIX12 ED GFP protein, GFP protein, NUDIX12-HIS protein, rice homologous protein OsNUDIX12 (OsJ05P0700)-HIS and HIS protein, 5 mM MgCl2, 1 mM dithiothreitol, 2 mM substrate (NADH), and 4 U alkaline phosphatase (sigma: P0114-10KU). In the TH287 treatment group, dimethyl sulfoxide (DMSO) was used as a control, and the concentrations of TH287 were 1 mM, 100 μM, and 10 μM, respectively.

[0036] After incubation at 37°C for 30 minutes, the reaction was terminated by adding 150 μL of 1N HCl, and then 100 μL of water and 700 μL of a mixed solution of 0.42% molybdate and 10% ascorbic acid (6:1) were added. The reaction tube was placed in a 45°C water bath for color development for 20 minutes, and then cooled to room temperature. The solution was measured at A820 using a spectrophotometer. The results are shown in Figure 2 and Figure 3

[0037] ​The research of the present inventors shows that the NUDIX12 protein expressed by Magnaporthe oryzae has NUDIX hydrolase activity. Compared with the control protein GFP (Green fluorescent protein), the NUDIX12 protein can more effectively hydrolyze intracellular dinucleotides, including reduced nicotinamide adenine dinucleotide (NADH), adenosine diphosphate ribose (ADPR) and flavin adenine dinucleotide (FLAVIN). The key site of NUDIX12 enzyme activity (deletion mutation of 104 to 111 amino acids to obtain NUDIX12 ED ), and the enzyme activity levels of the three substrates are reduced to 55.95%, 54.71% and 38.27% of the original activity, respectively Figure 2

[0038] The compound TH287 targets the NUDIX12 protein of Magnaporthe oryzae, specifically binds to it, and inhibits its hydrolase activity. Compared with the control group dimethyl sulfoxide (DMSO), after adding 1 mM, 100 μM and 10 μM of TH287, respectively, the hydrolase activity of NUDIX12 is significantly reduced, and the inhibition rates are 88.57%, 62.86% and 48.51%, respectively. The hydrolase activity of the homologous protein OsNUDIX12 of rice has no obvious difference Figure 3

[0039] Example 3: Specific binding of compound TH287 to pathogen MoNud5

[0040] This example is to verify that TH287 specifically binds to and inhibits the activity of NUDIX12 of Magnaporthe oryzae. The dissociation constant (Kd) values of TH287 and NUDIX12 and TH287 and OsNUDIX12 are determined by microscale thermophoresis (MST) experiment, wherein OsNUDIX12 is the highest homologous NUDIX hydrolase in rice. The compound TH287 is purchased from Merck (catalog number: HY-16965).

[0041] ​​1. The coding sequences for NUDIX12-HIS and OsNUDIX12-HIS proteins were constructed in the pET32a vector and transformed into *E. coli* BL21 to obtain prokaryotic expression strains. Prokaryotic expression induction conditions were as follows: 100 μmol of isopropyl-β-D-thiogalactoside (IPTG) was added to 200 mL of bacterial culture; induction temperature was 25°C; and induction time was 4 hours. After centrifugation, 1 mL of PBS buffer was added, and the cells were sonicated (20% power, 5 s disruption time, 5 s interval) until clear. The supernatant was collected after centrifugation at 3600 rpm for 15 minutes to obtain the proteins. The proteins were then purified using HIS beads (MCE: HY-K0210) to obtain NUDIX12-HIS and OsNUDIX12-HIS proteins.

[0042] 2. MST Experimental Method: NUDIX12-HIS and OsNUDIX12-HIS proteins were expressed and purified in *E. coli* BL21. The interaction strength between the compounds and NUDIX12 and OsNUDIX12 was determined using microthermophoresis with a Monolith NT.LabelFree (Nano Temper Technologies GMBH) instrument. First, the RED-NHS second-generation fluorescent probe was labeled onto NUDIX12-HIS and OsNUDIX12-HIS proteins using the Monolith™ RED-NHS second-generation protein labeling kit (Cat#MO-L011) and stored in a buffer containing 130 mM NaHCO3, 50 mM NaCl, and pH 8.2–8.3 at room temperature. Compound TH287 was diluted into different concentration gradients (1 mM as the highest concentration, with each sample diluted to half the previous concentration, for a total of 16 concentration gradients), and incubated with labeled NUDIX12-His and OsNUDIX12-His proteins for 10 minutes. The samples were then added to NT.Label Free labeled capillaries, and values ​​were measured at 20% LED and 40% MST parameters. Curves were plotted using the KD fitting function of Nano Temper analysis software (version 1.5.41), and the dissociation constant (Kd) value was calculated. A smaller Kd value indicates a stronger interaction.

[0043] The MST experiment results showed that the Kd value of compound TH287 with NUDIX12 protein was 7.36 μM, which was much lower than the Kd value of TH287 with rice OsNUDIX12 (73.86 μM), indicating that TH287 has a stronger binding ability to pathogen NUDIX12 compared with the NUDIX hydrolase OsNUDIX12 in rice. Figure 4 ).

[0044] Example 4: Inhibition of pathogenicity of Magnaporthe grisea in rice leaves

[0045] This example demonstrates the inhibition of pathogenicity of Magnaporthe grisea in rice leaves by compound TH287, which comprises the following steps:

[0046] The wild-type strain of Magnaporthe grisea Guy11 was used, which was preserved in our laboratory. The strain of Magnaporthe grisea was cultured on complete medium (CM) at 28°C in the dark. The strain was preserved on potato dextrose agar medium (PDA) at 10°C and subcultured every 3 months; the strain was long-term preserved on dry filter paper and stored in a refrigerator at -20°C.

[0047] The method of spraying spore solution of Magnaporthe grisea on rice leaves was used, which comprises the following steps:

[0048] (1) First, induce the production of conidia of Magnaporthe grisea, inoculate the mycelial block (2 mm x 2 mm) of Magnaporthe grisea strain Guy11 on CM medium on SDC medium, cultivate at 28°C in the dark for 4 days, then scrape off the surface mycelium, and induce for 3 days under a black light lamp to obtain conidia.

[0049] (2) The rice seedlings cultured in the greenhouse for 14 days were used for spraying inoculation experiment, 4 mL of conidia on the above SDC plate was collected, with a concentration of 1 x 10 5 / mL and containing 0.2% (w / v) gelatin with different concentrations (1 mM, 100 μM and 10 μM) of compound TH287. Spray on rice leaves, cultivate in the dark for 24 hours, then cultivate at 25°C with light and dark alternation for 5-7 days. Each treatment was repeated three times.

[0050] (3) Count the lesion area and calculate the inhibition rate, control effect (%) = (lesion area of control rice - lesion area of treated rice) / (lesion area of control rice) x 100.

[0051] The experimental results are shown in Figure 5, Figure 5 Part A of Figure 5 shows that the pathogenicity of Magnaporthe grisea was significantly reduced under the TH287 treatment compared with the control DMSO treatment; Figure 5 Part B of Figure 5 also shows that TH287 inhibits the pathogenicity of Magnaporthe grisea on rice using Fiji image J analysis of the lesion area of rice.

[0052] Method for preparing PDA medium: wash and peel potatoes, weigh 200 g and cut into small pieces, cook until soft (boil for 30 minutes), filter in a beaker with four layers of gauze, add 20 g of glucose and 15-20 g of agar powder, add water to 1 L, stir to dissolve thoroughly, then divide into triangular flasks, sterilize at 121°C for 20 minutes, and cool for use.

[0053] CM medium preparation method: 50 mL of 20x nitrate (120 g of sodium nitrate, 10.4 g of potassium chloride, 10.4 g of magnesium sulfate heptahydrate, 30.4 g of potassium dihydrogen phosphate, dissolved in 1 L of distilled water), 1 mL of 1000x trace elements (2.2 g of zinc sulfate heptahydrate, 1.1 g of boric acid, 0.5 g of manganese chloride tetrahydrate, 0.5 g of ferric sulfate heptahydrate, 0.17 g of cobalt chloride hexahydrate, 0.16 g of copper sulfate pentahydrate, 0.15 g of sodium manganate dihydrate, 5 g of EDTA tetrasodium, dissolved in 100 mL of distilled water), 1 mL of vitamin solution (0.01 g of biotin, 0.01 g of vitamin B6, 0.01 g of vitamin B1, 0.01 g of riboflavin, 0.01 g of p-aminobenzoic acid, 0.01 g of nicotinic acid, dissolved in 100 mL of distilled water), 10 g of glucose, 2 g of proteose peptone, 1 g of yeast extract, 1 g of casein amino acid, 15 g of agar powder, and distilled water to 1 L, and then sterilized at 121°C for 20 minutes. After cooling, it was ready for use.

[0054] Spore production medium preparation method: corn powder and rice straw were used to prepare the medium. 100 g of rice straw was added to 1 L of water and boiled for 30 minutes. Then, 40 g of corn powder and 15 g of agar powder were added and boiled for another 20 minutes. Finally, distilled water was added to make up to 1 L, and then sterilized at 121°C for 20 minutes. After cooling, it was ready for use.

[0055] SDC medium preparation method: 120 g of rice straw was cut into small pieces and boiled for 60 minutes. Then, it was filtered with double-layer gauze in a beaker. 40 g of corn powder and 15 g of agar powder were added and boiled for another 10 minutes. Distilled water was added to make up to 1 L, and then stirred to mix thoroughly. Then, it was sterilized at 121°C for 20 minutes. After cooling, it was ready for use.

[0056] Example 5 - Determination and results of compound TH287 inhibiting conidial production and appressorium formation of Magnaporthe grisea

[0057] This example verifies the inhibition of compound TH287 on conidial production and appressorium formation of Magnaporthe grisea, which includes the following steps:

[0058] First, conidial spores were induced. Mycelial plugs (2 mm x 2 mm) of Magnaporthe grisea strain Guy11 on CM medium were inoculated onto SDC medium, and different concentrations of TH287 (final concentrations of 1 mM, 100 μM, 10 μM, and 1 μM) were added. The culture was incubated at 28°C in the dark for 4 days, and then induced under a black light for 3 days to obtain conidial spores. The collected conidial spore solution was divided into two equal parts. One part was counted under a hemocytometer to directly count the conidial spore yield under different TH287 treatments. The results showed that TH287 at a concentration of 100 μM or higher significantly inhibited the production of conidial spores of Magnaporthe grisea (as shown in Table 1).

[0059] The spore solution was centrifuged at 4000 rpm for 3 minutes, washed with sterilized ddH2O for 3 times, and adjusted to a concentration of 2-5x10 4 The spore solution was centrifuged at 4000 rpm for 3 minutes, washed with sterilized ddH2O for 3 times, and adjusted to a concentration of 2-5x10

[0060] Table 1 Inhibitory activity of compound TH287 on spore germination of Magnaporthe grisea

[0061]

[0062] Note: Each treatment was repeated three times in the experiment, and the data in the table were the average of three repetitions.

[0063] Table 2 Inhibitory activity of compound TH287 on appressorium formation of Magnaporthe grisea

[0064]

[0065] Note: Each treatment was repeated three times in the experiment, and the data in the table were the average of three repetitions.

[0066] As can be seen from the above implementation, the TH287 screened by the present application has the characteristics of simple structure, convenient preparation, low cost, strong specificity, etc., and shows good inhibitory effect on Magnaporthe grisea, and has the value of further research and development.

Claims

1. A method for controlling or preventing rice blast disease, comprising applying a compound of formula (I), or an agriculturally active salt thereof, to rice, rice growing sites, or rice propagation material: Formula (I).

2. The method as described in claim 1, characterized in that, The agricultural chemically active salt is an inorganic or organic base addition salt.

3. The method as described in claim 2, characterized in that, The agricultural chemically active salt is a potassium salt, sodium salt, ammonium salt, or isopropylamine salt of the compound of formula (I).

4. The method according to claim 1, characterized in that, The compound of formula (I) is prepared into a drug in one of the following dosage forms: emulsifiable concentrate, suspension concentrate, powder, granule, aqueous solution, and poison bait.

5. The method according to claim 4, characterized in that, The compound of formula (I) is present in the drug at a content of 1 to 99% by weight.

6. The method according to claim 4, characterized in that, The compound of formula (I) is present in the drug at a content of 10 to 80% by weight.

7. The method according to claim 4, characterized in that, The compound of formula (I) is present in the drug at a content of 15 to 50% by weight.

8. The method according to claim 4, characterized in that, The effective dose concentration of the drug is 0.1 ug / ml to 100 ug / ml, and the dosage of the active ingredient is 10 to 500 g / ha.

9. The method according to claim 8, characterized in that, The effective ingredient dosage is 50-200g / ha.

10. The application of compound of formula (I) or its agrochemically active salt in the preparation of pesticides for controlling or preventing rice blast infection or disease caused by rice blast fungus. Formula (I).

11. The application as described in claim 10, characterized in that, The pesticide is formulated as an emulsifiable concentrate, suspension concentrate, powder, granule, aqueous solution, or poison bait; it also includes pesticide-acceptable auxiliary ingredients.

12. The application as described in claim 11, characterized in that, The pesticide is formulated for spray application.

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