Application of a heterocyclic compound to the prevention and treatment of plant root-knot nematodes

Abstract

The application discloses application of a heterocyclic compound to prevention and treatment of plant root-knot nematodes and belongs to the technical field of plant root-knot nematode prevention and treatment. The heterocyclic compound is 6-bromo-N-(pyridine-2-ylmethyl)benzo[D]thiazole-2-amine. It is found for the first time that 6-bromo-N-(pyridine-2-ylmethyl)benzo[D]thiazole-2-amine has strong activity to kill southern root-knot nematodes, Java root-knot nematodes and elephant-foot bean root-knot nematodes, has the characteristics of high efficiency and low toxicity, provides a new lead compound for research and development of plant root-knot nematode prevention and treatment pesticide preparations, and can be used as a main active ingredient or one of active ingredients of the plant root-knot nematode prevention and treatment pesticide preparations. It is also found that 6-bromo-N-(pyridine-2-ylmethyl)benzo[D]thiazole-2-amine has no insecticidal activity to non-plant pathogenic nematodes Caenorhabditis elegans.

Description

Technical Field

[0001] This invention belongs to the field of plant root-knot nematode control technology, specifically relating to the application of a heterocyclic compound in the control of plant root-knot nematodes. Background Technology

[0002] Root-knot nematodes are among the most destructive groups of plant parasitic nematodes. The larvae invade the roots of plants and induce abnormal cell division to form tumor-like "root knots," severely hindering the transport of water and nutrients. At the same time, the effectors they secrete regulate plant hormone pathways (such as cytokinins and auxins), forcing plants to establish "parasitic nutrient stores" for them to feed on. Root-knot nematodes have a wide range of hosts (covering more than 5,000 plant species), reproduce rapidly (2 to 10 generations per year), and can overcome some resistant varieties (such as the Mi-1 gene). They often co-infect with soil-borne fungi and bacteria, causing crop yield reductions of 20% to 80%, and are particularly serious in greenhouses and continuously cropped fields, posing a major threat to the sustainable development of global agriculture.

[0003] Currently, chemical control is the primary measure for controlling root-knot nematode disease. However, many existing chemical agents are banned or restricted due to their high toxicity, potential carcinogenic risks, and serious environmental pollution. Therefore, it is particularly urgent to develop compounds suitable for the control of root-knot nematodes. Summary of the Invention

[0004] The first objective of this invention is to provide an application of a heterocyclic compound in the control of plant root-knot nematodes, and the second objective is to provide a pesticide formulation for the control of plant root-knot nematodes.

[0005] The first objective of this invention is achieved through the application of the heterocyclic compound in the control of plant root-knot nematodes, wherein the heterocyclic compound is 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine, and its chemical structural formula is as follows:

[0006] ;

[0007] The plant root-knot nematode is one or more of *Symplocos septemblica*, *Symplocos javanica*, and *Eriocheir spp.*; the heterocyclic compound showed LC-reactivity against *Symplocos septemblica* at 24h and 48h. 50 The LC50 values ​​for *Zygophyllum javanicum* were 2.67 μg / mL and 2.44 μg / mL, respectively. 50 The LC50 concentrations for *Auricularia auricula-judae* were 18.73 μg / mL and 21.25 μg / mL, respectively. 50 The concentrations were 5.65 μg / mL and 5.74 μg / mL, respectively.

[0008] The second objective of this invention is achieved by the following: the pesticide formulation for controlling plant root-knot nematodes uses 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine as the main active ingredient or one of the active ingredients.

[0009] This invention is the first to discover that 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine has strong activity against root-knot nematodes. Studies have shown that, using DMSO aqueous solution as a solvent, the LC50 of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine at 24 h showed high activity against *Symplocos sulphureus*, *Jackie vulgaris*, and *Eriocheir heliotropium*. 50 The LC50 values ​​at 48 h were 2.67 μg / mL, 18.73 μg / mL, and 5.65 μg / mL, respectively, representing the median lethal concentrations (LC50) for *Strombus simonii*, *Strombus javanica*, and *Strombus elegans*. 50 The concentrations were 2.44 μg / mL, 21.25 μg / mL, and 5.74 μg / mL, respectively. It also has the characteristics of high efficiency and low toxicity, providing a new lead compound for the development of pesticide formulations for the control of plant root-knot nematodes. At the same time, it was found that 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine has no insecticidal activity against the non-plant pathogenic nematode Caenorhabditis elegans. Attached Figure Description

[0010] Figure 1 The chemical structural formula of the compound 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine of this invention is shown below.

[0011] Figure 2 The result of the toxicity test of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against southern root-knot nematodes in Experiment Example 1;

[0012] Figure 3 The result of the toxicity test of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against Javan root-knot nematode in Experiment Example 1;

[0013] Figure 4 The results of the toxicity test of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against *Auricularia auricula-judae* in Experiment Example 1 are as follows: Detailed Implementation

[0014] The present invention will be described in more detail below with reference to experimental examples and embodiments. However, the present invention is not limited thereto. Any modifications or improvements based on the present invention should be covered within the protection scope of the present invention.

[0015] The application of the heterocyclic compound described in this invention in the control of plant root-knot nematodes, wherein the heterocyclic compound is 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine, and its chemical structural formula is as follows:

[0016] ;

[0017] The plant root-knot nematode is one or more of *Symplocos septemblica*, *Symplocos javanica*, and *Eriocheir spp.*; the heterocyclic compound showed LC-reactivity against *Symplocos septemblica* at 24h and 48h. 50 The LC50 values ​​for *Zygophyllum javanicum* were 2.67 μg / mL and 2.44 μg / mL, respectively. 50 The LC50 concentrations for *Auricularia auricula-judae* were 18.73 μg / mL and 21.25 μg / mL, respectively. 50 The concentrations were 5.65 μg / mL and 5.74 μg / mL, respectively.

[0018] The pesticide formulation for controlling plant root-knot nematodes of the present invention uses 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine as the main active ingredient or one of the active ingredients.

[0019] Furthermore, the pesticide formulation is in the form of an aqueous solution, an emulsion, a wettable powder, a soluble powder, a soluble granule, a suspension concentrate, a tablet, or an effervescent granule.

[0020] Furthermore, the solvent for the aqueous solution or water emulsion is an aqueous solution of methanol, ethanol, or propanol with a mass concentration ≤ 5%.

[0021] The 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine described in this invention is derived from the 50K Diversity Library developed by MCE (MedChemExpress), with Catalog Number HY-W026215, Library ID 'HYCPK56337', CAS Number 941867-07-8, and a relative molecular mass of 320.2076. Its chemical structural formula is as follows: Figure 1 As shown.

[0022] The present invention performs the following tests on the 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine:

[0023] Experimental Example 1: Detection of nematicidal activity of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine

[0024] 1. Preparation of the test solution

[0025] 1-1. Test solutions for the experimental group: Accurately weigh 0.5~1.5 mg of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine (hereinafter referred to as technical grade) and dissolve it in DMSO (dimethyl sulfoxide). Dilute with aqueous solution to prepare test solutions with different technical grade concentrations. The final concentrations of technical grade in each test solution are 0.03 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, 10 μg / mL, 30 μg / mL, and 50 μg / mL, respectively. The concentration of DMSO in each test solution is 0.4% (mass fraction).

[0026] 1-2. Blank control group: test solution containing 0.4% (mass fraction) DMSO in aqueous solution.

[0027] 2. Preparation of nematodes for the experiment

[0028] 2-1. Southern root-knot nematode: Tomato roots infected with southern root-knot nematode were washed with clean water. Nematode egg sacs were picked up with surgical forceps and soaked in 0.4M KCl solution. Then, the nematode eggs were washed and disinfected sequentially with 4mL sterile water (three times), 1mL sterile penicillin-streptomycin solution, and 3mL 0.1% (w / w) NaClO solution. After each wash, the supernatant was removed by centrifugation at 8000rpm for 30s. The washed eggs were incubated in 0.4M ZnCl2 solution for 48h. The second-instar larvae of southern root-knot nematode were collected in 1.5mL centrifuge tubes by pipette and centrifuged at 5000rpm for 30s for enrichment.

[0029] 2-2. Javan root-knot nematode, elephant ear bean root-knot nematode, and Caenorhabditis elegans were obtained using methods available in the prior art.

[0030] 3. Test methods

[0031] 3-1. Southern root-knot nematode group: Take 100 μL of the test solution of different concentrations prepared in step 1-1 and place it in a 20-well plate. Add 50-80 live southern root-knot nematodes prepared in step 2-1 to each well. Place the plate at 25℃ and examine and calculate the nematode mortality rate using an optical microscope at 24h, 48h, and 72h. The method for judging the death of nematodes is as follows: add 1-5 drops of 5% (mass fraction) NaCl aqueous solution to the well of the treatment plate and observe after 2 minutes. If the nematode is stiff and does not respond to needle stimulation, it is dead. Live nematodes will curl or twist.

[0032] 3-2. For the Javan root-knot nematode group, the elephant bean root-knot nematode group, and the Caenorhabditis elegans group, follow the same procedure as in step 3-1, except that the live nematodes are replaced with Javan root-knot nematode, elephant bean root-knot nematode, and Caenorhabditis elegans, respectively.

[0033] 3-3. Blank control group: Follow the steps in steps 3-1 and 3-2 to set up blank control groups for the Southern Root-knot Nematode group, Javan Root-knot Nematode group, Elephant Ear Root-knot Nematode group, and Caenorhabditis elegans group, except that the test solution of the experimental group is replaced with the test solution of the blank control group.

[0034] 3-4. The entire experiment was repeated three times, and the average of the three trials was used to calculate the corrected mortality rate using the following formula:

[0035] Sample mortality rate % = (Number of dead nematodes / (Number of dead nematodes + Number of surviving nematodes)) × 100%

[0036] Corrected mortality rate % = Sample mortality rate – Blank control mortality rate

[0037] 4. Test Results

[0038] The insecticidal effects of 4-1,6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine on southern root-knot nematodes are shown in Table 1. Figure 2 As shown:

[0039] Table 1. Nematode-killing results of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against southern root-knot nematodes.

[0040]

[0041] After measurement (combined) Figure 2 The median lethal concentration (LC50) of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against southern root-knot nematodes at 24 h. 50 The median lethal concentration (LC50) for *Strombus haemolyticus* at 48 h was 2.67 μg / mL. 50 The concentration was 2.44 μg / mL. When applied, an effective concentration of ≥10 μg / mL can achieve a 100% mortality rate for southern root-knot nematodes.

[0042] The nematicidal effects of 4-2,6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against Javan root-knot nematodes are shown in Table 2. Figure 3 As shown:

[0043] Table 2. Nematode-killing results of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against Javan root-knot nematodes.

[0044]

[0045] After measurement (combined) Figure 3 The median lethal concentration (LC50) of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against Javan root-knot nematodes at 24 h. 50The median lethal concentration (LC50) for Javan root-knot nematodes at 48 h was 18.73 μg / mL. 50 The concentration was 21.25 μg / mL. When applied, an effective concentration of ≥50 μg / mL can achieve a 100% mortality rate for Javan root-knot nematodes.

[0046] The nematicidal effects of 4-3,6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine on *Auricularia auricula-judae* root-knot nematode are shown in Table 3. Figure 4 As shown:

[0047] Table 3. Nematode control results of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine against *Hemiberlesia lataniae* root-knot nematode.

[0048]

[0049] After measurement (combined) Figure 4 The median lethal concentration (LC50) of 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine at 24 h was determined for *Auricularia auricula-judae* root-knot nematode. 50 The median lethal concentration (LC50) of *Auricularia auricula-judae* nematode was 5.65 μg / mL at 48 h. 50 The concentration was 5.74 μg / mL. When applied, an effective concentration of ≥50 μg / mL can achieve a 100% mortality rate for the root-knot nematode of the bean weevil.

[0050] 4-4. Experiments showed that 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine did not exhibit insecticidal activity against Caenorhabditis elegans (a non-plant pathogenic nematode) at 24h, 48h, and 72h.

[0051] The above experimental results are sufficient to demonstrate that 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine has strong insecticidal activity against southern root-knot nematodes, Javan root-knot nematodes, and elephant bean root-knot nematodes, but has no insecticidal activity against the non-plant pathogenic nematode Caenorhabditis elegans. It can be used for the control of plant root-knot nematodes.

[0052] Example 1

[0053] The pesticide formulation for controlling plant root-knot nematodes described in this invention only requires 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine as the main active ingredient or one of the main active ingredients, and can be prepared into aqueous solutions, water-in-oil emulsions, wettable powders, soluble powders, soluble granules, suspensions, tablets, or effervescent granules according to conventional pesticide formulation preparation methods; the solvent for the aqueous solution or water-in-oil emulsion is selected as an aqueous solution of methanol, ethanol, or propanol with a mass ratio concentration ≤ 5%.

Claims

1. The application of a heterocyclic compound in the control of plant root-knot nematodes, characterized in that, The heterocyclic compound is 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazol-2-amine, and its chemical structural formula is as follows: ； The plant root-knot nematode is one or more of *Symplocos septemblica*, *Symplocos javanica*, and *Eriocheir spp.*; the heterocyclic compound showed LC-reactivity against *Symplocos septemblica* at 24h and 48h. 50 The LC50 values ​​for *Zygophyllum javanicum* were 2.67 μg / mL and 2.44 μg / mL, respectively. 50 The LC50 concentrations for *Auricularia auricula-judae* were 18.73 μg / mL and 21.25 μg / mL, respectively. 50 The concentrations were 5.65 μg / mL and 5.74 μg / mL, respectively.

2. A pesticide formulation for controlling plant root-knot nematodes, characterized in that, The pesticide formulation uses 6-bromo-N-(pyridin-2-ylmethyl)benzo[D]thiazole-2-amine as the main active ingredient as described in claim 1.

3. The pesticide formulation according to claim 2, characterized in that, The pesticide formulations are available in the form of aqueous solutions, emulsions, wettable powders, soluble powders, soluble granules, suspensions, tablets, or effervescent granules.

4. The pesticide formulation according to claim 3, characterized in that, The solvent for the aqueous solution or water emulsion is an aqueous solution of methanol, ethanol, or propanol with a mass ratio concentration ≤ 5%.

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