Application of methylmalonic acid in the preparation of nematode insecticides

Abstract

By determining the lethality rate to Meloidogyne incognita and Caenorhabditis elegans, it was found that the methylmalonic acid has a better nematicidal effect on the Caenorhabditis elegans, with the LC.sub.50 being 13.11 and 1.20 for the Meloidogyne incognita and the Caenorhabditis elegans, respectively. After compounding the methylmalonic acid with betaine, the LC.sub.50 was 2.85 and 0.27 for the Meloidogyne incognita and the Caenorhabditis elegans, respectively. Meanwhile, the methylmalonic acid also has an inhibiting effect on Pseudomonas solanacearum and Erwinia carotovora. The preparation of the methylmalonic acid provides a new choice for preparing novel biocontrol agents against the root-knot nematodes.

Claims

1. A nematode insecticide, comprising methylmalonic acid, and betaine, wherein the nematode insecticide is in a form of an aqueous composition; the methylmalonic acid and betaine are compounded; a concentration of the compounded methylmalonic acid and betaine in the aqueous composition is 0.25 μg/mL or more; and a mass ratio of the methylmalonic acid to the betaine is 1:1.

2. A method for treating and controlling nematode infection according to claim 1, comprising preparing the nematode insecticide according to claim 1, applying the nematode insecticide to a plant or soil infected with nematodes, and contacting and killing the nematodes with the nematode insecticide.

3. The method according to claim 2, wherein the nematode infection is caused by Meloidogyne incognita or Caenorhabditis elegans.

4. The method according to claim 2, wherein the concentration of the compounded methylmalonic acid and betaine in the aqueous composition is 2.85 μg/mL or more.

5. The method according to claim 2, wherein the concentration of the compounded methylmalonic acid and betaine in the aqueous composition is 0.27 μg/mL or more.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram of a chemical structure of methylmalonic acid.

(2) FIGS. 2A to 2C are diagrams showing microbicidal effects of the methylmalonic acid against bacterial plant pathogens in the present invention, where FIG. 2A shows the microbicidal effect against Ralstonia solanacearum; FIG. 2B shows the microbicidal effect against Erwinia carotovora; and FIG. 2C shows the microbicidal effect against black spot of walnut.

(3) FIGS. 3A to 3H are diagrams showing microbicidal effects of the methylmalonic acid against fungal plant pathogens, where FIG. 3A shows the microbicidal effect against Phytophthora capsici; FIG. 3B shows the microbicidal effect against Botrytis cinerea; FIG. 3C shows the microbicidal effect against Fusarium oxysporum; FIG. 3D shows the microbicidal effect on Alternaria solani; FIG. 3E shows the microbicidal effect against Corynespora cassiicola; F. microbicidal effect on Pestalotiopsis theae; and FIG. 3G shows the microbicidal effect against Rhizoctonia solani; H. Microbicidal effect against Sclerotinia sclerotiorum.

DETAILED DESCRIPTION OF THE INVENTION

(4) Unless otherwise stated, experimental methods in the embodiments below are all conventional microbiological operation methods that have been reported.

Example 1

(5) In the example, structural verification of nematicidal methylmalonic acid is conducted as follows: Methylmalonic acid was purchased from Sigma. Nuclear Magnetic Resonance (NMR) was used to detect the structure of the methylmalonic acid. Among others, hydrogen spectra (H NMR) and carbon spectra (.sup.13C NMR) are respectively determined by using deuterium water (D2O) as a solvent, with the NMR data shown in Table 1-1.

(6) TABLE-US-00001 TABLE 1-1 Two-dimensional NMR spectra data of methylmalonic acid No. δ.sub.H(J in Hz) δc 1 174.2 s 2 3.41(1H, dd, J = 13.7, 7.2) 45.9 d 3 174.2 s 4 ~1.19 (3H, overlapped) 12.8 q

Example 2

(7) Application of methylmalonic acid in the preparation of insecticides against root-knot nematodes, with an application process as follows:

(8) Roots and knots infected by the nematodes were picked from the roots of tomatoes. Egg masses were placed in a 96-well plate for incubation at 20° C. to observe the hatching of the root-knot nematodes. The nematodes were washed with sterile water; 20 μL of 1 mg/mL methylmalonic acid aqueous solution was added to the 96-well plate, and at the same time, purified water was added; the final concentrations of the methylmalonic acid in the well plate was adjusted to 1000, 100, 25, 16.67, 12.5, 10.1 m/mL in order; and 40 nematodes were added to each well; a bioassay system was 200 μL in total, with the purified water as a control, and 3 times repeated for each group. They were cultured in an incubator at 16° C. A LC.sub.50 value of the methylmalonic acid against the Meloidogyne incognita was 13.11 m/mL as shown in Table 2-1.

(9) TABLE-US-00002 TABLE 2-1 Insecticidal activity of methylmalonic acid against Meloidogyne incognita Medial Lethal Dose Lethality Logarithmic Probability Regression Confidence Concentration (pg/mL) Rate (%) Dose Unit Equation Interval (LC.sub.50, ug/mL) 1000 97.4 3.000 6.954 Y = 3.5248 + 1.3200X 0.88~194.67 13.11 100 97.5 2.000 6.965 (r = 0.8754) (a = 0.05) 50 80 1.699 5.840 25 81.1 1.398 5.883 16.67 76.3 1.222 5.719 12.5 29.3 1.097 4.458 10 12.5 1.000 3.850 1 8.1 0.000 3.598

(10) With the method above, the applicant tested the insecticidal effect of betaine, and based on test results, a LC.sub.50 value of the betaine against the Meloidogyne incognita was 75.22 m/ml.

Example 3

(11) Application of methylmalonic acid in the preparation of insecticides against Caenorhabditis elegans, with an application process as follows:

(12) 20 μL of 1 mg/mL methylmalonic acid aqueous solution was added to the 96-well plate, and at the same time, purified water was added; the final concentrations of the methylmalonic acid in the well plate was adjusted to 1000, 100, 25, 16.67, 12.5, 10.1 m/mL in order; 40 nematodes were added to each well; and a bioassay system was 200 μL in total, with the purified water as a control. The process was repeated 3 times for each group. They were cultured in an incubator at 16° C. A LC.sub.50 value of the methylmalonic acid against the Caenorhabditis elegans was 1.20 m/mL as shown in Table 3-1.

(13) TABLE-US-00003 TABLE 3-1 Insecticidal activity of methylmalonic acid against Caenorhabditis elegans Medial Lethal Dose Lethality Logarithmic Probability Confidence Concentration (pg/mL) Rate (%) Dose Unit Regression Equation Interval (LC.sub.50, ug/mL) 1000 97.5 3.000 6.965 Y = 4.9250 + 0.9343X 0.04~35.37 1.20 100 97.4 2.000 6.954 (r = 0.8103) (a = 0.05) 50 97.5 1.699 6.965 25 90.5 1.398 6.309 16.67 94.9 1.222 6.628 12.5 94.9 1.097 6.628 10 70.0 1.000 5.520 1 18.4 0.000 4.097

(14) With the method above, the applicant tested the insecticidal effect of betaine, and based on test results, a LC.sub.50 value of the betaine against the Caenorhabditis elegans was 14.75 μg/ml.

Example 4. Microbicidal Activity of Methylmalonic Acid Against Bacterial Plant Pathogens

(15) Ralstonia solanacearum, Erwinia carotovora and Xanthomonas campestris were activated through scribing to obtain single colonies, which were picked and inoculated into an LB liquid culture medium for activation. With an agar plate diffusion method, 1% pathogenic bacteria was added to the culture medium which was then poured into a culture dish; a piece of filter paper (with a diameter of 6 mm) was placed in the culture dish; 5 μL of 0.1 g/mL methylmalonic acid was added to the piece of filter paper, and then incubated at 30° C., with water as a negative control. The process was repeated 3 times. The results of microbicidal experiments showed that the methylmalonic acid showed a microbicidal activity against the Pseudomonas solanacearum and Erwinia carotovora, and the microbicidal activity against the Pseudomonas solanacearum was higher than that against the Erwinia carotovora (Table 4-1 and FIGS. 2A to 2C).

(16) TABLE-US-00004 TABLE 4-1 Microbicidal activity of methylmalonic acid against bacterial plant pathogens Diameter of Microbicidal Latin Name Diseases Caused Inhibition Zone Effect Ralstonia Bacterial wilt 12 mm ++ solanacearum of tomatoes Erwinia Hollow stalk 6.9 mm + carotovora of tomatoes Xanthomonas Black spot 0 − campestris of walnuts −: without microbicidal activity; +: with microbicidal activity, the diameter of an inhibition zone being less than 10 mm; ++ with microbicidal activity, and the diameter of an inhibition zone being more than 10 mm.

Example 5. Microbicidal activity of methylmalonic acid against fungal plant pathogens

(17) The mycelia of fungal pathogens were picked and inoculated to a PDA medium for activation. With a mycelium growth rate method, 200 μg/mL methylmalonic acid mother liquid was prepared using sterile water; 4.5 mL of sterile water was added to 0.5 mL of the mother liquid and evenly mixed; fungal masses were picked and placed in a PDA culture medium plate center, and then cultured at 28° C. to observe the results, with the sterile water as a negative control. After the plate was fully covered with the control mycelia, the diameters of the control and treatment colonies were counted with a crossing method. The results of microbicidal experiments showed that the methylmalonic acid showed no microbicidal activity against the fungal plant pathogens (Table 5-1 and FIGS. 3A to 3H).

(18) TABLE-US-00005 TABLE 5-1 Microbicidal activity of methylmalonic acid against fungal plant pathogens Microbicidal Latin Name Diseases Caused Effect Phytophthora capsici Phytophthora blight − on peppers Botrytis cinerea Grey mold of cucumber − Fusarium oxysporum Tomato wilt − Alternaria solani Potato early blight − Corynespora cassiicola Corynespora leaf spot − of cucumber Pestalotiopsis theae Pestalotiopsis leaf − spot of tea Rhizoctonia solani Rice sheath blight disease − Sclerotinia sclerotiorum Sclerotinia rot of colza − +: effective; −: ineffective.

Example 6

(19) Application of methylmalonic acid compounded with betaine in the preparation of insecticides against root-knot nematodes, with an application process as follows:

(20) Roots and knots infected by the nematodes were picked from the roots of tomatoes. Egg masses were placed in a 96-well plate for incubation at 20° C. to observe the hatching of the Meloidogyne. The nematodes were washed with sterile water; 20 μL of 1 mg/mL mixed aqueous solution of methylmalonic acid and betaine (mass ratio: 1:1) was added to the 96-well plate, and at the same time, purified water was added; the final concentrations of the compounded solution in the well plate was adjusted to 1000, 100, 25, 16.67, 12.5, 10, 1, 0.5, 0.25 μg/mL in order; and 40 nematodes were added to each well; a bioassay system was 200 μL in total, with the purified water as a control, and 3 times repeated for each group. They were cultured in an incubator at 16° C. A LC.sub.50 value of the compounded solution of methylmalonic acid and betaine against the Meloidogyne incognita was 2.85 μg/mL as shown in Table 6-1.

(21) TABLE-US-00006 TABLE 6-1 Insecticidal activity of methylmalonic acid compounded with betaine against Meloidogyne incognita Medial Lethal Dose Lethality Logarithmic Probability Regression Confidence Concentration (ug/mL) Rate (%) Dose Unit Equation Interval (LC.sub.50, ug/mL) 1000 97.4 3.000 6.943 Y = 4.5732 + 0.9387X 0.06~133.53 2.85 100 95.0 2.000 6.640 (r = 0.9210) (a = 0.05) 50 92.3 1.699 6.432 25 95.0 1.398 6.640 16.67 87.5 1.222 6.155 12.5 53.7 1.097 5.093 10 46.2 1.000 4.903 1 37.5 0.000 4.680 0.5 22.2 −0.301 4.237 0.25 13.2 −0.602 3.878

(22) The above results showed that a synergic nematode killing effect was achieved by mixing the methylmalonic acid and the betaine. As can be seen from Example 2, the single use of the methylmalonic acid resulted in an LC50 of 13.11 μg/mL against the Meloidogyne incognita; and the single use of the betaine resulted in an LC50 of 75.22 μg/ml against the Meloidogyne incognita. The compounded methylmalonic acid and betaine at the mass ratio of 1:1 resulted in an LC50 of 2.85 μg/mL against the Meloidogyne incognita, and the insecticidal efficiency was significantly increased.

Example 7

(23) Application of methylmalonic acid compounded with betaine in the preparation of insecticides against Caenorhabditis elegans, with an application process as follows:

(24) 20 μL of 1 mg/mL mixed aqueous solution of methylmalonic acid and betaine (mass ratio: 1:1) was added to the 96-well plate, and at the same time, purified water was added; the final concentrations of the compounded solution in the well plate was adjusted to 1000, 100, 25, 16.67, 12.5, 10, 1, 0.5, 0.25 μg/mL in order; and 40 nematodes were added to each well; a bioassay system was 200 μL in total, with the purified water as a control, and 3 times repeated for each group. They were cultured in an incubator at 16° C. A LC.sub.50 value of the methylmalonic acid against the Caenorhabditis elegans was 0.27 μg/mL as shown in Table 7-1.

(25) TABLE-US-00007 TABLE 7-1 Insecticidal activity of methylmalonic acid compounded with betaine against Caenorhabditis elegans Medial Lethal Dose Lethality Logarithmic Probability Regression Confidence Concentration (ug/mL) Rate (%) Dose Unit Equation Interval (LC.sub.50, ug/mL) 1000 97.5 3.000 6.965 Y = 5.4258 + 0.7483X 0.00~28.53 0.27 100 97.5 2.000 6.965 (r = 0.9014) (a = 0.05) 50 97.4 1.699 6.954 25 97.4 1.398 6.954 16.67 95.0 1.222 6.640 12.5 95.2 1.097 6.666 10 82.1 1.000 5.922 1 70.7 0.000 5.542 0.5 51.4 −0.301 5.037 0.25 30.0 −0.602 4.480

(26) The above results showed that a synergic nematode killing effect was achieved by mixing the methylmalonic acid and the betaine. As can be seen from Example 3, the single use of the methylmalonic acid resulted in an LC50 of 1.20 μg/mL against the Caenorhabditis elegans; and the single use of the betaine resulted in an LC50 of 14.75 μg/ml against the Caenorhabditis elegans. The compounded methylmalonic acid and betaine at the mass ratio of 1:1 resulted in an LC50 of 0.27 μg/mL against the Caenorhabditis elegans, and the insecticidal efficiency was significantly increased.

Example 8. Microbicidal Activity of Methylmalonic Acid Compounded with Betaine Against Bacterial Plant Pathogens

(27) Ralstonia solanacearum, Erwinia carotovora and Xanthomonas campestris were activated through scribing to obtain single colonies, which were picked and inoculated into an LB liquid culture medium for activation. With an agar plate diffusion method, 1% pathogenic bacteria was added to the culture medium which was then poured into a culture dish; a piece of filter paper (with a diameter of 6 mm) was placed in the culture dish; 5 μL of 0.1 mg/mL mixed aqueous solution of methylmalonic acid and betaine (mass ratio: 1:1) was added to the piece of filter paper, and then incubated at 30° C., with water as a negative control. The process was repeated 3 times. The results of microbicidal experiments showed that the compounded solution showed a microbicidal activity against the Pseudomonas solanacearum and Erwinia carotovora, but no microbicidal activity against the Xanthomonas campestris (Table 8-1).

(28) TABLE-US-00008 TABLE 8-1 Microbicidal activity of methylmalonic acid compounded with betaine against bacterial plant pathogens Diameter of Microbicidal Latin Name Diseases Caused Inhibition Zone Effect Ralstonia Bacterial wilt 6.5 mm + solanacearum of tomato Erwinia Hollow stalk 3.1 mm + carotovora of tomato Xanthomonas Black spot 0 − campestris of walnut −: without microbicidal activity; +: with microbicidal activity, the diameter of an inhibition zone being less than 10 mm.

Example 9. Microbicidal Activity of Methylmalonic Acid Compounded with Betaine Against Fungal Plant Pathogens

(29) The mycelia of fungal pathogens were picked and inoculated to a PDA medium for activation. With a mycelium growth rate method, 200 μg/mL mixed aqueous solution of methylmalonic acid and betaine (mass ratio: 1:1) was prepared using sterile water; 4.5 mL of sterile water was added to 0.5 mL of the mixed solution and evenly mixed; fungal masses were picked and placed in a PDA culture medium plate center, and then cultured at 28° C. to observe the results, with the sterile water as a negative control. After the plate was fully covered with the control mycelia, the diameters of the control and treatment colonies were counted with a crossing method. The results of microbicidal experiments showed that the compounded solution showed no microbicidal activity against the fungal plant pathogens (Table 9-1).

(30) TABLE-US-00009 TABLE 9-1 Microbicidal activity of methylmalonic acid against fungal plant pathogens Microbicidal Latin Name Diseases Caused Effect Phytophthora capsici Phytophthora blight − on peppers Botrytis cinerea Grey mold of cucumber − Fusarium oxysporum Tomato wilt − Alternaria solani Potato early blight − Corynespora cassiicola Corynespora leaf spot − of cucumber Pestalotiopsis theae Pestalotiopsis leaf − spot of tea Rhizoctonia solani Rice sheath blight disease − Sclerotinia sclerotiorum Sclerotinia rot of colza − +: effective; −: ineffective.