Use of ethyl cinnamate to kill pinewood nematodes

Abstract

The application is the application of ethyl cinnamate EC to pine wood nematode. Ethyl cinnamate has a killing effect on pine wood nematode, can reduce the survival rate, activity, egg laying rate, egg hatching rate and feeding and reproduction ability of pine wood nematode. In addition, EC can also cause lipid peroxidation of pine wood nematode, causing damage. This shows that ethyl cinnamate has a killing effect on pine wood nematode, and can be used as a promising compound for treating pine wood nematode disease.

Description

Technical Field

[0001] This invention relates to the effects of ethyl cinnamate on the growth, development, and oxidative damage of pine wood nematodes, and relates to the field of pine wood nematode prevention technology. Background Technology

[0002] Pine wilt nematode (Bursaphelenchus xylophilus) is the pathogenic nematode that causes pine wilt disease and is recognized internationally as an important quarantine pest. Currently, chemical control is still the primary method for controlling pine wilt nematode, but conventional nematicides are highly toxic, have a certain impact on the surrounding environment, and nematodes easily develop resistance. Therefore, research on the control of pine wilt disease both domestically and internationally is gradually shifting towards the development of novel, low-toxicity, and environmentally friendly nematicides. Ethyl cinnamate (EC) is a naturally derived compound used not only as a food additive and flavoring in daily life, but also has inhibitory effects on tumor cell growth and certain bactericidal effects. Therefore, it meets the current demand for the development of low-toxicity and environmentally friendly nematicides. This study aims to investigate whether ethyl cinnamate can affect the growth and development of pine wilt nematodes. Furthermore, there are currently no studies on the effects of ethyl cinnamate on the growth, development, and oxidative damage of pine wilt nematodes. Summary of the Invention

[0003] The purpose of this invention is to reveal the effects of ethyl cinnamate on the growth, development, and oxidative damage of pine wood nematodes, thereby providing a theoretical basis for the development and utilization of ethyl cinnamate as a nematicide.

[0004] The above-mentioned objective of the present invention is achieved by the following solution:

[0005] This invention uses pine wood nematode as the research subject. By feeding different concentrations of ethyl cinnamate, the survival rate, motility, oviposition rate, egg hatching rate, reproductive capacity, and oxidative damage were analyzed. The results showed that ethyl cinnamate can significantly inhibit the growth and development of pine wood nematode and cause oxidative damage. Attached Figure Description

[0006] Figure 1 shows the effect of ethyl cinnamate on the survival rate and activity of pine wood nematode.

[0007] Figure 2 shows the effect of ethyl cinnamate on the oviposition rate and egg hatching rate of pine wood nematode.

[0008] Figure 3 shows the effects of ethyl cinnamate on the feeding and propagation abilities of pine wood nematodes.

[0009] Figure 4 shows the detection of the damaging effect of ethyl cinnamate on pine wood nematodes. Detailed Implementation

[0010] The invention will be further illustrated below with reference to examples:

[0011] Experimental organisms: Bursaphelenchus xylophilus and Botrytis cinerca were both kindly donated by Professor Niu Ben of Northeast Forestry University.

[0012] Pine wood nematode culture: Botrytis cinerea was cultured on potato dextrose agar medium (PDA, 2.5% PDB, 1.7% Agar, 60 mm petri dish). After the Botrytis cinerea was fully grown, pine wood nematodes were inoculated onto the Botrytis cinerea and cultured in the dark at 25°C for 4-7 days.

[0013] Collection of pine wood nematodes: After the pine wood nematodes have covered the entire Botrytis cinerea agar plate, rinse the plate with double-distilled water (DDW), extract the pine wood nematodes from the plate into 50 mL centrifuge tubes, and then wash three times with DDW. Sterilize the nematodes by adding a final concentration of 40 μg / mL actinomycin solution and 200 μg / mL streptomycin sulfate solution, and wash three times with double-distilled water to prepare a sterile nematode suspension for later use.

[0014] Example 1: Test reagent and reagent preparation

[0015] Test reagents: Ethyl cinnamate was purchased from Shanghai Aladdin Company; Avermectin (ABA) was purchased from Sigma Company; Actinomycin and streptomycin sulfate were purchased from Beijing Bio-Top Technology Co., Ltd.; Dimethyl sulfoxide (DMSO) and anhydrous ethanol were purchased from Sigma Company.

[0016] Reagent preparation: Dissolve 25 μL of ethyl cinnamate in 975 μL of DMSO to prepare a 25 mg / mL stock solution. Then dilute the ethyl cinnamate in a 10% Tween-80 aqueous solution, and then dilute tenfold to obtain a 1 mg / mL EC stock solution for later use. Dissolve ABA in DMSO to prepare a 1 mg / mL ABA stock solution, and then gradually dilute with DDW to a 10 μg / mL ABA stock solution for later use. Prepare a 100 mg / mL anhydrous ethanol solution of actinomycete, filter sterilize using a 0.22 μm filter, and aliquot. Prepare a 50 mg / mL streptomycin sulfate solution with double-distilled water, filter sterilize using a 0.22 μm filter, and aliquot. ABA was used as a positive control, and DDW as a blank control.

[0017] Example 2: Effect of different concentrations of ethyl cinnamate on the survival rate of adult pine wood nematodes

[0018] Add 50 μL (approximately 50-100 adult nematodes) to a 96-well plate, and add ethyl cinnamate to achieve final concentrations of 0, 100, 200, and 400 μg / mL. Simultaneously, set up an ABA (5 μg / mL) positive control and a DDW blank control, with three replicates per group. Adjust the volume to 100 μL and incubate at 25°C in the dark. Record the number of dead nematodes every 12 h, and calculate the survival rate and median lethal concentration.

[0019] The results showed that ethyl cinnamate had a good nematicidal effect against pine wood nematodes, and the effect was positively correlated with concentration and exhibited a linear relationship. The dose-response relationship of ethyl cinnamate treatment for pine wood nematodes for 12 hours was calculated as follows: survival rate (%) = 164.41 - 48.82 × log10(concentration), R 2 = 0.926. The dose-response relationship after 24 h of treatment is: survival rate (%) = 115.48 - 31.82 × log10(concentration), R 2 = 0.891. The LC50 of ethyl cinnamate treatment for pine wood nematodes for 12 h was 220.41 ug / ml, and the LC50 for 24 h was 114.28 μg / ml. Figure 1 A).

[0020] Example 3: Effect of ethyl cinnamate on the locomotion ability of pine wood nematode

[0021] 100 μL of nematode suspension (approximately 100 nematodes) was added to 96-well plates and treated with ABA at a final concentration of 5 μg / mL, and EC solutions at concentrations of 0, 100, and 200 μg / mL in a constant temperature incubator at 25 °C for 12 h. Nematodes were randomly selected from the 96 wells, and the number of head movements of the nematodes within 30 s under an inverted microscope was recorded. Each treatment was repeated three times.

[0022] The results showed that EC could inhibit the motility of pine wood nematodes. The number of head swings in the CK group was 29.16, and in the ABA group it was 3.76, a decrease of 87.11% compared to the CK group, indicating a significant reduction in nematode motility. After treatment with 100 and 200 μg / mL EC, the number of head swings were 16.78 and 11.58, respectively, a decrease of 42.46% and 60.29% compared to the CK group. These results demonstrate that EC can reduce the motility of nematodes by inhibiting head swings. Figure 1 B).

[0023] Example 4: Effect of ethyl cinnamate on oviposition of pine wood nematode

[0024] 100 μL of sterile nematode suspension (approximately 100 nematodes) was added to a 96-well plate, followed by EC solution at final concentrations of 100 and 200 μg / mL. The 96-well plates were then incubated at 25°C in the dark. The number of eggs was recorded at 12 and 24 h using an inverted microscope. Each treatment was repeated three times. Double-distilled water and avermectin at a final concentration of 5 μg / mL were used as blank and positive controls, respectively.

[0025] The results showed that EC inhibited the oviposition capacity of pine wood nematodes. After 12 h of treatment with CK, 5 μg / mL ABA, 100, and 200 μg / mL EC, the oviposition numbers were 134, 17, 30, and 13, respectively. The nematodes in the CK group were able to lay a large number of eggs, while the 200 μg / mL EC treatment group was similar to the ABA group, showing reductions of 90.30% and 87.31% compared to the CK group, respectively; the 100 μg / mL EC treatment showed a reduction of 77.62% compared to the CK group. Figure 2 A, B).

[0026] Example 5: Effect of ethyl cinnamate on hatching rate of pine wood nematode eggs

[0027] 200 μL of nematodes (approximately 200 nematodes) were placed in a 24-well plate. After 24 h, the nematodes were removed by washing with DDW, and the eggs were collected. The eggs were then treated with DDW, 5 μg / mL ABA, 100 μg / mL EC, and 200 μg / mL EC for 48 h, respectively. The hatching rate of the eggs was recorded under a fluorescence microscope every 24 h.

[0028] The results indicated that EC significantly inhibited the hatching of pine wilt nematode eggs. The egg hatching rates at 24 h were 83.24%, 2.36%, 10.35%, and 4.80% for the CK, ABA, 100 μg / mL EC, and 200 μg / mL EC treatment groups, respectively. The egg hatching rates at 48 h were 88.97%, 12.99%, 14.37%, and 6.46%, respectively, with the 200 μg / mL EC treatment group showing the lowest rate, decreasing by 92.85% compared to the CK group. Figure 2 C, D).

[0029] Example 6: Effects of ethyl cinnamate on the feeding and reproductive capacity of pine wood nematodes

[0030] Pine wood nematodes were treated with DDW, 5 μg / mL ABA, and 100 and 200 μg / mL EC for 12 h to investigate the effects of different EC concentrations on the feeding and reproductive capacity of the nematodes. Fifty pairs of nematodes were randomly selected and added to PDA culture dishes contaminated with Botrytis cinerea. The experiment ended when the Botrytis cinerea mycelium in the control group was completely consumed. After the Botrytis cinerea was completely consumed, the nematodes were extracted and counted.

[0031] The results showed that EC significantly inhibited the reproduction of pine wilt nematode populations. On day 8, the mycelia of *Botrytis cinerea* in the control group were completely consumed. At this point, the feeding area in the ABA group was 71.11% lower than that in the CK group, and the EC concentrations of 100 and 200 μg / mL were 48.41% and 59.26% lower than those in the CK group, respectively.

[0032] Example 7: Effect of ethyl cinnamate on lipofuscin in pine wood nematodes

[0033] 1500 pine wood nematodes were extracted and treated in the dark with DDW, 5 μg / mL ABA, and 200 μg / mL EC at 25°C for 12 h. After washing three times with DDW, 20 nematodes were randomly selected, mounted, and photographed under a fluorescence microscope. Lipofuscin is a metabolic product of lipid peroxidation of intracellular polyunsaturated fatty acids under the action of ROS. Furthermore, lipofuscin itself exhibits blue fluorescence and can be directly observed under a fluorescence microscope.

[0034] The results showed that after treating pine wood nematodes with 200 μg / mL EC for 12 h, fluorescence imaging revealed that the blue fluorescence of the nematodes in the CK group was very weak, indicating a low content of lipofuscin, while the fluorescence of the 200 μg / mL EC and ABA groups was significantly enhanced, indicating a large accumulation of lipofuscin. Figure 4 A, B).

[0035] Example 8: Oxidative damage of ethyl cinnamate to pine wood nematode

[0036] 1500 pine wood nematodes were extracted and treated in the dark with DDW, 100, and 200 μg / mL EC at 25°C for 12 h. After washing three times with PBS, excess liquid was aspirated, and the mixture was flash-frozen in liquid nitrogen and then homogenized. 50 μL of PBS was added to homogenize the homogenate, which was then centrifuged at 2500 rpm for 10 minutes, and the supernatant was collected. The protein concentration in the supernatant was determined using the Coomassie Brilliant Blue assay (A045-2, Nanjing Jiancheng Biotechnology Institute), and the malondialdehyde (MDA) content was determined using the TBA colorimetric method (A003-2, Nanjing Jiancheng Biotechnology Institute). Malondialdehyde (MDA), a product of lipid peroxidation, is an important marker of the degree of oxidative damage in organisms, and the accumulation level of MDA directly reflects the severity of lipid peroxidation damage in nematodes.

[0037] The results showed that the MDA content in nematodes treated with 100 and 200 μg / mL EC increased by 177.46% and 338.70% respectively compared with the control group. This indicates that EC can induce oxidative damage in pine wood nematodes. Figure 4 C).

[0038] In summary, ethyl cinnamate can reduce the survival rate, activity, oviposition rate, egg hatching rate, feeding and reproductive capacity of pine wood nematodes. Furthermore, it can induce lipid peroxidation in pine wood nematodes. Ethyl cinnamate has significant nematicidal effects and is valuable for limiting the development of pine wood nematode disease.

Claims

1. The application of ethyl cinnamate in the insecticidal effect against pine wilt nematode, characterized in that: It can reduce the survival rate, mobility, and reproductive capacity of pine wood nematodes.

2. Ethyl cinnamate can cause oxidative damage to pine wood nematodes, characterized in that: It can cause the accumulation of lipofuscin in the body of pine wood nematodes and an increase in malondialdehyde content.

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