Methods for increasing pest resistance in crops, and their biological combination formulations and uses

Abstract

This invention discloses a method for enhancing the pest resistance of crops, as well as a biological combination formulation thereof and its use, belonging to the field of agricultural biotechnology. This invention is the first to improve the pest resistance of crops by utilizing α-linolenic acid seed coating treatment, which can significantly improve the resistance of crops to pests. Furthermore, it can be easily operated through seed coating treatment and intensive operations, saving labor and greatly alleviating the problem of difficulty in securing labor in pesticide application. This invention further provides a biological combination formulation containing α-linolenic acid and the pesticide chlorantraniliprole, which combines seed coating treatment using α-linolenic acid and chemical pesticides, reducing the amount of chemical pesticide used while still having a higher lethal effect against pests. This invention does not affect the seed germination rate after treatment, does not affect the growth and development of crops, reduces the amount of chemical agents used by half compared to conventional seed coating methods, is easily operated through intensive operations, saves labor, and has higher social and ecological effects.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to a method for enhancing the pest resistance of crops, as well as its biological combination formulations and uses.

Background Art

[0002] Rice is one of the important food crops. There are many pests inhabiting rice, among which the commonly seen ones are Chilo suppressalis, Sesamia inferens, Scirpophaga incertulas, Nymphula depunctalis, Nilaparvata lugens, Sogatella furcifera, etc. Except for Nymphula depunctalis, Nilaparvata lugens and Sogatella furcifera, the other pests are all boring pests, and the damage is hidden and it is difficult to effectively control them. In addition, because the growth of these pests is uneven, the generations of the population in the field overlap, making it difficult to grasp the effective control period, and the difficulty of control is further increased. Nymphula depunctalis, Nilaparvata lugens and Sogatella furcifera are migratory pests, and their control is more difficult due to the uncertainty of the flying period. Therefore, pesticides are used in large quantities and excessively, which not only causes environmental pollution problems, but also accelerates the formation and development of pest drug resistance.

[0003] Maize is also one of the important grains. There are many pests inhabiting maize, among which the commonly seen ones are Spodoptera frugiperda, Mythimna separata, Ostrinia furnacalis, Sesamia inferens, Peregrinus maidis, etc., and they are all migratory pests, and their control is more difficult due to the uncertainty of the flying period. Similarly, problems such as the large and excessive use of pesticides and environmental pollution are caused.

[0004] Crops are constantly susceptible to various diseases and pests during their growth process, and delays in control can lead to losses and crop failures. Effective control of crop diseases and pests is key to ensuring high and stable yields. Currently, crop disease and pest control still primarily relies on foliar spraying, which requires a significant amount of labor. While family farms, large-scale farmers, and new types of farmers' cooperatives are continuously growing and developing, the aging population and increasing urbanization are leading to a growing shortage of labor in rural areas. Farmers often cannot secure enough labor during the crop disease and pest control period. Furthermore, the frequent occurrence of extreme heat weather makes pesticide application work very difficult. Therefore, farmers urgently need new crop disease and pest control technologies that can not only reduce the number of pesticide applications but also achieve higher control efficacy. Furthermore, foliar application of pesticides has several disadvantages, including relatively low utilization rates, threats to human and animal safety due to spray particle drift, serious non-point source contamination, and significant impacts on non-target organisms. Developing new pest and disease control technologies that achieve higher control efficacy while simultaneously minimizing or avoiding the adverse effects of current foliar application of pesticides is a crucial need for the green development of agricultural production.

[0005] Seed treatment can, on the one hand, restore some vitality to seeds, increase germination rates, promote uniform seedling growth, and strengthen seedlings. On the other hand, by controlling crop diseases and pests in their early stages, it can promote healthy crop growth and development, and increase yields (Patent Publication No. CN107771811A, Publication Date March 9, 2018; Patent Publication No. CN114903041A, Publication Date August 16, 2022). Pest and disease control technology based on chemical seed coating is already being applied in production sites. According to research by Yu Julong et al., seed coating treatment using 5-10 mg of 20% chlorantraniliprole suspension per gram of dry rice seeds provided a control effect against rice leafminers that lasted for 110 days, reducing the number of pesticide applications by two. However, when the amount of pesticide used was less than 5 mg, the control effect clearly decreased (2019, Journal of Agrochemical Sciences, paper title: "Evaluation of the Control Efficacy and Safety of Seed Coating Treatment with Chlorantraniliprole against Rice Leafminers"). Developing new seed coating methods or technologies that reduce the use of chemical pesticides while still achieving higher pest and disease control effects plays an important role in protecting the ecological environment of farmland and promoting the green development of agricultural production. However, selecting and developing new seed coating agents and seed coating technologies requires a great deal of exploration and testing.

[0006] Research on controlling crop diseases and pests using substances related to the plant defense hormone pathway has already been reported. Methyl jasmonate is an important substance in the plant defense hormone pathway, and a patent (publication number CN111436453A, publication date July 24, 2020) discloses a "compound insecticide for controlling Cleontiades diltus." This patent describes a mixture of the chemical pesticide imidacloprid, methyl jasmonate, and Alternaria tenuissima protein to further control Cleontiades diltus. Based on this technology, the amount of chemical pesticides used can be reduced without compromising the control effect against Cleontiades diltus, resulting in greater social and ecological benefits.

[0007] Seed treatment-based crop pest and disease control technology fully implements the plant quarantine policy of "primarily prevention and integrated control." At the same time, this technology is easy to operate, allows for intensive treatment, reduces the number of pesticide applications, and achieves higher control efficacy, making it popular among large-scale farmers. Furthermore, seed treatment technology significantly avoids problems associated with pesticide application methods, such as reduced pesticide utilization rates, environmental pollution due to large-scale pesticide runoff, and poisoning of humans and animals. In recent years, there has been a constant push to reduce the use of chemical pesticides in crop production. Regarding seed coating technology, how to maintain high control efficacy against crop pests and diseases while reducing the use of chemical pesticides remains a hot topic of interest in the field of plant quarantine. For pest control of crops such as rice, corn, and cabbage, the development of green, highly efficient, simple, and intensive control technologies is urgently needed. [Overview of the project] [Problems that the invention aims to solve]

[0008] In response to problems such as the large amount of chemical pesticides used on crops and the difficulty in securing labor for pesticide application, this invention provides a method for increasing the pest resistance of crops. This provides a foundation for the development of green, highly efficient, simplified, and intensive crop pest control technologies, and effectively reduces the amount of chemicals used for controlling crop diseases and pests.

[0009] Another object of the present invention is to provide a biological combination formulation for controlling crop pests, which will provide important reference for the development of green, highly efficient, simplified, and intensive integrated control technologies for crop diseases and pests, significantly reduce the use of chemical pesticides for crop disease and pest control, alleviate the challenge of securing labor for pesticide application, and reduce non-point source contamination of pesticides in the field.

[0010] This invention further provides the use of biological combination formulations for controlling crop pests. [Means for solving the problem]

[0011] To achieve the above objective, the method for increasing the pest resistance of crops described in the present invention involves coating crop seeds with an alpha-linolenic acid solution, thoroughly and uniformly stirring them, and then sowing the seeds.

[0012] Dry crop seeds are coated with a 4-400 mg / L α-linolenic acid solution at a concentration of 0.5-1 mL per gram.

[0013] Preferably, 1 mL of a 4-400 mg / L α-linolenic acid solution is applied to 1 g of dried crop seeds as a seed coating.

[0014] Once the seed coating process is complete, leave the seedlings in a cool place for 12 to 24 hours before sowing. Sowing can be done by directly scattering the seeds in the field using a direct sowing method, or by growing the seedlings in a seedling tray before transplanting.

[0015] The present invention relates to a biological combination formulation for controlling crop pests, wherein the biological combination formulation comprises α-linolenic acid and the pesticide chlorantraniliprole as described in claim 1, and the mass ratio of the two is 0.004 to 0.4:0.5 to 5.

[0016] The aforementioned biological combination formulation comprises alpha-linolenic acid and a 20% chlorantraniliprole suspension, with a mass ratio of 0.004-0.4:2.5-5.

[0017] Preferably, the biological combination formulation comprises alpha-linolenic acid and a 20% chlorantraniliprole suspension, with a mass ratio of 0.004 to 0.4:2.5.

[0018] A method for increasing the pest resistance of the aforementioned crop or a biological combination formulation for controlling the aforementioned crop pests, wherein the aforementioned crop is rice, corn, or cabbage.

[0019] A method for increasing the pest resistance of the aforementioned crop or a biological combination formulation for controlling the aforementioned crop pests, wherein the aforementioned crop is paddy rice and maize.

[0020] A method for increasing the pest resistance of the crop or a biological combination formulation for controlling the crop pests, wherein the pests are the Japanese rice stem borer, the large rice stem borer, the rice leaf borer, Spodoptera fulgiperda, the corn borer, the red-eyed green moth, the armyworm, the diamondback moth, or the green worm.

[0021] Use of alpha-linolenic acid or a biological combination formulation for controlling crop pests as described in the present invention, in order to improve the pest resistance of crops or to control crop pests. The crop is rice, corn, or cabbage, and the pests are rice stem borer, large rice stem borer, red rice stem borer, rice leafhopper, brown planthopper, white-backed planthopper, Spodoptera fulgiperda, corn borer, armyworm, red-eyed green moth, or diamondback moth.

[0022] The alpha-linolenic acid or the biological combination formulation is used to improve the pest resistance of crops or to control crop pests by a seed coating method.

[0023] Furthermore, in the above-mentioned use, the seeds are coated with 0.5 to 1 mL of a 4 to 400 mg / L α-linolenic acid solution per gram of dry crop seeds before sowing, or the seeds are coated with 0.5 to 1 mL of a 4 to 400 mg / L α-linolenic acid solution and 2.5 to 5 mg of a 20% chlorantraniliprole suspension per gram of dry seeds before sowing.

[0024] Preferably, 1g of dry seeds is thoroughly and uniformly mixed with 1mL of 4-400mg / L α-linolenic acid solution and 2.5-5mg of 20% chlorantraniliprole suspension, and then the seeds are coated with powder before sowing.

[0025] Once the seed coating treatment is complete, leave the seeds in a cool place for 12-24 hours before sowing.

[0026] The aforementioned sowing method involves either directly scattering seeds in the field using a direct sowing method, or growing seedlings in seedling trays before transplanting.

[0027] The present invention first proposes to control crop pests and diseases by using α-linolenic acid for seed film coating treatment. Furthermore, by using α-linolenic acid for the first time in combination with chemical agents for seed film coating treatment, crop pests and diseases are controlled.

[0028] There are many related substances in the plant defense hormone pathway. To select substances that can promote the pest resistance of crops, it is necessary to prove through a large number of tests, and usually the selection method is the chemical spraying method. At present, it is difficult to obtain substances suitable for seed film coating treatment and having a higher insecticidal effect. The present invention has selected the plant resistance inducer "α-linolenic acid", and for the first time reveals that this substance can improve the pest resistance of crops by seed film coating. After treatment, it does not affect the germination rate of seeds, does not affect the growth and development of crops, and can greatly improve the resistance of crops to pests. In addition, the present invention first combines the inducer α-linolenic acid with chemical pesticides for seed film coating treatment. After treatment, it does not affect the germination rate of seeds, does not affect the growth and development of crops, reduces the usage amount of chemical pesticides, and still has a higher control effect on crop pests.

Effects of the Invention

[0029] Compared with the prior art, the present invention has the following advantages.

[0030] The present invention first uses α-linolenic acid to improve the pest resistance of crops. The specific feature is that by using α-linolenic acid at a specific concentration for seed film coating treatment, the pest resistance of crops is greatly improved. The method of the present invention does not affect the germination rate of seeds after treatment, does not affect the growth and development of crops, can greatly improve the resistance of crops such as rice to pests, effectively avoids problems such as environmental pollution caused by chemical spraying through seed film coating treatment, is intensively operated, can be easily operated, saves labor, and can greatly alleviate the problem of difficulty in ensuring labor force in chemical spraying. At the same time, α-linolenic acid is friendly to the environment and the human body, and effectively avoids the problem of environmental pollution.

[0031] This invention proposes a biological combination formulation that mixes alpha-linolenic acid with chemical pesticides to control crop pests. This invention is the first to utilize alpha-linolenic acid in conjunction with chemical pesticides for seed coating treatment, significantly reducing the amount of chemical pesticides used (by at least one time), while still maintaining a higher lethal effect against pests. Furthermore, alpha-linolenic acid is environmentally and human-friendly, significantly reducing environmental pollution caused by chemical pesticides.

[0032] This invention reduces the amount of chemical pesticides used while achieving a higher level of crop pest control by using a mixture of alpha-linolenic acid at a specific concentration and a reduced amount of 20% chlorantraniliprole suspension for seed coating treatment. The biological combination formulation of this invention does not affect seed germination rates or crop growth and development after treatment, reduces the amount of chemical agents used by half compared to conventional seed coating methods, is operated intensively and easily, saves labor, and has higher social and ecological benefits. [Brief explanation of the drawing]

[0033] [Figure 1] The table shows the height of rice seedlings after treating Nanryu 9108 seeds with alpha-linolenic acid. If the same lowercase letter is not present, it indicates a statistically significant difference, and P<0.05. [Figure 2] The table shows the height of rice seedlings after treating Shinryoyu No. 1 seeds with alpha-linolenic acid. If the same lowercase letter is not included, it indicates a statistically significant difference, and P<0.05. [Figure 3] The results of bioassays against rice stem borers after treating rice seeds with alpha-linolenic acid are shown. If the same lowercase letter is not present, it indicates a statistically significant difference, and P<0.05. [Figure 4] The results of measuring the hormone content after treating rice seeds with alpha-linolenic acid are shown. [Figure 5] The results of a bioassay for Spodoptera fulgiperda after treating corn seeds with alpha-linolenic acid are shown. [Figure 6] This image shows surviving individuals of maize plants treated with alpha-linolenic acid after being fed by Spodoptera fulgiperda. [Figure 7] The table shows the plant height after coating Nanryu 9108 seeds with a mixture of alpha-linolenic acid and chlorantraniliprole suspension. If the same lowercase letter is not included, it indicates a statistically significant difference, and P < 0.05. CK: Shimizu control group; T1: Group coated with a mixture of 4 mg / L alpha-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T2: Group coated with a mixture of 40 mg / L alpha-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with a mixture of 400 mg / L alpha-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension. [Figure 8] The results of bioassays against rice stem borers after coating rice seeds are shown, (A) Nanryu 9108 rice seeds; (B) Shinryoyu No. 1 rice seeds. If the same lowercase letters are not included, it indicates a statistically significant difference, and P < 0.05. CK: Shimizu control group; T1: Group coated with a mixture of 4 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T2: Group coated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with a mixture of 400 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T4: Group coated with 5 mg 20% ​​chlorantraniliprole suspension. [Figure 9] The table shows the height of corn seeds after seed coating. If the same lowercase letter is not included, it indicates a statistically significant difference, and P < 0.05. CK: Clean water control group; T1: Group coated with a mixture of 4 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T2: Group coated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with a mixture of 400 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T4: Group coated with 5 mg 20% ​​chlorantraniliprole suspension. [Figure 10] The bioassay results for Spodoptera fulgiperda after seed coating of corn seeds are shown. If the same lowercase letter is not included, it indicates a statistically significant difference, and P < 0.05. CK: Clean water control group; T1: Group coated with a mixture of 4 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T2: Group coated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with a mixture of 400 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T4: Group coated with 5 mg 20% ​​chlorantraniliprole suspension. [Figure 11] The table shows the height of cabbage seeds after seed coating. If the same lowercase letter is not included, it indicates a statistically significant difference, and P < 0.05. CK: Clean water control group; T1: Group coated with a mixture of 4 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T2: Group coated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with a mixture of 400 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T4: Group coated with 5 mg 20% ​​chlorantraniliprole suspension. [Figure 12] The bioassay results for diamondback moths after cabbage seeds were coated with seed powder are shown. If the same lowercase letter is not included, it indicates a statistically significant difference, and P<0.05. CK: Clean water control group; T1: Group coated with 40 mg / L α-linolenic acid; T2: Group coated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg 20% ​​chlorantraniliprole suspension; T3: Group coated with 5 mg 20% ​​chlorantraniliprole suspension. [Modes for carrying out the invention]

[0034] The present invention will be further described below with reference to the drawings and embodiments.

[0035] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods for which specific conditions are not described in the examples should generally be carried out under normal conditions or conditions recommended by the manufacturer.

[0036] The 20% chlorantraniliprole suspension (trade name: Coltar, manufactured by DuPont) was purchased directly from the market. Alpha-linolenic acid is a commercially available product, and any product can be used.

[0037] Example 1 Three concentrations of alpha-linolenic acid were established: 4 mg / L, 40 mg / L, and 400 mg / L. Nanryu 9108 (non-glutinous rice) and Shinryoyu No. 1 (Indica rice) were selected for seed treatment. Seeds were coated with 1 mL of alpha-linolenic acid solution of different concentrations (4-400 mg / L) per gram of dry seed. The weighed rice seeds were placed in plastic bags (small amounts of seeds were used in the laboratory test), then the alpha-linolenic acid solution was added, and the plastic bags were shaken vigorously for about 5 minutes to thoroughly and uniformly mix the contents. After leaving the mixture in a cool place for 24 hours, the seeds were sown in moisture-retaining petri dishes lined with absorbent paper. Seeds treated with clean water were used as a control (CK). Subsequently, germination rate and germination time were statistically analyzed.

[0038] According to the results of measurements of rice seed germination rate and germination time, when Nanryu 9108 seeds were treated with α-linolenic acid at different concentrations, there were some fluctuations in germination rate and germination time compared to the control (CK), but none were statistically significant (Table 1). When Shinryoyu No. 1 seeds were treated with α-linolenic acid at different concentrations, there was a tendency for the germination rate to increase, but it was not statistically significant compared to the control (CK). The germination time was significantly shorter when treated with a 4 mg / L concentration compared to the control, and was extended to some extent when treated with 40 mg / L and 400 mg / L concentrations compared to the control, but none were statistically significant (Table 2).

[0039] [Table 1] Note: If the same lowercase letter is not included, it indicates a statistically significant difference, and P<0.05.

[0040] [Table 2] Note: If the same lowercase letter is not included, it indicates a statistically significant difference, and P<0.05.

[0041] According to the results above, after treating rice seeds with alpha-linolenic acid at different concentrations, there were no overall significant adverse effects. In fact, some treatment concentrations showed some beneficial effects, including improved germination rates and reduced germination time. However, further increases in the concentration of alpha-linolenic acid did affect seed germination.

[0042] Example 2 Three concentration gradients of alpha-linolenic acid solution were established: 4 mg / L, 40 mg / L, and 400 mg / L. Nanryu 9108 (non-glutinous rice) and Shinryoyu No. 1 (Indica rice) were selected and treated as seeds. Seeds treated with clean water were used as the control (CK). The seed coating method was the same as in Example 1. Treated seeds were sown in disposable plastic cups, each with a volume of 750 mL, and 4 rice seeds were sown in each cup. After sowing, the plants were managed as usual. The soil in the plastic cups was a mixture of plant growth medium, vermiculite, and perlite in a ratio of 4:3:3 (V / V / V). The height of the rice seedlings in the plastic cups was measured on day 3, day 6, and day 9. 20 plants were randomly selected from each group for measurement.

[0043] According to the measurement results, after treating Nanryu 9108 seeds with α-linolenic acid at different concentrations, the height of the rice seedlings showed some variation compared to the control at different time points, but overall the height of the rice seedlings after seed coating was higher than the control, and at some concentrations it was significantly higher than the control (Figure 1). After treating Shinryoyu No. 1 seeds with α-linolenic acid at different concentrations, the height of the rice seedlings was lower than the control in the initial treatment group, but over time the height of the rice seedlings in the treatment group gradually caught up with and surpassed that of the control group (Figure 2).

[0044] According to the results above, after treating rice seeds with alpha-linolenic acid at different concentrations, there were no apparent adverse effects overall, and at some treatment concentrations, the height of the rice plants was significantly higher than that of the control.

[0045] Example 3 Three concentration gradients of alpha-linolenic acid solution were established: 4 mg / L, 40 mg / L, and 400 mg / L. Nanryu 9108 (non-glutinous rice) and Shinryoyu No. 1 (Indica rice) were selected and treated with seeds. Seeds treated with clean water were used as a control (CK). The seed coating method was the same as in Example 1. Treated seeds were sown in disposable plastic cups, each with a volume of 750 mL, and 4 rice seeds were sown in each cup. After sowing, the seedlings were managed as usual. The soil was the same as in Example 2. Newly hatched rice stem borer larvae were inoculated into the rice seedlings in the plastic cups 30 days after sowing. Ten test insects were inoculated per cup of rice seedlings, and each group was performed in 6 replicates. The number of surviving insects was checked 6 days after inoculation, and the survival rate of the test insects was statistically analyzed.

[0046] Data analysis showed that when Nanryu 9108 and Shinryoyu No. 1 seeds were treated with α-linolenic acid at different concentrations, the survival rate of rice stem borers gradually decreased as the α-linolenic acid concentration increased (Figure 3). These results indicate that the exogenous application of α-linolenic acid through seed coating can significantly improve the resistance of rice plants to rice stem borers.

[0047] Example 4 Rice seeds of the Nan-gyu variety 9108 were coated with 400 mg / L α-linolenic acid. Seeds treated with clean water were used as a control (CK). The seed coating method was the same as in Example 1. The coated seeds were sown in disposable plastic cups, each with a volume of 750 mL, and 10 rice seeds were sown in each cup. The soil was the same as in Example 2. Thirty days after sowing, the above-ground parts of the rice plants were collected, and the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) content were measured. The number of biological replicates was set to 4. The measurement method was liquid chromatography-mass spectrometry (HPLC-MS / MS). The chromatography-mass spectrometry data acquisition system mainly included ultra-high performance liquid chromatography (UPLC, SCIEX product) and tandem mass spectrometry (MS / MS, QTRAP 6500+, SCIEX product). Mobile phase: Ultrapure water containing 0.04% acetic acid (Phase A), 99.9% acetonitrile (Phase B). The Agilent C18 column (100 mm × 2.1 mm, 1.7 μm) was used at a column temperature of 40°C and an injection volume of 2 μL. Qualitative analysis of data detected by mass spectrometry was performed using the Metware Database (MWDB) database, which was constructed based on standards. Quantitative analysis was performed by integrating the chromatographic peaks of the target substance using the multiple reaction monitoring mode of triple quadrupole mass spectrometry, and then using standard curves.

[0048] According to the measurement results, 30 days after seed coating with α-linolenic acid, the content of both jasmonic acid and jasmonic acid-isoleucine complex in rice plants significantly increased. Jasmonic acid content increased by approximately 41% compared to the control, and jasmonic acid-isoleucine content increased by approximately 60% (Figure 4). These results indicate that seed coating with α-linolenic acid can significantly increase the content of protective hormones (JA and JA-Ile) in rice plants, thereby improving the resistance of rice to pests.

[0049] Example 5 Corn seeds (Sweet Star 221) were treated with a 400 mg / L concentration of alpha-linolenic acid. Seeds treated with clean water were used as a control (CK). The seed treatment method was the same as in Example 1. The treated seeds were sown in disposable plastic cups with a volume of 750 mL, and two seeds were sown in each cup. The soil was the same as in Example 2. Thirty days after sowing, newly hatched larvae of Spodoptera fulgiperda were inoculated. Ten newly hatched larvae were inoculated per seedling cup, and each group was performed with 7 replicates. The number of surviving insects was checked six days after inoculation, and the survival rate of the test insects was statistically analyzed.

[0050] According to the peeling test results, 30 days after seed coating with alpha-linolenic acid, the survival rate in the Spodoptera fulgiperda treatment group was significantly lower than in the control group (Figure 5), and the number of test insects surviving on maize in the treatment group was generally smaller than the number surviving on maize plants in the control group (Figure 6).

[0051] Example 6 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L, and a fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used. Nanryu 9108 seeds were selected for treatment. Each g of dry seed was coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Seeds treated with clean water were used as a control (CK). The seed coating method was the same as in Example 1. After leaving the seeds in a cool place for 24 hours, they were sown in moisture-retaining petri dishes lined with absorbent paper, and then germination rate and average germination time were statistically analyzed.

[0052] According to the results of measurements of rice seed germination rate and germination time, when Nanryu 9108 seeds were treated with a mixture of alpha-linolenic acid and chlorantraniliprole suspension at different concentrations, neither the germination rate nor the germination time showed a significant difference compared to the clean water control (CK) (Table 3).

[0053] [Table 3] Note: If the same lowercase letter is not included, it indicates a statistically significant difference, and P<0.05.

[0054] Example 7 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L, and a fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used. Rice variety Nanryu 9108 was selected for seed treatment. Each g of dry seed was coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Seeds treated with clean water were used as a control (CK). The seed coating method was the same as in Example 1. Treated seeds were sown in disposable plastic cups, each with a volume of 750 mL, and 4 rice seeds were sown in each cup. After sowing, the plants were managed in the usual manner. The soil in the plastic cups consisted of a mixture of plant growth medium, vermiculite, and perlite in a ratio of 4:3:3 (V / V / V). The height of the rice seedlings in the plastic cups was measured on days 3, 6, and 9. Twenty seedlings were randomly selected from each group for measurement.

[0055] According to the measurement results, after treating rice seeds with a mixture of α-linolenic acid and chlorantraniliprole suspension at different concentrations, the height of the rice seedlings showed some variation compared to the control at different measurement points. However, the height of the rice seedlings after seed coating was generally higher than that of the clean water control, and at some concentrations, it was significantly higher than that of the control (Figure 7). Based on the above results, treating rice seeds with a mixture of α-linolenic acid and chlorantraniliprole suspension at different concentrations did not have any apparent adverse effects on the growth of the rice plants overall, and at some treatment concentrations, the height of the rice plants was significantly higher than that of the control.

[0056] Example 8 Seeds were coated with a mixture of alpha-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of alpha-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L. A fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used, and one group was set up to be coated with 5 mg of 20% chlorantraniliprole suspension alone (existing reports clearly indicate that the control effect is significantly reduced when the amount of 20% chlorantraniliprole suspension used is below 5 mg / g of rice seeds). Seeds were treated using Nanryu 9108 (non-glutinous rice) and Shinryoyu No. 1 (Indica rice). Rice seeds were coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension per gram of dried seeds, and also with 5 mg of 20% chlorantraniliprole suspension alone. Seeds treated with clean water were used as the control (CK). The treated seeds were sown in disposable plastic cups. The volume of the plastic cups was 750 mL, and 4 rice seeds were sown in each cup. The soil in the plastic cups was a mixture of plant growth medium and vermiculite in a ratio of 6:4 (V / V). At different times after sowing, newly hatched rice stem borer larvae were inoculated into the rice seedlings in the plastic cups. Ten test insects were inoculated into each cup of rice seedlings, and each group was performed with 10 replicates. Five to seven days after inoculation, the number of surviving insects was checked, and the survival rate of the test insects was statistically analyzed.

[0057] According to the bioassay results for the Nanryu 9108 rice plants, the survival rate of test insects in each treatment group was 10% or less on days 22, 30, and 40, showing a significantly large difference compared to the control group (CK). Furthermore, the survival rate of test insects in the group treated with α-linolenic acid and chlorantraniliprole suspension in combination was not significantly different from that of the group treated with chlorantraniliprole suspension alone. On day 52, the survival rate of test insects in the group treated with α-linolenic acid and chlorantraniliprole suspension in combination increased slightly, but was still significantly larger than that of the control group (Figure 8A). When seeds were coated with 5 mg of 20% chlorantraniliprole suspension alone, the lethal effect on rice stem borers was not significantly different from that of the group treated with α-linolenic acid and reduced amounts of chlorantraniliprole (2.5 mg of 20% chlorantraniliprole suspension), regardless of whether the treatment was early (22d) or late (52d) (Figure 8A).

[0058] According to the bioassay results for the Shinryoyu No. 1 rice plants, at 23, 35, and 45 days after seed coating treatment, the survival rate in the control (CK) group was between 60-70%, while the survival rate in each treatment group was less than 10%. The difference from the control group was significant, and there were no significant differences between the treatment groups. At 62 days, although the survival rate of the test insects in each treatment group increased slightly, the difference from the control group was still significant, and there were no significant differences between the different treatment groups (Figure 8B). When seed coating was performed with 5 mg of 20% chlorantraniliprole suspension alone, at all detection time points (from 23 to 62 days), there was no significant difference compared to the lethal effect on rice stem borers in the group treated with a combination of alpha-linolenic acid and reduced amounts of chlorantraniliprole (2.5 mg of 20% chlorantraniliprole suspension) (Figure 8B).

[0059] The above test results show that when using seed coating technology to control pests in rice, the exogenous addition of alpha-linolenic acid can maintain a higher control effect against pests under conditions where the amount of chemical pesticides used is reduced.

[0060] Example 9 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L. A fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used for each concentration. Additionally, one group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Corn (variety: Sweet Star 221) was used for seed treatment. Each g of dry seed was coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Another group was coated with 5 mg of 20% chlorantraniliprole suspension alone. Seeds treated with clean water were used as the control (CK). The seed coating method was the same as in Example 1. After being left in a cool place for 24 hours, the seeds were sown in disposable plastic cups. Each cup had a volume of 750 mL, and three seeds were sown in each cup. After sowing, the plants were managed in the usual manner. The soil in the plastic cups was a mixture of plant growth medium, vermiculite, and perlite in a ratio of 4:3:3 (V / V / V). The height of the corn in the plastic cups was measured on days 3, 6, and 9. Twenty plants were randomly selected from each group for measurement.

[0061] According to the measurement results of corn plant height after seed coating treatment, on day 3, there was no significant difference in plant height between each treatment group and the clean water control (CK), nor was there a significant difference between the treatment groups. On day 6, the plant height in all treatment groups was higher than that of the CK group. The group treated with a mixture of 40 mg / L α-linolenic acid and 2.5 mg of 20% chlorantraniliprole suspension (T2) showed a significant difference compared to the CK group, while the remaining treatment groups showed no significant difference compared to the CK group. On day 9, the group treated with a mixture of 400 mg / L α-linolenic acid and 2.5 mg of 20% chlorantraniliprole suspension (T3) showed a significant difference compared to the T2 group, but none of the treatment groups showed a significant difference compared to the CK group (Figure 9). According to the results of this study, within a specific dose range, treating corn seeds with a mixture of alpha-linolenic acid at different concentrations and 20% chlorantraniliprole suspension did not have any significant adverse effects on corn growth overall. In fact, at some treatment concentrations, corn plant height was significantly higher than that of the control group. These results were essentially consistent with the results obtained in rice (Example 7).

[0062] Example 10 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L. A fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used for each concentration. Additionally, one group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Corn (variety: Sweet Star 221) was used for seed treatment. Each g of dry seed was coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Another group was coated with 5 mg of 20% chlorantraniliprole suspension alone. Seeds treated with clean water were used as the control (CK). The seed coating method was the same as in Example 1. After being left in a cool place for 24 hours, the seeds were sown in disposable plastic cups. Each plastic cup had a volume of 750 mL, and three seeds were sown in each cup. After sowing, the plants were managed in the usual manner. The soil in the plastic cups was a mixture of plant growth medium and vermiculite in a ratio of 6:4 (V / V). Thirty days after sowing, newly hatched larvae of Spodoptera fulgiperda were inoculated. Ten test insects were inoculated per cup of rice seedlings, and each group was used for 10 replicates. The number of surviving insects was checked five days after inoculation, and the survival rate of the test insects was statistically analyzed.

[0063] Bioassay results showed that the lethality against Spodoptera fulgiperda significantly increased after seed coating of corn. When the amount of chemical pesticide (chlorantraniliprole) used was halved and seed coating was performed in combination with α-linolenic acid and 20% chlorantraniliprole suspension (T1, T2, T3), a similarly higher insecticidal effect was obtained, and it was the same as the insecticidal effect when seed coating was performed with a high dose of 20% chlorantraniliprole suspension alone (T4) (Figure 10). These test results further indicate that when using seed coating technology for pest control in corn, the exogenous addition of α-linolenic acid can maintain a higher control effect against pests under conditions where the amount of chemical pesticide used is reduced. These results were consistent with the test results in rice (Example 8).

[0064] Example 11 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L. A fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used for each concentration. Additionally, one group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Cabbage (variety: Kyoho No. 1) was used for seed treatment. Per 1 g of dry seed, the seeds were coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Another group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Seeds treated with clean water were used as the control (CK). The seed coating method was the same as in Example 1. After being left in a cool place for 24 hours, the seeds were sown in disposable plastic cups. Each cup had a volume of 750 mL, and three seeds were sown in each cup. After sowing, the plants were managed in the usual manner. The soil in the plastic cups was a mixture of plant growth medium, vermiculite, and perlite in a ratio of 4:3:3 (V / V / V). The height of the cabbage plants in the plastic cups was measured on days 3, 6, and 9. Twenty plants were randomly selected from each group for measurement.

[0065] The measurement results showed that on day 3, the plant height in treatment groups T2 and T4 was significantly lower than that of the control group (CK), while the plant height in the remaining two treatment groups was not significantly different from that of the control group. On day 6, the plant height in each treatment group was not significantly different from that of the control group, nor were there any significant differences between the treatment groups. On day 9, the plant height in treatment group T3 was significantly higher than that of the control group, while the plant height in the remaining treatment groups was not significantly different from that of the control group (Figure 11). These test results indicate that, in the initial stages, chemical seed coating had a slight adverse effect on cabbage growth, but this adverse effect gradually disappeared over time. Furthermore, seed coating also had a certain promoting effect on cabbage growth.

[0066] Example 12 Seeds were coated with a mixture of α-linolenic acid and 20% chlorantraniliprole suspension. Three concentrations of α-linolenic acid were set: 4 mg / L, 40 mg / L, and 400 mg / L. A fixed dose of 2.5 mg of 20% chlorantraniliprole suspension was used for each concentration. Additionally, one group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Cabbage (variety: Kyoho No. 1) was used for seed treatment. Per 1 g of dry seed, the seeds were coated with a mixture of 1 mL of α-linolenic acid solution of different concentrations (4-400 mg / L) and 2.5 mg of 20% chlorantraniliprole suspension. Another group was treated with 5 mg of 20% chlorantraniliprole suspension alone. Seeds treated with clean water were used as the control (CK). The seed coating method was the same as in Example 1. After being left in a cool place for 24 hours, the seeds were sown in disposable plastic cups. Each plastic cup had a volume of 750 mL, and three seeds were sown in each cup. After sowing, the plants were managed in the usual manner. The soil in the plastic cups was a mixture of plant growth medium and vermiculite in a ratio of 6:4 (V / V). Thirty days after sowing, newly hatched diamondback moth larvae were inoculated. Ten test insects were inoculated per cup of rice seedlings, and each group was repeated for 10 replicates. The number of surviving insects was checked five days after inoculation, and the survival rate of the test insects was statistically analyzed.

[0067] According to the bioassay results for diamondback moth, seed coating with alpha-linolenic acid alone increased the resistance of cabbage to diamondback moths, and the exogenous addition of alpha-linolenic acid maintained a higher lethal effect against diamondback moths under conditions where the amount of chemical pesticides used was reduced (Figure 12). These results were consistent with the test results for paddy rice (Example 8) and corn (Example 10).

[0068] In short, the present invention proposes a biological combination formulation for controlling crop pests. When seed coating is performed using the biological combination formulation of the present invention, there are no apparent adverse effects on the germination and growth of crops (rice, corn, cabbage), and at some treatment concentrations, it shows a clear promoting effect. At the same time, even when the amount of chemical agents used is significantly reduced, it still has a higher lethal effect against the relevant pests. The present invention has an important guiding role in the development of greener and more efficient crop seed coating pest control technology.

Claims

1. A method for increasing the pest resistance of crops, A method for increasing the pest resistance of crops, characterized by treating crop seeds with an alpha-linolenic acid solution, thoroughly and uniformly mixing them, and then sowing them.

2. A method for increasing the insect pest resistance of crops according to claim 1, characterized by coating 1 g of dried crop seeds with 0.5 to 1 mL of a 4 to 400 mg / L α-linolenic acid solution.

3. The method for increasing the pest resistance of crops according to claim 1, characterized in that, after the seed coating operation is completed, the crops are left in a cool place for 12 to 24 hours before sowing, and the sowing is performed by directly scattering the seeds in the field using a direct sowing method, or by growing seedlings in a seedling tray and then transplanting them.

4. A biological combination formulation for controlling crop pests, The aforementioned biological combination formulation comprises alpha-linolenic acid and the pesticide chlorantraniliprole as described in claim 1, wherein the mass ratio of the two is 0.004 to 0.4:0.5 to 5, and is characterized as a biological combination formulation for controlling crop pests.

5. The biological combination formulation for controlling crop pests according to claim 4, characterized in that the biological combination formulation comprises alpha-linolenic acid and a 20% chlorantraniliprole suspension, and the mass ratio of the two is 0.004 to 0.4:2.5 to 5.

6. The method for increasing the pest resistance of a crop according to claim 1, characterized in that the crop is rice, corn, or cabbage, or the biological combination formulation for controlling crop pests according to claim 4.

7. The method for increasing the pest resistance of crops according to claim 1, characterized in that the crops are rice and corn, or the biological combination formulation for controlling crop pests according to claim 4.

8. The method for increasing the pest resistance of crops according to claim 1, characterized in that the aforementioned pests are the Japanese rice stem borer, the large rice stem borer, the rice leaf borer, the Spodoptera fulgiperda, the corn borer, the red-eyed green moth, the armyworm, the diamondback moth, or the cabbage worm, or the biological combination formulation for controlling crop pests according to claim 4.

9. Use of alpha-linolenic acid according to claim 1 or a biological combination formulation for controlling crop pests according to claim 5 in order to improve the pest resistance of crops or to control crop pests.

10. The use according to claim 9, characterized in that the crop is rice, corn, or cabbage, and the pest is rice stem borer, large rice stem borer, red rice stem borer, brown planthopper, white-backed planthopper, Spodoptera fulgiperda, corn borer, armyworm, red-eyed green moth, or diamondback moth.

11. The use according to claim 9, characterized in that the alpha-linolenic acid or the biological combination preparation is used to improve the resistance of crops to pests or to control crop pests by a seed coating method.

12. The use according to claim 9, characterized in that, in the use described above, 0.5 to 1 mL of a 4 to 400 mg / L α-linolenic acid solution is applied to 1 g of dried crop seeds before sowing, or 0.5 to 1 mL of a 4 to 400 mg / L α-linolenic acid solution and 2.5 to 5 mg of a 20% chlorantraniliprole suspension are applied to 1 g of dried seeds before sowing.

13. The use according to claim 12, characterized in that, after the seed powder coating treatment is completed, the seeds are left in a cool place for 12 to 24 hours before sowing.

14. The use according to claim 13, characterized in that the sowing is performed by directly scattering seeds in the field using a direct sowing method, or by raising seedlings in a seedling tray before transplanting.

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