Helicoverpa armigera larva feeding antifeedant and application thereof

Abstract

The invention discloses a cotton bollworm larva feeding antifeedant and application of special valeralose derived from plants in cotton bollworm larva feeding refusal, L-arabinose has antifeedant activity on cotton bollworm larvae, and the L-arabinose also has a function of inhibiting the sum of midgut digestive enzymes of the cotton bollworm larvae; l-arabinose is stable in chemical property and not easy to degrade in nature, not only can inhibit feeding of young larvae, but also can inhibit feeding of young cotton bollworm larvae. As a plant source antifeedant, the L-arabinose has a remarkable antifeedant effect on cotton bollworm larvae, is convenient to use and low in cost, has no harm to the environment and no influence on plant growth and development, is more suitable for being mixed with other types of plant source pesticides for application, and can be developed and applied as an efficient, economic, simple, convenient and novel plant source pesticide.

Description

Technical Field

[0001] The present invention relates to the field of green pesticides and plant protection, and in particular to a plant-derived substance L-arabinose as a feeding repellent for cotton bollworm larvae and an application thereof. Background Art

[0002] The cotton bollworm is a polyphagous pest that harms a variety of crops, including corn, wheat, cotton, rice, peanuts, and tomatoes. Although genetically modified bollworm-resistant crops are being gradually introduced in my country, bollworms have also developed resistance to genetically modified cotton. Currently, with the large-scale reduction in cotton acreage in central China, bollworm damage to crops such as corn, peanuts, tobacco, and vegetables has not decreased, but has instead increased.

[0003] Existing technologies for controlling cotton bollworms primarily include agricultural control, biological control, physical control, and chemical control. Agricultural control primarily involves deep plowing and irrigation to destroy the pupae in the soil. However, due to the bollworm's voluminous diet, crop rotation and other measures are less effective. Biological control primarily involves the application of natural enemies or the spraying of biological agents such as virus particles and diflubenzuron. Using bollworm sex pheromones to attract adult insects is also a biological control method. Physical control primarily exploits the phototaxis of adult insects. Chemical control primarily involves spraying chemical pesticides. Due to their rapid effectiveness and cost-effectiveness, chemical pesticides remain the primary method of cotton bollworm control in my country. Chemical control, however, continues to lead to increased resistance in the bollworm, environmental pollution, and pesticide residues in agricultural products.

[0004] Insect antifeedants are specific agents that prevent insects from feeding by interfering with or inhibiting the function of their taste receptors. These chemicals typically activate bitter taste neurons in insects, causing them to resist feeding and protect plants. Due to their low application concentrations, these agents generally do not directly kill insects. However, due to their green nature, pronounced antifeedant effects, and environmental friendliness, they hold great promise for application in agricultural pest control.

[0005] L-arabinose is a plant-specific aldopentose found widely in the husks of fruits and whole grains. Studies have shown that L-arabinose can inhibit the activity of sucrase and maltase in the digestive systems of mammals such as mice, pigs, and humans, thereby inhibiting their digestion and absorption of nutrients. Summary of the Invention

[0006] In order to solve the problems in the above-mentioned prior art of insufficient types of insect repellents and high cost of using existing types, the present invention provides a simple, low-cost, green and pollution-free insect repellent, which uses L-arabinose, a plant metabolite, as its core ingredient. It has a wide range of natural sources, is pollution-free to the environment, and is non-toxic to crops and leaves no residue.

[0007] A feeding antifeedant for cotton bollworm larvae, the effective active ingredient of which includes L-arabinose.

[0008] The cotton bollworm larvae antifeedant consists of L-arabinose and solvent water; The concentration of L-arabinose in the cotton bollworm larvae antifeedant is 66.6 mM to 500 mM; The preferred concentration of L-arabinose in the cotton bollworm larvae antifeedant is 199.8 mM to 500 mM; It also includes other active pesticide ingredients, wherein the other active pesticide ingredients are plant-derived pesticide substances such as matrine, azadirachtin and rotenone; The concentration of matrine in the cotton bollworm larvae antifeedant mixture is 100 mM; the concentration of azadirachtin is 0.001 mM to 0.1 mM; and the concentration of rotenone is 10 mM to 30 mM. Used as a feeding antifeedant for cotton bollworm larvae.

[0009] The cotton bollworm larvae antifeedant mixture is prepared by mixing L-arabinose and azadirachtin for application.

[0010] The invention relates to an application of L-arabinose in the cotton bollworm larvae antifeedant mixture in preparing the cotton bollworm larvae antifeedant.

[0011] The cotton bollworm larvae antifeedant can act as an insect antifeedant by inhibiting the activity of sucrase in the midgut of the cotton bollworm larvae.

[0012] The cotton bollworm larvae antifeedant of the present invention can be used as an insect antifeedant by activating the activity of bitter taste neurons of the cotton bollworm larvae.

[0013] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention has found that plant-derived L-arabinose can inhibit the activity of sucrase in the midgut of cotton bollworm larvae and activate the activity of bitter neurons of cotton bollworm larvae. It has excellent antifeedant activity on cotton bollworm larvae and is simple and convenient to use.

[0014] Positive and beneficial effects: The present invention dilutes plant-derived L-arabinose with water to produce an antifeedant effect on cotton bollworm larvae. L-arabinose is stable in nature, has a long antifeedant effect, is pollution-free, and has good social benefits in ensuring environmental safety. It is not easy to develop resistance and can be mixed with other biological pesticides or antifeedants. It is easy to use and does not require other pesticide adjuvants. L-arabinose dissolved in water can produce a significant antifeedant effect, which is easier to promote on a large scale. The antifeedant effect is fast and effective. L-arabinose can produce a significant antifeedant effect on cotton bollworm larvae within 4 hours, and has no effect on plant growth and development, providing a new method for preventing and controlling cotton bollworm larvae with high efficiency, simplicity, and low cost. In addition, compared with other plant-derived pesticides or antifeedants, L-arabinose is very low in cost and more readily available on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Comparison of the feeding refusal behavior of cotton bollworm larvae to leaf discs treated with different concentrations of L-arabinose; Figure 2 :L-arabinose can counteract the feeding effect of cotton bollworm larvae on sucrose; Figure 3 :Comparison chart of the effect of L-arabinose on larval feeding trajectory; Figure 4 :A comparative diagram of the effects of L-arabinose on the feeding trajectory of cotton bollworm larvae with a tendency to resist feeding; Figure 5 :Comparison chart of the effects of L-arabinose on midgut enzyme activities of cotton bollworm larvae; Figure 6 : Comparison of the effects of 199.8 mM L-arabinose mixed with three plant secondary metabolites on the feeding behavior of cotton bollworm larvae. DETAILED DESCRIPTION

[0016] The effects of the present invention are further described below by way of specific embodiments:

[0017] Example 1: Effect of L-arabinose on feeding behavior of cotton bollworm larvae 1. The cotton bollworms used in this experiment were collected from the Science and Education Park of Henan Agricultural University in Huiji District, Zhengzhou City, and were reared indoors. The larvae were fed an artificial diet (one serving consisting of 80 g soybean flour, 150 g wheat bran, 30 g yeast powder, 3 g sorbic acid, 3 g methylparaben, 20 g agar, 40 g casein, 3 g vitamin C, 0.8 g multivitamins, 10 g sucrose, 5 drops of linoleic acid, 2 mL formaldehyde, 4 mL acetic acid, and 1400 mL water) until they reached the fifth instar.

[0018] 2. The plant materials used in the experiment were pepper leaves, which were grown in the Plant Protection Laboratory of Henan Agricultural University.

[0019] 3. L-arabinose and sinigrin were produced and provided by Sigma; sucrose was produced and provided by Life Science; potassium chloride was purchased from Tianjin Beichen Fangzheng Reagent Factory; all compounds were of analytical grade.

[0020] 4. The feeding behavior test used a binomial leaf disc method: 5th-instar, second-day-old larvae of appropriate size were selected and placed in the center of a Petri dish lined with 12 cm diameter moist filter paper. Fresh, cleaned pepper leaves were then punched into leaf discs using a 1 cm diameter hole punch. Four treated leaf discs and four control leaf discs were placed alternately along the wall of the Petri dish in the order ABABABAB, with the distance between each pair of discs being equal. This arrangement ensured that the larvae had equal opportunities to feed on the treated and control leaf discs. Observations were conducted every 15 minutes. When a larva had consumed approximately half of the leaf disc area (i.e., two discs) on any given plant, it was removed, the experiment terminated, and the remaining leaf area was measured. Paired t-tests were used to analyze differences.

[0021] 5. This experiment used fresh pepper leaf discs as the medium and divided them into treatment and control groups. The control group consisted of discs treated with sterile distilled water, while the treatment groups consisted of discs treated with different concentrations of L-arabinose (6.6 mM, 66.6 mM, 199.8 mM, 333 mM, and 500 mM). Each concentration was replicated three times, with 50 insects in each test.

[0022] 6. The feeding selection index was used to analyze the feeding selection differences of cotton bollworm larvae on the treated and control leaf discs. The calculation formula of the feeding selection index is: The feeding selection index of the control leaf disc (P C ) = feeding area of ​​the control leaf disc in the dish / all feeding leaf areas in the dish; The feeding selection index of leaf discs treated with T ) = feeding area of ​​the treated leaf disc in the dish / total feeding area of ​​leaves in the dish.

[0023] 7. The results showed that low concentrations of L-arabinose could neither induce nor inhibit the feeding preference of cotton bollworm larvae. However, as the concentration of L-arabinose increased, cotton bollworms showed a significant aversion to L-arabinose-treated leaf discs. Figure 1As shown, A: Feeding preference index of cotton bollworm larvae on leaf discs treated with 6.6mM L-arabinose; B: Feeding preference index of cotton bollworm larvae on leaf discs treated with 66.6mM L-arabinose; C: Feeding preference index of cotton bollworm larvae on leaf discs treated with 199.8mM L-arabinose; D: Feeding preference index of cotton bollworm larvae on leaf discs treated with 333mM L-arabinose; E: Feeding preference index of cotton bollworm larvae on leaf discs treated with 500mM L-arabinose. Note: Control: distilled water; LA: L-arabinose; "*" indicates a significant difference in feeding preference index between treated and control leaf discs ( P <0.05), "***" indicates that the feeding selection index of the treated leaf disc and the control leaf disc is extremely significant ( P <0.01), “ns” means no significant difference ( P >0.05).

[0024] Example 2: Effect of L-arabinose on the sucrose feeding behavior of cotton bollworm larvae 1. The feeding choice behavior test used the binomial leaf disc method, which was the same as that in Example 1.

[0025] 2. Fresh pepper leaf discs were used as the medium and divided into control group and treatment group. The pepper leaf discs treated with 10mM sucrose solution were used as the control group, and the leaf discs treated with a mixed solution of 10mM sucrose and different concentrations (1mM, 10mM, 100mM, 200mM, 300mM, 500mM) of L-arabinose were used as the treatment group. The test of each concentration was repeated three times, and 50 insects were tested each time.

[0026] 3. The feeding selection index was used to analyze the feeding selection differences of cotton bollworm larvae on the treated leaf discs and the control leaf discs. The calculation formula of the feeding selection index was the same as that in Example 1.

[0027] 4. The results showed that L-arabinose could inhibit the feeding preference of cotton bollworm larvae for sucrose. Figure 2As shown in the figure: A: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 1mM L-arabinose and sucrose mixture; B: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 10mM L-arabinose and sucrose mixture; C: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 100mM L-arabinose and sucrose mixture; D: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 200mM L-arabinose and sucrose mixture; E: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 300mM L-arabinose and sucrose mixture; F: feeding choice behavior index of cotton bollworm larvae on leaf discs treated with 400mM L-arabinose and sucrose mixture. Note: S: sucrose; LA: L-arabinose; "**" indicates that the feeding choice index between the treated leaf discs and the control leaf discs is extremely significantly different ( P <0.01, “NS” means no significant difference ( P >0.05).

[0028] Example 3: Feeding trajectory of cotton bollworm larvae on L-arabinose within 2 hours 1. Determine the feeding trajectory of cotton bollworm larvae on L-arabinose. Petri dishes with a diameter of 12 cm were used. Leaf discs were divided into a control group and a treatment group. The control group was treated with 30 mL of distilled water, while the treatment group was treated with 30 mL of a 100 mM L-arabinose solution. Four leaf discs were placed in each dish: two from the treatment group and two from the control group. The two leaf discs were placed equidistantly along the edge of the filter paper in an A, B, and C pattern, and the positions of the different leaf discs were marked. Starved cotton bollworms were placed in the center of the filter paper, with one in each dish. The dishes were placed on a tabletop. Each larva feeding on a leaf disc was recorded. The feeding trajectory was observed and the feeding area was recorded. The number of times and the feeding area of ​​the leaf discs were counted within 1 hour and 2 hours. The experiment was terminated when the larvae had consumed approximately half the area of ​​any leaf disc (i.e., approximately one leaf disc). The experiment was repeated three times, with 20 insects per repeat.

[0029] 2. The feeding selection index was used to analyze the feeding selection differences between cotton bollworm larvae and control leaf discs. The feeding selection index was calculated as follows: The control leaf disc feeding selection index (P C ) = the feeding area of ​​the control leaf disc in the dish / the total feeding area of ​​all leaf discs in the dish Treatment leaf disc feeding selection index (P T ) = the feeding area of ​​the treated leaf disc in the dish / the total feeding area of ​​all leaf discs in the dish The leaf disc feeding selection index of the control (P C0 ) = the number of times the control leaf disc in the dish was fed / the total number of times all leaf discs in the dish were fed The leaf disc feeding selection frequency index (P T0 ) = Number of times the treated leaf disc in the dish was fed / Total number of times all leaf discs in the dish were fed 3. The results showed that there was no significant difference in the number of times larvae were exposed to L-arabinose between 1 hour and 2 hours compared with the control. However, the relative feeding preference index of larvae to L-arabinose within 2 hours was significantly lower than that of the control; Figure 3 As shown in the figure: A: Index of feeding selection times of cotton bollworm larvae on leaf discs treated with 100mM L-arabinose within 1 hour; B: Index of feeding selection times of cotton bollworm larvae on leaf discs treated with 100mM L-arabinose within 2 hours; C: Index of feeding selection times of cotton bollworm larvae on leaf discs treated with 100mM L-arabinose within 1 hour; D: Index of feeding selection times of cotton bollworm larvae on leaf discs treated with 100mM L-arabinose within 2 hours. Note: LA: L-arabinose; "*" indicates significant difference in feeding selection index between treated leaf discs and control leaf discs ( P <0.05, "**" indicates that the feeding selection index of the treated leaf disc and the control leaf disc was significantly different ( P <0.01, “ns” means no significant difference ( P >0.05).

[0030] Example 4: Feeding trajectory of L-arabinose in 2 hours of cotton bollworm larvae with a tendency to refuse feeding First, a two-way leaf disc assay was used to determine the feeding preference of cotton bollworm larvae for 100 mM L-arabinose. Fresh pepper leaves were used in the experiment, with the control and treatment groups divided into two groups. In the control group, eight leaf discs were placed per dish, all of which were treated with 30 mL of distilled water. In the treatment group, eight leaf discs were placed per dish: four control leaf discs treated with 30 mL of distilled water and four treated leaf discs treated with 30 mL of 100 mM L-arabinose solution. After 6 hours of feeding, larvae from the control and treatment groups that showed a difference in feeding were selected and their feeding patterns on 100 mM L-arabinose and water were observed.

[0031] 2. The feeding trajectory method and analysis method are the same as in Example 3.

[0032] 3. The results showed that there was no significant difference in the number of contacts between the two treatment groups in the control group larvae, but the feeding area of ​​LA by the larvae within 2 hours was significantly lower than that of the control group; there was also no significant difference in the number of contacts between the two treatment groups in the treatment group larvae, but the feeding area of ​​LA by the larvae within 1 hour was significantly lower than that of the control group, such as Figure 4As shown, A, B, C, and D are the control groups, and E, F, G, and H are the treatment groups. A, B: The feeding selection index of the cotton bollworm larvae in the control group on the leaf discs treated with 100mM L-arabinose within 1 hour and 2 hours, respectively; C, D: The feeding selection index of the cotton bollworm larvae in the control group on the leaf discs treated with 100mM L-arabinose within 1 hour and 2 hours, respectively; E, F: The feeding selection index of the cotton bollworm larvae in the treatment group on the leaf discs treated with 100mM L-arabinose within 1 hour and 2 hours, respectively; The feeding selection index of the cotton bollworm larvae in the treatment group on the leaf discs treated with 100mM L-arabinose within 1 hour and 2 hours, respectively. Note: LA: L-arabinose; "*" indicates that the feeding selection index of the treated leaf discs and the control leaf discs is significantly different ( P <0.05, “ns” means no significant difference ( P >0.05).

[0033] Example 5: Effect of L-arabinose on midgut digestive enzymes in cotton bollworm larvae 1. From the time of hatching, cotton bollworm larvae in the treatment group were fed an artificial diet supplemented with varying concentrations of L-arabinose (6.6 mM, 66.6 mM, 199.8 mM, 333 mM, and 500 mM). Larvae in the control group were fed a normal artificial diet. Fifth-instar larvae aged 24-48 hours were dissected and their midguts removed. The midguts of the second-day fifth-instar larvae were accurately weighed and added to six volumes of PBS at a weight (g) to volume (mL) ratio of 1:6. The mixture was mechanically homogenized in an ice-water bath and centrifuged at 2500 rpm for 10 minutes. The supernatant was then collected for analysis.

[0034] 2. The changes in sucrase and maltase in the midgut of fifth-instar larvae of cotton bollworm fed different concentrations of L-arabinose were tested using a kit produced by Nanjing Jiancheng Bioengineering Research Institute. The specific operation was carried out according to the instructions.

[0035] 3. The results showed that the activities of sucrase and maltase in the midgut of larvae fed with artificial diet containing low concentration of L-arabinose were not significantly different from those of control larvae; while the activities of sucrase and maltase in the midgut of larvae fed with artificial diet containing high concentration of L-arabinose were significantly lower than those of control larvae. Figure 5 As shown in the figure: A: Effects of different concentrations of L-arabinose on the activity of sucrase in the midgut of cotton bollworm larvae; B: Effects of different concentrations of L-arabinose on the activity of maltase in the midgut of cotton bollworm larvae. Note: LA: L-arabinose; error bars represent standard deviations, statistically significant differences ( P <0.05) are indicated by different letters.

[0036] Example 6: Effects of a mixture of L-arabinose and three plant secondary metabolites on the feeding behavior of cotton bollworm larvae 1. The feeding choice behavior test used the binomial leaf disc method, which was the same as that in Example 1.

[0037] 2. Fresh pepper leaf discs were used as mediators and divided into control and treatment groups. Based on the results of previous feeding behavior experiments, the treatment concentrations of the three compounds varied, with the optimal minimum concentration of L-arabinose being 199.8 mM. In the matrine feeding behavior experiment, the discs were treated with a mixture of 199.8 mM L-arabinose and 10, 30, and 100 mM matrine, while the controls were treated with distilled water (solvent). In the azadirachtin feeding behavior experiment, the discs were treated with a mixture of 199.8 mM L-arabinose and 0.001, 0.01, and 0.1 mM azadirachtin, while the controls were treated with 5.8% methanol (solvent). In the rotenone feeding behavior experiment, the discs were treated with a mixture of 199.8 mM L-arabinose and 1.0, 10, and 30 mM rotenone, while the controls were treated with 3% chloroform (solvent). The test of each concentration was repeated three times, with 50 insects in each test.

[0038] 3. The feeding selection index was used to analyze the feeding selection differences of cotton bollworm larvae on the treated leaf discs and the control leaf discs. The calculation formula of the feeding selection index was the same as that in Example 1.

[0039] 4. The results showed that L-arabinose mixed with high concentrations of matrine, azadirachtin, and rotenone could cause cotton bollworms to refuse to feed, while L-arabinose mixed with low concentrations of azadirachtin could also cause significant differences in the feeding behavior of cotton bollworms, and the differences were extremely significant at high concentrations of azadirachtin, indicating that L-arabinose is more suitable for mixing with azadirachtin in controlling cotton bollworm larvae. Figure 6Shown are: A: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 10 mM matrine; B: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 30 mM matrine; C: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 100 mM matrine; D: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 0.001 mM azadirachtin; E: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 0.01 mM azadirachtin; F: feeding selection index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 0.1 mM azadirachtin. G: Feeding preference index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 1mM rotenone; H: Feeding preference index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 10mM rotenone; I: Feeding preference index of cotton bollworm larvae on leaf discs treated with a mixture of L-arabinose and 30mM rotenone. Note: LA: L-arabinose; “*” indicates a significant difference in feeding preference index between treated and control leaf discs ( P <0.05, “****” indicates that the feeding selection index of the treated leaf disc and the control leaf disc is extremely significant ( P <0.01, “ns” means no significant difference ( P >0.05).

[0040] The present invention discovered that L-arabinose not only inhibits the activity of digestive enzymes in the midgut of cotton bollworm larvae but also exhibits significant antifeedant activity against them. Furthermore, while many current chemical pesticides are ineffective against older cotton bollworm larvae, L-arabinose can inhibit their feeding behavior, an advantage not possessed by many chemical pesticides. L-arabinose is stable under high temperature and humidity conditions, is non-toxic, and is non-polluting. Besides dissolving in water, it requires no additional additives, is easy to use, and is readily available on a large scale, demonstrating its potential for practical application in the control of cotton bollworm larvae.

Claims

1. A feeding antifeedant for cotton bollworm larvae, characterized in that: Its active ingredients include L-arabinose.

2. The feeding antifeedant for cotton bollworm larvae according to claim 1, characterized in that: The cotton bollworm larvae antifeedant consists of L-arabinose and solvent water.

3. The use according to claim 1 or 2, characterized in that: The concentration of L-arabinose in the cotton bollworm larvae antifeedant is 66.6 mM to 500 mM.

4. The use according to claim 2, characterized in that: The preferred concentration of L-arabinose in the cotton bollworm larvae antifeedant is 199.8 mM to 500 mM.

5. The feeding antifeedant for cotton bollworm larvae according to claim 1, characterized in that: Other pesticide active ingredients are also included.

6. The feeding antifeedant for cotton bollworm larvae according to claim 5, characterized in that: The other active pesticide ingredients are matrine, azadirachtin and rotenone plant-derived pesticide substances.

7. The feeding antifeedant for cotton bollworm larvae according to claim 6, characterized in that: In the cotton bollworm larvae antifeedant mixture, the concentration of matrine is 100 mM, the concentration of azadirachtin is 0.001 mM to 0.1 mM, and the concentration of rotenone is 10 mM to 30 mM.

8. Use of a feeding antifeedant for cotton bollworm larvae according to any one of claims 1 to 7, characterized in that: Used as a feeding antifeedant for cotton bollworm larvae.

9. The use of a feeding antifeedant for cotton bollworm larvae according to claim 8, characterized in that: The cotton bollworm larvae antifeedant mixture is prepared by mixing L-arabinose and azadirachtin for application.

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