KR101307626B1 - Method of Controlling Spodoptera exigua Using Stress Sound - Google Patents

Abstract

The present invention relates to a pest control method using stress sound waves. In the present invention, there is provided a method for controlling plant cultivation pests using stress sound waves, including treating sound waves at 100 to 5,000 Hz with insecticides in order to increase the insecticide sensitivity of the pests. In addition, in the present invention, the control method of each of the American leaf oyster ( Liriomyza trifolii ), peach aphid ( Myzus persicae ), scaboptera moth ( Spodoptera exigua ), which reduces the feeding amount , allegory rate, proliferation rate, egg laying rate, etc. using stress sound waves. Is provided. The present invention can be used as a control method that can effectively manage the plant cultivation pests without increasing the environmental pollution by increasing the effects of stress sonic treatment in combination with the existing chemical control method or biological control method without increasing the environmental pollution. Can be. In addition, the present invention can effectively manage the plant cultivation pests only by stress sound wave treatment can be used as a new control method without environmental pollution.

Description

Method for Control of Pabam Moth Using Stress Sound Waves {Method of Controlling Spodoptera exigua Using Stress Sound}

The present invention relates to a pest control method, and more particularly to a control method of pabam moth using stress sound waves.

Similar to odor communication, sound communication has a relatively wide range of use and superiority in scattering diversity and obstacle handling capability compared to contact or vision, but has a weak point of detecting stimulus sources and high energy cost (Alcock, 1979). In addition to cicadas and crickets that use sound communication, the noise signal of moth larvae (Brown et al ., 2006), the ultrasonic whistling of moth males (Nakano et al ., 2009), and the vibration sound of some stink bugs ( Cokl et al ., 2007) and sound signals are present in various insect species, such as friction sounds of extinct species. Except for the vibrational sound that strikes the surrounding medium, insect sounds are more common in relatively small body sizes, with frequency ranges of hundreds to thousands of Hz rather than low frequencies (Michelsen and Nocke, 1974). The sound of insects thus produced is used for intramural mating, warning, avoiding natural enemies, and for zone preference.

The study of stress sound waves focused on vertebrates. Acute stress waves induce stressant anociception, while long-term stress sound exposure induces chronic pain (fibromyalgin) or rheumatoid arthritis (Khasar et al ., 2005). For this reason, treatment of stress sound waves in experimental rats enhanced the activity of tryptophan hydroxylase, which is present in the brain and delivers the synthesis of neurotransmitters (Singh et al., 1990; Corley et al ., 1992). . In insects, the ultrasonic waves produced by bats can act as stress sound waves that induce evasive behavior of chestnut moths, and these stresses are associated with the activity variation of acetylcholine esterase in the insect nervous system (Zha et al ., 2008). .

Liriomyza trifolii is known as the native of Florida in the United States, and has spread around the world since the 1970s, causing damage to Korean crops (Parrella, 1987; Han et al., 1996). The damage of this pest can be divided into larvae cooling by forming tunnels in the lobular tissue and leaf damage caused by adult suckers (Parrella and Keil, 1984). The damage of American leaf oysters to these crop leaves causes photosynthesis deterioration and lowers the marketability of the crops, causing economic losses (Trumble et al., 1985). American leaf oyster flies, which occur mainly in facility cultivation, are the most suitable insects to adapt to high temperatures, with maximum adult catches in July and August, and generation in greenhouses even in winter (Park et al., 2000, 2001). . From egg to adult, the development period at 25 ° C is 16.3 days for gerbera and 12.3 days for tomato, causing multiple generations throughout the year, causing concern about the development of resistance to insecticides as well as damage (Bethke et al., 1987; Moon et. al., 2002).

Peach aphid ( Myzus persicae ) is a pest that infests fruits and vegetables. Larvae and adults live on leaves and new branches of peach trees, apricots, plums, cherry trees, etc. Causes minor harm. Occurs 10 times a year and winters as eggs near peach trees and winter snow. Ganmo, awakened from the egg in the spring, continues to unity until late autumn. From May, larvae appear and cause damage by switching to summer hosts such as radish, cabbage, cabbage, pepper, and potatoes. Ladybugs, flowers, dragonflies and parasitic bees are natural enemies and are distributed in Korea, Japan, China, India, Europe and the United States.

Spodoptera exigua is a major agricultural pest problem. The use of chemical pesticides, which are chemical control methods, is environmentally damaging, and the problem of weakness is also caused, and there are many difficulties in control. The result is biological control, which many farmers are interested in and continue to study. However, since biological control methods require a long time unlike chemical control methods, they do not get a great response from agricultural workers, and also have difficulty in effective control due to drug resistance.

[References]

Brown SG, Boettner GH, Yack JE, 2007. Clicking caterpillars: acoustic aposematism in Antheraea polyphemus and other Bombycidae . J. Exp. Biol. 210: 993-1005.

Cokl A, ZoroviC M, Millar JG. 2007. Vibrational communication along plants by the stink bugs Nezara viridula and Murgantia histrionica . Beh. Proc. 75: 40-54.

Cokl A, ZoroviC M, ZuniC A, Virant-Dobexlet M. 2005. Tuning of host plants with vibratory songs of Nezara viridula L. (Heteroptera: Pentatomidae). J. Exp. Biol. 208: 1481-1488.

Corley KC, Singh VB, Phan TH, Boadle-Biber MC. 1992. Effect of gepirone on increase in tryptophan hydroxylase in response to sound stress. Eur. J. Pharmacol. 31: 417-425.

Khasar SG, Green PG, Leine JD. 2005. Repeated sound stress enhances inflammatory pain in the rat. Pain 116: 79-86.

Nakano R, Takahashi T, Fuji T, Skals N, Surlykke A, Ishikawa Y. 2009. Moths are not silent, but whisper ultrasonic courtship songs. J. Exp. Biol. 212: 4072-4078.

Singh VB, Corley KC, Phan TH, Boadle-Biber MC. 1990. Increases in the activity of tryptophan hydroxylase from rat cortex and midbrain in response to acute or repeated sound stress are blocked by adrenalectomy and restored by dexamethasone treatment. Brain res. 516: 66-76.

Zha YP, Xu F, Chen QC, Lei CL. 2008. Effect of ultrasound on acetylcholinesterase activity in Helicoverpa armigera (Lepidoptere: Noctuidae). Can. Entomol. 140: 563-568.

American Leaf Oyster Fly ( Liriomyza) trifolii ), Peach aphid ( Myzus persicae , Parrot ( Spodoptera) Plant cultivation pests, such as exigua ), require effective and non-chemical pest management techniques to meet their reproductive capacity, drug resistance and production of safe produce under conditions of plant cultivation.

It is an object of the present invention to provide a new control method using stress sound waves against facility cultivation pests to improve the effectiveness of existing control methods and reduce side effects.

In addition, the present invention lowers the feeding amount , allegory rate, proliferation rate, egg laying rate, etc. using stress sound waves to control plant cultivation pests, in particular, American leaf oyster ( Liriomyza trifolii ), peach aphid ( Myzus persicae ) and pajamoth moth ( Spodoptera exigua ) It is an object of the present invention to provide a method for effectively controlling.

In the present invention,

A method for controlling pests of any one or more plant cultivated plants selected from American leaf oyster flies ( Liriomyza trifolii ), peach aphid ( Myzus persicae ), and parrot moth ( Spodoptera exigua ). There is provided a pest control method using stress sound waves, including processing sound waves at 5,000 Hz. The pesticides include both chemical pesticides and biological pesticides. If the pesticide is a chemical pesticide, it preferably treats sound waves of 2,000 to 5,000 Hz, and the pests in particular include pabam moths. In the case of biological pesticides, the sonic waves are preferably treated at 2,000 to 5,000 Hz, and the pesticides include Bacillus thuringiensis .

In the present invention,

Provided is a method for controlling American leaf oyster flies using stress sound waves, which includes treating a sound wave of 100 to 5,000 Hz to lower any one or more of feeding amount, allegory rate and scattering rate.

In the present invention,

Provided are methods for controlling peach aphids using stress sound waves, including treating sound waves at 100 to 5,000 Hz, thereby reducing development or growth rates.

In the present invention,

Provided is a method for controlling Pabam moths using stress sound waves, including the treatment of 100-5,000 Hz sound waves to lower one or more of feeding, digestive enzyme activity, and immunity.

According to the present invention, the use of stress sound waves can effectively manage facility cultivation pests such as American leaf oyster flies, peach aphids, and chestnut moths without increasing the environmental pollution and without the problem of drug resistance. In particular, the present invention can reduce the amount of side effects due to the chemical agent by reducing the amount of the conventional chemical agent by increasing the control effect by using the stress sound waves in combination with the conventional chemical control method or biological control method. In addition, despite the environmentally friendly advantages, the effective use of biological control can be promoted by increasing the effectiveness of biological agents such as Bt, which is less effective than chemicals and is less used. In addition, the present invention can effectively manage the plant cultivation pests by lowering the feeding amount, allegory rate, proliferation rate, spawning rate and the like only by stress sound treatment, it can be utilized as a new control method without environmental pollution.

1 is a result of the change in feeding amount of the American leaf oyster fly larva according to the stress sound processing, Figure 1a is the effect of the vibration on the feeding amount, Figure 1b is the effect of 24 hours sonic treatment at 5,000 Hz. The feeding tunnel length was measured by an image analysis program. Each treatment was repeated 10 times. The different letters on the standard deviation bar mean that there is a statistical difference between means at the significance (Type I error = 0.05, LSD test) level, which is the same below.
Figure 2 is the effect of the stress sound waves on the allegory of the American leaf oyster flies, after the exposure to the sound waves until the allegory by frequency was measured the fable rate and the duration of the fable. Fig. 2A is the allegorical rate and Fig. 2B is the allegorical period.
Figure 3 is an evaluation of the effect of the sonic treatment on the allegorical activity of American leaf oyster flies.
Figure 4a is to investigate whether the spawning occurs mainly in the near or distant part of the leaf of the leaf, Figure 4b is a measure of the number of spawning per female when treated at 25 ℃ for 5 days with sound waves of different frequencies . Ten or more females were used for the test.
FIG. 5 is a two-dimensional electrophoresis of protein changes after treatment with 95 dB, 5,000 Hz sound waves in chrysalis protein of American leaf oyster flies. Circles and triangles indicate specific proteins in the sonic and control groups. Each treatment used about 200 μg of protein extracted from more than 200 pupa.
Figure 6 analyzes the effect of stress sound on the insecticide susceptibility of American leaf oyster flies. Figure 6a is the insecticide rate according to the throughput of the spinosad (spinosad), 6b is the effect of sound waves on the insecticide. The sound waves were each treated at different frequencies and 10 larvae were used in the test. The test was conducted in three replicates and the insecticide rate was measured 5 days after treatment.
Figure 7 is the effect of stress sound on the development of peach aphid nymphs, nymphs waking up from the egg (25 °, 25 ℃, 95 dB conditions of various frequencies so that the treatment until the adult (adult) The number of days was measured. Treatments were carried out in three replicates, and 10 individuals were used for each treatment.
FIG. 8 shows the effect of sound waves of various frequencies on the proliferative capacity of female peach aphids. After the adult females were exposed to various sound waves at 95 dB, the number of scattered eggs during 25 days of treatment was measured. Treatments were carried out in three replicates, and 10 individuals were used for each treatment.
9 is a measure of protein changes in peach aphids under stress sound treatment. Figure 9a is divided into peach aphids treated group and untreated control group treated with 95 dB, 5,000 Hz sound waves for 24 hours after the protein change was measured by two-dimensional electrophoresis, circle and triangular display is a sound wave treatment Points to specific spots in the and controls respectively. 9B is a Venn diagram of protein expression patterns for total protein spots (N = 53).
Figure 10 analyzes the effect of stress sound on the insecticide susceptibility of peach aphids. Figure 10a is the insecticidal rate according to the throughput of imidacloprid without sonication, 10b is the effect of the sound waves on the insecticide susceptibility increase in the semi-numerical dose. The test was repeated in three replicates, with 10 larvae used in each test.
Figure 11 is the effect of the sound waves on the digestion of the night chestnut moth, Figure 11a is a feeding behavior when the sound waves of various frequencies at 95 ℃ for 24 hours, treated with 5 night insects at 25 ℃. Chinese cabbage was provided for the feed. Ten larvae were used for each treatment, and the treatment was repeated three times. 11b shows the effect of stress sound on digestive enzyme activity. After treatment at 5,000 Hz, the midgut of the larvae was extracted and separated to measure PLA ₂ activity in epithelial cells and lumens. Each treatment used 5 separate medium lengths, and the treatment was performed in three replicates.
12 is a result of evaluating the effect of sonication (95dB, 5,000 Hz, 24h) on the protein of the medium intestinal digestive juice of five chestnut moth. The triangle marks are specific luminal proteins in the control and treatment groups. Each treatment used about 200 μg of protein extracted from 50 larval tissues.
FIG. 13 shows the effect of sonication (95dB, 5,000 Hz, 24h) on the immune response of Pabamung moth nymphs. FIG. 13A shows Escherichia coli in the treated and untreated controls treated with sound waves at 25 ° C. for 8 hours. The vesicles formed after treatment with 5.0 × 10 ⁴ were measured. Ten larvae were used for each treatment. Figure 13b analyzes the PLA ₂ activity present in blood cells and plasma after sonication. Each treatment used 5 larvae and the treatment was performed in three replicates.
FIG. 14 analyzes the effects of various sonic treatments on the susceptibility of Pabam moths to two insecticides. Larvae were treated with 62.5 ppm fenvalerate or 125 ppm Bacillus thuringensis and then exposed to sound waves of various frequencies. Each treatment used 5 larvae and the treatment was performed in three replicates. Pesticides were measured 5 days after treatment.

In the present invention, the disturbance effect of the physiological process on stress sound waves was investigated in the American leaf oyster flies ( Liriomyza trifolii ), peach aphid ( Myzus persicae ), and night beetroot ( Spodoptera exigua ).

In the present invention, first to analyze the physiological changes of the leaves to suppress the viability of the American leaf oyster fly using stress sound waves. Sensory structures that respond to various chemical and physical stimuli were analyzed at the adult tactile sensation, suggesting the presence of centroid and foot sensations in response to longitudinal stimuli such as sound waves (Shin et al., 2006). . Therefore, the effects of larvae feeding, dragon development and adult spawning behaviors on the treatment of sound waves of various frequencies in American leaf dens were investigated. In addition, the physiological changes in the body according to sound waves were compared with the expression protein pattern patterns through two-dimensional electrophoresis. Finally, it was analyzed whether the suppression of viability caused by stress sound treatment was shown to improve the insecticide susceptibility.

In addition, the present invention analyzed the physiological changes appearing by processing sound waves of various frequencies in the peach aphid ( Myzus persicae ). The intensity of the sound was kept constant and frequency sounds from 0 to 5,000 Hz were exposed to the entire aphid development stage. The resulting nymph development rate and adult fertility were analyzed. In addition, the whole protein pattern analysis of the aphid response to stress sound waves was performed by using two-dimensional electrophoresis. Finally, the effect of this stress sound wave on the susceptibility of insecticides was analyzed.

In addition, the present invention analyzed whether sonic treatment affects the digestion and immune action of pabam moth. We analyzed the physiological changes of Pabam moth according to stress sound waves through protein analysis in digestive enzymes and digestive tract, and the effect on immunity was analyzed through vesicle formation and phosphatase activity analysis. In addition, this study analyzed whether there was a change in susceptibility to insecticides by the chestnut moth.

When feeding was analyzed through the feeding shaft length of American leaf larvae, larvae's feeding activity was significantly reduced by stress sound waves. Acoustic treatment prolonged the chrysalis period and suppressed allegory. The results of two-dimensional electrophoresis showed that stress sonication (5,000 Hz) caused changes in the protein pattern of pupa. Female scattering was also suppressed by sonication. However, adult daylight behavior was not affected by stress sound waves. These stress waves increased the susceptibility to insecticides of American leaf dens. Peach aphids with different frequencies delayed the development period from larvae to adult with increasing sonic frequency, and decreased female proliferation. In addition, aphids treated at 5,000 Hz sound waves showed changes in the protein in the body using two-dimensional electrophoresis. In the sound waves above 2,000 Hz, the sensitivity of the imidacloprid drug was improved. Sound waves of various frequencies had no effect on adult egg-laying rates of Pabam moths, but at 100-5,000 Hz frequencies reduced larval feeding and digestive enzyme activity. Two-dimensional electrophoresis showed that stress sound treatment disrupted protein patterns in the midgut lumen. At the frequency of 5,000 Hz, the larvae suppressed vesicle formation, an immune response to bacteria, and reduced PLA2 activity in blood cells and plasma. In addition, the caterpillars treated with the stress sound waves are more susceptible to insecticides. The present invention has confirmed that stress sound waves for insects are present and that such sound waves can be used to control pests.

[Example]

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these embodiments are only for describing the present invention more specifically, and the scope of the present invention is not limited by these embodiments.

Ⅰ. Physiological Disturbance and Control Effects of Stress Sound Waves on American Leaf Oyster Flies

Materials and methods

1. Breeding American Leaf Oyster Flies

The American Leaf Oyster Fly was distributed by the Department of Industrial Insects, Department of Biology, National Institute of Agricultural Science and Technology, RDA. During the entire breeding period, the temperature was maintained at a temperature of 25 ± 1 ° C., a relative humidity of 60 ± 10%, and a photoperiod of 16: 8 h (L: D). Kidney beans were used as host plants.

2. Sound wave processing

The sound wave equipment provided by Green Teco Co., Ltd. consists of a decibel measuring instrument, a sound wave processing tool (45 × 90 cm), and a sound wave transmitter. American leaf oyster flies were observed at physiological changes at room temperature of 25 ° C with a frequency of 95 dB and exposure for 24 hours in sound waves of 0, 100, 315, 630, 1000, 2000 and 5,000 Hz.

3. Fable Black

Ten leaves of American leaf oyster flies were repeated three times, each at 100, 315, 630, 1,000, 2,000, and 5,000 Hz for 24 hours, and the time period for the pupa to wake up for a certain period of time was investigated.

4. Scattering force test

25 eggs of adult leaf oyster flies are placed in the spawning container, and when the oysters begin to show on the kidney beans, the kidney leaves are divided into the middle and the end of the kidney. Recorded.

5. Two-dimensional Electrophoresis Analysis

Two-dimensional electrophoresis was analyzed using IEF (Isoelectric focusing) and Ettan IPGphor II IEF System (GE Healthcare, Uppsala, Sweden), protein content of 300 μg, running conditions for a total of 18 hours, 1 hour treatment at 100 V, 500 It consists of 8 steps: 1 hour at V, 1 hour at 1 kV, 2 hours at 2 kV, 2 hours at 4 kV, 1 hour at 6 kV, and 8 hours at 8 kV.

6. Insecticide Bioassay

The insecticides were analyzed by immersing the leaves in insecticides. Pesticides used were diluted in water with a formulation commercially available as Spinosad granulating agent (Dongbang Agro, Seoul, Korea). Repeated treatment of kidney bean leaves added to American leaf oyster fly larva with three repetitions of 10, 250, 125, 62.5, 31.25, 15.625 and 7.8125 ppm for each concentration to obtain a half dose (LC ₅₀ ). Each 10 animals were treated in three replicates. After treatment, the leaves were kept dry every day and the final pesticide rate was examined after observation for 5 days.

7. Statistical Processing

The test result was a percentage data, and after the arcsine conversion, ANOVA analysis was performed using SAS 'PROC GLM (SAS Institute, 1989). Half dose (LC ₅₀ ) was calculated using the PROBIT method (Raymond, 1985). Graphed using Sigma Plot 8.0.

result

1. Changes in Caterpillar Feeding Levels According to Stress Sonication

This study was conducted to investigate the difference in feeding amount of larvae of American leaf oysters according to sonication at various frequencies. The feeding amount was indicated by measuring the feeding tunnel length inside the leaf of the larva. In general, the amount of feeding decreased in swaying leaves compared to stationary leaves (FIG. 1A). This phenomenon meant that the larvae's feeding could be affected by the vibration of the leaves due to stress sound waves. When exposed to various frequencies, the feeding amount of American leaf oyster flies showed a significant difference, showing a difference in feeding amount compared to the control in the sound wave of 5,000 Hz or more (FIG. 1B).

2. The Effect of Stress Sound Waves on Allegory and Scattering Behavior of American Leaf Oyster Flies

In general, the allegory rate is high as about 90%, but the allegory rate is lowered according to the sonic treatment, and markedly lower than the control group at 1,000 Hz or more (FIG. 2A). In addition, stress sound waves were also affected during the fable period, and as the frequency was increased, the fable development period was delayed (FIG. 2B).

The effects on the allegorical activity of American leaf oyster flies through various sonic treatments were tested. The allegory of American leaf oyster flies showed daily rhythm that occurred only during the daytime (FIG. 3).

Stress sound influenced the scattering behavior of American leaf dens (FIG. 4). It was found to be close to the leaf foliage of the leaves (Fig. 4a), and one female American Leaf Oyster flies about three eggs per day and decreased as the frequency of stress sound increased (Fig. 4b). Up to 2 scattering numbers were recorded at 5,000 Hz exposure.

3. Protein Changes in American Leaf Oyster Flies According to Stress Sound Waves

In order to examine the bio-influence of the American leaf oyster flies according to the sound waves, 5,000 Hz was treated in the pupa, which was relatively sensitive, and the protein changed compared to the control was measured by two-dimensional electrophoresis (FIG. 5). A total of 25 proteins were detected in the control, and less than eight in the treatment. Specific proteins following sonication were detected.

4. Changes in insecticide susceptibility according to stress sound treatment

The effect of stress sound on insecticide susceptibility of American leaf oyster flies was analyzed (FIGS. 6a and b). When larvae were analyzed by leaf immersion, they showed a half dose at 15.62 ppm of the spinosad insecticide (FIG. 6A). When exposed to various sound waves based on this half-quantity dose, the American leaf oyster flies increased the insecticide susceptibility in proportion to the sound frequency (Fig. 6b).

Review

Physiological changes of American leaf oyster flies according to sonication were analyzed in the present invention. The effects of sound waves of different frequencies on the feeding behavior of larvae were investigated. The amount of feeding analyzed by the length of feeding tunnel decreased in proportion to the frequency of sound waves. Compared to the lowering of feeding due to vibrations, American leaf oyster flies can be regarded as inhibited by external physical fluctuations. It has been reported that leaf percussion of American leaf oyster flies affects crop production, resulting in 43% photosynthesis of damaged leaves by one larvae (Trumble, 1990). In addition, American leaf oyster flies are more damaging to mature leaves than new ones (Chaadler and Gilstray, 1986). Therefore, the decrease in feeding amount by sonication can have a direct effect on the control of this pest. In addition, the experimental results of the present invention indicate that the larvae in the leaf flesh detects these sound waves.

Acoustic treatment significantly degraded the development of the dragon. The container was extended and the allegory rate was lowered. The somatic protein patterns of dragons were also different compared to no treatment, indicating that the use of American leaf oyster flies was detected directly or indirectly. In vertebrates, stress sound is reported to alter the secretion of the neurotransmitter serotonin by altering the activity of tryptophan hydroxylase (Singh et al., 1990). In the present invention, the growth retardation and allegory lowering rate can be inferred as a change in hormonal signal. That is, abnormal secretion of glaze hormone or lowering of excisteroid can be controlled to delay the development of the dragon and lower the allegory.

During adulthood, stress sound had an inhibitory effect on female spawning power. There is evidence that the American Leaf Oyster flies are photogenic but have a preferred host (Wei et al., 2000) .Sensory level scattering tactile structures have observed various sensory structures that respond to chemical and physical signals (Shin et al. , 2006). Therefore, stress sound waves may have a negative effect on the scattering preference and behavior of female American leaf oyster flies.

The physiological changes of the above stress sound waves on American leaf oyster flies were expected to enhance the insecticide susceptibility, and synergistic effects were demonstrated for spinosad in the present invention. Pesticide-sensitive effects of stress sound waves can be inferred from the effect of nicotineic acetylcholine receptor (Nicotinic acetylcholine receptor), as well as the inhibition of drug metabolism. The present invention suggests that certain sound waves can act as stress in the development of American leaf oyster flies. It also shows that these stress waves can be used directly or as a cooperative control.

Ⅱ. Physiological Disturbance and Control Effects of Stress Sound Waves against Peach Aphids

Materials and methods

1. Peach aphid breeding

Peach aphids were distributed by the National Institute of Horticultural & Herbal Sciences, Rural Development Administration, and raised in the lab. Breeding conditions were raised at 25 ± 1 ℃, photoperiod 16: 8 h (L: D) conditions.

2. Sound wave treatment

The sound wave equipment provided by Green Teco Co., Ltd. consists of a decibel measuring instrument, a sound wave processing tool (45 × 90 cm), and a sound wave transmitter. The peach aphid was tested in 10 repetitions of 10 animals, and the frequency was fixed at 95 dB at room temperature of 25 ° C. Change was observed.

3. Insect development

Peach aphid nymphs were divided into three repetitions of 10 birds in the chalet and leaked at each frequency of 0, 100, 315, 630, 1000, 2000, and 5000 Hz.

4. Two-dimensional Electrophoresis Analysis

Two-dimensional electrophoresis was analyzed using Isoelectric focusing (IEF) and Ettan IPGphor II IEF System (GE Healthcare, Uppsala, Sweden), protein content of 300 μg, running condition at 100 V for 1 hour, and then 1 hour at 500 V. After 1 hour at 1 kV, 2 hours at 2 kV, 2 hours at 4 kV, 1 hour at 6 kV, 8 hours at 8 kV, and analyzed for 18 hours.

5. Insecticide Bioassay

The suspension was prepared by diluting at each concentration. Cabbage leaves (1 cm ² ) were immersed in this suspension for 10 minutes, and then dried in a container covered with filter paper for 5 minutes. Each leaf of Chinese cabbage was treated with three repetitions of 10 peach aphid nymphs, and the survival rate was examined up to 5 days by exposure to frequency (0, 100, 315, 630, 1000, 2000, 5000 Hz).

6. Statistical Analysis

The test results were ANOVA analysis using SAS PROC GLM (SAS Institute, 1989) after arcsine conversion as a percentage data. Half dose (LC ₅₀ ) was calculated using PROBIT analysis (Raymond, 1985). Sigma Plot 8.0 was used to graph.

Results and Discussion

1. Effect of stress sound waves on nymph development

Various sonic frequencies (0 to 5,000 Hz) were treated with aphid nymphs and affected the development (FIG. 7). As the processing frequency of sound waves increased, the developmental period to adult was delayed. This delay was more pronounced than the control at frequencies above 630 Hz. In addition, these stress sound waves also affect the growth rate of peach aphids, recording a significantly lower growth rate than the control at a frequency of 2,000 Hz or more (Fig. 8).

2. Protein changes due to stress sonication

To investigate the effect of stress sound waves on the body changes of peach aphids, 5,000 Hz sound waves were treated and compared with the control (FIGS. 9A and B). Protein analysis was performed using two-dimensional electrophoresis. In the control group, a total of 83 proteins were detected, whereas the sonic treatment group showed fewer than 80 proteins. In particular, sonication treatment showed a specific protein.

3. Changes in insecticide susceptibility according to stress sound treatment

The hypothesis that the effect of lowering the development of peach or aphids on stress sound waves may increase the susceptibility to insecticides (Figs. 10a and b). For imidacloprid insecticides, peach aphids showed a half dose of 125 ppm (75.77 <LC <148.41). This semi-numeric dose was used to test the sonic effect. Acoustic susceptibility to peach aphids was significantly enhanced with sound waves above 5,000 Hz for sonication.

Ⅲ. Physiological Disturbance and Control Effects of Stress Sound Waves on Parkan Moths

Materials and methods

1.Bamboo Moth Breeding

The artificial beetle (Gho et al., 1991) was used to raise the beetle moth larvae. All adults received 10% sugar water. During the entire breeding period, the temperature was maintained at 25 ± 1 ℃, relative humidity 60 ± 10%, and photoperiod 16: 8h (L: D). As a food for adults, 10% sugar water was supplied to the laying box.

2. Sound wave processing

The sound wave equipment provided by Green Teco Co., Ltd. consists of a decibel measuring instrument, a sound wave processing tool (45 × 90 cm), and a sound wave transmitter. Pabam moth was observed at physiological changes at room temperature of 25 ° C with a frequency of 95 dB and exposure for 24 hours in sound waves of 0, 100, 315, 630, 1,000, 2,000 and 5,000 Hz.

3. Feeding test according to sound wave treatment

The insects were examined at 5 h of insects with a fixed frequency of 95 dB and exposed for 24 hours in sound waves of 0, 100, 315, 630, 1,000, 2,000 and 5,000 Hz.

4. PLA ₂  Enzyme activity measurement

The medium intestine of the chestnut moth was extracted, PLA ₂ was measured by separating the digestive tract epidermis and the material in the digestive tract. When measuring PLA ₂ , add 20 µl of 10% bovine serum albumin, 1946 µl of 50 mM Tris-HCl buffer (pH 7.0), and 12 µl of 1M CaCl ₂ . Put and mix. 2 μl of pyrene labeled [1-hexadecanoyl-2- (1-pyrenedecanoyl) -sn-glycerol-3-phosphatidyl choline] was added and allowed to react for 10 minutes. PLA ₂ activity was measured using a spectrofluorometer.

5. Analysis of follicular response

Five-day-old two-day-old night beetle larvae were exposed to five (3 repeated) 5,000 Hz stress waves for 24 hours. Pambal moths exposed to sound waves were injected with ⁵ μl (10 ⁵ cfu / ml) after separating pathogenic fungus B. bassiana spores. The Pabamung moth not exposed to sound waves was observed immediately after 8 hours and maintained under the same temperature and humidity conditions. Pabamung moths exposed for 24 hours were observed after exposure to sound waves for 8 hours.

6. Insecticide Bioassay

The product components of chemical pesticides were fenvalerate and the biological ingredients of Bt were diluted to each concentration to prepare a suspension. Cabbage leaves (1 cm ² ) were immersed in this suspension for 10 minutes and dried in a container covered with filter paper for 5 minutes. Three cabbage leaves were treated with three repetitions of three insects of green chestnut moth, and the number of survivors was counted for 5 days in a 24-hour cycle every day. The control was treated in the same manner as above with sterile water.

7. Two-Dimensional Electrophoresis Analysis

Two-dimensional electrophoresis was analyzed using Isoelectric focusing (IEF) and Ettan IPGphor II IEF System (GE Healthcare, Uppsala, Sweden) .The protein content was 300 μg and running conditions for 18 hours in total, 1 hour treatment at 100 V, 500 It consists of 8 steps: 1 hour at V, 1 hour at 1 kV, 2 hours at 2 kV, 2 hours at 4 kV, 1 hour at 6 kV, and 8 hours at 8 kV.

8. Data Analysis

The test result was a percentage data, and after the arcsine conversion, ANOVA analysis was performed using SAS 'PROC GLM (SAS Institute, 1989). Half dose (LC ₅₀ ) was calculated using PROBIT analysis (Raymond, 1985). Sigma Plot 8.0 was used to graph.

result

1. Stress sound wave search

In order to determine stress sound waves that influence the development of PABAM moth and the immune response, sound waves ranging from 0 to 5,000 Hz were processed to 95 dB. Treatment with pabam moth eggs did not affect hatching rate according to sonic frequency. However, as a result of exposure to different sound waves for 24 hours in the three insects of Pabam moth, the feeding rate was induced in the sound waves of all frequencies (Fig. 11a).

2. Effect of Stress Sound Wave on Digestive Enzyme Activity

Digestive tracts were harvested after 24 hours exposure at 5,000 Hz for 5 night insects in Pabam moths and analyzed for medium intestinal PLA ₂ enzyme activity (FIG. 11b). At this time, the activity of the intestinal PLA ₂ present in the mesenteric cells was not changed by sonication, but the activity of PLA _{2 in} the intestinal lumen was reduced by sonication. Two-dimensional electrophoresis of proteins in the midgut lumen was used to compare the effects of sonication. The sonication showed a specific protein and also did not express the protein present in the control (FIG. 12).

3. The effect of stress sound waves on immunity

E. coli was injected into plasma to analyze cellular immune responses after 24 hours treatment with 5,000 Hz sonication in Pabam moth worms (FIG. 13). In the control group, about 35 vesicles appeared in the blood cavity, while only about 10 vesicles formed after sonication showed a markedly reduced cellular immune response (FIG. 13A). In order to determine the response difference of the vesicle formation, PLA ₂ activity in blood cells and plasma showed significantly lower activity in both blood and plasma than the control (FIG. 13B).

4. Effect of stress sound waves on insecticide susceptibility

In accordance with the hypothesis that digestion and immunophysiological degradation caused by stress sound waves may lead to a decrease in sensitivity to insecticides, changes in susceptibility due to treatment with different chemical pesticides (fenvalerate) and biopesticides (Bt) were investigated (FIG. 14). . Chemical pesticides showed higher sensitivity and lower sensitivity at 5,000 Hz than control, whereas biopesticides showed lower sensitivity than 2,000 Hz.

Review

In the present invention, the effects of sonic treatment at different frequencies on the digestive and immunophysiological effects of Pabam moths were analyzed. The 5 night insects of Pabam moth inhibited feeding activity at frequencies between 100 and 5,000 Hz in 95 dB intensity sound waves. Although tests of varying sonic intensities at the same frequency are required, the result is that Pabam moths have seen physiological changes in sonication. In particular, the treatment of the highest frequency, 5,000 Hz, decreased the phospholipid degradation ability of PLA ₂ , one of the digestive enzymes present in the mesenteric and mesenteric lumen. PLA ₂ mainly degrades phospholipids present in cell membranes and breaks them down into fatty acids and lysophospholipids (Valentin and lambeau, 2000). Free fatty acids are absorbed into the effect and lysophospholipids are broken down by other lipolytic enzymes. On the other hand, unlike other animals, it is estimated that lysophospholipid, a saponified substance, plays a role of bile juice and is involved in lipid digestion because it does not secrete specially differentiated bile juice (Stanley, 2000). Thus, degradation of PLA ₂ activity may lead to a decrease in overall lipid oxidation. As a direct example, the inhibition of PLA ₂ in the midgut of Pabam moth as a specific inhibitor resulted in larval growth and death (Kim and Kim, 2010). Therefore, the results of the present invention shows that the sound waves can act as a stress that adversely affects the physiological digestion of Pabam moth.

These stress waves suppressed the immune activity of pabam moths. Under sonic conditions, Pabam moth showed significantly lower follicle formation ability against bacterial invasion than control without sonic treatment. Hemocytic vesicle formation of these pabam moths is an immune entity and depends on the icosanoids (Park and Kim, 2000). Aicosanoids are polyunsaturated fatty acids with 20 carbon atoms, and the action of PLA ₂ in phospholipids results in the biosynthesis of a variety of icosanoids through various oxidases (Balsinde et al., 2002). Stress sound waves inhibited PLA ₂ activity of Pabam moth blood cells. In other words, stress sound resulted in the suppression of the icosanoid biosynthesis, which is thought to reduce the ability of vesicle formation dependent on the substance.

Under stressed sound waves, the degradation of pabam moth digestion and immune physiology may have influenced the viability of this pest and may also result in poor defenses against foreign substances such as insecticides. In order to prove this, the treatment of the insecticide susceptibility of Pabam moth was treated with penvalorate and the insecticide pesticide BT, which showed neurotoxic effects, which resulted in an increase in susceptibility. Fenvalerate is a pyrethroid insecticide, and Pabam moth possesses the ability to be released into the body by increasing its polarity by the body or monooxygenase. Although not analyzed in the present invention, the increase in susceptibility to penvalorate following sonication implies that stress sound waves inhibited the synthesis of single oxidase of Pabam moth. Alternatively, the increased sensitivity to Bt may be related to the immunodeficiency of Pabam moth due to the stress sonication described above. PLA ₂ for Pabam Lowering immunity with inhibitory factors resulted in increased Bt insecticide (Kim and Kim, 2008).

The above results show that a certain sound wave may act as a temporary stress against pabam moth, and this stress sound wave ultimately exacerbates the external resistance of pabam moth, thus acting as a cooperative agent to enhance the effectiveness of the insecticide.

The present invention can be used as a control method that can effectively manage the plant cultivation pests without increasing the environmental pollution by increasing the control effect by using the stress sound wave treatment combined with the existing chemical control method or biological control method. Can be. In particular, the present invention can be used as a control method for reducing the side effects due to the chemical agent by reducing the amount of the existing chemical agent, and can increase the use of the biological control method by increasing the effectiveness of the biological agent. In addition, the present invention can effectively manage the plant cultivation pests only by stress sound wave treatment can be used as a new control method without environmental pollution.

Claims (13)

delete delete A method for controlling Pabam moths using stress sound waves, including treatment of 5,000 Hz sound waves with fenvalerate. delete delete delete delete delete delete delete delete delete delete

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