KR100421779B1 - Korean entomopathogenic nematode, steinernema carpocapsae gsn 1 and control method for insect - Google Patents

Abstract

The present invention relates to a Korean insect pathogenic nematode and a method for controlling the same, and a method for controlling insects by a Steinernama carpocapsae GSN 1 and a Steinernama carpocapsae GSN 1 which are insect pathogenic nematodes against agricultural and forest pests. It is about. Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1) of the present invention has a very high lethal effect on pests can be used for the control of agricultural pests as well as a new environmentally friendly to overcome the disadvantages of chemical control Type is pest control factor.

Description

Korean insect pathogenic nematodes and methods for controlling them {KOREAN ENTOMOPATHOGENIC NEMATODE, STEINERNEMA CARPOCAPSAE GSN 1 AND CONTROL METHOD FOR INSECT}

The present invention relates to a Korean insect pathogenic nematode and a method for controlling the same, and more particularly, to an environmentally friendly biological control method for the important agricultural pests of our country by using a nematode isolated from the soil of our country.

To date, pest control in Korea has been mainly dependent on chemical pesticides, which has caused many side effects such as the emergence of resistant pests, pests of latent pests, destruction of ecosystems caused by environmental pollution, and impairment of public health. Therefore, new pest control methods are required.

The most effective method for controlling new pests is the microbial control of pests by insect pathogenic nematodes.

Insect pathogenic nematodes are highly available biopesticides that can cause sepsis on the host and kill the host in as little as 24 to 48 hours. In addition, entomopathogenic nematodes have unlimited potential as control methods due to their high pathogenicity, sustainability of effect, mass productivity, wide host range, safety for useful plants and animals, ease of spreading, and the possibility of mixing with other control factors. In particular, recently, the social interest in food safety and environmental pollution due to the improvement of the quality of people's life due to economic growth, the avoidance of pesticide application due to the aging of growers, the absence of effective pesticide against the invasive pests, and the excessive pesticide The emergence of resistant pests caused by abuse and the treatment of expensive pollinated insects caused by pesticide treatment are required to control them wisely, and pest control methods that can most realistically approach these times are biological pesticides. It can be called a biological control method using insect pathogenic nematodes. In addition, indigenous insect pathogenic nematodes distributed in a specific region has advantages in that they can not only coexist ecologically with local pests but also can be safe for useful organisms.

Currently, several foreign countries, including the United States, are studying insect pathogenic nematodes for the biological control of pests, some of which are already commercialized. The domestic application has a Bacillus papylia for the control of dorsal beetle larvae of the application No. 1999-015472 and the control method using the same, but there is almost no research on the biological control of pests using insect pathogenic nematodes.

Therefore, an object of the present invention is to provide a Korean insect pathogenic nematode.

It is another object of the present invention to provide a method for controlling insects using Korean insect pathogenic nematodes.

1 is an optical micrograph of the nematode ( Stainernema carpocapsae GSN 1) of the present invention became adult in the larvae after killing the macroscopic larvae,

Figure 2 is a photograph showing the appearance of the nematode propagated when the nematode ( Stainernema carpocapsae GSN 1) of the present invention treated small root flies larvae and then dissected the dead root flies,

Figure 3 confirms the ability to control small root fly larvae by the nematode ( Stainernema carpocapsae GSN 1) of the present invention, compared to the growth status of watermelon seedlings after treatment or not treated to the watermelon seedlings with small root fly larvae It is a photograph,

Figure 4 is a picture comparing the cultivated cultivation of healthy cultivars and the damaged cultivated oyster mushroom flies by killing nematode mushroom fly by nematodes after treatment of nematode ( Stainernema carpocapsae GSN 1) of the present invention.

In order to achieve the above object, the present invention provides a Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1), which is an insect pathogenic nematode GSN 1 KCTC 0981BP for agricultural and forest pests.

In another aspect, the present invention provides a pest control method using a Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1 KCTC 0981BP).

Hereinafter, the present invention will be described in detail.

The inventors separated and classified the new insect pathogenic nematodes from the soil of our country, and completed the present invention by confirming the control effect of pests using the same.

Insect pathogenic nematodes of the present invention have been separated from the soil, it was isolated by the honey bee moth larva as bait. In the present invention, nematodes have been deposited as KCTC 0981BP in the GenBank as a Steinerman carpocapsae GSN 1 ( teinernama carpocapsae GSN 1).

Steinermacarpocapus GSN 1 is used to grow in homeless larvae bee beetle moth culture conditions are 18 to 30 ℃, the storage method is refrigerated storage of about 8 ℃.

The Steinama carpocap vulture GSN 1 male adult has a morphological characteristic of 6 pairs of genital processes in the tail and a sharp end. The average length of the ligator is 75 μm or less, and the female has no double covering on the vulva. The life cycle of GSN 1 in Stenamacarpocapsae GSN 1 is at the stage of eggs, 1 worm, 2 worms, 3 worms, 4 worms, and adults. 2 to 3 generations after mating in the host insect body. It then takes 24 hours to 48 hours to kill the host and escapes from the cell to the soil and infects other hosts. The growth in the body afterwards depends on temperature and moisture conditions. It takes about 7 days.

Artificially, the dead worms infected with the Steinermacarcapac GSN 1 nematodes can be placed in a white trap, and nematodes can be obtained after 2 days at 25 ° C. The obtained adult insects can be harvested by inoculation into homeless larvae, beetle moths, which are bait insects for propagation.

Steinermacarpocapsa GSN 1 nematodes can be stored by the following method.

[Storage method of carpocapus GSN 1]

1. Refrigerated Storage: Refrigerate around 10 ℃ in the amount of 20-30ml (total nematode concentration less than 100,000) with sterile water and Triton X-100 in 100ml cell culture vessel.

2. Freeze liquid nitrogen: Inoculate 2 × 10 ⁵ ㎖ in 22% glycerol, maintain at 24 rpm for 24 hours in rotary shake at 25 ℃, centrifuge at 1000 rpm and put in 70% methanol (0 ℃) on ice. Hold for 10 minutes, and place it in 1 ml of 1.5 ml tube and freeze in -190 ℃ liquid nitrogen.

3. Refrigerated storage using a storage agent: Store with a solid storage agent refrigerated around 10 ℃.

Among the above methods, it is most preferable to store the Steinermacarcapac GSN 1 in the cold storage (1).

Steinermacarpocapsae GSN 1 (KCTC 0981BP) of the present invention is pathogenic to pests, and more specifically, invades the body of the target pests and causes septicemia to kill the pests. The pests are preferably agricultural and forest pests, and more preferably, small root flies, long mustard mushroom flies, silk snails, and castella moths.

In Figures 1 and 2, the Steinermacarpocapus GSN 1 (KCTC 0981BP) of the present invention showed the appearance of becoming an adult after killing the pest.

In another aspect, the present invention provides a pest control method using the Steinerman carpocapsae GSN 1. The pest control method is characterized in that the application of the Steinerman carpocap bird GSN 1 (KCTC 0981BP) to crops or agricultural pests. Stainernape Carcapacus GSN 1 (KCTC 0981BP) is sprayed onto the soil, seeds, seedlings, foliage, or roots of plants by ground spraying, water surface treatment or soil treatment, and the formulation is diluted or diluted with water in liquid form. It is preferable to use a solid dosage form. More preferably, Steinermacarcapac GSN 1 (KCTC 0981BP) is suspended in water and sprayed at 10 ⁵ to 10 ¹⁵ per hectare. Most preferably, 10 ^{8-10 10} grains / ha are processed.

Pest control using the Steinernacarcapacus GSN 1 is preferably carried out at the time of planting, rice transplanting, seedling, flowering, ripening, harvesting, resting, and most preferably at the seedling stage.

In addition, pest control using the stainer nacarcapacus GSN 1 is preferably performed on agricultural and forest pests, and more preferably, small root flies, long mustard mushroom flies, silk snails, and castella moths. Also applied are cabbages, mushrooms, grasses, and vegetable plants.

In addition, the pest control method can be used by further mixing pesticides or fertilizers with Stenamann carpocapsae GSN 1.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

EXAMPLES Isolation and Identification of Nematodes

Soil was harvested from the pine forests of Pocheon, Gyeonggi-do, Korea. The soil collected in a 300 플라스틱 plastic container was mixed with 200 토양 and 10 homeless larvae of bee-flying moths were bred from artificial feed. After 7 days, the presence of lethal nematodes was examined, and the nematode was re-inoculated with a sheet of filter paper in a Petri dish for pathogenicity test. This was harvested by installing a white trap (White trap) and stored in a refrigerator around 8 ℃.

The isolated nematodes are in the form of elongated threads as in FIG. 1. The nematode was identified as Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1) as a result of identification, and deposited as KCTC 0981BP in the GenBank.

Experimental Example

Experiment 1. Pathogenicity to Small Root Fly Larva

Small root fly larvae were used to collect the larvae in the top of the tray cell (tray cell) in the nursery of Jinju, Gyeongnam.

To determine the pathogenicity of small root flies of the insect pathogenic nematodes of the above embodiment₅₀) Was investigated. The circular elder mushroom mycelia were grown on a PDA in a plastic petri dish for 20 days, and then the PDA medium was removed by cork borer into a multiwell tissue culture plate having a diameter of 15 x 20 mm in height. . Here, small root fly larvae are divided into 2, 3 and 4 ages by age, and the nematodes of Examples were treated with 0, 5, 10, 20, 40, 80 and 160 concentrations of 0.5 ml each to 18, 24 and 30, respectively. Placed in a thermostat at < RTI ID = 0.0 > Nematodes were used diluted with sterile distilled water. After nematode treatment, the presence or absence of lethality was examined under a dissecting microscope at 24 hour intervals, and the results are shown in Table 1 below. The following half lethal concentration is the number of nematodes required to kill half the small root flies. Semi-lethal concentrations were investigated using a statistical program called PROBIT analysis, and a computer estimate of the concentration at which 50% could be killed. In addition, the 95% confidence limit is a range of statistically accepted probabilities in the natural sciences, which was analyzed in the 95% range to compare the results with other treatments.

Treatment temperature (℃) Developmental stage Half lethal concentration (95% confidence limit) 18 Two insects 342.2 (113.1-2800.6) 3 insects 18.4 (11.0-27.5) 4 insects 14.9 (8.4-22.5) 25 Two insects 73.1 (40.1-238.7) 3 insects 4.3 (1.9-6.8) 4 insects 4.2 (2.0-6.5) 30 Two insects 183.6 (86.5-1195.6) 3 insects 18.1 (11.9-25.6) 4 insects 12.7 (7.4-18.7)

The results of Table 1 show that the semi-lethal concentrations of the third and fourth insects at 18 ° C. differ between 18.4 and 14.9, but the 95% confidence limits are 11.0-27.5 and 8.4-22.5. Therefore, the nematode mortality rate can not be said to be significantly effective in three nymphs compared to four nymphs. However, in comparison between the second and third worms, the mortality rate for the third worm is significantly higher because the range of confidence limits does not overlap.

In addition, in order to determine the control rate of small root fly larvae in nursery nursery, the density of small root fly flies in each cell was investigated in a 32-hole tray cell of a greenhouse nursery where watermelon seedlings were planted. The nematodes of the examples were treated. After 1, 2 and 3 weeks of treatment, the density of small root flies was examined to determine the larval reduction rate, and is shown in Table 2 below.

process Caterpillar reduction rate (%) 7 days later 14 days later 21 days later No treatment 0 0 0 Example 1 Treatment 78.5 78.7 80.2

Figure 3 confirms the control of small-root fly larvae by the Steinama carpocap bird GSN 1, watermelon seedlings treated with the Steinerman carpocap bird GSN 1 (a) grows healthy, untreated watermelon seedlings (b) was not healthy.

To confirm the nematode viability of the examples in a plug tray, nematodes were sprayed with a hose sprayer at a concentration of 10 ⁸ birds / ha on a 1.5 × 40 m bench in a plug nursery, followed by 35 and 42 days after treatment. Topsoil was taken from each of the 10 trays. The survival rate of nematodes with or without lethal beetle moth larvae was put into the soil, and the results are shown in Table 3 below.

process Survival rate (%) 35 days later 42 days later Example 98.7 81.3

Experiment 2. Pathogenicity to Long-Beard Mushroom Flies

In order to determine the pathogenicity of the nematode of the nematode of the nematode of Example 1, the half lethal concentration (LC ₅₀ ) was investigated. As for the long beard mushroom fly, a medium in which a long beard mushroom fly was generated was removed from a mushroom cultivator near Jinju-si, Gyeongnam, and then separated into a 0.2 mm sieve. Growing circular hyphae mycelium for 20 days in a PDA of plastic petri dishes, and then placing the medium in a multiwell tissue culture plate 15 mm in diameter and 20 mm in height, and then distinguishing larvae of caterpillar fly larvae by age. Put it in. The nematodes were treated with 0.1 ml at concentrations of 0, 5, 10, 20, 40, 80 and 160, and placed in a thermostat at 20 and 25 ° C., respectively. After nematode treatment, the presence or absence of lethality under a dissecting microscope at 24 hour intervals is shown in Table 4 below.

Treatment temperature (℃) Developmental stage Half lethal concentration (95% confidence limit) 20 Two insects 511.1 (213.2-4695.4) 3 insects 38.2 (26.9-57.0) 4 insects 39.4 (26.7-62.2) 25 Two insects 584.7 (190.3-2217.8) 3 insects 20.0 (13.0-29.0) 4 insects 27.5 (19.3-39.2)

In addition, 1 × 1.5 m sized test plots were formed in the cultivated mushrooms of Chuncheon, Gangwon-do in five repeated complete randomization, and then 4 L was sprayed at a concentration of 2.25 × 10 ⁹ / ha and 4.5 × 10 ⁹ / ha. The untreated section was sprayed with 4 L of water only. On the 7th and 14th day of treatment, the density of larvae were examined in each sphere, and the reduction rate was examined.

Concentration Caterpillar reduction rate (%) In a week 2 weeks later 2.25 × 10 ⁵ 28.3 42.2 4.5 × 10 ⁵ 41.8 81.6

Figure 4 confirms the control of the long-bearing mushroom fly larva by the Steinerna carpocape GSN 1, the oyster mushroom treated with the Steinerma carpocape GSN 1 (a) grows healthy, but not Mushroom (b) was not healthy.

In addition, in order to examine the survival of nematodes in cultivated mushrooms, a 1 × 1.5 m-sized test plot was made with 5 repeated complete randomization methods in cultivated mushrooms in Chuncheon, Gangwon-do. Nematodes were sprayed 4 L at a concentration of 2.25 × 10 ⁹ birds / ha and 4.5 × 10 ⁹ birds / ha. The untreated section was sprayed with 4 L of water only. On the 7th and 14th day of treatment, the mushroom medium 10 × 10 × 10 cm 3 was sampled, brought to the laboratory, mixed well, and placed in a polyethylene cup of 9 cm in diameter and 7.5 cm in height. Five homeless larvae of bee-flying moths were added to it, and after 7 days, the presence of lethal was checked to confirm the survival rate of nematodes. The results are as shown below, and nematodes showed a survival rate of 100% both after 1 week and 2 weeks after treatment.

Concentration Survival rate (%) In a week 2 weeks later 2.25 × 10 ⁵ 100 100 4.5 × 10 ⁵ 100 100

Experiment 3. Silk Snail Control

The pathogenicity of nematodes to silk snails, a pest of vegetables, was confirmed. Silken snails were collected from the cabbage packaging of Gyeongnam Pearl. The sand was washed with tap water, dried for 48 hours, again sifted in a 850 μm sieve and sterilized twice. The sterilized sand was dried again for 48 hours, and then, 13% of water was included in sterilized water, and then 12 g each was placed in a 55 × 15 mm Petri dish. Cabbage leaves cut to 50 mm in diameter were placed therein, and nematodes were mixed with water at 20, 40, 80, 160, 320, and 640 concentrations to prepare a suspension, and inoculated by 1 ml each with a micropipette. Untreated was inoculated with only 1 ml of sterile water. After the treatment, one petiole and one silk silk snail were put into each Petri dish, and 7 days later, the nematode infection was examined under a dissecting microscope. Ten Petri dishes were repeated four times in one set. Experimental results showed that hemi-concentrations were 21.2 in milk and 24.1 in milk.

Developmental stage Half lethal concentration (95% confidence limit) Defeat 21.2 (15.9-26.3) Success 24.1 (14.9-33.4)

In addition, in order to examine the pathogenicity of the nematode's silkworm snail in the facility house, a 0.4 × 3 m sphere was set in a 25-day-old Chinese cabbage house in Jinju-si, Gyeongnam, and watered by 5 L at a concentration of 10 ⁹ birds / ha. Sprayed with iris. The experiment was carried out in three iterations, and only 5 L of water was sprayed with a water stopper. After 7 days, the snails were collected from each treatment and examined for mortality. As shown in Table 8, the mortality rate was 63% in the case of fat loss and 48% in the case of success.

Developmental stage % Mortality Defeat 63 Success 48

Experiment 4. Control effect of caterpillar moth

The pathogenicity of nematodes to grassworms and common vegetable pests, macroscopic larvae, was confirmed. Two or three insecticides, collected outdoors, were placed on a 9 cm Petri dish with a sheet of filter paper, and 10 animals were placed. Nematodes were inoculated 1 ml at a concentration of 100, 200, 400 / ml, sealed with polyethylene film, placed at 25 ° C. incubator, and examined for mortality after 7 days. The control group was inoculated with 1 ml of water only and tested three times.

Experimental results showed 76.7% mortality at 100 / ml concentrations and 100% mortality at 400 / ml concentrations (Table 9).

Concentration (mari / ml) % Mortality 0 0 100 76.7 200 100 400 100

In addition, experiments were conducted at the Dongnae Benest Golf Course in Busan to find out the control effect when nematodes were treated outdoors. Test specimens of 60 × 60 cm 2 size were placed in full randomization and the density of larvae in each bulb was examined. After density testing, the nematodes were sprayed with 3.74 L with a water stop at a concentration of 10 ⁹ / ha. No treatment was sprayed with 3.74 L of water alone. After one week of treatment, the density of castor larvae was examined and compared with the untreated group. As a result of the investigation, the nematode treatment group showed a 83% density reduction effect as shown in Table 10.

process Caterpillar reduction rate (%) Example 1 83

As mentioned above, the nematode of the present invention, Steinernama carpocapsae GSN 1 KCTC 0981BP ( Steinernama carpocapsae GSN 1) is very high lethal effect on pests can be used for the control of agricultural and forest pests. The present invention also overcomes the disadvantages of chemical control and provides new environmentally friendly pest control factors.

Claims (5)

Pest control against small root flies, silk snails or castor moths, including Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1), an insect pathogenic nematode against small root flies, silk snails or macropods . delete Steinernama carpocapsae GSN 1 KCTC 0981BP ( Steinernama carpocapsae GSN 1) is treated with 10 ⁵ to 10 ¹⁵ per hectare, Steinernama carpo, characterized in that to control small root flies, silk snails, or castella moth Control method of pests using the capsac GSN 1 KCTC 0981BP ( Steinernama carpocapsae GSN 1). delete delete