KR100450902B1 - Korean entomopathogenic nematode, heterorhabditis sp. gsn2 and control method for insect - Google Patents

Abstract

The present invention relates to an insect pathogenic nematode, Heterorhabditis sp. GSN2, and a method for controlling insect pests using the same, wherein the heterolabdytis GSN2 ( Heterorhabditis sp. GSN2) of the present invention is slugs. It is a new environmentally friendly pest control factor that can be used for the control of grass pests and other agricultural and forest pests as well as to overcome the disadvantages of chemical control.

Description

Korean entomopathogenic nematode heterolabditis WSN2 strain and pest control method using the same {KOREAN ENTOMOPATHOGENIC NEMATODE, HETERORHABDITIS SP. GSN2 AND CONTROL METHOD FOR INSECT}

The present invention relates to a heterogeneous GSN2 strain and a pest control method using the same in Korea, and more particularly, to environmentally friendly insects of golf grasses and slugs, which are important soil breeding pests, using nematodes isolated from Korean golf course grass soil. It relates to a biological control method.

Pest control in Korea has been mainly dependent on chemical insecticides, but recently, the demand for environmentally friendly agricultural production through harmonization and balance of agricultural ecosystem is increasing due to growing interest in the environment. In particular, leisure and recreational facilities such as golf courses, amusement parks, and sports stadiums are concerned about the use of chemical pesticides such as pesticides in terms of public health. In addition, there is an urgent need for new pest control methods to replace existing chemical control in water supply protected areas or in the production of high value-added agricultural products through organic farming. In order to prevent the disturbance of agricultural ecosystems and to stabilize the production of the world, there are in-depth interests and studies on biological control.

A new pest control method that can actively revive the domestic and international demand for such pest control is a microbial control method of pests by insect pathogenic nematodes. Insect pathogenic nematodes, along with their symbiotic bacteria, cause septicemia in the host, killing the host, especially when common biological control factors are slower in manifesting effects than chemical pesticides. This allows for rapid pest control. In addition, insect pathogenic nematodes have characteristics such as high pathogenicity, long-lasting in soil, recyclability, and wide host range depending on the type of host, and ease of spraying using general spraying equipment. Compared to this, it is a high control factor. In addition, it has an excellent effect on pests, has safety for human beings, useful animals and plants, and can be mixed with other control factors so that it can be actively used for comprehensive control of pests (IPM). Recently, increasing social interest in food safety and environmental pollution due to the improvement of the quality of people's life due to economic growth, avoiding the spraying of pesticides due to the aging of farmers, the absence of effective pesticides against invasive pests, and the abuse of pesticides There is a need for control measures that can wisely replace all negative factors, such as the emergence of resistant pests and the damage of expensive pollinated insects by pesticide treatment. The most realistic and effective approach to pest control is the biological control using insect pathogenic nematodes, which are biopesticides. In addition, indigenous insect pathogenic nematodes distributed in a specific region have advantages in that they can be coexisted and adapted ecologically with local pests, and can be safe for useful organisms.

Currently, insect pathogenic nematodes are used for biological control of pests in some countries, mainly in the United States and Europe, and are also commercially available for controlling pests. Domestically filed applications include Korean insect pathogenic nematodes and their control methods in Korean Patent Application No. 2001-0020122. However, because they differ in effectiveness depending on host insects, this is the purpose of using the Steinerma carpocaps mainly for the control of pests of lepidoptera and fly As yet, there has been no research on how to control biological agents using nematodes that have specificity to lepidopteran pests, as well as scarab larvae (slugs). Moreover, scarce beetle, green bean beetle, and orange are the most problematic problems in Korean golf courses. Nematodes that have host specificity to larvae such as long-legged beetle and snout-colored beetle and have high mortality are not known.

Accordingly, an object of the present invention is to provide a Korean insect pathogenic nematode heterolabditis GSN2 having a high mortality not only to lepidopteran pest but also to scarab larvae (slugs).

It is another object of the present invention to provide a biological control method using a Korean insect pathogenic nematode heterolabditis GSN2.

1 is an optical micrograph of a nematode ( Heterorhabditis sp. GSN2) infected larvae of the present invention,

Figure 2 is a fatality when treated with the present invention nematode ( Heterorhabditis sp. GSN2) to the honeybee moth larva,

Figure 3 is a photograph showing the appearance of a muzzle scarab beetle larva when treated with the present invention nematode ( Heterorhabditis sp. GSN2),

Figure 4 is a photograph showing a comparison of the healthy larvae of the dead beetle beetle larvae and untreated groups when treated with the present invention nematode ( Heterorhabditis sp. GSN2) to the golf course grass.

In order to achieve the above object, the present invention provides a Heterorhabditis sp. GSN 2 (KCTC 0991BP) heterolabditis GSN2 strain that is an insect pathogenic nematode against agricultural and forest pests.

The present invention also provides a method for controlling pests using Heterorhabditis sp. GSN 2 (KCTC 0991BP).

Hereinafter, the present invention will be described in detail.

The present inventors separated and classified the new insect pathogenic nematode heterolabditis GSN2 strain from the lethal insects of the dorsal beetle larvae of grassland golf course in Korea, and completed the present invention by confirming the control effect of the pests using the same.

Insect pathogenic nematode heterolabditis GSN2 strain of the present invention was isolated from the larvae beetle larvae, and was re-isolated using the larvae of the beetle moth ( Galleria Mellonella ) as bait. The isolated nematode was named Heterorhabditis sp. GSN 2 and deposited on April 16, 2001 as KCTC 0991BP in the GenBank.

Heterolabditis GSN 2 is used to grow in homeless larvae honeybee moth, the culture conditions are 18 ℃ to 30 ℃, the storage method is a refrigerated storage of about 15 ℃.

The life cycle of nematodes has the stages of eggs, larvae larvae, larvae larvae, larvae larvae, and four larvae. First-generation larvae are produced and later become adult adults of male and female larvae, 2-3 generations after mating. It then takes 24 hours to 48 hours to kill the host and escapes from the cell to the soil and infects other hosts. It takes about a day. Putting the white trap infected worms can harvest the nematodes escape after 2 days at 25 ℃. And when inoculated in the bee larva moth homegrown larvae for propagation, can be harvested around 200,000 worms per larvae. The harvested nematodes are stored in a refrigerator of around 10 ℃ in a quantity of 20-30ml (total concentration of 100,000 nematodes) together with sterile water and Triton X-100 in a 100ml cell culture vessel, or ②22% glycerol. After inoculation at 2 × 10 ⁵ ㎖ concentration at 25 ℃ rotary shake for 24 hours at 100rpm, centrifuged at 1000rpm and put in 70% methanol (0 ℃) for 10 minutes on ice, and 1ml each 1.5ml tube Put frozen in -190 ℃ liquid nitrogen or put it with a solid storage agent ③ stored in a refrigerator around 10 ℃, but ① method is most preferred.

The heterolabditis GSN2 strain (KCTC 0991BP) of the present invention is pathogenic to pests, and more specifically, enters the body of a pest of interest and commensal bacteria cause sepsis to kill pests. The pests are preferred for slugs, and more preferably have high effects on larvae such as scarab beetle, snout beetle, orange beetle beetle, and green beetle beetle.

In another aspect, the present invention provides a pest control method using a heterolabditis GSN2 strain (KCTC 0991BP). The pest control method is characterized by applying a heterolabditis GSN2 strain (KCTC 0991BP) to crops or grass pests. It is preferable to disperse the heterolabditis GSN2 strain (KCTC 0991BP) in the soil, seedlings, foliage, or roots of the plant by ground spraying or soil treatment, and the formulation is diluted in water in a liquid state or a water-soluble solid formulation. Do. More preferably, the heterolabditis GSN2 strain (KCTC 0991BP) is suspended in water and sprayed at 10 ⁵ to 10 ¹⁵ birds per hectare. Most preferably, 10 ^{8-10 10} grains / ha are processed.

Pest control using the heterolabditis GSN2 strain can be appropriately controlled according to the time of occurrence of the pest, and is preferably carried out at the time of planting, rice transplanting, seedling, flowering, ripening, harvesting, and resting. Most preferably, it is carried out during the seedling period.

In addition, pest control using a heterolabditis GSN2 strain is preferably carried out against agroforest pests, and more preferably scarabs such as scarab beetle, snout beetle, orange beetle beetle, and green beetle beetle. Also applied are grasses, cabbages, seedlings, and vegetable plants.

In addition, the pest control method can be used by mixing a pesticide or fertilizer with heterolabditis GSN2.

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.

Example: Isolation and Identification of Nematodes

After harvesting nematode beetle larvae were collected from the grass turf soil of the golf course near Daegu, Gyeongbuk, white traps were harvested and stored in a refrigerator at around 8 ℃. Thereafter, a filter paper was placed on a Petri dish in order to reassess the pathogenicity, and then a bee-larged moth larvae were put, and as a result of re-inoculation of the nematode, the larvae were confirmed to be pathogenic nematodes (FIG. 2). Again, white traps were installed to separate nematodes and stored in a refrigerator at around 8 ° C. The isolated nematodes are in the form of elongated threads as in FIG. 1. As a result of the identification of the nematode, the genus was classified into the genus heterolaviditis, which was named as heterolavidis genus GSN 2 ( Heterorhabditis sp. GSN 2), and deposited on the Gene Bank on April 16, 2001, accession no. Was granted.

In order to use the isolated and identified heterolabditis GSN 2 as a biological agent, inoculated into a bee larva moth homeless larvae and then grown at 24 ℃ and harvested by installing a white trap, sterilized in a 100ml cell culture vessel Water and Triton X-100 were stored in a refrigerator at around 15 ° C. in an amount of 20-30 ml (total nematode concentration of 100,000 or less).

Experimental Example 1: Pathogenicity to Scarab Beetle Larvae

The scarab beetle larvae were collected from the second-largest larvae on the grass at Dongnae Benest Golf Course in Dongnae-gu, Busan.

In order to determine the pathogenicity of the dorsal beetle 2 worms of the insect pathogenic nematodes of the above embodiment was tested using a 30g plastic cup. After the sterilized sand was dried, the humidity was adjusted to 12%, and 30 g was added to each cup. Then, two rattle beetles were placed in plastic cups, and 4-5 pieces of germinated rice seeds were fed for feeding. Thereafter, the concentration of 300 nematodes per ml was added to 2.9 ml of sterile water and inoculated with a pipette, and then the lid was covered and placed in a 25 ± 1 ° C. thermostat. Two weeks and four weeks after nematode treatment, the presence or absence of lethality by the nematode was confirmed under a dissecting microscope, and the results are shown in Table 1 below.

Pathogenicity Study on the Scarab Beetle Larvae of Heterolabditis GSN 2 process % Mortality after 2 weeks 4 weeks later No treatment 19.5 30.5 Heterablavitis GSN 2 treatment 97.2 100

In addition, the experiment was carried out in the same manner as above to determine the control rate of the dorsal beetle pupa, but the results of the lethality after 2 weeks of treatment are shown in Table 2 below.

Pathogenicity Study of Dorsal Scarab Pupa of Heterolabditis GSN 2 process % Mortality No treatment 3.3 Heterablavitis GSN 2 treatment 73.3

On the other hand, in order to increase the control effect of the rubella beetle three insects in the open air was mixed treatment experiment with pesticides. In the 8th hole tee of Dongnae Benest Golf Course in Geumjeong-gu, Busan, the treatment zone was set to 50 × 50cm, and the test zone was set with 4 repetition of the ingot method. I put in a pipe. Here, the grass that came with a hole cutter was reinserted. Eight beetle beetles were placed at a depth of 5 cm before insertion. And the nematodes of the above example was sprayed 6 l to each sphere at a concentration of 10 ⁹ / ha and the control was sprayed only water. Pesticide treatment was diluted with chrolopyrifos-methyl 1000 times and sprayed 6 liters each sphere. In the mixing treatment with nematodes, 1000 liters of nematodes and chrolopyrifos-methyl of the above examples were sparged by 3 l each. The results of survival after 4 weeks are shown in Table 3 below.

Pathogenicity of Heterorabbitis GSN 2 in Dorsal Beetle 3 Insects process Survival rate (%) No treatment 88.9 Heterolabeled GSN 2 Treatment 38.8 chrolopyrifos-methyl 21.9 Heterolabditis GSN 2 treatment + chrolopyrifos-methyl 12.5

Experimental Example 2: Pathogenicity of Green Bean Scarab Larvae

In order to examine the pathogenicity of green bean beetle larvae, a kind of grass pest of nematodes, green larva beetle 3 larvae were added to the field grass at Yongwon Golf Course in Jinhae, Gyeongnam.

In order to examine the pathogenicity of the green bean beetle three insects of the insect pathogenic nematodes of the above embodiment was tested using a 30g plastic cup. After the sterilized sand was dried, the humidity was adjusted to 12%, and 30 g was added to each cup. And one green bean beetle three insects were put in a plastic cup and 4-5 pieces of rice seed germinated as food. Thereafter, 300 ml of nematodes per ml were inoculated with 2.9 ml of sterile water and inoculated with a pipette, and then capped and stored in a 25 ± 1 ° C. thermostat. Four weeks after nematode treatment, the presence or absence of lethality by the nematode was confirmed under a dissecting microscope, and the results are shown in Table 4 below.

Pathogenicity Study of Heterorabbitis GSN 2 on Green Bean Beetle 3 Insects process % Mortality No treatment 0 Heterablavitis GSN 2 treatment 50

Experimental Example 3: Control of Orange Long-legged Beetle Larvae

In order to determine the pathogenicity of the orange larva beetle larvae, one of the grass pests of the nematode, the orange larvae beetle larvae 2 were collected and used at the Yongwon golf course in Jinhae, Gyeongnam.

In order to determine the pathogenicity of the orange long legs beetle insect worms of the insect pathogenic nematodes of the above embodiment was tested using a 30g plastic cup. After the sterilized sand was dried, the humidity was adjusted to 12%, and 30 g was added to each cup. And one orange beetle beetle 2 worms were placed in a plastic cup, and 4-5 pieces of germinated rice seeds for feeding. Thereafter, 300 ml of nematodes per ml were inoculated with 2.9 ml of sterile water and inoculated with a pipette, and then the lid was covered and placed in a 27 ± 1 ° C. thermostat. 4 weeks after nematode treatment, the presence or absence of lethality by the nematode was confirmed under a dissecting microscope, and the results are shown in Table 5 below.

Pathogenicity Study of Orange Larval Beetle Larvae of Heterolabditis GSN 2 process Corrected mortality rate (%) Heterablavitis GSN 2 treatment 90.9

Experimental Example 4: Control effect of snout chafer beetle

In order to determine the pathogenicity of the mule-colored beetle larvae, a kind of grass pest of the nematode, three-stage larvae, which were applied to the lice grass, were collected at a pearl golf course in Jinju, Gyeongnam.

In order to determine the pathogenicity of the worm-colored scarab beetle 3 insects of the insect pathogenic nematodes of the above embodiment was tested using a 30g plastic cup. After the sterilized sand was dried, the humidity was adjusted to 12%, and 30 g was added to each cup. And one orange beetle beetle three insects were placed in a plastic cup, and 4-5 pieces of germinated rice seeds for feeding. Thereafter, 300 ml of nematodes per ml were inoculated with 2.9 ml of sterile water and inoculated with a pipette, and then the lid was covered and placed in a 27 ± 1 ° C. thermostat. Four weeks after nematode treatment, the presence or absence of lethal death by the nematode was confirmed under a dissecting microscope.

As a result of the experiment, it was confirmed that when the nematode ( Heterorhabditis sp. GSN2) of the present invention was killed by the locust beetle beetle (Fig. 3), the results are shown in Table 6 below.

Investigation of the Pathogenicity of the Insect Pathogenic Nematodes to the Insects process % Mortality No treatment 15 Heterablavitis GSN 2 treatment 95

Experimental Example 5: the present invention nematode ( Heterorhabditis sp. Investigation of Pest Control Effects of GSN2) on Golf Course Turf

The Heterolabditis GSN2 strain (KCTC 0991BP) of the present invention was suspended in water at Jinju golf course in Jinju, Gyeongnam, and sprayed with 10 ⁵ birds per hectare. As a result, as shown in FIG. 4, it was confirmed that dorsal beetle larvae have a pest control effect.

As can be seen from the above, the nematode heterolabditis GSN2 strain of the present invention (Heterorhabditissp. GSN 2; KCTC 0991BP) is distinguished from the deadly beetle beetle larvae because it has an excellent effect on the control of pests such as the beetle larvae, green bean beetle larvae, orange long leg beetle larvae, snout beetle larvae.

As is apparent from the above Examples and Experimental Examples, the nematode, heterorabbitis GSN2 strain KCTC 0991BP of the present invention (Heterorhabditissp. GSN 2) has a very high lethal effect on pests, which can be used for controlling pests of forests and forests. It is a very useful invention in the pest control industry because it has the effect of overcoming and providing a new environmentally friendly pest control factor.

Claims (4)

Heterorhabditis sp. GSN2 strain (KCTC 0991BP), an entomopathogenic nematode against the scarab beetle, green bean beetle, orange beetle beetle, and mule beetle beetle. Heterorhabditis sp. GSN2 strain KCTC 0991BP from 10 ⁵ to ^{15 15} hectares alone or in combination with pesticides or fertilizers, including spotted beetle, green beetle and orange beetle And control method of muzzle scarlet beetle. delete delete