KR20120084162A - Korean entomopathogenic nematode, heterorhabditidoides chongmingensis cb and control method for insect - Google Patents

Abstract

PURPOSE: An insecticidal method using Korean entomopathogenic nematode is provided to eliminate Spodoptera exigua, Spodoptera litura, and Holotrichia morose and to enhance crop marketability and productivity. CONSTITUTION: A Heterorhabditidoides chongmingensis CBwhich is aKorean entomopathogenic nematode against Spodoptera exigua, Spodoptera litura, and Holotrichia morose is isolated from soil. An insecticidal agent against Spodoptera exigua, Spodoptera litura, and Holotrichia morose contains entomopathogenic nematode.

Description

Korean entomopathogenic nematode, Heterorhabditidoides chongmingensis CB and control method for insect}

The present invention is a Korean insect pathogenic nematode heterorabbitidedes chonmingensis The present invention relates to a CB and a method for controlling the same, and more particularly, to an insect pathogenic nematode isolated from domestic soil, and to a control agent for pabam moth, tobacco germweed moth and large black beetle using the same, and a method for controlling the same.

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 biological 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. Insect pathogenic nematodes of Steinernama carpocapsae GSN 1 (Stainerama carpocapsae GSN 1 KCTC 0981BP) in Korea Patent No. 0421779, Insect pathogenic nematodes of Heterorhabditis sp. Although there are studies on pest control methods, there are limitations in commercialization of biopesticides, which show widespread and excellent insecticidal susceptibility to insect pests due to differences in the effects of host insects due to host specificity, which is the most problematic problem in golf courses.

The present inventors have searched for new microorganisms from domestic soil microbial resources with the aim of developing a new biopesticide that exhibits broad and excellent insecticidal susceptibility to pests. As a result, Heterorhabditidoides chongmingensis CB insect hospital is the first in Korea. Nematodes were collected and identified, and the pest control effect was confirmed using the same, thereby completing the present invention.

It is an object of the present invention to provide Heterorhabditidoides chongmingensis CB, which is a novel entomopathogenic nematode.

Still another object of the present invention is to provide a pest control agent for a night moth, a tobacco spider web moth or a large black beetle including the insect pathogenic nematode and a method of controlling the same.

The present invention provides Heterorhabditidoides chongmingensis CB, which is an insect pathogenic nematode against green beetle moth, tobacco spiderworm moth and large black beetle isolated from soil.

More specifically, the insect pathogenic nematodes of the present invention are isolated from various farmland soils in Korea, and were isolated from three and five age silkworm larvae as bait. Spodoptera , a major pest that causes great damage to domestic crops by using isolated insect pathogenic nematodes exigua ), tobacco giant seminar ( Spodoptera) litura ), Black Scarab ( Holotrichia) morosa), such as a result of testing the lethal force to target a wide range of pests, third instar and fifth instar for larvae, there is lethal a half of posted after inoculation with time (LT ₅₀₎ of 20 to 108 time, the mortality is 60 to 100% was confirmed. See FIGS. 2 and 3.

In order to identify the entomopathogenic nematode isolated from the soil, the phylogenetic location was confirmed using 18S rDNA and 26S rDNA internal transcribed spacer (ITS) gene, and Heterorhabditidoides chongmingensis ) was identified as CB. See FIG. 5.

Heterorhabditidoides ( Heterorhabditidoides) chongmingensis ) Provides pest control against green chestnut moth, tobacco spiderworm moth or large black beetle containing CB.

As confirmed by the insecticidal test, Heterorhabditidoides ( Heterorhabditidoides) of the present invention chongmingensis ) CB can be used as an effective control against pests, including beetroot moth, tobacco germweed moth or large black beetle, and also can be used as a control against all pests exhibiting insecticidal insects in addition to the above pests.

In the present invention, the control agent is mainly treated to the soil or plants in which the beech moth, tobacco gerbera moth or large black beetle breeds, the applied plants are Chinese cabbage, mushrooms, grass, fruit vegetables.

In addition, the pest control agent may be used by further mixing the pesticide or fertilizer with Heterorhabditidoides chongmingensis ( Cheterorhabditidoides chongmingensis ) CB. Heterorhabditidoides ( Heterorhabditidoides) of the present invention chongmingensis ) CB may be used alone for pest control purposes or in combination with other biopesticides or chemical pesticides that are insecticidal. The method used together with other pesticides may be used to make a formulation in the form of a mixture of other pesticides and the present strain, or may be used in combination with the present strain and other pesticides.

The present invention is the heterolabdytides de Chonmingensis ( Heterorhabditidoides chongmingensis ) Provides a method for controlling the green beetle moth, tobacco germweed moth or large black beetle using CB.

The control method is Heterorhabditidoides ( heterorhabditidoides) chongmingensis ) CB is applied to crops or agricultural pests, characterized in that 10 ⁵ to 10 ¹⁵ birds per hectare, alone or in combination with pesticides or fertilizers.

In the present invention, the control method is Heterorhabditidoides chongmingensis ( Cheterorhabditidoides chongmingensis ) CB in the soil, seeds, seedlings, foliage, or roots of plants in the ground spray, water surface or soil treatment, immersion treatment, the formulation is Dilution with water in a liquid state or the use of a water-soluble solid formulation is preferred. More preferably, Heterorhabditidoides chongmingensis CB is suspended in water and sprayed at 10 ⁵ to 10 ¹⁵ per hectare. Most preferably 10 ⁸ to 10 ¹⁰ birds / ha.

Heterorhabditidoides chongmingensis ) PB control using CB is preferably carried out at the time of planting, rice transplanting, seedling, flowering, ripening, harvesting, and resting, and most preferably at seedling.

Insect pathogenic nematodes of the present invention not only have an excellent insecticidal effect on the major pests that cause great damage to domestic crops, such as pabam moth, tobacco gerbera moth, large black beetle, and economical Since the substitution effect is large and has a high performance, there is an effect that can increase the marketability and productivity of the crop.

1 is a diagram confirming the mortality of silkworms after 5 days by re-inoculation of insect-pathogenic nematodes isolated from soil to 5-year-old silkworm (silkworm),
Figure 2 is a result of confirming the lethality for the tobacco castoria moth using the insect pathogenic nematodes of the present invention,
(A: before inoculation, B: after inoculation)
Figure 3 is a result confirming the lethality for large black beetle using the insect pathogenic nematodes of the present invention,
(A: before inoculation, B: after inoculation)
Figure 4 is a result of confirming the appearance of a new insect pathogenic nematode nematode worms after the tobacco caster moth and large black beetle by the insect pathogenic nematodes of the present invention is killed,
(A: Tobacco grinder, B: Large black beetle)
5 is a sequencing result of 16S rDNA for the insect pathogenic nematodes of the present invention,
Figure 6 is a result confirming the phylogenetic location for the insect pathogenic nematodes of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

[ Example  1] Excellent insect pathogenicity Nematode  detach

After supplementing with 200g of soil samples collected from various farmland soils in Korea, spread them evenly, and then kill 10 silkworm larvae of 3rd and 5th ages, and incubate them at 25 ° C for 8 days to check the presence of lethal larvae. It was. The dead silkworms were washed with the surface, and white traps were used to isolate the entomopathogenic nematodes.

Thereafter, the isolated insect pathogenic nematodes were adjusted to a concentration of 200 / ml and re-inoculated to wild silkworms, and the mortality of the silkworms was examined 5 days later.

As a result of damaging the 3rd and 5th age silkworms in each soil sample, as shown in FIG. 1, it began to be killed from the third day, and all silkworm individuals showed a tendency to be killed from 6 to 8 days. The insect pathogenic nematode nematode began to separate from the third day after the white trap was installed on the dead silkworm individuals, and a number of insect pathogenic nematodes could be obtained. Using the insect pathogenic nematode CB obtained from each soil, the mortality rate was reinoculated to 5 year old wild silkworms, and the mortality was 100% on the 5th day.

Example 2 Lethality Assay of Insect Pathogenic Nematode CB for Major Pests

Using the insect pathogenic nematode CB isolated from Example 1, major insect pests ( Spodoptera exigua ), tobacco giant moth ( Spodoptera ) which are causing major damage to domestic crops litura ), Black Scarab ( Holotrichia) Mortality was tested against various pests, including morosa .

Third instar and fifth instar of the beet armyworm of (Spodoptera exigua), tobacco giant seminar room (Spodoptera litura) 10 larvae and third instar laid Chapter 1 filter paper in a petri dish and then inoculated with entomopathogenic nematodes CB with 200 / ㎖ concentration Ten large black beetles ( Holotrichia morosa ) were added to each, and the time required to kill half of the 10 individuals (LT ₅₀ ) and the final mortality after 7 days was confirmed, and are shown in Table 1 below.

As a result, as can be seen in Table 1, the night beetle ( Spodoptera) exigua ) 3 and 5 larvae were killed at 18 hours after inoculation, showing LT ₅₀ at 36 and 24 hours, respectively, and all three larvae of Pabam moth were killed within 7 days of inoculation. The mortality rate showed very good mortality against pabam moths. In case of 5 larvae, the mortality rate was 60%. From this, the mortality rate was slightly different according to the age of larvae.

Tobacco giant seminar room (Spodoptera litura ) 3 and 5 larvae showed LT ₅₀ at 84 and 108 hours, respectively, and had a longer mortality compared to Pabam moths. The final mortality after 7 days of inoculation showed that the mortality of 70 and 80% was observed in the 3 and 5 larvae, respectively.

Great Black Scarab ( Holotrichia) morosa ) LT ₅₀ took 60 hours and had a mortality rate of 80%.

In addition, as can be seen in Figures 2 and 3, 10 days after the inoculation of the insect pathogenic nematode CB tobacco gerbera moth showed a tendency to bend the body or to face up the stomach as the movement gradually slowed down, large black beetle Could identify the discoloration of the body.

Insect pathogenic nematode CB isolated and separated from Example 1 from the above results was found to be moth ( Spodoptera) exigua ), tobacco giant seminar ( Spodoptera) litura ) and Black Scarab ( Holotrichia) morosa ) was effective in controlling pests of major crops.

[ Example  3] Insect Pathogenic Nematodes CB Infectivity test of

With respect to the results of Examples 2 and 3, it was tested whether the cause of the pest killing was a simple lethal or an insect pathogenic nematode CB infection.

After inoculating the insect pathogenic nematode CB selected in Example 1, picking the deadly pests, washing the body surface with distilled water, and installing a white trap, after 7 days, confirming the appearance of a new insect pathogenic nematode, and then examining the infection rate. Table 2 shows.

As a result, as can be seen in Table 2, the infection rate of 3 and 5 year old larvae was 50 and 60%, respectively, showing a slightly lower infection rate. Infection rates of 75% and 100%, respectively, were found to be higher than those of pabam moths, and the killing effect of the insect pathogenic nematode was greater in tobacco germweed moths. In the black beetle, the infection rate of the three larvae was 75%, indicating that the black beetle had an excellent infection rate.

In addition, in Figure 4 it was confirmed that the infection with the new insect pathogenic nematode nematode worms are killed by tobacco castor moth and large black beetle.

[ Example  4] Excellent insect pathogenicity eelworm CB The identification of

(1) Gene Sequence Analysis of Insect Pathogenic Nematodes CB

Genomic DNA isolation from the entomopathogenic nematode larva isolated in Example 1 was carried out under a stereomicroscope of a larva isolated from protease K solution (10 mM Tris-HCl, 1% nonidet P-40, protease K 100 ng / ml) 5 DNA was extracted by adding 65 μl into a PCR tube and reacting at 65 ° C.-30 min, 94 ° C.-10 min, and 10 ° C.-5 min.

RDNA PCR amplification was performed using the extracted DNA as a template. 18S (5'-TTGATTACGTCCCTGCCCTTT-3 ') for the forward primer and 26S (5'-TTTCACTCGCCGTTACTAAGG-3') for the reverse primer were used as primers for amplifying the ITS region of rDNA (Vrain et al., 1992).

PCR conditions were initially denatured at 94 ° C for 5 minutes, followed by 35 times of denaturation (94 ° C, 1 minute), annealing (55 ° C, 1 minute), and extention (72 ° C, 2 minutes). 5 min at 72 ° C. PCR amplification products were subjected to electrophoresis (Mupid-21, Gel documentation system, Bio-Rad) for 30 minutes at 100 V in 0.5 × TAE buffer (0.045 M Tris-borate, 0.001 M EDTA) using 0.8% agarose gel. Afterwards, stained with Ethidium Bromide to confirm amplification under UV, and purified using Qiagen PCR Purification Kit (Qiagen Inc.,).

rDNA sequencing was performed using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), followed by ABI PRISM 310 Genetic Analyse (Applied Biosystems, Foster City, CA, USA). . After determining the nucleotide sequence (SEQ ID NO: 1) as shown in FIG. 5 through rDNA gene analysis, the phylogenetic relationship was analyzed using the BLAST program of the DDBJ / NCBI / RDP / GeneBank database. Alignment of each base sequence was aligned in parallel using the ClustalX algorithm, and phylogenetic location was determined based on the neighborhood binding method (Saitou et al., 1987).

As a result, as shown in Figure 6, it was confirmed that the insect pathogenic nematode CB was identified as Heterorhabditidoides chongmingensis CB.

Example 5 Control by Insect Pathogenic Nematode CB is Assayed

Heterorhabditidoides chongmingensis CB identified from Example 4 to investigate the control effect by inoculating 20 tobacco germweed moth larvae per week to the cabbage seedlings planted in the port for the control of the control of Heterorhabditidoides chongmingensis CB It was.

The test fixtures were placed in three repetition of the ingot method. After the incubation, the environmental conditions were set to 28 ° C, 24 ° C, 25 ° C, 72% relative humidity, and 16L: 8D. Investigations were carried out on the insect pest mortality of insect pest pathogenic nematodes in the second-largest larvae of tobacco gerbera moths by investigating the density of surviving tobacco germweed per port of cabbage before, after 3 days and 7 days after treatment. The effects were investigated and shown in Tables 3 and 4 below.

As a result, as can be seen in Table 3, the selected insect pathogenic nematode heterorabbitideide chonmingensis of Example 1 ( Heterorhabditidoides) The average viability of tobacco castor moth was 65% in the treatment group 3 days after the inoculation of CB, whereas it was 98.3% in the untreated group. Heterorhabditidoides chongmingensis ) CB treatment showed that the insecticidal rate of tobacco germweed moth decreased very much. As a result of the investigation of the control value by the insecticidal rate, 33.9% of the control value showed excellent control effect.

In addition, as can be seen in Table 4, the insect pest pathogenic nematode heterorabdytiides chongmingensis CB was treated on tobacco larva moth larvae 2 larvae, and the result was examined on the 7th day, and the result was 96.7% in the untreated group. 38.3% in the CB treatment group was found to significantly reduce the viability of tobacco castor moth.

As a result of reviewing the control value by virulence rate from the above results, the control value of 60.35% was shown, and the control effect of tobacco germweed moth was large by Heterorhabditidoides chongmingensis CB treatment. I could confirm it.

<110> CHO, Chun-Hwi          WHANG, Kyung-Sook <120> Korean entomopathogenic nematode, Heterorhabditidoides          chongmingensis CB and control method for insect <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 971 <212> DNA <213> Heterorhabditidoides chongmingensis <400> 1 tttcgagaag agtgaggact gctgtaaaga ggcttcggct tctctacgat ggaaatcatt 60 ttaatcgcaa tggcttgaac cgggcaaaag tcgtaacaag gtacctgtag gtgaacctgc 120 aggtggatca tcgctgattc cttacggaac tcgctagctt tgatctgtga atgctgcttg 180 gcgttatggg agtgggcctt ttctgtgtct gtctccttgt agcattcgct ycgtgtaaca 240 caacgtgagg tgttcgagca tcgatctgtg tagctttgtg tgactacngc acttcaatgt 300 gcgacagatt gatcacatgg cgctttcatt agtgctatgg tcgcgaactt aaggtatgac 360 ggcttcgtgt catcgcctca caaccttctt tgctggctat gagcgtgctt ctctgctcag 420 ttaccaccca ttgcgtgtgg aagccttaat gattcgtggc gctgcttctg cttctgcggt 480 tgcggtgctg tggacggcat cttcctccct tttcttaact tgtgttttgg cttgttcgag 540 agaacatctt actacccagc gggatggatc ggtcgattcg catatcgatg aacaacggag 600 caagctccgt tatttaccac gaattgcaga cgctttgagt ggtgaacttt ggaacgcata 660 gcgccgttgg gttttccctt cggcacgtct ggttcagggt tgttttcgtt actgtcaatg 720 cttcggcgtt ggcttgtcga gcggctatgt gaaataccat acccgttcta gttcagaatg 780 aaagcaacat tagatgtcgt gtgtcgttcg taaggacgac actgacatct tactgtaggt 840 gtgctcgtaa agcgtgtatc gccgtttgac tctcacgaat gcttctagtg agtttgtcga 900 gtggtggcct agtgataccg tttggagctt gcatcttgca ataagcaacc tgagctcagt 960 cgtgattacc c 971

Claims (3)

Heterorhabditidoides chongmingensis CB, an insect pathogenic nematode against green chestnut moths, tobacco castor moths, and black beetles isolated from soil. A pest control agent for night moths, tobacco spiderworm moths or large black beetles comprising the insect pathogenic nematode according to claim 1. The insect pathogenic nematode according to claim 2, 10 ⁵ to 10 ¹⁵ per hectare alone or mixed with pesticides or fertilizers, characterized in that the method of controlling the night moth, tobacco spider moth or large black beetle.

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