KR102231341B1 - Composition for controlling Haemaphysalis longicornis using Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 and method for preventing Haemaphysalis longicornisusing the same - Google Patents

Abstract

The present invention is Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) having a control effect on ticks, a composition for controlling ticks comprising the same, and a method for controlling ticks using the same About.

Description

Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 having a control effect on ticks, a composition for controlling ticks containing the same, and a method for controlling ticks using the same {Composition for controlling Haemaphysalis longicornis using Metarhizium anisopoliea JEF-279 Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 and method for preventing Haemaphysalis longicornisusing the same}

The present invention relates to a composition for controlling ticks including Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 having a control effect on ticks, and a tick control method using the same.

Small sophie true ticks are not insects, but taxonomically, the animal kingdom (Kingdom Animalia), Phylum Athropoda, Class Arachnida, Order Acari, Suborder Ixodida, and True tick family ( Ixodidae). It also falls into the category of Ixodid ticks, also commonly referred to as hard ticks. Depending on the life history, it grows in the order of eggs, larva, nymph, and adult. Adults are about 2-4mm, nymphs are about 1-2mm, and larvae are about 0.5mm, usually dark brown to yellowish brown. Small thorax mites, mainly distributed in temperate climate regions, are known to inhabit Japan, China, Far East Russia, Australia, New Zealand, Fiji, New Caledonia, and Hawaii, including all over Korea. In particular, Haemaphysalis longicornis Neumann (1901) is a major medium for Severe Fever with Thrombocytopenia Syndrome (SFTS) caused by a virus belonging to the bunyaviridae. It is the servant I received.

As interest in side effects such as emergence of resistant pests and environmental pollution due to the recent use and abuse of chemical pesticides has increased, the use of chemical pesticides is gradually restricted. Control methods are being actively studied. For example, it is a method of controlling by using natural enemies such as apexae (Orius sp.) and predatory mites, or by using insect pathogenic fungi that infect pests. More than 750 types of insect pathogenic fungi have been known to date, of which Beauveria bassiana, Metarhizium anisopliae, and Nomuraea rileyi are representative microbial insecticides. It has been developed and is used for controlling various pests.

With respect to the metarisodium anisopliae (Metarhizium anisopliae) strain, Korean Patent Laid-Open Publication No. 10-2015-0057124 discloses metarium anisopliae FT83 and a microbial preparation for controlling Pabam moth larvae containing the same. , Korean Laid-Open Patent No. 10-2019-0003133 discloses novel Metarhizium anisopliae JEF-197 and Metarhizium anisopliae JEF-279 strains having a control effect on brush beard sky cattle, a composition for controlling pests including the same, and a composition for controlling pests using the same. A method of controlling the is disclosed. However, there is still a need for new highly active microbial pesticides that are capable of controlling small petty mites with the same potency as chemical pesticides, and that are environmentally friendly and easy to handle and use.

Accordingly, the present inventors have been polite to discover new insect pathogenic fungi based on microorganisms that have a control effect on small phylum scabbards and establish an effective small scabie tufts mite management system while dramatically overcoming the problems of resistance and environmental residuals of existing synthetic pesticides. As a result of the effort, the present invention was completed by confirming the excellent insecticidal power of the excellent small Sophie Charm mite of the Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 strain.

The present invention was conceived to solve the above problems, the inventors of the present invention Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) of the strain excellent By confirming the insecticidal power of the tick, the present invention was completed.

It is an object of the present invention to provide Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) having a control effect on ticks.

Another object of the present invention is Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain having a control effect on ticks, spores of the strain or a culture medium of the strain It is to provide a composition for controlling ticks comprising a.

Another object of the present invention is to provide a method for preparing a tick control composition comprising the following steps:

Culturing Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain in a solid culture medium; And

Harvesting the culture.

Another object of the present invention is to provide a method for controlling mites by treating the composition for controlling mites on pests, their habitat, plants or soil to be protected from pests.

In order to achieve the above object, the present invention can provide Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) having a control effect on ticks.

According to a preferred embodiment of the present invention, the mite may be a small sophie true mite (Haemaphysalis longicornis), a vertical patterned house dust mite (Dermatophagoides pteronyssinus), or a large-legged house dust mite (Dermatophagoides farinae).

The present invention also provides a composition for controlling mites comprising Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P), spores of the strain or a culture solution of the strain. I can.

According to a preferred embodiment of the present invention, the mite may be a small sophie true mite (Haemaphysalis longicornis), a vertical patterned house dust mite (Dermatophagoides pteronyssinus), or a large-legged house dust mite (Dermatophagoides farinae).

According to a preferred embodiment of the present invention, the tick control composition may be formulated into any one selected from the group consisting of a liquid formulation, a powder formulation, and a granular formulation.

According to a preferred embodiment of the present invention, the composition for controlling ticks includes millet, white rice, bran, millet, Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P). , Sorghum, and a combination thereof may be obtained by culturing in any one solid culture medium selected from.

The present invention can also provide a method for preparing a tick control composition comprising the following steps:

Culturing Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain in a solid culture medium; And

Harvesting the culture.

The present invention can also provide a method for controlling mites by treating the above-described mite control composition on a pest, its habitat, a plant or soil to be protected from pests.

Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279 or Metarhizium anisopoliea JEF-290 strains of the present invention made spores in a solid medium, and the spore suspension was immersed in a small thorax mite to confirm the lethality. As a result, Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279, or Metarhizium anisopoliea JEF-290 strains showed a mortality rate of 50% 14 days after treatment, and had the effect of forming mycosis on small soficum ticks. As a result, Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279, or Metarhizium anisopoliea JEF-290 strains have a control effect on small saffron ticks. Furthermore, Metarhizium anisopoliea JEF-214, Metarhizium anisopoliea JEF-279, or Metarhizium anisopoliea JEF-290 strains showed high germination rate even after heat treatment and have high thermal stability, so the strains have the effect of settling well when treated in soil.

1 is a metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain observed with the naked eye (FIG. It shows the growth form (Fig. 1b).
Figure 2 shows the nucleotide sequence of ITS (Internal Transribed Spacer) of Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain.
Figure 3 is a result of comparative analysis of the relationship between the previously reported strains and Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain.
Figure 4 shows the insecticidal effect of small scabbard ticks by immersion treatment of Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain (short repetition) (Fig. 4a) And Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strains.
Figure 5 is a metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P), or Metarhizium anisopoliea JEF-290 (KFCC11794P) the insecticidal effect of the small thorax mites by spray treatment (semi-field) (Fig. ) And Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain.
Figure 6 is a confirmation of the spore productivity of the Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain, Metarhizium anisopoliea JEF-214 (FCCKea JEF-214) using Italian millet. Spore production results of Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain (Fig. (KFCC11794P) shows the number of spores of the strain (Fig. 6b).
Figure 7 shows the results of confirming the thermal stability of Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain.

Hereinafter, the present invention will be described in more detail.

As described above, it is possible to dramatically overcome the resistance problem and environmental residual problem of the existing synthetic pesticides, and a microbial base that improves the point that the control effect appears only when the pesticidal ingredient based on the existing plant extract directly contacts the mite. There is a need to discover new insecticidal fungi and to develop an effective tick management system.

The composition for controlling ticks using Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) according to the present invention forms spores and has high thermal stability after heat treatment. , The germination rate after heat treatment is also high, so it can be settled after treatment in the soil, and it shows a high insecticidal rate to the small Sophie Charms mites, so it is effective as a composition for controlling small Sophie Charms mites.

Accordingly, the present invention provides Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) having a tick control effect.

In a specific embodiment of the present invention, 5 to 10 cm of the surface layer was collected in order to collect a soil sample for strain isolation. A quarter of the collected soil sample was filled in a paper cup. Brown larvae (3-4 animals) were put, and 1 ml of distilled water was supplied every day to maintain high humidity conditions. Infection was induced by supplying moisture to the brown mealworm larvae treated in the soil for 5-10 days. The strain was isolated from the mycosis of the larva. The strain was isolated by culturing (25±1℃, dark condition) in 1/4 Sabouraud Dextrose Agar (1/4SDA; Difco, USA). A single colony was obtained through 2-3 separations. The acquired single colony was used to re-test the pathogenicity to the larvae. The strain was re-tested to confirm pathogenicity, and the gene was amplified using an ITS primer (ITS1F: 5'-TCCGTAGGTGAACCTGCGG-3'/ ITS4R: 5'-TCCTCCGCTTATTGATATGC-3'). BLAST was performed using the amplified gene information. The isolated and identified insect pathogenic fungi were stored at -70℃ with 15-20% glycerol stock for long-term storage.

In another specific embodiment of the present invention, the present inventors sequenced the strain obtained in Example 1 and finally identified the strain as Metarhizium anisopliae, and each of the strains Metarizium anisopliae ) JEF-214, Metarhizium anisopliae (Metarhizium anisopliae)-279, Metarhizium anisopliae (Metarhizium anisopliae)-290 were named (Fig. 2), and the Korea Microbiological Conservation Center November 1, 2017. Deposited and stored under the deposit number KFCC11746P (Metarhizium anisopoliea JEF-214) on June 13, 2017, KFCC11721P (Metarhizium anisopoliea JEF-279) on June 13, 2018 or KFCC11794P (Metarhizium anisopoliea JEF-290) on September 19, 2018.

Using the reported ITS sequence of the genus Metarhizium strain and various insect pathogenic fungi genus, the relationship was compared with M. anisopliae JEF-214, JEF-279, and JEF-290 (FIG. 3). As a result, it was found to be relatively high with most of the M. anisopliae strains. In addition, Metarhizium anisopliae JEF-214, Metarhizium anisopliae-279, and Metarhizium anisopliae-290 strains are inherited from existing strains. It was confirmed that it is a novel isolate with significant differences.

The mite may be a small physalis longicornis, a vertical patterned house dust mite (Dermatophagoides pteronyssinus), or a large-legged house dust mite (Dermatophagoides farinae), preferably, but may be limited to Haemaphysalis longicornis. It doesn't work.

The present invention provides a composition for controlling mites comprising Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P), spores of the strain or a culture solution of the strain.

The tick control composition is preferably prepared through solid culture by inoculating the suspension of the strain on a grain medium, more preferably millet, rice, Italian millet, barley, beans, It is to use a medium consisting of one or more grains selected from the group consisting of sorghum, crude, rice, and brown rice, of which it is most preferable to inoculate a millet medium and cultivate a solid (Fig. 6).

In the tick control composition of the present invention, the solid culture is after inoculating the suspension of the strain in a medium, preferably incubating for 14 to 20 days at 20 to 27°C, and then 3 to 7 at room temperature conditions. It can be dried for one day, more preferably after culturing at 25° C. for 14 days, and drying at room temperature for 2 days is preferable.

The composition for controlling ticks of the present invention contains the spores of the Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain having a tick control effect, After attaching to the host insect, it germinates, causing mycosis, etc., resulting in an insecticidal effect. In addition, in addition to the mites that directly contact this, even the mites that are not in direct contact with the composition may exhibit a control effect due to the transmission of spores.

The tick control composition of the present invention is preferably a liquid, powder or granular formulation. The powder formulation refers to a powder form, and the granular formulation refers to a form of grains or grains obtained by diluting a solid culture medium by 1 to 100 times the weight by using an extender, but is not limited thereto.

In the present invention, Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strains having a mite control effect exhibits excellent effects on the control of ticks on adult ticks.

The present invention can also provide a method of preparing a composition for mite barge comprising the following steps:

Culturing Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain in a solid culture medium; And

Harvesting the culture.

The solid culture medium is any one selected from the group consisting of crude, millet, bran, sorghum, cassia tora, oats, millet, mung beans, perilla, buckwheat, meso, barley, sorghum, perilla, sesame, whole wheat, rice, and combinations thereof. Can include.

The present invention can also provide a method for controlling mites comprising the step of treating the above-described mite control pests, their habitats, plants or soil to be protected from the pests.

The tick control composition can be prepared as a suspension.

The composition for controlling ticks may be subjected to contact treatment, spray treatment, and most preferably, immersion treatment.

The suspension of the present invention is preferably prepared by harvesting the spores of the cultured strain, which is a solid culture or composition for controlling ticks, counting with a hemocytometer, and then diluting according to the treatment concentration using various surfactants.

At this time, when the spores are counted with a hemocytometer after completion of solid culture, the number of spores may be 1x10 ¹ spores (conidia)/g to 1x10 ¹⁰ spores (conidia)/g, and more preferably 1* 10 ⁷ spores (conidia)/ml.

In the mite control method of the present invention, spray treatment on the tick means spraying 0.1 to 10 ml of the suspension per 100 mice on the tick, and preferably spray treatment with 1 ml of the suspension is preferred. When the suspension is treated in excess of the above capacity, it is difficult to secure the economy of control compared to the amount of treatment, and when the suspension is treated less than the above capacity, it is difficult to see high control activity.

Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strain spore suspension of the present invention is prepared and immersed in small scabies tick nymphs, and has high insecticidal activity. It was confirmed that it was visible (FIGS. 4 and 5). In addition, Metarhizium anisopoliea JEF-214 (KFCC11746P), Metarhizium anisopoliea JEF-279 (KFCC11721P) or Metarhizium anisopoliea JEF-290 (KFCC11794P) strains have a thermal stability effect (FIG. 6).

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Isolation and identification of strains

1-1. Isolation of strains

In order to collect soil samples for strain isolation, 5-10 cm of the surface layer was collected. A quarter of the collected soil sample was filled in a paper cup. Brown larvae (3-4 animals) were put, and 1 ml of distilled water was supplied every day to maintain high humidity conditions. Infection was induced by supplying moisture to the brown mealworm larvae treated in the soil for 5-10 days. The strain was isolated from the mycosis of the larva. The strain was isolated by culturing (25±2℃, dark condition) in 1/4 Sabouraud Dextrose Agar (1/4SDA; Difco, USA). A single colony was obtained through 2-3 separations. The acquired single colony was used to re-test the pathogenicity to the larvae. The strain was re-tested to confirm pathogenicity, and the gene was amplified using an ITS primer (ITS1F: 5'-TCCGTAGGTGAACCTGCGG-3'/ ITS4R: 5'-TCCTCCGCTTATTGATATGC-3'). BLAST was performed using the amplified gene information. The isolated and identified insect pathogenic fungi were stored at -70℃ with 15-20% glycerol stock for long-term storage.

1-2. Growth and morphology of the isolated strain

The agar block cultured with Metarhizium anisopliae strain was transferred to Sabraud Dextrose Agar (SDA) and incubated for 7 days (25°C, dark), and observed with the naked eye and an optical microscope (400 times) ( Fig. 1). Green spores and methods of sporulation, which are typical characteristics of the strain of the genus Metarhizium, were observed.

Example 2. DNA sequencing of the Metarhizium anisopliae strain of Example 1

The present invention identified Metarizium anisopliae of Example 1. Specifically, PCR was performed under the following conditions. 94°C for 5 minutes, 94°C for 60 seconds, 62°C for 30 seconds, and 72°C for 30 seconds were performed 30 times, and finally post maturation was performed at 72°C for 3 minutes. The sequence was performed using the ITS (internal transcribed spacer) primers shown in Table 1 below. PCR products were electrophoresed for 25 minutes at 140 V using 0.8% agarose gel and Eco dye. The finally obtained product was sequenced and finally identified as Metarhizium anisopliae (FIG. 2). The present inventors compared the Metarizium anisopliae of Example 1 to the Metarhizium anisopliae JEF-214, the Metarhizium anisopliae-279, and the Metarhizium anisopliae. They were named as Metarhizium anisopliae-290, respectively. In addition, the previously known Metarhizium To confirm the relationship with the genus strains, M using the ITS sequence of the genus Metarhizium and various insect pathogenic fungi reported to the National Center for Biological Information (http://www.ncbi.nlm.nih.gov/). Anisopliae JEF-214, JEF-279, JEF-290 and the relationship was compared (Fig. 3). As a result, it was found to be relatively high with most of the M. anisopliae strains.

primer direction order Sequence listing number ITS (internal transcribed spacer) Forward direction 5'-TCC GTA GGT GAA CCT GCG G- 3' One Reverse 5` -TCC TCC GCT TAT TGA TAT GC- 3` 2

Example 3. Confirmation of the control effect of small Sophie Charm mites of M. anisopoliea JEF-214, M. anisopoliea JEF-279, M. anisopoliea JEF-290 strain

In order to confirm the control effect of the M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 strains against small sofie charm mites, an experiment was performed under the following conditions. The M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 were cultured in 1/4 SDA at 25±2° C. for 14 days. The cultured strain was prepared as a spore suspension (1*10 ⁷ conidia/ml) using 0.03% siloxane solution. Filter paper (55 mm) was put on the top and bottom of Petri-dish (60x15mm). Filter paper-treated petri-dish was treated with 200 ul DW (top) and 200 ul SDB (bottom) to maintain moisture. Small Sophie Charm Mite (nymph) was immersed in the prepared spore suspension for about 10 seconds, and the Soaked Small Sophie Charm Mite was treated in the preceding petri-dish. As a control group, a small Sophie Charm mite immersed in 0.03% silwet was used. As a result, in the group treated with M. anisopoliea JEF-214, M. anisopoliea JEF-279 and M. anisopoliea JEF-290 strains, 50% mortality was confirmed on the 14th day of treatment, and other strains ( Ma JEF-206, -208 , -209, -210, -215, -216, -217, -218, -219, -220, -221, -222, -227, -230, -234, -237, If JEF-223, -225 , -228, -229, -233, Bb JEF-224, -226, -231, Pc JEF-236, La JEF-239). In addition, mycosis was confirmed in the bacteria treated with the bacteria (FIG. 4).

Example 4. Confirmation of the control effect of small sopi charm mites of M. anisopoliea JEF-214, M. anisopoliea JEF-279, M. anisopoliea JEF-290 strain (semi-field experiment)

Experiments were performed under the following conditions to confirm the effect of controlling small sophie charm mites in the field conditions of the M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 strains. The soil collected in the area where the small sophie true mite occurred was treated in petri-dish (100x40mm). The soil was treated with 20 nymph small phylum tick mites treated with fluorescent paint. M. anisopliae JEF-214, JEF-279, JEF-290 spore suspension (1*10 ⁸ conidia/ml) 1 ml, untreated group and positive control (2% cypermethrin) diluted 80 times and treated in small thorax mites I did. In order to prevent the escape of the small Sophie charm mite, petri-dish with soil was treated in a tray containing water. This experiment was carried out in 3 repetitions, and the number of viable insects was investigated for 30 days at intervals of 3 days. As a result of this experiment, an insecticidal rate of 60% was confirmed in the M. anisopoliea JEF-214 strain treatment group on the 30th day of treatment with M. anispilae JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290, and M. anisopoliea In the treatment of JEF-279 and M. anisopoliea JEF-290 strains, an insecticidal rate of more than 80% was confirmed. In the case of the M. anisopoliea JEF-290 strain treatment group, an insecticidal rate of about 50% appeared from the 6th day of treatment, indicating a high insecticidal effect. In addition, the mycosis of the small Sophie Charm mite was confirmed in the strain treatment (Fig. 5).

Example 5. Confirmation of spore productivity of M. anisopoliea JEF-214, M. anisopoliea JEF-279, M. anisopoliea JEF-290 strain

The spore productivity of M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 strains, which are important in producing granular strains using millet, was confirmed. The granular form was produced in the following way. After 200 g of millet was added to the polyvinyl bag, 0.16 ml of 50% citric acid was additionally added. Heat treatment was performed using a microwave for 3-4 minutes so that moisture was sufficiently absorbed in the millet. The inlet part of the polyvinyl bag was manufactured using a paper cup and sterile gauze for smooth oxygen supply. The polyvinyl bag containing the millet was sterilized for 15 min at 121°C. After cooling at room temperature, ^{1 ml of the strain suspension (1*10 7} conidia/ml) was inoculated with a polyvinyl bag. After culturing at 25°C for 14 days, it was dried at room temperature for 1 day to finally produce spores. 0.1 g of M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 strains cultured in millet were placed in a 1.5 ml micro test tube, and 0.03% silwet, a surfactant, was added to 1 ml micron for spore harvesting. After putting it in the test tube, it was vortexed for 1-2 minutes. The harvested spores were confirmed using a hemocytometer. As a result, it was confirmed that all three strains ^{produced spores of about 6*10 8} conidia/g (FIG. 6).

Example 6. Confirmation of thermal stability of M. anisopoliea JEF-214, M. anisopoliea JEF-279, M. anisopoliea JEF-290 strain

When the strain was treated in natural soil, thermal stability, which is one of the conditions for the strain to settle well, was evaluated. Thus, the strains of M. anisopoliea JEF-214, M. anisopoliea JEF-279, and M. anisopoliea JEF-290 cultured in the tank were put into a 1.5 ml micro test tube. They were exposed to heat for 30, 60, and 90 minutes in an incubator at 35, 40, and 45°C. 200 ul of 0.03% silwet was added to the exposed strain. Spores were harvested by vortexing for 1-2 minutes. 1/4 Sabouraud Dextrose Agar (SDA) was dispensed in 2 µl medium and cultured in an incubator at 24°C. After 18 hours, the germination rate of spores was investigated. As a result, in the heat treatment at 35℃, all three strains had a germination rate of 90% or more, and in the heat treatment at 40℃, M. anisopoliea JEF-214 and M. anisopoliea JFE-279 strains had relatively high germination rates of 80% or more in 90 minutes of heat treatment. Confirmed. In the heat treatment at 45°C, a relatively high germination rate of 40% or more was observed in all three strains in 30 minutes (FIG. 7).

[Microorganism deposit]

Depositary institution name: Korea Microorganism Conservation Center (domestic)

Accession number: KFCC11721P

Consignment Date: 20170613

Depositary institution name: Korea Microorganism Conservation Center (domestic)

Accession number: KFCC11746P

Consignment Date: 20171101

Depositary institution name: Korea Microorganism Conservation Center (domestic)

Accession number: KFCC11794P

Consignment Date: 20180919

Claims (9)

It has the control effect of Haemaphysalis longicornis,
Metarhizium anisopliea JEF-214 (KFCC11746P), which has thermal stability at 35°C to 45°C for 90 minutes. delete Metarhizium anisopliea JEF-214 (KFCC11746P), which has a control effect of Haemaphysalis longicornis, and has thermal stability at 35°C to 45°C for 90 minutes, containing the spores of the strain or the culture medium of the strain, A composition for controlling ticks (Haemaphysalis longicornis). delete The method of claim 3, wherein the composition for controlling Haemaphysalis longicornis ticks is formulated with any one selected from the group consisting of a liquid formulation, a powder formulation, and a granular formulation, and is formulated with Haemaphysalis longicornis tick control. Dragon composition. The method of claim 5, wherein the composition for controlling Haemaphysalis longicornis ticks comprises Metarhizium anisopoliea JEF-214 (KFCC11746P) as any one solid culture medium selected from millet, white rice, bran, crude, sorghum, and combinations thereof. A composition for controlling ticks obtained by culturing in Small Sophie Charms (Haemaphysalis longicornis). Culturing the Metarhizium anisopliea JEF-214 (KFCC11746P) strain in a solid culture medium; And
Harvesting the culture;
Small Sophie Charm mite (Haemaphysalis longicornis) method for producing a control composition comprising a. The group consisting of crude, millet, bran, sorghum, cassia tora, oats, millet, mung bean, perilla, buckwheat, meso, barley, sorghum, perilla, sesame, whole wheat, rice, and combinations thereof. A method for preparing a composition for controlling small Sophie Charm mite (Haemaphysalis longicornis) comprising any one selected from. The method of controlling small sophie salis longicornis by treating the composition for control of Clause 3 on plants or soil to be protected from Haemaphysalis longicornis, its habitat, and small sophie salis longicornis. .

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