KR20200112083A - Composition for controlling thrips using Beauveria bassiana JEF-350 and method for preventing thrips using the same - Google Patents

Abstract

The present invention relates to Beauveria bassiana JEF-350 (KFCC11820P) having a thrips control effect, a thrips control composition using the same, and a thrips control method using the same.

Description

Beauveria bassiana JEF-350 having thrips control effect, composition for controlling thrips containing same, and method for controlling thrips using the same {Composition for controlling thrips using Beauveria bassiana JEF-350 and method for preventing thrips using the same}

The present invention relates to a Beauveria bassiana JEF-350 having a thrips control effect, a composition for controlling thrips including the same, and a method for controlling thrips using the same.

Thrips spp. is an insect belonging to the order Thrips spp. It is a microscopic pest that occurs year-round in all flowering crops such as horticulture and flowers and causes enormous damage. In particular, the yellow flower thrips (Frankliniella occidentalis) and the cucumber thrips (Thrips palmi Karny) are invasive pests that have recently spread nationwide, causing serious damage to various crops, and mediating viruses such as tomato spot counterfeit virus (TSWV). Therefore, control is particularly required. On the other hand, thrips have strong proliferation and develop resistance to chemical insecticides faster than other pests. In addition, thrips are considered to be relatively difficult pests to be controlled by pesticides because a generation elapses after about a month after invading into the facility, and then occurs as a mixture of various age groups such as eggs, larvae, pupae, and imago.

For the control of thrips, about 220 kinds of chemical insecticides such as pyriproxyfen and pinetoram have been used so far. However, thrips are resistant to most of these chemical insecticides, and are also resistant to recently researched and developed chemical insecticides such as spinosad, acetamiprid, and denotfuran. Carbamite insecticides (bendiocarb, carbosulfan, methiocarb, etc.), organophosphate insecticides (methomyl, clopyrophos, diazino, etc.), synthetic pyrethroid insecticides (acrinathrin, benpetrin, etc.) Halothrin, etc.), neonicotinoid insecticides (imidacloprid, acetapride, dinotefuran, etc.), microbial-derived biochemicals (spinosad), mectin insecticides (abamectin, milbamectin, etc.), etc. It is reported as a resistant chemical pesticide.

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, and the use of chemical pesticides can be replaced and environmentally safe biological Control methods are being actively studied. For example, it is a method of controlling by using natural enemies such as aeko norin (Orius sp.) and predatory mites, or by using insect pathogenic fungi that infect pests. More than 750 species of insect pathogenic fungi have been known to date, and among them, Beauveria bassiana, Metarhizium anisopliae, Nomuraea rileyi, etc. are representative microbial insecticides. It has been developed and is used for controlling various pests.

Regarding the Boberia bassiana strain, Korean Patent Publication No. 10-2016-0000537 discloses the control activity against the Boberia bassiana FG274 strain and its Pabam moth larvae, and Korean Patent Publication No. 10-2016-0139521 discloses Control activity against Boberia bassiana SD15 strain and its spotted mites or peach aphids, Korean Patent Publication No. 10-2016-0084968 is a control activity against Boberia bassiana JEF007 strain and its saw-legged ant spermatozoa, domestic patent Publication No. 10-2011-0011239 is Boberia bassiana MaW1 strain and its control activity against blackheads or hairy toads, and Korean Patent Publication No. 10-2011-0094749 is Boberia bassiana MsW1 strain and its control activity against Northern Beards. , Korean Patent Publication No. 10-2015-0113255 discloses a control activity against water pests such as Boberia bassiana ERL 836 strain and apex or rice weevil. However, there is still a need for new highly active microbial pesticides that can control thrips with the same potency as chemical pesticides, and that are environmentally friendly and easy to handle and use.

Accordingly, the present inventors have made great efforts to discover new insect pathogenic fungi based on microorganisms that have a control effect on thrips, and to establish an effective thrips insect management system while dramatically overcoming the resistance problem and environmental residual problem of the existing synthetic insecticides. As a result, the excellent thrips of the JEF-350 strain of Beauveria bassiana (Beauveria bassiana) confirmed the insecticidal power of thrips, particularly cucumber thrips, and completed the present invention.

The present invention was conceived to solve the above problems, the present inventors confirmed the excellent insecticidal power of the Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain, and completed the present invention.

An object of the present invention is to provide a Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain having a control effect on thrips.

Another object of the present invention is to provide a composition for controlling thrips worms comprising a strain of Boberia bassiana JEF-350 (KFCC11820P), a spore of the strain or a culture solution of the strain.

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

In order to achieve the above object, the present invention can provide a Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain having a thrips control effect.

According to a preferred embodiment of the present invention, the thrips may be a flower yellow thrips, a cucumber thrips, a Taiwanese thrips or a green onion thrips.

The present invention may also provide a composition for controlling thrips worms comprising a Boberia bassiana JEF-350 (KFCC11820P) strain, a spore of the strain or a culture solution of the strain.

According to a preferred embodiment of the present invention, the thrips may be a flower yellow thrips, a cucumber thrips, a Taiwanese thrips or a green onion thrips.

According to a preferred embodiment of the present invention, the composition for controlling thrips 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 thrips is any one selected from millet, white rice, bran, crude, sorghum, and combinations thereof, using a strain of Boberia bassiana JEF-350 (KFCC11820P). It may be obtained by culturing in a solid culture medium of.

The present invention can also provide a method for controlling thrips by treating the above-described composition for controlling thrips on pests, their habitat, plants or soil to be protected from pests.

The Boberia bassiana JEF-350 (KFCC11820P) strain of the present invention was cultured in a solid medium containing millet to create spores, and the spore suspension was sprayed on cucumber leaves for 6 days at 25° C. The number of dead insects was confirmed. In the untreated group, a viability of 100% was confirmed, whereas the strain of Boberia bassiana JEF-350 (KFCC11820P) of the present invention was found to have a viability of 0%, and other Boberia bassiana ( Beauveria bassiana) showed a lower viability than the strain. Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain of the present invention has a significantly higher control effect of thrips than the previously reported Boberia bassiana (Beauveria bassiana) strain. Furthermore, Beauveria bassiana JEF-350 (KFCC11820P) strain has an effect of having high thermal stability compared to other strains, so when treated in the soil, the strain has the effect of settling well.

Figure 1 is a boberia bassiana ( Beauveria bassiana ) JEF-350 (KFCC11820P) strain observed with the naked eye (Fig. 1a) and the growth form of mycelium and spores observed with a stereomicroscope (Fig. 1b) is shown.
Figure 2 shows the base sequence of the ITS (Internal Transribed Spacer) of the Boberia bassiana ( Beauveria bassiana ) JEF-350 (KFCC11820P) strain.
FIG. 3 is a result of comparing and analyzing the relationship between the previously reported strains and the Beauveria bassiana JEF-350 (KFCC11820P) strain.
Figure 4 shows the results of spore production of the Boberia bassiana JEF-350 (KFCC11820P) strain using an Italian millet.
Figure 5 shows the viability of cucumber thrips (single repetition) (Figure 5a) and Boberia bassiana (Beauveria bassiana) JEF-350 ( KFCC11820P) Cucumber thrips killed by the strain (Day 6) (Fig. 5B) is shown.
Figure 6 shows the viability of cucumber thrips (3 repetitions) (Figure 6a) and Boberia bassiana JEF-350 (Beauveria bassiana) JEF-350 (KFCC11820P) strain separation treatment KFCC11820P) Cucumber thrips killed by the strain (Day 6) (Fig. 6b) is shown.
Figure 7 shows the results of confirming the thermal stability of the Boberia bassiana ( Beauveria bassiana ) JEF-350 (KFCC11820P) strain and other Boberia bassiana (Beauveria bassiana) strains.

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

As described above, microorganisms that can dramatically overcome the resistance problem and environmental residual problem of existing synthetic pesticides, and that the control effect appears only when the pesticidal component based on the existing plant extract directly contacts thrips. There is a demand for the discovery of new insecticidal fungi and the development of an effective thrips insect management system.

The composition for controlling thrips using the Boberia bassiana JEF-350 (KFCC11820P) strain according to the present invention provides excellent insecticidal power and has thermal stability, so it is effective as a composition for controlling thrips.

Accordingly, the present invention can provide a strain of Boberia bassiana JEF-350 (KFCC11820P) having a thrips 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. The collected soil samples were filled in a paper cup about 1/4. Brown larvae (3-4 animals) were placed, 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 brown mealworm larva. The strain was re-tested to confirm pathogenicity and the gene was amplified using 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 it as a Boberia bassiana JEF-350 strain (FIG. 2), and was sent to the Korea Microbiology Conservation Center in 2019. It is deposited and is being kept under the deposit number KFCC11820P on February 12th. By comparing the relationship between the previously reported 15 strains of the genus Beauveria and the ITS (internal transcribed spacer) sequence (Figure 3), the JEF-350 strain of Beauveria bassiana is inherited from the existing strain. It was confirmed that it is a novel strain with significant differences.

The thrips may be flower yellow thrips, cucumber thrips, Taiwan thrips or leeks thrips, and preferably may be cucumber thrips, but is not limited thereto.

The present invention provides a composition for controlling thrips, including Boberia bassiana JEF-350 (KFCC11820P), spores of the strain or a culture solution of the strain.

The composition for controlling thrips is preferably prepared through solid culture by inoculating the suspension of the strain on a grain medium, more preferably millet, rice, Italian millet, barley, and soybeans. , Sorghum, millet, rice and brown rice is to use a medium consisting of one or more selected from the group consisting of grains, among which, it is most preferable to inoculate a millet medium and culture the solid (Fig. 4).

In the composition for controlling thrips worms of the present invention, the solid culture is after inoculating the suspension of the strain in a medium, preferably incubating at 20 to 30° C. for 5 to 20 days, and then at room temperature conditions of 1 to It can be dried for 30 days, more preferably after incubation for 7 days at 25° C., and drying at room temperature for 1 day is preferable.

Since the composition for controlling thrips of the present invention contains spores of the Beauveria bassiana JEF-350 (KFCC11820P) strain, the spores attach to the host insect and germinate to cause mycosis, so that the insecticidal effect appears. do. In addition, in addition to the thrips that have directly contacted them, even thrips that are not in direct contact with the composition may exhibit a control effect due to the transmission of spores.

The composition for controlling thrips 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, Beauveria bassiana JEF-350 (KFCC11820P) strain causes mycosis in adult cucumber thrips and exhibits excellent effects on thrips.

The present invention can also provide a method for controlling thrips by treating the above-described composition for controlling thrips on pests, their habitat, plants or soil to be protected from pests.

The composition for controlling thrips can be prepared as a suspension.

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

The suspension of the present invention is preferably prepared by harvesting spores of the cultured strain as a solid culture medium or composition for controlling thrips, counting with a hemocytometer, and 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, preferably 1x10 ³ spores (conidia) It may be from /g to 1x10 ⁷ spores (conidia)/g, more preferably 1x10 ⁷ spores (conidia)/ml.

In the method for controlling thrips of the present invention, spray treatment on thrips means spraying 0.1 to 10 ml of suspension per 100 heads of thrips, preferably spraying with 1 ml of suspension. . If the suspension is treated in excess of the above capacity, it is difficult to secure economic efficiency of control compared to the treatment amount, and if the suspension is treated less than the above capacity, it is difficult to obtain high control activity.

When a spore suspension of the Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain of the present invention was prepared and spray-treated on adult cucumber thrips, it was confirmed that it showed high insecticidal activity (FIGS. 5 and 6). It was confirmed that the Boberia bassiana JEF-350 strain has superior thermal stability than other Boberia bassiana strains (FIG. 7).

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

The present inventors isolated insect pathogenic fungi from soil samples under the following conditions. In order to collect a soil sample for strain isolation, 5-10 cm of the surface layer was collected. The collected soil samples were filled in a paper cup about 1/4. Brown larvae (3-4 animals) were placed, 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 brown mealworm larva. The strain was re-tested to confirm pathogenicity and the gene was amplified using 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 in which the Beauveria bassiana JEF-350 strain was cultured was transferred to Sabraud Dextrose Agar (SDA) and incubated for 7 days (25℃, dark), and observed with the naked eye and an optical microscope (400 times) ( Fig. 1). White hyphae, grayish white spores, and spore formation, which are typical of the strains of the genus Beauveria, were observed.

Example 2. DNA sequencing of the Beauveria bassiana strain of Example 1

The present invention identified the Beauveria bassiana 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 carried out using the ITS primers in Table 1 below. PCR products were electrophoresed for 25 minutes at 140 V using 0.8% agarose gel and Eco dye. As a result of sequencing the finally obtained product, it was identified as B. bassiana (FIG. 2). The present inventors named the Boberia bassiana of Example 1 as the Boberia bassiana JEF-350 (KFCC11820P) strain. The ITS (internal transcribed spacer) sequence of the Beauveria bassiana JEF-350 strain was described in SEQ ID NOs: 1 and 2. In addition, in order to confirm the relationship with the known strains of the genus Beauveria , 10 strains of the genus Beauveria and two strains of the genus Isaria reported to the National Center for Biological Information (http://www.ncbi.nlm.nih.gov/) Using the ITS sequence of the genus strain, two kinds of Metarhizium genus strain and one kind of Pochonia genus strain, the relationship with B. bassiana JEF-350 was compared (Fig. 3). As a result, it was confirmed that the relationship between most B. bassiana strains was relatively high, and that the relationship between Isaria, Metarhizium, and Pochonia strains was relatively low.

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. Production of spores under solid culture conditions using millet

After 200 g of millet was added to the polyvinyl bag for culture, 0.16 ml of 50% citric acid was additionally treated. Heat treatment was performed at 90° C. for 1 hour using a water bath so that moisture was sufficiently absorbed into the millet. The entrance 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 at 121°C for 15 minutes. After cooling at room temperature, 1 ml of B. bassiana JEF-350 strain suspension (1*10 ⁷ conidia/ml) was inoculated into a polyvinyl bag. After culturing at 25° C. for 7 days, dried for 1 day at room temperature, B. bassiana JEF-350 spores were finally produced (FIG. 4).

Example 4. Control effect of JEF-350 strain of Beauveria bassiana against cucumber thrips (spray treatment-single repeat)

A spore suspension (1x10 ⁷ conidia/ml) was prepared using B. bassiana JEF-350 cultured in millet, and then spray-treated on cucumber leaves (provided as a food source). Please tell us the type and concentration of the positive control. It is 5% spinetoram, the active ingredient of the positive control, and was diluted 2,000 times. The number of viable and dead insects was investigated for 6 days at 25℃. Death worms were identified in the treated groups treated with JEF-350 from day 1 after treatment, and on the 6th day of treatment, 100% viability was found in Control (untreated group), whereas B. bassiana JEF-350 had 0% viability. Loyalty was confirmed. B. bassiana strains other than the strain of the present invention (JEF-006, -091, -105, -148, -163, -331, -347, -388, -479, -501, -509, -527) Day 6 The viability of 11 to 50% was confirmed (Fig. 5A). It was confirmed that the B. bassiana JEF-350 strain of the present invention exhibits a higher lethal effect than other strains. After 6 days of treatment, as a result of confirming mycosis in adult worms of each treatment group (FIG. 5B), it was confirmed that mycosis appeared in each strain treatment group, and in the case of untreated group, it was confirmed that most of the individuals were not killed.

Example 5. Control effect of JEF-350 strain of Beauveria bassiana against cucumber thrips (spray treatment-3 repetitions)

A spore suspension (1x10 ⁷ conidia/ml) was prepared using B. bassiana JEF-350 cultured in millet, and then sprayed onto cucumber leaves (provided as a food source). The experiment was carried out in 3 repetitions. The number of viable and dead insects was investigated for 6 days at 25℃. Death worms were identified in the treatment group treated with JEF-350 from the first day after treatment, and the viability of Control 100% was confirmed on the 6th day of treatment, and 0% viability was confirmed in B. bassiana JEF-350. In addition to the present strain, other B. bassiana strains were also tested, and a viability of 20 to 70% was confirmed on the 6th day of treatment (FIG. 6A). It was reconfirmed that the B. bassiana JEF-350 strain showed a higher lethal effect than other B. bassiana strains. In addition, after 6 days of treatment, as a result of confirming mycosis in adults of each treatment group (FIG. 6B), it was confirmed that mycosis appeared in each strain treatment group, and in the case of untreated group, most of the individuals were not killed.

Example 6. Confirmation of thermal stability of B. bassiana JEF-350 strain

When the strain was treated in natural soil, thermal stability, which is one of the conditions for the strain to settle well, was confirmed. A spore suspension was prepared at a concentration of 1×10 ⁷ conidia/ml using a strain cultured in Sabouraud Dextrose Agar (SDA) medium. Heat treatment was performed at 45° C. for 0, 30, 60, 90, and 120 minutes in an incubater. After dropping the heat-treated spore suspension from SDA medium by 5 μl, it was cultured in a 24°C incubator. After 18 hours, the germination rate of spores was investigated. As a result, it was confirmed that the germination rate of the JEF-350 strain was 77±1% under heat treatment conditions at 45° C. for 120 minutes. High thermal stability was confirmed by comparing the thermal stability with other strains (FIG. 7).

[Accession number]

Depositary institution name: Korea Microbial Conservation Center (domestic)

Accession number: KFCC11820P

Consignment Date: 20190212

Claims (7)

Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain having a control effect on thrips. The method according to claim 1, wherein the thrips are flower yellow thrips, cucumber thrips, Taiwan thrips or leeks thrips, Beauveria bassiana JEF-350 (KFCC11820P) strain. Boberia bassiana (Beauveria bassiana) JEF-350 (KFCC11820P) strain, a composition for controlling thrips, including the spores of the strain or the culture medium of the strain. The composition for controlling thrips according to claim 3, wherein the thrips are flower yellow thrips, cucumber thrips, Taiwan thrips or green thrips. The composition for controlling thrips according to claim 3, wherein the composition for controlling thrips is formulated with any one selected from the group consisting of a liquid formulation, a powder formulation, and a granular formulation. The method of claim 5, wherein the composition for controlling thrips is any one solid culture medium selected from millet, white rice, wheat bran, crude, sorghum, and combinations thereof, using the Boberia bassiana JEF-350 (KFCC11820P) strain. A composition for controlling thrips obtained by culturing in. A method of controlling thrips by treating the composition for controlling of claim 3 on a pest, its habitat, a plant or soil to be protected from pests.

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