KR101680638B1 - New microorganism Beauveria bassiana FG274 and Microbial control agent for the prevention of Spodoptera exigua larva - Google Patents

Abstract

The present invention relates to a novel microorganism, Beauveria bassiana , FG274 and a microorganism preparation for controlling larvae of the present invention.
The novel microorganism, Beauveria bassiana , The strain FG274 (KACC93200P) is represented by SEQ ID NO: 1 and is characterized in that it has a controlling effect against the parasitic larvae.
According to the present invention, a new microorganism, Beauveria bassiana FG274, which has a controlling effect against the parasitic larvae, and a microorganism preparation for controlling parasitic larvae containing the same are provided.

Description

(New microorganism Beauveria bassiana FG274 and Microbial control agent for the prevention of Spodoptera exigua larvae)

The present invention relates to a novel microbial bovaria barbiana FG274 and a microorganism preparation for controlling the larvae of the present invention. More particularly, the present invention relates to a novel microorganism, Beauveria bassiana , FG274 strain and a microorganism preparation containing the same.

Insect pathogenic fungi are mostly infectious fungi (Hyphomycetes). They are harmless to humans, plants, and plants. They are a pathogenic fungus that is specific to the target insect pests, and play a role in regulating host density in the natural state.

Insect pathogenic fungi The invasive mechanism against the insect host is different from insect pathogenic bacterium or virus which is infected through host feeding. The fungus spore begins to adhere to the host's epidermis, penetrates the epidermis physically and chemically, To block the immune function and secrete toxic substances to kill the host.

When these insect pathogenic fungi are used for pest control, there is no indirect risk factor such as accumulation of dangerous substances directly in human, higher organisms, fish, and grains, and stability is high, and selectivity that can selectively remove only the desired insect pests And the persistence of the insecticidal effect is maintained so that the application of the repetitive microorganism preparation can be minimized.

To date, more than 750 insect pathogenic fungi have been known, among which Beauveria bassiana , Metarhizium anisopliae , Nomuraea rileyi ) have been developed as microbial insecticides and have been used in various pest control.

Spodoptera exigua is a pest insect belonging to Lepidoptera, Noctuidae, and is a typical insect pest which is causing economic damage by adding almost all agricultural crops including vegetables, flowers and fruit trees. It is relatively insensitive to insecticides. It enters into the stem at the early stage of insect repellent, and there is less chance of exposure to the medicines due to the harmful effect from the inside. When the insecticidal activity of the third instar larva is over, resistance to organic synthetic insecticide becomes strong, It is considered a pest.

Until recently, chemical insecticides such as organophosphorus, carbamate and pyrethroid have been mainly used for the control of pestilence, but they are resistant to existing insecticides due to unreasonable abuse of insecticides, There is increasing interest in environmentally friendly methods of control due to problems, pesticide resistance, and concerns about people's health.

Related prior arts are disclosed in Korean Patent Laid-Open Publication No. 10-2011-0005355 (Method of Controlling and Controlling Paralichthys olivaceus using Antifungal Insecticidal Fungi) and Patent Application No. 10-2008-0006679 A microflora fungus and a microbial pesticide composition using the same).

It is an object of the present invention to provide a new microorganism, Beauveria bassiana , FG274 strain and a microorganism preparation for controlling the larvae of Paralichthys olivaceus, which contains the same.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

To attain the above object, the present invention provides a novel microorganism, Beauveria bassiana , The strain FG274 (KACC93200P) is represented by SEQ ID NO: 1 and is characterized in that it has a controlling effect against the parasitic larvae.

The strain is characterized in that the strain is inoculated in the soil and separated from the infected bee moth larvae.

The strain is characterized in that it is used as an active ingredient of any one of an insecticidal microbial pesticide, a foliar spraying agent or a conventional spraying agent.

The microorganism preparation for controlling parasitic fly larva according to the present invention is characterized by comprising the microorganism strain of the above-mentioned new microorganism Beauveria bassiana FG274 (KACC93200P) or a culture thereof as an active ingredient.

The culture solution is added to the microbial medium in the presence of the above-mentioned Beauveria bassiana , And FG274 (KACC93200P) are cultured at 20 to 30 DEG C for 5 to 15 days.

The microorganism medium is a PDA (Potato dextrose agar) medium.

The culture broth contains the above-mentioned Beauveria bassiana (1 × 10 ^{6 to} 1 × 10 ⁸ spores per ml) FG274 (KACC93200P).

The method for controlling the parasitic larva of the present invention is characterized in that the microorganism preparation is spray-treated on a plant in which the parasitic larva is present.

The spraying treatment is performed at a temperature of 20 to 30 캜.

As described above, the microorganism preparation containing Beauveria bassiana FG274 or its culture of the present invention has pathogenicity to the parasitic larvae, is environmentally friendly and environmentally friendly, It can be used effectively as microbial pesticide for control.

Brief Description of the Drawings Fig. 1 is a graph showing the results of a comparison between the fresh microorganism Beauveria bassiana isolated in Example 1, FG274. ≪ / RTI >
Fig. 2 is a graph showing the results of a comparison between the fresh microorganism Beauveria bassiana isolated in Example 1, FG274. ≪ / RTI >
Fig. 3 is a graph showing the activity of the new microorganism Beauveria bassiana isolated in Example 1, FIG. 5 is a diagram showing the insecticidal status of the larvae of the parasitic larvae when treated with FG274. FIG.
Fig. 4 is a graph showing the relationship between the new microorganism Beauveria bassiana isolated in Example 1, The insecticidal effect on the larvae of the parasitic larvae according to the treatment concentration of FG274 is shown in FIG.
Fig. 5 is a graph showing the results of a comparison with the fresh microorganism Beauveria bassiana isolated in Example 1. Fig. FG274 is a pest insecticidal effect on the larvae according to the treatment temperature.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

The novel microorganism, Beauveria bassiana , FG274 is represented by SEQ. ID. NO. 1 and is isolated from the infested beeswax moth larvae inoculated in soil, and has a controlling effect against Spodoptera exigua larva .

In order to select a new strain exhibiting a high control effect on the larvae of Bombyx mori, the soil was collected, and the bees were then cultivated for a certain period of time, Insect pathogenic strains were selected.

When the selected strain was inoculated into a PDA medium and cultured, it was observed to be in the form of a grape cluster, and the color of the mycelium was white at first, and became milky white with time, so Beauveria sp. Respectively.

As a result, the 18S rRNA analysis revealed that the strain was a new strain of the genus Beauveria sp. And named this strain as Beauveria bassiana FG274. This strain was deposited at the National Institute of Agricultural Science, And received KACC93200P as deposit number on May 22, 2014.

The strain of Beauveria bassiana FG274 is composed of the entire nucleotide sequence shown in SEQ ID NO: 1 or a part thereof and is composed of an open reading frame (ORF) of 530 bp.

The new microorganism Beauveria bassiana FG274 of the present invention can be industrially used because it can be mass-produced in a short time when it is cultured under the following culture conditions.

The new microorganism grows well on the PDA medium as a culture medium. Especially when cultivated at 20 to 30 DEG C for 5 to 15 days, the most optimal strain growth is achieved (as shown in Table 1 below).

That is, when the culture is performed at a temperature lower than 20 ° C, the temperature is too low to slow the growth rate of the strain, and if it exceeds 30 ° C, the temperature is too high to slow the growth rate of the strain.

The microorganism strain of the new microorganism Beauveria bassiana FG274 of the present invention or a culture solution thereof is used as an effective ingredient of a microorganism preparation for controlling the larvae of Paralichthys olivaceus.

Such microbial agents are excellent in the survival period of the active fungi, germination rate during the survival period, and ability to maintain the insecticidal activity, and thus can be used for insecticidal microbial pesticides, foliar spraying agents,

Preferably, the microorganism preparation of the present invention is subjected to spray treatment by concentrating and spraying a strain of Beauveria bassiana FG274 on a crop attached to larva or larva of Paralichthys olivaceus, and the effective amount of the microorganism is determined by microorganism per unit volume (ml) Is preferably sprayed at about 1 x 10 ^{6 to} 1 x 10 ⁸ spores. At this time, when spraying is carried out at a temperature of 20 to 30 ° C., the activity of the FG274 strain of Beauveria bassiana is high, and thus it exhibits the most excellent insecticidal effect against the larvae of Paralichthys olivaceus.

The present invention will be described in more detail with reference to the following examples and experimental examples, but the scope of protection is not limited to the following examples and experimental examples.

< Example  1> Isolation of a New Microorganism of the Present Invention

In order to isolate the pathogenic microorganisms against parasitoids, 500 ml of soil was collected from four sites.

The collected samples were dropped into 2 mm sieve, and 20 ml of each sample was placed in a plastic container, and 10 mice of 1.5 cm sized bee-bearing moth larvae were administered.

After incubation at 25 ℃ for 7 ~ 14 days, the insect pathogenic strain (FG274) was isolated from the surface of infected bee moth larvae.

< Example  2> Morphological Characteristics and Sequence Analysis of Isolated New Microorganisms

The strain (FG274) isolated in Example 1 was inoculated into a PDA (Potato dextrose agar) medium, cultured at 25 캜 for 7 days, and then observed with an optical microscope.

The observed strain is as shown in Fig. 1 and morphologically looks like grape clusters. The color of the mycelium is white in the beginning, and the color becomes milky white . Respectively.

Sequencing of the ITS portion of the 18s rRNA from the strain isolated in Example 1 confirmed that the strain was 100% homologous with the genus Beauveria sp. , And the analyzed Fragments of Beauveria bassiana FG274 The 18s RNA base sequence is shown in FIG.

<Experimental Example 1> Determination of the effect of the selected bobeliabassiana FG274 on the larvae

1. Experimental Method

The conidia of the strain (FG274) isolated in Example 1 were cultured in a PDA (Potato dextrose agar) medium for 14 days.

The culture broth was sprayed on a second instar larvae of a Chinese cabbage on a leaf of Chinese cabbage with a diameter of 90 mm, and the state was confirmed on days 3, 5, and 7.

2. Experimental results

As shown in FIG. 3, it was confirmed that the fungus larvae of Beauveria bassiana FG274, which was isolated in Example 1, died out over time.

<Experimental Example 2> Determination of optimum germination temperature of the selected strains of bobeliabassiana FG274

1. Experimental Method

In order to determine the optimum germination temperature of the selected strain of Beauveria bassiana FG274, the strain FG274 selected in Example 1 was cultured on the PDA medium for 24 hours at each temperature.

2. Experimental results

The results of the experiment are shown in Table 1 below.

Temperature (℃) Germination rate (%) average 15 18.4 ± 2.3 20 71.9 ± 1.8 25 75.2 ± 2.1 30 40.6 ± 1.1 35 3.4 ± 0.5

As shown in Table 1, the strain FG274 of Beauveria bassiana selected in Example 1 of the present invention was found to be well grown at 20 to 30 ° C.

< Experimental Example  3> Identification of the control effect of the selected bovisia barbiana FG274 strain on the larvae of the moth,

1. Experimental Method

Conidia of the strain (FG274) isolated in Example 1 were cultured in a PDA (Potato dextrose agar) medium for 14 days to collect spores.

The collected spores were suspended in a 0.01% Tween 80 solution, and then the spore concentration was measured using a hemocytometer. Then, the spores were suspended in a spore suspension of 1 × 10 ⁶ , 1 × 10 ⁷ , 1 × 10 ⁸ spores / .

The prepared spore suspension was sprayed on the front and back sides of the cabbage leaves having a diameter of 90 mm, into which 700 μl of the larvae had been sprayed, and then kept at a relative humidity of 95% or more at 25 ° C.

The insecticidal activity was investigated daily until 6 days after the treatment, and 30 replicates were repeated 3 times.

2. Experimental results

The results of the experiment are shown in Table 2 and FIG.

Treatment concentration
(Ml) Mortality rate
(%) LT ₅₀
(Day, half lethal time) 1 x 10 &lt; ⁶ &gt; / ml 90.8 ± 7.1 4.13 1 x 10 &lt; ⁷ &gt; / ml 90.0 + - 10.0 3.68 1 x 10 &lt; ⁸ &gt; / ml 100.0 0.0 3.06

As shown in Table 2 and FIG. 4, when the spore suspension of the new microorganism, Beauveria bassiana FG274 strain of the present invention was sprayed at a concentration of 1 × 10 ⁶ to 1 × 10 ⁸ spores, a high insecticidal rate . Especially, it was confirmed that the spraying rate was 100% at 1 × 10 ⁸ spore concentration.

< Experimental Example  4> Indoors by temperature of selected boveriac basiana strain FG274 Mortality rate  black

1. Experimental Method

Conidia of the strain (FG274) isolated in Example 1 were cultured in a PDA (Potato dextrose agar) medium for 14 days to collect spores.

The collected spores were suspended in a 0.01% Tween 80 solution, and then the spore concentration was measured using a hemocytometer, and a spore suspension was prepared so as to have a concentration of 1 × 10 ⁸ spores / ml.

The prepared spore suspension was sprayed with 700 μl of each of the front and back of the cabbage leaf having a diameter of 90 mm attached to a second instar larva (4 days after hatching), and maintained at a relative humidity of 95% or more at 15 ° C. and 20 ° C. , 25 ℃ and 30 ℃, respectively.

The insecticidal activity was investigated daily for 6 days after the treatment, and the repetition of the insecticidal activity was repeated twice with 20 animals per repetition.

2. Experimental results

The results of the experiment are shown in Table 3 and FIG.

Temperature
(° C) Mortality rate
(%) LT ₅₀
(Day, half lethal time) 15 50.0 + - 10.0 6.7 20 100 ± 0.0 4.0 25 100 ± 0.0 2.9 30 100 ± 0.0 3.9

As shown in Table 3 and FIG. 5, when the spore suspension of Beauveria bassiana FG274 isolated in Example 1 of the present invention was tested for insecticidal activity against P. chinensis at different temperature conditions, ℃, respectively.

As described above, the novel microorganism of the present invention, Beauveria bassiana FG274, exhibits excellent control effect against larvae of Paralichthys olivaceus, and thus the microorganism preparation containing the same is environmentally friendly and environmentally friendly, It can be used as a microbial pesticide.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

National Institute of Agricultural Science KACC93200P 20140522

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> New microorganism Beauveria bassiana FG274 and Microbial control          agent for the prevention of Spodoptera exigua larva <130> P2014-0063 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 530 <212> DNA <213> Beauveria sp. <400> 1 gtttactccc acccttctgt gacctaccta tcgttgcttc ggcggactcg ccccagcccg 60 gacgcggaca ggaccagcgg cccgccgggg acctcaaact cttgtattcc agcatcttct 120 gaatacgccg caaggcaaaa caaatgaatc aaaactttca acaacggatc tcttggctct 180 ggcatcgatg aagaacgcag cgaaatgcga taagtaatgt gaattgcaga atccagtgaa 240 tcatcgaatc tttgaacgca cattgcgccc gccagcattc tggcgggcat gcctgttcga 300 gcgtcatttc aaccctcgac ctccccttgg ggacgtcggc gttggggacc ggcagcacac 360 cgccggccct gaaatggagt ggcggcccgt ccgcggcgac ctctgcgtag taatacagct 420 cgcaccggaa ccccgacgcg gccacgccgt aaaacaccca acttctgaac gttgacctcg 480 aatcaggtag gactacccgc tgaacttaag catatcaata agcggaggaa 530

Claims (9)

delete delete delete A novel microorganism, Beauveria bassiana , which has a controlling effect against the parasitic larvae, FG274 (KACC93200P) strain or a culture of a strain as an active ingredient,
The strain is characterized in that it is isolated from a honeybee larva, which is cultivated in soil,
The culture broth was prepared by adding the above-mentioned Beauveria bassiana to the PDA medium, FG274 (KACC93200P) is cultured at 20 to 30 DEG C for 5 to 15 days.
The culture broth contains the above-mentioned Beauveria bassiana , (1 × 10 ^{6 to} 1 × 10 ⁸ spores per 1 ml of FG274 (KACC93200P) strain)
Microorganism preparation for control of larvae of Bombyx mori.
delete delete delete Characterized in that the microbial formulation of claim 4 is spray-applied to a plant in which the second instar larva of Paralichthys olivaceus is present.
9. The method of claim 8,
Wherein the spraying treatment is performed at 20 to 30 占 폚.

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