KR101680639B1 - New microorganism Isaria fumosorosea FG340 and Microbial control agent for the prevention of Spodoptera exigua larva - Google Patents

Abstract

The present invention relates to a novel microorganism Isaria fumosorosea , FG340 and a microorganism preparation containing the same.
The novel microorganism Isaria fumosorosea FG340 (KACC93199P) of the present invention 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, Isaria fumosorosea FG340 strain, which is a novel microorganism having a controlling effect against larvae of Paralichthys olivaceus, and a microorganism preparation for controlling paralichia larvae containing the same are provided.

Description

[0001] The present invention relates to a new microorganism Isaria fumosorose FG340 and a microorganism preparation for controlling the larvae of Spodoptera exigua larvae containing the same,

The present invention relates to a novel microbial isolia fumoroside FG340 and a microorganism preparation for controlling the larvae of the present invention. More particularly, the present invention relates to a novel microorganism Isaria fumosorosea , FG340 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, including 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 novel microorganism Isaria fumosorosea , FG340 strain and a microorganism preparation containing 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.

In order to achieve the above object, the novel microorganism Isaria fumosorosea , The strain FG340 (KACC93199P) 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 containing the above-mentioned microorganism Isaria fumosorosea FG340 (KACC93199P) strain or a culture thereof as an active ingredient.

The culture broth was cultured on Isoba fumosorosea , FG340 (KACC93199P) is 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 Isaria fumosorosea at a concentration of 1 × 10 ^{6 to} 1 × 10 ⁸ spores per ml, FG340 (KACC93199P).

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 Isaria fumosorosea FG340 or a culture thereof of the present invention has pathogenicity to the parasitic larvae and is environmentally friendly because it has no environmental pollution and human toxicity It can be used effectively as a microbial pesticide for the control of larvae of the insects.

Fig. 1 is a graph showing the results of the isolation of the novel microorganism Isaria fumosorosea , FG340 < / RTI >
Fig. 2 is a graph showing the results of the isolation of the novel microorganism Isaria fumosorosea isolated in Example 1. Fig. 16S rRNA base sequence of FG340.
Fig. 3 is a graph showing the results of the isolation of the novel microorganism Isaria fumosorosea , FIG. 6 is a diagram showing the insecticidal status of the larvae of the paralichthys olivaceus during the treatment with FG340. FIG.
Fig. 4 is a graph showing the relationship between the new microorganism Isaria fumosorosea isolated in Example 1, FIG. 5 is a diagram showing the insecticidal effect against the larvae of the parasitic larvae according to the treatment concentration of FG340. FIG.
Fig. 5 is a graph showing the activity of the new microorganism Isaria fumosorosea isolated in Example 1. Fig. FIG. 5 is a diagram showing an insecticidal effect on the larvae of the parasitic larvae according to the treatment temperature of FG340. FIG.

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 Isaria fumosorosea of the present invention, FG340 contains the nucleotide sequence of SEQ ID NO: 1 and shows a control effect against Spodoptera exigua larva , which has been identified through the following examples and experimental examples.

In order to select a new microorganism exhibiting a high control effect against the parasitoid larvae, 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 the PDA medium and cultured, the color of the mycelium changed from white to gradually light brown at first, and thus Isaria sp. Respectively.

As a result of 16S rRNA analysis, it was identified as a new strain of Isaria sp., Named as Isaria fumosorosea FG340 strain, and this strain was designated as the National Agricultural Research Service Agricultural Genetic Support Center On May 22, 2014 to receive the deposit number KACC93199P.

The Isaria fumosorosea FG340 strain consists 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 571 bp.

The new microorganism Isaria fumosorosea FG340 of the present invention can be industrially used as it can be mass produced in a short time when it is cultured through 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 Isaria fumosorosea FG340 strain of the present invention or the culture thereof is used as an effective ingredient of a microorganism preparation for controlling B. larvae.

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 Isaria fumosorosea FG340 strain in a crop having larva or larva of Paralichthys olivaceus, and the effective amount of the microorganism is expressed in unit dose (ml) It is preferable that the number of sugar microorganisms is sprayed at about 1 × 10 ^{6 to} 1 × 10 ⁸ spores. At this time, when spraying is carried out at a temperature of 20 to 30 ° C., the activity of Isaria fumosorosea FG340 is high, and thus it exhibits the most excellent insecticidal effect against the parasitoid larvae.

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, insect pathogenic strain (FG340) was isolated from the surface of infected bee moth larvae.

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

The strain (FG340) isolated in Example 1 was inoculated into a PDA (Potato dextrose agar) medium, cultured at 25 ° C for 7 days, and then observed with an optical microscope.

The observed strain is as shown in Fig. 1, and the color of the mycelium is initially white and gradually becomes light brown . Respectively.

In addition, the strain isolated in Example 1 was found to be 100% homologous to Isaria sp. By sequencing the ITS portion of 16S rRNA . Isaria fumosorosea , which was analyzed, The 16S RNA base sequence of FG340 is shown in Fig.

<Experimental Example 1> Determination of the effect of the selected Isaria fumorosa FG340 on the larvae

1. Experimental Method

Conidia of the strain (FG340) 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 Chinese cabbage on a leaf of Chinese cabbage having a diameter of 100 mm, and the state was confirmed on days 3, 5, and 7.

2. Experimental results

As shown in FIG. 3, as time elapsed, Isaria fumosorosea FG340 isolated from Example 1 was found to be killed by the moth larva.

<Experimental Example 2> Determination of mycelial growth of the selected Isaria fumorosa FG340 strain by optimum germination temperature and temperature

1. Experimental Method

To determine the optimum germination temperature of the selected Isaria fumosorosea FG340 strain, the FG340 strain selected in Example 1 was cultured on the PDA medium for 24 days at each temperature.

2. Experimental results

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

Temperature (℃) Mycelial growth for 7 days
(mm, mean 占 SE) Germination rate (%) average 15 11.5 ± 0.5 16.6 ± 0.5 20 22.2 ± 0.8 91.8 ± 0.2 25 32.4 ± 2.0 88.3 ± 1.1 30 28.1 ± 0.7 93.4 ± 0.7 35 7.0 ± 0.0 1.8 ± 0.4

As shown in Table 1, Isaria fumosorosea FG340 strain 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 Isaria fuomorosse FG340 strain on the larvae of the moth larvae by the spore spray treatment concentration

1. Experimental Method

Conidia of the strain (FG340) 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 100 mm and the back surface of the cabbage larvae, respectively, at 700, and maintained at a temperature of 25 캜 while maintaining a relative humidity of 95% or more.

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 86.7 8.3 3.69 1 x 10 &lt; ⁷ &gt; / ml 100.0 + - 10.0 2.71 1 x 10 &lt; ⁸ &gt; / ml 100.0 0.0 2.47

As shown in Table 2 and FIG. 4, the spore suspension of Isaria fumosorosea FG340, a novel microorganism of the present invention, was sprayed at a concentration of 1 × 10 ⁶ to 1 × 10 ⁸ spores, And it was confirmed that the insecticidal rate was 100% when sprayed at a concentration of 1 × 10 ⁷ to 1 × 10 ⁸ spores.

< Experimental Example  4> Indoors by temperature of selected Isaria furomorose FG340 strain Mortality rate  black

1. Experimental Method

Conidia of the strain (FG340) 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 leaves having a diameter of 100 mm and 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 14 ± 3.4 15.0 20 100 ± 0.0 3.0 25 100 ± 0.0 2.5 30 100 ± 0.0 2.0

As shown in Table 3 and FIG. 5, when the spore suspension of Isaria fumosorosea FG340 isolated from Example 1 of the present invention was tested for insecticidal activity against P. chinensis at different temperature conditions, It was confirmed that the insecticidal rate was about 30 ℃.

As described above, Isaria fumosorosea FG340, a novel microorganism of the present invention, exhibits excellent controlling effect against larvae of Bacillus thuringiensis, and thus the microorganism preparation containing the same is environmentally friendly and environmentally friendly, It can be used effectively as a pesticide for controlling microorganisms.

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 KACC93199P 20140522

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> New microorganism Isaria fumosorosea FG340 and Microbial control          agent for the prevention of Spodoptera exigua larva} <130> P2014-0064 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 571 <212> DNA <213> Isaria sp. <400> 1 tccgtaggtg aacctgcggg ntttttactc cctacccttt gtgacatacc tatcgttgct 60 tcggcggact cgccccggcg tccggacggc cctgcgccgc ccgcgacccg gacccaggcg 120 gccgccggag acccacaaat tctgtttcta tcagtctttc tgaatccgcc gcaaggcaaa 180 acaaatgaat caaaactttc aacaacggat ctcttggttc tggcatcgat gaagaacgca 240 gcgaaatgcg ataagtaatg tgaattgcag aattcagtga atcatcgaat ctttgaacgc 300 acattgcgcc cgccagcatt ctggcgggca tgcctgttcg agcgtcattt caaccctcga 360 caccccttcg ggggagtcgg cgttggggac cggcagcata ccgccggccc cgaaatacag 420 tggcggcccg tccgcggcga cctctgcgta gtactccaac gcgcaccggg aacccgacgc 480 ggccacgccg taaaacaccc aacttctgaa cgttgacctc ggatcaggta ggactacccg 540 ctgaacttaa gcatatcaat aagcggagga a 571

Claims (9)

Comprising a nucleotide sequence of SEQ ID NO: 1 and having a controlling effect against parasitic larvae,
A new microbial isolate , Isaria fumosorosea , FG340 (KACC93199P) strain.
delete The method according to claim 1,
Wherein the strain is used as an active ingredient of any one of an insecticidal microbial pesticide, a leaf side spraying agent,
A new microbial isolate , Isaria fumosorosea , FG340 (KACC93199P) strain.
A culture medium obtained by cultivating the Isaria fumosorosea FG340 (KACC93199P) strain of the novel microorganism of claim 1 or a microorganism medium thereof at 20 to 30 DEG C for 5 to 15 days As an active ingredient,
Microorganism preparation for control of larvae of Bombyx mori.
delete 5. The method of claim 4,
Characterized in that the microorganism medium is a PDA (Potato dextrose agar)
Microorganism preparation for control of larvae of Bombyx mori.
delete Characterized in that the microbial formulation of claims 4 or 6 is spray-applied to a plant in which the larvae are present.
9. The method of claim 8,
Wherein the spraying treatment is carried out at a temperature of 20 to 30 占 폚.

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