KR102145620B1 - Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media - Google Patents

Abstract

The present invention relates to a microbial preparation for controlling cabbage moth larvae containing Isaria fumosorosea FT337 strain ( Isaria fumosorosea FT337, KACC 93317P) strain or the same, and controlling the larvae of cabbage moth, which is a pest, to produce excellent and safe agricultural products. Can be produced. In addition, the development of pest control technology that can be used in eco-friendly cultivation areas is effective in reducing the control cost and control power of farmers, and it is expected to increase farm household income by increasing the quantity and marketability of cultivated crops.

Description

The microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media}

The present invention relates to a microbial preparation for controlling larvae of Isaria fumosorosea FT337 strain ( Isaria fumosorosea FT337, KACC 93317P) or Chinese cabbage moth containing the same ( Plutella xylostella ) larvae.

Insect pathogenic fungi are mostly incomplete fungi (Hyphomycetes) and are harmless to humans, animals and plants, but are specifically pathogenic to target pests, and play a role in controlling the density of hosts in nature. Unlike insect pathogenic bacteria or viruses that are infected through host feeding, the mechanism of invasion of insect pathogenic fungi into the insect host begins with the attachment of fungal spores to the host epidermis, penetrates the epidermis through physical and chemical reactions, and invades the insect. It blocks the immune function of and kills the host by secreting toxic substances. When these insect pathogenic fungi are used for pest control, there is no direct or indirect hazard such as accumulation of dangerous substances such as humans, higher organisms, fish, grains, etc., so stability is high, and selectivity to selectively remove only the intended pests In addition, there is an advantage in that the persistence of the insecticidal effect is maintained, so that repeated application of the microbial agent can be minimized.

More than 750 types of insect pathogenic fungi are known to date, and among them, Beauveria bassiana , Metarhizium anisopliae , and Nomuraea rileyi are representative microbial insecticides. It has been developed and is used for controlling various pests.

Plutella xylostella is a pest belonging to the Lepidoptera family, Yponomeutoidea , which causes the most damage in various forms by binge eating the leaves and veins of cruciferous crops, including Chinese cabbage, among plant-grown leaf vegetables. It is widely distributed and its damage has been reported in more than 80 countries, causing serious economic damage to farms in the facility cultivation area.

Until recently, the control of Chinese cabbage moths mainly depended on chemical pesticides, and thus, more than 1 billion dollars a year was spent on control. The continued use of chemical pesticides, along with enormous control costs, has led to the development of resistance to the cabbage moth. Since it was reported that the Chinese cabbage moth had resistance to DDT, resistance to teflubenzuron and chlorfluazuron of insect growth regulators such as organophosphorus, carbamate, synthetic pyrethrod, etc. Such resistance increased the concentration and frequency of application of chemical pesticides, and caused bioaccumulation to adversely affect human life.

Meanwhile, the present inventors completed the present invention by confirming that a new strain of Isaria fumo-sorosea effectively controls Chinese cabbage moth larvae while conducting research on insect pathogenic microorganisms.

Korean Patent Registration No. 10-1680639 (Name of the invention: New microbial Isaria fumosorose FG340 and microbial preparation for controlling larvae of Pabam moth containing it, Applicant: Republic of Korea, registration date: 2016.11.23.)

Xiao-ge Nian, Yu-rong He, Li-hua Lu, Rui Zhao, Evaluation of the time-concentration-mortality responses of Plutella xylostella larvae to the interaction of Isaria fumosorosea with the insecticides, beta-cypermethrin and Bacillus thuringiensis, Pest Management Science, 2015, 71(2):216-224.

An object of the present invention is to provide a microbial preparation for controlling pests of Isaria fumosorosea FT337 strain ( Isaria fumosorosea FT337, KACC 93317P) or Chinese cabbage moth ( Plutella xylostella ) larvae using the same.

The present invention relates to a novel insect pathogenic microorganism Isaria fumosorosea FT337 ( Isaria fumosorosea FT337, KACC 93317P) strain.

The strain is characterized in that it has a control effect against the cabbage moth larvae. In addition, the strain is characterized in that it contains the nucleotide sequence of the 18S rRNA gene represented by SEQ ID NO: 1.

The present invention also provides a microbial preparation for controlling Chinese cabbage moth larvae containing the bacteria of the Isaria fumosorosea FT337 ( Isaria fumosorosea FT337, KACC 93317P) strain or a culture solution thereof as an active ingredient.

The present invention relates to an insecticidal microbial pesticide, foliar spray or drench spray comprising the microbial preparation.

In addition, the present invention is a step of diluting the microbial preparation so that the concentration of spores is 1×10 ⁴ to 1×10 ⁷ per 1 ml to obtain a dilution solution; And spraying the diluted solution onto the plant to be controlled. It provides a method for controlling Chinese cabbage moth larvae, comprising:

Hereinafter, the present invention will be described in detail.

The present invention relates to a new insect pathogenic microorganism Isaria fumo-sorosea FT337 strain having a control effect against Chinese cabbage moth larvae, wherein the strain or a culture solution thereof, and a microbial preparation containing them are used as a control agent for Chinese cabbage moth larvae. I can.

The culture medium may preferably be a culture medium in which the Isaria fumo-sorosea FT337 strain is cultured in a microbial medium. The microbial medium is PDA (potato dextrose agar), PDB (potato dextrose broth), SDA (sabouraud dextrose agar), SDAY (sabouraud dextrose agar with yeast extract), AD in which Isaria fumo sorosea FT337 strain can grow. (Modified adamek's media) A medium such as a liquid medium or an AD solid medium may be used, and a PDA medium may be preferably used.

The PDA medium may be prepared by mixing 4g/L potato starch, 20g/L dextrose, and 15g/L agar, but is not limited thereto. PDB medium is a liquid medium prepared without mixing agar in PDA. AD liquid medium can be prepared by mixing 20g/L glucose, 20g/L yeast extract, and 30g/L corn steep liquor by modifying the basic composition of Adameks media. AD solid medium can be prepared by mixing 15g / L agar in the AD liquid medium. SDA can be prepared by mixing 40g/L dextrose, 10g/L mycological peptone, and 15g/L agar. SDAY can be prepared by mixing 10g/L yeast extract with SDA.

In addition, in the present invention, Isaiah fumo-sorosea FT337 strain or a culture solution thereof for effectively controlling Chinese cabbage moth larvae is diluted and preferably 1×10 ⁴ to 1×10 ⁷ conidia/ml.

The germination condition of the Isaria fumo-sorosea FT337 strain of the present invention is preferably 24 hours at 15 to 35°C, the pH is preferably 6.8 to 7.2, and more preferably pH 7.0.

The culture medium of the Isaria fumo-sorosea FT337 strain of the present invention is characterized in that it is a culture medium obtained by culturing the strain at 15 to 30° C. for 5 to 15 days. The culture conditions of the strain are suitable for 5 to 15 days at 15 to 30 ℃, more preferably it is good to culture for 14 days at 20 to 30 ℃. At this time, when the culture temperature is less than 15°C, the culture temperature is too low to slow the growth rate of the strain, and when the culture temperature exceeds 30°C, the culture temperature is too high to limit the growth of the strain. In addition, when culturing the strain, the pH is preferably pH 6.8 to 7.2, more preferably pH 7.0.

On the other hand, it is preferable that the control conditions for the cabbage moth larvae of the Isaria fumo-sorosea FT337 strain of the present invention are 15-35°C at 44-98% relative humidity. In the culture and control conditions, the mycelial growth of the strain may vary depending on temperature or humidity.

In the present invention, in order to control the Chinese cabbage moth larvae, a microbial preparation containing the Isaria fumo-sorosea FT337 strain or culture medium is spray-treated on a plant in which the Chinese cabbage moth larvae are present. At this time, preferably, diluting the microbial agent so that the spore concentration is 1×10 ⁴ to 1×10 ⁷ per 1 ml to obtain a dilution solution; And, spraying the diluted solution on the plant to be controlled; it can control the cabbage moth larvae. The spray treatment may be performed under a temperature of 15 ~ 30 ℃.

Plants to which the strain of the present invention and its culture solution, and the microbial agent containing the same can be applied are crops such as Chinese cabbage ( Bassica rapa ), cabbage ( Bassica oleracea ), radish ( Raphanus sativus ), and rapeseed ( Brassica napus ), The plant is not limited thereto.

The microbial preparation of the present invention can be used for insecticidal microbial pesticides, foliar sprays, drench sprays, etc., since the active strain spores have excellent germination rate and insecticidal power during the survival period and the survival period.

The present invention relates to a microbial preparation for controlling cabbage moth larvae containing Isaria fumosorosea FT337 strain ( Isaria fumosorosea FT337, KACC 93317P) or the same, and to produce excellent and safe agricultural products by controlling the larvae of cabbage moth, which is a pest. I can. In addition, the development of pest control technology that can be used in eco-friendly cultivation areas is effective in reducing the control cost and control power of farmers, and it is expected to increase farm household income by increasing the quantity and marketability of cultivated crops.

1 is a photograph of Isaiah Fumo Sorosea FT337 strain of the present invention observed with a microscope.
Figure 2 is a photograph of the larvae infected with microorganisms after inoculation of Isaria fumo-sorosea FT337 strain to the cabbage moth larvae.
Figure 3 is a graph showing the indoor insecticidal rate by temperature of Isaria fumo-sorosea FT337 strain against the cabbage moth larvae.
4 is a graph showing the insecticidal rate of Chinese cabbage moth larvae according to the concentration of spore spray treatment of Isaria Fumo Sorosea FT337 strain.
Figure 5 is a photograph of the state of cabbage seedlings according to the spore spray treatment concentration of Isaria Fumo Sorosea FT337 strain.
6 is a graph showing the insecticidal rate of Chinese cabbage moth larvae according to the concentration of spore spray treatment of Isaria fumo-sorosea FT337 strain during Chinese cabbage packaging cultivation.
7 is a photograph of the state of cabbage according to the concentration of spore spray treatment of Isaria fumo-sorosea FT337 strain during packaging cultivation of cabbage.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

<Example 1. Isolation and identification of Isaria fumo-sorosea FT337 strain from soil>

Example 1-1. Isolation of strains

Among the insect pathogenic fungi collected from the soil using the insect-bait method (bait insects: brown larvae), strains having pathogenicity to the Chinese cabbage moth larvae were selected. In more detail, 500 ㎖ of soil was collected from 4 locations of 20㎡ of uncultivated land in Damyang, placed in a zipper bag, and mixed well. The soil brought to the laboratory was placed on a 2mm sieve, 20ml was placed in a plastic bucket, and 10 brown larvae of 1.5cm size were added. After culturing at 25° C. for 14 days, an insect pathogenic strain was isolated from the surface of the infected brown mealworm larva.

Example 1-2. Identification of strains

Among the fungi purely isolated from the surface of the larvae of Brown by the method of Example 1-1, the strain with the greatest antibacterial activity was identified through microscopic observation, and the DNA sequence of the 18S rRNA gene of this strain was determined. Extraction and ITS analysis were performed. As a result, it was finally confirmed that the strain is Isaria fumosorosea and 18S rRNA match, and has the DNA nucleotide sequence of the 18S rRNA gene of SEQ ID NO: 1 below, Isaria fumosorosea Named FT337 ( Isaria fumosorosea FT337) and deposited with the National Academy of Agricultural Sciences (KACC 93317P).

DNA nucleotide sequence of 18S rRNA gene-SEQ ID NO: 1:

Example 2. Preparation of Spore Suspension of Isaria Fumo Sorosea FT337 Strain

Purely isolated conidia of Isaria fumosorosea FT337 strain ( Isaria fumosorosea FT337) were inoculated in PDA medium, cultured at 28°C for 14 days, and the spores were collected and suspended in 0.01% tween 80 solution, followed by a hemocytometer ( hemocytometer) was used to prepare a spore suspension having a spore concentration of 1×10 ⁴ , 1×10 ⁵ , 1×10 ⁶ , and 1×10 ⁷ conidia/ml.

Experimental Example 1. Confirmation of germination rate and mycelial growth by temperature of Isaria fumo-sorosea FT337 strain

Experimental Example 1-1. Germination rate of Isaria fumo-sorosea FT337 strain by temperature

In order to check the germination rate of the selected Isaria fumosorosea FT337 strain by temperature, the suspension of the strain (1×10 ⁶ conidia/ml) was spread on a water agar medium (water agar) with 100 µl each and 15 After incubation at ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ for 24 hours, Lactophenol cotton blue solution (Lactophenol cotton blue solution) was dropped to investigate the germination rate of spores.

Isolates Temperature(℃) 15℃ 20℃ 25℃ 30℃ 35℃ FT337
(24 hours germination rate, %) 61.9±3.1 94.9±1.1 96.3±0.5 94.6±0.7 45.7±4.4

As shown in Table 1, it was confirmed that the Isaria fumo-sorosea FT337 strain of the present invention had good spore germination rates at 15 to 35°C, particularly at 20 to 30°C.

Experimental Example 1-2. Mycelial Growth of Isaria Fumosorosea FT337 Strains by Temperature

The selected cells of Isaria fumosorosea FT337 ( Isaria fumosorosea FT337) were placed in the center of PDA medium and cultured at 15°C, 20°C, 25°C, 30°C and 35°C for 14 days to investigate the growth of the mycelium.

Isolates Temperature(℃) 15℃ 20℃ 25℃ 30℃ 35℃ FT337
(Mycelial growth for 14 days, mm) 13.1±0.5 10.9±0.4 10.6±1.8 15.1±0.3 0.0±0.0

As shown in Table 2, the Isaria fumo-sorosea FT337 strain of the present invention has good growth conditions at 15 to 30°C, and it was confirmed that it grows best at 30°C.

In addition, it was confirmed that the appropriate pH for the growth of the Isaria fumo-sorosea FT337 strain was 6.8 to 7.2, and most appropriately pH 7.0.

Experimental Example 2. Control effect of Isaiah fumo-sorosea FT337 strain against temperature-dependent Chinese cabbage moth larvae

A spore suspension (1×10 ⁷ conidia/ml) of the Isaria fumosorosea FT337 strain prepared in Example 2 was prepared. In order to test the insecticidal power of cabbage moth larvae by temperature, spray the prepared spore suspension to the front and back of 90 mm diameter cabbage leaves into which the cabbage moth larvae (3 in., 5 days after hatching) were injected, and then 95% or more. They were reared under different temperature conditions of 15℃, 20℃, 25℃, 30℃, and 35℃ while maintaining the relative humidity. Investigation of insecticidal power was carried out every day until the 6th day after spray treatment, and the results were shown in FIG. 3 by repeating each of 10 animals 5 times.

Referring to FIG. 3, in cabbage leaves spray-treated with the spore suspension of the strain, cabbage moth larvae are all insecticides on the 6th day despite different temperature conditions of 15°C, 20°C, 25°C, 30°C, and 35°C. The rate was 100%. In particular, in the 25°C temperature condition, the insecticide rate was 100% on the 5th day, so it could be determined that the control effect was the best. Through this, it was confirmed that the Isaria fumo-sorosea FT337 strain of the present invention has an excellent control effect against Chinese cabbage moth larvae regardless of temperature.

Experimental Example 3. Control effect of Isaiah fumo sorosea FT337 strain against spore spray treatment concentration against Chinese cabbage moth larvae

A spore suspension (1×10 ⁴ , 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ conidia/ml) of the Isaria fumo-sorosea FT337 strain prepared in Example 2 was prepared.

Next, the prepared spore suspension (1×10 ⁴ , 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ conidia/ml) was added to each of 10 Chinese cabbage moth larvae (3 instars, 5 days after hatching). The seedlings were sprayed with 30 ml each, and then put in an acrylic cage and reared at a temperature of 25°C while maintaining a relative humidity of 80% or more, preferably 95% or more. Investigation of insecticidal power was carried out every day until the 5th day after spraying treatment, and each of 10 animals was repeated twice, and the results are shown in a graph in FIG. 4 and the state of cabbage seedlings on the 5th day is shown in FIG.

Referring to FIG. 4, the spore suspension of the novel microorganism of the present invention, Isaria fumo-sorosea FT337 strain, exhibited the highest insecticidal rate when sprayed at a concentration of 1×10 ⁷ conidia/ml, and was about 80% on the 5th day. It was confirmed that the spore suspension of the new strain had excellent insecticidal effect at a concentration of 1×10 ⁷ conidia/ml.

In addition, referring to FIG. 5, it can be seen that the spore suspension of the novel strain of the present invention is spray-treated at a concentration of 1×10 ⁷ conidia/ml in the cabbage seedling, which has the most excellent control effect against the cabbage moth larvae.

Experimental Example 4. Controlling effect of Isaria fumo-sorosea FT337 strain against Chinese cabbage moth larvae during packaging cultivation

A spore suspension (1×10 ⁶ , 1×10 ⁷ conidia/ml) of the Isaria fumo sorosea FT337 strain prepared in Example 2 was prepared. In order to check the control effect on Chinese cabbage moth larvae during packaging cultivation, 20 Chinese cabbage moth larvae (3 years old, 5 days after hatching) were added to the prepared spore suspension (1×10 ⁶ , 1×10 ⁷ conidia/ml). The injected cabbage was sprayed with 30 ml each, and the insecticidal waters of the cabbage moth larvae were investigated for 6 days. In the experiment, 20 weeks of cabbage seedlings were used per treatment, and the temperature, humidity, and light intensity of the package were investigated every 30 minutes. The experimental results are shown in a graph in FIG. 6 and the state of packaged cabbage on the 6th day is shown in FIG. 7.

* Packaging environment

-Average temperature 23.7℃ (maximum 35.0℃, lowest 16.5℃)

-Humidity 82.3% (maximum 97.4%, lowest 44.5%)

-Light intensity 179.4 lm/ft ² (maximum 1619.0 lm/ft ² , minimum 0.4 lm/ft ² )

Referring to FIG. 6, when the spore suspension (1×10 ⁶ , 1×10 ⁷ conidia/ml) of the strain was treated on the packaged cabbage, the insecticidal rate for the cabbage moth larvae was about 80 from the second day after treatment. % Or more, and from the 4th day, it showed an insecticidal rate of 90% or more regardless of the concentration of the treated spore suspension. In particular, after 5 days of treatment, the insecticidal rate of 92.6±2.5% was shown, indicating that the Isaria fumo-sorosea FT337 strain had excellent control effects against the Chinese cabbage moth larvae.

In addition, referring to FIG. 7, the packaged cabbage treated with the spore suspension (1×10 ⁶ , 1×10 ⁷ conidia/ml) of the strain has little damage to the cabbage moth larvae compared to the packaged cabbage of the untreated group. I could confirm.

National Academy of Agricultural Sciences KACC93317P 20181112

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microbial agent for control of Plutella xylostella larva using Isaria fumosorosea FT337 and its culture media <130> 0364 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 523 <212> DNA <213> Unknown <220> <223> Isaria fumosorosea <400> 1 accctttgtg aacataccta tcgttgcttc ggcggactcg ccccggcgtc cggacggccc 60 tgcgccgccc gcgacccgga cccaggcggc cgccggagac ccacaaattc tgtttctatc 120 agtctttctg aatccgccgc aaggcaaaac aaatgaatca aaactttcaa caacggatct 180 cttggttctg gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa 240 ttcagtgaat catcgaatct ttgaacgcac attgcgcccg ccagcattct ggcgggcatg 300 cctgttcgag cgtcatttca accctcgaca ccccttcggg ggagtcggcg ttggggaccg 360 gcagcatacc gccggccccg aaatacagtg gcggcccgtc cgcggcgacc tctgcgtagt 420 actccaacgc gcaccgggaa cccgacgcgg ccacgccgta aaacacccaa cttctgaacg 480 ttgacctcgg atcaggtagg actacccgct gaacttaagc ata 523

Claims (5)

Chinese cabbage moth ( Plutella xylostella ) larvae control microorganism Isaria fumosorosea FT337 ( Isaria fumosorosea FT337, KACC 93317P) strain. Chinese cabbage moth ( Plutella xylostella ) larvae control microorganism of claim 1 Isaria fumosorosea FT337 ( Isaria fumosorosea FT337, KACC 93317P), characterized in that it contains the cells of the strain or a culture medium thereof as an active ingredient ( Plutella xylostella ) Microbial preparation for controlling larvae. An insecticidal microbial pesticide comprising the microbial preparation of claim 2. A foliar spray agent comprising the microbial agent of claim 2. Diluting the microbial preparation of claim 2 so that the concentration of spores is 1×10 ⁴ to 1×10 ⁷ per 1 ml to obtain a diluted solution; And,
Spraying the diluted solution onto the plant to be controlled;
Chinese cabbage moth ( Plutella xylostella ), characterized in that it comprises a method for controlling larvae.

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