KR20230058891A - Metarhizium anisopliae 432 strain for controlling bulb mite and uses thereof - Google Patents

Abstract

The present invention relates to: a Metarhizium anisopliae 432 strain (Accession number: KACC 83053BP), which has mite control ability and excellent spore heat stability; a composition for controlling mites comprising the strain or a culture as an active ingredient; and a mite control method comprising a step of treating the composition on plants, plant seeds, or plant planting soil.

Description

Metarhizium anisopliae 432 strain having root mite control ability and its use {Metarhizium anisopliae 432 strain for controlling bulb mite and uses its}

The present invention relates to a Metarhizium anisopliae 432 strain having root mite control ability and uses thereof.

Various crop protection agents are used to control pests and plant diseases, which are pointed out as the biggest obstacles to plant growth, and research and development for the development of more effective protection agents are being actively conducted worldwide. Crop protection agents for pest control are almost indispensable for growing plants for economic purposes such as food production or for landscaping purposes, and the economic cost accordingly is also very high. Until now, almost all of these crop protection agents have been developed and used using chemicals, and thus, in recent years, crop protection agents based on these chemicals have been followed by various side effects such as harm to humans and animals, as well as destruction of the ecosystem. . In addition, in the case of insecticides using chemical substances, many pests develop resistance, making the control more difficult, and resistance of pests to these chemical pesticides is particularly severe in sucking micropests that occur many generations throughout the year. In order to solve the side effects of these crop protection agents, various eco-friendly control methods have been developed, and one of the methods that is attracting attention is the development and use of microbial insecticides using entomopathogenic microorganisms.

Microorganisms that can be used as materials for microbial insecticides include various microorganisms such as bacteria, viruses, and molds, and among them, insect pathogenic fungi have the longest history. Entomopathogenic fungi are microorganisms that cause disease and death to insects. In the process of invading and proliferating in insect hosts, entomopathogenic fungi produce various extracellular enzymes, toxins, and secondary metabolites to overcome the host insect's immune response or kill the host. It is also being used for competition with other microorganisms. Using these characteristics, research and development on antibacterial and antifungal activities by various metabolites of insect pathogenic fungi have also been actively conducted recently.

The entomopathogenic fungi most often used as microbial insecticide materials are fungi belonging to the genus Beauveria and Metarhizium . Insecticides against a wide variety of pests, such as lepidopteran pests and coleopteran pests, have been developed and marketed using these fungal strains. Since pesticides using these insect pathogenic fungi are developed using living fungi as materials, stability during storage, distribution, and treatment of the developed pesticides is evaluated as very important. Among various environmental factors that can affect the activity of fungi, heat stability and UV stability are considered to be very important factors. Therefore, in recent years, in the development of insecticides using fungi, studies to use these fungal strains with high heat stability and UV stability have been actively conducted.

Root mite ( Rhizoglyphus robini Claparede ) is a pest belonging to the Acarina family Acaridae and is widely distributed in the soil. do damage Root mite causes primary damage by physically attacking the roots in the soil, and then causes secondary damage such as decay and infection by pathogens as fungus or bacteria invade through wounds caused by the injury. In order to control root mites, synthetic pesticides such as bifenthrin, deltamethrin, promecarb, and diazinon are mainly used, but resistance to root mites is acquired and environmental pollution problems, etc. Because of this problem, it is considered a pest that is very difficult to control.

On the other hand, Korean Patent Publication No. 2016-0140560 discloses 'entomopathogenic fungus Metarhizium anisopleae SD4-2 strain and Beauveria bassiana SD15 strain having pest control and antibacterial activity', and Korean Patent Publication No. No. 2021-0012341 discloses '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', but this There is no description about the 'Metharia anisopleae 432 strain having root mite control ability and its use' of the present invention.

The present invention has been derived from the above needs, and the present inventors show high insecticidal resistance to sucking pests such as Rhizoglyphus robini Claparede and control using insect pathogenic fungi, which is a biological control method with little resistance. In order to develop a method, 342 insect pathogenic fungal strains isolated and secured in Korea were selected to select fungal strains with high killing power against root mites, and Metarhizium anisopliae 432 strains were found. The present invention was completed by confirming that the thermal stability of the spores was very high in addition to the high killing power against the root mite.

In order to solve the above problems, the present invention provides a metarhizium anisopliae 432 strain (accession number: KACC 83053BP) having mite control ability and excellent spore heat stability.

In addition, the present invention provides a composition for controlling mites comprising the strain or its culture as an active ingredient.

In addition, the present invention provides a mite control method comprising the step of treating plants, plant seeds or plant planting soil with the composition.

Since the Metarhidium anisopleae 432 strain of the present invention shows a specific insecticidal effect only against root mites, it can be used as a very safe pest control source for humans and animals and the environmental ecosystem, and the strain of the present invention has very high spore heat stability. It is characterized by high stability in distribution and storage during formulation, so it can be used industrially usefully.

1 is a photograph showing dead root mite caterpillars infected with insect pathogenic fungal strains.
Figure 2 is a graph showing the killing power for 7 days for 11 insect pathogenic fungal strains ( Rhizoglyphus robini Claparede). The control was treated only with Tween80, and the vertical line represents the standard error. 111, Metarhizium pemphigi 111; 1101, M. pemphigi 1101; 312, M. pingshaense 312; 441, M. pingshaense 441; 322, M. anisopliae 322; 432, M. anisopliae 432; 481, M. anisopliae 481; 4141, M. anisopliae 4141; 4183, M. anisopliae 4183; 423, M. anisopliae 423; 4312, M. anisopliae 4312.
Figure 3 is a graph showing the spore germination rate after treating the spores of each strain at 45 ℃ to evaluate the thermal stability of 11 insect pathogenic fungal strains.

In order to achieve the object of the present invention, the present invention provides a metarhizium anisopliae 432 strain (accession number: KACC 83053BP) having mite control ability and excellent spore heat stability.

The Metarhizium anisoflea strain of the present invention is an entomopathogenic fungus, and the entomopathogenic fungus causes fungal diseases in invertebrates including insects to kill the host, thereby controlling the density of the host in a natural state. Since insects that can cause disease are limited according to the method, it only damages the target pest and is not toxic to the environment or other animals, and it is reported that the resistance mechanism by insects is hardly expressed, so it is difficult to control pests. A lot of research and development is being done for control.

The present inventors isolated fungi with high killing power against Rhizoglyphus robini Claparede using 342 insect pathogenic fungal strains, and as a result, 11 strains belonging to the genus Metarhizium spp. It was confirmed that it has. In addition, as a result of evaluating the spore thermal stability of the 11 fungal strains in order to increase the usefulness of the marketability as an insecticide, finally, Metatridium anisopliae 432 strain has the highest acaricidal activity and high spore stability It was confirmed that it has. Accordingly, the Metarhidium anisopleae 432 strain was deposited with the Research Institute of Agricultural Biotechnology (KACC) on August 20, 2021 and was given the accession number KACC 83053BP.

The Metarhizium anisoplea 432 strain of the present invention is obtained by identifying the ITS region sequence composed of the nucleotide sequence of SEQ ID NO: 1 and the EF-1α region sequence composed of the nucleotide sequence of SEQ ID NO: 2. strain was identified.

The present invention also provides a composition for controlling mites comprising the strain or its culture as an active ingredient.

The mite control composition of the present invention contains Metarhizium anisopliae 432 strain (Accession Number: KACC 83053BP) or its culture as an active ingredient, which has high acaricidal activity against Rhizoglyphus robini Claparede Thus, there is a mite control effect.

In the composition of the present invention, the 'culture' may include a culture of the Metarhidium anisopleae 432 strain, a concentrate of the culture, or a dried product of the culture, but is not limited thereto.

In addition, the culture may be used in the form of the culture itself containing the strain of the present invention, or may be subjected to centrifugation or filtration to remove the culture medium in the culture medium and recover only the concentrated cells, such Steps can be performed as needed by those skilled in the art. Concentrated cells can be frozen or lyophilized according to conventional methods to preserve their activity.

The culture can be obtained by culturing in large quantities by a conventional microbial culture method, and a medium containing a carbon source, a nitrogen source, vitamins and minerals can be used as the culture medium, and the carbon source includes starch, sucrose, Citrate, maltose, glucose, mannitol, glycerol or xylose may be used, but is not limited thereto.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a particular method.

When using the Metarhidium anisopleae 432 strain or its culture obtained in the step of culturing the strain of the present invention as an additive, the strain or the culture of the strain may be added as it is or used together with other additives, It can be used appropriately according to a conventional method. The mixing amount of the components can be suitably determined depending on the intended use thereof.

The mite control composition according to the present invention may be prepared, for example, in the form of directly sprayable solutions, powders and suspensions or as highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering materials or granules. It may, but is not limited thereto. In addition, the composition for controlling plant diseases may be used by spraying, spraying, spraying, scattering or pouring. The form of use depends on the intended purpose; in all cases, it should be such that the distribution of the composition according to the invention is as fine and uniform as possible.

In addition, the composition for controlling mites of the present invention can be formulated in various forms. The formulation can be prepared, for example, by adding a solvent and/or a carrier. Often, inactive additives and surface-active substances such as emulsifiers or dispersants are mixed into the formulation. Suitable surface-active substances are aromatic sulfonic acids (eg lignosulfonic acids, phenol-sulfonic acids, naphthalene- and dibutylnaphthalenesulfonic acids), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkalis of fatty alcohol sulfates. Metals, alkaline earth metals, ammonium salts, sulfated hexa-, hepta- and octadecanols, salts of fatty alcohol glycol ethers, sulfonates naphthalene and its derivatives, condensates of formaldehyde, naphthalene or naphthalenesulfonic acids, phenols and formaldehyde Condensates, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonylphenols, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohols, fatty alcohol/ethylene oxide condensates , ethoxylated castor oil, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquid or methyl cellulose, but is not limited thereto.

Suitable solid carrier materials are, in principle, all porous, agriculturally acceptable carriers, for example mineral earths (such as silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, boulders, loess, clays, Dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetics), fertilizers (e.g. ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), vegetable products (e.g. grain flour, bark meal, wood meal) and nut shell powder) or cellulose powder, but is not limited thereto. In addition, one type or a mixture of two or more types may be used for the above solid carrier.

The mite control composition of the present invention may be used alone as an active ingredient, or in combination with two or more other acaricide substances.

In addition, the method for preparing the mite control composition may use any method known in the art, and is not particularly limited to a specific method.

The composition for controlling mites of the present invention can be mixed with a spreading agent, a penetrating agent, or a surfactant in order to enhance plant absorption and effectiveness.

The present invention also provides a mite control method comprising the step of treating plants, plant seeds or plant planting soil with the composition.

In the mite control method according to the present invention, the composition is Metarhizium anisopliae 432 strain (Accession Number: KACC 83053BP) or a culture thereof having high acaricidal activity against Rhizoglyphus robini Claparede contains as an active ingredient.

The mite control method of the present invention may be performed by immersing or drenching, ie, spraying, an effective amount of a composition containing the Metarhidium anisopleae 432 strain or a culture thereof as an active ingredient. In the case of the immersion method, an effective amount of the composition may be poured into the soil around the plant or the seed may be immersed in the effective amount of the composition.

The 'effective amount' of the present invention is an amount sufficient to produce a beneficial or desired result, in particular, to control the mite, especially the root mite ( R. robini Claparede), by uniformly adding the Metarhidium anisopleae 432 strain or its culture to water. After diluting to a certain extent, it can be sprayed on plants and farmland using an appropriate spraying device such as a power sprayer.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

spotted mite

Root mite ( R. robini ) was used in experiments while supplying onions and breeding under laboratory conditions at a temperature of 25 ° C, a relative humidity of 80-90%, and a light condition of 12L: 12D.

Entomopathogenic fungal strains

1) Cultivation of fungal strains

342 fungal strains (Shin et al., Biocontrol Science and Technology 2013, 23(3):288-304) isolated from national soils in Korea were used (Table 1).

342 strains of entomopathogenic fungi isolated in Korea mold type number of strains mold type number of strains Acremonium spp. One Metarhizium lepidiotum One Aspergillus spp. 8 Metarhizium pemphigi 16 Beauveria bassiana 110 Mucoromycotina spp. One Beauveria brongniartii 8 Myrothecium spp. 5 Beauveria pseudobassiana 8 Paecilomyces carneus 3 Bionecria spp. 7 Paecilomyces marquandii 5 Chaunopycnis spp. 23 Paraconiothyrium spp. 2 Clonostachys rosea One Phialocephala spp. One Fusarium spp. 2 Pochonia spp. 17 Isaria farinosa 12 Purpureocilium lilacinum 4 Isaria fumosorosea 6 Simplicillium spp. One Isaria spp. 22 Tolypocladium spp. 3 Lecanicillium spp. 8 Verticillium spp. 3 Metarhizium anisopliae 64

The fungal strain was cultured on a potato dextrose agar (PDA) plate at 25°C for 14 days, then the 14-day-old fungal conidia were scraped off the PDA plate and the material was placed in a 0.05% Tween-80 (Difco, USA) solution. Collected by resuspension. The conidial suspension was vigorously stirred and filtered through cotton gauze to remove mycelial debris. The concentration of filtered spores was measured using a hemocytometer.

2) Identification of fungal strains

Identification of the strains confirmed to be pathogenic to root mite was performed by molecular biological identification along with morphological identification through spore shape and color through microscopic observation. For molecular biological identification, genomic DNA extraction of fungi was carried out by using fungal DNA extraction buffer (0.2 M Tris-Cl (pH 7.5), 0.5 M Nacl, 10 nM EDTA (pH 8.0) and 1% ( w/v) SDS) and centrifuged with phenol-chloroform-isoamyl alcohol (25:24:1) to purify DNA. The purified DNA solution was precipitated using cold ethanol, centrifuged, and dissolved in sterile distilled water for use in the experiment.

PCR primers for molecular biological identification of the isolated strain are ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO: 3) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3) for amplification of the internal transcribed spacer (ITS1-5.8S-ITS2) part. ', SEQ ID NO: 4) primers were used, and for amplification of the elongation factor-1 aplha part, 1577F (5'-CARGAYGTBTACAAGATYGGTGG-3', SEQ ID NO: 5) and 2218R (5'-ATGACACCRACRGCRACRGTYTG-3', SEQ ID NO: 6) Primer was used. The PCR reaction was performed using AccuPower PCR PreMix (Bioneer Co., Korea) under conditions of denaturation at 94°C for 5 minutes, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute 35 times in a Thermal Cycler. (TaKaRa, Japan).

After the PCR reaction, the amplification product was analyzed by electrophoresis using a 1.0% agarose gel and purified using a Power Gel Extraction Kit (Dyne Bio Inc., Korea). The purified PCR products were sequenced by requesting direct sequencing to Macrogen (Korea). The base sequence was aligned using Multalin (http://multalin.toulouse.inra.fr/multalin/) and then compared and analyzed with other previously reported fungal sequences using the BLAST search tool.

Bioassay

1) Isolation of root mite pathogenic fungi

In order to isolate fungi pathogenic to root mite, 14-day-old fungal spores were used for bioassay. Spore suspensions with viability greater than 90% were used for bioassays. Spore viability was determined on SDAY+B medium (Saboraud dextrose agar, 1% Yeast extract, 0.05% benomyl, pH 5.6).

342 fungal strains were randomly divided into 4 groups, and spore suspensions of each group of fungal strains were combined into one and used for bioassay. In the bioassay, 50 adult root mites were inoculated with the fungal spore suspension of each group by immersion, and the inoculated root mites were observed every day for 10 days, and each larvae were collected. Root mite larvae were recognized as death caused by fungi only when the occurrence of mold on the epidermis was visually confirmed.

In order to isolate pathogenic fungi from dead root mite caterpillars, fungal spores were collected from caterpillars with a sterilized toothpick, suspended in 0.02% Tween-80 solution, and then SDA-D50 (sabouraud dextrose agar, 100 μg /ml chloramphenicol, 50 μg/ml streptomycin, 50 μg/ml dodine). Thereafter, colonies formed while culturing at 25 ° C. for 7 days in the dark were isolated, inoculated into PDA medium to isolate pure fungi, and after completing morphological and molecular biological identification, they were used in the next experiment.

2) Acaricidal activity of fungi

Fungal strains confirmed to be pathogenic for root mites were cultured in PDA (potato dextrose agar) medium for 2 weeks to induce sporulation, and then the resulting spores were tested using 0.05% Tween-80. A spore suspension was prepared, counted using a hemocytometer, and used in the experiment. Before the bioassay, all spores with a survival rate of 90% or more were used. The spore concentration of each fungal strain was 1 x 10 ⁷ spores/ml and 30 root mites were immersed and inoculated. Larvae were observed and collected every day for 7 days after inoculation, and death caused by fungi was recognized only when fungi were visually confirmed on the surface of the larvae. As a control, only 0.05% Tween-80 solution was treated with root mites.

Thermal stability assay of spores

In order to evaluate the thermal stability of spores of fungal strains, 100 μl (5 x 10 ⁶ conidia/ml) of spore suspension was placed in a 0.5 ml PCR tube and treated at 45° C. for 1 hour or 2 hours using a Thermal Cycler (Takara), respectively. . 20 μl of the heat-treated spore suspension was added to the SDYA+B medium and incubated for 24 hours at 25° C. in the dark, and then the spore germination rate was randomly examined using a 400X phase contrast microscope.

statistical analysis

The evaluation of acaricidal activity and thermal stability was repeated three times, and the result value was tested for significance at a significance level of 0.05 or less using the SPSS statistical program. Data are expressed as mean ± standard error (SE).

Example 1. Isolation of the spotted mite pathogenic fungus

1) Isolation of pathogenic fungi

342 strains of entomopathogenic fungi were grouped into 4 groups, and spore suspensions were prepared for each group to test pathogenicity against root mites. As a result, among 200 root mites, 38 mites were killed by fungi.

As a result of isolating fungi from root mite caterpillars whose death by fungi was confirmed, 57 strains were isolated. For the isolated strains, molecular biological identification of the ITS region and Elongation factor-1 aplha region was performed along with morphological identification based on spore shape and color. As a result, Beauveria bassiana ( Beauveria bassiana ) 29 strains, Metarhizium anisopliae ( Metarhizium anisopliae ) 19 strains, Metarhizium pemphigi ( M. pemphigi ) 5 strains, Metarhizium pingchaens ( M. pingshaense ) Two strains, Akanthomyces attenuatus ( Akanthomyces attenuatus ) One strain and Akanthomyces besicolor ( A. versicolor ) One strain was identified (see Tables 2 to 3).

As a result of re-examination of pathogenicity for Rhizoglyphus robini using 57 identified strains, pathogenicity was reconfirmed only in 15 strains, among which strains with 100% matching molecular biological identification results were judged as the same strain Therefore, only one strain was used in the next experiment. Among the finally isolated strains, Metatridium anisopliae ( M. anisopliae ) 7 strains, Metatridium pemphigi ( M. pemphigi ) 2 strains, Metatridium pingsharens ( M. pingshaense ) 2 strains was selected.

Example 2. Killing power against spotted mites

The viability of 11 strains whose pathogenicity was confirmed for root mite was compared and evaluated (FIG. 1). As a result, the killing power of the 11 strains against root mites varied depending on the strain, and ranged from 38% to 100% at 7 days after treatment. Among them, Metarhizium anisopleae 432 and 4312 strains, and Metarhizium pemphigi 111 strains showed 100% killing power. Among them, Metarhizium anisoplea 432 and Metarhizium pempygi 111 strains showed high killing power of 99% or more even 5 days after treatment.

Example 3. Thermal stability of fungal spores

The thermal stability of spores of 11 strains whose killing power was confirmed against root mite was compared and evaluated. As a result of heat treatment at 45 ° C. for 1 hour, 8 strains, including 7 strains of Metarhidium anisoplea and 1 strain of Metarhidium pingchaens, showed a spore germination rate of 97% or higher and showed high stability. As a result of heat treatment at 45 ° C. for 2 hours, most of the strains showed low heat stability, but four strains of Metarhidium anisopleae 322, 432, 481, and 4312 still showed a spore germination rate of 60% or more. In particular, the Metarhidium anisopleae 432 strain showed the highest heat stability by showing a spore germination rate of about 81%.

Summarizing the above results, it was confirmed that the M. anisopliae 432 strain is the most useful strain for root mite control because it has the highest viability and thermal stability of spores against root mites. The Metarhizium Anisoflea 432 strain was deposited with the Korean Agricultural Culture Collection (KACC) on August 20, 2021, and was given the accession number KACC 83053BP from the depository institution.

Agricultural Biotechnology Research Institute KACC83053BP 20210820

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Claims (5)

Metarhizium anisopliae 432 strain (accession number: KACC 83053BP) with mite control ability and excellent spore heat stability. The method of claim 1, wherein the mite is a root mite ( Rhizoglyphus robini Claparede), characterized in that Metarhizium anisopleae 432 strain. A composition for controlling mites comprising the strain or culture thereof of claim 1 as an active ingredient. A method for controlling mites, comprising the step of treating plants, plant seeds or plant planting soil with the composition of claim 3. The method of claim 4, wherein the plant is garlic, green onion, chives, onion, leek, potato, sweet potato, lily, tulip, gladiolus, daffodil, caladium, iris, hyacinth, crocus, dahlia or tamarisk, characterized in that, How to get rid of mites.

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