WO2024085482A1 - Novel metarhizium pinghaense 15r strain with insecticidal and bactericidal activity and use thereof - Google Patents

Abstract

The present invention relates to a novel Metarhizium pinghaense 15R strain with insecticidal and bactericidal activity, and a use thereof. Specifically, the present invention relates to a novel Metarhizium pinghaense 15R strain that exhibits control activity against both plant pests and plant pathogens, a composition containing the Metarhizium pinghaense 15R strain as an active ingredient for the control of both harmful insects and plant diseases, and a method for simultaneously controlling harmful insects and plant diseases using the Metarhizium pinghaense 15R strain. The Metarhizium pinghaense 15R strain according to the present invention is a pest control source safe for humans and animals and the ecological environment and can simultaneously control pests harmful to crops and various plant pathogens, thus finding advantageous applications as a new biological agent.

Description

Novel Metarhizium pinghans 15R strain with insecticidal and bactericidal activity and uses thereof

The present invention relates to a novel strain of the fungus Metarhizium pinghans 15R, which has bactericidal activity against plant pests and plant disease pathogens, and its use.

Various crop protection agents are being used to control pests and plant diseases, which are pointed out as the biggest obstacles to plant growth, and research to develop more effective protection agents is actively underway around the world. Crop protection agents for controlling pests and plant diseases are essential not only for economic purposes such as food production but also for landscaping purposes, and the resulting economic costs are also very high. To date, most of these crop protection agents have been developed using chemicals, and as a result, crop protection agents based on these chemicals have recently caused various side effects, such as risk to human populations and destruction of the ecosystem. To solve the side effects of these crop protection agents, various eco-friendly control methods are being developed, and one of them that is attracting attention is the development and use of microbial pesticides using entomopathogenic microorganisms.

There are various microorganisms that can be used as materials for microbial pesticides, including bacteria, viruses, and molds, and among them, entomopathogenic fungi have the longest history. Entomopathogenic fungi are microorganisms that cause disease and death in insects. During the process of invading and proliferating in insect hosts, they produce various extracellular enzymes, toxins, and secondary metabolites to overcome the host insect's immune response or kill the host. It can also be used to compete with other microorganisms.

Meanwhile, in order to effectively control pests and diseases that damage plants, the use of crop protection agents for each individual control is essential. However, crop protection agents that can effectively control plant pathogens as well as various pests harmful to plants at the same time are essential. Development is insufficient.

Accordingly, the present inventors isolated and identified Metarhizium pinghaense 15R, a novel entomopathogenic fungal strain, from the peach aphid, and the spores and metabolites of the strain were found to be effective against the peach aphid, cotton aphid, and borderless aphid. As it was confirmed that it not only has an insecticidal effect against plant pests such as spotted mites and root-knot nematodes, but also has high antibacterial activity against various plant pathogens, it can be used as a new crop protection agent for simultaneous control of pests and plant pathogens. By confirming this, the present invention was completed.

Therefore, the purpose of the present invention is to provide Metarhizium pinghaense 15R strain (Accession Number: KACC83065BP), which simultaneously has control activity against novel pests and plant pathogens.

Another object of the present invention is to use Metarhizium pinghaense 15R strain (accession number: KACC83065BP), its spores, the strain culture, the concentrate of the strain culture, or the dried product of the strain culture as an active ingredient. To provide a composition for simultaneous control of pests and plant diseases, including:

Another object of the present invention is to provide a method for simultaneously controlling pests and plant diseases, comprising spraying the composition for simultaneously controlling pests and plant diseases of the present invention in areas where pests and plant diseases have grown.

In order to achieve the object of the present invention as described above, the present invention provides Metarhizium pinghaense 15R strain (Accession Number: KACC83065BP), which has both pest control and plant pathogenic activity.

In one embodiment of the present invention, the pests may be peach aphids, cotton aphids, borderless aphids, spotted mites, or root-knot nematodes.

In one embodiment of the present invention, the control activity against plant pathogens may be antifungal or antibacterial activity against fungi or bacteria that cause plant diseases.

In one embodiment of the present invention, the fungus may be Botrytis cinerea T3-4, Colletotrichum acutatum 15AS32, or Colletotrichum fructicola CGF160401. there is.

In one embodiment of the present invention, the bacterium may be Clavibacter michiganensis (C. michiganensis subsp. michiganensis).

In addition, the present invention includes Metarhizium pinghaense 15R strain (accession number: KACC83065BP), its spores, a culture of the strain, a concentrate of the culture of the strain, or a dried product of the culture of the strain as an active ingredient. , provides a composition for simultaneous control of pests and plant diseases.

In one embodiment of the present invention, the pests may be peach aphids, cotton aphids, borderless aphids, spotted mites, or root-knot nematodes.

In one embodiment of the present invention, the plant disease is Botrytis cinerea T3-4, Colletotrichum acutatum 15AS32, Colletotrichum fructicola CGF160401, or It may be caused by Clavibacter michiganensis (C. michiganensis subsp. michiganensis).

Furthermore, the present invention provides a method for simultaneously controlling pests and plant diseases, comprising the step of spraying the composition for simultaneous control of pests and plant diseases of the present invention to areas where pests and plant diseases multiply.

In one embodiment of the present invention, the area may be soil or crops.

In one embodiment of the present invention, the crops include eggplant, pepper, tomato, strawberry, cabbage, pear, apple, cucumber, rose, cherry, money tree, persimmon, plum, chinensis tree, tangerine, potato, sweet potato, tobacco, and cotton. , it may be aperitia, oleander, harpoon tree, wolfberry tree, paulownia tree, apricot tree, plum tree or coriander tree.

The Metarhizium pinghans 15R strain of the present invention (Accession number: KACC83065BP) has excellent insecticidal effects against peach aphids, cotton aphids, borderless aphids, spotted mites, and root-knot nematodes, and also has bactericidal activity against various plant pathogens. There is a characteristic. In addition, the Metarhizium pinghans 15R strain of the present invention is a pest control source that is safe for human livestock and the environmental ecosystem, and can simultaneously control pests harmful to crops and various plant pathogens, so it can be usefully used as a new biological agent, improving the economic efficiency of farms. can contribute greatly to

Figure 1 compares the results of evaluating the insecticidal power against peach aphids for spores of the Metarhizium pinkhans strain 15R, germinating spores of the strain, a culture solution from which cells were removed from the strain culture, a mixture of spores and the culture solution, and a mixture of germinating spores and the culture solution. This is one graph.

Figure 2 is a graph confirming the insecticidal effect on pests of the liquid culture of the Metarhizium pinkhans 15R strain, and comparing the results of the evaluation of insecticidal power against peach aphid, cotton aphid, and borderless aphid.

Figure 3 is a graph comparing the results of evaluating the insecticidal power of the liquid culture of the Metarhizium pinghans 15R strain against spotted mites.

Figure 4 is a graph comparing the results of evaluating the insecticidal power against root-knot nematodes of the liquid culture of the Metarhizium pinghans 15R strain.

Figure 5 is a graph comparing the results of antifungal activity against Botrytis cinerea by liquid culture of the Metarhizium pinkhans 15R strain.

Figure 6 is a graph showing the results of antifungal activity against Colletotrichum acutatum (A) and Colletotrichum fruticola (B) by liquid culture of the Metarhizium pinkhans 15R strain.

Figure 7 is a graph showing the results of antibacterial activity against Clavibacter misiganensis by liquid culture of Metarhizium fingerhans 15R strain.

The present invention is characterized by providing a novel Metarhizium pinghaense 15R strain (Accession Number: KACC83065BP) that simultaneously has control activity against plant pests and plant pathogens.

As used herein, the term “control” refers to the ability of a substance to significantly increase the mortality rate or inhibit the growth rate of crop pests or plant pathogens.

Representative obstacles to growing plants include various pests and plant diseases, and pesticides, fungicides, fungicides, and virus agents are used to solve these problems. To control these various pests and plant diseases, each pest control method is required. The reality is that pesticides must be used, and there are almost no effective pesticides that can control them simultaneously.

Accordingly, while the present inventors were researching to develop a microbial agent capable of controlling more effective pests and plant diseases at the same time, the novel Metarhizium pinghaense 15R strain, isolated during the breeding process of peach aphids, was used to treat pests. It was confirmed that it has excellent killing power and high antibacterial activity against various plant pathogens.

Therefore, the present inventors deposited the Metarhizium pinghaense 15R strain isolated and identified in the present invention to the Microbial Bank (KACC) of the National Academy of Agricultural Sciences of the Rural Development Administration on April 15, 2022, and received the deposit number KACC83065BP.

Furthermore, the present inventors conducted an experiment to confirm the acaricidal activity of the Metarhizium pinghans 15R strain of the present invention against pests, including the Metarhizium pinghans 15R strain, budding spores obtained by culturing the strain, and its culture solution (culture solution containing metabolites secreted from the strain from which spores and hyphae were removed) was analyzed for its insecticidal power against pests.

As a result, it was confirmed that the spores, budding spores, and culture medium of the Metarhizium pinghaense 15R strain of the present invention all have high insecticidal activity against peach aphids, and in particular, it was confirmed that the insecticidal effect of the culture medium was very excellent. did.

Furthermore, the present inventors performed a pot test on the insecticidal activity of the culture medium of the Metarhizium pinghans 15R strain of the present invention, and as a result, the culture medium of the strain was found to be about 80% effective against cotton aphids and borderless aphids in addition to peach aphids. It showed high insecticidal activity, and both adults and nymphs of spotted mites showed a high killing power of over 97% 2 days after treatment. High insecticidal activity against root-knot nematodes was confirmed at about 94% on the 1st day after treatment with the strain culture and about 99% on the 5th day after treatment.

Therefore, the present inventors found that the spores, budding spores, and culture medium of the Metarhizium pinghaense 15R strain can all be usefully used as a control agent to control various pests.

In addition, in order to confirm the antifungal or antibacterial activity of the strain of the present invention, the present inventors used strawberry anthrax pathogen Colletotrichum fructicola, pepper anthrax pathogen Colletotrichum acutatum, The antibacterial activity was analyzed against Botrytis cinerea, a gray mold pathogen, and Clavibacter michiganensis subsp. michiganensis, a plant ulcer pathogen. As a result, the strain of the present invention was found to be the plant pathogen. It was confirmed that it had excellent control activity against field.

Therefore, the present invention can provide Metarhizium pinghaense 15R strain (accession number KACC83065BP), which simultaneously has control activity against pests and plant pathogens.

The pests that can be controlled by the strain according to the present invention are not limited thereto, but may be peach aphid, cotton aphid, borderless aphid, spotted mite, or root-knot nematode.

In addition, the strain according to the present invention has the characteristic of having both antifungal or antibacterial activity against fungi or bacteria that cause plant diseases.

In the present invention, the type of fungus is not limited thereto, but includes Botrytis cinerea T3-4, Colletotrichum acutatum 15AS32, or Colletotrichum fructicola CGF160401. It can be.

In the present invention, the bacterium may be Clavibacter michiganensis (C. michiganensis subsp. michiganensis).

In addition, the present invention can provide a composition for simultaneous control of pests and plant diseases comprising the strain of the present invention, its spores, a culture of the strain, a concentrate of the culture of the strain, or a dried product of the culture of the strain as an active ingredient. .

The culture can be obtained by culturing in large quantities using a conventional microorganism culture method. A culture medium containing a carbon source, a nitrogen source, vitamins, and minerals can be used. The carbon source may include starch, sucrose, Citrate, maltose, glucose, mannitol, glycerol, or xylose can be used.

In addition, the culture can be used in the form of the culture itself containing the strain of the present invention, or the cells can be removed and only the culture solution can be used, or the culture medium in the culture solution can be removed and only the concentrated cells can be recovered. It may be subjected to centrifugation or filtration, and these steps can be performed by those skilled in the art as needed. Culture medium or concentrated bacterial cells can be frozen or lyophilized according to conventional methods to preserve their activity.

For the purpose of producing a stable formulation suitable for actual use, the composition of the present invention is supplemented with water, carrier, diluent, in addition to the active ingredients (strain, strain culture, strain culture, concentrate of strain culture, or dried product of strain culture) described above. It may further contain one or more substances such as surfactants, drug efficacy enhancers, inorganic salts, auxiliaries, binders, and extenders.

The surfactant is an amphiphilic substance that has both hydrophilic and lipophilic molecular groups in the molecule, and is understood to have the characteristics of excellent cleaning power, dispersing power, emulsifying power, solubilizing power, wetting power, sterilizing power, foaming power, and penetration power. , It can be understood that the active ingredient (strain or culture thereof) in the mosquito insecticidal composition according to the present invention acts to hydrate, suspend, and disperse so that the medicinal effect is effectively expressed.

The surfactant includes sodium salts of sulfonates such as alkylbenzenesulfonate, alkylnaphthalenesulfonate, dialkylsulfosuccinate, lignin sulfonate, alkylnaphthalenesulfonate formalin condensate, polyoxyalkylenealkylphenylsulfonate, or Sodium salt of sulfate such as calcium salt, alkyl sulfate, polyoxyalkylene alkyl sulfate, polyoxyalkylene alkylphenyl sulfate or sodium salt of succinate such as calcium salt, naphthalene sulfosuccinate, polyoxyalkylene succinate, or Anionic surfactants such as calcium salts, nonionic surfactants such as ethoxylated alkyl ethers, polyoxyalkylene alkylphenyl polymers, and polyalcohols may be used alone or in a mixture of two or more types, all of which are listed by way of example. It will be easily understood by those skilled in the art that surfactants other than these may be used.

The extender is a material that can be used with a surfactant to adsorb and disperse the surfactant, for example, starch, soybean meal, wheat bran, granular fiber, oil, diatomaceous earth, zeolite, bentonite, talc, kaolin, pyro. Fillite, white carbon, etc. may be used alone or in a mixture of two or more types.

The binder is a substance that binds the components in the composition together. For example, water-soluble starch, dextrin, carboxymethyl cellulose, sodium polyacrylate, polyvinyl alcohol, gum arabic, or xanthan gum can be used alone or in a mixture of two or more types. there is.

In addition, the present invention provides a method for simultaneously controlling pests and plant diseases, comprising spraying the composition for simultaneously controlling pests and plant diseases of the present invention in areas where pests and plant diseases have grown.

The area where the composition of the present invention is sprayed may be soil or crops, and the types of crops are not limited thereto, but the crops include eggplant, pepper, pear, apple, cucumber, rose, cherry, money tree, flower, It may be a plum, apricot tree, a tangerine, a potato, a tobacco tree, a cotton tree, a cypress tree, an oleander tree, a harpoon tree, a wolfberry tree, a paulownia tree, an apricot tree, a plum tree, or a cypress tree.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

<Preparation example and experiment method>

Pest breeding conditions

The peach aphid (Myzus persicae) and the borderless aphid (Lipaphis eryimi) were supplied with spring cabbage, and the cotton aphid (Aphis gossypii) was supplied with cucumber leaves in a laboratory under temperature conditions of 25°C, relative humidity of 70%, and light conditions of 12L:12D. While reared under conditions, they were separated into art brushes and used in experiments. Spotted mites (Tetranychus urticae) were used in experiments by feeding them kidney beans and raising them under laboratory conditions at a temperature of 25°C, relative humidity of 70%, and light conditions of 12L:12D. The root-knot nematode (Meloidogyne incognita) was raised in a greenhouse under room temperature conditions by inoculating 20,000 to 50,000 second-instar nymphs on tomatoes grown in a 1:1 ratio of topsoil and sand to prevent overly humid environments. When used in an experiment, infected host roots were washed, cut with scissors, mixed with 1 liter of 1% sodium hypochlorite (NaOCl), ground finely in a blender, filtered through a sieve, washed with tap water, and the number of nematodes was counted in a 24-well plate. .

solid culture

Solid culture of the fungus Metarhizium pinghaense 15R isolated and identified in the present invention was performed as follows. The strain was cultured on potato dextrose agar (PDA) plates at 25°C for 14 days, then 14-day-old fungal conidia were scraped from the PDA plates and the material was reproduced in 0.01% Tween-80 (Difco, USA) solution. It was collected by turbidity. The conidial suspension was stirred vigorously and filtered through cotton gauze to remove mycelial debris. Spore concentration was measured using a hemocytometer.

For the grain medium, millet was placed in a plastic bag and water containing 0.15% citric acid was poured to half the volume of grain. After treatment at 95°C for 15 minutes, high-pressure sterilization at 121°C for 30 minutes and cooled, liquid-cultured mold was inoculated and cultured at 25°C for 14 days. Grains cultured with mold were placed in 0.02% Tween-80 solution and stirred vigorously, conidia were collected, and spore concentration was measured using a hemocytometer.

liquid culture

The spore suspension obtained through solid culture was grown on SDAY+B medium with 0.05% benomyl (95% active ingredient) added to SDAY (Saboraud dextrose agar with 1% yeast extract, pH 5.6) medium before inoculation into liquid medium. Germination was checked, and only those with a survival rate of 90% or more were used. The spore suspension was inoculated into PDB (potato dextrose broth, pH 5.6) medium and cultured for 14 days at 25°C, 150 rpm, and dark conditions. The culture product cultured for 14 days was centrifuged to separate the bacterial cells and the culture medium. The obtained culture medium was first filtered with filter paper (ADVANTEC, No. 2), and then the culture medium was adjusted to pH 5.6 for antifungal activity testing. did. After adjusting pH, pure culture solution obtained through secondary filtration using a 0.45 um filter was used in the experiment. To collect budding spores, the separated cells were washed twice with sterilized distilled water after centrifugation to remove the culture medium. Distilled water was added as much as the original culture medium and then homogenized to prepare a suspension. Afterwards, the bacterial cells were removed with sterilized gauze, and only the budding spores were collected and used in the experiment.

Fungal genomic DNA extraction

Extraction of the fungal genomic DNA was performed by partially modifying the chemical digestion method. The fungal cells grown in the medium were ground using liquid nitrogen and a mortar, and 400 ul of lysis buffer (0.2 M Tris-HCl (pH 7.5), 0.5 M NaCl, 10mM EDTA (pH 8.0) was added to about 1 g of the cell sample. , 1% w/v SDS) and an equal amount of phenol-chloroform-isoamylalcohol (25:24:1) were mixed and stirred vigorously for 5 minutes. Afterwards, centrifugation was performed at 9.800 After the reaction, the same amount of phenol-chloroform-isoamyl alcohol (25:24:1) was added again, centrifuged under the same conditions as above, and the supernatant was collected. After adding 100% cold alcohol equivalent to 2.5 times the supernatant, centrifugation was performed at 16.000x g for 10 minutes at 4°C to obtain DNA precipitate. The obtained DNA precipitate was washed with 70% alcohol, dried, and dissolved in 50 ul of sterile water.

Molecular biological identification

For molecular biological identification, the genomic DNA of fungi was extracted by extracting some fungal cells grown on PDA for 2 weeks in 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 then centrifuged with phenol-chloroform-isoamyl alcohol (25:24:1) to purify DNA. The purified DNA solution was precipitated using cold ethanol, centrifuged, dissolved in sterile distilled water, and used in experiments.

PCR primers for molecular biological identification of the isolated strain are EF1-983F (5' - GCYCCYGGHCAYCGTGAYTTYAT - 3') and EF1-2218R (5' - ATGACACCRACRGCRACRGTYTG - 3 for amplification of the translation elongation factor 1-alpha (EF-1a) portion. ') primer was used. The PCR reaction was performed using AccuPower PCR PreMix (Bioneer Co., Korea) using a Thermal Cycler (TaKaRa, Japan) under the conditions of denaturation at 94˚C for 30 seconds, annealing at 55˚C for 30 seconds, and extensiometry at 72˚C for 1 minute and 30 cycles. carried out.

After the PCR reaction, the amplification product was analyzed by electrophoresis using a 1.0% agarose gel and purified using the Power Gel Extraction Kit (Dyne Bio Inc., Korea). The purified PCR product was subjected to direct sequencing by Macrogen (Korea) and its base sequence was analyzed. The base sequences were 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.

plant pathogenic fungi

The plant pathogenic fungi used in the antifungal activity test were the gray mold fungus Botrytis cinerea T3-4, the pepper anthracnose Colletotrichum acutatum 15AS32, and the strawberry anthracnose fungus Colletotrichum fruiticola. (Colletotrichum fructicola CGF160401) was used, purchased from the Plant Mycology Laboratory of Chungbuk National University, and the strains were inoculated into PDA medium and cultured at 22°C for Botris cinerea and 25°C for the remaining strains and used in the experiment.

plant pathogenic bacteria

Clavibacter michiganensis subsp. michiganensis LMG 7333, a phytopathogenic bacterium used in the antibacterial activity test, was purchased from the Plant Bacteriology Laboratory of Chungbuk National University and grown in King's B medium (2% Proteose peptone no.3). , 1% glycerol, 0.15% K ₂ HPO ₄ , 0.15% MgSO _4· 7H ₂ O, pH 7.4) and used in the experiment while culturing at 27°C.

Bioassay

Fungal spores formed on grain media, budding spores obtained through liquid culture, and culture medium from which hyphae and budding spores were removed were used in bioassays, respectively. Spore suspensions with a viability exceeding 90% were used in the bioassay, and spore viability was determined in SDAY+B medium.

① Insecticidal power test

In the peach aphid bioassay using spores formed on grain media or budding spores formed in liquid culture, spore suspension was inoculated into 50 peach aphids by spraying, and dead worms were collected while observing daily for 7 days. Dead worms were recognized as being killed by mold only when the growth of mold on the epidermis was observed with the naked eye. The bioassay was performed three times.

For the peach aphid bioassay using a culture medium from which budding spores and hyphae of fungi cultured in a liquid medium have been removed, cabbage leaves cut to a diameter of 25 mm are placed in a 60 mm Petri dish containing overmoistened cotton wool, and then placed on top. After placing 20 adult aphids, a method of dipping 5 uL of the strain culture medium containing 0.02% Tween-80 into the worms using a pipette was used. Aphids were maintained at a temperature of 25°C, light conditions of 12L:12D, and humidity of 70% by removing the culture medium after inoculation and observed daily at 24-hour intervals. As a control, only 0.02% Tween-80 solution was treated, and the bioassay was repeated 3 times. did.

In an indoor drug efficacy test against root-knot nematodes, 30 animals were treated per plate well, the culture medium was treated, placed in a dark room, and the number of live and dead insects was examined.

Pot tests for peach aphids, cotton aphids, borderless aphids, and spotted mites are performed by injecting each pest into a pot planted with cabbage, cucumbers, or tomatoes, then processing the culture medium and examining the number of live insects to determine the control agent. decided.

② Antifungal activity analysis of fungal culture medium

The antifungal activity assay of the fungal strain culture was performed as follows. Botrytis cinerea (B. cinerea) was cultured in PDA medium to induce spore formation, and spores harvested by scraping the surface of the fungus were used to create a spore suspension using 10% PDB medium. The mycelium of Botrytis cinerea (B. cinerea) was removed from the collected spore suspension using sterilized gauze, and the spores were counted using a hemocytometer and incubated at about 2 using 2 After adjusting to × 10 ⁴ spores/ml, 100 ul was inoculated into a 96 well plate. After inoculation, 100 ul each of 100% entomopathogenic fungal culture and 1% and 10% entomopathogenic fungal culture diluted with sterilized water were inoculated, and after culturing for 48 hours at 22°C in the dark, the absorbance was measured at 595 nm for botry. The growth of B. cinerea was observed. At this time, 100 ul of sterilized water was used as the control instead of culture medium. In addition, antifungal activity against other plant pathogenic fungi, such as pepper anthracnose Colletotrichum acutatum 15AS32 and strawberry anthracnose Colletotrichum fructicola CGF160401, was performed in the same manner.

③ Antibacterial activity analysis

The antibacterial activity test of the entomopathogenic fungal strain culture of the present invention is performed by measuring the optical density (OD) at 650 nm in KB medium (pH 7.4) while culturing Clavibacter michiganensis (C. michiganensis subsp. michiganensis) to determine the number of bacteria. After culturing to about 1 × 10 ⁸ CFU/ml, it was adjusted to about 5 × 10 ⁵ CFU/ml using 2 After inoculation, 100 ul each of 100% entomopathogenic fungal culture adjusted to pH 7.4 and 1% and 10% culture diluted with sterilized water were inoculated, and the absorbance was measured at 650 nm after culturing at 27°C for 36 hours. The control group was 100 ul of sterile water was used instead of culture medium.

Statistical analysis

The evaluation of insecticidal activity and antibacterial activity was repeated three times, and the results were tested for significance using the SPSS statistical program at a significance level of 0.05 or less. Data were expressed as mean ± standard error (SE).

<Example 1>

Selection of pathogenic strains with control activity against pests and plant pathogens

A fungal disease was observed in peach aphids reared indoors, and the fungus was isolated from it. Fungal spores were isolated from peach aphid larvae covered with fungal spores, inoculated into SDAY+B medium, a selective medium for entomopathogenic fungi, and cultured at 25°C in the dark for 7 days. The fungi grown in the selective medium were again inoculated with PDA medium and cultured at 25°C in dark conditions for 14 days.

For molecular biological identification of the isolated fungus, fungal cells were collected, DNA was extracted, and the nucleotide sequence was determined after PCR amplification of the EF1-a region. As a result of comparative analysis of the determined base sequence with previously reported strains, it was confirmed to be a strain of the genus Metarhizium pinghaense. Accordingly, the present inventors named the isolated strain as “Metarhizium pinghaense 15R” and transferred the Metarhizium pinghaense 15R strain of the present invention to the Microbial Bank of the National Academy of Agricultural Sciences of the Rural Development Administration (KACC). ) on April 15, 2022, and was given the deposit number KACC83065BP.

<Example 2>

Evaluation of the insecticidal power of the Metarhizium pinghans 15R strain of the present invention against pests

The present inventors analyzed the insecticidal effect of the Metarhizium pinghans 15R strain isolated and identified in the present invention on the following pests.

<2-1> Insecticidal power against peach aphids

Insecticidal activity against peach aphids was evaluated using spores produced in grain media, budding spores as a liquid culture product, and culture medium (culture medium with mycelia and spores removed).

As a result of the analysis, the spores and culture medium produced in the grain medium were treated with peach aphids and the insecticidal power was compared 7 days later. The spores showed an insecticidal power of over 98%, and the culture medium showed a high insecticidal power of over 96%. In addition, on the 3rd day after treatment, the insecticidal activity of about 72% was confirmed in the culture solution-treated group, and the insecticidal activity of approximately 22% was confirmed in the group treated with spores, showing that the insecticidal activity of the culture solution was better than that of spores (Figure 1).

In addition, the effect of the culture medium showed a marked increase on the 2nd to 4th day after treatment when treated with spores. In addition, in the case of budding spores, when treated alone, the insecticidal power was about 84% at 7 days after treatment, but when treated with the culture medium, it showed a high insecticidal power of almost 100%. It was confirmed that when germinating spores and culture medium were mixed, very fast and high insecticidal power was observed.

Through the above results, the present inventors were able to confirm that the spores, budding spores, and culture medium of the Metarhizium pinghaense 15R strain according to the present invention have high insecticidal activity against peach aphids. In particular, it was found that the insecticidal activity of the culture medium was superior to that of spores or budding spores.

<2-2> Evaluation of insecticidal power of culture medium against various pests

Through the results of <2-1> above, it was confirmed that the culture medium of the Metarhizium pinkhans 15R strain of the present invention has excellent insecticidal power against peach aphids, and therefore, the culture medium was effective against peach aphids, cotton aphids, and borderless aphids. The insecticidal effect was analyzed through a pot test.

As a result, it was found that a high control value of more than 80% was shown for all peach aphids, cotton aphids, and borderless aphids on the 3rd day after treatment (Figure 2).

In addition, an analysis of the insecticidal power against spotted mites was performed, and the results showed that both adults and nymphs showed a high insecticidal power of more than about 97% in just 2 days after treatment (Figure 3), and the insecticidal power against root-knot nematodes was evaluated through indoor tests. The results also showed a high insecticidal effect of about 94% on the 1st day after treatment, and a high insecticidal effect of about 99% on the 5th day after treatment (Figure 4).

Through the above results, the present inventors found that the Metarhizium pinghaense 15R strain isolated and identified in the present invention had excellent insecticidal activity against various plant pests in both the spores and culture medium of the strain.

<Example 3>

Evaluation of antibacterial activity of Metarhizium pinghans 15R strain of the present invention against plant pathogens

Furthermore, the present inventors evaluated the antibacterial activity against various plant pathogens in the culture medium of Metarhizium pinghans 15R strain.

<3-1> Analysis of antifungal activity against plant pathogenic fungi

To confirm the antifungal activity of the Metarhizium pinghans 15R strain culture medium against Botrytis cinerea (B. cinerea), the Metarhizium pinghans 15R strain culture was obtained after liquid culture in PDB medium (pH 5.6) for 2 weeks. Bacterial cells were removed from the product, and the culture medium was adjusted to pH 5.6 ± 0.2. Antifungal activity against Botrytis cinerea (B. cinerea) was analyzed using the obtained pure culture solution.

As a result, the Metarhizium pinghans 15R strain culture medium was treated at different concentrations (100% (v/v), 10% (v/v), 1% (v/v)), and 100% and 10% treatments were obtained. High antifungal activity was observed in the group (Figure 5).

In addition, the present inventors, in order to confirm whether the Metarhizium pinghaense 15R strain of the present invention has antifungal activity against other plant pathogenic fungi, Colletotrichum acutatum 15AS32 and Colle Colletotrichum fructicola CGF160401 was analyzed using the same method as above.

As a result, it was shown that the culture medium of the Metarhizium pinghans 15R strain of the present invention had antifungal activity against both of these two types of plant pathogenic fungi. Specifically, against Colletotrichum acutatum, the culture medium of the strain 15R was found to have antifungal activity. Antifungal activity was shown even at % (v/v) dilution, and relatively high antifungal activity was shown even at 40% (v/v) dilution against Coletotrichum fruticola (Figure 6).

Through these results, the present inventors have demonstrated that the Metarhizium pinghaense 15R strain of the present invention is not only B. cinerea but also Colletotrichum acutatum 15AS32, a pepper anthracnose fungus, and It was confirmed that it had excellent antifungal activity against both the strawberry anthracnose bacteria, Colletotrichum fructicola CGF160401.

<3-2> Analysis of antibacterial activity against plant pathogenic bacteria

Furthermore, the present inventors analyzed the antibacterial activity of the culture medium of Metarhizium pinghans 15R strain against Clavibacter michiganensis subsp. michiganensis, a plant ulcer pathogen.

As a result, it was found that there was antibacterial activity depending on the concentration of the treated culture medium (Figure 7). These results mean that the Metarhizium pinghaense 15R strain of the present invention has antibacterial activity even against plant pathogenic bacteria.

Summarizing the above results, the Metarhizium pinghaense 15R strain according to the present invention, all culture products including strains and spores, are peach aphid, cotton aphid, borderless aphid, spotted mite, and sweet potato root-knot nematode. It was found to have excellent insecticidal power against plant pests, and in particular, the culture medium of the strain was used against the plant pathogenic fungi Botrytis cinerea (B. cinerea), Colletotrichum acutatum 15AS32 and Colleto. It was found that it not only has high antifungal activity against Colletotrichum fructicola CGF160401, but also has antibacterial activity against Clavibacter michiganensis subsp. michiganensis, a plant pathogenic bacterium.

Therefore, it was confirmed that the Metarhizium pinghaense 15R strain of the present invention is a useful strain that can effectively control not only pests but also plant pathogens at the same time.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Name of depository institution: National Academy of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC)

Accession number: KACC83065BP

Trust date: 20220415

[Correction 12.10.2023 pursuant to Rule 91]

Claims (11)

1. Metarhizium pinghaense 15R strain (Accession number: KACC83065BP), which has simultaneous control activity against pests and plant pathogens.
2. According to paragraph 1,

   The pest is Metarhizium pinghaense 15R strain (Accession number: KACC83065BP), which is characterized in that it is peach aphid, cotton aphid, borderless aphid, spotted mite or root-knot nematode.
3. According to paragraph 1,

   Metarhizium pinghaense 15R strain (Accession number: KACC83065BP), which has antifungal or antibacterial activity against fungi or bacteria that cause plant diseases.
4. According to paragraph 3,

   The fungus is Metarhizium pinghans, characterized in that it is Botrytis cinerea T3-4, Colletotrichum acutatum 15AS32, or Colletotrichum fructicola CGF160401. (Metarhizium pinghaense) strain 15R (accession number: KACC83065BP).
5. According to paragraph 3,

   The bacterium is a Metarhizium pinghaense 15R strain (accession number: KACC83065BP), characterized in that it is C. michiganensis subsp. michiganensis.
6. A composition for simultaneously controlling pests and plant diseases, comprising the strain of claim 1, its spores, a culture of the strain, a concentrate of the culture of the strain, or a dried product of the culture of the strain as an active ingredient.
7. According to clause 6,

   A composition for simultaneously controlling pests and plant diseases, wherein the pests are peach aphids, cotton aphids, borderless aphids, spotted mites, or root-knot nematodes.
8. According to clause 6,

   The plant disease is caused by Botrytis cinerea T3-4, Colletotrichum acutatum 15AS32, Colletotrichum fructicola CGF160401, or Clavibacter misiganensis (C. A composition for simultaneous control of pests and plant diseases caused by michiganensis subsp.
9. A method for simultaneously controlling pests and plant diseases, comprising the step of spraying the composition for simultaneous control of pests and plant diseases of claim 6 in an area where pests and plant diseases have grown.
10. According to clause 9,

    A method for simultaneously controlling pests and plant diseases, wherein the area is soil or crops.
11. According to clause 10,

    The above crops include eggplant, pepper, tomato, strawberry, Chinese cabbage, pear, apple, cucumber, rose, cherry, money tree, apricot flower, plum, chinensis tree, tangerine, potato, sweet potato, tobacco, cotton, perennial tree, oleander, harpoon tree, A method for controlling pests or plant diseases, characterized in that it is a wolfberry tree, a paulownia tree, an apricot tree, a plum tree, or a plum tree.

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