KR101836007B1 - Entomopathogenic fungi Metarhizium anisopliae SD4-2 having antimicrobial activities and insectcide - Google Patents

Abstract

The present invention relates to a biological agent using an insect pathogenic fungus Metarhizium anisopliae strain SD4-2 having pest control and antimicrobial activity. The strain of the present invention is excellent in the effect of controlling the spotted mite It also has an effect of controlling microorganisms.
Thus, the Metarhizium < RTI ID = 0.0 > anisopliae ) The strain SD4-2 shows high pathogenicity to the plantation crops, and it can be used as a biological agent to control plant pathogens at the same time as pest control, which is very useful for environmental industry.

Description

Entomopathogenic fungi Metarhizium anisopliae SD4-2 having antimicrobial activities and insectcide which is an insect pathogenic fungus having pest control and antimicrobial activity.

The present invention relates to a biological agent using an insect pathogenic fungus methyrizium anisopria SD4-2 strain having pest control and antimicrobial activity.

Barriers to plant growth include environmental factors such as water, temperature, and nutrients, as well as microorganisms, which are insects and diseases that affect plants as biological barriers. Among these obstacles, especially those who raise plants from an economic point of view, the most important concern is to solve these problems as botanicals and pests. In order to solve these problems, disinfectants, fungicides, viral agents, The research and development and use of pesticides have been actively carried out all over the world. In order to protect plants from diseases and pests, it is essential that crop protection agents for plant diseases and pest control are essential for growing plants for various purposes such as agriculture, forest, landscape, and grass for food production. And costs are incurred. In particular, recently, the importance of the production of safe agricultural products along with the environmental risk of these crop protection agents has been emphasized, and the interest in the environment friendly control method of plant diseases and pests has increased, and such control methods are actually being expanded.

Insect pathogenic fungi are microorganisms that cause insect diseases and kill them and have been widely used as pesticide materials for over 100 years. Recently, a variety of extracellular enzymes, protein toxins, and secondary metabolites produced during the intrusion and proliferation of these fungi have been used to overcome the host immune response, to be used for host defense, and to compete with other microorganisms And various other substances are being studied for the use of antibacterial and antifungal agents. Currently, crop protection agents for individual control are essential for the control of plant diseases and insect pests. However, if these two purposes can be achieved at the same time as one drug, especially as an environmentally friendly control agent, It is believed that the development of a control agent that can satisfy both convenience and safety is considered to be possible.

Therefore, it is urgent to develop environmentally friendly materials that can control both plant diseases and pests simultaneously.

Korean Patent No. 10-2011-0004731 Korean Patent No. 10-0404767

Therefore, in the present invention, Metarhizium < RTI ID = 0.0 > anisopliae ) SD4-2 strain has excellent pest control ability and at the same time can control microorganisms. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a strain of Metarhizium anisopliae SD4-2 (accession number KACC93228P) having pest control ability.

In addition, another object of the present invention is to provide a process for the preparation of anispliae) SD4-2 strain, a culture of the strain, a concentrate of the cultured product, and a dried product of the culture, as an active ingredient.

Another object of the present invention is to provide a pest control method comprising the step of spraying the insect pest control composition of the present invention on a region where pest is propagated.

In order to achieve the above object, the present invention provides a strain of Metarhizium anisopliae SD4-2 (accession number KACC93228P) having pest control ability.

In one embodiment of the present invention, the strain can prevent spotted mites.

In one embodiment of the present invention, the strain may have antifungal or antibacterial activity.

In one embodiment of the present invention, it may be isolated from the insect pathogenic fungus.

The present invention also relates to the use of the Metarhizium anispliae) SD4-2 strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture as an active ingredient.

The present invention also provides a pest control method comprising the step of spraying the insect pest control composition according to the present invention in a region where pests have been propagated.

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

In one embodiment of the present invention, the crop is selected from the group consisting of eggplant, pepper, pear, apple, cucumber, rose, cherry, , Gujuga tree, paulownia tree, apricot tree, plum tree and cypress tree.

The present invention is not predetermined erase Metairie having a debugging function Philippe Ke (Metarhizium anisopliae) SD4-2 strain, and the strain of the present invention is excellent in the effect of controlling the spotted mite and has an effect of controlling microorganisms.

Thus, the Metarhizium < RTI ID = 0.0 > anisopliae ) The strain SD4-2 shows high pathogenicity to the plantation crops, and it can be used as a biological agent to control plant pathogens at the same time as pest control, which is very useful for environmental industry.

FIG. 1 shows the result of the test for antifungal activity against insect pathogenic fungi (right part) and B. cinerea (left part) according to the present invention.
A) L. attenuatum SD4-1 with strong antifungal activity
B) B. bassiana SD7 showing intermediate antifungal activity
C) M. flavoviride without antifungal activity SD4
Fig. 2 shows the result of the test for the antibacterial activity against the insect pathogenic fungus (middle part) and the bacterium B. cereus (whole part of the medium) according to the present invention.
A) Strong anti-bacterial activity of B. bassiana SD5-1
B) B. bassiana showing intermediate antibacterial activity SD14
C) L. attenuatum SDMp1 without antibiotic activity
FIG. 3 is a graph showing antimicrobial activity of the insect pathogenic fungus culture of the present invention.
A) Antibacterial activity against B. cereus
B) Antifungal activity against mold B. cinerea

The present invention relates to a method for controlling insect pests, which is excellent in pest control ability and at the same time can control microorganisms, such as Metarhizium anisopliae ) SD4-2 strain, and is characterized by providing a method for controlling pests using the strain.

In order to solve these problems, there is a need for a pesticide for control of plant diseases and a pesticide for controlling pests such as a bactericide, a fungicide, and a virus agent. Research has been actively carried out, but there is a problem in that it is costly and time consuming.

Therefore, although a crop protection agent for each individual control is essentially used to control pests and microorganisms, there is a problem that two purposes can not be achieved at the same time.

Therefore, the present inventors have isolated a strain that exists in a natural environment having excellent insect pest and microbial control ability in order to develop a method for simultaneously controlling insects and microorganisms.

As a result, the strain isolated by the present inventors was Metarhizium anisopliae SD4-2, and it was confirmed that the strain was the most excellent insect pest and microorganism control ability than the other strains isolated together. Thus, the strain was deposited with the National Institute of Agricultural Science and Technology (KACC) on May 22, 2015, and was deposited with Metarhizium anisopliae ) SD4-2 strain Accession No. KACC93228P.

Thus, the inventors of the present invention have found that Metarhizium anisopliae ). In order to investigate whether the SD4-2 has excellent pest control ability, in one embodiment of the present invention, a biosynthetic test was carried out on spotted mites. Specifically, about 300 insect pathogenic fungi isolated from soil in Korea were analyzed by PDA After culturing in the medium and inducing spore formation, spore suspension was prepared, sprayed on 50 adult spider mites and observed daily for 7 days to collect the larvae. As a result, Aspergillus flavus SD7; B. bassiana SD5-1, SD6, SD2R-3-3-1, SD2R-4-5, SD2R-4-7, SDTu; C. rosea SD5-2; L. attenuatum SD4-1; M. anisopliae SD2-2, SD4-2; 12 strains of P. lilacinus SD2R-4-6 were found to be effective in controlling pests. In the spotted mite, Metarhizium anisopliae ) SD4-2 showed excellent efficacy (see Examples 1 and 2).

Therefore, the inventors of the present invention have found that the above-mentioned strain can be used for controlling pests.

Furthermore, the present inventors have conducted experiments to confirm the antifungal or antibacterial activity of the above-mentioned strains, and found that the bacterium B. bassiana SD2R-4-5, M. anisopliae SD4-2, Aphid pathogenic fungi B. bassiana SD15, L. attenuatum SDMp2 was selected. As a result, all of the four strains ( B. bassiana SD2R-4-5, M. anisopliae SD4-2, B. bassiana SD15, and L. attenuatum SDMp2) , And the strain used in this experiment was confirmed to produce an antimicrobial active substance in each microorganism, and it was confirmed that various microbial control can be practically practiced using the microorganism (see Examples 3 to 6).

Therefore, the present invention can provide a strain of Metarhizium anisopliae SD4-2 (accession number KACC93228P) having pest control ability.

In addition, the insect pests that can be controlled by the strain according to the present invention are not limited thereto, but may be spotted mites.

In addition, the strain according to the present invention can provide antifungal or antibacterial activity.

The present invention also relates to the use of the above Metarhizium anisopliae) SD4-2 strain, the culture, the culture of a concentrate with water, and at least one member selected from the group consisting of the dry matter of water culture of the strain can be provided for the pest control composition comprising, as an active ingredient.

The culture can be obtained by culturing a large amount by a conventional culture method of microorganisms. As the culture medium, a medium containing a carbon source, a nitrogen source, a vitamin and a mineral can be used. Examples of the carbon source include starch, sucrose, Citrate, maltose, glucose, mannitol, glycerol or xylose can be used.

In addition, the culture can be used in the form of a culture containing the strain of the present invention, or can be subjected to centrifugation or filtration to remove the culture medium from the culture and recover only concentrated cells. The steps may be carried out as required by those skilled in the art. The concentrated cells may be frozen or lyophilized according to a conventional method so as not to lose their activity.

Thus, the Metarhizium < RTI ID = 0.0 > anisopliae ) The SD4-2 strain has all the characteristics that can control microbes as well as the activity to control pests.

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

≪ Example 1 >

Screening of spotted mite pathogenic strains

<1-1> Pest conditions

The present inventors used Tetranychus urticae for the experiment while feeding the kidney beans and cultivating them at a temperature of 25 ° C, a relative humidity of 70%, and a light condition of 12L: 12D in order to examine the pest control effect of the pathogenic strain.

<1-2> Selection of pathogenic strains for pest control

Collection of soil samples to isolate insect pathogenic fungi from soil in Korea was conducted in winter from 2007 to 2009. Approximately 50 g of soil samples were collected in plastic packs at depths of 5 to 10 cm from various soil (rice field, field, orchard, tree, mountain, riverbank) in 9 provinces nationwide and taken to the laboratory for isolation of fungi.

To isolate insect pathogenic fungi from soil samples, 1.3 ml of 0.02% tween 80 and 0.2 g of soil sample were added to a 1.5 ml microcentrifuge tube and mixed vigorously and diluted to 10 ^-1 . 150 μl of the diluted sample was plated on an insect pathogenic fungus selective medium SDA-D50 (sabouraud dextrose agar, 100 μg / ml chloramphenicol, 50 μg / ml streptomycin, 50 μg / ml dodine) Respectively. After cultivation, colonies showing an insect pathogenic fungus-specific proliferation phase (color, size, texture) were transferred to a PDA medium using a sterilized toothpick and cultured at 25 ° C for 7 days under dark conditions. After cultivation, 300 isolated insect pathogenic strains were used for the selection of the spider mite pathogenic strain (see Table 1).

The fungal pathogenic strain used in the present invention Isolates Number of isolates Acremonium spp. One Aspergillus spp. 8 Beauveria bassiana 109 Beauveria brongniartii 8 Beauveria pseudobassiana 8 Bionectria spp. 7 Chaunopycnis spp. 23 Clonostachys rosea One Fusarium spp. 2 Isaria farinosa 12 Isaria fumosorosea 6 Isaria spp. 22 Lecanicillium spp. 8 There is Metarhizium anisopliae . anisopliae 65 I have M. anisopliae . leptidium One I have M. flavoviride . pemphigum 16 Mortierella spp. One Mucoralean One Myrothecium spp. 5 Paecilomyces carneus 3 P. lilacinus 3 P. marquandii 5 Paraconiothyrium spp. 2 Phialocephala spp. One Pochonia spp. 17 Simplicillium spp. One Tolypocladium spp. 3 Verticillium spp. 3

In order to select the pathogenic strains of spotted mites, the insect pathogenic fungus strains of Table 1 were cultured in PDA (potato dextrose agar) medium for 2 weeks to induce spore formation. Then, spores of the fungi were randomly divided into groups of 100, It was used for the selection of pathogenic fungi in each pest. The bioassay for the selection of pathogenic fungi for each insect was carried out by inoculating the prepared spore suspension with 50 sprayed adult mite adults and collecting the larvae daily for 7 days. In order to isolate the pathogenic fungus from the killed dead larvae, the spores were picked up with a sterilized toothpick in a fungal sickle, and a 0.02% tween 80 solution (SDA-D50, Sabouraud dextrose agar, 100 μg / ml chloramphenicol, 50 μg / ml streptomycin, and 50 μg / ml dodine) after the suspension. Then, colonies formed by culturing at 25 ° C for 7 days in dark condition were separated and again inoculated into PDA medium.

Spore suspension was prepared by treating the spore suspensions prepared for the pathogenic fungi of spotted mites. Each spore was picked from sterilized mites by a sterilized toothpick and suspended in a 0.02% tween 80 solution. The insect pathogenic fungus selective medium SDA-D50 (sabouraud dextrose agar , 100 μg / ml chloramphenicol, 50 μg / ml streptomycin, and 50 μg / ml dodine), and the results were as follows: Aspergillus flavus SD7; B. bassiana SD5-1, SD6, SD2R-3-3-1, SD2R-4-5, SD2R-4-7, SDTu; C. rosea SD5-2; L. attenuatum SD4-1; M. anisopliae SD2-2, SD4-2; P. lilacinus SD2R-4-6 Twelve strains were isolated and used in the following experiments.

&Lt; Example 2 >

Inactivation of insect pathogenic fungi

In order to quantitatively test the pathogenicity of the spore mite pathogenic fungus strain obtained in Example 1, spore formation was induced by culturing the fungal strains of each of the spotted mite species selected in the above for 2 weeks in a PDA (potato dextrose agar) medium, Spore suspension was prepared using 0.05% tween-80 and counted using a hemocytometer. Survival rates of all the spores were 90% or more.

The bioassay for spotted mite was carried out by placing a bean leaf cut to a diameter of 60 mm on a 90 mm patridish containing a humidified cotton wool, carefully placing 10 spotted mite adults thereon, and then spraying 1 ml of spore suspension of each strain prepared 1 × 10 ⁸ conidia / ml) were inoculated using an SD tower sprayer. After the inoculation, mushroom water was observed daily for 5 days at 24 hour intervals.

The isolates were tested for biomarkers of spotted mites and adults, and the mortality rate of each strain varied from 10 to 100% on the 5th day. Specifically, the order of the strains having high pathogenicity among the spot-borne pathogenic strains was B. bassiana SD2R-4-7, B. bassiana SD2R-3-3-1, L. attenuatum SD4-1, M. anisopliae SD4-2, P. lilacinus SD2R-4-6, B. bassiana SD6, M. anisopliae SD2-2, and C. rosea SD5-2 (Table 2).

As a result of this bioassay, it was confirmed that the spores of the isolate of the spider mite pathogenic fungus can be used as a spore mite biological control agent by itself.

The percentage of the spider mite pathogenic fungi used in the present invention to the spotted mite Strain name Survival rate (%) LT50 (days) Aspergillus flavus SD7 59 4.2 B. bassiana SD5-1 55 4.1 SD6 67 3.7 SD2R-3-3-1 75 2.7 SD2R-4-5 67 4.0 SD2R-4-7 82 2.6 SDTu 59 4.7 C. rosea SD5-2 60 3.8 L. attenuatum SD4-1 100 2.8 M. anisopliae SD2-2 69 3.8 SD4-2 100 2.9 P. lilacinus SD2R-4-6 84 3.1

&Lt; Example 3 >

Antifungal activity of insect pathogenic fungi

The antifungal activity of insect pathogenic strains was evaluated by inoculating 5 μl (1 × 10 ⁶ conidia / ml) of each spore suspension of spotted mite pathogenic fungus and fungal Botrytis cinerea in PDA medium at 30 mm intervals for 4 days, The clear zone of the two fungi was measured.

As a result, it was observed that about 67% of the twelve insect pathogenic fungi that were tested showed that they inhibited the growth of B. cinerea with different ability to form a clear zone 1).

Among the strains showing eight antifungal activities, two strains ( B. bassiana SD2R-4-5, L. attenuatum SD4-1) showed high activity of 3 mm or more, and the remaining strains showed activity between 1 and 3 mm.

<Example 4>

Antibacterial activity of insect pathogenic fungi

The antibiotic activity test was performed by inoculating 5 μl (1 × 10 ⁶ conidia / ml) of the spore suspension of the spider mite pathogenic fungus to the center of the PDA medium for 3 days, 4 ml of Bacillus cereus suspension (1 × 10 ⁶ cfu / ml with 0.5% agar) cultured in 10 g peptone in 1000 ml distilled water, pH 7.4) medium was inoculated in the PDA medium as a whole. Then, after culturing at 25 DEG C for 24 hours, a clear zone of growth was measured.

As a result, it was observed that about 10% of the 12 insect pathogenic fungi tested had different ability to grow B. cereus and formed a clear zone (Table 3 and Fig. 2).

Of B. cereus Ten strains that inhibit growth by different abilities and form a clear zone Strain name A. flavus SD7 B. bassiana SD5-1, SD6, SD2R-3-3-1, SD2R-4-5, SD2R-4-7, SDTu L. attenuatum SD4-1 M. anisopliae SD2-2, SD4-2

Of the 10 strains showing antibacterial activity, 5 strains corresponding to 50% showed high activity of 20 mm or more, and 3 strains corresponding to 30% showed intermediate activities between 10 and 20 mm. Finally, 20% Two of the corresponding strains showed low activity (see Table 4). The detailed results are shown in Table 4 below.

Strain according to degree of activity Strain name High antimicrobial activity B. bassiana SD5-1, SD2R-3-3-1, SD2R-4-5, SDTu M. anisopliae SD2-2 Intermediate antibacterial activity B. bassiana SD6 M. anisopliae SD4-2 L. attenuatum SD4-1 Low antimicrobial activity A. flavus SD7 B. bassiana SD2R-4-7

&Lt; Example 5 >

Antimutagenic and Antimicrobial Activity of Insect Pathogenic Fungi

As a result of selection of the strains having both saliva and antimicrobial activity by integrating the results of the antimicrobial activity test of the insect pathogenic fungi and the results of the inactivation of the existing target insect pests, eight spider mite pathogenic fungi strains were selected (see Table 5) ).

Based on these results, B. bassiana SD2R-4-5 and M. anisopliae SD4-2, which are highly pathogenic and highly antimicrobial, were selected for the next experiment.

Antibacterial and Antifungal Activity against Spotted Mite of Spotted Mature Pathogenic Fungi Strain name Survival rate (%) LT ₅₀ (days) Antimicrobial activity (Clear zone, mm) Antibacterial (± SE) Antifungal (± SE) Aspergillus flavus SD7 59 4.2 7.3 ± 0.2 0 ± 0 B. bassiana SD5-1 55 4.1 21 ± 0.8 2.6 ± 0.4 SD6 67 3.7 16.4 ± 0.2 2.1 ± 0.1 SD2R-3-3-1 75 2.7 21 ± 0.3 1.3 ± 0.3 SD2R-4-5 67 4.0 25.7 ± 0.5 3.3 ± 0.3 SD2R-4-7 82 2.6 6.5 ± 0.3 0 ± 0 SDTu 59 4.7 20.3 ± 0.2 2.4 ± 0.6 C. rosea SD5-2 60 3.8 N / D N / D L. attenuatum SD4-1 100 2.8 12.6 ± 0.3 4.1 ± 0.5 M. anisopliae SD2-2 69 3.8 23.2 ± 0.4 2.0 ± 0.9 SD4-2 100 2.9 14.7 ± 0.4 1.8 ± 0.3 P. lilacinus SD2R-4-6 84 3.1 0 ± 0 0 ± 0 N / D, not determined

&Lt; Example 6 >

Antimicrobial activity of insect pathogenic fungi

In order to confirm the antimicrobial activity of the insect pathogenic fungus culture liquid selected in Example 5, spores harvested by scraping the fungus surface cultured for 2 weeks were counted using a hemocytometer, and the resulting spore suspension was inoculated into 30 ml of PDB dextrose broth) inoculated in ^{100 ml flask (1.5 × 10 3} conidia / ml) containing culture medium and 25 ℃, after 10 days incubation at 150 rpm and was centrifuged filtering. The culture broth was used for the antifungal activity test before the experiment. The culture solution used for the antifungal activity assay was adjusted to pH 5.6, and the culture solution used for the antibacterial activity assay was adjusted to pH 7.4.

The antifungal activity of B. cinerea sp. Was adjusted to about 2 × 10 ⁴ conidia / ml by using PDB (pH 5.6) medium, and then 100 μl . Subsequently, 100 μl of a culture of the fungal strain diluted with 1%, 10%, 20%, 40%, 60%, 80% and 100% was inoculated into the PDB medium and cultured at 22 ° C. for 48 hours and then the absorbance was measured at 595 nm Respectively. The control was sterilized with 1%, 10%, 20%, 40%, 60%, 80% and 100% diluted in 100 μl of PDB medium.

The bacterial activity of B. cereus was adjusted to about 5 × 10 ⁵ CFU / ml in BG medium and 100 μl was inoculated in a 96-well plate. Subsequently, 100 μl of a culture solution of fungal strains diluted with 1%, 10%, 20%, 40%, 60%, 80% and 100% was inoculated into the PDB (pH 7.4) medium and cultured at 25 ° C for 16 hours. The absorbance was measured. The control was sterilized with 1%, 10%, 20%, 40%, 60%, 80% and 100% diluted in 100 μl of PDB medium.

As a result, the two strains used in the experiment (the spider mite pathogenic fungus Vervia barbiana SD2R-4-5, SD4-2) inhibited the growth of microorganisms with different abilities, and it was confirmed that the strains used in this experiment produced antimicrobial active substances in the respective microorganisms. It was judged that various microbial control could be practically practiced (see Fig. 3).

According to the results of the above examples, insect pathogenic fungi Metarhizium , which has high virulence to spotted mites and has antibacterial activity against bacteria and fungi, anisopliae SD4-2 (accession number: KACC93228P) deposited on May 22, 2015 with the National Institute of Agricultural Science and Technology Center for Agricultural Genetic Resources.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Agricultural Biotechnology Research Institute KACC93228 20150522

Claims (8)

Metarhizium anisopliae SD4-2 strain (Accession No. KACC93228P), which has antimicrobial activity against Bacillus cereus or Botrytis cinerea and has an effect of preventing spots mite. delete delete delete A pharmaceutical composition comprising at least one member selected from the group consisting of Metarhizium anisopliae SD4-2 strain of claim 1, a culture of the strain, a concentrate of the culture and a dried product of the culture, A composition for preventing spotted mites and plant diseases. A method for the prevention of spotted mites and plant diseases, comprising the step of spraying the composition for preventing spotted mites and plant diseases according to claim 5 in a region where pests and plant diseases have propagated. The method according to claim 6,
Wherein said area is a soil or a crop. 8. The method of claim 7,
The above crops may be selected from the group consisting of eggplant, pepper, pear, apple, cucumber, rose, cherry, pinna, japanese, plum, cherry, tangerine, potato, tobacco, cotton, Characterized in that it is a tree, a plum tree or a gomoma tree.

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