KR102126582B1 - Microbial agent for simultaneous control of both Colletotrichum acutatum and Thrips spp. using Isaria javanica FT333 and its culture media - Google Patents

Abstract

The present invention relates to a microbial agent for the simultaneous control of Isaria javanica FT333 strain ( Isaria javanica FT333, KACC 93316P) or thrips and plant anthrax ( Colletotrichum acutatum ) using the same. It is possible to produce excellent and safe agricultural products by simultaneously controlling thrips and pepper anthracnose, which are heating pests, and it is effective in reducing the control cost and control power of farmers through the development of simultaneous pest and disease control technology that can be used in eco-friendly cultivation areas.

Description

Isaaria Javanica FT333 strain or a microbial agent for simultaneous control of thrips and plant anthrax containing the same {Microbial agent for simultaneous control of both Colletotrichum acutatum and Thrips spp. using Isaria javanica FT333 and its culture media}

The present invention relates to a microbial agent for the simultaneous control of Isaria javanica FT333 strain ( Isaria javanica FT333, KACC 93316P) or thrips and plant anthrax ( Colletotrichum acutatum ) using the same.

Thrips spp. is an insect belonging to the thrips family and is a micro-pest that occurs all year round and causes enormous damage to all flowering crops such as horticulture and flowers. In particular, the yellow flower thrips ( Frankliniella occidentalis ) and the cucumber thrips ( Thrips palmi Karny) are foreign infested pests that have recently spread throughout the country, causing serious damage to various crops. In plant cultivation cucumbers, peppers, melons and other fruits and vegetables, before flowering, shoots are mainly added to cause deformed leaves or yellowing, and after flowering, flowers are added to form malformation, reducing the productability. On the other hand, thrips are highly proliferative and have faster development of resistance to chemical pesticides than other pests. In addition, thrips are considered to be relatively difficult to control by insecticides, because after a month after invasion into the facility, a generation passes and various ages such as eggs, larvae, pupae, and adult insects occur.

To control thrips, about 220 chemical pesticides such as pyriproxyfen and pinetoram have been used. However, thrips are resistant to most of these chemical pesticides, and are also resistant to recently developed chemical pesticides such as spinosad, acetamiprid, and denotefuran. Carbamite-based insecticides (bendiocarb, carbosulfan, methiocarb, etc.), organic phosphate-based insecticides (methomil, clopyriphos, diazino, etc.), synthetic pyrethroid-based insecticides (acrinathrin, benpetrin, ci Halotrine, etc.), neonicotinoid-based insecticides (imidacloprid, acetaprid, dinotefuran, etc.), microorganism-derived biochemicals (spinosad), mectin pesticides (abamectin, milbamectin, etc.) It has been reported as a resistant chemical pesticide.

Recently, as the interest in side effects such as the emergence of resistant pests and environmental pollution due to the use and abuse of chemical pesticides has increased, the use of chemical pesticides is gradually limited, and these chemical pesticides can be replaced and biologically safe. Control methods are actively being studied. For example, it is a method of controlling by using natural enemies such as Arius spp. and predatory mites, or by using insect pathogenic fungi that infect pests. To date, more than 750 insect pathogenic fungi have been known, among which are the microbial insecticides such as Beauveria bassiana , Metarhizium anisopliae , and Nomuraea rileyi. It has been developed and used for controlling various pests.

Pepper anthracnose usually occurs in fruits. In the fruit, the infected area initially appears as a slightly recessed circular spot with an acupuncture needle, and as it progresses, the lesion expands to a circular to irregular double pattern. A pale yellow to yellowish brown spore mass is formed in the lesion area, and the severely diseased fruit is twisted and dried. Some lesions with maturation sometimes have a black double pattern, and some may appear during drying after harvest. Red pepper anthrax is caused by Colletotrichum acutatum , and this strain overwinters in the form of amethyst and hyphae in seeds or diseased areas to become the primary source of infection. The first half of the disease is mainly caused by conidia spores, and the outbreak is more severe in the open field packaging than in the facility cultivation packaging. In the noji pavement, during the summer rainy season, conidia spores are mainly caused by rain and wind. In the green pepper of the open field packaging, the disease starts to develop from the beginning of July and continues to occur until the harvest season.

Pepper anthracnose reported in Korea is difficult to control, but not very effective. In addition, the cost of control is expensive and resistance to the drug may occur when used repeatedly. Vegetables that develop anthrax anthrax are mainly consumed directly as raw food, so the safety problem of pesticides continues to emerge from consumers, and the use of chemical pesticides is also avoided. In addition, the control method using chemical pesticides has a fatal disadvantage that resistance to chemicals may occur over time, and environmental hazards caused by chemical residues have been raised.

Accordingly, the present inventors can adapt well to the domestic environment, and as a result of diligent efforts to develop a microbial agent comprising an indigenous insect-pathogenic fungal strain that exhibits rapid insecticidal or microbial effects, against thrips and plant anthrax in the domestic soil The present invention was completed by separating Isaria javanica FT333, a new strain having excellent simultaneous control effect.

Korean Registered Patent No. 10-1773339 (Invention name: Isaria fumosoracea Pf212 strain or composition for simultaneous control of aphids, jalococcus and pepper anthrax using the same, Applicant: Republic of Korea, Date of registration: 2017.08.25.) Korean Registered Patent No. 10-1785098 (Invention name: Isaria Javanica Pf185 strain or composition for simultaneous control of aphids and jalococcus bacteria using the same, Applicant: Republic of Korea, Date of registration: 2017.09.28.)

Steven Paul, Arthurs, Luis Fernando Aristizabal, Pasco Bruce Avery, Evaluation of Entomopathogenic Fungi Against Chilli Thrips, Scirtothrips dorsalis, Journal of Insect Science, 2013, 13(31):1-16.

An object of the present invention is to provide a microbial agent for the simultaneous control of thrips and plant anthrax using Isaria javanica FT333 strain ( Isaria javanica FT333, KACC 93316P).

The present invention relates to the Isaria javanica FT333 strain ( Isaria javanica FT333, KACC 93316P) having the control ability of thrips or plant anthrax ( Colletotrtichum acutatum ).

The Isaria Javanica FT333 strain is characterized by having the simultaneous control ability of thrips and plant anthrax.

The present invention also provides a microbial agent for the control of thrips or plant anthracnose, which contains the cells of the Isaria japonica FT333 strain or a culture medium thereof as an active ingredient.

Accordingly, the present invention provides a method for controlling the thrips or plant anthracnose by treating the plant with a method for culturing the Isaria japonica FT333 strain and a microbial agent containing the cells of the Issarial javnica FT333 strain or a culture medium thereof. do.

Hereinafter, the present invention will be described in detail.

The present invention relates to a strain of Isaria javanica FT333 ( Isaria javanica FT333, KACC93316P) that has the control ability of thrips or plant anthracnose, the strain or a culture medium thereof, the microbial agent containing them control the thrips or plant anthracnose It can be used as a zero.

The culture medium may be a culture medium in which the Isaaria javanica FT333 strain is cultured in a microbial medium. As the microbial medium, Isaaria Javanica FT333 can grow, PDA (potato dextrose agar), PDB (potato dextrose broth), SDA (sabouraud dextrose agar), SDAY (sabouraud dextrose agar with yeast extract), AD (Modified) adamek's media) Medium such as liquid medium and AD solid medium may be used, and preferably PDA medium.

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

When using the culture medium as a microbial agent, after culturing the Isaria Javanica FT333 strain, it may be used while containing the cells of the Isaria Javanica FT333 strain, and may be used as a culture filtrate state in which the cells are removed.

The cultivation method of the Isaaria Javanica FT333 strain may include: (a) inoculating spores of Isaaria Javanica FT333 into a microbial medium; And (b) culturing the inoculum at 25 to 30°C for 7 to 20 days. The microbial medium of step (a) may preferably use AD medium or PDA medium. When inoculating the Isaria javanica FT333 strain in the step (a), the initial inoculation concentration may be preferably 5×10 ⁵ ~5×10 ⁶ conidia/ml, more preferably 1×10 ⁶ conidia/ May be ml. The step (b) may further include the step of removing the cells for the culture medium. Therefore, the microbial agent of the present invention containing a culture medium of the Isaaria Javanica FT333 strain can be prepared through the above culturing method.

The strain of the present invention and the culture medium thereof, the thrips which can control the microbial agent containing the thrips, Megalurothrips distalis , Thrips hawaiiensis , Thrips hawaiiensis , Microcephalothrips abdominalis , Chinese thrips Haplothrips chinensis ), convex thrips ( Scirotothrips dorsalis ), thrips palmi Karny, flower thrips ( Frankliniella occidentalis ), rice thrips ( Baliothrips biformis ), etc., preferably cucumber thrips May be, but the thrips are not limited thereto.

In addition, the strain of the present invention and a culture medium thereof, a plant that can be applied to a microbial agent containing the cucumber ( Cucumis sativus ), pepper ( Capsicum annuum ), rice ( Oryza sativa ), soybean ( Glycine max ), strawberry ( Fragaria x crops such as ananassa ), chrysanthemum ( Chrysanthemum morifolium ), persimmon tree ( Diospyros kaki ), plum tree ( Prunus mume ), peach tree ( Prunus persica Batsch), citrus tree ( Citrus unshiu ), apple tree ( Malus pulila var. dulcissima KOIDZ) , Chestnut ( Castanea crenata var. dulcis), but may be fruit trees, but the plant is not limited thereto.

Microbial preparations for simultaneous control of thrips and pepper anthracnose of the present invention are used and sprayed with the composition according to the physiological state of the plant, the type of insect, the degree and concentration of pathogen infection, season, humidity, the age and growth stage of the plant, and the plant treatment method It can be considered in deciding how.

Isaria Javanica FT333 contained in the microbial agent for controlling thrips or pepper anthracnose of the present invention should be in a viable state, which may be mycelial and spore-shaped cells.

Isaria javanica FT333 strain of the present invention is an insect pathogenic strain, the term'insect pathogenicity', when fungi, that is, spores of the fungus attach to the outer shell of the host insect and germinate, chitinase (chitinase), protease (protease) , Lipase (lipase), esterase (esterase) to produce and penetrate the cuticle layer (cuticle) of the insect, and then invade, then proliferates in the body of the insect to produce a toxic substance means the effect of killing host insects, It can be used interchangeably with the'insecticide effect'.

Microbial preparations prepared by culturing the Isaaria javanica FT333 strain of the present invention can be prepared using solid or liquid culture technology. The culture temperature of the Isaria japonica FT333 strain is 10 ~ 30 ℃, preferably 25 ~ 30 ℃, it is preferable that the proper pH when cultured is 6.8 to 7.2, pH 7.0. In addition, the culture period is preferably 7 to 20 days, more preferably 10 to 15 days, most preferably 14 days to culture to obtain a culture solution or culture.

The microbial agent of the present invention may include agriculturally acceptable carriers, fillers, solvents, excipients, surfactants, suspending agents, spreaders, adhesives , Antifoaming agents, dispersants, wetting agents, drift reduction agents, auxiliaries, adjuvants or mixtures thereof.

The microbial formulations of the present invention can be formulated in the form of concentrates, solutions, sprays, aerosols, immersion baths, dips, emulsions, suspension concentrates, gels and granules.

The microbial agent of the present invention, the microbial agent alone or other agricultural agents, pesticide microbial pesticides, insecticides (insecticides), acaricides, fungicides (fungal agents harmless to the fungus of the present invention), bactericides, herbicides, Foliar sprays, antibiotics, antimicrobial agents, nematocides, rodenticides, entompathogens, pheromones, attractants, plant growth regulators, plant hormones ), combined with insect growth regulators, chemosterilants, microbial pest control agents, repellents, viruses, feeding stimulants, plant nutrients, plant fertilizers and biological control agents Alternatively, it may be used sequentially, and specific examples thereof are obvious to a person having ordinary skill in the art and can be easily obtained.

The present invention relates to a microbial agent for the simultaneous control of the Isaria javanica FT333 ( Isaria javanica FT333, KACC 93316P) strain or thrips and plant anthrax ( Colletotrichum acutatum ) using the same, the heating pests of thrips and pepper anthracnose At the same time, it can be controlled to produce excellent and safe agricultural products. In addition, the development of pest and pest control technologies that can be used in eco-friendly cultivation areas is effective in reducing the control cost and control power of farmers, and it can be expected to increase farm income by increasing the quantity and productability of cultivated crops.

On the other hand, Korean Registered Patent No. 10-1773339 (Isaaria Fumosorose Pf212 strain or a composition for simultaneous control of aphids, jalocococcus and pepper anthracnose using the same) or Korean Registered Patent No. 10-1785098 (Isaria Javanica Pf185 strain or a composition for the simultaneous control of aphids and locusts using the same) is described for the genus Isaria or the strain Isarian Javanica, but the content of the simultaneous control effect of thrips and plant anthrax is not described.

Figure 1 is a photograph of the present invention Isaaria Javanica FT333 strain observed under a microscope.
Figure 2 is a photograph showing the control effect on the cucumber thrips of the Isaaria Javanica FT333 strain of the present invention.
Figure 3 is a graph showing the insecticidal rate of cucumber thrips over time after treatment with the Isaaria javanica FT333 strain of the present invention.
Figure 4 is a photograph showing the antimicrobial activity effect on the plant anthrax bacterium ( Colletotrichum acutatum ) of the Isaaria japonica FT333 strain of the present invention.
Figure 5 is a photograph showing the control effect on the anthrax anthrax strain of the present invention Isaaria Javanica FT333 strain.

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

<Example 1. Isolation and identification of Isaaria japonica FT333 strain from soil>

Example 1-1. Isolation of strain

Among insect pathogenic fungi collected by using insect-bait method (bait insect: brown larva larva) from soil, strains having pathogenicity against thrips were selected. More specifically, 500 ml of soil was collected from 4 locations on 20 m2 of the Guryong Yongmyeon and mixed in a zipper bag. The soil brought to the laboratory was lowered to a 2 mm sieve, and 20 ml was placed in a plastic container, and 10 brown larvae of 1.5 cm in size were added. After incubation at 25°C for 14 days, the insect pathogenic strain was isolated from the surface of the infected brown fly larva. The isolated insect pathogenic strain is treated with cucumber leaves inoculated with Thrips palmi Karny, and the cucumber ginseng beetle of the untreated group and the untreated untreated group 7 days after the treatment are shown in FIG. 2. Did. Referring to Figure 2, it can be confirmed that the cucumber pathogen that is 7 days after the treatment of the insect pathogenic strain is infected with the insect pathogenic strain and dies.

Example 1-2. Identification of strain

Among the fungi isolated from the surface of the brown larvae by the method of Example 1-1, the strain having the greatest antibacterial activity was identified through microscopic observation, and the DNA sequencing of the 18S rRNA gene of this strain was obtained. ITS analysis was performed by extraction. As a result, finally, the strain was confirmed that the Isaria javanica ( Isaria javanica ) and 18S rRNA match, and has the DNA base sequence of the 18S rRNA gene of SEQ ID NO: 1, Isaria javanica FT333 ( Isaria javanica) FT333) and deposited with the National Academy of Agricultural Science (KACC 93316P).

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

Example 2. Preparation of spore suspension of Isaria japonica FT333 strain

Conidia spores of pure isolated Isaria javanica FT333 strain ( Isaria javanica FT333) were cultured in PDA medium for 14 days at 28° C., spores were collected, suspended in a 0.01% tween80 solution, and then used with a hemocytometer. Spore suspensions were prepared so that the spore concentrations were 1×10 ⁵ , 1×10 ⁶ , and 1×10 ⁷ conidia/mL.

Experimental Example 1. Control effect of Isaria japonica FT333 strain on cucumber thrips

The following experiment was carried out to test the control effect on the thrips palmi Karny of the Isaaria javanica FT333 strain of the present invention. The spore suspension prepared in Example 2 (1×10 ⁷ conidia/ml) was sprayed 1 ml on the front and back of cucumber leaves of about 150 mm in length with 10 cucumber thrips adult injected, and the relative humidity was at least 95%. It was kept under the temperature condition of 25 ℃ while maintaining. Insecticidal investigation was conducted daily until the 5th day after spraying, and the control effect test was repeated 3 times and averaged, and the results are shown in FIG. 3.

Referring to FIG. 3, when the spore suspension of the Isaria japonica FT333 strain was treated, it was confirmed that the insecticidal rate of cucumber thrips increased over time. In particular, on the fifth day, the insecticidal rate was 80% or more. Through this, it was found that the Isaria japonica FT333 strain of the present invention has excellent control effect against cucumber thrips.

Experimental Example 2. Control effect of plant anthrax bacterium of Isaria japonica FT333 strain

Experiments were conducted to see if the Isaria japonica FT333 strain of the present invention has a control effect against Colletotrichum acutatum , a plant anthracnose.

Plant anthracnose (diameter 5 mm, 1×10 ⁶ conidia/ml) in the center of PDA medium, and isaria janicanica FT333 strain (diameter 5 mm, 1×10 ⁶ conidia/ml) at a position 30 mm away from both sides After incubation, the cells were cultured at 25°C for 14 days to investigate antibacterial activity. It is shown in Table 1 and Figure 4 below.

Antibacterial strain Zone of inhibition (㎜) Colletotrichum acutatum Isaria javanica FT333 21.2

Referring to Table 1 and FIG. 4, it was confirmed that the Isaria Javanica FT333 strain exhibited an inhibitory ring of 21.2 mm in size, and that the colletotricum aquattum did not grow toward the tooth of the Isaria Javanica FT333 strain. Could. Through this, it was found that the Isaria japonica FT333 strain of the present invention has excellent control effect against the plant anthracnose, Colletotricum aquatumum.

Experimental Example 3. Assessing the control effect of plant anthrax by Isaria japonica FT333 strain

Spore suspension (1×10 ⁶ conidia/) of Colletotrichum acutatum , which is wound with a pin on pepper to test the control effect of plant anthracnose of Isaria japonica FT333 strain of the present invention ML) were inoculated 5 μl each. After drying the pathogen suspension, ⁵ μl each of the spore suspensions (1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ conidia/ml) of the selected Isaaria javanica FT333 strain was inoculated into the wound. After inoculation, the relative humidity of 95% or more was maintained at 25°C, and the onset or absence was investigated for 14 days, and the results are shown in FIG. 5.

Referring to FIG. 5, it was confirmed that the anthracnose developed in the portion inoculated with the plant anthracnose in the untreated group not treated with the Isaria japonica FT333 strain. On the other hand, it was confirmed that the outbreak does not progress due to the plant anthracnose in peppers treated with the Isaaria Javanica FT333 strain. In addition, it was confirmed that anthrax did not develop in the spore suspensions of 1×10 ⁵ , 1×10 ⁶ and 1×10 ⁷ conidia/mL concentration.

National Academy of Agricultural Science KACC93316P 20181112

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microbial agent for simultaneous control of both Colletotrichum acutatum and Thrips spp. using Isaria javanica FT333 and its culture media <130> 0366 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 522 <212> DNA <213> Unknown <220> <223> Isaria javanica <400> 1 accctttgtg aacataccta tcgttgcttc ggcggactcg ccccggcgtc cggacggccc 60 tgcgccgccc gcgacccgga cccaggcggc cgccggagac ccacaaattc tgtttctatc 120 agtctttctg aatccgccgc aaggcaaaac aaatgaatca aaactttcaa caacggatct 180 cttggttctg gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa 240 ttcagtgaat catcgaatct ttgaacgcac attgcgcccg ccagcattct ggcgggcatg 300 cctgttcgag cgtcatttca accctcgaca ccccttcggg ggagtcggcg ttggggaccg 360 gcagcatacc gccggccccg aaatacagtg gcggcccgtc cgcggcgacc tctgcgtagt 420 actccaacgc gcaccgggaa cccgacgcgg ccacgccgta aaacacccaa cttctgaacg 480 ttgacctcgg atcaggtagg actacccgct gaacttaagc at 522

Claims (7)

delete Isaria javanica FT333 strain comprising the 18S rRNA gene represented by the nucleotide sequence of SEQ ID NO: 1, and having simultaneous control in thrips palmi and Colletotrichum acutatum KACC 93316P). Claim 2, Isaria javanica FT333 strain ( Isaria javanica FT333, KACC 93316P) microbial agent for the control of thrips or plant anthrax ( Colletotrichum acutatum ), characterized in that it contains a cell or a culture medium thereof as an active ingredient. A method of culturing the Isaria javanica FT333 strain of claim 2 (KACC 93316P). Method for simultaneous control of thrips or plant anthrax ( Colletotrichum acutatum ) by treating the microbial agent of claim 3 to a plant. A pesticidal microorganism pesticide comprising the microbial agent of claim 3. Foliar spraying agent comprising the microbial agent of claim 3.

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