KR20080019831A - Microbial agents against ginseng plant pathogens that contains induced systemic resistant bacillus megaterium hk-isr1 strain and cultured solution therefrom and prevention methods for ginseng plant disease using the same - Google Patents

Abstract

An induced systemic resistant Bacillus megaterium HK-ISR1 strain is provided to induce the induced systemic resistance in ginseng plants while it does not have antagonistic activity against ginseng plant pathogenic bacteria, so that it is useful for controlling ginseng plant disease. An induced systemic resistant Bacillus megaterium HK-ISR1 strain(KACC 91261P) is isolated from the roots and aerial part of ginseng and controls ginseng plant disease caused by a ginseng plant pathogenic bacterium selected from Cylindrocarpon destructans, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium solani, Botrytis cinerea, Phytophthora cactorum, Pythium ultimum, Alternaria panax and Colletotrichum gloeosporioides. A microbial agent contains the induced systemic resistant Bacillus megaterium HK-ISR1 strain(KACC 91261P) and is formulated as particle, wettable powder or capsule. Further, a concentration of microbe in microbial agent is 1Î10^6 to 1Î10^9 cfu/ml.

Description

Microbial agents against ginseng plant pathogens that contains induced systemic resistant Bacillus megaterium HK-ISR1 strain and cultured solution therefrom and prevention methods for ginseng plant disease using the same}

1 is a Bacillus megaterium ( Bacillus megaterium ) 16S RNA sequence of HK-ISR1 strain (hereinafter referred to as "HK-ISR1").

Figure 2 is a gas chromatogram showing the fatty acid content of the HK-ISR1 strain.

FIG. 3 is a graph showing healthy seedlings inoculated with HK-ISR1 strain suspension in soil and inoculated with spores of late blight pathogen Phytophthora cactorum on the ground.

Figure 4 is a photograph showing the effect of lignin accumulation of the epidermal cell wall by inoculation of Cylindrocarpon destructans in seedlings root tissues treated with Bacillus megaterium.

FIG. 5 is a graph showing the seedling rate when seedlings immersed in HK-ISR1 strain were transplanted into soil contaminated with ginseng root rot ( Cylindrocarpon destructans ).

FIG. 6 is a graph showing the root disease morbidity when seedlings immersed in HK-ISR1 strain were transplanted into soil contaminated with ginseng root rot ( Cylindrocarpon destructans ).

Figure 7 is a graph showing the ratio of whole seedlings of treated, untreated control, antagonistic Bacillus subtilis and conventional pesticide cultivation seeded by dipping the ginseng seeds soaked in HK-ISR1 strain culture.

Figure 8 is a graph showing the number of whole seedlings of treated, untreated control, antagonistic Bacillus subtilis and conventional pesticide cultivation seeded by dipping the ginseng seeds soaked in HK-ISR1 strain culture.

The present invention relates to a ginseng pathogen microbial agent comprising the induced systemic resistance Bacillus megaterium HK-ISR1 strain, more specifically Bacillus megaterium HK-ISR1 strain having induced systemic resistance against ginseng root and ground pathogens It relates to a microbial agent containing the culture solution thereof as an active ingredient or a method for controlling ginseng disease using the microbial agent.

Korean ginseng is the king of herbal medicine that has been used for the health of mankind from prehistoric times to the present day as Korea's natural, exclusive and historical biological resources. As Western medicine has shown limitations in overcoming the side effects of synthetic drugs and adult diseases, alternative medicines that are expected for biopharmaceuticals and natural products including ginseng are being developed day by day. As ginseng's efficacy, cancer prevention, aging control, liver protection, diabetes control, fatigue recovery, and brain function enhancement have already been revealed. Recently, it prevents the damage of environmental hormones, prevents obesity, and has an effect on AIDS patients. It is known that it is losing.

The efficacy of Korean ginseng is detailed in the Tong-Nong Herbal Medicine, the best document of the East (4000 years ago), and Korean ginseng, which has been proven as a Chinese herbal medicine for thousands of years, has been proven to be of the highest quality. In 2003, ginseng exports in Korea increased 30% compared to 2002 and 2004 increased 40% compared to 2003. However, the condition of cultivation of ginseng in Korea is longer than 6 years, so there are many root rot diseases, and more than half are combined and once planted, it is impossible to grow due to root rot disease even after 20 years (Dae Hwi Cho: Pest Control for Production of Clean Raw Ginseng) 19-30, Technical Approach Strategies for High Quality Ginseng Production 2004. Rural Development Administration, Crop Science Institute, Publication No. 11-1390625-060010-01) 10 to 15 years after covering soil Often planted again. In this case, due to many diseases, pesticides are excessively used. Therefore, there is a lot of pesticide residues in ginseng, which are exported to Europe and the Americas and returned to Korea (Park, Hyung-Dal: Status and Direction of Pesticide Stability Survey of Ginseng. 47-56. Ginseng Production, Rural Development Administration, Crop Science Institute, Publication No. 11-1390625-060010-01).

As the agricultural market is opened globally, Korean agriculture, which has a narrow territory, must find a new path from ginseng cultivation, a monopolistic resource, and for this, it is urgent to develop environmentally friendly ginseng disease control method that can replace chemical pesticides with residual amount. .

Although chemical control methods using fumigants such as pyrons and bassamides have been recommended for the regeneration of cropland, success rate is unstable depending on soil factors, and it is expected to cease use in years due to residual problems and environmental problems. I can't do it.

Recently, the method of utilizing antagonistic microorganisms is actively progressed and several species are commercially available. However, there are many cases of soil factors that are ineffective depending on the field. This is mainly due to the different degrees of rooting settlement of antagonistic microorganisms. To be effective, antagonist microorganisms must be distributed evenly in the ginseng root zone with effective density so that the pathogens cannot be approached. Even if the whole root zone is settled evenly, it is not easy to maintain effective density continuously. This is because the antagonistic bacteria cause a difficult environment to grow continuously by causing adverse environmental changes such as lack of nutrients during the long-term settlement. In other words, the effects of antagonistic microbial agents are extremely difficult to achieve the full effect due to the property that the pathogen must be closely within the range of action of the antagonists in time and space. Recently, research on controlling plant diseases by using induced systemic or induced systemic microorganisms to activate the defense mechanisms of plants has been actively conducted. In other crops, induced systemic resistance and microorganisms are commercially available. (Park, Kyung-Seok: Environmentally Friendly Disease Control Technology Using ISR. 69-103. Development of Environmentally Friendly Plant Disease Control Technology. 2004 Academic Symposium. Rural Development Administration. Publication No. 11-390093-000103-01, Ray Hammerschmidt: Chap6; Induced Plant Resistance to Fungal Pathogens: Mechanisms and Practical Applications.179-200. 2004. Food Product Press).

Therefore. The inventors of the present invention, Bacillus megaterium ( Bacillus) in the process of selecting microorganisms with root disease control ability in healthy ginseng soil megaterium ) HK-ISR1 was identified and selected, and although the strain showed no antagonism against ginseng pathogens through in-flight culture experiments, it showed a high pathogenic effect when directly treated with ginseng (Induced Systemic Resistance). The present invention was completed by confirming that there is an action and verifying the effect by field experiment.

An object of the present invention is to provide a microorganism for controlling ginseng disease, a microorganism preparation including the microorganism and a method for controlling ginseng disease.

In order to achieve the above object, the present invention is a novel Bacillus megaterium ( Bacillus) causing the induced systemic resistance of ginseng itself against ginseng disease megaterium ) provides an HK-ISR1 strain.

In addition, the present invention provides a microbial preparation for ginseng disease control containing the Bacillus megaterium HK-ISR1 strain or its culture as an active ingredient.

In addition, the present invention provides a method for controlling ginseng disease using the microbial agent.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Bacillus megaterium HK-ISR1 strain that induces induced systemic resistance of ginseng itself against ginseng disease.

The inventors have isolated 16S rRNA from strains to classify and identify the primary screened antagonistic strains, and disclose using a BLAST search program of the National Center for Biotechnology Information (NCBI) based on the nucleotide sequence (see FIG. 1). As a result of comparing the genetic similarity with the strain, Bacillus megaterium megaterium ) identical to the bases of AY030338, DQ105968, AY505510 (100% consensus). Subsequently, by using the MIDI Shertork microorganism identification system, the constituent properties of the unique fatty acids of the strains of the present invention were examined and compared with the test strains, and showed 75% similarity to Bacillus megaterium (see FIG. 2). Therefore, the present inventors classified the strain into Bacillus megaterium based on the above results, and named it Bacillus megaterium HK-ISR1, and Korea Agricultural Microbial Resources located at 225 Seodun-dong, Gwonseon-gu, Suwon-si, Korea on July 21, 2006. Deposited to the center (accession number: KACC 91261P).

The inventors of the present invention to investigate the physiological characteristics of the Bacillus megaterium strain HK-ISR1 (hereinafter referred to as "HK-ISR1") of the present invention, HCN (hydrogen cyanide), proteinase, cellulase (Cellulase), peck Pectinase, Gluconase, and IAA (indole acetic acid) production were investigated. As a result, the HK-ISR1 strain did not produce HCN, proteinase, cellulase, pectinase, glucosease and IAA.

The present inventors examined the antagonistic ability of the HK-ISR1 strain against ginseng pathogens. As a result, the mycelial growth of ginseng pathogens was not inhibited at all, and it was confirmed that the HK-ISR1 strain of the present invention had no antagonism directly inhibiting the growth of ginseng pathogens (see Table 1). The present inventors then irradiate the culture medium of the HK-ISR1 strain to the soil around the ginseng seedling stem, and the pestilence spores of the late blight ( Phytophthora) cactorum ) was inoculated to prevent direct contact between the HK-ISR1 strain and the pathogen. As a result, the average healthy seedling number was 0.8 in the treatment of only late blight pathogens, whereas 1.5 was significantly different in the treatment of the late blight pathogen and the culture medium of HK-ISR1 (see FIG. 3). Through this, pathogens were not directly inhibited by the HK-ISR1 strain when invaded, but it was found that ginseng suppresses the disease by inducing the expression of defense-related genes and inducing systemic resistance.

Subsequently, the present inventors observed a change in the ginseng root tissue that induced resistance by HK-ISR1. In this experiment, ginseng roots showing systemic resistance were collected and examined for lignin accumulation in the root cell wall. As a result, it was confirmed that lignin was clearly accumulated in the cell wall of epidermal tissue cells so that the HK-ISR1 strain induced systemic resistance in ginseng (see FIG. 4).

In order for disease to develop when plant pathogens invade, the primary fallopian tube must progress through the cell wall (1994, Schafer, W. Annual). Review of Phytopathology 32: 461-477). Resistant plants, however, respond to cell wall degrading enzymes produced by pathogens by causing changes in the cell walls upon invading pathogens and increasing mechanical strength (1978, Ride, JP, Annals). of Applied Biology 89: 302-306). One type of cell wall change is lignin accumulation or phenolic compounds, which are closely related to plant resistance (1992, Nicholson, RL, Hammerschmidt, R. Annual). Review of Phytopathology 30: 369-389). Plant cells that are in contact with pathogens early in the invasion of phytopathogens into resistant plants produce large amounts of lignin or phenolic compounds, and these reactions function to block and sequester the migration of new pathogens to new cells (1993, Stein, BD, K). lomparens , K., Hammerschmidt , R. Journals of Phytopathology 137: 177-188). Therefore, it can be seen that the increased lignin accumulation due to the induction resistance by HK-ISR1 of the present invention increases the resistance to pathogens of ginseng itself.

The present inventors investigated whether the HK-ISR1 strain culture medium had an inhibitory effect on root rot in seedlings. After immersing seedlings in HK-ISR1 culture, Cylindrocarpon destructans ) mixed soil and peat moss, and seedlings were grown. After that, the seedling rate of seedlings was measured and the degree of root rot was measured to investigate the morbidity of root rot. As a result, in the seedling rate, the HK-ISR1 strain culture treated group was 79%, which was about 7 times higher than the untreated group 11%, and there was no statistical difference between 86% of healthy soil seedlings (see FIG. 5). In the case of near-morbidity, there was a statistically significant difference of 20% in the HK-ISR1 culture treatment group and 50% in the untreated group, and a 60% control effect (see FIG. 6). It was confirmed that HK-ISR1 strain inhibits ginseng root disease.

The present inventors investigated the effect of HK-ISR1 strain culture on root rot inhibitory effect and yield in farm packaging. When irradiated, the ginseng seed was soaked in culture medium and sown, and divided into untreated, known antagonistic treatment, HK-ISR1 strain treatment and conventionally used pesticide treatment. As a result, the percentage of whole seedlings was the highest in the HK-ISR1 strain treatment group (47.3%), more than twice the effect of conventional pesticide treatment. It also showed a higher effect than Bacillus subtilis, an antagonistic microbial agent (see FIG. 7 and Table 2). In the case of whole seedling yield, HK-ISR1 strain increased 2.6 times compared to pesticide practice, and higher than that of antagonist microorganisms (see FIG. 8 and Table 3). The high percentage of whole seedlings is due to germs occurring in seeds during freezing and after freezing after sowing, and after the germination, during the spring, summer, and autumn. Yields seem to have increased because they survived root rot bacteria. The higher yield ratio than the difference in seedling ratio is not due to the increase of the seedling ratio alone, but it can be considered that the induced resistance microorganism promoted seedling root growth. In general, plant growth promoting rhizobacteria are known to produce induced systemic resistance. Through the above results, the HK-ISR1 strain of the present invention is a root disease, Cylindrocarpon destructans ( Cylindrocarpon). destructans ), as well as the phytophthora cactorum , which is a late blight bacterium, exhibits the ability to inhibit induction resistance and prevents the onset of late blight. It can be seen that it has the ability to suppress induction resistance against pathogens and above-ground pathogens.

In addition, the present invention provides a microorganism preparation for ginseng disease control containing the HK-ISR1 strain and its culture as an active ingredient.

The microorganism preparation for ginseng disease control of the present invention contains the above-mentioned HK-ISR1 strain and its culture solution having induced systemic resistance against ginseng root rot and late blight.

Pathogens exhibiting systemic resistance of ginseng disease control microorganisms of the present invention is Cylindrocarpon ( Cylindrocarpon) destructans ), Rhizoctonia solani ), sclerotinia sclerotinia sclerotiorum ), Fusarium solani ), Botrytis cinerea ), Phytophthora cactorum , Pythium ultimum ), Alternaria panax , Colletotrichum gloeosporioides , and the like.

The microbial preparations of the invention may be prepared in the form of hydrates, granules or encapsulations for the purpose of stable formulation of the microorganisms. In the microbial preparation of the present invention, the HK-ISR1 strain or its culture solution may be supplied as contained in the composition for controlling ginseng disease, or may be stored separately for long-term storage and mixed before use. When the microorganism is supplied separately for long term preservation, the microorganism may be stored at -70 ° C. or lower in a glycerol storage solution or may be used by lyophilization at −20 to −80 ° C.

Hydrating agents of the present invention are prepared by drying and pulverizing a solid medium inoculated with microorganisms, and then adding and mixing a surfactant and a extender / nutrient. In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene One or two or more selected from the group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkelone alkylphenyl ethers, polyoxyethylene nonyl phenyl ethers, Triton 100 and Tween 80 And extenders and nutrients include one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch.

The granules of the present invention are prepared by drying and grinding a solid medium inoculated with microorganisms and then adding a surfactant, an extender / nutrient and a disintegrant. In the above surfactants, polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene One or two or more selected from the group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkelone alkylphenyl ethers, polyoxyethylene nonyl phenyl ethers, Triton 100 and Tween 80 As the extender and nutrient, one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch are used, and bentonite, talc as a disintegrant (talc), dialite, kaolin and calcium carbonate It uses more than one or two selected from the group. In addition, the granules of the present invention may be one or two or more selected from the group consisting of surface active agents, inert carriers, preservatives, wetting agents, feed promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectors, buffers and flow agents to microorganisms. It can be prepared by addition.

Application of the microbial preparation of the present invention is preferably sprayed by concentrating the HK-ISR1 strain in the roots or the soil close to the ginseng or sprayed from the ground part at any time. In addition, the application of the microbial agent is milliliter ^{(㎖) 1 × 10 6 ~} 1 × 10 9 of biomass concentration directly spraying the solution to the root of ginseng was diluted with or millimeters per liter of ^{1 × 10 6 ~ 1 × 10} 9 of microorganisms per Ginseng can be immersed in a solution diluted to a concentration. As a ginseng pathogen that can obtain a control effect by using the microbial preparation of the present invention, Cylindrocarpon destructans ), Rhizoctonia solani ), Sclerotinia sclerotiorum , Fusarium solani ), Botrytis cinerea ), Phytophthora cactorum ), Pythium ultimum ), Alternaria panax ), Colletotrichum gloetosporioides gloeosporioides ).

Therefore, the microbial preparation of the present invention has excellent control ability by inducing systemic resistance against the upper pathogens including ginseng root rot and late blight, and there is no environmental pollution and human toxicity, so it is environmentally friendly ginseng root rot and ginseng ground. It can be usefully used as a microbial agent for disease control.

In addition, the present invention provides a method for controlling ginseng disease using the microbial agent.

The present invention is to control the ginseng pathogen by applying the HK-ISR1 strain or its culture solution to the ginseng or the soil around the ginseng disease control effect, wherein the HK-ISR1 strain is applied directly to the root or soil or prepared above It can be applied in the form of one microbial agent. In addition, the control method of the present invention can be sprayed by concentrating the solution diluted with the HK-ISR1 strain in the root or soil of ginseng, or immersed ginseng in the microbial preparation for 1-2 hours.

It is preferable to spray the microorganism of the present invention directly to the ginseng or the soil around it or to the ground in the form of a microbial preparation, the number of microorganisms applied to the microbial preparation is 1 × 10 ⁴ ~ 1 × 10 per milliliter (ml) Dilution to a microbial concentration of ⁹ cfu / ml is preferred.

Hereinafter, the present invention will be described in detail by examples and formulation examples.

However, the following Examples and Preparation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Preparation Examples.

< Example  1> Ginseng pathogen  Separation

The present inventors collected ginseng showing diseased ginseng root and ground and cut the boundary area of ginseng and healthy part of ginseng into 3 mm size, immersed in sodium hypochlorite for about 30 seconds, washed with sterile water and then filter paper After removing the water, put on agar plate. This was incubated for 7 days in a thermostat at 20 ℃ was identified based on the morphological characteristics on the microscope.

As a result, the microorganisms are Cylindrocarpon destructans , Rhizoctonia solani ), sclerotinia sclerotinia sclerotiorum ), Fusarium solani ), Botrytis nerea ), Phytophthora cactorum ), Pythium ultimum ), Alternaria panax ) and Colletotriccum gloeosporioides .

< Example  2> Explored  Identification of induced telesis resistant microorganisms

Selection of the strain is a Bacillus (Bacillus) in 106 bacteria strain isolated from the rhizosphere of ginseng and other crops were selected through ginseng seed and seedling test experiment.

The soil used for the bioassay of ginseng root rot was collected from two bottles of KT &G's raw material research institute in Suwon. The plastic pot (20 × 30 × 20 cm, W × L × D) was filled with sterilized peat moss up to 15 cm and then covered with 1 cm of soil soil and filled with sterilized peat moss up to 20 cm. Bacillus 106 strains were cultured in TSA medium for 2 days to prepare a bacterial suspension (1 × 10 ⁸ cfu / ml), and the ginseng seeds treated with Gaegi were immersed in the bacterial suspension for 1 hour. The soaked ginseng seeds were sown in prepared pots and placed in plastic containers with sterile water and watered. The water is taken out of the port with a ginseng seed after reaching the planting soil surface from the lower end port were located in the interior (about 20 ℃) water was the water cycle in the same way every week. Thirty days after seeding, ginseng seeds and roots were examined and retested one or more times to select antagonistic microorganisms with root rot control.

The seedling experiment was prepared by mixing the diseased soil that had been stored at low temperature with pit moss (morbid soil: pitmos, 1: 3 w / w) in a round plastic pot (20 × 20 cm). The first selected antagonist microorganisms were cultured in TSA medium for 2 days to prepare a bacterial suspension (1 × 10 ⁸ cfu / ml), and 1 year old seedlings were immersed in the bacterial suspension for 1 hour. Soaked seedlings were transplanted into prepared pots for 10 weeks and grown in the growth chamber. Watering was done by filling a plastic container with water soaking from the bottom of the pot, dipping the pot with seedlings transplanted, and watering once a week. Germination and root rot disease rates were visually examined 30 days after treatment.

16S rRNA was isolated and identified using the BLAST search program of the National Center for Biotechnology Information (NCBI) based on the nucleotide sequence (FIG. 1 and SEQ ID NO: 1) for the identification of the induced induced systemic microorganisms. Similarity was analyzed. As a result, all of the strains of Bacillus metaterium AY030338, DQ105968, AY505510 and 1401 of 1401 bases were identical (100% consensus).

Shertork Microbial Identification System (1988, Norman, DJ, Yuen, JMF Can.J. Plant Pathol . 20: 171-175, 2000, Walcott, RR et al . Phytopathology 90: 191-196) was used to compare and analyze the constituent characteristics (FIG. 2) of the unique fatty acids of the induced systemic resistant strains, and then classified and identified them based on the results. The results showed 74.5% similarity with Bacillus megaterium.

The inventors classified the Bacillus megaterium on the basis of 16S rRNA sequence comparison data and the characteristics of the fatty acid composition of the strain for the identification of the induced systemic strain, and named it Bacillus megaterium HK-ISR1. As of March 21st, it was deposited with the Korea Agricultural Microbiological Resource Center located at 225 Seodun-dong, Gwonseon-gu, Suwon-si, Korea (accession number: KACC 91261P).

In order to investigate the physiological characteristics of the identified Bacillus megaterium HK-ISR1 strain (hereinafter referred to as "HK-ISR1"), the present inventors have identified HCN (hydrogen cyanide), proteinase, cellulase, and peck. The production of tinases (Pectinase), Gluconase (Gluconase), IAA (indole acetic acid) was investigated.

HCN activity was determined by plating the above-identified HK-ISR1 strain in Petri dishes on TSA (Tryptic soy agar: Becton, Dickinson and company, USA) to which glycine (4.4 g / l) was added, and picric acid solution. The filter paper strip (Whateman Co. USA) soaked in a Petri dish lid was incubated for 2-4 days in a 28 ℃ incubator to investigate the HCN production by changing the color of the filter paper. Protease activity was established after 48 hours by inoculating HK-ISR1 strain into Scheme milk medium (Skim milk 100 g, yeast extract 1.5 g, Bacterium agar 15 g / 1 L, distilled water, DIFCO, USA). Was determined. Cellulase activity was assessed by the Teather and Wood methods (1982, Applied and Environmental Microbiology 43: 777-780), and 10 μl of a suspension of the HK-ISR1 strain incubated in LB medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g, DIFCO, USA) for 24 hours was added to the prepared medium at 25 ° C. After incubation for 48 hours, it was determined by adding Congo red (1 mg / ml) solution (Sigma, USA) for 15 minutes. Pectinase activity was determined by media containing apple pectin [(NH ₄ ) ₂ SO ₄ 2.0 g, KH ₂ PO ₄ 4.0 g, Na ₂ HPO ₄ 6.0 g, FeSO ₄ .7H ₂ O 1 mg, MgSO ₄ 0.2 g, CaCl ₂ 1 mg, H ₃ BO ₃ 10 ug, MnSO ₄ 10 ug, ZnSO ₄ 70 ug, CuSO ₄ 50 ug, MoO ₃ 10 ug, Apple pectin 5 g, Yeast extract 1 g, Agar 15 g, DW 1000 ml) ] Was cultured by HK-ISR1 strain, 1% hexadecyl trimethyl ammonium bromide (HTAB) solution was added to the plate, left for 10 minutes, and then observed by inhibiting the band. The production of IAA was determined by adding 5 mM 1-treptophan to TSA medium, placing a nitrocellulose membranse disk on the medium, inoculating HK-ISR1 strain, and incubating at 28 ° C for 3 days. Salkowski reagent (1951, Plant Physiology 26: 192-195) to determine the production.

As a result, the HK-ISR1 strains identified in the present invention did not produce HCN, proteinase, cellulase, pectinase, gluconase and IAA.

< Example  3> HK - ISR1  Strain To ginseng pathogen  Antagonistic ability and induced telescopic resistance Induced Systemic Resistance ) Research

The present inventors performed replacement culture to investigate the effect of the HK-ISR1 strain on the growth of the ginseng pathogen isolated in Example 1. Specifically, HK-ISR1 strain was incubated for 48 hours at 28 ℃ using TSA medium. PDK medium (10 g of Potato Dextrose Broth, 5 g of Peptone, 15 g of Agar, 1 L of distilled water, DIFCO, USA) was used as a medium for the replacement culture experiment. 70 μl of the HK-ISR1 strain suspension was inoculated on a sterile 8 mm diameter paper disk, and then aliquoted into Petri dishes and placed on a plate of prepared PDK medium. The ginseng pathogens isolated in Example 1 for replacement culture were incubated in PDA medium [38 g of Potato Dextrose Agar, 5 g of Agar, 1 L of distilled water, DIFCO, USA] for 7 days. Then, using a sterile kolkborough with a diameter of 6 mm, the mycelia grown medium was detached and replaced by HK-ISR1 strain on the PDK medium plate. HK-ISR1 strains and ginseng pathogens were wounded and incubated in a thermostat at 25 ° C. for 10 days, and the growth inhibition of pathogens was examined.

  As a result, the mycelial growth of ginseng pathogens was not inhibited at all, and it was confirmed that the HK-ISR1 strain had no antagonism directly inhibiting the growth of ginseng pathogens (Table 1).

Subsequently, irrigation of the suspension of the HK-ISR1 strain in the soil around the ginseng seedling stems, and at the ground level, the spores of late blight ( Phytophthora) cactorum ) was inoculated to treat HK-ISR1 strain and pathogens. The average dry seedling count was 0.8 in the only disease-treated pathogen, but the HK-ISR1 strain showed a significant difference as 1.5 (FIG. 3). Through this, pathogens were not directly inhibited by the HK-ISR1 strain when invading, but it was confirmed that ginseng inhibits the disease by inducing the expression of defense-related genes and inducing systemic resistance.

Bacillus Megaterium  Strain HK - ISR1 Antimicrobial activity against ginseng pathogens Pathogens Antimicrobial Activity ^＊ Rodrigo published Carphone Destruction Tansu (Cylindrocarpon destructans ) - Rhizoctonia Solar Age (Rhizoctonia solani ) - Sclerotinia Sclerotinia sclerotiorum ) - Fuzzium Solai ( Fusarium) solani ) - Botrytis cinerea ) - Phytophthora cactorum ) - Fish help stop Ultima (Pythium ultimum ) - Alternaria panax ) - Colletotrichum gloetosporioides gloeosporioides ) -

＊ antibacterial activity; -: Restraining  none.

< Example  4> Changes in Ginseng Root Tissue HK - ISR1  Induction Resistance of Strains

Roots were collected from the seedlings treated in Example 3 and examined for lignin accumulation in the root cell wall. 30-day-old roots were thinned, fixed with formalin-acetic acid-alcohol, dehydrated with ethanol and tertiary butyl alcohol (TBA), and stained with rootoglucinol-hydrochloric acid solution (Sigma, USA) for 1 minute. Accumulated lignin was observed.

  As a result, lignin was clearly accumulated in the cell wall of epidermal tissue cells, and it was confirmed that the HK-ISR1 strain induced systemic resistance to ginseng (FIG. 4).

< Example  5> HK - ISR1  Of strain culture Ginseng root rot  Control effect investigation

The present inventors confirmed whether the HK-ISR1 strain has an effect of inhibiting root rot in seedlings. Specifically, HK-ISR1 strains were incubated in TSA (Tryptic soy agar, Becton, Dickinson and company, USA) for 2 days to prepare a suspension of 1 × 10 ⁸ cfu / ml concentration, and the seedlings were soaked in the suspension for 1 hour. Soil and Pitmoss mixed with Cylindrocarpon destructans are mixed 1: 3 (w / w) in plastic pots (20 cm × 20 cm × 20 cm), and 10 roots of seedlings per pot are transplanted and grown. chamber). After 30 days, the number of stems sprouted was measured to measure the seedling rate, and the degree of root rot was examined to determine the prevalence of root rot.

As a result, in the case of seedling rate, the treatment of HK-ISR1 strain culture medium was 79%, 7 times higher than the 11% untreated group, and there was no statistically significant difference with the seeding rate of healthy soil (85%) (FIG. 5). In the case of root disease (total ratio of root rot disease by individual), 20% were treated with HK-ISR1 strain culture, and 50% was untreated with 60% control. The effect was shown (FIG. 6).

[Control Effect = (Non-Treatment Incidence-Treatment Incidence / Non-Treatment Incidence) × 100]

This confirmed that the HK-ISR1 strain inhibits ginseng root disease.

< Example  6> HK - ISR1  Ginseng yield and treatment of strain culture Whole seedling ratio  Field test of the effect on the increase

The present inventors confirmed whether the HK-ISR1 strain culture medium has an effect on the inhibitory effect and yield of root rot in farm packages. Specifically, the suspension was prepared by culturing the HK-ISR1 strain of the present invention in TSA medium for 2 days. In November 2004, 1 × 10 ⁸ cfu / ml HK-ISR1 strain suspension was diluted 100-fold with sterile water in ginseng seed, ginseng seeded in ginseng, and sprinkled on pit moss by 3 l per compartment to mix the topsoil. Thereafter, the seeds were soaked in HK-ISR1 strain suspension (1 × 10 ⁸ cfu / ml) for 30 minutes before planting and sown. Meanwhile, the antagonist treatment group The antagonistic suspension of 1 × 10 ⁷ cfu / ml concentration was diluted 100 times with sterile water and sprinkled onto pit moss 3 l per compartment to mix the topsoil. Thereafter, the seeds were soaked in antagonistic suspension (1 × 10 ⁷ cfu / ml) for 30 minutes before planting, and then sown. Conventional pesticide cultivation was immersed in ginseng seeds in a 1000-fold dilution of Toros hydrate. The four treatment groups (non-treated areas, gilhanggyun treatments, HK-ISR1 strain sphere and pesticide practice balls) was placed in three repeated on experimental (Ⅰ, Ⅱ, Ⅲ). According to the processing of seedling production in March 2006, the percentage of whole seedlings (healthy seedlings to sowing seeds) was 47.3%, which was the highest in the HK-ISR1 strain treatment group, which was more than 21.7% of pesticide practices. Also, 11.1% was higher than 36.2% of the antagonistic treatment group, that is, Bacillus subtilis, which is an antagonistic microbial agent (FIG. 7 and Table 2). The yield of whole seedlings was 2.6 times higher in the HK-ISR1 strain treatment group than in the pesticide practice group (p = 0.01) and 16% more than the antagonist microorganisms (Fig. 8 and Table 3).

Through the above results, it was confirmed that the HK-ISR1 strain can sufficiently inhibit the root rot pathogens in the general packaging.

* There is no significant difference between mean values of the same character.

* There is no significant difference between mean values of the same character.

< Formulation example  1> Preparation of Microbial Agents

<1-1> Preparation of Hydrating Agent

The inventors of the present invention using the HK-ISR1 strain or culture medium thereof confirmed to have inhibitory activity against the ginseng root rot pathogen in Example 3, to prepare a microbial preparation for the control of ginseng root rot pathogen, as a powder for the purpose of stable formulation Hydrating agents were prepared which were hydrated when diluted in water. Specifically, the hydrated powder was ground by drying for 2 hours in a dryer at 70 ℃. Then surfactant and extender were added and mixed. The mixture was ground by passing through a Jet-O-mill (Aljet) and the ground powder was tested for hydration to prepare a microbial formulation in the form of a hydrate.

<1-2> Granular  Produce

The present inventors prepared granules that can be used as they are in the particle state for the purpose of stable formulation of microbial preparations using the HK-ISR1 strain. Specifically, the granules were ground and dried for 2 hours in a dryer at 70 ° C. for pulverization. The pulverized powder is pulverized again through a jet-O-mill, and then the microbial pulverized powder, surfactant, extender / nutrient and disintegrant are 50% spores of the strain, 3% polycarboxylate, and 3% sodium lignosulfonate. , Sodium dialkyl sulfosuccinate 1%, starch 10%, bentonite 20%, talc 13% of the mixture was mixed with about 35% of the total powder of the powder mixture and kneaded. After the dough was finished, it was granulated using a granulator (1 mm in diameter, 1-5 mm in length) and dried in a dryer at 70 ° C. The dried product was passed through a 16-30 mesh sieve to remove the powder to prepare granules.

<1-3> Preparation of Encapsulated Formulations

The present inventors intended to prepare an encapsulated formulation for the purpose of stable formulation of the microbial formulation using the HK-ISR1 strain. Specifically, 11% soybean meal, 6% green beans, 4% rice flour, 4% potato starch, 4% rye flour, and 3% ocher were mixed under pressure sterilization at 121 ° C. for 20 minutes, and then mixed again before cooking to cool in cold water. . After thoroughly mixing for 10 minutes or more in a stirrer (d = 5 cm, 200 rpm or more), the microbial sterilization encapsulation formulation was prepared by thoroughly adding the additive and Bacillus megaterium strain HK-ISR1 while stirring was continued.

<1-4> Production of microorganism organic material (compost)

Microbial formulations were incubated at 30 ° C by dry heat or steam sterilization of coffee foil, coir, peat moss, organic fertilizer and the like.

The microbial agent containing Bacillus megaterium HK-ISR1 strain or its culture medium as an active ingredient against ginseng roots and ground pathogens has no antagonistic ability against ginseng pathogens, but causes induction systemic resistance in plants themselves. Because of its ability to inhibit, it can be usefully used as an environmentally friendly microbial pesticide.

<110> Herbking <120> Microbial agents against ginseng plant pathogens that contains          induced systemic resistant Bacillus megaterium HK-ISR1 strain and          cultured solution of therefrom and prevention methods for ginseng          plant disease using the same <130> 6p-06-50 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1402 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of Bacillus megaterium HK-ISR1 <400> 1 acgagcgaac tgattagaag cttgcttcta tgacgttagc ggcggacggg tgagtaacac 60 gtgggcaacc tgcctgtaag actgggataa cttcgggaaa ccgaagctaa taccggatag 120 gatcttctcc ttcatgggag atgattgaaa gatggtttcg gctatcactt acagatgggc 180 ccgcggtgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcatagccga 240 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360 aaggctttcg ggtcgtaaaa ctctgttgtt agggaagaac aagtacaaga gtaactgctt 420 gtaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480 tacgtaggtg gcaagcgtta tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct 540 taagtctgat gtgaaagccc acggctcaac cgtggagggt cattggaaac tggggaactt 600 gagtgcagaa gagaaaagcg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660 gaacaccagt ggcgaaggcg gctttttggt ctgtaactga cgctgaggcg cgaaagcgtg 720 gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780 tagagggttt ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta 840 cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900 ggtttaattc gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaactcta 960 gagatagagc gttccccttc gggggacaga gtgacaggtg gtgcatggtt gtcgtcagct 1020 cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1080 gcatttagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1140 cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gatggtacaa 1200 agggctgcaa gaccgcgagg tcaagccaat cccataaaac cattctcagt tcggattgta 1260 ggctgcaact cgcctacatg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1320 tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1380 gaagtcggtg gagtaaccgt aa 1402  

Claims (9)

Bacillus megaterium ( Bacillus) has the following bacteriological characteristics and induces the induced systemicity of ginseng itself against ginseng disease megaterium ) HK-ISR1 strain: Iii) bacilli that are positive upon Gram staining; Ii) inert to hydrogen cyanide, proteinase, cellulase, pectinase, gluconase, indium acetic acid (IAA); And V.) Has homology with fatty acids of Bacillus megaterium. The Bacillus megaterium HK-ISR1 strain according to claim 1, which is described by accession number KACC 91261P. A microbial agent for controlling ginseng disease, comprising the Bacillus megaterium HK-ISR1 strain of claim 1 or a culture thereof as an active ingredient. The method according to claim 1 or 3, wherein the ginseng disease is Cylindrocarpon destructans ), Rhizoctonia solani ), sclerotinia sclerotinia sclerotiorum ), Fusarium solani ), Botrytis cinerea ), Phytophthora cactorum ), Pythium ultimum ), and Alternaria panax ), Colletotrichum gloetosporioides gloeosporioides ) is a microbial agent, characterized in that caused by one or more ginseng pathogens selected from the group consisting of. 4. The microbial preparation of claim 3, wherein the microbial preparation is a granule, a hydrating agent or a capsule. 4. The microbial preparation of claim 3, wherein the microbial preparation is prepared by adding a surfactant, extender or nutrient to Bacillus megaterium strain HK-ISR1 or its culture. The microorganism preparation according to claim 3, wherein the microorganism included in the microbial preparation is 1 × 10 ⁶ to 1 × 10 ⁹ cfu / ml per milliliter (ml). Method of controlling ginseng disease using the microbial agent of claim 3. The method of claim 10, wherein the microbial agent is sprayed on the roots or adjacent soils of ginseng, or sprayed frequently on the ground, and soaking seedlings or seeds.

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