KR20150011066A - Microbial agent for the prevention of disease of ginseng containing Bacillus amyloliquefaciens strain - Google Patents

Abstract

The present invention relates to a microbial agent for preventing root rot pathogens of ginseng, containing a Bacillus amyloliquefaciens strain. According to the present invention, the microbial agent for preventing root rot pathogens of ginseng comprises Bacillus amyloliquefaciens GR4-5 strain KACC91825P having antagonism against root rot pathogens of ginseng or a culture medium thereof as an active ingredient. The microbial agent containing the Bacillus amyloliquefaciens GR4-5 strain KACC91825P or the culture medium thereof has excellent antagonism against root rot pathogens of ginseng, and has no environmental pollution and human toxicity, thereby being used as an eco-friendly microbial pesticide for preventing root rot pathogens of ginseng.

Description

Technical Field [0001] The present invention relates to a microbial agent for inhibiting roots of roots of Bacillus amyloliquefaciens strain containing ginseng root of Bacillus amyloliquefaciens,

The present invention relates to a microbial agent for inhibiting root rot of a ginseng comprising a Bacillus amyloliquefaciens strain , and more particularly, to a microbial agent for inhibiting root rot of Bacillus amyloliquefaciens having an antagonistic action against major ginseng root rot disease bacteria , And a microbial agent for controlling ginseng root rot caused by ginseng, which contains strain GR4-5 as an active ingredient.

It is also reported that ginseng prevents cancer, inhibits aging, protects the liver, strengthens fatigue and brain function, and recently prevents environmental hormone damage. Ginseng, the first of all herbal medicines, is a plant that grows in an extremely limited environment. It has a longer growth period than other crops, and the cultivation method is also special and difficult.

Korea is a ginseng suzerain producing climatic ginseng, which is excellent in its medicinal effect due to its natural climate and cultivation area. However, many countries are increasingly interested in the ideal condition of ginseng as a health food or health care drug. In addition, existing ginseng producers as well as non-producers are actively promoting policies that encourage research and production of ginseng.

In Korea, China, Japan, the United States, and some parts of Europe, ginseng has been cultivated in Korea, China, Japan, Taiwan and Southeast Asia. Is expanding.

The ginseng production and consumption market in the world is difficult to accurately collect statistical data due to the characteristics of ginseng distribution structure in each country, diversity of product types and security measures of recent data. However, the export and import volume of Hong Kong, which is the center of the world ginseng sales market, , Ginseng production statistics of each country show that Korea, which maintained 46% of ginseng production in the world until the late 1980s, had a share of 39% in the 1990s, accounting for 0.5% China, which occupied 43% of the world production in the 1980s, grew by more than 50% in the 1990s, showing a high annual growth rate of more than 7.2%.

As such, Korean Ginseng has weakened international competitiveness in terms of price, and in order to enhance international competitiveness, it is necessary to study the development of cultivation technology to improve the quality of ginseng as a raw material. Especially, the most urgent problems in cultivation of ginseng in recent years are the absolute lack of proper place, high incidence due to worsening of soil conditions in the country, and pesticide residue due to increase of pesticide usage. There is an urgent need for intensive research on

In the meantime, many researchers have been focusing on the development of scientific ginseng cultivation techniques such as early establishment of ginseng production base, improvement and management of ginseng seeds, improvement of agricultural structure, improvement of cultivation method and promotion of mechanization. In particular, ginseng is cultivated under a sunshade structure unlike other crops, so the variation in quality and yield varies depending on environmental factors such as planting location, rainfall, temperature, wind strength, soil fertility, moisture content, The cultivation technology and safety are inferior to the main crops.

On the other hand, ginseng root rot symptoms (ginseng root symptoms), which are important in ginseng cultivation, are observed not only in aged paddy field but also in superpowder. Root symptoms are caused by dropping of the emergence and residual rate . ≪ / RTI > In fact, it is common for the seedlings that have been consumed at the farmhouse to have a survival rate of about 50% or less at harvest time after 5 years. This is because the root pathogens live in the area where the ginseng root rotates during the primary cultivation.

When ginseng was cultivated as a result of ginseng damage after root damage caused by root pathogens in the supercollade, the thick spores of residual root pathogens in the soil were uniformly distributed in the soil by 7-10 times tillage and rotary work It is thought that it will cause damage to ginseng.

Indeed, root rot is caused by Cylindrocarpon destructans , Fusarium solani , Pythium sp. , Phytophthora cactorum , Erwinia carotovora , ( Erwinia carotovora ) have been reported to be involved either singly or in combination (ginseng cultivation, standard farming manual p 103, Rural Development Administration, 2000).

In addition, the causes of the giant ginseng groundworm include Rhizoctoniasolani , Pythium sp. , And Alternaria panax , which cause root canals on the ground surface of seedlings . It is problematic because it is damaged by.

In order to reduce the density of pathogens in the soil or to control the disease, chemical control using soil fumigant is being tried through the use of organic materials to control the disease. .

However, the above-mentioned control method is difficult to obtain a clear effect, and soil-infectious pathogens such as ginseng root rot disease cause a disease by forming a thick spore in the soil, so that it is almost impossible to control. The use of soil fumigants has the potential to dominate the pathogen in five years due to the destruction of the soil ecosystem.

In particular, the major soil diseases of ginseng are infected at various temperature ranges, so once infected, the disease progresses slowly but it is damaged throughout the year. Therefore, in order to effectively control soil disease, the biological control of ginseng cultivation is considered to be the most suitable for the control of ginseng disease, which is a continuous and environmentally stable disease control effect on the nature of ginseng cultivation. In this regard, the ginseng root rot disease disease ( Cylindrocarpon destructans ) The identification and antagonistic action of Streptomyces species has been reported (2nd World Ginseng Symposium, Jung Hoon Jung, Chung Hee Chung, p. 67-74, 1978, Korea Ginseng Research Institute).

So far, a method of biological control using antagonistic microorganisms has been used as a method to control ginseng root rot disease, and in particular, a chemical control method using a soil fumigant such as a sand theory, a rosemide, and a rice paddy cultivation method The method is not systematized. In particular, the biological control studies on ginseng soil disease have been conducted mainly on the selection of antagonistic microorganisms and the effect of the treatment of the pesticides. However, there is a lack of understanding of the various rhizosphere soil environments, the antimicrobial activity of the selected strain against the pathogenic microorganisms is not high, Or antimicrobial activity of ginseng root rot caused by the inferiority of bactericidal ability in soil or seedling due to insufficient treatment methods.

On the other hand, the Bacillus subtilis B-4228 strain is ginseng enemy sides causative agents of penicillin PARFUMER rose over (Penicillium purpurogenum) and Trichoderma irregularities di that the effect of suppressing the growth of (Trichoderma viride) reported (Republic of Korea Patent No. 312 731 number).

However, there have been no studies on bacteria that exhibit the controlling effect against ginseng root rot disease among Bacillus amyloliquefaciens strains so far.

It is an object of the present invention to provide a microbial agent for inhibiting root rot of ginseng root rot, comprising as an active ingredient Bacillus amyloliquefaciens strain GR4-5 having an antagonistic action against the major ginseng root rot disease bacteria.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

In order to achieve the above object, the microorganism preparation for controlling the root rot of Ginseng according to the present invention is a Bacillus amyloliquefaciens GR4-5 strain KACC91825P or a culture thereof having an antagonistic action against the root rot- It is characterized by containing it as an active ingredient.

The ginseng root rot fungus may be selected from the group consisting of Cylindrocarpon destructans , Fusarium oxysporum , Fusarium solani , Rhizoctonia solani , Sclerotinia nivalis. ≪ / RTI >

The culture broth is a culture solution obtained by culturing Bacillus amyloliquefaciens GR4-5 strain KACC91825P at 25 to 30 DEG C for 3 to 5 days in a microorganism culture medium.

The microorganism culture medium is TSB (Trypticase Soy Broth) culture medium.

The microbial agent is characterized by being a granule, a wetting agent or a capsule.

The microorganism preparation is characterized by being prepared by adding a surfactant, an extender, and a nutrient to the Bacillus amyloliquefaciens GR4-5 strain KACC91825P or a culture thereof.

The microorganism contained in the microorganism preparation is characterized by having 1 × 10 ^{6 to} 1 × 10 ⁹ cells per milliliter (ml).

A method for producing a microorganism preparation for controlling root rot of a ginseng according to the present invention comprises the steps of: (a) adding a spore of Bacillus amyloliquefaciens GR4-5 strain KACC91825P to a TSB (Trypticase Soy Broth) medium in a concentration of ¹ x ¹⁰ conidia / ml Lt; / RTI > (b) culturing the inoculum at 25 DEG C for 3 to 5 days; And (c) removing impurities of the culture obtained in step (b).

The method of controlling ginseng root rot disease according to the present invention is characterized by using the microorganism preparation described above. Preferably, the microorganism preparation is concentrated by spraying or immersing the microorganism preparation in the roots of ginseng or the soil adjacent thereto.

As described above, the microorganism preparation containing the Bacillus amyloliquefaciens strain GR4-5 strain KACC91825P of the present invention or the culture thereof has excellent antagonistic ability against the germinacea root rot disease bacteria, and the environmental pollution and human toxicity It can be used as an environmentally friendly ginseng microbial pesticide for controlling root rot disease.

Figure 1 is a molecular system diagram based on the analyzed 16S rRNA gene sequence of the selected strain (GR4-5).
2 is a diagram showing growth inhibition against Cylindrocarpon destructans KACC44656 and KACC41077 of the selected strain (GR4-5).
3 is a diagram showing genes involved in biosynthesis of secondary metabolites produced by the selected strain (GR4-5).

Hereinafter, the present invention will be described in detail.

The microorganism preparation for controlling ginseng root rot disease according to the present invention contains a strain having an antagonistic ability against ginseng root rot fungi. At this time, the strain must be in a viable state, which may be in the form of mycelial and spore.

In the present invention, to isolate strains having antagonistic ability against ginseng roots rot root disease ginseng root rot and root pathogens, a total of 439 strains were isolated from ginseng cultivated soil in Gangwon province, A good GR4-5 strain is isolated from the soil.

The isolated GR4-5 strain is a major pathogen of ginseng root rot, Cylindrocarpon destructans), Fusarium oxy's forum (Fusarium oxysporum), Fusarium solani (Fusarium solani , Rhizoctonia solani and Sclerotinia nivalis , which are known to have a very high antimicrobial activity.

The starting strain is the GR4-5 16S rRNA gene sequences bar subjected to genetic analysis system with Bacillus amyl quinone Lowry Pacific Enschede (Bacillus amyloliquefaciens subsp . plantarum FZB42T ). As a result, it was confirmed that the GR4-5 strain having an antagonistic ability against the ginseng root rot rotavirus was Bacillus amyloliquefaciens GR4-5 strain.

Accordingly, the present inventor named this strain as " Bacillus amyloliquefaciens GR4-5 strain" and deposited this strain on the National Agricultural Research Service Agricultural Genetic Resources Support Center, and deposited the accession number KACC91825P on June 17, 2013 .

The Bacillus amyloliquefaciens GR4-5 strain KACC91825P consists of the entire nucleotide sequence shown in SEQ ID NO: 1 or a part thereof and is composed of an open reading frame (ORF) of 1457 bp.

In addition, the microorganism preparation for controlling ginseng root rot disease of the present invention may contain Bacillus amyloliquefaciens strain GR4-5 in the form of a culture solution.

The term " culture medium " in the present invention means a microorganism which is inoculated with a certain concentration of spores in a medium suitable for microorganism growth, inducing the microorganisms to grow for a certain period of time in an incubator, , And a nutrient component necessary for growth, and can be produced by a process of filtering off impurities.

The term " control " of the present invention generally includes preventing, reducing or eradicating plant pathogenic infections, wherein the prevention or reduction in infection means that there is a statistically significant difference for untreated host plants.

Therefore, when the Bacillus amyloliquefaciens GR4-5 strain is contained in the microorganism preparation for controlling root rot disease of the present invention in the form of a culture solution, the microorganism culture medium is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) A TSB (Trypticase Soy Broth) medium in which GR4-5 strain can be grown is used, and the culture broth of Bacillus amyloliquefaciens GR4-5 isolated as described above is added to the microbial culture at 25 to 30 DEG C For 3 to 5 days.

The microorganism preparation of the present invention can be prepared in the form of a wettable powder, granules or encapsulation for the purpose of stable formulation of microorganisms.

In the microorganism preparation of the present invention, the microorganism or the culture solution may be supplied in a composition for controlling germplasm of root rot of rot germs, may be separately stored for long-term storage, and may be mixed immediately before use. When the microorganism is separately supplied for long-term storage, it may be stored in glycerol stock solution at -70 ° C or lower or freeze-dried at -20 to -80 ° C.

The wettable powder of the present invention is prepared by drying and pulverizing a solid medium inoculated with a microorganism, and then adding and mixing a surfactant and an extender / nutrient. Examples of the surfactant include a surfactant such as polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Polyoxyethylene alkylarylpolyether, polyoxyethylene nonylphenyl ether, sodium sulphonate naphthalene formaldehyde, Triton 100 and Tween 80. The composition of claim 1, And one or more selected from the group consisting of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose, and starch is used as the extender and the nutrient.

In addition, the granule of the present invention is prepared by drying and pulverizing a solid medium inoculated with a microorganism, and then adding a surfactant, an extender / nutrient and a disintegrant. Examples of the surfactant include a surfactant such as polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Polyoxyethylene alkylarylpolyether, polyoxyethylene nonylphenyl ether, sodium sulphonate naphthalene formaldehyde, Triton 100 and Tween 80. The composition of claim 1, And one or more selected from the group consisting of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose, and starch is used as the extender and nutrient, and the disintegrant includes bentonite bentonite, talc, dialite, Use De (kaolin), calcium carbonate and at least one or two selected from the group consisting of (calcium carbonate).

In addition, the granule of the present invention may contain one or more than one selected from the group consisting of a surface active agent, an inert carrier, a preservative, a wetting agent, a feed accelerator, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protecting agent, And the like.

In the application of the microorganism preparation of the present invention, Bacillus amyloliquefaciens GR4-5 strain is concentratedly applied to the roots of ginseng root or the soil adjacent thereto, and the effective amount of the microorganism is 1 (microorganism) per 1 acre area X 10 < ⁷ > or more.

In addition, the application of the microorganism preparation can be carried out by directly spraying a solution diluted with a concentration of 1 × 10 ^{6 to} 1 × 10 ⁹ microorganisms per milliliter in the roots of ginseng, or 1 × 10 ^{6 to} 1 × 10 ⁹ per liter The ginseng can be immersed in the diluted solution for 1 to 2 hours.

Thus, the microorganism preparation of the present invention has an excellent antagonistic ability against the ginseng root rot disease bacteria and is useful as a microbial agent for controlling environment-friendly ginseng root rot disease because of environmental pollution and human toxicity.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples and Experimental Examples.

Example 1 Isolation and Selection of Strain

In May 2012, soil was collected from the ginseng pavement of Gangwon-do Agricultural Research and Extension Service in Cheolwon-gun, Gangwon-do to isolate microorganisms having pathogenic forces against major pathogens causing ginseng root rot disease.

The soil samples were prepared by diluting 2 g of soil sample with sterile physiological saline to make a dilution suspension (10 ^-1 ~ 10 ^-6 ), and 0.1 ml of TSA (tryptase-soy agar; BBL) And R2A (Difco) medium.

A total of 439 strains were isolated from 228 strains and 221 strains from TSA medium and R2A medium, respectively, for 5 days at 30 ℃.

The first experimental strain was Rhizoctonia ( Rhizoctonia) , which is relatively sensitive to antagonistic microorganisms solani ) KACC40123, and 31 isolates among the selected strains were observed to inhibit mycelial growth of pathogenic bacteria.

In addition, strains first selected were further tested for mycelial growth inhibition of pathogenic bacteria against Sclerotinia nivalis KACC45152.

In the second experiment, 6 strains showed mycelial growth inhibition, and 3 strains (GP4-1, GR2-2, GR4-5) showing the greatest activity were finally selected.

The culture medium of the strain was cultured in a 30 ° C agitator for 3 days using TSB (Trypticase Soy Broth; Difco co.) Medium.

Example 2 Identification of the Selection Strain (GP4-1, GR2-2, GR4-5) of the Present Invention

The identification of the strain selected in Example 1 was carried out by 16S rRNA gene sequencing.

(1) 16S rRNA sequencing

The 16S rRNA gene was analyzed to identify the three selected strains (GP4-1, GR2-2, GR4-5).

3 strains (GP4-1, GR2-2, GR4-5) were inoculated into NB (Nutrient Broth; Difco) and cultured at 30 ° C for 24 hours before the 16S rRNA gene was analyzed. Respectively.

Harvested cells are using the 27F (AGAGTTTGATCCTGGCTCAG) and ^{1492R (GGTTACCTTGTTACGACTT) G-Spin TM} Genomic DNA Extraction Kit (Intron, Korea) Universal primer (Universal primer) to amplify the 16S rRNA gene was extracted DNA using , PCR reaction was performed.

The amplified PCR products were purified using QIAquick PCR Purification kit (QIAGEN), and sequenced by Genentech (Korea). Seqman (DNASTAR, USA) program was used for analysis.

The nucleotide sequences of the comparative standard strains were downloaded through the EzTaxon-e server (http://www.eztaxon.org), and the nucleotide sequences were aligned with the Clustal W program. The aligned nucleotide sequences were analyzed using the MEGA 3.0 program et al., 2004).

Using the Neighbor-joining algorithm (Saitouand Nei, 1987), bootstrapping (Felsenstein, 1985) was performed 1000 times to confirm the robustness of the system. The analyzed nucleotide sequences were registered in the GenBank online database.

(2) Results of 16S rRNA sequencing

It was created a molecular flow diagram as shown in Figure 1 based on the analysis of 16S rRNA gene sequences, selected strain (GP4-1, GR2-2, GR4-5) both in Bacillus amyl quinone Pacific yen (Bacillus amyloliquefaciens ) subsp. and showed the highest homology with plantarum FZB42 ^T (99.9%). We also found that Bacillus sp. M27 (GenBANK NO.AMPK00000000) strain.

<Experimental Example 1> Antimicrobial activity test of the selected strain (GP4-1, GR2-2, GR4-5)

(1) Preparing the experiment

The isolated strains (GP4-1, GR2-2, GR4-5) of Example 1 were treated with 5 kinds of ginseng roots rot fungi ( Cylindrocarpon destructans) KACC44656 KACC41077 and Fusarium oxy's forum (Fusarium oxysporum ) KACC40037, Fusarium solani KACC44891, Rhizoctonia solani ) KACC40123, Sclerotinia ( Sclerotinia nivalis KACC45152) were purchased from the Korean Agricultural Culture Collection (KACC) of RDA.

Each isolated pathogen was cut with an 8 mm cork borer and pinched in the center of PDA (Potato Dextrose Agar; Difco) medium. The isolated strain (GP4-1, GR2-2, GR4-5) .

Replacing the optimum for each pathogen culture growth temperature (cylinder throw car phone des truck Tansu (C. destructans), Klee's Loti California you balise (S.nivalis) is 21 ℃, Fusarium solani (F. solani), Fusa After incubation for 4 to 5 days at 25 ° C in F. oxysporum and R. solani, the mycelial growth inhibition ability of the selected strains was examined.

(2) Experimental results

As a result of examining the mycelial growth inhibition ability of the three selected strains (GP4-1, GR2-2, GR4-5) against 5 kinds of ginseng root rot disease fungi, .

Strain C. destructans F. oxysporum F. solani R. solani S. nivalis GP4-1 ++ ^* ++ ++ +++ ++ GR2-2 ++ ++ ++ +++ +++ GR4-5 +++ +++ +++ +++ +++

* The inhibition of growth of 5 species of ginseng root rot disease by 3 strains selected at last

+ Is inhibition zone 1 ~ 3mm, ++ 4 ~ 10mm, +++ 10mm or more

The three selected strains (GP4-1, GR2-2, GR4-5) showed comparable antibacterial activity, but the GR4-5 strain showed stronger activity than the other two strains.

In particular, the apparent path drag the draw Carphone Death trucks Tansu (C. destructans) KACC44656 and KACC41077 appeared as 2 proves that these strains are greater excellence as a microbial agent for the control of ginseng root rot.

Check the secondary metabolite gene of <Experiment 2> selected strain (GP4-1, GR2-2, GR4-5)

(1) Preparing the experiment

PCR assays were used to identify genes involved in the biosynthesis of secondary metabolites produced by selected strains (GP4-1, GR2-2, GR4-5).

Bacillus (Bacillus) For detection of bacillomycin D, fengycin, iturin A, surfactin and zwittermicin A, which strains generally produce, bmyA (875 bp), fend (964 bp), ituA (647 bp), srfA (441 bp) and zwiA (779 bp).

The sequences of the primers used in the PCR are shown in Table 2 below.

Antibiotic Name of the primer
(primer name) Primer sequence
(Primer sequence 5'-3 ') Bacillomycin D
BACC1F GAA GGA CAC GGC AGA GAG TC BACC1R CGC TGA TGA CTG TTC ATG CT Fengycin
FEND1F TTT GGC AGC AGG AGA AGT TT FEND1R GCT GTC CGT TCT GCT TTT TC Iturine
ITUD1F GAT GCG ATC TGG TTG GAT GT ITUD1R ATC GTC ATG TGC TGC TTG AG Surfactin
SUR3F ACA GTA TGG AGG CAT GGT C SUR3R TTC CGC CAC TTT TTC AGT TT Zwittermicine
ZWITF2 TTG GGA GAA TAT ACA GCT CT ZWITR1 GAC CTT TTG AAA TGG GCG TA

Reaction mixture (2X TOPsimple ^™ DyeMIX-Tenuto; Enzymonics Co., Korea) was used for the PCR reaction. 1 μl of genomic DNA (50 ng) and 10 pmol primers were added. The PCR reaction temperature was determined by denaturation (95 ° C., 30 seconds), annealing (annealing, 55 ° C., 45 seconds), initial denaturation (95 ° C., Elongation (72 ° C, 1 minute) was performed, and the final elongation reaction was carried out at 72 ° C for 5 minutes.

(2) Experimental results

As shown in Fig. 3, iturin A and surfactin biosynthetic genes were detected in the selected strains (GP4-1, GR2-2, and GR4-5) No biosynthetic genes for fengycin and zwittermicin A were detected. However, bacillomycin D biosynthesis gene was detected only in GR4-5 strain. These results suggest that GR4-5 strain may additionally produce bacillomycin D. Actually, in the antimicrobial activity test, this strain (GR4-5) has slightly higher activity than the other two strains (GP4-1, GR2-2) The results are consistent with what is shown.

Therefore, antagonistic action against ginseng rot rot caused by GR4-5 among several selected strains can be presumed to be caused by antibiotic substances of nonribosomal lipopeptide series such as iturin A and surfactin.

<Experimental Example 3> Biochemical characteristics of the selected strain (GP4-1, GR2-2, GR4-5)

(1) Experimental method

To investigate the biochemical activity of the selected strains (GP4-1, GR2-2, GR4-5), the size of the clear zone was examined after inoculation of the separated bacteria into each test medium. LB (Luria-Bertani) medium supplemented with 0.5% (w / v) skim milk (Difco) was used to confirm protease activity. LB (Luria-Bertani) medium supplemented with 0.5% (w / v) CMC (carboxylmethyl-cellulose) was used to confirm cellulase activity.

Fibrinolytic clearing The clear zone was washed with 0.5% Congo red solution for 30 minutes, washed with sterile distilled water and reacted for 15 minutes in 1M NaCl solution to confirm the presence of a transparent ring. In order to confirm chitinase activity, a clear zone was observed by incubating in R2A medium (Difco) supplemented with chitin 1% (w / v).

In addition, we observed phosphoric acid solubilization ability (phosphatase), which is in an insoluble state in the soil and promotes plant growth by increasing the soluble phosphoric acid content in the soil.

(2) Experimental results

Cellulase, chitinase and protease were investigated in order to examine cell wall degradation characteristics of selected strains (GP4-1, GR2-2, GR4-5). The results were as follows: Table 3 shows the enzymatic activity of the fibrinolytic, chitinolytic and proteolytic enzymes in the case of a transparent ring of 1 to 3 mm +, a transparent ring of 4 to 10 mm of ++, and a transparent ring of 11 mm or more of +++.

Strain Fibrin resolution
(Cellulase) Chitin resolution
(chitinase) Protein resolution
(protease) Phosphoric acid solubilization ability
(phosphatase) GP4-1 ++ - ++ - GR2-2 ++ - ++ - GR4-5 ++ - ++ -

As shown in Table 3, it was confirmed that all the strains had the ability of resolving fibrin and protein, and the chitin resolving ability was not confirmed. These enzymatic features are consistent with similar biochemical metabolic pathways and similar taxonomic locations. These biochemical features are also expected to be involved in the infiltration of crops and pathogens.

As a result of confirming the phosphatase, all strains (GP4-1, GR2-2, GR4-5) showed negative results.

Thus, the microorganism preparation containing the Bacillus amyloliquefaciens strain of the present invention or the culture thereof has excellent antagonistic ability against the ginseng root rot disease bacteria, and is environmentally friendly and does not have human toxicity. Thus, the ginseng root rot disease It can be used effectively as microbial pesticide for control.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Agricultural Biotechnology Research Institute KACC91825 20130617

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microbial agent for the prevention of disease of ginseng          containing Bacillus amyloliquefaciens strain <130> p2013-0089 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1457 <212> DNA <213> Bacillus amyloliquefaciens GR4-5 <400> 1 ctcaggacga acgctggcgg cgtgcctaat acatgcaagt cgagcggaca gatgggagct 60 tgctccctga tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact 120 gggataactc cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga 180 cataaaaggt ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt 240 gaggtaacgg ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac 300 tgggactgag acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg 360 gacgaaagtc tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc 420 tgttgttagg gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca 480 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 540 cggaattatt gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc 600 ggctcaaccg gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga 660 attccacgtg tagcggtgaa atgcgrtaga gatgtggagg aacaccagtg gcgaaggcga 720 ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag 840 tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg 1020 gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 1140 tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta 1260 agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag 1320 ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc ngggccttgt 1380 acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgag gtaaccttta 1440 tggagccagc cgccgaa 1457

Claims (10)

Bacillus amyl Lori rake Pacifico Enschede (Bacillus having antagonistic activity against root rot of ginseng Won Gyun amyloliquefaciens GR4-5 strain KACC91825P or a culture thereof as an active ingredient,
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
The ginseng root rot fungus may be selected from the group consisting of Cylindrocarpon destructans , Fusarium oxysporum , Fusarium solani , Rhizoctonia solani , &Lt; / RTI &gt; Sclerotinia nivalis ,
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
Wherein the culture medium is a culture obtained by culturing Bacillus amyloliquefaciens GR4-5 strain KACC91825P at 25 to 30 DEG C for 3 to 5 days in a microorganism culture medium.
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
Wherein the microbial medium is TSB (Trypticase Soy Broth)
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
Wherein the microbial agent is a granule, a wetting agent or a capsule.
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
Characterized in that the microorganism preparation is prepared by adding a surfactant, an extender and a nutrient to the Bacillus amyloliquefaciens GR4-5 strain KACC91825P or a culture thereof.
Microorganism preparation for controlling root rot rot fungus of ginseng.
The method according to claim 1,
Characterized in that the microorganism contained in the microorganism preparation is 1 x 10 ^{6 to} 1 x 10 ⁹ per milliliter (ml)
Microorganism preparation for controlling root rot rot fungus of ginseng.
A method for producing a microorganism preparation for inhibiting root rot of a ginseng root comprising the steps of:
comprising the steps of: (a) the spores of the TSB (Trypticase Soy Broth) culture medium for Bacillus amyl quinone Lowry Pacific Enschede (Bacillus amyloliquefaciens) GR4-5 strain KACC91825P inoculation such that 1 × 10 ⁶ conidia / ml;
(b) culturing the inoculum at 25 to 30 DEG C for 3 to 5 days; And
(c) removing the impurities of the culture obtained in the step (b).
A method for controlling ginseng root rot disease using a microorganism preparation according to any one of claims 1 to 7.
10. The method of claim 9,
Characterized in that the microbial agent is concentrated, sprayed or immersed in the roots of ginseng or the soil adjacent thereto.

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