KR100518776B1 - Microbial agent for the prevention of disease of ginseng containing Bacillus subtilis strain - Google Patents

Abstract

The present invention relates to a microbial preparation for controlling ginseng pathogens, and more particularly, ginseng containing Bacillus subtilis strain ( Bcillus subtilis ) B-4228 or the microbial culture medium as an active ingredient having an antagonistic ability against ginseng root rot pathogens. It relates to a microbial agent for controlling root rot disease and a method for controlling ginseng root rot disease using the microbial agent. The microbial agent of the present invention can be usefully used as an environmentally friendly microbial pesticide with antagonistic ability against phytopathogenic fungi.

Description

Microbial agent for the control of ginseng pathogens, including Bacillus subtilis strains

The present invention relates to a microbial preparation for controlling ginseng pathogens, and more particularly, to a microbial agent containing Bacillus subtilis strain B-4228 or a microbial culture medium as an active ingredient having an antagonistic ability against ginseng root rot pathogens. It is about.

Korean ginseng is Korea's unique natural resource, and it is a representative herbal medicine of Korea, which has been widely used for thousands of years as an oriental medicine or medicine for the health of mankind. As Western medicine has shown limitations in overcoming the side effects of synthetic drugs and intractable diseases, the value of herbal medicines and natural products including ginseng is emerging. Ginseng prevents cancer, inhibits aging, protects the liver, strengthens fatigue and improves brain function, and recently, it has been reported to prevent the damage of environmental hormones. Ginseng, the chief of all herbal medicines, is a plant that grows in extremely limited environments. It has a longer growth period than other crops, and its cultivation method is special and difficult, and has been produced only in some countries of the world.

Korea is a ginseng seeding country that produces Korean ginseng with excellent medicinal effects due to its climate and climate suitable for its natural growth and cultivation. However, many countries are interested in the ideal condition that ginseng has as a health food or health drug, and not only existing ginseng producing countries but also non-producing countries are actively promoting policies to encourage the research and production of ginseng. Ginseng growers include Korea, China, Japan, the United States, and parts of Europe. In the past, most of the countries that consumed ginseng were Confucian culture, including Korea, China, Japan, Taiwan, and Southeast Asia. This is expanding. Although the size of ginseng production and consumption market in the world is difficult to collect statistical data accurately due to the characteristics of ginseng distribution structure of each country, variety of product types and security measures of recent data, it is centered on import and export of Hong Kong, the center of global ginseng sales market According to the ginseng production statistics of each country, Korea, which maintained 46% of global ginseng production by the late 80s, decreased its share to around 39% in the 90s, 0.5% of the world's average production growth rate. In contrast, China, which accounted for 43% of the world's production in the 1980s, increased to more than 50% in the 1990s, showing a high production growth rate of more than 7.2% per year. As such, Korean ginseng is weakening its international competitiveness in terms of price, and in order to strengthen its international competitiveness, research on the development of cultivation technology to improve the quality of fresh ginseng is needed. In particular, the most urgent problem in the recent ginseng cultivation is the lack of proper scheduled land, high incidence due to deteriorated soil conditions, and many pesticides cannot be exported due to residual pesticides due to the increase of pesticide use. Intensive research is urgently needed.

Many researchers have been concentrating on the development of scientific ginseng cultivation technology, including the establishment of ginseng production base, the improvement and management of seed, improvement of agricultural structure, improvement of cultivation methods and promotion of mechanization. In particular, ginseng is cultivated under the inverted structure unlike other crops, so the quality and yield varies depending on environmental factors such as planting location, rainfall, temperature, wind strength, soil fertility, moisture content, breathability and acidity. It is inferior in cultivation technology and safety compared to major crops.

On the other hand, ginseng root rot symptoms (ginseng root symptom), which are important for ginseng cultivation, have been observed not only in arable land but also in cropland. Root symptom is grown as old root after seedling planting. It is thought to be one of. In fact, seedlings planted at farms usually have a residual rate of less than about 50% when harvested after 5 years. This is because root pathogens inhabit the roots of ginseng during herbivore cultivation. In this way, when ginseng is damaged by root pathogens in cropland, when the ginseng is grown as a series, the thick film spores of remaining root pathogens in the soil are distributed evenly in the soil by 7-10 times tiller and rotary operation when managing the planned site. If it is more likely to cause damage to ginseng. The root causes of root rot are actually Cylindrocarpon destructans , Fusarium solani , Pythium sp., Phytophthora cactorum , and Erwinia carotobora . ( Erwinia carotovora ) has been reported to be largely involved alone or in combination (Ginseng cultivation, Standard Agricultural Manual p 103, Rural Development Administration, 2000). In addition, the ground diseases of ginseng include Rhizoctonia solani , Pythium sp., Which causes mozzarlock disease, which occurs on the ground of seedlings, and Alternaria panax , which causes spots . It is a problem because it is damaged by the back. In order to control these diseases, management of planned sites through the use of organic substances to reduce the density of the pathogens in the soil or to suppress the onset and chemical control using soil fumigants are being attempted. It is becoming. However, it is difficult to achieve a clear effect by the above control methods, and soil infectious pathogens such as ginseng root rot are almost impossible to control because they form a thick spore in the soil. The use of soil fumigants also threatens to prevail in pathogens for 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 they are infected, they will be damaged year-round although the disease will progress slowly. Therefore, for effective soil disease control, it is considered that biological control is most suitable as a countermeasure for continuous ginseng disease that is effective in the nature of ginseng cultivation and is stable for the environment, and shows antagonistic activity against Cylindrocarpon destructans in Korea. The identification and antagonism of Streptomyces species has been reported (Second World Ginseng Symposium, Huh-Sup Chung, Chung-Hee Kim, p 67-74, 1978, Korea Ginseng Research Institute).

Until now, as a means to control the ginseng root rot disease, biological control methods using antagonistic microorganisms and chemical control methods using soil fumigants such as cylon and bassamide, especially in arable lands, and seedling methods such as paddy cultivation of answer Gyunhwan, have been used. The method is not organized. In particular, the biological control research on ginseng soil disease has been mainly conducted for the selection of antagonism microorganisms and the evaluation of pavement treatment effects, but there is a lack of understanding of the various root zone soil environment, the antibacterial activity of the selection bacteria against target pathogens, Or antagonistic microorganisms with excellent control effect against ginseng root rot have not been found due to poor treatment methods, such as poor ability to settle bacteria in soil and seedlings.

On the other hand, Bacillus subtilis B-4228 strain has been reported to have the effect of inhibiting the growth of Penicillium purpurogenum and Trichoderma viride , the causative agent of ginseng reddening (Korea Patent Registration No. 312731 number).

The present inventors completed the present invention by revealing that the Bacillus subtilis strain has an antagonistic action against ginseng root rot, while continuing research on the microbial agent having a control effect against ginseng root rot.

An object of the present invention relates to a microbial preparation for controlling ginseng root rot and a method for controlling ginseng root rot using the microbial agent.

In order to achieve the above object, the present invention provides a microbial preparation for controlling ginseng root rot disease.

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

Hereinafter, the present invention will be described in detail.

The present invention provides a microbial preparation for controlling ginseng root rot disease.

The microbial preparation for ginseng root rot disease control of the present invention contains a strain having a antagonistic ability against the ginseng root rot pathogen or a culture thereof.

In the present invention, while inhibiting the ginseng root rot pathogens, in particular, strains with antagonistic ability against root pathogens and terrestrial pathogens of ginseng inhibit the red pigment production of the red ginseng causative bacteria in the existing patent (Korean Patent Registration No. 10-0312731) It was confirmed that Bacillus subtilis B-4228 (Accession Number; KCTC 0657BP) and its culture, which have been reported to inhibit growth, also exhibit antagonism against ginseng root rot pathogens (see FIGS . 1 and 2 ). .

Ginseng root rot pathogens showing the antagonism of the strain of the present invention include Alternaria panax , Phytophthora cactorum , Rhizoctonia solani , Botrytis cineraria ( Botrytis) cinerea ) and Cylindrocarpon destructans .

Accordingly, the present invention provides a microorganism preparation for controlling ginseng root rot disease containing Bacillus subtilis strain B-4228 or a culture solution of the microorganism having an antagonistic ability against ginseng root rot pathogen. 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 microorganism or the culture medium is ginseng root rot pathogen It may be supplied as contained in the composition for control, 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 glycerol stock solution or may be used by freeze-drying at -20 to -80 ° C.

The hydrating agent of the present invention is prepared by drying and pulverizing a solid medium inoculated with microorganisms, followed by mixing by adding a surfactant and an 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 Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 At least one selected from the group consisting of one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch.

In addition, 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 Group consisting of alkyl aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80 One or two or more selected from the group consisting of one or two or more selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch, and bentonite as a disintegrant. (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 granules of the present invention may include 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 for microorganisms. It can be prepared by further addition.

Application of the microbial agent of the present invention is concentrated by spraying Bacillus subtilis B-4228 on the roots or adjacent soil of ginseng, the effective amount of microorganisms is sprayed to the number of microorganisms per 1 (10 m2) of arable land more than 1 × 10 ⁷ It is desirable to.

In addition, the microbial agent is applied miririteo ^{(㎖) 1 × 10 6 ~1} × 10 9 mari a solution diluted with a biomass concentration directly sprayed on the roots of the ginseng, or the advance per liter of 1 × 10 ⁶ ~1 × 10 ⁹ pigs per in Ginseng may be immersed in a solution diluted to a microbial concentration of 1 to 2 hours.

In a preferred embodiment of the present invention, Bacillus subtilis strain B-4228 was immersed in seedlings (1 year old) after culturing, and 0.5 liter (L) per 1 a (A) was used. The microorganisms of the present invention were treated with soil taken from the Cylindrocarpon distractances and Fujurium genus, which causes certified extreme root rot. As a result, the soil treated with the microbial agent of the present invention showed a healthy root ratio (ratio of disease free roots) of more than 82%, and the untreated soil showed a healthy root ratio of 20% and the morbidity rate (root rot by individual). The total ratio of disease degree was 6.0% in the soil treated with microbial agents and 50.4% in the untreated soil (see FIG. 4 ). Ginseng root rot pathogens that can obtain the control effect using the microorganism of the present invention include the root pathogen or ground pathogen of ginseng, Alternaria panax , Phytophthora cactorum , lysatonia sorani ( Rhizoctonia solani ), Botrytis cinerea and Cylindrocarpon destructans .

Therefore, the microbial agent of the present invention has excellent antagonistic ability against ginseng root rot pathogen and can be usefully used as an environmentally friendly microorganism agent for controlling ginseng root rot disease because it does not have environmental pollution and human toxicity.

The present invention also provides a method for controlling ginseng root rot using the microbial agent.

The present invention is to control the ginseng root rot disease by applying the Bacillus subtilis B-4228 or its culture solution having a ginseng root rot pathogen control effect to the ginseng or the surrounding soil, wherein the Bacillus subtilis B-4228 is rooted It may be applied directly to the soil or in the form of a microbial agent prepared above.

Application of the microbial agent of the present invention can be concentrated by spraying the diluted solution of Bacillus subtilis B-4228 in the root or adjacent soil of ginseng, or the ginseng can be immersed in the microbial preparation for 1-2 hours.

When spraying microorganisms directly or applying them in the form of microbial preparations, the effective amount of microorganisms is preferably sprayed with more than 1 × 10 ⁷ microorganisms per cultivated area (㎡), and the number of microorganisms applied to microbial preparations is Dilution to a concentration of 1 × 10 ^{6 to} 1 × 10 ⁹ per ml (ml) is preferred.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Identification of Ginseng Root Pathogens

The present inventors cut the border between the diseased and healthy parts of ginseng showing root rot to a size of about 3 mm and then immersed in sodium hypochlorite for about 30 seconds, washed with sterile water, and then removed from the filter paper to remove the agar. Placed on a plate, this was incubated at 24 ° C. for 3 days and identified based on morphological features on a microscope.

As a result of the isolation of the strains causing root rot, that is, root disease and ground disease, from the ginseng of the cultivation process, the microorganisms are alternaria panax, phytophthora mectorum, lysatonia sorani, botrytis cineri and It was confirmed that the strain was a cylinder carcap distractance.

Example 2 Investigation of Antagonistic Ability of Ginseng Root Pathogens of Bacillus Subtilis Strains

The present inventors have previously reported that the Bacillus subtilis strain B-4228 (Accession Number; KCTC 0657BP), which has been reported to antagonize the causative agent of ginseng redness in the existing patent (Korean Patent Registration No. 312731), isolated from Example 1 above. To investigate the effect on the growth of ginseng root rot pathogens were replaced. Specifically, PDK medium (Potato Dextrose Broth) 10g, Peptone (5g), Agar (15g), 1L of distilled water, DIFCO) was used as a medium for the replacement culture experiment. 70 μl of Bacillus strain B-4228 suspension was inoculated into a sterile 8 mm diameter paper disk, and then dispensed into Petri dishes and placed on a plate of PDK medium prepared. Ginseng root rot pathogens isolated in Example 1 for replacement culture were incubated in PDA (Potato Dextrose Agar) 39g, Agar 5g, distilled water 1ℓ, DIFCO medium for 7 days, respectively, and diameter 6mm. Using a sterilized kolkborough of the mycelia grown medium was removed and replaced with the Bacillus strain on the plate of PDK medium was haejugo. Bacillus strains and ginseng root rot pathogens were healed and incubated in a thermostat at 28 ° C. for 7 days.

As a result, mycelial growth of ginseng pathogens was inhibited, so that the mycelia grew only in the opposite direction to the Bacillus strain, or the growth was inhibited as a whole. Therefore, the Bacillus strains are not only the ginseng root cause bacteria, but also the ginseng root rot pathogens, in particular, the above-mentioned Alternaria panax, phytophthora factoraum, lysatonia sorani, botrytis ceria, and silindrocapon distractance It was confirmed that the growth inhibitory effect on the ( FIG. 1 and 2 ).

From the above results, it was found that Bacillus subtilis strain B-4228 was a microorganism having growth inhibition activity against ginseng root rot pathogen.

<Example 3> Experiment on the control effect of ginseng root rot disease of the culture

The present inventors confirmed whether Bacillus subtilis strain B-4228 has a ginseng root rot control effect directly on seedlings. Specifically, after dipping seedlings in the culture medium of Bacillus subtilis strain B-4228 1.2 × 10 ⁹ CFU / ㎖, and collected the soil of alveolar soil with Cylindrocarpon destructant ( Cylindrocarpon destructant ) and 50:50 The mixture was grown for 10 weeks per pollen for 2 months. The ginseng root pathogen is a pathogen that is known to gradually increase during the period of ginseng cultivation even after a small package (圃 場), and especially rapidly increase after 4 years to cause rotting roots of ginseng. After the cultivation, the root vegetation of ginseng that was not diseased was counted, and the root vein rate was measured, and the root rot rate was measured to analyze the root disease morbidity.

As a result, it was found that the survival rate of ginseng was significantly higher than that of the control group treated with the bacteria. In the case of pollen survival rate, the healthy rooting rate (% of rootless disease) was significantly higher in the untreated group but 82% in the fungus group, and the morbidity rate (total percentage of root rot disease per individual) was not treated. In the case of 50.4%, the fungus treatment was 6.0%, it was found that the effect of inhibiting the onset of the fungus treatment was high ( Table 1, Figures 3 and 4 ). The statistical results of the experiment showed more than 1% level of significance. In other words, the F test showed 21.20 references and this was more than 1% level. Table 2 ), it was confirmed that the root disease morbidity reduction effect has a higher significance (303) ( Table 3 ).

Inhibitory Effect of Bacillus Strains repeat One 2 3 4 5 Average Health ratio (%) No treatment 10 30 20 20 20 20 B-4228 80 80 60 90 100 82 Morbidity ^* (%) No treatment 44 54 52.5 51 50.5 50.4 B-4228 4 11 14 0.5 0 6.0

Root rot

Variance analysis of the effect of increasing the healthy muscle Sum of squares Degrees of freedom Average of square F black all 10690 9 - repeat 540 4 - process 9610 One 9610 ** 71.18 (21.20) error 540 4 135

**: significance above 1%

Variance Analysis of Root-infective Disease Reduction Sum of squares Degrees of freedom Average of square F black all 5167 9 - repeat 152 4 - process 4950 One 4950 ** 303 (21.20) error 65 4 16.3

 **: significance above 1%

Therefore, Bacillus subtilis strain B-4228 or its culture medium of the present invention has a high antagonism to inhibit pathogens by increasing the survival rate in the alveolar pathogen alveolar alveoli, and from this gradually increasing ginseng in general packaging It was confirmed that the root rot pathogens can be sufficiently suppressed.

Preparation Example 1 Preparation of Microbial Preparation

<1-1> Preparation of Hydrating Agent

The inventors of the present invention using a Bacillus subtilis strain B-4228 or a culture solution thereof confirmed that the growth inhibitory activity against the ginseng root rot pathogen in Example 3, or a culture solution thereof to prepare a microbial preparation for the control of ginseng root rot pathogen, stable formulation For the purpose of preparing a hydrating agent that is hydrated when diluted in water as a powder phase. Specifically, the hydrate was pulverized by adding a surfactant and a extender after heating, drying the solid medium. That is, the raw powder was dried and pulverized for about 2 hours in a dryer at 70 ° C. Then the coarsely crushed powder, surfactant and extender were mixed evenly and mixed. The mixture was ground by passing through a Jet-O-mill (manufactured by Aljet) and the ground powder was tested for hydration to prepare a microbial formulation in the form of a hydrate.

<1-2> Preparation of granules

The present inventors prepared granules which can be used as they are in granular form for the purpose of stable formulation of microbial preparations using Bacillus subtilis strain B-4228. Specifically, the granules were pulverized by drying the raw powder for about 2 hours in a dryer at 70 ° C. The pulverized powder was pulverized through a jet-O-mill, and then the microbial pulverized powder, surfactant, extender / nutrient and disintegrant were spores of the strain 50%, polycarboxylate 3%, sodium lignosulfonate 3%, 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 (diameter 1 mm, length 1-5 mm) 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 a granule.

<1-3> Preparation of Encapsulated Formulations

The inventors intended to prepare encapsulated formulations for the purpose of stable formulation of microbial formulations using Bacillus subtilis strain B-4228. Specifically, 11% soy flour, 6% green beans, 4% rice flour, 4% potato starch, 4% rye flour, 3% ocher soil, and autoclaved at 121 ° C. for 20 minutes, then thoroughly mixed before cooking and cooled in cold water. I was. After thoroughly mixing for 10 minutes or more in a stirrer (d = 5cm, 200 rpm or more), the microbial sterilization encapsulation formulation was prepared by completely mixing the additive and Bacillus subtilis strain B-4228 while stirring was continued.

As described above, the microbial agent containing Bacillus subtilis strain B-4228 of the present invention or its culture solution has excellent antagonistic ability against ginseng root rot pathogens, and is environmentally friendly and does not have human toxicity. It can be usefully used as a microbial pesticide for controlling rot disease.

1 is Bacillus subtilis strain B-4228 (middle) of the present invention, the pathogens of ginseng lysatonia sorani (upper), Cylindrocarpon distractance (lower), Alternaria panax ( Left), the photo shows the result of the replacement culture for Botrytis Ceria (right),

Figure 2 is a photograph showing the results of replacing culture of Bacillus subtilis B-4228 (left) of the present invention to cause growth inhibition against the pathogen of Phytophthora myeongtorum (right),

Figure 3 is a photograph of the comparison of the seedlings treated with Bacillus subtilis B-4228 of the present invention compared to the seedlings (control) without any treatment when grown in soil contaminated with Cylindrocarpon distractance,

4 is a graph showing the healthy muscle and morbidity when treated with Bacillus subtilis B-4228 of the present invention and no treatment.

Claims (8)

Bacillus subtilis strain B-4228 (Accession Number: KCTC 0657BP) or a microbial agent for the control of ginseng root rot disease containing the culture medium thereof as an active ingredient. The method of claim 1, wherein the ginseng root rot is one or more selected from the group consisting of alternaria panax, phytophthora fetorum, lysatonia sorani, botrytis cineria and cylindrocarpon distractance. Microbial agent characterized in. The method of claim 1, wherein the microbial agent is a microbial agent, characterized in that the granules, hydrates or capsules. According to claim 1, wherein the microbial agent is prepared by adding a surfactant, extender and nutrient to Bacillus subtilis strain B-4228 or its culture. The microorganism preparation according to claim 1, wherein the microorganism included in the microorganism preparation is 1 × 10 ^{6 to} 1 × 10 ⁹ per ml (ml). 2. The microbial preparation according to claim 1, wherein the effective amount of the microorganism included in the microbial preparation is sprayed with at least 1 × 10 ⁷ microorganisms per cultivated land area (m 2). Method for controlling ginseng root rot disease using the microbial agent of claim 1. 8. The method of claim 7, wherein the microbial agent is sprayed or immersed in concentrated roots or soils close to the ginseng.