KR101389975B1 - culture medium for preventing disease and promoting growth of plant, manufacturing method of microbial agent using the same - Google Patents

Abstract

The present invention relates to a culture medium for the cultivation of Bacillus subtilis and a method for producing microorganism preparation using the same, and more particularly, to a culture medium for cultivation of Bacillus subtilis, The present invention relates to a culture medium for Bacillus subtilis culture which is economical and has excellent effect of controlling the disease and promoting growth of crops, and a method for producing microorganism preparation using the same.
The medium for cultivation of Bacillus subtilis for controlling the disease and promoting the growth of the crops of the present invention contains a lung ginseng culture medium used for culturing wild ginseng and a carbon source.

Description

TECHNICAL FIELD The present invention relates to a culture medium for culturing Bacillus subtilis and a method for producing microbial agents using the same,

The present invention relates to a culture medium for the cultivation of Bacillus subtilis and a method for producing microorganism preparation using the same, and more particularly, to a culture medium for cultivation of Bacillus subtilis, The present invention relates to a culture medium for Bacillus subtilis culture which is economical and has excellent effect of controlling the disease and promoting growth of crops, and a method for producing microorganism preparation using the same.

The use of pesticides is inevitable in order to increase yields because there is a problem that crop yields decrease from 30% to 100% when pesticides are not used when cultivating crops. However, due to misuse of synthetic pesticides, which constitute the majority of pesticides, soil, water and agricultural product pollution, toxicity, ecosystem disturbance, and the emergence of resistant strains have been raised. Research on the development of a biopesticide using an antagonistic microorganism, which is one of the measures to solve such a problem, has been actively carried out in the world in recent years.

Biopesticides are largely divided into plants, microorganisms, natural enemies, natural biologically active substances, and genetically modified organisms (GMOs). Research on the development of microbial agents, especially of biological pesticides, has been steadily progressing as a major field of plant pathology for the past 70 years and more than 40 products have been developed over the past decade.

Especially, among various microorganisms, various soil microorganisms which exist in plant rhizosphere and have many useful traits are being developed and commercialized. Such useful traits are the secondary metabolites, antimicrobial active substances, , Chitin material decomposition, etc. Directly to control the disease or to indirectly promote the growth of plants. In recent years, studies have been actively conducted to induce resistance to diseases by interacting with these plant microorganisms, and microbial agents of new materials have been commercialized accordingly.

Gram-positive bacteria belonging to the genus Bacillus are the next most widely studied and commercialized bacteria following the genus Pseudomonas. They produce various secondary metabolites that exist in large numbers in the soil and have biological activity. They are resistant to heat and resistant to harsh environment It has the characteristic of forming endospore.

The Bacillus subtilis strain having these characteristics was developed in 1994 as a seed and furrow treatment agent of cotton and peanut by Gustafson under the trade name Kodiak (Backman et al., Improving Plant Productivity with Rhizosphere Bacteria, pp 3-8). In 2001, Bacillus subtilis and Bacillus amyloliquefaciens were mixed to release a microbicide called BioYield. In Germany, Bayer has developed a soil-borne disease control agent that uses Bacillus FZB 24, and Taensa in the US has introduced a microbicide using the same bacteria. AgraQuest in the US has developed Serenade ), A microbial fungicide using Bacillus subtilis strain QST713.

However, most of the microbial preparations developed so far have effective mechanisms, yet they are reluctant to use them practically in the farmhouse. This is because the control of microbial agents is lower than that of chemical pesticides, the effects are variable, This is due to the lack of accurate information on pests. In addition, conventional microorganism preparations have a problem in that the production cost is high because an expensive culture medium is used to mass-culture microorganisms.

On the other hand, Panax ginseng ) is a traditional herbal medicine that has been used for a long time and has been scientifically recognized as a pharmacological efficacy. However, the older the wild ginseng is, the more rare it is, the more expensive it is, and it is difficult to supply it more stable than the demand.

A way to solve this problem is to cultivate wild ginseng through biotechnological methods. Prior art related to this is Korean Patent Application No. 2001-0003285 and 2001-0031029.

In order to grow large amounts of wild ginseng, cultivation of wild ginseng culture is necessary. Typically, the wild ginseng cultures are mB5 (modified Gamborg's B5), Durzan, MS (Murashige-Skoog), DKW (Driver-Kuniyuk-Walnut), GD (Gresshoff and Doy), SH (Schenk and Hildebrandt) and Lepiovre), WPM (Lloyd & McCown Woody Plant Medium) medium.

The ginseng culture grown after cultivation of wild ginseng contains a large amount of useful components such as growth promoting hormone, saponin naturally released in the culture medium and various secondary metabolites, but it is discarded after use.

The present invention has been made in order to overcome the above-mentioned problems, and it is an object of the present invention to provide a microorganism which is produced by cultivating a Bacillus subtilis microorganism using a germ- It is an object of the present invention to provide a culture medium for culturing Bacillus subtilis and a method for producing microorganism preparation using the culture medium.

To achieve the above object, the present invention provides a culture medium for culturing Bacillus subtilis for controlling diseases and promoting growth of agricultural crops, which comprises a lung ginseng culture medium used for culturing wild ginseng and a carbon source.

The carbon source is malic acid.

The crop disease is caused by Botrytis cinerea , which is a pathogenic fungus such as gray mold, Fulvia fulva is a pathogenic fungus, and Colletotrichum coccodes is an anthracnose pathogen.

In order to accomplish the above object, the present invention provides a method for producing a microorganism preparation for controlling diseases and promoting growth of a crop, comprising the steps of: preparing a culture medium for microorganisms containing lung ginseng culture liquid and carbon source, ; And culturing the Bacillus subtilis strain by inoculating the culture medium.

The medium composition step comprises the steps of: a) filtering the lung ginseng culture solution; b) adding the carbon source to the ginseng culture solution; and c) sterilizing the lung ginseng culture solution.

As described above, according to the present invention, various useful components contained in the ginseng culture can be reused by providing a culture medium for culturing Bacillus subtilis microorganism using the lung ginseng culture solution.

Also, the cost of culturing the microorganism can be greatly reduced by using the lung ginseng culture liquid to be discarded after being used for the cultivation of wild ginseng.

In addition, the present invention provides a microorganism preparation excellent in controlling disease and promoting growth of crops.

Figs. 1 to 7 are photographs showing the effect of inhibiting pathogenic bacteria growth,
8 is a graph showing the results of an experiment to control pepper gray mold,
9 to 12 are graphs showing the results of growth experiments of red pepper and tobacco,
13 is a photograph showing the growth state of the pepper seedlings,
14 is a photograph showing the growth state of the tobacco seedlings.

Hereinafter, a culture medium for culturing Bacillus subtilis for cultivated disease control and promotion of growth and a method for producing a microbial preparation using the culture medium for the cultivation of Bacillus subtilis according to a preferred embodiment of the present invention will be described in detail.

The microbial agent produced according to the present invention has crop pest control and growth promoting effect. Here, crops refer to crops and vegetables that are planted in rice fields or fields, and crop disease refers to the damage of crops caused by pathogens.

The microbial agent produced according to the present invention prevents the disease that may occur in the leaves, roots, stems, and fruits of crops. In particular, the microbial agent is excellent in the effect of controlling the disease that may occur in the leaves of crops. These diseases include Botrytis cinerea , a gray mold fungus that is a pathogenic fungus, a leaf mold fungus that Fulvia fulva is a pathogen, and Colletotrichum coccodes , anthracnose which is a pathogenic fungus .

The culture medium for culturing Bacillus subtilis of the present invention for producing the microorganism preparation contains a ginseng culture medium and carbon source.

The lung ginseng culture liquid means a culture solution of wild ginseng which is used for culture of wild ginseng and discarded. The composition of the lung ginseng culture may be different depending on the kind of the medium used as the wild ginseng culture liquid for culturing wild ginseng. The medium used as the wild ginseng culture medium is usually mB5 (modified Gamborg's B5), Durzan, MS (Murashige-Skoog), DKW (Driver-Kuniyuk-Walnut), GD (Gresshoff and Doy), SH (Schenk and Hildebrandt) , LP (Quoirin and Lepiovre), and WPM (Lloyd & McCown Woody Plant Medium).

In addition to the substances constituting the above-described medium, the cultured lung ginseng culture medium may contain growth promoting hormone, saponin released during cultivation of ginseng, and various secondary metabolites.

Malic acid, glucose, sucrose, glycerol, mannitol and the like can be used as the carbon source added to the lung ginseng culture solution, and the preferable carbon source is malic acid. When malic acid is used as a carbon source, the effect of controlling crop diseases is better than that of using other carbon sources.

Hereinafter, a method for producing a microorganism preparation using the culture medium for culturing Bacillus subtilis will be described step by step.

1. Medium composition step

In the medium composition step, a culture medium for microorganisms containing lung ginseng culture medium and carbon source is prepared.

The medium composition step may include, for example, a) filtering a lung ginseng culture solution, b) adding a carbon source to the lung ginseng culture, and c) sterilizing the lung ginseng culture.

First, the lung ginseng culture liquid is collected. After cultivation of wild ginseng, the ginseng culture liquid stored in the incubator is discharged and collected. During collection, filtration can be performed at the same time in order to remove the remaining ginseng culturing roots remaining in the lung ginseng culture solution. It is needless to say that the lung ginseng culture solution after collection can be filtered. Filtration can be performed in a variety of ways. For example, the collected lung ginseng culture liquid can be filtered using a sieve, a filter cloth, gauze, or the like.

Next, a carbon source is added to the lung ginseng culture liquid. As the carbon source, malic acid, glucose, sucrose, glycerol, mannitol and the like can be used. The carbon source may be added in an amount of 0.1 to 0.5 parts by weight based on 100 parts by weight of the ginseng culture liquid.

Next, the lung ginseng culture liquid is sterilized. Sterilization can be carried out by applying steam at 121 ° C for 30 minutes. It is needless to say that various known sterilization techniques can be applied.

2. Culture step

When the culture medium for Bacillus subtilis culture is formed, a culture step of culturing the microorganisms by inoculating the Bacillus subtilis strain is carried out.

Bacillus subtillus , a microorganism to be applied to the present invention, is a well-known microorganism that has been widely studied as Gram-positive bacteria and has been widely used as a commercially available microorganism, and produces a variety of secondary metabolites with high biological activity. And endospore resistant to harsh environments.

Bacillus subtilis can be purchased or sold separately from the soil.

To inoculate Bacillus subtilis strains into the sterilized lung ginseng culture, seed culture is first performed. For example, the strain of Bacillus subtilis is inoculated into 500 ml of LB (Luria Bertani) medium and incubated for 24 hours at 30 ° C with shaking. Then, inoculated on 50 L of LB medium, cultured for 24 hours at 30 DEG C with shaking, and cultured.

The cultured seeds are inoculated into a medium for cultivation of Bacillus subtilis and cultured at 30 ° C for 36 hours to obtain a Bacillus subtilis culture for use as a microorganism preparation.

In the present invention, the microorganism preparation may be the Bacillus subtilis culture itself or the Bacillus subtilis strain itself isolated and extracted from the culture broth. The microorganism preparation may be prepared in the form of a wetting agent, a granule or an encapsulation by adding a known formulating substance for the purpose of stable formulation of the microorganism.

Microbial agents can be used to control the disease of the crops and promote growth by treating the leaves, roots, etc. of the growing soil or growing crops. The microorganism preparation may be treated with an amount of 1.0 × 10 ⁴ cells / ml to 1.0 × 10 ¹⁰ cells / ml, preferably 1.0 × 10 ⁶ cells / ml to 1.0 × 10 ⁹ cells / ml to the target crop.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by these examples.

(Example)

<1. Isolation of strains>

The soil was collected, suspended in sterilized water, plated on LB (Luria Bertani) agar medium, cultured at 30 ° C for 24 hours, and colonies having respective characteristics were separated into a single colony.

Each of the isolated strains was shake cultured in LB liquid medium at 28 DEG C for 24 hours at a rotation speed of 200 revolutions per minute. Then, the strain was recovered by centrifugation and suspended in sterilized water. Then, each strain was treated with Vortisitis The strain with the strongest antimicrobial activity was selected through confluent culture with Botrytis cinerea .

The 16S rRNA sequence was analyzed and the strain was identified to be 99% homologous to Bacillus subtilis. The strain was identified as Bacillus subtilis strain It was identified.

<2. Preparation of microbial preparation >

WPM medium (distilled water per _{100ℓ K 2 SO 4 99g, CaCl} 2 · 2H 2 O 9.6g, Ca (NO 3) 2 · 4H 2 O 38.6g, NH 4 NO 3 40g, MgSO 4 · 7H 2 O 40g, MnSO 4 _{1.992g, KH 2 PO 4 17g,} FeNa-EDTA 3.67g, ZnSO 4 · 7H 2 O 0.86g, CuSO 4 · 5H 2 O 0.025g, Na 2 MoO 4 · 2H 2 O 0.025g, H 3 BO 3 0.62g , Vitamins (0.31 g), MYO-INOSITOL (10 g) and IBA (0.5 g)) were cultured for 45 days and then filtered using gauze. 0.3 parts by weight of malic acid, glucose, sucrose, glycerol, and mannitol as carbon sources were added to 100 parts by weight of the ginseng culture broth and sterilized by steam at 121 ° C for 30 minutes to obtain a culture medium for Bacillus subtilis Test medium) were prepared.

The Bacillus subtilis (hereinafter referred to as Bacillus subtilis HN) strain isolated above was inoculated into 500 ml of LB (Luria Bertani) medium, shake cultured at 30 ° C for 24 hours at 200 revolutions per minute, and then cultured in LB medium 50 L The seeds were inoculated and cultured for 24 hours at 30 ° C with shaking at 200 revolutions per minute. The cultured seed bacterium was inoculated into each test medium and cultured for 36 hours at 30 DEG C at a rotation speed of 200 revolutions per minute to prepare a microbial preparation.

&Lt; 3.

Bacillus subtilis HN culture experiments were performed using the lung ginseng culture medium and a conventional LB (Luria Bertani) medium for culturing the microorganisms. The results are shown in Table 1 below.

division Primary culture Secondary culture Tertiary culture Lung ginseng culture 3.14 x 10 &lt; ⁹ &gt; cfu / ml 2.87 × 10 ⁹ cfu / ml 3.32 × 10 ⁹ cfu / ml LB medium 2.82 × 10 ⁹ cfu / ml 4.27 x 10 &lt; ⁹ &gt; cfu / ml 2.55 x 10 &lt; ⁹ &gt; cfu / ml

Referring to the results of Table 1, it can be seen from Table 1 that there is no significant difference in the number of cultured bacterium when comparing the LB (Luria Bertani) medium, which is commonly used for culturing microorganisms, there was. Therefore, it is expected that the cost of cultivation can be greatly reduced if the microorganism preparation is prepared using the lung ginseng culture solution.

<4. Experiment of growth inhibition against pathogenic bacteria of gray mold>

In order to examine the antimicrobial activity of the microorganism preparation, Botrytis cinerea , which causes gray mold disease as a plant pathogen, was used to test the growth inhibition of pathogenic bacteria.

Agar was added to the test medium containing malic acid, glucose, sucrose, glycerol, and mannitol as a carbon source for growth of pathogens, and then the mixture was poured into a Petri dish and completely hardened. When the pathogen was inoculated in the center of the petridish and the mycelial growth of the pathogenic bacteria was about half of the diameter of the petri dish, Bacillus subtilis HN and the Pseudomonas chlorampis were inoculated at a distance of about 3 cm from the mycelium to be replaced . The effect of inhibiting growth of pathogens according to the type of carbon source after incubation at 25 캜, which is a proper temperature for growing the fungus, is shown in Table 2 and Figs. For comparison, the inhibition of pathogen growth was also carried out in Petri dishes containing agar in germinated ginseng culture medium and PDA (Potato Dextrose Agar) medium containing no carbon source, respectively.

division
Diameter of support for growth by carbon source (mm) Bacillus Subtilus HN The control (Pseudomonas chlorampis) Malic acid supplemented test media (Figure 1) 14 12 glucose supplemented test medium (Figure 2) 5 0 The sucrose addition test medium (Fig. 3) 5 0 glycerol supplemented test media (Figure 4) 5 0 Mannitol addition test media (Figure 5) 6 4 PDA (Figure 6) 4 6 The lung ginseng culture (Fig. 7) 5 6

 As shown in FIG. 1 and FIG. 2, it was confirmed that growth of Bacillus subtilis HN in the test medium inhibited the growth of pathogenic bacteria of the gray mold.

Especially, when the malic acid was added as a carbon source, the growth inhibition effect of the pathogen was increased about 3 times as compared with other carbon sources. This suggests that when malic acid is added to lung ginseng culture medium, a large amount of antimicrobial active substance is synthesized against pathogenic bacteria.

<5. Experiment on growth inhibition against various pathogens>

In the above experiment, growth inhibition experiment for various pathogenic microorganisms including malic acid as a carbon source, which showed the greatest inhibition effect on growth, was examined.

Pathogen
Diameter of support for growth by pathogen (mm) Microbial agent The control (E. coli) Botrytis cinerea 14 0 Fuarium oxysporum 2 0 Rhizoctonia solani 2 0 Fulvia fulva 10 0 Colletotrichum coccodes 11 0

As shown in Table 3 above, the antimicrobial effect of the microbial agent of the present invention and the plant pathogenic microorganism was examined. As a result, the microbial agent of the present invention was found to be a pathogenic microorganism that causes gray mold fungi with a diameter of 10 mm or less Botrytis cinerea ), a pathogenic fungus causing leaf mold disease, Fulvia fulva , and anthracnose-causing pathogens such as collectothrium cocodes . In addition, Fusarium oxysporum and Rhizoctonia solani have antimicrobial activity but their effect is low. Thus, it was confirmed that the microbial agent of the present invention is excellent in inhibiting the growth of pathogenic bacteria occurring on the leaves, rather than the pathogens occurring in the roots.

<6. Control of Pepper gray mold disease>

After 1 week of seeding, the microorganism preparation (2.4 × 10 ⁸ CFU / ㎖) was diluted 100 times with the control, and only the water was used as the control (untreated). After 5 days of culture , Botrytis cinerea spore suspension (1 × 10 ⁵ spores / ml), a causative agent of gray mold, was sprayed on the leaves of pepper. The condition was kept at 100% humidity for 24 hours and maintained at 25 캜 for 5 days, and the degree of disease was evaluated after 5 days. The results are shown in FIG. In FIG. 8, glucose, glycerol, malic acid, sucrose, and mannitol in the abscissa indicate the microorganism preparation containing each carbon source, and the PDA means the culture liquid obtained by inoculating Bacillus subtilis HN into PDA (Potato Dextrose Agar) And the culture solution of wild ginseng refers to a culture solution obtained by inoculating Bacillus subtilis HN into a lung ginseng culture solution.

Referring to FIG. 8, malic acid-added microbial agent as a carbon source showed the lowest effect, showing a high control effect. The culture medium containing Bacillus subtilis HN in the culture medium without added carbon source showed higher control effect than the control with the carbon source except malic acid.

<7. Plant Growth Promotion Experiment>

The effect of microbial agent containing malic acid as a carbon source on pepper and tobacco was investigated in the roots of seedlings. Red pepper and tobacco were cultivated for 2 weeks in the pots, and were divided into two groups: a stock solution of microorganism, a 100-fold dilution, a 1000-fold dilution, and a water-free treatment. Three replicate experiments were performed with 10 replicates. Experimental results are shown in Figs. 9 to 14. Fig.

FIG. 9 is a graph showing fresh weight of the pepper seedlings, FIG. 10 is a graph showing the buildings of the pepper seedlings, FIG. 11 is a graph showing the live weight of the tobacco seedlings, and FIG. 12 is a graph showing the buildings of the tobacco seedlings. 13 is a photograph showing the growth state of the pepper seedlings, and FIG. 14 is a photograph showing the growth state of the tobacco seedlings.

9 to 14, it was confirmed that the plants treated with the undiluted solution or 100-fold dilution of the microbial agent did not exhibit a large difference in the effect of untreated control and the growth effect in both red pepper and tobacco. However, in 1000 times diluted treatments, the growth of pepper was promoted by 18% compared to untreated control, and the live weight was increased by 8% and the increase by 26% in the building. The growth of tobacco was about 9%, and the live weight was increased by 31% in the 8% building.

The microorganism preparation using the waste ginseng culture liquid which has been discarded through the above-mentioned experiments remarkably reduced the cost of culturing the microorganism, and showed the excellent effect of controlling the disease and promoting the growth of the crops.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

Claims (5)

The ginseng culture used in cultivation of wild ginseng contains malic acid as a carbon source,
Crop disease is characterized by Botrytis cinerea as a gray mold fungus, Fulvia fulva as a pathogenic fungus, and Colletotrichum coccodes as an anthracnose pathogen. And a culture medium for culturing Bacillus subtilis for promoting growth control. delete delete A culture medium for culturing a lung ginseng culture used for culturing wild ginseng and a culture medium for culturing a microorganism containing malic acid as a carbon source;
Culturing the Bacillus subtilis strain in a culture medium,
Crop disease is characterized by Botrytis cinerea as a gray mold fungus, Fulvia fulva as a pathogenic fungus, and Colletotrichum coccodes as an anthracnose pathogen. Wherein the microbial agent is used for controlling crop diseases and promoting growth. The method of claim 4, wherein the culture medium comprises the steps of: a) filtering the lung ginseng culture solution; b) adding the carbon source to the lung ginseng culture liquid; and c) sterilizing the lung ginseng culture liquid Wherein the microbial agent is used for controlling crop diseases and promoting growth.

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