KR101175532B1 - Novel burkholderia sp., and media for mass culture of burkholderia for increasing antifungal effect and method for mass production thereof using the same - Google Patents

Abstract

PURPOSE: A novel Burkholderia pyrrocinia LA101 with excellent antifungal property is provided to effectively prevent plant diseases in strawberries, ginseng, garlic, cucumber, watermelon, and banana. CONSTITUTION: A microorganism medium for culturing Burkholderia pyrrocinia LA101(deposit number KACC91663P) contains soy peptone, SIP(isolated soy protein), corn steep liquor, glucose, chitin, and water. A microorganism agent contains a culture of Burkholderia pyrrocinia, prepared in a medium containing A101 soypeptone, SIP, corn steep liquor, glucose, chitin, and water. The microorganism formulation has antibacterial effects against Alternaria panax, Fusarium oxysporum, Cylindrocarpon destructans, Botrytis cinerea, Colletotrichum gloeosporioides, Sclerotinia cepivorum, Sclerotinia sclerotiorum, and Penicillium hirsutum.

Description

Novel Burkholderia sp., And media for mass culture of Burkholderia for increasing antifungal effect and method for mass production according using the same}

The present invention is a novel Burkholderia pyrrocinia LA101 strain having excellent antifungal properties ( Burkholderia pyrrocinia LA101, Microorganism Accession No. KACC91663P), Burkholderia spp . It relates to a microbial medium containing soy peptone, soybean isolate, corn leachate, glucose, chitin and water useful for mass cultivation of microorganisms and increase antifungal properties.

In the cultivation of crops, in order to control the diseases of the crops, up to now, methods such as land soil, freshwater immersion, and lime disinfection of cultivated land, or methods of spraying chemical insecticides or fungicides have been used. However, it was difficult to expect a satisfactory effect only in the case of arable land or fresh water immersion, and the drug effect was reduced by the emergence of pathogenic strains resistant to chemical insecticides and fungicides. In addition, chemical insecticides or fungicides are recognized as harmful to the environment, animals and humans, and their use is being avoided.

Meanwhile, as interest in environmentally friendly agricultural products has increased, interest in biological control agents has recently increased. Biological control agents respond to the growing demand for safe agricultural products by consumers, and use microorganisms or plant extracts instead of chemical pesticides that are harmful to the environment, thereby selectively controlling only plant diseases without affecting the environment, other plants, and killing. can do.

Of biological control agent, a control agent using a microorganism, Bacillus (Bacillus) spp, Pseudomonas microorganism that has efficacy in the tiny-creatures-killing or sterilizing, such as (Pseudomonas, Burkholderia) sp, Streptomyces, alkali jeneseu (Alcaligenes) spp, Pseudomonas ( Microorganisms with antifungal activity , such as Pseudomonas ) strain and Burkholderia strain, are used.

In the present invention, by providing a microorganism that can be used as a biological control agent as described above and a microbial medium that can be cultured in large quantities, it was possible to culture the microorganism with high efficiency.

It is an object of the present invention to provide a novel Burkholderia pyrrocinia LA101 strain ( Burkholderia pyrrocinia LA101, microorganism accession number KACC91663P) and a microorganism medium useful for mass cultivation of microorganisms.

Still another object of the present invention is to provide an antifungal enhanced microbial agent comprising a culture obtained by culturing Berkholderia pyrosinia as the active ingredient.

In the present invention, the novel Burkholderia strain Burkholderia pyrosinia LA101 strain ( Burkholderia) pyrrocinia LA101, microorganism accession number KACC91663P).

The present invention also provides a microbial medium containing soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water.

Burkholderia Pyrosinia LA101 strain of the present invention ( Burkholderia pyrrocinia LA101) was able to grow within 15 ℃ ~ 42 ℃, pH 5.0 ~ 7.5 range, the optimum pH is 6.5 ~ 7.0, most preferably pH 6.8 is the most appropriate.

The Berkholderia Pyrosinia LA101 has a circular milky white, double cross-section (umbonate), cellobiose, trehalose, paratinose and mannitol. Sorbitol (Sorbitol), sucrose (Sucrose), glucose (Glucose) can be used to proliferate.

The Berkholderia Pyrosinia LA101 is Alternaria panax ), Botrytis cineraria ( Botrytis cinerea ), Colletotrichum gloeosporioides ), Sclerotinia cepivorum ), Sclerotinia sclerotiorum ), Penicillium hirsutum ), Fusarium oxysporum ), Cylindrocarpon destructans ) and the like have an antimicrobial effect.

Soy peptone used in the microbial medium of the present invention is a soy-derived protein, mainly used as a nutrient for the microbial medium, and the protein content of the soybean isolated protein is about 90%. Contains the most. It is one of the representative soy protein foods along with concentrated soy protein and tissue-based soy protein.

When corn is immersed in water for corn starch extraction, various soluble components (inorganic components, reducing sugars, amino acids) are leached into the immersion water, and the dark brown viscous liquid is concentrated to 50% water content. Corn steep liquor (CLS), and is mainly used as a source of liquid protein.

Glucose is a monosaccharide that plays an important role in living organisms. It is contained in sweet fruits and honey, and is also contained in blood and lymph. It is a small sugar, polysaccharide and glycoside. It is also a component such as 體). Industrially, starch is produced by hydrolysis with dilute acid.

Chitin is a polysaccharide composed of amino sugars (amino derivatives of sugars) in which N-acetylglucosamine is polymerized by β-1 and 4-bonds. White powder, insoluble in water and weak in reactivity. When you get a large amount of chitin, you can dip the shell of shrimp or crab in hydrochloric acid, dissolve the calcium carbonate, boil it with alkali to remove the protein, wash the remaining precipitate well and dry it. Chitin is an important component of the cell walls of fungi, as well as building the hard epidermis or shell of arthropods.

The microbial medium of the present invention containing the soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water has an effect of increasing the number of microorganisms, and when culturing the microorganisms with antifungal It is confirmed that the antifungal activity is increased, compared to the culture cultured in the medium used normally.

In addition, the microbial medium containing the soy peptone, soybean isolate, corn leachate, glucose, chitin and water of the present invention, Burkholderia pyrosinia LA101 strain of the present invention ( Burkholderia pyrrocinia LA101 or Burkholderia Cultures of pyrrocinia sp.) can be used as antifungal microbial agents. When the strains of the genus Berkholderia are cultured in the microbial medium, it is confirmed that the antifungal activity is increased, compared to the culture cultured in the medium used normally. In addition to the strains belonging to the genus Berkholderia, other antifungal strains may be cultured, and the antifungal properties of the strains may be increased compared to cultures cultured in a medium that is commonly used.

Preferably, Burkholderia in the microbial medium containing the soy peptone, soybean separation protein, corn leachate, glucose, chitin and water The culture of the pyrrocinia strain was a plant pathogen, Fusarium oxysporum. oxysporum , wilting disease), Cylindrocarpon destructans, root rot disease, Botrytis cineraria cinerea , gray mold causing bacteria, Colletotrichum gloeosporioides , anthrax causative), Sclerotinia cepivorum , black rot bacillus causative bacteria, Sclerotinia sclerotiorum , or penicillium hirstum hirsutum , which can be used as a control against blue fungi.

In addition, the microbial control agent may be used as a control agent for wilting disease, root rot disease, black rot fungal disease, gray mold, anthrax, fungal disease, blue mold disease and the like of strawberry, ginseng, garlic, cucumber, watermelon or banana.

The microbial medium containing soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water, based on 100 parts by weight of soy peptone, 20 to 80 parts by weight of soybean isolate protein, 100 to 200 parts by weight corn extract, glucose 30 ˜70 parts by weight, chitin 1-5 parts and water 500-800 parts by weight.

Burkholderia in the microbial medium containing the soy peptone, soybean separation protein, corn leachate, glucose, chitin and water The culture cultured pyrrocinia strain,

Based on 100 parts by weight of soy peptone, 20 to 80 parts by weight of soybean separation protein, 100 to 200 parts by weight of corn leachate, 30 to 70 parts by weight of glucose, 1 to 5 parts by weight of chitin and 500 to 800 parts by weight of water;

Adjusting the mixture to pH 6.5-7.0;

Autoclaving the mixture whose pH is adjusted;

Burkholderia on the autoclaved medium Burkholderia inoculating a pyrrocinia strain; And

Incubating the medium inoculated with the Berkholderia pyrosinia at 20-30 ° C. for 4-6 days;

It can be cultured to include.

As described in detail above, Burkholderia pyrosinia LA101 strain of the present invention ( Burkholderia pyrrocinia LA101 is Alternaria panax and Botrytis cinerea ), Colletotrichum gloeosporioides , Sclerotinia cepivorum , Sclerotinia sclerotiorum , Penicillium hirsutum , Fusarium oxysporum and Cylindrocarpon destructans have been found to have antimicrobial effects.

In addition, the microbial medium containing soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water of the present invention can be effectively used in the mass growth of microorganisms by effectively increasing the number of microorganisms in microbial culture. Confirmed.

In addition, the culture cultured Burkholderia pyrrocinia strain in the microbial medium containing the soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water, wilted rot, root rot, black rot It has been confirmed that it can be used as a control agent to effectively control various plant pathogens including fungal nucleus, gray mold, anthrax, fungal nucleus, and blue mold disease. Especially, plants such as strawberries, ginseng, garlic, cucumber, watermelon, banana, etc. It was effective in controlling the disease.

Accordingly, the microbial medium of the present invention and Burkholderia Using pyrrocinia ) cultured cultures, it is considered that it is possible to mass-produce environmentally friendly plant disease control agents with excellent plant disease control effects.

1 is a gene sequence table of 16s rRNA of a Burkholderia pyrrocinia LA101 strain isolated in Example 1 of the present invention, wherein the Burkholderia pyrosinia LA101 strain is a Burkholderia pi. It is 98% consistent with Lossia.
Figure 2 is a Burkholderia Pyrosinia LA101 ( Burkholderia) isolated in Example 1 of the present invention DNA sequence of the 16s rRNA of the pyrrocinia LA101 strain.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

Example 1 Preparation of Microbial Medium Containing Soy Peptone, Soybean Isolate Protein, Corn Leachate, Glucose and Chitin>

In order to prepare a microbial medium according to the present invention, the pH was adjusted to 6.8 by mixing the medium composition by the weight shown in Table 1, and then autoclaved. The weight of the composition is based on 100 parts by weight of soy peptone, 20 to 80 parts by weight of soybean separation protein, 100 to 200 parts by weight of corn leachate, 30 to 70 parts by weight of glucose, 1 to 5 parts by weight of chitin and 500 to 800 parts by weight of water. The weight corresponding to the condition. In addition, in the composition, water was mixed at different weights in each example so that the final volume of the mixture was 1000 ml. Soybean isolate protein (Chinese) in the composition was purchased from Sinwon Trading Co., glucose was used as Q1 products. Soy peptone was purchased from Megafeed, corn leach was purchased from Samyang, and chitin was purchased from General Chemicals.

Example 2. Preparation of Microbial Cultures

The microbial medium of Examples 1-1 to 1-9 was inoculated with the Berkholderia pyrosinia LA101 strain and incubated at 26 ° C. for 3 days, and the pH of the medium was adjusted to 6.8 using phosphate buffer. The inoculation amount of the Berkholderia pyrosinia LA101 strain was inoculated to the microorganism medium to 0.5% (v / v) of the precultured strain culture. The conditions of the microbial medium used for the culture are shown in Table 2 below.

Comparative Example 1. Preparation of Control Microorganism Medium

In order to compare the effects of the microbial medium of Examples 1-1 to 1-9, by mixing the medium composition according to the weight shown in Table 3 to adjust the pH to 6.8, it was autoclaved (Comparative Examples 1-1 to 1) -14). In addition, in the medium composition of Comparative Examples 1-1 to 1-14, water was mixed in different weights for each Comparative Example so that the final volume of the mixture was 1000 ml.

In the medium of Comparative Examples 1-15, trypticase soy broth was used as a control for the microorganism medium that is commonly used. Trypticase Soy medium is prepared by adding water to a total volume of 1000 ml in 17 g of pancreatic digest of casein, 3 g of papaic digest of soybean meal, 5 g of sodium chloride, 2.5 g of dipotassium phosphate, and 2.5 g of dextrose (pH 7.3).

Comparative Example 2. Preparation of Control Microorganism Cultures

The microbial medium of Comparative Examples 1-1 to 1-15 was inoculated with Berkholderia pyrosinia LA101 strain and incubated at 26 ° C. for 5 days, and the pH of the medium was adjusted to 6.8 using phosphate buffer (comparatively). Example 1-15 shows pH 7.3). The inoculation amount of the Berkholderia pyrosinia LA101 strain was inoculated to the microorganism medium so that 0.5% (v / v) of the precultured strain culture (same as the culture used in Example 2). The conditions of the microbial medium used for the culture are shown in Table 4 below.

Experimental Example 1: Isolation and Antimicrobial Activity of Antagonist Microorganisms>

Antagonist microorganisms of the present invention was isolated from the soil of Chungnam smoke group. The soil samples were mixed well and diluted in sterile water and microorganisms were separated by dilution plate method in NA (Nutrient Agar) medium and incubated at 27 ° C. for 5 days, and the soil microorganisms separated therefrom were transferred to NA medium for pure separation. It was. Subsequently, the phytopathogens Alternaria panax , Botrytis cinerea , Colletotrichum gloeosporioides , and sclerotidium were used by paper disc diffusion method. Sclerotinia cepivorum , Sclerotinia sclerotiorum , Penicillium hirsutum , Fusarium oxysporum and Cylindrocarpon destructans antagonistic microorganisms that inhibit mycelial growth were selected by replacing the destructans ) with the soil microorganisms. The most antagonistic strain among the antagonists was selected and named Burkholderia pyrrocinia LA101. Antagonists of the Burkholderia pyrrocinia LA101 according to the paper disc diffusion method are shown in Table 5.

Experimental Example 2. Characteristics of Antagonistic Microorganism Berkholderia Pyrosinia LA101

In this experiment, the analysis of microbial properties was performed using commonly known microbial identification methods.

Physiological and biochemical analysis of Berkholderia pyrosinia LA101 revealed that it is a gram-negative bacterium, an aerobic bacilli, that is motility and oxidase positive.

In order to know the optimal growth temperature of the Berkholderia pyrosinia LA101 was used NB (nutrient broth) and NA (nutrient agar) medium generally used in bacterial culture. The growth temperature of the antagonist Burkholderia Pyrosinia LA101 strain of the present invention was investigated using a temperature gradient device (Temperature Gradient Incubator TN2148, Advantec.), And the optimum growth temperature was 26 ° C. It did not appear to grow below 15 ° C or above 42 ° C.

Berkholderia pyrosinia LA101 was able to grow in the pH range of 5.0-7.5, with an optimum pH of 6.5-7.0, most preferably pH 6.8.

After inoculating the selected antagonist Berkholderia pyrosinia LA101 in NA medium and incubating at 28 ° C. for 2 days, the colony characteristics were observed. The milky white circumference had a double umbonate. The sugar availability of the strain is shown in Table 6.

When confirming the results of Table 6, the Berkholderia Pyrosinia LA101 is a cellobiose (Cellobiose), Trehalose (Trehalose), Paratinose (Paratinose), Mannitol (Mannitol), Sorbitol (Sorbitol), Sucrose (Sucrose), Glucose (Glucose) has been shown to have the property of proliferation. The glycolysis effect was confirmed using a microorganism identification kit commonly used.

<Experimental Example 3 16S ribosomal RNA (rRNA) gene sequencing analysis of the antagonist Berkholderia pyrosinia LA101>

Was identified to be antagonistic microorganism of the present invention 16s rRNA gene sequence analysis results when a buck holder Ria pie California (Burkholderia pyrrocinia) (figure 1), when in this buck holder Ria pie California (Burkholderia pyrrocinia ) LA101.

To this end, the DNA of the strain was isolated and the nucleotide sequence of the 16S rRNA was followed to identify the phylogenetic location. 27F (5′-GTG CTG CAG AGA GTT TGA TCC TGG CTC AG-3 ′) and 1492r (5'-CAC GGA TCC TAC GGG TAC CTT GTT ACG ACT T-3 ') primers to amplify antagonist 16S rRNA Was used. The base sequence of the 16S rRNA gene was searched for homology with the DNA database of NCBI GeneBank, and the phylogenetic position was confirmed using the CLUSTAL X program and the PHYLIP program. Molecular phylogenetic analysis based on the nucleotide sequence of 16S rRNA revealed a 98% flexible relationship with Burkholderia pyrrocinia , a strain belonging to a systematic group including the species of the genus Burkholderia. It was confirmed (see FIG. 1). According to the above results, the antagonist microorganism of the present invention was identified as Burkholderia pyrrocinia and deposited with the accession number KACC91663P to the National Institute of Agricultural Science, Agricultural Genetic Resource Center microbial bank.

Experimental Example 4. Preparation of Microbial Culture and Number of Cells

In order to confirm the effect of increasing the number of cells of the microbial medium of the present invention, Burkholderia pi in the cultures of Examples 2-1 to 2-9, and the cultures of Comparative Examples 1-1 to 1-15 The number of Rossini LA101 cells was confirmed. The number of cells of the Berkholderia pyrosinia LA101 is shown in Table 7 below.

Checking the results of Table 7 it can be seen that the number of buckholderia pyrosinia LA101 cells in the culture of Examples 2-1 to 2-9 corresponding to the present invention is 1.5 ~ 2.3 × 10 ⁷ cfu / ㎖ .

On the other hand, cultures of Comparative Example 2-1 to Comparative Example 2-12 and Comparative Example 2-15 with or without Soy Peptone, Soybean Isolate, Corn Leachate or Glucose [Comparative Example 2-1 (No Soy Peptone), Comparison Example 2-2 (small amount of soy peptone), Comparative Example 2-3 (excessive soy peptone), Comparative Example 2-4 (no soybean isolate protein), Comparative Example 2-5 (small amount of soybean isolate protein), Comparative Example 2-6 (Excess of soybean separation protein), Comparative Example 2-7 (no corn leach), Comparative Example 2-8 (small amount of corn leach), Comparative Example 2-9 (excess of corn leach), Comparative Example 2-10 (no glucose), In the cultures of Comparative Example 2-11 (small amount of glucose), Comparative Example 2-12 (excess glucose), Comparative Example 2-15 (trypticase soy medium)] It can be seen that it is significantly less than in the culture of -1 to 2-9.

 On the other hand, the number of cells of Burkholderia pyrosinia LA101 in the culture of Comparative Example 2-13 without chitin and Comparative Example 2-14 with excess chitin was different from Examples 2-1 to 2-9. It did not appear that chitin did not affect the increase in cell counts.

Experimental Example 5 Confirmation of Antifungal Increasing Effect of Microbial Medium Containing Soy Peptone, Soybean Isolate Protein, Corn Leachate, Glucose and Chitin>

5-1. Control effect of wilted ill disease (cause: Fuziium oxyporum)

In order to confirm the antifungal increase effect of the microbial culture of the present invention, the effect of wilting disease (caused fungus: Fuxium oxysporum) on strawberry (cancer varieties) in seedling packaging was investigated. The cultures of Examples 2-1 to 2-9 and Comparative Examples 2-1 to 2-15 were adjusted to 1.0 × 10 ⁷ cfu / ml to control the number of microorganisms, which were then irrigated three times at 10-day intervals (100 One month after the ml / strawberry), the morbidity rate was examined, and the control value was confirmed at the harvest time. Copper hydroxide (77%) was used as a control agent and the morbidity and control values are shown in Table 8.

Based on the results of Table 8, the cultures of Examples 2-1 to 2-9 showed better control value than the Cooper hydrating agent, a control agent, it can be substituted for the chemical pesticides disclosed in strawberry wilted bottle.

The cultures of Comparative Examples 2-1 to 2-15 were found to exhibit a control value similar to that of Cooper Watering Agent, although they were less effective than those of Examples 2-1 to 2-9.

Therefore, the microbial culture of the present invention, when culturing the Berkholderia pyrosinia strain, it can be confirmed that there is an effect to increase the antifungal properties of the strain to the maximum.

On the other hand, the culture of Comparative Example 2-13 appears to have a lower control effect than other cultures. The culture corresponds to a culture of culturing Berkholderia pyrosinia LA101 using a medium that does not contain chitin, through which the chitin increases the antifungal effect on the medium of the present invention. It was confirmed.

Therefore, it can be seen that chitin is a medium composition that affects antifungal rather than microbial cell growth.

5-2. Root rot disease (causative bacillus: Cylindrocarpon destructans)

In order to confirm the antifungal increase effect of the microbial culture of the present invention, the ginseng root rot inhibitory activity was investigated in ginseng farmland.

The presence of root rot bacteria was checked in the ginseng arable land under a fall, and 2 year old ginseng was transplanted into one of the 5000 arable land (Note: The size of one cell is 1.8 × 0.9m and the number of ginseng used for the test is about 500 roots. ). The cultures of Examples 2-1 to 2-9 and the cultures of Comparative Examples 2-1 to 2-15 were adsorbed on vermiculite and the like, and the adsorbed material was evenly sprayed on the cultivated soil in which root rot bacteria were present. After that, the ginseng was grown. The untreated group was not treated with the culture of the present invention. After three months, ginseng was excavated, and the improvement of root rot disease of ginseng for each culture treatment was shown in Table 9.

Referring to the results of Table 9, the roots of the untreated group were rotting intact, and the roots treated with the cultures of Examples 2-1 to 2-9 improved root rot disease and confirmed that the growth state was very good. there was. On the other hand, the roots treated with the cultures of Comparative Examples 2-1 to 2-15 had improved root rot than the untreated group, but compared to the culture treated groups of Examples 2-1 to 2-9, the roots. Rot disease was in progress. On the other hand, the culture of Comparative Example 2-13 using a medium containing no chitin was found that the control effect against root rot disease is better than other conditions, as in strawberry wilting disease.

 National Academy of Agricultural Science KACC91663P 20110812

Attach an electronic file to a sequence list

Claims (9)

delete delete delete A medium for culturing Burkholderia pyrrocinia LA101 (Microorganism Accession No. KACC91663P), a microbial medium containing soy peptone, soybean isolate, corn leachate, glucose, chitin and water. Contains as an active ingredient a culture of Burkholderia pyrrocinia LA101 strain (KACC91663P) in a microbial medium containing soy peptone, soybean isolate protein, corn leachate, glucose, chitin and water Microbial agent, characterized in that. The method of claim 4, wherein
The medium, based on 100 parts by weight of soy peptone, 20 to 80 parts by weight of soybean separation protein, 100 to 200 parts by weight of corn leachate, 30 to 70 parts by weight of glucose, 1 to 5 parts by weight of chitin and 500 to 800 parts by weight of water Microbial medium, characterized in that. The method of claim 5, wherein
The medium, based on 100 parts by weight of soy peptone, 20 to 80 parts by weight of soybean separation protein, 100 to 200 parts by weight of corn leachate, 30 to 70 parts by weight of glucose, 1 to 5 parts by weight of chitin and 500 to 800 parts by weight of water Microbial agent, characterized in that. The method according to claim 5 or 7,
The microbial agent may include Alternaria panax , Fusarium oxysporum , Cylindrocarpon destructans , Botrytis cinerea , and cholorthrite gloeos. At least one selected from the group consisting of Colletotrichum gloeosporioides , Sclerotinia cepivorum , Sclerotinia sclerotiorum and Penicillium hirsutum Microbial agent, characterized in that the control effect on the strain. A method of controlling plant pathogens by treating the plant with the microbial agent of claim 5.

Images