KR100769360B1 - 37-2 Bacillus subtilis S 37-2 and Microbial fertilizer using the same - Google Patents

Abstract

A novel Bacillus subtilis S 37-2 strain and a microorganism fertilizer are provided to excellently inhibit plant pathogenic fungi such as Fusarium Wilt, Fusarium oxysporum, and Sclerotium disease, promote the plant growth excellently, be effectively used as an environment-friendly microorganism fertilizer having both functions of pesticide and fertilizer and be adequate for house soil and salt-accumulated soil requiring heat-resistance and salt-resistance. A Bacillus subtilis S 37-2 strain inhibiting the growth of plant pathogenic fungi and promoting the growth of the plant is deposited as a deposition number of KACC 91281P. A microbial agent for controlling the plant pathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum or a microorganism fertilizer comprises the Bacillus subtilis S 37-2 strain or a culture solution thereof as an effective ingredient.

Description

Novel Bacillus subtilis S37-2 strain and microbial fertilizer using the same {Bacillus subtilis S 37-2 and Microbial fertilizer using the same}

1 is Fusarium oxysporum which is a phytopathogenic fungus of the present invention Bacillus subtilis S37-2 strain oxysporum , KACC 40037), Rhizoctonia solani , KACC 40140) and Sclerotinia sclerotirum (KACC 40457), which inhibit the growth of

Figure 2 shows a micrograph (size: length 2.1-5.2 × diameter 1.1 ㎛) of Bacillus subtilis S 37-2.

Figure 3 shows the results identified by 16S rRNA gene sequence analysis of Bacillus subtilis S 37-2.

Figure 4 shows a phylogenetic analysis using 16S rRNA gene sequence of Bacillus subtilis S 37-2.

The present invention relates to a microbial fertilizer using Bacillus subtilis S37-2 to inhibit the growth of phytopathogenic fungi. In more detail, the present invention relates to a microbial fertilizer containing Bacillus subtilis S37-2 which inhibits the growth of phytopathogenic fungi and promotes the growth of plants.

During the year-round cultivation of high-yielding economic crops, the concentration of salt in the soil due to over-application of chemical fertilizers or organic fertilizers increases the salt concentration of soil solution, imbalance of soil nutrients, crop growth defects, pest occurrence, serial disturbance Many problems have occurred, and the income of farmers is lowered, and the quality of products is markedly reduced. In order to solve such a problem, as a means of replacing chemical pesticides and chemical fertilizers, biological methods of solving safe foods while minimizing agricultural environment and ecosystem burden are being actively researched. There is a growing interest in and need for microbial fertilizers, which also promote crop growth.

The effects of plant growth-promoting microorganisms are well known VA mycorrhizal fungi, including mycorrhizal fungi that have been found by many researchers, as well as Pseudomonas (Kloepper, Nature, 1980; Dey, Microbiological Research 2004, 159). Azotobacter (Abbass, Soil Biology & Biochemistry , 1993, 25 (8))], Bacillus (Provanza, Applied Soil Ecology , 2002, 20; Cakmakci, Soil Biology & Biochemistry , 2005)], Azospirrilum (Fallik, Soil Biology & Biochemistry , 1995, 28 (1)] are known to help plant growth and are still limited to the use of some microorganisms compared to the presence of various microorganisms.

Microbial fertilizers have the advantage that they do not cause environmental pollution problems such as salt accumulation and soil pollution.The use of microbial fertilizer does not cause salt accumulation unlike chemical fertilizers, so it is excellent for maintaining the safety of the ecosystem and continuously improves crop productivity. There is this. Using these advantages, many researchers have attracted much attention in the development of microbial fertilizers.

One of the important determinants of success or failure in inhibiting phytopathogens and promoting plant growth is how long the treated strain maintains its effective density in the plant root zone (Park, Symposium). on utilization of micro - organisms resources for the secure and safe agricultural production , 1989). In the early stages of treatment, even though the effect is excellent, the treatment effect of the strain quickly disappears or becomes low over time. Therefore, although a formulation or a supplement is developed and supplied, a large amount is required to supply the whole soil. It is also difficult to maintain more than a certain level of density that is effective due to the forces trying to equilibrate soil microorganisms even with large amounts of treatment (Park, Phytopathology . 1989, 78). In addition, even though the strain having excellent function, there are also strains with low salt resistance according to species, and there are many strains whose activity is decreased in soils with high salt concentration (Kwon, Journal of Soil Fertilizer Society , 1998, 31 (3)). In light of this, microorganisms with high activity and resistance are advantageous in various soil environment conditions for maximizing utilization of useful functional bacteria. In other words, microorganisms with excellent viability are required in salt-integrated soils requiring flame resistance, house-cultivated soils requiring heat resistance, and poor soil environments in which crop failure occurs frequently.

To this end, the present invention is to isolate a novel strain having an inhibitory ability against phytopathogenic fungi and to establish its culture method, and to provide a microbial fertilizer containing the same. The novel strain of the present invention is characterized by excellent salt and heat resistance and excellent activity in various soil environmental conditions. In addition, by producing a cell suspension using the novel microorganism of the present invention, effective strains can be effectively settled throughout the roots and effective density of bacteria can be settled in a short time, effectively inhibiting plant pathogens invading roots. Growth promoting effect is also remarkably superior to the conventional effect can be usefully used.

As described above, in order to develop a microorganism having excellent flame resistance and heat resistance, and to develop a fertilizer using the same, the present invention provides a microbial fertilizer using Bacillus subtilis S37-2 strain and its preparation method, and the microbial fertilizer of the present invention is Bacillus. Characterized by subtilis S37-2 strains, and to continuously settle in the plant roots to suppress phytopathogens, and at the same time to provide a microbial fertilizer to promote the growth of plants.

The present invention is to settle the Bacillus subtilis S37-2 strain in the root zone of the plant Fusarium oxysporum which is a phytopathogenic fungus ( Fusarium oxysporum ), Rhizoctonia solani ), Sclerotinia sclerotirum , and Bacillus subtilis S37-2, which is a strain for inhibiting the growth of plants and promoting the growth of plants, and a microbial fertilizer containing the same. Hereinafter, the present invention will be described in detail.

The isolate of 373 strains was isolated from the rhizome samples of crops. Among them, strains that best inhibited phytopathogenic fungi and were effective in promoting plant growth were separated and analyzed by molecular sibling analysis and biochemical characteristics. The strain was identified as Tilis S37-2 strain, and was deposited by the applicant to the Korea Agricultural Microbiological Conservation Center (KACC 91281P) on November 14, 2006.

Hereinafter, the manufacturing process of the present invention will be described in detail. The primary culture was inoculated with Bacillus subtilis S37-2 strain in TSB (Tryptic Soy Broth) liquid medium solution and incubated at 30 ° C for 24 hours. Secondary culture is prepared by incubating 1 to 2% of the culture medium precultured in the same medium for 3 to 4 days at 30 ℃.

The primary culture is composed of 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 5 g of sodium chloride (NaCl), and 2.5 g of potassium phosphate (K ₂ HPO ₄ ) in 1 liter of water. .

In addition, when culturing the Bacillus subtilis S37-2 in large quantities, rice bran, bran, bean curd, soybean meal and the like can be used as a culture substrate. The Bacillus subtilis S37-2 can be cultured by appropriately adding a carbon source, a nitrogen source, an inorganic salt, or the like to the culture solution. The carbon source can be glucose or sugar. Mixing with by-product compost can effectively induce the growth of microorganisms. The amount used may vary depending on the type of crop, the amount of fertilization and the time of use.

The microbial fertilizer of the present invention can be irrelevant to the foliage spray or plant root zone of the plant when the plant is growing. In the case of a culture solution, it is appropriately diluted, and spraying or irrigation time is possible from the time of planting to growth stage. Spraying and irrigation may vary depending on the condition of the plant, but can be done at intervals of three or five days. In the embodiment of the present invention, only collected cells were suspended and 10 ^{7-10 8} cells / ml were irrigated 1-2 times every 7 days. The irrigation concentration of the collected cell suspension was shown to be effective to contain 1 × 10 ⁸ ~ 1 × 10 ⁹ cells / ㎖ or more Bacillus subtilis S37-2 strain.

In addition, the mass-produced culture medium is properly diluted and passed through a 100 μm → 50 μm → 25 μm sieve so that it can be effectively used using an automatic irrigation system.

In addition, the microbial fertilizer of the present invention can be prepared in the form of a liquid, granules, hydrates.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the protection scope of the present invention is not limited only to the examples.

Example  One. : Bacillus Subtilis S37 Separation of -2

Collect roots and root soil of crops, shake the sample with sterilized physiological saline at 240 rpm for 10 minutes, take 1 ml and dilute step by step at 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ It was. Take 0.2 ml of each diluent and add 1 g of TSATryptic Soy Agar-water to 17 g of tryptone, 3 g of soytone, 2.5 g of glucose, 5 g of sodium chloride (NaCl), and potassium phosphate (K ₂ HPO). ₄ ) Inoculate 2.5 g, 15 g of agar (Agar) and smear and incubate at 30 ° C. to show bacterial colonies (colony) on the medium. Platinum was used for each colony and the microbial cells were buried and then suspended in sterile water. After diluting the solution into TSA medium, colonies appeared and repeated subcultured 2-3 times to separate pure colonies.

Example  2.: present invention Bacillus Subtilis S37 Identification of -2 Strains

The identified strains were analyzed for 16S rRNA gene sequencing and morphology and biochemical properties.

(1) 16S rRNA sequencing

16S rRNA gene sequencing was used to identify bacteria. In other words, the DNA of the isolated strain was extracted with a DNA extraction kit (Toyobo, Japan), and PCR of the 16S rDNA gene was performed using the universal primers fD1 (5'-AGAGTTTGATCCTGGCTCAG-3 ') and rP2 (5'-ACGGCTACCTTGTTACGACTT-3'). Amplified by The PCR product thus obtained was reacted using a DNA sequencing kit (BigDye terminator Cycle Sequencing Ready Reacions v3.1; Applied Biosystem), and then sequenced by 3100 Genetic Analyser (Applied Biosystems). The sequence was identified to the genus through the BLAST program of NCBI server. To analyze the relationship between the strains, the 16S rDNA sequences were aligned with those of the standard strains using the CLUSTAL W program (Thompson et al., 1994). The evolutionary tree of the dataset was created using the MEGA version 3.1 (Kumar et al., 2004) program, and the stability of the branch (bootstrap value) was investigated by 1,000 resampling.

As a result of phylogenetic analysis, Bacillus 37-2 strains were shown as B. mojavensis , B. subtilis , B. amyloliquefaciens and related species as shown in FIG. 4. According to Bacillus subtilis was identified. The strain according to the present invention was named Bacillus subtilis S37-2.

 (2) Morphological and biochemical characterization

Morphology and Biochemical Properties of Bacillus Subtilis S37-2 Strain division characteristic division characteristic Gram Dyeing + Hydrolysis of Starch + Strain size Length 2.1-5.2 × diameter 1.1 μm Hydrolysis of Casein + Sporulation U Hydrolysis of Gelatin + Nitrate Reduction + Hydrolysis of Esculin + Citrate - Hydrolysis of Urea + Catalase Activity + CM-cellulose hydrolysis + Oxidase Activity - Hydrogen sulfide (H ₂ S) generation - Arginine dihydrolase + Acetoin production + β-galactosidase - Indole generation - Lysine decarboxylase - Sodium chloride (NaCl) growth 2% + Ornithine decarboxylase +        〃 5% + Tryptophane deaminase -        〃 7% + Anaerobic Growth -        〃 10% +

The strain isolated in Example 1 grows thin and sticky in tryptic soy agar medium and the shape of bacteria is rod-like and forms spores as shown in phase contrast micrographs (FIG. 2). Biochemical properties are shown in Table 1 above. The catalase test was positive and negative in the oxidase test. Starch, casein, gelatin, esculin, urea, and siem- Cellulose (CM-cellulose) was hydrolyzed. It also has the property of growing at 10% sodium chloride (NaCl). Carbon source availability is shown in Table 2 below. Glucose, Arabinose, Mannose, Mannitol, Maltose, Enacetyl, except Caprate, Adipate, and Phenyl-Acetate Sugars such as glucosamine (N-Acetyl-Glucosamine), gluconate, malate, citrate, and the like were used.

Carbohydrate Availability of the Bacillus Subtilis S37-2 Strain division characteristic division characteristic Glucose + Gluconate + Arabinose + Caprate - Mannose + Adipate - Mannitol + Malate + Maltose + Citrate + Acetylglucosamine (N-Acetyl-Glucosamine) + Phenyl-Acetate -

Example  3: Bacillus Subtilis S37 Selection of -2 Strains

The strain isolated in Example 1 was replaced with a culture to see the inhibitory ability against three phytopathogenic fungi. Phytopathogenic fungi were distributed from Korea Agricultural Microbial Preservation Center (KACC). In other words, the Fusarium oxysporum was pre-cultured with paper disks on both sides of the V8 badge and PDA badge. oxysporum , KACC 40037), Rhizoctonia solani , KACC 40140) and Sclerotinia sclerotirum (KACC 40457) were divided into 50 μl of each of the cultures, and the selected isolates made a cell suspension and absorbed into a cotton swab. The inoculum was inoculated in the center between the two paper disks inoculated with the fungus and inoculated, and the low support of the phytopathogenic fungi was compared and finally selected while incubating for 2 weeks at 28 ° C. Fusarium oxy Spokane Room (Fusarium on V8 medium oxysporum , KACC 40037), Rhizoctonia solani , KACC 40140), Sclerotinia sclerotirum , KACC 40457) shows the magnitude of the low support for the inhibition. Fusarium oxysporum on PDA oxysporum ), Rhizoctonia solani ), Sclerotinia sclerotirum ) is shown in Table 3 and FIG. 1.

Growth Inhibition of Phytopathogenic Fungi of Bacillus subtilis S37-2 Strain Isolated from Soil Selection strain badge Growth inhibitory activity of phytopathogenic fungi (low support: mm) Fusarium oxysporum Rhizoctonia solani Sclerotinia sclerotirum S 37-2 V8 8.3 ± 0.6 11.0 ± 1.0 23.0 ± 0.0 PDA 10.2 ± 0.8 9.0 ± 0.7 18.0 ± 0.7

As shown in Table 3 and Figure 1 Bacillus subtilis S37-2 strain Fusarium ( Fusarium) oxysporum , KACC 40037), Rhizoctonia solani (KACC 40140), Sclerotinia sclerotirum (KACC 40457) and the like were strongly inhibited.

Example  4. : Bacillus Subtilis S37 -2 plant growth promotion assay

In order to evaluate the plant growth of the Bacillus subtilis S37-2 strain of the present invention, the growth amount of the root of cucumber and lettuce was treated by treating the cell suspension. The treated cell suspension was incubated in TSB (Tryptic Soy Broth) culture for 3 ~ 4 days, and the supernatant was discarded, and only the cells were suspended in water and 5.6 × 10 ⁷ cells / ml in cucumbers. Was treated with 6.7 × 10 ⁸ cells / ml for artificial soil cultivation and 8.7 × 10 ⁸ cells / ml for pot soil cultivation, 50 ml per week, respectively. The results are shown in Tables 4, 5 and 6.

Cucumber Growth during Root Treatment of Cell Suspension of Bacillus Subtilis S37-2 Strain (Artificial Cultivation)  cucumber Cucumber growth % Increase contrast S 37-2  Extra long (cm) 15.6 ± 0.5 17.6 ± 0.4 12.8  Foliage weight (g) 5.4 ± 0.1 7.0 ± 0.4 29.6  (G) in root building 0.12 ± 0.01 0.17 ± 0.02 41.7

Effect of Cell Suspension Treatment of S37-2 Strain on Artificial Clay Cultivation of Lettuce Growth ratio(%) contrast S 37-2 Extra long (cm) 10.1 ± 1.0 14.6 ± 0.6 44.6 Leaves () 10.8 ± 1.0 12.6 ± 0.9 16.7 Leaf weight (g) 4.3 ± 1.5 10.8 ± 1.1 151.2 In root building (mg) 238 ± 79 420 ± 80 76.5

Growth Promoting Effect of Lettuce Root Soil Treatment by Cell Suspension Concentration of Bacillus Subtilis S37-2 (Soil Cultivation) Inoculation bacteria count (cfu / mL) Lettuce Growth Effect Leaf weight (g) % Increase (G) in root building % Increase 0 7.8 ± 0.4 - 0.300 ± 0.015 - 10 ⁵ 8.1 ± 1.0 3.8 0.319 ± 0.099 6.3 10 ⁶ 8.5 ± 0.6 8.9 0.367 ± 0.072 22.3 10 ⁷ 9.0 ± 0.7 15.4 0.377 ± 0.062 25.7 10 ⁸ 11.6 ± 0.7 48.7 0.427 ± 0.089 42.3

As shown in Table 4, in the case of cucumber, the growth of cucumber was 12.8%, 29.6% in foliage, 41.7% in roots, and 41.7% in roots, respectively. In Table 5, 50 ml of the suspension of Bacillus subtilis S37-2 strain was added to artificial clay (Pitmos + Vermiculite), and the leaf weight was increased by 151.2% and 76.5% in root. The effect was shown. Table 6 shows the growth promoting effect of the bacterial suspension concentrations of Bacillus subtilis S37-2 strains in soil, showing a 48.7% increase in the leaf weight of lettuce and 42.3% in the roots, respectively. It was shown to effect.

Example  5: Bacillus Subtilis S37 -2 strains Phytopathogenic fungi  Deterrent

To see the inhibitory effect of the phytopathogenic fungi of the present invention to strains of lettuce seedlings tea room in the gyunhaekbyeong's Crescent Loti Nias Crescent (Sclerotinia sclerotirum , KACC 40457) was treated artificially and then treated with the Bacillus subtilis S37-2 strain of the present invention and the control group not inoculated to investigate the inhibition of the onset.

The inoculation method is Sclerotinia , a phytopathogenic fungus. sclerotirum , KACC 40457) All mycelial suspensions were spray-inoculated to the same leaves, and then the Bacillus subtilis S 37-2 strain, the test strain of the present invention, was spray-inoculated, and the inoculated and non-inoculated controls. Separated.

In the lettuce growing period, the main leaves were seedlings of 1-2 leaf seedlings and 4-5 leaf seedlings. Inoculated gyunhaekbyeong bacteria PDB (Potato Dextrose Broth) 3 ilgan liquid after the culture was centrifuged to insert the saline to the resulting mycelium by Homo to homogenization in homogenizer to create a mycelial suspension, this 1 × 10 ⁶ suspension was diluted to cell / ㎖ concentration Was used. Bacillus subtilis S37-2 strains were collected from the TSA (Tryptic Soy Agar) medium and suspended in sterile water, the suspension was diluted with 1 × 10 ⁸ cells / ㎖ of bacteria, lettuce seedlings (main leaf) 1 ~ 2 leaves) were sprayed twice every 3 days, and the regular seedlings (4 ~ 5 leaves) were sprayed three times every 5 days. Seedlings were examined 3, 6, 9 days after inoculation, and regular seedlings were examined 7 days, 14 days, 21 days, 28 days after the infection of the bacterial pathogens, and Table 7 and Table 8 show the inhibitory effect of mycobacterial disease.

Inhibitory Effects of Lettuce Seedling Mycobacterium Tuberculosis on Bacillus Subtilis S37-2 Strains Strain Byeong stock rate (%) 3 days later 6 days later 9 days later contrast 0.0 75.0 100 S 37-2 0.0 8.3 16.7

Inhibitory Effect of Bacillus Subtilis S37-2 on Lettuce Nucleus Diseases Strain Byeong stock rate (%) 7 days later 14 days later 21 days later 28 days later contrast 2.8 22.2 30.5 38.9 S 37-2 2.8 2.8 2.8 5.6

Lettuce seedlings showed 100% morbidity on day 9 after inoculation, whereas the fungus suspension of Bacillus subtilis S 37-2 showed 16.7% morbidity, showing a marked inhibitory effect. In the control group, 38.9% and Bacillus subtilis S37-2 strain were treated with 5.6% of the morbidity.

In order to see the inhibitory effect of the Fusarium oxysporum (KACC 40037) strain of the present invention, after treatment with the Fusarium spores in the soil treated with the strain Bacillus subtilis S 37-2 of the present invention and control was not treated Compared. First, Fusarium oxysporum (KACC 40037) was inoculated with a 10-fold dilution of PDB (Potato Dextrose Broth) medium, which was incubated in PDA medium at 30 ° C. for 3 days and left at room temperature for 10 days. Spore suspension inoculation concentration was 3.1 × 10 ⁷ cfu / ㎖, inoculation solution was treated in the same manner in the test zones in a controlled amount close to about 7.5% of dry soil weight was treated with the strain Bacillus subtilis S 37-2 of the present invention. The treatment method of the strain of the present invention was cultured in TSB (Tryptic Soy Broth) for 3 days, the cells obtained by centrifugation was suspended in 10-fold dilution of TSB medium and treated with an adjusted amount of approximately 7.5% of dry weight, and the control was free of bacteria. The 10-fold dilution of TSB medium was treated in the same way to compare the inhibitory effect of Fusarium bacteria in soil by the invention strain.

Inhibitory effect of Bacillus subtilis S37-2 strain on treated soil Fusarium bacteria Strain Fusarium Density (× 10 ⁴ cfu / g) 0 3 days later 6 days later 9 days later contrast 104 ± 12 292 ± 15 324 ± 12 367 ± 16 S 37-2 102 ± 6 127 ± 16 145 ± 7 158 ± 17

To see the inhibitory effect of Fusarium oxysporum (KACC 40037) strains of the present invention, Fusarium spores were treated with soil treated with Bacillus subtilis S 37-2 strain after soil treatment. As a result, as shown in Table 9, the untreated control group showed more than twice the number of Fusarium bacterium compared to the treated group of the present invention strain, and showed a tendency to increase significantly as the seal passed. As described above, the strain of the present invention showed a significant suppression of Fusarium bacteria in the soil.

The novel Bacillus subtilis S37-2 strain to be provided by the present invention, the microbial fertilizer containing it as an active ingredient has an excellent effect of promoting plant growth and inhibition of phytopathogenic fungi, such as wilt disease, stem rot disease, fungal nucleus disease, It can be effectively used as an environmentally friendly microbial fertilizer that has the function of pesticide and fertilizer at the same time. In particular, it is suitable for house soils and salt integrated soils that require heat resistance and flame resistance.

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Claims (6)

Bacillus subtilis S 37-2 (KACC 91281P) strain inhibits the growth of phytopathogenic fungi and promotes plant growth. A microbial agent for controlling phytopathogenic fungi using the bacterium of Bacillus subtilis S 37-2 strain or its culture medium as an active ingredient. The method of claim 2, wherein the phytopathogenic fungi are Fusarium oxysporum , Rhizoctonia solani ) and Sclerotinia sclerotirum . A microbial fertilizer which promotes plant growth by using the bacterium of Bacillus subtilis S 37-2 strain or its culture medium as an active ingredient. A method for controlling phytopathogenic fungi by spraying or irrigation of the strain of claim 1 or the microbial agent of claim 2 on a plant. A method of promoting plant growth by spraying or irrigation of the strain of claim 1 or the microbial fertilizer of claim 4 to the plant.

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