KR102525319B1 - Manufacturing method of Bacillus licheniformis JWS22003 strain that promotes fermentation of by-products and plant growth and produces auxin, method for fermentation of by-products using the same, method for improving plant growth - Google Patents

Abstract

The present invention discloses a method for promoting fermentation of by-products by using a 100-fold dilution of a Bacillus licheniformis JWS22003 strain (accession No.: KACC 92452P) and a Bacillus licheniformis JWS22003 strain, which promote fermentation of by-products, generation of auxin, and the growth of plants, a method for promoting the growth of plants; a method for generating auxin by a Bacillus licheniformis JWS22003 strain; and a method for producing a Bacillus licheniformis JWS22003 strain, which is produced by treating agricultural by-products with ethyl methane sulfonate (EMS) that induces chemical mutations in the Bacillus licheniformis JWS22003 strain collected from neighboring soil.

Description

Manufacturing method of Bacillus licheniformis JWS22003 strain that promotes fermentation of by-products, promotes plant growth, and produces auxin, and a by-product fermentation method and plant growth promotion method using the same products and plant growth and produces auxin, method for fermentation of by-products using the same, method for improving plant growth}

The present invention is a Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) characterized by promoting fermentation of by-products, auxin production and plant growth, a method for fermenting by-products using the same, and a method for promoting plant growth It relates to, more specifically, a method for promoting fermentation of by-products using a 100-fold dilution of the Bacillus licheniformis JWS22003 strain, a method for promoting plant growth using a 100-fold dilution, Bacillus Method for producing auxin by Bacillus licheniformis JWS22003 strain and ethyl methane sulfonate (EMS) causing chemical mutagenesis in Bacillus licheniformis strain collected from crop by-products and nearby soil It relates to a method for preparing a Bacillus licheniformis JWS22003 strain by treating Bacillus licheniformis JWS22003.

Currently , B. subtilis, B. thuringiensis, and B. licheniformis are attracting attention as organic agricultural materials of the Bacillus genus. Compared to other microorganisms, the aforementioned B. subtilis and B. thuringiensis are used in various industrial fields as agricultural antibiotics, microbial fertilizers, and transgenic genetic materials. On the other hand, analysis techniques suitable for culturing and identifying B. licheniformis have not been established, and thus research and industrialization of useful strains are relatively insufficient.

Bacillus licheniformis (hereinafter referred to as B. licheniformis ) is a Gram-negative, mesophilic bacterium commonly found in soil or crop residues. Depending on the strain, it can survive at a temperature higher than 50℃ or higher, so it is attracting attention for agricultural use recently because it has low concerns about contamination, deterioration, and death during self-manufacturing or production and distribution as agricultural materials in farms.

Among various microorganisms, ecologically, there are cases in which various useful functions are exhibited by interaction with plant pathogens or plants. The principle is, firstly, suppressive antibiotic action (antibiosis) through antimicrobial antibiotics against Streptomyces blastmyces , S. griseus , Peicillium nigricans , Bacillus subtilis , etc., and secondly, fungal outer membrane hydrolysis, such as chitinase and β-1,3-glucanase. Third, plant growth-promoting rhizobacteria (PGPR); Fourth, it promotes fermentation of plant residues or by-products to produce high-efficiency, high-quality fermented compost. There are cases where the secretion of enzymes such as cellulase and chitinase is excellent. In particular, B. licheniformis is known to make various enzymes to ferment by-products, promote plant growth, and biosynthesize auxin.

This technology pays attention to the efficiency of agricultural material production and plant growth promotion (PGPR) through the possibility of culturing B. licheniformis at high temperatures, which has been known so far, to search for new B. licheniformis with high vitality, and to transform it into EMS (Ethyl Methane Sulfonate) chemical mutation. The purpose is to develop a new strain using this strain, and to use this strain for fermentation of by-products and promotion of crop growth.

Registered Patent Publication No. 10-1500930 (Bacillus licheniformis strain having antibacterial activity)

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An object of the present invention is to prepare a strain of Bacillus licheniformis JWS22003 and to ferment by-products using the strain, improve plant growth, and produce auxin.

In addition, an object of the present invention is to prepare a Bacillus licheniformis JWS22003 strain having high vitality in plant growth promotion (PGPR) and efficiency in production of agricultural materials through the possibility of culturing B. licheniformis at high temperature.

In order to achieve the above object, the Lactobacillus paracasei strain according to the present invention promotes fermentation of by-products and plant growth and produces auxin, Bacillus licheniformis JWS22003 (Accession Number: KACC) 92452P).

In addition, the method for accelerating the fermentation of by-products according to the present invention is characterized by promoting the fermentation of by-products by utilizing a 100-fold dilution of the Bacillus licheniformis JWS22003 strain (Accession Number: KACC 92452P).

In addition, the plant growth promotion method according to the present invention is characterized by foliar fertilization of a 100-fold dilution of the Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) to crops to promote plant growth. .

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In addition, Bacillus licheniformis JWS22003 ( Bacillus licheniformis JWS22003) strain (Accession Number: KACC 92452P) manufacturing method according to the present invention is a chemical mutation in Bacillus licheniformis JWS22003 ( Bacillus licheniformis ) strain collected from crop by-products and nearby soil It is characterized in that it is a method manufactured by treating ethyl methane sulfonate (EMS) that causes.

The present invention has the effect of securing Bacillus licheniformis JWS22003 biological resources by using ethyl methane sulfonate (EMS) chemical mutation.

In addition, the present invention has an effect that compost fermentation can be improved through the Bacillus licheniformis JWS22003 strain.

In addition, the present invention has the effect of improving crop growth by the Bacillus licheniformis JWS22003 strain.

In addition, the present invention has the effect that auxin can be produced by the Bacillus licheniformis JWS22003 strain.

1 is a view showing in vitro culture of B. licheniformis JWS22003 strain.
Figure 2 is the genetic sequence information of B. licheniformis J WS22003 strain.
Figure 3 is the result of genetic relationship analysis of B. licheniformis J WS22003 strain.
Figure 4 is the result of HPLC analysis of auxin production of B. licheniformis J WS22003 strain.

The Bacillus licheniformis JWS22003 strain (Accession No.: KACC 92452P) promotes fermentation of by-products, plant growth, and produces auxin.

Bacillus licheniformis JWS22003 ( Bacillus licheniformis JWS22003 ) strain (accession number: KACC 92452P) manufacturing method is EMS (ethyl methane sulfonate).

When foliar application of a 100-fold dilution of Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) to crops in order to promote plant growth, it is preferable to apply foliar application three times.

Hereinafter, the present invention will be described in detail with reference to the drawings.

1. Development and analysis method

B. licheniformis was isolated based on samples collected from the residue left as a by-product after harvesting rice and from the soil nearby. Purely isolated B. licheniformis strains that grew vigorously in vitro were selected in a nutritional agar medium. The selected B. licheniformis strain NA was cultured for 48 hours at a culture temperature of 28 ° C. The selected B. licheniformis strain was dispensed in 10 repetitions of 0.9 ml each, and then treated with 100 μl/ml EMS for 10 minutes. 100 μl of the above suspension was plated on NA medium and cultured at 28° C. for 48 hours. Among them, the strain with the most vigorous in vitro growth was selected and named as B. licheniformis JWS22003.

Named B. licheniformis JWS22003 was applied to wood by-product compost, crops (Hwaseong rice, Daewon bean, Cheongchima lettuce, Chunwang cabbage, Grand pepper, Hongro apple) in the control group untreated, treatment No. 1 diluted 10 times, treatment No. 2 diluted 100 times, treatment Three 500-fold dilutions were performed. The physicochemical analysis of this material was analyzed according to the AOAC (Association of Official Agricultural Chemists) standard analysis method.

2. Development results

B. licheniformis was selected as a single colony with strong in vitro growth on NA medium. The selected single colony strain is subjected to EMS mutation, and among these, the dead or poor in vitro growth strains are culled, and one EMS mutant strain with vigorous in vitro growth in NA medium is finally selected to be B. licheniformis JWS22003. named (Fig. 1).

The gene sequence of the selected EMS mutant B. licheniformis JWS22003 strain was aligned and analyzed (Fig. 2).

Based on this genetic information, a comparative analysis of the ITS16s rRNA gene base sequence with the existing Bacillus subtilis registered in the National Center for Biotechnology Information of the United States and a phylogenetic tree were performed to identify the same species that have been identified so far. It was confirmed that it was different compared to Bacillus subtilis ( B. licheniformis ) (FIG. 3).

3. Assay results

In the present invention, when the new B. licheniformis J WS22003 strain was treated with compost fermentation, all treatments showed better effects than untreated control. In treatment group 1, the ammonia nitrogen content was 78.99 mg/L at 20.41 mg/L and the nitrate nitrogen content was 182.13 mg/L at 76.04 mg/L. In treatment group No. 2, the ammonia nitrogen content was 63.46 mg/L at 21.15 mg/L and the nitrate nitrogen content was 173.80 mg/L at 80.28 mg/L. In two treatments of treatment groups 1 and 2, the effect was three times higher than that of the control group. In particular, treatment group 2 was economical because the dose of B. licheniformis J WS22003 strain was 1/10 compared to treatment group 1.

Table 1 shows the nitrogen content change after treatment in compost fermentation of B. licheniformis J WS22003 strain.

Sample before treatment (mg/L) Sample after treatment (mg/L) NH ₄ ⁺ NO ₃ ^- NH ₄ ⁺ NO ₃ ^- control 19.25 75.58 44.48 61.86 treatment area 1 20.41 76.04 78.99 182.13 treatment area 2 21.15 80.28 63.46 173.80 treatment zone 3 21.49 74.99 54.46 86.68

HPLC analysis was performed to determine the amount of auxin produced by the B. licheniformis J WS22003 strain. As a result, it was confirmed that auxin was biosynthesized by the B. licheniformis J WS22003 strain (FIG. 4).

Here, auxin is one of several regulators for the germination and growth of plants from seeds, and is a kind of plant growth hormone that is particularly involved in the elongation of stems. In addition, auxin is produced at the tips of stems and roots where growth is vigorous, and promotes length growth by elongating cell walls. Accordingly, the growth of the plant can be promoted due to the production of auxin.

B. licheniformis J WS22003 strain culture was foliarly applied to rice (Hwaseong rice) three times, and it was confirmed that plant height, tillering number, and chlorophyll were improved throughout the entire growth period compared to the control group in all treatments (Table 2). In particular, plant height of No. 2 in the 100-fold treatment group was 62.46 cm on 30 days, 93.64 cm on 60 days, 106.73 cm on 90 days, number of tillers 10.96/week on 30 days, 13.30/week on 60 days, 13.77/week on 90 days, chlorophyll 41.82 on 30 days. , 37.73 at 60 days and 35.48 at 90 days, showing that the B. licheniformis J WS22003 strain improves rice growth.

Table 2 shows the growth improvement effect of rice (Hwaseong rice) after foliar fertilization of B. licheniformis J WS22003 strain.

pasture
(cm) Tiller water
(ea/plant) chlorophyll
(SPAD) 30 days 60 days 90 days 30 days 60 days 90 days 30 days 60 days 90 days control 56.58 84.13 102.27 7.45 10.98 12.63 34.02 31.32 32.91 treatment area 1 59.52 85.95 100.41 9.78 11.55 11.13 39.64 32.57 34.03 treatment area 2 62.46 93.64 106.73 10.96 13.30 13.77 41.82 37.73 35.48 treatment zone 3 59.11 91.95 105.05 10.91 13.07 13.66 41.39 36.38 33.82

B. licheniformis J WS22003 strain culture was foliarly applied to soybean (Daewon bean) three times, and it was confirmed that all treatments improved stellate length, branch number, seed number, white grain weight, yield, and production rate compared to the control group (Table 3). In particular, treatment No. 2 with 100 times the liquid was the most excellent with long length 74.97cm, branching number 82.94 pieces/week, seed number 250.60 grains/week, white grain weight 27.88g, yield 259.56kg/10a, and production rate 128% . B. licheniformis J WS22003 The strain was shown to enhance soybean growth.

Table 3 shows the growth improvement effect of soybean (Daewon bean) after foliar fertilization of B. licheniformis J WS22003 strain.

light coat
(cm) branching index
(recast) seed count
(lips/week) Baekgripjung
(g) output
(kg/10a) production rate
(%) control 67.40 74.36 215.52 24.06 203.25 100 treatment area 1 72.38 75.18 230.40 26.03 228.69 113 treatment area 2 74.97 82.94 250.60 27.88 259.56 128 treatment zone 3 72.24 71.82 243.18 25.22 231.71 114

B. licheniformis J WS22003 strain culture was foliarly applied to lettuce (cheongchima) three times, and in all treatments, it was confirmed that the above-ground live weight and dry weight ratio, underground root length, underground live weight, and underground dry weight ratio were improved compared to the control ( Table 4). In particular, treatment No. 2 with 100 times the liquid was the most excellent with 41.35g of dry weight in the aboveground part, 3.74% of dry weight, 35.76cm of root length in the underground part, 4.78g of live weight in the underground part, and 3.72% of dry weight in the underground part. has been shown to improve.

Table 4 shows the growth improvement effect of lettuce (cheongchima) after foliar fertilization of B. licheniformis J WS22003 strain.

above ground underground live weight
(g) dry weight ratio
(%) root length
(cm) live weight
(g) dry weight ratio
(%) control 33.46 3.58 30.73 3.39 3.24 treatment area 1 38.07 3.59 31.00 4.69 3.61 treatment area 2 41.35 3.74 35.76 4.78 3.72 treatment zone 3 37.50 3.61 34.44 3.80 4.75

B. licheniformis J WS22003 strain culture was foliarly applied to Chinese cabbage (Chungwang) three times, and it was confirmed that all treatment groups improved the number of bulbs, bulbs, corms, leaves, and chlorophyll compared to the control group (Table 5). In particular, treatment group No. 2 with 100 times the solution was the most excellent with bulb weight of 2.95kg, bulb height of 27.72cm, diameter of 17.60cm, number of leaves of 41.95, and chlorophyll of 35.02.

Table 5 shows the growth improvement effect of Chinese cabbage (Chunkwang) after foliar fertilization of B. licheniformis J WS22003 strain.

courtesy
(kg) Gugo
(cm) caliber
(cm) ground game
(hawk) chlorophyll
(SPAD) control 2.26 24.86 16.51 33.70 32.29 treatment area 1 2.76 27.66 17.25 35.11 33.17 treatment area 2 2.95 27.72 17.60 41.95 35.02 treatment zone 3 2.79 26.31 17.12 39.04 34.36

B. licheniformis J WS22003 strain culture was foliarly applied to pepper (Geojanghan) three times, and it was confirmed that all treatments improved plant height, number of leaves, leaf area, live weight, dry weight, fruit tree, weight, and dry weight compared to the control group (Table 6). In particular, plant length 152.81cm, number of leaves 2,088.34 sheets, leaf area 25,592.94cm ² /week, live weight 489.13g/week, dry weight 105.31g/week, fruit tree 46.17ea/week, fruit weight 537.52g/week, dry weight 84.24 g/week was the most excellent, indicating that the B. licheniformis J WS22003 strain improved the growth of pepper.

Table 6 shows the growth improvement effect of red pepper (Geojanghan) after foliar fertilization of B. licheniformis J WS22003 strain.

pasture
(cm) ground game
(hawk) leaf area
(cm ² /week) live weight
(g/week) in the building
(g/week) fruit tree
(recast) overload
(g/week) dry weight
(g/week) control 148.45 2,033.75 24,822.59 471.32 101.35 40.94 490.07 81.73 treatment area 1 151.24 2,074.60 25,445.81 482.34 103.98 43.01 515.65 83.92 treatment area 2 152.81 2,088.34 25,592.94 489.13 105.31 46.17 537.52 84.24 treatment zone 3 150.70 2,071.78 25,310.70 484.97 103.54 45.24 517.04 82.07

B. licheniformis J WS22003 strain culture was foliarly applied to apples (hongro) three times, and it was confirmed that the amount of shoots, shoots, fruit trees, and chlorophyll were improved in all treatments compared to the control (Table 7). In particular, in treatment group No. 2 with 100 times the liquid, shoot weight 64.70cm, shoot 49.17mm, fruit tree 36.09/stock, and chlorophyll 35.97 were the most excellent, indicating that B. licheniformis J WS22003 strain improved apple growth.

Table 7 shows the growth improvement effect of apple (red roe) after foliar fertilization of B. licheniformis J WS22003 strain.

Shinchoryang
(cm) motorboat
(mm) fruit tree
(recast) chlorophyll
(SPAD) control 59.23 42.30 32.88 30.05 treatment area 1 61.61 44.50 34.15 33.11 treatment area 2 64.70 49.17 36.09 35.97 treatment zone 3 63.13 45.98 35.65 33.43

As a result of testing the improvement effect by treating the above B. licheniformis JWS22003 strain with wood by-product compost and six types of horticultural crops,

In the case of by-products, as a result of treating the B. licheniformis J WS22003 strain with compost fermentation, the growth was the best when treated with 10-fold solution compared to the control without any treatment . Since the dosage of the licheniformis J WS22003 strain is 1/10 level, it is economical, so the growth is the best, and it was shown that the B. licheniformis J WS22003 strain improves the growth.

In addition, in the case of plants, as a result of foliar fertilization of B. licheniformis J WS22003 strain culture medium to crops three times, it was the best to treat B. The strain licheniformis J WS22003 has been shown to improve growth.

In addition, as a result of HPLC analysis to determine the amount of auxin produced by the B. licheniformis J WS22003 strain, it was confirmed that auxin was biosynthesized by the B. licheniformis J WS22003 strain.

Accordingly, the B. licheniformis J WS22003 strain has high vitality by paying attention to the efficiency of agricultural material production and plant growth promotion (PGPR) through the possibility of high-temperature cultivation of B. licheniformis, so it can be used for fermentation of by-products and promotion of crop growth. there is.

Rural Development Administration National Academy of Agricultural Sciences Microorganism Bank (KACC) KACC92452P 20220922

Claims (5)

A Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) characterized by promoting fermentation of by-products and growth of plants and producing auxin.
A method for accelerating the fermentation of by-products by utilizing a 100-fold dilution of the Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) according to claim 1.
A method for promoting plant growth, characterized in that foliar fertilization of a 100-fold diluted solution of the Bacillus licheniformis JWS22003 strain (accession number: KACC 92452P) according to claim 1 is applied to crops to promote plant growth.
delete The Bacillus licheniformis JWS22003 strain (Accession Number: KACC 92452P) according to claim 1 is a strain that induces chemical mutations in Bacillus licheniformis strains collected from crop by-products and nearby soil. Bacillus licheniformis JWS22003 ( Bacillus licheniformis JWS22003) strain production method, characterized in that prepared by processing EMS (ethyl methane sulfonate).

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