KR100963694B1 - Bacillus amyloliquefaciens KJ-6 and the method for decomposing to citrus tree sawdust using it - Google Patents

Abstract

The present invention provides a Bacillus amyloliquefacien s ( B. amyloliquefacien s) KJ-6 (KACC 91260P) having a fibrinolytic and antimicrobial activity and a method for composting citrus by-products using the same.

According to the present invention, it is possible to stably compost citrus fruit by-products using Bacillus amyloliquisis KJ-6, thereby recycling the resources of citrus fruit by-products.

Description

Bacillus amyloliquefaciens KJ-6 and a method for composting citrus by-products using the same {Bacillus amyloliquefaciens KJ-6 and the method for decomposing to citrus tree sawdust using it}

The present invention relates to a new microorganism having Bacillus amyloliquipeciency KJ-6 (KACC 91260P) having fibrinolytic and antimicrobial activity and a method for composting citrus fruit by-products using the same.

Due to the abuse of chemical synthetic agricultural materials, farmland is contaminated and interest in food safety is increasing, and the necessity of eco-friendly agriculture is required for safe agricultural food production and sustainable agriculture. To this end, many efforts are being made for the use of environmentally friendly agricultural materials and the recycling of agricultural by-products.

For the recycling of agricultural by-products, the function of microorganisms, which play an important role in the decomposition and circulation of materials, is emphasized. Recently, a variety of products have been produced and used, such as microbial decomposition using microorganisms, promoting crop growth, and antagonists.

In the development of pest control technologies intended for environmental conservation, the function of antagonism microorganisms, which are the constituents of the ecosystem, or the inhibited soils that are the result of antagonism are reexamined and obtained by replacing current control measures, or as one of such means. The development of usable control methods is expected.

Antagonists are involved in many cases. Its use is to inhibit the activity of soil pathogens or to reduce the incidence by reducing the number of bacteria by activating indigenous microorganisms originally inhabiting the soil and by culturing once isolated strains and propagating them into the soil. . In the case of growing healthy crops by crop rotation or proper pavement management, the soil pathogen count is suppressed to a low level, and it is estimated that the activity of soil pathogens is also suppressed by the activity of indigenous microorganisms. Control tests using organic materials were actively conducted in Japan in the 1960s and 1970s in order to activate indigenous microorganisms and to inhibit the activity of pathogens.

Soil-inhibiting soil against soil diseases is a good example of spontaneous biocontrol, and the mechanism is being energized. Both of these naturally use indigenous antagonists. From there, a strain of promising antagonistic microorganisms was isolated and the development of biological control methods by the introduced microorganisms was studied. Since all soil diseases originate from the bottom of the soil, they do not feel early in the onset of disease, and when symptoms appear, they are often late for handwriting. Therefore, preventive measures such as control using antagonistic microorganisms that make up these ecosystems are needed.

Therefore, in order to resource localized by-products, it is necessary to select and use indigenous strains that have excellent viability and function well under local environmental conditions. Many microbial products are commercialized and used in farms, so the function of the strain is much lowered and the effect is not being obtained.

Moreover, citrus by-products have excellent fertilization value, but when they are not composted due to the residual of citrus pathogens in the material, there is a fear of spreading pathogens when supplied to farmland.

In order to solve the above problems, an object of the present invention is to select strains with excellent environmental stress, high antibacterial activity and high fibrinolytic ability to supply the farmland after composting citrus by-products.

In addition, it is an object to compost the citrus by-products using the strain or microbial agent comprising the same.

The present invention provides B. amyloliquefacien s KJ-6 (KACC 91260P) having fibrinolytic and antimicrobial activity.

The present invention also provides a method for composting citrus fruit by-products using B. amyloliquefacien s KJ-6 (KACC 91260P).

According to the present invention, it is possible to stably compost citrus fruit by-products using Bacillus amyloliquisis KJ-6 having fibrinolytic and antimicrobial activity, thereby recycling the resources of citrus fruit by-products.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are only presented to aid the understanding of the present invention, but the present invention is not limited thereto.

Example 1 Isolation and Identification of Bacillus amyloliquefacien s KJ-6 (KACC 91260P)

(1) Isolation of B. amyloliquefacien s KJ-6

For the purpose of developing highly functional biofertilizer using citrus sawdust and livestock manure, 30 g of soil was collected from barns, citrus tree shredded orchards, soil, and foliar soil to be used as soil samples. The sample was added to sterile water and shaken at 150 rpm for 30 minutes, and then diluted to 10 ⁵ levels. Selection medium (yeast extract 3.0 g, glucose 1.0 g, K ₂ HPO ₄ 0.3 g, KH ₂ PO ₄ 0.2 g, MgSO _{4 ·} 7H ₂ O 0.2g, agar (agar) 15.0g, distilled water 1000ml, pH 6.8) was used to select a good bacteria growth at high temperature. That is, bacteria having excellent fibrinolytic ability were isolated and selected by culturing carboxymethylcellulose (hereinafter referred to as “CMC”) and cellulose at 30 ° C. as a carbon source.

(2) Identification by B. amyloliquefacien s ( B. amyloliquefacien s) 16S rDNA sequencing of KJ-6

To identify the selected strains, fatty acids present in the cell wall were extracted and identified using a microorganism identification system (Sherlock Microbial Identification System, MIDI Inc.), and the species name having a similarity index value of 0.5 or more was used as the strain name. Cultivation of isolated strains for identification and Gas Chromatography (GC (HP 6990)) conditions were referred to the MIDI Operation Manual Version 6, and the saponification, fatty acid The extract was analyzed by gas chromatography through esterification, extraction of fatty acid methylester (FAME), and pre-treatment of base washing. The base sequence (approximately 1500 bp) of 16S rDNA of B. amyloliquefaciens ( B. amyloliquefaciens ) was determined, the bacterial reference stock database and the American GenBank, Japan's Japan DNA Data Bank (DDBJ; DNA Data Bank of Japan and European Molecular Biology Laboratory (EMBL) have searched for the homology of the base sequence. As a result, MIDI analysis of Bacillus subtilis group showed Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens; Bacillus). subtilis In accordance with group ), Bacillus amyloliquefaciens KJ-6 was named. Figure 1 shows the results of observing the form of the strain with an electron microscope.

(3) B. amyloliquefaciens KJ-6 culture method

100 µl of the cryopreserved cell suspension of B. amyloliquefaciens of the strain B. amyloliquefaciens of the present invention was selected (3.0 g of yeast extract, 1.0 g of glucose, 0.3 g of K ₂ HPO ₄ ). , 0.2 g of KH ₂ PO _4, 0.2 g of MgSO _{4 ·} 7H ₂ O, 15.0 g of agar (agar), 1000 ml of distilled water, pH 6.8).

Example 2 Evaluation of Fertilizer Value of Citrus Tree By-Products

In order to improve the quality of the tangerines and to control the yield, thinning and partial waste are underway every year. The by-products of citrus crush and sawdust are about 38,500 tons per year. Physicochemical components of citrus sawdust were analyzed in order to use citrus crush and sawdust. The results are shown in Table 1.

[Table 1] Physicochemical Components of Citrus Sawdust (%)

division T-C T-N T-P K Ca Mg C / N Density (g / cm 3) Cedar sawdust 48.1 0.15 0.02 0.09 0.27 0.03 320.6 0.16 Citrus Sawdust 48.7 0.49 0.06 0.22 0.66 0.04 99.4 0.27

As shown in Table 1, citrus trees have a higher total nitrogen, potassium, calcium content and carbonaceous ratio (C / N ratio) than three times as compared to conventional cedars, which are easy to compost and have excellent fertilizer value. Therefore, when mixed with livestock manure, well-adjusted by microorganisms, the recycling of resources and manure can be properly handled, and the cost of livestock manure and marine waste can be reduced, resulting in the use of by-product compost by bad materials. It can reduce the damage of farms.

Example 3 Evaluation of Fibrinolytic Activity of B. amyloliquefacien s KJ-6 (KACC 91260P)

(1) measurement of fibrinolytic activity

1) Determination of the activity of Carboxymethylcellulase (hereinafter referred to as "CMCase")

CMC plate medium (CMC 10g, NaNO ₃ 2g, K ₂ HPO ₄ 1g, MgSO ₄ · 7H ₂ O 0.5g, KCl 0.5g, FeSO _{4 ·} 7H ₂ O 0.5g, Agar 15g, Distilled water 1L, pH 7.0 ) Was inoculated with B. amyloliquefaciens KJ-6 and incubated at 28 ° C. for 5-7 days. Then, stained with 0.1% Congo-red for 15 minutes, washed twice with 1M NaCl, and then decomposed by the size of the clear zone formed as the CMC decomposed at the end of growth of the inoculated bacteria. Was investigated. The results are shown in Table 2 and FIG. 2A.

2) Determination of Cellulase Activity

Cellulose flat medium (cellulose powder 1g, yeast extract 1g, (NH ₄ ) ₂ SO ₄ 2.5g, K ₂ HPO ₄ 0.25g, NaCl 0.1g, MgSO _{4 ·} 7H ₂ O _{0.125g, FeSO 4 · 7H 2 O} 0.0025g, MnSO 4 · 4H 2 O 0.0025g, distilled water 1L, agar (agar) 10g, pH 7.2) of Bacillus amyl Lowry kwipe KJ-6 sieon's (B. amyloliquefacien s) in Inoculation was incubated for 5-7 days at 28 ℃. After staining with 0.1% Congo-red for 15 minutes, washing twice with 1M NaCl, and then degrading activity due to the size of the clear zone resulting from the decomposition of cellulose at the end of growth of the inoculated bacteria Was investigated. The results are shown in Table 2 and FIG. 2B.

[Table 2] Fibrinolytic Activity

Strain
CMCase Cellulase 5 days 7 days 5 days 7 days KJ-6 ++ +++ ++++ ++++

* Transparent ring formation: ++++: 18-19 mm, +++: 16-17 mm, ++: 14-15 mm, +: 12-13 mm

3) Determination of peroxydase activity

Agar-malt plate medium (malt extract 20g, agar 20g, distilled water 1000ml) was inoculated with B. amyloliquefacien s KJ-6 at 28 ° C. Incubated for 3-5 days. Incubate for 4 hours with the addition of O-dianisidine solution (O-dianisidine 0.1g, pH 4.4) and peroxidase with red-brown color. (peroxydase) activity was measured. Figure 2c is a photograph measuring the peroxydase (peroxydase) activity.

4) Determination of Amylase Activity

Starch-ammonia plate medium (starch water-soluble starch 10g, (NH ₄ ) ₂ SO ₄ 2g, NaCl 1g, CaCO ₃ 3g, K ₂ HPO ₄ 1g, MgSO ₄ 1g, agar 20g, B. amyloliquefaciens KJ-6 was inoculated in 1000 ml of distilled water) and incubated at 28 ° C. for 3-5 days. Lugol solution (Lugol solution) (I ₂ 1g, KI 3g, distilled water 300ml) was added to measure the activity by the size of the transparent ring after staining. Figure 2d is a photograph measuring the activity of amylase.

(2) results

As shown in Table 2 and Figures 2a to 2d, the selection strain ( B. amyloliquefacien s KJ-6) is an indigenous microorganism isolated from the citrus fruit source citrus tree with excellent environmental stress and fibrinolytic activity By accelerating the decomposition of by-products can shorten the composting period.

(3) Comparison of Fibrinolytic Activity According to Strains

B. amyloliquefacien s ( B. amyloliquefacien s) To evaluate the fibrinolytic activity of KJ-6 was composted with different fibrinolytic microorganisms, micromono sporarosaria RK-2 and RC-8, respectively.

[Table 3] Humic acid content according to composting inoculation strains

process
Humic acid C (g / kg) E4 / E6 contrast 14.5 11.0 KJ-6 13.5 11.6 RK-2 13.5 10.3 RC-8 11.7 11.3

As shown in Table 3, the values of E4 / E6, an indicator of the maturity of 50-day-compost compost, were 11.6 in KJ-6 (11.0) and RK-2 (10.30 and RC-8 (11.3). Higher than that, it can be seen that the housing is progressing smoothly.

[Table 4] Changes in fibrin content of 50-day-old compost according to strain inoculation

process
cellulose hemicellulose Building quantity Decrease Building quantity Decrease contrast 13.49 23.2 5.68 9.0 KJ-6 9.79 44.2 5.68 25.0 RK-2 13.18 24.9 5.18 17.0 RC-8 10.16 42.1 5.80 7.0

In addition, as shown in Table 4, the cellulose of the 50-day-old compost was 44.2% KJ-6, 42.1% RC-8, which was much lower than the control 23.2%. And hemicellulose (KJ-6 25%, RK-2 17%) was 2 ~ 3 times higher than control 9%.

This is believed to be reduced by the degradation of the fiber in the compost by these strains in the composting process. In terms of the amount of fiber reduction in the compost, KJ-6 had the greatest effect of fibrinolysis.

Example 4 Evaluation of Antimicrobial Activity of B. amyloliquefacien s KJ-6

(1) Antibacterial activity measurement method

1) Test Pathogen

Citrus, Diaporthe citri and Fusarium on Potato dextrose agar (PDA) media oxysporum ) and phytophthora sp. were implanted and cultured in a thermostat at 25 ° C. for 5 days, followed by removal of a 5 mm diameter pathogen from the center of a 87 mm diameter disposable Petri dish dispensed with PDA medium.

2) Mycelial growth inhibition test

Bacillus amyloliquipeciency KJ-6 was incubated for 24 hours at 28 ℃, 150rpm using nutrient agar (NA) medium. Thereafter, inoculated strains at regular intervals in petri dishes with pathogens and allowed to incubate at 30 ° C. for 7 days and then examined the inhibition zone of the pathogens.

Table 5 Antimicrobial Activity against Plant Pathogens

Strain plague Citrus Black Spot Wilted KJ-6 +++ +++ +

As shown in Table 5, KJ-6 has antimicrobial activity against plant pathogens and wilted ills, which are phytopathogens, and have an antagonistic effect against citrus black spot pattern pathogens (FIG. 3). It is thought that it is possible to reduce the density and to reduce the pathogen density of the citrus source when fed as compost. In addition, as shown in Figure 3, it can be seen that there is an antagonistic effect against wilted disease when growing tomatoes in compost using citrus sawdust.

(2) Antagonistic Evaluation of Plant Pathogenic Bacteria (Tomato wilt, Fusarium oxysporum)

We investigated whether KJ-6 inoculation showed direct antimicrobial activity against wilted bacteria of tomato. The test method involves filling garden pots with plastic pots and removing the fungus ( Fusarium). oxysporum ), after seeding the tomatoes and growing them in a greenhouse for a certain period of time was measured plant weight and root weight per week (Table 6).

[Table 6] Antagonistic Evaluation of Plant Pathogenic Bacteria in Strain Inoculation Compost

process
Growth amount (g) Plant weight Root weight contrast 0.6 ± 0.32 0.2 ± 0.09 N-P-K Standard Fertilization 3.4 ± 0.63 0.5 ± 0.30 KJ-6 1.4 ± 0.77 0.5 ± 0.30 RK-2 0.5 ± 0.21 0.2 ± 0.08 RC-8 0.6 ± 0.19 0.2 ± 0.06

As a result of tomato cultivation test using Fusarium oxysporum , the plant weight was 0.6 ± 0.32g in control, 3.4 ± 0.63g in chemical fertilizer and 1.4 ± 0.77g in KJ-6. As shown in Table 6, KJ-6 had better growth than RK-2 and RC-8.

In addition, as shown in Figure 4, withering strain pathogenic bacteria ( Fusarium) oxysporum ), KJ-6 was 15%, RK-2 49%, and RC-8 30%. KJ-6 had three times higher antimicrobial activity than RK-2.

Example 5 Growth Range of B. amyloliquefacien s KJ-6

As shown in FIGS. 5 and 6, B. amyloliquefacien s KJ-6 has a wide growth range with a growth potential of 4-7 and a growth temperature of 4-70 ° C. In addition, Bacillus amyloliquefaciensis ( B. amyloliquefacien s) KJ-6 as an indigenous strain has excellent settlement ability.

Example 6 Evaluation of Compost Using B. amyloliquefacien s KJ-6

Bacillus amyloliquefacien s ( B. amyloliquefacien s) according to Example 1 150-250 kg of citrus tree shreds, citrus tree sawdust 150-250 kg, 300-500 kg of livestock manure was mixed and stirred while supplying moisture so that the water content was contained in 50 to 60% of the total weight of the mixture. At this time, the stirring was further stirred 3 to 4 times while fermenting for 150 days.

As shown in Fig. 7 and 8, as a result of measuring the maturity of compost and other products using citrus sawdust, the seed germination rate was 96% higher, the root length is 11.4% than the control compost in the 50-day compost The high weight of 28% makes it possible to produce compost with high toxicity and low antibacterial activity.

B. amyloliquefaciens KJ-6, which has excellent fibrinolytic and antimicrobial activity, was deposited with the Agricultural Biotechnology Research Institute under the microbial accession number KACC 91260P.

1 is an electron micrograph of B. amyloliquefacien s KJ-6.

2a, 2b, 2c and 2d are photographs showing CMCase, cellulose, peroxidase and amylase enzyme activity of Bacillus amyloliquipeciency KJ-6, respectively.

Figure 3 is a photograph showing the antimicrobial activity against citrus black spot disease (left) and tomato wilted disease (right).

Figure 4 is a chart showing the damage rate by strain for tomato wilt.

5 is a chart showing the growthable pH of Bacillus amyloliquipeciency KJ-6.

6 is a chart showing the growth temperature of Bacillus amyloliquipeciency KJ-6.

7 is a chart showing seed germination rate using compost and prototype according to the present invention.

8 is a chart evaluating the toxicity of 50-day-old compost.

Claims (2)

B. amyloliquefacien s KJ-6 (KACC 91260P) having fibrinolytic and antimicrobial activity against plant pathogens. Method for composting citrus sawdust using B. amyloliquefacien s KJ-6 (KACC 91260P).

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