KR100747701B1 - 11 Antagonistic soil bacterium Bacillus Licheniformis K11 and biological control and plant growth promotion using the same - Google Patents

Abstract

An antagonistic microorganism Bacillus licheniformis K11 and a method for biologically controlling plant pathogenic fungi and promoting plant growth by using the same microorganism are provided to inhibit growth of plant pathogenic fungi by producing antifungal substance, and siderophore, degrade cell wall of the plant pathogenic fungi by producing cellulase, and promote growth of plant by producing auxin. The antagonistic microorganism Bacillus licheniformis K11(KACC 91206P) producing at least one material selected from an antifungal substance, siderophore, cellulase and auxin is isolated from the soil. A composition for biologically controlling plant pathogenic fungi and promoting plant growth comprises Bacillus licheniformis K11(KACC 91206P) and further comprises at least one material selected from an antifungal substance, siderophore, cellulase and auxin, wherein the plant pathogenic fungi include Phytophtora capsici and Fusarium oxysporum. The cellulase is produced by culturing Bacillus licheniformis K11(KACC 91206P) at 20-70 deg.C and pH 4-10.

Description

{Antagonistic soil bacterium Bacillus licheniformis K11 and biological growth control using plant growth promotion using the same,

Fig. 1 shows the growth inhibition ability of K11 against Fusarium oxysporum and Phytophthora capsici in a confluent culture experiment.

FIG. 2 shows the production of antifungal antibiotics against wilt fungus and pepper spp.

On the left side, the production of antifungal antibiotics against pepper rot fungi was measured. On the right side, the production of antifungal antibiotics against tomato wilt fungi was measured.

FIG. 3 shows the production ability of K11 fungal cell wall cellulose by the congo red test.

FIG. 4 is a graph showing the side-pore productivity of antagonistic Bacillus licheniformis K11 of the present invention by CAS (chlome azurol S) blue agar.

FIG. 5 shows the taxonomic identification of the Bacillus licheniformis K11 strain finally identified using the Bergey's Manual of Systematic Bacteriology index.

FIG. 6 shows the optimum culture time of antagonist Bacillus licheniformis K11 by growth and 3,5-dinitrosalicylic acid (hereinafter referred to as 'DNS method').

FIG. 7 shows the optimum culture temperature of antagonist Bacillus licheniformis K11 by growth and DNS method.

FIG. 8 shows the optimum culture pH of antagonist Bacillus licheniformis K11 by growth and DNS method.

FIG. 9 is a graph showing the optimum reaction pH of a cellulase produced by antagonist Bacillus licheniformis K11 by the DNA method.

Fig. 10 shows the optimum reaction temperature of the cellulase produced by the antagonistic strain Bacillus licheniformis K11 by DNS.

Fig. 11 shows the optimum reaction time of the cellulase produced by the antagonistic strain Bacillus licheniformis K11 by DNS.

12 shows an active fraction obtained by gel filtration of a crude enzyme solution of cellulase produced by the antagonistic strain Bacillus licheniformis K11 of the present invention with Sephadex G-75.

FIG. 13 shows the activity fraction of the crude enzyme solution of cellulase produced by the antagonistic strain Bacillus licheniformis K11 of the present invention by SDS-PAGE.

FIG. 14 is a photograph of the auxin produced by the antagonistic strain Bacillus licheniformis K11 of the present invention by TLC (Thin Layer Chromatography).

15 shows the effect of the auxin produced by the antagonistic strain Bacillus licheniformis K11 of the present invention on the pea.

16 shows the effect of the auxin produced by the antagonistic strain Bacillus licheniformis K11 of the present invention on green beans.

17 shows the effect of the auxin produced by the antagonistic strain Bacillus licheniformis K11 of the present invention on the Chinese cabbage.

FIG. 18 is a graph showing the plant control ability of Bacillus licheniformis K11 of the present invention through pot soil experiments on P. aeruginosa.

19 is a diagram showing the results of 16 rRNA sequence analysis for the taxonomic identification of the Bacillus licheniformis K11 strain of the present invention.

20 is a graph showing the cellulase production ability of CMC and avicell.

[TECHNICAL FIELD]

The present invention relates to an antagonistic strain, Bacillus licheniformis K11, and a biological control method using the same. More particularly, the present invention relates to an antifungal antibiotic substance which inhibits the growth of phytopathogenic fungi, The present invention relates to a novel antagonistic strain K11 which produces cellulosic material, which is a decomposition substance, to effectively control plant pathogenic bacteria, and a biological control method using the same.

BACKGROUND ART [0002]

It is true that organic chemical pesticides, which have been developed rapidly since the early 1950s, have greatly contributed to the world's food production. However, serious abuses such as destruction of ecosystems due to their abuse have been taking place. It is being strengthened day by day. Under these regulatory conditions, the growth trend of the chemical pesticide market is gradually declining, while the market of new bio pesticides and microbial products is brisk.

Most of agricultural antibiotics using actives secreted by microorganisms were actinomycetes in soil microorganisms and they were developed relatively late compared to antibiotics for medicines. Among them, Blastin for rice blast disease control in 1958 is known as the first practical product.

In Korea, biocontrol methods using some imported microbial agents have been tried, but most of them have failed to show their activity due to lack of adaptability to climate and soil environment in Korea. Therefore, it is required to select the indigenous antagonistic microorganisms in our soil which can be easily adapted to the environment of our region and to be restored in the soil, and biocontrol method using them.

The loss of various crops due to plant insect damage accounts for 12% of the total crop production worldwide, only 20% of which is caused by soil nematodes and bacteria and most of the rest is caused by phytopathogenic fungi. Therefore, it is more urgent to control fungal pathogens than to control plant diseases caused by bacteria. So far, organic chemical pesticides for fungal control have been mainly used, but there is a limit to continue use.

In addition, the microbial control method is advantageous in that there is no environmental pollution, low toxicity, low production cost and low investment in production facility, and the research and development cost is about 1/10 to 1/20 of the existing pesticide development cost, The period is also about 1/3.

Sideropore, known as a causative agent of antagonism, is a soluble, low molecular weight ferric iron-chelating compound, usually a yellowish green fluorescent chromophore linked to a peptide chain. It is divided into two structural types depending on the structure and size of the peptide chain. The derivative of catechol (o-dihydroxybenzene) is a derivative of catechol amides (phenolates, catecholates) and hydroxamic acid hydroxamic acid (RCONHOH), which is hydro-xamates. The siderophore in the catechol form is enterobactin, parabactin, acrobactin, anguibactin, vibriobactin, azotobactin, and the like. , Side-ropors in the hydroxamate form include ferrichrome, ferrioxamine, coprogen, nocardamine, and the like. Recently, siderophores in the form of citrate-hydroxamate have been known as arthrobactin, aerobactin, schizokinen and the like, and quinoline- The siderophore is known as pseudo-bactin or pyoverdin.

The amount of iron (Fe) required by the microorganism in a natural environment is about 0.4 to 4.0 μM in the case of gram-positive bacteria and fungi, and about 0.3 to 1.8 μM in the case of gram-negative bacteria. Most of these iron are present as ferric-oxyhydroxide polymer (Fe (OH ₃ )) with a solubility constant of 10 ^-38.7 at neutral or alkaline pH under aerobic conditions, The free ferric iron concentration is generally very low at a pH of 7 and a solubility constant of about 10 ^-18 .

Although the iron content in the soil is rich in 1 to 6% of total inorganic ions, the amount of iron ions (Fe ³⁺ ) needed by the microorganisms is not sufficient to sustain their growth and is a competitive factor among all soil microorganisms There is no other choice. Therefore, in order to overcome this low-iron stress, many aerobic and anaerobic microorganisms require a specific iron regulated outer membrane protein (IROMP) in order to obtain a sufficient amount of iron necessary for growth (Mainly 400 to 2000 daltons), which is highly compatible with iron ions and exhibits fluorescence, and has a high affinity iron transport system. Therefore, siderophore is secreted by iron ion Complex, and then bound to a recongnate-specific receptor in the outer membrane, which is subsequently taken up into the cell through the cytoplasmic membrane by an energy-dependent process. The biosynthesis of the side pores and the outer membrane protein associated with this high affinity iron transfer system are controlled by iron ions.

Iron is an essential factor in the spore germination and germ tube elongation of plant pathogens. If iron ion concentration is not restricted, the growth of plant pathogens becomes active, resulting in plant root infiltration and infection It causes. Therefore, PGPR in the rhizosphere lives on the roots of plant roots and inhibits the growth of plant pathogens by predominating in the competition of iron absorption, mostly as a side secretion of fluorescent pseudomonads The iron around the root is chelated by the siderophore to inhibit spore germination and germ tube elongation of the plant pathogen. Thus, this competitive antagonism is a very effective biological control method that can reduce the soil infectious disease and enhance the crop growth by promoting the rhizosphere settlement ability of the host plant, thereby increasing the harvesting.

In addition, the pathogenic fungus is composed of chitin, cellulose, and beta-1,3-glucan. Cellulase, an enzyme that degrades cellulose, hydrolyzes the beta-1,4-glycoside bond of cellulose. Cellulase is present in fungi, bacteria, higher plants and molluscs. In particular, celluloses produced by germs can produce cellulase with high activity for the purpose of controlling phytopathogenic fungi because of short incubation time, The development of germs is advantageous over using other organisms.

Auxin, a plant growth-promoting hormone, is an endogenous growth regulator of plants, and has important donations for cell division, growth, and loss of growth and differentiation of many horticultural crops. The rhizosphere microorganisms constituting the rhizosphere of plants may produce plant growth materials such as auxin, solubilize insoluble phosphorus, directly affect plants through nitrogen fixation, inhibit phytopathogenic microorganisms, And indirectly affect the effect of the plant can have a big impact.

The use of organic synthetic chemical pesticides for control requires the development of new pesticides or microbial control agents in order to prevent the occurrence of harmful effects such as destruction of ecosystems, and it is necessary to develop microorganisms including plant growth promoting ability as well as controllability It is true.

The present invention relates to an antagonistic strain, Bacillus licheniformis K11, capable of effectively controlling plant pathogens and producing plant growth promoting substances by producing antifungal antibiotics, cellulases, seed pores and the like, and biological control It is intended to provide a method.

The present invention provides an antifungal antibiotic substance, a seed pore, a cellulase, and an auxin productivity antagonistic strain Bacillus licheniformis K11 (accession number 9206P) to achieve the above object.

Also provided is a biocontrol method characterized by treating the plant with the antagonistic strain Bacillus licheniformis K11 to inhibit the growth of the plant pathogenic fungus.

Hereinafter, the present invention will be described in detail.

Although plants can synthesize auxin in the body, they can grow physiologically to exogenously supplied auxin under cultivation conditions. Many microorganisms capable of supplying auxin from outside have been reported, and they can promote the growth of plants by supplying auxin, a hormone, from outside the plant.

As such, rhizosphere communities inhabiting rhizosphere communities and plant-growth promoting rhizosphere (PGPR) are known to have a great influence on plant growth and crop growth.

The rhizospheric microorganisms have the ability to produce various chemical groups having various characteristics and thus produce plant growth promoting hormone. In addition, antibiotic substances which inhibit phytopathogenic fungi, extracellular wall hydrolase, siderophore, Can be used to promote plant growth through the control of soil infectious disease.

The present inventors have found that Bacillus licheniformis K11 has such an effect while observing the rhizosphere microorganisms producing the antifungal antagonistic substance having plant growth promoting ability and inhibiting plant pathogenic fungi as described above.

The present invention also relates to a composition for controlling plants comprising a Bacillus licheniformis K11 antagonistic strain, as well as a plant growth promoting substance, as well as an antifungal antibiotic substance for inhibiting the growth of phytopathogenic fungi, A cell wall degrading substance of the present invention, and the like. The Bacillus licheniformis K11 antagonistic strain according to the present invention is characterized in that the productivity of cellulase is highest among the antagonistic substances.

The plant growth promoting substance may be an auxin, and the antagonistic substance of the plant pathogenic microorganism may be one or more selected from the group consisting of antifungal antibiotics, sidelophore and cellulase, Cellulase is a cellulase that decomposes cellulosic material. Antifungal antibiotics inhibit the cell wall and DNA and RNA synthesis of phytopathogenic fungi. Seedropore inhibits the iron synthesis of phytopathogenic fungi and inhibits the growth of phytopathogenic fungi. .

The method for separating the antagonistic microorganism according to the present invention will be described in detail. The method for isolating the antagonistic microorganism according to the present invention comprises: selecting an antagonistic strain producing plant growth promoting substance, antifungal antibiotic substance, seed pore, and cellulase; Identifying said antagonistic strain with Bacillus licheniformis k11 using a morphological and biochemical property testing and identification system; And culturing the identified Bacillus licheniformis K11 in a medium at a constant temperature, pH and time condition to establish a production condition for a substance capable of hydrolyzing the plant pathogenic fungus cell wall and purifying the cellulase, a plant produced by the strain Identifying the auxin, a growth promoting substance, and examining the effect on actual plants.

More specifically, as a method for controlling plant diseases by selecting Bacillus licheniformis according to the present invention, oxine is produced from antagonistic bacteria and an antifungal antagonistic substance is produced to inhibit the growth of tomato wilt disease bacteria and pepper root rot bacteria Gt; antagonist < / RTI > Identifying that the antagonistic substance produced by Bacillus licheniformis K11 of the present invention is an antifungal antibiotic, a side lobe, or a cellulase; The morphological characteristics of the selected antagonistic strain Bacillus licheniformis K11 of the present invention were observed with a microscope and the antagonistic activity of the antagonistic strain of the present invention was determined using a biochemical property test and a Biolog's identification system, Identifying Kenny Formis K11; Controlling the pH stability of the cellulase produced by the antagonist of the present invention by adjusting the amount of the cellulase control agent obtained from the antagonistic strain Bacillus licheniformis K11 of the present invention to a pH of 4 to 10; The cellulosic adjuvant solution obtained from the antagonistic strain Bacillus licheniformis K11 of the present invention was treated at a temperature range of 30 to 70 캜 and then subjected to heat treatment of the cellulase produced by the present antagonistic strain Bacillus licheniformis K11 Examining stability; Culturing the antagonistic strain Bacillus licheniformis K11 of the present invention in King's B broth to obtain a plant growth promoting material and then confirming the characteristics of the plant growth promoting material; The antifungal antagonistic substance produced by the antagonistic antibiotic Bacillus licheniformis K11 of the present invention is tested through port experiments to confirm whether pepper and tomato wilt disease are effectively controlled in pepper and tomato.

Hereinafter, preferred embodiments of the present invention will be described. However, the following examples are only a preferred embodiment of the present invention, and the present invention is not limited by the following examples.

Example  One: Antagonism  detach

1-1: Auxin  Productivity and Plant disease The ability to control  Selection of microbial strains having

The K11 strain of Table 1 isolated from the pepper and tomato cultivation soil of Gyeongbuk Province was inoculated into King's B broth medium supplemented with 0.1% L-Tryptophane and cultured at 30 ° C for 3 days. 2 ml of Salkowski reagent was added to 1 ml of the culture supernatant , Reaction for 30 minutes, measurement at 535 nm using a spectrophotometer, and production of oxine were quantified in comparison with a standard verifying line prepared with IAA.

Also, in order to select a plant-growth-promoting rhizobacteria (hereinafter referred to as PGPR) having a plant growth promoting ability and a plant disease controlling ability simultaneously, the strains identified as auxin productivity were tested for potato dextrose agar (Fusarium oxysporum and Phytophtora Capsici) were used as a test organism in a potato dextrose agar (PDA) medium. The inhibition was tested by the pairing plate culture test 1 together.

% Inhibition Auxin productivity ^* (Ab 535 nm) P. capsici F. oxysporum PLP4059 75 25 0.166 K11 83 75 0.249 KH1 39 40 0.021

^* Measurement by Salkowski test

As shown in Table 1, the K11 strain was found to be excellent in auxin productivity, and also had excellent growth inhibitory ability against tomato wilt disease and pepper sprout disease. As shown in FIG. 1,

1-2: Confirmation of control mechanism of auxin productivity antagonism

In order to determine whether the plant pathogenic antagonist produced by K11 selected as a microbial strain having excellent auxin productivity and plant disease control ability in Example 1-1 is an antibiotic substance or an enzyme protein, the following experiment was conducted.

K11 was cultured in a nutrient broth with shaking. Each culture supernatant was heat-treated at 80 ° C for 2 minutes. Residual antibiotic resistance was confirmed by a dermal disc method, and antagonistic substance was transferred to n-butanol, The results are shown in Fig.

As shown in FIG. 2, when antibiotics were separated from the culture supernatant of K11 strain by using n-butanol and subjected to a disk disc method, it was found that antifungal substances with pathway resistance were produced in both P.capsici and F.oxysporum I could.

In order to investigate the production ability of fungal cell wall hydrolase from auxotrophic antagonistic fungi, first, the cellulase production ability was determined in CMC nutrient medium containing 1% carboxymethylcellulose (CMC) in nutrient agar The strain was inoculated with a sterilized toothpick, cultured at 30 ° C. for 2 days, and the resolution of fungal cell wall cellulose was confirmed using a congo red indicator, and it is shown in FIG.

In addition, the productivity of two cellulases, CMCase and Avicellase, was confirmed by DNS method using CMC and Avicell as substrates, respectively.

In addition, a K11 antagonistic strain was inoculated on a CAS (chlome azurol S) blue agar medium, which is a separation medium of a sidropore-producing strain, and cultured at 28 ° C, The production ability of the safflower, which is widely known as a plant pathogenic antagonist, was observed in FIG. 4 using the discoloration rate of CAS according to the sidelobe activity by the CAS liquid analysis method.

As shown in FIG. 3 and FIG. 4, in the investigation of the productivity of the outer wall hydrolase, the K11 strain formed a clear zone in the Congo Red experiment, indicating that cellulase, an extracellular wall hydrolase, was produced from K11. In addition, the productivity of side rope was examined by the multiple control mechanism of the auxin productivity antagonist. As a result, the K11 strain produced orange rings due to pore production on the CAS medium.

Therefore, the antagonistic mechanism of K11 selected as auxin productivity multifunctional plant growth promoter is to produce siderophora, an iron-binding substance, and to produce cellulase which is a plant pathogen cell outer wall hydrolase, and also to produce multiple PGPR And the value as a biocontrol agent was high.

Example  2: Antagonism  Sympathy

For the taxonomic identification of the Bacillus licheniformis K11 strain isolated and isolated in Example 1, various biochemical and morphological tests were performed and then Bergey's Mannual of Systematic Bacteriology index was used based on Biolog TM System 4.0 and 16S rRNA analysis And finally identified as shown in Fig.

The identified auxin-producing PGPR strain K11 of the present invention was 99% homologous to Bacillus licheniformis and identified as Bacillus licheniformis K11. The strain was deposited at the Institute of Agricultural Biotechnology in November 2005 And deposited with KACC 91206P on the 25th.

Example  3: Antagonist stock price  Production conditions of producing cellulase

3-1: Culture time, temperature and pH on productivity of cellulase

To confirm the growth of selected strain K11 and cellulase productivity according to the incubation time, the K11 pre-culture medium was inoculated into the nutrient both medium, and the bacterial growth rate was measured by absorbance at 600 nm every 24 hours. The cellulase productivity The measurement was carried out by using the culture supernatant and measuring the activity of 3,5-dinitrosalicylic acid (hereinafter referred to as 'DNS method'), which is a method for measuring the activity of the fungal cell wall cellulolytic enzyme, and is shown in FIG.

As shown in FIG. 6, the growth of the strain and the productivity of cellulase were examined according to the incubation time of K11. As a result, the bacterial growth was most proliferated on the fourth day, and the productivity of cellulase was also highest Respectively.

In order to investigate the influence of temperature on the growth of the strain and the productivity of cellulase, the K11 pre-culture broth was inoculated into the nutrient broth and cultured at 20 ° C, 30 ° C, 40 ° C and 50 ° C for 3 days, The culture supernatant was measured by cellulase activity with crude enzyme solution and shown in Fig.

As shown in FIG. 7, the growth at 30 ° C was the best, but the activity of the cellulase was the highest at 40 ° C.

In addition, the nutrient broth was titrated to a pH of 4 to 10, followed by inoculation with a K11 pre-culture for 3 days. The growth of the strain and the productivity of the strain according to the pH change were examined in the same manner as described above.

As shown in FIG. 8, the highest growth was observed at pH 5.0, while the cellulase was the most productive at pH 8.0, and the productivity was similar at pH 5, pH 6, pH 7 and pH 9.

3-2: Composition of the medium on the productivity of cellulase

1) Effect of carbon source

Arabinose, which is a 10 carbon source instead of a carbon source, was added to the David minimal medium (David minimal medium; 0.1% glucose, 0.7% K2HPO4, 0.2% KH2PO4, 0.1% (NH4) 2SO4, 0.05% sodium citrate, 0.01% MgSO4.7H2O) 0.1% each of fructose, xylose, glucose, CMC, maltose, saccharose, glycerol, and galactose was added thereto, followed by inoculation with K11 pre-culture and incubation at 37 ° C for 3 days. After the culture, the cell mass was measured at 600 nm, and the cell supernatant was measured for cellulase activity by the DSN method with the enzyme solution, and is shown in Table 2 below.

Carbon source Cell growth (Cell / ml) Inhibition rate (%) - 0.056 0.013 Arabinose 0.111 0.05 Fructose 0.111 0.164 Lactose 0.079 0.142 Xylose 0.216 0.07 Glucose 0.078 0.157 CMC 0.125 0.049 Maltose 0.083 0.119 Sakaros 0.099 0.149 Glycerol 0.161 0.125 Galactose 0.233 0.094

As shown in Table 2, it was found that the growth of K11 strain and the productivity of cellulase were the best as a carbon source for fructose.

2) Influence of nitrogen source

Nitrogen sources (NH4) 2O, NH4H2PO4, KNO3, NaNO3, (NH4) 2SO4, yeast extract, beef extract, malt extract, protecheptone NO3 (0.1%) of protosepeptone NO3, bactopeptone and urea were added to each culture medium, followed by incubation at 37 ° C for 3 days. Growth and enzyme activity of the bacteria were examined in the same manner as in the carbon source, Respectively.

Nitrogen source Cell growth (600 nm) Cellulase activity (540 nm) - 0.028 0.030 (NH4) 2O 0.138 0.005 NH4H2PO4 0.102 0.025 KNO3 0.100 0.045 NaNO3 0.081 0.02 (NH4) 2SO4 0.091 0.018 Enzyme extract 0.125 0.038 Beef extract 0.121 0.120 Malt 0.025 0.096 Protoss peptone NO3 0.205 0.015 Pak Tote Lipton 0.195 0.113 Urea 0.108 0.097

As shown in Table 3, when the beef extract was used as a nitrogen source, it was known that the growth of K11 strain and the productivity of cellulase were the most excellent.

3) Influence of inorganic salts

The optimum medium of carbon and the source of nitrogen were included in the minimum medium of David and 13 kinds of inorganic salts FeCl _{3`</sub> 6H <sub>2</sub> O, BaCl <sub>2`} 2H ₂ O, RbCl ₂ , Na _{2`</sub> HPO <sub>4</sub> , CaCO <sub>3</sub> , ZnSO <sub>4</sub> , <sub>MgSO 4` 7H 2 O, KCl,} LiCl, K 2 HPO 4, NaCl, CaCl 2, Pb (CH 3 COO) 2` was added to 3H ₂ O was inoculated with the pre-culture K11 cultured for 3 days at 37 ℃, gyuncheryang And enzyme activity were measured in the same manner as in 1) above and shown in Table 3.

3-3: Investigation of enzymatic properties of cellulase

1) Investigation of optimum pH, temperature and time

A) Experimental method

In order to investigate the optimum reaction pH of the cellulase, the pH was adjusted to 1 / 15M phosphoric acid by pH 5 to 8 with 1M HCl-soduim acetate at pH 2 to 5 and by using 1 / 20M Na2B4O7- After different adjustments, the reaction was carried out at 37 ° C for 90 minutes, and enzyme activity was measured and shown in FIG.

Further, 1 ml of 0.1% CMC solution, 1 ml of 1 / 15M phosphoric acid (pH 7.0) buffer and 0.1 ml of the enzyme solution were reacted at a temperature of 30 to 70 캜 for 10 minutes at an interval of 10 캜 for 90 minutes, 10.

The activity was measured at 37 ° C for 60 minutes to 200 minutes at 20 minute intervals using 1 ml of 0.1% CMC solution, 1 ml of 1 / 15M phosphoric acid (pH 7.0) buffer and 0.1 ml of the enzyme solution, .

B) Results

As shown in FIGS. 9 to 11, the cellulase produced by Bacillus licheniformis K11 showed the maximum activity at pH 8.5 (FIG. 9) and the maximum activity at 60 ° C (FIG. 10). In addition, it was found that the cellulase undergoes reaction of 90% or more after 2 hours and 20 minutes (140 minutes) with respect to the substrate carboxylmethylcellulose (FIG. 11).

 2) Effect of metal ion or inhibitor

The effect of metal ions on enzyme activity was investigated by measuring the residual activity of the enzyme after pretreatment at 4 ° C for 12 hours by adding the enzyme solution to various metal salts such as MgSO4 and CoCl2 dissolved in 2X10-3M. The inhibitor was added to the enzyme solution and the effect of the inhibitor on the activity of the enzyme was investigated in the same manner and shown in Table 4 together.

Metal ion Residual activity (%) Inhibitor Residual activity (%) FeSO ₄ 90 Urea 79 KCI 86 Thiourea 91 AgNO ₃ 63 Sodium azide 99 ZnSO ₄ 79 CDTA 54 CuSO ₄ 85 Iodoacetate 77 MnSO ₄ 94 p-CMB 83 CaCl ₂ 59 EDTA 82 MgSO ₄ 83 BaCI ₂ 65

As shown in Table 4, the activity of metal ions was inactivated by Ag +, Ca + and Ba + ions by 40% or more, and the activity of K11-produced cellulase was decreased by 40% or more by the chemical inhibitor CDTA Most of the inhibitors, except thiourea and sodium azide, showed a decrease of activity by 15% or more.

Mineral salt Cell growth (600 nm) Cellulase activity (540 nm) - 0.070 0.001 FeCl ₃ 6H ₂ O 0.028 0.004 BaCl _{2 ·} 2H ₂ O 0.098 0.023 RbCl ₂ 0.091 0.007 Na _{2 ·} HPO ₄ 0.082 0.084 CaCO ₃ 0.324 0.064 ZnSO ₄ 0.010 0.005 MgSO _{4 ·} 7H ₂ O 0.178 0.040 KCl 0.127 0.017 LiCl 0.141 0.020 K ₂ HPO ₄ 0.106 0.073 NaCl 0.110 0.006 CaCl ₂ 0.170 0.113 _{Pb (CH 3 COO) 2 ·} 3H 2 O 0.177 0.020

As shown in Table 5, when CaCl ₂ was added as the inorganic ion, it was found that the productivity was the best.

Example  4: Antagonist stock price  Producing Cellulase Purification

Bacillus sp. For the purification of cellulase of K11, Bacillus sp. The K11 pre-culture was inoculated into the above cellulase productivity medium (0.1% fructose, 0.1% beef extract, 5 mM CaCl _{2 ,} pH 6) supplemented with 0.1% CMC and cultured for 4 days with shaking. Then, the culture supernatant was recovered by centrifugation, saturated with ammonium sulfate 75%, dissolved in 1 / 15M phosphate buffer (pH 7.0), dialyzed with distilled water, and lyophilized and used as crude enzyme solution.

The crude enzyme solution was subjected to gel filtration with a 1/15 phosphate buffer on a Sephadex G-75 column (15 x 750 mm) at 4 ° C, and the fraction having the enzyme activity was collected and concentrated. The cellulase activity of each fraction was measured and shown in FIG. Further, the active fraction was lyophilized and confirmed by SDS-PAGE and shown in FIG.

As shown in FIG. 13, the cellulase produced by Bacillus subtilis K11 of the present invention was estimated to be about 30 kDa.

Example  5: Antagonist stock price  Produce Auxin  Chemical characteristic

For the auxin purification of the multifunctional PGPR strain B. licheniformis K11, the culture supernatant was adjusted to pH 2.8 with phosphoric acid, extracted twice with ethyl acetate, and then concentrated under reduced pressure at 37 ° C. The concentrate was subjected to HPLC analysis using a C18 column at a flow rate of 1 min / ml with the adjuster oxine sample, and Rf value was confirmed by TLC together with IAA (indole-3-acetic acid) used as a standard.

At this time, as a developing solution of TLC, ethyl acetate, 1-propanol: ammonia: water were mixed at a ratio of 6: 3: 1, and Ehrlich reagent was sprayed with a coloring reagent.

The stasis time of the Bacillus licheniformis K11 antagonist of the present invention was 42 minutes, and the IAA stagnation time used as a standard was 50 minutes, and the auxin produced by the antagonistic strain K11 of the present invention was estimated to be similar to IAA. However, TLC analysis showed that the Bacillus licheniformis K11 was not the same as the IAA and Rf values.

Example  6: Antagonist  Produced by Auxin Plant growth ability  Research

6-1: Investigation by mung bean root biopsy

The auxin productivity of the selected antagonistic strain K11 was confirmed by using the mild root biopsy method, which is a test method using the rooting promoting action, which is the main action of auxin. Mungbean seeds were immersed in 0.3% sodium hypochlorite solution for 3 minutes and then immersed in running tap water for 24 hours, seeded in aseptic soil, and cultured for 5-7 days in a plant culture room under 28 ℃ and 5000 lux natural light. The cotyledons were removed from the mongolian seedlings of a uniform size of the first three leaflets of the foliage of the first three leaflets. The cotyledons were cut with a sharp knife having a length of 3 cm under the cotyledon, and then seeded with King's B broth supplemented with 0.1% tryptophan One of the seedlings cut into a small bottle filled with 50 占 퐇 of culture supernatant of cultured auxin productivity antagonist and 5 ml of sterilized distilled water was added. Thereafter, the solution was replenished to a certain level with distilled water every 24 hours so that the volume of the solution was made equal, and after 6 days of rooting with continuous illumination, the number of roots having a length of 1 mm or more was counted on the 6th day.

sample Number of roots (ea) 0.1 ppm 0.5 ppm 1 ppm 5 ppm 10ppm IBA 3.2 8 10.6 18.9 1.2 IAA 7.6 9.9 12 12.6 14 K11 7.9 10.8 0.5 0 0

As can be seen from Table 6, commercially available IAA and IBA showed average rooting of 18.9 and 12.6 at 5 ppm concentration, but auxin _K11 showed 10.8 rooting at 0.5 ppm, indicating strong plant growth even at lower concentrations than existing commercial products It showed. Therefore, in this study, we found that the plant has much more plant growth promoting ability than the existing auxin.

6-2: Identification of plant growth promoting ability of pea, mung bean and Chinese cabbage

The seeds of the plants described in the following Table 6 were immersed in water at 25 캜 under a dark condition for 24 hours and inoculated with 10 ⁵ CFU / g of auxin productivity antagonist in sterilized soil (manufactured by Dongbu Chemical Co., Ltd.) Respectively. Thereafter, seeds sown with seeds were irradiated with light at 28 ° C for 12 hours, and cultured for 3 weeks. Roots were weighed and measured in the following Table 6, and the degree of growth of each plant is shown in FIG. 15 to FIG. As a control, seeds seeded in the soil without the auxin - producing antagonist were used.

Plant Type Control group (mg) Experimental group (㎎) Experimental / Control (%) Mungbean root 75.2 122.2 160 Pea root 93.3 145.2 150 Chinese cabbage root (Chinese cabbage) 13.1 18.1 130

As shown in the above Table 7 and FIGS. 15 to 17, the auxin produced by the antagonistic strain K11 of the present invention was significantly higher than the cabbage, pea and mung bean in the control group, compared with the cabbage, pea, mung bean Increased the weight by 30%, 60% and 50%, respectively.

Example  7: Through pot test Auxin productivity Antagonism Biological control  Confirm

Red pepper and tomatoes as host plants were inoculated with potato antagonist Phytophthora capsici in a warm room kept at 28 ℃ and 70% humidity, respectively. 70% humidity). The ovine productivity antagonistic bacterium was treated, and periodic onset was observed while growing in a constant temperature and humidity chamber at 28 ° C and 70%, and the degree of onset at day 7 was shown in FIG.

As shown in Fig. 18, the antagonistic strain K11 of the present invention showed strong antifungal activity in in vitro and in vivo pot experiments. Therefore, it was confirmed that the selected PGPR strain of the present invention is a multifunctional PGPR strain having not only plant growth promotion but also plant disease control ability.

The antagonistic strain Bacillus licheniformis K11 of the present invention inhibits plant pathogenic growth by producing an antifungal antibiotic substance and a side lore and inhibits the growth of plant pathogenic bacteria by producing a cellulase which is a decomposing substance of plant cell wall, To an antagonistic strain having excellent plant growth promoting ability and a biological control method using the same.

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Claims (6)

Bacillus licheniformis strain, which is deposited at KACC 91206P, which produces at least one substance selected from the group consisting of antifungal antibiotics, sidelopore, cellulase, and auxin. delete A composition for controlling Phytophthora Capsici or Fusarium Oxysporum comprising the strain of claim 1. A plant growth promoting composition comprising the strain of claim 1. The composition according to claim 3, wherein the composition further comprises at least one antagonist selected from the group consisting of antifungal antibiotics, siderophores, and cellulases. A method for producing a cellulase agent by culturing the strain of claim 1 at a temperature of 20 to 70 占 폚 and a pH of 4 to 10.

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