KR100747700B1 - Antagonistic soil bacterium bacillus subtillis and biological control and plant growth promotion using the same - Google Patents

Abstract

An antagonistic microorganism Bacillus subtilis and a method for biologically controlling plant pathogenic fungi and promoting plant growth by using the same microorganism are provided to inhibit growth of pathogenic fungi by producing antifungal materials, siderophore and cellulase, and promote plant growth by producing a plant growth stimulating hormone, auxin. The antagonistic microorganism Bacillus subtilis(KACC 91205P) produces siderophore, cellulase or auxin and is isolated from the soil. A composition for controlling Phytophtora capsici or Fusarium oxysporum or promoting plant growth comprises Bacillus subtilis(KACC 91205P) and further comprises at least one of antagonistic materials selected from siderophore and cellulase.

Description

Antagonistic strain Bacillus subtilis and plant growth promoting and biological control using the same {ANTAGONISTIC SOIL BACTERIUM BACILLUS SUBTILLIS AND BIOLOGICAL CONTROL AND PLANT GROWTH PROMOTION USING THE SAME}

1A shows the fungal cell wall cellulose resolution of AH18 by congo red test, and FIG. 1B shows the antagonist strain Bacillus subtilis by DNS method using avicell, filter paper, PNPG and carboxymethyl cellulose as substrates. The cellulase production capacity of AH18 was measured.

Figure 2 is a measure of the siderophore production capacity of the antagonistic bacterium Bacillus subtilis AH18 of the present invention by the CAS (chlome azurol S) blue agar method.

Figure 3 shows the taxonomic identification of the Bacillus subtilis AH18 strain finally identified using Bergey's Manual of Systematic Bacteriology index.

Figure 4 shows the optimal incubation time and siderophore production capacity of the antagonist Bacillus subtilis AH18 by growth and CAS liquid analysis method.

5 is a sidelopor production capacity of the antagonistic strain Bacillus subtilis AH18 with temperature.

6 is a sidelopor production capacity of the antagonistic strain Bacillus subtilis AH18 according to pH conditions.

Figure 7 shows the HPLC results in the process of purifying auxin after extraction with a ethyl acetate layer from a culture cultured in King's broth by adding Bacillus subtilis AH18 strain.

FIG. 8 shows the results of GC-MS analysis on the fractions obtained from the Bacillus subtilis AH18 strain during instrumental analysis.

9 is a measure of mung bean germination confirming the plant growth promoting ability of the Bacillus subtilis AH18 strain.

Figure 10 is a measure of the effect of the Bacillus subtilis AH18 strain on tomato wilt Fusarium oxysporum, A was treated with the AH18 strain, B is not treated with the AH18 strain.

Figure 11 is a measure of the effect of the Bacillus subtilis AH18 strain on the pepper bacterium Phytophthora capsici, where A is not treated with AH18, B is treated with AH18 strain.

FIG. 12 shows 16rRNA sequencing results for taxonomic identification of Bacillus subtilis of the present invention. FIG.

[Technical Field]

The present invention relates to an antagonist Bacillus subtilis AH18 and a biological control method using the same, and more particularly, to produce a plant growth promoting substance, to produce cellulase, a cell wall degrading agent of phytopathogenic fungi, and to metabolize phytopathogenic fungi. The present invention relates to a novel antagonistic strain AH18 for selectively controlling phytopathogens by selectively binding iron ions to be used for and biological control methods using the same.

[Private Technology]

Everyone has acknowledged that organic synthetic chemical pesticides, which have been rapidly developed since the early 1950s, have contributed greatly to the world's food production, but serious damages such as the destruction of ecosystems caused by its abuse are continuing. The center is strengthening day by day. Under these regulatory conditions, the growth trend of the chemical pesticide market is gradually decreasing, while the market for new biopesticides and microbial products is becoming active.

Most agricultural antibiotics using active substances secreted by microorganisms use actinomycetes among soil microorganisms and were developed relatively late compared to medicinal antibiotics. Among them, the most developed in Japan, Blastin for control of rice fever in 1958 is known as the first commercial product.

In Korea, biocontrol methods using some imported microorganisms have been attempted, but most of them have not been able to perform their activities due to lack of adaptability to the climate climate and soil environment. Therefore, there is a need for a biocontrol method that selects and utilizes indigenous pathogenic microorganisms of Korean soil that can be easily adapted to the environment of our region and become a predominant restoration in the soil.

The loss of crop production by plant pests accounts for 12% of the world's crop production, but only 20% of them are damaged by soil nematodes and bacteria, most of which are caused by phytopathogenic fungi. Therefore, it is much more urgently necessary to control fungal phytopathogens than to control plant diseases caused by bacteria. Until now, such fungicides have been used mainly for organic chemical pesticides, but there are limitations to continue using them.

In addition, the control method using microorganisms has the advantages of no environmental pollution, low toxicity, low production cost, and low investment in production equipment, and the R & D cost is about 1/10 to 1/20 of the existing pesticide development cost. There is an advantage that it takes about 1/3.

Siderophores, known as antagonists of antagonism, are water-soluble, low-molecular ferric iron-chelating compounds, generally yellow-green fluorescent chromophores linked by peptide chains. It is divided into two structural forms according to the composition and size of the peptide chain. It is a derivative of catechol (catechol; o-dihydroxybenzene), which is a form of catechol amides (phenolates, catecholates) and hydroxyxamic acid ( A derivative of hydroxamic acid (RCONHOH) is in the form of hydro-xamates. Siderophores in the catechol form include enterrobactin, parabactin, acrobactin, anguibactin, vibriobactin and azotobactin. Hydroxamate-type sidelopores include ferrichrome, ferrioxamine, coprogen, and nocardamine. In recent years, citrate-hydroxamate form of siderophores include athrobactin, aerobactin, schizokinen, and quinoline. Siderophores have revealed pseudo-bactin or pyoverdin.

The amount of iron (Fe) required by microorganisms in the natural environment is about 0.4 to 4.0 μM for Gram-positive bacteria and fungi, and about 0.3 to 1.8 μM for Gram-negative bacteria. Most of this iron is under aerobic environment and insoluble in neutral ferric extent dissolution constant of 10 ^-38.7 In the alkaline pH - is present in oxyhydroxide polymer (ferric-oxyhydroxide polymer [Fe ( OH 3)]), the free ferric Iron concentrations (free ferric iron) is generally very low at a pH of about ^10-18 dissolution constant.

Although iron is abundant in the soil with 1 to 6% of the total inorganic ions, the amount of iron ions (Fe ³⁺ ) required by microorganisms is not sufficient to sustain their growth and will be a competitive factor among all soil microorganisms. There is no choice but to. Therefore, in order to overcome this low-iron stress, many aerobic and breathable anaerobic microorganisms have been added to a special iron regulated outer membrane protein (IROMP) to effectively obtain sufficient iron for growth. It has a high affinity iron transport system, and secretes low-molecular weight (mainly 400-2000 daltons) soluble siderophore that has high affinity with iron ions and fluorescent color. After the complex is formed, it is bound to a recongnate-specific receptor on the outer membrane, which is sequentially introduced into the cell through the cytoplasmic membrane by an energy-dependent process. Sidepore biosynthesis and outer membrane proteins associated with these high affinity iron transport systems are controlled by iron ions.

Iron is generally an essential factor for spore germination and germ tube elongation of phytopathogens. If iron ions are not constrained, the growth of phytopathogens will be active, resulting in plant root infiltration and infection. Cause. Therefore, PGPR in the rhizosphere suppresses the growth of phytopathogens by living in clusters on the surface of plant roots and giving an advantage in the competition of iron ions absorption, which is mostly a side that secretes fluorescent pseudomodans. Siderophore inhibits spore germination and germ tube elongation of phytopathogens by chelating iron around the roots. Therefore, this competitive antagonism is a very effective biological control method that can reduce soil infectious diseases and promote the rooting ability of host plants to improve crop growth and increase cropping.

In addition, pathogenic fungi have a cell wall composed of chitin, cellulose and beta-1,3-glucan. Cellulase, an enzyme that breaks down cellulose, hydrolyzes beta-1,4-glycosidic bonds of cellulose. Cellulase exists in fungi, bacteria, higher plants, mollusks, etc. Especially, cellulose produced by bacteria has a short incubation time, low nutritional requirements, and easy genetic manipulation, so it can produce highly active cellulase for the purpose of controlling phytopathogenic fungi. The development of bacteria is advantageous over the use of other organisms.

Auxin, a plant growth-promoting hormone, is an endogenous growth regulator of plants and has important donations for cell division, growth, and deletion of many horticultural crops. The rhizosphere microorganism that constitutes the rhizosphere of the plant produces plant growth substances such as auxin, solubilizes insoluble phosphorus, directly affects plants through nitrogen fixation, inhibits plant pathogenic microorganisms or interacts with other rhizosphere microorganisms. Can indirectly affect the plant's impact.

The present invention provides an ability to produce auxin, a plant growth promoting substance, and an antagonistic strain Bacillus subtilis AH18, which can effectively control phytopathogens, and a biological control method using the same.

The present invention provides an antagonist strain Bacillus subtilis AH18 (Accession No. KACC 91205P), which produces seedlopoa and cellulase that inhibit the growth of phytopathogenic fungi in order to achieve the above object.

In addition, the antagonistic strain Bacillus subtilis AH18 is treated with plants to provide a biocontrol method characterized in that the growth of phytopathogens is suppressed.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Although plants can synthesize auxin (auxin) in the body, they can grow by causing physiological reactions to externally supplied auxin under growing conditions. Many microorganisms that can supply auxin from the outside have been reported, they can promote the plant growth by producing a hormone called auxin from outside the plant.

As such, the rhizosphere microorganisms inhabiting the rhizosphere community of plants and having a great influence on plant growth and crop growth are called plant growth promoting rhizosphere (PGPR).

The rhizosphere microorganism has the ability to produce various groups of chemicals with various characteristics, and in addition to the production of plant growth promoting hormones, the production of extracellular wall hydrolases and siderophores that inhibit phytopathogenic fungi. It can promote plant growth by controlling soil infectious diseases.

The present inventors have discovered that Bacillus subtilis AH18 has such efficacy while observing myosphere microorganisms having plant growth promoting ability and inhibiting phytopathogenic fungi as described above.

In addition, the present invention can provide a control composition for producing plant growth promoting material as a control composition comprising a Bacillus subtilis AH18 antagonist strain, as well as for producing a antagonist of phytopathogens and degradation of phytopathogens. Bacillus subtilis antagonist strain according to the present invention is characterized by the highest siderophore productivity among the antagonists.

The plant growth promoting material may be auxin, and the antagonist of the phytopathogen may be one or more selected from the group consisting of sideropores and celluloses, and the phytopathogen antagonist is synthesized for the main phytopathogen growth. By selectively binding to the iron ions may be a siderophore (siderophore) inhibiting the growth of phytopathogens by inhibiting the iron ion synthesis of phytopathogens.

In addition, the present invention comprises the steps of selecting a strain antagonizing plant growth promoting substances and phytopathogenic fungi; Identifying the antagonistic strain as Bacillus Subtilis AH18 using a morphological, biochemical test and identification system: and identifying the Bacillus subtilis AH18 in a medium at a constant temperature, pH and time. It provides a biocontrol method comprising the step of producing an antagonist against phytopathogenic fungi by culturing under conditions.

More specifically, the plant control method using Bacillus subtilis AH18 of the present invention produces auxin from indigenous antagonistic bacteria isolated from red pepper and tomato arable soil in Gyeongsan-si, Gyeongsangbuk-do, Finally selecting antagonistic strains that inhibit the growth of the germs; Confirming that the red pepper antagonist and tomato wilted antagonist mechanism of the antifungal antagonist produced by the final selected strains are siderophore and cellulase; The selected antagonist strain Bacillus subtilis AH18 of the present invention was observed under a microscope to investigate the morphological characteristics and biochemical properties and biologous identification system of the antagonist strain of the present invention Bacillus subtilis Identifying as AH18; Culturing the antagonistic bacterium Bacillus subtilis AH18 of the present invention in King's B Broth to obtain a side fungus as an antifungal antagonist, and then examining the antifungal activity of the sidelopores; Investigating the pH stability of the siderophore produced by the antagonist strain of the present invention by adjusting the antibiotic solution obtained from the antagonist strain Bacillus rickenformis AH18 to pH 4 to 10; After treating the crude liquid of Sideropore obtained from the present invention antagonist Bacillus rickenformis AH18 at a temperature range of 20 ℃ to 70 ℃, the antifungal activity against the phytopathogenic bacteria was measured to determine the present invention antagonist Bacillus rickenformix Examining the thermal stability of the sidelopores produced by AH18; Purifying auxin, which is a plant growth promoting hormone produced by the Bacillus subtilis AH18 strain, to confirm its properties; It includes the step of confirming the control ability in the actual pepper and tomato through the pot experiment of the antifungal antagonist produced by the Bacillus subtilis AH18 strain.

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred embodiments of the present invention, and the present invention is not limited to the following examples.

Example  One: Antagonistic  detach

1-1: Auxin  Productivity and Plant disease Control  Selection of Microbial Strains Having

The bacteria isolated from the cultivated soil were inoculated in King's B broth medium containing 0.1% L-Tryptophan and incubated at 30 ° C. for 3 days. After 2 ml of Salkowski reagent was added to 1 ml of the culture supernatant, it was reacted for 30 minutes. Using a spectrophotometer measured at 535nm compared to the standard verification line made with IAA quantitative productivity of auxin is shown in Table 1.

In addition, potato dextrose agar was selected from the strains for which auxin productivity was identified in order to select a multifunctional plant-growth-promoting rhizobacteria (PGPR) having both plant growth promoting ability and plant disease control ability. Potato dextrose agar (PDA) medium with two kinds of tomato wilted fungus ( Fusarium) Oxysporum ) and red pepper bacterium ( Phytophtora Capsici ) were used as test bacteria, and the inhibitory ability was examined by pairing plate culture test.

% Inhibition Auxin Productivity ^* (Ab 535nm) P. capsici F. oxysporum PLP4059 75 25 0.166 AH18 80 78 0.263 K11 43 45 0.049 KH1 39 40 0.021

^* Measurement by Salkowski test

As shown in Table 1, the AH18 strain was not only excellent in auxin productivity, but also excellent in inhibiting growth against tomato wilt and pepper germ disease.

1-2: Auxin Productivity Antagonistic Confirmation of control mechanism

In order to determine whether the phytopathogen inhibitor produced by AH18 selected as a microbial strain having excellent auxin productivity and phytopathogenic activity in Example 1-1 is an antibiotic or an enzyme protein, as follows.

After shaking AH18 with nutrient liquid medium, each culture supernatant was heat-treated at 80 ° C. for 20 minutes, and the remaining antibiotic was checked by the free disk method, and the antagonist was transferred to n-butanol by transferring the antagonist to n-butanol. The ability was examined by bacterial cell mass test. In addition, to investigate the fungal cell wall hydrolase production capacity of auxin-producing multifunctional antagonist strains, cellulase production capacity was first selected in CMC nutrient medium containing 1% carboxymethyl cellulose (CMC) in nutrient agar. After inoculating the strain using a sterilized toothpick, the strain was incubated at 30 ° C. for 2 days, and the resolution of cellulose, which is a fungal cell wall component, was determined using a congo red indicator and is shown in FIG. 1A.

In addition, by using the substrate as a CMC, Avicell, filter paper or PNPG confirmed the production capacity of the cellulase by DNS method, respectively, shown in Figure 1B.

As shown in FIG. 1A, it was found that cellulase, which is an extracellular wall hydrolase, was greatly produced by forming a clear zone in the Congo red test. In addition, as shown in FIG. 1B, the cellulase produced by Bacillus subtilis AH18 was capable of decomposing insoluble cellulose, avicell and filter paper, and decomposing CMC, soluble cellulose. In particular, the activity of CMCase was the highest among the cellulase produced by Bacillus subtilis AH18.

In addition, AH18 antagonist was inoculated into CAS (chlome azurol S) blue agar medium, which is a separation medium of the siderophore-producing strain, and cultured at 28 ° C., and the sideropore-producing bacterium was produced by the generation of orange halo zone. Firstly, by using a CAS liquid assay, the decolorization rate of CAS according to the siderophore activity was used to measure the production capacity of the sideropores, which are widely known as phytopathogen antagonists, and are shown in FIG. 2.

As shown in Figure 2, the AH18 antagonist strain produced an orange ring by clear siderophore production on CAS medium.

Therefore, AH18 selected in the present invention is an iron ion binding agent of siderophore and phytopathogenic fungi which prevents metabolism of iron ions by selectively binding iron ions used for metabolism by phytopathogenic fungi. It is a multi-PGPR with potent plant growth promoting ability to simultaneously produce cellulase that breaks down cellulose, a major component of cell walls.

Example  2: Antagonistic  Sympathy

The biochemical and morphological tests of the Bacillus subtilis AH18 strain isolated in Example 1 were performed for various biochemical and morphological tests, and then Bergberg's Manual of Systematic Bacteriology index was used based on Biolog TM System 4.0 and 16S rRNA analysis. The final identification was shown in FIG.

As shown in FIG. 3, the AH18 strain showed 99% homology with Bacillus subtilis and was identified as Bacillus subtilis AH18.

The identified Bacillus subtilis AH18 strain of the present invention was deposited with KACC 91205P on November 25, 2005 to the Institute of Agricultural Biotechnology.

Example  3: of the present invention Antagonist strain  Characteristics of Producing Antifungal Antagonists

3-1: Stability according to culture time and culture composition

In order to investigate the optimal production medium conditions for the production of the side fungi, the antifungal antagonist produced by the antagonistic bacillus subtilis AH18, pre-culture of AH18 (precultured in small amounts prior to the main culture) King B, After inoculation into Alexander, sucrose asparagines medium (SAM), and succinnate minimal medium (SMM), the growth and siderophore productivity were investigated every 24 hours and shown in FIG. 4.

As shown in FIG. 4, Bacillus subtilis AH18 was found to have the best siderophore productivity when inoculated with preculture in SAM medium (pH 6.0).

3-2: according to temperature Sidelopor Productivity

Antagonist strain AH18 was inoculated in a SAM medium and incubated at 10 ° C., 20 ° C., 30 ° C., 40 ° C., and 50 ° C. for 3 days, respectively, and the production capacity of sideropores according to temperature was measured and shown in FIG. 5. In addition, by inoculating the pre-culture of AH18 in the SAM medium adjusted to pH 3 to 11 was measured the effect of pH on the side ropore production was shown in Figure 5.

As shown in FIG. 5, the Bacillus subtilis AH18 strain had the highest siderophore production capacity when cultured at 30 ° C., and the sideropore production capacity was the highest when cultured at pH 6.0 as shown in FIG. 6.

4 and 5 show cell growth and antagonist production according to temperature and pH. Only when cells grow at the appropriate (or optimal) temperature and pH is the antagonist productive. In other words, it is an experiment to determine the optimal (titration) temperature and pH for the production of siderophore of the strain of the present invention.

Example  4: Bacillus Subtilis AH18 of Auxin  refine

Each of the selected AH18 strains was cultured at 30 ° C. for 3 days in King B broth (0.1% tryptophan addition), which is an optimum medium for auxin production, to obtain a culture supernatant. The culture supernatant was titrated to pH 2.8 with 30% phosphoric acid, and then used as a sample for auxin purification. Equivalent amount of ethyl acetate was slowly stirred in a stirrer for 15 minutes, and auxin of the culture supernatant was extracted with an ethyl acetate layer. The aqueous layer was discarded, and only the ethyl acetate layer was taken. Then, ethyl acetate was added with 1/2 amount of distilled water and stirred for 15 minutes. Small amounts of auxin and impurities that were not extracted into the layer were transferred to the aqueous layer and completely removed. The procedure was repeated three times. Thus obtained ethyl acetate layer was used as a sample of auxin purification by removing the ethyl acetate through a vacuum condenser and dissolved in 50% methanol.

Purification proceeded in the order of gel filtration chromatography, HPLC, GC-MS. First, the auxin adjusted as described above was loaded on a Sephadex LH-20 column equilibrated with 50% methanol, and each fraction received 5 ml at a flow rate of 0.5 ml / min. The active fractions were taken by reloading on a Sephadex LH-20 column.

The active fraction obtained in Sephadex LH-20 column chromatography fractionated fractions with similar retention times as compared to IAA, a standard by HPLC, and the HPLC results are shown in FIG. HPLC used a C18 column with reverse-phase HPLC and the flow rate was 1 ml / min.

As shown in FIG. 7, the Rt value of IAA was 17 minutes, and the peak of 22 minutes in the HPLC fraction of Auxin AH18 showed the most similar peak to IAA. The results on HPLC indicated that no single peak was formed, suggesting that other materials besides IAA might be present.

Fractions aliquoted during the above HPLC experiments were used as samples for GC-MS analysis. Fractions having an Rt value similar to that of IAA on HPLC were concentrated using nitrogen gas, and GC-MS analysis performed at Daegu Branch, Korea Research Institute of Basic Science, is shown in FIG. 8.

As shown in FIG. 8, it was found that IBA (indole-3-betric acid) and IPA (indole-3-pronionic acid) exist in addition to IAA in GC-MS analysis. On GC-MS, the Rt values were 14.56 minutes for IAA, 15.54 minutes for IBA, and 16.62 minutes for IPA. The molecular weights were 175 and 203 for IAA and IBA, and 189 for IPA. The auxin produced by Bacillus subtilis AH18 was IAA, IBA, IPA in the ratio of 1: 1.5: 2.6.

Therefore, the AH18 strain of the present invention was confirmed to promote the growth of various crops by producing auxin mixed with IAA, IBA, IPA.

Example  5: For auxin production  To promote plant growth

5-1: AH18 Plant growth Facilitating  Confirm

Seeds of Spinach, Pea, Chinese cabbage, and Cucumber were immersed in water at 25 ° C. for 24 hours under dark conditions, and 10 ⁵ CFU of AH18 was added to sterilized soil (Seoul). After inoculation at / g, 100 seeds were each sown. As a control, seeds planted in the soil not inoculated with AH18 were used. The experimental and control groups in the same manner as shown in Table 2 to measure the root weight when sowing by incubating for 3 weeks by irradiating light at 28 ℃ 12 hours cycle.

Control (mg) Experimental group (mg) Experiment / Control Spinach (spinach) 15.0 42.3 160 Pea 19.1 25.2 150 Chinese cabbage 16.6 31.6 130 Cucumber 9.5 12.8 130

 As shown in Table 2, the root of the spinach by Bacillus subtilis AH18 the weight of 60%, cabbage and cucumber 30%, peas increased by 50%.

Seeds of plants listed in Table 3 were immersed in water at 25 ° C. under dark conditions for 24 hours, inoculated with 10 ⁵ CFU / g of auxin-producing antagonist strain in sterilized soil (manufactured by Dongbu Chemical) and 100 seeds each. Sowing was carried out. Thereafter, the seeded seeds were irradiated with light at 28 ° C. for 12 hours and incubated for 3 weeks to measure the weight of the roots, which are described in Table 6 below. As a control, seeds planted in the soil not inoculated with auxin antagonistic strains were used.

In addition, 0.1 mg / d of auxin AH18, a regulator of Bacillus subtilis AH18, and 1000 ppm of standard IAA (indole-3-acetic acid) in a sterile glass dish were used to observe the germination rate of seeds ( green onion, spinish and radish ) of each plant. The stock solution was prepared by mixing with water in a ml concentration. Equivalent amount of each stock solution was added to a plate coated with sterile filter paper, and 50 grains per plate were placed on the sterile filter paper, respectively, and the germination rate was investigated daily while incubating at 28 ° C. and 70% humidity. This experiment also showed that auxin AH18 showed a greater effect at a lower concentration than the standard.

5-2: Auxin AH18 of Plant growth promoting ability  Verification

The effect of the adjusted auxin of Bacillus subtilis AH18 strain on the germination of vegetable seeds and the elongation of plant roots and stems was investigated using seeds of mung bean, radish and green onion.

In order to observe the germination rate, preservative solution was prepared by mixing auxiliary auxin AH18 of Bacillus subtilis AH18 and 1000 ppm of IAA (indole-3-acetic acid) standard with water at a concentration of 1 μl, 5 μl and 10 μl, respectively. It prepared. Each stock solution is added to the plate with sterile filter paper and the amount of radish and dug is 50 grains per plate, and 20 grains are incubated at 28 ℃ and 70% humidity, considering that the seeds are green. Daily germination rates were examined and shown in Table 3 and FIG. 9. In Table 3, the germination rate was counted by treating 1, 5, and 10 ppm of auxin AH18 in each of the radish and onion seeds and the same concentration of IAA (standard). Auxin AH18 has more germination at the same concentration than IAA.

Botanical name Processing days Control Auxin _AH18 (ppm) IAA (ppm) One 5 10 One 5 10 Radish One 24 39 25 43 41 42 39 2 45 49 45 49 18 49 49 Spring onion One 0 3 6 2 One 2 One 2 3 15 22 16 10 15 10

As shown in Table 3, the _{heat radish} and _{green onions} had a germination rate higher than or similar to that of IAA-treated plates in a plate to which 5 mg and 10 ppm of auxin _AH18 was added after 2 days, but higher than the germination rate of the untreated plate. The germination rate was more than 2 times higher, and after 3 days, the germination rate was 1.5 times higher than that of the IAA and no treatment.

In addition, as shown in Figure 9, the green bean treated with auxin AH18 all germinated after 2 days in both IAA, untreated, and the green bean treated with auxin AH18 did not germinate more than 80% in the pot on the other than no germination. there was.

In addition, 30 grains of thermal radish seeds were immersed in a sample made by mixing 1 ml and 2 ml of auxin AH18 and 10 ppm of standard IAA in 20 ml sterile distilled water, and shaking at 160 rpm in a shaker for 1 hour. Sowing and incubation in a constant temperature and humidity room at 28 ℃, 70% humidity to investigate the root and stem elongation is shown in Table 4.

sample Elongation (cm) Root stem AH18 5.2 13.2 IAA 5.0 12.7 Control 3.8 6.4

As shown in Table 4, the stem and root elongation of the radish treated with auxin AH18 produced by Bacillus subtilis AH18 showed significantly higher elongation compared to the untreated control, and showed an elongation similar to that of IAA. .

In addition, the adjusted auxin was treated in various concentrations in the rust, and the optimum concentration was measured through Mung bean root biopsy and shown in Table 5.

sample Mung bean contingency root number (ea) 0.1 ppm 0.5 ppm 1 ppm 5 ppm 10 ppm IBA 3.2 8 10.6 18.9 1.2 IAA 7.6 9.9 12 12.6 14 AH18 8.2 12 16.6 0.8 0

As shown in Table 5, IAA and IBA showed an average of 18.9 and 12.6 rooting at a concentration of 5 ppm, but auxin AH18 and silver showed 16.6 rooting at 1 ppm, showing a strong plant promoting ability at a lower concentration than the conventional purified product. .

Thus, when Bacillus subtilis AH18 is used as a microbial agent, it will be a powerful plant growth promoter capable of high efficiency in a small amount.

Example  6: In vivo  Port Control Experiment

In order to verify whether the selected antagonistic strain AH18 exerts effective plant disease control, plant control experiments were carried out using red pepper and tomato as host plants. In a constant temperature and humidity room maintained at 28 ℃ and 70% humidity, phytophthora capsici, a pepper germ bacterium, and Fusarium oxysporum, a tomato wilting disease, were inoculated in pots in which host and tomato, respectively, were transplanted. Humidity), and treated with auxin-producing antagonist bacteria and grown in a constant temperature and humidity chamber at 28 ° C. and 70%, and confirmed the onset periodically. The results are shown in FIGS. 10 and 11. A in FIG. 10 is a case where AH18 is processed, B is a case where AH18 is not processed, A in FIG. 11 is a case where AH18 is not processed, and B is a case where AH18 is processed.

10 and 11, it was found that AH18, an antagonist strain, exhibited sufficient control ability even in an in vivo pot experiment.

The antagonistic bacterium Bacillus subtilis AH18 of the present invention produces sideropores to selectively bind iron ions in the soil to inhibit the metabolism of phytopathogenic fungi and to decompose cellulase that decomposes cellulose which is a major component of phytopathogenic fungal cell walls. To inhibit the growth of phytopathogenic fungi, and to produce auxin, a plant growth promoting hormone, relates to an antagonistic strain having excellent plant production promoting ability and a biological control method using the same.

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Claims (5)

Bacillus subtillis strain having accession number KACC 91205P, which produces one or more substances selected from the group consisting of siderophores, cellulases, and auxins. delete Comprising the strain of claim 1, the composition for the control of the pepper rot (Phytophtora Capsici) or tomato wilt (Fusarium Oxysporum). Claim 1 comprising a strain, plant growth promoting composition. The composition of claim 3, wherein the composition further comprises at least one antagonist selected from the group consisting of sideropores and cellulase.

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