KR20090116242A - Chryseobacterium indologenes strain ise14, an antagonistic rhizobacterium for controlling phytophthora blight of pepper - Google Patents

Abstract

PURPOSE: An eco-friendly Chryseobacterium indologenes ISE14 is provided to ensure an effect of suppressing Phytophthora blight of pepper. CONSTITUTION: A Chryseobacterium indologenes ISE14(deposit number KACC 91370P) has antagonist effect to plant pathogen. The plant pathogen is Phytophthora capsici. Plant disease is prevented by treating fungicide containing Chryseobacterium indologenes ISE14 or residual culture medium of the strain to a plant. The plant disease is plant disease caused by Phytophthora capsici.

Description

Chryseobacterium indologenes strain ISE14, an antagonistic rhizobacterium for controlling Phytophthora blight of pepper

In the present invention exhibit an antagonistic effect on the chilli pepper blight soil having a quantity promoting Rhizobacteria active Cry Seo tumefaciens indole jeneseu (Chryseobacterium indologenes) Relates to strain ISE14. More specifically, it has excellent biological control against plant diseases including pepper blight caused by Phytophthora capsici , which causes severe damage to pepper production, and also is an environmentally friendly biological control bacterium that increases the yield of pepper. Inn Chryseobacterium Indologenes indologenes ) Strain ISE14, a fungicide for controlling plant diseases and a method for controlling plant diseases using the same.

Modern agriculture relies on the use of organic synthetic agricultural fungicides to protect crops from disease. These agricultural fungicides are inevitably used for stable agricultural production at this time, but the emergence of virulent and drug resistant pathogen strains. And the adverse effects on humans and the environment such as pesticide residues in the soil have reached a serious level causing social problems.

For this reason, many researchers at home and abroad are trying to develop new and safe microbial pesticides for humans and the environment. However, microorganisms with antagonistic ability selected under the limited conditions of the laboratory do not settle and survive in the actual field or antagonistic effects because they select antagonistic bacteria only under the limited conditions of the laboratory.

In particular, antagonistic microorganisms that have excellent effects in foreign countries may not be effective in Korea. In addition, imported antagonistic microorganisms and the environment of Korea do not match each other, so it is difficult to establish and survive in the root zone, and does not exhibit antagonistic effects. Because. In addition, the application of antagonistic microorganisms for biological control agents tends to rely mainly on the antibiotics secreted by the strains, and therefore, in various environmental conditions such as packaging, it does not often have the same inhibitory effect as the limited laboratory conditions. Therefore, it is desirable to use microorganisms that have various disease suppression mechanisms indigenously existing in Korea.

In addition, as modern agriculture proceeds to intensive agriculture, in order to increase yields, excessive amounts of fertilizer are applied to the soil, and this high amount of nutrients accumulates in the soil, causing the soil environment to become acidic. As an alternative to this, research has been conducted to promote plant growth using microorganisms, but it is still insufficient to directly increase the yield of plants. In addition, there are various problems such as changes in the domestic soil environment and ineffectiveness in Korea.

Overall, domestic research institutes are searching for antagonism microorganisms and using them to control plant diseases using bacteria and fungi like advanced foreign countries, but they are still conducting basic research and controlling plant diseases using antagonism microorganisms in crop cultivation. Although research on university is being conducted mainly by universities, national research institutes, government-funded research institutes, etc., the accumulation of agricultural biopesticides that is effective for crop production is not sufficient as domestic research manpower and technology accumulation in foreign countries and applied to domestic cultivation conditions. Use and practical use is insignificant.

On the other hand, red pepper accounts for about 24% of the total vegetable growing area and about 49% of seasoned vegetable growing area in Korea as of 2005. In addition, the household income contribution is more than 850 billion won per year, which is one of the main crops of the farm economy. However, pepper pestilence, anthrax, and bacterial spot disease cause serious damage in pepper cultivation, especially pepper pest disease, which reduced the yield of about 25% of pepper production in 2006.

In order to control the late blight which causes the biggest economic loss in pepper cultivation, it mainly relies on excessive amounts of organic synthetic pesticides. As described above, the misuse of organic synthetic pesticides causes problems such as the emergence of resistant strains, destruction of the ozone layer, and residues in soil and crops, and many studies such as biological control using antagonistic microorganisms have been conducted to replace them. Although it has been made, there is still a small amount of effective antagonistic microorganisms against pepper blight at home and abroad, and thus there is no biological control against pepper blight.

In particular, there is a need for intensive biological control research of pepper blight in relation to environmentally sustainable sustainable agriculture and high value-added pesticide-free industries. The securing of antagonistic microorganisms is insignificant.

Furthermore, the phenomenon caused by the excessive fertilizer application of the above-mentioned modern agriculture is gradually accumulating, and thus, there is an urgent need for an alternative to increase yield while preventing soil contamination caused by excessive use of organic synthetic fertilizers.

Therefore, an object of the present invention is to provide a myobacterial strain having excellent antagonistic effect even in the packaging of bacteria separated into myosphere soil in order to environmentally control plant diseases, including pepper pestilence.

It is also an object of the present invention to provide a non-toxic and environmentally friendly microbial fungicide and plant disease control method that can be used in plant diseases, including pepper blight, using the Root Bacterial strain.

Another object of the present invention is to provide a microbial fertilizer that can promote plant growth.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, the inventors of the present invention, Chryseobacterium Chryseobacterium Indoroseneus indologenes ) The strain ISE14 was isolated, and the present invention was completed by confirming that the strain is excellent in inhibiting pepper late blight in packaging as well as in laboratory conditions and does not affect indigenous microorganisms, and can promote plant growth.

Therefore, the present invention, Chryseobacterium indologenes ( Chryseobacterium) showing an antagonistic effect against phytopathogens including pepper blight bacteria indologenes ) Strain ISE14 (Accession No. KACC 91370P) is provided.

In order to achieve another object of the present invention, the present invention provides the novel Chryseobacterium indologenes ( Chryseobacterium) indologenes ) It provides a non-toxic and environmentally friendly fungicide that can be used for the control of plant diseases including strain ISE14 or pepper blight, including the culture filtrate of this strain.

In another aspect, the present invention Chryseobacterium Indologenes ( Chryseobacterium) indologenes ) Also provided is a method for controlling plant diseases including pepper blight using strain ISE14 or a culture filtrate of this strain.

Furthermore, the present invention provides the novel Cryseobacterium indologenes ( Chryseobacterium). indologenes ) Provided is a microbial fertilizer comprising strain ISE14 or a culture filtrate of the strain as an active ingredient and a method for promoting plant growth using the same.

Hereinafter, the configuration of the present invention with reference to the accompanying drawings in detail as follows.

The present inventors collected plant and root zone soils of various crops in the country in order to select and secure soil root zone bacteria having an antagonistic effect against plant diseases including pepper blight, and secured ISE14 strain among them.

ISE14 strain according to the present invention is a resource collected in the soil environment of Korea isolated from the root of cucumber, and excellent pepper blight in the sequential verification of pepper seed seed (radicle), seedling and adult plants. There was an inhibitory effect (see Tables 1-4). In addition, the myobacterial bacteria ISE14 strain according to the present invention was very stably inhibited pepper blight, even in three years of packaging verification by various treatments, it was confirmed that does not affect the total population (Table 5 to Table 9) Reference). Furthermore, the ISE14 strain according to the present invention had the effect of enhancing the yield of red pepper only by treating the strain itself without using fertilizer (see Table 10).

ISE14 strain according to the present invention is Chryseobacterium indologenes ( Chryseobacterium) through 16S rRNA sequence analysis, morphological characteristics, etc. was identified as a new strain belonging to indologenes , and Chryseobacterium indologenes ) The strain was named ISE14 and deposited (Accession Number KACC 91370P).

More specifically, Chryseobacterium indologenes ( Chryseobacterium) according to the present invention indologenes ) Strain ISE14 was analyzed by 16S rRNA sequence (SEQ ID NO: 1), Chryseobacterium indologenes ( Chryseobacterium) indologenes ) and 99% homology (see FIGS. 1A and 1B). Therefore, the ISE14 strain is Chryseobacterium indologenes ) is a new strain.

In addition, Chryseobacterium indologenes according to the present invention Strain ISE14 is a rod-like, non-uniform, numerous pili on the surface of bacteria and a large amount of viscous material on the cell surface (see FIGS. 2A and 2B). Results The most similar to Chryseobacterium indologenes (see FIG. 3).

Furthermore, Chryseobacterium indologenes according to the present invention Strain ISE14 was analyzed by fatty acid methyl ester (FAME), Chryseobacterium indologenes ) and was found to be the most significant (Figure 4).

Therefore, through the 16S rRNA analysis, morphological observation by electron microscope, the degree of carbon source utilization by Biolog analysis, fatty acid analysis of bacteria, ISE14 strain according to the present invention is Chryseobacterium indologenes ( Chryseobacterium) indologenes ) It can be identified as strain ISE14 (FIGS. 1A-4).

Chryseobacterium indologenes isolated according to the present invention Strain ISE14 and the filtrate of the strain are both naturally-derived substances that can be easily degraded by microorganisms in the natural environment. Therefore, the strain and the culture filtrate according to the present invention can be prepared as a pollution-free, environmentally friendly plant disease control fungicide composition by mixing the fillers or the like commonly used in the art belonging to the present invention, to control plant diseases such as pepper blight It can be very useful.

Furthermore, Chryseobacterium indologenes according to the present invention Strain ISE14 can increase the yield of pepper by promoting the growth of the plant, it will be very useful for increasing the yield of plants such as pepper.

Therefore, according to the present invention, the soil root zone bacteria ISE14 strain may be utilized as a biological control and plant growth promoting bacteria having antagonistic effect against pepper blight and yield increase effect by plant growth promotion.

As described above, Chryseobacterium indologenes ( Chryseobacterium) according to the present invention indologenes ) Strain ISE14 is a Korean soil rhizosphere bacterium, which shows excellent antibacterial effect against red pepper blight on the pavement. Therefore, it is possible to reduce the amount of previously used agricultural fungicide.

In addition, the ISE14 strain according to the present invention can be usefully used in the field of biological pesticides as an environmentally friendly biological control agent because it does not affect the number of soil microorganisms by the soil treatment of the strain.

Furthermore, Chryseobacterium indologenes according to the present invention Since strain ISE14 has an effect of promoting plant growth, it is possible to increase the yield of crops such as pepper.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. will be.

[ Example  1] Antagonist Soil Root Bacteria Against Red Pepper Plague ISE14  Isolation of Strains

In order to select antagonistic rhizome bacteria against pepper blight, sampled cucumbers were grown in the packaging of Iksan, Jeollabuk-do, Korea in 2002 (Table 1). After soaking in NaOCl solution for 90 seconds to sterilize the surface several times with sterile distilled water. The surface sterilized cucumber roots were completely ground with a sterilized grinder, and the ground roots were put in 100 ml of sterilized water and shaken at 28 ° C. 160 rpm for 30 minutes. The suspension was plated in TSA (tryptic soy agar, Difco, Detroit, USA) medium containing cyclohexamide at a concentration of 50 mg / L, incubated for 48 hours, and then morphologically isolated from other bacteria. Among them, yellow ISE14 strain was isolated. The isolated ISE14 strain was incubated in NB (Nutrient broth, Difco, Detroit, USA) medium mixed with 20% glycerol and stored at -70 ℃. Table 1 shows information such as the isolation region, crops, and timing of the root antagonist ISE14 strain against pepper blight.

area Collection year Separation Host Iksan In 2002 Inside the roots cucumber

[ Example  2] ISE14  Antagonistic Effects of Strains on Pepper Plague Erosion ( radicle ) Verification

It was evaluated in the pepper roots whether the bacterial ISE14 strain isolated from the inside of the cucumber root had the effect of reducing the infection rate caused by pepper blight. In order to evaluate the effect of reducing pepper infection in pepper roots, pepper seeds were tested in three replicates of 10 grains, and the pepper seeds were surface sterilized for 1 minute and 30 seconds in 1% NaOCl and washed 5 times with sterile distilled water. It was. Thus, the surface sterilized pepper seeds were inoculated on sterilized filter paper and incubated at 28 ° C. for 3 days to use pepper seeds with oily roots.

Pepper toxin ( Phytophthora capsici ) is a corkborer (0.5 cm in diameter) of the mycelium edge of the incubated for 3 days in V-8 juice agar medium, and then added with WA2 (0.02% glucose) water agar) was inoculated in the center of the medium and incubated at 28 ℃ for 5 days.

The ISE14 strain was incubated for 48 hours in NA, harvested with ₁₀ mM MgSO ₄ buffer, and the surface sterilized seeds were immersed in this bacterial bacterial suspension for 3 hours. Thereafter, the roots of the seed treated with the ISE14 strain were placed on the hyphae of the late blight bacteria cultured for 5 days, and then cultured at 28 ° C. for 2 to 3 days.

Infection rate of 10 seeds was evaluated by assessing that infection caused by the invasion of pepper seeds and causing brown symptom as pepper blight bacteria grew. The ISE14 strain was infected about 53% of the root infection caused by pepper blight when the control was infected by 100% pathogen in Experiment 1, and in the experiment 2, the root treated with ISE14 strain was about 57% when the control was infected by about 97%. % Infected had about 44% control value overall (Table 2). Table 2 shows the results of evaluation of the inhibitory effect of the phytophthora capsici , a pepper late blight bacterium, on the roots of peppers by the antagonist ISE14 strain.

process Perilla infection rate (%) Experiment 1 Experiment 2 Average No treatment 100.0 ± 0.0 96.7 ± 3.3 98.3 ± 2.4 ISE14 53.3 ± 8.8 56.7 ± 14.5 55.0 ± 10.8

[ Example  3] ISE14  Antagonistic Effects of Strains on Pepper Plague Seedling  Verification( seedling assay )

In the radicle assay, the ISE14 strain, which showed excellent anti-inflammatory effect against pepper blight, was sequentially tested for pepper blight control in pepper seedlings.

ISE14 strains were incubated at 28 ° C. for 48 hours in NA medium, and a single flora was precultured in NB 5 ml for 24 hours, and then precultured ISE14 strains were inoculated in 500 ml of NB and incubated at 160 rpm at 28 ° C. for 48 hours. It was. The cultured ISE14 strain was centrifuged at 5000 rpm for 15 minutes to harvest the cells of the ISE14 strain, and then a bacterial suspension was prepared with 10 mM MgSO ₄ buffer. Bacterial suspension was measured by spectrophotometer and adjusted to a concentration of OD = 0.5 (about 10 ⁸ cells / ml) in A600 with 10 mM MgSO ₄ buffer.

Red pepper seeds were seeded in a 128-neck tray containing soil mixed with 1.5 grains (v / v) of TKS2: peat moss in three repetitions of 8 grains, and treated with ISE14 bacterial suspension at a concentration of 1 ml per 1 g of soil. . At this time, a control treated with ₁₀ mM MgSO ₄ buffer was used. The red peppers sown were grown for two weeks in the culture room under 16 hours of light conditions, and two weeks after sowing, they were artificially inoculated with the red pepper bacterium.

Pepper germ was cultured in oatmeal medium at 7O < 0 > C for 7 days, and then irrigated with 15 ml of sterile distilled water and then cultured for 7 days in a light condition for 24 hours to form a vesicle sac. After that, in order to form the seedling spores, which are the main inoculum of red pepper bacterium, were treated at 30 ° C. for 30 minutes at 4 ° C. and for 30 minutes at room temperature, and only the seedling spores were harvested by gauze. The harvested seedling spores were adjusted to a concentration of 12,000 seedling spores / ml using a hemocytometer.

Yuju spores, the inoculation source of red pepper bacterium, were inoculated with red pepper seeds grown for 2 weeks at a concentration of 2,000 gsp spores / soil g at a depth of 1 cm and continued soil moisture until disease occurred after inoculation for uniform disease occurrence. Maintained.

The incidence rate was assessed based on mozzarlock symptoms occurring in pepper seedlings 4 days after vaccination. In this way, eight plants were repeated twice in three replicates. As a result, when about 95% of the disease occurred in the control, about 30-46% of the disease occurred in the pepper seedlings treated with the ISE14 strain. Since the disease is reduced by%, it has a control value of about 60% (Table 3). Table 3 shows the results of evaluation of the inhibitory effect of pepper blight on pepper seedlings by the GS309 strain.

process Seedlings (% disease incidence) Experiment 1 Experiment 2 Average No treatment 95.8 ± 4.2 95.8 ± 4.1 95.8 ± 2.6 ISE14 45.8 ± 4.2 29.2 ± 4.2 37.5 ± 4.6

[ Example  4] ISE14  Antagonistic Effects of Strains on Pepper Plague plant ) Verification

In order to verify the effects of pepper blight control by the ISE14 strains sequentially, the antagonistic effects of pepper blight were evaluated in pepper adult plants following the validation of radicle and seedling.

For the verification of adult pepper plants, red pepper seeds were sown in 128 spheres containing top soil, and then grown for three weeks in a culture room under 16 hours of light conditions, and three week red pepper seedlings were transplanted into pasteurized field soil in pots (9 cm in diameter). One week after transplanting, the ISE14 strain suspension was prepared by irrigation at the concentration of 25 ml / pot according to the method described above, and a 10 mM MgSO ₄ buffer was used as a control. After 1 week of treatment with the bacterial suspension and buffer, the seed spores of Capsicum pneumoniae bacillus were formed as described above, and were inoculated into the pot at a concentration of 25 spores / g soil.

After inoculation, soil moisture was maintained for uniform incidence, and disease progression was evaluated every day, starting at about 4-5 days after the onset of disease. For the disease evaluation, the degree of disease was evaluated on the basis of 0 (healthy plant) to 5 (dead plant), and finally evaluated by disease severity and translational curve (AUDPC). This pepper adult plant experiment was repeated twice with 15 plants for each treatment. As a result, treatment of ISE14 strain reduced the incidence to less than 2 when about 4 incidences were induced in the control. In the case of AUDPC, ISE14 strain treatment was 8.2 and 9.0, respectively, when control was 17.8 and 19.5, respectively. As a result, ISE14 bacteria have a disease suppression effect against pepper blight, in order to pepper's radicle, seedling and adult plant (Table 4). Table 4 shows the results of the evaluation of inhibition of pepper blight caused by the ISE14 strain in adult pepper plants.

process Disease severity Translation Curve (AUDPC) Experiment 1 Experiment 2 Experiment 1 Experiment 2 No treatment 4.3 ± 0.2 4.1 ± 0.2 18.8 ± 1.9 19.5 ± 1.4 ISE14 1.9 ± 0.5 1.9 ± 0.4 8.2 ± 2.6 9.0 ± 2.0

[ Example  5] ISE14  Validation of Antagonistic Effects on Strains of Pepper Plague

To evaluate whether the antagonistic bacterium ISE14 strain, which has excellent inhibitory effect against red pepper late blight on red pepper, seedling and adult plants, has the anti-inflammatory antagonistic effect against the same red pepper late blight on actual field, Namyangju-si, Gyeonggi-do This was verified through three years of field trials from 2005 to 2007 at the affiliated farm in Korea University in Deokso.

In 2005 and 2006, packaging verification was performed by irrigation of pepper plants with ISE14 strains on inoculated packages with pathogens, and in 2006 and 2007, bacterial suspensions of ISE14 strains when peppers were settled in packages where pepper blight occurs naturally. After dipping red pepper seedlings in the formulation, the experiment was applied effectively to the cultivation method. In addition, the yield of pepper was evaluated in 2007 in order to evaluate the effect of improving pepper yield by ISE14 strain treatment.

Experimental Example  1. According to the present invention ISE14 Of 2005 ~ 2006 Human Vaccination  Packaging verification

ISE14 bacteria, which are effective in controlling red pepper, seedling, and adult plants, were subjected to three-year field trials from 2005 to 2007 at an affiliated farm in Korea University, located in Deokso, Namyangju, Gyeonggi-do. Antagonistic effects against pepper blight were verified.

To verify the antagonistic effect of ISE14 strains against pepper blight in Deokso field in 2005 and 2006, pepper seeds were sown on March 26, 2005 and March 16, 2006. It was established on May 10, 2006. The treatments were randomly placed in the package and 4 repetitions of 40 plants per treatment were used. As a control, 10mM MgSO ₄ buffer and metalaxyl, the most widely used disinfectant to control pepper blight, were recommended by the manufacturer. Treated according to.

The antagonist ISE14 was cultured as described above, and then made a bacterial suspension twice in a pepper plant planted on the packaging on July 23 and August 6, 2005 and July 26 and August 2, 2006. 100 ml each was irrigated. Then, as the above-described method was formed in the yeast of the pepper germ bacillus was artificially inoculated on August 11, 2005 and August 7, 2006 at the concentration of 10 ⁵ seed spores / pepper plants.

In 2005 and 2006, the whole disease occurred in Dokso packaging, which was tested for pepper blight. In 2005 and 2006, 40% of peppers treated with ISE14 strains were treated with 65% and 69% of control disease. The disease was significantly reduced by 26% compared to the control, and metalaxyl, which is used as agrochemicals, reduced red pepper disease to a level similar to that of 25.6% in 2005 and 16.6% in 2006 (Table 5). ). Table 5 shows the results of red pepper plague pathogenic packaging by bacterial suspension irrigation of the ISE14 strain from 2005 to 2006.

Treatment Incidence Rate (%) 2005's 2006's No treatment 65.4 ± 8.0 69.0 ± 6.8 Metalaccil (Fungicide) 25.6 ± 3.8 16.6 ± 6.8 ISE14 39.9 ± 4.9 25.6 ± 9.8

In order to confirm that the ISE14 strain also reduced the invasion rate of the roots caused by late blight, the pepper roots were randomly sampled by four repetitions of four plants in 2005 and four repetitions of five plants in 2006. Was cut to 1cm size and placed on PARPH (Pimaricin-ampicilin-rifampicin-PCNB-hymexazol) medium, which is a selective bacterium bacterium bacterium, and incubated at 28 ° C.

After confirming that the late blight was cultured in the roots infected by the pepper blight, the number of late blights from the roots of the pepper was evaluated. In other words, the infected pepper root pieces per sample / pepper root pieces per each sample x 100 was evaluated, and the experiment was repeated three times 100 roots in one plant. As a result, in ISE14 strains, untreated control had 82% infection rate in 2005 compared to control group, metalaccil was 61%, ISE14 strain had infection rate of 72%, and in 2006, untreated control group showed 78% infection rate. ISE14 strain reduced the disease to 37%. This phenomenon showed an inhibitory effect on infection rate when treated with ISE14 strains in 2005 and 2006, similar to the metalaccil drug commonly used for controlling late blight (Table 6). Table 6 shows the results of the pepper root infection rate of pepper blight caused by bacterial suspension irrigation of the strain ISE14 from 2005 to 2006.

Treatment Root Infection Rate (%) 2005's 2006's No treatment 82.4 ± 1.3 78.2 ± 7.0 Metalaccil (Fungicide) 60.7 ± 4.2 24.9 ± 2.9 ISE14 72.2 ± 4.1 36.7 ± 2.8

To examine whether the ISE14 strains affect the indigenous microorganisms, red pepper root soils were randomly sampled from the package, and 10 g of the sampled soil was suspended in 100 ml of sterile water. The total number of bacteria present in the soil was evaluated by smearing in TSA (Tryptic soy agar) medium mixed at the concentration of / L, and OAES medium (agar 20g, sucrose 5g, yeast extract 2g, NaNO ₃ 1g, MgSO ₄ 7H). 0.5 g of ₂ O, 1 g of KH ₂ PO _4, 1 g of Na propionate, 1 g of Oxgall, 50 mg of streptomycin sulfate, and 50 mg / L of chloramphenicol) were used to evaluate the total number of fungi present in the soil. Compared to the control, ISE14 strain was confirmed that does not affect the total number of indigenous soil bacteria and fungi (Table 7). Table 7 shows the total soil and fungal numbers of soil in artificial pathogenic packaging by bacterial suspension irrigation of ISE14 strains from 2005 to 2006.

process Total bacterial count (Log ₁₀ CFU / g during soil building) Total mold count (Log ₁₀ CFU / g soil building) 2005's 2006's 2005's 2006's No treatment 6.62 ± 0.11 6.23 ± 0.16 4.45 ± 0.05 3.38 ± 0.01 Metalaccil (Fungicide) 6.52 ± 0.05 6.00 ± 0.08 4.31 ± 0.08 3.27 ± 0.06 ISE14 6.50 ± 0.06 5.93 ± 0.08 4.49 ± 0.03 3.28 ± 0.07

Experimental Example  2. According to the present invention ISE14 Of 2006 ~ 2007 Natural Vaccination  Packaging verification

For 2006 and 2007 packaging experiments, bacterial suspensions of rhizosphere bacterium ISE14 strains were prepared as described above, and the red pepper seedlings were added to the antagonist suspension 30 immediately before planting peppers on the packaging. After soaking for a minute, the bacteriostatic effect was evaluated by planting red pepper germ into the soil already present in the soil. As a result, in 2006, 11-80% of the disease occurred in the whole package. In 2006 and 2007, ISE14 decreased by 26% and 38%, respectively, compared to the control (Table 8).

Also, in order to confirm the infection rate due to pepper blight in real pepper roots, pepper roots were cut into 1cm size on PARPH medium, which was selected as pepper blight selective medium, and the infection rate was evaluated. Infection was reduced by 52% in 2006 and 37% in 2007 (Table 8). Table 8 shows the results of inhibition of spontaneous pepper blight disease caused by antagonistic ISE14 strains in the plague spontaneous pneumonia from 2006 to 2007 and the effect of inhibiting root infection rate by Phytophthora capsici , the pepper blight bacterium. .

 process Disease Incidence (%) Root Infection Rate (%) 2006's 2007 2006's 2007 No treatment 87.3 ± 5.6 65.3 ± 2.3 86.0 ± 2.8 65.7 ± 2.9 Metalaccil (Fungicide) - 22.5 ± 3.1 - 40.5 ± 8.8 ISE14 25.5 ± 8.6 38.3 ± 2.6 24.2 ± 2.9 46.6 ± 1.6

As described above, the 2006 and 2007 pavement verification showed that the artificially treated antagonist myobacterial ISE14 strain did not affect the indigenous bacteria and fungi, as it was described above. Table 9). Table 9 shows the total soil and fungal numbers of soils in natural pathogenic packaging by ISE14 strain bacterial suspension irrigation for 2006-2007.

 process Total bacterial count (Log ₁₀ CFU / g during soil building) Total mold count (Log ₁₀ CFU / g soil building) 2006's 2007 2006's 2007 No treatment 5.72 ± 0.01 6.46 ± 0.12 3.08 ± 0.07 3.12 ± 0.10 Metalaccil (Fungicide) - 6.63 ± 0.14 - 3.30 ± 0.17 ISE14 5.75 ± 0.12 6.56 ± 0.09 3.15 ± 0.09 3.08 ± 0.04

[ Example  7] antagonist myobacteria ISE14 Yield of Red Pepper by Plant Growth Promotion

It was confirmed in 2007 packaging experiments that pepper yield increased by antagonistic bacteria ISE14. ISE14 bacteria were immersed for 30 minutes in pepper seedlings to be settled in the bacterial suspension when the pepper was settled, and then packaged in a package and harvested more than 8cm of red pepper having a commercial value three times on August 13, August 27, September 22, 2007 The number and weight of peppers were measured by dividing them into blue peppers and red peppers. The measured plants were repeated 4 experiments of 40 plants, the average for each iteration. Since ISE14 strain increases the number or weight of green peppers and red peppers, it has an effect of increasing pepper yield by promoting plant growth (Table 10). Table 10 shows the results of the increase in pepper yield by the antagonistic bacteria ISE14 strain in 2007.

process Pepper can Chili Pepper Live Weight (g) Green pepper Red pepper Sum Green pepper Red pepper Sum No treatment 287 ± 14 150 ± 8 437 ± 16 2423 ± 215 1728 ± 124 4151 ± 216 Metalaccil (Fungicide) 324 ± 37 138 ± 16 462 ± 46 2621 ± 391 1674 ± 234 4295 ± 585 ISE14 395 ± 60 220 ± 37 616 ± 86 3417 ± 557 2761 ± 575 6178 ± 1076

[ Example  7] About pepper plague Disease suppression  Effective Root Antagonist ISE14  Strain Molecular , Identification of bacteria using physiological characteristics

DNA of ISE14 strain was isolated to amplify 16S rRNA sequences of bacteria commonly used for bacterial identification. After incubating the ISE14 strain, the bacteria were harvested by centrifugation at 3000 rpm for 10 minutes, and mixed with 300 μl of SET buffer. Lysozyme (lysozyme) was made to a concentration of 50mg / ml and mixed 6μl and incubated for 1 hour at 37 ℃. After incubation, 1/10 volume of SDS was added, followed by mixing proteinase K at a concentration of 0.5 mg / ml, incubating at 55 ° C. for 2 hours, and then processing at 75 ° C. for 20 minutes. Proteinase K was inactivated. Thereafter, RNase was added thereto, followed by incubation at 37 ° C. for 30 minutes, followed by mixing 1/3 volume of 5M NaCl and 1 volume of chloroform (chloroform) for 30 minutes at room temperature. After incubation, only the supernatant obtained by centrifugation at 10000rpm for 10 minutes was collected and the same amount of isopropanol was mixed to harvest DNA. The harvested DNA was washed twice with 70% ethanol.

PCR was performed using the isolated DNA of ISE14 using primers fd1 (AGAGT TTVAT CCTGGG; SEQ ID NO: 2) and rp2 (ACGGC TACCT TGTTA CGACTT; SEQ ID NO: 3), which are universal primers. As a condition for amplifying 16S rDNA by PCR, it was repeated 4 times at 95 ° C, 4 minutes at 95 ° C, 1 minute at 58 ° C, and 2 minutes at 72 ° C. extension step) for 10 minutes at 72 ℃. PCR products were electrophoresed on 0.8% agarose gel and then purified. To purify the PCR product, electrophoresis was performed to isolate the PCR amplified band identified in the UV and placed for 15 minutes at -20 ° C. Then, 50 µl of the liquid portion containing the PCR amplified band was harvested by parafilm and 1.5. It was placed in a sterile tube and mixed with 400 μl of TE buffer. Thereafter, 450 μl of phenol: chloroform (24: 1) solution was added thereto, mixed for 3 minutes, and centrifuged at 13,000 rpm for 7 minutes. After adding 400 µl of the supernatant centrifuged to a new tube, 44 µl of 3M sodium acetate (pH 5.2) was added and mixed. After adding 1 ml of 99% ethanol, the mixture was placed at -20 ° C for 15 minutes. Thereafter, after centrifugation at 13,000 rpm for 15 minutes, the liquid portion was removed, and 500 µl of 70% ethanol was further mixed and centrifuged at 13,000 rpm for 5 minutes to obtain a pure PCR product.

In this way, the purified PCR products were sequenced with fd1 (AGAGT TTVAT CCTGGG) and rp2 (ACGGC TACCT TGTTA CGACTT) primers. About 1,400 nucleotide sequences were analyzed and blasted in NCBI. . Analysis of the 16S rDNA sequence (SEQ ID NO: 1) showed 99% homology with Chryseobacterium indologenes (FIGS. 1A and 1B).

In addition, in order to observe the flagella and bacterial morphology of the ISE14 strain through a transmission electron microscope, the ISE14 strain was incubated for 24 hours in a NA medium, and the ISE14 bacterial suspension of a single flora was immobilized on a gold coater and then the transmission electron. Observation was made under a microscope, and as a result, it was rod-shaped, flagella was not observed, numerous pili were present on the bacterial surface, and a large amount of viscous material was observed on the cell surface (FIGS. 2A and 2B).

To determine whether ISE14 strains could use 96 different sugars, we analyzed the GN2 Biolog system. In order to use the Biolog system, the ISE14 strain was incubated in TSA medium for 24 hours, and the bacterial suspension was prepared with 0.8% physiological saline to adjust the bacterial concentration to OD = 0.5 in A600 with a spectrophotometer. The bacterial suspension was put in 100 μl of GN2 Biolog 96 well plate and incubated at 28 ° C. for 24 hours. The cultured GN2 Biolog 96 well plate was reacted according to the degree of sugar utilization and analyzed by Microlog 3 database release 4.01A (Biolog, Hayward, CA) in the Miolog GN Microplate system. As a result use of capacity per ISE14 strain in Cry Seo tumefaciens indole jeneseu (Chryseobacterium indologenes ) (FIG. 3).

In order to classify and identify bacteria according to the fatty acid content of bacteria, fatty acid methyl ester (FAME) portion of ISE14 bacteria was extracted and identified by gas chromatography using MIDI system. Fatty acid metyl ester (FAME) portion of ISE14 was found to be most significant with Chryseobacterium indologenes (FIG. 4).

Therefore, through 16S rRNA analysis, morphological observation by electron microscopy, carbon source utilization by Biolog analysis, fatty acid analysis of bacteria, ISE14 strain according to the present invention is Chryseobacterium indologenes ( Chryseobacterium) indologenes ) was identified as ISE14 (FIGS. 1A-4).

In summary, the Chryseobacterium indologenes ( Chryseobacterium) according to the present invention isolated from Korean rhizosphere soil indologenes ) Strain ISE14 is a new antagonist that can reduce the incidence of pepper blight, and will be of value as an environmentally friendly biological control bacterium.

Furthermore, Chryseobacterium indologenes according to the present invention Strain ISE14 is a very useful invention that can be used commercially as it can contribute to increase pepper yield by promoting plant growth.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1A and 1B are diagrams illustrating a 16S rDNA base sequence and a schematic diagram for molecular identification of an ISE14 strain according to the present invention.

Figure 2a and Figure 2b is a view showing the bacterial morphology by transmission electron microscopy for bacterial identification of ISE14 strain according to the present invention.

Figure 3 is a view showing the results of physiological sugar BIOLOG analysis for bacterial identification of ISE14 strain according to the present invention.

4 is a view showing the results of analysis of fatty acid (fatty acid methyl ester, FAME) of bacteria for bacterial identification of ISE14 according to the present invention.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Chryseobacterium indologenes strain ISE14, an antagonistic          rhizobacterium for controlling Phytophthora blight of pepper <130> P11-080416-02 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1392 <212> DNA <213> Chryseobacterium indologenes ISE14 <400> 1 catgcaagcc gagcggtaga gatctttcgg gatcttgaga gcggcgtacg ggtgcggaac 60 acgtgtgcaa cctgccttta tctgggggat agcctttcga aaggaagatt aataccccat 120 aatatactgg atggcatcat tcggtattga aaactccggt ggatagagat gggcacgcgc 180 aagattagat agttggtgag gtaacggctc accaagtcta cgatctttag ggggcctgag 240 agggtgatcc cccacactgg tactgagaca cggaccagac tcctacggga ggcagcagtg 300 aggaatattg gacaatgggt gagagcctga tccagccatc ccgcgtgaag gacgacggcc 360 ctatgggttg taaacttctt ttgtataggg ataaacctac tctcgtgaga gtagctgaag 420 gtactatacg aataagcacc ggctaactcc gtgccagcag ccgcggtaat acggagggtg 480 caagcgttat ccggatttat tgggtttaaa gggtccgtag gcggatgtgt aagtcagtgg 540 tgaaatctca cagcttaact gtgaaactgc cattgatact gcatgtcttg agtgttgttg 600 aagtagctgg aataagtagt gtagcggtga aatgcataga tattacttag aacaccaatt 660 gcgaaggcag gttactaagc aacaactgac gctgatggac gaaagcgtgg ggagcgaaca 720 ggattagata ccctggtagt ccacgccgta aacgatgcta actcgttttt ggagcgcaag 780 cttcagagac taagcgaaag tgataagtta gccacctggg gagtacgttc gcaagaatga 840 aactcaaagg aattgacggg ggcccgcaca agcggtggat tatgtggttt aattcgatga 900 tacgcgagga accttaccaa ggcttaaatg ggaaatgaca ggtttagaaa tagacttttc 960 ttcggacatt tttcaaggtg ctgcatggtt gtcgtcagct cgtgccgtga ggtgttaggt 1020 taagtcctgc aacgagcgca acccctgtca ctagttgcca tcattaagtt ggggactcta 1080 gtgagactgc ctacgcaagt agagaggaag gtggggatga cgtcaaatca tcacggccct 1140 tacgccttgg gccacacacg taatacaatg gccagtacag agggcagcta cacggtgacg 1200 tgatgcaaat ctcgaaagct ggtctcagtt cggattggag tctgcaactc gactctatga 1260 agctggaatc gctagtaatc gcgcatcagc catggcgcgg tgaatacgtt cccgggcctt 1320 gtacacaccg cccgtcaagc catggaagtc tggggtacct gaagtcggtg accgtaacag 1380 gagctgccta gg 1392 <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 2 agagtttvat cctggg 16 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PCR PRIMER <400> 3 acggctacct tgttacgact t 21  

Claims (8)

Chryseobacterium indologenes with an antagonistic effect on plant pathogens ISE14 , Accession No. KACC 91370P). 2. The cryosebacterium indologenes strain ISE14 according to claim 1, wherein the plant pathogen is Phytophthora capsici. A fungicide for controlling plant diseases, comprising the strain of claim 1 or a culture filtrate of the strain. The method of claim 3, wherein the plant disease is a plant disease control plant disease characterized in that the plant disease caused by Phytophthora capsici (Phytophthora capsici). A plant disease control method for controlling plant diseases by treating a plant with a bactericide comprising the strain according to claim 1 or a culture filtrate of the strain. The method of claim 5, wherein the plant disease is a plant disease characterized in that the plant disease caused by Phytophthora capsici (Phytophthora capsici). A microorganism fertilizer capable of promoting the growth of plants comprising the strain of claim 1 or the culture filtrate of the strain as an active ingredient. 8. The microorganism fertilizer according to claim 7, wherein the plant is red pepper.

Images