KR20090116245A - Pseudomonas corrugata strain ccr80, an antagonistic rhizobacterium for controlling phytophthora blight of pepper - Google Patents

Abstract

PURPOSE: A Pseudomonas corrugate CCR80 with activity of promoting plant growth is provided to prevent Phytophthora blight of pepper and enhance the yield of Capsicum annuum. CONSTITUTION: A Pseudomonas corrugate CCR80(deposit number KACC 91367P) has an effect of antagonist to plant pathogen. The plant pathogen is Phytophthora capsici. Plant disease is caused by Phytophthora capsic.

Description

Pseudomonas corrugata strain CCR80, an antagonistic rhizobacterium for controlling Phytophthora blight of pepper

The present invention exhibits an antagonistic effect against pepper blight and has a root yield bacterium Pseudomonas korugata ( Pseudomonas corrugata ) strain Relates to the CCR80. More specifically, it has excellent biological control against plant diseases, including pepper blight caused by Phytophthora capsici , which causes severe damage to pepper production. Control bacteria Pseudomonas korugata ( Pseudomonas corrugata ) strain CCR80, 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 unavoidable for stable agricultural production at present, but the emergence of virulent, 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, antagonistic microorganisms selected under the limited conditions of the laboratory are often not antagonistic in the actual field, which means that the microorganisms treated in the root zone of the plant do not settle and survive, or antagonize only in the limited conditions of the laboratory. This is because bacteria are selected.

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, research has been conducted to promote the growth of plants using microorganisms, but there is still little protection of microorganisms that directly increase the yield of plants, and royalties are paid when foreign bacteria are introduced. 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 residuals in soil and crops. Although it has been made, there is still a small amount of effective antagonistic microorganisms against pepper blight, both domestic and foreign, and thus, biological control against pepper blight has not been achieved.

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

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 present inventors have proposed a novel soil rhizosphere bacteria Pseudomonas korugata from Korean soil. ( Pseudomonas corrugata ) strain Isolation of CCR80, and confirmed that the strain is excellent in inhibiting pepper late blight in the packaging as well as laboratory conditions, does not affect the indigenous microorganisms, and can promote the growth of plants to complete the present invention.

Thus, the present invention is Pseudomonas Korugata strain CCR80 exhibiting an antagonistic effect against phytopathogens including pepper blight bacteria ( Pseudomonas corrugata strain CCR80; Accession number KACC 91367P).

In order to achieve the other object of the present invention, the present invention is a novel Pseudomonas Korugata ( Pseudomonas corrugata ) strain Provided is a non-toxic and environmentally friendly fungicide that can be used for controlling plant diseases, including red pepper blight, containing CCR80 or the filtrate of this strain.

In addition, the present invention is the novel Pseudomonas Korugata ( Pseudomonas corrugata ) strain There is also provided a method for controlling plant diseases including pepper blight using CCR80 or a culture filtrate of this strain.

Furthermore, the present invention is the novel Pseudomonas korugata ( Pseudomonas corrugata ) strain Provided is a microbial fertilizer comprising CCR80 or a culture filtrate of this 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.

In order to select and secure soil rhizosphere bacteria having an antagonistic effect against plant diseases including pepper blight, the present inventors collected rhizosphere soil of various crops in the country, and secured CCR80 strain among them.

CCR80 strain according to the present invention is a resource collected in the soil environment of Korea isolated from the rhizosphere soil of cucumber, and excellent red pepper plague in the sequential verification of seed seed radicle, seedling and adult plant of red pepper There was an inhibitory effect (see Tables 1-4). In addition, the myobacterial bacterium CCR80 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 CCR80 strain according to the present invention had the effect of enhancing the yield of pepper even by treating the strain itself without using fertilizer (see Table 10).

CCR80 strain according to the present invention Pseudomonas Korugata through 16S rRNA sequence analysis, morphological characteristics, etc. ( Pseudomonas corrugata ), a strain belonging to Pseudomonas corugata It was named CCR80 and deposited (Accession Number KACC 91367P).

More specifically, Pseudomonas corugata according to the present invention ( Pseudomonas corrugata ) strain CCR80, Pseudomonas corugata, was analyzed by 16S rRNA sequence (SEQ ID NO: 1). ( Pseudomonas corrugata ) shows 99% homology (see FIGS. 1A and 1B).

In addition, Pseudomonas Korugata according to the present invention ( Pseudomonas corrugata ) strain CCR80 is a simple type, similar to other Pseudomonas corugata, and has 4 to 5 flagella on one side (see FIGS. 2A and 2B). ( Pseudomonas corrugata ) was found to be the most similar (see Figure 3).

Furthermore, Pseudomonas korugata according to the present invention ( Pseudomonas corrugata ) strain CCR80 is a fatty acid (Fatty acid methyl ester, FAME) analysis results Pseudomonas Korugata ( Pseudomonas corrugata ) was found to be the most significant (Fig. 4).

Therefore, through the 16S rRNA analysis, morphological observation by electron microscope, the degree of carbon source utilization by Biolog analysis, the fatty acid analysis of bacteria, the CCR80 strain according to the present invention is Pseudomonas korugata It was confirmed that it is a new strain belonging to ( Pseudomonas corrugata ) (Fig. 1a to Fig. 4).

Isolated Pseudomonas corugata according to the present invention ( Pseudomonas corrugata ) strain Both CCR80 and the filtrate of this strain are naturally derived materials, which are 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, Pseudomonas korugata according to the present invention ( Pseudomonas corrugata ) strain CCR80 can increase the yield of crops by promoting plant growth, and can be used as a pollution-free, environmentally friendly microbial fertilizer for increasing the yield of plants such as pepper.

Therefore, the soil root zone bacteria CCR80 strain according to the present invention can be utilized as a biological control and plant growth promoting bacteria having the antagonistic effect against pepper blight and the yield increase effect by plant growth promotion at the same time.

As described above, Pseudomonas corugata according to the present invention ( Pseudomonas corrugata ) strain CCR80 is a Korean soil rhizosphere bacterium, which shows excellent anti-bacterial effect against red pepper late blight on the pavement. Therefore, it can reduce the amount of agricultural fungicide used previously.

In addition, since the CCR80 strain according to the present invention does not affect the number of soil microorganisms by soil treatment of the strain, it can be usefully used in the field of biological pesticides as an environmentally friendly biological control agent.

Furthermore, Pseudomonas Korugata according to the present invention ( Pseudomonas corrugata ) strain Since CCR80 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. .

[ Example  1] Antagonist Soil Root Bacteria Against Red Pepper Plague CCR80  Isolation of Strains

In order to select antagonist rhizome bacteria against red pepper plague, the present invention sampled the rhizosphere soil of cucumber in Chuncheon region of Korea as shown in Table 1 (Table 1), and then isolates bacteria from cucumber rhizome soil. 10 g of the sampled soil was placed in 100 ml of sterile water and shaken at 28 ° C. 160 rpm for 30 minutes to form a soil suspension. 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, light brown CCR80 strain was isolated. The isolated CCR80 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 CCR80 strain of rhizome antagonistic bacteria against pepper blight.

area Collection year Separation Host Chun Cheon 2003's Root surface cucumber

[ Example  2] CCR80  Antagonistic Effects of Strains on Pepper Plague Erosion ( radicle ) Verification

We evaluated the effect of CCR80 strain isolated from the rhizosphere of cucumber soil to reduce the rate of infection caused by pepper blight in pepper radicle. In order to evaluate the effect of reducing pepper infection in pepper roots, pepper seeds were surface sterilized in 1% NaOCl for 1 minute and 30 seconds, and then washed 5 times with sterile distilled water. Thus, the surface sterilized pepper seeds were inoculated on sterilized filter paper and incubated at 28 ° C. for 3 days to induce the root formation of red pepper. Ten seeds were repeated three times for this rooting test.

Phytophthora capsici was incubated for 3 days in V-8 juice agar medium. After inoculation, glucose was inoculated in the center of WA (water agar) medium containing 0.02% of glucose and incubated at 28 ° C. for 5 days.

The CCR80 strain was incubated for 48 hours in NA, harvested with ₁₀ mM MgSO ₄ buffer, and then made a bacterial suspension, and the surface sterilized seeds were immersed in the bacterial suspension of the CCR80 strain for 3 hours. The pepper seeds immersed in the bacterial suspension as described above was placed on the mycelial edge of the late blight bacteria incubated for 5 days in WA so that the end of the pepper seed's roots was incubated for 2 to 3 days.

The infection rate was evaluated as the percentage of seeds infected with pepper blight with respect to 10 seeds, which were infected by the invasion of pepper seeds with roots as the pepper blight bacteria grew. As a result, the CCR80 strain reduced the infection rate to 53% when the untreated control was 100% infected with pepper root disease caused by pepper blight in Experiment 1 and 37% when the control was about 97% in Experiment 2. In two replicates, the CCR80 strain exhibited approximately 59% control value compared to the untreated control, significantly reducing infection rates (Table 2). Table 2 shows the results of evaluating the inhibitory effect of phytophthora capsici , a pepper late blight bacteria Phytophthora capsici in the roots of peppers by the antagonist CCR80 strain.

process Perilla infection rate (%) Experiment 1 Experiment 2 Average No treatment 100.0 ± 0.0 96.7 ± 3.3 98.3 ± 2.4 CCR80 53.3 ± 8.8 37.3 ± 12.7 40.0 ± 8.2

[ Example  3] CCR80  Antagonistic Effects of Strains on Pepper Plague Seedling  Verification( seedling assay )

CCR80 strains showing effective reduction of pepper blight from red pepper radicle were sequentially tested for pepper blight control in pepper seedlings.

For this pepper seedling assay (seedling assay), CCR80 strains were incubated for 48 hours at 28 ° C. in NA (Nutrient broth, Difco, USA) medium, and after pre-cultivation of a single flora in 5 ml of NB (Nutrient broth, Difco, USA), 500 ml of CCR80 strain was pre-cultured. Inoculated to NB and incubated at 160rpm at 28 ℃ for 48 hours. The cultured CCR80 strain was centrifuged at 5000 rpm for 15 minutes to harvest the cells of the CCR80 strain, followed by bacterial suspension 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 a mixture of TKS2: peat moss 1.5: 1 (v / v) in three repeats of 8 grains, and the CCR80 bacterial suspension was treated with 1 ml of 1 g of clay. . At this time, a seed immersed in 10mM MgSO ₄ buffer was used as a control. 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.

Hot pepper blight was incubated in oatmeal medium for 7 days at 28 ° C., and then irrigated with 15 ml of sterile distilled water and incubated for 24 hours under light conditions for 7 days. After that, in order to form the seedling spores, which are the main inoculation of red pepper germ bacterium, 30 minutes at 4 ° C. and 30 minutes at room temperature, 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, which were inoculated with red pepper germ bacterium, were inoculated 1 cm deep into red pepper seeds grown for 2 weeks.

The incidence rate (%) for healthy plants was evaluated based on the mozalloc symptoms occurring in pepper seedlings 4 days after vaccination. In the above experiment, eight plants were repeated three times in a total of three repetitions. As shown in Table 3, only about 34% of the pepper seedlings treated with CCR80 strains occurred in the control group when about 96% of the disease occurred in Experiment 1 and about 31% when 96% of the disease occurred in Control 2 in Experiment 2. Reduced the disease. In experiments 1 and 2 above, when the control group generated 96% disease, the CCR80 strain reduced the disease to about 32%, so there was about 66% control (Table 3). Table 3 shows the results of the evaluation of the inhibitory effect of pepper blight on pepper seedlings by the CCR80 strain.

process Seedlings (% disease incidence) Experiment 1 Experiment 2 Average No treatment 95.8 ± 4.2 95.8 ± 4.1 95.8 ± 2.6 CCR80 33.9 ± 14.6 30.6 ± 6.9 32.2 ± 7.3

[ Example  4] CCR80  Antagonistic Effects of Strains on Pepper Plague plant ) Verification

Pepper plant test was performed to evaluate whether the CCR80 strain, which has a significant anti-inflammatory effect against pepper blight in pepper radicle and seedling, has an antagonistic effect on pepper blight in five-week-old pepper adult plants. Was performed. To test pepper adult plants, pepper seeds were sown in 128 spheres containing top soil, and then cultured under light conditions for 16 hours for 3 weeks. Three-week-old pepper seedlings were transplanted into pasteur-sterilized field soil in pots of 9 cm diameter. One week after the transplant, bacterial suspensions of the CCR80 strain were prepared and irrigated at a concentration of 25 ml / pot according to the method described above, and a 10 mM MgSO ₄ buffer was used as a control. One week after treatment of the bacterial suspension, the spores of Capsicum B. pneumoniae were formed as indicated above, and were inoculated at a concentration of 25 spores / g soil.

After inoculation, soil moisture was maintained for uniform incidence, and the disease was evaluated as disease severity from about 4-5 days later. In order to evaluate the disease severity by the time of the symptom occurred on the basis of 0 (healthy plants) to 5 (dead plants), the degree of disease was evaluated every day, and 15 plants were repeated twice. As in Table 4, CCR80 bacteria significantly reduced the incidence to less than 1.9 and 1.5 when the control in the experiments 1 and 2 caused about 4 out of the incidences of 0-5. . In addition, in the AUDPC, which shows the pattern of disease development over time, CCR80 strains were 6.1 and 4.2, respectively, when the control group was 18.8 and 19.5 in Experiments 1 and 2, which significantly reduced disease progression over time (Table 4). ). Table 4 shows the results of the evaluation of inhibition of pepper blight caused by the CCR80 strain in adult pepper plants.

process Disease severity Translation Curve (AUDPC) Experiment 1 Experiment 2 Experiment 1 Experiment 2 No treatment 4.2 ± 0.2 4.1 ± 0.2 17.8 ± 1.9 19.5 ± 1.4 CCR80 1.9 ± 0.3 1.5 ± 0.2 6.1 ± 1.3 4.2 ± 1.0

[ Example  5] CCR80  Validation of Antagonistic Effects on Strains of Pepper Plague

In order to evaluate whether the antagonistic bacterium CCR80 strains, which have excellent inhibitory effect against pepper blight, in pepper radicle, seedling, and adult plants, have the effect of inhibiting antagonism against the same pepper blight on actual field, Namyangju, 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 validation was performed by irrigation of pepper plants with CCR80 strains on inoculated packages with pathogens, and in 2006 and 2007, bacterial suspensions of CCR80 strains when peppers were settled in packaging where pepper blight occurs naturally. After dipping red pepper seedlings in the formulation, the experiment was applied effectively to the cultivation method.

Experimental Example  1. According to the present invention CCR80 Of 2005 ~ 2006 Human Vaccination  Packaging verification

To verify the inoculation packaging in 2005 and 2006, red pepper seeds were sown on March 26, 2005 and March 16, 2006, respectively, and on packaging on June 1, 2005 and May 10, 2006, respectively. It was formal. 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 CCR80 is cultured as described above, and then a bacterial suspension is prepared and placed on two pepper plants planted on the packaging twice 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 entire bottle was inoculated in Dokso inoculated. In 2005 and 2006, when 65.4% and 69% of the untreated controls were found, 13.9% and 29.2 of peppers treated with CCR80 strains were treated. The percentage of disease was significantly reduced compared to the control. Antagonist Bacterial CCR80 strains had a control value of about 79% in 2005 and about 58% in 2006 compared to the untreated control (Table 5). Table 5 shows the results of red pepper plague pathogenic packaging by bacterial suspension irrigation of the CCR80 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 CCR80 13.9 ± 1.6 29.2 ± 7.4

In order to evaluate whether the antagonistic CCR80 strain actually reduced the infection rate of pepper roots, pepper roots were randomly sampled by repeating four plants in 2005 and four plants in 2006 in four packages. Chili roots are cut into 1cm pieces, and the PARPH (Pimaricin-ampicilin-rifampicin-PCNB-hym)

-exazol) was placed on the medium and incubated at 28 ℃. PARPH medium was autoclaved 17g / L of Cornmeal agar (CMA), followed by pimaricin 5mg, sodium ampicillin 250mg, rifampicin 10mg, PCNB 100mg, Hymexazol, 70% ai) were prepared by mixing at a concentration of 50 mg / L.

The infection rate of late blight in the roots was 1cm in size in PARPH medium. After checking that the late blight was cultivated in the roots infected by the late blight, we evaluated the number of late blight from the red pepper roots. 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, the antagonist CCR80 reduced root infection rates to 64.8% and 42.1%, respectively, in 2005 and 2006 when the untreated control showed 82.4% and 78.2% root infection rates, respectively. This resulted in a reduction of root infection caused by red pepper blight, with a control rate of about 21-46% compared to the untreated control, and similar to the treatment of metalaccil, a fungicide, reduced root infection caused by red pepper blight (Table 6). Table 6 shows the results of the pepper root infection rate of pepper blight caused by bacterial suspension irrigation of the CCR80 strain 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 CCR80 64.8 ± 4.3 42.1 ± 2.2

To evaluate whether the antagonistic bacteria CCR80 strain affects indigenous microorganisms by artificially treating the package, the total number of bacteria and fungi present in the root zone package was quantitatively evaluated. In 2005, 4 plants were repeated 4 times and in 2006, 5 plants were repeated 4 times, and pepper roots were randomly sampled. 10 g of the sampled soil was suspended in 100 ml of sterile water, and then made a soil suspension, which was plated in a TSA (Tryptic soy agar) medium containing cyclohexamide at a concentration of 50 mg / L. The number of bacteria was evaluated, OAES medium (agar 20g, sucrose 5g, yeast extract 2g, NaNO ₃ 1g, MgSO ₄ 7H ₂ O 0.5g, KH ₂ PO ₄ 1g, Na propionate 1g, Oxgall 1g, streptomycin sulfate 50mg, chloramphenicol 50 mg / L) was used to assess the total number of fungi present in the soil. As a result, it was confirmed that the antagonistic bacteria CCR80 strain did not affect the total number of bacteria and fungi indigenously compared to the untreated control and metalaccil treatment (Table 7). Table 7 shows the total number of bacteria and fungi in soil pathogenic packaging by bacterial suspension irrigation of CCR80 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 CCR80 6.57 ± 0.03 6.10 ± 0.08 4.48 ± 0.03 3.33 ± 0.04

Experimental Example  2. According to the present invention CCR80 Of 2006 ~ 2007 Natural Outbreaks  Packaging verification

Bacterial suspensions of myobacterial CCR80 strains were prepared for the 2006 and 2007 spontaneous outbreak packaging experiments. For this packaging experiment, pepper seedlings were soaked for 30 minutes in the bacterial suspension of the antagonist CCR80 strain prepared on May 10, 2006 and just before June 1, 2007. The soil in the soil was formally formulated. In 2006 and 2007, there was an overall epidemic on the packaging, and in 2006 and 2007, when 87% and 65% of the disease occurred in the untreated control, the antagonistic CCR80 strains were about 11% and 31%, respectively. The disease was significantly reduced compared to the control (Table 8).

In addition, in order to confirm the infection rate due to pepper blight in real pepper roots, pepper roots were cut into 1cm size and placed on PARPH medium, which was selected as a pepper blight selective medium, and the infection rate was evaluated. %) Also decreased the infection rate to about 41% in the treatment of antagonist CCR80 strains, compared with 86% and 66% in the untreated control in 2006 and 2007, respectively (Table 8). The antagonist CCR80 strain effectively reduced the incidence of pepper blight and root infection rate in the field to a similar degree to the fungicide metalaccil. Table 8 shows a 2006-2007 epidemic in the pie when naturally occurring bacteria Saratov pepper Station Torah kaepsi by antagonistic bacteria strains CCR80 disease incidence in the field and inhibition of root infection (Phytophthora capsici) effectiveness research.

 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 CCR80 11.4 ± 4.5 30.6 ± 1.9 40.9 ± 6.1 41.2 ± 1.7

As described above, the 2006 and 2007 field validation also quantitatively assessed whether artificially treated antagonist myobacterial CCR80 strains affect indigenous bacteria and fungi. The treatment of diseased packaging does not affect the total number of indigenous bacteria and fungi compared to the untreated control, suggesting less risk of ecosystem disturbances (Table 9). Table 9 shows the total soil and fungal numbers of soils in natural pathogenic packages by 2006-2007 CCR80 strain bacterial suspension irrigation.

 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 CCR80 5.73 ± 0.09 6.69 ± 0.12 3.19 ± 0.15 3.23 ± 0.13

[ Example  7] antagonist myobacteria CCR80 Yield of Red Pepper by Plant Growth Promotion

It was confirmed in 2007 packaging experiments that pepper yield increased by antagonistic bacteria CCR80. CCR80 strain was immersed for 30 minutes in pepper seedlings to be settled in bacterial suspension when the pepper is settled, and then packaged in a package, and harvested more than 8cm of red pepper commercially 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.

When the untreated control produced 287 green peppers, 2423g and 150 red peppers, and 1728g, the peppers treated with CCR80 strains produced 433 3480g blue peppers, 221 red peppers, and 2646g. The total number and weight of peppers produced 654 6126g of peppers from the CCR80 strain when the untreated control produced 437g of 4151g, and the total number of peppers was 51%. There is an increase in quantity (Table 10). Therefore, the CCR80 strain has the effect of increasing the yield of pepper by promoting the growth of pepper in addition to the disease suppression effect of pepper blight. Table 10 shows the results of the increase in pepper yield by the antagonist CCR80 strain in 2007.

process Pepper can Chili Pepper Live Weight (g) Green pepper Red pepper Sum Green pepper Red pepper Sum No treatment 287 ± 28 150 ± 16 437 ± 32 2423 ± 430 1728 ± 248 4151 ± 431 Metalaccil (Fungicide) 324 ± 74 138 ± 31 462 ± 93 2621 ± 782 1674 ± 467 4295 ± 1171 CCR80 433 ± 73 221 ± 16 654 ± 63 3480 ± 629 2646 ± 236 6126 ± 772

[ Example  7] About pepper plague Disease suppression  Effective Root Antagonist CCR80  Strain Molecular , Identification of bacteria using physiological characteristics

DNA of the CCR80 strain was isolated to amplify the 16S rRNA sequence of bacteria commonly used for bacterial identification. After incubating the CCR80 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.

The PCR of the CCR80 DNA thus isolated was carried out using the universal primers fd1 (AGAGT TTVAT CCTGGG; SEQ ID NO: 2) and rp2 (ACGGC TACCT TGTTA CGACTT; SEQ ID NO: 3) 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. . As a result of analyzing 16S rDNA sequence (SEQ ID NO: 1), CCR80 strain was Pseudomonas korugata. ( Pseudomonas corrugata ) and showed 99% homology (FIGS. 1A and 1B).

In addition, in order to observe the flagella and bacterial morphology of CCR80 strain through transmission electron microscope, CCR80 strain was incubated for 24 hours in NA medium, and the CCR80 bacterial suspension of a single flora was immobilized on a gold coater and then transmitted electron. It was observed under a microscope, and as a result, the CCR80 strain was rod-shaped, and Pseudomoans Similar to that observed in corrugata ), 4-5 flagellae were observed at one end (FIGS. 2A and 2B).

In order to check whether 96 strains of CCR80 strains could use different sugars, the GN2 Biolog system was analyzed. In order to use the Biolog system, CCR80 strains were incubated in TSA medium for 24 hours, and the bacterial suspension was prepared with 0.8% physiological saline to adjust the bacterial concentration to A600 OD = 0.5 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, the CCR80 strain was found to be most similar to Pseudomonas corrugata in the sugar utilization capacity (FIG. 3).

In order to classify and identify bacteria according to the fatty acid content of bacteria, fatty acid methyl ester (FAME) portion of CCR80 bacteria was extracted and identified by gas chromatography using a MIDI system. Analysis of fatty acid methyl ester (FAME) portion of CCR80, Pseudomonas Korugata ( Pseudomonas corrugata ) and was found to be the most significant (Figure 4).

Therefore, through 16S rRNA analysis, morphological observation by electron microscope, degree of carbon source use by Biolog analysis, and fatty acid analysis of bacteria, CCR80 strain according to the present invention is Pseudomonas korugata ( Pseudomonas corrugata ) strain It can be identified by CCR80 (Figs. 1A to 4).

Summarizing the above, Pseudomonas korugata according to the present invention isolated from Korean rhizosphere soil ( Pseudomonas corrugata ) strain CCR80 is a new antagonistic bacterium that can reduce the incidence of pepper blight and is worth being used as an environmentally friendly biological control bacterium.

Furthermore, Pseudomonas Korugata according to the present invention ( Pseudomonas corrugata ) strain CCR80 is a very useful invention that can be used commercially as it can contribute to the increase in 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 showing a 16S rDNA base sequence and a schematic diagram for molecular identification of a CCR80 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 CCR80 strain according to the present invention.

3 is a view showing the results of physiological sugar BIOLOG analysis for bacterial identification of CCR80 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 CCR80 strain according to the present invention.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Pseudomonas corrugata strain CCR80, an antagonistic          rhizobacterium for controlling Phytophthora blight of pepper <130> P11-080416-05 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1400 <212> DNA <213> Pseudomonas corrugata CCR80 <400> 1 gtcgagcggt agagaggtgc ttgcacctct tgagagcggc ggacgggtga gtaatgccta 60 ggaatctgcc tggtagtggg ggataacgct cggaaacgga cgctaatacc gcatacgtcc 120 tacgggagaa agcaggggac cttcgggcct tgcgctatca gatgagccta ggtcggatta 180 gctagttggt gaggtaatgg ctcaccaagg cgacgatccg taactggtct gagaggatga 240 tcagtcacac tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata 300 ttggacaatg ggcgaaagcc tgatccagcc atgccgcgtg tgtgaagaag gtcttcggat 360 tgtaaagcac tttaagttgg gaggaagggc attaacctaa tacgttagtg ttttgacgtt 420 accgacagaa taagcaccgg ctaactctgt gccagcagcc gcggtaatac agagggtgca 480 agcgttaatc ggaattactg ggcgtaaagc gcgcgtaggt ggtttgttaa gttggatgtg 540 aaagccccgg gctcaacctg ggaactgcat tcaaaactga caagctagag tatggtagag 600 ggtggtggaa tttcctgtgt agcggtgaaa tgcgtagata taggaaggaa caccagtggc 660 gaaggcgacc acctggactg atactgacac tgaggtgcga aagcgtgggg agcaaacagg 720 attagatacc ctggtagtcc acgccgtaaa cgatgtcaac tagccgttgg gagccttgag 780 ctcttagtgg cgcagctaac gcattaagtt gaccgcctgg ggagtacggc cgcaaggtta 840 aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 900 caacgcgaag aaccttacca ggccttgaca tccaatgaac tttccagaga tggattggtg 960 ccttcgggaa cattgagaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1020 gggttaagtc ccgtaacgag cgcaaccctt gtccttagtt accagcacgt tatggtgggc 1080 actctaagga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat 1140 ggcccttacg gcctgggcta cacacgtgct acaatggtcg gtacagaggg ttgccaagcc 1200 gcgaggtgga gctaatccca caaaaccgat cgtagtccgg atcgcagtct gcaactcgac 1260 tgcgtgaagt cggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg 1320 ggccttgtac acaccgcccg tcacaccatg ggagtgggtt gcaccagaag tagctagtct 1380 aaccttcggg gggacggtac 1400 <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)

Pseudomonas corrugata Pseudomonas corrugata strain having an antagonistic effect on plant pathogens strain CCR80; Accession number KACC 91367P). The method of claim 1, wherein the plant pathogen is Phytophthora capsici ( Phytophthora capsici ) Pseudomonas Korugata strain CCR80. A fungicide for controlling plant diseases, comprising the strain of claim 1 or a culture filtrate of the strain. According to 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. 6. The method of claim 5, wherein the plant disease is a plant disease caused by 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.

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