KR20110092617A - Composition for controlling anthracnose and promoting fruit yield and ripening of plants comprising flavobacterium sp. strain gse09 and controlling method using the same - Google Patents

Abstract

PURPOSE: A composition containing Flavobacterium sp. GSE09 is provided to enhance productivity and to promote maturation in plants. CONSTITUTION: A composition for preventing plant anthracnose contains Flavobacterium sp. GSE09(deposit number: KACC 91368P) or metabolites thereof. The anthracnose is caused by Colletotrichum sp. The metabolite is a volatile material. A microorganism fertilizer composition for enhancing yield contains the strain or the metabolites. A method for promoting maturation of Capsicum annuum comprises a step of treating the metabolites to green Capsicum annuum.

Description

Composition for controlling anthracnose and promoting fruit yield and ripening of plants comprising Flavobacterium sp. strain GSE09 and controlling method using the same}

The present invention relates to an anthracnose control and yield increasing and maturation promoting composition comprising the soil rhizosphere bacteria Flavobacterium strain GSE09, and an anthracnose control method using the same. More specifically, the present invention has an excellent biological control effect against anthracnose, which occurs in red pepper fruit during packaging and planting of red pepper, which causes severe damage to red pepper production, and at the same time promotes yield and maturation of red pepper. The present invention relates to plant anthracnose control, including an environmentally friendly biological control bacterium, GSE09, which promotes the yield of high-value red pepper, and a composition for promoting yield and maturation, and a method for controlling anthrax using the same.

Modern agriculture relies on the use of agricultural fungicides to protect crops from disease, and these agricultural fungicides are unavoidable for stable agricultural production despite the negative effects on humans and the environment.

For this reason, many researchers at home and abroad are trying to develop new and safe microbial pesticides for humans and the environment, but it is still insufficient to secure microorganisms with antagonistic effects against plant diseases at home and abroad.

Antagonistic microorganisms selected under the limited conditions of the laboratory may not survive or exhibit antagonistic effects in various environmental conditions of the actual packaging, and for this reason, excellent antagonistic microorganisms in foreign countries are effective in the Korean environment. Many times you can't.

In addition, when cultivating high value-added crops such as red pepper, excessive amounts of fertilizer are used to increase the yield, and in some farms, chemicals such as growth promoters are used, which causes environmental pollution and toxicity. Serious problems can arise with the production of safe peppers.

Although domestic research institutes are searching for antagonism microorganisms and using them to control plant diseases using bacteria and fungi as in advanced countries, they are still conducting basic research. Although research is being conducted mainly by universities, national research institutes, and government-funded research institutes, there are not enough domestic research personnel and technologies as foreign countries, and they are applied to domestic cultivation conditions, which are effective for producing agricultural pesticides and fertilizers that are effective for crop production. Use and practical use is insignificant.

On the other hand, anthrax in plants is caused by fungi such as genus Colletotrichum. Most of the plants on our table are affected by the pathogen. Plant anthrax destroys leaves, stems, fruits, and flowers. It is especially severe in eggplants and gourds and crops such as pepper, a seasoning vegetable that is indispensable to Korean food, and watermelon, a summer fruit.

These peppers, which have been severely damaged by anthrax, account for about 24% of the total vegetable cultivation area in Korea in 2005, about 49% of the seasoned vegetable cultivation area, and contribute more than 850 billion won to farmers annually. One of the major crops. In particular, the consumption of red pepper is markedly high due to the characteristics of the Korean diet, and the market size for processed products using the same is increasing.

However, pepper anthrax, late blight, and bacterial spot disease cause serious damage in pepper cultivation. Especially, the damage caused by pepper anthracnose on pepper fruits is serious, especially in relation to the production of red pepper fruits. Rely on synthetic pesticide spraying. The misuse of organic synthetic pesticides has caused 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. To date, biological control using effective antagonistic microorganisms against pepper anthrax, which is a disease of pepper that occurs frequently during warm and rainy seasons and as the harvest season is due to airborne diseases, is insignificant. In recent years, there has been a need for intensive biocontrol studies of plant anthrax in relation to environmentally sustainable sustainable agriculture and pesticide-free high value-added industries, but the lack of understanding of airborne diseases and diseases occurring on the ground of plants It is still insufficient to secure effective antagonistic microorganisms compared to conventional agricultural fungicides even under the packaging conditions. At the same time, microorganisms having the effect of promoting the yield of pepper and the ripening of pepper are not secured.

Therefore, the present invention is for the control of plant anthrax, including the endogenous rhizosphere bacteria Flavobacterium strain GSE09 isolated from the rhizosphere soil of Korea, which can environmentally and effectively control an anthropogenic anthrax, which causes serious problems when growing plants including red pepper. It is an object to provide a composition.

In addition, another object of the present invention is to provide a method for controlling plant anthrax using the Flavobacterium strain GSE09.

Still another object of the present invention is to provide a composition for increasing yield and promoting maturation, including an endogenous rhizosphere bacterium Flavobacterium strain GSE09 isolated from the rhizosphere soil of Korea that can promote plant yield and promote maturation.

It is also an object of the present invention to provide a method for promoting yield and maturation of plants using the Flavobacterium strain GSE09.

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

In order to select and secure soil rhizosphere bacteria having an antagonistic effect against anthrax, the present inventors collected rhizosphere soils of various crops from all over the country. It was identified as Bacterium ( Flavobacterium sp.) Strain. GSE09 strains were evaluated at the control plant for pepper anthrax caused by growing peppers in packaging and facilities for two years in 2008 and 2009 at Korea University affiliated farm located in Deokso, Namyangju-si, Gyeonggi-do. Enhancement and pepper maturation promoting effect was verified. As a result, the GSE09 strain was confirmed to have excellent effects on biological control and plant growth to maturation promoting pepper anthrax, and completed the present invention.

Therefore, the present invention is the Flavobacterium sp. Provided is a non-toxic and environmentally friendly fungicide composition that can be used to control plant anthrax containing GSE09 (Accession No. KACC 91368P) or metabolites produced from this strain.

In addition, the present invention is Flavobacterium sp. Also provided is a method for controlling plant anthrax, comprising treating a plant with a composition containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain.

The present invention is Flavobacterium sp. Provided is a microbial fertilizer composition for increasing plant yield and a composition for promoting maturation containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain.

In addition, the present invention is Flavobacterium sp. Also provided is a method for promoting plant yield and maturation comprising treating a plant with a composition containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain.

In the compositions and methods according to the invention, the strain or metabolite is a cell of the strain, a suspension of the strain, a culture of the strain, a concentrate of the culture, a concentrate of the volatiles of the culture and the culture of the culture. It can be used in a state of dry matter. Cells of the strain is meant to include both the body and spores of the bacteria itself, the suspension means a state in which the cells are suspended in distilled water, physiological saline, buffer, etc., the culture is a culture containing cells and cells without cells It includes all, and the concentrate and dried means to be concentrated or dried according to the method commonly used in the art to which this invention belongs. Preferably said strain metabolite is a volatile substance.

In the composition and control method according to the invention, the anthrax is characterized in that anthrax is caused by a pathogen of Colletotrichum sp. For example, the plant anthrax may include colletotrichum acutatum , colletotrichum coccodes , colletotrichum gloeosporioides , colletotriccum dematium ( Colletotrichum dematium ), Glomerella cingulata and Colletotrichum lagenarium may be anthrax caused by one or more pathogens selected from the group consisting of, but not limited to.

In the compositions and methods according to the present invention, the applicable plant is not particularly limited and may be applied to at least one plant selected from the group consisting of, for example, red pepper, paprika, strawberry, tomato, watermelon, cucumber and apple, It is not limited to this.

In the compositions and methods according to the present invention, there is no particular limitation on the method of treating the anthracnose control or yield increasing and maturation-promoting compositions to plants, for example root-dipping or irrigation of plants before planting. It can be processed and used. In the case of the dipping method, the composition may be poured around the plant or soaked seedlings when formulated.

Plant maturation promotion method according to the present invention is characterized in that the metabolite is a volatile substance and can promote the maturation with red pepper even when treated not only pepper plant but also harvested blue pepper.

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

The present inventors collected the rhizosphere soils of various crops in the country in order to select and secure soil rhizosphere bacteria having an antagonistic effect against pepper blight, and obtained the GSE09 strain among them and deposited with the accession number KACC 91368P. The present invention has been confirmed to be effective in controlling anthrax, increasing yield, and promoting maturation.

GSE09 strain according to the present invention is a resource collected in the soil environment of Korea isolated from the rhizosphere soil of cucumber, and had an excellent inhibitory effect on the growth of anthrax pathogens (see Table 10). In addition, the rhizosphere bacterium GSE09 strain according to the present invention was confirmed to suppress the pepper anthrax very stably even in facility cultivation and packaging cultivation for two years by various treatments (see Table 3 and Table 4). Furthermore, the GSE09 strain according to the present invention was found to be effective in promoting pepper yield and promoting pepper maturity in facility cultivation and packaging cultivation for two years by various treatments (see Tables 5 to 9).

The GSE09 strain according to the present invention was identified as belonging to Flavobacterium sp. Through 16S rRNA sequencing, morphological characteristics, etc., and was named and deposited Flavobacterium strain GSE09 (Accession No. KACC 91368P).

More specifically, Flavobacterium sp. Strain according to the present invention GSE09 shows 96% homology with Flavobacterium sp. As a result of analyzing the 16S rRNA sequence (SEQ ID NO: 1) (see FIGS. 1A and 1B). Thus, the GSE09 strain belongs to the genus of Flavobacterium and is new at the species level because it shows less than 97% homology in 16S rRNA analysis.

In addition, Flavobacterium strain (Flavobac terium sp.) Strain according to the present invention GSE09 is rod-shaped, has no flagella, viscous material is formed around bacteria (see FIGS. 2A and 2B), and 96 kinds of sugar utilization have been investigated most recently with Photobacterium logei . Since the similarity is very low at 0.239, the GSE09 strain cannot be said to belong to Photobacterium , and it was identified as a new strain belonging to the genus Flavobacterium sp. (FIG. 3).

Furthermore, Flavobacterium sp. Strain according to the present invention GSE09 was found to be most significant with Flavobacterium sp. (Fatty acid methyl ester) analysis (FIG. 4).

As a result of comparing the physiological and biochemical properties of other bacteria of the genus Flavobacterium ( Flavobacterium ), the GSE09 strain according to the present invention forms yellow colonies, and is mobile and Gram-negative bacteria. The GSE09 strain was able to survive both TSA (tryptic soy agar) and NA (nutrient agar), and was able to grow under 25C conditions. Flexirubin type pigments, which are also formed by the bacteria in Flavobacterium, did not form in the GSE09 strain, and were capable of absorbing congo red. In addition, it did not form urease, an enzyme capable of degrading urea, it could decompose gelatin and casein, and could not decompose carboxymethyl cellulose, agar and pectin. The ability to decompose chitin was moderate, with the ability to degrade alginate, starch, DNA and tyrosine and to precipitate in egg-yolk medium. While it did not respond to ONPG. When compared with the physiological biochemical properties compared to the strains, it was confirmed that the congo red (congo red) absorbency, urea (urea) and other properties such as substrate degradation ability (Table 2).

Therefore, through the 16S rRNA analysis, morphological observation by electron microscopy, biolog analysis, carbon source utilization, bacterial fatty acid analysis and physiological biochemical characteristics comparative analysis, the GSE09 strain according to the present invention is Flavobacterium sp. It was confirmed to be a strain of a new species belonging to (FIGS. 1A-4, Table 2).

Flavobacterium sp. Isolated strain according to the present invention GSE09 and its metabolites are all naturally derived substances that can be readily degraded by microorganisms in their natural environment. Therefore, the strain and the culture filtrate according to the present invention can be prepared alone or by mixing a filler or the like commonly used in the art belonging to the present invention as a pollution-free, environmentally friendly plant disease control fungicide composition and a composition for promoting quantity and maturity promotion In addition, it can be very useful for controlling plant anthrax including peppers, increasing yield and promoting maturity.

Therefore, the soil rhizosphere bacterium GSE09 strain according to the present invention may be utilized as a biological control and plant growth or maturation promoting bacteria having an antagonistic effect on plant anthrax such as pepper.

As described above, Flavobacterium sp. Strain according to the present invention GSE09 is a Korean soil rhizosphere bacterium, which shows excellent disease suppression effect against plant anthrax on the pavement. Therefore, it can promote the effect of environmentally friendly plant anthrax control. In addition, it is possible to reduce the amount of agricultural fungicides used in the past can solve problems such as environmental pollution and the emergence of resistant strains.

Also, Flavobacterium sp. Strain according to the present invention Compositions containing GSE09 can be environmentally friendly to increase the yield of plants, including pepper, and at the same time can promote the effect of promoting maturation. In particular, the composition according to the present invention can be used as a bio fertilizer to promote the maturation of pepper from red pepper to commercially high red pepper can solve the problem of chemical fertilizer.

1A and 1B are diagrams showing a 16S rDNA base sequence and a schematic diagram for molecular identification of a GSE09 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 the GSE09 strain according to the present invention.
Figure 3 is a view showing the results of physiological sugar BIOLOG analysis for bacterial identification of GSE09 strain according to the present invention.
Figure 4 is a view showing the results of the analysis of fatty acid (fatty acid methyl ester, FAME) of bacteria for bacterial identification of GSE09 strain according to the present invention.
5 is a diagram showing the nucleotide sequence and the classification position for molecular identification of plant anthrax pathogens.
Figure 6 is a photograph showing the pepper maturation promoting phenomenon during facility cultivation by GSE09 strain according to the present invention.
Figure 7 is a photograph showing the pepper maturation promoting effect by the volatile substances formed by the GSE09 strain according to the present invention.

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 Isolation of Antagonistic Soil Root Bacteria GSE09 Strain against Anthrax

In order to select antagonist rhizome bacteria against anthrax, the present invention sampled cucumbers grown in the pavement of Gunsan, Jeollabuk-do, Korea in 2002, and to isolate bacteria inside the roots of cucumbers. 10 g of roots were soaked in 1% NaOCl solution for 90 seconds for surface sterilization and then washed 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 GSE09 strain was isolated. The isolated GSE09 strain was cultured 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 rhizome antagonist GSE09 strain against anthrax.

area
Collection year
Separation
Host
Gunsan
2003's
Inside the roots
cucumber

Example 2 Identification of Bacteria Using Molecular and Physiological Characteristics of Root Antagonist Bacteria GSE09 Strain with Inhibitory Effect on Anthrax

Root antagonist Bacteria GSE09, which has an anti-inflammatory effect against anthrax, was identified using molecular and physiological characteristics. DNA of the GSE09 strain was isolated to amplify the 16S rRNA sequence of bacteria commonly used for bacterial identification. After incubating the GSE09 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 DNA of GSE09 thus separated was subjected to PCR using 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.

The PCR product purified in this manner was sequenced with fd1 (AGAGT TTVAT CCTGGG: SEQ ID NO: 2) and rp2 (ACGGC TACCT TGTTA CGACTT: SEQ ID NO: 3) primers. Blast (blast) at and analyzed. Analysis of the 16S rDNA sequence (SEQ ID NO: 1) showed that the GSE09 strain showed 96% homology with Flavobacterium sp. (FIGS. 1A and 1B). Therefore, the GSE09 strain belongs to the genus of Flavobacterium , and may be called a new strain at the species level because it shows less than 97% homology in 16S rRNA analysis.

In addition, in order to observe the flagella and bacterial morphology of the GSE09 strain through a transmission electron microscope, the GSE09 strain was incubated for 24 hours in NA medium, and the GSE09 bacterial suspension of a single flora was fixed on a gold coater and permeated. Observation was carried out by electron microscopy. As a result, the GSE09 strain was observed to have a rod-like bacterium shape, no flagella was observed, and a viscous substance was observed around the bacteria (FIGS. 2A and 2B).

GSE09 strains were analyzed using the GN2 Biolog system to determine whether 96 different sugars could be used. In order to use the Biolog system, the GSE09 strain was incubated in TSA medium for 24 hours, and a 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, the GSE09 strain was most recently associated with Photobacterium logei in sugar utilization, but since the similarity was very low at 0.239, the GSE09 strain could not be said to belong to Photobacterium (Fig. 3), and 16S. A new strain of Flavobacterium genus based on rRNA and electron microscopy, fatty acid content and physiological and biochemical properties. It was confirmed (FIGS. 1A-4).

In order to classify and identify bacteria according to the fatty acid content of bacteria, fatty acid methyl ester (FAME) portion of GSE09 bacteria was extracted and identified by gas chromatography using a MIDI system. Fatty acid methyl ester (FAME) portion of GSE09 was analyzed and found to be the most significant with Flavobacterium (Flavobacterium sp.) (Fig. 4).

Therefore, through 16S rRNA analysis, morphological observation by electron microscopy, the degree of carbon source use by Biolog analysis, fatty acid analysis of bacteria, GSE09 strain according to the present invention is Flavobacterium (Flavobac terium sp.) Strain Can be identified by GSE09 (Fig. 1a ~ 4).

The 16S rDNA sequencing, biolog, and fatty acid analysis confirmed that the GSE09 strain belongs to the genus of Flavobacterium , but the result of the analysis showed that the strain was classified as the most similar to the GSE09 strain. Because of less similarity, different bacteria from the genus Flavobacterium were distributed and compared with their physiological and biochemical properties.

To compare these physiological biochemical characteristics, 11 species of Flavobacterium were distributed from the Korea Agricultural Research Institute. As shown in Table 2 below, 1; In comparison with the GSE09 pre-sorted strains 2; Flavobacterium johnsoniae , 3; Flavobacterium pectinovorum , 4; Flavobacterium saccharophilum , 5; Flavobacterium hydatis , 6; Flavobacterium psychrolimnae , 7; Flavobacterium daejeoneanse , 8; Flavobacterium sundheonense , 9; Flavobacterium flevense , 10; Flavobacterium calogense , 11; Flavobacterium aguatile , 12; Flavobacterium branchiophilum .

The colony color and mobility gram reactions of the above strains were compared with the growth, enzyme secretion, nutrient use and oxidation of the medium, and were tested as positive (+), negative (-) and moderate (+ w). Evaluated. GSE09 strains formed yellow colonies, were mobile and Gram-negative bacteria. And it was able to survive in both TSA (tryptic soy agar) and NA (nutrient agar), it was possible to grow in the conditions of 25C. Flexirubin type pigments, which are also formed by the bacteria in Flavobacterium, did not form in the GSE09 strain, and were capable of absorbing congo red. In addition, it did not form urease, an enzyme capable of degrading urea, it could decompose gelatin and casein, and could not decompose carboxymethyl cellulose, agar and pectin. The ability to decompose chitin was moderate, with the ability to degrade alginate, starch, DNA and tyrosine and to precipitate in egg-yolk medium. While it did not respond to ONPG. When compared with the strains that received the physiological biochemical properties, it was confirmed that the congo red (congo red) absorbency, urea (urea) and other properties such as substrate degradation ability (Table 2). Table 2 summarizes the results of comparison of physiological and biochemical characteristics between Flavobacterium sp. GSE09 strain and type strain.

characteristic
(Characteristics) 1 ^a
2
3
4
5 6
7
8
9
10
11
12 Color of colonies Y ^b Y Y Y W Y W Y Y Y W Y
Motility + + - + + + + + + + + +
Gram staining
-
-
+
-
-
-
-
-
-
-
-
-
Growth on












Tryptic soy agar
+
+
+
+
+
+
+
+
+
+
+
+
Nutrient agar
+
+
+
+
+
+
+
+
+
+
+
+
Growth at 25C
+
+
+
+
+
+
+
+
+
+
+
+
Flexirubin-type pigment
-
-
+
+
+
+
-
+
+
+
+
-
Congo red absorption
+
-
+
+
+
+
-
+
+
+
+
-
Urease
-
+ w
-
-
-
-
-
-
+ w
-
-
+
Degradation of:












Gelatin
+
+
+
+
+
-
-
+
+
-
-
-
Casein
+
+
+
-
-
-
+
+
-
-
+
+
Carboxymethyl cellulose
-
-
+
+
+
-
-
-
+
+
+
+
Agar
-
-
-
+
+
+
+
+
+
+
+
-
Pectin
-
-
-
+
+
+
+
+
+
+
+
-
Chitin
+ w
-
+
+
+
+
+
+
-
-
-
-
Alginate
+
+
+
+
+
+
-
-
-
-
-
-
Starch
+
+
+
+
+
-
+
+
+
+
+
+
DNA
+
+
+
+
+
-
-
+
+
+
+ w
-
Tyrosine
+
+
+
+
-
-
-
+
+
+
+ w
-
Precipitation on egg-yolk agar -
-
+
-
-
+
+
+
+
-
+
+
ONPG - - - - + + + + + - - -

Therefore, through 16S rRNA analysis, morphological observation by electron microscopy, biolog analysis, carbon source utilization, bacterial fatty acid analysis and physiological biochemical characteristics comparison analysis, GSE09 strain according to the present invention is a genus of Flavobacterium . (genus) was identified as a strain of new species (speices) (Fig. 1a to 4, Table 2).

Example 3 Plant Cultivation Validation Effect of GSE09 Strain on Pepper Anthrax

In 2008 and 2009, the antagonistic bacteria GSE09 strains were tested to reduce the incidence of pepper anthrax.

GSE09 strains were incubated for 48 hours at 28 ° C. in NA (nutrient agar, Difco, USA) medium for pre-cultivation assays. , The pre-cultured GSE09 strain was inoculated into 500 ml of NB and incubated at 160 rpm at 28 ° C. for 48 hours. The cultured GSE09 strain was centrifuged at 5000 rpm for 15 minutes to harvest the cells of the GSE09 strain, and then bacterial suspension was made 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.

The bacterial suspension thus produced was irrigated 100 ml of pepper seedlings on April 19, 2008 and April 18, 2009, and was planted on April 22, 2008 and April 20, 2009 at the facility house planting facility. Disease evaluation was performed on 4 repetitions of 15 plants on 2 August 2008 and 4 repetitions of 20 plants on 29 July 2009. Table 3 shows the results. In other words, Table 3 shows the results of testing the disease suppression effect of pepper anthrax caused by the antagonistic GSE09 strain in the facility cultivation (farm, affiliated to Korea University, Deokso University, Namyangju-si, Gyeonggi-do).

Year and treatment
Incidence Rate (%)

Green pepper Red pepper Blue + Red Pepper 2008
   No treatment 1.7 ± 1.2 8.3 ± 1.4 4.1 ± 1.4
GSE09
0.8 ± 0.3 4.3 ± 0.3 2.1 ± 0.3 2009
No treatment
5.5 ± 1.1 8.6 ± 0.7 7.3 ± 0.4 GSE09
2.3 ± 1.0 3.3 ± 1.4 2.8 ± 0.8

As shown in Table 3, the incidence of anthrax was decreased in the green peppers and red peppers treated with GSE09 in both green and red peppers compared to the untreated control in both 2008 and 2009. In 2008, when the untreated control had 1.7, 8.3, and 4.1% anthrax in blue, red, and blue + red peppers, 0.8, 4.3, and 2.1% anthrax were produced in the GSE09 treatment, respectively. In 2009, similar to 2008, GSE09 reduced anthrax compared to the untreated control, reducing the disease by 49-62%.

Example 4 Investigation of the Packaging Inhibition Effect of GSE09 Strain on Pepper Anthrax

Antagonistic bacteria GSE09 was prepared as in the verification of plant cultivation, and the bacterial suspension was prepared by irrigating 100 ml of pepper seedlings on June 13, 2008 and May 14, 2009, and the pepper fruit was September 2, 2008. Seven plants were harvested and 4 repetitions were carried out. In 2009, harvest was performed on August 5 and 10 plants were repeated 4 times and the occurrence of anthrax was evaluated. Table 4 shows the results. In other words, Table 4 shows the results of testing the inhibitory effect on pepper anthrax caused by the antagonistic GSE09 strain in the field of cultivation (2008-2009, farms affiliated to Korea University, Deokso University, Namyangju, Gyeonggi-do).

Year and treatment
Incidence Rate (%)

Green pepper Red pepper Blue + red pepper 2008
No treatment
9.3 ± 1.7 45.3 ± 4.6 15.9 ± 1.5 GSE09
3.3 ± 0.2 10.5 ± 2.7  4.6 ± 0.5 2009
No treatment
8.9 ± 0.6 21.9 ± 3.8 12.8 ± 1.4 GSE09
2.4 ± 0.7 12.2 ± 5.5 5.8 ± 2.1 b

As shown in Table 4, the incidence of anthrax was significantly reduced in packaged peppers in 2008 and 2009 when treated with GSE09 compared to untreated control in both 2008 and 2009. In 2008, anthrax was reduced by 65% in blue peppers, 77% in red peppers, and 71% in blue and red peppers compared to untreated controls.In 2009, blue in packaged peppers treated with GSE09 was reduced compared to untreated controls. Anthrax was reduced by 73% in peppers, 44% in red peppers and 55% in blue + red peppers (Table 4).

Example 5 Yield Enhancement Effect in Plant Cultivation of GSE09 Strains

To determine if pepper production was enhanced in peppers grown in cultivated antagonistic bacteria GSE09, bacterial suspensions were prepared as described above and irrigated with 100 ml of pepper seedlings on April 19, 2008 and April 18, 2009. On April 22, 2008 and April 20, 2009, the plant house plant was planted. The yield improvement evaluation was carried out in four repetitions of 15 plants on August 2, 2008, and in four repetitions of 20 plants on July 29, 2009. Tables 5 and 6). Table 5 shows the results of the pepper growth enhancement effect by the antagonistic GSE09 strain in the facility cultivation, Table 6 shows the results of the pepper yield improvement effect by the cultivation of the antagonistic GSE09 strain in the facility cultivation (2008 ~ 2009, Farm, affiliated with Korea University, Deokso, Namyangju-si, Gyeonggi-do).

Year and treatment
Pepper can
First harvest
Secondary harvest
Green pepper Red pepper Blue + red
pepper Green pepper Red pepper Blue + red
pepper 2008
No treatment
171.0 ± 12.6 302.3 ± 22.3 473.3 ± 28.0 234.5 ± 19.7 223.0 ± 14.2 457.5 ± 32.1 GSE09
218.5 ± 13.7 408.5 ± 35.7 627.0 ± 45.1 310.5 ± 6.7 224.8 ± 2.3 535.3 ± 5.0 2009
No treatment
142.8 ± 11.3 209.8 ± 4.8 352.5 ± 15.6 62.5 ± 9.0 b 88.0 ± 13.9 150.5 ± 20.2 GSE09
182.0 ± 19.0 234.5 ± 16.8 416.5 ± 35.7 112.8 ± 14.1 136.0 ± 5.6 248.8 ± 14.6

Year and treatment
Red pepper weights
First harvest
 Secondary harvest 2008
No treatment
 3.4 ± 0.4  4.6 ± 0.5 GSE09
 4.0 ± 0.3  5.5 ± 0.2 2009
No treatment
 2.9 ± 0.4  1.2 ± 0.1 GSE09
 4.8 ± 0.8  2.8 ± 0.5

As shown in Tables 5 and 6, in 2008 and 2009, when the GSE09 strains were treated during the first and second harvests, the number of peppers increased in the range of about 10-70% compared to the untreated control, and the pepper weight was 20 ~ 130% increase. In particular, the production of red peppers in facility cultivation increased about 20% by GSE09 strains in the first and second harvests in 2008. In 2009, the red pepper production was 70% and the second harvest in the first harvest. Increased by 130% (Table 6).

Example 6 Yield Enhancement Effect in Packaging Culture of GSE09 Strains

When the antagonist GSE09 was treated in peppers, the number and yield of peppers produced were measured to determine whether pepper production increased when planted in the field, similar to that in plant cultivation (Tables 7 and 8). Table 7 shows the results of testing the pepper growth by the antagonist GSE09 strain in the packaging cultivation, Table 8 shows the results of testing the pepper yield improvement effect by the antagonistic GSE09 strain in the packaging cultivation (2008 ~ 2009, Affiliated Farm, Korea University, Deokso, Namyangju-si, Gyeonggi-do).

Year and treatment
Harvested Pepper Count
Green pepper
 Red pepper  Blue + Red Pepper 2008
No treatment
 99.3 ± 0.8  21.3 ± 5.3  120.7 ± 5.5 GSE09
 114.5 ± 19.1  32.3 ± 3.1  146.8 ± 19.1 2009
No treatment
 43.8 ± 7.8  17.3 ± 4.6  61.0 ± 5.5 GSE09
 58.0 ± 9.1  27.3 ± 3.0  85.3 ± 11.0

Year and treatment
Harvested Pepper Weight (kg)
Green pepper
 Red pepper  Blue + Red Pepper 2008
No treatment
 0.98 ± 0.01  0.22 ± 0.06  1.20 ± 0.17 GSE09
 1.63 ± 0.30  0.44 ± 0.05  2.07 ± 0.30 2009
No treatment
 0.60 ± 0.08  0.30 ± 0.10  0.90 ± 0.12 GSE09
 1.00 ± 0.21  0.42 ± 0.08  1.42 ± 0.26

As shown in Tables 7 and 8, the harvest of red pepper during packaging by GSE09 strain treatment ranged from about 20 to 60% of the number of red peppers in 2008 and 2009 compared to the untreated control during the first and second harvests. Pepper weight increased by 40-100%. The number of green peppers in the package increased by 20% and red peppers by 50% compared to the control in 2008 by GSE09 strain treatment. In 2009, the green peppers increased by 30% and the red peppers by 60% (Table 7). . In addition, the weight of the produced red pepper was increased by 70% in the green pepper and 100% in the red pepper by the GSE09 strain treatment in 2008 compared to the control, and increased by 70 and 40% in 2009 (Table 8 and Fig. 6). ).

Example 7 Verification of Pepper Maturation Promotion Effect of GSE09 Strains

Based on the observation that the red pepper yield was increased in the packaging and facility cultivation verification by the treatment of the GSE09 strain, the GSE09 strain showed the effect of promoting the maturation of the red pepper from the green pepper to the red pepper. To verify.

For this purpose, in order to verify that pepper maturation is promoted by volatile substances formed by GSE09 strain, GSE09 strain is cultured in 5cm diameter Petri dish NA medium and put into blue pepper in 12cm x 12cm square Petri dish and sealed for 16 hours. It was incubated for 7 days in an incubator at 28 ℃ under light and 8 hours dark. After 7 days of cultivation, the degree of maturation of green peppers into red peppers is% and 0 ~ 4 (0: blue peppers, 1: 0-10% red peppers, 2: 10-50% red peppers, 3: 50-90% red Pepper, 4: 90 to 100% red pepper) was evaluated in units. At this time, 10mM MgSO ₄ buffer was used as a control, blue pepper was used by surface sterilization with 1% NaOCl solution, and the experiment was repeated twice with 10 repetitions. The results are shown in Table 9. That is, Table 9 shows the results of verifying the pepper maturation promoting effect from blue pepper to red pepper by the antagonistic GSE09 strain.

process
 Chili maturity (%)  Pepper maturity index (0 ~ 4) No treatment
 2.5 ± 1.6  0.3 ± 0.2 GSE09
 81.0 ± 5.4  3.3 ± 0.1

As shown in Table 9, when the untreated control matured from blue pepper to red pepper, 2.5% after 7 days of treatment, when the GSE09 strain was treated, it matured to about 81% red pepper. When expressed as an index of the range (4) of the non-treated control was 0.3 when the GSE09 treatment was effective to promote the maturation to red pepper markedly to 3.3 (Fig. 7).

Example 8 Verification of Growth Inhibition Effect of Red Pepper Anthrax Pathogen by GSE09 Strains

Anthrax in pepper was reduced by GSE09 strain, and the yield of pepper was increased, based on the observation that the formation of complex volatiles promotes the maturation of red pepper into red pepper. We evaluated whether volatile substances secreted by GSE09 affect the growth of pepper anthrax.

For this purpose, a partition is formed in the middle of 9cm diameter Petri dish, so that the GSE09 strain and anthrax pathogens do not meet directly, but only Petri dishes (I-plate) can evaluate the effects of volatile substances formed by the GSE09 strain. It was. Half of this Petri dish was made with NA medium for culturing GSE09, and the other half was made with PDA (potato dextros agar, Difco, USA) for culturing pepper anthrax. The GSE09 strain prepared a bacterial suspension in the same manner as described above and smeared 200 μl onto the NA medium, and pepper anthrax was used to cork (5 mm in diameter) the mycelial edge of the culture medium in the PDA medium for 5 days. Mycelia plugs were made and incubated in PDA. Treated petri dishes were sealed with parafilm and cultured in a 28 ° C. incubator for evaluation.

The pepper anthracnose fungus used at this time was used to separate the pepper anthracnose disease generated during packaging and plant cultivation in 2008 and 2009. In order to isolate the pepper anthrax pathogen from the lesion, the mycelium was surface-sterilized with 1% NaOCl, followed by incubation with pure hyphae obtained by culturing in PDA containing streptomycin at a concentration of 50 μg / ml. Among the cultured pepper anthrax pathogens, three morphologically most common strains were selected, and DNA was isolated by CTAB method. After amplification by PCR, the nucleotide sequences were analyzed and all were identified as Colletotrichum acutatum. This was then used (FIG. 5, SEQ ID NOs: 4-6). After incubation, the length of the hyphae was measured, and after the measurement, the spores were harvested with 1 ml primary sterilized distilled water, and the amount of spores was measured using a hemocytometer under a 200-fold microscope, and then calculated for the mycelial growth. It was. In addition, the harvested spores were placed on a slide glass 20μl each, incubated for 24 hours, and then evaluated for the germination rate and the adherence formation rate to evaluate the growth of pepper anthrax. Table 10 shows the result. That is, Table 10 shows the results of verifying the inhibitory effect of growth (hyphae growth, spore formation amount, germination rate, adherence formation rate) of pepper anthrax pathogen by the antagonistic GSE09 strain.

process
Mycelial length (mm) Spore Formation (%) Germination Rate (%) Adhesion Formation Rate (%) No treatment
23.90 ± 0.78 5.46 ± 0.07 88.51 ± 1.50 86.19 ± 1.53 GSE09
20.02 ± 0.98 5.00 ± 0.07 69.26 ± 1.92 53.14 ± 3.76

As shown in Table 10, mycelial growth was reduced by 16%, spore formation rate was 8%, germination rate was 22%, and attachment group formation rate was decreased 38% by the volatile substances formed by GSE09. In particular, the germination rate and the formation rate of adhesion group were remarkable, and it can be seen that volatile substance of GSE09 influences the invasion mechanism after surviving on the surface of pepper of pepper anthrax pathogens to form the attachment group.

Institute of Agricultural Biotechnology KACC91368 20080317

<110> Korea University Industrial and Academic Collaboration Foundation <120> Composition for controlling anthracnose and promoting fruit yield          and ripening of plants comprising Flavobacterium sp. strain GSE09          and controlling method using the same <130> P11-091230-03 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 1395 <212> DNA <213> Flavobacterium sp. GSE09 <400> 1 ccagttttac cctaggcagc tccttgcggt caccgacttc aggcaccccc agcttccatg 60 gcttgacggg cggtgtgtac aaggcccggg aacgtattca ccggatcatg gctgatatcc 120 gattactagc gattccagct tcacggagtc gagttgcaga ctccgatccg aactgtgacc 180 ggctttatag attcgctccc cctcgcgggg tggctgctct ctgtaccggc cattgtagca 240 cgtgtgtagc ccaaggcgta agggccgtga tgatttgacg tcatccccac cttcctcaca 300 gtttgcactg gcagtcttgc tagagttccc gacatgactc gctggcaact aacaacaggg 360 gttgcgctcg ttataggact taacctgaca cctcacggca cgagctgacg acaaccatgc 420 agcaccttgt agaccgtctt gcgaaaggtc tgtttccaaa ccggtcagtc tgcatttaag 480 ccttggtaag gttcctcgcg tatcatcgaa ttaaaccaca tgctccaccg cttgtgcggg 540 cccccgtcaa ttcctttgag tttcaaactt gcgttcgtac tccccaggtg ggatacttat 600 cactttcgct tagccactga acttgcgccc aacagctagt atccatcgtt tacggcgtgg 660 actaccaggg tatctaatcc tgttcgctac ccacgctttc gtccatcagc gtcaatccat 720 tggtagcaac ctgccttcgc aattggtatt ccatgtaatc tctaagcatt tcaccgctac 780 actacatatt ctagttgctt cccaataatt caagtccagc agtatcaatg gccgttccac 840 cgttgagcga tgggctttca ccactgactt acaagaccgc ctacagaccc tttaaaccca 900 atgattccgg ataacgcttg gatcctccgt attaccgcgg ctgctggcac ggagttagcc 960 gatccttatt cttacggtac cgtcaagctc ctacacgtag gagggtttct tcccgtacaa 1020 aagcagttta caatccatag gaccgtcatc ctgcacgcgg catggctggt tcaggcttgc 1080 gcccattgac caatattcct cactgctgcc tcccgtagga gtctggtccg tgtctcagta 1140 ccagtgtggg ggatctccct ctcaggaccc ctacccatcg tagccttggt aagccgttac 1200 cttaccaact agctaatggg acgcatgctc atcttccacc gttgtgactt taatcatatc 1260 ccgatgccgg gtcatgatgc tatgaggtat taatccaaat ttctctgggc tatccctctg 1320 tggaaggcag attgcatacg cgttacgcac ccgtgcgccg gtctcaaggc ccgaaagcct 1380 ctacccctcg acttg 1395 <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 <210> 4 <211> 300 <212> DNA <213> Colletotrichum acutatum <400> 4 ggtggggaac ccacgccacg acgcggcgat ttcgacattt gacacgatct cgtactgacc 60 tcggtacagg caaaacattt ctggcgagca cggcctcgac agcaatggcg tgtatgtcac 120 ttgtcctcca gtgtggcttc ctcatggacc cagcagctaa tcataccaca ggtacaacgg 180 cacttccgag ctccagctcg agcgcatgag cgtctacttc aacgaagttt gttatcctag 240 tcccccagtg tgcaggcaat cctattgaca aatgctgacc ttctcaccca aataggcccc 300                                                                          300 <210> 5 <211> 419 <212> DNA <213> Colletotrichum acutatum <400> 5 ggtaaccaga ttggtgctgc cttctggtgc gtagccaacc gccaacgacg cggcgatttc 60 gacatttgac acgatctcgt actgacctcg gtacaggcaa aacatctctg gcgagcacgg 120 cctcgacagc aatggcgtgt atgtcacttg tcctccagtg tggcttcctc atggacccag 180 cagctaatca taccacaggt acaacggcac ttccgagctc cagctcgagc gcatgagcgt 240 ctacttcaac gaagtttgtt atcctagtcc cccagtgtgc aggcaatcct attgacgaat 300 gctgaccttc tcacccaacc aggcctccgg caacaagtac gttccccgcg ccgtcctcgt 360 cgacttggag cccggtacca tggacgccgt ccgtgccggt ccctttggcc agctttccg 419 <210> 6 <211> 430 <212> DNA <213> Colletotrichum acutatum <400> 6 cgccacgacg cggcgatttc gacatttgac acgatctcgt actgacctcg gtacaggcaa 60 aacatctctg gcgagcacgg cctcgacagc aatggcgtgt atgtcacttg tcctccagtg 120 tggcttcctc atggacccag cagctaatca taccacaggt acaacggcac ttccgagctc 180 cagctcgagc gcatgagcgt ctacttcaac gaagtttgtt atcctagtcc cccagtgtgc 240 aggcaatcct attgacaaat gctgaccttc tcacccaacc aggcctccgg caacaagtac 300 gttccccgcg ccgtcctcgt cgacttggag cccggtacca tggacgccgt ccgtgccggt 360 ccctttggcc agcttttccg ccccgacaac ttcgtctttg gccagtccgg tgccggcaac 420 aactgggcca 430

Claims (9)

Flavobacterium sp. A composition for controlling plant anthrax, comprising GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain. The composition of claim 1, wherein the plant anthrax is anthrax caused by a pathogen of Colletotrichum sp. Flavobacterium sp. A method of controlling plant anthrax, comprising treating a plant with GSE09 (Accession No. KACC 91368P) or a composition containing a metabolite produced from the strain. 4. The method of claim 3, wherein the plant anthrax is anthrax caused by a pathogen of Colletotrichum sp. The method for controlling plant anthrax according to claim 3 or 4, wherein the metabolite is a volatile substance. Flavobacterium sp. A microbial fertilizer composition for increasing yield of a plant containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain. Flavobacterium sp. A composition for promoting plant maturation containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain. Flavobacterium sp. A method of promoting plant maturation comprising treating a plant with a composition containing GSE09 (Accession No. KACC 91368P) or a metabolite produced from the strain. The method of claim 8, wherein the metabolite is a volatile substance and is treated with the harvested green peppers to promote maturation with red peppers.

Images