KR20140011918A - Composition for preventing or controlling bacterial brown blotch of mushroom using bacteriophages - Google Patents

Abstract

The present invention relates to novel bacteriophages specifically killing Pseudomonas tolaasii, a composition and a method for controlling brown blotch disease of mushroom using the same. The bacteriophage selected from the group consisting of Φ-6264(deposition number: KACC97018P), Φ-119(deposition number: KACC97016P), Φ-36(deposition number: KACC97012P), Φ-554(deposition number: KACC97017P), Φ-74(deposition number: KACC97015P), Φ-70(deposition number: KACC97014P), Φ-48(deposition number: KACC97013P), and Φ-69(deposition number: KACC97019P) has an ability to kill Pseudomonas tolaasii specifically, and the 8 species of bacteriophages are isolated from various kinds of Pseudomonas tolaasii mutants, sorted by host ranges, and then selected by toxicity against each mutant in a wide host range. The composition containing all the 8 species of bacteriophages has an effect in completely killing all kinds of bacteria causing brown blotch disease, thereby being useful as a composition for controlling plant diseases caused by Pseudomonas tolaasii.

Description

Composition for preventing or controlling bacterial brown blotch of mushroom using bacteriophages

The present invention relates to a novel bacteriophage having a specific killing ability against Pseudomonas tolaasii , a composition for controlling mushroom branch disease, and a method for controlling mushroom branch disease.

Pleurotus eryngii is one of the most profitable crops per unit area, and it is the main crop in mushroom cultivation in Korea, occupying the first place in cultivation area and second place in cultivation. Pleurotus eryngii is an excellent ingredient with excellent taste and aroma and rich in various nutrients. It is known to be effective in antitumor effect, tumor suppression, hypertension and diabetes.

Therefore, oyster mushroom is a favorite crop of many farms and is grown all over the country. Cultivation of Pleurotus eryngii has been done from early on, and high-quality mushrooms with high yield and selling price are obtained through the cultivation of fungi using rice straw, sawdust and cotton.

However, the production of oyster mushrooms has been decreasing for the last decade. There are many reasons for this, but one of the important reasons is the frequent occurrence of bacterial crab disease. Although cultivation of oyster mushrooms guarantees high profitability, it is very carefully done due to the occurrence of crab disease.

Calvary disease also causes serious damage to mushrooms, mushrooms, mushrooms, and enoki mushrooms, as well as soft mushrooms such as various edible mushrooms that have been cultivated recently. Calvary disease is called bacterial brown pattern disease, mushroom browning disease, bacterial calving disease, brown spot pattern disease in Korea, and it is known as brown blotch disease abroad and is doing great damage in cultivated mushrooms including mushrooms in the world.

Prevention and control methods for these diseases are not yet developed despite many studies. As a means to prevent gallbladder, it is made by sterilization of groundwater, fumigation of planter, vinyl mulching cultivation, bag cultivation, bottle cultivation method, but there is no countermeasure in case of onset. However, there are some examples of the use of antibiotics such as Agreptoburamycin to sterilize pathogens on bacteria that have been experimentally removed. However, mushrooms are fresh crops and are not allowed to use drugs such as antibiotics or fungicides. .

Recently, cultivation of oyster mushroom has been cultivated by means of bottle cultivation or bag cultivation method, which is relatively safe against bran disease. Through this, the outbreak of bran disease has been greatly reduced, but serious disease has been reported in large-scale cultivation plants. In addition, cultivation methods other than normal cultivation cannot produce high-quality mushrooms, and many farmers' incomes are low, and they are in a stage to consider whether to continue management. In other words, the farmhouse is a result of choosing the bottle cultivation and bag cultivation method with less disease in spite of the disadvantage in terms of price and productivity because the damage to pontoon cultivation due to Calvary disease is very serious. This is also related to the decrease in production since 2000. The oyster mushroom produced by the bottle cultivation method has thinner stems and less color compared to the conventional cultivation method, and the cost of delivery is one-third of the average cultivation product. This is serious.

Therefore, the development of eco-friendly and high-efficiency new technologies is urgently needed in cultivation for safe and excellent food production, and biological control techniques are also emerging as a countermeasure. As a biological control technique, a competitive microorganism, such as an antagonist, is isolated and cultured against a pathogen, and then treated directly with soil, seeds, or plants to compete with the pathogen or another strain that secretes bactericidal substances. However, the expected effect was not obtained. In addition, some methods using a bacteriophage virus, which attack and lyse bacteriophage, have been reported, but a single phage could not prevent various bacteriophage-induced mutants. Nevertheless, such biological control is considered to be very desirable in that there is no problem of environmental pollution, weakness and residual toxicity of organic synthetic pesticides.

On the other hand, bacteriophage (bacteriophage) is a virus that specifically lysates or lysates bacteria by infecting only bacteria, and infects only prokaryotic bacteria and viruses with high selectivity to bacterial hosts compared to viruses of animals or plants. Therefore, bacteriophages have been active in the late 2000s, when antibiotic-resistant bacteria are frequently reported as an alternative method for controlling bacteria instead of antibiotics.

Recently, attempts to control Listeria monocytogenes , a food-polluting bacterium, with bacteriophages rather than antibiotics have been approved by the US Food and Drug Administration (Federal Register, vol 71. No. 160 page 47729-47731). , 2006). In the case of plant bacterial diseases, attempts are being made to use bacteriophages instead of antibiotics because bacteriophages have shorter development and production periods and are much less expensive than antibiotics.

In addition, the use of bacteriophage in food has already been allowed in developed countries, and starting in 2006, the US FDA has approved the use of bacteriophage in various foods.

Therefore, by using high-toxic bacteriophage to lower the density and killing of pathogens in the flora and surrounding environment during plant cultivation can be a way to prevent the development of plant diseases. However, although bacteriophage therapy has many advantages as described above, it also has a disadvantage in that it does not exert any effects on various strain pathogens due to its very high host specificity.

Therefore, the present inventors have isolated a variety of pathogens that have a high host specificity that can cause gallbladder disease, classify them according to the bacteriophage host area, and then use a mixture of eight bacteriophage screens that show toxicity in a wide host range for each strain. As a result, the present invention was completed by confirming the effect of completely killing all the causative bacteria.

Korean Patent Publication No. 10-2011-0034178 Korean Patent Publication No. 10-2012-0073724

It is therefore an object of the present invention to provide novel bacteriophages with specific killing ability against Pseudomonas tolaasii .

In addition, another object of the present invention is an environmentally friendly and effective Pseudomonas It is to provide a composition for controlling the tolaasii ).

In addition, another object of the present invention is an environmentally friendly and effective Pseudomonas Tolaasii ) to provide a control method that can control.

In order to achieve the object of the present invention as described above, the present invention Pseudomonas Pseudomonas having a specific killing capability for tolaasii), Φ-6264 (Accession No: KACC97018P), Φ-119 (accession number: KACC97016P), Φ-36 (Accession No: KACC97012P), Φ-554 (accession number: KACC97017P) At least one bacteriophage selected from the group consisting of Φ-74 (Accession Number: KACC97015P), Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P). To provide.

In addition, the present invention is Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number : KACC97015P), Φ-70 (Accession No: KACC97014P), Φ-48 (Accession No .: KACC97013P) and Φ-69 (Accession No: Pseudomonas toll Lashio comprising a bacteriophage of 8 consisting KACC97019P) species as an active ingredient (Pseudomonas tolaasii ) Provides a composition for controlling induced plant diseases.

In one embodiment of the present invention, the plant disease may be mushroom brown blotch disease.

In one embodiment of the present invention, the mushroom may be selected from the group consisting of oyster mushroom, mushroom mushroom, matsutake mushroom and enoki mushroom.

In another aspect, the present invention Pseudomonas toll Lashio (Pseudomonas including the step of processing the composition to a plant tolaasii ) Provides a method of controlling the induced plant diseases.

Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number: Any one or more bacteriophages selected from the group consisting of KACC97015P), Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P) are Pseudomonas tolaasii As a novel bacteriophage having specific killing ability, in particular, the eight species were isolated from various kinds of Pseudomonas tolasi strains, which were classified according to the bacteriophage host region, and then showed toxicity in a wide host range for each strain. As a screening method, the composition of the present invention comprising all eight of these bacteriophages has the effect of completely killing all the causative agents causing Pseudomonas Pseudomonas tolaasii ) It can be useful for the control of inducing plant diseases.

Figure 1 shows the phylogenetic tree through the sequencing of the representative strains (P1a-6246, P1β-36, P1γ-69) of the Pseudomonas tolasi bacteria by group (P1a, P1β, P1γ).
Figure 2 is to test whether the formation of the branch through the mushroom depression test to confirm the pathogenicity of Pseudomonas Tolasi bacteria by group (P1a, P1β, P1γ) strains.
Figure 3 shows the hemolytic activity of the Pseudomonas tolassi bacteria group by group (P1a, P1β, P1γ) strains.
Figure 4 by mixing all 20 strains of Pseudomonas Tolasi bacteria group (P1a, P1β, P1γ) by dropping on the mushroom surface and causing the galvanic, control effect of the eight bacteriophage mixed solution of the present invention treatment The experiment was conducted by mushroom tissue depression assay.

The present invention relates to a bacteriophage having a specific killing ability against Pseudomonas tolaasii , and more particularly, to a novel bacteriophage capable of killing Pseudomonas tolaci bacteria that cause crab disease in plants. It is characterized by providing a microbial material that can be usefully used for the prevention (control) of gallbladder disease caused by Pseudomonas tolasi by using this microorganism.

Bacteriophage discovered plant and animal viruses, and in 1915 Twart observed the lytic phenomena of Staphylococcus aureus, and in 1917, the Herler of Pasteur Institute observed the same phenomenon in the bacterium. Found out that Since then, a virus that infects bacteria has been named bacteriophage in the sense of "Phage" and is simply called "phage."

The phage is classified into a lytic and a phage phage, and a virulent phage and a phosphorus cycle are repeated as a temperate phage. The lysogenic phage proliferates when the host bacterium proliferates by inserting its own gene into the host bacterium gene. This long-term condition persists as long as the host bacterium is in good environmental conditions, and when the host bacterium's growing environment deteriorates, the phage gene in the host bacterium is converted into a lysis cycle, creating new phages to dissolve the host bacterium and release it out of the host cell. .

In the case of lytic phage, the gene is actively expressed after infection and 60-100 new phages are made after about 30 minutes, and they break out of the cell membranes of the bacteria and lysate. The phage growth rate is much higher than that of bacteria. It is so fast that eventually all bacteria are infected and killed by lytic phage. Therefore, toxic lytic phage may be an important factor limiting the growth of bacteria.

These phages were limited in their use during the 1910s to 1930s, when they were found to be unsolvable at the time, such as host specificity problems and endotoxins and exotoxins of host bacteria in the manufacture of phages for therapeutic purposes. . These days, the penicillins discovered by Flaming in 1929 and Streptomycin in 1943 by Works only had broad antibacterial effects against phages, which were effective against multiple infections with a single drug, and because there were no resistant bacteria at first. Chemotherapeutic agents have become widely used.

On the other hand, phage method, which is uncertain in effect, has gradually disappeared, and (1) the need for various phages with different infectivity due to the sensitivity of the phage to the host, (2) the phagocytosis of phage is difficult to occur in animals, And (3) the phage law was declared invalid by the WHO in the 1950s because phage-resistant bacteria existed in the same bacterial population, and only in Eastern Europe and the former Soviet Union.

Nevertheless, some scholars continued to study phage treatments and used them to treat actual diseases. Phage research in Eastern Europe has been known in recent years, and the interest in phage therapy has increased again due to the depletion of new antibiotic resources and the emergence of antibiotic resistant bacteria that are resistant to antibiotics. In addition, in the case of plant bacterial diseases, attempts are being made to use bacteriophages instead of antibiotics because bacteriophages have shorter development or production periods and are much less expensive than antibiotics. In addition, the US ΦDA authorizes the use of bacteriophages in various foods, and the use of bacteriophages has been confirmed to be scientifically and hygienic.

Therefore, the present inventors use bacteriophage, which is a bacterial virus of Pseudomonas tolaasii as a host bacterium, as a host bacterium, to remove tollosis bacteria present in mushroom cultivators or mushroom individuals, or to reduce the density of the bacterium. It was intended to prepare a way to suppress the

The bacteriophage of the present invention was obtained by using agar bilayer method to obtain a sample of the sewage stream of Cheongju.

In detail, each stream was inoculated with sewage fluid and host bacteria in a 50 soft agar tube at a ratio of 1: 2, and then poured into hard agar medium that had been hardened beforehand to prepare an agar double layer (hard agar layer was 1.5% agar). And soft agar layers contain 0.7% agar respectively). Agar bilayer medium was cultured, one bacteriophage plaque was selected using a sterile toothpick for amplification of bacteriophage, isolated, suspended in PAF medium and co-inoculated with host bacteria and incubated overnight. At this time, the host bacterium is killed by lytic phage and the rapid growth of phage. NaCl was added to the obtained lysate to 10%, incubated at 0 for 1 hour, and centrifuged at 8,000 rpm for 10 minutes. After the addition of polyethylene glycol 6000 to 10% in the supernatant obtained and incubated. Then, the bacteriophage precipitated by ultra-high speed centrifugation was resuspended in the bacteriophage buffer and purified.

The isolated bacteriophages were obtained from 41 species of 20 strains of Streptococcus spp., And the number of phages isolated from each host bacteria is shown in Table 3 below. In addition, they carried out bacteriophage typing to examine the infectivity of 20 host strains, and again selected 8 bacteriophages showing toxicity in a broad host range for each strain. The inventors have arbitrarily named each of these as Φ-6264, Φ-119, Φ-36, Φ-554, Φ-74, Φ-70, Φ-48 and Φ-69, and the novel bacteriophage of the present invention Deposited to KACC as of April 11, 2013, the deposit numbers Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number: KACC97015P), Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P) Given (see Tables 4 and 5 below).

The present invention also provides Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number : KACC97015P), Φ-70 (Accession No: KACC97014P), Φ-48 (Accession No .: KACC97013P) and Φ-69 (Accession No: Pseudomonas toll Lashio comprising a bacteriophage of 8 consisting KACC97019P) species as an active ingredient (Pseudomonas tolaasii ) Provides a composition for controlling induced plant diseases.

Of the eight novel bacteriophages of the present invention, Φ-6264 can control all P1a group mutants among a total of 20 Pseudomonas tolasi strains (P1a, P1β, P1γ) (see Table 2); A combination of Φ-119, Φ-36, Φ-554-2, Φ-74, Φ-70, and Φ-48 may control the P1β group; Since Φ-69 can control the P1γ group, consequently, when a combination of the above eight kinds of bacteriophages is treated, there is an effect capable of controlling all strains of Pseudomonas tolasi Streptococcus bacteria discovered to date.

In the following experiment of the present invention by mixing 20 species of Pseudomonas tolasis bacteria and dropping on the surface of the mushrooms, causing the galvanic, by confirming that the treatment of the mixture of the eight bacteriophages of the present invention to completely suppress the formation of the galvanic formation, This was demonstrated experimentally (see FIG. 4).

Therefore, Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number: KACC97015P) The composition of the present invention comprising 8 bacteriophages consisting of Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P) as an active ingredient is Pseudomonas tolasi ( Pseudomonas tolaasii ) can effectively control or prevent induced plant diseases.

Pseudomonas tolaasii ( Pseudomonas tolaasii ) is a pathogenic bacterium that causes branching in plants, especially when growing mushrooms, it causes infection by groundwater or air and causes branching disease, and the occurrence of branching disease destroys young mushrooms and dissolves or forms branching in fruiting bodies. Eliminates the merchandise of.

The composition according to the present invention can be used for the purpose of controlling the plant diseases caused by Pseudomonas tolaasii ( Pseudomonas tolaasii ), and thus the plant diseases of the present invention may be mushroom bran disease.

The mushroom may be selected from the group consisting of oyster mushroom, mushroom mushroom, matsutake mushroom and enoki mushroom, but is not limited thereto.

The composition of the present invention is an active ingredient described above for the purpose of stable formulation suitable for use in actual packaging (Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P) ), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number: KACC97015P), Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P) In addition to)) in addition to the surfactants for foods that can neutralize the toxin secreted by the gallbladder bacteria can be prepared in the form of a hydrating agent, granular water soluble, liquid hydrated, liquid, water-soluble, water-soluble granules or encapsulating agent have.

The food surfactant may be a polyglycerin fatty acid ester compound or a sucrose fatty acid ester compound; For example, the polyglycerol fatty acid ester compound is a diglycerin ester or decaglycerol monomyristate, and the sucrose fatty acid ester compound is sucrose myristate ester and sucrose stearate. Sucrosestearate ester, sucrose laurate ester, or sucrose palmitate ester.

According to a preferred embodiment of the present invention, the concentration of the food surfactant may be 1 μM to 10 mM, more preferably 5 μM to 1 mM, and most preferably 10 μM to 100 μM.

The present invention also provides Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number : A composition of the present invention containing eight kinds of bacteriophages consisting of KACC97015P), Φ-70 (Accession Number: KACC97014P), Φ-48 (Accession Number: KACC97013P), and Φ-69 (Accession Number: KACC97019P) as an active ingredient. Pseudomonas toll Lashio comprising the step of processing the plant (Pseudomonas tolaasii ) Provides a method of controlling the induced plant diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Crab disease  Pseudomonas causing Tolasi ( Pseudomonas tolaasii Isolation of bacteria

Pseudomonas tolasis was isolated from the mushrooms infected with crab disease in mushroom cultivation farms with crab disease in Chungcheong and Gyeongsang areas. First, cut about 1 cm ² from the fresh tissue of Oyster mushrooms infected with Calvary disease with a sterile knife, put it in 10 mL of sterile water, roughly grind it with a mixer, and then use the sterile distilled water to prepare 10 ^-3 -10 ^-10 . Diluted. Dilute about 200 μL of Pseudomonas Agar F (PAF; (10 g Bacto-peptone 10 g, Bacto-tryptone 10 g, K2HPO4 1.5 g, MgSO4 1.5 g, glycerol 10 mL, agar 15 g per liter) on a plate medium, and then 25 Bacteria were obtained by culturing for 1 day at ° C. Fluorescent strains were selected from various types of bacteria grown in the medium using ultraviolet rays, etc. The pathogenicity of the selected strains was evaluated by white sedimentation generation assay and mushroom tissue depression assay. The isolated bacteria were suspended in 1.5 mL of PAF bacterial storage medium containing 20% glycerol and stored at -80 ° C.

< Example  2>

Pathogenic Pseudomonas Tolacitic  Genetic Analysis and Identification

In order to measure the cytotoxicity of about 100 species of the strain isolated in Example 1, a mushroom tissue depression test (pitting test) using a mushroom mushroom was carried out, and 40 strains having high pathogenicity were isolated among them. As a result of performing sequencing of the 16S rRNA gene for these, and using the gene sequence to identify strains using GenBank data, 20 kinds of bacteria were identified as Pseudomonas tolasis bacteria as shown in Table 2 below. Results of subdividing them based on the phylogenetic tree analysis (see FIG. 1) Among the strains belonging to the P1a group belong to the same classification as that of the Tolaxi strain 6264, which is used as a standard pathogen in our laboratory, and has a record of many accidents. The roots were very close to the strains. One strain belonged to the P1γ classification and was separated from the 6264 strain and the phylogenetic tree, but it was also classified as tolasis. Figure 2 is a phylogenetic taxonomy of gene sequences prepared using GenBank's database. Pseudomonas tolasi P1a, P1β, and P1γ are located close to each other in the phylogenetic tree, and various characteristics are considered to be very similar.

Nucleotide Sequences of 16S rRNA Gene of Representative Pseudomonas Tolasis Identified in the Invention Pseudomonas tolasi bacteria of the present invention SEQ ID NO: Pseudomonas tolaasii 6264 (P1α) SEQ ID NO: 1 Pseudomonas tolaasii 36 (P1β) SEQ ID NO: 2 Pseudomonas tolaasii 69 (P1γ) SEQ ID NO: 3

Subclassification on the basis of the genetic lineage of 20 species of Pseudomonas tolasis identified in the present invention Subdivision Pseudomonas tolasi strain P1α 6264, 32, 87, 101, 125, 152 P1β 36, 37, 46, 47, 48, 54, 67, 70, 71, 74, 93, 119, 554 P1γ 69

< Example  3>

Branch disease To the causative strain  Mushroom tissue depression test Hemolytic activity  Measure

In order to confirm the pathogenicity of the Pseudomonas tolaci pathogens belonging to the three subclasses in Example 2, mushroom tissue depression assay using the fruiting body of the mushroom mushroom was performed.

10 μL of P1a (6264), P1β (36), and P1γ (69) cultures (10 ⁸ -10 ⁹ cfu / ml) of Pseudomonas tolasi strains were absorbed onto horizontal sections of fresh mushrooms of mushrooms. In a square dish containing filter paper soaked in sterile water, sealed and incubated at 25 ° C. for 12-15 hours, discoloration and depression of the drop site were observed.

As a result, as shown in Figure 2, all three of these species showed that the formation of the galvan, which means that they have a high pathogenicity.

In addition, Pseudomonas tolasi bacteria secrete the peptide tolasin (tolaasin) to destroy mushroom cells, and the toxicity of tolacin toxin can be measured by the hemolytic activity that destroys red blood cells.

The hemolytic activity of tolacin was measured using red blood cells of rats. Erythrocytes used for hemolytic activity were diluted 10-fold with sterile HEPES-buffered saline (HBS; 150 mM NaCl, 5 mM KCl, 5 mM HEPES, 1 mM MgSO4, pH 7.4) buffer immediately before use. Add to 10%. Toxin was used for the measurement of hemolytic activity by 100 μL of each bacterial culture supernatant. Hemolytic activity was measured by absorbance change at 600 nm using a spectrophotometer.

As a result, as shown in Figure 3, the representative Streptococcus bacteria Pseudomonas Tolasi 6264 (P1a) and 69 (P1γ) all showed more than 90% hemolytic activity 30 minutes after incubation, Pseudomonas Tolasi 36 (P1β) and all P1β strains showed no or very low hemolytic activity. This shows that the P1a and P1γ strains secrete toxic peptides such as tolasin, but the P1β strains do not destroy red blood cells. However, the P1β strains may also cause cravings in mushroom tissues, confirming that they have different pathogenesis mechanisms from P1a and P1γ strains.

< Example  4>

Isolation of Bacteriophage and Host  Research

Bacteriophages were sampled from the sewage streams of Cheongju-si and separated using the agar bilayer method. At this time, the hard agar layer contains 1.5% agar and the soft agar layer contains 0.7% agar, respectively. Sewage and host bacteria in each stream were inoculated in 50 soft agar tubes at a ratio of 1: 2, and then poured into hard agar medium that had been hardened beforehand to create an agar bilayer. Agar bilayer media was incubated at 25 for 15 hours. For bacteriophage amplification, one bacteriophage plaque was selected using a sterile toothpick, isolated, suspended in 5 ml of PAF medium and co-inoculated with 1% of the host bacteria and incubated overnight. NaCl was added to the obtained lysate to 10%, incubated at 0 for 1 hour, and centrifuged at 8,000 rpm for 10 minutes. After adding polyethylene glycol 6000 to 10% in the supernatant obtained, and incubated at 0 for 1 hour. The bacteriophage precipitated by PEG 6000 was then centrifuged at 8,000 rpm for 10 minutes in bacteriophage buffer (50 mM Tris-HCl, pH 7.3, 150 mM NaCl, 20 mM, NH4Cl, 10 mM MgCl2, 1 mM CaCl2, 0.2%). resuspended and stored in gelatin).

Table 3 below lists the number of strain-specific bacteriophages isolated from Pseudomonas tolasi strains, P1a, P1β, and P1γ in parentheses. Among the 41 bacteriophages isolated, 31 bacteriophages of Pseudomonas tolasi P1a host bacteria were isolated, and 8 bacteriophages of Pseudomonas tolasi P1α were isolated, and two bacteriophages comprising Pseudomonas tolaci P1γ as host bacteria were isolated. Although it was difficult to isolate bacteriophages from the P1β group as a host among the P1β group among the pathogens that caused the accident, it was successful to isolate bacteriophages for all P1β group pathogens, but it was safe to isolate additional bacteriophages from P1β group strains. Required for control.

Number of Bacteriophage Specific to Each Strain of 20 Pseudomonas Tolacis Identified in the Invention Subdivision Pseudomonas tolasi strains (number of bacteriophages that are hosts) P1α 6264 (20), 32 (6), 87 (2), 101 (3), 125 (0), 152 (0) P1β 36 (1), 37 (1), 48 (1), 70 (1), 74 (1), 119 (1), 554 (2), 46 (0), 47 (0), 54 (0), 67 (0), 71 (0), 93 (0) P1γ 69 (2)

< Example  5>

Classification of Bacteriophage by Host

If you want to suppress the early stage of the disease caused by the onset of mushroom farms or spray the bacteriophage mixture as a preventive measure, you must separate the bacteriophages specific to each strain and make a mixed solution to control all the strains that may cause the disease. . Because bacteriophages are highly specific to the host, they cannot attack other strains within the same species. However, if Pseudomonas tolassis is genetically closely related to the cell wall structure, the bacteriophage may attack several strains simultaneously. Bacteriophage typing was performed to reveal the range of host sites of bacteriophages.

Pseudomonas tolaasii ) There are 41 bacteriophages isolated from P1 type strains including 6264. These were subjected to bacteriophage typing to investigate the infectivity of 20 host bacteria.

As a result, as shown in Table 4 below, the pathogens of the P1a group were classified into three classes according to their sensitivity to bacteriophages. In the case of Φ-6264 bacteriophages, all three P1a subclass strains were attacked. In case of Φ-32, two of three strains could be attacked, and in case of Φ-87, only one of 6264 strains was attacked. Among the bacteriophages isolated using the P1a strain as a host, no bacteriophage attacking the P1β strain or the P1γ strain was found. Therefore, the pathogens of the P1a group could be controlled by one bacteriophage of Φ-6264 type.

Meanwhile, in the bacteriophage host station analysis of the P1β group, six types of bacteriophages that do not overlap with 13 pathogens belonging to this group were used. In Table 3, the P1β strains were divided into 10 types according to the host regions of the bacteriophages, and none of the 6 types of bacteriophages attacked all of these 10 types of host bacteria. In order to control all P1β group strains, it was determined that 6 bacteriophages were used.

In addition, P1 type selected Φ-69 which is highly toxic among two bacteriophages isolated as one strain. Among the bacteriophages isolated, no bacteriophage capable of attacking beyond the boundaries of the P1a, P1β and P1γ groups was not found.

In conclusion, a total of 20 Pseudomonas tolasi strains that have caused crab disease have been divided into three subclasses, P1a, P1β, and P1γ by genetic analysis, and the bacteriophage host regions were analyzed to control all strains. One bacteriophage was required for the P1a group, six bacteriophages for the P1β group, and one bacteriophage for the P1γ strain. Judging from these results, a total of eight bacteriophage treatments could be used to control all strains of Pseudomonas tolaci bacillus.

Therefore, the present inventors have identified 8 types of isolated bacteriophages having specific killing ability against Pseudomonas tolaasii , Φ-6264, Φ-119, Φ-36, Φ-554, Φ-74, and Φ-70. , Φ-48 and Φ-69, respectively, and the novel bacteriophage of the present invention was deposited on April 11, 2013 to the Institute of Agricultural Biotechnology (KACC), the deposit number was given as shown in Table 5.

A total of eight bacteriophages with specific killing ability against Pseudomonas tolaci Strain name Accession number φ-6264 KACC97018P φ-119 KACC97016P φ-36 KACC97012P φ-554 KACC97017P φ-74 KACC97015P φ-70 KACC97014P φ-48 KACC97013P φ-69 KACC97019P

< Experimental Example  1>

Eight kinds of bacteriophage mixed solution of the present invention Branch disease  Control experiment

In order to confirm the results obtained above, Pseudomonas tolasi strains were combined and the effect of the bacteriophage mixture was measured using the mushroom tissue depression assay.

As a result, as shown in Fig. 4, when 20 species of Pseudomonas tolacis were mixed and dropped onto the surface of the mushrooms and caused cravings, P1a-specific bacteriophages, P1β-specific bacteriophages, and P1γ-specific bacteriophages were treated separately. In one case, the formation of gallbladder could not be prevented. However, it was shown that the treatment of the mixed bacteriophage of the present invention completely suppressed the formation of the branch. This confirmed that 8 minimum bacteriophage combinations could completely suppress all the strains of the disease.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Agricultural Biotechnology Research Institute KACC97012P 20130411 Agricultural Biotechnology Research Institute KACC97013P 20130411 Agricultural Biotechnology Research Institute KACC97014P 20130411 Agricultural Biotechnology Research Institute KACC97015P 20130411 Agricultural Biotechnology Research Institute KACC97016P 20130411 Agricultural Biotechnology Research Institute KACC97017P 20130411 Agricultural Biotechnology Research Institute KACC97018P 20130411 Agricultural Biotechnology Research Institute KACC97019P 20130411

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> Composition for Preventing or controlling bacterial brown blotch          of mushroom using bacteriophages <130> PN1207-367 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1501 <212> DNA Pseudomonas tolaassi 6264 (P1alpha) 16S rRNA gene nucleotide sequence <400> 1 gttcaggctc agattgaacg ctggcggcag gcctaacaca tgcaagtcga gcggtagaga 60 gaagcttgct tctcttgaga gcggcggacg ggtgagtaat gcctaggaat ctgcctggta 120 gtgggggata acgtccggaa acggacgcta ataccgcata cgtcctacgg gagaaagcag 180 gggaccttcg ggccttgcgc tatcagatga gcctaggtcg gattagctag ttggtgaggt 240 aatggctcac caaggcgacg atccgtaact ggtctgagag gatgatcagt cacactggaa 300 ctgagacacg gtccagactc ctacgggagg cagcagtggg gaatattgga caatgggcga 360 aagcctgatc cagccatgcc gcgtgtgtga agaaggtctt cggattgtaa agcactttaa 420 gttgggagga agggcagttg cctaatacgt aactgttttg acgttaccga cagaataagc 480 accggctaac tctgtgccag cagccgcggt aatacagagg gtgcaagcgt taatcggaat 540 tactgggcgt aaagcgcgcg taggtggttt gttaagttgg atgtgaaatc cccgggctca 600 acctgggaac tgcattcaaa actgactgac tagagtatgg tagagggtgg tggaatttcc 660 tgtgtagcgg tgaaatgcgt agatatagga aggaacacca gtggcgaagg cgaccacctg 720 gactaatact gacactgagg tgcgaaagcg tggggagcaa acaggattag ataccctggt 780 agtccacgcc gtaaacgatg tcaactagcc gttggaagcc ttgagctttt agtggcgcag 840 ctaacgcatt aagttgaccg cctggggagt acggccgcaa ggttaaaact caaatgaatt 900 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgaagaacct 960 taccaggcct tgacatccaa tgaactttcc agagatggat tggtgccttc gggaacattg 1020 agacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgta 1080 acgagcgcaa cccttgtcct tagttaccag cacgttatgg tgggcactct aaggagactg 1140 ccggtgacaa accggaggaa ggtggggatg acgtcaagtc atcatggccc ttacggcctg 1200 ggctacacac gtgctacaat ggtcggtaca gagggttgcc aagccgcgag gtggagctaa 1260 tcccataaaa ccgatcgtag tccggatcgc agtctgcaac tcgactgcgt gaagtcggaa 1320 tcgctagtaa tcgcgaatca gaatgtcgcg gtgaatacgt tcccgggcct tgtacacacc 1380 gcccgtcaca ccatggggag tgggttgcac cagaagtagc tagtctaacc ttcgggagga 1440 cggttaccac ggtgtgattc atgactgggg tgagtcagng gggaaggcgg aacaaaaaaa 1500 a 1501 <210> 2 <211> 1512 <212> DNA <213> Pseudomonas tolaassi 36 (P1beta) 16S rRNA gene nucleotide sequence <400> 2 gtaagatttt ttttttcagg ctcagattga acgctggcgg caggcctaac acatgcaagt 60 cgagcggtag agagaagctt gcttctcttg agagcggcgg acgggtgagt aatgcctagg 120 aatctgcctg gtagtggggg ataacgttcg gaaacggacg ctaataccgc atacgtccta 180 cgggagaaag caggggacct tcgggccttg cgctatcaga tgagcctagg tcggattagc 240 tagttggtgg ggtaatggct caccaaggcg acgatccgta actggtctga gaggatgatc 300 agtcacactg gaactgagac acggtccaga ctcctacggg aggcagcagt ggggaatatt 360 ggacaatggg cgaaagcctg atccagccat gccgcgtgtg tgaagaaggt cttcggattg 420 taaagcactt taagttggga ggaagggcag ttgcctaata cgtaactgtt ttgacgttac 480 cgacagaata agcaccggct aactctgtgc cagcagccgc ggtaatacag agggtgcaag 540 cgttaatcgg aattactggg cgtaaagcgc gcgtaggtgg tttgttaagt tggatgtgaa 600 atccccgggc tcaacctggg aactgcattc aaaactgact gactagagta tggtagaggg 660 tggtggaatt tcctgtgtag cggtgaaatg cgtagatata ggaaggaaca ccagtggcga 720 aggcgaccac ctggactaat actgacactg aggtgcgaaa gcgtggggag caaacaggat 780 tagataccct ggtagtccac gccgtaaacg atgtcaacta gccgttggaa gccttgagct 840 tttagtggcg cagctaacgc attaagttga ccgcctgggg agtacggccg caaggttaaa 900 actcaaatga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 960 acgcgaagaa ccttaccagg ccttgacatc caatgaactt tccagagatg gattggtgcc 1020 ttcgggaaca ttgagacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gtaacgagcg caacccttgt ccttagttac cagcacgtta tggtgggcac 1140 tctaaggaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa gtcatcatgg 1200 cccttacggc ctgggctaca cacgtgctac aatggtcggt acagagggtt gccaagccgc 1260 gaggtggagc taatcccata aaaccgatcg tagtccggat cgcagtctgc aactcgactg 1320 cgtgaagtcg gaatcgctag taatcgcgaa tcagaatgtc gcggtgaata cgttcccggg 1380 ccttgtacac accgcccgtc acaccatggg gagtgggttg caccagaagt agctagtcta 1440 accttcggga ggacggttac cacggtgtga ttcatgactg gggtgagtct aaggggaagg 1500 agcccacaaa aa 1512 <210> 3 <211> 1511 <212> DNA Pseudomonas tolaassi 69 (P1gamma) 16S rRNA gene nucleotide sequence <400> 3 tttttttttg cggctccttc ctctagctcc acccagtcat gatccacacg gtgtacccgt 60 cctcccgaag gttagactag ctacttctgg tgcaacccca ctcccatggt gtgacgggcg 120 gtgtgtacaa ggccccggaa cgtattcacc gcgacatttt gattcgcgat tactagcgat 180 tccgacttca cgcagtcgag ttgcagactg cgatccggac tacgatcggt ttatgggatt 240 agctccacct cgcggcttgg caaccctctg taccgaccat tgtagcacgt gtgtagccca 300 ggccgtaagg gccatgatga cttgacgtca tccccacctt cctccggttt gtcaccggca 360 gtctccttag agtgcccacc ataacgtgct ggtaactaag gacaagggtt gcgctcgtta 420 cgggacttaa cccaacatct cacgacacga gctgacgaca gccatgcagc acctgtctca 480 atgttcccga aggcaccaat ctatctctag aaagttcatt ggatgtcaag gcctggtaag 540 gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg cccccgtcaa 600 ttcatttgag ttttaacctt gcggccgtac tccccaggcg gtcaacttaa tgcgttagct 660 gcgccactaa aagctcaagg cttccaacgg ctagttgaca tcgtttacgg cgtggactac 720 cagggtatct aatcctgttt gctccccacg ctttcgcacc tcagtgtcag tatcagtcca 780 ggtggtcgcc ttcgccactg gtgttccttc ctatatctac gcatttcacc gctacacagg 840 aaattccacc accctctacc atactctagt cagtcagttt tgaatgcagt tcccaggttg 900 agcccgggga tttcacatcc aacttaacaa accacctacg cgcgctttac gcccagtaat 960 tccgattaac gcttgcaccc tctgtattac cgcggctgct ggcacagagt tagccggtgc 1020 ttattctgtc ggtaacgtca aaacactaac gtattaggtt aatgcccttc ctcccaactt 1080 aaagtgcttt acaatccgaa gaccttcttc acacacgcgg catggctgga tcaggctttc 1140 gcccattgtc caatattccc cactgctgcc tcccgtagga gtctggaccg tgtctcagtt 1200 ccagtgtgac tgatcatcct ctcagaccag ttacggatcg tcgccttggt gagccattac 1260 ctcaccaact agctaatccg acctaggctc atctgatagc gcaaggcccg aaggtcccct 1320 gctttctccc gtaggacgta tgcggtatta gcgttcgttt ccgaacgtta tcccccacta 1380 ccaggcagat tcctaggcat tactcacccg tccgccgctc tcaagagaag caagcttctc 1440 tctaccgctc gacttgcatg tgttaggcct gccgccagcg ttcaatctga gcctgtacaa 1500 aaaacctaca t 1511

Claims (5)

Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (with specific killing ability against Pseudomonas tolaasii ) Accession number: KACC97017P), Φ-74 (Accession number: KACC97015P), Φ-70 (Accession number: KACC97014P), Φ-48 (Accession number: KACC97013P), and Φ-69 (Accession number: KACC97019P) Any one or more bacteriophages. Φ-6264 (Accession Number: KACC97018P), Φ-119 (Accession Number: KACC97016P), Φ-36 (Accession Number: KACC97012P), Φ-554 (Accession Number: KACC97017P), Φ-74 (Accession Number: KACC97015P), Φ-70 (Accession No: KACC97014P), Φ-48 (Accession No: KACC97013P) and Φ-69: Pseudomonas toll Lashio containing bacteriophage consisting of 8 (Accession No KACC97019P) species as an active ingredient (Pseudomonas tolaasii ) Composition for the control of inducible plant diseases. 3. The method of claim 2,
The plant disease is characterized by a mushroom brown blotch disease ( Pseudomonas) tolaasii ) Composition for the control of inducible plant diseases. The method of claim 3,
The mushrooms are Pseudomonas toll Lashio (Pseudomonas, characterized in that is selected from the group consisting of oyster mushroom, Agaricus bisporus, Pleurotus eryngii and enoki tolaasii ) Composition for the control of inducible plant diseases. The Pseudomonas toll Lashio to the composition of claim 2 comprising the step of treating the plant (Pseudomonas tolaasii ) A method for controlling induced plant diseases.

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