KR101185843B1 - Formulation for controlling vegetable soft rot containing bacteriophage PPP1 - Google Patents

Abstract

The present invention relates to a preparation for control of vegetable softening disease comprising bacteriophage PPP1, and the bacteriophage PPP1 of the present invention has a control effect on the purifying disease of fresh vegetables, so that it can be used as a microbial pesticide for the control of vegetable vegetables, and can be used instead of antibiotics. This may reduce the likelihood of antibiotic resistant bacteria and increase environmental safety.

Description

Formulation for controlling vegetable soft rot containing bacteriophage PPP1 including bacteriophage PPC1

The present invention relates to a vegetable soft drink control formulation comprising bacteriophage PPP1.

Innocent disease refers to the disease of a decaying plant that has a peculiar smell and is often called soft disease. When the disease occurs, the pectin in the middle layer of the cell wall melts, and at first the water becomes visible but gradually recedes and rots and becomes mushy. Softworms are a common disease in vegetables, but also in tomatoes, sweet potatoes, and potatoes.

Peck tobak causing the rot of vegetables, including cabbage Te Karo Solarium Sat borum (Pectobacterium carotovorum subsp. carotovorum ) grows at a temperature of 26 ~ 28 ℃, but unlike other plant pathogens, it grows well at about 36 ℃, which is the biggest obstacle to summer cabbage production.

Bacillus bacteria can invade and infect almost any plant, including cabbage, and once invaded, they move to other vegetables around them to spread the outbreak. Recently, the spread of well-being culture has led to a sharp increase in the consumption of fresh vegetables. In particular, the fresh vegetables displayed on the shelves continue to supply moisture to maintain freshness, even through the moisture spreads.

Once a disease occurs on cabbage or fresh vegetables, it is very difficult to control because consumers do not cook cabbage or vegetables, and most of them eat it as it is. In addition, the pathogen Pectobacterium ( Cartoborum) carotovorum subsp. carotovorum ) is able to survive in soil for a relatively long time, and pesticide control effects are generally known to be low.

Therefore, it is urgent to develop an eco-friendly and effective control technique. Until now, disease control of various crops has largely depended on the development of resistant varieties and chemical agents by using pesticides. Among them, the development of resistant varieties is subject to a lot of technical, economic and time constraints, and even when selecting a new resistant varieties, the ability to adapt quickly to the fast breeding bacteria grows the ability to lose the resistance. In addition, antibiotics, known as chemical agents, also work on animal bacteria, causing problems that can encourage antibiotic-resistant bacteria.

As a countermeasure against this, biological control techniques are currently emerging, and antagonists and competition microorganisms are isolated and cultured, and then treated directly with soil, seeds or plants to prevent invasion of pathogens or secrete germicides to secrete pathogenic bacteria. A method of suppressing the proliferation of is known, and a method of using a bacteriophageran virus that specifically attacks only bacteria to lyse is known. 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.

The virus was discovered independently by Twert (1915) and Herler (1917) at the beginning of the 20th century, and the virus that infects the bacteria was called "bacteriophage" in the sense of Phage. It is also called simply "phage".

Phage from 1910 to 1930 when phages were discovered, problems that could not be solved scientifically at that time, such as endotoxins and exotoxins of host bacteria, and penicillin discovered by flaming in 1929 and streptotoxicity by Worksman in 1943 The discovery and discovery of a chemotherapeutic agent (a drug), which is represented by mycin, has slowed its use and development.

On the other hand, there were some problems with using phage as a therapeutic agent at that time. Among them, the specific problem was that the high specificity acts only on a few strains and the resistance was high, so that resistant bacteria appeared immediately after use and the effect as a therapeutic agent disappeared immediately. On the other hand, chemotherapy has been widely used because of its broad antibacterial properties and no resistance to resistant bacteria at the time of its first application, resulting in almost no phage therapy. Since then, in the 1950s, phage therapy has been declared invalid by the WHO and has only been maintained in the Eastern bloc and the Soviet Union.

The idea of phage therapy changed in the 1980s, however, and in 1982, British researchers groups Smith and Huggins experimented with a mouse model of meningitis. Injecting the bacteria into the subcutaneous muscle of the mouse and the specific subcutaneous phage of the bacterium in the other subcutaneous, the control group that did not inject phage survived 100% when the phage was injected (experimental group). It was confirmed that it was superior to the effects of antibiotics (streptomycin, etc.). At this time, the problem of phage resistance could also be solved by using various types of phage as a cocktail.

Phage size is about 1/10 of the bacteria and its shape can not be observed under the electron microscope. However, when phage lysates bacteria, transparent spots (plaques) are formed on bacterial colonies that have grown on agar medium, so that the presence of phage can be visually confirmed.

Phage produce about 100 to 200 new phages about 30 minutes after bacterial infection and break out of the bacterial membrane. The bacterium multiplies in two divisions, the fastest one being two individuals in about 20 minutes, four in 40 minutes, and eight in 60 minutes. In the case of phage, more than 100 new phages are produced in about 30 minutes, which is much superior to bacteria in the rate of proliferation. If a particular bacterium grows indefinitely once every 20 minutes, a bacterium starting from one cell takes only 36 hours to weigh like the earth, so phage can be considered as an important candidate to suppress the growth of bacteria. have.

Phage are classified into lytic phages and lysogenic phages. Viral phage is defined as repeating the lysis cycle only and temperate phage is repeated only the lysis cycle. Because weak phage inserts phage gene information into the genome of the host bacterium, the phage disappears but the host bacterium is not destroyed. This condition continues as long as the host bacterium is in good environmental conditions. When the host bacterium's environment deteriorates, the phage gene in the host bacterium gene produces phage particles, which are then converted into a lysis cycle to lyse the host bacterium and host cells. To escape out.

Meanwhile, the phages have several advantages over antibiotics. First, phage can be replicated only in the presence of a host. Second, phages exist in their natural state, wherever they are, they are everywhere. Third, phage is safe for any eukaryotic organism. Fourth, its specificity is safe for other beneficial bacteria. Fifth, the gripping is very easy to prepare.

Due to various advantages as described above, more and more phages are being developed recently.

Accordingly, an object of the present invention is to provide a composition for inhibiting or killing bacterial growth, which is isolated from a new bacteriophage capable of inducing the death of the bacteria.

In order to achieve the above object, the present invention provides a bacteriophage PPP1 (KACC97005P) that exerts a control against vegetable purifying disease.

The present invention provides a vegetable softening control agent comprising bacteriophage PPP1 (KACC97005P) as an active ingredient.

In addition, the present invention is a bacteriophage PPP1 (KACC97005P) Pectobacterium Cartoborum, characterized in that it comprises as an active ingredient ( Pectobacterium carotovorum subsp. carotovorum ) provides an agent for inhibiting or killing proliferation.

Hereinafter, the means for solving the problems of the present invention will be described in detail.

The present invention isolates the bacteriophage PPP1 exerts control against the vegetable blight bacteria from Pyeongchang soil, and deposited it in the KACC (Agricultural Microorganism Resource Center) was given the accession number KACC97005P. Bacteriophage PPP1 (KACC97005P) has a very short tail that does not shrink and thus belongs to Podoviridae and can be classified as a type 1 virus with two strands of DNA.

Bacteriophage PPP1 (KACC97005P) of the present invention was confirmed in the following experimental example that exhibits host susceptibility to vegetable rot fungi, can dissolve the rot fungi broadly, lysing all rot fungi with different host in all regions of Korea It can be confirmed through the experimental example that it can.

On the other hand, bacteriophage PPP1 (KACC97005P) is particularly specifications tobak Te Solarium Caro sat borum (Pectobacterium carotovorum subsp. carotovorum ) It was confirmed through the experimental example that the proliferation inhibitory and killing effect is excellent.

On the other hand, vegetable softening control agent and pectobacterium cartoborum of the present invention ( Pectobacterium carotovorum subsp. carotovorum ) Any one selected from the agent for inhibiting or killing growth may be prepared by using a surfactant or extender which can be used as a biochemical pesticide for the purpose of a stable formulation suitable for use in actual packaging. It may be prepared in the form of a water-soluble granule or an encapsulating agent. Examples of the surfactant include alkyl (C ₈ -C ₁₂ ) arylsulfonate, dialkyl (C ₃ -C ₆ ) arylsulfonate, dialkyl (C ₈ -C ₁₂ ) sulfosuccinate, ligninsulfonate, naphthalene Sodium salts of sulfonate compounds such as sulfonate condensates, naphthalenesulfonate formalin condensates, alkyl (C ₈ -C ₁₂ ) naphthalenesulfonate formalin condensates, polyoxyethylene alkyl (C ₈ -C ₁₂ ) phenylsulfonates or Calcium salts, alkyl (C ₈ -C ₁₂ ) sulfates, alkyl (C ₈ -C ₁₂ ) arylsulfates, polyoxyethylene alkyl (C ₈ -C ₁₂ ) sulfates, polyoxyethylene alkyl (C ₈ -C ₁₂ ) phenylsulfates Sodium salts or calcium salts of sulfate compounds, such as sodium salts or calcium salts of succinate compounds such as polyoxyalkylene succinates, anionic surfactants such as calcium benzoate, alkylcarboxylates, polyoxyethylene alkyls (C ₁₂ ~ C ₁₈₎ ether, poly As when the ethylene-alkyl (C ₈ ~ C ₁₂₎ phenyl ether, polyoxyethylene alkyl (C ₈ ~ C ₁₂₎ phenyl polymer and ethylene oxide propylene-non-ionic surfactants, polycarboxylate, Triton-100 and Tween 80, such as oxide copolymer One or two or more selected from the group consisting of may be used in combination, and other surfactants may be used. It will be readily understood by those skilled in the art. Extenders include bentonite, talc, clay, kaolin, calcium carbonate, silica sand, pumice, diatomaceous earth, acidic clay, zeolite, pearlite, white carbon, ammonium sulfate, urea, glucose, dextrin, soy flour, rice, wheat It may be easily understood by those skilled in the art that ocher, glucose and starch, water, and the like may be used alone or in combination of two or more thereof, and other extenders may be used. will be.

As described above, the bacteriophage PPP1 newly isolated in the present invention has a control effect on the softening of fresh vegetables to prevent loss due to decay during distribution and storage of agricultural products after harvesting, and also as a microbial pesticide for controlling softening of crops. Can be utilized.

In addition, the bacteriophage having bacterial growth inhibition or killing ability of the present invention can be used instead of antibiotics, thereby reducing the possibility of antibiotic resistant bacteria through reducing antibiotic use and increasing environmental safety.

1 is a diagram showing an electron micrograph of a bacteriophage isolated in the present invention.
Figure 2 is a diagram of the genetic analysis of proteins affecting the bacteriophage of the present invention to broaden the range of the host.
Figure 3 is a diagram showing the results of the experiment to inhibit the phage of the present invention inoculated fungus inoculated on lettuce.

Hereinafter, the content of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

Example  1: Bacteriophage Separation

The purpose of this study was to isolate bacteriophage against Chinese cabbage fungus from the soil in which the cabbage fom disease-producing plants are grown.

The soil was placed in a 50 mL tube to about 30 mL, and 10 mM MgSO ₄ was added to make the total 45 mL. Host Pectobacterium carotovorum subsp. Carotovorum Pcc3 was incubated for 16 hours and 1 mL was added to each soil sample. After incubation at 160 rpm for 16 hours at 160 rpm, the mixture was allowed to stand for 30 minutes to obtain a supernatant. After adding chloroform to the separated supernatant, the residue was removed by centrifugation at 3800 rpm for 10 minutes. Pipette a suspension of 0.1 mL of the sample and 1 mL of Chinese cabbage bruises incubated for 12 hours or more, and then mixed. The mixture was mixed with agar (0.6%) medium, poured into a plate containing 1.5% agar medium, and then incubated overnight at 28 ° C.

When the desired bacteriophage appeared on the plate, independent and transparent plaques were observed. The plaque region was separated using a platinum loop and then placed in a new bacterial culture, which observed clearing of the culture.

Thereafter, the culture solution was centrifuged, and only the supernatant was taken out to recover a filtrate containing only bacteriophage. The recovered bacteriophage filtrate was inoculated differently on agar medium, and then pure bacteriophage was obtained by re-separation of the plaque three times.

The bacteriophage thus obtained was named bacteriophage PPP1, and was deposited on March 12, 2010 at the National Institute of Agricultural Science, Agricultural Genetic Resource Center, and was given accession number KACC97005P.

Example  2: shape and classification of acquired phage

One species was selected from the bacteriophages obtained in Example 1, and electron micrographs of the phages were analyzed (FIG. 1).

The electron microscope was an Energy-Filtering Transmission Electron Microscope. The bacteriophage sample to be observed was placed on a carbon coated copper grid and negatively stained with 2% liquid uranyl acetate. Stained samples were observed using an energy filtered transmission electron microscope (LIBRA 120, Carl Zeiss) at a voltage of 80 kV.

The observed bacteriophage belongs to Podoviridae, which has a short tail as a sorting criterion, and could be classified as a type 1 virus according to Baltimor classification.

Example  3: sequencing of the obtained phage

Phage gene was isolated for sequencing the isolated phage. 0.5 M EDTA (pH 8.0), proteinase K, and SDS were added to 1 mL of purified bacteriophage, but the concentrations after adding the reagents were 20 mM, 50 ug / mL, and 0.5%, respectively. The mixed solution was kept at 56 ° C. for 2 hours and then cooled at room temperature. The same amount of phenol was added and centrifuged at 3000 g for 5 minutes. Only the supernatant was obtained and treated with PC (phenol-chloroform) once again, and centrifuged under the same conditions. The same amount of chloroform was added to the supernatant obtained above, followed by centrifugation. This process was repeated and centrifuged for 15 minutes by adding 50 mL of 1 mL of 95% ethanol and 3 M sodium acetate (pH 5.2), followed by centrifugation once more by adding 70% ethanol. After ethanol was removed and the DNA was dried, the DNA was dissolved in IX TE buffer. The separated DNA was cut with the restriction enzyme EcoRI, and the degree of isolation of the gene was confirmed.

The nucleotide sequence of the pure genes isolated was determined and the determined nucleotide sequence was analyzed using 'Blast program'. As a result, the open reading frame representing each gene was as shown in Figure 2, the estimated function of each gene was as shown in Table 1 below.

ORF CDS Start Position CDS stop position CDS length
(nucleotides) Protein length (amino acids) Start codon Stop codon Remarks One 1929 2903 975 324 ATG TAG DNA ligase 2 3244 3729 486 161 ATG TAA GNAT 3 3976 5502 1527 508 ATG GGA head portal protein 4 5502 6410 909 302 ATG TAA putative scaffolding 5 6514 7731 1218 405 ATG TAA major capsid protein 6 7784 8521 738 245 ATG TAA tail tubular protein A 7 8521 10911 2391 796 ATG TAA tail tubular protein B 8 10872 11612 741 246 ATG TAA putative internal virion protein 9 11622 14597 2976 991 ATG TAG lysozyme like domain 10 14639 18454 3816 1271 ATG TAA internal virion like protein 11 18454 19422 969 322 ATG TGA tail fiber like protein 12 19450 19602 152 49 ATG TGA putative Holin 13 19583 19897 315 104 ATG TAA putative small terminase subunit 14 19897 21795 1899 632 ATG TAA putative large terminase subunit 15 21961 22251 291 96 ATG TAA 심포치 16 22261 22539 279 92 ATG TAA 심포치 17 22552 22833 282 93 ATG TAG 심포치 18 22973 23320 348 115 ATG TAA peptidase M15-like protein 19 23645 25447 1803 600 ATG TAA hydrolase related protein 20 28194 28610 417 138 ATG TAA hydrolase related protein 21 30122 32743 2622 873 ATG TAA RNA polymerase 22 33507 35495 1989 662 ATG TAG DNA primase / helcase like protein 23 37071 39584 2514 837 ATG TAA DNA polymerase A 24 39598 40059 462 153 ATG TAA 심포치 25 40302 40844 543 180 ATG TAA 심포치 26 40932 41309 378 125 ATG TAA 심포치 27 41495 42301 807 268 ATG TAG 심포치 28 42856 43227 372 123 ATG TAA 심포치 29 43352 43651 300 99 ATG TAG 심포치 30 43561 44592 1032 343 ATG TAG similar to endonuclease 31 44577 223 411 136 ATG TAA endonuclease VII

Example  4: for isolated bacteriophages Host Range ( host range ) decision

The host range for the isolated bacteriophage was determined by the dropping method.

After mixing the culture medium and cultured 0.6% agar medium for 3 hours, poured into a plate and dried for 15 minutes at 28 ℃, drop the diluted phage on it, dried at room temperature and incubated at 28 ℃ for 10 hours. If the strain is susceptible to phage, it shows a clear area, or plaque, on the plate, proving that it is the result of complete lysis of bacterial cells. If the sensitivity is weak, a cloudy area is formed or an independent bold spot appears.

Table 2 shows the Pectobacterium cartoborum species of bacteriophage PPP1 of the present invention ( Pectobacterium) carotovorum subsp. Carotovorum species and other bacteria belonging to the genus Pectobacterium were examined.

Seed bacteria P120301-4 P. carotovorum subsp. carotovorum Pcc1 + P. carotovorum subsp. carotovorum Pcc3 + P. carotovorum subsp. carotovorum Pcc4 + P. carotovorum subsp. carotovorum Pcc5 + P. carotovorum subsp. carotovorum Pcc8 + P. carotovorum subsp. carotovorum Pcc9 + P. carotovorum subsp. carotovorum Pcc10 + P. carotovorum subsp. carotovorum Pcc11 + P. carotovorum subsp. carotovorum Pcc12 + P. carotovorum subsp. carotovorum Pcc13 + P. carotovorum subsp. carotovorum Pcc14 + P. carotovorum subsp. carotovorum Pcc15 + P. carotovorum subsp.carotovorum Pcc17 + P. carotovorum subsp. carotovorum Pcc21 + P. carotovorum subsp. carotovorum Pcc22 + P. carotovorum subsp. carotovorum Pcc25 + P. carotovorum subsp. carotovorum Pcc26 + P. carotovorum subsp. carotovorum Pcc27 + P. carotovorum subsp. carotovorum Pcc29 + P. carotovorum subsp. carotovorum Pcc49 + P. carotovorum subsp. carotovorum Pcc87 + P. carotovorum subsp. carotovorum Pcc88 + P. carotovorum subsp. carotovorum Pcc97 + P. carotovorum subsp. carotovorum Pcc99 + P. carotovorum subsp. carotovorum Pcc101 + P. carotovorum subsp. carotovorum Pcc102 + P. carotovorum subsp. carotovorum Pcc103 + P. carotovorum subsp. betabasculrorum KACC10056 - P. carotovorum subsp. wasabiae KACC10061 - P. chrysanthemi KACC10062 - P. carotovorum subsp. wasabiae KACC10406 - P. carotovorum subsp. atrocepticum KACC10478 - P. carotovorum subsp. atrocepticum KACC10480 - P. carotovorum subsp. odovirerum KACC10486 - P. carotovorum subsp. betabasculrorum KACC10538 - P. carotovorum subsp. wasabiae KACC10869 -

Experimental results (Table 2), Pectobacterium Cartoborum carotovorum subsp. carotovorum ) It was confirmed that the strain can be widely dissolved.

Example  5: measurement of cabbage softening incidence caused by phage

Wide range of Pectobacterium cartoborum in greenhouse carotovorum subsp. We evaluated the alleviation of bacteriophage PPP1 against insomnia caused by invasion of carotovorum ).

Lettuce seeds were sown in horticultural soil and grown for about 3 weeks. Pectobacterium CartoboroomPectobacterium carotovorum subsp.carotovorum) Bacterium precipitate obtained by preculturing Pcc3 through preculture and after culture and centrifuged at 5000 rpm for 20 minutes for 10 mM MgCl₂ Suspended in a buffer, Tween20 was added as an electrodeposition agent and well mixed, and then inoculated fully into the lettuce by a spray method. Plants inoculated with bacteria were wet treated with vinyl.

0.1 mL of the filtrate obtained in Example 1 was inoculated into 1 mL of the host culture incubated for 16 hours, and then grown for 3 hours to confirm that the culture became clear. Chloroform was added and the supernatant obtained by centrifugation at 3800 rpm for 10 minutes was inoculated again in 10 mL of the host culture, and the above procedure was repeated. In this same manner, 10% polyethylene-glycol 8000 (PEG, mw 7000-9000) and 2.9% NaCl were completely dissolved in a phage broth obtained by incubating with 100 mL and incubating again with 1000 mL. The mixture was allowed to stand at 4 ° C. for 16 hours. Gripping the PEG and NaCl dissolved (phage) was centrifuged at 8000 rpm for 1 hour, the culture supernatant was discarded pellets (pellet) is SM buffer _{(NaCl 5.8g, MgSO4? 7H 2} O 2.0g, pH7.4 50 ml of 1M Tris-Cl). Dissolve the CsCl in SM buffer to make the CsCl density 1.3 g / mL, 1.45 g / mL, 1.5 g / mL, 1.7 g / mL, respectively, into four stages of solution. The solution was added so as not to mix the layers by 7 mL in turn. SM buffer washed phage pellets were placed in a tube containing CsCl solution, and centrifuged at 25,000 rpm for 3 hours. After centrifugation, the phage separated according to the density was extracted using a syringe, put into a membrane, and soaked in dialysis buffer (10 mM NaCl, 50 mM Tris-Cl, 10 mM MgCl ₂ ) at 4 ° C. Sufficient dialysis to remove CsCl yielded only pure phage. 10 mM MgSO ₄ and phage were mixed to make a phage suspension. After inoculating the bacteria into the lettuce, Tween20 was added to the phage suspension and sprayed again on the lettuce, and the lettuce was wetted. Over time, the development of the disease was continuously observed.

The inoculation of the phage inoculation group, the bacterial inoculation group, and the bacterium-phage inoculation group was observed on the sixth day (FIG. 3). I could confirm it.

National Institute of Agricultural Science KACC97005P 20100312

Claims (5)

Bacteriophage PPP1 (KACC97005P) Exerts Control Against Vegetables
Vegetable softening control agent comprising bacteriophage PPP1 (KACC97005P) as an active ingredient.
The method of claim 2,
The vegetable,
Vegetable softening control agent characterized in that the Chinese cabbage or lettuce.
The method of claim 2,
The vegetable softener,
Pectobacterium carotovorum subsp.carotovorum ( Vectobacterium cartovorum ), characterized in that the disease caused by vegetable softening control agent.
Peck tobak comprises a bacteriophage PPP1 (KACC97005P) as an active ingredient Te Solarium Caro sat borum (Pectobacterium carotovorum subsp. carotovorum ) proliferation inhibitor or killing agent

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