KR101481897B1 - Streptomyces sp. BS063 and biological control of plant pathogenic fungi by using the same - Google Patents

Abstract

The present invention relates to a strain Streptomyces sp. BS063 which shows excellent effect on a gray mold disease ( Botrytis cinerea ) occurring in various crops. More particularly, the present invention relates to a composition for controlling a gray mold infectious disease which contains as an active ingredient a culture of Streptomyces sp. BS063 or a culture filtrate, which has antibacterial effect on the gray mold fungus.

Description

Streptomyces sp. BS063 and plant disease control using the same. BS063 and biological control of plant pathogenic fungi by using the same}

The present invention relates to a Streptomyces sp. BS063 ( Streptomyces sp. The present invention relates to a composition capable of effectively preventing and controlling a gray mold disease caused by cultivation of ginseng and other crops using a culture of a strain or culture filtrate.

Botrytis cinerea , a pathogen of gray mold that causes root rot of ginseng, is used in almost all vegetable products including strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin, cabbage, lettuce, asparagus, potato, kidney bean, It is known to be an unstable strain of polygamous which causes corruption in fruits. Gray mold disease can be controlled by controlling with resistant cultivars, cultivating environment control, alien control by removing diseased plants, chemical control using bactericides, and biological control using microorganisms. However, since the use of resistant cultivars and vigorous control have little effect, the control of gray mold depends mainly on chemical control. In recent years, resistance to chemical pesticides has been frequently observed in conventional chemical pesticides, and the effectiveness of the chemical treatment has been deteriorating. In the case of pesticides having some effects, in case of strawberries or cucumbers which are to be harvested due to residual toxicity, The use thereof is strictly limited. Therefore, related researches are being actively carried out to develop a low toxicity, environmentally friendly biocide pesticide that can replace chemical pesticides.

As biocidal pesticides for controlling gray mold, methods using natural resources such as plants and microorganisms have been proposed (Korean Patent Laid-Open No. 10-2010-0132861, Korean Patent Laid-Open No. 10-2004-0069366) ), Antifungal agents, siderophore, NH ₃ enzyme and hydrogen cyanide have been reported as microbial metabolites inhibiting disease. They are known to exhibit antagonistic activity. In the case of foreign countries, Bio-save 10, a product formulated with Pseudomonas syringae , has been put to practical use. In Japan, Bacillus sp. ( Bacillus sp.) Was used in 2003 for the control of gray mold and powdery mildew. Are commercialized and used. Thus, more than 40 kinds of microorganisms are registered in the world, and these are mainly bacteria such as Pseudomonas sp., Bacillus spp., Streptomyces sp., Burkholderia , tri coder equine (Trichoderma spp.), Fusarium (Fusarium) known to be derived from fungal and yeast (yeast), etc. and the like. In Korea, since the early 1970s, biological control of ginseng root rot disease has been studied, and biological control of gray mold has been studied. Recently, Bacillus licheniformis ( Bacillus licheniformis) , which inhibits mycelial growth and spore germination of gray mold, ) A study on the control of gray mold disease using strain N1 has been reported. However, despite many research examples, there is no established biocide for the control of gray mold disease, which is effective, inexpensive and safe. There have been many attempts to directly use the isolated functional strains or to directly use the metabolites of the strains. However, attempts to isolate and apply the mutants resulting from the changes in the functions from the active strains have been few.

Accordingly, the present inventors have found that cultures and strains of Streptomyces sp. BS063, which has excellent antimicrobial activity against gray mold fungi, can prevent the occurrence of gray mold disease, which is caused by the growth of ginseng and other crops, And can be used for purposes such as affinity-type biologics or soil-improving agents.

The present invention relates to a Streptomyces sp. Strain BS063, a microorganism having excellent antimicrobial activity, and a composition showing a gray mold fungicide inhibitory effect of various crops containing the culture and the culture filtrate as an effective ingredient. The culture and culture filtrate can be used as a composition for an environmentally friendly biological agent or a soil conditioner which can prevent the occurrence of gray mold due to its excellent antimicrobial effect against gray mold fungi.

In order to achieve the above object, the present invention provides Streptomyces sp. BS063 (Accession number: KACC 91715P).

In one embodiment of the present invention, the strain is characterized by having an antibacterial effect against a plant pathogenic bacterium.

In order to achieve the above object, the present invention also provides a culture or a culture filtrate of Streptomyces sp. BS063 (fungal accession number: KACC 91715P).

In order to achieve the above object, the present invention also provides a composition for controlling plant pathogenic bacteria comprising the culture or the culture filtrate as an active ingredient.

In one embodiment of the present invention, the plant pathogenic bacterium is characterized by being a gray mold germ ( Botrytis cinerea ). The plant pathogenic bacterium is characterized in that it is at least one selected from the group consisting of Magnaphorthe grisea , Fulvia fulva , and Colletotrichum gloeosporioides .

The culture or culture filtrate of Streptomyces esp. BS063 strain of the present invention has an excellent antimicrobial effect against Botrytis cinerea which is a gray mold fungus, and thus is environmentally friendly which can effectively prevent the gray mold of various crops Or as a composition for a soil improvement agent.

FIG. 1 shows the antifungal activity of Streptomyces sp. BS063.
FIG. 2 is a graph showing the antifungal activity of Streptomyces sp. BS063.
FIG. 3 is a graph showing changes in the culture day-by-day antifungal activity of Streptomyces sp. BS063.
FIG. 4 is a graph comparing antimicrobial activities of strains of Streptomyces sp. BS063 according to dilution drainage and concentration.

The present invention provides Streptomyces sp. BS063 ( Streptomyces sp. BS063, strain accession number: KACC91715P) strain, which is an actinomycetes having excellent antifungal activity.

The present invention provides a composition for inhibiting plant pathogenic bacteria comprising the culture of the strain BS063 or the culture filtrate as an active ingredient. More specifically, the composition according to the present invention can be used for the control of gray mold caused by Botrytis cinerea such as ginseng, cucumber, tomato, and strawberry.

In addition, the present invention relates to a method for producing a fungus such as Magnaphorthe grisea , Fulvia fulva , Colletotrichum gloeosporioides , and the like, in addition to a gray mold fungus ( Botrytis cinerea ) Lt; RTI ID = 0.0 > antimicrobial < / RTI >

Hereinafter, the present invention will be described with reference to the following examples and test examples. The following examples and test examples are only illustrative of the present invention, and the scope of the present invention is not limited to these examples.

The strain Streptomyces esp BS063 of the present invention is a mutant produced by irradiation of ultraviolet light of a Streptomyces espí BS062 strain. BS062 strains were diluted to 1000 colonies, and irradiated with ultraviolet light in a dark room. BS063 strains were selected which did not undergo mutation in the subculture and liquid cultures and maintained the increased activity. As a result of the antimicrobial activity test against the gray mold pathogens, it was confirmed that the BS063 strain had an increased antimicrobial activity as compared with the BS062 strain, and the incubation time was shortened sharply (FIGS. 1 to 3).

In addition, it was confirmed that the antimicrobial activity of BS063 and BS062 cultures was diluted by concentration and the antimicrobial activity of BS063 culture was increased about 100 times as compared with BS062 (FIG. 4).

As a result, the composition according to the present invention is excellent in antibacterial effect against Botrytis cinerea , a gray mold fungus, and thus is suitable for controlling a gray mold disease. The gray mold pathogen is a pathogenic bacterium causing almost all the vegetables and fruits such as root rot disease of ginseng, strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin, cabbage, lettuce, asparagus, potato, kidney bean and pea. The culture can be used as a composition for environmentally friendly biological agents or soil improvement agents which can prevent the occurrence of gray mold disease.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are provided only for easy understanding of the present invention, and the technical content of the present invention is not changed or reduced.

Example 1: Preparation of Streptomyces espi BS063 strain

For the present invention, the spore suspension of Streptomyces sp. BS062 strain was diluted to set the number of the average number of colonies to 1000, and UV light was irradiated in the dark room condition. At this time, the UV lamp was irradiated at a height of 15 cm, and UV irradiation was performed at 99% lethal dose in the UV irradiation. Colonies formed after UV irradiation were cultured on a 96 well plate and then examined for antimicrobial activity by agar plug method. Only colonies with increased antifungal activity were selected compared to wild type strains. Experiments for setting the conditions for UV-induced strain improvement UV irradiation at 254 nm for 15 minutes at a height of 1 minute and 30 seconds was found to be the most suitable for UV mutagenesis (Table 1). BS062, which was confirmed to have increased antimicrobial activity as a result of UV mutation The microorganisms grown on MBA (modified bennett 's agar) solid medium were suspended and stored in sterilized water.

Table 1 below shows the survival rate of the strains according to the UV irradiation time.

UV irradiation time (min) Number of colonies formed Survival rate (%)           0     1504.3 ± 81.0          -          1.0      827.5 ± 48.4        54.98          1.5       16.8 ± 2.3         1.11          2.0        5.6 ± 1.8         0.37          2.5          0         0

Example 2: Streptomyces espe Culture of BS063

Streptomyces esp BS063 culture of the present invention was performed as follows. 100 ml of the preculture medium (Table 2) was placed in a 500 ml Erlenmeyer flask, and platelets were plated in platelets. After inoculation (7 mm diameter agar plugs), the cells were incubated for 2 days at 28 ° C with shaking at 150 rpm And then used as a seed inoculum. The culture broth (Table 2) was prepared by dispensing 200 ml each in a 1 L Erlenmeyer flask and autoclaving it at 1.5 atm and 121 ° C for 20 minutes. The bacterial culture seeded in the above-prepared culture medium was inoculated in an amount of 3% (v / v) per flask and cultured at 28 ° C with shaking at 200 rpm for 7 days.

Table 2 below shows the composition of the medium.

Medium component density Pre-culture medium (g / 0.5L) The culture broth (g / L, pH 7.5) Soluble starch 10 70 Glycerol 7 Bacto peptone 3 Soytone peptone 5 anti-form 0.5 ml Soybean flour 5 Yeast extract 3 17 Ammonium sulfate One Cornsteep liquor 5 ml Calcium carbonate One

Example 3: Antimicrobial activity of BS063 strain against a gray mold fungus as compared with the original strain

BS063 strain and original strain BS062 according to the present invention were cultured in the present culture broth for 7 days and then centrifuged to separate the culture broth and the culture filtrate, and the culture filtrate was dropped on a paper disk by 50 점 and air-dried. This paper disk was prepared by pre-fabricating a gray mold test plate for 2 ~ 3 days. As a result, it was confirmed that the culture of strain BS063 exhibited a high inhibitory activity against the gray mold pathogen as compared with the strain BS062 (FIGS. 1 and 2). Also, as a result of comparing the antimicrobial activities of BS063 and original strain BS062, the maximum antimicrobial activity of BS063 was superior to that of BS062 and the incubation time was shortened sharply (FIG. 3). The BS063 and BS062 cultures were diluted by concentration and the antimicrobial activities were compared. As a result, it was confirmed that the BS063 culture increased the antimicrobial activity about 100 times as compared with BS062 (FIG. 4).

Example 4: Antimicrobial spectrum of BS063

The BS063 strain according to the present invention was cultured in a GSS liquid medium for 7 days, and then centrifuged to separate the cells and the culture filtrate. The cells were extracted with an 80% acetone solution and concentrated under reduced pressure. 50 [mu] l was added dropwise to a paper disk and air-dried. This paper disk was placed on a pre-prepared plate for pathogen examination and cultured for 2-3 days, and the size of the generated hypocotyl was examined.

Table 3 below shows the antimicrobial spectrum of the BS063 culture.

Bottle fungus Antimicrobial activity ^a (mm) Bacillus subtilis 0    Salmonella typhimurium 0 Candida albicans 0 Botrytis cinerea 52 Magnaporthe grisea 52 Fusarium oxysporum 20 Fulvia fulva 45 Aspergillus niger 0 Alternaria porri 0 Alternaria mali 0 Colletotrichum gloeosporiodes 20 Alternaria panax 0 Pythium sp. 0 Cylindrocarpon destructans 20 Rhizoctonia solani 0 Trichoderma sp. 52

^a Paper disk method

As can be seen from the present Example, the culture filtrate of the strain according to the present invention can be used for the production of Botrytis cinerea , Magnaphorthe grisea , Fulvia fulva , Trichoderma sp. Lt; RTI ID = 0.0 > antibacterial < / RTI >

Example 5 Biological Control of Gray Mold Fungi Using Streptomyces espe BS063

Test Example I. Effect of Controlling Gray Mold Fungus on Cucumber Leaf Disk

Cucumber seedlings cultivated under greenhouse conditions were tested for the control effect against gray mold. Cucumber seedlings were used in one or two seedlings of the main stem. Unless otherwise noted, the experiment was carried out in a thermostat at a relative humidity of 90% at 20 ° C, 12 hours: 12 hours / dark condition.

The treatment solution was cultured as in Example 1, and then the whole culture was centrifuged, and the culture filtrate obtained by dividing the cells by the culture solution and the culture filtrate and diluting them to a predetermined concentration was used.

The treated liquid diluted to a predetermined concentration was sprayed onto cucumber seedlings and air dried. Then, a leaf disk having a diameter of 3 to 4 cm was made, and the suspension of the pathogen spore suspension was inoculated with the suspension. The incubation was carried out at 20 ° C in a thermostat, The control effect was checked by examining the incidence rate.

(1)

Gray mold pathogen spores in 0.1 molar potassium phosphate (0.1M KH ₂ PO ₄₎ is added to 20% v arm (V8) was suspended in 1 ㅧ 10 ⁶ spore / ㎖ concentration jyuwooseu leaf disk 4 conventional ipdang by 100 ㎕ Inoculation method.

Test Example I-1 : Effect of the treatment time of BS063 strain

In order to investigate the effect of Streptomyces esp BS063 on the control of gray mold disease, leaf discs of cucumber seedlings growing in the greenhouse were treated 24 hours before (after pretreatment) and after 24 hours (posttreatment) Simultaneous treatment) was carried out to investigate the disease occurrence and the control effect of BS063 culture filtrate according to the present invention.

As a result, 100% control effect was obtained at the pretreatment and simultaneous treatment, and the control effect was over 95.5% at the posttreatment.

Table 4 shows the effect of controlling the gray mold fungus disease according to the treatment time of BS063 strain

Processing time Outbreak rate (%) ^a Control (%) Pretreatment 0 ± 0.0 100 Concurrent processing 0 ± 0.0 100 After treatment 0 ± 0.0 100 Control 90.5 ± 2.4 -

^a Results of treatment after 6 days of bacterial culture filtrate treatment and pathogen inoculation

Test Example I-2 : Effect according to dilution drainage of culture filtrate

The culture filtrate was diluted 100, 200 times with sterilized distilled water and the effect of dilution drainage on the control of gray mold was examined. Pathogens were inoculated after the culture filtrate was sprayed and air dried. As a result, 90% control effect was observed at 200 times dilution, and it was found that the 20-fold dilution treatment completely prevented the gray mold disease.

Table 5 shows the effect of controlling the gray mold fungus by dilution concentration of BS063 culture filtrate.

Processing contents Outbreak rate (%) Control (%) tomato tomato   Culture filtrate stock solution 0 100            100 times 0 100            200 times 100.5 89   500 times of dichroic wetting agent 0 100   Control 924 -

After 6 days of inoculation,

Test Example II. Effect of controlling gray mold disease on strawberry

To investigate the effect of BS063 strain on the control of strawberry gray mold, cultivars cultivated in Nonsan strawberry field in Nonsan, Chungcheongnam - Detailed tests were conducted as described in Test Example I above and in the following Test Examples.

Test Example II-1 : Effect of controlling the treatment time of the bacterial culture

The BS063 culture according to the present invention was immersed in strawberry for 15 seconds, sprayed in the same manner as in Test Example I before and after air drying, and the infection rate and control effect were examined after 7 days of spraying . As a result, both the pretreatment, the simultaneous treatment and the posttreatment showed high control effect.

Table 6 shows the effect of treating BS063 strain with the treatment time of strawberry gray mold.

Processing time Disease rate (%) Control (%) BS063 Dieterpen CAP is a 1000 watt solution Distilled water BS063 Dieterpen CAP is a 1000 watt solution Distilled water Pretreatment 2 7 42 95 83 - Concurrent processing 2 5 44 95 89 - After treatment 5 25 47 89 47 -

Test Example III. Effect of BS063 culture medium on the control of ginseng gray mold

Gray mold fungus is very common during storage, especially in ginseng. In this experimental example, the control of gray mold fungus in ginseng using BS063 culture was tested.

Test Example III-1 : Effect of Controlling Gray Mold Fungi Occurring During Storage of Seedlings

In order to investigate the effectiveness of controlling the gray mold fungus during the storage of the seedlings by BS063 strain, the seedlings were immersed in the culture solution for 30 seconds, and then air - dried, packed in a plastic bag, refrigerated (4-7 ℃) And the length of post - emergence stem were investigated. As can be seen from the results shown in Table 6 below, Streptomyces sp. BS063 showed an excellent effect in controlling the gray mold of Ginseng.

Table 7 relates to the control effect of the BS063 strain on the control of ginseng gray mold.

Processing contents Dry rate
(%) Rate of decay (%) After childbirth
Stem length (cm) The control
(%) Crotch + epidermis BS063 84 16 3.8 82 Dietopen capph is a 500-fold diluted solution 80 20 3.6 77 No treatment 13 87 - -

After the seedlings were immersed in the culture filtrate, they were placed in a plastic bag, and after 30 days of refrigerated storage,

Director, Center for Agricultural Genetic Resources, National Institute of Agricultural Science and Technology KACC91715P 20120410

Claims (6)

Streptomyces, characterized in that it has an antibacterial effect against plant pathogenic processes SP BS063 (Streptomyces sp BS063, strain deposit number:. KACC 91715P) in the said plant pathogens are gray mold pathogen (Botrytis cinerea), rice blast fungus (Magnaphorthe Streptomyces sp. BS063 (strain accession number: KACC 91715P), which is at least one selected from the group consisting of grisea , Fulvia fulva , and Colletotrichum gloeosporioides . . delete A culture or culture filtrate of the strain according to claim 1. A plant pathogen inhibiting composition comprising the culture or the culture filtrate according to claim 3 as an active ingredient, wherein the plant pathogen is selected from the group consisting of Botrytis cinerea , Magnaphorthe grisea , Fulvia fulva ) And Colletotrichum gloeosporioides. ≪ Desc / Clms Page number 24 > delete delete

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