KR20020031961A - Streptomyces kasugaensis GBA-0927 - Google Patents

Abstract

PURPOSE: An antagonist microorganism, Streptomyces kasugaensis GBA-0927, a microbial preparation containing it, and a method for controlling Fusarium oxysporum and Cladosporium cucumerinum using the microbial preparation are provided, therefore Fusarium oxysporum and Cladosporium cucumerinum can be effectively controlled. CONSTITUTION: Streptomyces kasugaensis GBA-0927(KFCC-11218) capable of inhibiting Fusarium oxysporum and Cladosporium cucumerinum is isolated by the steps of: collecting soils from various regions; cultivating the soil samples in an agar medium; inoculating plant disease-causing bacteria and cultivating; and selecting a sample producing transparent circles. The method for controlling Fusarium oxysporum and Cladosporium cucumerinum comprises the steps of: cultivating Streptomyces kasugaensis GBA-0927(KFCC-11218) in a medium containing 15g of soy bean powder, 200g of glucose, 105g of ammonium sulfate, 5g of potassium phosphate and 25g of magnesium sulfate in 1 liter of distilled water under conditions of pH 7.0, 30 deg.C, 150 rpm and 0.5 vvm for 168 to 180 hours; preparing a microbial preparation containing an effective amount of the cultivated Streptomyces kasugaensis GBA-0927(KFCC-11218) medium; and treating plants with the microbial preparation.

Description

Antagonist microorganism Streptomyces kashugensis GBI-0927 strain, microbial preparation comprising the same, and a method for controlling egg wilt disease and cucumber melanoma disease using the same {Streptomyces kasugaensis GBA-0927}

The present invention relates to the isolation and cultivation of microorganisms having excellent antagonism against specific phytopathogens.

About 4,000 kinds of pests in crops and flowers are known in Korea, and not only directly reduce the yield of crops, but also deteriorate the quality and reduce the value of commodities.

In order to control these pests, organic synthetic pesticides have been used indispensably, and in fact, organic synthetic pesticides are very effective as a means of protecting crops, and play an important role in increasing food production, stable supply, and reducing labor force of agricultural workers. Has been in charge. However, in recent years, the negative perspectives of overuse, misuse, toxicity, environmental pollution, and adverse effects on the natural ecosystem and poor drug effects due to the emergence of drug resistance have gradually increased. Increasingly, social demands for natural orientation are becoming increasingly strict in social regulation of synthetic pesticides that have played an important role in efficient food production. Therefore, much attention has been focused on the development of biopesticides as an alternative to supplement the disadvantages of such organic synthetic pesticides.

Biological pesticides (microbial pesticides) are microorganisms (e.g., fungi, bacteria, viruses) that exist in nature and active substances produced by these microorganisms. The fields of application are bactericides, insecticides, herbicides and growth regulators. And anti-injury agents have been put into practical use. Compared with organic synthetic pesticides, these microbial pesticides have low toxicity, have little effect on the ecosystem, and do not show drug resistance or resistance. Therefore, until recently, the proportion of microbial pesticides in the overall pesticide market is very weak compared to organic synthetic pesticides. However, the rapid development of the biotechnology sector today greatly brightens the prospects for microbial pesticides as new alternatives, and further interest in research into the development of economical, pollution-free and low-toxic microbial pesticides compared to organic synthetic pesticides. have.

In foreign countries, research into microbial control methods was the first to use actinomycetes for the control of potato beetle disease in the United States in 1927, and since 1960, it has been put into practical use as a pesticide. Many products have been developed from the beginning of Trichoderma and probiotics, and most of the practical products have been developed for the control of mozzarlock disease occurring in seedlings. Currently, about 70 kinds of biopesticides using microorganisms in foreign countries have been put into practical use.

In addition, there are active substances produced by the microorganisms, and the like, which are metabolic active substances produced by the microorganisms in culture, which are separated and purified and used as antibiotics for medical and / or agricultural purposes. Among them, about 20 kinds are used for agriculture, and about 6 kinds are mainly used for pest control.

Most agricultural antibiotic-producing bacteria are Streptomyces strains among Actinomycetes, which are widely distributed in soil, and the world's first agricultural antibiotic, blastidine, which was identified as an antibiotic for control of blast disease in 1958. Kasugamycin, Polyoxin, and Validamycin are in use, leading Blasticidin S. Since these agricultural antibiotics use the products of microorganisms, not microorganisms themselves, the safety evaluation as pesticides is evaluated at the same level as organic synthetic pesticides, but is known to be very low toxicity compared to chemical pesticides.

In Korea, basic research was conducted in the 1980s, and there are not many products currently in practical use, but recently, the research level has been improved much compared to the research history according to systematic laboratories and experts. It has been studied for controlling diseases using microorganisms. Since 1985, pathologists of national research institutes and universities have been focusing on mosaic mosaic of tobacco (Bacterial wilt), cucumber widow (cucumber) and pepper Research has been reported on Phytophthora blight, strawberry wilting disease, Rhizoctoina bud rot, beet-damping-off, rice blast, paperback disease control.

Of these plant diseases, the magnitude of damage caused by black diseases in cucumber is less than other major diseases such as ash fungus, anthrax, and powdery mildew, but it is still one of the major diseases affecting the crop of cucumber. Along with soft disease, white disease, and black disease, it is an important disease on the root side, and it is damaging to the cultivation of eggs, which is popular as a high value-added product. In particular, in case of egg production, about 300 ha of cultivation area is currently formed in Korea, but it is expected that its cultivation area and output will gradually increase due to the expansion of domestic and export markets. It is increasing.

Accordingly, the present inventors have tried to find strains that are effective in controlling the disease of wilt and cucumber black disease caused by soil microorganisms in Korea, and as a result, isolates of actinomycetes Streptomyces Kashgensis gibi-0-0 strains, The present invention was completed by assaying the growth inhibitory effect on Fusarium oxyporum and Cucumber Cladosporium cucumerium in the culture of microorganisms.

On the other hand, various methods for use in agriculture using other strains belonging to the same species as the strain of the present invention have already been known, these methods are mainly related to the production of agricultural antibiotics Kasugamycin (Kasugamycin), other bacteria And it does not disclose information on the control of wilted ill by molds, etc., and the action on the control of wilted ill and cucumber black disease has not been reported yet.

In addition, the activity against various pathogenic fungi and bacteria of the antagonist produced by the streptomyces Kashgensis Zbiei-0927 strain of the present invention as an antibacterial material produced by Actinomycetes Streptomyces Kashgensis. Comparative assays have shown that only strains of the present invention have antagonistic activity against wilted wilt disease and cucumber melanoma.

An object of the present invention is to provide an actinomycetes Streptomyces Kashgensis ZBIA-0927 strain which is an antagonistic microorganism for controlling plant diseases caused by wilting disease of eggs and blackness disease of cucumber.

Another object of the present invention relates to a microbial agent for controlling egg wilt and cucumber melanoma, which contains the antagonist microorganisms as an active ingredient, and a method for controlling the disease using the same.

In order to achieve the above object, the present invention provides a actinomycetes Streptomyces Kashgensis ZBIA-0927 strain as an antagonist microorganism, and also contains a microbial agent for the control of egg wilt disease and cucumber melanoma containing it as an active ingredient, and It provides a method for controlling the disease used.

Streptomyces kashugensis GBI-0927 strain contained as an active ingredient in the microbial preparation of the present invention is a bacterium isolated from natural soils in Korea, and has been confirmed to be non-pathogenic as a result of pathogenicity tests on various plants. The microbial agent has a wide growth inhibitory effect against pathogens such as fungal diseases of crops such as Jalok disease, wearing disease, wilted disease and brown patch, large patch and rice fever. In particular, the microorganism that is a major cause of egg wilted disease and cucumber melanoma disease It has been found to have a very strong antagonistic effect against them.

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

Example 1 Screening and Screening of Antagonist Microorganisms

Phytopathogens include botrytis cineria , Fusarium sp., Pythium sp., Phytophtora capsici , and Black fungus Alternaria. citri , Cladosporium cucumerium , Pyricularia oryzae , Brown patch ( Rhizoctonia solani MAFF 511103), and large patch ( Rhizoctonia solani MAFF 305245) were used.

In order to isolate the microorganisms having antagonistic activity against the disclosed pathogenic microorganisms, soil samples from field soils and forest soils in Gyeonggi and Gangwon-do were collected seasonally. In order to isolate antagonistic microorganisms, a conventional triple layer flat method was used. 250 g of high quality soil soil was added with 2 times distilled water and autoclaved, and filtered using gauze. The filtrate was adjusted to 1 L with distilled water, and then 15 g of agar was dissolved, and 10 ml of the agar was dispensed into a 30 ml test tube, and then sterilized by a conventional method and stored in a constant temperature water bath at 50 ° C. Subsequently, 1 g of the collected soil sample was suspended in 100 ml of sterile physiological saline, and 2-3 drops of the suspension were mixed in the test tube, mixed well, and immediately poured into a petri dish to solidify. 5 ml of agar solution was dispensed on the solidified Petri dish to solidify. Next, a spore suspension of phytopathogenic microorganisms (1 × 10 ⁵ cfu / mL) was prepared from a subcultured medium under passage, added 2-3 drops to 10 ml of potato agar medium prepared in a 50 ° C. thermostat, and mixed well. Dispensing again in Petri dishes to prepare a triple layered medium. The prepared plate medium was incubated for 3-7 days in a 30 ℃ thermostat, and examined the antagonism (determined by the formation of a transparent ring) to the target phytopathogenic microorganisms, and then the excellent microorganisms were separated purely in a conventional manner. After culturing using a liquid culture medium according to the selected strains, the conventional symmetrical culture and the transparent ring diameter were investigated, and the antagonistic microorganisms with the highest antagonism were selected.

Example 2: Identification of antagonistic microorganisms against mold causing major diseases of crops

Antagonist microorganisms selected in Example 1 were identified as Streptomyces kasugaensis as a result of 16S rDNA sequencing, which was designated as GBI-0927 (GBA-0927). The strain was deposited with the Korean spawn association on September 25, 2000 under accession no. KFCC-11218.

Example 3 Assay of Growth Inhibitory Effect on Various Plant Pathogens

Phytopathogens include botrytis cineria , pathogenic bacteria (Fusarium sp.), Pythium sp., Phytophtora capsici , and black fungus ( Alternaria). 9 species of citri), bacterial heukseongbyeong (Cladosporium cucumerium), rice blast fungus (Pyricularia oryzae), brown patch (Rhizoctonia solani MAFF 511103), large patch (Rhizoctonia solani MAFF 305245) was used.

After sterilizing the potato agar medium, plate the mycelium, cells, and spores of each pathogen in a medium of 50 ℃, and after the medium is completely hardened, the medium cultured with antagonistic microorganisms is buried on a paper disc (diameter 8 mm), and then the pathogens are mixed. Placed in Petri dishes and incubated for 3 to 4 days at a temperature well grown various pathogens. The effect of antagonistic microorganisms on various pathogens was investigated with a low support diameter, which is shown in Table 1 below.

As can be seen in Table 1, as a result of the antagonistic effect of the antagonistic microorganism Streptomyces Kashgensis Zbiei-0927 strain of the present invention and the phytopathogen, the strain of the present invention is a cause of wilting disease Fusarium ( Fusarium sp.) And Cladosporium cucumerium , the causative agent of black nephropathy , have high inhibitory effects, and Pythium sp., The causative agent of gray and fungal diseases Inhibitory effects were also observed for Botrytis cineria , Brown Patch ( R. solani MAFF 511103) and Large Patch ( R. solani MAFF 305245).

Table 1. S treptomyces kasugaensis

Antimicrobial Spectrum of GBA-0927 Strains

Target bacteria Active size mold Botrytis cineria + Cladosporium cucumerium ++++ Fusarium oxysporum ++++ Phytopthora capsici + Pythium aphanidermatum + Pythium ultimum + Rhizoctonia solani AG1-1A ++ Rhizoctonia solani AG2-2IV ++ Pyricularia oryzae ++

* +: 10 ~ 15mm size of clear zone

++: low support size 16-20mm

+++: Low support size 21-25mm

++++: size 25mm or more of low support

Example 4 Culture of Antagonist Microorganisms

Antagonist microorganism Streptomyces Kashgensis ZBIA-0927 strain was cultured using a 500L fermenter. The production medium used at this time was 15g soy flour, 200g glucose, 105g ammonium sulfate, 5g calcium phosphate, 25g magnesium sulfate and 1L distilled water. After rotting in to adjust the pH to 7.0 was used to prepare a liquid medium. Species culture was a triangular flask using yeast extract 0.1%, juicy extract 0.1%, glucose 1.0%. 1L of seed culture cultured at 30 ° C. at 150 rpm for 120 hours was inoculated into a 50 L fermenter containing 30 L of production medium, pre-incubated at 30 rpm for 150 hours at 150 rpm and 1vvm, and then containing 400 L of production medium. It was inoculated in a 500 L fermenter and incubated under the conditions of 30 ° C., 150 rpm, and 0.5 vvm until the highest antagonistic activity (168-180 hours from the start of fermentation).

Example 4 Antimicrobial Spectrum Comparison of Antagonist Streptomyces Kashgensis gibiae-0927 and Kasugamycin

The antimicrobial spectrum of the antagonist Streptomyces kashugensis ZBi-0927 strain and Kashugamycin known as antibiotics produced by Streptomyces kasugaensis were compared. Fusarium oxyporum , C. ladosporium cucumerium , and Pyricularia oryzae were used as the pathogens to be compared.

After sterilizing the potato agar medium, plate the mycelium, cells, and spores of each pathogen on a medium of 50 ° C, and after the medium is completely hardened, the paper disc (diameter 8 mm) is immersed in the culture medium in which the antagonist microorganism is cultured, Placed on a petri dish and incubated for 3 to 4 days at a temperature well grown various pathogens. The effect of the antagonistic microorganisms on various pathogens was investigated with a low support diameter, which is shown in Table 2.

As can be seen in Table 2, as a result of comparing the antagonistic effect of the antagonist Streptomyces Kashgensis gibi-0-0 strain and the antibiotic kasugamycin, Streptomyces Kashgen of the present invention Sisgei-0927 strain showed high inhibitory effects against Fusarium sp., Which causes wilting disease, and Cladosporium cucumerium , which causes black nephropathy , whereas antibiotic Kashgamycin has an inhibitory effect. Not shown at all. On the other hand, the antibiotic Kkasugamycin has a high inhibitory activity against Pyricularia oryzae .

Table 2.Antimicrobial Spectrum Comparison of Antagonist Streptomyces Kashgensis ZEI-0927 and Antibiotic Kashgamycin

Target bacteria Active size Streptomyces kasugaensis GBA-0927 Kasugamycin Fusarium oxysporum ++++ - Cladosporium cucumerium ++++ - Pyricularia oryzae ++ ++++

* +: Size of low support 10-15mm

++: low support size 16-20mm

+++: Low support size 21-25mm

++++: size 25mm or more of low support

As described above, the Streptomyces Kashgensis gibiae-0927 strain of the present invention has a wide range of activities against plant diseases, including wilting disease of the egg and melanoma disease of the cucumber, so that the microbial agent comprising the same Withered disease and black diseases of cucumber can be effectively controlled.

Claims (3)

Actinomycetes Streptomyces kasugaensis , an antagonistic microorganism with plant disease control ability GBA-0927 KFCC-11218 . Streptomyces kasugaensis GBA-0927 KFCC-11218 was mixed with 15 g of soy flour, 200 g of glucose, 105 g of ammonium sulfate, 5 g of calcium phosphate, and 25 g of magnesium sulfate in distilled water, and then in a liquid medium adjusted to pH 7.0. , A culture obtained by culturing under the conditions of 30 ℃, 150rpm, 0.5vvm from the start of fermentation to 168-180 hours. S treptomyces kasugaensis GBA-0927 KFCC-11218 or an antimicrobial agent for astigmatism and cucumber necrosis, which is an active ingredient thereof.