WO2002034884A1

WO2002034884A1 - Streptomyces kasugaensis inhibiting the fungal pathogens of plant

Info

Publication number: WO2002034884A1
Application number: PCT/KR2001/001799
Authority: WO
Inventors: Jae Ho Lee; Ki Hyeon Choi; Sung Won Choi; Ji Tae Kim; Ki Suk Doh; Jae Seong Yang
Original assignee: GREEN BIOTECH CO Ltd
Current assignee: GREEN BIOTECH CO Ltd
Priority date: 2000-10-25
Filing date: 2001-10-24
Publication date: 2002-05-02
Anticipated expiration: 2003-04-25
Also published as: KR20020031961A; AU2002211050A1

Abstract

The present invention relates to Streptomyces kasugaensis GBA-0927 which is an antagonist microorganism isolated from soils and show strong antifungal activity against plant fungal pathogens, wherein said plant fungal pathogens comprise Fusarium oxyporum, Cladosporium cucumerium or Botrytis cineria.

Description

STREPTOMYCES KASUGAENSIS INHIBITING THE FUNGAL PATHOGENS OF PLANT

Field of the Invention The present invention relates to a microorganism which is effective against plant fungal pathogens and a preparation containing the microogranism. The present invention also relates to Streptomyces kasugaensis GBA-0927 that exhibits antagonistic activity against plant pathogens, and a preparation containing the same.

Background of the Invention

About 4,000 kinds of various disease and insect pest which directly reduces yield and quality of crops and flowering plants, have been known in Korea.

In order to control such disease and insect pest, organosynthetic agrocultural chemicals have been inevitably used up to the present; actually, organo-synthetic agrochemicals as being very efficient means for crop protection, have played an important role for increasing the amount of food resources, stable supply of the products, and reduction of labor of farmers.

However, recently, negative insight on abuse, misuse, toxicity, environmental pollution and bad influence on ecosystem, and side effect such as decrease of efficiency according to appearance of tolerant-strain against pesticide, has been gradually increased, thereby promoting social requisite for intending safety, healthy and nature with advanced nations as leader. The social circumstance around organosynthetic agrocultural chemicals that played an important role in effective food production is getting stricter. Thus, much attention is paid for developing biopesticide as an alternative means which can overcome the disadvantage of such organosynthetic agricultural chemicals.

Biopesticide use microorganism (fungi, bacteria, virus) as natural resources, or active substances produced by microorganism. They are practically applied to the plant an effective amount of the fungicide, insecticide, herbicide, growth regulator and injury-control. Such a biopesticide, when compared with organosynthetic agricultural chemicals, shows low toxicity, gives less influence on ecosystem, and shows no tolerance and resistance to preparation, however, it occasionally has disadvantages of slow revelation of effect, low product stability or difficult process for preparation. Thus, up to recent days, the ratio of biopesticide to organosynthetic agricultural chemicals in the whole pesticide market has been extremely feeble. However, as described above, for the purpose of preventing environmental pollution caused by abusing of organosynthetic agricultural chemicals, much attention has been paid these days for developing economical biopesticide with high effect, low toxicity and no pollution. In foreign countries, the research for preventing disease and insect pest by the use of microorganism was firstly tried in the United States by using Actinomvcetes for controlling potato scab in 1927, and then microorganism has been practically used as biopesticide since 1960. In Japan, various products have been developed starting from the living microorganism and Trichoderma preparation to treat tobacco stem wilt in 1962, while most of the practically used products are developed to control damping- off on seedlings.

At present, about 70 kinds of biological control agent using microorganism itself are practically used in foreign countries. Besides, there are biopesticide using active substances produced by microorganism. Active substances produced during cultivation of microorganism are used as antibiotics for medicine and agriculture after isolation and purification. About twenty kinds among them are apply to use for agriculture, and six kinds among them are widely used for controlling of disease and insect pest. Most of the microorganisms producing antibiotics for agriculture are strains belonging to Streptomyces genus among Actinomycetes. Starting from the first agricultural antibiotic Blasticidin S for controlling rice blast, in 1958, Kasugamycin, Polyoxin, Validamycin or the like have been applied. Those antibiotics for agriculture take advantage of metabolite produced by microorganism, not the microorganism itself, so that the evaluation of safety as a pesticide is performed in the equivalent level of organosynthetic agricultural chemical, but showing very low toxicity.

In Korea, basic researches on the biopesticide have been performed since 1980's, and few products are practically used at present. Recently, continuous efforts of experts in laboratories have given good results of highly enhanced research level as compared to short research history. The researches on biopesticide for disease and insect pest by the use of microorganism itself include researches on tobacco mosaic virus and bacterial wilt in tobacco, Fusarium wilt in cucumber, Phytophthora blight in pepper, wilt disease in strawberry, Rhizoctoina bud rot, damping-off in sugar beet, rice blast, or the like, by the pathologist of National Research Institutes or University since 1985.

A few conventional domestic or foreign Laid-Open patents similar to the present invention exist; among them there is a patent concerning the production of Kasugamycin as antibiotic for agriculture, and products for the control of wilt disease caused by bacteria or fungi. However, there was not report relates to the control of disease caused by pathogens Fusarium oxyporum, Cladosporium cucumerium or Botrytis cineria by the use of Streptomyces kasugaensis GBA-0927. Though it is widely known that the antibiotics Kasugamycin is produced by Streptomyces kasugaensis, it is confirmed that metabolite produced by Streptomyces kasugaensis GBA0927 was different from Kasugamycin according to the metabolites produced by Streptomyces kasugaensis GBA0927 shows only antagonism against the Fusarium oxysporum, Cladosporium cucumerium or Botrytis cinerea.

In case of cucumber, the damage level caused by Cladosporium cucumerium is relatively feeble as compared to those of important disease such as gray mold, anthracnose or powdery mildew. However, once it is contracted, the disease is very difficult to treat, so that the prevention is very important. In case of orchid, the wilt disease caused by Fusarium oxyporum is one of the important root diseases including root rot, whiteness and dark rot, thus gives much damage on orchid that is very popular with high added value. In case of tomato, the gray mold is one of the important disease to cause much damage to the grower farm household.

Summary of the Invention The present invention is intended to solve the problems described above, and designed according to the requisites mentioned above. The object of the present invention is to provide a microorganism showing antifungal effect against significant plant fungal pathogens. In order to achieve above-mentioned object, the present invention provides Streptomyces kasugaensis GBA-0927 (KCCM- 10326) as a microorganism to control plant fungal pathogen.

The microorganism according to the present invention is effective on diseases caused by Fusarium oxyporum, Cladosporium cucumerium or Botrytis cinerea.

Further, the present invention provides pure culture of above strains, and a composition of microorganism preparation comprising pure culture of the strains.

For the process for preparing the composition of microorganism preparation according to the present invention, conventional methods in the technical field to which the invention belongs were employed, while various salts or the like may be added to enhance the activity.

Detailed Description of the Invention

Now, the present invention is described in more detail.

The present invention relates to antagonistic microorganism for controlling plant pathogens. The crops and pathogens to be applied is Cladosporium cucumerium of cucumber, Fusarium oxyporum of orchid or Botrytis cinerea of tomato.

The present inventors made intensive effort to find a microorganism having an antifungal activity against plant pathogens from soils. As a result, they isolated antagonistic microorganism Streptomyces kasugaensis GBA-0927, and completed the present invention by showing the effective control of important disease and damage of crops caused by fungal pathogens using the microorganism preparation comprising the antagonistic microorganism. The active ingredient of the present microorganism preparation, Streptomyces kasugaensis GBA-0927, is a microorganism isolated from domestic natural soil, that is confirmed to be non-pathogenic as a result of pathogenic tests on various plants.

The microorganism preparation according to the present invention has antagonistic effect on the fungal pathogens such as Pythium sp., Fusarium sp., Botrytis cinerea and brown patch, large patch, or the like, however, it showed particularly strong antagonistic effect against important plant pathogen causing Fusarium wilt of orchid, rice blast, anthracnose of cucumber or gray mold of tomato.

[Example]

The invention is described in more detail by referring to the examples below, but it should be noticed that those examples are described only to specifically describe the present invention, so that the present invention is not restricted to the examples by any means.

Example 1 : Detection and isolation of antagonistic microorganism

As the plant pathogens, used were 12 kinds of pathogens which do much harm to plants, which include Botrytis cinerea, Fusarium sp., Pythium sp., Phytophtora capsici, Aiternaria citri, Cladosporium cucumerium, Phytophthora infestans, Puccinia recondite, Erysiphe graminis f. sp. hordei, Magnaporthe grisea, Rhizoctonia solani MAFF 511103 (brown patch), Rhizoctonia solani MAFF 305245 (large patch).

In order to isolate the microorganism having antagonistic activity against above publicly known pathogenic microorganism, soil samples were taken from farm and forest soils for every season in Kyunggi-do and Kangwon-do area. To isolate the antagonistic microorganism, conventional triple plate method was applied. To 250 g of fertile soil, twice a volume of distilled water was added. After sterilizing at high pressure, the soil was filtered through gauze. Volume of the filtrate was adjusted to 1 L by using distilled water, and agar (15 g) is dissolved therein. The solution was added by 10 ml to 30 ml test tubes, which were then sterilized at high pressure according to conventional method, and stored in an incubator at 50°C.

The soil sample (1 g) taken as above was well suspended in 100 ml of sterile physiological saline, and 2~3 drops of the suspension were added to the test tube to be well mixed, and the mixture was immediately poured into a petri dish to be solidified. On the solidified petri dish, agar solution (5 ml) was added to be solidified. Then, spore suspension of plant pathogens (1 x 10^s cfu/ml) was prepared from the subculture medium. To potato-agar medium (10 ml) prepared in 50°C incubator, 2^3 drops of the suspension were added and well mixed. The mixture was added to the above petri dish again to prepare triple plate culture medium. The prepared plate medium was cultured in an incubator at 30°C for 3~7 days, and the antagonistic effect (evaluated by formation of clear ring) against the object plant pathogen was examined, and excellent microorganism was purely isolated by using conventional method. According to the selected strain, the microorganism was cultured by using liquid culture medium, and the diameter of clear ring was examined by using conventional symmetric culture method. The antagonistic microorganism GBA-0927 having the highest antagonistic activity was selected.

Example 2 : Cultivation of antagonistic microorganism The antagonistic microorganism GBA-0927 was cultured by using 500 L fermenter. Liquid culture medium comprising corn starch 1%, glucose 0.5%, calcium carbonate 0.02%, yeast extract 0.5%, corn steep liquer 0.5%, of which pH was adjusted to 6.8, was employed in seed culture and main culture. The seed culture (5 L) cultured at 30°C, 200 rpm for 72 hours was inoculated in a 500 L fermenter containing 300 L of production medium, and cultured under the condition at 30°C, 120 rpm, 1 vvm until the point of highest antagonistic effect (168 hours from initiation of fermentation).

Example 3 : Identification of antagonistic microorganism against the fungi causing significant disease of crops

As a result of 16S rDNA base sequence analysis, the strain of the antagonistic microorganism was identified as Streptomyces kasugaensis and named as GBA-0927. The strain was deposited with KFCC located in Hongje-dong, Seodaemun-gu, Seoul, Korea as deposit No. KFCC-10326 on October 4, 2000 (see Identification of Strain attached).

Example 4 : Effect of growth control against various plant pathogens

As the plant pathogens, used were 12 kinds of pathogens which do much harm to plants, which include Botrytis cinerea, Fusarium sp., Pythium sp., Phytophtora capsici, Alternaria citri, Cladosporium cucumerium, Phytophthora infestans, Puccinia recondite, Erysiphe gram in is f. sp. horde! , Magnaporthe grisea, Rhizoctonia solani MAFF 511103 (brown patch), Rhizoctonia solani MAFF 305245 (large patch) . To sterilized potato-agar medium at 50°C, hyphae, cells or spores of each pathogen was mixed and plated. After the medium was completely solidified, the cultured solution of antagonistic microorganism was applied on a paper disc (diameter 8 mm), and placed on a petri dish wherein the pathogen was mixed. It was cultivated at optimal temperature of each pathogen for 3^4 days. The effect of the antagonistic microorganism on each pathogen was examined by diameter of the inhibition zone diameter (Table 1).

As can be seen from Table 1, as a result of examining antagonism of the antagonistic microorganism Streptomyces kasugaensis GBA-0927 against plant pathogens, the antagonistic microorganism showed antifungal activity against Fusarium sp. (which causes Fusarium wilt), Cladosporium cucumerium, Magnaporthe grisea (which causes rice blast), Botrytis cinerea, Pythium sp., Phytophthora infestans (which causes Phytophthora blight), Rhizoctonia solani MAFF 511103 (brown patch), and Rhizoctonia solani MAFF 305245 (large patch).

[Table 1] Antifungal spectrum of antagonistic microorganism Streptomyces kasugaensis GBA-0927

* + : Size of clear zone : 10^15 mm 4-+ : Size of clear zone : 16~20 mm + + + : Size of clear zone : 21~25 mm + + + + : Size of clear zone : 25 mm or more

Example 5 : Prevention effect against various plant pathogens As the plant pathogens, used were 9 kinds of pathogens, which include Botrytis cinerea, Fusarium sp., Cladosporium cucumerium, Phytophthora infestans, Puccinia recondite, Erysiphe graminis f. sp. hordei, Magnaporthe grisea, Rhizoctonia solani MAFF 511103 (brown patch), Rhizoctonia solani MAFF 305245 (large patch). Preparation of microorganism treatment was performed by placing each 3 pots of plants for each plant disease on a turn table, and evenly spraying the preparation on the whole body of the plant by using spray gun (1 kg/cm²) with turning the table round. The treated plant was stored indoor for 1 day, and pathogen was inoculated to measure the control value.

[Table 2] Control value of antagonistic microorganism

Streptomyces kasugaensis GBA-0927

* + : Control value 25% or less + + : Control value 26~>50% + + + : Control value 51^75% ++ + + : Control value 76% or more

Example 6: Comparison of antifungal spectum of antagonistic microorganism Streptomyces kasugaensis GBA-0927 with Kasugamycin

Antifungal spectum of antagonistic microorganism Streptomyces kasugaensis GBA-0927 was compared with that of kasugamycin which had been already known as antibiotics produced by Streptomyces kasugaensis. Target pathogens to be compared were Fusarium oxyporum, Cladosporium cucumerium and Botrytis cinerea. To sterilized potato-agar medium at 50°C, hyphae, cells or spores of each pathogen was mixed and plated. After the medium was completely solidified, the cultured solution of antagonistic microorganism was applied on a paper disc (diameter 8 mm), and placed on a petri dish wherein the pathogen was mixed. It was cultivated at a temperature of good growth of each pathogen for 3~4 days. The effect of the antagonistic microorganism on each pathogen was examined by diameter of the prevention circle (Table

3).

As can be seen from Table 3, as a result of comparing antagonistic effect of the antagonistic microorganism Streptomyces kasugaensis GBA-0927 with that of antibiotic kasugamycin, antagonistic microorganism Streptomyces kasugaensis GBA-0927 showed excellent antifungal effect against Fusarium sp. Cladosporium cucumerium and Botrytis cinerea.

[Table 3] Comparison of antifungal spectrum of antagonistic microorganism Streptomyces kasugaensis GBA-0927 with that of kasugamycin

* - : Size of clear zone : 0mm 4- : Size of clear zone : 10~15 mm + + : Size of clear zone : 16~20 mm +++ : Size of clear zone : 21^25 mm + + + + : Size of clear zone : 25 mm or more

As described above, the strain Streptomyces kasugaensis

GBA-0927 has broad spectrum against important fungal diseases including Fusarium wilt in orchid, scab in cucumber or gray mold in tomato, so that a preparation comprising the microorganism can effectively prevent the plant damage caused by fungal diseases.

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

To Jae-ho Lee

45-70 Yadang-π, Gyoha-myeon, Paju, RECEIPT IN THE CASE OF AN ORIGINAL

Gyeonggi-do 413-830, issued pursuant to Rule 7 1 by the

Korea INTERNATIONAL DEPOSITARY AUTHORITY identified at the bottom of this page

Claims

What is claimed is:

1. Streptomyces kasugaensis GBA-0927[KCCM- 10326] as a microorganism against fungal pathogen.

2. The microorganism according to claim 1, wherein the microorganism is effective on plant disease caused by Fusarium oxyporum, Cladosporium cucumerium or Botrytis cinerea.

3. Pure culture of the microorganism according to claim 1.

4. A composition of microorganism preparation comprising pure culture of the microorganism according to claim 1 as an active component.