KR20050038662A - Antagonistic bacteria for protection of club root disease and method for produciting biopesticide by using the bacteria - Google Patents

Abstract

The present invention relates to a antagonist for the control of Chinese cabbage wart disease and a method for preparing the antagonist using the same, the present invention provides a bacterium Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) for the control of Chinese cabbage wart disease, In addition, the step Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) by aerobic culture in a nutrient medium to prepare an antagonist culture medium; Adsorbing the antagonist culture broth to an adsorbate and drying to prepare an antagonist dry matter; And it provides a method for producing antagonistic bacteria for the control of Chinese cabbage wart disease comprising the step of mixing the antagonist food ingredient to the antagonist dry.

Description

Antagonists for the control of Chinese cabbage wart disease and a method for producing the antagonists using the same {ANTAGONISTIC BACTERIA FOR PROTECTION OF CLUB ROOT DISEASE AND METHOD FOR PRODUCITING BIOPESTICIDE BY USING THE BACTERIA}

The present invention relates to an antagonist for the control of Chinese cabbage wart disease and a method for preparing the antagonist using the same.

Warty disease is a disease caused by a soil pathogen called Plasmodiophora brassicae, which causes fatal damage by adding cruciferous crops such as radish, cabbage, and cabbage. Pathogens are soil phagocytosis, invading through the roots of the roots to form cancerous nodules on the roots, and these humps squeeze the conduits, causing the leaves to wither due to inability to increase moisture from the roots. Rooted by a second invasion, the entire plant eventually dies. Warts create large amounts of dormant spores in the lesion area, and these dormant spores can survive in the soil for about 5 to 6 years, so once the warts have been infested, they are inevitably damaged each year. Thus, wart disease is the most frightening disease in the cultivation of cruciferous vegetables. Wart disease did not cause any damage to farmers until the end of the 1980s, but it started in 1991 in Goyang, Gimpo, Pyeongtaek, and Muju, resulting in a decrease of cabbage yield of about 30% in Goyang, Gyeonggi-do in 1994. In Hoengseong-gun in 1998, about 61% of the total cultivation area of 408 ha of Chinese cabbage, 250 ha, was impossible to harvest due to wart disease.

In wart disease, the biggest problem is that pathogens are present in the soil, and the density increases as the year progresses. Therefore, Gangwon-do, the main producer of high-fried cabbage, is proposing measures by declaring "War against Warriors", and in Taebaek, more than 30% of the total budget of the Agricultural Technology Center is devoted to combating warts, but with satisfactory results. The situation is not coming out. Radish and cabbage surges are known to be due to rainy seasons, natural disasters such as floods, and wart disease.

Conventional chemical pesticides have been used as a method for controlling warts disease.

At present, such control agents are known as Fluajinam (Fluajinam, 40 kg / 10a) and Fulsululfamide (Flusulfamide, 20 kg / 10a). It is known to suppress dormant spore germination, myometrial infection and skin infection, but the control value is very low at 50-60%, so the cabbage cultivation farmers are unstable throughout the growing season. The economic loss is very large.

As a means for controlling soil infectious diseases that are difficult to control by drug spraying such as wart diseases, biological control methods using antagonists have recently been studied, which causes environmental pollution, weakness, and residual toxicity. In addition to its advantages, it is considered to be a very desirable control method in that the control effect is long lasting.

The present invention is to solve the above problems, the object of the present invention is to find an antagonist having excellent control effect on wart disease, and to provide a method for producing an antagonist using the same.

The present invention provides Bacillus subtilis KB-291 (KFCC 11324), which is very effective in controlling cabbage wart disease.

In addition, the present invention comprises the steps of producing a antagonist culture medium by aerobic culture in Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) in a nutrient medium; Adsorbing the antagonist culture medium to an adsorbate such as degreasing steel, zeolite and vermiculite and drying to prepare an antagonist dry matter; And it provides a method for producing antagonists for the control of Chinese cabbage wart disease comprising the step of mixing the antagonist food components such as glucose, molasses, soy flour, jelly, fish meal and blood powder to the antagonist dry.

Bacillus subtilis KB-291 of the present invention (Bacillus subtilis KB-291, KFCC 11324) and antagonist using the same has an excellent effect in the control of cabbage wart disease.

Hereinafter, the present invention will be described in detail.

The present inventors conducted a study for about 3 years to establish a biological control method of cabbage wart disease by antagonists, and found an antagonist for the control of cabbage wart disease that is excellent in practical use in farming while the cabbage wart control effect is excellent. Based on the discovery, the present invention has been completed.

The present inventors isolated a total of 967 microorganisms in the cabbage root soil of 12 domestic cabbage jusan area to evaluate the antibacterial activity against pythium bacteria in the room and pot, safety evaluation of cabbage seeds and seedlings, artificial culture evaluation, As a result of evaluation of the ability to form a durable body, one kind of bacteria having the best effect was finally selected.

The main bacteriological characteristics of the selected bacteria are as follows.

[Characteristics in NA Medium]

1.Color: Light Yellow

2. Balance total form: round

3. Balance total surface: Convex type

4. Bacterial total size: 0.5∼2.0um

[Physiological characteristics]

Gram Reaction

2. Formation of resistant spore: presence

3. Cell type: Liver type

4. Mobility: U

5. Arc, Anaerobic: Aerobic

6. Anaerobic Growth: No

7. Catalase Production: U

8. Anaerobic Growth: No

9.VP test: positive

10. Oxidation

     Glucose: U

     Xylose: U

     Mannitol: U

     Arabian: U

11. Gas generation from glucose: no

12. Hydrolysis

     Casein: positive

     Gelatin: positive

     Rust: positive

13. Citrate usability: yes

14. Propionate Availability: No

15. Tyrosine resolution: no

16. Nitrate Reducing Ability

17. Indole formation ability: no

18. Salinity Requirements: No

19. Growth at 7% salinity:

20. Urea Hydrolysis: No

21. Growth by temperature

     5 ℃: Not available

    30 ℃: Available

    40 ℃: Available

    55 ℃: Not available

The present inventors named the bacterium of the present invention Bacillus subtilis KB-291 in consideration of the above physiological characteristics.

Antagonist Bacillus subtilis KB-291 of the present invention is It was deposited with the accession number of KFCC 11324 to the Korea Center for Microbiological Conservation on October 15, 2003.

The antagonistic bacillus subtilis KB-291 of the present invention has the activity of significantly inhibiting mycelial growth and spore germination of Psethium spp. Of cabbage wart fungi, and does not adversely affect crops or the environment without affecting crops or the environment. (For reference, Plasmodiophora brassicae, a cabbage wart fungus, is a parasitic bacterium and cannot be artificially cultured. Therefore, the antimicrobial activity was tested against Pythium, the most physiologically similar bacterium.)

Method of producing an antagonist for the control of Chinese cabbage anti-Witch disease, Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) by culturing aerobic in a nutrient medium to prepare an antagonist culture medium; Adsorbing the antagonist culture broth to an adsorbate and drying to prepare an antagonist dry matter; And mixing the antagonist food ingredient with the antagonist dry matter.

The adsorbate is preferably at least one selected from the group consisting of degreasing steel, zeolite and vermiculite, and preferably 10 to 200 times by weight relative to the antagonist culture medium.

The antagonist food ingredient is preferably at least one selected from the group consisting of glucose, molasses, soy flour, jelly, fish meal and blood meal, and preferably 0.1 to 20% by weight relative to the antagonist dry matter.

The antagonistic bacillus subtilis KB-291 of the present invention is used in the cabbage cultivation soil as a formulation containing a feed ingredient for breeding in the soil, specifically, after the cabbage seedlings evenly sprayed on the soil before the cabbage and cabbage It will grow cultivated. Antagonists applied to the soil will increase in density by breeding by using food ingredients such as rice bran, soy flour, salt, fish meal, blood meal contained in the formulation. Since the antagonistic bacteria have excellent antagonistic ability against WART pathogens, they inhibit the reproduction of WART pathogens in cabbage cultivation soil, weaken the root invasion activity, and cause the density of pathogens in the soil to drop rapidly. It is to suppress the onset of the disease.

Hereinafter, the present invention will be described in detail through Examples and Experimental Examples.

Experimental Example 1 Evaluation of Antimicrobial Activity of Antagonistic Bacillus Subtilis KB-291

In order to evaluate the antimicrobial activity of Bacillus subtilis KB-291 of the present invention, the antimicrobial activity against phytopathogens was evaluated along with several other antagonists. That is, the 0.45um filtration membrane filtrate of the antagonist NA culture medium was prepared and the results of the evaluation of the antimicrobial activity against three plant pathogens (lysatonia, pisium, fusidium) according to Paper Disc method are shown in Table 1 below.

Evaluation of Antimicrobial Activity against Three Plant Pathogens of Antagonistic Cultures Target pathogen Growth inhibitory activity of pathogens of antagonists (length of growth inhibitory ring, mm) KB-291 KB-14 KB-714 Raiztonia 18.4 14.6 2.5 Fishium 21.1 11.7 4.1 Fusedum 15.8 11.8 1.5

 As shown in Table 1, it can be seen that the antibacterial activity against phytopathogens of the antagonistic bacillus subtilis KB-291 of the present invention is superior to other antagonists (KB-14, KB-714), Bacillus subtilis KB-291 has excellent antimicrobial activity against three plant pathogens.

Experimental Example 2 Pot Test for the Simple Verification of the Control Effect of Cabbage Wart on the Antibacterial Bacillus Subtilis KB-291

   This test was conducted in a pot using soil that had previously caused severe wart disease, and investigated soil microbial density, wart disease incidence, and cabbage growth. That is, after treating 2.5ml of the antagonist culture medium in the pot (inner volume 2.5 liters) and cultivating cabbage seedlings for 60 days, the incidence and growth rate were investigated and the results are shown in Table 2 below. The control group is a case where the Chinese cabbage is grown without any treatment on the disease-free healthy soil, and the untreated group is a case where the cabbage is grown without any treatment on the wart-developed soil. Chinese cabbage is grown.

Pot Test Results Treatment result Days to First Development (Days) Final Incidence (%) Chinese cabbage rate (commodity rate,%) Soil microbial density after 60 days (cfu / g) Germ (X100,000) Mold (X10,000) Control 57 2 100 548 64 No treatment 15 98 17 193 163 Processing Unit-1 47 18 92 582 47 Processing Zone-2 33 34 78 427 82 Processing Zone-3 25 55 62 345 139

Processing Zone-1: KB-291 Processing, Processing Zone-2: KB-14 Processing, Processing Zone-3: KB-714 Processing

As shown in Table 2, when treated with the antagonistic Bacillus subtilis KB-291 of the present invention (treatment-1), the first onset took 47 days, the final incidence of cabbage harvest is 18%, The cabbage product rate is 92%, which is much better than the untreated or other treatment. In addition, when treated with KB-291, the density of bacteria was much higher than that of other treatments, and the density of mold was lower.

Example 1 Preparation of Antagonist (Prototype) Containing Antagonist

 Antagonist KB-291 was cultured in a liquid aerobic culture in NA medium for 5 days to prepare a culture solution, and then mixed in 50 times the defatted steel by weight ratio and dried at 40 degrees to prepare an antagonist dry matter. In order to promote the early propagation of antagonistic bacteria in soil, glucose, molasses, and soybean powder were added to the antagonist dried by weight ratio of 1%, 3%, and 10%, respectively, to prepare antagonistic agents. Subsequent tests were carried out using the formulations thus prepared.

Experimental Example 3 Storage Stability Test of Antagonist

The survival of the antagonist KB-291 of the antagonist preparation prepared in Example 1 was analyzed over time, and the results are shown in Table 3 below.

Time-lapse Survival Results of Antagonists in Antagonist Preparations division Early 30 days later 60 days later 100 days later 150 days later 200 days later 250 days later 300 days later 360 days later Survival rate (%) 100 100 100 99 100 100 100 100 100

 As shown in Table 3, the antagonistic KB-291 was confirmed to survive for at least one year in the product.

Experimental Example 4 Effects of Antagonist Drugs on Cabbage Wart Disease (Greenhouse Test)

 The greenhouse test was carried out by cultivating 50 cabbages at an area of 5 pyeong per test area in a plastic house of Chinese cabbage cultivation farmer in Jeongnam-myeon, Hwaseong-si, Gyeonggi-do using the antagonist preparation prepared in Example 1. First, a sufficient amount of cabbage root humps with wart disease was applied to the soil and mixed with soil, and after treating the antagonists and the control pesticides of the present invention, seedlings were formulated and 60 days of cultivation of Chinese cabbage was used. It was. The test results are shown in Table 4 below.

Effect of Antagonist Drug on the Control of Chinese Cabbage Wart Disease (Greenhouse Test) Treatment result Days to elapse before first outbreak Final Incidence (%) Chinese cabbage rate (commodity rate,%) Soil microbial density after 60 days (cfu / g) Germ (X100,000) Mold (X10,000) No treatment 13 96 14 368 137 Control-1 36 18 82 86 61 Control-2 39 16 84 116 88 Treatment 43 12 92 496 115

  * Control-1: Chemical pesticide (trade name "Anna") treatment

  * Control-2: Chemical pesticide (trade name "Fronside") treatment

 As shown in Table 4, when treated with pesticides it takes 36 to 39 days to the initial onset and the final incidence was found to be 16 to 18% level, when treated with the formulation of the present invention 43 days until the first onset , The final incidence is 12%, it can be seen that the antagonist of the present invention exhibits a control effect over the control pesticide. In addition, in the case of cabbage product rate, the pesticide treatment group was 82-84%, the formulation treatment group of the present invention appeared 92%, it was confirmed that the antagonist agent of the present invention also increases the productivity of the Chinese cabbage. In addition, as a result of the investigation of bacterial density in the soil, the control pesticide was shown to adversely affect the soil microorganisms by drastically lowering the bacterial density in the soil, but the antagonist agent of the present invention improves the soil microorganism by increasing the bacterial density rather than Appeared to.

Experimental Example 5 Effects of Antagonist Drugs on Cabbage Wart Disease (Farm Farming Test-1)

  Using the antagonist preparation prepared in Example 1 was carried out at the site 1,200 pyeong of cabbage cultivation farm in Jinwi-myeon Pyeongtaek-si, Gyeonggi-do. The cabbage field used for the test is a soil that causes severe wart disease every year, and this test is commonly used for controlling wart disease in farms as well as the formulation of the present invention from April 2, 2002 to June 8, 2002. Investigate the incidence of wart disease and Chinese cabbage product while growing Chinese cabbage (type name: Bulam No. 3) by treating each company's antagonistic bacteria and chemical pesticides in 300 pyeong each, and the results are shown in Table 5 below.

Effect of Antagonist Drug on the Control of Chinese Cabbage Wart Disease (Farm Packaging Test-1) Treatment result Days to First Development (Days) Final Incidence (%) Chinese cabbage rate (commodity rate,%) Soil bacterial density at the end of the test (cfu / g) No treatment 17 99 12 132 Control-1 38 16 88 35 Control-2 31 36 72 102 Treatment 44 10 96 328

* Control-1: Chemical pesticide (trade name "Anna") treatment

* Control-2: Treatment with a commercially available antagonist (trade name "Sagumatan")

As shown in Table 5, the antagonist treatment treatment of the present invention is compared to the case of treatment with commercially available antagonists or chemical pesticides in all sectors, such as the number of days until the first onset, final incidence rate, cabbage product rate, bacterial density in the soil, etc. It shows an excellent effect. That is, in the case of the preparation treatment of the present invention, by antagonistic Bacillus subtilis KB-291 multiply vigorously by feeding the nutrients in the preparation in the soil to form a constant force to suppress the growth of Warts pathogen near the root of Chinese cabbage Because of the prevention of root invasion of pathogens, it is thought to have excellent wart control effect.

Experimental Example 6 Evaluation of Wart Disease Control Effect and Chinese Cabbage Growth by Antagonist (Agricultural Packaging Test-2)

   By using the antagonist preparation prepared in Example 1 was carried out at the cabbage cultivation farm site in Shangmyeon-myeon, Haenam-gun, Jeollanam-do. The cabbage field used for the test is soil that causes severe wart disease every year, and this test was conducted from September 6, 2002 to November 10, 2002 with 500 pyeong of each of the preparations and chemical pesticides (or Anna) of the present invention. The incidence of WART disease and the growth of Chinese cabbage were investigated while growing Chinese cabbage (type name: CR cabbage). The results are shown in Table 6. The cabbage ground reproductive weight and root weight were estimated by randomly collecting 10 healthy disease-free cabbages from the test paper.

Effect of Antagonists on Wart Disease Control and Evaluation of Chinese Cabbage Growth (Farm Packaging Test-2) Treatment result Final Incidence (%) Chinese cabbage (product rate,%) Chinese cabbage ground weight (g) Chinese cabbage root weight (g) Control 18 86 2,283 15.6 Treatment 12 92 2,582 17.8

* Control: Chemical pesticide (trade name "Anna") treatment

As shown in Table 6, the preparation treatment of the present invention shows a more excellent effect than the control (chemical pesticide) in the final incidence rate, cabbage product rate and the like. In addition, the antagonistic bacillus subtilis KB-291 also appeared to be heavier in the cabbage ground portion and root weight, the preparation of the present invention was confirmed to enhance the growth of Chinese cabbage as well as the control of Chinese cabbage wart disease. This is because Bacillus subtilus KB-291 has been actively fed by feeding the nutrients in the preparation in the soil, solubilizing insoluble nutrients in the soil, or antagonists secreted growth promoting ingredients necessary for the growth of cabbage, thereby increasing the growth of cabbage. It is considered to be.

Bacillus subtilis KB-291 of the present invention (Bacillus subtilis KB-291, KFCC 11324) has an excellent control effect against cabbage wart disease, Bacillus subtilis KB-291 antagonist using the same as can be seen in the experimental example It has been shown to be very effective in controlling Chinese cabbage wart disease than conventionally disclosed chemical pesticides and other antagonist agents.

Claims (3)

Bacillus subtilis KB-291, KFCC 11324 for the control of Chinese cabbage wart disease. Antagonist culture prepared by culturing the Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) of claim 1. Claim 1 Bacillus subtilis KB-291 (Bacillus subtilis KB-291, KFCC 11324) by aerobic culture in a nutrient medium to prepare an antagonist culture medium; Adsorbing the antagonist culture broth to an adsorbate and drying to prepare an antagonist dry matter; And Method for producing antagonistic bacteria for the control of Chinese cabbage wart disease comprising the step of mixing the antagonist food ingredient to the antagonist dry.