KR20030087289A - The antagonistic microorganism bacillus sp. big21003 and the preparation method of pelleted seed including thereof - Google Patents

Abstract

PURPOSE: An antagonistic microorganism Bacillus sp. BIG21003 and a preparation method of a pelleted seed including the same are provided, thereby effectively controlling the infection of Plasmodiophora brassicae in plants. CONSTITUTION: An antagonistic microorganism Bacillus sp. BIG21003(KCTC 10236BP) to soil pathogenic bacteria is provided. A pelleted seed including the antagonistic microorganism Bacillus sp. BIG21003(KCTC 10236BP) is provided. A method for preparing the pelleted seed including the antagonistic microorganism Bacillus sp. BIG21003(KCTC 10236BP) comprises the steps of: culturing Bacillus sp. BIG21003(KCTC 10236BP) to produce spores; mixing the spores with a weight increasing agent consisting of calcium carbonate, kaolin, surfactant, silica and clay to prepare the weight increasing mixture; dissolving one or more materials selected from methyl cellulose, ethyl cellulose, dextran, Arabic gum, xanthan gum, paraffin oil, gelatin, starch, polyvinyl alcohol, polyethyleneglycol and carboxymethyl cellulose in water to prepare slurry; spraying the weight increasing mixture and the slurry to seeds in a pellet making machine to produce the sphere pelleted seed; and selecting and drying the seed.

Description

Antagonist microorganism Bacillus biy 21003 and method for producing pellet seeds containing the same {THE ANTAGONISTIC MICROORGANISM BACILLUS SP. BIG21003 AND THE PREPARATION METHOD OF PELLETED SEED INCLUDING THEREOF}

The present invention relates to a microbial Bacillus genus BIG21003 showing excellent antagonistic effects on wart bacteria and a method for producing pellet seeds containing the same.

Plsamodiophora brassicae is one of the soil infectious diseases of cruciferous crops. Plants infected with this germ show normal vitality at first but gradually wither to the ground. Young plants die in a short period of time, and delayed onset of plants that have continued to grow, but are not alive, so the value of the product is greatly reduced.

Even if the wart germs survive in the soil for more than 10 years, the cultivation of cruciferous crops in soil contaminated with this pathogen should be avoided.

Warts are causing great damage in all parts of the world where cruciferous crops are grown, such as Europe, North America, and Asia.In Korea, cabbage warts caused by Warts have been used in cabbage cultivation complexes in Gyeonggi-do, Gangwon-do and Jeonbuk since 1990. And causing massive damage to cabbage farmers.

As a method of controlling wart germs, the control of soil pH through lime application is known, but these germs have been reported to cause disease in a wide pH spectrum, and the control of soil pH alone is limited.

In addition, there is a soil fumigation method using methyl bromide, chloropicrin, dazomet, etc. to control the warts, but in this case, there are problems such as destruction of the soil ecosystem and environmental pollution due to excessive use of pesticides.

In addition, the soil treatment of Fluazinam or Flusulfamide powder is expensive to control, exerting a wide range of bactericidal action to useful microorganisms, there is a problem that causes the soil degradation by soil ecosystem destruction.

The present invention relates to antagonistic microorganisms for controlling soil diseases, Korean Patent Application No. 10-1999-0043807 (Bitty-containing microbial insecticide), Korean Patent Application No. 10-1999-0002239 (Pesticide composition comprising a microorganism having an antagonistic action to pests), Korean Patent Application 10-2000-0041858 (New Bacillus sp. Strain and soil microbial agent using the same), Korean Patent Application 10-1999-0034872 (Plant antagonist microbial agent), Korean Patent Application 10-1988-0001416 Antagonist microorganisms and the method of controlling pepper blight using this antagonist microorganism), but the antagonistic bacteria have a weak antagonistic effect.

On the other hand, when pelleting the cabbage seeds and the like, there is a problem that the germination rate and germination is lower than the seed without pelleting.

The present invention is to provide a novel microorganism Bacillus genus BIG21003 having a high antagonistic effect on warts.

It is also an object of the present invention to provide pellet seeds containing Bacillus genus BIG21003 of the present invention with high germination rate and high germination.

1 is a photo showing the antagonistic effect of the bacterium BIG21003 of the AC-1 strain against the bactericidal bacterium

Figure 2 is a graph showing spore production according to Glucose concentration in culture of Bacillus BIG21003.

The present invention relates to a microbial Bacillus genus BIG21003 showing excellent antagonistic effects on wart bacteria and a method for producing pellet seeds containing the same.

According to the present invention, Bacillus genus BIG21003 is provided that exhibits an antagonistic effect on soil pathogens such as late blight, wearing disease, and wilt disease, and particularly exhibits excellent antagonistic effect on wart disease.

In addition, there is provided a method of producing pellet seeds having a high germination rate and a high germination rate, while exhibiting excellent antagonistic effects on wart germs.

A slurry solution prepared by culturing Bacillus BIG21003 to make a foaming agent, mixing the foaming agent with an extender made of calcium carbonate, kaolin, surfactant, diatomaceous earth, and clay, and dissolving the binder gum in water. Then, the seed in the pelleting machine was repeatedly sprayed with a bulking agent and a slurry solution to make a spherical pellet seed, and then screened to a predetermined size and dried at low temperature, and pellet seeds containing Bacillus BIG21003 of the present invention. Manufacture.

On the other hand, in the preparation of the pellet seed of the present invention, the antagonistic microorganisms include Bacillus BIG21003, as well as Agrobacterium sp., Bacillus sp., Pseudomonas sp., Stereptomyces sp., Burkholderia sp. Bacteria, Gliocladium sp., Trichoderma sp. And antagonistic microorganisms derived from non-pathogenic Fusarium and Pythium . Moreover, synergistic effect can also be improved by adding 1 type, or 2 or more types of antagonistic microorganisms simultaneously.

In addition, in the preparation of the pellet seed of the present invention, as seeds, as well as cabbage seeds, radish, carrot, cabbage, spinach, lettuce, lettuce, watermelon, melon, cucumber, pumpkin, tomato, pepper, onion, green onion, deodeok, grass, Tobacco, wild vegetables, flowers, tree seeds, grass seeds, etc. may also be used.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but these do not limit the scope of the present invention.

Example 1 Selection and Identification of Antagonistic Microorganism Bacillus BIG21003 from Soil

Soil samples from rice fields, fields, and forests were collected from 20 regions of the country.

100 species of bacteria showing antifungal activity were first selected using various media (Nutrient agar, Psedomonas separation medium: EMB, actinomycetes separation medium) at a culture temperature of 30 ° C. and pH 6.5.

Among the 100 species selected primarily, 13 species were selected by selecting the strains which are particularly excellent against late blight, wearing disease and withering disease.

13 selected strains were treated on the soil and seeded with Chinese cabbage, and then inoculated with the wart germ. One strain having excellent growth promoting effect and excellent wart control effect was selected and named BIG21003.

The selected BIG21003 strain was identified as a G (+) strain by Gram staining, and has been characterized as endogenous spores when the carbon source is depleted and identified as Bacillus.

The ecological and biochemical properties of Bacillus BIG21003 are shown in Table 1 below.

<Table 1> Morphological and biochemical characteristics of Bacillus BIG21003 strain

Morphological characteristics result shape Bacillus Gram Dyeing positivity Sporulation Resistant sporulation motility has exist Biochemical properties Growth temperature range 6-40 Optimum growth temperature 30 ℃ Growth pH Range 5.0 to 8.0 Optimal growth pH 6.5 Oxygen composition Aerobic

Experimental Example 1 Investigation of Antimicrobial Activity of Bacillus BIG21003 Against Soil Infectious Pathogens

In a medium treated with Bacillus BIG21003, representative plant pathogens PG: Pyricularia grisea , PU: Pythium ultimum , RS: Rhizoctonia solani , BD: Botryosphaeria dothidea , BC: Botrytis cinerea , CG: Colletotrichum gloeosporioides , MM: Mycosphaerella Phytophthora capsici , AS: Alternaria solani and FO: Fusarium oxysporum were inoculated to investigate the antimicrobial activity.

As a result, Bacillus genus BIG21003 of the present invention showed very good antimicrobial activity against 9 species including PG among 10 plant pathogens, and showed excellent antimicrobial activity against the other one (PU).

Experimental Example 2 Investigation of Antagonistic Effect of Bacillus BIG21003 against Red Pepper Bacteria

The antagonistic effect of Bacillus genus BIG 21003 of the present invention was investigated against a representative soil pathogen, Phytophthora capsisi (PC), which has a similar ecology to wart disease.

Developed by the Institute of Agricultural Science and Technology as a control bacterium, AC-1 strain having a broad spectrum against soil infectious pathogens was used.

Bacillus genus BIG21003 and bacteriostatic bacterium and AC-1 strains were inoculated and cultured in a medium mixed with PDA and NA 1: 1 to confirm the mycelial growth inhibition range and the results are shown in the photograph in FIG.

Fig. 1A is a bacterium BIG21003 and pepper bacillus and control bacterium AC-1 at the same time inoculated and cultured.

It can be seen that Bacillus genus BIG21003 of the present invention grows vigorously and spreads to the pepper germ disease.

Figure 1 B is inoculated and cultured control bacterium AC-1 and Capsicum B. pneumoniae at the same time. It can be seen that the control bacterium AC-1 is antagonistic due to the bacteriostatic bacterium.

As described above, the Bacillus genus BIG21003 of the present invention showed excellent antagonistic effects against the causative bacterium bacterium, which is a representative soil pathogen.

Bacillus sp. BIG21003 ( Bacillus sp. BIG21003) of the present invention was deposited with the accession number KCTC 10236BP to the Gene Bank (KCTC), an international microbial deposit institution for the application of April 20, 2002.

On the other hand, pellet seeds generally tend to have lower germination rates compared to seeds without pelleting.

Germination rate of the pellet seed is greatly affected by the extender, the present inventors repeated a number of experiments as shown in Experiment 3 to find the optimum component and composition ratio of the extender.

Experimental Example 3 Investigation of Germination Rate According to the Components and Composition of the Extender

In the production of pellet seeds, germination rate of the cabbage seeds was investigated to determine the optimum composition and proportion of the extender.

Seed samples were from mountain village cabbage sold by Eastern Hannong.

Pellet seeds of Chinese cabbage were prepared by varying the composition and composition ratio of the extender as shown in Table 2 below, and remaining conditions and methods in the same manner as in Example 2 described below.

<Table 2> Extender Composition Ratio Manufacturing Table

Composition ratio (%) Calcium carbonate Vermiculite Limestone Bentonite Wood powder kaoline Surfactants Carbonate Diatomaceous earth clay Talc ocher ceramic One 30 20 20 30 2 30 10 10 30 20 3 30 10 30 10 20 4 30 20 20 10 20 5 10 10 50 10 20 6 10 20 10 10 50 7 30 40 10 10 10 8 40 10 10 20 20 9 20 30 20 20 10 10 30 20 20 20 10 11 20 20 40 20 12 20 20 30 20 20

The germination rate of each prepared cabbage seed pellets was investigated and the results are shown in Table 3 below.

Table 3 Results of germination rate of Chinese cabbage pellet seeds according to the composition of the extender

Creation costs Germination rate (%) 2 repetitions No treatment Pellet seed One 100 60 2 98 86 3 98 70 4 98 88 5 96 65 6 96 50 7 100 98 8 100 90 9 96 56 10 98 92 11 100 66 12 98 62

As shown in Table 3, 2,4,7,8,10 showed good germination rate and the best germination rate was 7 times.

In addition, in terms of strength and dust, 1 and 6 were the best, but germination rate was not high, and the condition for making shape was 2 and 7 conditions.

As a result, it was found that the composition ratio No. 7, which is easy to make a shape and shows good germination rate, was the best.

The production process of the pellet seeds containing Bacillus BIG21003 of the present invention is as follows.

<Step 1> Preparation of Bacillus BIG21003 foaming agent

The temperature is 30 ℃, pH 6.5 and Bacillus genus BIG21003 is incubated while adjusting to maintain at least 20% dissolved oxygen in the fermentor.

Cultured using GYS medium, the medium composition is as follows.

Glucose 0.15%, Yeast extract 0.2%, K ₂ HPO ₄ 0.05%, (NH ₄ ) ₂ SO ₄ 0.2%, MgSO ₄ 0.002%, MnSO ₄ 4H ₂ O 0.005%, CaCl ₂ 0.008%

After 5-6 hours of incubation, Glucose, a carbon source in the medium, was exhausted and spores began to form. After 30 hours of fermentation, all strains were converted into spores (see FIG. 2).

After 30 hours of incubation, the supernatant was removed by centrifugation of the culture medium, and 1% of Sucrose and 0.2% of CaCl ₂ were added, followed by in let temp. 110 ℃ and out let temp. Spray drying at 95 ℃ to produce a foaming agent.

<Step 2> Preparation of the Extending Agent

An extender is prepared by mixing 30% by weight calcium carbonate, 40% by weight kaolin, 10% by weight surfactant, 10% by weight diatomaceous earth and 10% by weight clay.

The bulking agent is prepared by mixing 0.1 to 10.0% by weight of the foaming agent prepared in the first step (10 ¹¹ cfu / g or more) and preferably 1.0 to 3.0% by weight of the prepared extender.

On the other hand, in the manufacture of extenders, talc, calcium carbonate, limestone, slaked lime, wood flour, vermiculite, bentonite, clay, mica, titanium oxide, calcium carbonate, zeolite, peat, clay may be used in combination.

In addition, when the extender is prepared, it may be prepared by adding a growth regulator, a growth promoter, an algal repellent, and the like, which are functional substances.

The growth regulator added at this time, IAA, NAA, kinetin, cytokinin, gibberellin, ethylene, absic acid. Ethephon, acetamide, maleic acid, hydrazide, diminozide and the like can be added.

As growth promoters, carbohydrates, essential amino acids, vitamins, chitin, chitosan, steroate saponins, immunostimulants and the like can be used.

Since the physiological characteristics of the crops vary, the ingredients and blending ratios of the extenders are selectively applied for each crop.

<3rd process> Preparation of the slurry liquid for binders

For the preparation of the slurry liquid for the binder, one or more selected from methyl cellulose, ethyl cellulose, dextran, arabic gum, xanthan gum, paraffin oil, gelatin, starch, polyvinyl alcohol, polyethylen glycol, carboxymethyl cellulose, etc. are used. Other polymers and copolymers may also be used.

Arabic gum used as the binder is dissolved in water to prepare a slurry solution of 3%.

<Step 4> Pellet Seed

Seeds are placed inside the pelleting machine, the extender is sprayed, and the slurry solution is sprayed repeatedly to form spherical pellets.

Select to size.

The seed pellet of the present invention is prepared by drying at low temperature.

Example 2 Preparation of Chinese Cabbage Pellets Seed Containing Bacillus BIG21003

1. The temperature of 30 ℃, pH 6.5 was adjusted to maintain the dissolved oxygen in the fermenter 20% or more while BYS21 BIG21003 was incubated using a GYS medium.

After 30 hours of incubation, the supernatant was removed by centrifugation.

As a preservative, 1% of Sucrose and 0.2% of CaCl ₂ were added.

in let temp. 110 ° C., out let temp. Spray drying at 95 ° C. produced a foaming agent.

The foaming agent was 10 ¹¹ cfu / g or more.

2. 10 kg of extender was prepared by mixing 3 kg of calcium carbonate, 4 kg of kaolin, 1 kg of surfactant, 1 kg of diatomaceous earth, and 1 kg of clay.

An extender was prepared by mixing 0.03 kg of a foaming agent in 10 kg of the prepared extender.

3. Arabic gum was dissolved in water to prepare a slurry solution of 3% to prepare 5L.

4. 1 liter (200,000 grains) of cabbage seed was placed inside the pelleting machine.

After spraying the extender, the process of spraying the slurry solution was repeated to form spherical pellets.

190,000 pellets 3.5 mm in diameter were selected.

Cabbage seed pellets containing Bacillus BIG21003 of the present invention were prepared by drying at low temperature.

<Experiment 4> Investigation of rooting ability of Bacillus BIG21003

Root-settlement ability of Bacillus BIG21003 of the cabbage pellet seed containing Bacillus BIG21003 of the present invention prepared in Example 2 was investigated.

(1) Two filter papers were laid on a 10 cm diameter Petri dish, and 4 ml of Heungnong Biotope extract was sprinkled.

^Five cabbage pellet seeds (5.6 × 10 ⁵ cfu / seed) containing Bacillus BIG 21003 of the present invention prepared in Example 2 were placed on the edge of the filter paper.

It was incubated for 5 days in the dark 28 degreeC incubator.

(2) Heungnong Biosoil contained about 3/4 of the 16 ports.

Chinese cabbage pellet seeds (2.8 × 10 ⁴ cfu / seed) containing Bacillus BIG21003 prepared in Example 2 were sown in two grains.

The seedlings were seeded at 25 ° C. (16 hours light, 8 hours dark) for 7 days, and washed 1 week after 3 days of sowing.

(1) In vivo and (2) in vitro cut the cabbage roots into 6 sites, then gathered the same sites for 6 weeks, shaken for 30 minutes in 10 ml sterile water, and then 100 ml of nutritious agar The number of colonies was examined by spreading on nutrient agar medium.

The investigation results are shown in Table 4 below.

<Table 4> Results of Root Settlement Capacity of Bacillus BIG21003

Root In vitro (5 days after sowing) In vivo (7 days after sowing) cfu / root root weight (mg) cfu / root root weight (mg) One 2.0 * 10 ⁴ 0.40 2.7 * 10 ³ 3.9 2 5.9 * 10 ³ 0.30 1.0 * 10 ³ 1.9 3 4.3 * 10 ³ 0.23 4.2 * 10 ² 1.3 4 6.8 * 10 ³ 0.20 8.3 * 10 ² 0.9 5 1.0 * 10 ⁴ 0.19 2.9 * 10 ² 1.0 6 3.4 * 10 ⁴ 0.14 2.5 * 10 ³ 0.4

In general, soil microorganisms having excellent rooting ability tend to have a high density of bacteria at the proximal end of plant parts and roots, and low density at intermediate sites.

As shown in Table 4, the Bacillus genus BIG21003 also tends to increase the density of bacteria in the root and proximal roots.

Experimental Example 5 Packaging Experiments of Bacillus BIG21003 Chinese Cabbage Pellets Seeds

Pellet seeds containing Bacillus BIG21003 prepared by changing the varieties of Chinese cabbage, but prepared as in Example 2 were subjected to packaging experiments.

The training was conducted in Pyeongchang, Gangwon-do and Gyeonggi-do.

The test of Pyeongchang, Gangwon-do, used mountain village cabbage of eastern Hanjong seedlings.

It was planted after 20 days seedling by sowing using Heungnong Biosoil, a commercial seedling soil.

The total test area was 150 pyeong, and the cultivation method was in accordance with farm practices.

The disease was investigated 4 weeks after the meal.

For the test of Gyeonggi-do, black pearl cabbage of eastern Hanjong seedlings was used.

It was planted after 20 days seedling by sowing using Heungnong Biosoil, a commercial seedling soil.

The total test area was 50 pyeong, and the cultivation method was in accordance with farm practices.

The disease was investigated 4 weeks after the meal.

The test results of PyeongChang and Positive are shown in Table 5 below.

<Table 5> Outbreak Test Results of Chinese Cabbage Pellets

division Incidence (%) Control price (%) No treatment Treatment Pyeongchang-gun 83.3 19.4 77 positivity 82 18 78

The pellet seeds containing Bacillus BIG21003 of the present invention in both Pyeongchang and positive test cloths exhibited a high control effect against cabbage wart disease.

The present invention provides a microorganism Bacillus genus BIG21003 exhibiting an excellent antagonistic effect on soil pathogens such as warts and bacteria.

In addition, pellet seeds containing Bacillus BIG21003, which have a high germination rate, a high rhizome fixation ability, and a large control effect against soil pathogens, are provided.

Claims (7)

Antagonism against soil pathogens Bacillus sp. BIG21003 ( Bacillus sp. BIG21003) (Accession No. KCTC 10236BP). A pellet seed containing the antagonistic microorganism Bacillus BIG21003 of claim 1. Culture of antagonistic microorganisms to make a foaming agent, mixed with a foaming agent in an extender made of calcium carbonate, kaolin, surfactants, diatomaceous earth, clay, to make a bulking agent, methyl cellulose, ethyl cellulose, dextran, arabic gum, One or more selected from xanthan gum, paraffin oil, gelatin, starch, polyvinyl alcohol, polyethylene glycol, and carboxymethyl cellulose are dissolved in water to make a slurry liquid, and then the seed agent in the pelletizing machine Method of producing a pellet seed containing antagonistic microorganisms consisting of repeatedly spraying to make a spherical pellet seed, then sorting to a predetermined size, and drying at a low temperature. The method according to claim 3, wherein the antagonistic microorganism has an antagonistic effect on phytopathogens , Agrobacterium sp., Bacillus sp., Pseudomonas sp., Stereptomyces sp., Burkholderia sp., Gliocladium sp., Trichoderma sp. And antagonistic microorganisms derived from the non-pathogenic Fusarium, Pythium . The seed according to claim 3, wherein the seeds are radish, carrot, cabbage, cabbage, spinach, lettuce, lettuce, watermelon, melon, cucumber, pumpkin, tomato, pepper, onion, leek, duck, grass, tobacco, wild vegetables, flower, tree seed A method for producing pellet seeds containing antagonistic microorganisms, characterized in that the species is selected from grass seed. Bacillus BIG21003 was cultured to make a foaming agent, and a bulking agent was mixed with an extender made of calcium carbonate, kaolin, surfactant, diatomaceous earth, and clay to make a bulking agent, and the arabic gum as a binder was dissolved in water to make a slurry solution. Next, a method for producing pellet seeds containing Bacillus BIG21003, comprising seeding in a pelleting machine, followed by repeated spraying of the bulking agent and the slurry solution to make spherical pellet seeds, and then sorting them to a predetermined size and drying them at low temperature. The method according to claim 3 to 6, The bulking agent is characterized in that the mixture is prepared by mixing 30% by weight of calcium carbonate, 40% by weight of kaolin, 10% by weight of surfactant, 10% by weight of diatomaceous earth, 10% by weight of clay, pellet seed production method.