KR100506721B1 - Bacillus licheniformis N1 and microbial agent for preventing plant-pathogenic fungi containing the same - Google Patents

Abstract

The present invention relates to Bacillus licheniformis having a antagonistic ability against fungi and a microbial agent comprising the same, more specifically Bacillus rickeniformis having antibacterial or antifungal activity, plant pathogenic comprising the same It relates to a microbial agent for controlling fungi and a method for producing the same. The microbial agent of the present invention is an environmentally friendly microbial agent that minimizes the effect on crops and is effective in controlling diseases of plants including ash fungus, and can be produced at low cost, and thus, perilla, tomato, cucumber and strawberry, etc. It can be usefully used to control crops of plants.

Description

Bacillus licheniformis N1 and microbial preparations for controlling phytopathogenic fungi comprising the same {Bacillus licheniformis N1 and microbial agent for preventing plant-pathogenic fungi containing the same}

The present invention relates to a Bacillus sp. Strain having an antagonistic ability against fungi or fungi and a microbial agent using the same . More specifically, Bacillus licheniformis having an antagonistic ability against phytopathogenic fungi And a microbial agent for controlling fungi of plants using the same.

Gray fungal pathogens grow by-products not only in flowers and fruit trees, but also in a variety of living crops such as cucumbers, tomatoes, eggplants, strawberries, watermelons, melons, pumpkins, peppers, and lettuces, as well as plant horticultural crops. It is a damaging pathogen. The pathogen is a versatile bacterium, and the host range is very wide, the variation is severe, and the emergence of drug-resistant bacteria due to the large amount of spraying of pesticides is not only difficult to control, but also causes residual toxicity and environmental pollution problems. As the social side effects of chemical pesticides are emerging around the world, the growth of organic synthetic pesticides has slowed significantly. On the other hand, research on the development of environmentally friendly microbial pesticides has been vigorous, and many antagonistic microorganisms and microbial agents have been developed. It is becoming. For example, about 40 kinds of microbial pesticide products are marketed worldwide for the control of plant diseases. In Korea, the development of microbial pesticides of strawberry gray fungus using Bacillus subtilis has been reported. Items are not disclosed as pesticides.

A report on the biological control of Botrytis cinerea , a report using Bacillus pumilus and B. amyloliquefaciens for the biological control of hypotonic pear gray mold. (Nari et al., 1996, Biological control 7: 30-37) and control of vine and soybean mildew disease using Trichoderma harzianum (Zimand et al., 1996, Phytopathology 86: 1255-). 1260; Harman et al., 1996, Biological control 7: 259-266), Gliocladium roseum , Ulocladium atrum , control of gray mold of cyclamen (Kohl et al. , 1998, Phytopathology 88: 568-575), control of apple gray mold by yeast (Filonow et al., 1996, Biological control 7: 212-220), blood by Pseudomonas fluorescens Tunier Gray Mold Control (Gould, etc., 1996, Plant Dis 80:. 1029-1033), Pseudomonas Sepharose cyano (P. cepacia) gray mold of apple, pear and rose with an antibiotic of pyrrole knitted Lin (Pyrrolnitrin) away from the control ( Janisiewicz et al., 1988, Phytopathology 78: 1697-1700; Hammer et al., 1993, Plant Dis. 77: 283-286). In addition, Gliocladium roseum , Penicillium sp. For the control of gray mold disease in strawberry. Trichoderma viride was used (Sutton et al., 1993, Phytopathology 83: 615-621), and there is a report using Bacillus spp. For gray mold disease of lettuce and apples (Choi, YC Control of fungal diseases with antagonistic bacteria, Bacillus sp.AC-1.Proc.Int.Symp.on Biological control of plant diseases : 50-61; Li, Debao, Biological control of plant diseases with Bacillus species.Proc.Int . Symp.on Biological control of plant diseases.pp . 75-85).

Bio-save 110 using Pseudomonas syringae for control of citrus trees and fruits as a fungus for controlling Botrytis cineraria, a bacterium of gray mold fungus. Trichoderma harzianum and other products such as Trichodex (Trichodex) are commercially available, but these are microbial pesticides for the control of hypotonic diseases, and crop spreading is rarely developed. In particular, among the genus Bacillus, Bacillus licheniformis strains were released from other studies as antibiotics, such as Microsporum canis , Mucor mucedo , Muco plumbeus Although it has been reported to antagonize Corynebacterium glutamicum , there are no studies on the biological control of gray mold by Bacillus licheniformis .

Accordingly, the present inventors selected strains having excellent antagonistic ability against fungi or fungi, and identified the strains as a novel Bacillus rickenomyces strain, the microbial agent using the disease of the plant, including gray mold The present invention has been completed by revealing its effectiveness in controlling.

An object of the present invention is to provide a bacterium or fungal control microorganisms for the fungus or fungi of plants comprising the strain and the antibacterial or antifungal activity.

In order to achieve the above object, the present invention provides a novel Bacillus rickeniformis having antifungal activity. The present invention also provides a microbial agent for controlling phytopathogenic fungi comprising the Bacillus rickeniformis and a preparation method thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides Bacillus rickeniformis having antifungal activity.

In the present invention, among the microorganisms isolated from the soil, the strains with the greatest mycelial growth and conidia spore germination inhibitory effect were selected first, and the strains with the greatest suppression effect were assayed by assaying the inhibitory effect of the antagonists with gray fungus disease. The final antagonists were selected (see Table 5 , Table 6, Table 7 ). As a result of examining the bacteriological properties of the strain for the identification of the antagonistic strain, it was confirmed that the morphological, physiological and biochemical characteristics as shown in Table 1 below.

Test N1 strain Bacillus rickeniformis Gram dyeing + ^a + Endospore + + Cell diameter> 1.0 μm - - Anaerobic Growth + + Growth in NaCl 2% + + Grow at 5% NaCl + + Growth in NaCl 7% + + Grow at 5 ℃ + - Grow at 10 ℃ - - Grow at 30 ℃ + + Grow at 40 ℃ + + Grow at 50 ℃ + + Hydrolysis of casein + + Hydrolysis of gelatin + + Hydrolysis of starch + + Nitrate reduction + + Utilization of Citrate + + Vogues-prosakauer test + +

^a +: 90% or more mismatch,-: 90% or more mismatch

In addition, as a result of analyzing the 16S rRNA sequence of the strain, the nucleotide sequence showed more than 99% homology with Bacillus licheniformis , the base sequence is shown in SEQ ID NO: 1 attached As described. From the above results, the antifungal active strain isolated in the present invention was identified as Bacillus rickeniformis. In the present invention, the strain was named "Bacillus rickeniformis N1", which was deposited on July 31, 2002 to the Korea Institute of Biotechnology Gene Bank (Accession Number: KCTC 10319BP).

In addition, the present invention provides a microbial agent for controlling phytopathogenic fungi comprising the Bacillus rickeniformis N1 and a method for producing the same.

Bacillus rickenformis N1 is Bacillus rickeniformis having antifungal activity, specifically, Bacillus rickenformis N1 (Accession Number: KCTC 10319BP) is preferred.

Since Bacillus rickenomyces used in the microbial preparations of the present invention have an antagonistic ability against fungi, microbial preparations using the strains can be usefully used for controlling plant diseases. The microbial agent of the present invention shows a control effect against phytopathogenic fungi, specifically, the control effect against fungi such as gray mold fungi, fungal nucleus, rot, black pattern disease and wilt disease. The gray fungal pathogen is Botrytis cinerea . Crops to which the microbial agent of the present invention can be applied are not particularly limited, and can be applied to flowers or special crops in addition to economic crops such as perilla, cabbage lettuce (strawberry), strawberries, tomatoes and cucumbers.

In addition, the microbial agent of the present invention may be prepared in various forms, and specifically, may be prepared in the form of a hydrate, liquid or emulsion.

The Bacillus rickeniformis N1 is added to a carbon source selected from the group consisting of glucose, starch, sorbitol and inositol, tryptone, beef extract, yeast extract and malt extract The antifungal activity is excellent when cultured in a medium containing a nitrogen source selected from the group consisting of (see Table 7 and Table 8 ).

In addition, when culturing the Bacillus rickenformis N1 in large quantities, farm by-products and farm wastes such as bean, wheat bran, soybean meal, jelly, rice straw and the like can be used as a substrate for mass culture. In a preferred embodiment of the present invention, Bacillus rikenipo in a medium comprising one or more selected from the group consisting of sesame, fish cake, wheat bran, soybean meal, barley malt, rice cake, rice bran, teacup, rice flour and rice straw Miss N1 was incubated. Among the media components, the medium using the busy medium has a high density of antagonistic microorganisms in mass culture, is relatively easy to obtain on the market, and is inexpensive, thereby lowering the price of the medium, as well as nutrients to plants and environment such as residual toxicity of pesticides. There is no concern about contamination, which is advantageous in terms of environmental protection (see FIG. 6 ).

In addition, the microbial agent of the present invention may further include a biopolymer, a natural polymer or a mineral in the culture medium of Bacillus rickeniformis N1.

In the above, the biopolymer is preferably at least one selected from the group consisting of rice flour, soy flour, amber flour, pine needle powder, spinach powder, flour, and the like.

In addition, the natural polymer is preferably at least one selected from the group consisting of starch, gelatin, collagen, casein, albumin, fibrinogen, elastin, fat, cellulose, cellulose derivatives, ethers.

In addition, the mineral is preferably at least one selected from the group consisting of vermiculite, earth, ceramic and alginate.

In addition, the microbial agent of the present invention may further include glucose or minerals, and as minerals, CaCO ₃ , FeSO ₄ · 7H ₂ O or MnCl ₂ · 4H ₂ O may be used, but is not limited thereto.

The soy formulation, a microbial agent of the present invention, may be well mixed with a soybean powder, rice flour, glucose or tryptone, which is a natural polymer, in a culture solution of Bacillus rikenimoform N1 strain, and may be prepared in the form of a hydrate, granule, or emulsion. In a preferred embodiment of the present invention, the mixture was dried and crushed to prepare a hydrate.

In addition, the microbial agent of the present invention preferably further comprises soy flour, rice flour, glucose and minerals in the culture medium of Bacillus rikenimoform N1 strain, 200-300 g soybean powder with respect to 1 L of Bacillus rikenimoform culture. More preferably, 200 to 300 g of rice flour, 4.0 to 5.0 g of glucose, 0.1 to 0.3 g of FeSO ₄ 7H ₂ O, and 0.02 to 0.01 g of MnCl ₂ H ₂ O.

In addition, the microbial agent of the present invention preferably contains at least 2 × 10 ⁹ cells, preferably 2 × 10 ⁹ ~ 2 × 10 ¹¹ cells / ㎖ Bacillus liqueniformis per ml of the liquid microbial delivery medium. More preferably, it contains at 2 * 10 <^11> -9 * 10 <^12> cells / mL.

The microbial agent of the present invention may be treated by coating, mixing or spraying the microbial agent on at least one of the seed, plant, and soil in which the plant is grown. The microbial agent of the present invention can be sprayed on the whole plant or soil as a foliar spray.

In addition, in addition to the composition of the microbial agent of the present invention, an excipient, diluent, diatomaceous earth, zeolite, white carbon and other mineral carriers, various additives or surfactants may be added.

As a result of assaying the control effect on the gray mold of the microbial agent of the present invention, it was confirmed that the control effect is higher than the conventional chemical pesticides (see Table 10 ). In addition, the microbial agent of the present invention has a high stability, and even when stored for a long time, the control effect does not change.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Experimental Example 1 Gray Mold Pathogen and Pathogenicity Assay

The inventors of the present invention are Botrytis cinerea , a gray fungal pathogen. LVF12 strain (hereinafter abbreviated as "LVF12") was used, and the strain was purely isolated from perilla with perilla symptom and leaf tip at the perilla cultivation house in Gangdong, Busan, in 1997 and 1998. It is a strain identified as perilla ash fungal pathogen through the characteristics of pathogens and pathogenicity assay ( FIG. 1 ).

In order to develop an effective spore suspension in pathogenic assays using conidia spores against perilla perilla of the LVF12 strain, PDA in bactericidal water, PDB (potato dextrose broth, Difco, USA) and 10% tomato juice (trade name: Gaya) Condensed spores of the LVF12 strain formed by culturing for three weeks in a medium (potato dextrose agar, Difco, USA) were suspended and inoculated in perilla with spore suspension adjusted to a concentration of 10 ⁶ per ml. Five weeks per treatment group was sprayed on perilla leaves and stored at 90% relative humidity and 20 ± 2 ℃ growth. The incidence was investigated 7 days after treatment.

As a result, no outbreak occurred in spore suspension using sterilized water and 76.0% incidence was observed in spore suspension using PDB, whereas a high incidence of 85.0% was observed in spore suspension using 10% tomato juice. It was found to be the most effective to use ( Table 2 ).

Conidia Suspension Incidence (%) Sterilization Water (SW) 0 Potato Liquid Medium (PDB) 76.0 10% Tomato Juice (Tomato) 85.0 Control (no treatment) 0

Experimental Example 2 Perilla variety test

The present inventors cultivated 11 varieties of perilla, such as Gwangyang, Gurye 3-13, Goseong native species, Pearl native species, Perilla perilla No. 1-1, Milyang, Chubujong, Okdong, Kyungsin, Hadong, Gupo The incidence by LVF12 strain was investigated. Specifically, 1 × 10 ⁶ per the suspended for three weeks and cultured formed disclosure pathogens 10% confirmed that the suspension effective spores conidia of LVF12 strain pathogenicity test tomato juice on PDA medium ㎖ to test the resistance level between breeds Dogs were inoculated in the perilla seedlings for 3 weeks and then stored at 20 ± 2 ℃ relative humidity and 20 ± 2 ° C. The incidence was examined 8 days after treatment.

As a result, all 11 varieties disclosed showed high incidence, and the resistance between varieties to gray mold was not found to be high. Among them, wheat sheep was 46.7%, showing the lowest incidence ( Table 3 ).

Perilla variety test kind Incidence (%) Okdong 61.7 Gupo 53.3 Kwangyang 83.3 Perilla 1-1 (small) 76.7 Milyang 46.7 Chubu 75.0 Gurae 3-13 80.0 Kyungsin 58.3 Gosung jerae 78.3 Hadong 56.7 Jinju jerae 78.3

Example 1 Selection of Antagonistic Microorganisms against Aseptic Fungal Pathogens

The present inventors disclosed 250 microorganisms isolated from perilla leaves and rhizosphere soils of Gimhae, Gyeongnam province, and selected the strains with the greatest mycelial growth and conidia spore germination inhibitory effects of L.A. fungal pathogen LVF12 strains. The inhibitory effect was assayed and strains with a high oncogenic effect were selected as final antagonists. Mycelial growth inhibitory effect was determined by investigating the microorganisms and LVF12 strain on potato agar plate (PDA) medium to investigate the size of lowland. Specifically, the effect of inhibiting spore germination was cultured by shaking the cultured microorganisms (NB, nutrient broth, Difco, USA) at 30 ° C. for 5 days and centrifuging the supernatant with a 0.22 μm filter (Millipore). After mixing the conidia of LVF12 strain 1 ml in water agar medium (Water agar, Difco, USA) and spore germination rate was investigated after 12 and 48 hours. Mixed with sterilized water instead of the culture filtrate was used as a control. In addition, after the culture filtrate filtered in the same manner as above on the PDA medium, the mycelial fragments of the pathogenic pathogens were dentured and the effects on mycelial growth were also investigated. To primarily the onset inhibitory effect with the gilhanggyun selected as to port black in saengyuksang, potato agar plates (PDA) by using three weeks cultured on a culture medium suspend the conidia formed LVF12 strain in 10% tomato juice ㎖ per 10 ⁷ After adjusting the concentration of spores suspended in dogs and the cultivated antagonists shaken for 5 days in a nutrient medium to a concentration similar to that of pathogens, the same amount was mixed and sprayed inoculated on perilla for 5 weeks. The incidence was investigated after 7 days of treatment while stored at 2 ° C. and compared to the control group treated with the pathogen.

As a result, six strains having antagonistic ability against the gray fungal pathogen LVF12 were selected and named as N1, N2, N3, N4, N5 and N6, respectively. All of the strains showed excellent mycelial growth inhibitory effect, and among them, the highest inhibitory effect by the N1 and N4 strains ( Table 4 and FIG. 2 ).

Growth inhibitory effect of pathogens by antagonistic microorganisms Antagonist Growth Low Zone (mm) ^a  N1 35.0  N2 24.0  N3 22.0  N4 37.0  N5 25.0  N6 27.0

^a growth zone; Irradiation after 7 days of culture

The present inventors mixed conidia of pathogen LVF12 strain with culture filtrates of N1 and N4 strains and examined the spore germination rate.In the case of sterile water without antagonistic bacteria, the conidia germination rate was 90% after 48 hours of treatment. Or germination was inhibited 100% when the N4 strain was treated ( Table 5 ). In addition, mycelial growth was very slow on the potato agar plate (PDA) medium in which the culture filtrate was used, and the mycelia were also severely modified ( FIG. 3 ). As a result of pot test on the inhibition of onset by 6 bacterial strains selected primarily because of high mycelial growth and spore germination effects, all 6 strains showed more than 70% control and 95.3 control against N1 strain. %, N4 strain showed a high control value of 90.8% ( Fig. 4 ). Therefore, the present inventors finally selected the N1 strain showing the highest control effect as an antagonist against gray mold.

Inhibitory Effect of Pathogenic Germ on Conidia Spore Germination by Antagonistic Bacteria Antagonistic bacteria Conidia Germination Rate (% ± SD) ^a 12 hours later 48 hours later N1 0 0 N4 0 3 ± 2 water 72 ± 4 90 ± 4

^a conidia germination rate; Microscopic observation on agar medium

Example 2 Prevention of Onset and Treatment Effect of Assay Fungal Disease by Selected Antagonists

Condensed spores of the LVF12 strain formed by culturing for 3 weeks on potato agar plate (PDA) medium were suspended in 10% tomato juice and adjusted to 10 ⁷ concentrations per ml and N1 and N4 antagonists cultured for 5 days in NB medium. It was adjusted to the same concentration as the pathogen, and the effect of pathogen treatment by antagonists was investigated by inoculating the pathogen first and inoculating the antagonists at daily intervals for 3 days and examining the incidence. In addition, after inoculating the antagonists first, the inoculation pathogens were inoculated at 1 day intervals for 3 days and the incidence was investigated to investigate the prevention of the onset. After treatment with perilla for 5 weeks each, the relative humidity was 90% or more, stored at 20 ± 2 ℃ growth in 7 days after treatment to investigate the incidence was converted to the control value, the control value was calculated as Equation 1 below.

As a result, when the N1 and N4 antagonists and the pathogens were simultaneously inoculated, the N1 and N4 antagonists were treated 100% of the N1 strains and N4 strains even when the N1 and N4 antagonists were treated 1 to 3 days before and 1 to 3 days after the pathogens. In the range of 100-87.3% ( Table 6 ). From the above results, it was confirmed that both the N1 and N4 strain was excellent in the prevention and treatment effect of gray mold.

Prevention of onset and treatment effect by selected antagonists Elapsed time after vaccination         Incidence (%)        Control price (%) N1 ^a + Pa Pa ^b alone N1 + Pa 3 days ago 0.0 - 100 2 days ago 0.0 - 100 1 day ago 0.0 - 100 Simultaneous vaccination 0.0 73.3 100 1 day later 0.0 - 100 2 days later 0.0 - 100 3 days later 0.0 - 100

^a N1: antagonistic bacterium Bacillus licheniformis

^b Pa alone: Pathogen treatment alone ( Botrytis cinerea LVF12)

Example 3 Identification of Antagonist Bacteria

The inventors of the present invention in Example 1 and Example 2 to identify the N1 antagonists confirmed the prevention and treatment effect of gray mold, strains with an optical microscope (x1,000) and electron microscope (SEM, Hitachi, Japan) The biochemical, cultural and physiological properties were examined according to the Burgy's manual (Bergey's manual of Systematic Bacteriology. 1986. Williams & Wilkins) ( Table 1 ). It was identified using the 50CHB kit (bioMerieux, France).

As a result, looking at the morphological, physiological and biochemical properties of the N1 strain, the strain is Gram-positive, forms an endospores, the cell diameter is 1 μm or less, and grows in anaerobic, 2-7% NaCl It also grew in concentration. In addition, casein, gelatin and starch were hydrolyzed and positive in nitrate reduction, citric acid utilization and Bolz-Prosacauur tests, appearing to have the same characteristics as Bacillus rickeniformis (see Table 1 ).

In order to confirm the accuracy of the identification, the N1 strain was identified by the API system, which was 88.8% similar to that of Bacillus rickeniformis, which was consistent with the identification by the buggy's manual. In addition, as a result of 16S rRNA analysis, the strain has a nucleotide sequence of 1,508 bp, and the nucleotide sequence shows more than 99% homology with Bacillus licheniformis , and the N1 strain is Bacillus rickeniformis. I was identified.

The inventors named N1 strain selected and identified above as "Bacillus rickeniformis N1" and deposited it on July 31, 2002 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10319BP).

Example 4 Influence of Carbon Sources on Antagonistic Activity of Bacillus rickenomyces N1 Strain

We investigated carbon sources that influence the antagonism of Bacillus rickeniformis N1 strain. In order to formulate antagonists to make microbial preparations, we first need to investigate carbon sources and nitrogen sources that can enhance the antagonism of the strains. First, which carbon source has the effect of inhibiting the mycelial growth of gray fungus bacteria caused by Bacillus rikenimomis N1? Was investigated. Specifically, 1% soy flour, FeSO ₄ · 7H ₂ O 0.05%, MnCl ₂ · 4H ₂ O 0.005% as a base medium and sterilized by adding 1% of the carbon source, and inoculated with antagonistic bacteria and inoculated at 5 The cells were shaken and centrifuged daily. After filtering the supernatant with a 0.22 μm filter (Millipore), 50 μl was soaked in a 5.0 mm paper disk, the pathogen and the paper disk were soaked in PDA medium, and replaced with culture to inhibit the pathogen. . The carbon sources disclosed were glucose, fructose, lactose, maltose, galactose, starch, sorbitol, inositol and glycerol, and each of them was inoculated with antagonistic bacteria by adding 1% of each to 30 ° C., Shake culture at 160 rpm. After 48 hours of incubation, the supernatant was filtered by centrifugation at 3,000 rpm for 15 minutes (Milipore filter, 0.22 µm, USA), 50 µl of the filtrate was treated on a sterile paper disk (5.0 mm in diameter), and naturally dried. The diameter of mycelial growth zones of gray fungal pathogens were examined by comparing them with pathogen fragments (8.0 ㎜ in diameter).

As a result, when cultured using glucose as a carbon source, it showed the lowest effect of 5.3 mm low zone, and showed an inhibitory effect when cultured using starch, sorbitol, and inositol as carbon sources. However, when incubated with fructose, lactose, maltose and galactose as carbon sources, there was no inhibitory effect at all ( Table 7 ).

Effect of Carbon Sources on Antagonistic Activity of Bacillus Rickenomyces N1 Strain Carbon source (1.0%) Growth basin (㎜) Glucose 5.3 Fructose - Lactose - Maltose - Galactose - Starch 3.2 Sorbitol 2.3 Inosyltol 2.1 Glycerol -

Example 5 Influence of Nitrogen Sources on Antagonistic Activity of Bacillus rickeniformis N1 Strain

We investigated nitrogen sources that influence the antagonism of Bacillus rickeniformis N1 strain. Specifically, in order to investigate which nitrogen source influences the mycelial growth inhibition effect of gray fungus bacteria, 1% glucose, FeSO ₄ · 7H ₂ O 0.05%, and MnCl ₂ · 4H ₂ O 0.005% are used as the basic medium. After sterilization by adding 0.5% of the nitrogen source, inoculated with the strain of N1 of the present invention and filtered by shaking culture for 30 days at 30 ℃. Lowland investigation was carried out in the same manner as in Example 4. Nitrogen sources disclosed were (NH ₄ ) ₂ SO ₄ , casamino acid, trypsin, beef extract, malt extract, yeast extract, and 0.5% of each was added.

As a result, among the nitrogen sources disclosed, the most effective was the low-lying zone of 4.5 mm in trypsin, followed by beef extract, yeast extract and malt tablets, but (NH ₄ ) ₂ SO ₄ and kazamic acid had no inhibitory effect ( Table 8 ).

Effect of Nitrogen Sources on Antagonistic Activity of Bacillus Rickenomyces N1 Nitrogen source (0.5%) Growth low zone (mm) (NH ₄ ) ₂ SO ₄ - Casamino acid - Trypsin 4.5 Beef Extract 2.9 Malt extract 2.3 Yeast extract 2.8

Example 6 Mass Cultivation of Bacillus rickeniformis

The inventors of the present invention have selected a medium in which Bacillus rickenformis N1 strain is cultivated in large quantities to optimize production of antifungal actives. Specifically, 100 ml of distilled water is placed in a 250 ml flask, and 5% of farm wastes and farm wastes, such as inexpensive bean curd, bran, soybean meal, soybean paste, rice straw, etc., which are readily available on the market as a bulk culture medium, are added. Was inoculated with 2 ml of N1 strain (1 × 10 ⁷ cells / ml) cultured in nutrient medium (Nutrient broth, Difco, USA) for 24 hours. Cultivation was carried out at 30 ° C. and 160 rpm for 72 hours, and the density of bacteria was measured by an absorbance spectrometer (spectrophotometer, 660 nm) at 4 hour intervals. Bees powder was obtained by discarding wastes from the market and dried by completely removing moisture from the oven. Corn starch, rice flour, barley malt, and bran were purchased and sold on the market. Rice bran, rice plant, soybean meal, etc. Collected from and used imports. As a by-product of soy sauce, leftover residue was used to prepare soy sauce, and fish paste fish paste was used. All substrates except for singlet were dried at 60 ° C. for 24 hours and then ground using a blender (Blender, Waring, USA).

As a result, among the 14 media reported, bacterial density was the highest at 10 ¹¹ cells / ml in busy media, followed by fish paste, bran, soybean meal, barley malt, salt, rice bran, rice wine, rice flour and It was also effective in rice straw ( FIG. 6 ).

Example 7 Formulation of Bacillus rickeniformis N1 Strain

The present inventors have prepared a microbial agent for the control of gray mold disease using Bacillus rickeniformis N1 strain. Specifically, inoculated N1 strain of the present invention in a nutrient broth added with 1.0% glucose and 0.5% trypsin, which were found to be effective in improving antagonism, and cultured with shaking at 30 ° C. and 200 rpm for 5 days, and then cultured 1 l. In the preliminary experiment, add soy flour, rice flour, glucose, FeSO ₄ · 7H ₂ O and MnCl ₂ · 4H ₂ O that were found to be effective in controlling various types of polymer materials such as soy flour, rice flour, corn starch, and flour. It was lightly coated, dried at 55 ° C. for 48 hours, and then pulverized with a blender to prepare a hydrated microbial agent containing the antagonistic bacteria, which was designated as a “soy preparation”. The components of the formulations are summarized in Table 9 below.

Constituents of Soy Formulation for Control of Gray Mold Disease  Target crop  Formulation name Ingredient / culture medium 1 L Perilla Soy preparation Soy flour 200 g Rice flour 200 g Glucose 4.0 g FeSO ₄ 7H ₂ O 0.1 g MnCl ₂ 4H ₂ O 0.02 g

<Example 8> Control effect of gray mold fungus by Soy formulation

The present inventors assayed the control effect of perilla ash fungus disease by Soy formulation of Bacillus rickeniformis N1 strain prepared in Example 7 in house. Specifically, the viable cell suspension (10 ⁷ cells / ml) suspended in live water of Bacillus rickenomyces N1 strain, culture medium of the N1 strain (10 ⁷ cells / ml) cultured for 5 days in nutrient broth (NB broth) , Benzymidazole-based, Benomiyl Hydrate 2000 times (Pesticide Instructions, 2000) and Soy preparation 50 times (4 × 10 ⁸ cells / ml) of Bacillus liqueniformis N1 strain They were sprayed on the front and back of 10 perilla perilla leaves, which were pot-cultivated in each house, and dried, and then condensate suspension (adjusted to 10 ⁷ cells / ml in 10% tomato juice) of the gray fungal pathogen LVF12 strain. It was evenly sprayed on, and the relative humidity was more than 90%, stored in a house of 20 ± 2 ℃ 7 days after the incidence was investigated and converted to the control value. The controlling value was calculated by the following equation. In addition, perilla was used as a Gwangyang variety confirmed to have high sensitivity.

As a result, in the control of the soy formulation in going 93.1% was higher than the culture process (90.4%) and benomil wettable powder (86.1%), Bacillus Lee Kenny Po Ms bacteria suspension processed in the N1 strain showed a control of 70.4% of the (Table 10 and 7 ).

Control of Soy Formulation Processing example Incidence (%) Control is (%) B suspension ^a 24.3 70.4b ^f N suspension ^b 7.9 90.4a Benomil (chemical pesticide) ^c 11.4 86.1a Soy formulation ^d 5.7 93.1a Pathogen alone treatment ^e 82.1 0c

^a bacterial suspension prepared with sterile water (1 × 10 ⁷ cells / ml)

^b Bacterial broth cultured for 5 days (1 × 10 ⁷ cells / ml)

^c Chemical pesticides Benomimilizing agent sprayed by diluting 2,000 times

^d Soy preparations: Hydration formulations (4 × 10 ⁸ cells / ml) prepared with Bacillus rickeniformis N1 strain.

^e Pa: pathogen alone treatment (Botritis Ceria LVF12)

DMRT at ^f 0.05 level

As described above, the Bacillus rikenimoform of the present invention has excellent antifungal activity against gray fungal pathogens, and microbial preparations using the strains include gray fungal disease, fungal nucleus, rot, black pattern and wilted disease. Not only is it effective in controlling crop diseases that cause the disease, and microorganisms can be prepared at low cost, and thus can be useful for controlling diseases of crops such as perilla, tomatoes, cucumbers and strawberries.

1 is a photograph showing the symptoms of perilla ash fungus disease caused by the pathogen Botrytis cinerea .

A, B, C: Symptoms of spontaneous gray mold disease

D, E: Symptoms of artificially inoculated gray mold disease

Figure 2 is a photograph of the growth of gray mold pathogens inhibited by the antagonistic bacteria N1 and N4 strains selected in the present invention.

A: N1 strain, B: N4 strain

Figure 3 is a photograph observing with a microscope that the gray mold pathogen modified by the antagonistic bacteria N1 strain selected in the present invention.

A: Mycelia of normal gray fungal pathogen (X 400),

B: Mycelia of gray mold pathogen modified by N1 strain (X 400)

Figure 4 is a photograph showing the control effect of gray mold according to the type of antagonistic strains in perilla seed culture.

N1, N2, N3, N4, N5, N6: antagonistic strains selected in the present invention,

Pa: Botrytis cineria

5 is a photograph of the Bacillus rickenformis N1 according to the present invention observed with an electron microscope.

Figure 6 is Bacillus rickenformis N1 of the present invention rice flour (A), rice bran (B), bran (C), soybean meal (D), jelly (E), barley malt (F), rice straw (G), busy (H), fish paste (I), Dansul (J), soybean production byproduct (K), apple peel (L), tangerine peel (M) or a culture medium in a culture medium (N) .

Figure 7 is a photograph showing the control effect of gray mold by the soy formulation of the present invention.

A: Perilla leaf treated with Bacillus rickeniformis N1 of the present invention and gray fungal pathogen Botrytis cieria

B: Perilla leaves treated only with Botrytis cineraria

<110> Dong A university <120> Bacillus licheniformis N1 and microbial agent for preventing plant-pathogenic fungi containing the same <130> 2p-07-11 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1508 <212> DNA <213> Bacillus licheniformis N1 <400> 1 agagtttgat tggctcaggc gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga 60 cagatgggag cttgctccct gatgttagcg gcggacgggt gagtaacacg tgggtaacct 120 gcctgtaaga ctgggataac tccgggaaac cggggctaat accggatagt tgtttgaacc 180 gcatggttca gacataaaag gtggcttcgg ctaccactta cagatggacc cgcggcgcat 240 tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt 300 gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 360 tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttcgg 420 atcgtaaaac tctgttgtta gggaagaaca agtgccgttc caatagggcg gcaccttgac 480 ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540 gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct taagtctgat 600 gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa 660 gaggagagtg gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt 720 ggcgaaggcg actctctggt ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac 780 aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt tagggggttt 840 ccgcccttta gtgctgcagc taacgcatta agcactccgc ctggggagta cggtcgcaag 900 actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960 gaagcaacgc gaagaacctt accaggtctt gacatcctct gacaacccta gagatagggc 1020 ttccccttcg ggggcagcgt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc attcagttgg 1140 gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc 1200 atgcccctta tgacctgggc tacacacgtg ctacaatgga cagaacaaag ggcagcgaaa 1260 ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc ggatcgcagt ctgcaactcg 1320 actgcgtgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc 1380 cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgag 1440 gtaacctttt ggagccagcc gccgaaggtg ggacagatga ttggggtgaa gtcgtaacaa 1500 ggtagccg 1508

Claims (13)

Bacillus licheniformis N1 (Accession No .: KCTC 10319BP) with antifungal activity. delete A microbial agent for controlling plant pathogenic fungi, comprising Bacillus rickeniformis N1 of claim 1. The method of claim 3, wherein the fungus is a microbial agent, characterized in that the gray fungal pathogen. The microorganism preparation according to claim 4, wherein the gray fungal pathogen is Botrytis cinerea . 4. The microorganism preparation according to claim 3, wherein the plant is selected from the group consisting of perilla, tomato, cucumber, strawberry and lettuce. 4. The microbial preparation according to claim 3, wherein the microbial preparation is a hydrating agent. 4. The medium according to claim 3, wherein the Bacillus liqueniformis comprises a carbon source selected from the group consisting of glucose, starch, sorbitol and inositol and a nitrogen source selected from the group consisting of tryptone, beef extract, yeast extract and malt tablets. Microbial agent, characterized in that cultured in. 4. The microbial agent according to claim 3, comprising one or more selected from the group consisting of sesame, fish cake, wheat bran, soybean meal, barley malt, rice cake, rice bran, rice wine, rice flour and rice straw. The method of claim 3, wherein the microbial agent is a microbial agent, characterized in that the biopolymer, glucose, FeSO ₄ 7H ₂ O and MnCl ₂ H ₂ O is added to the culture medium of Bacillus rickeniformis N1. The microbial agent according to claim 10, wherein the biopolymer is one or more selected from the group consisting of rice flour, soy flour, amber flour, pine needle powder, spinach powder and flour. According to claim 10, wherein the microbial agent is 200-300 g soy flour, 200-300 g rice flour, 4.0-5.0 g glucose, 0.1-0.3 g FeSO ₄ 7H ₂ O and MnCl per 1 liter of the culture medium of Bacillus rickeniformis microbial agent comprising a ₂ H ₂ O 0.02~0.01 g. The microorganism for controlling pathogenic fungi of the plant of claim 3, comprising culturing the Bacillus rickeniformis N1 of claim ₁ and adding a biopolymer, glucose, FeSO ₄ 7H ₂ O and MnCl ₂ H ₂ O to the culture medium. Method of Preparation of the Formulation.