KR20110120748A - Bacillus amyloliquefaciens cp1 and contro method of strawberry anthracnose using the same - Google Patents

Abstract

PURPOSE: A Bacillus amyloliquefaciens CP1 having an antifungal activity to Colletotrichum gloeosporioides and to suppressing growth of the fungus. CONSTITUTION: A Bacillus amyloliquefaciens CP1(deposit number:KCTC 11676BP) has an antifungal activity to Colletotrichum gloeosporioides. A culture of Bacillus amyloliquefaciens CP1 prevents Colletotrichum gloeosporioides. Powdery mildew of strawberry is prevented using the culture by culturing at 10-50°C and pH 5-8. A method for promoting strawberry growth comprises a step of diluting the culture in a ratio of 1/10-1/2000 and treating the diluted culture to strawberries.

Description

Bacillus amyloliquefaciens CP1 and contro method of strawberry anthracnose using the same

Strawberry is a perennial crop of the Rosaceae family and grows well in various soils and environments and is widely distributed in Africa, Asia, and Europe, as well as in North America and Oceania. Strawberry varieties cultivated a lot these days include scent, falcon, janghui, yukbo, etc. It is an important crop that accounts for 9% of the domestic vegetable production value. Strawberry cultivation is mainly focused on productive and productive facility cultivation, and is popular as a high-income crop. However, due to the cultivation of these facilities, the crop growth environment is poor in many cases, and the density of pathogens increases, so that many diseases occur.

Currently, about 30 kinds of strawberry diseases reported in Korea are known. Major diseases include powdery mildew, wilting disease, anthrax, gray mold, among which are seriously damaged by soil infection or anthrax.

Strawberry anthrax is mainly spread during the rainy season and is easily invaded the longer the leaves are wet, the more severe the soil moisture. Asymptomatic is present in the soil and occurs a lot during serial cultivation is very difficult to control. In addition, the remnants of diseased plants become an important first infectious agent of anthrax, causing wilting and killing of seedlings and seedlings in plant seedlings, such as creases and crowns, rather than fruits. The damage caused by strawberries is serious.

The general control method of strawberry anthracnose in Korea uses a lot of cultivation control and chemical control such as seedling or snow cultivation in house. However, effective control is difficult due to the characteristics of anthrax or soil propagation. Chemical control is more effective when treated prophylactically than treatment. Drugs reported in Korea include dithianon, carbendazim, chlorothalonil, azoxystrobin, and mancozeb. However, soil diseases such as anthrax are very difficult to control, and these chemical pesticides require safe control measures for farmers and consumers due to the big problems such as ecosystem destruction, environmental pollution and residual toxicity of pesticides.

In order to solve the problems of the prior art as described above, the present invention focuses on the pathogen, which is a pathogen that causes strawberry anthrax, and Bacillus amyloliqueur, which can produce metabolites having mycelial growth and spore germination inhibitory activity of the pathogen. The purpose of the present invention is to select a strain of Bacillus amyloliuefaciens CP1 and to provide a method for controlling strawberry anthrax using the same.

It is another object of the present invention to reduce the anthrax disease caused by strawberries using the Bacillus amyloliqueposis CP1 strain as an environmentally friendly agricultural material, and to reduce the harmfulness of the currently used organic synthetic pesticides and ultimately to improve the marketability and productivity of strawberries. To increase.

Research into the development of biopesticides, which attempt to use microorganisms themselves or secondary metabolites produced by microorganisms as an alternative to chemical pesticides, has been recognized since the 1980s and has attracted much attention until today. Since it turns out, many excellent achievements have been made. For example, Pseudomonas sp . , Streptomyces sp., Gliocladium virens, Trichoderma spp . Etc. Therefore, it is a very urgent problem to select and improve pollution-free antagonism microorganisms that can separate effective antagonistic bacteria and maintain environmental sterilization effect.

The antifungal antibiotics produced by the bacteria of the genus Bacillus are known to include 10 kinds of mycosubtilin, eumycin, bacillomycin, surfactin and iturin cyclic polypeptides, aminopolyols such as zwittermicin, lipoprotein, and B. subtilis , B. cereus , B. Polymixa , B. amyloliquefaciens and B. thuringiensis are typical strains used.

Accordingly, in the present invention, Bacillus , the most stable bacterium that has been widely used for biological control of crop disease as an antagonist. amyloliquefaciens was isolated from healthy strawberry plants, and the major pathogens of strawberry, Colletotrichum gloeosporioides and Botrytis cinerea , Fusarium oxysporum , Phytophthora High endogenous bacteria were selected by checking the antagonism of cactorum . Using this, we tried to develop an eco-friendly biological control method that can help farmers by demonstrating the disease control effect on the anthrax disease that causes the most damage during the recent growth of strawberry as well as promoting plant growth.

The present invention is a plant endogenous bacterium represented by the genus Bacillus among the bacteria used for biological control is colonized in the tissues of the plant, including the seed and the seed of the plant, fruit, stems, roots and tubers, that is, conduit and cortical tissues. However, it does not cause disease and increases the plant's disease resistance and has a growth promoting effect, so it is widely used for biological control.

Bacillus genus, which has been spotlighted as a microorganism for biological control, has been studied for the production of enzymes since the 1940's, but recently, researches on plant disease control and plant growth promotion have been conducted. It has been reported to antagonize pathogenic fungi by secreting antimicrobial or toxic substances out of cells. In addition, it is excellent in ability to withstand the stimulation of heat, drying, pH, sunlight, etc., so it is not easily killed in crop cultivation and nature, and it can survive for a long time by forming endogenous spores. Also advantageous.

1 is an optical micrograph after gram staining Bacillus amyloliqueposis CPI 1 strain (CP1)
Figure 2 is a scanning electron micrograph of Bacillus amyloliqueposis CPI 1 strain (CP1)
Figure 3 is a photograph showing the antimicrobial mechanism of Bacillus amylolytic partition C1 strain (CP1)
Figure 4 is a photograph showing the effect of mycelial growth and spore germination inhibited by the metabolite of Bacillus amyloliqueposis C1 strain (CP1)
Figure 5 is a photograph of the incidence of the control when treated with diluting the bacterial suspension and culture filtrate of Bacillus amyloliqueposis C1 strain (CP1) after cultivating strawberries in pots
Figure 6 is a photograph of an anthrax control experiment of strawberry as a culture filtrate of Bacillus amyloliqueposis C1 strain (CP1) in the open field packaging
Figure 7 is a photograph showing the growth promoting effect of strawberries using Bacillus amyloliqueposis C1 strain (CP1)

The present invention relates to a novel microbial Bacillus amyloliqueposis CP1 (Accession No .: KCTC 11676BP) having antifungal activity against strawberry anthrax and a method for controlling strawberry anthrax using the same. Hereinafter, a detailed technical configuration for carrying out the invention will be described with reference to experimental examples and drawings.

In order to select useful microorganisms for the control of anthrax, the major disease of strawberries, healthy strawberry plants were cultivated and divided into three parts: leaf, stem, and root.

Surface disinfected sites were divided into Nutrient agar (NA, Difco), DRBC agar (DRBC, Difco), Kings'B agar, V-8 juice agar, and Potato Dextrose agar, respectively. After incubation for a time, the first letter of the medium and the three parts of the plant in order to separate the leaves (L), stem (S), roots (R) in order in order to give the numbers.

A single colony of bacteria from the medium was passaged back to Nutrient agar, followed by culturing against gray mold and anthrax, and re-isolating bacteria forming low support by antagonism. The re-isolated bacteria were suspended in 20% glycerol solution and stored in deep-freezer (-70 ℃).

The endogenous bacteria were isolated by dividing the healthy strawberry plants into roots, stems, and leaves. As a result, 66 strains were obtained from the roots, 35 strains from the stems, and 17 strains from the leaves. For the identification of these endogenous bacteria, strains belonging to the Bacillus group were tentatively estimated according to Bergey's Manual (1984) by observing the characteristics of color, clarity, height, shape, motility, gram staining and size of colonies.

In order to test the antagonism of 118 isolated endogenous bacteria, the cultures were replaced with strawberry gray fungus and anthrax, and after 7-10 days, the size of low support was selected to be 20mm or more. As a result, three strains (NR32, NR81, NR83) isolated from the root, one strain selected from the stem (VS25), and one strain isolated from the leaf (KL78) were all identified as Bacillus group by morphological characteristics. Strains.

Among these five strains, the KL 78 strain maintained the strong antimicrobial activity to maintain low support for up to 2 months and finally selected the strain because it did not induce resistance to the antimicrobial agent of the pathogen (Table 1). This strain was named CP1.

Bacillus analysis of 16S rDNA of CP1 strain 99% similarity with velezensis (Table 2).

In the neighbor-joining tree based on the nucleotide sequence of the Gyrase A gene, the CP1 strain was B. velezensis. And belonged to one group with 100% similarity (Table 3). Therefore, B. velezensis is B. CP1 has been reported as B homologous to amyloliquefaciens . velezensis or B. Amyloliquefaciens was identified.

Morphological characteristics of strains CP1, and finally B through use pattern and 16S rDNA analysis, such as sequencing and gyr Agene per Using API kit. Amyloliquefaciens was identified.

As a result of identification of Bacillus amyl liqueur par syeonseu ssipi 1 (Bacillus Morphological and culture characteristics of amyloliquefaciens CP1) (Accession No .: KCTC 11676BP) were as shown in Table 4 and Table 5 below.

Morphological features shape Simple type Cell size 0.5 ~ 1.0㎛ motility + Gram Dyeing + Sporulation Oval Spore location center

Culture characteristics Colony form circle Colony surface lubricity Colony corners wave Colony Evolution protrusion Colony Blur Opacity Colony color cream color Colony gloss Not shining

The Bacillus amyl to extract DNA of a selected endogenous bacterial liqueur wave syeonseu ssipi 1 (Bacillus After inoculating amyloliquefaciens CP1) strain into LB medium, 1ml of cultured cells were placed in an Effendorf tube and centrifuged at 13,000rpm to obtain pure cells, and DNA was obtained using DNeasy Blood & Tissue Kit (QIAGEN. Cat. No. 69504). Obtained and then used while stored at -20 ° C. For cloning 16S rDNA, PCR amplification was performed using Primer 5'-AGAGTTTGATCMTGGCTCAG-3 '(Forward) and 5'-ACGGGCGGGTGTGTRC-3' (Reverse).

PCR amplification was performed using 100 mg template DNA, 0.5 μm primer, 0.2 mM dNTPs, 10X Taq buffer (10X Ex Taq buffer containing 20mM magnesium, Takara Co. Japan), and 0.025U / μl of Taq polymerase. A 35 cycle reaction was performed at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, annealing at 72 ° C. for 1 minute 30 seconds, and amplified by a Biometra thermocycler (Tampa, Florida, USA). PCR products were identified by 1% agarose gel electrophoresis. The identified PCR products were purified using a PCR Purification kit (Bioneer, Korea), and then the purified PCR products were ligation to pGEM T-easy vector (Promega, Madison, WI, USA) using T4 ligase and 16s rDNA sequencing Through OK1-4 and OK1-6 full sequence data were obtained.

gyr The final primer was adjusted to 50 μm by adding 10 pmol of p-gyrA-f and p-gyrA-r primer, 10XTaq buffer, 10 mM dNTP-Mix, 50Unit Taq and 50ng of template DNA. DNA amplification was performed for 5 minutes at the initial denaturation at 94 ℃, 30 cycles with denaturation 40 seconds (94 ℃), annealing 40 seconds (55 ℃), and extension 1 minute (72 ℃), followed by 15 minutes at final extension 72 ℃. It was.

As a result of identification of Bacillus bacteria amyl liqueur par syeonseu ssipi 1 (Bacillus amyloliquefaciens CP1) Botrytis cinerea , Colletotrichum gloeosporioides , Fusarium Bacillus from KACC to compare antagonism against oxysporum and Phytophthora cactorum amyloliquefaciens ATCC 23843, Bacillus velezensis CCUG 50740 strain was pre-cultured and compared with the antimicrobial activity after incubation under the same conditions.

Antagonists of CP1 strains were assayed using starters of cucumber (Bacdadagi), red pepper (red pepper) and strawberries (red fur).

At this time, the PDA plate medium was sterilized and cooled (50 ° C.), and the culture filtrate was added thereto for each dilution fold (10, 20, 40, 60, 80, 100, 1,000, 2,000 times) to make a drug flat medium. The mycelial diameter of 8 mm in diameter was removed from the mycelial tips of four pathogens cultured for 7 days in the center of the drug plate medium, and cultured at 25 ° C. Mycelial growth inhibition was calculated by the following equation.

Anthracnose spores of strawberries were suspended in sterile water to prepare a final spore concentration of 1 × 10 ⁶ / ml. Put the culture filtrate in PDB by diluting fold in 1.5ml tube, add 100µl of spore suspension, and incubate at 28 ℃ incubator for 3, 8, 12 hours, and spore germination with optical microscope (× 400). Inhibition rate and germination tube length were investigated.

After 24 hours of spraying the culture solution, the culture filtrate, and the bacteria by dilution factor, the control value was calculated by spraying 1 × 10 ⁶ spore / ml of the spore concentration of the anthrax bacterium. The control value was calculated at two days after 28 days of treatment with Difenoconazole, a culture filtrate, and a control drug, three times at weekly interval.

Bacillus in Strawberry Anthrax Control Investigation When the concentration of amyloliuefaciens CP1 strain was treated with 10 ⁷ spores / ml, the growth of strawberries was observed and sprayed once a week. After 4 weeks, the leaf area and stem length were measured.

Isolated Endogenous Bacillus After spraying and spraying the culture medium of amyloliuefaciens CP1 strain, surface sterilization of leaves, stems, and roots in order to rediscover the presence of the genus Bacillus in the plant, and then finely crushed with Homogenizer (IKA, 25 ㎛) and uniform particles. The impurities were filtered out using. Afterwards, Bacillus selective medium (MYP Agar base, OXOID) was plated three times by diluting fold (10 ^-1 ~ 10 ^-7 ) and observed for 1 to 2 days at 30 ℃ incubator to count single spore colony.

Bacillus The antimicrobial material produced by amyloliuefaciens CP1 was mainly present in the culture filtrate and showed strong antimicrobial activity in the butanol layer after fractionation with organic solvent. The active fraction of each step of the antimicrobial activity was identified as antimicrobial activity against anthrax. First, Bacillus for the production of antimicrobial substances Amyloliuefaciens CP1 was inoculated into Molasses broth, shaken and cultured at 30 ° C. for 4 days, and 4L of the culture was centrifuged at 1,000 rpm for 40 minutes to obtain a culture filtrate and cells. The culture filtrate was mixed with 100% Acetone in a 1: 3 ratio and left for 24 hours and concentrated using a Rotary Evaporator. The material extracted by the four solvent fractions was observed through antibacterial activity against anthrax.

The culture filtrate was passed through Diaion HP-20 resin without pH adjustment, washed with 50% hydrous methanol and eluted with 30% hydrous methanol. The 30% hydrous methanol fraction showing activity was concentrated under reduced pressure and freeze-dried to obtain a light brown powder.

When the antibacterial substance was separated by fraction using C18 reverse column, the fraction showing antimicrobial activity against anthrax was identified and the substance was determined by measuring the molecular weight using MALDI-TOF mass spectrum.

An antifungal substance which is a metabolite of the novel microbial Bacillus amyloliquefaciens CP1 (Accession No .: KCTC 11676BP) strain of the present invention is called iturin A2.

Inoculation of iturin A2, a metabolite of Bacillus amyloliquefaction CPI1 strain, into the starters (cucumbers, peppers, strawberries) showed that growth inhibition, abnormal growth, and the like were observed.

Therefore, Bacillus amyloliquefaction CPI1 of the present invention was found that it can be used as a biological control agent by inhibiting the mycelial growth of major pathogens without harming the starter.

Ie B. amyloliquefaciens in Molasses medium After culturing CP1, the filtrates were mixed on the PDA medium, and four major pathogens of strawberries were cultured to examine mycelial growth. The culture filtrate showed strong mycelial growth inhibition against B. cinerea and C. gloeosporioides .

In addition, B. cinerea , C. gloeosporioides in a medium containing 1/10 of the Bacillus amyloliquefaction CPI 1 culture filtrate of the present invention The mycelial growth rate was over 90%, and the effect was not significantly reduced even when diluted to 1/2000.

In particular, Bacillus amyloliquefaciens CP1 showed a 72% inhibition rate against anthrax, even when diluted 1/2000. In addition, when the Bacillus amyloliquefaction CPI 1 culture was diluted to a ratio of 1/10 to 1/2000 and treated to strawberries, the growth of strawberries was healthy and showed growth activity.

In the study of mycelial growth inhibition, Bacillus amyloliquefaction CPI1 culture filtrate inhibited B, cinerea and C. gloeosporioides among four major pathogens of strawberry, and the inhibition of spore germination of C. gloeosporioides was higher than that of control. Germination tube length and spore germination rate were very low. At 10, 20-fold dilution, 3, 8, and 12 hours of observation showed that the spores remained intact and the shape of anthrax was maintained at 3 hours, and spores were hardly observed from 12 hours. This is because spores were dissolved in the antimicrobial activity of the culture filtrate.

Bacillus amyloliqueursion CPI 1 strain of the present invention significantly reduced the disease incidence of strawberries, there was a promoting effect of plant growth to grow strawberry seedlings healthy.

When the strawberries were grown in pots and diluted with the bacterial suspension and culture filtrate of Bacillus amyloliqueposis C1 strain, respectively, the incidence of the control group was effective in controlling the disease to 75%. Bacillus Treatment of amyloliuefaciens CP1 strain with 10 and 20-fold cultures showed more than 70% higher control value and 100-fold showed less than 50% control value. This is due to the gradual but persistent release of antimicrobial substances by the bacteria due to the treatment of the suspension.

Also Bacillus Amyloliquefaciens CP1 was also effective in promoting plant growth. That is, as shown in Fig. 5, the area of the leaves and the length of the stems were much better by the treatment of the bacterial suspension.

Bacillus in open field The control of strawberry anthrax with culture filtrate of amyloliquefaciens CP1 strain showed a high 92% control effect. Therefore, the secondary metabolite (iturin A2) of the CP1 strain may be used as a microbial pesticide that can replace the chemical pesticide.

In addition, itruin A2 was found to be significantly suppressed powdery mildew in addition to anthrax.

<Experimental Example 1> Bacillus according to the culture conditions of the medium Growth test of amyloliquefaciens CP1 strain

Bacillus in 5 kinds of medium mainly used for bacterial culture including nutient broth for mass culture of CP1 strain amyloliquefaciens After culturing the CP1 strain under the same conditions, the absorbance (OD. 600 nm) was measured for each time period, and the number of cells was confirmed.

Bacillus CP1 amyloliquefaciens strain showed the highest antibacterial activity when cultured in broth Molasses, Bacillus, as shown in Table 6 The amyloliquefaciens CP1 strain is most suitable for the mass cultivation of Bacillus amyloliquefaciens CP1 strain because it is most proliferated in Molasses broth medium.

Bacillus Amyloliquefaciens CP1 strains showed the highest cell counts after 72 hours of culture, whereas B. atrophaeus showed the highest density at 24 hours and B. amyloliquefaciens and Burkholderia pyrrocinia at 30 hours, respectively. The different density of cells is shown to be due to not only the physiological characteristics of the strain, but also the difference between the spectrophotometer for measuring the number of cells and the type of culture medium and the concentration and dose of the inoculum.

Experimental Example 2 Bacillus According to Culture Temperature Growth test of amyloliquefaciens CP1 strain

Conditional Search for Growth Temperature of Bacillus in 500ml Erlenmeyer Flask Amyloliquefaciens CP1 strains were inoculated in Molasses broth medium and incubated with a shacking incubator at 10 ° C. The growth was excellent at 30 ° C. when the cell count was measured using absorbance (OD. 600 nm) during incubation (Table 7). However, at 50 ° C, the proportion of endogenous spores was higher than that at 30 ° C. This induces the generation of endospores and is considered to be highly applicable as a biological control agent.

Generally, strains of the genus Bacillus produce dipicolinic acid when spores are produced. Endogenous spores are very useful for making biological control agents, but media that can enhance the sporulation rate of Bacillus genus strains known to date are mainly used to increase the production of secondary metabolites such as increased production of crytstal toxin protein or increased production of antibiotics. Most of the medium for increasing the sporulation rate and no medium for the production of dry spores.

Experimental Example 3 Bacillus According to pH Growth test of amyloliquefaciens CP1 strain

Bacillus by Differentiating Initial pH in Optimal Medium Molasses broth When the growth of amyloliquefaciens CP1 strain was measured, it grew well from pH 5 to pH 8 (Table 8), especially at pH 6, and the shortest generation time was selected as the optimal condition.

Experimental Example 4 Bacillus According to Mass Culture Growth test of amyloliquefaciens CP1 strain

Bulk cultures when formulated antagonistic microorganism culture obtained from a mass-production is easy to optimize the production process simplified and used as the biological control, it is advantageous to produce economical products from the above experimental Bacillus Optimal Culture Conditions of Amyloliquefaciens CP1 Strains 4L volume culture using Jar-fermenter (BIOTRON, LiFlis GX, 5L vessel) under conditions of medium: Molasses broth, temperature: 30 ° C, pH: 6 resulted in a concentration of 3 On the first day, the cell count reached the highest density, and since then, it has become increasingly dead. The antimicrobial activity was also the strongest on day 4 (Table 9).

Gram staining was performed periodically to determine whether there was contamination, and gram-positive rods were observed. On the third day, spontaneous endospores were produced.

On the other hand, in carrying out mass cultivation of useful microorganisms such as Bacillus and Pseudomonas strains using Jar-fermenter, the optimal conditions such as growth factors, inorganic salts, nitrogen sources, and carbon sources, which are components of the medium, are selected and the maximum number of live cells Density and antimicrobial activity are considered important factors.

Claims (7)

The Bacillus amyl having antifungal activity effect on strawberry anthracnose fungus liqueur wave syeonseu ssipi 1 (Bacillus amyloliquefaciens CP1) (Accession Number: KCTC 11676BP)
The method of claim 1,
Strawberry anthracnose fungus collection Sat tree glutamicum (Colletotrichum gloeosporioides) germs characteristics of the Bacillus amyl liqueur wave syeonseu ssipi 1 (Bacillus as the amyloliquefaciens CP1)
A method for controlling anthrax bacteria of strawberries using Bacillus amyloliquefaciens CP1 culture according to claim 1 or 2.
A method for controlling powdery mildew of strawberries using Bacillus amyloliquefaciens CP1 culture according to claim 1 or 2.
According to claim 3, Bacillus amyloliquefaciens CP1 (Bacillus amyloliquefaciens CP1) culture is a method of controlling the anthrax in strawberry by culturing in large quantities at a temperature of 10 ~ 50 ℃.
According to claim 3, Bacillus amyloliquefaciens CP1 (Bacillus amyloliquefaciens CP1) culture is a method of controlling anthrax in strawberry by culturing in a large amount in the range of pH 5-8.
A method for promoting the growth activity of a strawberry, wherein the Bacillus amyloliquefaciens CP1 culture of claim 1 or 2 is diluted to a ratio of 1/10 to 1/2000 and treated to strawberries.

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