KR101716136B1 - Method of purification and production of bioactive compound for biocontrol of plant diseases from Bacillus amyloliquefaciens subsp. plantarum EML-BC32-1 - Google Patents

Abstract

The invention amyl quinone Bacillus Lowry ocher origin having anti-fungal activity Patience plan tareom subspecies (Bacillus amyloliquefaciens subsp. plantarum ) EML-BC32-1. It is possible to obtain a variety of plant disease control agents through the purification and production method of the antifungal active substance using EML-BC32-1, and thus it can be used for increasing the commerciality and productivity of crops It is expected.

Description

FIELD OF THE INVENTION [0001] The present invention relates to an active substance of Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 for the control of plant mold fungi, a method for purifying and producing the same, and a method for producing the active substance of Bacillus amyloliquefaciens subsp. plantarum EML-BC32-1}

The present invention relates to a plant disease fungus control using a Bacillus amyloliquefaciens subspecies plantarum EML-BC32-1 strain isolated from loess, and more specifically, to a plant disease fungus control agent having a Bacillus amyloliquefaciens subsp. Plantarum EML -BC32-1 and a production method thereof.

Worldwide, phytopathogenic diseases such as wilt, anthrax and damping-off are serious diseases of various crops. Researches using biologically active substances produced by microorganisms for controlling these plant diseases actively proceed . Microorganisms can produce specific substances depending on the environment in which they live, especially those inhabited by harsh environments are more likely to produce certain substances.

Hwangto, which accounts for about 30% of Korea, has a less favorable environment than general soils, and the number of microbial populations is less than that of ordinary soils. Although the number of populations is small, the microorganisms inhabiting the loess are living in a harsh environment and are likely to produce certain substances that are distinct from microorganisms inhabiting common soils. Therefore, there is a need to find useful microorganisms derived from loess and to secure biological resources through the production of these biologically active substances.

In the Republic of Korea Registered Patent No. 1181756 Bacillus bacterium having an antifungal activity, and organic resolution (Bacillus and Korean Patent No. 0132218 discloses a novel antifungal antibiotic substance produced from the genus Bacillus sp. and a biological control agent using microorganisms such as Bacillus sp. The invention of the subject is continuously carried out. However, few microorganisms have been isolated in a particular environment, especially in harsh conditions.

The present invention relates to a bacterium belonging to the genus Bacilus , which is expected to be available for the control of plant diseases in the course of studying domestic loess inhabiting microorganisms. Bacillus spp. Isolated from loess has exhibited antifungal activity against phytopathogenic strains, and it is possible to suggest the possibility of development of environmentally friendly and high value - added biological control agent suitable for crop plant disease prevention.

Korea Patent No. 0132218 Korean Patent No. 1181756

The present invention is Bacillus amyl quinone Lori Patience subspecies tareom plan showing the antifungal for controlling plant diseases (Bacillus amyloliquefaciens subsp. plantarum ) EML-BC32-1 (accession number: KCTC12729BP), and a method for producing the biologically active substance.

The present invention provides an antifungal agent for controlling plant diseases, comprising Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 exhibiting antifungal activity and a culture thereof.

The present invention provides a method for purifying and producing an antifungal active substance of Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1.

In the present invention, the Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 or the culture thereof exhibits an antifungal effect, which is useful for controlling various plant diseases and for producing a composition containing an active substance capable of an antifungal action effective against pathogenic fungi The productivity and productivity of the crop can be effectively increased.

Figure 1 shows that the isolated microorganism is a bacillary amyloliquefaciens subplanter through gyrase A ( gyrA ) gene sequencing.
Figure 2 shows the antifungal activity of Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1.
Figure 3 shows the morphology of Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1.
Figure 4 shows the effect of Fusarium oxysporum (Bacillus amyloliquefaciens subsp. ≪ RTI ID = 0.0 > f. sp. radicis - for lycopersici (KACC 40031)) (A ) is 30 ℃, (B) shows the anti-fungal activity at 37 ℃, left on each agar plate (agar plate) is EML-BC32-1 supernatant, the right and TSB Lt; / RTI >
Figure 5 is a Bacillus amyl quinone Lori Patience subspecies plan eluted tareom the EML-BC32-1 culture medium with 80% ethanol in the HP-20 resin shows a mycelial growth inhibition amount of a culture fluid fraction, (A) is Hussain volume (Fusarium oxysporum f. sp. radicis - lycopersici (KACC 40031)) and (B) is the degree of inhibition against Botrytis cinerea (KACC 40574), and (C) anthrax ( Colletotrichum gloeosporioides (KACC 40003), which is the activity of the EML-BC32-1 culture eluted with 80% ethanol using an 8 mm paper disc.
Figure 6 shows the reverse phase-HPLC results in the analysis of the antifungal active substance.
Figure 7 shows (A) UV results and (B) FT-IR results in the analysis of antifungal active substances.
Fig. 8 is a graph showing the effect of Fusarium oxysporum f. Sp. On the antifungal active substances sufectin, penicillin and iturin. radicis - lycopersici (KACC 40031)).
Figure 9 shows the gyr gene A nucleotide sequence of Bacillus amyl quinone Lori Patience subspecies plan tareom.

Hereinafter, the present invention will be described in detail. The terms used in the present specification should be construed as generally understood by a person having ordinary skill in the art unless otherwise defined. It is to be understood that the drawings and embodiments are not intended to limit the scope of the present invention, which is not intended to limit the scope of the present invention. It is not.

The present invention relates to Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) , which is derived from loess and exhibits antifungal properties and can be used for the control of plant diseases subsp. plantarum ) EML-BC32-1.

In order to achieve the object of the present invention, Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 was deposited on December 5, 2014 (Accession No .: KCTC 12729BP) at the Genetic Resource Center of Korea Biotechnology Research Institute (KCTC).

The EML-BC32-1 strain of the present invention corresponds to a strain belonging to Bacillus amyloliquefaciens subsp. Plantarum as a result of the identification.

The Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 of the present invention is an antifungal strain derived from loess, inhibits the growth of bacteria and fungi, and is particularly effective in controlling plant diseases. The plant disease control effect can be achieved by inhibiting plant pathogen growth by various antifungal substances produced by Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1.

The antifungal substance produced by Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 corresponds to lipopeptides of various types. Specifically, cyclic lipopeptide (iturin-type), which is a cyclic lipopeptide, , A fengycin-type, and a surfactin-type lipid peptide. The iturin type means that iturine includes intrinsic amino acids such as A, C, L, N, and O, and intrinsin having a fatty acid chain of different lengths to intrinsin. The term "penicillin" refers to the inclusion of penicillin having a different length of fatty acid chain in the penicillin and penicillin, and "suipectin" refers to a protein having a fatty acid chain of different length in the penicillin, Pectin. ≪ / RTI > The antifungal agent can be fractionated and purified from Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 by RP-HPLC and LC / MS (FIGS. 6 and 7) Various homologous molecules have different lengths of fatty acid chains.

Of the antifungal substances produced by Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1, the penile-type lipid peptides have an antifungal activity such as penile ischemia, which is an iturin-type lipid peptide with excellent antifungal activity. . On the other hand, su- pectin type lipid peptides have biosurfactant rather than antifungal activity. The sufectin form may be less effective than other lipid peptides in terms of antifungal activity, but exhibits a more elevated antifungal effect when acting with other lipid peptides having antifungal activity. Specifically, the antifungal activity of penicillin type is the most excellent among the above lipid peptides, and the antifungal effect is enhanced when iturin or sufectin acts together with the pengiin type. In particular, the antifungal effect is further enhanced when sufectin acts with iturin. In addition, sufectin can be expected to have a synergistic effect of antifungal activity by inhibiting the growth of the strain because it is an important factor for inhibiting the formation of biological membranes of other bacteria and fungi.

In the present invention, Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 is particularly effective against wilt, scleroderma, anthracnose and gray mold in plant diseases, but can also be used for controlling plant diseases other than these plant diseases.

Among the antifungal pathogens of Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 in the present invention, Fusarium < RTI ID = 0.0 > oxysporum , Sclerotinia < RTI ID = 0.0 > sclerotiorum , Colletotrichum gloeosporioides and Botrytis < RTI ID = 0.0 > cinerea ), but can also be used for inhibiting the growth of other pathogens and for preventing plant diseases caused by them.

The plant disease control agent according to the present invention utilizes an antifungal active substance produced by Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1, and is used for controlling plant diseases. Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 Or a control agent containing the culture medium obtained by culturing Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 may be used as an effective ingredient. The active ingredient contained in the culture liquid is at least one kind of lipid peptide selected from iturin type, punggi type, and sufectin type.

The culture supernatant of Bacillus amyloliquefaciens subsp. Subplantarum EML-BC32-1 was cultured by culturing Bacillus amyloliquefaciens subsp. Subplantarum plantarum EML-BC32-1. The culture supernatant was dissolved in ethanol ≪ / RTI > in the presence of a base. In this case, the ethanol is more than 60 to 90% (v / v), 70 to 90% (v / v), preferably 75 to 85% (v / v) Lt; RTI ID = 0.0 > pathogenic < / RTI >

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited by the following Examples.

Gwangju metropolitan city, Naju city, Muan county, Haenam county, Yeongam county. The collected loess samples (1 g each) were suspended in 9 ml of sterilized water, diluted 10-fold, and then evenly distributed in TSA (Tryptic soy agar) medium and cultured at 37 ° C for one day. Store the microorganisms in TS broth at -70 ° C in 20% glycerol-supplemented medium.

Genomic DNA extraction was performed using HiGene ™ Genomic DNA Prep Kit (Biofact Co., Daejeon, South Korea). The PCR reaction consisted of 2 μl of genomic DNA, 1.5 μl (5 pmol) of each primer, 14 μl of distilled water, Mu] l PCR premix (Bioneer Co., Daejeon, South Korea). Gyrase A gene (Chun and Bae 2000) analysis was used for amplification of gyr AF (5'-CAGTCAGGAAATGCGTACGTCCTT-3 ') and gyr AR (5'-CAAGGTAATGCTCCAGGCATTGCT-3') set primers. PCR was carried out under the following conditions. The initial denaturation step was 5 min at 95 ° C and 30 cycles at 95 ° C for 1 min followed by annealing at 53 ° C for 40 sec or 1 min and final elongation step after 1 min at 72 ° C Followed by reaction at 72 ° C for 10 minutes. After PCR, the purified PCR product was sequenced using an ABI 3700 automated DNA sequencer. The gene sequence derived from this strain and GenBank DB was clustal X windows interface v. 2.1 (Larkin et al. 2007) and BioEdit software v. And analyzed using 7.2.5. Gene sequencing was performed using MEGA v. 6.0 (Tamura et al., 2013) and using Kimura's two-parameter distance model.

The gyrase A gene ( gyrA ) was sequenced from the isolated DNA and identified by molecular systematic taxonomy. As a result, Bcillus amyloliquefaciens subsp. plantarum and 99.6% (885/889 bp), respectively. Bacillus amyl quinone Lori Patience plan equal to the tareom EML-BC32-1 gyr A nucleotide sequence of Figure 9 (SEQ ID NO: 3). As a result of identification of this strain, Bacillus amyloliquefaciens subsp. plantarum ) (Fig. 1). The identified microorganism was named Bacillus amyloliquefaciensplantera EML-BC32-1 and deposited with the Genetic Resource Center (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (Accession No .: KCTC 12729BP).

Bacillus amyloliquefaciens plantarum EML-BC32-1 identified single colonies showing only morphologic differences in TSA (Tryptic soy agar) and LB (Luria-Bertani) medium among many complete mediums, And morphology was observed by streaking in TSA (Tryptic soy agar) medium (FIG. 3).

Bacillus amyloliquefaciens plantarum EML-BC32-1 was cultured in LB medium (10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract and 1 L of distilled water) at 180 rpm and 37 ° C. for 24 hours. The strains BC32-2, BC32-3 and BC32-4 were also cultured. After culturing, each of the above Bacillus amyloliquefaciens planarum was treated with Fusarium < RTI ID = 0.0 > oxysporum f. sp . radices - lycopersici ) culture medium. As a result of the inoculation, only EML-BC32-1 in the Bacillus amyloliquefacillus plantarum showed antifungal activity (FIG. 2).

To determine the optimal growth conditions of Bacillus amyloliquefaciens platamum EML-BC32-1, cultivation was carried out at 30 ° C (A) and 37 ° C (B), respectively, in TSB (Tryptic soy broth). The supernatant (left) was centrifuged at 15,000 rpm for 10 minutes and filtered at 0.45 ㎛. The supernatant (left) was filtered through a 8 mm paper disc using Fusarium oxysporum f. sp. As a result, 100 μl of the same amount of radix - lycopersici (KACC 40031) culture medium was used as the control (right) TSB medium. As a result, it was confirmed that the clear zone was larger at 37 ° C and 37 ° C was the optimum temperature (Fig. 4).

For purification of the antifungal active substance of Bacillus amyloliquefaciens plantarum EML-BC32-1, the cells were cultured in LB medium (10 g of tryptone, 10 g of sodium chloride, 5 g of yeast extract and 1 L of distilled water) at 180 rpm and 37 캜 for 24 hours, After the incubation, the supernatant was obtained by centrifugation at 7,000 rpm, 4 ° C for 10 minutes. The resulting supernatant was eluted with alcohol (0%, 20%, 40%, 60%, 80%, and 100% ethanol (v / v) using Diaion HP-20 resin manufactured by Mitsubishi Chemical). After that, it was measured as a degree of inhibition of mycelial growth of phytopathogenic fungi by a method of floc substitution culture of eluted supernatant for Fusarium , Botrytis and Colletotrichum , X when it hardly occurs, and ○ when inhibited by 0.5 mm or more are shown in Table 1 below.

Ethanol (v / v) Antifungal activity 0% EtOH × 20% EtOH × 40% EtOH × 60% EtOH × 80% EtOH ○ 100% EtOH ×

As a result of checking the antimicrobial activity, the antifungal activity was exhibited in the 80% ethanol fraction (FIG. 5).

The antifungal active substance of Bacillus amyloliquefaciens platamum EML-BC32-1 was purified from the supernatant fraction eluted with 80% ethanol exhibiting the antifungal activity. The mixture was completely evaporated using an evaporator, dissolved in a minimal amount of methanol, and filtered through a 0.20 μm 2 mm non-sterile hydrophilic syringe filter (MACHEREY-NAGEL). After filtration, the solution was dissolved in a minimal amount of methanol and dissolved in 50% acetonitrile containing 0.1% TFA (trifluoroacetic acid). The solution was filtered through a 0.2 μm non-sterile single use syringe filter (RC-membrane, PP-housing, Sartorius). The filtrate contained C ₁₈ And purified by reversed-phase HPLC (Shimadzu 10AD, Japan) using a column (Waters μBondapak C ₁₈ 3.9 × 300 mm). Separation and purification were performed by flowing 5 ml of acetonitrile containing 0.1% TFA and 95% acetonitrile containing 0.1% TFA at a concentration of 1 ml per minute for 50 minutes. The result of the purification was confirmed by the change of absorbance at 230 nm, and the elution of the sample was confirmed. The materials were collected by each fraction, lyophilized, dissolved in a minimum amount of metalol, and 100. As a result, a single peak with an activity was obtained and it was found to be well purified. Analysis of the antifungal active substance of Bacillus amyloliquefaciens planarum EML-BC32-1 was performed by LC / MS (API 2000, USA) and FT-IR spectroscopy (FIGS. 6 and 7). The results showed that the active substances were the lipopeptides inturin, fengycin and surfactin, and the measured values were slightly different depending on the number of carbon atoms in each substance (Table 2).

The lipopeptides produced in Bacillus amyloliquefaciens subsp. Plantarum EML-BC32-1 Lipopeptide Residence time
(min) Molecular Weight
[M + H] < ⁺ >
Molecular Weight
[M + Na] < ⁺ > #One Iturin
(iturin)

28.7 1,031.9 1,054.2 #2 30.5 1,046.1 1,068.9 # 3 32.0 1,059.7 1,082.8 #4 Pen machine
(fenchycin)

33.5 1,463.8 1,486.9 # 5 34.3 1,477.6 1,499.4 # 6 35.5 1,505.4 1,527.5 # 7 Water pectin
(surfactin)

40.5 1,008.8 1,031.1 #8 41.5 1,023.1 1,044.9 # 9 45.0 1,037.2 1,058.8

The antifungal activity of each lipopeptides shown in Table 2 Hussain volume (Fusarium oxysporumf f sp radices -. .. Lycopersici) inoculation experiments lipopeptide discarded divine activity in the culture medium more excellent than yitu lean and the number of pectin (Fig. 8).

In addition, in the packaging experiment, this strain exhibited the same disease control activity spectrum for the above pathogens.

Korea Biotechnology Research Institute KCTC12729BP 20141205

<110> CHONNAM NATIONAL UNIVERSITY <120> Method of purification and production of bioactive compound for          biocontrol of plant diseases from Bacillus amyloliquefaciens          subsp. plantarum BC32-1 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> gyre forward primer <400> 1 cagtcaggaa atgcgtacgt cctt 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> gyr reverse primer <400> 2 caaggtaatg ctccaggcat tgct 24 <210> 3 <211> 900 <212> DNA <213> Bacillus amyloliquefaciens subsp. plantarum BC32-1 gyrase A <400> 3 tgaagccggt tcacagacgg attttgtacg caatgaatga tttaggcatg accagtgaca 60 aaccatataa aaagtctgcc cgtatcgtcg gtgaagttat cggtaagtac catccgcacg 120 gtgactcagc ggtttacgaa tcaatggtca gaatggcgca ggattttaac taccgctaca 180 tgcttgttga cggacacggc aacttcggtt cggttgacgg cgactcagcg gccgcgatgc 240 gttacacaga agcgagaatg tcaaaaatcg caatggaaat tctgcgtgac attacgaaag 300 acacgattga ctatcaagat aactatgacg gttcagaaag agagcctgcc gtcatgccat 360 cgagatttcc gaatctgctc gtaaacgggg ctgccggtat tgcggtcgga atggcgacaa 420 acattcctcc gcatcagctt ggagaagtca ttgaaggcgt gcttgccgta agtgagaatc 480 ccgagattac aaaccaggag ctgatggaat acatcccggg cccggatttt ccgactgctg 540 gtcagatttt gggccggagc ggcatccgca aggcatatga atccggacgg ggatcaatca 600 caatccgggc taaggctgaa atcgaagaga catcatcagg aaaagaaaga attattgtta 660 cggaacttcc ttatcaggtg aacaaagcga gattaattga aaaaatcgca gatcttgtcc 720 gagacaaaaa aatcgaagga attaccgacc tgcgagacga atccgaccgt aacggaatga 780 gaatcgtcat tgagatccgc cgtgacgcca atgctcacgt cattttgaat aacctgtaca 840 aacaaacggc cctgcagacg tctttcggaa tcaacctgct ggcgctcgtt gacggacagc 900                                                                          900

Claims (7)

It is possible to produce iturin, penicillin and sufectin as antifungal active substances,
Which exhibit antifungal activity against at least one strain selected from the group consisting of Fusarium oxysporum , Colletotrichum gloeosporioides , and Botrytis cinerea .
Loess Bacillus amyl Laurie for control of plant diseases, microbial-derived quinone Patience subspecies plan tareom (. Bacillus amyloliquefaciens subsp plantarum) EML -EML-BC32-1 ( accession number: KCTC 12729BP). The method according to claim 1,
The plant disease wilt, gyunhaekbyeong, anthracnose and gray mold is at least one disease selected from the group consisting of, amyl quinone Bacillus Lowry for controlling plant diseases Patience plan tareom subspecies (Bacillus amyloliquefaciens subsp. Plantarum) EML -EML-BC32-1 ( Depositary No. KCTC 12729BP). delete A microorganism culture medium containing the microorganism culture solution of claim 1 as an active ingredient,
A plant exhibiting antifungal activity against at least one strain selected from the group consisting of Fusarium oxysporum , Colletotrichum gloeosporioides , and Botrytis cinerea . Antifungal Control Agent for Bottle Control. delete 5. The method of claim 4,
Wherein said culture medium is a culture medium eluted with 70 to 90% alcohol (v / v). delete

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