KR101656230B1 - Brevibacillus R1a13 and its use - Google Patents

Brevibacillus R1a13 and its use Download PDF

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KR101656230B1
KR101656230B1 KR1020140149505A KR20140149505A KR101656230B1 KR 101656230 B1 KR101656230 B1 KR 101656230B1 KR 1020140149505 A KR1020140149505 A KR 1020140149505A KR 20140149505 A KR20140149505 A KR 20140149505A KR 101656230 B1 KR101656230 B1 KR 101656230B1
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fusarium
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aspergillus
r1a13
penicillium
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KR20160050715A (en
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이데레사
주해진
이수형
류재기
김미자
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대한민국
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Abstract

The present invention breather with Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in growth inhibition and Fusarium (Fusarium) spore germination inhibitory activity on the in fungi for such fungi Brevibacillus genus R1a13 (KCTC18310P), and a method for inhibiting the growth and spore germination of Fusarium spp. In agricultural products using such a strain. The prophylactic or therapeutic composition containing the Brevibacillus sp. R1a13 (KCTC18310P) of the present invention and the prophylactic agent or composition containing the same has a remarkable effect on the growth of fungi belonging to the genus Fusarium, genus Aspergillus or Penicillium, It is excellent.

Description

Brevibacillus R1a13 and its uses {Brevibacillus R1a13 and its use}

The present invention relates to novel strains (strain), and relates to their use, in particular Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in the growth of such fungal inhibition and Fusarium ( The present invention relates to a novel strain having a spore germination inhibiting activity against a fungus belonging to the genus Fusarium and to a method for inhibiting the growth and spore germination of a fungus belonging to the genus Fusarium using such a strain.

Mycotoxins are secondary metabolites produced by contaminated fungi during the growth, storage, and distribution of crops and cause acyclovir and chronic mycotoxicoses in humans and livestock (Marasas, 1984; Desjardins, 2006). Crop disease and toxin pollution caused by toxin-producing fungi The toxic damage caused by the ingestion of agricultural products and foodstuffs is constantly occurring worldwide every year. Therefore, WHO, FAO and other advanced countries regard fungal toxins as dangerous substances in agricultural products and foods, And regulates the generation and propagation through the setting.

Toxins that cause serious problems not only in Korea but also all over the world are toxins produced by Aspergillus / Penicillium fungi and Fusarium fungi respectively. However, these toxins occur in different regions. The fungal toxins produced by Aspergillus / Penicillium fungi are Aflatoxin, Ochratoxin and Patulin. These toxins are mainly found in high temperature and high humidity tropical or subtropical grains Occurs. However, since the latter fungi toxin is common in cereals produced in temperate regions such as Korea, the importance of Fusarium fungi and toxin contamination is emerging from the perspective of domestic agro-food industry and public health hygiene (Lee et al., 2010; Lee et al., 2011; Lee et al., 2012). Toxin Generation From the viewpoint of damaging plant diseases caused by Liarium fungi, the genus Gibberella ( genus Gibberella ) is distributed throughout the world, and is known to cause red fungus disease, ear acacia, It causes diseases such as ear rot, stem rot, and root rot disease, resulting in a decrease in yield and quality of crops, as well as toxicosis by producing fungal toxins. Disease outbreaks The severity of the damage caused by the latter disease is increasing worldwide, not only because of the year but also because of the tendency to recur for many years depending on environmental conditions.

Fusarium fungus toxin, unlike aspergillus / penicillium toxin, occurs during the growth of crops, and the major secondary toxins are trichothecene and fumonisin. The tricothecenotoxin is produced mainly by Fusarium species such as F. graminearum, F. culmorum, F. poae, F. crookwellense, and F. sporotrichioides , and is produced mainly in various pulses and corn. Bleeding in circulatory organs, diarrhea, vomiting, neurological disorders, refusal to feed (Marasas, 1984; Desjardins, 2006). In Korea, Deoxynivalenol and Nivalenol toxins are typical tricothecene fungi toxins which are detected in rice, barley and corn. These toxins are mainly F. graminearum (complete generation: Gibberella zeae ) (Desjardins, 2006). Fu monitor Territories mycotoxins are Gibberella fujikuroi complex [F. verticillioides (= F. moniliforme, fully three: Gibberella moniliformis), F. fujikuroi (full generation: G. fujikuroi), F. proliferatum (full generation: G. intermedia)] , F. nygami , F. oxysporum, and the like, which cause cerebral leukodystrophy in horses, mules, and donkeys, pulmonary edema syndrome in pigs, liver cancer, hepatotoxicity and renal toxicity in experimental animal rats. It is also estimated that there is a strong relationship between the incidence of esophageal cancer in humans (Marasas, 2001).

As described above, in Korea, the toxicity of the latter fungus causes the disease to be caused not only in rice and corn, but also in the toxicity caused by ingestion of the fungus poisonous plant.

To minimize these damages, it is necessary to develop eco - friendly technology that reduces the toxin produced by fusarium fungi, which is dominant in cereals, but it is still not enough.

Thus germination of growth and spore, such aspect is harmful fungi, in particular Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in fungi that produce toxins another object of the present invention In an effective and environmentally friendly manner, and a method for inhibiting the growth of harmful fungi and germination of spores in agricultural products and foods using such strains.

In order to accomplish the above object, the present invention provides a method for effectively and efficiently cultivating harmful fungi producing toxins, particularly fungus of the genus Fusarium , genus Aspergillus , or genus Penicillium , Brevibacillus genus R1a13 (KCTC18310P), which is a novel strain that inhibits the growth of the plant.

The present inventors isolated 300 kinds of microorganisms in the cultivated soils and then cultivated 8 kinds of fusarium toxin fungi ( Fusarium graminearum PH-1, Fusarium graminearum H- 11, Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium graminearum , Fusarium proliferatum , and Fusarium verticillioides ). As a result, a strain having resistance to the fungus of the latter genus was selected (Fig. 1), and the selected strain R1a13 The 16S rRNA gene was identified in Brevibacillus through sequencing (FIG. 2).

The strain Brevibacillus strain R1a13 (KCTC18310P) according to the present invention can be cultured in a large amount by a conventional culture method of Brevibacillus sp. Strain. As the culture medium, a medium composed of carbon source, nitrogen source, vitamins and minerals can be used. For example, NB (nutrient broth), TSB (tryptic soy broth) liquid medium, or TSA (tryptic soy agar) solid medium can be used. The culture can be carried out under normal culture conditions, for example, at about 37 ° C for about 10-72 hours. The culture medium in the culture broth can be removed and centrifuged or filtered to recover only the concentrated cells, and these steps can be carried out according to the needs of a person skilled in the art. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.

In addition, the Brevibacillus genus R1a13 (KCTC18310P) strain according to the present invention may be modified or improved to have an activity equivalent thereto by a conventional physicochemical mutation method or the like known in the art or may have an activity superior thereto .

The present invention also provides a probiotic having an antifungal activity comprising a strain of the genus Brevibacillus R1a13 (KCTC18310P).

In addition, the present invention provides a composition for inhibiting noxious fungi comprising Brevibacillus genus R1a13 (KCTC18310P) strain.

The Brevibacillus genus R1a13 (KCTC18310P) strain is not limited to the strain itself, but may be a crushed cell wall fraction, a live cell, a dead bacterium, a dried bacterium, or a culture or culture thereof of R1a13 strain, or Aspergillus spp., Penicillium spp . , Alternaria spp. , Epicoccum sp. Spp. , Escherichia spp . , Fusarium spp. Isolated from its culture or culture, Or an antagonist against Epicoccum nigrum . In addition, the probiotics or compositions may further comprise suitable excipients or carriers. The culture medium may be a culture medium itself obtained by culturing the strain according to the present invention in a suitable liquid medium, a filtrate (filtrate or centrifuged supernatant) obtained by removing the strain by filtration or centrifugation of the culture medium, ultrasonication of the culture medium or dissolution in the culture medium A cell lysate obtained by treating an enzyme (lysozyme), and the like. The prophylactic or therapeutic composition may contain, but is not limited to, a solid culture broth or a culture such as a dried or extract of the culture broth.

Bracelet according to the present invention the non-Bacillus (Brevibacillus) in R1a13 (KCTC18310P) as a harmful fungi strains showing the antagonistic effects specifically to the Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, penny room Solarium (Penicillium) And may be any one selected from the group consisting of fungi, Alternaria sp. , Epicoccum sp. , Epicoccum nigrum , and the like.

The fungi belonging to the genus Fusarium are Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminearum H7-4, Fusarium graminearum , California Grammy Room (Fusarium graminearum) H7-11, Fusarium Asia tikum (Fusarium asiaticum) SCK04, Fusarium Asia tikum (Fusarium asiaticum) R308, Fusarium program Raleigh Blow Tomb (Fusarium proliferatum), dE (Fusarium Fusarium Bertie Sicily cucumber verticillioides , Fusarium oxysporum , Fusarium armeniacum , Fusarium anthophilum , Fusarium avenaceum , Fusarium babinda , Fusarium remarks to you (Fusarium begoniae), Fusarium stood natum (Fusarium circinatum), Fusarium outlet rikum (Fusarium concentricum), Fusarium Crooked wells Lawrence (Fusarium crookwellense), then Leeum ekwi Cassetti (Fusarium equiseti), Fusarium Fuji Kuroishi (Fusarium fujikuroi), Fusarium Globo Island (Fusarium globosum), Fusarium articles Lamy num / hetero's forum (Fusarium graminum / heterosporum), Fusarium konjum (Fusarium konzum Fusarium lactis , Fusarium langsethiae , Fusarium napiforme , Fusarium nisikadoi , Fusarium nygamai , and the latter, Fusarium species such as Fusarium phyllophilum , Fusarium poae , Fusarium pseudoanthophilum , Fusarium pseudograminearum , Fusarium ramigenum , Fusarium species such as Fusarium redolens , Fusarium sacchari , Fusarium sambucinum , Fusarium scirpi , Fiumarium sprotriquioide ( F usarium sporotrichioides), Fusarium Ste Lily Hippo Island (Fusarium sterilihyphosum), Fusarium sub-article Ruti nonce (Fusarium subglutinans), Fusarium Ladies ticker (Fusarium succisae), Fusarium tapsi num (Fusarium thapsinum), Fusarium tree sintum (Fusarium tricinctum , and Fusarium venenatum .

The Aspergillus (Aspergillus) in fungi Aspergillus cancer seutelro Damiani (Aspergillus amstelodami), Aspergillus Fu fumigatus (Aspergillus fumigatus), Aspergillus Klee as Tio Room (Aspergillus schlerotiorum), and Aspergillus Tree Tee And Aspergillus tritici .

The penny room Solarium (Penicillium) in fungi and the like penny room Solarium brother is thio draw-raised (Penicillium aurantiogriseum), Penny room Solarium sheet rinum (Penicillium citrinum), and Penny room Solarium irregularities dicarboxylic Tomb (Penicillium viridicatum).

The present invention also provides a method for inhibiting harmful fungi that is treated with a strain of Brevibacillus genus R1a13 (KCTC18310P). Preferably, the method for inhibiting the noxious fungus of the present invention is a method for suppressing harmful fungi of the genus Fusarium , the genus Aspergillus , the genus Penicillium , the genus Alternaria sp. , Epicoccum sp. , Or Epicoccum nigrum , and for the suppression of spore germination against the fungi of the genus Fusarium . The method comprising culture medium or separated from the culture Fusarium (Fusarium) genus Aspergillus (Aspergillus), A penny room Solarium (Penicillium), An Alterna Ria species (Alternaria sp.), Epi kokum species (Epicoccum sp. ), And Epicoccum nigrum and the like.

The composition for inhibiting harmful bacteria according to the present invention, which comprises the Brevibacillus genus R1a13 (KCTC18310P), a culture or a culture thereof, or an antagonistic substance isolated from the culture or the culture thereof, Or a detergent additive.

Of the present invention breather ratio Bacillus (Brevibacillus) in R1a13 (KCTC18310P), probiotic or compositions containing them are Fusarium (Fusarium) in fungi, Aspergillus (Aspergillus) in fungi, or penny room Solarium (Penicillium) in fungi of the like The inhibition of growth and the inhibition of spore germination for the fungi belonging to the genus Fusarium are remarkably excellent.

Fig. 1 shows eight Fusarium spp. Fungi belonging to the genus Brevibacillus This is a photograph showing the growth inhibitory effect of the fungi of the genus Fusarium at the time of confluence with the strain R1a13.
Fig. 2 is a flow diagram showing that strain R1a13 is deficient in Brevibacillus.
FIG. 3 shows that the strain R1a13 was cultured in a nutrient medium and then mixed with a fungal spore suspension (10 4 / ml) (culture filtrate 900ul + spore suspension 100ul) and cultured at 25 ° C to observe germination of spores at 10 days Results.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

[Example 1] Brevibacillus  Screening of R1a13

Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminarium ( Fusarium graminearum ), Fusarium graminearum , Fusarium graminearum , Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium proliferatum , , Fusarium verticillioides ) at 25 ° C for 5 days. Fusarium proliferatum and Fusarium verticillioides were distributed at Soonchunhyang University and the remaining fungi were retained by the National Institute of Agricultural Science Were used.

As a result of the experiment, a strain having the ability to inhibit the growth of Fusarium spp. Was selected as shown in Fig.

Based on the molecular sequence analysis based on the 16s rDNA nucleotide sequence described below, the strain was identified as Brevibacillus and designated as R1a13. On July 17, 2014, the Korea Research Institute of Bioscience and Biotechnology (KCTC) And received the deposit number KCTC18310P.

[Example 2] Brevibacillus  Identification of strain R1a13

16S rRNA sequencing was performed to identify selected strains of R1a13 and 16S rRNA sequences were compared and analyzed using EzBioCloud ( http://www.ezbiocloud.net/ ) to confirm genetic lineage of R1a13 strain .

As shown in FIG. 2, the 16S rRNA sequence of strain R1a13, which is a selective strain of the present invention, showed considerable homology with the genus Brevibacillus , and the strain R1a13 was identified as Brevibacillus .

[Example 3] Brevibacillus  Evaluation of spore germination inhibitory activity of strain R1a13

Brevibacillus R1a13 according to the present invention The strain was examined for the germination inhibitory activity against the spores of the fungi belonging to the genus Fusarium . R1a13 strain was cultivated in a nutrient medium for 3 days, and then mixed with a suspension of a fungal spore suspension (10 4 / ml) (culture filtrate, 900 μl + spore suspension, 100 μl) Respectively. As a result of observation, as shown in Fig. 3, on the 10th day of observation, eight kinds of Fusarium spp. ( Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminearum H7-4, Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium proliferatum ), And Fusarium verticillioides ), respectively. This spore germination inhibition effect lasted to 21 days.

[Example 4] Brevibacillus  Evaluation of growth inhibitory activity of strain R1a13

In order to evaluate the growth inhibiting activity of the Brevibacillus R1a13 strain, Example 1 Fusarium genus fungi 8 species other than the Fusarium genus, Aspergillus genus, Penny room Solarium in, Alterna Ria species, and epi kokum species like Brevibacillus R1a13 shown in And the growth inhibitory activity was evaluated.

As a result, Brevibacillus R1a13 Wherein the strain is selected from the group consisting of Fusarium oxysporum , Fusarium armeniacum , Fusarium anthophilum , Fusarium avenaceum , Fusarium albinum , Fusarium species , such as Fusarium babinda , Fusarium begoniae , Fusarium circinatum , Fusarium concentricum , Fusarium crookwellense , Fusarium equiseti , , Fusarium Fuji Kuroishi, Fusarium lactis (Fusarium fujikuroi), Fusarium Globo island (Fusarium globosum), Fusarium articles Lamy num / hetero's forum (Fusarium graminum / heterosporum), Fusarium konjum (Fusarium konzum) ( Fusarium lactis , Fusarium langsethiae , Fusarium napiforme , Fusarium nisikadoi , Fusarium nygamy, amai), Fusarium Philo pilrum (Fusarium phyllophilum), the Fusarium PO (Fusarium poae), Fusarium pseudo St pilrum (Fusarium pseudoanthophilum), Fusarium pseudo Graeme California room (Fusarium pseudograminearum), Fusarium lamina genum (Fusarium ramigenum) , Fusarium redolens , Fusarium sacchari , Fusarium sambucinum , Fusarium scirpi , Fusarium sporotrichioides , and the latter. Fusarium sterilihyphosum , Fusarium subglutinans , Fusarium succisae , Fusarium thapsinum , Fusarium tricinctum , and the latter. It was confirmed that Fusarium venenatum showed growth inhibitory activity.

In addition, Brevibacillus R1a13 Strain Aspergillus cancer seutelro Damiani (Aspergillus amstelodami), Aspergillus Fu fumigatus (Aspergillus fumigatus), Aspergillus Klee as Tio Room (Aspergillus schlerotiorum), Aspergillus Tree Tea City (Aspergillus tritici), Penny Room Solarium Such as Penicillium aurantiogriseum , Penicillium citrinum , Penicillium viridicatum , Alternaria sp. , Epicoccum sp. , Pseudomonas spp. And Epicoccum nigrum ( Epicoccum nigrum ).

Korea Biotechnology Research Institute KCTC18310P 20140717

Claims (9)

  1. Brevibacillus genus R1a13 (KCTC18310P).
  2. Brevibacillus genus R1a13 (KCTC18310P) strain,
    Toxins produced Fusarium (Fusarium) genus Aspergillus (Aspergillus), A penny room Solarium (Penicillium), An Alterna Ria species (Alternaria sp.), Epi kokum species (Epicoccum sp.), And epitaxial kokum you Groom ( Epicoccum nigrum ). ≪ / RTI >
  3. Brevibacillus genus R1a13 (KCTC18310P) strain,
    Toxins produced Fusarium (Fusarium) genus Aspergillus (Aspergillus), A penny room Solarium (Penicillium), An Alterna Ria species (Alternaria sp.), Epi kokum species (Epicoccum sp.), And epitaxial kokum you Groom ( Epicoccum nigrum ). ≪ / RTI >
  4. delete
  5. 4. The method according to claim 3, wherein the toxin-producing fungus is Fusarium graminearum PH-1, Fusarium graminearum H-11, Fusarium graminearum H7-4 , Fusarium graminearum H7-11, Fusarium asiaticum SCK04, Fusarium asiaticum R308, Fusarium proliferatum , Fusarium graminearum , de (Fusarium verticillioides), Fusarium oxy sports rooms (Fusarium oxysporum), Fusarium Armenia glutamicum (Fusarium armeniacum), Fusarium St pilrum (Fusarium anthophilum), Fusarium Abe and erecting (Fusarium avenaceum), Good Fusarium bar (Fusarium babinda , Fusarium begoniae , Fusarium circinatum , Fusarium concentricum , Fusarium crooi, kusense , Fusarium equiseti , Fusarium fujikuroi , Fusarium globosum , Fusarium graminum / heterosporum , Fusarium spp. konjum (Fusarium konzum), Fusarium lactis (Fusarium lactis), the Fusarium ditch Shetty (Fusarium langsethiae), Fusarium or pipom (Fusarium napiforme), Fusarium West kadoyi (Fusarium nisikadoi), Fusarium you're my (Fusarium nygamai), Fusarium Philo pilrum (Fusarium phyllophilum), the Fusarium PO (Fusarium poae), Fusarium pseudo St pilrum (Fusarium pseudoanthophilum), Fusarium pseudo Graeme California room (Fusarium pseudograminearum), Fusarium lamina genum (Fusarium ramigenum) , Fusarium redol Lawrence (Fusarium redolens), Fusarium four Kari (Fusarium sacchari), Fusarium Samburu when num (Fusarium sambucinum), silpi Fusarium (Fusarium scirpi), Fusarium Spokane Lot Key cucumber DE (Fusarium sporotrichioides), Fusarium Ste Lily Hippo Island (Fusarium sterilihyphosum), Fusarium sub-article Ruti nonce (Fusarium subglutinans), Fusarium Ladies ticker (Fusarium succisae), Fusarium tapsi num (Fusarium thapsinum), Fusarium tree sintum (Fusarium tricinctum), and Fusarium Venetian natum (Fusarium venenatum) in the composition, characterized in that any one selected from the group consisting of.
  6. 4. The method of claim 3, wherein the Aspergillus genus is selected from the group consisting of Aspergillus amstelodami , Aspergillus fumigatus , Aspergillus schlerotiorum , Wherein the composition is selected from the group consisting of Aspergillus tritici .
  7. 4. The method of claim 3 wherein the penny room Solarium genus penny room Solarium brother is thio draw-raised (Penicillium aurantiogriseum), Penny room Solarium sheet rinum (Penicillium citrinum), and Penny room Solarium irregularities dicarboxylic Tomb selected from the group consisting of (Penicillium viridicatum) ≪ / RTI >
  8. Aspergillus genus, Penicillium genus, Alteraria genus (genus Fusarium genus) produced by the toxin-producing Fusarium genus, Brevibacillus genus R1a13 (KCTC18310P) Wherein the composition is selected from the group consisting of Alternaria sp. , Epicoccum sp. , And Epicoccum nigrum .
  9. delete
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100475559B1 (en) 2003-07-08 2005-03-14 (주)바이오쉴드 A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation thereof
US20070060477A1 (en) 2004-03-31 2007-03-15 Pedersen Hans C Process

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005045005A1 (en) * 2003-11-11 2005-05-19 Higeta Shoyu Co., Ltd. Novel brevibacillus choshinensis and process for producing protein with use of the microbe as host

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100475559B1 (en) 2003-07-08 2005-03-14 (주)바이오쉴드 A microbes pesticide active against brevibacillus compost rot boil effect and process for preparation thereof
US20070060477A1 (en) 2004-03-31 2007-03-15 Pedersen Hans C Process

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
The Korean Journal of Microbiology, Vol.42, pp.135-141(2006.06.)*

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