KR20080037849A - Novel streptomyces sp. bacteria and biopesticide using the same - Google Patents

Abstract

A novel Streptomyces sp. strain is provided to improve antibacterial activity against various plant pathogenic fungi. A biopesticide containing the same strain is also provided to inhibit growth of plant pathogenic fungi without harmful effects on human, domestic animals and plants or remaining toxicity in environment. A novel Streptomyces sp. strain having antifungal activity is isolated from the agricultural soil, has 16S rRNA(ribosomal RNA) having the nucleotide sequence of SEQ ID NO:1, and inhibit growth of plant pathogenic fungi including Colletotrichum coccodes, Botrytis cinerea, Cladosporium cucumerinum, Didymella bryoniae, Aspergillus niger, Fusarium solani, Paecilomyces sp., and Trichophyton mentagrophytes. A biopesticide contains Streptomyces sp. strain, its cultured medium or filtered solution of the cultured medium.

Description

Novel Streptomyces sp. Novel Streptomyces sp. bacteria and Biopesticide using the same}

1 is a photograph observing the form of the colonies formed by culturing the strain Streptomyces strain Actino-H1 of the present invention with an optical microscope.

Figure 2 is a photograph of the Streptomyces genus strain Actino-H1 of the present invention observed with an optical microscope.

Figure 3 is a photograph of the Streptomyces genus strain Actino-H1 of the present invention observed with a scanning electron microscope.

Figure 4 is a phylogenetic tree based on the 16s rRNA gene of Streptomyces sp. Actino-H1 of the present invention.

5 is a UV spectrum analysis result of the antifungal active substance produced by Streptomyces sp. Strain Acitno-H1 of the present invention.

Figure 6 is a graph showing the pH stability of the antifungal active substance produced by the Streptomyces genus strain Actino-H1 of the present invention.

Figure 7 is a graph showing the cell mass (◆) of the Streptomyces sp. Strain of the present invention according to the culture time (◆) and the production amount (■) of the antifungal active substance produced thereby.

The present invention relates to a novel radiation flux strain and a microbial pesticide preparation using the strain. More specifically, the present invention relates to a microbial pesticide preparation using the above-described radiation strain having antifungal activity, and the strain useful for the prevention and treatment of plant diseases caused by fungi.

Streptomyces sp . ) Is a gram-positive bacterium that exists in various soils and is an important industrial microorganism used for the production of antibiotics, bioactive substances, vitamins, and various bioenzymes (Chang, Jin-Jin et al., 1995; 23 (1): 36-42 ). So far, about 2/3 of the 100,000 kinds of bioactive substances detected from microorganisms are found in Streptomyces spp., And these bacteria occupy a very high proportion in the search for various bioactive substances.

Today's agriculture faces the urgent task of resolving the food shortage by exponentially increasing population. Therefore, it is important to safely cultivate the crops, and stable and maximal yield can be expected only when the pests, insects and weeds that are damaging during the cultivation and growth of the crops are removed. As one of the solutions to increase the yield, the amount of chemical pesticides has been increased.

However, side effects such as toxicity, environmental pollution and adverse effects of natural ecosystems, resistance to drugs, and resistance emergence due to the use of indiscriminate and excessive chemical pesticides are contaminating soil, rivers, and groundwater, resulting in the destruction of environmental ecosystems. In addition, residual toxicity and pesticide poisoning of agricultural products have a significant direct impact on human health. Therefore, research on biological control is being actively conducted as a part of reducing the severe damage of such chemical pesticides.

Biological control can be divided into microorganisms (fungus, bacteria, viruses) that exist in nature or by using active substances produced by microorganisms. Such biological control prevents pests by using substances derived from animals, plants, microorganisms and natural products. Unlike chemical pesticides, the effect is delayed, and the effective cultivation of crops is not a simple control to solve the whole with just one effective pesticide. It is a comprehensive control method that is applied to fully understand and observe the characteristics and the process of occurrence of pests. Biological control has the disadvantage of developing various kinds of biological pesticides for different pests depending on the type of harmful pests, but on the contrary, it is an environmentally friendly control that does not destroy the ecosystem because it selectively controls only specific pests. In addition, since biological control agents are harmless to humans and can be used at harvest time, it is expected that the development of these eco-friendly biological pesticide control technologies will be actively promoted and their use will increase. It is expected to expand into pesticides.

In biological control, microbial pesticides and antibiotics or toxins are used to kill pathogens and pests. Microbial fertilizers that promote growth.

Used as a microbial pesticide, antibiotics produced as metabolites from living organisms have the advantage of showing antimicrobial activity only against target pathogens and pests, and having little effect on humans, livestock, and plants, and do not remain in the environment. . Antibiotics produced from these microorganisms are emerging as an important means of biological control in that they are environmentally friendly and have no residual toxicity. In particular, environmental damage and health damage caused by the indiscriminate use of chemical pesticides have become serious, and development of microbial pesticide preparations using microorganisms is required.

Accordingly, the present inventors have isolated a strain of Streptomyces genus having antifungal activity against fungi causing disease in crops from soil samples, and developed a microbial pesticide preparation using the same.

It is an object of the present invention to provide a novel Streptomyces spp. Strain with antifungal activity.

Still another object of the present invention is to provide a microbial pesticide preparation containing the strain of Streptomyces sp., Which has antifungal activity, its culture solution, or its culture filtrate as an active ingredient.

In order to achieve the above object, the present invention is Streptomyces genus having antifungal activity sp . Strains belonging to).

In one embodiment of the invention, the strain Streptomyces genus having antifungal activity is characterized by having the 16S rRNA sequence of SEQ ID NO: 1.

Streptomyces genus strains of the present invention were isolated from soil samples of low-ply arable land, Streptomyces albogrisus of Streptomyces genus by morphological characteristics (Figures 1 to 3) and 16S rRNA analysis (see Figure 4) ( Streptomyces albogriseus ).

The strain Streptomyces genus of the present invention produces an antifungal active substance.

In one aspect of the invention, the antifungal activity of the genus Streptomyces strains collect rikum kokko des (Collectrichum coccodes ), Botrytis cinerea cinerea ), Cladosphorium cucumerinum ), Didymella bryoniae and Aspergillus niger niger ), Fusarium solani solani ), Paecilomyces sp . ), And Trichophyton metagrophytes , against one or more plant pests selected from the group consisting of:

In another aspect, the present invention provides a microbial pesticide preparation containing at least one selected from the group consisting of Streptomyces sp. Strain having antifungal activity, its culture solution and its culture filtrate as an active ingredient.

Antifungal active substances produced from the Streptomyces sp. Strain of the present invention can be secreted extracellularly. Therefore, the strain of Streptomyces sp. Of the present invention, its culture solution or its culture filtrate can be used as an active ingredient of a microbial pesticide preparation.

The culture solution may be obtained by incubating the Streptomyces sp. Strain of the present invention in a liquid medium at 25 to 30 ° C. for 10 to 14 days, and may be included in the microbial pesticide preparation in a liquid or dried solid phase. Preferably, the culture solution can be obtained by culturing the radiation flux strain of the present invention in a liquid medium (including glucose, yeast extract and malt extract) at pH 8.0 for 14 days.

The culture filtrate is a supernatant from which the cells are removed from the culture solution, and can be obtained by centrifugation or filtration. The culture filtrate may be included in the microbial pesticide formulation in a liquid or dried solid phase.

The microbial pesticide preparation of the present invention may further include a carrier according to the use, purpose and method in addition to such an active ingredient.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention and are not intended to limit the present invention.

Example  One: New Streptomyces  genus( Streptomyces sp .) Isolation and Identification of Strains

1. Strain Isolation

Soil samples were collected from low disease farmland in Chungcheongnam-do and North Korea. The collected soil samples were heat-treated at 100 ° C. for 60 minutes and then suspended in sterile saline solution and diluted in an appropriate ratio to remove Bennet's agar, 2 g of yeast extract; 1 g of cassamino; 2 g of dextrose 10 g; 15 g of agar, 1 L of distilled water; pH 7.0 ± 0.2) and Bacto-Actinomycete Isolation Agar (AIA) (2 g of sodium caseinate; 0.1 g of asparagine; 4 g of sodium propionate; 0.5 g of dipotassium phosphate; 0.1 g of ferrous sulfate; 0.1 g of agar) 15 g; 1 L of distilled water; pH 8.1 ± 0.2), and then incubated at 25 ± 1 ° C. Colonies of the filamentous Streptomyces genus as a result of culture were isolated and isolated on Potato Dextrose Agar plate (potato extract 200g; Bactobacillus dextrose 20g; Bacto agar 26g; distilled water 1L; pH 5.6 ± 0.2) Inoculated strains of Streptomyces genus were examined for the presence and size of growth hypoxia. The strain of Streptomyces genus showing the highest antifungal activity was isolated and named as Actino-H1.

2. Strain Identification

(1) morphological characteristics

Optical microscopy of morphology and strains of colonies formed by incubating the selected Streptomyces sp. Actino-H1 in YEME medium (4 g of yeast extract; 10 g of malt extract; 4 g of glucose; 1 l of distilled water; pH 7.0 ± 0.2) at 25 ° C for 72 hours As a result, as shown in Figures 1 and 2, the typical Streptomyces genus morphology was shown. The spores were found to be smooth in spore form and spore chains were rectiflexible. The constituents of diaminopimelic acid (DAP), a cell wall component in Streptomyces sp., Consisted of LL-DAP. In addition, as a result of investigating the characteristics of intracellular sugars by the method of Lechevalier (Lechevalier et al., Actinomycete taxonomy, special publication 6.Socoety for Industrial microbiology, Arlington. 1980), the characteristic sugar of Streptomyces genus was identified as C. The results were consistent with the characteristics of Streptomyces spp.

Inorganic salts starch agar medium (soluble starch 10g; (NH ₄ ) ₂ SO ₄ 2g; CaCl ₂ 2g; K was used as a medium for sporulation and morphology of Streptomyces spp. ₂ HPO ₄ 1g; agar 15g; distilled water 1L; pH 7.0 ± 0.2) was incubated at 28 ℃ for 5 days and observed with a scanning electron microscope (SEM) as shown in FIG.

(2) 16S rRNA sequence identification

After culturing Streptomyces spp. Of the present invention in YMEM medium, chromosomal DNA was isolated and then denatured at 94 ° C. for 1 minute using primers of SEQ ID NO: 2 and SEQ ID NO: 3 used for 16S rRNA sequencing, at 60 ° Amplification was carried out by PCR in xx cycles ( desired for cycle counts ) under conditions of annealing for 1 minute and synthesis for 1 minute and 30 seconds at 72 ° C. 0.8% agarose gel electrophoresis was performed on the amplified PCR result, and the DNA encoding 16S rRNA of about 1400 bp was separated and purified. The nucleotide sequence of SEQ ID NO: 1 was confirmed by analyzing the gene sequence of the isolated 16S rRNA using an ABI PRISM 3700 DNA Analyzer. The analyzed sequence was compared with the rRNA sequence of the BLAST program and RDP (RNA database project) in GenBank to obtain a schematic as shown in FIG. 4. As shown in Figure 4, the strain Actino-H1 isolated from the present invention was identified as a new strain belonging to Streptomyces albogriseus ( Streptomyces albogriseus ).

Example  2: Isolation and Characterization of Antifungal Active Substances

1.Isolation and Purification of Antifungal Active Substances

In order to isolate and purify the antifungal active substance produced by the Streptomyces genus strain Actino-H1 isolated in the present invention, the strain was incubated in YEME medium for 25 ± 1 ° C., 150 RPM, 72 hours. The culture solution was centrifuged to separate the supernatant and the cells, and the supernatant (culture filtrate) was filtered with Whatman No. 2, and the secondary metabolite was extracted with an organic solvent, ethyl acetate, to obtain a yellow substance. The ethyl acetate layer was concentrated using an evaporator and the concentrate was dissolved in distilled water and stored at 4 ° C. for one day. Thereafter, the precipitate obtained by centrifugation was dissolved in methanol and developed in a silica gel column chromatography (Merck Co, 70-230 mesh, 4 × 20 cm) as a solution of methanol: H ₂ O = 7: 3 ratio. Subsequently, the mixture was eluted with methanol using Sephadex LH-20 column chromatography (Pharmacia, 4 × 20 cm), and then concentrated and purified. As a result of confirming the purity of the isolated and purified antifungal active material using an ultraviolet spectrophotometer (UV), as shown in Figure 5, the antifungal active material produced by the strain Streptomyces genus isolated in the present invention has an absorbance at 340nm It was found to be a substance.

2. Characterization of antifungal active substances

 (1) pH stability of antifungal active substance

The antifungal actives produced by Actino-H1 were adjusted to pH 1-13 using 1N HCl and 1N NaOH solution, pretreated for 17 hours at 4 ° C, neutralized to pH 7.0, and the remaining antifungal activity was measured by viable cell mass measurement. Investigate. As shown in Figure 6, the antifungal active material produced by this strain is relatively stable in acidic conditions than in alkaline conditions, and thus it was investigated that it can be used for practical control in acidified Korean soil.

(2) thermal stability of antifungal active substances

The antifungal activity produced by Actino-H1 was heat treated at 40 ° C., 60 ° C., 80 ° C., 100 ° C., and 121 ° C. for 30 minutes. As a result, the antifungal active material was relatively stable to heat, and even after heat-treating for 30 minutes at 80 ° C., it was investigated that the antibacterial activity was maintained at 50% or more.

(3) Productivity of antifungal active substance according to incubation time

In order to investigate the optimal production condition of antifungal active substance according to the incubation time of Actino-H1 strain, 1% of strains were inoculated to investigate the production of antifungal active substance according to the incubation time. As a result, as shown in Figure 7 (◆: cell weight, ■: antifungal active material), the production rate of the antifungal active material increased more than 90% when Actino-H1 strains were cultured for 14 days or more. The growth rate of the cells was the highest in the dry weight of the cells when cultured for 10 days. Comparing the growth rate of the cells with the production of antifungal active substances, the growth of the cells was the highest at 10 days after cultivation, whereas the production of the antifungal actives was the highest at 14 days. It is thought that antifungal actives were produced by post secondary metabolism.

Example  3: antifungal spectrum investigation

In this example, Potato Dextrose agar (potato extract 200g; bactodextrose 20g; bacto agar 26g) was tested to examine whether the secondary metabolite of the isolated Streptomyces sp. After inoculating crop pathogens in 1 L of distilled water; pH 5.6 ± 0.2), the growth inhibitory effect on the crop pathogenic fungi was evaluated using a culture medium of the strain Actino-H1 of Streptomyces spp. The crop pathogenic fungus Collectrichum coccodes , used for the evaluation of growth inhibitory effects, develops black dot root rot on branches, anthracnose on peppers, and anthrax and black spots on tomatoes. It is a causative agent of rot, and Botrytis cinerea is a causative agent of gray mold in potatoes, sweet potatoes, kidney beans, ginseng, eggplants, and flowers. Cladosporium cucumerinum is the causative agent of Scab in cucumbers, melons, cantaloupe, pumpkins, etc. It was a crop pathogenic fungus that causes Gummy stem rot in crops.

The paper disc method was used to identify the size and size of growth-lowering ring that inhibits the growth of pathogenic fungi around the disc. Collectrichum coccodes, a fungal pathogenic fungus, using the secondary metabolite of Streptomyces sp. Actino-H1 isolated from the present invention KACC 40227, Botrytis cinerea KACC 40573, Cladosporium cucumerinum KACC 40576, Didymella bryoniae The growth inhibitory effect on KACC 40900 showed that Actino-H1 had very good antifungal activity. In addition, the secondary metabolite of Actino-H1 is Aspergillus , a common pathogenic fungus. niger ATCC 31656, Fusarium solani KACC 40384, Paecilomyces sp . Trichophyton . metagrophytes also showed antifungal activity to ATCC 9129.

Example  4: Comparison of efficacy with conventional biopesticides

In this example, the antifungal activity of commercially available biochemical pesticides and Streptomyces sp. Strain of the present invention was compared. Commercial pesticides to be compared had the composition shown in Table 1. Collectrichum coccodes KACC 40227, Botrytis cinerea KACC 40573, Cladosporium cucumerinum KACC 40576, Didymella bryoniae KACC 40900 and Aspergillus niger ATCC 31656, Fusarium solani KACC 40384, Paecilomyces sp . Comparison of antifungal activity against Trichophyton metagrophytes ATCC 9129 It is shown in Table 2. As shown in the results, the antifungal titer of the company A was Collectrichum coccodes , Didymella bryoniae , Aspergillus niger, Trichophyton metagrophytes and B and excellent four products Collectrichum coccodes , Botrytis cinerea , and Fusarium Excellent in solani , C company products Botrytis cinerea , Didymella bryoniae , and Paecilomyces sp . Company D's product is Collectrichum coccodes , Cladosporium cucumerinum , Paecilomyces antifungal activity against sp ., and Trichophyton . On the other hand, Actino-H1 showed excellent antifungal activity against all strains. Table 3 below summarizes the antifungal activity spectrum of the Streptomyces sp. Actino-H1 isolated in the present invention.

Table 1.Components of Commercial Biopesticide Formulations Compared in the Examples

product ingredient Formulation Volume A B 0.05%; Mo 0.0005% Liquid 1L B Paenibacillus polymyxa 1.0 × 106 cfu / g Liquid 1L C Lacto bacillus confusa 1.5 × 106 cfu / ml, Pichia anomalla 1.5 × 106 cfu / ml Liquid 1L D Bacillus sp. Ac-1 3.5 × 105 cfu / g powder 250 g E Streptomyces Actino-H1 Liquid 1L

Table 2. Comparison of Antifungal Activity

Fungus A B C D Actino-H1 C. coccodes ++ + - + +++ B. cinerea - ++ + - ++ C. cucumeriunm - - - + ++ D. bryoniae + - + - ++ Aspergillus niger + - - - +++ Paecilomyces sp . - - + + ++ Fusarium solani - + - - ++ T. metagrophytes + - - - +

Table 3. Antifungal activity spectrum of Actino-H1 against various pathogenic fungi

Pathogenic fungi Major host disease % Inhibition Collectrichum coccodes Eggplant, pepper Black Spot Rot Disease, Anthrax 95 Botrytis cinerea chives Gray mold 90 Cladosporium cucumerinum cucumber Potato beetle disease 95 Didymella bryoniae watermelon Black pattern 100 Aspergillus niger pepper Black mold 100 Fusarium solani sweet potato Rot 100 Paecilomyces sp . Larva Insect Pathogenicity 95 Trichophyton metagrophytes Animal skin Dermatosis 90

Strains of the genus Streptomyces of the present invention have a wide range of antifungal activity against various crop pathogenic fungi, and biological control can be achieved using a microbial pesticide preparation containing the strain, its culture solution or its culture filtrate as an active ingredient.

<110> KNAG, Byeungkon <120> Novel Streptomyces sp. bacteria and Biopesticide using the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 807 <212> DNA <213> 16S rRNA gene sequence of Streptomyces Actino-H1 <400> 1 ctaccatgca agtcgaacga tgaacctcct tcgggagggg attagtggcg aacgggtgag 60 taacacgtgg gcaatctgcc cttcactctg ggacaagccc tggaaacggg gtctaatacc 120 ggatatgaca cggggtcgca tgatctccgt gtggaaagct ccggcggtga aggatgagcc 180 cgcggcctat cagcttgttg gtgaggtagt ggctcaccaa ggcgacgacg ggtagccggc 240 ctgagagggc gaccggccac actgggactg agacacggcc cagactccta cgggaggcag 300 cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgagggatga 360 cggccttcgg gttgtaaacc tctttcagca gggaagaagc gagagtgacg gtacctgcag 420 aagaagcgcc ggctaactac gtgccagcag ccgcggtaat acgtagggcg cgagcgttgt 480 ccggaattat tgggcgtaaa gagctcgtag gcggcttgtc acgtcggatg tgaaagcccg 540 gggcttaacc ccgggtctgc attcgatacg ggcaggctag agttcggtag gggagatcgg 600 aattcctggt gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg 660 atctctgggc cgatactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 720 ccctggtagt ccacgccgta aacgttggga actaggtgtg ggcgacattc cacgtcgtcc 780 gtgccgcagc taacgcatta agttccc 807 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 agagtttgat catggctcag 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ggataccttg ttacgactt 19  

Claims (4)

Streptomyces genus with antifungal activity sp . ) Strains. The strain according to claim 1, which has a 16S rRNA nucleotide sequence of SEQ ID NO: 1. According to claim 1, wherein the antifungal activity is Collectrichum coccodes ( Collectrichum coccodes ), Botrytis cinerea ( Botrytis cinerea ), Cladosphorium cucumerinum ), Didymella bryoniae ), Aspergillus niger ), Fusarium solani solani ), Paecilomyces sp . ), And Trichophyton metagrophytes , wherein the strain acts on one or more plant pests selected from the group consisting of: Microbial pesticide preparation containing at least one selected from the group consisting of the strain of any one of claims 1 to 3, its culture solution and its culture filtrate as an active ingredient.

Images