KR20150044831A - New strain of Streptomyces sp. DUCC501 having antifungal activity and a composition for plant disease control including the strain - Google Patents

Abstract

The present invention relates to a Streptomyces sp. strain having antimicrobial activity and, more particularly, to a novel strain of Streptomyces sp., which is DUCC501, and a plant disease controlling composition and an advantageous insect disease controlling composition including the novel strain, wherein the strain shows antifungal effects on a broad range of fungal species including insect pathogenic fungi and also plant pathogenic fungi.

Description

A composition for controlling plant diseases comprising the new strain DUCC501 of the genus Streptomyces having antifungal activity and the new strain {New strain of Streptomyces sp. DUCC501 having antifungal activity and a composition for plant disease control including the strain}

The present invention relates to a strain of the genus Streptomyces having antimicrobial activity, and more specifically, a new strain of the genus Streptomyces that exhibits antifungal effects against a wide range of fungal species including phytopathogenic fungi as well as insect pathogenic fungi ( Streptomyces sp. ) About DUCC501.

One of the decisive factors affecting crop yield is plant fungal disease. Representative phytopathogenic fungi include Penicillium spp., which causes blue mold and tangerine rot in fruit trees, Botrytis cinera, which causes gray fungal diseases of various plants, and Fossa, which causes crop wilting and rot. And the Fusariun oxysporum. In particular, Fusarium Oxysforum bacteria is a phytopathogenic fungus that causes a lot of damage to expensive varieties when cultivating oriental and western eggs.

As described above, damage to crops caused by phytopathogenic fungi causes a problem of reducing crop production along with pests and weeds. The control of phytopathogenic fungi mainly includes a chemical method using a fungicide, a physical method to improve the cultivation environment, and a microbiological method using microorganisms. Physical methods alone are not effective, and chemical methods cause problems such as the appearance of drug-resistant bacteria, toxicity to humans, and environmental pollution due to residual pesticides.

In Korea, the concept of biological pesticides was practically insufficient, and soil microbial agents have been mainly used for fertilizer addition. However, in June 2000, the registration standards and methods of microbial pesticides were notified, providing institutional support to ensure that products that are recognized for sterilization and insecticidal effects among soil microbial agents are included in the category of microbial pesticides.

Since then, research on microbial products has been actively studied. In particular, control using Trichoderma bacteria, or control using microorganisms of the genus Streptomyces and many types of physiologically active substances derived therefrom are being studied.

Currently, as microbial pesticides used in agriculture, Blaticidine S, Polyoxin, Kasugamycin, Validamycine, Mildiomycin, etc. produced by Streptomyces are commercialized and used as antifungal agents. Avermectin and Milbemtcin are pesticides, Bialaphos, Alteichin, Hydantocidin, Herboxidine, etc. are herbicides Was developed as. And Bacillus sp. has been reported to produce antibiotics such as iturin, amicoymacin C, aurantinin A, butirosin A, bacillomycin Fa, bacillomycin L, prumycin, and polypeptin.

As such, antifungal agents currently used for agriculture include validamycin, kasugamycin, and streptomycin, but microbial agents used as biological pesticides (biocontrol agents) that can control phytopathogenic fungi. Is inadequate.

Prior art literature information

1.Patent registration No. 10-1005484

As a result of research efforts to solve the above-described problems, the present inventors completed the present invention by isolating and identifying new strains of the genus Streptomyces that exhibit antibacterial activity against a wide range of fungal species including phytopathogenic fungi as well as insect pathogenic fungi. .

Accordingly, an object of the present invention is to provide a novel strain of Streptomyces DUCC501 having antibacterial activity against a wide range of fungal species including phytopathogenic fungi as well as insect pathogenic fungi.

Another object of the present invention is to provide an eco-friendly and economical biocontrol agent, that is, a composition for controlling plant diseases and a method for controlling plant diseases, since it is possible to control plant diseases caused by a wide range of phytopathogenic fungi.

Another object of the present invention is to provide an eco-friendly and economical composition for controlling useful insect diseases and a method for controlling useful insect diseases capable of controlling useful insect diseases caused by insect pathogenic fungi.

The object of the present invention is not limited to the above-mentioned object, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a Streptomyces sp. DUCC501 strain (KCTC 18259P), characterized in that it has antibacterial activity against insect pathogenic fungi and phytopathogenic fungi.

In a preferred embodiment, the phytopathogenic fungi are Phialophora fastigiata , Phytophthora , Sclerotinia minor , Fusarium oxysporum , and Cole Colletotrichum coccodes , Botrytis cinerea , Alternaria solani , Colletotrichum acutatum , Colletotrichum gloeosporioides ( Colletotrichum gloeosporioi des ), Corynespora cassiicola , Fusarium oxysporum f. sp.lycopersici , Fusarium proliferatum, Mucor racemo It is one or more selected from the group consisting of Mucor racemosus , Nectria haematoccoa , and Phialophora cinerescens.

In a preferred embodiment, the insect pathogenic fungus is one selected from the group consisting of Cordyceps pruinosa , Cordyceps militaris , and Cordyceps bassiana. That's it.

In addition, the present invention provides a composition for controlling plant diseases comprising the above-described strain or a culture solution thereof as an active ingredient.

In a preferred embodiment, the plant disease is Phialophora fastigiata , Phytophthora , Sclerotinia minor , Fusarium oxysporum , Collecto Colletotrichum coccodes , Botrytis cinerea , Alternaria solani , Colletotrichum acutatum , Colletotrichum Gloeosporioides gloeosporioide s ), Corynespora cassiicola , Fusarium oxysporum f. sp.lycopersici , Fusarium proliferatum, Mucor racemosus It is caused by one or more phytopathogenic fungi selected from the group consisting of (Mucor racemosus ), Nectria haematoccoa , and Phialophora cinerescens.

In addition, the present invention provides a method for controlling plant diseases comprising the step of treating the above-described strains and their culture solutions, or the above-described composition for controlling plant diseases on soil, plants, or seeds of plants.

First, the novel strain of the genus Streptomyces DUCC501 of the present invention has antibacterial activity against a wide range of fungal species including phytopathogenic fungi as well as insect pathogenic fungi.

In addition, the composition and control method for controlling plant diseases of the present invention is an eco-friendly and economical biocontrol agent because it can control plant diseases caused by a wide range of phytopathogenic fungi.

In addition, the composition and method for controlling useful insect diseases of the present invention are environmentally friendly and economical because they can control useful insect diseases caused by insect pathogenic fungi.

The effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a photograph showing the appearance of a novel strain of Streptomyces genus DUCC501 of the present invention.
Figure 2 is a schematic diagram showing the phylogenetic relationship between the novel Streptomyces DUCC501 strain and similar species of the present invention.
3 is a photograph showing the results of antagonistic tests against various phytopathogenic fungi of the novel Streptomyces DUCC501 strain of the present invention.
Figure 4 is a photograph showing the results of the antagonism test against various phytopathogenic fungi of the culture medium extract of the novel Streptomyces genus DUCC501 strain of the present invention.

Terms used in the present invention have selected general terms that are currently widely used as possible while taking functions of the present invention into consideration, but this may vary according to the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. The same reference numerals used to describe the present invention throughout the specification denote the same elements.

The present invention provides an insect pathogenic fungus and Streptomyces sp. DUCC501 strain (KCTC 18259P) having antibacterial activity against phytopathogenic fungi.

In the present invention, it was attempted to search for new microorganisms for controlling plant diseases caused by phytopathogenic fungi as well as useful insect diseases.It was collected from a forest with sea pine forests and caused various phytopathogenic fungi as well as useful insect diseases by using a multi-stage dilution method. A new strain, Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501), which exhibits strong antibacterial activity against insect pathogenic fungi, was isolated and identified.

The isolated strain of Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC) on October 4, 2013, and the accession number was assigned KCTC 18259P.

Antagonistic experiments were performed on various phytopathogenic fungi as well as insect pathogenic fungi on the strain of Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain and strain culture solution extract.

As a result, Phialophora fastigiata , Phytophthora , Sclerotinia minor , Fusarium oxysporum , Colletotrichum coccodes ), Botrytis cinerea , Alternaria solani , Colletotrichum acutatum , Colletotrichum gloeosporioides , Corynespora Cassii Corynespora cassiicola , Fusarium oxysporum f. sp.lycopersici , Fusarium proliferatum , Mucor racemosus , under Nectria Matokoa ( Nectria haematoccoa ), Phialophora cinerescens (Pialophora cinerescens), etc. were confirmed to significantly inhibit the effect.

In addition, it was also confirmed that the effect of remarkably inhibiting cordyceps pruinosa , Cordyceps militaris , Cordyceps bassiana, and the like.

Example. Isolation and identification of novel Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain

1. Isolation of strains

Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain of the present invention was isolated as follows. That is, the strain Streptomyces sp. which shows strong activity against various phytopathogenic fungi by using a multi-stage dilution method in soil samples collected from forests with Haesong forest. DUCC501 was selected. At this time, the selected strains were inoculated in ISP2 (malt extract 1%, yeast extract 0.4%, glucose 0.4%, agar 1.5%) medium for pre-culture, and then cultured at 28°C for 5 days. For mass cultivation, spores were inoculated in ISP2 liquid medium and rotated at 28° C. and 200 rpm for 7 days. The pH of all media was adjusted to 7 and used.

2. Identification of strains

(1) Fatty acid analysis

For fatty acid analysis of cell wall components to identify the selected strains, 40 mg of DUCC501 cells cultured for 3 days in ISP2 plate medium were put into a screw cap test tube for reaction and an alkali sensitized solution (NaOH 15 g, methanol 50 ml, distilled water 50 ml) 1 After adding ml, the lid was tightly closed and reacted at 100° C. for 5 minutes. After shaking well for 5 to 10 seconds, it was reacted again at 100°C for 25 minutes. After contacting with flowing water to lower the temperature of the test tube, 2 ml of methylation reagent (6N HCl 130 ml, methanol 110 ml) was added and sealed, followed by reaction at 80° C. for 10 minutes. After contacting with flowing water to lower the temperature of the test tube again, 1.25 ml of an extraction solvent (equal mixture of hexane-tert-butylmethyleter) was added and slowly mixed for 10 minutes. Water in the lower layer was removed using a mechanical pipette, and 3 ml of a washing solution (1.2 g of NaOH, 100 ml of distilled water) was added to the remaining solvent layer, and the lid was closed, followed by stirring for 5 minutes to mix. 2/3 of the solvent layer was taken and used as a sample for analysis, and gas chromatography was analyzed by the ACTIN6 method.

(2) Observation of morphological characteristics of strains

In order to investigate the morphological characteristics of the selected strain, the selected strain was cultured in ISP2 medium for 5 days, and then observed with a scanning electron microscope, and a photograph of the result is shown in FIG. 1.

(3) 16S rDNA sequence investigation

For the 16S rDNA sequence investigation, the strain was cultured in ISP2 liquid medium at 28° C. for 24 hours and then colony PCR was performed. To amplify the 16S rDNA gene, primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGYTACCTTGTTACGACTT-3') were used. The base sequence was determined by requesting Macrogen (SEQ ID NO: 1). The base sequence of the sample was analyzed using the Basic Local Alignment Search Tool (BLAST) of National Center Biotechnology Information (NCBI).

The system diagram was prepared based on the determined 16S rDNA using the Mega 4 program, and is shown in FIG. 2.

(4) Results of strain identification

As shown in FIG. 1, the DUCC501 strain of the present invention has a gray spore in ISP2 medium, exhibits a spiral shape, and 30 or more spores are connected. The surface of the spores is smooth, and the width is 0.4 ?? 0.6 μm, length 0.6 ?? It has an elliptical shape of 0.8 μm.

The DUCC501 strain 16S rDNA sequence (SEQ ID NO: 1) of the present invention showed a high degree of similarity with Streptomyces costaricanus and Streptomyces griseofuscus. costaricanus) was the highest.

As a result of the analysis of the phylogenetic diagram shown in FIG. 2, the DUCC501 strain was divided into different clades, but the branching map was low. However, as shown in Table 1 below, the major fatty acids of the DUCC501 strain are anteiso-C15:0 and iso-C16:0, and the major fatty acids of the Streptomyces costaricanus standard strain are anteiso-C15:0 and anteiso. There was a big difference with -C17:0.

Type of fatty acid Fatty acid content (%) Streptomyces sp. DUCC501 Streptomyces costaricanus CR-43 ^T anteiso-C _{15 : 0} 26.34 25.2 iso-C _{16 : 0} 28.13 8.1 anteiso-C _{17 :} 5.79 20.3 iso-C _{15 : 0} 6.87 8.4 iso-C _{16 :1} H 3.53 0.1 iso-C _{14 : 0} 13.28 0.6 C _{16 : 0} 11.81 24.2

As described above, the fatty acid composition is largely different from the Streptomyces costaricanus strain, so it was judged as a new strain, named Streptomyces sp. DUCC501, and the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center (KCTC) It was deposited on October 4, 2013 (Accession No. KCTC 18259P).

Experimental Example 1.

In order to test the antibacterial activity of the novel Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain, 23 fungal strains including phytopathogenic fungi and insect pathogenic fungi were tested as follows.

The antimicrobial activity assay of the strain was performed using a replacement culture method in ISP2 plate medium. At this time, the fungal strain used in the test was cultured on potato agar medium at 25° C. for 5 days and used. After inoculation and incubation at 25° C. for 7 days, growth inhibition was measured and evaluated, and the results are shown in Table 2 and Fig. 3 (the number of Petri dishes is the same as the fungal species number in Table 2).

Kinds Species Mycelial growth
Inhibition rate (%) plant
Pathogenicity
mold One Alternaria solani KACC40570 86.3% 2 Botrytis cinerea KACC40574 89.8% 3 Colletotrichum acutatum KACC40805 78.4% 4 C. coccodes KACC40802 99.0% 5 C. gloeosporioides CGF 75.2% 6 C. gloeosporioides CNKJ1 82.1% 7 Corynespora cassiicola DUCC 3-1 62.1% 8 Fusarium oxysporum DUCC FO-2 78.0% 9 F. oxysporum f. sp . lycopersici KACC40047 70.3% 10 F. oxysporum f. sp . lycopersici KACC40044 71.7% 11 F. oxysporum f. sp . lycopersici KACC40032 75.8% 12 F. oxysporum DUCC FO-1 73.6% 13 F. proliferatum DUCC FC 70.1% 14 Mucor racemosus KACC41012 67.3% 15 M. racemosus f. racemosus KACC41332 71.2% 16 Nectria haematoccoa DUCC FB 70.0% 17 Phialophora cinerescens MAFF425142 87.4% 18 Phialophora fastigiata MAFF425144 94.2% 19 Phytophthora sp. KACC40401 90.6% 20 Sclerotinia minor KACC41068 98.4% insect
Pathogenicity
mold 21 Cordyceps pruinosa 84.2% 22 Cordyceps militaris 87.5% 23 Cordyceps bassiana 90.5%

From Table 2 and FIG. 3, it can be seen that the novel Streptomyces DUCC501 (Streptomyces sp. DUCC501) strain of the present invention has very excellent antibacterial activity not only against various phytopathogenic fungi, but also insect pathogenic fungi that cause diseases in useful insects.

Experimental Example 2.

In order to test the antibacterial activity of the novel Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain culture, 10 fungal strains including phytopathogenic fungi and insect pathogenic fungi were tested as follows.

The antimicrobial activity assay of the culture medium of the strain of Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) was performed using a general paper-disc method. The mold strains used in the test were molds 1 to 5, molds 9, 11, 14, 20, and 21 of the mold species shown in Table 2, and were cultured on potato agar medium at 25° C. for 5 days and used. After adsorbing 10 μl of the culture medium onto a paper-disc, 3 pieces were placed on the solid medium inoculated with each test mold. After inoculation and culture at 25° C. for 7 days, the growth inhibition was measured and evaluated, and shown in FIG. 4 (the number of Petri dishes is the same as the fungal species number in Table 2). In FIG. 4, the upper part is a control group using only ethyl acetate on the disc, and the lower part is an experimental group using the culture solution of the strain of Streptomyces DUCC501 of the present invention.

From Figure 4, it can be seen that the culture medium of the novel Streptomyces sp. DUCC501 (Streptomyces sp. DUCC501) strain also has excellent antimicrobial activity not only against various phytopathogenic fungi, but also insect pathogenic fungi that cause diseases in useful insects.

From these experimental results, few strains having antimicrobial activity against a wide range of fungal species were known, but the novel Streptomyces DUCC501 strain of the present invention is a broad range of fungal species including phytopathogenic fungi as well as insect pathogenic fungi. It can be seen that the antibacterial activity is excellent.

Therefore, the novel strain of the genus Streptomyces DUCC501 of the present invention and its culture solution can control plant diseases caused by a wide range of phytopathogenic fungi, and thus are eco-friendly and economical biocontrol agents, as well as insect pathogenicity. It can be widely applied to useful insect diseases caused by fungi, in particular, insect pathogenic fungi that infect bees in the beekeeping industry.

As described above, the present invention can be applied not only to control fungi using a recent biological control method, but is also predicted to have a greater effect on greenhouse cultivation, and control using the novel Streptomyces DUCC501 strain culture filtrate of the present invention Method would be possible too.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Korea Research Institute of Bioscience and Biotechnology KCTC18259P 20131004

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> New strain of Streptomyces sp. DUCC501 having antifungal activity and a composition for plant disease control including the strain <130> DPP-2014-0505-KR <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1486 <212> DNA <213> Streptomyces sp. DUCC501 <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgaagccc ttcggggtgg attagtggcg aacgggtgag taacacgtgg gcaatctgcc 120 ctgcactctg ggacaagccc tggaaacggg gtctaatacc ggatatgacc atcttgggca 180 tccttgatgg tgtaaagctc cggcggtgca ggatgagccc gcggcctatc agcttgttgg 240 tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggcg accggccaca 300 ctaggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 360 gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct 420 ctttcagcag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg gctaactacg 480 tgccagcagc cgcggtaata cgtagggcgc aagcgttgtc cggaattatt gggcgtaaag 540 ggctcgtagg cggcttgtca cgtcgattgt gaaagctcgg ggcttaaccc cgagtctgca 600 gtcgatacgg gctagctaga gtgtggtagg ggagatcgga attcctggtg tagcggtgaa 660 atgcgcagat atcaggagga acaccggtgg cgagggcgga tctctgggcc attactgacg 720 ctgangagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780 acggtgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa 840 gttccccgcc tggggagtac ggccgcaagg ctaaacctca aaggaattga cgggggcccg 900 cacaagcggc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta ccaaggcttg 960 acatacaccg gaaagcatta gagatagtgc cccccttgtg gtcggtgtac aggtggtgca 1020 tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tgtcccgtgt tgccagcagg cccttgtggt gctggggact cacgggagac cgccggggtc 1140 aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtct tgggctgcac 1200 acgtgctaca atggccggta caatgagctg cgataccgtg aggtggagcg aatctcaaaa 1260 agccggtctc agctcggatt ggggtctgca actcgacccc atgaagtcgg agtcgctagt 1320 aatcgcagat cagcattgct gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380 acgtcacgaa agtcggtaac acccgaagcc ggtggcccaa ccccttgtgg gagggagctg 1440 tcgaaggtgg gaccagcgat tgggacgaag tcgtaacaag gtaacc 1486

Claims (6)

Streptomyces sp. DUCC501 strain (KCTC 18259P), which has antimicrobial activity against insect pathogenic fungi and plant pathogenic fungi.
The method according to claim 1,
The phytopathogenic fungi may be selected from the group consisting of Phialophora fastigiata , Phytophthora , Sclerotinia minor , Fusarium oxysporum , Collectotrichum cocodes ( Colletotrichum coccodes , Botrytis cinerea , Alternaria solani , Colletotrichum acutatum , Colletotrichum gloeosporioides , Correnes spp . ( Corynespora cassiicola ), Fusarium oxysporum f. Lycopersici , Fusarium proliferatum , Mucor racemosus , Neck, Nectaria haematoccoa , Phialophora cinerescens , and the like, which is at least one selected from the group consisting of Streptomyces sp. Strain DUCC501 (KCTC 18259P).
The method according to claim 1,
Wherein the insect pathogenic fungus is at least one selected from the group consisting of Cordyceps pruinosa , Cordyceps militaris , Cordyceps bassiana , Streptomyces sp. Strain DUCC501 (KCTC 18259P).
A plant disease controlling composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
5. The method of claim 4,
The plant disease can be selected from the group consisting of Phialophora fastigiata , Phytophthora , Sclerotinia minor , Fusarium oxysporum , Colletotrichum cucumber, for example , coccodes , Botrytis cinerea , Alternaria solani , Colletotrichum acutatum , Colletotrichum gloeosporioides , ( Corynespora cassiicola ), Fusarium oxysporum f. Lycopersici , Fusarium proliferatum , Mucor racemosus , Neptria sp. & Lt ; RTI ID = 0.0 &gt; under Mato core (Nectria haematoccoa), Fora Le Cine sense one or more plant pathogenic fungi are selected from the group consisting of (Phialophora cinerescens) to PR For controlling plant diseases, it characterized in that the composition caused by.
A method for controlling plant diseases, comprising the step of treating the strain of claim 1 or 2 with a culture solution thereof, or a composition for controlling plant diseases according to claim 4 to a soil, a plant or a plant seed.

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