KR20230089201A - Novel Streptomyces collinus Inha504 for controlling against plant pathogenic fungi and use thereof - Google Patents

Abstract

The present invention relates to a novel strain of Streptomyces collinus Inha504 for controlling plant pathogenic fungi and uses thereof. In the present invention, actinomycetes that produce polyene compounds with excellent antifungal activity are selected using genome search technology, and by decoding the genome based on NGS, the characteristics of the relevant bioactive substances are identified at the molecular level. Optimization of the culture process was performed to commercialize these useful actinomycetes (or bioactive substances derived from actinomycetes) as biopesticides. Accordingly, the Streptomyces Inha504 strain of the present invention has the potential to be useful as a biopesticide because it effectively controls plant pathogenic fungi.

Description

Novel Streptomyces collinus Inha504 for controlling plant pathogenic fungi and its use {Novel Streptomyces collinus Inha504 for controlling against plant pathogenic fungi and use thereof}

The present invention relates to a novel strain Streptomyces collinus Inha504 for controlling plant pathogenic fungi and uses thereof.

The need for microbial-based eco-friendly biopesticides is so desperate that no further explanation is needed. Currently, as biopesticides, various microbial species, including Bacillus -based insecticides, are used, but as antifungal biopesticides for agricultural crops, actinomycetes, particularly Streptomyces species, occupy the majority.

According to the registration status of domestic biological pesticides (aka natural plant protection agents) (as of the end of December 2012), two types of actinomycetes of KIBC Co., Ltd. (Streptomyces goshikiensis WY 324 liquid formulation) are actinomycetes-based biological pesticides. , Streptomyces columbiensis WY 20 liquid) has been registered and used.

Actinomyces-based biological pesticides are being used more actively overseas, especially Streptomyces from Novozyme in the US. lydicus WYEC108 (trade name Actinovate) and Streptomyces K61 (trade name Mycostop) from Verdera, Finland have actinomycetes as their main components. In the case of China, the actinomycete Streptomyces , which produces polyene-based Kendicin and Natamycin, is used as a biological pesticide to control fungal infections and fungal symbiotic pests in an eco-friendly manner.

Korean Registered Patent No. 10-0512331 (registered on 2005.08.26)

An object of the present invention is Streptomyces collinus having antifungal activity against phytopathogenic fungi ( Streptomyces collinus ) Inha504 strain (KCTC14683BP) and to provide a composition for controlling phytopathogenic fungi containing the strain or its culture medium as an active ingredient.

In order to achieve the above object, the present invention is deposited as KCTC14683BP, and provides a Streptomyces collinus Inha504 strain having antifungal activity against phytopathogenic fungi.

In addition, the present invention provides a composition for controlling phytopathogenic fungi comprising the strain or its culture medium as an active ingredient.

The present invention relates to a novel strain for controlling plant pathogenic fungi, Streptomyces collinus Inha504, and its use. Based on this, the genome was decoded to identify the characteristics of the physiologically active substance at the molecular level, and the cultivation process optimization was performed to commercialize these useful actinomycetes (or physiologically active substances derived from actinomycetes) as biological pesticides. Accordingly, Streptomyces collinus of the present invention ( Streptomyces collinus ) Inha504 strain is likely to be usefully used as a biological pesticide because it effectively controls plant pathogenic fungi.

1 is a result of confirming the antifungal activity of actinomycete culture samples.
Figure 2 shows the predicted polyene compound biosynthetic gene group of Streptomyces collinus Inha504 strain.
Figure 3 shows the structure of the polyene compound expected to be produced by the Streptomyces collinus Inha504 strain.
Figure 4 shows the antifungal activity results of the Streptomyces collinus Inha504 strain against various phytopathogenic fungi.

The present invention is deposited as KCTC14683BP, and has antifungal activity against phytopathogenic fungi Streptomyces collinus ( Streptomyces collinus ) Inha504 strain is provided.

Preferably, the strain is Candida albicans, Aspergillus niger , Fusarium oxysporum, Fusarium oxysporum , Fusarium solani , Fusarium verticillioi de ( Fusarium verticillioides ), Fusarium semitectum ( Fusarium semitectum ), Botrytis cinerea ( Botrytis cinerea ) and Curvularia lunata ( Curvularia lunata ) It may have antifungal activity against any one or more fungi selected from the group consisting of , but is not limited thereto.

Preferably, the strain may include the 16S rRNA nucleotide sequence represented by SEQ ID NO: 1 or the ropB nucleotide sequence represented by SEQ ID NO: 2, but is not limited thereto.

Preferably, the strain may produce a polyene compound, and more preferably, the polyene compound may be represented by Formula 1 below, but is not limited thereto.

[Formula 1]

In addition, the present invention provides a composition for controlling phytopathogenic fungi comprising the strain or its culture medium as an active ingredient.

In the present invention, the composition for controlling phytopathogenic fungi contains an effective amount of the strain according to the present invention and an agrochemically acceptable carrier. The composition according to the present invention is prepared according to a method of formulating microbial pesticides known in the art, for example, powder, pellets or granules, microcapsules, etc. It can be formulated and used as is. The composition includes a surface active agent, an inert carrier, a preservative, a wetting agent, a supply promoter, an attractant, an encapsulant, a binder, an emulsifier, a dye, a U.V. It can be obtained by adding other components that are easy to apply according to the target pathogen and crop, such as a protective agent and a buffer. The composition of the present invention may be diluted with an appropriate amount of water or other diluent prior to application and may be in a form suitable for direct application or in a concentrate or primary composition. It will be clear to those skilled in the art that antifungal concentrations, particularly concentrated or used directly, may vary depending on the nature of the particular formulation, the type of plant to be applied, the soil and climate of the growing region, and the like.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Sample collection and separation

In order to select strains with antifungal activity, an antifungal test was conducted using 2419 actinomycete cultures from Dr. Kim Chang-jin's laboratory at the Korea Research Institute of Bioscience and Biotechnology. Among them, a Streptomyces collinus Inha504 (AN090291) strain identified from an organic green tea field in Boseong in 2009 was distributed. Biological characteristics include Fusarium oxysporum (KCTC 46453), Candida It was confirmed as a strain having antifungal activity against albicans (ATCC 14053).

2. Growth conditions and media

Streptomyces collinus Inha504 (AN090291) strain is M3 agar (soluble starch 20 g/L, agar 20 g/L, soytone 10 g/L, glucose 10 g/L, CaCO ₃ 3 g/L, FeSO ₄ 7H ₂ O 0.2 g/L) at 30℃ for 7 days, pre-culture was performed through solid static culture, and GSS agar (soybean flour 25 g/L, glucose 20 g/L, soluble starch 10 g/L, yeast extract 4 g/L, NaCl 2 g/L, CaCO ₃ 2 g/L, Beef extract 1 g/L, K ₂ HPO ₄ 0.25 g/L) at 30° C. for 7 days, and production culture is performed through solid stationary culture. Plant pathogenic 7 species [ Fusarium oxysporum (KACC 40051), Fusarium solani (KACC 44891), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Botrytis cinerea (KACC 40574) , Curvularia lunata (KACC 40861), Aspergillus niger (KCTC46204)] is subjected to solid static culture at 30℃ for 3 to 7 days in PDA (potato starch 4 g/L, glucose 2 g/L, agar 15 g/L). Candida albicans (ATCC 14053) strain was grown on YM agar (yeast extraction 3 g/L, malt extraction 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L) at 30℃, 220 rpm, 12 hours. culture while shaking.

3. Antifungal assay by plate-to-plate assay

Prepare a circular agar plug with a diameter of 0.5 cm from the GSS agar plate where production culture was performed through solid stationary culture. In order to prepare a PDA agar plate containing the fungus to be tested for inhibitory activity, scrape the spores of the fungus that had undergone solid static culture to a size of 2 cm × 2 cm, mix them with 1% agarose gel, and place them on the PDA agar plate. Pour over to cover and set aside for 30 minutes. After placing the circular agar plug on the PDA agar plate containing the fungus in an appropriate position, observe the inhibition ring while performing solid stationary culture at 30 ° C for 3 to 7 days and record the size.

4. Whole Genome Sequencing and Cluster Prediction

The entire sequence of Streptomyces collinus Inha504 (AN090291) strain was outsourced to Korean macrogen for analysis using PacBio RSII, Illumina platform and De novo assembly. The biosynthetic gene cluster of secondary metabolites in the entire sequence was analyzed using antiSMASH bacterial version 5.0 ((https://antismash.secondarymetabolites.org/#!/start).

5. Evaluation of applicability as plant fertilizer under greenhouse conditions

After requesting a service from the Korea Plant Environment Research Institute in Suwon, a confirmation experiment was conducted (pot test).

go. Target disease: Wilt disease ( Fusarium oxysporium , KACC40051)

me. Test crops: Red pepper (Dokyacheong), Tomato (Superculant), Strawberry (Seolhyang)

※ Target disease occurrence status

The target pathogen, Fusarium oxysporium , was distributed from the Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration, and the inoculum was prepared by culturing on PDA (Potato Dextrose Agar) medium, and seedlings were inoculated to induce the occurrence of wilt disease.

all. Details of treatment: soil drench treatment - diluted culture medium 1L/m ² (dilution factor: 500 times), number of treatment pots: 5

la. Seedling outline: Cultivation type (facility cultivation, pot cultivation), pot size: 6cm in diameter

mind. Investigation method and criterion: Drug efficacy test (number of sicknesses, degree of disease occurrence by crop after 7 days of drug treatment), drug toxicity test (appearance of drug damage, appearance check after 3, 5, or 7 days of drug treatment)

< Example 1> having antifungal activity Streptomyces collinus Inha504 (AN090291) actinomycetes Selection

An antifungal activity comparison experiment was conducted using C. albicans , which is often used to check antifungal activity, and F. oxysporum , a phytopathogenic fungus, by receiving aliquots of actinomycete cultures from Dr. Kim Chang-jin's laboratory at KRIBB.

Antifungal activity was confirmed by showing an inhibition zone in 149 samples out of 2419 samples (FIG. 1).

Streptomyces collinus Inha504 (AN090291) strain, which was considered a new strain, was selected through sequence comparison of specific genes such as 16s rRNA sequence and RNA polymerase B.

< Example 2> Streptomyces collinus Inha504 (AN090291) Whole genome sequencing of strain and characterization of antifungal active substances

Streptomyces through whole-genome sequencing It was confirmed that it was a new strain of the series (Table 1).

Using the antiSMASH program, clusters producing secondary metabolites were found in a total of 36 regions, and the 34th region was predicted to be a biosynthetic gene group producing polyene compounds (FIG. 2). This showed high similarity with the previously known natamycin cluster (Fig. 3).

< Example 3> Confirmation of antifungal activity

As a result of confirming the antifungal activity against various phytopathogenic fungi below, the activity was confirmed in all the confirmed fungi (FIG. 4).

Fusarium oxysporum (KACC 40051), Fusarium solani (KACC 44891), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Botrytis cinerea (KACC 40574) , Curvularia lunata (KACC 40861), Aspergillus niger (KCTC46204).

< Example 4> Confirmation of antifungal activity at pollen level to confirm potential as a microbial agent

The Korea Plant Environment Research Institute (Suwon) commissioned the service and proceeded. As a result of the drug efficacy test, the control rates of red pepper (50.1%), strawberry (53.9%), and tomato (63.6%) were confirmed. As a result of the drug toxicity test, it was confirmed that the culture solution of Streptomyces collinus Inha504 (AN090291) did not harm the plants (Table 2).

In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC14683BP 20210826

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Novel Streptomyces collinus Inha504 for controlling against plant pathogenic fungi and their use <130> ADP-2021-0518 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 1514 <212> RNA <213> Streptomyces collinus Inha504 <400> 1 agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgaaccac ttcggtgggg attagtggcg aacgggtgag taacacgtgg gcaatctgcc 120 cttcactctg ggacaagccc tggaaacggg gtctaatacc ggatatgagc ctccaccgca 180 tggtggaggc tgtaaagctc cggcggtgaa ggatgagccc gcggcctatc agcttgttgg 240 tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggcg accggccaca 300 ctgggactga 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 agctcgtagg cggcttgtca cgtcgattgt gaaagcccga ggcttaacct cgggtctgca 600 gtcgatacgg gctagctaga gtgtggtagg ggagatcgga attcctggtg tagcggtgaa 660 atgcgcagat atcaggagga acaccggtgg cgaaggcgga tctctgggcc attactgacg 720 ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa 780 acggtgggaa ctaggtgttg gcgacattcc acgtcgtcgg tgccgcagct aacgcattaa 840 gttccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cgggggcccg 900 cacaagcggc ggagcatgtg gcttaattcg acgcaacgcg aagaacctta ccaaggcttg 960 acatacaccg gaaacgtctg gagacaggcg cccccttgtg gtcggtgtac aggtggtgca 1020 tggctgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct 1080 tgttctgtgt tgccagcatg cccttcgggg tgatggggac tcacaggaga ccgccggggt 1140 caactcggag gaaggtgggg acgacgtcaa gtcatcatgc cccttatgtc ttgggctgca 1200 cacgtgctac aatggccggt acaatgagct gcgataccgt gaggtggagc gaatctcaaa 1260 aagccggtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagtcgctag 1320 taatcgcaga tcagcattgc tgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacgtcacga aagttggtaa cacccgaagc cggtggccca accccttgtg ggagggagct 1440 gtcgaaggtg ggactagcga ttgggacgaa gtcgtaacaa ggtagccgta ccggaaggtg 1500 cggctggatc acct 1514 <210> 2 <211> 3486 <212> DNA <213> Streptomyces collinus Inha504 <400> 2 ttggccgcct cgcgcaacgc ctcgaccgcg aatacgaaca acggcgccag caccgccccg 60 ctgcgcatct cctttgcaaa gatcaaggag cctctcgagg ttccgaacct gctcgcgctg 120 cagaccgaga gcttcgactg gctgctcggc aacaccgcct ggcagagtcg ggtcgaggag 180 gctcttgaga acggtcagga cgtccccacc aagtccggtc tggaggagat cttcgaggag 240 atctccccga tcgaggactt ctccgggtcg atgtccctga ccttccggga ccaccgcttc 300 gagccgccga agaactccat cgacgagtgc aaggaccgcg acttcacgta cgccgccccg 360 ctcttcgtca ccgccgagtt caccaataac gagaccggtg agatcaagtc ccagacggtc 420 ttcatgggcg acttcccgct catgacgaac aagggcacct tcgtcatcaa cggcaccgag 480 cgtgtcgtgg tgtctcagct ggtccgttcc cccggtgtct acttcgattc ctcgatcgac 540 aagacgtccg acaaggacat cttctccgcc aagatcatcc cgtcccgggg tgcctggctg 600 gagatggaga tcgacaagcg tgacatggtc ggtgtccgca tcgaccgcaa gcgcaagcag 660 720 ggcgactacg agtcgatgcg cgccaccctg gagaaggacc acacccaggg ccaggacgac 780 gcgctgctcg acatctaccg caagctgcgt ccgggcgagc cccccacgcg tgaggccgcg 840 cagacgctgc tcgagaacct gtacttcaac cccaagcgct acgacctggc caaggtcggc 900 cgctacaagg tcaacaagaa gctgggcgcc gacgctccgc tcgacgcggg catcctgacc 960 gtcgaggaca tcatctcgac gatcaagtac ctggtgcagc tgcacgccgg cgagaccgag 1020 accaccggcg acaacggcga cagcatcgtt gtcgagacgg acgacatcga ccacttcggc 1080 aaccggcgcc tgcgcagcgt cggcgagctg atccagaacc aggtccgtac gggtctcgcc 1140 cgtatggagc gcgtcgtgcg cgagcgcatg acgacccagg acgtcgaggc gatcacgccg 1200 cagaccctga tcaacatccg gccggtcgtc gcctccatca aggagttctt cggcaccagc 1260 cagctgtcgc agttcatgga ccagaacaac ccgctgtcgg gtctcaccca caagcgccgc 1320 ctgtcggccc ttggcccggg tggtctctcc cgtgagcggg ccggcttcga ggtccgtgac 1380 gtgcacccct cgcactacgg ccgcatgtgc ccgatcgaga cgcccgaagg cccgaacatc 1440 ggtctgatcg gctcgctcgc ctcctacggc cgggtcaacg cgttcggttt cgtcgagacc 1500 ccgtaccgca aggtcaacga cggccaggtc accgacgagg tcgactacct gaccgccgac 1560 gaggaggacc gcttcgtcat cgcgcaggcc aacgccacgc tcaacgacga actgcggttc 1620 gccgaggcgc gtgtcctggt ccgccgccgt ggcggtgagg tcgactacgt cagccccgag 1680 gacgtcgact acatggacgt ctcgccgcgc cagatggtgt cggtcgcgac cgccatgatc 1740 ccgttcctcg agcacgacga cgccaaccgt gccctcatgg gcgcgaacat gatgcgccag 1800 gccgtgccgc tcatcaagtc cgaggcgccg ctcgtcggca ccggcatgga gtaccgctcc 1860 gcggtcgacg ccggcgacgt ggtcaaggcc gagaaggacg gtgtggtcca ggaggtctcc 1920 gcggactaca tcaccacggc gaacgacgac ggcacgtaca tcacgtaccg cctgcacaag 1980 ttctcccggt ccaaccaggg cacctcggtc aaccagaagg tcgtcgtcga cgagggcgac 2040 cggatcatca cgggccaggt gctcgcggac ggtccggcca ccgagaacgg cgagatggcc 2100 ctgggcaaga acctgctcgt ggcgttcatg ccgtgggagg gtcacaacta cgaggacgcg 2160 atcatcctgt cgcagcgcct cgtgcaggac gacgtcctct cctcgatccca catcgaggag 2220 cacgaggtcg acgcccgtga caccaagctg ggccccgagg agatcacccg ggacatcccg 2280 aacgtctccg aggaggtcct cgccgacctc gacgagcgcg gcatcatccg tatcggtgcc 2340 gaggtcgtcg ccggtgacat cctcgtcggc aaggtcacgc ccaagggtga gaccgagctg 2400 acaccggagg agcgcctgct gcgcgcgatc ttcggtgaga aggcccgtga ggtccgtgac 2460 acctccctga aggtgccgca cggcgagatc ggcaaggtca tcggcgtccg cgtcttcgac 2520 cgcgaggagg gcgacgagct gccgccgggc gtgaaccagc tggtccgcgt ctacgtcgcg 2580 cagaagcgca agatcaccga cggtgacaag ctcgccggcc gtcacggcaa caagggtgtc 2640 atctccaaga tcctgccgat cgaggacatg ccgttcctgg aggacggcac cccggtcgac 2700 atcatcctca acccgctggg tgtgccgtcc cgaatgaacc cgggacaggt cctggagatc 2760 cacctcggct ggctcgccag ccgtggctgg gacgtctccg gtctcgccga cgagtgggcg 2820 cagcgcctgc aggtcatcgg cgccgaccag gtcgacccgg gcacgaacgt cgcgaccccc 2880 gtcttcgacg gtgcgcgcga ggacgagctc gcgggtctgc tgcagcacac catcccgaac 2940 cgcgacggcg agcgcatggt gctcccgtcc ggcaaggcgc ggctgttcga cggccgcagc 3000 ggtgagccgt tcccggagcc gatctcggtc ggctacatgt acatcctgaa gctgcaccac 3060 ctggtcgacg acaagctgca cgcccgctcg accggtccgt actcgatgat cacccagcag 3120 ccgctgggtg gtaaggcgca gttcggtggc cagcggttcg gcgagatgga ggtgtgggcc 3180 ctcgaggcct acggcgccgc ctacgcgctg caggagctgc tgacgatcaa gtccgacgac 3240 gtgaccggcc gcgtgaaggt ctacgaggcc atcgtcaagg gcgagaacat ccccgagccc 3300 ggcatccccg agtccttcaa ggtgctcatc aaggagatgc agtcgctctg cctgaacgtg 3360 gaggtgctgt ccagcgacgg tatgtccatc gagatgcgcg acaccgacga ggacgtcttc 3420 cgcgctgcgg aggagctcgg tatcgacctg tcccggcgcg agccgagcag cgtcgaagag 3480 gtctga 3486

Claims (6)

Streptomyces collinus deposited as KCTC14683BP and having antifungal activity against phytopathogenic fungi ( Streptomyces collinus ) Inha504 strain. The method of claim 1, wherein the strain is Candida albicans ( Candida albicans ), Aspergillus niger ( Aspergillus niger ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium solani ( Fusarium solani ), Fusarium verticillioides ( Fusarium verticillioides ), Fusarium semitectum ( Fusarium semitectum ), Botrytis cinerea , and Curvularia lunata , characterized in that it has antifungal activity against any one or more fungi selected from the group consisting of Streptomyces collinus ( Streptomyces collinus ) Inha504 strain. The strain according to claim 1, wherein the strain comprises the 16S rRNA nucleotide sequence represented by SEQ ID NO: 1 or the ropB nucleotide sequence represented by SEQ ID NO: 2. Streptomyces collinus Inha504 strain. The strain according to claim 1, wherein the strain produces a polyene compound. Streptomyces collinus strain Inha504. According to claim 4, wherein the polyene compound is Streptomyces collinus, characterized in that represented by the formula (1) ( Streptomyces collinus ) Inha504 strain.
[Formula 1]

A composition for controlling phytopathogenic fungi comprising the strain of any one of claims 1 to 5 or its culture medium as an active ingredient.

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