KR102175138B1 - Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof - Google Patents

Abstract

The present invention relates to a strain of Chryseobacterium indologenes having antibacterial activity against Pseudomonas syringae .
In addition, the present invention is a Pseudomonas syringae ( Pseudomonas syringae ) containing any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof. Pseudomonas syringae ) control method.
Therefore, the Cryseobacterium indologenes strain has excellent antibacterial activity against plants, particularly Pseudomonas shiringe strains that cause leaf spot disease of tomatoes, and thus can be effectively used in compositions or methods for controlling Pseudomonas shiringe. In particular, the above effect can be obtained most significantly by the Cryseobacterium indologenes PgBE177 (accession number: KACC 92247P) of the present invention.

Description

Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof, which has antibacterial activity against plant pathogen Pseudomonas syringae

The present invention relates to a strain of Chryseobacterium indologenes having antibacterial activity against Pseudomonas syringae .

In addition, the present invention is Pseudomonas siringe ( Pseudomonas) containing any one or more selected from the group consisting of the strain, its culture medium and a mixture thereof syringae ) Controlling composition to Pseudomonas syringae comprising the step of treating the composition ( Pseudomonas syringae ) to a control method.

Bacterial speck disease is a crop disease caused by a parasitization of bacteria. A small pale brown or brown spot appears on leaves, stems, or fruits. In the case of leaves, angular lesions may occur only in leaf veins, and in severe cases, lesions fuse and leaves wither. Unlike spot diseases caused by filamentous fungi, fungi cannot be found in the lesion, but when the humidity is high, bacteria (bacterial mass) ooze around the lesion in the form of pus. Pathogens vary depending on the type of crop, but Pseudomonas ( Pseudomonas ) caused by bacteria occurs in 18 kinds of crops such as cucumbers, beans, carnations, especially house cultivation crops are very damaging.

Pseudomonas syringae ( Pseudomonas syringae pv. tomato ; Pst) strain caused by bacterial speck disease (bacterial speck disease) is known to reduce the production of tomatoes in greenhouse cultivation. In particular, Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) is Pseudomonas Shiringe pv. It is a kind of tomato (P. syringae pv. tomato ), and is known as a representative pathogen of tomato spot bacterial disease. Although it has been studied that spraying of chemicals is effective against bacterial spotting disease caused by the Pseudomonas Shiringe strain, the use of such pesticides is not environmentally friendly, increases the resistance of pathogens, and is harmful to useful organisms.

Endophytic bacteria inhabit and interact with plants without causing disease symptoms. They are ubiquitous in plant species, inhabit a variety of plant tissues, and can have beneficial effects such as plant growth, suppression of plant pathogens, improvement of plant nutrition and antibiotic production. Because of the stable habitat provided by the host plant, endogenous bacteria have little competition for nutrients and are protected from various external stresses. Thus, these bacteria can exert a more consistent effect than phyllosphere or rhizosphere bacteria that exist outside the plant.

Republic of Korea Patent Publication No. 10-2009-0058106 relates to a microbial preparation for controlling bacterial spotting disease of tomatoes, including 20 strains of Methylobacterium oriza CBMB, wherein the strain inhibits the activity of the Pseudomonas Shiringe strain. It is disclosed that it is possible to effectively suppress bacterial spotty disease. However, among ginseng endogenous bacteria, studies or descriptions on whether the Cryseobacterium indologenes of the present invention has a control effect against Pseudomonas shiringe strains or tomato bacterial spots have not been disclosed.

Accordingly, the present inventors have made diligent efforts to provide a biological control agent that can be used as a control agent for Pseudomonas syringae , as a result of a novel Chryseobacterium indologenes strain among ginseng endogenous strains. It was confirmed that the Cryseobacterium indologenes PgBE177 strain exhibited antimicrobial activity against Pseudomonas syringe, and the present invention was completed.

Accordingly, an object of the present invention is to provide a Chryseobacterium indologenes strain having antibacterial activity against Pseudomonas syringae .

Another object of the present invention is a composition for controlling Pseudomonas syringae comprising any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof, or a Pseudomonas syringe comprising the step of treating the same ( Pseudomonas syringae ) to provide a control method.

In order to achieve the above object, the present invention provides a Chryseobacterium indologenes strain having antimicrobial activity against Pseudomonas syringae .

According to a preferred embodiment of the present invention, the Pseudomonas Shiringe is Pseudomonas Shiringe pv. It may be a tomato ( Pseudomonas syringae pv. tomato ).

According to a preferred embodiment of the present invention, the Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) may be any one or more selected from the group consisting of.

According to a preferred embodiment of the present invention, the Pseudomonas syringe may cause bacterial speck disease in plants.

According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.

According to a preferred embodiment of the present invention, the strain may include the nucleotide sequence of SEQ ID NO: 1.

According to a preferred embodiment of the present invention, the strain may be a strain endogenous at any one or more sites selected from the group consisting of roots, stems, leaves, flowers, flower stems, and outposts of ginseng.

According to a preferred embodiment of the present invention, the strain may be a Cryseobacterium indologenes PgBE177 strain of accession number KACC 92247P.

The present invention also provides a composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof.

According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.

The present invention also provides a Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the control composition.

According to a preferred embodiment of the present invention, the plant may be any one or more selected from the group consisting of peppers, Arabidopsis and tomatoes.

Since the Cryseobacterium indologenes strain has excellent antibacterial activity against plants, particularly Pseudomonas shiringe strains that cause leaf spot disease of tomatoes, it can be effectively used in compositions or methods for controlling Pseudomonas shiringe. In particular, the above effect can be obtained most significantly by the Cryseobacterium indologenes PgBE177 (accession number: KACC 92247P) of the present invention.

1 is a Cryseobacterium indologenes PgBE177 strain of the present invention is Pseudomonas Shiringe pv. It shows that it has antibacterial activity in tomato DC3000 strain.
Figure 2 shows the genomic map of the Cryseobacterium indologenes PgBE177 strain of the present invention.

Hereinafter, the present invention will be described in more detail.

As described above, in the prior art, chemical control methods were mainly used to control leaf spots of plants, especially tomatoes, but the effect was not sustained, and the soil ecosystem was destroyed due to the generation of resistant bacteria and toxicity, or toxicity remained in the plants. It has problems such as the possibility of being toxic to mammals that consume it. Although biological control methods were also being studied, their effects were insignificant.

Chryseobacterium indologenes strain according to the present invention, in particular, Cryseobacterium indologenes PgBE177 is an eco-friendly ginseng endogenous strain, and Pseudomonas syringae has a significant antibacterial effect against Pseudomonas siringae . It can be effectively used in the composition or control method for controlling Ringae.

Accordingly, the present invention can provide a Chryseobacterium indologenes strain having antimicrobial activity against Pseudomonas syringae .

The Pseudomonas syringae of the present invention is Pseudomonas syringae pv. actinidiae , Pseudomonas syringae pv. syringae , Pseudomonas syringae pv. lachrymans , Pseudomonas syringae pv. tabaci , Pseudomonas syringae pv. glycines and Pseudomonas syringae pv. It is preferably any one or more selected from the group consisting of tomato , more preferably Pseudomonas Shiringe is Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is preferred.

The Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) is Pseudomonas syringae pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) is preferably any one or more selected from the group consisting of, more preferably Pseudomonas syringae pv. Tomato DC3000 is preferred.

The Pseudomonas Siringe pv. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) is known as a pathogen that causes bacterial leaf spot disease in crops such as pepper, Arabidopsis or tomato.

The Pseudomonas syringe of the present invention is preferably to cause bacterial speck disease in plants, but is not limited thereto.

The plant of the present invention is preferably any one or more selected from the group consisting of peppers, Arabidopsis, and tomatoes, most preferably tomatoes.

It is preferable that the strain of the present invention contains the nucleotide sequence of SEQ ID NO: 1, and more preferably, it is more preferably a Cryseobacterium indologenes PgBE177 strain of accession number KACC 92247P.

The strain of the present invention is preferably a strain endogenous at any one or more sites selected from the group consisting of ginseng roots, stems, leaves, flowers, flower stems and outposts, more preferably ginseng roots. . The ginseng is preferably American ginseng ( Panax quinquefolius ), but is not limited thereto.

The present invention can also provide a composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain, its culture medium, and a mixture thereof.

The culture solution is obtained after inoculating a certain concentration of spores in a medium suitable for growth of microorganisms to induce the growth of microorganisms for a certain period in an incubator, and a mixture of the cells, substances secreted while the cells are growing, and nutrients necessary for growth As, it means that it is manufactured through a process of filtering out impurities. Specifically, the Cryseobacterium indologenes strain of the present invention was used as LB (Luria Bertani) medium, NB (nutrient broth) medium, BHI (brain heart infusion) medium, KB (King's medium B) or TSB (Tryptic Soy Broth) It is preferable to mean that it is produced by culturing in any one or more media selected from the group consisting of media, but is not limited thereto.

The plant of the present invention is preferably any one or more selected from the group consisting of pepper, Arabidopsis and tomato, but is not limited thereto.

The Pseudomonas syringae ( Pseudomonas syringae ) is the same as the strain of the Chryseobacterium indologenes ( Chryseobacterium indologenes ) strain of the present invention having antibacterial activity, so the description will be replaced with the description.

The composition for control is not particularly limited as long as it is a conventional control agent used for the prevention, reduction or eradication of diseases caused by plant pathogens, and the prevention or reduction has a statistically significant difference with respect to the host plant in which the composition is untreated. Means there is. Preferably, it is selected from the group consisting of microbial pesticides, seed coating agents, microbial pesticides for controlling Pseudomonas shiringe, additives for producing a non-sterile medium for crop cultivation, biotop soil, microbial nutrients, soil improvers, composting agents, foliar sprayers or irrigation sprayers It may be any one or more. In particular, Pseudomonas Shiringe pv, a pathogen of tomato leaf spot disease. Tomato ( Pseudomonas syringae pv. tomato ) has an excellent effect on growth inhibition, so it can be used for microbial pesticides for controlling leaf spots on tomatoes, additives for the production of non-sterile medium for tomato cultivation, and also for the production of biotope. The microbial pesticide is not particularly limited as long as it is prepared by a conventional method, including the Cryseobacterium indologenes strain of the present invention. For example, it may include a Cryseobacterium indologenes strain and a bio-matrix that is a microbial carrier, and the bio-matrix may include a plant resource polymer material, a microbial-derived biopolymer material, a nutrient, an auxiliary material, and an additive. have. In addition, the plant resource polymer material may include potato powder, soybean powder, starch powder, and the like, and the microbial-based biopolymer may include mucopolysaccharide, poly-betahydroxy-alkanoate, and a coagulant. Further, the nutrient may include glucose, peptone and inorganic salts, and the auxiliary material may include vermiculite, rice husk burned powder, and the like, and the additive may include an extender, a UV blocker, and a surfactant. The microbial pesticide may be used as an antifungal microbial pesticide, a seed coating agent, a microbial nutrient agent, a soil conditioner, a composting agent, a foliar spray agent or a drench spray agent.

The present invention may also provide a Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the control composition.

The plant of the present invention is preferably any one or more selected from the group consisting of pepper, Arabidopsis and tomato, but is not limited thereto.

The control method of the present invention may be a method of controlling tomato leaf spots by treating the control composition to soil, plants, or plant seeds. At this time, treatment methods are generally used, namely, spraying (e.g. spraying, misting, atomizing, powder spraying, granule spraying, water surface application, regular use, etc.), soil application (e.g. mixing, irrigation, etc.) , Surface use (for example, coating, smearing, coating, etc.), immersion, poisoning, smoking, etc. can be performed. The amount of use can be appropriately determined depending on the formulation, damage situation, application method, application location, and the like. In the present invention, the effective amount of microorganisms contained in the formulation treated according to the above method may be included in the number of microorganisms of 1 to 1 × 10 ¹⁰⁰ per cultivated land area (m 2 ). In addition, the effective amount of microorganisms contained in the formulation treated by spraying among the above methods may be included in a concentration of microorganisms of 1 to 1×10 ¹⁰⁰ per ml, and the effective amount of microorganisms contained in the composition treated by immersion is 1 per ml. It may be included in a microbial concentration of 1 × 10 ¹⁰⁰ .

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

Isolation and identification of endogenous bacteria

National Horticultural Research Institute teukjak ginseng teukjak Part 4 - year - old ginseng ginseng packaging (Panax quinquefolius ) roots were secured. The root sample was washed with tap water to remove soil and foreign substances on the root surface, sterilized with 12% Lax and 70% ethanol, and then rinsed with sterile water. 500 mg of the ground sample was mixed with 1 ml of sterilized water to take 100 µl and then spread on an LB (Luria-Bertani) plate. A single colony that appeared after 2 days incubation at room temperature was subcultured and then 16S rRNA gene sequence analysis was performed. The obtained nucleotide sequence was identified as similar species through BLAST search of NCBI.

As a result, it was confirmed that PgBE177 among the strains whose base sequence was confirmed is a species of Chryseobacterium indologenes . It was deposited with GenBank under accession number MH211268.

Plant pathogen inhibition activity in-flight assay

In order to find out the usefulness of the isolated strain, the antibacterial activity against plant pathogens was tested in-flight targeting the endogenous bacterial strain.

The target pathogen was Pseudomonas Shiringe pv. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) was used. Pathogens were tagged with GFP protein, and were used to monitor the growth of pathogens by using the properties that the fluorescence intensity increases when pathogens grow and the fluorescence decreases when suppression. PgBE177 and pathogens were incubated with shaking at 28°C and 200 rpm for 20 hours, and then the concentration of each culture solution was adjusted to OD ₆₀₀ = 0.1, mixed at 1:1, and then dropped on an LB (Luria-Bertani) plate. After 1-2 days, the inhibitory activity was confirmed by comparing the fluorescence of the pathogen with the control.

As a result, as shown in [Fig. 1], the PgBE177 of the present invention exhibited inhibitory activity against the pathogen.

Total base sequence analysis of PgBE177

In order to clarify the functional importance of PgBE177 of the present invention, the total base sequence was analyzed using the PacBio RS II system (Chunlab, Inc.).

Specifically, for genomic DNA extraction, bacterial cells were incubated at 65° C. for 30 minutes with Chelex 100 resin (Cat. No. 143-2832, Bio-Rad, CA, USA) and proteinase K, and then at 100° C. Boiled for 10 minutes. Centrifugation at 10,000X for 10 minutes to obtain a supernatant containing bacterial genomic DNA. For sample preparation, SMRTbell template preparation kit (Pacific Biosciences, Menlo Park, CA, USA) was used according to the manufacturer's protocol. 20-kb DNA templates were selected using the BluePippin size selection system. After DNA size selection, the library was sequenced using a PacBio RS II instrument with P6-C4 chemistry (Pacific Biosciences) in 8-well single molecule real-time (SMRT) cells (version 3). The assembly process was performed with PacBio SMRT Analysis 2.3.0 using the Hierarchical Genome Assembly Process 2 (HGAP2) pipeline.

For further analysis, genome sequence analysis was performed using RAST and antiSMASH 4.2.0 programs.

As a result, the PgBE177 of the present invention was composed of one contig, the total genome size was 5,008,467 bp, the GC content was 36.4%, and the depth of coverage was 211.23×. PgBE177 had 9 gene clusters, a bacteriocin cluster involved in plant pathogen inhibitory activity, and a siderophore related cluster involved in plant growth promotion.

As a result, through the genomic analysis of PgBE177 of the present invention, a trait expected to be useful in plants could be identified, and [Fig. 2] shows a genomic map of the Chryseobacterium indologenes PgBE177 of the present invention. .

[Consignment institution]

Name of deposit institution: Institute of Agricultural Biotechnology

Accession number: KACC92247

Consignment Date: 20181023

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Chryseobacterium indologenes strain possessing antifungal activity against pathogenic bacteria Pseudomonas syringae and use thereof <130> 1065775 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> PgBe177 16S ribosomal RNA gene <400> 1 gatgaacgct agcgggaggc ctaacacatg caagccgagc ggtagagatc tttcgggatc 60 ttgagagcgg cgtacgggtg cggaacacgt gtgcaacctg cctttatctg ggggatagcc 120 tttcgaaagg aagattaata ccccataata tttttgatgg catcattaga aattgaaaac 180 tccggtggat agagatgggc acgcgcaaga ttagatagtt ggtgaggtaa cggctcacca 240 agtctacgat ctttaggggg cctgagaggg tgatccccca cactggtact gagacacgga 300 ccagactcct acgggaggca gcagtgagga atattggaca atgggtgaga gcctgatcca 360 gccatcccgc gtgaaggacg acggccctat gggttgtaaa cttcttttgt atagggataa 420 acctactctc gtgagagtag ctgaaggtac tatacgaata agcaccggct aactccgtgc 480 cagcagccgc ggtaatacgg agggtgcaag cgttatccgg atttattggg tttaaagggt 540 ccgtaggcgg atctgtaagt cagtggtgaa atctcacagc ttaactgtga aactgccatt 600 gatactgcag gtcttgagtg ttattgaagt agctggaata agtagtgtag cggtgaaatg 660 catagatatt acttagaaca ccaattgcga aggcaggtta ctaagcaaca actgacgctg 720 atggacgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgctaactc gtttttggtt tttcggaatc agagactaag cgaaagtgat aagttagcca 840 cctggggagt acgttcgcaa gaatgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggattatg tggtttaatt cgatgatacg cgaggaacct taccaaggct taaatgggaa 960 atgacaggtt tagaaataga cttttcttcg gacatttttc aaggtgctgc atggttgtcg 1020 tcagctcgtg ccgtgaggtg ttaggttaag tcctgcaacg agcgcaaccc ctgtcactag 1080 ttgccatcat taagttgggg actctagtga gactgcctac gcaagtagag aggaaggtgg 1140 ggatgacgtc aaatcatcac ggcccttacg ccttgggcca cacacgtaat acaatggccg 1200 gtacagaggg cagctacaca gcgatgtgat gcaaatctcg aaagccggtc tcagttcgga 1260 ttggagtctg caactcgact ctatgaagct ggaatcgcta gtaatcgcgc atcagccatg 1320 gcgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcaagccatg gaagtctggg 1380 gtacctgaag tcggtgaccg taacaggagc tgcctagggt aaaac 1425

Claims (12)

Pseudomonas syringae ( Pseudomonas syringae ) of the accession number KACC 92247P, which has antibacterial activity, Chryseobacterium indologenes PgBE177 strain.
The method of claim 1, wherein the Pseudomonas Shiringe is Pseudomonas Shiringe pv. Tomato ( Pseudomonas syringae pv. tomato ) of the accession number KACC 92247P, characterized in that the Chryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain.
The method of claim 2, wherein the Pseudomonas Siringe pv. Tomato is Pseudomonas Shiringe pv. Tomato DC3000 (P. syringae pv. tomato DC3000), Pseudomonas shiringe pv. Tomato LMG5093 (P. syringae pv. tomato LMG5093) and Pseudomonas shiringe pv. Tomato OH314 (P. syringae pv. tomato OH314) Cryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain of accession number KACC 92247P, characterized in that any one or more selected from the group consisting of.
The strain of claim 1, wherein the Pseudomonas syringe causes bacterial speck disease in plants. Chryseobacterium indologenes PgBE177 of accession number KACC 92247P.
According to claim 4, The plant is Chryseobacterium indologenes ( Chryseobacterium indologenes ) PgBE177 strain of accession number KACC 92247P, characterized in that any one or more selected from the group consisting of pepper, Arabidopsis and tomato.
The strain of claim 1, wherein the strain comprises the nucleotide sequence of SEQ ID NO: 1, Chryseobacterium indologenes PgBE177 of accession number KACC 92247P.
delete delete The composition for controlling Pseudomonas syringae ( Pseudomonas syringae ) comprising any one or more selected from the group consisting of the strain of claim 1, its culture medium and a mixture thereof.
delete Pseudomonas syringae ( Pseudomonas syringae ) control method comprising the step of treating the composition for control of claim 9.
delete

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