KR102299675B1 - Plant growth promoting novel phototrophic bacterium Porphyrobacter sp. COR-2 and composition comprising the same - Google Patents

Abstract

The present invention relates to a novel photosynthetic bacterium Porphyrobacter sp. COR-2 strain with plant growth promotion and a composition comprising the same and, more specifically, relates to a growth promoting ability of a cucumber plant using a novel photosynthetic bacterium Porphyrobacter sp. COR-2 strain culture having a plant growth promoting ability. The Porphyrobacter sp. COR-2 strain, which is a novel plant growth promoting photosynthetic bacterium, according to the present invention has significantly excellent plant growth promoting ability compared to strains belonging to the same genus and is particularly effective in terms of vitality index (SVI) and rooting ability by more than 60%, respectively.

Description

A novel photosynthetic bacterium Porphyrobacter sp. COR-2 strain and composition comprising the same {Plant growth promoting novel phototrophic bacterium Porphyrobacter sp. COR-2 and composition comprising the same}

The present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) relates to a strain and a composition comprising the same, and more particularly, Porphyrobacter sp. It relates to the growth promoting ability of cucumber plants using COR-2 strain culture.

The demand for agricultural microbial agents, an alternative means, is increasing due to the policy to reduce the use of chemical fertilizers and pesticides and the increase in the market size of eco-friendly agricultural products. Currently there are various types of functionality useful microorganisms in the environment-friendly agricultural sector is being used, have been reported typically Bacillus subtilis, yeast, lactic acid bacteria, photosynthetic bacteria, Pseudomonas (Pseudomonas), A fungus (Trichoderma, Aspergillus). Among them, photosynthetic bacteria secrete proteins, essential amino acids, natural pigments, vitamins and physiologically active substances among the cell components to help the growth of beneficial bacteria in the soil, agglomerate the soil and increase fertility to improve soil and prevent acidification, promote crop growth, etc. As it contributes greatly to the production of high-quality, eco-friendly agricultural products in the agricultural field, it is expected as an eco-friendly agricultural material.

The photosynthetic bacteria currently used as eco-friendly agricultural materials are Rhodobacter capsulatus , Rhodobacter sphaeroids , and Rhodopseudomonas palustris . The types are very limited, and there is an urgent need to discover new photosynthetic bacteria materials.

Furuhata K et al., Porphyrobacter colymbi sp. Nov. isolated from swimming pool water in Tokyo, Japan. J. Gen. Appl. Microbiol., 59, 245-250 (2013).

In the present invention, in order to solve the above problems, a novel photosynthetic bacterium Porphyrobacter sp. It was intended to provide a COR-2 strain culture and a multifunctional composition for promoting plant growth containing the same.

The composition for promoting plant growth according to the present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P).

In the composition for promoting plant growth according to an embodiment of the present invention, the plant growth promotion may be measured by measuring vitality index (SVI) or rooting ability.

In the composition for promoting plant growth according to an embodiment of the present invention, the vitality index value (SVI) can be obtained through the following Equation I.

[Equation I]

Vitality index value (SVI) = [length of root (RL) + length of stem (SL)] * germination rate (%)

In the composition for promoting plant growth according to an embodiment of the present invention, the rooting force can be obtained by measuring the number of lateral roots.

In addition, the present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P) is provided.

In addition, the present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P), the novel photosynthetic bacterium Porphyrobacter sp. Provided is a microbial preparation comprising a part or all of a culture solution of COR-2.

A novel photosynthetic bacterium Porphyrobacter sp with excellent plant growth promoting ability according to the present invention. COR-2 ( Porphyrobacter sp. COR-2) strain has significantly superior plant growth promoting ability compared to strains belonging to the same genus. confirmed that there is.

1 is a novel photosynthetic bacterium Porphyrobacter sp. The results are about the genome tree (Phylogenomic tree) of the COR-2 strain.
2 is a novel photosynthetic bacterium Porphyrobacter sp. This is a comparison result regarding the plant growth promoting ability (SVI, rooting power) of the COR-2 strain.

With reference to the accompanying tables or drawings, the novel photosynthetic bacteria Porphyrobacter sp. The COR-2 strain and a composition comprising the same will be described in detail.

When drawings are described, they are provided as examples so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art. Therefore, the present invention is not limited to the drawings presented and may be embodied in other forms, and the drawings may be exaggerated to clarify the spirit of the present invention.

At this time, if there is no other definition in the technical terms and scientific terms used, it has the meaning commonly understood by those of ordinary skill in the art to which this invention belongs, and the summary of the present invention in the following description and accompanying drawings Descriptions of known functions and configurations that may be unnecessarily obscure will be omitted.

In addition, the singular form used in the specification of the present invention may also be intended to include the plural form unless the context specifically dictates otherwise.

In addition, in the specification of the present invention, the unit used without special mention is based on the weight, for example, the unit of % or ratio means weight % or weight ratio.

In addition, in the specification of the present invention, the expression "comprising" is an open-ended description having an equivalent meaning to expressions such as "having", "containing", "having" or "characterized by", and additionally listed It does not exclude elements, materials or processes that do not exist. Also, “really… The expression "consisting of" means that other elements, materials, or processes not listed together with the specified element, material or process do not unacceptably significantly affect at least one basic and novel technical idea of the invention. It means that it can exist in quantity. Also, the expression “consisting of” means that only the elements, materials, or processes described are present.

As used herein, the terms "ingredient", "composition", "composition of a compound", "compound", "drug", "pharmaceutically active agent", "active agent", "healing" "therapeutic" "treatment" or "agent" is used interchangeably to mean a compound or composition of compound(s) or substances that, when administered to a subject (human or animal), induces a desired pharmaceutical and/or physiological effect by local and/or systemic action. used interchangeably.

In the present invention, “sample” or “sample” refers to a subject for analysis, and is used in the same sense throughout the specification.

Hereinafter, the novel photosynthetic bacteria Porphyrobacter sp of the present invention. A composition for promoting plant growth comprising COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P) will be described in detail.

The composition for promoting plant growth according to the present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P).

In the composition for promoting plant growth according to an embodiment of the present invention, the plant growth promotion can be measured by measuring vitality index (SVI) or rooting force.

In the composition for promoting plant growth according to an embodiment of the present invention, the vitality index value (SVI) can be obtained through the following Equation I.

[Equation I]

Vitality index value (SVI) = [length of root (RL) + length of stem (SL)] * germination rate (%)

In the composition for promoting plant growth according to an embodiment of the present invention, the rooting force can be obtained by measuring the number of shoots, for example, but the scope of the present invention is not limited thereto, and can be generally recognized. It can be measured using any method.

The present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P) is provided, and the strain was deposited with the National Academy of Agricultural and Life Sciences (KACC), Rural Development Administration.

Deposit No.: KACC 92315P

Deposit date: 20200917

Name of deposit institution: National Academy of Agricultural Life Science, Rural Development Administration

Porphyrobacter sp of the present invention. The COR-2 strain is not particularly limited in terms of isolation and identification, but a paddy soil sample was collected from a rice cultivation test field at the Chungbuk Agricultural Research & Extension Services and the strain was isolated using a mixed culture method, and the isolated Porphyrobacter sp. After DNA of the COR-2 strain was extracted, the whole genome sequence was obtained through whole genome analysis (WG) and phylogenetic analysis was performed.

In addition, the present invention is a novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P), the novel photosynthetic bacterium Porphyrobacter sp. It provides a plant biopromoting microorganism preparation comprising a part or all of the culture solution of the COR-2 strain.

In addition to some or all of the culture medium, the microorganism preparation may be used in various fields as a solution in which the filtrate of the culture medium, the freeze-dried powder of the culture medium, the freeze-dried powder or the freeze-dried powder of the culture filtrate is dissolved. In addition, the culture medium may include a material produced by the cells in a medium, the cells themselves, and the medium, and the culture filtrate may include a supernatant obtained by centrifuging the culture medium, a liquid obtained by filtering the supernatant, etc. can

Hereinafter, the content of the present invention will be described in more detail through examples. The examples are only for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

[Test materials, reagents and equipment]

- The test crop, Cucumber (Baekbong Dadagi), was purchased from an agricultural material store near Yuseong and used.

- The control Porphyrobacter colimbi KCTC 32078 strain was used as a standard strain from KCTC, Korea Research Institute of Bioscience and Biotechnology.

- The disclosed chemicals were purchased from reagent companies such as Sigma and Merck.

- Other measuring instruments and equipment were purchased and used in Korea and overseas sales companies.

<Test Example 1> Novel photosynthetic bacterium Porphyrobacter sp. Identification of COR-2

For paddy soil samples, the soil environment team of the Environmental Research Division of Chungcheongbuk-do Agricultural Research and Extension Services collected samples by dividing the soil into oxidized soil and reduced soil for rice cultivation test fields that have been managed for more than 20 years.

Weigh 10 g each of non-soil samples (oxidized soil, reduced soil), put them in 90 ml of sterile water, and disperse treatment at 30 w for 2 minutes with an ultrasonicator (VCX750; SONIC Vibra-Cell), and then place 1 ml of soil sample in PSBA medium. It was mixed with (Photosynthesis Selected Bacteria Agar) and cultured for 2 weeks at 28°C under light conditions (8,000 lux). Among the purple colonies formed on the PSBA plate medium, 500 strains of purple photosynthetic bacteria were obtained through pufLM gene amplification and photosynthetic dye analysis.

As a result of examining the production ability of indole-3-acetic acid (IAA), a plant growth hormone, of 500 strains of obtained purple photosynthetic bacteria, 38 strains with an IAA production amount of 30 ug/ml or more were selected, and plant growth promoting ability (PGPR traits) , Porphyrobacter sp. through the seedling vigor index and plant growth promotion effect test (plant bioassay). The COR-2 strain was finally selected as the best strain of the PGPR microbial gene resource.

Porphyrobacter sp. For Whole Genome analysis (WG) of the COR-2 strain, the strain was inoculated on a PSBA plate medium and cultured at 30°C for 5 days, and then DNA was extracted using Genomic DNA Prep Kit for Bacterium (Biofact). . Sequence data was acquired using Illumina Miseq 300 platform (Thermo Fisher Scientific) and SPAdes 3.13.0 (Center for Algorithmic Biotechnology, St. Petersburg State University, https://cab.spbu.ru/software/spades/) As a result of completing the full-length genome assembly using this method, a genome with a size of about 3.5 Mb was obtained, and the GC content was analyzed to be 67.7%. This genome contained 3,351 CDSs, 3 rRNA genes, and 44 tRNA genes. Porphyrobacter sp. The whole genome results of the COR-2 strain are shown in Table 1.

[Table 1] Novel photosynthetic bacteria Porphyrobacter sp. ( Porphyrobacter sp.) Genome Interpretation of the COR-2 Strain

To compare the similarity between Porphyrobacter relatives, the average nucleotide identity (ANI) value and digital DNA-DNA hybridization (dDDH) were analyzed. When the ANI is less than 95% and dDDH less than 70%, it can be restricted to new microorganisms. . Porphyrobacter sp. As a result of comparative analysis of the whole genome sequence between the COR-2 strain and its relatives, it showed an ANI value of 79.8~85.2%, and dDDH analysis showed a low homology of 21.8-29.1%, which was confirmed as a new species belonging to the genus Porphyrobacter. became The similarity between Porphyrobacter relatives and the results of phylogenetic analysis are shown in Table 2 and FIG. 1, respectively.

[Table 2] Novel photosynthetic bacteria Porphyrobacter sp. Comparison of genomic similarity and homology of COR-2 strains

<Test Example 2> Novel photosynthetic bacterium Porphyrobacter sp. Comparison of genes related to plant growth promotion and antibacterial activity between COR-2 and related species

The Porphyrobacter colimbi KCTC 32078 strain, a strain sold by the Korea Research Institute of Bioscience and Biotechnology, and the Porphyrobacter sp. The COR-2 strain was compared with the presence of genes related to plant growth promotion and physiological/biochemical characteristics, and the results are shown in Tables 3 and 4 below, respectively.

[Table 3] Comparison of possessing genes related to plant growth promotion

Porphyrobacter sp. As a result of evaluating the genetic characteristics of the COR-2 strain, it contains phosphate solubilizing enzyme, auxin generation, nitrogen fixation, siderophore and antibacterial activity genes involved in plant growth promoting ability, and a related species, Porphyrobacter colimbi KCTC. As a result of comparison and review with strain 32078, it possesses tryptophan synthetase trpA and trpB related to the auxin biosynthesis metabolic pathway. It was confirmed that it has FixG, FixS, and cpdR, a nitrogen assimilation regulatory protein.

From this, even in two strains that are phylogenetically similar, which are strains included in the same genus, the genes possessed as described above in metabolic properties are different from each other, and thus the types of proteins expressed are also different, so that the physiological properties of the individual and / or Biological characteristics can also be inferred to be different.

[Table 4] Novel photosynthetic bacteria Prophyrobacter sp. Physiological and biochemical characteristics of COR-2 strain

Based on the above speculation, Porphyrobacter sp of the present invention. COR-2 was compared by measuring and comparing the physiological and biochemical properties of the Porphyrobacter colimbi KCTC 32078 strain, a strain belonging to the same genus, through a commonly known measurement method, as shown in Table 4 above, plant growth promoting rhizosphere bacteria ( Plant Growth-Promoting Rhizobacterium ), there is a difference in the characteristics, it is different in antifungal activity, lipopeptide antibiotic synthesis gene, etc., and it was confirmed that there is a difference in various genomic characteristics such as enzyme activity.

[Example 1] Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) Plant growth promoting ability of the strain

Porphyrobacter sp of the present invention. For the measurement of the plant growth promoting activity of the COR-2 strain, cucumbers (Baekbongdagi) purchased from Yuseong Agricultural Materials Store were used as test crops.

The purchased cucumbers were untreated group, Porphyrobacter colimbi KCTC 32078 treated comparative experimental group, Porphyrobacter sp of the present invention. The experiment was performed by dividing the COR-2 strain into three experimental groups in the experimental group of the example.

First, the sterilized cucumber seeds were fertilized in 3 repetitions of 5 grains each in MS medium added at a concentration of 5% after filtering the culture medium of each strain. Then, when the length of the root from the cucumber seed was 1 mm or more, the germination rate (%) was investigated.

After germination was completed, each experimental group was cultured for 2 weeks under conditions of 28°C, 8,000 lux, and 60% humidity to induce the development of true leaves and roots.

After two weeks, the root length and the shoot length were measured, respectively, and then, the vitality index (SVI) was calculated using the following Equation I.

[Equation I]

Vitality index value (SVI) = [root length + shoot length] × germination (%)

The rooting power of cucumber seedlings was measured by examining the average number of lateral roots.

The growth results for each experimental group through the above process are shown in Tables 5 and 6 below.

[Table 5] Novel photosynthetic bacteria Porphyrobacter sp. Comparison result on plant growth promoting ability (SVI, Rooting ability) of COR-2 strain

[Table 6] Plant growth promoting ability (SVI, measurement of rooting power)

As can be seen from the above results, the photosynthetic bacteria according to the present invention, Porphyrobacter sp. In the case of the COR-2 strain, Porphyrobacter colimbi KCTC 32078 strain, a comparative experimental group of the same genus, expected to show similar characteristics, with a whopping 121% and 108% better rooting power compared to the untreated group, which is a negative control group. It was confirmed that both the vitality index value (SVI) and rooting power were more than 60% superior to that of . From this, the photosynthetic bacteria of the present invention, Porphyrobacter sp. It was determined that the COR-2 strain could be used as a fertilizer and eco-friendly organic agricultural material having a plant growth promoting effect.

The cause of such a result is unknown, but in the characteristics of the genome analyzed through the test example, the presence or absence of a gene expressing a specific protein or enzyme, which is an individual characteristic of the strain, may affect, but must be caused by It may not be an effect, and it is presumed that it may be the result of a complex action of various differences between strains.

Rural Development Administration National Academy of Agricultural Sciences KACC92315P 20200917

Claims (6)

A novel photosynthetic bacterium Porphyrobacter sp. As a composition for promoting plant growth, comprising COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P),
The Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P) is
Catalase, antibiotic biosynthesis gene (PKS-I), Esterase (C4), Esterase Lipase (C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, Trypsin, α different from photosynthetic bacteria belonging to Genus PorPhyrobacter -chymotrypsin, α-galactosidase, β-glucuronidase, including α-glucosidase,
tryptophan synthetase TrpA and TrpB involved in the auxin biosynthetic metabolic pathway; electron transfer flavoproteins related to nitrogen immobilization FixB; nitrogen immobilized proteins FixH, FixG and FixS; and nitrogen assimilation regulatory protein CpdR;
A composition for promoting plant growth, characterized in that it generates sideropores and exhibits antibacterial activity against pathogens of rice sheath pattern blight. The method of claim 1,
The plant growth promotion is to be measured by measuring the vitality index value (SVI) or rooting power, the composition for plant growth promotion. 3. The method of claim 2,
The vitality index value (SVI) is obtained through the following formula (I), the composition for promoting plant growth.
[Equation I]
Vitality index value (SVI) = [Root length (RL) + Stem length (SL)] * Germination rate (%) 3. The method of claim 2,
The rooting force is obtained by measuring the number of lateral roots, the composition for promoting plant growth. delete A novel photosynthetic bacterium Porphyrobacter sp. COR-2 ( Porphyrobacter sp. COR-2) (Accession No.: KACC 92315P), the novel photosynthetic bacterium Porphyrobacter sp. As a microbial preparation for plant growth promotion, comprising some or all of the culture solution of COR-2 (Accession No.: KACC 92315P),
Catalase, antibiotic biosynthesis gene (PKS-I), Esterase (C4), Esterase Lipase (C8), Leucine arylamidase, Valine arylamidase, Crystine arylamidase, Trypsin, α different from photosynthetic bacteria belonging to Genus PorPhyrobacter -chymotrypsin, α-galactosidase, β-glucuronidase, including α-glucosidase,
tryptophan synthetase TrpA and TrpB involved in the auxin biosynthetic metabolic pathway; electron transfer flavoproteins related to nitrogen immobilization FixB; nitrogen immobilized proteins FixH, FixG and FixS; and nitrogen assimilation regulatory protein CpdR;
A microbial agent for promoting plant growth, characterized in that it generates sideropores and exhibits antibacterial activity against the rice sheath pattern pathogen.

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