KR102663790B1 - Pseudoxanthomonas sp. JBCE485 and Variovorax paradoxus JBCE486 strins in promoting plant growth tolerance and resistance to salt stress and composition comprising the same - Google Patents

Abstract

The present invention relates to strains of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486, which are excellent in promoting plant growth and salt resistance, and to a composition containing the same. When the JBCE486 strain is mixed and treated, it efficiently settles on the outside and inside of the roots, respectively, increasing the effect of promoting the growth of the roots and stems of the plant. It is possible to increase biomass by promoting healthy seedlings and growth, and has excellent salt resistance. It can be usefully used as an eco-friendly preparation using microorganisms.

Description

Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains excellent in promoting plant growth and salt resistance and compositions containing the same {Pseudoxanthomonas sp. JBCE485 and Variovorax paradoxus JBCE486 strins in promoting plant growth tolerance and resistance to salt stress and composition comprising the same}

The present invention relates to strains of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486, which are excellent in promoting plant growth and salt resistance, and to a composition containing the same.

In response to issues such as anxiety about chemical pesticides, environmental pollution, and persistence, much research is being done on eco-friendly farming methods, and plants are improving their tolerance to stresses such as dryness and salt damage, which are increasing due to global warming and rapid environmental changes. Interest in eco-friendly functional microbial materials that can improve the growth and health of microorganisms is increasing. In particular, the development of various eco-friendly farming methods using agricultural useful microorganisms is progressing, and the number of farms producing crops in an eco-friendly manner by reducing the use of chemical agents is increasing.

Ulleungsan garlic, also called Myeongi or mountain garlic, is a perennial plant belonging to the Liliaceae family. It is a wild vegetable that smells like garlic throughout the plant. In Korea, it grows wild in the highlands of Jiri Mountain, Odaesan Mountain, and Seorak Mountain and on Ulleungdo Island, and is also distributed in Japan, China, and Siberia. Mountain garlic has scale leaves similar to green onions, which are 4 to 7 cm long and have a slightly curved lanceolate shape. There are 2 to 3 leaves, 20 to 30 cm long and 3 to 10 cm wide, with a long oval shape, and the lower part of the leaf has a flower stalk wrapped in leaf sheath. A flower stalk emerges from the leaf stem and blooms white from May to July. The lower part of the flower has egg-shaped involucre, has 6 long oval petals, has 6 stamens, and the pistil and stamen are longer than the petals, and the anthers are yellow-green. After the flowers fade, small capsules develop and bear fruit, and the seeds are black and round. There are five species centered on the inland Baekdudaegan and the Ulleung species native to island regions, and it is reported that they were derived through different evolutionary processes in different climatic and ecological environments. Ulleung species has wide, oval leaves with 2 to 3 leaves. In addition, the scale leaves of the underground part are almost white and plump, and the roots are light brown and generally close to erect. The flowers are white, have a large amount of seed production, and have a slightly garlicky scent, and the range of cultivation adaptation is wide, from flat areas to highland areas. Research using the plant biome (phytobiome) to preserve and utilize Ulleungsan garlic is still insufficient.

Accordingly, the present inventor increased tolerance to stresses such as dryness and salt damage, which are increasing due to environmental changes due to climate warming, and developed eco-friendly functional microbial materials that can promote plant growth and health. As a result of analyzing strains in soil, two new types of microorganisms were obtained, and the present invention was completed by confirming that a mixture of two types of strains was more effective in promoting plant growth than treatment alone.

(001) Republic of Korea registered patent KR 10-1972079 (002) Republic of Korea registered patent KR 10-1972068

The present invention provides a composition for promoting the growth or salt resistance of plants containing Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains. It's about .

The present invention provides a composition for promoting the growth or salt resistance of plants containing Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains. provides.

In addition, the Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains of paragraph 1, cultures of the strains, and enrichment of the cultures Provided is a microbial preparation for promoting plant growth or salt resistance, comprising as an active ingredient at least one selected from the group consisting of water, dried product of the culture, and combinations thereof.

In addition, a method for promoting plant growth or salt resistance is provided, which includes treating plant seeds or plant planting soil with a microbial agent.

When the Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains of the present invention are mixed and treated, they efficiently settle on the outside and inside of the roots, respectively, increasing the growth promotion effect of the roots and stems of the plant, thereby promoting healthy seedlings and growth. It is possible to increase biomass through biomass, and because it has excellent salt resistance, it can be usefully used as an eco-friendly agent using microorganisms.

Figure 1 shows Pseudoxanthomonas sp. This is a graph showing the growth rate of Arabidopsis thaliana by treatment concentration of JBCE485 and Variovorax paradoxus JBCE486 strains.
Figure 2 shows Pseudoxanthomonas sp. This photo shows the growth rate of Arabidopsis thaliana by treatment concentration of JBCE485 and Variovorax paradoxus JBCE486.
Figure 3 shows Pseudoxanthomonas sp. This is a graph showing the growth of Arabidopsis thaliana in (A) regular MS medium or (B) MS medium supplemented with 100 mM NaCl by treatment with JBCE485 and Variovorax paradoxus JBCE486 strains (1 × 10 ⁷ cfu/ml).
Figure 4 shows Pseudoxanthomonas sp. This is a photograph showing the growth of Arabidopsis thaliana in (A) regular MS medium or (B) MS medium supplemented with 100mM NaCl by treatment with JBCE485 and Variovorax paradoxus JBCE486 strains (1× ¹⁰⁷ cfu/ml).
Figure 5 shows Pseudoxanthomonas sp. This is a graph showing the effect of reducing salt damage (175 mM NaCl, B) stress in chives by treatment with JBCE485 and Variovorax paradoxus JBCE486 strains (1 × 10 ⁷ cfu/ml) (A group without salt treatment).
Figure 6 shows Pseudoxanthomonas sp. This photo shows the effect of reducing salt damage (175mM NaCl, B) stress in chives by treatment with JBCE485 and Variovorax paradoxus JBCE486 strains (1× ¹⁰⁷ cfu/ml) (A group without salt treatment).
Figure 7 shows Pseudoxanthomonas sp. This is a photo confirming the compatibility (whether growth is inhibited) between JBCE485 and Variovorax paradoxus JBCE486 strains.
Figure 8 shows Pseudoxanthomonas sp. isolated from the rhizosphere and roots of Ulleungsan garlic plants. This is a photo confirming the growth promotion activity of JBCE485 and Variovorax paradoxus JBCE486 strains: (A) Siderophore production changes the color of CAS medium from blue to orange (B) Phosphate solubilization induces a transparent area around the colony to change the Pikovskaya agar medium. Used.
Figure 9 shows Pseudoxanthomonas sp. This is a photograph confirming the population dynamics that settled inside and outside the roots for 28 days after treating leek seeds with JBCE485 and Variovorax paradoxus JBCE486 strains: (A) Pseudoxanthomonas sp. Change in population density on the root surface after treatment with JBCE485 and Variovorax paradoxus JBCE486 strains individually or in combination (B) Change in population density inside the root (C) Total number of bacteria inside and outside the root.

Hereinafter, the present invention will be described in detail.

The present inventors collected Ulleungsan garlic and the surrounding soil from samples collected from two places: a wild habitat (Ulleungdo, Ulleung-gun, Gyeongsangbuk-do) and a cultivation area (Jinan-gun, Jeollabuk-do) during the growing season of Ulleungsan garlic (June-July 2020). Fractionation into the rhizosphere and roots was performed to analyze plant-related microorganisms using metagenome, and microbial analysis and microbial resources were explored using cultural methods.

As a result of testing the growth-promoting effect of bacteria isolated from the roots or rhizosphere of Ulleungsan garlic, strains JBCE485 and JBCE486 increased the growth of Arabidopsis thaliana. Using 16rDNA, JBCE485 and JBCE486 were each identified as Pseudoxanthomonas sp. and Variovorax paradoxus .

It was confirmed that the combined treatment of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 was excellent in alleviating salt stress in Arabidopsis thaliana (model plant).

In addition, since experiments using seeds were not possible for Ulleungsan garlic, chive seeds from the same Allium genus as Ulleungsan garlic were used to confirm the salt stress reduction and growth promotion effects of Pseudojantomonas JBCE485 and Varioborax paradoxus. It was confirmed that mixing and treating the JBCE486 strain increased the effect of promoting the growth of plant roots and stems, enabling biomass to be increased by promoting healthy seedlings and growth, and was highly effective in improving tolerance to salt stress.

The present invention provides a composition for promoting the growth or salt resistance of plants containing Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains. provides.

In addition, the Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains of paragraph 1, cultures of the strains, and enrichment of the cultures Provided is a microbial preparation for promoting plant growth or salt resistance, comprising as an active ingredient at least one selected from the group consisting of water, dried product of the culture, and combinations thereof.

In addition, a method for promoting plant growth or salt resistance is provided, which includes treating plant seeds or plant planting soil with a microbial agent.

According to the above, the present invention promotes plant growth and/ Alternatively, a microbial preparation for controlling plant diseases can be provided, and a method for promoting plant growth and/or controlling plant diseases can be provided, including the step of treating the microbial preparation on soil, plants, or plant seeds.

The microbial preparation may be included as an active ingredient in microbial pesticides and/or fertilizers in accordance with its intended use, and when the microbial preparation contains the above strains, the Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains are 10 ⁶ It is preferably included at a concentration of 10 ⁸ CFU/ml, but is not limited thereto.

In addition, the culture of the strain that can be included in the microbial preparation is preferably obtained by separating it from LB (Luria Bertini) medium or TS (Trypic Soy) medium in which the strain was cultured, but is not limited thereto.

The microbial preparation can be formulated for plant growth promotion or antibacterial purposes by conventional methods, and can be prepared in dry powder form or liquid form. Specifically, the microbial preparation composition according to the present invention can be prepared in a liquid form, and can be used in the form of powder by adding an extender, or can be formulated and granulated. However, there is no particular limitation to the formulation. The microbial preparation can be manufactured by adding additives such as additives, extenders, and nutrients to the strain or its culture. At this time, the additives include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl. The group consisting of aryl phosphoric esters, polyoxyethylene alkyl aryl ethers, polyoxyethylene alkyl aryl polymers, polyoxyalkylone alkyl phenyl ethers, polyoxyethylene nonyl phenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100, and Tween 80. One or more selected from the group may be used, and the extender and nutrient include skim milk (medium), soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, starch, and combinations thereof. You can use one or more of your choice.

In addition, the microbial preparation provides a method of promoting plant growth and inhibiting the growth of pathogens by treating soil or plants. At this time, the treatment methods include commonly used methods, namely spraying (e.g. spraying, misting, atomizing, powder spraying, granule spraying, surface application, etc.), soil application (e.g. mixing, irrigation, etc.), and surface use. It can be done by (e.g. application, smearing, coating, etc.), immersion, etc., and the amount used can be appropriately determined depending on the formulation, damage situation, application method, application location, etc.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

<Example 1> Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 Selection and identification of strains

Ulleungsan garlic ( Allium ulleungense HJ Choi & N. Friesen) and the surrounding soil were collected from two locations: a wild habitat (Ulleungdo, Ulleung-gun, Gyeongsangbuk-do) and a cultivation site (Jinan-gun, Jeollabuk-do) during the growing season of Ulleungsan garlic (June-July 2020). Sampled. Samples collected from each region were fractionated into bulk soil, rhizosphere, and endophyte.

Specifically, the collected soil and plants were placed in separate plastic bags and stored at 4°C. After transferring these samples to the laboratory, within 2 to 3 days, they were fractionated into bulk soil, rhizosphere, and endophyte to separate microorganisms and DNA. First, the roots were carefully shaken to collect the loose soil, filtered through a 2 mm sieve and considered as normal soil. The soil still attached to the roots was shaken vigorously to remove the attached soil, and then the remaining soil was brushed off with a sterilized brush. After collection, it was considered root zone soil. Endophytic bacteria were isolated from the roots. The roots were disinfected with 70% (v/v) ethanol for 1 minute, surface disinfected with a 3% (v/v) sodium hypochlorite solution for 3 minutes, and then washed 5 times with sterile distilled water. . To check sterilization, 100 μl of the last washed sterilized water was plated on LB medium and incubated at 30°C for 48 hours to check for the appearance of bacterial colonies. General soil, rhizosphere, and endophytic bacteria (roots) were each investigated three times.

After collection, DNA was isolated from repeated samples of each isolated plant using the GeneAll Exgene ^TM Soil DNA isolation kit (GeneAll, South Korea). The purity of the isolated DNA sample was confirmed using a BioTek, Epoch ^TM Spectrometer (USA), and the state of the DNA was confirmed through 1% agarose gel electrophoresis. In order to create a bacterial 16S rRNA gene library, first, the V3-V4 region of the 16S rRNA gene was cloned into 341F (5′-CCT ACG GGN GGC WGC AG-3′ (SEQ ID NO. 1)) and 805R (5′-GAC TAC). It was amplified using primers (HVG GGT ATC TAA TCC -3′ (SEQ ID NO. 2)), which combined the Nextera consensus and adapter sequence with the forward primer (5′-TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG-target sequence-3′ (SEQ ID NO. 3)). and reverse primer (5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-target sequence-3′ (SEQ ID NO. 4)). The PCR amplification product was purified and used as a template to use the forward index i5 primer (5′-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXXTCGTCGGCAGCGTC-3′ (SEQ ID NO. 5)) and reverse i7 primer (5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXGTCTCGTGGGCTCGG) with Illumina dual indices and sequencing adapters. It was amplified using -3′ (SEQ ID NO: 6). The previous two PCRs were processed at 94℃ for 3 minutes, followed by 25 cycles of denaturation (94℃, 30s), annealing (55℃, 30s), and extension (72℃, 30s), followed by extension at 72℃ for 5 minutes. It was carried out. The quantity of the PCR product was measured using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, USA), and the quality was confirmed using the Agilent Bioanalyzer 2100 system. After collecting the purified libraries, ChunLab Inc. (South Korea) sequenced them with the Illumina MiSeq system using MiSeq Reagent Kit v2 (Illumina Inc., USA).

Meanwhile, to identify the strains isolated and stored above, sequencing was performed using primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 7)) and primer 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 8)). .

Sequence material was provided by ChunLab Inc. The analysis was conducted using EzBioCloud (https://www.ezbiocloud.net/), developed in-house by (Seoul, Korea). Sequencing data was removed using the Trimmomatic v0.32 program with an average quality value of 25 or less. Sequences were combined using PandaSeq, primer sequences were removed using the EzBioCloud program with a similarity cut-off value of 0.8, and sequence errors were cleaned up using DUDE-Seq software. The analysis pipeline used the EzBioCloud 16S rRNA database, and chimeric sequences were removed through UCHIME. Microbiological classification was searched in the EzBioCloud database using the USEARCH program. Sequence similarity was calculated through pairwise alignment. The query sequence showing more than 97% similarity to the reference sequence of EzBioCloud was considered to be identified at the species level.

Sequences not found in the database data were searched with a similarity of 97% using the Cluster Database at High Identity with Tolerance (CD-HIT) or UCLUST tool. The genera identified in the EzBioCloud database and OTUs obtained through CD-HIT and UCLUST were used to determine the final OTUs. The reference sequence criteria used for classification are as follows; x = similarity: species (x ≥ 97%), genus (97% > x ≥ 94%), family (94% > x ≥ 90%), order (90% > x ≥ 85%), class (85% > x ≥ 80%) and phylum (80 > x ≥ 75%). If the similarity was lower than the above standard value, it was classified as unclassified.

In addition, if the scientific name is not exactly known, the known name is written first, and the rest are written with additional words such as the following (e.g. If the name of the class is not known, type a 'c' after the name of the phylum. It is written as follows, name. 'Acidobacteria_c'; c = class, o = order, f = family, g = genus, and s = species). Microbial diversity was analyzed and compared using CLcommunity software (Chunlab, Inc., Seoul, South Korea). To compare alpha diversity, abundance-based coverage-ACE, Chao1, Shannon, Simpson, and rarefaction curves were analyzed. Beta diversity, including principal coordinate analysis (PCA), was analyzed based on Fast UniFrac. Differences in alpha diversity, including the number of OTUs, species richness, and diversity, were compared according to the sampled area, and differences in microbiota of the isolation area, i.e., general soil, rhizosphere, and roots, and differences according to location were compared between species in the phylum ( Comparisons were made up to the level of species.

<Example 2> Growth promotion analysis in model plants of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains

In Example 1, Pseudoxanthomonas sp. isolated from the roots or rhizosphere of Ulleungsan garlic. The growth promotion effect of Arabidopsis thaliana was tested using JBCE485 and Variovorax paradoxus JBCE486 strains.

Specifically, bacteria isolated from Ulleungsan garlic and stored at -80°C were cultured at 28°C for 48 hours and then diluted in sterilized distilled water containing 0.2% sterilized carboxylmethyl cellulose (CMC). Arabidopsis seeds were surface disinfected with 70% (v/v) ethanol for 90 seconds, 1% (v/v) sodium hypochlorite for 10 minutes, and then washed with sterile distilled water. Sterilized seeds ^were placed in each bacterial suspension (1 Seeds coated with bacteria were placed on sterilized filter paper and allowed to dry on the surface for 30 minutes at room temperature (25°C). Meanwhile, bacteria treated with sterilized distilled water (0.2% CMC) were used as a control. Seeds treated with bacteria were sown on the surface of a medium hardened by adding half-strength Murashige and Skoog (1/2MS) solution containing 1.5% sucrose and 0.8% (w/v) agar to a Petri dish. The plate on which the seeds were sown was sealed with parafilm and placed in a plant growth bed at an angle of approximately 70°, under 23±1°C and light (16-h light/8-h dark; 100 μmol m ^-2 s ^-1 ) conditions. It was cultured in . After 10 days of culture, the length of roots and stems was determined.

As a result, Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 The strain significantly increased the growth of Arabidopsis thaliana. As a result of identification by comparing the base sequences of NCBI using 16rDNA sequenced using primers in Example 1 (SEQ ID NO: 7 and SEQ ID NO: 8), JBCE485 and JBCE486 were identified as Pseudoxanthomonas sp. and Variovorax paradoxus .

Therefore, Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains were identified from Ulleungsan garlic and deposited in a seed bank (KACC, Korean Agricultural Culture Collection, National Institute of Agricultural Sciences, Rural Development Administration).

Additionally, in order to find the optimal concentration for treatment, each strain was treated on Arabidopsis seeds at concentrations of 1×10 ⁶ , 1×10 ⁷ and 1×10 ⁸ cfu/ml.

As a result, it was confirmed that growth increased when treated at a concentration of 1×10 ⁷ or 1×10 ⁸ cfu/ml. Considering the degree of growth increase and dilution factor, 1×10 ⁷ cfu/ml was selected as the optimal treatment concentration. was determined (Figures 1 and 2).

<Example 3> Analysis of salt stress reduction in model plants of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains

Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains were treated with Arabidopsis thaliana to analyze the degree of inhibition of salt stress.

Specifically, to investigate the effect of alleviating abiotic (salt damage) stress, a suspension (1 × 10 ⁷ cfu/ml) of each strain (JBCE485, JBCE486) or a mixed strain (JBCE485+JBCE486) was treated with Arabidopsis seeds. Then, the seeds were sown on 1/2 MS medium or 1/2 MS medium supplemented with 100 mM NaCl, and the growth rate was examined.

As a result, it was confirmed that root length, number of secondary roots, and live weight increased in the medium without adding NaCl, and there was a difference in the phenotype of Arabidopsis when treated with individual bacteria and when treated with mixed strains. There was. When each or mixed strains were treated, the number of secondary roots tended to increase similarly, but live weight increased significantly when mixed strains were treated. When treated with the JBCE485 strain, the roots were long and thin, but when treated with the JBCE486 strain, the main and lateral roots tended to become thicker. On the other hand, in the case of MS medium under salt stress with the addition of NaCl, when each bacteria was treated, the root length increased compared to the untreated. When mixed strains were treated, the root length tended to be similar to the untreated, but each The number of entourage tended to increase compared to the treated strain. Treatment of the strain led to an increase in root length and number of lateral roots, which resulted in an overall increase in live weight compared to untreated. The number of secondary roots was the largest, the fresh weight was similar to that treated with JBCE486 alone, and the number of roots increased. When the JBCE485 strain was treated, the roots were thin and long, but when the JBCE486 strain was treated, the roots were thick and the number of lateral roots was relatively large, which was similar to that in MS medium without salt stress (Figures 3 and 4).

<Example 4> Analysis of growth promotion and salt stress reduction in chives of Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains

Since experiments using seeds were not possible for Ulleungsan garlic, the growth promotion effect and salt stress reduction of selected strains were investigated using chive seeds, which are of the same Allium genus as Ulleungsan garlic.

Specifically, chive seeds treated with each Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strain or a suspension of mixed bacteria (1× ¹⁰⁷ cfu/ml) were grown in normal soil and 175 mM NaCl (soil/water, 2 :1 v/v) was sown in soil and growth was examined.

As a result, it was observed that the emergence speed of seeds treated with the JBCE486 strain increased. When treated with mixed bacteria, the length of stems and roots increased similarly to when treated with JBCE485 and JBCE486 strains, respectively. Meanwhile, compared to individual treatments, treatment with mixed bacteria induced an increase in stems and roots, and this increase significantly increased the overall live weight of chives (Figure 5).

Meanwhile, in soil with 175mM NaCl added, it was observed that the germination of chive seeds was delayed compared to normal soil, the tips of leaves dried and yellowed, and growth was delayed. It was confirmed that the leaves were green and looked healthier. Treatment with the JBCE486 strain increased the length of stems and roots, and treatment with the JBCE486 strain mainly led to an increase in live weight. When treated with the JBCE486 strain or mixed bacteria, the number of leaves per plant increased compared to untreated. Based on the above results, treatment with each strain or mixed bacteria increased the growth of chives in different ways and also increased resistance to salt stress (Figure 6).

<Example 5> Compatibility analysis between Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains

Pseudoxanthomonas sp. To confirm whether mixed use between JBCE485 and Variovorax paradoxus JBCE486 strains was possible, the two strains were cross-cultured at a 90-degree angle or stratified culture to investigate whether growth was inhibited or the formation of support was examined.

As a result, no growth inhibition or inhibitory support was formed around the intersection or double culture, and it was confirmed that this result was possible even by mixing the two strains (Figure 7).

<Example 6> Analysis of growth promotion relationship between Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains

Pseudoxanthomonas sp. The characteristics related to plant growth promotion were investigated and compared for JBCE485, Variovorax paradoxus JBCE486, and mixed strains. Both strains (JBCE485 and JBCE486) were confirmed to produce siderophores by forming an orange halo zone in CAS medium, but no difference in the degree of siderophore production by mixing the two strains could be confirmed. Strain JBCE485 produced protease, but when strains were mixed, the level of protease production tended to be similar or slightly decreased.

Meanwhile, as shown in Table 1 below, the JBCE485 strain produced IAA, and when the strains were mixed, the production of IAA tended to increase significantly. When the mixed strains were treated, the increase in IAA production promoted the growth of Arabidopsis thaliana and chives. It was a key factor in improving salt tolerance. Meanwhile, both strains did not produce cytokinin (Figure 8).

<Example 7> Population density inside and outside the roots when Pseudojantomonas JBCE485 and Varioborax paradoxus JBCE486 strains were inoculated into chive seeds separately or simultaneously

Strains JBCE485 and JBCE486 were treated with chive seeds and sown in the soil to examine the degree of establishment inside and outside the roots.

As a result, when each fungus was treated and sown on chive seeds, the JBCE485 and JBCE486 strains settled well and survived on the surface of the chive roots for up to 28 days (Figure 9A). When the two strains were inoculated separately or simultaneously, the JBCE485 strain tended to exist at a higher density than the JBCE486 strain. Meanwhile, when inoculated at the same time, the total population number 7 days after treatment was similar to that when the JBCE485 strain was treated alone, but afterwards the density was lower than when the JBCE485 strain was treated alone, but the density when the JBCE486 strain was treated alone remained similar to .

In addition, as a result of examining the bacterial settlement and survival density inside the roots of chives, the JBCE486 strain was present at a higher density than the JBCE485 strain both when the two strains were treated separately and simultaneously (Figure 9B). When both strains were treated simultaneously, the total number of bacteria inside the roots was lower than when the JBCE486 strain was treated alone, but was higher than when the JBCE485 strain was treated alone. On the other hand, when the two strains were treated simultaneously, the bacterial density 7 days after treatment was higher than when treated individually, but thereafter it was similar to the density when the JBCE485 strain was treated alone (Figure 9C). Based on the above results, it is believed that strain JBCE485 mainly settles on the surface of chive roots, while strain JBCE486 settles and resides inside the roots. These results suggest that when two strains are inoculated simultaneously, they can successfully colonize the inside and outside of the roots without competing with each other, showing high efficacy.

strain production or active Sideophore production Phosphate Solubilization Protease activity IAA production Cytokinin production Pseudoxanthomonas sp. JBCE485 + - + ++(3.16) - Variovorax paradoxus JBCE486 + - - +(1.79) - Pseudoxanthomonas sp.JBCE485 + Variovorax paradoxus JBCE486 + - + +++(3.75) - +++; very high, ++; high, +; low, -; no production or activity

Name of depository institution: National Institute of Agricultural Sciences

Accession number: KACC92411P

Trust date: 20220318

Name of depository institution: National Institute of Agricultural Sciences

Accession number: KACC92412P

Trust date: 20220318

<110> CHONBUK NATIONAL UNIVERSITY <120> Pseudoxanthomonas sp. JBCE485 and Variovorax paradoxus JBCE486 strins in promoting plant growth tolerance and resistance to salt stress and composition comprising the same} <130>DHP22-132 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> 341F <400> 1 cctacgggng gcwgca 16 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 805R <400> 2 gactachvgg gtatctaatc c 21 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Nextera consensus and adapter sequence forward primer <400> 3 tcgtcggcag cgtcagatgt gtataagaga cag 33 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Nextera consensus and adapter sequence reverse primer <400> 4 gtctcgtggg ctcggagatg tgtataagag acag 34 <210> 5 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> forward index i5 primer <400> 5 aatgatacgg cgaccaccga gatctacacn nnnnnnntcg tcggcagcgt c 51 <210> 6 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> reverse i7 primer <400> 6 caagcagaag acggcatacg agatnnnnnn ngtctcgtgg gctcgg 46 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 7 agagtttgat cctggctcag 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 8 ggttaccttg ttacgactt 19

Claims (6)

Composition for promoting growth and salt resistance of wild garlic or chives containing Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains . delete Pseudoxanthomonas sp. JBCE485 (accession number KACC 92411P) and Variovorax paradoxus JBCE486 (accession number KACC 92412P) strains of claim 1, cultures of the strains, and concentrates of the cultures, A microbial preparation for promoting the growth and salt resistance of wild garlic or chives, comprising as an active ingredient at least one selected from the group consisting of the dried product of the culture and combinations thereof. A method for promoting the growth and salt damage resistance of wild garlic or chives, comprising the step of treating plant seeds or plant planting soil with the microbial agent of claim 3. delete The method of claim 4, wherein the treatment is immersion or spraying.