Rhodopseudomonas telluris daeji-NZ23 strain having greenhouse gas reduction and plant growth-promoting activity and use thereof

Abstract

The present invention relates to a (Rhodopseudomonas telluris DAEJI-NZ23 strain with accession number KACC81302BP, which exhibits nitrogen fixation capability, produces indole acetic acid (IAA), and has activities of reducing greenhouse gas, promoting plant growth, and secreting extracellular enzymes, and uses thereof. The strain of the present invention can be advantageously utilized in the technical field of greenhouse gas reduction, and is expected to increase crop yield while reducing excessive use of agricultural chemicals.

Description

Rhodopseudomonas telluris DAEJI-NZ23 strain having greenhouse gas reduction and plant growth promotion activity and its use

[0001] The present invention relates to a Rhodopseudomonas telluris DAEJI-NZ23 strain having a deposit number of KACC81302BP and having greenhouse gas reduction and plant growth promotion activities, and to a use thereof.

[0002] Climate change, caused by the increase in greenhouse gases since the Industrial Revolution, has emerged as a global problem threatening the Earth's ecosystem. Since the United Nations Framework Convention on Climate Change, various international joint measures and policies, such as carbon emissions trading and carbon taxes, have been established and implemented, and research on greenhouse gas reduction is actively underway. Furthermore, the need and demand for greenhouse gas emissions reduction is expanding beyond industrial facilities to include agriculture, livestock, and waste management.

[0003] The genus Rhodopseudomonas was proposed in 1937 by Czurda and Maresch based on their analysis of morphological and physiological characteristics using a culture of purple non-sulfur bacteria. There was controversy over the establishment of the genus itself due to the absence of a standard strain, but Rhodopseudomonas palustris was newly proposed as the standard strain for the genus Rhodopseudomonas by van Niel in 1944, and it became the standard strain (van Niel CB. Bacteriol Rev. 1944, 8(1):1-118). It is a genus representing purple non-sulfur bacteria in the class Alphaproteobacteria, and many strains have been reported as new strains in this genus.

[0004] Rhodopseudomonas bacteria possess carotenoids and bacteriochlorophyll a, enabling oxygenic photosynthesis. They are Gram-negative rod-shaped bacteria with flagella for mobility, and are characterized by budding, asymmetrical divisions that form rosettes. Due to their high metabolic flexibility, Rhodopseudomonas is widely used as a model for studying bacterial physiology.

[0005] Meanwhile, Korean Patent Publication No. 2009-0063515 discloses 'Rhodopseudomonas faecalis KL9 KCTC 11243BP and its culture solution and microbial preparation containing the same', and Korean Patent Publication No. 2001-0050310 discloses 'Microbial complex preparation containing Rhodopseudomonas bacteria and use thereof', but 'Rhodopseudomonas telluris DAEJI-NZ23 strain having greenhouse gas reduction and plant growth promotion activity and use thereof' of the present invention is not described.

[0006] The present invention was derived from the above-mentioned needs, and the inventors of the present invention sought to secure highly efficient carbon dioxide reduction microorganisms and to elucidate the plant growth promotion characteristics utilizing the strains, thereby developing eco-friendly landscaping microbial materials for achieving carbon neutrality.

[0007] To solve the above problem, the present invention provides a Rhodopseudomonas telluris DAEJI-NZ23 strain, accession number KACC81302BP, which has nitrogen fixation ability, produces indole acetic acid (IAA), reduces greenhouse gases, promotes plant growth, and secretes extracellular enzymes.

[0008] In addition, the present invention provides a microbial preparation for reducing greenhouse gases containing the strain or a culture solution thereof as an effective ingredient.

[0009] In addition, the present invention provides a microbial preparation for promoting plant growth containing the strain or a culture solution thereof as an effective ingredient.

[0010] In addition, the present invention provides a method for promoting plant growth, comprising a step of treating an effective amount of the strain or a culture solution thereof to a plant, a plant seed, or a plant cultivation site.

[0011] The strain of the present invention is expected to be useful in the field of technology related to greenhouse gas reduction due to its excellent ability to reduce nitrous oxide (N2O), methane (CH4), and carbon dioxide (CO2), and is also expected to have a positive effect of increasing crop yield while reducing excessive use of pesticides due to its excellent activity in promoting crop growth.

[0012] Figure 1 summarizes the results of a plant growth promotion activity test of strains isolated from peat.

[0013] Figure 2 is a transmission electron microscope image observing the cell morphology of the strain Rhodopseudomonas telluris DAEJI-NZ23 of the present invention.

[0014] Figure 3 shows the results of phylogenetic analysis using 16S rRNA of Rhodopseudomonas telluris DAEJI-NZ23 strain.

[0015] Figure 4 shows an experiment to evaluate the greenhouse gas reduction ability of the Rhodopseudomonas telluris DAEJI-NZ23 strain.

[0016] Figure 5 shows the reduction rates of nitrous oxide (A), methane (B), and carbon dioxide (C) in air by the Rhodopseudomonas telluris DAEJI-NZ23 strain.

[0017] In order to achieve the purpose of the present invention, the present invention provides a Rhodopseudomonas telluris DAEJI-NZ23 strain, having a deposit number of KACC81302BP, which has nitrogen fixation ability, produces indole acetic acid (IAA), and has greenhouse gas reduction, plant growth promotion, and extracellular enzyme secretion abilities.

[0018] In the present invention, a Rhodopseudomonas strain that does not produce siderophores, has no phosphate solubilizing ability, but has nitrogen fixation ability, produces IAA (indole acetic acid), has excellent greenhouse gas reduction ability, and exhibits plant growth promoting activity was isolated and identified from peat soil of Daeji Development Co., Ltd., and the 16S rRNA of the strain was analyzed to name the strain Rhodopseudomonas telluris DAEJI-NZ23 and deposited with the Agricultural Microbial Genetic Resource Center (KACC), National Institute of Agricultural Sciences, on June 25, 2024 (Accession Number: KACC92584P).

[0019] In the present invention, the greenhouse gas may preferably be nitrous oxide (N2O), methane (CH4), and carbon dioxide (CO2), but is not limited thereto.

[0020] In addition, in the present invention, the extracellular enzyme may be, but is not limited to, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase.

[0021] The present invention also provides a microbial preparation for reducing greenhouse gases containing, as an active ingredient, the Rhodopseudomonas telluris DAEJI-NZ23 strain having the deposit number KACC81302BP or a culture solution thereof.

[0022] In the microbial preparation for reducing greenhouse gases according to the present invention, the greenhouse gas may preferably be nitrous oxide (N2O), methane (CH4), or carbon dioxide (CO2), but is not limited thereto.

[0023] In the present invention, the term "culture solution" may include, but is not limited to, a culture solution of the Rhodopseudomonas telluris DAEJI-NZ23 strain having the deposit number KACC81302BP, a concentrate of the culture solution, or a dried product of the culture solution.

[0024] The above culture solution can be obtained by culturing in large quantities using a conventional microbial culture method, and a culture medium containing a carbon source, a nitrogen source, vitamins, and minerals can be used.

[0025] The method for culturing the strain of the present invention can be performed according to a method commonly used in the art, and is not limited to a specific method.

[0026] The present invention also provides a microbial preparation for promoting plant growth containing, as an active ingredient, the Rhodopseudomonas telluris DAEJI-NZ23 strain having the deposit number KACC81302BP or a culture solution thereof.

[0027] The plant growth promoting microbial preparation of the present invention may be prepared in the form of, for example, a directly sprayable solution, powder, and suspension, or a highly concentrated aqueous, oily, or other suspension, dispersion, emulsion, oily dispersion, paste, dust, dustable material, or granule, but is not limited thereto.

[0028] The plant growth-promoting microbial preparation of the present invention can be formulated in various forms. These preparations can be prepared, for example, by adding a solvent and / or carrier. Often, inert additives and surface-active substances, such as emulsifiers or dispersants, are mixed into the preparation. Suitable surface-active substances are aromatic sulfonic acids (e.g. lignosulfonic acid, phenolsulfonic acid, naphthalenesulfonic acid and dibutylnaphthalenesulfonic acid), fatty acids, alkyl- and alkylarylsulfonates, alkali metal, alkaline earth metal and ammonium salts of fatty alcohol sulfates, sulfated hexa-, hepta- and octadecanol, salts of fatty alcohol glycol ethers, sulfonated naphthalene and derivatives thereof, condensates of formaldehyde, naphthalene, phenol, polyoxyethylene octylphenyl ether, ethoxylated isooctyl-, octyl- or nonylphenol, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohol, fatty alcohol / ethylene oxide condensates, ethoxylated castor oil, polyoxyethylene alkylethers or polyoxypropylene, lauryl alcohol polyglycol ether acetate, It may be, but is not limited to, sorbitol ester, lignin-sulfite waste liquor or methylcellulose.

[0029] Suitable solid carrier materials are, in principle, all porous and agriculturally acceptable carriers, such as, but not limited to, mineral earths (e.g., silica, silica gel, silicates, talc, kaolin, limestone, lime, chalk, boll, loess, clays, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, perlite, vermucurite, dredged soil, ground synthetic materials), fertilizers (e.g., ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), plant products (e.g., cereal flour, bark flour, cocopeat, peat moss, humic acid, potting soil, potting soil, wood meal and nut shell flour) or cellulose powder. In addition, the solid carriers may be used alone or in combination of two or more.

[0030] The plant growth promoting microbial preparation of the present invention can be mixed with a dispersing agent, a penetrating agent, or a surfactant to increase plant absorption and effectiveness.

[0031] The present invention also provides a method for promoting plant growth, comprising the step of treating an effective amount of Rhodopseudomonas telluris DAEJI-NZ23 strain having a deposit number of KACC81302BP or a culture solution thereof to a plant, a plant seed, or a plant cultivation site.

[0032] In the method for promoting plant growth according to the present invention, the "effective amount" is an amount sufficient to cause beneficial or desired results, and in order to promote plant growth, the Rhodopseudomonas telluris DAEJI-NZ23 strain or its culture solution can be uniformly diluted with water or an appropriate solvent and then sprayed onto plants and plant cultivation areas using an appropriate spraying device such as a power sprayer.

[0033] The method for promoting plant growth according to the present invention can be carried out by immersing or irrigating, i.e., spraying, an effective amount of the Rhodopseudomonas telluris DAEJI-NZ23 strain or its culture solution onto the plant. In the case of the immersion method, an effective amount of the Rhodopseudomonas telluris DAEJI-NZ23 strain or its culture solution can be poured onto the soil around the plant, or seeds can be soaked in an effective amount of the Rhodopseudomonas telluris DAEJI-NZ23 strain or its culture solution.

[0034]

[0035] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0036]

[0037] Example 1. Isolation of representative bacteria from peat

[0038] In this study, 10g of peat soil samples 1 and 2 from Daeji Development Co., Ltd. were each dissolved in sterilized water. -4 ~10 -6 After diluting with tea and inoculating it on a general PSBA medium, it was cultured at 28℃ for 5 days, and a total of 24 strains were isolated based on colony morphology.

[0039] PSBA mediumPSBA medium composition (based on 1L)L-Pyroglutamic1.1gIron(Ⅱ) Sulfate heptahydrate (FeSO4·7H2O)0.012gEDTA0.02gPotassium phosphate dibasic (K2HPO4)1gPotassium dihydrogen phosphate (KH2PO4)0.5gMagnesium sulfate heptahydrate (MgSO4· 7H2O)0.2gCalcium chloride dihydrate (CaCl2·2H2O)0.075gYeast extract2gAgar15gD.W1LpH 7.5 ~ 8.0

[0040]

[0041] Example 2. Test for plant growth promotion activity of strains isolated from peat

[0042] The 24 strains isolated in Example 1 were evaluated for siderophore production ability, nitrogen fixation ability, phosphorus solubilization ability, and indole acetic acid (IAA) production ability.

[0043] Siderophore production ability was measured using the chrome azurole S (CAS) blue agar plate assay method (Int. J. Curr. Microbiol. App. Sci. 2013, 2(12):303-312), a differentiation medium for siderophore-producing isolates. Each strain was inoculated onto a CAS plate medium, cultured at 28°C for 48 hours, and then the change in medium color was observed.

[0044] Nitrogen fixation ability was determined by inoculating each strain on nitrogen-free bromothymol blue (NFB; 0.5% (HO2CCH2CH(OH)CO2H, 0.05% K2HPO4, 0.001% MgSO4·7H2O, 0.002% NaCl, 0.005% FeSO4·7H2O, 0.0002% Na2MoO4, 0.001% MnSO4·7H2O, 0.001% CaCl2, 0.4% KOH, 0.2% bromothymol blue (in 0.5% alcohol), 0.175% agar, pH 6.8) medium and culturing at 28℃ for 7 days. If the medium color turned blue, it was judged to be positive. NFB medium is a selective medium that only grows strains capable of fixing atmospheric nitrogen as it is a medium deficient in nitrogen sources.

[0045] To investigate the phosphate solubilization ability, each isolated strain was activated in LB medium and placed separately on Monkina inorganic phosphorus solid medium by the dropwise addition method and cultured at 28°C for 10 days. The ability of the strain to solubilize inorganic phosphorus was measured by calculating the ratio of the diameter of the phosphate ring to the colony diameter (D / d).

[0046] To evaluate IAA production ability, each strain was inoculated into LB liquid medium (containing 200 mg / L L-trytopan) and cultured with shaking at 28°C and 180 rpm for 4 days. Then, 50 μl of Salkowski's reagent (35% HClO450 ml, 0.5 M FeCl31 ml) and 50 μl of strain culture were added to a white ceramic plate and reacted at 25°C for 30 minutes. If the color turned red, it was judged to be positive for IAA production.

[0047] As a result of the analysis, siderophore production ability, nitrogen fixation ability, phosphate solubilization ability, and IAA (indole acetic acid) production ability were confirmed for the 24 isolated strains. As a result, 5 strains had phosphate solubilization ability, 23 strains produced siderophore, 21 strains were confirmed to have nitrogen fixation ability, and 19 strains showed IAA production ability (Fig. 1).

[0048] 16S rRNA gene analysis was performed on 24 strains isolated from peat soil, and a total of 11 genera were identified: Brevundimonas (1 strain), Sphingomonas (1 strain), Achromobacter (1 strain), Burkholderia (3 strains), Variovorax (2 strains), Janthinobacterium (2 strains), Ideonella (1 strain), Pseudomonas (4 strains), Pedobacter (7 strains), Pseudarthrobacter (1 strain), and Rhodopseudomonas (1 strain). Among these, Rhodopseudomonas belongs to photosynthetic bacteria and can convert carbon dioxide into organic matter in the process of obtaining energy through photosynthesis, which can contribute to the carbon cycle and environmental purification. In addition, some species have nitrogen fixation capabilities and are considered beneficial microorganisms in the agricultural field.

[0049] In the present invention, strain DAEJI-NZ23 belonging to the genus Rhodopseudomonas was finally selected as a greenhouse gas reduction microorganism, and the characteristics of the strain were analyzed.

[0050]

[0051] Example 3. Characteristic evaluation of selected strains

[0052] The selected DAEJI-NZ23 strain was confirmed to be rod-shaped when observed using a transmission electron microscope (Fig. 2). The physiological and biochemical characteristics of the DAEJI-NZ23 strain were evaluated using API 50CH, 20NE, and ZYM.

[0053] As a result of enzyme activity analysis of the DAEJI-NZ23 strain using API zym kit (BioMerieux Vitek Inc.), it was confirmed that it produces fat-decomposing enzymes esterase (C4) and esterase lipase (C8); protein-decomposing enzymes leucine arylamidase, valine arylamidase, cystine arylamidase, and trypsin; and phosphatase enzymes alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase (Table 2).

[0054] API zym kit results of DAEJI-NZ23 strain Enzyme activity Results Enzyme activity Results Alkaline phosphatase + Naphthol-AS-BI-phosphohydrolase + W Esterase (C4)+ W a-galactosidase-Esterase Lipase (C8)+ W b-galactosidase-Lipase (C14)-b-glucuronidase-Leucine arylamidase+a-glucosidase-Valine arylamidase+b-glucosidase-Cystine arylamidase+N-acetyl-b-glucosaminidase-Trypsin+ W a-mannosidase-a-chymotrypsin-a-fucosidase-Acid phospatase+ W W ​: weak

[0055] In addition, carbohydrate metabolism of the DAEJI-NZ23 strain was examined using API 50CH strips. The analysis results showed positive fermentation / oxidation reactions for glycerol, D-arabinose, D-ribose, D-xylose, L-xylose, methyl-β D-xylopyranoside, D-galactose, D-glucose, D-fructose, L-sorbose, L-rhamnose, dulcitol, and inositol (Table 3).

[0056]

[0057] In addition, the biochemical characteristics of the DAEJI-NZ23 strain were confirmed using API 20NE, and the DAEJI-NZ23 strain had the ability to reduce nitrate to nitrite and showed the characteristics of utilizing D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-glucosamine, D-maltose, potassium gluconate, adipic acid, malic acid, and trisodium citrate (Table 4).

[0058] API 20NE results of DAEJI-NZ23 strainReactions / EnzymesResultsReactions / EnzymesResultsreduction of nitrates to nitrites+assimilation (ManNosE)+indole production (Tryptophane)+ W assimilation (MANnitol)+fermentation D-glucose -assimilation (N-Acetyl-Glucosamine)+Arginine DiHydrolase-assimilation (MALtose)+UREase-assimilation(potassium GlucoNate)+hydrolysis (β-glucosidase) (ESCulin)-assimilation (CAPric acid)-hydrolysis (protease) (GELatin)-assimilation (ADIpic acid)+β-galactosidase (Para-NitroPhenyl-ßD-Galactopyranosidase) - assimilation (MaLaTe)+assimilation (GLUcose)+assimilation(trisodium CITrate)+assimilation (ARAbinose)+assimilation(PhenylACetic acid)-

[0059] In order to identify the DAEJI-NZ23 strain, the 16S rRNA gene base sequence (sequence number 1) was determined and analyzed using the blast program of NCBI DB. As a result, it was confirmed to have 100% homology with Rhodopseudomonas telluris UT3615 AB498822 (Fig. 3).

[0060]

[0061] Example 4. Evaluation of the greenhouse gas reduction capacity of selected strains

[0062] The carbon dioxide reduction ability of the DAEJI-NZ23 strain was evaluated by inoculating the strain with 4,000 ppm or more of carbon dioxide (CO2) in PSBA liquid medium and measuring the levels of nitrous oxide (N2O), methane (CH4), and carbon dioxide during culturing for 7 days to analyze the greenhouse gas reduction ability.

[0063] The analysis results showed that the DAEJI-NZ23 strain reduced nitrous oxide in the air by about 7% (Fig. 5A), methane in the air by about 53.7% (Fig. 5B), and carbon dioxide in the air by about 51% (Fig. 5C).

[0064] Whole-genome sequencing of high-quality DNA extracted from the DAEJI-NZ23 strain, which has carbon dioxide reduction potential, revealed that the genome of the DAEJI-NZ23 strain is 5,576,623 bp in total length, and is composed of 4,962 CDSs, 55 tRNA genes, and a GC content of 65.6%. In addition, screening of biosynthetic gene clusters (BGCs) using antiSMASH and identification of useful secondary metabolite biosynthetic genes revealed that numerous genes capable of biosynthesizing enzymes involved in carbon dioxide fixation and numerous genes capable of biosynthesizing enzymes involved in the nitrogen cycle, such as nitrogen fixation and denitrification.

[0065]

[0066] Example 5. Evaluation of the plant growth promotion ability of selected strains

[0067] The inventors of the present invention conducted a lettuce cultivation test to evaluate the plant growth promotion ability of the DAEJI-NZ23 strain, which has greenhouse gas reduction ability.

[0068] The experiment was conducted in a glass greenhouse at the College of Agriculture and Life Sciences, Chungnam National University (Daejeon, Korea). The lettuce used was the Jeokchima cultivar (Kwonnong Seedlings), and the soil used was sandy loam. The experimental groups were composed of a control (untreated with DAEJI-NZ23), a reference plot (treated with 7 ml of DAEJI-NZ23 culture medium), and a drainage plot (treated with 14 ml of DAEJI-NZ23 culture medium) with a completely randomized distribution and three replicates. The control group was treated with 7 ml of PSBA, a culture medium for the DAEJI-NZ23 strain.

[0069] Characteristics of sandy soil used in lettuce cultivation test Texture pH (1:5) EC (dS / m) TN (%) OM (%) Ava.-P2O5 (mg / kg) Ex.-cations (cmol) c / ㎏)CEC(cmol c / ㎏)Ca 2+ Mg 2+ K + Na + Sandy loam 5.731.350.112.213335.551.460.500.539.04

[0070] The fertilization method of the microbial agent (DAEJI-NZ23 culture solution) was as follows: before lettuce planting (March 26, 2024), DAEJI-NZ23 culture solution was applied to the soil of the pots by drenching. After planting, DAEJI-NZ23 culture solution was applied to the soil of each treatment group at 7-day intervals. DAEJI-NZ23 culture solution was applied a total of 5 times during the test period. Growth and yield were investigated 30 days after planting.

[0071] Looking at the final growth results according to the microbial agent treatment, no difference (fertilizer damage) in lettuce was found due to microbial agent fertilization throughout the entire test period, and the treatment groups (reference group, multiplied group) fertilized with microbial agents showed better results in all growth items than the control group that was not treated with microbial agents, and in particular, it was confirmed that fresh weight increased by 15-17% in the microbial agent reference and multiplied treatment groups compared to the control group.

[0072] Results of lettuce growth survey 30 days after transplanting Treatment group Leaf length Leaf width Chlorophyll vitality Weight length (cm) Index (%) Width (cm) Index (%) Number of ea Index (%) Content (mg / 100cm) 2 ) Index (%) Weight (g / pot) Index (%) Control group 15.9 a* 10015.2 a 10014.0 a 1002.30 b 10046.3 b 100 standard 16.3 a 10315.5 a 10216.0 a 1142.55ab 11153.1 a 115x16.8 a 10615.9 a 10416.0 a 1142.63 a 11454.3 a 117* Duncan's multiple range test (DMRT) 5% level

[0073] Through the above results, it was confirmed that the DAEJI-NZ23 strain, which has greenhouse gas reduction activity, also has an excellent plant growth promotion effect. The inventors of the present invention named the DAEJI-NZ23 strain, a Rhodopseudomonas strain with excellent greenhouse gas reduction activity and plant growth promotion effect, as Rhodopseudomonas telluris DAEJI-NZ23 based on the 16S rRNA identification results and deposited it with the Agricultural Microbial Genetic Resources Center (KACC), National Institute of Agricultural Sciences.

[0074] [Accession number]

[0075] Name of depositor: National Institute of Agricultural Sciences, Rural Development Administration, Microbial Bank (KACC)

[0076] Accession number: KACC81302BP

[0077] Date of acceptance: 20240625

[0078]

Claims

1. Rhodopseudomonas telluris DAEJI-NZ23 strain, accession number KACC81302BP, which has nitrogen fixation ability, produces indole acetic acid (IAA), reduces greenhouse gases, promotes plant growth, and secretes extracellular enzymes.

2. Rhodopseudomonas telluris DAEJI-NZ23 strain, characterized in that the greenhouse gas in paragraph 1 is nitrous oxide (N2O), methane (CH4), and carbon dioxide (CO2).

3. Rhodopseudomonas telluris DAEJI-NZ23 strain, characterized in that in paragraph 2, the extracellular enzyme is esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase.

4. A microbial preparation for reducing greenhouse gases containing the strain of paragraph 1 or its culture solution as an active ingredient.

5. A microbial preparation for promoting plant growth containing the strain of paragraph 1 or its culture solution as an effective ingredient.

6. A method for promoting plant growth, comprising a step of treating an effective amount of the strain of paragraph 1 or a culture solution thereof to a plant, a plant seed, or a plant cultivation site.

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