Microbial materials, plant cultivation methods, and bacterial strains

Abstract

The present invention addresses the problem of providing a bacterial strain having exceptional nitrogen-fixing capabilities and N2O-reducing capabilities, and a microbial material including said bacterial strain.　The problem can be solved by a microbial material including a bacterial strain belonging to the Bradyrhizobium ottawaense clade that has nitrogen-fixing capabilities and N2O-reducing capabilities.

Description

[Technical Field]

[0001] The disclosure in this application relates to microbial materials and methods for cultivating plants, as well as bacterial strains. [Background technology]

[0002] Bradyrhizobium rhizobia are plant-associated microorganisms that form nodules on soybean roots and fix atmospheric nitrogen. Many of these microorganisms also possess denitrification ability. Among these microorganisms, B. diazoefficiens possesses the N2O reductase gene (nos), which is responsible for the final denitrification reaction, N2O → N2 reduction. N2O produced during the denitrification process has a greenhouse effect approximately 300 times greater than CO2, making its reduction an important challenge. Inoculating soybean fields with N2O reductase-containing B. diazoefficiens can reduce N2O emissions from the soybean rhizosphere. It is also known that the N2O reduction activity of B. diazoefficiens can be enhanced using genetic engineering (see Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] K. Minamisawa et al., “Regulation of nitrous oxide reductase genes by NasT-mediated transcription antitermination in Bradyrhizobium diazoefficiens”, Environ Microbiol Reports, 2017, 9(4), p389-396 [Non-patent document 2] K. Minamisawa et al., “The nitrate-sensing NasST system regulates nitrousoxide reductase and periplasmic nitrate reductase in Bradyrhizobium japonicum”, Environmental Microbiology, 2014, doi:10.1111 / 1462-2920.12546 Summary of the Invention [Problem to be solved by the invention]

[0004] The nos-enhanced mutant strains described in Non-Patent Documents 1 and 2 have stronger NO reduction ability than wild-type strains. However, international frameworks have been established for the use of genetically modified organisms to prevent adverse effects on biodiversity. In Japan, the "Act on the Conservation of Biological Diversity through Regulations on the Use of Genetically Modified Organisms, etc." (commonly known as the "Cartagena Protocol") also regulates the use of genetically modified organisms. Therefore, the nos-enhanced mutant strains described in Non-Patent Documents 1 and 2 cannot be used in natural environments. Therefore, there is a need to discover bacterial strains that have nitrogen fixation ability, which can promote plant growth, and have excellent NO reduction ability.

[0005] The disclosure of the present application has been made to solve the above problems, and as a result of extensive research, it has been newly discovered that there exists a bacterial strain of the genus Bradyrhizobium ottawaense that has excellent nitrogen fixation and NO reduction abilities, and that this bacterial strain is useful as a microbial material.

[0006] That is, the purpose of the present disclosure is to provide microbial materials and methods for cultivating plants, as well as bacterial strains. [Means for solving the problem]

[0007] (1) Microbial materials containing bacterial strains belonging to the Bradyrhizobium genus ottawaense clade that have nitrogen fixation and NO reduction capabilities. (2) A microbial material according to (1) above, wherein the bacterial strain belongs to a clade containing the B. ottawaense type strain OO99 in an evolutionary tree analysis that includes the B. ottawaense type strain OO99 and one or more Bradyrhizobium species in its OTU (operational taxonomic unit). (3) Phylogenetic tree analysis is performed using AMPHORA to extract amino acid sequences encoded by the dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, and tsf genes, to create a concatenated sequence for each OTU, and then analyze the concatenated sequence. (4) A microbial material according to (1) or (2) above, wherein the bacterial strain further has an ANI value of 95% or more in ANI analysis against the B. ottawaense type strain OO99. (5) A microbial material according to any one of (1), (2), and (4) above, wherein the bacterial strain further has an ITS (16S-23S rRNA intergenic region) base sequence that is 97% or more identical to the ITS base sequence of a Bradyrhizobium bacterium. (6) The microbial material according to any one of (1) to (5) above, wherein the bacterial strain is SG09 (accession number: NITE BP-03361). (7) The microbial material according to any one of (1) to (5) above, wherein the bacterial strain is SF21 (Accession No.: NITE BP-03552) or SH12 (Accession No.: NITE BP-03553). (8) A microbial material according to any one of (1) to (7) above, which functions as a plant growth promoter. (9) The microbial material according to (8) above, wherein the plant is a legume. (10) A method for cultivating a plant, comprising the step of contacting the microbial material according to any one of (1) to (9) above with the seeds or roots of a plant, or causing the microbial material to be present in the vicinity of the roots of a plant. (11) It belongs to the Bradyrhizobium ottawaense clade, which has nitrogen fixation and NO reduction capabilities. A bacterial strain that belongs to a clade that includes the B. ottawaense type strain OO99 in an evolutionary tree analysis that includes the B. ottawaense type strain OO99 and one or more Bradyrhizobium species in an OTU (operational taxonomic unit). (12) The bacterial strain according to (11) above, wherein the bacterial strain is SG09 (accession number: NITE BP-03361). (13) The bacterial strain according to (11) above, wherein the bacterial strain is SF21 (Accession No.: NITE BP-03552) or SH12 (Accession No.: NITE BP-03553). [Effects of the Invention]

[0008] The microbial material disclosed in the present application has the ability to fix nitrogen and reduce N2O, thereby promoting plant growth and suppressing emissions of N2O, a greenhouse gas. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1A shows an evolutionary tree of Bradyrhizobium genus bacterial strains based on AMPHORA (housekeeping gene), and FIG. 1B shows the functions of each bacterial strain. [Figure 2] FIG. 2 is a graph showing the occupancy rate of the nodules formed in Example 1. [Figure 3] 3A is a photograph of soybeans grown in Example 2, and FIG. 3B is a photograph of soybeans grown in Comparative Example 1. [Figure 4]FIG. 4 is a graph showing the difference in N2O reduction ability between a wild-type strain belonging to the B. ottawaense clade and a wild-type strain belonging to the B. diazoefficiens clade and a mutant strain with enhanced N2O reduction ability. [Figure 5] FIG. 5A shows an evolutionary tree of Bradyrhizobium bacterial strains based on AMPHORA (housekeeping gene), and FIG. 5B shows the functions of each bacterial strain. [Figure 6] FIG. 6 is a photograph in place of a drawing, showing soybeans grown in Example 3 (SG09, SF21, SH12), Comparative Example 2 (USDA110), and Comparative Example 3 (no inoculation). [Figure 7] FIG. 7 shows the nodules dry weight of soybeans grown in Example 3 and Comparative Example 2, and the nodules number of soybeans grown in Example 3 and Comparative Example 2. [Figure 8] FIG. 8 is a graph showing the difference in N2O reduction ability between wild-type strains belonging to the B. ottawaense clade and wild-type strains belonging to the B. diazoefficiens clade. [Figure 9] FIG. 9 is a graph showing the relative N2O flux measurement values ​​of Example 4 (SG09) and Comparative Example 5 (110ΔH1) when the N2O flux measurement value of Comparative Example 4 (USDA110) is set to 1. DETAILED DESCRIPTION OF THE INVENTION

[0010] The microbial material and plant cultivation method disclosed in the present application are described in detail below. Note that in this specification, numerical values, abbreviations, etc. are interpreted as follows. (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field.

[0011] (Embodiments of Microbial Materials) The microbial material disclosed in this application includes bacterial strains (hereinafter sometimes simply referred to as "bacterial strains") belonging to the ottawaense clade of the genus Bradyrhizobium that have the ability to fix nitrogen and reduce NO. In this specification, the term "microbial material" refers to the microorganism (bacterial strain) itself that is inoculated into soil or crop seeds for the purpose of improving plant growth and health, or the microorganism to which other components have been added.

[0012] The new bacterial strain disclosed in this application was isolated from sorghum roots in a field in Nihonmatsu, Fukushima, and belongs to the Bradyrhizobium genus. The genus Bradyrhizobium includes two known species: the diazoefficiens clade, which has NO reduction ability, and the japonicum clade, which does not. The new bacterial strain disclosed in this application belongs to the B. ottawaense clade, a highly effective soybean root nodule bacterium isolated in Canada. After examining the activity of numerous bacterial strains, the inventors found that some strains in the ottawaense clade have NO reduction ability but not nitrogen fixation ability. Therefore, the use of bacterial strains belonging to the ottawaense clade with nitrogen fixation and NO reduction ability as microbial materials can promote plant growth and reduce NO emissions from fields. As used herein, "nitrogen fixation ability" refers to the ability to convert stable atmospheric N into other nitrogen compounds, such as highly reactive NH. Furthermore, "N2O reduction ability" refers to the ability to reduce N2O, which is generated during the nitrification and denitrification of NH3, to N2. Furthermore, when a bacterial strain "has nitrogen fixation ability and N2O reduction ability," it means that it possesses genes that exert the above abilities.

[0013] The bacterial strain disclosed in the present application is not particularly limited as long as it belongs to the ottawaense clade and has the ability to fix nitrogen and reduce NO. As described below, the presence or absence of nitrogen fixation and NO reduction abilities can be determined from among the screened bacterial strains.

[0014] The type strain of the B. ottawaense clade is OO99, collected in Canada (Xiumei Yu et al., "Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen-fixing bacterium from root nodules of soybeans in Canada", International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3202-3207).

[0015] As described in the Examples below, the B. ottawaense type strain OO99 is a wild-type strain that has excellent NO reduction ability. In a phylogenetic tree analysis that includes the B. ottawaense type strain OO99 and one or more Bradyrhizobium species as operational taxonomic units (OTUs), the bacterial strain disclosed in the present application belongs to a clade that includes the B. ottawaense type strain OO99.

[0016] The OTUs mentioned above are units obtained by classifying the base sequences of essential bacterial genes (generally 16S ribosomal RNA genes) on a computer using their similarity as an index. If the OTUs are the same, they can be said to be composed of the same evolutionarily species of bacteria.

[0017] Phylogenetic tree analysis is performed by creating and analyzing a concatenated sequence for each OTU consisting of the amino acid sequences encoded by the dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, and tsf genes extracted using AMPHORA.

[0018] Details of AMPHORA are described in (1) Martin Wu et al., "A simple, fast, and accurate method of phylogenomic inference," Genome Biology 2008, 9:R151, and (2) Martin Wu et al., "Phylogenomic analysis of bacterial and archaeal sequences with AMPHORA2," BIOINFORMATICS APPLICATIONS NOTE, Vol. 28, no. 7, 2012, pp. 1033-1034 (hereinafter referred to as "Wu and Scott, 2012").

[0019] The bacterial strain may also have an average nucleotide identity (ANI) value of 95% or higher compared to the B. ottawaense type strain OO99 in ANI analysis. An ANI value of 95% or higher indicates that the strain is of the same species. ANI analysis may be performed by a known method.

[0020] The bacterial strain may further have an ITS (16S-23S rRNA intergenic region) nucleotide sequence that is 97% or more identical to the ITS nucleotide sequence of a Bradyrhizobium bacterium. The ITS is a region used in molecular phylogenetic analysis, and a homology of 97% or more indicates that the strain is of the same species.

[0021] Examples of bacterial strains include, but are not limited to, SG09, SG11, SF21, and SH12.

[0022] Of the bacterial strains disclosed in this application, SG09 was received by the National Institute of Technology and Evaluation Patent Microorganisms Depositary on January 5, 2021, with accession number "NITE P-03361." SG09 was transferred to the National Institute of Technology and Evaluation Patent Microorganisms Depositary as an international deposit under the Budapest Treaty and received on November 8, 2021, with accession number "NITE BP-03361."

[0023] "SF21" and "SH12" were received as an international deposit under the Budapest Treaty at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation on November 8, 2021. The accession numbers are as follows: ·"SF21": "NITE BP-03552" ·"SH12": "NITE BP-03553"

[0024] Any known medium can be used to culture the bacterial strain disclosed in the present application. In addition to liquid media, solid media such as agar-containing slants and plates can also be used. By using these media, the bacterial strain can be grown to a desired bacterial cell mass.

[0025] Any carbon source that can be assimilated by the bacterial strain can be used as the carbon source for the medium, and examples thereof include sugars such as glucose, galactose, lactose, arabinose, mannose, and malt extract starch hydrolysate.

[0026] Similarly, various synthetic or natural sources that can be utilized by bacterial strains can be used as nitrogen sources, such as peptone, meat extract, yeast extract, etc.

[0027] Furthermore, inorganic salts such as salt and phosphates, salts of metals such as calcium, magnesium and iron, vitamins, amino acids and other trace nutrient sources can also be added as needed, according to common methods for microbial cultivation.

[0028] Other components contained in the microbial material are not particularly limited as long as they are components commonly contained in microbial materials, such as carriers such as porous materials for adsorbing and stabilizing microorganisms, various organic materials used as nutrient sources for microbial growth, fertilizer components, other minerals, diluents, dispersants, etc.

[0029] As described above, the bacterial strain disclosed in the present application has the ability to fix nitrogen and reduce NO. Therefore, a microbial material containing the bacterial strain functions as a plant growth promoter and also as an agent for suppressing NO emissions from farm fields. Examples of plants whose growth is promoted include legumes (soybeans, peanuts, mung beans, etc.) and grasses (sorghum, corn, wheat, barley, etc.).

[0030] (Embodiment of Plant Cultivation Method) The plant cultivation method disclosed in the present application includes a step of contacting the microbial material with the seeds or roots of a plant, or causing the microbial material to be present in the vicinity of the roots of a plant. This step promotes the growth of the plant. In this specification, "roots" refers to the part of a plant that is in the soil or hydroponic solution when cultivated and absorbs water and nutrients. Furthermore, "promoting growth" means promoting the growth of a host plant by nitrogen fixation, regardless of whether or not nodule formation occurs.

[0031] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. [Example]

[0032] [Isolation of bacterial strains] A bacterial extract was prepared from surface-sterilized sorghum roots, and the extract was inoculated onto soybean seeds. Bradyrhizobium species were isolated from the resulting nodules. The specific procedure is as follows.

[0033] Approximately 30 g of sorghum roots harvested from a field in Nihonmatsu, Fukushima Prefecture, were washed with 70% ethanol and then surface-sterilized by immersion in 2.5% NaOCl at room temperature for 10 minutes. After 10 washes with sterile distilled water, the roots were frozen in liquid nitrogen and crushed using a sterilized mortar and pestle. Approximately 200 mL of Tris-HCl buffer (50 mM, pH 7.5) was added to the crushed material, mixed thoroughly, and filtered through Miracloth (Milipore) to remove plant debris. The filtrate was centrifuged (9,876 × g, 10 min), and the precipitate was suspended in Tris-HCl buffer (50 mM, pH 7.5) and the volume was adjusted to 10 mL. Five surface-sterilized soybean seeds (Glycine max cv. Enrei) were placed spaced apart in a Leonardo jar (Inaba et al., "N2O Emission from Degraded Soybean Nodules Depends on Denitrification by Bradyrhizobium japonicum and Other Microbes in the Rhizosphere", Microbes Environ., 2012, December;27(4):470-476), and 1 mL of bacterial extract was added dropwise to each seed. Five seeds were sown per pot and grown in a climate chamber (Koito Electric Industries, 23°C, 16 hours light, 8 hours dark) for three weeks. The nodules were then collected. The nodules were washed with 70% ethanol for one minute and then immersed in 0.5% NaOCl for 10 minutes for surface sterilization. The nodules were cut with a flame-sterilized cutter, and the inside of the nodule cross section was touched with a sterilized toothpick and streaked onto a 100-fold diluted NA agar plate (Difco®, Nutrient Broth). After culturing at 28°C for 10 days, the emerged colonies were further purified into single colonies on a 100-fold diluted NA agar plate to obtain isolates.

[0034] Next, the analytical methods and measurement methods used in the examples will be described. [ITS array] The 16S-23S rRNA intergenic region (ITS) of the isolates was amplified by PCR and sequenced by the Sanger method. To improve performance, PCR was performed using Blend taq®-plus (TOYOBO CO., LTD., Okasa) with primers ITS-F and ITS-R described in Saeki et al., "Grouping of Bradyrhizobium USDA Strains by Sequence Analysis of 16S rDNA and 16S-23S rDNA Internal Transcribed Spacer Region," Soil Sci. Plant Nutr., 50(4), 517-525, 2004. The PCR reaction mixture composition is shown in Table 1, and the reaction conditions are shown in Table 2.

[0035] [Table 1]

[0036] [Table 2]

[0037] The determined ITS sequences were subjected to a BLAST search at NCBI (https: / / www.ncbi.nlm.nih.gov / ), and it was confirmed that SG09 and SG11 obtained in this study belong to the genus Bradyrhizobium.

[0038] [AMPHORA phylogenetic tree] The draft genome sequences of the isolates were uploaded to DFAST (https: / / dfast.nig.ac.jp / ) and converted to amino acids. To analyze phylogenetic relationships, the amino acid sequences of the 31 housekeeping genes listed above were extracted using AMPHORA (Wu and Scott, 2012). The extracted gene sequences were concatenated and a phylogenetic tree was constructed using MEGA v.7.0 (Sudhir Kumar et al., "MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets", Mol. Biol. Evol. 33(7):1870-1874, 2016) using the neighbor-joining method (Naruya Saitou et al., "The Neighbor-Joining Method: A New Method for Reconstructing Phylogenetic Trees", Mol. Biol. Evol. 4(4):406-425, 1987) with 1000 bootstraps.

[0039] [Phylogenetic tree of Bradyrhizobium isolates based on ITS sequences] OTUs were constructed based on ITS sequences in 97% of cases, and the isolates were identified as multiple bacterial strains belonging to the ottawaense, diazoefficiens, and japonicum clades.

[0040] [Measurement of NO reduction activity] N2O reduction activity was measured by observing the reduction of added N2O. First, the target strain was inoculated into 15 mL of HMM medium (Sameshima-Saito et al. 2006) in a 75 mL test tube and pre-cultured at 30°C under aerobic conditions for approximately 6 hours. Next, a butyl rubber stopper was attached to the test tube containing the bacterial suspension, and the gas phase was replaced with 4.98% N2O gas (95.02% N2). The culture was then shaken for 12–14 hours to induce N2O reduction. The culture solution was mixed with sterilized medium and the absorbance was adjusted to approximately 0.05 (light path = 10 mm). To maintain anaerobic conditions, the medium and the gas phase in the test tube were degassed with N2 gas beforehand, and the dead space of the plastic syringe and needle used was also washed three times with N2 gas. Ten mL of the prepared bacterial solution was transferred to a sterile test tube filled with 100% N2 gas, and the bacterial solution and gas phase volumes were standardized for each strain. The test tube volume was 73.8 ± 0.2 mL with a butyl rubber stopper attached, and the gas phase volume was 63.8 mL. 0.65 mL of 100% N2O gas was introduced into the test tube to adjust the final concentration to approximately 1%. This time point was set to 0 hours, and the decrease in N2O concentration was measured over time. N2O concentration was measured using gas chromatography (Shimazu, GC2014) under the conditions shown in Table 3 below.

[0041] [Table 3]

[0042] Figure 1A shows the evolutionary tree of Bradyrhizobium strains based on AMPHORA (housekeeping genes), and Figure 1B shows the function of each strain. For N2 fixation (nitrogen fixation) and nodulation (nodulation), a solid square indicates the presence of the corresponding gene cluster, while a square indicates the absence of the corresponding gene cluster. For denitrification, a solid square indicates the presence of the corresponding gene cluster, while a solid circle indicates the absence of the nos gene cluster. In addition to the SG09 and SG11 strains obtained in this study, Figure 1 also includes other known Bradyrhizobium strains for comparison. Among these, OO99, a known soybean rhizobia belonging to the ottawaense clade, is the type strain for the ottawaense species. Details of TM102, TM233, and TM239, which are included in the ottawaense clade, are described in Shintaro Hara et al., “Identification of Nitrogen-Fixing Bradyrhizobium Associated With Roots of Field-Grown Sorghum by Metagenome and Proteome Analyses,” March 2019 | Volume 10 | Article 407.

[0043] Furthermore, when the obtained full-length genomes of the SG09 strain and the OO99 strain were subjected to ANI analysis using a known method, the SG09 strain showed an ANI value of 99.08% of that of the OO99 strain.

[0044] From these results, we confirmed that (1) the SG09 strain obtained in this study is a different bacterial strain from OO99, a known soybean rhizobia belonging to the ottawaense clade, although it is the same species, and (2) the bacterial strains belonging to the ottawaense clade have the ability to reduce NO, but some have the ability to fix nitrogen and some do not.

[0045] [Preparation of microbial materials and confirmation of nodule formation] Example 1 The SG09 strain isolated in the above [Isolation of bacterial strains] was suspended in sterile water to prepare a microbial material. Next, 0.5 × 10 SG09 strain and USDA122 (BCRC number: 13533), a known soybean rhizobia that belongs to the same cluster as B. diazoefficiens USDA110 in 16S ribosomal RNA gene phylogenetic analysis, were mixed at 0.5 × 10 7 A microbial material was prepared by mixing the strains at a 1:1 ratio based on the density of 122 cells / mL. Details of USDA122 are described in Masayuki Sugawara et al., "Complete Genome Sequence of Bradyrhizobium diazoefficiens USDA122, a Nitrogen-Fixing Soybean Symbiont," Genome Announc. 2017, doi:10.1128 / genomeA.01743-16. Four soybean seeds (Glycine max, cv. Enteri) were sown in a Leonardo jar pot with vermiculite soil, and 1 mL of the prepared microbial solution (microbial material) was inoculated into the pot in four separate inoculations. The microbial solution was dripped from a distance of approximately 1 cm to avoid direct contact with the soybean seeds. After the soybeans germinated, three of the four seeds were thinned out, and one individual plant was grown per pot. The plants were cultivated in an artificial climate chamber (Koito Electric Industries, 23°C, 16 hours light, 8 hours dark) for 26 days.

[0046] Figure 2 is a graph showing the occupancy rate of the formed nodules. As is clear from Figure 2, even though the number of bacterial cells of the inoculated bacterial strains was the same, the dominance rate of SG09 in soybean nodules after 26 days was approximately 74%. These results demonstrate that the competitive nodule formation ability of SG09 is superior to that of B. diazoefficiens USDA122, a known soybean rhizobia.

[0047] [Confirmation of the growth-promoting ability of SG09] <Example 2> The microbial material containing only the SG09 strain prepared in Example 1 (density of the SG09 strain: 0.5 × 10 7 cells / mL) and soybeans were grown in the same manner as in Example 1. Figure 3A shows a photograph of soybeans grown in Example 2 on the 26th day after inoculation.

[0048] <Comparative Example 1> Except for not inoculating the microbial material, the experiment was carried out in the same manner as in Example 2. Figure 3B shows a photograph of the soybeans grown in Comparative Example 1.

[0049] As is clear from Figures 3A and 3B, no signs of nitrogen deficiency were observed in the soybeans inoculated with SG09 in Example 2. On the other hand, the soybeans in Comparative Example 1, which were not inoculated with SG09, grew poorer than those in Example 2 and exhibited symptoms of nitrogen deficiency, such as yellowing of the lower leaves. These results confirmed that SG09, despite being isolated from sorghum roots, coexists with soybeans and exhibits normal nitrogen fixation ability.

[0050] [Comparison of NO reduction ability] Next, we compared the NO reduction activity of wild-type strains belonging to the B. ottawaense clade with that of strains belonging to the B. diazoefficiens clade. Figure 4 shows the difference in NO reduction activity between wild-type strains (SG09, OO99) belonging to the B. ottawaense clade, a wild-type strain (USDA110, JCM No.: 10833) belonging to the B. diazoefficiens clade, and genetically engineered mutant strains (USDA110ΔH1, USDA110ΔnasS) with enhanced NO reduction activity. USDA110ΔH1 is a genetically engineered mutant strain according to the procedure described in Non-Patent Document 1, and USDA110ΔnasS is a genetically engineered mutant strain according to the procedure described in Non-Patent Document 2. The values ​​shown in Figure 4 were measured using the "NO reduction activity measurement" method described above.

[0051] As shown in Figure 4, the wild-type strains SG09 and OO99, which belong to the B. ottawaense clade, had approximately 5.4-fold higher NO reduction activity than the wild-type strain USDA110, which belongs to the B. diazoefficiens clade, and were confirmed to have similar NO reduction activity to the mutant strains USDA110△H1 and USDA110△nasS, which have enhanced NO reduction activity. Furthermore, a Tukey's HSD test confirmed that a and b were significantly different at p<0.05.

[0052] [Search for strains other than SG09] Using the same procedures as those described above for [Isolation of bacterial strains], [ITS sequences], [Evolutionary tree by AMPHORA], [Phylogenetic tree of Bradyrhizobium isolates based on ITS sequences], and [Measurement of N2O reduction activity], we searched for other bacterial strains in the ottawaense clade.

[0053] Figure 5A shows the evolutionary tree of Bradyrhizobium genus strains based on AMPHORA (housekeeping gene), and Figure 5B shows the functions of each bacterial strain. The symbols (■, □, ●, ◯) in the "N2 fixation," "Nodulation," and "Denitrification" columns in Figure 5B are the same as those in Figure 1B.

[0054] In addition, the full-length genomes of the SF21 and SH12 strains and the full-length genome of the OO99 strain were subjected to ANI analysis using known techniques. The SF21 strain showed an ANI value of 99.1% of that of the OO99 strain, and the SH12 strain showed an ANI value of 99.1% of that of the OO99 strain.

[0055] These results confirmed that the newly obtained strains SF21 and SH12 are homologous but distinct from OO99, a known soybean rhizobia belonging to the ottawaense clade.

[0056] [Preparation of microbial materials and confirmation of growth-promoting ability] Example 3 The isolated strains SG09, SF21, and SH12 were cultured at 1 × 10 9 The microbial material was prepared by suspending the seeds in sterile water to a density of 1000 cells / mL. Five soybean seeds (Glycine max, cv. Enteri) surface-sterilized with 0.5% sodium hypochlorite were sown per pot in Leonardo jar pots filled with sterilized vermiculite and inoculated with 1 mL of the microbial material. The seeds were grown in a greenhouse at 25°C under a 16-hour light / 8-hour dark cycle. Three days after sowing, only three well-germinated seeds were thinned out, and the seeds were grown for the next 27 days. The pots were periodically watered with nitrogen-free hydroponic solution. After cultivation, nodules were collected from each pot. The number of nodules per pot was counted, and the nodules were then dried at 80°C for 48 hours, after which the dry weight was immediately measured.

[0057] <Comparative Example 2> The experiment was carried out in the same manner as in Example 3, except that B. diazoefficiens USDA110 (JCM number: 10833) was used as the strain.

[0058] <Comparative Example 3> The experiment was carried out in the same manner as in Example 3, except that no microbial material was inoculated.

[0059] Figure 6 shows photographs of soybeans grown in Example 3, Comparative Example 2, and Comparative Example 3. As is clear from Figure 6, the growth condition of the soybeans inoculated with the microbial material of Example 3 was slightly inferior to that of the microbial material of Comparative Example 2, which used known soybean rhizobia, but was better than that of Comparative Example 3, which was not inoculated with the microbial material.

[0060] Figure 7 shows the nodules dry weight of soybeans grown in Example 3 and Comparative Example 2, and the nodules number of soybeans grown in Example 3 and Comparative Example 2. As is clear from Figure 7, it was confirmed that when the SG09, SF21, and SH12 strains were used as the microbial material, the number and amount of nodules were greater than in Comparative Example 2 (USDA110).

[0061] [Comparison of NO reduction ability between SF21 and SH12] Next, we used the bacterial strains USDA110, SG09, SF21, and SH12 to compare their NO reduction abilities using the same procedure as described above in [Comparison of NO reduction ability]. The results are shown in Figure 8. As shown in Figure 8, SG09, SF21, and SH12, which are wild-type strains belonging to the B. ottawaense clade, all had higher NO reduction abilities than USDA110, a wild-type strain belonging to the B. diazoefficiens clade. Furthermore, a Tukey's HSD test confirmed that a and b were significantly different at p<0.05.

[0062] [Measurement of NO flux in senescent root nodules] Example 4 Next, we measured NO flux in senescent nodules of the SG09 strain. The procedure is as follows.

[0063] (Soybean cultivation) The isolated SG09 strain was diluted to 1 × 10 8 The microbial material was prepared by suspending soybean seeds (Glycine max, cv. Enteri) in sterile water to a density of 1000 cells / mL. Three soybean seeds (Glycine max, cv. Enteri) surface-sterilized with 0.5% sodium hypochlorite were sown per pot in Leonardo jar pots filled with sterilized vermiculite, and 1 mL of the microbial material was inoculated. The seeds were grown in a greenhouse at 25°C under a 16-hour light / 8-hour dark cycle. Three days after sowing, the seeds were thinned out to one with good germination, and then grown for the next 27 days. The pots were periodically supplied with nitrogen-free hydroponic solution.

[0064] (Nodule aging treatment) Soil from the Kashimadai field at Tohoku University was used for the nodule senescence treatment. The soil was sieved through a 2 mm sieve and placed in 10 g portions into 50 ml centrifuge tubes, the same number as the number of pots. To wash the soil, 30 ml of distilled water was added to the centrifuge tube, followed by shaking for 10 minutes and then centrifuging at 5000 g for 10 minutes, and the supernatant was discarded. This process was repeated three times, after which 30 ml of distilled water was added to the centrifuge tube to thoroughly suspend the soil. To artificially accelerate soybean nodule senescence, the above-ground parts were removed, and 10 g of soil suspended in 30 ml of distilled water was added to the pots with the remaining root system (including nodules). The pots were then placed at 25°C for 20 days under a 16 h light / 8 h dark cycle to promote nodule senescence.

[0065] (N2O flux measurement) The root systems were collected from the pots, placed in 60 mL glass vials, sealed, and incubated at 25°C for 3 hours. Gas samples were taken before and after incubation, and the N2O concentration in the gas phase was measured using an ECD gas chromatograph (Shimazu, G-C2014) to determine the N2O flux under atmospheric conditions.

[0066] <Comparative Example 4> Senescent nodule N2O flux measurements were carried out in the same manner as in Example 4, except that the USDA110 strain was used instead of the SG09 strain.

[0067] <Comparative Example 5> Senescent nodule N2O flux measurements were carried out in the same manner as in Example 4, except that the USDA110△H1 strain was used instead of the SG09 strain.

[0068] 9 is a graph showing the relative NO flux measurements of Example 4 and Comparative Example 5, with the NO flux measurement of the USDA110 strain (a wild-type strain of B. diazoefficiens, a known soybean rhizobia) of Comparative Example 4 set at 1. As is clear from FIG. 9, it was confirmed that the wild-type SG09 strain (Example 4) was able to reduce the NO flux of the wild-type USDA110 by approximately 50%, and was able to reduce the NO flux to approximately the same level as that of Comparative Example 5, which is a mutant strain (USDA110ΔH1) whose NO reduction ability has been enhanced by genetic engineering.

[0069] These results confirmed that bacterial strains belonging to the B. ottawaense clade (1) have high NO reduction capacity even in wild-type strains, (2) some strains have nitrogen fixation capacity and some do not, and (3) the strains with nitrogen fixation capacity coexist with soybean and promote its growth. Therefore, the use of microbial materials containing bacterial strains belonging to the B. ottawaense clade with nitrogen fixation and NO reduction capacity in the field can promote plant growth and reduce NO emissions into the atmosphere. [Industrial Applicability]

[0070] The microbial material disclosed in the present application can promote plant growth and reduce NO emissions into the atmosphere, and is therefore useful in the agricultural field.

[0071] TIFF0007808855000004.tif214150TIFF0007808855000005.tif72153

Claims

1. Nitrogen fixation capacity and N 2 A microbial material comprising a bacterial strain belonging to the O-reducing clade of the genus Bradyrhizobium, A microbial material, wherein the bacterial strain is SF21 (accession number: NITE BP-03552) or SH12 (accession number: NITE BP-03553).

2. The microbial material of claim 1, which functions as a plant growth promoter.

3. 3. The microbial material of claim 2, wherein the plant is a legume.

4. A method for cultivating a plant, comprising the step of contacting the microbial material according to any one of claims 1 to 3 with the seeds or roots of a plant, or causing the microbial material to be present in the vicinity of the roots of a plant.

5. Nitrogen fixation capacity and N 2 It belongs to the Ottawaense clade of the genus Bradyrhizobium, which has the ability to reduce oxygen. In an evolutionary tree analysis including the B. ottawaense type strain OO99 and one or more Bradyrhizobium species in the OTU (operational taxonomic unit), a bacterial strain belonging to a clade including the B. ottawaense type strain OO99, The bacterial strain is SF21 (accession number: NITE BP-03552) or SH12 (accession number: NITE BP-03553).

Images