Rice growth promoter, growing medium for rice, rice seeds, rice seedlings, and method of cultivating rice.

Abstract

It promotes the growth of rice. [Solution] The rice growth promoter contains at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207. The culture medium for growing rice contains at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207. The method for cultivating rice involves treating rice seeds or seedlings with at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

Description

Technical Field

[0001] The technology disclosed in this specification relates to a rice growth promoter, a medium for growing rice, rice seeds, rice seedlings, and a rice cultivation method.

Background Art

[0002] Research and development has been conducted to promote the growth of rice seedlings by administering OsPep, an immune regulatory peptide of rice, to rice seedlings (see Patent Document 1). In addition, a method of inoculating plants with a preparation containing an endophytic strain such as Achromobacter xylosidans has also been proposed to promote the growth of plants such as rice and increase resistance to biotic / abiotic stresses (see Patent Document 2).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] There was room for improvement in the above methods for promoting plant growth.

Means for Solving the Problems

[0005] The technology disclosed in this specification can be realized, for example, in the following forms. (1) The rice growth promoter disclosed by this specification contains at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

[0006] (2) The rice growth promoter described in (1) above may further contain Achromobacter microorganisms deposited under accession number NITE-BP-03938.

[0007] (3) The rice growth promoter described in (1) or (2) above may further contain Sphingomonas microorganisms deposited under accession number NITE-BP-03939.

[0008] (4) The growing medium for rice disclosed herein comprises any one of the rice growth promoters described in (1) to (3) above.

[0009] (5) The rice seeds disclosed herein are seeds treated with any one of the rice growth promoters described in (1) to (3) above.

[0010] (6) The rice seedlings disclosed herein are seedlings grown in the culture medium described in (4) above.

[0011] (7) The rice seedlings mentioned in (6) above may be paddy rice seedlings.

[0012] (8) The method of cultivating rice disclosed herein involves treating rice seeds or seedlings with at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

[0013] The technologies disclosed herein can be implemented in various forms, for example, as rice growth promoters, growing media for rice seedlings, methods for producing the same, and rice seedling cultivation using these growing media or rice growth promoters. [Brief explanation of the drawing]

[0014] [Figure 1]Figure 1 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not the S5 and S6 strains were provided. The upper row shows the fresh weight of the above-ground part of the seedlings after 7 days of cultivation, and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where no strain was provided, "S5" represents the group where the S5 strain was provided, and "S6" represents the group where the S6 strain was provided. "*" indicates that a statistically significant difference was found in the t-test with p<0.05. [Figure 2] Figure 2 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple strains of fungal bacteria were provided. The upper row shows the fresh weight of the above-ground part of the seedlings 4 weeks after cultivation (3 weeks after provision), and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where no fungal strains were provided, and "NB7+R2A7+S5+S6" represents the group where strains NB7, R2A7, S5, and S6 were provided, respectively. "*" indicates that a statistically significant difference was found with p<0.05 in a t-test. [Figure 3] Figure 3 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple fungal strains and OsPep-3 were provided. The graph shows the weight of the seedling roots 4 weeks after cultivation (3 weeks after provision). In the figure, "mock" represents the untreated control group where neither fungal strain nor OsPep-3 was provided, "OsPep" represents the group where OsPep-3 was provided, "NB7+R2A7+S5+S6" represents the group where NB7 strain, R2A7 strain, S5 strain, and S6 strain were provided, and "NB7+R2A7+S5+S6+OsPep" represents the group where NB7 strain, R2A7 strain, S5 strain, S6 strain, and OsPep-3 were provided. "*" indicates that a statistically significant difference was found with p<0.05 in the t-test. [Figure 4]Figure 4 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple fungal strains were provided. The upper row shows the fresh weight of the above-ground part of the seedlings 4 weeks after cultivation (3 weeks after provision), and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where no fungal strains were provided, and "NB7+R2A7+S5+S6" represents the group where strains NB7, R2A7, S5, and S6 were provided, respectively. "**" indicates that a significant difference was found with p<0.01 in the t-test, and "***" indicates that a significant difference was found with p<0.001 in the t-test. [Figure 5] Figure 5 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple fungal strains and OsPep-3 were provided. The upper row shows the fresh weight of the above-ground part of the seedlings 4 weeks after cultivation (3 weeks after provision), and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where neither fungal strain nor OsPep-3 was provided, "NB7+OsPep" represents the group where NB7 strain and OsPep-3 were provided, "R2A7+S5+S6" represents the group where R2A7 strain, S5 strain, and S6 strain were provided, and "NB7+R2A7+S5+S6" represents the group where NB7 strain, R2A7 strain, S5 strain, and S6 strain were provided. "*" indicates that a statistically significant difference was found with p<0.05 in the t-test. [Figure 6] Figure 6 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple strains of fungal bacteria were provided. The upper row shows the fresh weight of the above-ground part of the seedlings 4 weeks after cultivation (3 weeks after provision), and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where no fungal bacteria were provided, "NB7+S5+S6" represents the group provided with NB7, S5, and S6 strains, "NB7+R2A7+S5+S6" represents the group provided with NB7, R2A7, S5, and S6 strains, and "S5+S6" represents the group provided with S5 and S6 strains. The letters in the graph indicate that, in Tukey's multiple testing, there was no significant difference between groups with the same letter (p<0.05), while there was a significant difference between groups with different letters. [Figure 7]Figure 7 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple strains of fungal agents were provided. The upper row shows the fresh weight of the above-ground part of the seedlings 4 weeks after cultivation (3 weeks after provision), and the lower row shows the weight of the roots. In the figure, "mock" represents the untreated control group where no fungal agents were provided, "NB7+S5+S6" represents the group provided with NB7, S5, and S6 strains, and "NB7+R2A7" represents the group provided with NB7 and R2A7 strains. The letters in the graph indicate that, in Tukey's multiple testing, there was no significant difference between groups with the same letter (p<0.05), while there was a significant difference between groups with different letters. [Figure 8] Figure 8 is a graph showing the results of an investigation into the differences in rice seedling growth depending on whether or not multiple fungal strains were provided. The graph shows the fresh weight of the roots of the seedlings 4 weeks after cultivation (3 weeks after provision). In the figure, "mock" represents the untreated control group where no fungal strains were provided, and "4M" - "3M-4" represents the groups where fungal strains were provided in the combinations shown in Table 4. The letters in the graph indicate that, in Tukey's multiple testing, there was no significant difference between groups with the same letter (p<0.05), while there was a significant difference between groups with different letters. [Figure 9] Figure 9 is a graph showing the temperature (in °C) and humidity (in %) inside the outdoor glass enclosure during the test period of "Effect Confirmation Test under High Temperature (Fluctuating) Conditions (1)," from July 18 to August 22, 2024. [Figure 10]Figure 10 is a graph showing the results of an investigation into the differences in rice seedling growth under high-temperature (fluctuating) conditions, depending on whether or not multiple fungal strains and OsPep-3 were provided. The graph shows the fresh weight of the seedling roots 4 weeks after cultivation (3 weeks after provision). In the figure, "mock" represents the untreated control group where neither fungal strain nor OsPep-3 was provided, "NB7+OsPep" represents the group where NB7 strain and OsPep-3 were provided, "R2A7" represents the group where R2A7 strain was provided, "S5" represents the group where S5 strain was provided, "S6" represents the group where S6 strain was provided, and "NB7+R2A7+S5+S6" represents the group where NB7 strain, R2A7 strain, S5 strain, and S6 strain were provided. In the graph, the letters indicate that, in Tukey's multiple tests, there is no significant difference between items with the same letter (p<0.05), while there is a significant difference between items with different letters. [Figure 11] Figure 11 is a graph showing the temperature (in °C) and humidity (in %) inside the outdoor glass enclosure during the test period of "Effect Confirmation Test under High Temperature (Fluctuating) Conditions (2)," from August 29 to September 18, 2024. [Figure 12] Figure 12 is a graph showing the results of an investigation into the differences in rice seedling growth under quasi-steady-state conditions, depending on whether or not multiple strains of fungi and OsPep-3 were provided. The graph shows the fresh weight of rice roots 4 weeks after cultivation (3 weeks after provision). In the figure, "mock" represents the untreated control group where neither fungal strain nor OsPep-3 was provided, "NB7+OsPep" represents the group where NB7 strain and OsPep-3 were provided, and "2M-5", "3M-3", "3M-4", and "4M" represent the groups where the fungal strains were provided in the combinations shown in Table 4. "*" indicates a statistically significant difference with p<0.05 in the t-test, and "**" indicates a statistically significant difference with p<0.01 in the t-test. [Figure 13]FIG. 13 is a graph showing the results of examining the differences in the growth status of rice seedlings depending on the presence or absence of donation of a plurality of strains and OsPep-3 when cultivation is carried out under high-temperature (fluctuating) conditions. It shows the fresh weight of the roots of the seedlings 4 weeks after cultivation (3 weeks after donation). In the figure, "mock" is a non-treated control group where neither the strain nor OsPep-3 was donated, "NB7+OsPep" is a group where the NB7 strain and OsPep-3 were donated, and "2M-5", "3M-3", "3M-4", and "4M" respectively show groups where the strain was donated in the combinations shown in Table 4. "*" indicates that a significant difference was recognized by t-test with p < 0.05.

MODE FOR CARRYING OUT THE INVENTION

[0015] (Embodiment) The rice growth promoter of the present embodiment contains at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

[0016] The Sphingomonas microorganism contained in the rice growth promoter of this embodiment is at least one of the strains deposited under accession number NITE-BP-04206 (depositary: Patent Microorganism Depositary Center (NPMD), National Institute of Technology and Evaluation, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, deposit date: November 15, 2024) and NITE-BP-04207 (depositary: Patent Microorganism Depositary Center (NPMD), National Institute of Technology and Evaluation, address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, deposit date: November 15, 2024). In this specification, the strain deposited under accession number NITE-BP-04206 is referred to as "S5 strain," and the strain deposited under accession number NITE-BP-04207 is referred to as "S6 strain." "Containing at least one of S5 strain and S6 strain" may mean including S5 strain and not including S6 strain, including S6 strain and not including S5 strain, or including both S5 strain and S6 strain. The rice growth promoter may be, for example, a suspension obtained by suspending one or both of S5 strain and S6 strain in water. The suspension may further contain components such as nutrients necessary to keep the strain alive.

[0017] The rice growth promoter may further contain an Achromobacter microorganism deposited under accession number NITE-BP-03938 (depositary: National Institute of Technology and Evaluation Patent Microorganism Depository Center (NPMD), address: Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan, deposit date: July 7, 2023). In this specification, this strain will be referred to as "strain R2A7".

[0018] The rice growth promoter may further contain a Sphingomonas microorganism deposited under NITE-BP-03939 (depositary: National Institute of Technology and Evaluation Patent Microorganism Depository Center (NPMD), address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, deposit date: July 7, 2023). In this specification, this strain will be referred to as "NB7 strain".

[0019] The growing medium for rice in this embodiment includes at least one of the S5 strain and the S6 strain. The growing medium may further include the R2A7 strain or the NB7 strain. The growing medium may be an agar medium, a hydroponic solution, or soil. The growing medium may further include nutrients and other components necessary for growing rice seedlings.

[0020] The rice seeds in this embodiment may be seeds treated with the above-mentioned rice growth stimulant. "Treatment with a rice growth stimulant" means, for example, applying the rice growth stimulant to the seeds. Applying the rice growth stimulant to the seeds can be done, for example, by soaking the seeds in the rice growth stimulant for a certain period of time.

[0021] The rice seedlings in this embodiment may be seedlings grown in the above-mentioned rice growing medium. "Grown in the above-mentioned rice growing medium" may mean, for example, sowing rice seeds in the above-mentioned rice growing medium, allowing them to germinate and grow, or sowing rice seeds in another growing medium, raising the seedlings to a certain extent, and then transplanting them to the above-mentioned rice growing medium for further growth.

[0022] The rice cultivation method of this embodiment includes treating rice seeds or seedlings with at least one of the S5 strain and S6 strain. "Treating rice seeds" with at least one of the S5 strain and S6 strain may, for example, involve attaching at least one of the S5 strain and S6 strain to the seeds, sowing rice seeds in a culture medium containing at least one of the S5 strain and S6 strain, or dropping a suspension containing at least one of the S5 strain and S6 strain onto a culture medium on which rice seeds have been sown. Attaching at least one of the S5 strain and S6 strain to the seeds may, for example, involve dropping a suspension containing at least one of the S5 strain and S6 strain onto the seeds, or immersing the seeds in a suspension containing at least one of the S5 strain and S6 strain for a certain period of time. "Treating rice seedlings with at least one of the S5 and S6 strains" may mean, for example, planting rice seedlings in a culture medium containing at least one of the S5 and S6 strains, or dropping a suspension containing at least one of the S5 and S6 strains onto a culture medium in which rice seedlings have been planted.

[0023] According to the above-mentioned rice growing medium, rice growth promoter, rice seedlings, rice seeds, and rice cultivation method, the growth of rice seedlings is promoted. This promotes the establishment of seedlings immediately after transplanting, when they are most susceptible to environmental changes, stress, and disease-causing fungi during the rice cultivation period. Subsequently, rice growth in the field is stabilized, and yields are increased.

[0024] The rice growth promoter, rice growing medium, and rice cultivation method of this embodiment can be applied to rice seeds of paddy rice, that is, rice seeds for cultivation under flooded conditions in a paddy field. The same applies to rice seedlings. Conventionally, although mycorrhizal symbiosis can occur under flooded conditions (paddy rice), it is known that the symbiotic effect (promotion of rice growth and carbon-phosphorus exchange between rice and fungi) is reduced compared to conditions with low water levels. By applying strain S5 or S6, a high rice growth promotion effect can be obtained due to the symbiotic effect between rice seedlings and microorganisms.

[0025] (Examples) 1. Isolation and identification of strains S5, S6, and R2A7 1) Plant materials and cultivation conditions The rice cultivar Nipponbare (Oryza sativa L. cv. Nipponbare) was used as the wild type. After harvesting and drying, the seeds were stored at 4°C and deflowered immediately before use. For microbial analysis, Nipponbare seeds harvested from a greenhouse at Nara Institute of Science and Technology (25-30°C, 10 hours of light / 14 hours of darkness) and from field fields throughout Japan (Kyoto, Miyagi, and Shizuoka prefectures) were used. For seeds from Kyoto Prefecture, seeds from fertilized and unfertilized fields (where rice had been continuously cultivated without fertilizer for over 20 years) were harvested from adjacent fields where rice had been grown. Rice roots were sampled and used as the target for symbiotic microbial community investigation. The surface of the sampled rice was sterilized, and only symbiotic fungi residing inside the plants were targeted.

[0026] 2) Hydroponic cultivation of rice For sowing in the presence of seed microorganisms, detached seeds were immersed in a 0.25% GF Benlate wettable powder solution (Sumitomo Chemical, active ingredient: benomyl), a fungicide, and allowed to absorb water for 24 hours in the dark at 28°C. For completely sterilized sowing, the seeds were sterilized with 20% sodium hypochlorite for 1 hour, thoroughly washed with sterile water, and allowed to absorb water for 23 hours in the dark at 28°C. Table 1 shows the composition and final concentration of the hydroponic solution used in hydroponic cultivation.

[0027] [Table 1]

[0028] Hydroponic solution was placed in 50 mL tubes, and a horticultural pot bottom net was floated on the surface of the water. The hydroponic solution used was either phosphorus-sufficient (+Pi; 200 μmol / L) or phosphorus-deficient (-Pi; 20 μmol / L) KH2PO4. Five water-absorbing seeds were placed on top of the pot bottom net in each tube, and rice was cultivated with the above-ground parts remaining in the air and only the roots extending into the water. The tops of the tubes were sealed with plastic wrap, and the rice was cultivated in an artificial climate chamber (32°C, 14 hours of light / 10 hours of darkness) for 4 or 7 days.

[0029] 3) Inoculation test of bacterial strains Bacterial strains isolated from rice roots cultivated in paddy fields were used in inoculation experiments. After surface sterilization of the roots, tissue samples were cultured in R2A solid medium. Glycerol stocks of the isolated bacteria were inoculated into 2 mL of NB or R2A solid medium and cultured. After 1-2 days, the formed single colonies were inoculated into 4 mL of NB or R2A liquid medium and cultured with shaking at 28°C for approximately 24 hours (pre-culture). 100 μL of the culture solution was added to 5 mL of fresh medium and cultured overnight (main culture). After the main culture, the culture solution was centrifuged (2000 × g, 15 minutes, 20°C), the supernatant was discarded, and sterile water was added to prepare a bacterial suspension with an OD600 of 0.1.

[0030] The seeds used for inoculation were sterilized with 20% sodium hypochlorite for 1 hour. They were then immersed in sterile water for 2 days (with one water change during this time). For the growing pots, autoclaved plant culture boxes (75 x 75 x 100 mm, VWR) were stacked upside down. A growing agar medium was prepared by adding 9 g / L of agar to the hydroponic solution, and 100 mL of this was added to the plant boxes after autoclaving. After the agar had completely solidified, 9 seeds were sown in each box. The plants were then cultivated for 7 days in an artificial climate chamber (30°C, 14 hours light / 10 hours dark). After surface sterilization of the roots, the number of bacteria (CFU) contained within the tissue was measured. The roots were crushed with 10 mmol / L-MgCl2, and dilutions were prepared from the crushed solution by serial dilution. The dilutions were spread on R2A agar medium and cultured at 28°C for 3 days, after which the number of colonies formed (CFU) was measured.

[0031] 4) Meta-16s sequencing analysis Wild-type rice seeds treated with a fungicide were sown in hydroponic solution. Four days later, the roots were rinsed with sterile water, separated from the plant, and three or more roots were collected as a sample and flash-frozen with liquid nitrogen. The sample was freeze-disrupted, and microbial DNA was extracted using NucleoSpin® Soil (MACHERRY-NAGEL). Subsequently, the 16S-rRNA gene region of bacteria and archaea was amplified by touchdown PCR using KOD FXNeo (TOYOBO) and 515 forward and 806 reverse primers (known). The primers and PCR cycle were based on previously reported data (Edwards et al. 2015. Structure, variation, and assembly of the root-associated microbiomes of rice. Proc Natl Acad Sci 112:E911-E920. doi:10.1073 / pnas.1414592112). The target DNA band was excised by agarose gel electrophoresis and purified using a gel / PCR extraction kit (Fast Gene). In the following procedure, sample DNA was prepared according to the Illumina Miseq protocol (https: / / support.illumina.com / documents / documentation / chemistry_documentation / 16s / 16s-metagenomiclibrary-prep-guide-15044223-b.pdf), and paired-end sequencing was performed on MiSeq using the MiSeq Reagent Kit V3.

[0032] Sequences sequenced by Miseq were analyzed using the method described in a previous report (Utami et al. (2018) Phylogenetic diversity and single-cell genome analysis of "melainabacteria", anon-photosynthetic cyanobacterial group, in the termite gut. Microbes Environ 33:50-57. doi: 10.1264 / jsme2.ME17137). The DADA2 v1.10.0 package (Callahan et al. 2016. DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods 13:581-583. doi: 10.1038 / nmeth.3869.DADA2) was used for trimming and filtering, and the sequences were classified based on amplification-consequence variants (ASVs). The obtained ASV sequences were systematically classified based on homology of 80% or more using SINA v1.2.11 (Pruesse et al. 2012. SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28:1823-1829. doi:10.1093 / bioinformatics / bts252) and the database SILVA SSU Ref NR99 release 132 (Quast et al. 2012. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41: D590-D596. doi:10.1093 / nar / gks1219). Sequences derived from eukaryotes, mitochondria, and plastids, as well as sequences that could not be matched with database sequences, were excluded from the analysis.For the analysis of microbiota diversity, we used QIIME v.1.9.1 (Caporaso et al. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335-336. doi: 10.1038 / nmeth.f.303.QIIME) and the R packages vega and ggplot2. Although meta-16S-rRNA analysis includes bacterial and archaeal sequences, for convenience, we will refer to it as microbiota analysis, following common terminology.

[0033] 5) Analysis using bioinformatics Using bioinformatics, we performed machine learning on the microbial community data with the nucleotide sequences obtained above to identify symbiotic fungi that are important for the health and growth of rice plants.

[0034] 6) Isolation of symbiotic bacteria Using the method described above, symbiotic bacteria residing inside rice roots were isolated. More than 600 symbiotic bacterial strains were obtained and inoculated into rice seedlings to verify their plant growth-promoting effects. First, bacterial strain inoculation tests were conducted using method 3) above (primary screening), followed by inoculation tests using seedlings grown in soil (secondary screening). A brief explanation is as follows:

[0035] Seeds with hulls were used in the inoculation test after being immersed in a 0.25% GF Benlate wettable powder solution (Sumitomo Chemical, active ingredient: benomyl, used as a fungicide) in 70% ethanol for 1 minute, allowed to absorb water at room temperature for 1 day, and then allowed to absorb sterile water for 1 day. Seeds were directly sown in soil in seedling trays (Yanmar vegetable transplanter trays, 25cm square). The seedling trays were placed in plastic fruit packs (strawberry sales cases) and grown in a greenhouse (25-30°C, 10 hours light / 14 hours dark) for 30 days while being moistened with tap water. The soil used was a low-nutrient soil (50% soil) mixed with Kumiai Ube soil (Ube Industries): Akadama soil (Plantation Iwamoto; extra-fine grain): Kanuma soil in a volume ratio of 5:4:1 (soil:nutrient-poor soil = 1:1). Next, single colonies cultured under the isolation and culture conditions described above were cultured in liquid culture, diluted with sterile water to prepare a bacterial suspension with an OD600 of 0.1. This suspension was inoculated at the base of each seedling (1 plant) in one plot, 7 days after sowing, by 1 mL. In contrast, the uninoculated plot (control plot) was treated with the same amount of sterile water. Thirty days after sowing, the fresh weight of rice plants in both the uninoculated and inoculated plots was measured.

[0036] 7) Bacterial genome analysis DNA Sanger sequencing analysis of isolated bacterial strains Bacterial DNA was prepared from bacterial suspension cultured in liquid under the isolation and culture conditions described above. The 16s-rRNA gene (region from residue 27 to 907) of the isolated strain was amplified by touchdown PCR using KOD FX Neo (TOYOBO) and 27 forward and 907 reverse primers (Table 2). The target DNA band was excised by agarose gel electrophoresis and purified using a gel / PCR extraction kit (Fast Gene). The purified DNA was amplified in both directions by cycle PCR using Big Dye and primers, and then purified by ethanol precipitation for sequencing analysis. Based on BLAST analysis of the sequencing data, rough classification information of the isolated strains was obtained, revealing that strains S5 and S6 are species of the genus Sphingomonas, and strain R2A7 is a species of the genus Achromobacter.

[0037] [Table 2]

[0038] Draft genome analysis of isolated bacterial strains Bacteria were inoculated from a glycerol stock into 2 mL of R2A liquid medium and cultured with shaking at 28°C for approximately 24 hours (pre-culture). 100 μL of the culture solution was added to 5 mL of fresh medium and cultured overnight (main culture). 1.5 mL of bacterial turbidity was centrifuged (2000 × g, 15 min, 20°C), and genomic DNA was extracted from the precipitated cells using NucleoSpin® Micronial DNA (MACHERRY-NAGEL). The extracted DNA was subjected to draft genomic DNA sequencing analysis, and the full-length contig sequence was obtained by assembly using Illumina (short reads). Subsequently, a search for closely related species based on Average nucleotide identity (ANI) was conducted from bacterial species registered in NCBI. As a result, strain R2A7 is considered to be the same species as Achromobacter ruhlandii.

[0039] Strains S5 and S6 formed yellow colonies characteristic of the Sphingomonas genus. Furthermore, both strains S5 and S6 possessed some genes related to phosphate metabolism and the production of the plant hormone IAA.

[0040] Furthermore, while Achromobacter xylosidance, reported in Patent Document 2 above to promote plant growth, possesses nitrogen-fixing ability and the nitrogenase nifH gene, strain R2A7 did not possess nitrogenase. Therefore, it is possible that strain R2A7 promotes rice growth through a mechanism different from nitrogen fixation.

[0041] 2. Efficacy confirmation study using S5 strain and S6 strain alone 1) Preparation of the suspension i) Glycerol stocks of strain S5 were streaked onto R2A solid medium (9 cm plate) and cultured at 25°C for 3 days. The formed single colonies were inoculated into approximately 1-2 mL of R2A liquid medium and cultured with shaking at 28°C and 180 rpm for approximately 24 hours (pre-culture). An appropriate amount (e.g., 100 μL) of this culture solution was added to 10 mL of fresh R2A medium and cultured with shaking at 28°C and 180 rpm for approximately 24 hours (main culture). After the main culture, the culture solution was centrifuged (2000 × g, 10 min, room temperature), the supernatant was discarded, and the precipitated pellet (bacterial cells) was collected. The collected bacterial cells were suspended and washed with an equal volume of sterile MilliQ® water used during culture, and then centrifuged again (2000 × g, 10 min, 20°C) to collect the remaining cells. This process was repeated, and sterile water was added to the recovered bacterial cells to prepare a suspension of strain S5 so that the OD600 was 0.1.

[0042] ii) A bacterial suspension was prepared using strain S6 in the same manner as in 2.1)i) above.

[0043] 2) Cultivation i) Seeds of the Japanese variety "Nihonbare" were used. Dehulled seeds were immersed in 70% ethanol for 1 minute, then immersed in 20% sodium hypochlorite and shaken for about 1 hour to completely sterilize them. After sterilization, the seeds were thoroughly washed with sterile water, immersed in sterile water, and allowed to absorb water while being shaken for 2 days to induce germination.

[0044] A hydroponic solution with the composition shown in Table 3 was adjusted to pH 6.4. 100 mL of this hydroponic solution was poured into a plant culture box (65 × 65 × 100 mm), and a horticultural pot bottom net was floated on the water surface. Nine germinated seeds were placed on top of the pot bottom net, 500 μL of a suspension of strain S5 was dropped onto them, and the plant was then covered with the plant box and cultivated for 7 days in an artificial climate chamber (28°C, 14 hours of light / 10 hours of darkness).

[0045] [Table 3]

[0046] ii) Instead of using a bacterial suspension of strain S5, a bacterial suspension of strain S6 was used, and cultivation and measurement of seedling weight were carried out in the same manner as in 2.2)i) above.

[0047] iii) For comparison, cultivation and measurement of seedling weight were carried out in the same manner as in 2.2)i) above, except that the fungal suspension was not added.

[0048] 3) Results The results are shown in Figure 1. In the upper part of Figure 1, the measured fresh weight of the above-ground part of each seedling is shown as dots, and the median for each group is shown as a bar. In the lower part of Figure 1, the measured fresh weight of the roots of each seedling is shown as dots, and the median for each group is shown as a bar. From Figure 1, the groups that were given S5 and S6 plants had higher above-ground weight and root weight compared to the group that was not given S5 and S6 plants (mock). It is thought that providing S5 and S6 plants individually promotes rice growth.

[0049] 3. Efficacy confirmation study by co-vaccination (1) 1) Preparation of bacterial suspension Bacterial suspensions of strains S5 and S6 were prepared in the same manner as described in 2.1) above. Similarly, bacterial suspensions were prepared using strains NB7 and R2A7. Equal volumes of the four bacterial suspensions were mixed together to create a four-component mixed bacterial solution.

[0050] 2) Cultivation i) Seeds of the Japanese 'Nipponbare' variety were used. Dehulled seeds were immersed in a 0.25% GF Benlate wettable powder solution (Sumitomo Chemical, active ingredient: benomyl), a fungicide, and surface-sterilized by shaking at 80-100 rpm at room temperature for about 24 hours. After sterilization, the Benlate solution was discarded, and the seeds were rinsed 3-5 times with sterile MilliQ water. After rinsing, the seeds were immersed in sterile MilliQ® water and allowed to absorb water while shaking at 80-100 rpm at room temperature for about 24 hours to induce germination.

[0051] 25mm square cells were filled to the brim with growing medium. The growing medium used was a nutrient-poor mixture of Kumiai Ube Growing Medium No. 2, Akadama soil (very fine grain), and Kanuma soil in a mass ratio of 5:4:1. The growing medium was moistened by bottom watering. Using tweezers, holes were dug to bury the seeds, and seeds with a pigeon chest shape were selected and sown one by one. The plants were then cultivated for one week in an artificial climate chamber (25-30°C, 14 hours of light / 10 hours of darkness). After that, 1 ml of a mixed bacterial solution of four types was dripped onto the surface of the growing medium per cell, and the plants were cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured four weeks after cultivation (three weeks after the dripping of the bacterial solution).

[0052] ii) For comparison, cultivation and measurement of seedling weight were carried out in the same manner as in 3.2)i) above, except that the mixed bacterial solution of the four types was not dropped onto the growing medium.

[0053] 3) Results The results are shown in Figure 2. The upper part of Figure 2 shows the median fresh weight of the above-ground parts for each group as a bar graph. The lower part of Figure 2 shows the median fresh weight of the roots for each group as a bar graph. From Figure 2, it can be seen that the groups supplied with the four types of fungi had higher weights of both above-ground parts and roots compared to the groups that were not supplied. It is thought that supplying rice seedlings with strains S5, S6, NB7, and R2A7 promotes rice growth.

[0054] 4. Efficacy confirmation study by co-vaccination (2) 1) Preparation of bacterial suspension A mixture of four types of bacteria was prepared in the same manner as described in 3.1) above.

[0055] 2) Preparation of OsPep aqueous solution An aqueous solution of 1 mmol / L OsPep-3 was prepared. The amino acid sequence of the OsPep-3 used and its acquisition method are described, for example, in Patent Document 1 and previously reported ( This is publicly known, as described in Tomonori Shinya et al. Integration of danger peptide signals with herbivore-associated molecular pattern signaling amplifies anti-herbivore defense responses in rice (The Plant Journal (2018) 94, 626-637 https: / / doi.org / 10.1111 / tpj.13883).

[0056] 3) Cultivation i) Seeds of *Nipponbare* were sown in growing medium in the same manner as in 3.2) above, and cultivated for one week in an artificial climate chamber (25-30°C, 14 hours of light / 10 hours of darkness). After that, 1 ml of a mixed bacterial solution of four types was dropped onto the surface of the growing medium per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0057] ii) Seeds of Nipponbare were sown in growing medium in the same manner as in 3.2) above, and cultivated for one week in an artificial climate chamber (25-30°C, 14 hours of light / 10 hours of darkness). After that, 100 μl of OsPep aqueous solution was dropped onto the surface of the growing medium per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0058] iii) Seeds of Nipponbare were sown in growing medium in the same manner as in 3.2) above, and cultivated for one week in an artificial climate chamber (25-30°C, 14 hours of light / 10 hours of darkness). Then, 1 ml of a mixed bacterial solution of four types and 100 μl of OsPep aqueous solution were dropped onto the surface of the growing medium per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0059] iv) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 4.3)i) above, except that neither the bacterial suspension nor the OsPep aqueous solution was dropped onto the growing medium.

[0060] 4) Results The results are shown in Figure 3. For each group, n≧8 was used, and the measured fresh root weight for each seedling is shown as dots, while the median for each group is shown as a bar graph. In the groups supplied with OsPep-3, and in the groups supplied with both OsPep-3 and the four fungal strains, the root weight was equivalent to or slightly greater than that of the control group (mock). In the groups supplied with NB7 strain, R2A7 strain, S5 strain, and S6 strain, the root weight increased even further. Based on these results, it is considered that supplying NB7 strain, R2A7 strain, S5 strain, and S6 strain can provide a growth-promoting effect on rice that exceeds that of OsPep-3 administration.

[0061] 5. Efficacy confirmation study by co-vaccination (3) Instead of using Nihonbare seeds, we used seeds of Hinohikari, a variety recommended for cultivation, and performed cultivation and measured the weight of seedlings in the same manner as described in 3. above.

[0062] The results are shown in Figure 4. In the upper part of Figure 4, the measured fresh weight of the above-ground part of each seedling is shown as dots, and the median for each group is shown as a bar graph. In the lower part of Figure 4, the measured fresh weight of the roots of each seedling is shown as dots, and the median for each group is shown as a bar graph. From Figure 4, it can be seen that the groups that were supplied with the four fungal strains had higher weights of both the above-ground part and the roots compared to the groups that were not supplied. It is thought that in Hinohikari rice, supplying NB7 strain, R2A7 strain, S5 strain, and S6 strain can also promote rice growth.

[0063] 6. Efficacy confirmation study using co-vaccination (4) 1) Preparation of bacterial suspension and OsPep aqueous solution Suspensions of bacterial strains NB7, R2A7, S5, and S6 were prepared in the same manner as described in 3.1) above. Equal volumes of the four bacterial suspensions were mixed to make a four-component mixed bacterial solution. Equal volumes of the bacterial suspensions of R2A7, S5, and S6 were also mixed to make a three-component mixed bacterial solution. In addition, an OsPep aqueous solution was prepared in the same manner as described in 4.2) above.

[0064] 2) Cultivation i) Seeds of *Nipponbare* were sown in potting soil in the same manner as in 3.2) above, and cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of a bacterial suspension of strain NB7 and 100 μL of OsPep aqueous solution were dropped onto the surface of the potting soil per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0065] ii) Seeds of Nipponbare were sown in potting soil in the same manner as in 3.2) above, and cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of a mixed bacterial solution of three types was dropped onto the surface of the potting soil per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0066] iii) Instead of the three-component mixed bacterial solution, a four-component mixed bacterial solution was used, and cultivation and measurement of seedling weight were carried out in the same manner as in 6.2)ii) above.

[0067] iv) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 6.2)i) above, except that neither the bacterial suspension nor the OsPep aqueous solution was dropped onto the growing medium.

[0068] 3) Results The results are shown in Figure 5. The upper part of Figure 5 shows the median fresh weight of the above-ground parts for each group as a bar graph. The lower part of Figure 5 shows the median fresh weight of the roots for each group as a bar graph. From Figure 5, it can be seen that in the group given four strains (NB7, R2A7, S5, and S6), the group given three strains (R2A7, S5, and S6), and the group given NB7 and OsPep-3, both the above-ground weight and root weight were higher compared to the control group (mock). It is thought that providing three strains (R2A7, S5, and S6) or four strains (NB7, R2A7, S5, and S6) can produce a growth-promoting effect equivalent to or greater than that of the combination of NB7 and OsPep-3.

[0069] 7. Efficacy confirmation study by co-vaccination (5) 1) Preparation of bacterial suspension Suspensions of NB7 strain, R2A7 strain, S5 strain, and S6 strain were prepared in the same manner as described in 3.1) above. Equal volumes of the four bacterial suspensions were mixed to make a four-component mixed bacterial solution. Equal volumes of the R2A7 strain, S5 strain, and S6 strain suspensions were mixed to make a three-component mixed bacterial solution. Equal volumes of the S5 strain and S6 strain suspensions were mixed to make a two-component mixed bacterial solution.

[0070] 2) Cultivation i) Seeds of *Nipponbare* were sown in potting soil in the same manner as in 3.2) above, and cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of a mixed bacterial solution of four types was dropped onto the surface of the potting soil per cell, and cultivated for another three weeks. The fresh weight of the above-ground parts and roots of the seedlings was measured after four weeks of cultivation.

[0071] ii) Instead of the four-component mixed bacterial solution, a three-component mixed bacterial solution and a two-component mixed bacterial solution were used, and cultivation and measurement of seedling weight were carried out in the same manner as in 7.2)i) above.

[0072] iii) For comparison, cultivation and measurement of seedling weight were carried out in the same manner as in 7.2)i) above, except that the fungal suspension was not dropped onto the growing medium.

[0073] 3) Results The results are shown in Figure 6. In the upper part of Figure 6, the measured fresh weight of the above-ground part of each seedling is shown as dots, and the median for each group is shown as a bar. In the lower part of Figure 6, the measured fresh weight of the roots of each seedling is shown as dots, and the median for each group is shown as a bar. From Figure 6, in the group provided with four varieties (NB7, R2A7, S5, S6), the group provided with three varieties (NB7, S5, S6), and the group provided with two varieties (S5, S6), both the above-ground weight and root weight were higher compared to the control group (mock), confirming the effect of promoting rice growth.

[0074] 8. Efficacy confirmation study using co-vaccination (6) 1) Preparation of bacterial suspension Suspensions of bacterial strains NB7, R2A7, S5, and S6 were prepared in the same manner as described in 3.1) above. Equal volumes of the bacterial suspensions of NB7, S5, and S6 were mixed to make a three-component mixed bacterial solution. Equal volumes of the bacterial suspensions of NB7 and R2A7 were mixed to make a two-component mixed bacterial solution.

[0075] 2) Cultivation Using the three-component mixed bacterial solution and the two-component mixed bacterial solution prepared in 8.1) above, cultivation and measurement of seedling weight were carried out in the same manner as in 7.2)i). For comparison, cultivation and measurement of seedling weight were also carried out in the same manner as in 7.2)i), except that the bacterial suspension was not dropped onto the growing medium.

[0076] 3) Results The results are shown in Figure 7. In the upper part of Figure 7, the measured fresh weight of the above-ground part for each seedling is shown as dots, and the median for each group is shown as a bar. In the lower part of Figure 7, the measured fresh weight of the roots for each seedling is shown as dots, and the median for each group is shown as a bar. From Figure 6, in the group provided with three types of plants (NB7, S5, and S6) and the group provided with two types of plants (NB7 and R2A7), both the above-ground weight and root weight were higher compared to the control group (mock), confirming the effect of promoting rice growth.

[0077] 9. Efficacy confirmation trials using co-vaccination (7) 1) Preparation of bacterial suspension Suspensions of bacterial strains NB7, R2A7, S5, and S6 were prepared in the same manner as described in 3.1) above. Equal volumes of the bacterial suspensions were mixed together in the combinations shown in Table 4 to prepare mixed bacterial solutions.

[0078] [Table 4]

[0079] 2) Cultivation i) Koshihikari rice seeds were used instead of Nipponbare rice seeds. The seeds were sown in the growing medium in the same manner as in 3.2) above, and then cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of mixed bacterial solution was dropped onto the surface of the growing medium per cell, and the plants were cultivated for another three weeks. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0080] ii) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 9.2)i) above, except that the fungal suspension was not mixed into the growing medium.

[0081] 3) Results The results are shown in Figure 8. In Figure 8, the measured values ​​for the fresh root weight of each seedling are shown as dots, and the median values ​​for each group are shown as bar graphs. From Figure 8, it can be seen that in all groups provided with multiple strains of fungal agents, the root weight was greater compared to the control group (mock), and a significant growth-promoting effect on rice was confirmed, particularly in the 4M, 2M-1, 2M-3, 2M-4, 3M-1, 3M-3, and 3M-4 groups.

[0082] 10. Effectiveness confirmation test under high temperature (fluctuating) conditions (1) A cultivation experiment was conducted from July 18 to August 22, 2024, as described below. Figure 9 shows graphs of the temperature and humidity inside the outdoor glass enclosure during this period.

[0083] 1) Preparation of bacterial suspension and OsPep aqueous solution Suspensions of bacterial strains NB7, R2A7, S5, and S6 were prepared in the same manner as described in 3.1) above. Equal volumes of the four bacterial suspensions were mixed to form a four-component mixed bacterial solution. In addition, an OsPep aqueous solution was prepared in the same manner as described in 4.2) above.

[0084] 2) Cultivation i) Koshihikari seeds were sown in growing medium in the same manner as in 9.2)i) above, and then cultivated for one week in an outdoor glasshouse under conditions without temperature control or supplemental lighting. After that, 1 ml of NB7 strain suspension and 100 μL of OsPep aqueous solution were dropped onto the surface of the growing medium per cell, and the plants were cultivated for another three weeks. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0085] ii) After sowing Koshihikari seeds in the same manner as in 9.2)i) above, the seedlings were cultivated for one week in an outdoor glasshouse under conditions without temperature control or supplemental lighting. Subsequently, 1 ml of a suspension of the R2A7 strain was dropped onto the surface of the growing medium per cell, and the seedlings were cultivated for another three weeks. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0086] iii) Instead of the bacterial suspension of strain R2A7, the bacterial suspension of strain S5, the bacterial suspension of strain S6, and a mixed bacterial solution of the four strains were used, and cultivation was carried out in the same manner as in 10.2)ii) above, and the fresh weight of the roots of the seedlings was measured after 4 weeks of cultivation.

[0087] iv) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 10.2)i) above, except that neither the bacterial suspension nor the OsPep aqueous solution was dropped onto the growing medium.

[0088] 3) Results The results are shown in Figure 10. In Figure 10, the measured fresh root weight for each seedling is shown as dots, and the median value for each group is shown as a bar graph. In the group given NB7 strain and OsPep-3, the root weight was smaller compared to the control group (mock), indicating inhibition of rice growth due to high temperature. In the groups given R2A7 strain, S5 strain, and S6 strain individually, the root weight was similar to or slightly greater than that of the control group (mock). These strains are thought to be able to mitigate the inhibition of rice growth due to high temperature, even when administered individually. In the group given all four strains (NB7 strain, R2A7 strain, S5 strain, and S6 strain), the root weight was greater than that of the control group (mock). It is thought that providing these four strains can promote rice growth even under high temperature conditions.

[0089] 11. Effectiveness confirmation test under high temperature (fluctuating) conditions (2) A cultivation experiment was conducted between August 29 and September 18, 2024, as described below. Figure 11 shows graphs of the temperature and humidity inside the outdoor glass enclosure during this period.

[0090] 1) Preparation of bacterial suspension and OsPep aqueous solution Mixed bacterial solutions were prepared in the same manner as in 9.1) above, as shown in Table 4 for combinations 2M-5, 3M-3, 3M-4, and 4M. OsPep aqueous solutions were also prepared in the same manner as in 4.2) above.

[0091] 2) Cultivation under quasi-steady state i) Koshihikari seeds were sown in potting soil in the same manner as in 9.2)i) above, and cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of NB7 strain suspension and 100 μL of OsPep aqueous solution were dropped onto the surface of the potting soil per cell, and cultivated for another three weeks. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0092] ii) Koshihikari seeds were sown in potting soil in the same manner as in 9.2)i) above, and cultivated in a general greenhouse at 28°C under natural light + supplemental light (8:00-18:00) for one week. After that, 1 ml of 2M-5 fungal suspension per cell was dropped onto the surface of the potting soil, and the plants were cultivated for another three weeks. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0093] iii) Instead of the 2M-5 bacterial suspension, 3M-3, 3M-4, and 4M bacterial suspensions were used, respectively, and cultivation was carried out in the same manner as in 10.2)ii) above. The fresh weight of the roots of the seedlings was measured after 4 weeks of cultivation.

[0094] iv) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 10.2)i) above, except that neither the bacterial suspension nor the OsPep aqueous solution was dropped onto the growing medium.

[0095] 3) Cultivation under high temperature (fluctuating) conditions i) Koshihikari seeds were sown in potting soil in the same manner as in 9.2)i) above, and cultivated in a general greenhouse at 28°C with natural light + supplemental light (8:00-18:00) for one week. Then, 1 ml of NB7 strain suspension and 100 μL of OsPep aqueous solution were dropped onto the surface of the potting soil per cell, and the plants were moved to an outdoor glasshouse the day after inoculation and cultivated for a further three weeks under conditions without temperature control or supplemental light. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0096] ii) Koshihikari seeds were sown in growing medium in the same manner as in 9.2)i) above, and cultivated in a general greenhouse at 28°C with natural light + supplemental light (8:00-18:00) for one week. Then, 1 ml of 2M-5 fungal suspension per cell was dropped onto the surface of the growing medium, and the next day the cells were moved to an outdoor glasshouse where they were cultivated for a further three weeks under conditions without temperature control or supplemental light. The fresh weight of the roots of the seedlings was measured after four weeks of cultivation.

[0097] iii) Instead of the 2M-5 bacterial suspension, 3M-3, 3M-4, and 4M bacterial suspensions were used, respectively, and cultivation was carried out in the same manner as in 10.3)ii) above. The fresh weight of the roots of the seedlings was measured after 4 weeks of cultivation.

[0098] iv) For comparison, cultivation and measurement of seedling weights were carried out in the same manner as in 10.3)i) above, except that neither the bacterial suspension nor the OsPep aqueous solution was dropped onto the growing medium.

[0099] 4) Results The results under quasi-steady-state conditions are shown in Figure 12, and the results under high-temperature (fluctuating) conditions are shown in Figure 13. In Figures 12 and 13, the measured values ​​of the fresh root weight for each seedling are shown as dots, and the median values ​​for each group are shown as bar graphs. From Figure 12, under quasi-steady-state conditions, the root weight was higher in both the group supplied with NB7 strain and OsPep-3, and the groups supplied with the combinations of strains shown in Table 4 (2M-5, 3M-3, 3M-4, 4M) compared to the control group (mock). It is thought that, under conditions that are not extremely high in temperature, rice growth promotion can be achieved by supplying a combination of several strains from among NB7 strain, R2A7 strain, S5 strain, and S6 strain.

[0100] Under high-temperature (fluctuating) conditions, the group supplied with NB7 strain and OsPep-3 had smaller root weights compared to the control group (mock), indicating inhibition of rice growth due to high temperature. In the groups supplied with the strain combinations shown in 2M-5, 3M-3, and 3M-4, the root weight was similar to or slightly greater than that of the control group (mock). It is thought that supplying a combination of several strains from among NB7, R2A7, S5, and S6 can mitigate the inhibition of rice growth due to high temperature. In the group supplied with the strain combination shown in 4M, the root weight was greater than that of the control group (mock). It is thought that supplying NB7, R2A7, S5, and S6 strains can promote rice growth even under high-temperature conditions.

[0101] In the efficacy verification test described above, rice seedlings were treated with the fungal strain to confirm its growth-promoting effect. However, previous studies of the same type have not shown a significant difference in effect between treating seeds and seedlings with the fungal strain. Therefore, it is thought that treating rice seeds with the fungal strain would also yield a similar growth-promoting effect.

[0102] Furthermore, in the efficacy confirmation tests using the S5 and S6 strains individually as the growing medium for the rice plants, hydroponic solution was used. In the efficacy confirmation tests using co-inoculation (1)-(7) and the efficacy confirmation tests under high temperature (fluctuating) conditions (1)(2), soil was used. However, since no significant difference in efficacy due to the difference in growing medium was observed in previous studies of the same type, it is thought that a similar growth-promoting effect can be obtained regardless of the type of growing medium.

[0103] In the efficacy testing described above, when multiple bacterial strains were provided, the suspensions of the multiple strains were mixed and added dropwise. However, it is believed that a similar growth-promoting effect can be obtained even if the suspensions of each strain are added separately without mixing. In that case, whether the timing of adding the suspensions of the multiple strains is simultaneous or slightly staggered, as long as multiple bacterial strains ultimately coexist in the culture medium, a similar growth-promoting effect can be obtained.

Claims

1. A rice growth promoter comprising at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

2. A rice growth promoter according to claim 1, Further containing Achromobacter microorganisms deposited under accession number NITE-BP-03938, A growth stimulant for rice plants.

3. A rice growth promoter according to claim 2, Further including microorganisms of the genus Sphingomonas deposited under accession number NITE-BP-03939, A growth stimulant for rice plants.

4. A growing medium for rice, comprising a rice growth promoter according to any one of claims 1 to 3.

5. Rice seeds treated with a rice growth promoter according to any one of claims 1 to 3.

6. Rice seedlings grown in the culture medium described in claim 4.

7. Rice seedling according to claim 3, The aforementioned rice is paddy rice. Rice seedlings.

8. A method for cultivating rice, comprising treating rice seeds or seedlings with at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04206 and at least one of the Sphingomonas microorganisms deposited under accession number NITE-BP-04207.

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