Rice growing medium, rice seedlings, rice growth promoter, and rice cultivation method.

Abstract

It promotes the growth of rice. [Solution] The culture medium for growing rice contains Achromobacter microorganisms deposited under accession number NITE-BP-03938 and zinc. The rice growth promoter contains Achromobacter microorganisms deposited under accession number NITE-BP-03938 and zinc. The method for cultivating rice involves treating Achromobacter microorganisms deposited under accession number NITE-BP-03938 with a zinc-containing solution, and then inoculating rice seeds or seedlings with the solution.

Description

Technical Field

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

Background Art

[0002] For example, in order to improve the growth characteristics of rice crops such as germination, sprouting, establishment, and growth rate, an oxygen generator selected from magnesium peroxide (MgO2), strontium peroxide (SrO2), zinc peroxide (ZnO2), and calcium peroxide (CaO2), a seed mass increasing agent selected from iron (Fe), quartz sand, barium sulfate, calcium carbonate, zinc oxide, and iron oxide (Fe2O3), and a method including treating rice seeds with a rice seed treatment composition containing a water-insoluble binder, sowing the treated seeds, and growing a rice crop has been proposed (see Patent Document 1).

[0003] Also, a method of inoculating plants with a preparation containing an endophytic strain such as Achromobacter xylosidans has been proposed for promoting the growth of plants such as rice and increasing resistance to biotic / abiotic stresses (see Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] There was room for improvement in the above methods for promoting the growth of plants.

Means for Solving the Problems

[0006] The technologies disclosed herein can be implemented, for example, in the following forms: (1) The culture medium for growing rice disclosed herein comprises Achromobacter microorganisms deposited under accession number NITE-BP-03938 and zinc.

[0007] (2) The rice seedlings disclosed herein are seedlings grown in the culture medium described in (1) above.

[0008] (3) The rice seedlings mentioned in (2) above may be paddy rice seedlings.

[0009] (4) The rice growth promoter disclosed herein comprises an Achromobacter microorganism deposited under accession number NITE-BP-03938, and zinc.

[0010] (5) The method of cultivating rice disclosed herein involves treating Achromobacter microorganisms deposited under accession number NITE-BP-03938 with a zinc-containing solution and then inoculating them into rice seeds or seedlings.

[0011] The technologies disclosed herein can be implemented in various forms, for example, in the form of a culture medium for rice seedlings, a rice growth promoter, a method for producing the same, and rice seedlings using these culture media or rice growth promoters. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a graph showing the results of an investigation into the differences in rice seedling growth depending on the concentration of zinc in the culture medium and whether or not the R2A7 strain was 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 phosphorus (P) content per 1 mg of above-ground part. In the figure, "*" indicates that a significant difference was found with p<0.05 in the t-test, and "***" indicates that a significant difference was found with p<0.001 in the t-test. [Figure 2] Figure 2 is a graph showing the results of an investigation into the differences in rice seedling growth depending on the concentration of zinc in the culture medium and whether or not the R2A7 strain was 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 fresh weight of the roots. In the figure, "*" indicates a significant difference with p<0.05 in the t-test, "**" indicates a significant difference with p<0.01 in the t-test, and "***" indicates a significant difference with p<0.001 in the t-test. [Figure 3] Figure 3 is a graph showing the results of an investigation into the differences in rice seedling growth depending on the concentration of zinc in the culture medium and whether or not the R2A7 strain was provided (+) or not (-). 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 fresh weight of the roots. In the figure, "*" indicates a significant difference with p<0.05 in the t-test, "**" indicates a significant difference with p<0.01 in the t-test, and "***" indicates a significant difference with p<0.001 in the t-test. [Figure 4] Figure 4 is a graph showing the results of an investigation into the differences in rice seedling growth due to differences in the preparation conditions of the R2A7 strain suspension added to zinc-deficient agar medium. The upper panel shows the fresh weight of the above-ground part of the seedlings after 7 days of cultivation, and the lower panel shows the fresh weight of the roots. In the figure, "mock" represents the untreated control group that was not given the R2A7 strain suspension, and M1-M4 represents the groups that were given the R2A7 strain suspension prepared under conditions M1-M4, respectively. "*" indicates that a statistically significant difference was observed with p<0.05 in the t-test. [Figure 5] Figure 5 is a graph showing the results of an investigation into the differences in rice seedling growth due to differences in the preparation conditions of the R2A7 strain suspension added to zinc-containing agar medium. The upper panel shows the fresh weight of the above-ground part of the seedlings after 7 days of cultivation, and the lower panel shows the fresh weight of the roots. In the figure, "mock" represents the untreated control group that was not provided with the R2A7 strain suspension, and M1-M4 represents the groups that were provided with the R2A7 strain suspension prepared under conditions M1-M4, respectively. "*" indicates that a significant difference was found with p<0.05 in the t-test, and "**" indicates that a significant difference was found with p<0.01 in the t-test. [Modes for carrying out the invention]

[0013] (Embodiment) The culture medium for growing rice in this embodiment contains Achromobacter microorganisms deposited under accession number NITE-BP-03938, and zinc.

[0014] The Achromobacter microorganism contained in the culture medium of this embodiment is a strain deposited under accession number NITE-BP-03938 (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: July 7, 2023). In this specification, this strain is referred to as "strain R2A7".

[0015] The culture medium may be an agar medium, a hydroponic solution, or soil. Zinc may be included as a water-soluble zinc compound, for example, as zinc sulfate (ZnSO4) or zinc chloride (ZnCl2). Alternatively, zinc may be included as a lysate of zinc-containing biological cells. In addition to zinc, the culture medium may contain other components necessary for growing rice seedlings.

[0016] The rice growth promoter of this embodiment comprises the R2A7 strain and zinc. The rice growth promoter may be, for example, a suspension obtained by suspending the R2A7 strain in water containing zinc. The zinc may be included as a water-soluble zinc compound, for example, as zinc sulfate (ZnSO4) or zinc chloride (ZnCl2). Alternatively, it may be included as a lysate of biologically derived cells containing zinc. In addition to zinc, the suspension may contain other components such as nutrients necessary to keep the R2A7 strain alive.

[0017] The rice seedlings of this embodiment may be seedlings grown in the above-mentioned medium for growing rice. "Growing in the medium for growing rice" may mean, for example, sowing rice seeds in the above-mentioned medium for growing rice, germinating them, and growing them, or sowing rice seeds in another medium, raising seedlings to a certain extent, and then transplanting them to the above-mentioned medium for growing rice and growing them.

[0018] The rice cultivation method of this embodiment includes treating the R2A7 strain with a solution containing zinc and then inoculating rice seeds or seedlings. The solution containing zinc may be an aqueous solution obtained by dissolving a zinc compound soluble in water in water, for example, an aqueous solution of zinc sulfate (ZnSO4) or zinc chloride (ZnCl2). Alternatively, the solution containing zinc may be a solution containing a disrupted product of zinc-containing biological cells. "Treating with a solution containing zinc" may mean, for example, adding a solution containing zinc to the cells obtained by centrifuging the culture solution of the R2A7 strain and holding it for a predetermined time, or adding a solution containing zinc to the culture solution after culturing the R2A7 strain for a certain time and holding it for a predetermined time. "Inoculating the R2A7 strain into rice seeds" may mean attaching the R2A7 strain to the seeds, sowing rice seeds in a medium added with the R2A7 strain, or dropping a suspension of the R2A7 strain onto the medium in which rice seeds are sown. Attaching the R2A7 strain to the seeds may mean, for example, dropping a suspension of the R2A7 strain onto the seeds or soaking the seeds in a suspension of the R2A7 strain for a certain time. "Inoculating the R2A7 strain into rice seedlings" may mean, for example, planting rice seedlings in a medium added with the R2A7 strain or dropping a suspension of the R2A7 strain onto the medium in which rice seedlings are planted.

[0019] According to the above-mentioned medium for growing rice, rice growth promoter, and rice cultivation method, the growth of rice seedlings is promoted. As a result, the survival of the seedlings immediately after transplanting, which is most susceptible to environmental changes, stress, and pathogenic bacteria during the rice cultivation period, is promoted, and the subsequent growth of rice is stabilized in the field, and the yield also increases.

[0020] The medium for cultivating rice, the rice growth promoter, and the seeds or seedlings of rice to which the rice cultivation method of this embodiment is applied may be seeds or seedlings of paddy rice, that is, seeds or seedlings of rice for cultivation under flooded conditions in paddy fields. Conventionally, although mycorrhizal symbiosis can occur even under flooded conditions (paddy rice), it is known that the symbiotic effect (promotion of rice growth and carbon-phosphorus exchange amount between rice and fungi) decreases compared with the condition of less water volume. By using the R2A7 strain and zinc in combination, a high rice growth promoting effect due to the symbiotic effect between rice seedlings and the R2A7 strain can be obtained.

[0021] (Example) 1. Isolation and identification of the R2A7 strain 1) Plant materials and culture conditions Oryza sativa L. cv. Nipponbare, a rice cultivar, was used as the wild type. After harvesting and drying, it was stored at 4°C and dehulled immediately before use. For mycoflora analysis, seeds of Nipponbare harvested in the greenhouse of Nara Institute of Science and Technology (25 - 30°C, light period 10 hours / dark period 14 hours) and field plots in various parts of Japan (Kyoto Prefecture, Miyagi Prefecture, Shizuoka Prefecture) were used. Among the seeds produced in Kyoto Prefecture, those harvested from adjacent fields in fertilized fields and unfertilized fields (continuous cultivation of rice without fertilization for 20 years or more) were used. The roots of rice were sampled and used as the object of investigation of the symbiotic mycoflora. The surface of the sampled rice was sterilized, and only the symbiotic fungi inside the plant were targeted.

[0022] 2. Hydroponic cultivation of rice Sowing in the presence of seed microorganisms was carried out by immersing the dehulled seeds in a 0.25% GF benomyl wettable powder solution (Sumitomo Chemical, active ingredient: benomyl), a fungicide, and allowing them to absorb water at 28°C in a 24-hour dark period. For completely sterilized sowing, after sterilizing with 20% sodium hypochlorite for 1 hour, they were thoroughly washed with sterilized water and allowed to absorb water at 28°C in a 23-hour dark period. Table 1 shows the composition and final concentration of the hydroponic solution used for hydroponic cultivation.

[0023] [Table 1]

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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 the previously published 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.

[0028] 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.

[0029] 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.

[0030] 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:

[0031] 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.

[0032] 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 strain was obtained, and it was found that strain R2A7 is a species of the genus Achromobacter.

[0033] [Table 2]

[0034] 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.

[0035] 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.

[0036] 2. Efficacy confirmation test of R2A7 strain (1) 1) Preparation of a suspension of the R2A7 strain Isolated bacteria were inoculated from glycerol stocks onto R2A solid medium and cultured. After 1-2 days, the formed single colonies were inoculated into 4 mL of R2A liquid medium and cultured with shaking at 28°C for approximately 24 hours (pre-culture). 1 mL of this culture solution was added to 4 mL of fresh medium and cultured overnight (main culture). After the main culture, the culture solution was centrifuged (2000 × g, 15 min, 20°C), the supernatant was discarded, and the precipitated pellet (bacterial cells) was collected. The collected bacterial cells were suspended and washed with 4 mL of sterile water, and then centrifuged again (2000 × g, 10 min, 20°C) to collect the cells. This washing process was repeated twice, and sterile water was added to the collected bacterial cells to prepare a bacterial suspension with an OD600 of 0.1.

[0037] 2) Cultivation The seeds of the Japanese 'Nipponbare' variety were sterilized with 20% sodium hypochlorite for 1 hour, then immersed in sterile water for 2 days to allow them to absorb water. The water was changed once during this time. For the growing pots, autoclaved plant culture boxes (75 x 75 x 100 mm, VWR) were stacked on top of each other.

[0038] Three types of hydroponic solutions were prepared, based on the phosphorus-deficient (-Pi) composition shown in Table 1 above, with ZnSO4·7H2O concentrations (hereinafter referred to as "zinc concentration") of 0, 2, and 4 μmol / L. 9 g / L of agar was added to each hydroponic solution to create agar growing media, which were then autoclaved and placed in 100 mL of a plant box. Just before the agar solidified, 1 mL of bacterial suspension was mixed in. After the agar had completely solidified, nine seeds were sown in each box and cultivated for seven days in an artificial climate chamber (30°C, 14 hours light / 10 hours dark). The fresh weight of the above-ground parts of the seedlings and the phosphorus content per 1 mg of above-ground parts were measured after seven days of cultivation. For comparison, cultivation was carried out in the same manner as above, except that the bacterial suspension was not mixed into the growing agar media. The fresh weight of the above-ground parts of the seedlings and the phosphorus content per 1 mg of above-ground parts were measured after seven days of cultivation.

[0039] 3) Results The results are shown in Figure 1. Data were collected for n < 9 for each group. In the upper part of Figure 1, the measured values ​​of the fresh weight of the above-ground part of each seedling are shown as dots, and the median value for each group is shown as a bar. In the lower part of Figure 1, the measured values ​​of the phosphorus (P) content per 1 mg of above-ground part of each seedling are shown as dots, and the median value for each group is shown as a bar.

[0040] As shown in Figure 1, when agar medium prepared using a hydroponic solution with a zinc concentration of 0 μmol / L was used as the culture medium, no significant difference was observed between the group inoculated with the R2A7 strain and the group that was not inoculated, in terms of both the weight of the above-ground parts and the phosphorus (P) content per 1 mg of above-ground parts. When the zinc concentration was 2 and 4 μmol / L, the group inoculated with the R2A7 strain had a greater weight of the above-ground parts and a higher phosphorus content per 1 mg of above-ground parts than the group that was not inoculated. In particular, at a zinc concentration of 4 μmol / L, the weight of the above-ground parts of the group inoculated with the R2A7 strain increased significantly. From these results, it is thought that the promotion of rice growth by the R2A7 strain is dependent on zinc, and that rice growth is promoted by using a culture medium containing a zinc concentration slightly higher than the typical zinc concentration (2 μmol / L) used in rice cultivation media.

[0041] 3. Efficacy confirmation test of R2A7 strain (2) Five types of hydroponic solutions with zinc concentrations of 0, 2, 4, 20, and 100 μmol / L were prepared for the cultivation agar medium. Cultivation was carried out in the same manner as described in section 2 above, and the fresh weight of the above-ground parts and roots of the seedlings was measured after 7 days of cultivation. Figure 2 shows the results. Each group had n=18 data points. The upper panel of Figure 2 shows the measured fresh weight of the above-ground parts for each seedling as dots, and the median for each group as a bar graph. The lower panel of Figure 2 shows the measured fresh weight of the roots for each seedling as dots, and the median for each group as a bar graph.

[0042] As shown in Figure 2, when agar medium prepared using a hydroponic solution with a zinc concentration of 0 μmol / L was used as the culture medium, no significant difference was observed in either the weight of the above-ground parts or the weight of the roots between the group inoculated with the R2A7 strain and the group that was not inoculated. When the zinc concentration was 2 μmol / L, the group inoculated with the R2A7 strain had a greater root weight than the group that was not inoculated. When the zinc concentration was 4 μmol / L, both the weight of the above-ground parts and the weight of the roots increased in the group that was not inoculated with the R2A7 strain compared to the case where the zinc concentration was 2 μmol / L, but both the weight of the above-ground parts and the weight of the roots increased even further in the group that was inoculated with the R2A7 strain. When the zinc concentration was 20 μmol / L or higher, the weight of the roots decreased in the group that was not inoculated with the R2A7 strain compared to the case where the zinc concentration was 2 μmol / L, but the decrease in root weight was smaller in the group that was inoculated with the R2A7 strain. Based on these results, it is considered that even when the zinc concentration in the culture medium fluctuates over a wide range, the growth-promoting effect on rice by inoculating with the R2A7 strain can be reliably obtained. In particular, when the zinc concentration is 20 μmol / L or higher, root growth is inhibited, but it is thought that inoculation with the R2A7 strain mitigates this inhibition of root growth. In other words, it is considered that rice can be grown even in culture media where the zinc concentration is high for some reason by inoculating with the R2A7 strain.

[0043] 4. Efficacy confirmation test of R2A7 strain (3) Five types of hydroponic solutions with zinc concentrations of 2, 4, 6, 8, and 10 μmol / L were prepared for the cultivation agar medium. Cultivation was carried out in the same manner as described in section 2 above, and the fresh weight of the above-ground parts and roots of the seedlings was measured after 7 days of cultivation. The results are shown in Figure 3. Data were collected for n=18 for each group. In the upper part of Figure 3, the measured values ​​of the fresh weight of the above-ground parts for each seedling are shown as dots, and the median value for each group is shown as a bar graph. In the lower part of Figure 3, the measured values ​​of the fresh weight of the roots for each seedling are shown as dots, and the median value for each group is shown as a bar graph.

[0044] As shown in Figure 3, when agar media prepared using hydroponic solutions with zinc concentrations of 2, 4, 6, 8, and 10 μmol / L were used as the culture medium, the weight of both the above-ground parts and roots increased in the group inoculated with the R2A7 strain compared to the group that was not inoculated. Furthermore, no inhibition of seedling growth by zinc was observed. Compared to cultivation without inoculation with the R2A7 strain at a typical zinc concentration (2 μmol / L), the weight of both the above-ground parts and roots increased in the groups inoculated with the R2A7 strain at zinc concentrations of 4, 6, 8, and 10 μmol / L. From these results, it is considered that if the zinc concentration is in the range of 4-10 μmol / L, a high growth-promoting effect can be obtained by inoculating with the R2A7 strain without experiencing growth inhibition due to excessively high zinc concentrations.

[0045] 5. Efficacy confirmation test of R2A7 strain (4) 1) Preparation of a suspension of the R2A7 strain i) Condition M1 A bacterial suspension was prepared in the same manner as described in 2.1) above.

[0046] ii) Condition M2 Pre-culture and main culture of strain R2A7 were performed in the same manner as described in 2.1) above. After the main culture, the culture medium was centrifuged (2000×g, 15 min, 20°C), the supernatant was discarded, and the bacterial cells were collected. A 4 μmol / L aqueous solution of ZnSO4·7H2O was added to the bacterial cells, and the mixture was held at 28°C for 1 hour. Then, it was centrifuged (2000×g, 15 min, 20°C), the supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were suspended and washed with 4 mL of sterile water, and then centrifuged again (2000×g, 10 min, 20°C) to collect the bacterial cells. This washing process was repeated twice, and sterile water was added to the collected bacterial cells to prepare a bacterial suspension with an OD600 of 0.1.

[0047] iii) Condition M3 The bacterial suspension was prepared in the same manner as in condition 2 above, except that a 10 μmol / L aqueous solution of ZnSO4·7H2O was used instead of a 4 μmol / L aqueous solution of ZnSO4·7H2O as the solution added to the bacterial cells.

[0048] iv) Condition M4 Pre-culture of strain R2A7 was performed in the same manner as described in 2.1) above. 1 mL of this culture solution was added to 3 mL of new culture medium, and then ZnSO4·7H2O aqueous solution was added to a final concentration of 4 μmol / L, and the culture was incubated overnight (main culture). After the main culture, the culture solution was centrifuged (2000 × g, 15 min, 20°C), the supernatant was discarded, and the recovered precipitate pellet (bacterial cells) was suspended and washed with 4 mL of sterile water, and then centrifuged again (2000 × g, 10 min, 20°C) to recover the bacterial cells. The same washing process was repeated twice, and sterile water was added to the recovered bacterial cells to prepare a bacterial suspension with an OD600 of 0.1.

[0049] 2) Cultivation i) Cultivation in zinc-deficient medium A hydroponic solution with the same composition as the phosphorus-deficient (-Pi) solution in Table 1 above was prepared, except that it did not contain ZnSO4·7H2O. 9 g / L of agar was added to this hydroponic solution to create a growing agar medium, which was then autoclaved and placed in 100 mL of a plant box similar to the one described in 2. Just before the agar solidified, 1 mL of the bacterial suspension prepared under the conditions M1 was mixed in. After the agar had completely solidified, nine seeds treated in the same manner as in 2. above were sown per box and cultivated for 7 days in an artificial climate chamber (30°C, 14 hours light / 10 hours dark). The fresh weight of the above-ground parts and roots of the seedlings was measured after 7 days of cultivation.

[0050] Similarly, cultivation agar plates were prepared using the bacterial suspensions prepared under the above conditions M2-4, rice seeds were sown, and cultivation was carried out. The fresh weight of the above-ground part and the fresh weight of the roots of the seedlings were measured after 7 days of cultivation. For comparison, cultivation and measurement of seedling weights were carried out in the same manner as above, except that the bacterial suspension was not mixed into the cultivation agar plate.

[0051] The results are shown in Figure 4. For each group, n=9 was used. The upper panel of Figure 4 shows the measured fresh weight of the above-ground part of each seedling as dots, and the median for each group as a bar graph. The lower panel of Figure 4 shows the measured fresh weight of the roots of each seedling as dots, and the median for each group as a bar graph.

[0052] ii) Cultivation in a zinc-containing culture medium Cultivation and measurement of seedling weights were carried out in the same manner as in 5.2)i) above, except that the hydroponic solution used for preparing the growth agar medium had the same composition as the phosphorus-deficient (-Pi) solution in Table 1 above, i.e., a zinc concentration of 2 μmol / L. The results are shown in Figure 5. For each group, n=9 was used. In the upper part of Figure 5, the measured values ​​of the fresh weight of the above-ground part of each seedling are shown as dots, and the median value for each group is shown as a bar graph. In the lower part of Figure 5, the measured values ​​of the fresh weight of the roots of each seedling are shown as dots, and the median value for each group is shown as a bar graph.

[0053] 3) Results As shown in Figure 4, when cultivation was carried out under zinc-deficient conditions, the group using the fungal suspension prepared under condition M3 had higher weights of both above-ground parts and roots compared to the control group (mock) and the group using the fungal suspension prepared under condition M1.

[0054] As shown in Figure 5, when cultivated in a zinc-containing medium, the groups using the bacterial suspensions prepared under conditions M1-4 had higher weights of both above-ground and above-ground parts compared to the control group (mock). In particular, the group using the bacterial suspension prepared under condition M3 showed a significant increase in both root and above-ground weights.

[0055] Based on these results, it is considered that the growth-promoting effect of the R2A7 strain on rice can be obtained by inoculating rice seeds with the R2A7 strain treated with an aqueous solution containing zinc, or with the R2A7 strain cultured in a zinc-containing culture medium. In particular, it is considered that the growth-promoting effect of the R2A7 strain on rice can be obtained by inoculating rice seeds with the R2A7 strain treated with an aqueous solution containing a relatively high concentration of zinc, under a wide range of zinc concentration conditions, including zinc-deficient conditions in which the culture medium does not contain zinc.

[0056] Furthermore, as shown in Figure 5, when cultivated in a zinc-containing medium, the group using the fungal suspension prepared under condition M4 showed an increase in root weight. From these results, it is considered that when cultivating in a zinc-containing medium, inoculating rice seeds with the R2A7 strain treated with a zinc-containing aqueous solution can promote rice growth.

[0057] In the efficacy confirmation tests (1)-(3) described above, the growth-promoting effect was confirmed under a wide range of zinc concentration conditions by inoculating rice seeds with zinc and the R2A7 strain. However, since no significant difference in effect was observed between inoculating seeds and seedlings with the fungal strain in previous studies of the same type, it is thought that a similar growth-promoting effect can be obtained under a wide range of zinc concentration conditions by inoculating rice seedlings with zinc and the R2A7 strain. Similarly, in the efficacy confirmation test (4) described above, the R2A7 strain treated with a zinc-containing aqueous solution was inoculated into rice seeds. It is thought that a similar growth-promoting effect can be obtained by inoculating rice seedlings with the R2A7 strain treated with a zinc-containing aqueous solution and growing them.

[0058] Furthermore, in the above efficacy confirmation tests (1)-(4), agar medium was used as the growing medium for rice. However, since no significant difference in effect due to differences in the growing medium was observed in previous studies of the same type, it is thought that similar growth-promoting effects can be obtained even when using a different growing medium, such as hydroponic solution or soil, instead of agar medium.

Claims

1. A culture medium for growing rice, containing Achromobacter microorganisms deposited under accession number NITE-BP-03938, and zinc.

2. Rice seedlings grown in the culture medium described in claim 1.

3. Rice seedling according to claim 2, The aforementioned rice is paddy rice. Rice seedlings.

4. A rice growth promoter containing Achromobacter microorganisms deposited under accession number NITE-BP-03938, and zinc.

5. A method for cultivating rice, comprising treating Achromobacter microorganisms deposited under accession number NITE-BP-03938 with a zinc-containing solution, and then inoculating rice seeds or seedlings with them.

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