Microorganisms for promoting plant growth

Abstract

To provide symbiotic microorganisms that can provide rice growth-promoting effect throughout a cultivation period by enhancing the growth and stress resistance of rice seedlings in the seedbed and stabilizing and increasing the rooting and growth of seedlings immediately after transplanting, which are vulnerable to cold damage and disease.SOLUTION: This is achieved by a microorganism that promotes the growth of Poaceae plants, which is an Achromobacter genus microorganism deposited under accession number NITE BP-03938. This is also achieved by an agricultural composition that contains an Achromobacter genus microorganism and has a plant growth-promoting effect and is characterized in that the agricultural composition contains a 16S rRNA gene that has a homology of 95.0% or more with the base sequence represented by SEQ ID NO:1.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microorganism that promotes plant growth. [Background technology]

[0002] It should be noted that the use of chemical fertilizers to grow plants requires a large amount of energy to produce and results in large greenhouse gas emissions. For this reason, reducing the use of chemical fertilizers is an urgent issue for realizing sustainable agriculture. One solution that has been proposed is to reduce the use of chemical fertilizers by utilizing symbiotic microorganisms. In particular, in the cultivation of rice, since the raised seedlings are transplanted into the field, it is essential to raise healthy seedlings in order to achieve stable and high yields. In particular, the period immediately after rice planting is a time when rice is susceptible to various biological and environmental stresses, and smooth establishment and growth of the seedlings is important for increasing the stress resistance of rice and leading to stable and high yields. It is known that various symbiotic microorganisms contribute to the promotion of plant growth, environmental stress resistance, and disease resistance, and there is a demand for the implementation of symbiotic microorganisms in agriculture in order to promote sustainable agriculture. For example, research and development is being conducted to stabilize rice production using symbiotic microorganisms of rice (Patent Documents 1 to 3). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Retable 2005-040358 [Patent Document 2] Japanese Patent Application Publication No. 11-089563 [Patent Document 3] Special table 2023-522007 Summary of the Invention [Problem to be solved by the invention]

[0004] However, at present, there is little knowledge about useful symbiotic microbial resources, and even when significant effects have been observed in laboratory environments, there are many cases where the symbiotic effects are unstable in the field. As a result, potentially useful microbial resources cannot be utilized and their implementation has not progressed. Therefore, it is also necessary to develop methods for stably expressing the symbiotic effects of microorganisms in outdoor environments. The present invention has been made in consideration of the above-mentioned circumstances, and its object is to provide a symbiotic microorganism that can promote rice growth throughout the cultivation period by enhancing the growth and stress resistance of rice seedlings in the seedbed and stabilizing and increasing the rooting and growth of seedlings immediately after planting, which are vulnerable to cold damage, disease, and the like. [Means for solving the problem]

[0005] In view of the above circumstances, the present inventors have made extensive research and have completed the present invention. The microorganism according to the invention for achieving the above object promotes the growth of grasses and is a microorganism of the genus Achromobacter deposited under accession number NITE BP-03938. A plant refers to an organism that has roots and lives in a fixed location, such as grass or trees. In addition, an agricultural composition according to another invention preferably contains an Achromobacter microorganism, has a growth-promoting effect on grasses, and contains a 16S rRNA gene having 95.0% or more (preferably 97.0% or more) homology with the base sequence having SEQ ID NO: 1. In this case, the Achromobacter microorganism is preferably the microorganism having the accession number NITE BP-03938.

[0006] A method for cultivating a plant according to another aspect of the present invention is characterized in that it comprises a step of treating plant seeds with the agricultural composition of the above-mentioned invention. Specifically, for example, the microorganisms can be inoculated or administered simultaneously to rice seeds or seedlings that have been treated with an antifungal agent in the usual manner. Effect of the Invention

[0007] The present invention relates to an agricultural composition using symbiotic microorganisms of plants (especially rice) and a plant cultivation technique, which contributes to the promotion of fertilizer-saving agriculture. In particular, by promoting the growth of seedlings, it is possible to promote the establishment of seedlings immediately after transplanting, which is the stage during the rice cultivation period when they are most susceptible to environmental changes, stress, and pathogenic bacteria, and to stabilize the subsequent growth of rice in the field and increase the yield. In particular, the inoculation and symbiotic effects of this strain were observed remarkably both under conditions of sufficient fertilization and under conditions of low nutrient fertilization, and its effectiveness has been confirmed even in outdoor non-fertilized fields, contributing to the promotion of fertilizer-saving cultivation. [Brief description of the drawings]

[0008] [Figure 1] This is a photograph (left) and a bar graph (right) showing the results of investigating the effects of R2A7 in low-nutrient soil (50% culture soil). The left side of the photograph (A) shows representative photographs of rice grown in mock and +R2A7, the upper right (B) shows representative photographs of mock, and the lower right (C) shows representative photographs of +R2A7. The upper side of the graph (D) shows the total weight of rice, and the lower side (E) shows the weight of rice roots. Mock indicates the control group, and R2A7 indicates the group to which the microorganisms were administered. In the figure, "*" indicates that a significant difference was observed at p<0.05 compared to the control (mock). [Diagram 2] This is a photograph (left) and a bar graph (right) showing the results of investigating the plant growth conditions of wild-type rice (WT) and ccamk mutant rice (ccamk) in the presence (ccamk + R2A7) and absence (ccamk) of the symbiotic bacterium R2A7. The left side of the photograph (A) shows representative photographs of ccamk and ccamk + R2A7, the upper right (B) shows representative photographs of ccamk, and the lower right (C) shows representative photographs of ccamk + R2A7. In the figure, "*" indicates a significant difference compared to the control (WT) at p<0.01. [Diagram 3] This is a graph showing the results of investigating the effect of R2A7 on the growth of Hinohikari. "Mock" indicates the control group, and "+R2A7" indicates the group to which R2A7 was administered. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Next, the embodiments of the present invention will be described with reference to the drawings. The technical scope of the present invention is not limited to these embodiments, and the present invention can be embodied in various forms without changing the gist of the invention. <Test method and test results> 1. Plant Materials and Culture 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 hulled just before use. For the bacterial flora analysis, Nipponbare seeds harvested in the greenhouse of the Nara Institute of Science and Technology (25-30°C, 10 hours light / 14 hours dark) and in outdoor fields across Japan (Kyoto, Miyagi, and Shizuoka prefectures) were used. For the seeds from Kyoto prefecture, those from fertilized and unfertilized fields (where rice had been cultivated continuously without fertilization for more than 20 years) were harvested from adjacent fields. Rice roots were sampled and used as the subject of the symbiotic bacterial flora investigation. The surface of the sampled rice was sterilized, and only the symbiotic bacteria present inside the plant were examined. The ccamk mutant (ccamk-2) was prepared by cultivating and harvesting seeds of a TOS17 insertion line (provided by Dr. Minamizawa Kiwamu, Tohoku University) in an unfertilized field in Kyoto Prefecture.

[0010] 2. Hydroponic rice cultivation Sowing in the presence of seed microorganisms was performed by immersing dehulled seeds in a 0.25% GF Benlate wettable powder solution (Sumitomo Chemical, active ingredient: benomyl), an antifungal agent, and allowing them to absorb water at 28°C under a 24-hour dark period. Completely sterilized sowing was performed by sterilizing the seeds with 20% sodium hypochlorite for 1 hour, washing thoroughly with sterile water, and allowing them to absorb water at 28°C under a 23-hour dark period. Table 1 shows the medium composition and final concentration used for hydroponic cultivation.

[0011] [Table 1]

[0012] Hydroponic solution was placed in a 50mL tube, and a horticultural pot bottom net was floated on the water surface. Phosphorus-sufficient (+Pi; 200μM) or phosphorus-deficient (-Pi; 20μM) KH2PO4 was used as the hydroponic solution. Five water-absorbed seeds from each tube were placed on the pot bottom net, and rice was grown with the aboveground part remaining in the air and only the roots extending into the water. The top of the tube was sealed with plastic wrap, and the tubes were cultivated in an artificial climate chamber (32°C, 14 hours light / 10 hours dark) for 4 or 7 days.

[0013] 3. Bacterial strain inoculation test Bacterial strains isolated from surface-sterilized rice roots grown in a paddy field were used in the inoculation experiments. Glycerol stocks of the isolated bacteria were inoculated into 2 mL of NB or R2A liquid medium and cultured with shaking at 28°C for approximately 24 hours (preculture). 100 μL of the culture was added to 5 mL of fresh medium and cultured overnight (main culture). The bacterial suspension was centrifuged (2000 × g, 15 min, 20°C), the supernatant was discarded, and sterile water was added to prepare a bacterial suspension with an OD600 of 0.1. The seeds used for inoculation were treated with a fungicide or 20% sodium hypochlorite according to the above method. After that, they were immersed in sterilized water and allowed to absorb water for 2 days (water was changed once during the period). Autoclaved plant boxes (75 x 75 x 100 mm, VWR) were stacked on top of each other as growth pots. Agar 9 g / L was added to the hydroponic solution to prepare a growth agar medium, and 100 mL was placed in the plant box after autoclaving. After the agar had completely solidified, nine seeds were sown per box. Then, the seeds were grown in an artificial climate chamber (30°C, 14 hours light / 10 hours dark) for 7 days. Furthermore, the bacterial count (CFU) was measured using the roots of rice grown for 7 days. The rice roots were crushed with 10 mM MgCl2, and dilutions were prepared by serial dilution from the crushed solution. The diluted solution was spread on R2A agar medium and cultured at 28°C for 3 days to measure the number of colonies formed (CFU).

[0014] [Table 2]

[0015] 5. Meta 16s Sequence Analysis Wild-type rice seeds treated with fungicide were sown in hydroponic solution. After 4 days, the roots were rinsed with sterile water, separated from the plant body, and three or more roots were collected as one sample and flash frozen in liquid nitrogen. The samples were freeze-shattered and microbial DNA was extracted using NucleoSpin® Soil (MACHERRY-NAGEL). Then, the bacterial and archaeal 16S rRNA gene regions were amplified by touchdown PCR using KOD FXNeo (TOYOBO) and 515 forward and 806 reverse primers (Table 2). The primers and PCR cycles were as previously reported (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 band of the target DNA 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 the Miseq using the MiSeq Reagent KitV3.

[0016] Sequences sequenced by Miseq were analyzed according to a previously published method (Utami et al. (2018) Phylogenetic diversity and single-cell genome analysis of “melainabacteria”, anon-photosynthetic cyanobacterial group, in the termite gut. Microbes Environ33:50-57. doi: 10.1264 / jsme2.ME17137). Sequences were trimmed, filtered, and classified based on ampliconsequence variants (ASVs) using 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). The ASV sequences were phylogenetically classified based on 80% or higher identity 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 SILVASSURef NR99 release 132 (Quast et al. 2012. The SILVA ribosomal RNA gene database project: improved dataprocessing and web-based tools. Nucleic Acids Res 41:D590-D596.doi: 10.1093 / nar / gks1219). ASV sequences derived from eukaryotes, mitochondria, and plastids, as well as sequences that could not be matched to database sequences, were excluded from the analysis.For the analysis of the diversity of the microbiota, we used QIIME v.1.9.1 (Caporaso et al. 2010. QIIME allows analysis of highthroughput community sequencing data. Nat Methods 7:335-336. doi: 10.1038 / nmeth.f.303.QIIME) and the R packages vega and ggplot2. Although the meta-16S rRNA analysis includes sequences of bacteria and archaea, for convenience, we refer to this as a microbiota analysis in accordance with the common name.

[0017] 6. Bioinformatics Analysis Using bioinformatics, we performed machine learning on the bacterial flora data from the base sequences obtained above to identify symbiotic bacteria that are important for rice health and growth. 7. Isolation of Commensal Bacteria Based on the results of the analysis above, we isolated symbiotic bacteria that are thought to be important for rice growth. At this stage, we obtained more than 600 strains. The obtained symbiotic bacteria were subjected to rice inoculation tests to confirm the plant growth promoting effect of each bacteria. The soil inoculation test method was used for the test. The results are briefly explained as follows. Rice seeds were sterilized with 70% alcohol for 1 minute and then allowed to absorb water at room temperature for 2 days. The seeds were germinated at 32°C for 2 days and sown in a tray of test soil (a mixture of culture soil, Akadama soil, and Kanuma soil in a ratio of 5:4:1, nutrient-poor conditions). After 7 days, 500 μL of bacterial solution (OD600=0.1) was inoculated and the seeds were grown for 4 weeks at 28°C under conditions of 14 hours light and 10 hours dark. After growth, the fresh weight of the rice was measured and photographed.

[0018] 8. Bacterial Genome Analysis The R2A7 strain was identified by the above tests. The glycerol stock of the R2A7 strain was inoculated into 2 mL of R2A liquid medium and cultured at 28 ° C for about 24 hours with shaking (preculture). 100 μL of the culture solution was added to 5 mL of fresh medium and cultured overnight (main culture). 1.5 mL of the bacterial suspension was centrifuged (2000 × g, 15 minutes, 20 ° C), and genomic DNA was extracted from the precipitated bacterial cells using NucleoSpin (registered trademark) Micronial DNA (MACHERRY-NAGEL). The extracted DNA was subjected to genome analysis (Tokyo University of Agriculture and Life Sciences Genome Analysis Center), and a full-length contig sequence was obtained by assembly using Illumina (short read). To obtain information on the bacterium R2A7, a DNA sequence including approximately 760 bp of the V3-V4 region of 16s rRNA was determined by Sanger sequencing. The result is shown in SEQ ID NO: 1. A BLAST search revealed that R2A7 belongs to the genus Achromobacter.

[0019] 9. R2A7 Efficacy Verification Test (1) Some Achromobacter species are known to promote rice growth, but their detailed information is not fully understood. Therefore, R2A7 was inoculated and administered simultaneously to rice seeds or seedlings approximately one week after germination that had been treated with a fungicide in the usual manner. 50 μL of the bacterial strain culture suspension was administered per seedling cell. Figure 1 shows the results of growing rice in low-nutrient soil (50% culture soil) without the addition of microorganisms (mock) or with the addition of microorganisms (R2A7). As can be seen from the photograph on the left, qualitatively, inoculation with R2A7 was observed to improve rice growth. Quantitatively, the results of examining the total weight and root weight of rice showed that the R2A7-inoculated group grew significantly (p<0.05) better than the control.

[0020] 10.R2A7 Efficacy Verification Test (2) Next, we investigated the CCaMK-dependence of R2A7 in wild-type and ccamk mutant rice plants. The test method was the same as Method 9 above, and the effects of inoculation with or without R2A7 on wild-type rice (WT) and ccamk mutant rice (ccamk) were examined. Figure 2 shows the results of investigating the growth of wild-type rice (WT) and ccamk-deficient mutant rice (ccamk) plants with (ccamk + R2A7) or without (ccamk) the symbiotic bacterium R2A7. As shown in the figure, when ccamk mutant rice was inoculated with R2A7, its growth was significantly (p<0.01) suppressed compared to wild-type rice. CCaMK (Calcium- and calmodulin-dependent protein kinase) is known as a symbiosis regulator and is known to be required for both mycorrhizal symbiosis and the nodule symbiosis characteristic of legumes. However, the role of CCaMK in controlling the symbiotic microbiota is not fully understood. The results of this study showed that the rice growth-promoting effect of R2A7 depends on the rice CCaMK gene.

[0021] 11.R2A7 Efficacy Verification Test (3) Next, we investigated the effect of R2A7 on plant growth using Hinohikari. A control group (mock) and an R2A7-inoculated group (+R2A7) were used as evaluation groups. The test method was as described in Method 9 above. Figure 3 shows the results of investigating the growth of Hinohikari in low-nutrient soil (50% culture soil) four weeks after inoculation. Data is n = 18 for each group, and is shown as total data and average values ​​(horizontal bars). In addition, a t-test was performed between groups, and significant differences at a risk level of less than 5% (p < 0.05) are indicated with an *. The R2A7 strain identified in this study was found to have a significant growth-promoting effect compared to the control group. As a result of the above test, R2A7 showed a remarkable growth-promoting effect on rice seedlings immediately after transplanting from the seedbed in nutrient-poor soils such as phosphorus-deficient soils. Furthermore, when the symbiotic seedlings were cultivated in an unfertilized paddy field, the growth of rice was promoted from immediately after transplanting until the ripening period, without any negative effects on yield, etc.

[0022] As described above, according to the present embodiment, the growth and stress resistance of rice seedlings in seedbeds are enhanced, and the rooting and growth of seedlings immediately after transplanting, which are vulnerable to cold damage and disease, are stabilized and increased, thereby providing a microorganism that can bring about a rice growth-promoting effect throughout the cultivation period. The R2A7 strain promoted seedling growth regardless of whether nutrients were sufficient or deficient. As a result, it was found to promote the establishment of seedlings immediately after transplanting, the period during the rice cultivation period when rice is most susceptible to environmental changes, stress, and disease pathogens, and to have an effect of stabilizing rice growth in the field. The inoculation and symbiotic effects of this strain have also been confirmed in non-fertilized paddy fields, and it is believed that this strain will contribute to the promotion of reduced fertilizer cultivation.

Claims

1. A microorganism that promotes the growth of grasses, which is an Achromobacter genus microorganism, deposited under accession number NITE BP-03938.

2. An agricultural composition comprising an Achromobacter genus microorganism, having the effect of promoting the growth of grasses, and containing a 16S rRNA gene having a homology of 95.0% or more with the base sequence represented by sequence number 1.

3. The agricultural composition according to claim 2, wherein the Achromobacter microorganism is that according to claim 1.

4. A method for cultivating a plant, comprising a step of treating plant seeds with the agricultural composition according to claim 2 or 3.

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