Microorganism that promotes plant growth

Abstract

[Problem] To provide a symbiotic microorganism capable of promoting the growth and stress resistance of rice seedlings in a plant bed, and stably expediting the root taking and growth of seedlings immediately after planting, which are fragile with respect to cold weather and diseases, etc., thereby providing a rice growth-promoting effect throughout the cultivation period. [Solution] The problem is solved by a microorganism that promotes the growth of gramineous plants and is deposited under accession number NITE BP-03938, the microorganism belonging to the genus Achromobacter. The problem is also solved by an agricultural composition that contains a microorganism belonging to the genus Achromobacter and that has a plant growth promoting effect, the composition being characterized by comprising 16SrRNA gene, which is at least 95.0% homologous with the base sequence represented by SEQ ID NO: 1.

Description

Microorganisms that promote plant growth

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

[0002] It should be noted that the use of chemical fertilizers for plant growth requires a large amount of energy to produce and results in significant greenhouse gas emissions. Therefore, reducing the use of chemical fertilizers is an urgent issue for achieving sustainable agriculture. One proposed solution is to reduce chemical fertilizer use by utilizing symbiotic microorganisms. In particular, in rice cultivation, since seedlings are transplanted into fields, raising healthy seedlings is essential to achieving stable, high yields. The period immediately after transplanting is particularly susceptible to various biological and environmental stresses. Therefore, smooth establishment and growth of seedlings is crucial for enhancing rice's stress tolerance and leading to stable, high yields. Various symbiotic microorganisms are known to contribute to plant growth promotion, environmental stress tolerance, and disease resistance. Therefore, the implementation of symbiotic microorganisms in agriculture is required to promote sustainable agriculture. For example, research and development is being conducted to stabilize rice production using rice symbiotic microorganisms (Patent Documents 1 to 3).

[0003] Re-table 2005-040358 JP-A-11-089563 Special table 2023-522007

[0004] However, currently, there is little knowledge about useful symbiotic microbial resources. Even when significant effects are observed in laboratory environments, there are many cases where the symbiotic effects are unstable in the field. This has resulted in the inability to utilize potentially useful microbial resources and limited progress in their implementation. Therefore, there is a need to develop strategies for stably manifesting the symbiotic effects of microorganisms in outdoor environments. The present invention was made in light of the above circumstances. Its purpose is to provide symbiotic microorganisms 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 transplanting, which are vulnerable to cold damage and disease.

[0005]

[0006] Another aspect of the present invention is a method for cultivating plants, which comprises the step of treating plant seeds with the agricultural composition of the present invention. Specifically, for example, the microorganisms can be inoculated and administered to rice seeds or seedlings that have been treated with an antifungal agent in the usual manner.

[0007] The present invention relates to agricultural compositions and plant cultivation techniques that use symbiotic microorganisms of plants (especially rice), and contributes to the promotion of fertilizer-reduced agriculture. In particular, promoting seedling growth promotes the establishment of seedlings immediately after transplanting, which is the stage during rice cultivation when they are most susceptible to environmental changes, stress, and pathogenic bacteria, thereby stabilizing subsequent rice growth in the field and increasing yields. In particular, the inoculation and symbiotic effects of this strain are remarkable both under conditions with sufficient fertilization and under low-nutrient, reduced-fertilization conditions. The effect has also been confirmed in outdoor, unfertilized fields, contributing to the promotion of fertilizer-reduced cultivation.

[0008] The photograph (left) and bar graph (right) show the results of an investigation into the effects of R2A7 on nutrient-poor soil (50% compost). The left side of the photograph (A) shows representative photographs of rice plants grown under mock and +R2A7 conditions, while the upper right (B) shows representative photographs of mock and +R2A7 conditions, respectively. The upper right (C) shows representative photographs of the mock and +R2A7 conditions, respectively. The upper (D) and lower (E) graphs show the total weight of rice plants and the root weight, respectively. Mock indicates the control group, and R2A7 indicates the group treated with the microorganisms. In the figure, an asterisk (*) indicates a significant difference (p<0.05) compared to the control (mock). The photograph (left) and bar graph (right) show the results of an investigation into the plant growth 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. This graph shows the results of investigating the effect of R2A7 on the growth of Hinohikari. "Mock" indicates the control group, and "+R2A7" indicates the group given R2A7.

[0009] Next, embodiments of the present invention will be described with reference to the accompanying drawings. The technical scope of the present invention is not limited to these embodiments, and various embodiments can be implemented without departing from the spirit and scope of the invention. <Test Methods 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 dehulled immediately before use. For the bacterial flora analysis, Nipponbare seeds were harvested in the greenhouse at 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). For the seeds from Kyoto Prefecture, those from fertilized and unfertilized fields (where rice had been cultivated continuously without fertilization for over 20 years) were harvested from adjacent fields. Rice roots were sampled and used for symbiotic bacterial flora analysis. The surface of the sampled rice plants was sterilized to target only the symbiotic bacteria present inside the plants. The ccamk mutant (ccamk-2) was grown and harvested from seeds of a TOS17 insertion line (kindly provided by Dr. Kiwamu Minamizawa of Tohoku University) in an unfertilized field in Kyoto Prefecture.

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

[0011]

[0012] Hydroponic solution was placed in 50 mL tubes, with a horticultural potting net floating on the water surface. The hydroponic solution used was either phosphorus-sufficient (+Pi; 200 μM) or phosphorus-deficient (-Pi; 20 μM) KH2PO4. Five absorbed seeds per tube were placed on top of the potting net, and the rice was grown with the aboveground 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 plants were grown in an artificial climate chamber (32°C, 14 hours light / 10 hours dark) for 4 or 7 days.

[0013] 3. Bacterial Inoculation Tests. Bacterial strains isolated from surface-sterilized rice roots grown in a paddy field were used in the inoculation experiments. A glycerol stock of the isolated bacteria was inoculated into 2 mL of NB or R2A liquid medium and cultured at 28°C for approximately 24 hours with shaking (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 minutes, 20°C), the supernatant was discarded, and sterile water was added to an OD600 of 0.1 to prepare a bacterial suspension. Seeds used for inoculation were treated with a fungicide or 20% sodium hypochlorite as described above. They were then immersed in sterile water for 2 days to absorb water (with one water change). Autoclaved plant culture boxes (75 × 75 × 100 mm, VWR) were stacked on top of each other to form the growth pots. Agar growth medium was prepared by adding 9 g / L of agar to the hydroponic solution. After autoclaving, 100 mL of the medium was placed in a plant box. After the agar had completely solidified, nine seeds were sown per box. The plants were then grown in a climate chamber (30°C, 14 hours light / 10 hours dark) for 7 days. The bacterial count (CFU) was then measured using rice roots grown for 7 days. Rice roots were crushed with 10 mM MgCl2, and serial dilutions were prepared from the crushed roots. The diluted solution was applied to R2A agar medium and incubated at 28°C for 3 days to measure the colony-forming units (CFU).

[0014]

[0015] 5. Meta-16s Sequencing Analysis. Wild-type rice seeds treated with fungicide were sown in hydroponic solution. After 4 days, roots were rinsed with sterile water, detached from the plant, and samples of three or more roots were collected and flash-frozen in liquid nitrogen. The samples were freeze-fractured, and microbial DNA was extracted using NucleoSpin® Soil (MACHERRY-NAGEL). Subsequently, 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). Primers and PCR cycles were based on a previous publication (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 DNA bands of interest were excised from agarose gel electrophoresis and purified using a gel / PCR extraction kit (Fast Gene). 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 Kit V3.

[0016] Sequences sequenced by Miseq were analyzed based on a previously published method (Utami et al. (2018) Phylogenetic diversity and single-cell genome analysis of “melainabacteria”, an anon-photosynthetic cyanobacterial group, in the termite gut. Microbes Environ 33:50-57. doi: 10.1264 / jsme2.ME17137). 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), sequences were trimmed, filtered, and classified based on ampliconsequence variants (ASVs). The resulting ASV sequences were phylogenetically classified based on 80% or greater 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 data processing 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 did not match any database sequences, were excluded from the analysis.To analyze the diversity of the microbiota, 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 the meta-16S rRNA analysis includes bacterial and archaeal sequences, we refer to this analysis as a microbiota analysis for convenience.

[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 important to rice health and growth. 7. Isolation of Symbiotic Bacteria: Based on the analytical results obtained above, symbiotic bacteria thought to be important for rice growth were isolated. At this stage, more than 600 strains were obtained. Rice inoculation tests were conducted on the obtained symbiotic bacteria, and the plant growth-promoting effect of each strain was confirmed. A soil inoculation test was used for the test. Briefly, the procedure was as follows: Rice seeds were sterilized in 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 then sown in test soil (a mixture of culture medium, Akadama soil, and Kanuma soil in a 5:4:1 ratio, under nutrient-poor conditions) in a tray. After 7 days, 500 μL of bacterial solution (OD600 = 0.1) was inoculated and grown for 4 weeks at 28°C under a 14-hour light / 10-hour dark condition. After growth, the rice plants were weighed and photographed.

[0018] 8. Bacterial Genome Analysis The R2A7 strain was identified through the above tests. A glycerol stock of the R2A7 strain was inoculated into 2 mL of R2A liquid medium and cultured at 28°C for approximately 24 hours with shaking (preculture). 100 μL of the culture 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® Micronial DNA (MACHERRY-NAGEL). The extracted DNA was subjected to genome analysis (Tokyo University of Agriculture, Bioresources Genome Analysis Center), and a full-length contig sequence was obtained by assembly using Illumina (short read). To obtain information on the R2A7 bacterium, a DNA sequence containing approximately 760 bp of the V3-V4 region of the 16s rRNA was determined by Sanger sequencing. The results are shown in SEQ ID NO: 1. A BLAST search revealed that R2A7 was a species of the genus Achromobacter.

[0019] 9. Efficacy Test of R2A7 (1) Some Achromobacter species are known to promote rice growth. However, detailed information is not yet fully understood. Therefore, R2A7 was inoculated and administered to rice seeds or seedlings approximately one week after germination, which had been treated with a fungicide according to standard procedures. 50 μL of bacterial culture suspension was administered per seedling cell. Figure 1 shows the results of growing rice in low-nutrient soil (50% potting soil) without or with R2A7 microorganisms. As can be seen from the photograph on the left, qualitatively, inoculation with R2A7 improved rice growth. Quantitatively, the R2A7-inoculated group exhibited significantly better growth than the control group (p<0.05) when measured for total rice weight and root weight.

[0020] 10. Effect of R2A7 (2) Next, we conducted a test using wild-type rice and ccamk mutant rice to examine the dependency of R2A7 on CCaMK. The test method was the same as described in 9 above, and the effects of R2A7 inoculation on wild-type rice (WT) and ccamk mutant rice (ccamk) were examined. Figure 2 shows the results of plant growth analysis of wild-type rice (WT) and ccamk-deficient mutant rice (ccamk) with (ccamk + R2A7) and without (ccamk) the symbiotic bacterium R2A7. As shown in the figure, inoculation of ccamk mutant rice with R2A7 significantly suppressed growth compared to wild-type rice (p < 0.01). CCaMK (Calcium- and calmodulin-dependent protein kinase) is known to be a symbiotic regulator and is known to be required for both mycorrhizal symbiosis and the root nodule symbiosis characteristic of legumes. However, the role of CCaMK in regulating the symbiotic microbiota is not fully understood. The results of this study showed that the growth-promoting effect of R2A7 on rice depends on the CCaMK gene in rice.

[0021] 11. Effect of R2A7 (3) Next, the effects of R2A7 on plant growth were examined using Hinohikari rice. A control group (mock) and an R2A7-inoculated group (+R2A7) were used as evaluation groups. The test method followed the method described in Section 9 above. Figure 3 shows the results of Hinohikari rice growth 4 weeks after inoculation in low-nutrient soil (50% potting soil). Data are presented as total data and average values ​​(horizontal bars), with n = 18 for each group. 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 asterisk (*). The R2A7 strain identified in this test demonstrated significant growth-promoting effects compared to the control group. The results of the above test showed that R2A7 significantly promoted the growth of seedlings immediately after transplanting from the seedbed in low-nutrient soil, such as phosphorus-deficient soil. Furthermore, when the symbiotic seedlings were cultivated in an unfertilized paddy field, the growth of rice plants was promoted from immediately after transplanting until the ripening period, without any negative impact on yield, etc.

[0022] As described above, this embodiment provides a microorganism that can promote rice growth throughout the cultivation period by enhancing the growth and stress tolerance 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. The R2A7 strain promoted seedling growth regardless of whether nutrient levels were sufficient or deficient. As a result, the strain was found to promote the rooting of seedlings immediately after transplanting, which is the time during the rice cultivation period when rice is most susceptible to environmental changes, stress, and disease pathogens, and to stabilize rice growth in the field. The inoculation and symbiotic effects of this strain have also been confirmed in unfertilized paddy fields, suggesting its potential for promoting reduced fertilizer cultivation.

[0023] Information on the deposited microorganisms is shown in Table 3.

Claims

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

2. An agricultural composition containing 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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