Microorganism that promotes growth of gramineous plants

Abstract

[Problem] To provide a symbiotic microorganism that can elicit a rice growth promoting effect throughout a cultivation period by enhancing the growth and stress resistance of rice seedlings in a seed bed, and stabilizing and increasing the rooting and growth of seedlings, which are vulnerable to cold-weather damage, disease injury, or the like, immediately after rice planting. [Solution] The problem is solved by a microorganism which is deposited under accession number NITE BP-03939 and is a microorganism belonging to the genus Sphingomonas. Moreover, the problem is solved by an agricultural composition containing a microorganism belonging to the genus Sphingomonas and having a rice growth promoting effect, the agricultural composition being characterized by containing a 16S rRNA gene having a homology of at least 95.0% with a base sequence including rRNA of SEQ ID NO: 1.

Description

Microorganisms that promote the growth of grass plants

[0001] The present invention relates to a microorganism that promotes the growth of grass plants.

[0002] It should be noted that the use of chemical fertilizers for plant growth not only incurs environmental pollution and soil degradation costs, but also requires a large amount of energy for production and contributes to greenhouse gas emissions. For this reason, reducing the use of chemical fertilizers is an urgent issue for achieving sustainable agriculture. One solution proposed is to utilize symbiotic microorganisms to reduce chemical fertilizer use. In particular, in rice cultivation, where seedlings are transplanted into fields, raising healthy seedlings is essential to achieving stable, high yields. Immediately after transplanting, seedlings are susceptible to various biological and environmental stresses. Therefore, smooth establishment and growth of seedlings is crucial for enhancing rice stress tolerance and leading to stable, high yields. In general, many symbiotic microorganisms in plants are known to contribute to 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 Re-table 2016-143458

[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 methods for stably expressing 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 temperature stress and disease.

[0005] The present inventors conducted research into the function of the rice immunomodulatory peptide OsPep and found that this system possesses both plant immunity-enhancing and root system expansion functions (Patent Document 4). Subsequent research revealed that OsPep promotes root system expansion through the control of symbiotic bacteria. Based on this finding, the present inventors predicted the existence of beneficial microorganisms whose symbiosis is promoted by OsPep. They isolated and identified beneficial microorganisms from rice grown in an unfertilized field and demonstrated their functionality through seedling inoculation tests, thereby completing the present invention. The microorganisms described in this invention that achieve the above-mentioned objectives are microorganisms that promote the growth of grass plants and are Sphingomonas microorganisms deposited under Accession Number NITE BP-03939. Another agricultural composition according to the present invention contains a microorganism of the genus Sphingomonas and has the effect of promoting rice growth. The agricultural composition is characterized by containing a 16S rRNA gene that has 95.0% or more (preferably 97.0% or more) homology to the nucleotide sequence of SEQ ID NO: 1. More precisely, a portion of the rRNA sequence described in SEQ ID NO: 1 is highly conserved and may be completely identical even between different microorganisms with different functionality. Therefore, strains can be identified by comparing sequence homology (average nucleotide identity) across the entire genome.

[0006] In the above invention, it is preferable that the composition further contains OsPep. Another invention relates to a method for cultivating rice, characterized by comprising a step of treating plant seeds with the agricultural composition of the above invention. At this time, NB7 and OsPep are simultaneously inoculated and administered to the rice seeds or seedlings. It is also preferable that the rice seeds or seedlings have been treated with a fungicide in accordance with conventional methods. OsPep is a plant immunoregulatory peptide known to induce defense response genes. Furthermore, the inventors have discovered that it also has the function of promoting root growth and modifying symbiotic microflora. In the present invention, it is preferable that the composition further contains at least one OsPep selected from the OsPep family having the amino acid sequence described below. In this invention, we have demonstrated the effectiveness of OsPep3-C, a member of the OsPep family, as a peptide that promotes rice growth in cooperation with NB7. However, any OsPep that activates the OsPep receptor OsPEPR1, which is necessary for the rice growth-promoting effect of NB7-OsPep, or an activator of the receptor (e.g., a peptide, protein, or low-molecular-weight compound) may also be used. Another invention provides a method for identifying OsPEPR1 activators, which involves assessing whether a candidate substance induces an OsPEPR1-dependent physiological response when applied to plant tissues or plant cells expressing the OsPep receptor OsPEPR1.

[0007] The present invention provides a microorganism that can promote rice growth throughout the cultivation period by enhancing rice seedling growth and stress tolerance in the seedbed and stabilizing and increasing the establishment and growth of seedlings immediately after transplanting, which are vulnerable to temperature changes and disease. In particular, inoculating seeds or seedlings with the rice immune-activating peptide OsPep promotes seedling growth both under nutrient-sufficient and nutrient-deficient conditions (although this effect is particularly pronounced under phosphorus-deficient conditions). As a result, the establishment of seedlings immediately after transplanting, which is the period most susceptible to environmental changes, stress, and pathogens during the rice cultivation period, is promoted, thereby stabilizing rice growth in the field. The inoculation and symbiotic effects of this strain are particularly evident under low-nutrient, low-fertilization conditions, and have also been confirmed in unfertilized paddy fields, which is expected to contribute to the promotion of low-fertilization cultivation.

[0008] This figure shows a phylogenetic tree indicating the position of NB7 in Sphingomonas bacteria. This figure shows a photograph (top) and a bar graph (bottom) depicting the results of an investigation into the growth of wild-type rice (A) and a mutant rice (B) lacking the rice symbiotic regulator CCaMK gene, in the presence (+) or absence (-) of the symbiotic bacterium NB7, and in the presence (+) or absence (-) of OsPep3C. This figure shows a photograph (top) and a bar graph (bottom) depicting the results of an investigation into the growth of wild-type rice (A) and a mutant rice (B) lacking the OsPep receptor OsPEPR1 gene, in the presence (+) or absence (-) of the symbiotic bacterium NB7, and in the presence (+) or absence (-) of OsPep3C. This figure shows the results of an investigation into the effects of NB7 and OsPep on the growth of Hinohikari rice. "Mock" indicates the untreated control group, and "NB7+pep" indicates the group inoculated with NB7 and OsPep3-C.

[0009] Next, 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 various embodiments can be implemented without departing from the spirit and scope of the invention. The inventors analyzed rice symbiotic fungi using the following procedure. Briefly, the procedure is as follows. <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 dehulled immediately before use. For the bacterial flora analysis, Nipponbare seeds 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) were used. For the seeds from Kyoto Prefecture, seeds harvested from fertilized and unfertilized fields (where rice had been continuously cultivated without fertilization for over 20 years) were harvested from adjacent fields. Rice roots were sampled and used to investigate the symbiotic microbiota. The surface of the sampled rice plants was sterilized, allowing us to study only the symbiotic microbiota present inside the plants. Transformants (OsPEPR1 overexpressing plants and OsPEPR1 CRISPR-Cas9 knockout plants) were generated by infecting scutellum-derived callus of Nipponbare with Agrobacterium carrying the respective binary vectors, with the cooperation of collaborator Dr. Takuma Ishizaki (JIRCAS). The ccamk-deficient mutant (ccamk-2) was cultivated and harvested from seeds of the TOS17 insertion line (kindly provided by Dr. Kiwamu Minamizawa, Tohoku University) in an unfertilized field in Kyoto Prefecture.

[0010] 2. Experiments administering peptides to hydroponically grown rice Sowing in the presence of seed microorganisms was performed 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 performed 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] Table 2 shows the amino acid sequences and alignments of the 14 OsPep family peptides. Note that, throughout this specification, "OsPep" refers to either the entire OsPep family or individual peptides. OsPep numbering has been subject to confusion depending on the date of discovery and the author (the same peptides are sometimes given different names). Therefore, OsPep names follow the OsPROPEP numbers published in 2020 (Plant J 2020. doi: 10.1111 / tpj.15022). A 50 mL tube was filled with hydroponic solution containing 1 μM OsPep (individual peptides), and a horticultural potting net was placed on the surface. The hydroponic solution used was either phosphorus-sufficient (+Pi; 200 μM) or phosphorus-deficient (-Pi; 20 μM) KH2PO4. Five seeds per tube were placed on a net at the bottom of a pot, and the rice plants were grown with the roots submerged in water and the aboveground parts remaining in the air. 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. The results showed that OsPep3-C and OsPep5-C had a significant growth-promoting effect.

[0013]

[0014] 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. Growth agar medium was prepared by adding 9 g / L agar to the hydroponic solution. After autoclaving, 100 mL of the mixture was placed in a plant box. Just before the agar solidified, 1 mL of bacterial suspension and 1 μM OsPep3-C were mixed. After the agar solidified completely, nine seeds were sown per box. The plants were then grown in a climate chamber (30°C, 14 h light / 10 h dark) for 7 days. Furthermore, bacterial counts (CFU) were 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 then spread on R2A agar medium and incubated at 28°C for 3 days to measure colony-forming units (CFU).

[0015] 4. Meta-16S Sequence Analysis. Wild-type rice seeds treated with fungicides were sown in a hydroponic solution containing 1 μM OsPep3-C. After 4 days, roots were rinsed with sterile water, detached from the plant, and samples of three or more roots were pooled 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 the 515 forward and 806 reverse primers listed in Table 3.

[0016]

[0017] 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 by 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 a MiSeq using the MiSeq Reagent Kit V3. MiSeq sequences were analyzed using 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). 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 obtained ASV sequences were analyzed using SINA v1.2.11 (Pruesse et al. 2012 ).ASVs were phylogenetically classified based on 80% or greater identity using the SINA (Accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28:1823-1829.doi:10.1093 / bioinformatics / bts252) and the database SILVA SSURef 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 vegan and ggplot2. Although the meta-16S rRNA analysis includes bacterial and archaeal sequences, we refer to this analysis as a microbiota analysis for convenience.

[0018] 5. 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. 6. Isolation of Symbiotic Fungi: Based on the analytical results obtained above, symbiotic fungi 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 strains, and the plant growth-promoting effects of each strain were 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 the rice was grown for 4 weeks at 28°C under a 14-hour light / 10-hour dark condition. After growth, the rice was weighed and photographed.

[0019] 7. Bacterial Genome Analysis. A glycerol stock of the NB7 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 (Seibitsu Giken Co., Ltd.), and the full-length genome sequence was obtained by hybrid assembly using GridION (long reads) and DNBSEQ (short reads). Subsequently, a search for closely related species based on average nucleotide identity (ANI) was conducted among bacterial species registered with NCBI.

[0020] To obtain taxonomic information for the bacterium NB7, we determined the DNA sequence of approximately 760 bp of the V3-V4 region of 16S rRNA by Sanger sequencing. The result is shown in SEQ ID NO: 1. BLAST search indicated that NB7 is a species of Sphingomonas. NB7 formed yellow colonies characteristic of Sphingomonas on R2A agar medium. NB7 was also isolated from rice roots grown in a high-quality, unfertilized field in Uji City, Kyoto Prefecture. This bacterium was also detected in meta-16S sequencing of rice roots from adjacent unfertilized and fertilized fields, and its relative abundance in the unfertilized field was higher during the early to mid-stages of rice growth. Furthermore, full-length genome sequencing was performed to obtain the full-length genome sequence of NB7. The National Center for Biotechnology Information (NCBI) database contains 601 genomes registered under the genus Sphingomonas, and a search for closely related species was conducted based on average nucleotide identity (ANI). A widely used method for determining ANI identity is 95-96% or higher. As shown in Table 4, S. azotifigens, S. trueperi, S. pituitosa, and S. elodea were identified as closely related species to NB7. Even S. azotifigens NBRC 15497, which had the highest ANI, only had an ANI of 89.03%.

[0021]

[0022] Since the genomes of all currently reported Sphingomonas species share less than 90% of the NB7 and ANI ratios, the NB7 strain is considered to be a new species. Furthermore, a phylogenetic tree was constructed using RaxML-NG v1.1.0 for NB7, 27 Sphingomonas species, and Zymomonas mobilis as an outgroup, for a total of 29 strains. 235 marker genes common to the 29 strains were selected, and amino acid sequences were used to construct the tree. Branch support values ​​were calculated using 1,000 bootstrap runs. The results are shown in Figure 1. The NB7 branch is indicated by a square and an asterisk (*). NB7 lacks nitrogenase-related genes such as nifH, suggesting that it does not fix nitrogen itself. These findings demonstrate that NB7 is a novel symbiotic bacterial strain of the Sphingomonas genus that plays a role in the growth-promoting function of OsPep.

[0023] 8. NB7 Efficacy Test (1) Next, rice seeds or seedlings approximately 1 week after germination, treated with antifungal agents according to standard procedures, were inoculated with NB7 and simultaneously treated with OsPep. 50 μL of OsPep solution (1 mM) was administered per seedling cell along with the bacterial culture suspension. This test was performed using wild-type rice and ccamk-deficient rice mutants to examine the dependence of the symbiotic regulator CCaMK. Figure 2 shows the results of plant growth in wild-type rice (A) and ccamk-deficient rice mutants (B) in the presence (+) or absence (-) of the symbiotic bacterium NB7 and the presence (+) or absence (-) of OsPep3-C. Photographs of representative rice plants are shown on the top panel, and a graph comparing plant weights (mg) is shown on the bottom panel (data are shown as mean ± SE, N ≥ 11). In the graph, an asterisk (*) indicates a significant difference at a level of less than 5% (p < 0.05).

[0024] In wild-type rice, inoculation with the symbiotic fungus NB7 tended to improve rice growth. Furthermore, the addition of OsPep3-C significantly improved rice growth. On the other hand, inoculation with the symbiotic fungus NB7 did not significantly improve rice growth in the ccamk-deficient mutant. Although there was a slight decrease in growth, no significant difference was observed between NB7(-) OsPep3-C(-) and NB7(+) OsPep3-C(-). Furthermore, administration of OsPep3-C did not promote rice growth. OsPep3-C, one of the OsPeps, is known to activate rice immunity, and its other functions are also being investigated. OsPep3-C, acting in concert with NB7, significantly promoted rice growth. CCaMK (Calcium- and calmodulin-dependent protein kinase) is known to be a symbiotic regulator and is required for both root nodule and mycorrhizal symbiosis. However, the role of CCaMK in regulating the symbiotic microbiota is not fully understood. By comparing ccamk-deficient mutants with wild-type strains, it is possible to evaluate whether the physiological effects of specific factors (e.g., symbiotic microorganisms) depend on CCaMK. Our results indicate that the growth-promoting effect of NB7 and / or OsPep3-C on rice plants is dependent on CCaMK.

[0025] 9. NB7 Efficacy Test (2) Next, rice seeds or seedlings approximately 1 week after germination were inoculated with NB7 and treated with OsPep simultaneously. 50 μL of 1 mM OsPep solution was administered per seedling cell along with the bacterial culture suspension. This test was performed using wild-type rice and ospepr1-deficient rice to examine the relationship with OsPep3C. Figure 3 shows the results of plant growth in wild-type rice (A) and ospepr1-deficient rice (B) in the presence (+) or absence (-) of the symbiotic bacterium NB7 and the presence (+) or absence (-) of OsPep3C. Photographs of representative rice plants are shown on the top panel, and a graph comparing plant weights (mg) is shown on the bottom panel (data are shown as mean ± SE, N ≥ 11). In the graph, an asterisk (*) indicates a significant difference at a level of less than 5% (p < 0.05).

[0026] In wild-type rice, inoculation with the symbiotic fungus NB7 tended to improve rice growth. Addition of OsPep3C to this mixture significantly improved rice growth. In contrast, inoculation with the symbiotic fungus NB7 did not have a growth-promoting effect on ospepr1-deficient mutants. Furthermore, administration of OsPep3C did not promote rice growth. OsPEPR1 is an OsPep receptor, activated by binding to OsPep and transmitting signals. These results suggest that inoculation with NB7 did not promote rice growth in the ospepr1-deficient mutant because OsPep3C is unable to activate OsPEPR1. This suggests that inoculation with NB7 of substances that activate OsPEPR1 (proteins, amino acids, and low-molecular-weight compounds) may enhance rice growth. Several members of the OsPep family have been identified. OsPep is known to be expressed as a precursor (OsPROPEP) and then cleaved at an appropriate site to become OsPeP. Table 5 shows the gene names, common names, number of OsPep motifs, database URLs, and OsPROPEP sequences of the OsPep peptide precursor family, as disclosed in Plant J 2020. doi: 10.1111 / tpj.15022. The amino acid sequences in Table 5 are listed in the sequence listing as SEQ ID NOs: 18 to 25, in order.

[0027]

[0028] 10. NB7 Effect Confirmation Test (3) Next, the effects of NB7 and OsPep3-C on plant growth were examined using Hinohikari rice. The evaluation groups were a control group (mock), an NB7 and OsPep3-C treatment group (NB7+pep), and an Achromobacter group (Achromobacter sp.). Achromobacter is known to promote rice growth. The test method was the same as described in Section 9 above. Figure 4 shows the results. Data were n = 18 for each group, and the total data and mean values ​​(horizontal bars) are shown. 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 NB7 and OsPep3-C treatment group also demonstrated significant growth-promoting effects compared to the control group. The results of the above test showed that NB7 and / or OsPep significantly promoted the growth of 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, rice growth was promoted from immediately after transplanting through to the ripening stage, without any negative impact on yield, etc.

[0029] Thus, this embodiment provides a microorganism that can promote rice growth throughout the cultivation period by enhancing rice seedling growth and stress tolerance in the seedbed and stabilizing and increasing the establishment and growth of seedlings immediately after transplanting, which are vulnerable to cold damage and disease. In particular, inoculating seeds or seedlings with the rice immune-activating peptide OsPep promoted seedling growth even under nutrient (especially phosphorus)-deficient conditions. As a result, the microorganism promoted the establishment of seedlings immediately after transplanting, which is the period most susceptible to environmental changes, stress, and disease during the rice cultivation period, and stabilized rice growth in the field. The inoculation and symbiotic effects of this strain were particularly evident under low-nutrient, low-fertilization conditions, and its effectiveness has also been confirmed in unfertilized paddy fields, which is expected to contribute to the promotion of low-fertilization cultivation.

[0030] [Rule 26, amended 25.12.2023]

Claims

1. A microorganism that promotes the growth of grasses, which is a Sphingomonas microorganism and has been deposited under Accession No. NITE BP-03939.

2. An agricultural composition containing a Sphingomonas microorganism and promoting the growth of grasses, the agricultural composition comprising a 16S rRNA gene having a homology of 95.0% or more with the base sequence of SEQ ID NO:

1.

3. The agricultural composition according to claim 2, wherein the Sphingomonas microorganism is a microorganism according to claim 1.

4. The agricultural composition according to claim 2 or 3, further comprising OsPep.

5. The agricultural composition according to claim 2 or 3, further comprising an OsPEPR1 activator that activates the OsPep receptor OsPEPR1.

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

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

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