WO2021066115A1 - 土壌の植物生産性を改善する微生物の使用 - Google Patents
土壌の植物生産性を改善する微生物の使用 Download PDFInfo
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- WO2021066115A1 WO2021066115A1 PCT/JP2020/037476 JP2020037476W WO2021066115A1 WO 2021066115 A1 WO2021066115 A1 WO 2021066115A1 JP 2020037476 W JP2020037476 W JP 2020037476W WO 2021066115 A1 WO2021066115 A1 WO 2021066115A1
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- bacteria belonging
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- soil
- compost
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions
- the present invention uses bacteria belonging to the order Rhizobiales to improve soil plant productivity, compositions, methods, and composts, as well as compositions, microorganisms, to increase the antioxidant activity of plants. And compost.
- Ammonia and lime nitrogen produced by industrial nitrogen fixation are nitrogenous chemical fertilizers and their raw materials, and are widely used for crop cultivation in modern agriculture.
- industrial nitrogen fixation depends on fossil resources for a part of the reaction substrate and the energy required for the reaction, and its sustainability is limited.
- the large-scale use of fossil carbon is one of the factors that impair the stability of the global environment. Therefore, since the technology to promote the agricultural use of biological nitrogen fixation is indispensable for a sustainable agricultural system, research on microorganisms that supply nitrogen to plants and promote their growth has been widely conducted. There is.
- Non-Patent Document 1 a method for artificially manipulating the flora of soil has not been established, and therefore it has been difficult to improve the plant productivity of soil using such a method.
- the problem to be solved by the present invention is to improve the plant productivity of the soil by manipulating the flora of the soil, and to increase the antioxidant activity of the plant to obtain the productivity of the plant and its harvest. It is to improve the quality of things.
- the inventors of the present application changed the flora of the soil by adding a bacterium belonging to the order Rhizobiales to the soil, thereby changing the flora of the plant.
- the inventors of the present application have found the surprising finding that bacteria belonging to the genera Bacillus, Promicromonospora, or Olivibacter increase the antioxidant activity of plants. Based on these findings, the present invention has been completed.
- the present invention is as follows. [1] A composition for improving plant productivity of soil, which comprises a bacterium belonging to the order Rhizobiales. [2] A method for improving plant productivity of soil, which comprises adding a bacterium belonging to the order Rhizobiales to the soil. [3] The method according to 2, wherein the plant is a cruciferous plant. [4] The method according to 3, wherein the cruciferous plant is Camelina or Komatsuna. [5] The method according to any one of 2 to 4, wherein the number of pods per plant, the number of seeds per plant, and / or the weight of the plant body is increased by improving the plant productivity of the soil.
- Bacteria belonging to the order Rhizobiales Bacteria belonging to the order Actinomycetales, bacteria belonging to the order Bacillales, bacteria belonging to the order Gaiellales, belonging to the order Myxococcales. Bacteria, Bacteria belonging to the order iii1-15, Bacteria belonging to the order Solilbrobacterales, Bacteria belonging to the order Xanthomonadales, Bacteria belonging to the order Burkholderiales, and Bacteria belonging to the order Gemmatales. Compost containing bacteria belonging to. [7] The abundance ratio of bacteria belonging to the order Rhizobiales in the compost is 9% or more, and the abundance ratio of bacteria belonging to the order iii1-15 is 6% or less, according to 6. compost.
- a composition for increasing the antioxidant activity of a plant which comprises a bacterium belonging to the genus Bacillus, Promicromonospora, or Olivibacter.
- the bacterium belonging to the genus Bacillus is Bacillus cereus, and the bacterium belonging to the genus Promicromonospora is Promicromonospora citrea.
- the composition according to 10, wherein the bacterium belonging to the genus Olivibacter is a species of Olivibacter (Olivibacter sp.).
- Bacillus cereus is a strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-02974, and Promicromonospora citrea is deposited at the Patent Microorganisms Depositary Center. 11. The composition according to 11, wherein the strain is deposited under the number NITE BP-03025 and the Olivibacter sp. Is a strain deposited at the Patent Microorganisms Depositary Center under the accession number NITE BP-03026. [13] A bacterial strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-02974. [14] A bacterial strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-03025. [15] A bacterial strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-03026. [16] A compost containing the bacterial strain according to any one of 13 to 15.
- the plant productivity of the soil is improved, and the yield of the plant is significantly improved by increasing the number of sheaths per plant, the number of seeds per plant, and / or the weight of the plant body.
- the antioxidant activity of plants it is possible to significantly improve the productivity of plants related to oxidative stress and the quality of their crops (for example, functionality, disease resistance, storage stability, etc.). Become.
- (A) It is a photograph of a camelina cultivated using the compost according to the present invention.
- composition for improving plant productivity of soil which comprises a bacterium belonging to the order Rhizobiales, is provided.
- "improving the plant productivity of soil” means promoting the plant growth ability inherent in the soil, whereby the yield of the plant, for example, the number of sheaths per plant, the number of seeds per plant, etc. And / or means to increase the weight of the plant.
- Rhizobiales is an eye belonging to the class Alphaproteobacteria, and is a large eye with more than 130 genera in 17 families.
- the order Rhizobiales contains a wide variety of species. Some species that coexist with plants form nodules, which are agriculturally important because they supply nitrogen to the plants.
- Agriculturally important Rhizobium species include, for example, the N 2 fixed bacterial Rhizobium species, which can form nodules on the roots of legumes via symbiotic nitrogen fixation. Converts atmospheric N 2 to ammonia, which can be used as a nitrogen source by plants in contrast to atmospheric N 2.
- Concentration of Rhizobium (Rhizobiales) belonging to the eye bacteria contained in the composition of the present invention typically, 10 6 CFU / g or more, preferably, 10 7 CFU / g or higher, and optimally, 10 8 CFU / G or more.
- composition of the present invention may be solid or liquid and imparts various properties such as increased stability, wettability, or dispersibility in addition to the active ingredient Rhizobiales. It may contain a carrier to be used.
- the carrier is typically an agricultural carrier, including soil, plant growth medium, water, fertilizers, plant oils, wetting agents, or combinations thereof.
- a method for improving plant productivity of soil which comprises adding a bacterium belonging to the order Rhizobiales to the soil.
- Addition of bacteria belonging to the order Rhizobiales to soil is carried out, for example, by adding the composition of the present invention to the soil prior to planting the desired plant or by mixing it with the soil at the time of planting. be able to.
- the plants whose productivity is improved by this method are not particularly limited, and are typically agricultural products, such as Brassicaceae, Gramineae, Leguminosae, Asteraceae, Solanaceae, Solanaceae, and Morningglories.
- Examples include plants of the family Asteraceae, preferably cruciferous plants.
- Examples of cruciferous plants include camelina or komatsuna.
- camelina is one of the crops that has been cultivated in Europe for a long time to obtain oil, has high oil production capacity, short-term maturity, low demand for water and nutrients, resistance to pathogens and pests. It has some agricultural advantages such as, and in recent years, the oil has been attracting attention as a raw material for biofuels.
- a bacterium belonging to the order Rhizobiales a bacterium belonging to the order Actinomycetales, a bacterium belonging to the order Bacillales, a bacterium belonging to the order Gaiellales, mixococcus.
- Rhizobiales Bacteria belonging to the order Rhizobiales are typically bacteria belonging to the genera Rhodoplanes, Bradyrhizobium, Pedomicrobium, such as Rhodoplanes elegans, Methylobacterium adhaesivum.
- Bacteria belonging to the order Actinomycetales are typically bacteria belonging to the genera Terracoccus, Mycobacterium, Streptomyces, such as Actinomadura vinacea, Rathayibacter caricis, Actinoallomurus iriomotensis.
- Bacteria belonging to the order Bacillus are typically bacteria belonging to the genera Bacillus, Rummeliibacillus, Planifilum, such as Bacillus cereus, Paenibacillus chondroitinus, Bacillus clausii.
- Bacteria belonging to the order Gaiellales are typically bacteria belonging to the family Gaiellaceae, AK1AB1 02E.
- Bacteria belonging to the order Myxococcales are typically bacteria belonging to the genera Sorangium, Plesiocystis, Nannocystis, such as Sorangium cellulosum.
- Bacteria belonging to the order iii1-15 are typically bacteria belonging to the RB40 family and the mb2424 family.
- Bacteria belonging to the order Solirubrobacterales are typically bacteria belonging to the genus Conexibacter.
- Bacteria belonging to the order Xanthomonadales are typically bacteria belonging to the genera Stenotrophomonas, Luteimonas, Dokdonella, such as Stenotrophomonas acidaminiphila, Pseudoxanthomonas mexicana.
- Bacteria belonging to the order Burkholderiales are typically bacteria belonging to the genera Burkholderia, Polaromonas, and Methylibium.
- Bacteria belonging to the order Gemmatales are typically bacteria belonging to the genus Gemmatas.
- the abundance ratio of each bacterium in the compost is not particularly limited as long as the plant productivity is improved, but the abundance ratio of the bacterium belonging to the order Rhizobiales is 9% or more and belongs to the order iii1-15.
- the abundance ratio of bacteria is preferably 6% or less.
- Composting can be carried out using methods customary to those skilled in the art, and generally, composting raw materials such as sludge, livestock manure, straw, and dead grass are mixed with aerobic microorganisms that decompose them. It is carried out by fermentation under aerobic conditions. In composting, water content, pH, carbon-to-nitrogen ratio (C / N ratio), temperature, oxygen, etc. affect the rate of decomposition of organic matter and cause nitrogen starvation of crops. It is important to adjust.
- composting raw materials such as sludge, livestock manure, straw, and dead grass are mixed with aerobic microorganisms that decompose them. It is carried out by fermentation under aerobic conditions.
- water content, pH, carbon-to-nitrogen ratio (C / N ratio), temperature, oxygen, etc. affect the rate of decomposition of organic matter and cause nitrogen starvation of crops. It is important to adjust.
- the moisture content of the compost is typically about 30-60%, preferably about 25-55%.
- the pH of the compost is preferably about 5.5 to 8.5.
- the amount (EC) of ions such as potassium, sodium, chlorine and nitric acid contained in compost is preferably low. It is preferably about 3.0 dS / m or less for bark compost and about 5.0 dS / m or less for livestock manure.
- the ratio of carbon to nitrogen (C / N ratio) in compost is preferably about 10 to 40 because if the value is too large, the soil may cause nitrogen starvation.
- the C / N ratio measures carbon and nitrogen of easily decomposable organic matter and persistent organic matter at the same time, for example, the C / N ratio of sawdust is very high, about 340 to 1250, and sawdust is mixed as an auxiliary material. The C / N ratio of the compost produced tends to increase.
- nitrate nitrogen is formed by nitrifying ammonia, and this reaction occurs mainly during secondary fermentation.
- the fertilizer component balance in compost (the ratio of potassium when the total amount of nitrogen is 1) is preferably low, and the appropriate value is 5 or less.
- heavy metals especially copper and zinc
- the appropriate value of heavy metal concentration in compost is 300 ppm or less for copper and zinc. It is 900 ppm or less.
- the compost of the present invention has extremely high productivity with respect to the above-mentioned plants.
- compositions for increasing the antioxidant activity of a plant which is a bacterium belonging to the genus Bacillus, Promicromonospora, or Olivibacter. Including, the composition is provided.
- Oxidative stress due to accumulation of reactive oxygen species (ROS) is related to the cause of plant death due to such environmental stress.
- ROS reactive oxygen species
- changes in the intracellular redox state that depend on the balance between ROS production and elimination act as a signal, and the expression of the defense system during environmental stress response, as well as the physiology of programmed cell death and growth / development.
- Bacteria belonging to the genus Bacillales, Promicromonospora, or Olivibacter which are known to be involved in the control of the phenomenon, are now plants. We have found a surprising finding that it increases the antioxidant activity of.
- the bacterium belonging to the genus Bacillus is preferably Bacillus cereus, and most preferably a bacterial strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-02974.
- the bacterium belonging to the genus Promicromonospora is preferably Promicromonospora citrea, and most preferably a bacterial strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-03025.
- the bacterium belonging to the genus Olivibacter is preferably an Olivibacter species (Olivibacter sp.), And most preferably a strain deposited at the Patent Microorganisms Depositary Center under the accession number NITE BP-03026.
- the compost used for camelina cultivation was produced as follows. For the compost used in the test plot, pig droppings were used as a raw material, sawdust was used as an auxiliary material, and a bacterium belonging to Rhizobiales, which was independently isolated and cultured, was used as a bacterium to be further added. Composting was started by mixing pig droppings, fungal culture solution, and sawdust. The water content was adjusted to about 60% with sawdust, and the mixture was stirred as needed to make compost over 3 months. The compost used in the control group was also produced by the same method except that no bacteria were added.
- the primary PCR targets the variable region V4 (about 250 bp) of the 16S rRNA gene, and the primers 1st_515F_MIX (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-GTGCCAGCMGCCGCGCGGTAA-3': SEQ ID NO: 1) and 1st_806R_MIXTG. 3': SEQ ID NO: 2) was used.
- the primers used in the sequence analysis were mixed primers into which random sequences of different lengths of 0 to 5 bases were inserted for the purpose of improving quality.
- primer 2nd F (5'-AATGATACGGCGACCACCGAGATCTACAC-Index2 (TATAGCCT)-ACACTCTTTCCCTACACGACGC) that binds to the common sequences at both ends of the primary PCR product and has a sample identification index added.
- the primary PCR product was amplified using -3': SEQ ID NO: 3) and 2nd R (5'-CAAGCAGAAGACGGCATACGAGAT-Index1 (GCAGCGTA) -GTGACTGGAGTTCAGACGTGTG-3': SEQ ID NO: 4).
- Each PCR product was purified by Agencourt AMPure XP (BECKMAN COU-LTER). Sequence analysis of the obtained library was performed using MiSeq (Illumina).
- the nucleotide sequence data obtained by sequencing was analyzed as follows. Using fastq_barcode_spliltter of Fastx toolkit, only the sequences whose sequence reading start exactly matches the primer used were extracted. The primer sequence of the extracted sequence was deleted. Then, the sequence having a quality value of less than 20 was removed, and the sequence having a length of 40 bases or less and its pair sequence were discarded. Arrays that have undergone quality filtering have been merged using the pair-end merge script FLASH. The conditions for merging were a fragment length of 260 bases after merging, a read fragment length of 230 bases, and a minimum overlap length of 10 bases. The merged sequences were filtered by fragment length and only 246 to 260 bases were used for subsequent analysis.
- Chimeric sequences were checked for sequences that passed all filtering using usearch's uchime algorithm.
- the database was 97% OTU of Greengene attached to the bacterial flora analysis pipeline Qime, and all the sequences that were not judged to be chimeric were extracted and used for the subsequent analysis.
- Qiime's workflow script was used for OTU creation and system estimation.
- FIG. 1 shows the state of camelina being cultivated and the strains of the test plot and the control plot after harvesting.
- FIG. 2 and Table 1 show the results of comparing the cultivated amount according to the present invention with the cultivated amount in Japan and overseas. From this result, it can be confirmed that the number of pods per plant is several times higher than that of other cultivation except for the cultivation example in India. Since the number of species per Pod in the present invention was medium, it is considered that the number of seeds per plant increased. In addition, since the seed weight was not significantly different from other cultivation results, it is considered that the seed yield per plant increased. This is clear from the photograph of FIG.
- Soil flora analysis The flora in the soil was analyzed by subjecting the DNA extracted from the soil to the next-generation sequence. In the test plot, the fungus Rhizobiales added this time was detected most frequently, accounting for 9.42% of the total. Other than Rhizobiales, the most detected bacteria are Actinomycetales (7.61%), Bacillales (7.02%), and Gaiellales (6.58%). , Myxococcales (4.89%), iii1-15 (3.32%), Solirubrobacterales (3.09%), Xanthomonadales (2.). 40%), Burkholderiales (2.33%), Gemmatales (2.30%).
- Rhizobiales eyes detected in the control group was 5.75% of the total, which was less than that in the test group.
- Other bacteria with high detection levels are Actinomycetales (7.52%), Gaiellales (7.21%), Bacillales (6.01%), and Myxococcales. ) Eyes (5.11%), iii1-15 eyes (3.26%), Solirubrobacterales eyes (3.25%), Burkholderiales eyes (2.53%) , Gemmatales (2.45%) and Nitrososphaerales (2.42%).
- Non-Patent Document 5 It has been found that in camelina cultivation, the number of pods per plant and the seed weight tend to decrease as the cultivation density increases (Non-Patent Document 5).
- the yield per plant shows a very high value in the cultivation example in India, but this is the result of cultivation at an extremely low density (17.6 plants / m 2 ), and the yield per unit area is It is as low as 1.31 t / ha.
- the number of pods per plant was high even though the cultivation density was medium.
- the yield per unit area was 6.75 t / ha, which was very high.
- the yield per unit area of the control plot was not significantly different from that of the existing cultivation examples.
- Rhizobiales was detected most frequently at 9.42% of the total, and it increased even when compared with the control group. From this result, it is presumed that the addition of bacteria to the compost increased the number of Rhizobiales in the soil and contributed to the improvement of soil productivity.
- Non-Patent Document 1 Focus on the positive correlation between Rhizobiales and iii1-15, and the negative correlation between Acidobacteriales and Solibacterales. Comparisons were made with the proportions of bacteria in the test plots and control plots, as well as at 12 sites reported in Non-Patent Document 1. Table 2 shows the results. In addition, Table 3 is a correspondence table of the code representing the sampling point of each soil and the latitude and longitude in Non-Patent Document 1.
- Rhizobiales is the second most common (9.42%) and iii1-15 is the eighth (3.32%).
- Acidobacteriales was the third lowest (0.82%), and Solibacterales was the second lowest (1.02%).
- the soil of the control plot had the highest number of Rhizobiales (5.75%), which was less than that of the test plot.
- the abundance rate of bacteria other than Rhizobiales is 9th (3.26%) in iii1-15, 4th (1.10%) in Acidobacteriales, and sori.
- the order of Rhizobiales (Solibacterales) was the third lowest (1.26%). In any case, the soil in the test plot is considered to have an extremely productive bacterial composition.
- camelina was cultivated using compost containing bacteria, and it was confirmed that a yield of 2.4 times or more per unit area was obtained compared to existing reports in Japan and overseas.
- a yield of 2.4 times or more per unit area was obtained compared to existing reports in Japan and overseas.
- the possibility of a method of manipulating the flora of the cultivated soil and increasing the yield of the product by adding a specific bacterium at the time of compost production has been found.
- Example 2 The compost used for Komatsuna cultivation was produced as follows. For the compost used in the test plot, pig droppings, sawdust, and a culture solution of a bacterium belonging to the order Rhizobiales, which was independently isolated and cultured, were used as raw materials. Composting was started by mixing pig droppings, fungal culture solution, and sawdust. The water content was adjusted to 60% with sawdust, and the mixture was stirred as needed to make compost over 3 months. The compost used in the control group was produced by the same method except that no bacteria were added.
- the produced compost was used for the Komatsuna (Brassica rapa var. Perviridis) pot cultivation test.
- Komatsuna pot cultivation test soil in which production compost and Akadama soil were mixed at a ratio of 3: 7 was used. Further, 0.84 g of a chemical fertilizer containing 8% each of ammoniacal nitrogen, soluble phosphoric acid and water-soluble potassium was used with respect to 1 L of soil. Cultivation was carried out from seeds at room temperature under fluorescent light, and their growth was observed.
- Example 3 Evaluation of antioxidant power of Komatsuna 3.1 Bacillus cereus Komatsuna was inoculated with 2764-01-S16 (accession number NITE BP-02974), which is a strain of Bacillus cereus isolated by the applicant, and a cultivation test of Komatsuna was conducted. Specifically, the fungus was inoculated by immersing the fungus suspension in the roots of Komatsuna seedlings cultivated with vermiculite on the 10th day for about 30 seconds. Sterilized water was used for the control group. Cultivation was carried out using a soil in which steam-sterilized field soil and vermiculite were mixed at a ratio of 1: 1 and liquid fertilizer was given about once a week. Cultivation was carried out in a vinyl house.
- the edible part of Komatsuna cultivated above is used as an analysis sample, cut into 1 cm squares, mixed with 4 times the weight of water, destroyed with a juicer, heated at 80 ° C. for 30 minutes, cooled and then filtered. Then, a sample solution was prepared. Mix 25 ⁇ L of sample solution, 44.4 mM 2,2,6,6-tetramethyl-4-piperidone (TMPD) 50 ⁇ L, 2.5 mM dimethyl sulfoxide (DMSO) 100 ⁇ L, and 55.5 mM riboflavin 50 ⁇ L into the mixed solution.
- TMPD 2,2,6,6-tetramethyl-4-piperidone
- DMSO dimethyl sulfoxide
- ESR electron spin resonance
- the intensity of radicals trapped in TMPD which is a supplement, can be measured.
- the actual amount of substance can be estimated based on this intensity from a calibration curve prepared in advance using histidine as a standard substance for observing singlet oxygen scavenging activity (antioxidant power).
- the higher the antioxidant power of the sample the more radicals are eliminated and not captured by the supplement, resulting in a smaller signal. Comparing the weight and antioxidant power of the Komatsuna strain that was not inoculated with the bacterium, the weight of the Komatsuna strain that was not inoculated with the bacterium was 3.15 g, and the antioxidant power was 1560 ⁇ mol histidine / g. Since the weight of the strain was 4.07 g and the antioxidant power was 1890 ⁇ mol histidine / g, a significant improvement in weight and antioxidant power was observed in the Komatsuna strain inoculated with the bacterium.
- Promicromonospora citrea Komatsuna was inoculated with 27624-02-C06 (accession number NITE BP-03025), which is a strain of Promicromonospora citrea isolated by the applicant, and a cultivation test of Komatsuna was conducted. Specifically, the fungus was inoculated by immersing the fungus suspension in the roots of Komatsuna seedlings cultivated with vermiculite on the 10th day for about 30 seconds. Sterilized water was used for the control group. Cultivation was carried out using a soil in which steam-sterilized field soil and vermiculite were mixed at a ratio of 1: 1 and liquid fertilizer was given about once a week. Cultivation was carried out in a vinyl house.
- the edible part of Komatsuna cultivated above is used as an analysis sample, cut into 1 cm squares, mixed with 4 times the weight of water, destroyed with a juicer, heated at 80 ° C. for 30 minutes, cooled and then filtered. Then, a sample solution was prepared. Mix 50 ⁇ L of sample solution, 5.7 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO) 20 ⁇ L, and 2.5 mM hydrogen peroxide 90 ⁇ L, irradiate the mixed solution with ultraviolet rays for 30 seconds, and then measure conditions.
- DMPO 5,5-dimethyl-1-pyrroline N-oxide
- the intensity of radicals trapped in DMPO which is a supplement, can be measured.
- the actual amount of substance can be estimated based on this intensity from a calibration curve prepared in advance using DMSO as a standard substance for observing hydroxyl radical scavenging activity (antioxidant power).
- the higher the antioxidant power of the sample the more radicals are eliminated and not captured by the supplement, resulting in a smaller signal.
- the weight and antioxidant power of the Komatsuna strain not inoculated with the bacterium were compared, the weight of the Komatsuna strain not inoculated with the bacterium was 3.51 g and the antioxidant power was 1670 ⁇ mol DMSO / g. Since the weight of the strain was 4.29 g and the antioxidant power was 2350 ⁇ mol DMSO / g, a significant improvement in weight and antioxidant power was observed in the Komatsuna strain inoculated with the bacterium.
- the edible part of Komatsuna cultivated above is used as an analysis sample, cut into 1 cm squares, mixed with 4 times the weight of water, destroyed with a juicer, heated at 80 ° C. for 30 minutes, cooled and then filtered. Then, a sample solution was prepared.
- the superoxide radical scavenging activity was measured by the ESR method set to 250, and the antioxidant power of Komatsuna was evaluated.
- the intensity of radicals trapped in DMPO which is a supplement, can be measured.
- the actual amount of substance can be estimated based on this intensity from a calibration curve prepared in advance using superoxide gymstase as a standard substance for observing superoxide radical scavenging activity (antioxidant power).
- the higher the antioxidant power of the sample the more radicals are eliminated and not captured by the supplement, resulting in a smaller signal.
- the weight and antioxidant power of the Komatsuna strain not inoculated with the bacterium were compared, the weight of the Komatsuna strain not inoculated with the bacterium was 3.51 g and the antioxidant power was 122 units SOD / g, and the bacterium was inoculated. Since the weight of the Komatsuna strain was 4.47 g and the antioxidant power was 190 units SOD / g, a significant improvement in weight and antioxidant power was observed in the Komatsuna strain inoculated with the bacterium.
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Abstract
Description
[1] リゾビウム(Rhizobiales)目に属する細菌を含む、土壌の植物生産性を改善するための組成物。
[2] 土壌の植物生産性を改善する方法であって、前記土壌にリゾビウム(Rhizobiales)目に属する細菌を添加することを含む、方法。
[3] 前記植物がアブラナ科植物である、2に記載の方法。
[4] 前記アブラナ科植物がカメリナ又はコマツナである、3に記載の方法。
[5] 前記土壌の植物生産性が改善することにより、植物あたりの鞘数、植物あたりの種子数、及び/又は植物体の重量が増大する、2~4のいずれかに記載の方法。
[6] リゾビウム(Rhizobiales)目に属する細菌、ならびに、アクチノミセス(Actinomycetales)目に属する細菌、バシラス(Bacillales)目に属する細菌、ガイエラレス(Gaiellales)目に属する細菌、ミクソコッカス(Myxococcales)目に属する細菌、iii1-15目に属する細菌、ソリルブロバクテラレス(Solirubrobacterales)目に属する細菌、キサントモナス(Xanthomonadales)目に属する細菌、バークホルデリア(Burkholderiales)目に属する細菌、及びゲルマタレス(Gemmatales)目に属する細菌を含む堆肥。
[7] 前記堆肥中の前記リゾビウム(Rhizobiales)目に属する細菌の存在比率が9%以上であり、かつ、前記iii1-15目に属する細菌の存在比率が6%以下である、6に記載の堆肥。
[8] アブラナ科植物を栽培するための、6又は7に記載の堆肥。
[9] 前記アブラナ科植物がカメリナ又はコマツナである、8に記載の堆肥。
[10] 植物の抗酸化活性を増大させるための組成物であって、バシラス(Bacillales)属、プロミクロモノスポラ(Promicromonospora)属、又はオリビバクター(Olivibacter)属に属する細菌を含む、組成物。
[11] 前記バシラス(Bacillales)属に属する細菌が、バシラス・セレウス(Bacillus cereus)であり、プロミクロモノスポラ(Promicromonospora)属に属する細菌が、プロミクロモノスポラ・シトレア(Promicromonospora citrea)であり、オリビバクター(Olivibacter)属に属する細菌が、オリビバクター種(Olivibacter sp.)である、10に記載の組成物。
[12] バシラス・セレウス(Bacillus cereus)が、特許微生物寄託センターに受託番号NITE BP-02974で寄託されている株であり、プロミクロモノスポラ・シトレア(Promicromonospora citrea)が、特許微生物寄託センターに受託番号NITE BP-03025で寄託されている株であり、オリビバクター種(Olivibacter sp.)が、特許微生物寄託センターに受託番号NITE BP-03026で寄託されている株である、11に記載の組成物。
[13] 特許微生物寄託センターに受託番号NITE BP-02974で寄託されている細菌株。
[14] 特許微生物寄託センターに受託番号NITE BP-03025で寄託されている細菌株。
[15] 特許微生物寄託センターに受託番号NITE BP-03026で寄託されている細菌株。
[16] 13~15のいずれかに記載の細菌株を含む堆肥。
リゾビウム(Rhizobiales)目に属する細菌は、典型的には、Rhodoplanes属、Bradyrhizobium属、Pedomicrobium属、例えば、Rhodoplanes elegans、Methylobacterium adhaesivumに属する細菌である。アクチノミセス(Actinomycetales)目に属する細菌は、典型的には、Terracoccus属、Mycobacterium属、Streptomyces属、例えば、Actinomadura vinacea、Rathayibacter caricis、Actinoallomurus iriomotensisに属する細菌である。バシラス(Bacillales)目に属する細菌は、典型的には、Bacillus属、Rummeliibacillus属、Planifilum属、例えば、Bacillus cereus、Paenibacillus chondroitinus、Bacillus clausiiに属する細菌である。ガイエラレス(Gaiellales)目に属する細菌は、典型的には、Gaiellaceae科、AK1AB1 02E科に属する細菌である。ミクソコッカス(Myxococcales)目に属する細菌は、典型的には、Sorangium属、Plesiocystis属、Nannocystis属、例えば、Sorangium cellulosumに属する細菌である。iii1-15目に属する細菌は、典型的には、RB40科、mb2424科に属する細菌である。ソリルブロバクテラレス(Solirubrobacterales)目に属する細菌は、典型的には、Conexibacter属に属する細菌である。キサントモナス(Xanthomonadales)目に属する細菌は、典型的には、Steroidobacter属、Luteimonas属、Dokdonella属、例えば、Stenotrophomonas acidaminiphila、Pseudoxanthomonas mexicanaに属する細菌である。バークホルデリア(Burkholderiales)目に属する細菌は、典型的には、Burkholderia属、Polaromonas属、Methylibium属に属する細菌である。ゲルマタレス(Gemmatales)目に属する細菌は、典型的には、Gemmata属に属する細菌である。
1.1 栽培条件
カメリナ(Camelina sativa)の栽培は、2018年3月25日から6月24日にかけて群馬県渋川市(36.53N、139.01E)の畑にて行った。カメリナ品種はカレナ(Calena)を用いた。畑には後述の方法により製造した堆肥を1.5kg/m2撒き、その他の化学肥料は使用しなかった。収穫時の鞘数、種子収量を測定し、他の栽培収穫量と比較した。試験区は15m2、対照区は1m2で栽培を行った。試験区の栽培密度は167株/m2、対照区の栽培密度は169株/m2であった。
カメリナ栽培に用いた堆肥は、以下のとおり製造した。試験区に用いた堆肥には原料として豚糞を、副資材としておが屑を、更に添加する菌として、独自に単離・培養したリゾビウム(Rhizobiales)に属する菌を用いた。豚糞、菌培養液、及びおが屑を混合することで堆肥化を開始した。含水率はおが屑により約60%に調整し、混合物は随時撹拌を行い、三か月かけて堆肥とした。対照区に用いた堆肥も菌の添加を行わなかった点を除き同様の方法で製造した。
土壌成分はJapan Soil Association(2010)を参照し、それぞれ次に示す方法により分析した。窒素全量(N):マクロコーダー(JM1000CN)、リン酸全量(P):硝酸-過塩素酸分解、バナドモリブデン酸アンモニウム法、カリウム全量(K):硝酸-過塩素酸分解、原子吸光測光法、石灰全量(Ca):硝酸-過塩素酸分解、原子吸光測光法、マグネシウム全量(Mg):硝酸-過塩素酸分解、原子吸光測光法。
VD-250Rフリーズドライヤー(TAITEC)を用いて、栽培土壌サンプルを凍結乾燥した。Shake Master Neo(bms)を用いて、凍結乾燥サンプルを粉砕した。その後、粉砕サンプルよりMPure Bacterial DNA Extraction Kit(MP Bio)を用いて、DNAを抽出した。ライブラリー作製は2 step tailed PCR法により実施した。1次PCRは16S rRNA遺伝子の可変領域V4(約250bp)を標的として、プライマー1st_515F_MIX(5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-GTGCCAGCMGCCGCGGTAA-3’:配列番号1)及び1st_806R_MIX(5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GGACTACHVGGGTWTCTAAT-3’:配列番号2)を用いた。シーケンス解析時に使用したプライマーは、品質向上を目的として、0~5塩基の異なる長さのランダム配列が挿入された混合プライマーであった。1次PCR産物の精製後、2次PCRでは、1次PCR産物の両末端にある共通配列と結合し、サンプル識別用インデックスを付加したプライマー2nd F(5’-AATGATACGGCGACCACCGAGATCTACAC-Index2(TATAGCCT)-ACACTCTTTCCCTACACGACGC-3’:配列番号3)及び2nd R(5’-CAAGCAGAAGACGGCATACGAGAT-Index1(GCAGCGTA)-GTGACTGGAGTTCAGACGTGTG-3’:配列番号4)を用いて1次PCR産物を増幅した。各PCR産物は、Agencourt AMPure XP(BECKMAN COU-LTER)により精製した。MiSeq(Illumina)を用いて、得られたライブラリーのシーケンス解析を実施した。
土壌生産性と相関を示す菌であるリゾビウム(Rhizobiales)目を添加した堆肥を用いて栽培を行った。図1に栽培中のカメリナの様子及び収穫後の試験区の株及び対照区の株を示す。図2と表1は、本発明による栽培量と国内外における栽培量を比較した結果である。この結果から、植物当たりの鞘数は、インドの栽培例を除き他栽培の数倍多い結果であることが確認できる。本発明でのPod当たりの種数については中程度であったことから、植物当たりの種子数が多くなったと考えられる。また、種子重量については他栽培結果と大きな差は見られないことから、植物当たりの種子収量が多くなったと考えられる。このことは図1(B)の写真からも明確である。栽培密度は中程度であることから、最終的な面積当たりの収量は6.75t/haと増大したことが分かる。図2から、この値は、国内外の栽培例の何れよりも多く、最も収量が多いフランスの2.8t/haと比較しても2.4倍以上であった。
土壌から抽出したDNAを次世代シークエンスに供することで、土壌中の菌相を分析した。試験区において今回添加した菌であるリゾビウム(Rhizobiales)目は最も多く検出され全体の9.42%であった。リゾビウム(Rhizobiales)目以外の検出量が多い菌として、アクチノミセス(Actinomycetales)目(7.61%)、バシラス(Bacillales)目(7.02%)、ガイエラレス(Gaiellales)目(6.58%)、ミクソコッカス(Myxococcales)目(4.89%)、iii1-15目(3.32%)、ソリルブロバクテラレス(Solirubrobacterales)目(3.09%)、キサントモナス(Xanthomonadales)目(2.40%)、バークホルデリア(Burkholderiales)目(2.33%)、ゲルマタレス(Gemmatales)目(2.30%)が挙げられる。
コマツナ栽培に用いた堆肥は、以下のとおり製造した。試験区に用いた堆肥には原料として豚糞、おが屑、更に独自に単離、培養したリゾビウム(Rhizobiales)目に属する菌の培養液を用いた。豚糞、菌培養液、及びおが屑を混合することで堆肥化を開始した。含水率はおが屑により60%に調整し、混合物は随時撹拌を行い、三か月かけて堆肥とした。対照区に用いた堆肥は菌の添加を行わなかった点を除き同様の方法で製造した。
3.1 バシラス・セレウス(Bacillus cereus)
コマツナに対して、出願人が単離したバシラス・セレウス(Bacillus cereus)の菌株である2764-01-S16(受託番号NITE BP-02974)を接種し、コマツナの栽培試験を行った。具体的には、菌の接種はバーミキュライトで栽培した10日目のコマツナ苗の根に菌の懸濁液を約30秒間浸漬し行った。なお対照区には滅菌水を用いた。栽培は蒸気滅菌した圃場土とバーミキュライトを1:1で混合した土を用い、液体肥料を約1週間に1回与えて行った。なお、栽培はビニールハウス内で実施した。
菌を接種しなかったコマツナ株と重量及び抗酸化力を比較したところ、菌を接種しなかったコマツナ株の重量は3.15g、抗酸化力は1560μmol ヒスチジン/gであり、菌を接種したコマツナ株の重量は4.07g、抗酸化力は1890μmol ヒスチジン/gであったことから、菌を接種したコマツナ株において、重量及び抗酸化力の有意な改善が見られた。
コマツナに対して、出願人が単離したプロミクロモノスポラ・シトレア(Promicromonospora citrea)の菌株である27624-02-C06(受託番号NITE BP-03025)を播種し、コマツナの栽培試験を行った。具体的には、菌の接種はバーミキュライトで栽培した10日目のコマツナ苗の根に菌の懸濁液を約30秒間浸漬し行った。なお対照区には滅菌水を用いた。栽培は蒸気滅菌した圃場土とバーミキュライトを1:1で混合した土を用い、液体肥料を約1週間に1回与えて行った。なお、栽培はビニールハウス内で実施した。
菌を接種しなかったコマツナ株と重量及び抗酸化力を比較したところ、菌を接種しなかったコマツナ株の重量は3.51g、抗酸化力は1670μmol DMSO/gであり、菌を接種したコマツナ株の重量は4.29g、抗酸化力は2350μmol DMSO/gであったことから、菌を接種したコマツナ株において、重量及び抗酸化力の有意な改善が見られた。
コマツナに対して、出願人が単離したオリビバクター種(Olivibacter sp.)の菌株である27624-02-C07(受託番号NITE BP-03026)を播種し、コマツナの栽培試験を行った。具体的には、菌の接種はバーミキュライトで栽培した10日目のコマツナ苗の根に菌の懸濁液を約30秒間浸漬し行った。なお対照区には滅菌水を用いた。栽培は蒸気滅菌した圃場土とバーミキュライトを1:1で混合した土を用い、液体肥料を約1週間に1回与えて行った。なお、栽培はビニールハウス内で実施した。
菌を接種しなかったコマツナ株と重量及び抗酸化力を比較したところ、菌を接種しなかったコマツナ株の重量は3.51g、抗酸化力は122ユニット SOD/gであり、菌を接種したコマツナ株の重量は4.47g、抗酸化力は190ユニット SOD/gであったことから、菌を接種したコマツナ株において、重量及び抗酸化力の有意な改善が見られた。
NITE BP-03025
NITE BP-03026
Claims (16)
- リゾビウム(Rhizobiales)目に属する細菌を含む、土壌の植物生産性を改善するための組成物。
- 土壌の植物生産性を改善する方法であって、前記土壌にリゾビウム(Rhizobiales)目に属する細菌を添加することを含む、方法。
- 前記植物がアブラナ科植物である、請求項2に記載の方法。
- 前記アブラナ科植物がカメリナ又はコマツナである、請求項3に記載の方法。
- 前記土壌の植物生産性が改善することにより、植物あたりの鞘数、植物あたりの種子数、及び/又は植物体の重量が増大する、請求項2~4のいずれか1項に記載の方法。
- リゾビウム(Rhizobiales)目に属する細菌、ならびに、アクチノミセス(Actinomycetales)目に属する細菌、バシラス(Bacillales)目に属する細菌、ガイエラレス(Gaiellales)目に属する細菌、ミクソコッカス(Myxococcales)目に属する細菌、iii1-15目に属する細菌、ソリルブロバクテラレス(Solirubrobacterales)目に属する細菌、キサントモナス(Xanthomonadales)目に属する細菌、バークホルデリア(Burkholderiales)目に属する細菌、及びゲルマタレス(Gemmatales)目に属する細菌を含む堆肥。
- 前記堆肥中の前記リゾビウム(Rhizobiales)目に属する細菌の存在比率が9%以上であり、かつ、前記iii1-15目に属する細菌の存在比率が6%以下である、請求項6に記載の堆肥。
- アブラナ科植物を栽培するための、請求項6又は7に記載の堆肥。
- 前記アブラナ科植物がカメリナ又はコマツナである、請求項8に記載の堆肥。
- 植物の抗酸化活性を増大させるための組成物であって、バシラス(Bacillales)属、プロミクロモノスポラ(Promicromonospora)属、又はオリビバクター(Olivibacter)属に属する細菌を含む、組成物。
- 前記バシラス(Bacillales)属に属する細菌が、バシラス・セレウス(Bacillus cereus)であり、プロミクロモノスポラ(Promicromonospora)属に属する細菌が、プロミクロモノスポラ・シトレア(Promicromonospora citrea)であり、オリビバクター(Olivibacter)属に属する細菌が、オリビバクター種(Olivibacter sp.)である、請求項10に記載の組成物。
- バシラス・セレウス(Bacillus cereus)が、特許微生物寄託センターに受託番号NITE BP-02974で寄託されている株であり、プロミクロモノスポラ・シトレア(Promicromonospora citrea)が、特許微生物寄託センターに受託番号NITE BP-03025で寄託されている株であり、オリビバクター種(Olivibacter sp.)が、特許微生物寄託センターに受託番号NITE BP-03026で寄託されている株である、請求項11に記載の組成物。
- 特許微生物寄託センターに受託番号NITE BP-02974で寄託されている細菌株。
- 特許微生物寄託センターに受託番号NITE BP-03025で寄託されている細菌株。
- 特許微生物寄託センターに受託番号NITE BP-03026で寄託されている細菌株。
- 請求項13~15のいずれか1項に記載の細菌株を含む堆肥。
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JP2018502869A (ja) * | 2015-01-16 | 2018-02-01 | バレント・バイオサイエンシーズ・リミテッド・ライアビリティ・カンパニーValent BioSciences LLC | 植物の害虫防除のための相乗的バチルス・チューリンゲンシス亜種アイザワイおよびクロラントラニリプロール混合物 |
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AU2016219488A1 (en) * | 2015-02-09 | 2017-09-14 | Bioconsortia, Inc. | Agriculturally beneficial microbes, microbial compositions, and consortia |
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2020
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- 2020-10-01 CN CN202080069782.8A patent/CN115843310A/zh active Pending
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AU2020360215A1 (en) | 2022-05-12 |
NO20220473A1 (en) | 2022-04-27 |
KR20220070224A (ko) | 2022-05-30 |
EP4039095A1 (en) | 2022-08-10 |
CN115843310A (zh) | 2023-03-24 |
US11963532B2 (en) | 2024-04-23 |
CA3155834A1 (en) | 2021-04-08 |
US20220361505A1 (en) | 2022-11-17 |
BR112022005696A2 (pt) | 2022-06-21 |
JPWO2021066115A1 (ja) | 2021-10-21 |
EP4039095A4 (en) | 2024-03-27 |
MX2022003839A (es) | 2022-06-08 |
JP6986807B2 (ja) | 2021-12-22 |
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