The use of microorganisms that improve soil plant productivity

Abstract

Soil plant productivity can be improved by manipulating the soil microbial community. This is achieved by adding bacteria belonging to the order Rhizobiales to the soil.

Description

Technical Field

[0001] This invention relates to compositions, methods, and composts for improving plant productivity in soil using bacteria belonging to the order Rhizobiales, as well as compositions, microorganisms, and composts for increasing the antioxidant activity of plants. Background Technology

[0002] Ammonia and calcium cyanamide, produced through industrial nitrogen fixation, are nitrogen-based chemical fertilizers or their raw materials, widely used in crop cultivation in modern agriculture. However, industrial nitrogen fixation relies on fossil resources for a portion of its reaction substrates and the energy required for the reaction, thus limiting its sustainability. Furthermore, the large-scale use of fossil carbon is one of the main causes of environmental instability. Therefore, since technologies that enhance the agricultural use of biological nitrogen fixation are indispensable for sustainable agricultural systems, extensive research has been conducted to date on microorganisms that supply nitrogen to plants and promote plant growth.

[0003] It is known that soil microbial community affects soil plant productivity. Specifically, the presence rate of bacteria belonging to the orders Rhizobiales and iii1-15 is positively correlated with soil plant productivity. Conversely, the presence rate of bacteria belonging to the orders Acidobacteriales and Solibacterales has been reported to be negatively correlated with soil plant productivity (Non-Patent Literature 1). However, to date, no method for artificially manipulating soil microbial community has been established, making it difficult to improve soil plant productivity using such methods.

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: Wang et al., PLoS One, 13, e0204085 (2018)

[0007] Non-patent literature 2: Fujita et al., Soil Science and Plant Nutrition, 60, 156-161 (2014)

[0008] Non-patent literature 3: Kumar et al., BMC Research Notes, 5, 137 (2012)

[0009] Non-patent literature 4: Mohammed et al., Agronomy Journal, 109, 309-316 (2017)

[0010] Non-patent literature 5: Czarnik et al., Emirates Journal of Food and Agriculture, 29, 988-993 (2017)

[0011] Non-patent literature 6: Waraich et al., Australian Journal of Crop Science, 7, 1551-1559 (2013) Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The problem this invention aims to solve is to improve soil plant productivity by manipulating the soil microbial community, and to improve plant productivity and the quality of its harvest by increasing the antioxidant activity of plants.

[0014] Methods for solving problems

[0015] To address the aforementioned issues, the inventors conducted in-depth research and repeated experiments, discovering the surprising fact that adding bacteria belonging to the order Rhizobiales to the soil alters the soil microbial community, thereby significantly improving plant productivity. Furthermore, the inventors discovered the surprising fact that bacteria belonging to the genus Bacillales, the genus Promicromonospora, or the genus Olivobacter increase the antioxidant activity of plants. Based on this knowledge, the present invention was completed.

[0016] The present invention is as follows.

[0017] [1] A composition for improving the plant productivity of soil, comprising bacteria belonging to the order Rhizobiales.

[0018] [2] A method for improving the plant productivity of soil, the method comprising adding bacteria belonging to the order Rhizobiales to the soil.

[0019] [3] According to the method described in 2, the plant is a cruciferous plant.

[0020] [4] According to the method described in 3, the cruciferous plant is flax or komatsuna.

[0021] [5] The method according to any one of 2 to 4 improves the plant productivity of the soil, thereby increasing the number of sheaths per plant, the number of seeds per plant, and / or the weight of the plant body.

[0022] [6] Compost contains bacteria belonging to the order Rhizobiales, Actinomycetales, Bacillales, Gaiellales, Myxococcales, iii1-15, Solirubrobacterales, Xanthomonadales, Burkholderiales, and Gemmatales.

[0023] [7] According to the compost of 6, the presence rate of bacteria belonging to the order Rhizobiales in the compost is more than 9%, and the presence rate of bacteria belonging to the orders iii1-15 is less than 6%.

[0024] [8] The compost described in 6 or 7 is used for the cultivation of cruciferous plants.

[0025] [9] According to the composting described in 8, the cruciferous plant is flax or komatsuna.

[0026]

[10] A composition for increasing the antioxidant activity of plants, the composition comprising bacteria belonging to the genus Bacillales, bacteria belonging to the genus Promicromonospora, or bacteria belonging to the genus Olivibacter.

[0027]

[11] In the composition according to 10, the bacterium belonging to the genus Bacillales is Bacillus cereus, the bacterium belonging to the genus Promicromonospora is Promicromonospora citrea, and the bacterium belonging to the genus Olivibacter is Olivibacter sp.

[0028]

[12] According to the composition described in 11, Bacillus cereus is a strain deposited at the Patent Microbial Collection Center with accession number NITE BP-02974, Promicromonosporacitrea is a strain deposited at the Patent Microbial Collection Center with accession number NITE BP-03025, and Olivebacter sp. is a strain deposited at the Patent Microbial Collection Center with accession number NITE BP-03026.

[0029]

[13] The strain is deposited at the Patent Microbial Collection Center with accession number NITE BP-02974.

[0030]

[14] The strain deposited at the Patent Microbial Collection Center with accession number NITE BP-03025.

[0031]

[15] The strain is deposited at the Patent Microbial Collection Center with accession number NITE BP-03026.

[0032]

[16] Compost containing any of the strains described in any of 13 to 15.

[0033] The effects of the invention

[0034] According to the present invention, plant yield is significantly increased by improving soil plant productivity, increasing the number of sheaths per plant, increasing the number of seeds per plant, and / or increasing the weight of the plant body, and plant productivity associated with oxidative stress and the quality of the harvest (e.g., functionality, disease resistance, preservation, etc.) are significantly improved by increasing the antioxidant activity of the plant. Attached Figure Description

[0035] Figure 1 (A) is a photograph of flaxseed cultivated using the compost involved in this invention. (B) is a photograph of harvested flaxseed. The left side is a photograph of the experimental area, and the right side is a photograph of the control area.

[0036] Figure 2 : This indicates a comparison of the yield of flaxseed in the test area and control area, as well as in the countries disclosed in the following documents: Japan (Non-Patent Document 2), India (Non-Patent Document 3), the United States (Non-Patent Document 4), Poland (Non-Patent Document 5), Canada, France, Australia, Germany, and Chile (Non-Patent Document 6).

[0037] Figure 3 This indicates a comparison of plant weight of *Komatsuna* in the experimental and control areas. Error bars show the standard error (n=6). The two intervals were found to be significantly different at the 1% level using a Student's t-test. Detailed Implementation

[0038] In this invention, a composition for improving soil plant productivity is provided, comprising bacteria belonging to the order Rhizobiales.

[0039] In this invention, "improving the plant productivity of soil" means promoting the plant growth capacity inherent in the soil, thereby increasing plant yield, such as the number of sheaths per plant, the number of seeds per plant, and / or the weight of the plant body.

[0040] Rhizobiales is an order belonging to the class Alphaproteobacteri, a large order comprising over 17 families and 130 genera. Rhizobiales includes numerous species. Among the species that live in symbiosis with plants, some form root nodules, supplying nitrogen to the plants and playing an important role in agriculture. Examples of agriculturally important species in the order Rhizobium include, for instance, species of the genus *Rhizobium*, N2-fixing bacteria that form nodules on the roots of leguminous plants through symbiotic nitrogen fixation, converting atmospheric N2 into ammonia, which can be used by plants as a nitrogen source.

[0041] The concentration of bacteria belonging to the order Rhizobiales contained in the compositions of the present invention is typically 10. 6 CFU / g or higher, preferably 10 7 CFU / g or higher, preferably 10. 8 CFU / g or higher.

[0042] The compositions of the present invention can be either solid or liquid, and in addition to bacteria belonging to the order Rhizobiales as active ingredients, they may also contain a carrier that imparts various properties such as increased stability, wettability, or dispersibility. The carrier is typically an agricultural carrier, and examples include soil, plant growth medium, water, fertilizer, plant-based oil, humectant, or combinations thereof.

[0043] In other embodiments of the invention, a method for improving the plant productivity of soil is provided, comprising adding bacteria belonging to the order Rhizobiales to the soil.

[0044] The addition of bacteria belonging to the order Rhizobiales to the soil can be achieved, for example, by adding the composition of the present invention described above before planting the desired plant, or by mixing it with the soil during planting.

[0045] Plants whose productivity is improved through this method are not particularly limited, but are typically crops, including those from the Brassicaceae, Poaceae, Fabaceae, Asteraceae, Solanaceae, Rosaceae, Cucurbitaceae, and Convolvulaceae families, with Brassicaceae plants being preferred. Examples of Brassicaceae plants include shepherd's purse or komatsuna. Shepherd's purse, in particular, has been cultivated in Europe since ancient times for oil production. It possesses several agricultural advantages, including high oil production capacity, short maturity, low water and nutrient requirements, and tolerance to pathogens and pests. In recent years, its oil has attracted attention as a biofuel feedstock. In fact, multiple test flights of fighter jets and passenger aircraft using bio-jet engine fuel made from shepherd's purse oil have been conducted, and performance has been reported to be satisfactory. To increase shepherd's purse yield, cultivation trials and gene recombination experiments targeting soil composition and sowing time have been conducted in various countries, and shepherd's purse oil is considered a strong candidate for a biofuel feedstock.

[0046] Improving soil plant productivity can increase the number of sheaths per plant, the number of seeds per plant, and / or the weight of the plant body.

[0047] In further embodiments of the present invention, compost is provided comprising bacteria belonging to the orders Rhizobiales, Actinomycetales, Bacillales, Gaiellales, Myxococcales, iii1-15, Solirubrobacterales, Xanthomonadales, Burkholderiales, and Gemmatales.

[0048] Bacteria belonging to the order Rhizobiales are typically those belonging to the genera *Rhodoplanes*, *Bradyrhizobium*, and *Pedomicrobium*, such as *Pedomicrobium* and *Methylobacterium adhaesivum*. Bacteria belonging to the order Actinomycetales are typically those belonging to the genera *Terracoccus*, *Mycobacterium*, and *Streptomyces*, such as *Actinomaduravinacea*, *Rathayibacter caricis*, and *Actinoallomurus iriomotensis*. Bacteria belonging to the order Bacillales typically include bacteria belonging to the genera *Bacillus*, *Rummeliibacillus*, and *Planifilum*, such as *Bacillus cereus*, *Paenibacillus chondroitinus*, and *Bacillus clausii*. Bacteria belonging to the order Gaiellales typically belong to the families Gaiellaceae and AK1AB1 02E. Bacteria belonging to the order Myxococcales typically include bacteria belonging to the genera *Sorangium*, *Plesiocystis*, and *Nannocystis*, such as *Sorangium cellulosum*. Bacteria belonging to the orders iii1-15 typically belong to the families RB40 and mb2424. 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 Steroidobacter, Luteimonas, Dokdonella, such as Stenotrophomonas acidaminiphila and Pseudoxanthomonas mexicana.Bacteria belonging to the order Burkholderiales are typically bacteria belonging to the genera *Burkholderia*, *Polaromonas*, and *Methylibium*. Bacteria belonging to the order Gemmataes are typically bacteria belonging to the genus *Gemmata*.

[0049] The presence ratio of each bacteria in the compost is not particularly limited as long as it improves plant productivity, but preferably the presence ratio of bacteria belonging to the order Rhizobiales is 9% or more, and the presence ratio of bacteria belonging to the orders iii1-15 is 6% or less.

[0050] Composting can be carried out using methods familiar to those skilled in the art, generally by mixing aerobic microorganisms that decompose materials such as sludge, livestock manure, straw, and hay into composting materials, and then fermenting them under aerobic conditions. During composting, moisture content, pH, carbon to nitrogen ratio (C / N ratio), temperature, and oxygen affect the rate of organic matter decomposition and are also major causes of nitrogen starvation in crops; therefore, adjusting these conditions is important.

[0051] If the moisture content of the compost is above approximately 60%, its apparent specific gravity and adhesion increase, making bagging and transportation difficult. Conversely, if the moisture content falls below approximately 30%, dust will be generated. Furthermore, if incompletely composted compost is dried, decomposition stops, resulting in unequal compost. Therefore, drying is preferably carried out after composting is fully completed. The typical moisture content of compost is approximately 30–60%, preferably approximately 25–55%.

[0052] When compost is acidic, excessive damage to minerals, fixation of phosphate, and absorption obstacles can occur. The optimal pH for compost is approximately 5.5 to 8.5.

[0053] The amount (EC) of ions such as potassium, sodium, chloride, and nitrate contained in compost is preferably low. For bark compost, it is preferably below about 3.0 dS / m, and for livestock manure, it is preferably below about 5.0 dS / m.

[0054] Regarding the carbon-to-nitrogen ratio (C / N ratio) in compost, the higher the value, the more likely it is to cause nitrogen starvation in the soil; therefore, a value of about 10 to 40 is preferred. However, since the carbon and nitrogen content of both readily decomposable and poorly decomposable organic matter are measured simultaneously, the C / N ratio of sawdust, for example, is very high, around 340 to 1250, and the C / N ratio of compost mixed with sawdust as a by-product also tends to be high.

[0055] Ammonia is produced in the early stages of composting, contributing to foul odors and hindering crop growth; therefore, a lower proportion of ammonia nitrogen in compost is preferable. Conversely, a higher proportion of nitrate nitrogen, which constitutes the inorganic nitrogen content of compost, is preferable. Nitrate nitrogen is produced by the nitrification of ammonia, a reaction that primarily occurs during secondary fermentation.

[0056] The fertilizer composition balance in compost should ideally be low (the proportion of potassium when the total nitrogen content is 1), with an appropriate value below 5. Additionally, heavy metals (especially copper and zinc) are essential micronutrients for crops, but excessive amounts can be harmful. Therefore, the appropriate heavy metal concentration in compost is below 300 ppm for copper and below 900 ppm for zinc.

[0057] The compost of the present invention has extremely high productivity for the aforementioned plants.

[0058] In a further embodiment of the invention, a composition for increasing the antioxidant activity of plants is provided, the composition comprising bacteria belonging to the genus Bacillales, bacteria belonging to the genus Promicromonospora, or bacteria belonging to the genus Olivibacter.

[0059] Plant productivity can be significantly reduced by abiotic environmental stresses such as strong light, dryness, temperature, salt, heavy metals, and ozone, or biotic environmental stresses such as diseases. The reason these environmental stresses cause plant death is related to oxidative stress (damage) caused by the accumulation of reactive oxygen species (ROS). It is known that under oxidative stress, changes in the intracellular redox state, dependent on the balance between ROS production and scavenging, act as signals, represented by the expression of defense systems in response to environmental stress, and participate in the control of physiological phenomena such as programmed cell death, growth / development. However, the inventors have now discovered the surprising fact that bacteria belonging to the genera *Bacillales*, *Promicromonospora*, or *Olivibacter* increase the antioxidant activity of plants.

[0060] The bacteria belonging to the genus *Bacillales* are preferably *Bacillus cereus*, and most preferably a strain deposited at the Patent Microbial Collection Center under accession number NITE BP-02974. The bacteria belonging to the genus *Promicromonospora* are preferably *Promicromonosporacitrea*, and most preferably a strain deposited at the Patent Microbial Collection Center under accession number NITE BP-03025. The bacteria belonging to the genus *Olivibacter* are preferably *Olivibacter sp.*, and most preferably a strain deposited at the Patent Microbial Collection Center under accession number NITE BP-03026.

[0061] By increasing the antioxidant activity of plants, it is possible to meaningfully improve the productivity of plants associated with oxidative stress and the quality of their harvests (e.g., functionality, disease resistance, preservation).

[0062] The following embodiments illustrate the present invention in further detail. However, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications.

[0063] Example

[0064] Example 1: Cultivation experiment of flaxseed.

[0065] 1.1 Cultivation conditions

[0066] Cultivation of Camelina sativa was conducted from March 25th to June 24th, 2018, in dryland fields in Shibukawa City, Gunma Prefecture (36.53N, 139.01E). The cultivar used was Calena. The planting density in the dryland fields was 1.5 kg / m². 2 Compost prepared using the method described later was sown without the use of other chemical fertilizers. The number of sheaths and seed yield at harvest were measured and compared with yields from other cultivation methods. The experimental plots were 15m². 2 Cultivation was carried out, with the control area at 1m 2 Cultivation was carried out. The planting density in the experimental area was 167 plants / m². 2 The planting density in the control area was 169 plants / m². 2 .

[0067] 1.2 Compost Production

[0068] The compost used in flaxseed cultivation was prepared as follows. In the experimental area, pig manure was used as the raw material, sawdust as a by-product, and separately isolated / cultured bacteria belonging to the order Rhizobiales were used as the added microorganisms. Composting was initiated by mixing pig manure, microbial culture, and sawdust. The moisture content was adjusted to approximately 60% using sawdust, and the mixture was stirred continuously for three months to produce compost. The compost used in the control area was prepared using the same method, except that no microorganisms were added.

[0069] 1.3 Analysis of soil composition after cultivation

[0070] Soil composition was analyzed according to the Japan Soil Association (2010) using the methods shown below. Total nitrogen (N): Macrocord (JM1000CN); Total phosphoric acid (P): Nitric acid-perchloric acid decomposition, ammonium molybdate-vanadate method; Total potassium (K): Nitric acid-perchloric acid decomposition, atomic absorption spectrometry; Total lime (Ca): Nitric acid-perchloric acid decomposition, atomic absorption spectrometry; Total magnesium (Mg): Nitric acid-perchloric acid decomposition, atomic absorption spectrometry.

[0071] 1.4 Microbial community analysis of cultivation soil using next-generation sequencers

[0072] Cultivation soil samples were freeze-dried using a VD-250R freeze dryer (TAITEC). The freeze-dried samples were then pulverized using a Shake Master Neo (bms). DNA was then extracted from the pulverized samples using the MPure Bacterial DNA Extraction Kit (MP Bio). Library preparation was performed using a two-step tailed PCR method. The first PCR targeted the variable region V4 (approximately 250 bp) of the 16S rRNA gene, using primers 1st_515F_MIX (5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-GTGCCAGCMGCCGCGGTAA-3': SEQ ID NO. 1) and 1st_806R_MIX (5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GGACTACHVGGGTWTCTAAT-3': SEQ ID NO. 2). To improve quality, primers used in sequence analysis were mixed primers containing random sequences of varying lengths, ranging from 0 to 5 bases. After purification of the first PCR product, in the second PCR, primers 2nd F (5'-AATGATACGGCGACCACCGAGATCTACAC-Index2(TATAGCCT)-ACACTCTTTCCCTACACGACGC-3': SEQ ID NO. 3) and 2nd R (5'-CAAGCAGAAGACGGCATACGAGAT-Index1(GCAGCGTA)-GTGACTGGAGTTCAGACGTGTG-3': SEQ ID NO. 4) were used to amplify the first PCR product. All PCR products were purified using an Agencourt AMPure XP (BECKMAN COULTER). Sequence analysis of the resulting libraries was performed using MiSeq (Illumina).

[0073] The sequence data obtained through sequencing were analyzed as follows. Using the Fastx toolkit's fastq_barcode_splitter, only sequences whose read start region (reading start) was extracted and matched the primers used. Primer sequences from the extracted sequences were then removed. Sequences with a quality value less than 20 were then removed, and sequences shorter than 40 bases and their paired sequences were destroyed. The pair-endo merge script FLASH was used to merge sequences that passed the quality filter. The merging criteria were a merged fragment length of 260 bases, a READ fragment length of 230 bases, and a minimum overlap of 10 bases. Mergeable sequences were then filtered based on fragment length, using only sequences between 246 and 260 bases for subsequent analysis. The sequences that passed all filtering were then checked for chimerism using Usearch's uchime algorithm. The database is 97% OUT of Greengene, which is part of the Qiime microbial community analysis pipeline. All sequences not identified as chimeras were extracted for subsequent analysis. OTUs were generated and system inferences were performed using Qiime's workflow script.

[0074] 2.1 Cultivation Results

[0075] Cultivation was carried out using compost containing Rhizobiales, which are bacteria associated with soil productivity. Figure 1 The image shows the appearance of flax during cultivation and the plants in the experimental and control areas after harvest. Figure 2 Table 1 compares the cultivation yields produced by this invention with those of domestic and international cultivation practices. These results confirm that the number of sheaths per plant is several times greater than in other cultivations except for the Indian example. Since the number of seeds per pod in this invention is moderate, it is considered that the number of seeds per plant has increased. Furthermore, since the seed weight did not show significant differences compared to other cultivation results, it is considered that the seed yield per plant has increased. This is from... Figure 1 Photo (B) also clearly shows that, due to the medium planting density, the final yield per unit area increased to 6.75 tons / hectare. Figure 2 It can be seen that this value is higher than any cultivation example at home and abroad, and more than 2.4 times higher than the 2.8 tons / hectare of France, which has the highest yield.

[0076] Table 1

[0077] Table 1 Comparison of cultivation results of flaxseed burdock

[0078]

[0079] 2.2 Soil microbial community analysis

[0080] The microbial community in the soil was analyzed by providing DNA extracted from the soil to a next-generation sequencer. Rhizobiales, the most abundant order of bacteria added to the experimental area, was detected, accounting for 9.42% of the total. Other orders of bacteria with high detection rates besides Rhizobiales include Actinomycetales (7.61%), Bacillales (7.02%), Gaiellales (6.58%), Myxococcales (4.89%), iii1-15 (3.32%), Solirubrobacterales (3.09%), Xanthomonadales (2.40%), Burkholderiales (2.33%), and Gemmatales (2.30%).

[0081] On the other hand, the Rhizobiales order detected in the control area accounted for 5.75% of the total, less than in the test area. Other bacteria detected in large quantities included Actinomycetales (7.52%), Gaiellales (7.21%), Bacillales (6.01%), Myxococcales (5.11%), iii1-15 (3.26%), Solirubrobacterales (3.25%), Burkholderiales (2.53%), Gemmatales (2.45%), and Nitrososphaerales (2.42%).

[0082] It was determined that in *Capsella bursa-pastoris* cultivation, the number of sheaths and seed weight per plant tend to decrease with increasing planting density (Non-Patent Literature 5). Regarding yield per plant, Indian cultivation examples show very high values, but this is at extremely low densities (17.6 plants / m²). 2 Under the cultivation conditions described above, the yield per unit area was as low as 1.31 tons / ha. On the other hand, in this experiment, despite a moderate planting density, the number of sheaths per plant was high. This result was a very high yield of 6.75 tons / ha. Meanwhile, the yield per unit area in the control area was not significantly different from that in existing cultivation examples.

[0083] Microbial analysis of the soil in which compost was applied showed that Rhizobiales were the most abundant, at 9.42%, an increase compared to the control area. This result suggests that the addition of microorganisms to the compost increases the number of Rhizobiales in the soil, thus contributing to improved soil productivity.

[0084] Focusing on four bacterial orders—Rhizobiales and iii1-15—which showed a positive correlation with soil productivity, and Acidobacteriales and Solibacterales, which showed a negative correlation, the proportions of bacteria in the experimental and control areas, as well as at 12 locations reported in Non-Patent Literature 1, were compared. Table 2 presents the results. Furthermore, Table 3 shows the correspondence between the codes for soil sampling locations and their latitude and longitude as described in Non-Patent Literature 1.

[0085] Table 2

[0086] Table 2. Correlation between bacterial presence ratio and soil productivity

[0087]

[0088] Table 3

[0089] Table 3 Soil sampling locations

[0090]

[0091] The presence rates of four bacterial orders were compared in the soils of the 12 locations, the experimental area, and the control area. In the experimental area, Rhizobiales were the second most abundant (9.42%) among the 14 soil samples, while iii1-15 were the eighth most abundant (3.32%). On the other hand, Acidobacteriales were the third least abundant (0.82%), and Solibacterales were the second least abundant (1.02%). In contrast, the control area had the eighth most abundant Rhizobiales (5.75%) among the 14 soil samples, less abundant than the experimental area. Regarding the presence rates of bacteria other than Rhizobiales, iii1-15 were the ninth most abundant (3.26%), Acidobacteriales were the fourth least abundant (1.10%), and Solibacterales were the third least abundant (1.26%). In any case, it can be assumed that the soil in the experimental area has a highly productive microbial composition.

[0092] In the above experiments, flaxseed cultivation was carried out using compost with added microorganisms, confirming a yield more than 2.4 times higher per unit area compared to existing reports both domestically and internationally. Analysis of the soil microorganisms revealed a high concentration of the added microorganisms. This demonstrates the potential to increase crop yields by manipulating the microbial community of the cultivation soil through the addition of specific microorganisms during compost production.

[0093] Example 2. Cultivation experiment of Komatsuna

[0094] The compost used in Komatsuna cultivation was prepared as follows. In the experimental area, pig manure, sawdust, and a culture solution of separately isolated and cultured bacteria belonging to the order Rhizobiales were used as raw materials. Composting was initiated by mixing the pig manure, bacterial culture solution, and sawdust. The moisture content was adjusted to 60% with sawdust, and the mixture was stirred continuously for three months to produce compost. The compost used in the control area was prepared using the same method, except that no bacteria were added.

[0095] Compost was prepared and used in pot cultivation experiments of Komatsuna (Brassica rapa var. perviridis). The potting mix used in these experiments was a mixture of compost and Akadama soil in a 3:7 ratio. Additionally, 0.84 g of a chemical fertilizer containing 8% each of ammoniacal nitrogen, soluble phosphoric acid, and water-soluble potassium was applied per liter of soil. Cultivation began from seed and was conducted at room temperature under fluorescent lighting, with growth observed.

[0096] In this experiment, compost was made using bacteria from the order Rhizobiales and then cultivated in Komatsuna, a cruciferous vegetable similar to flaxseed. The results showed that Komatsuna plants using compost supplemented with these bacteria experienced a 1.7-fold increase in plant weight.

[0097] Example 3. Evaluation of the antioxidant capacity of Komatsuna (Japanese mustard greens)

[0098] 3.1 Bacillus cereus

[0099] For Komatsuna (Japanese amaranth), a cultivation experiment was conducted by inoculating the roots of Komatsuna seedlings isolated by the applicant with strain 2764-01-S16 (accession number NITE BP-02974). Specifically, inoculation was performed by soaking the roots of Komatsuna seedlings grown in vermiculite on the 10th day of cultivation in a suspension of the bacteria for approximately 30 seconds. Sterilized water was used in the control area. Cultivation was carried out using soil obtained by mixing steam-sterilized farm soil with vermiculite in a 1:1 ratio, and liquid fertilizer was applied approximately once a week. Furthermore, cultivation was conducted in a plastic greenhouse.

[0100] The edible parts of the cultivated Komatsuna were used as the analytical sample, cut into 1 cm cubes, mixed with 4 times their weight of water, crushed using a juicer, heated at 80°C for 30 minutes, cooled, and filtered to prepare the sample solution. 25 μL of the sample solution, 50 μL of 44.4 mM 2,2,6,6-tetramethyl-4-piperidine (TMPD), 100 μL of 2.5 mM dimethyl sulfoxide (DMSO), and 50 μL of 55.5 mM riboflavin were mixed, and the mixture was irradiated with ultraviolet light for 20 seconds. The singlet oxygen scavenging activity of the sample solution was then determined by electron spin resonance (ESR) under the following conditions: Field = 336.4 ± 5 ​​mT (magnetic field range), Power = 3 mW, Modulation Width = 0.1 mT, Sweep Time = 1 minute, Time Constant = 0.1 seconds, and Amplify = 250, to evaluate the antioxidant capacity of Komatsuna.

[0101] The signal obtained from the above settings can be used to determine the strength of free radicals replenished by TMPD as a supplement. The actual amount of free radicals can be estimated based on this strength using a pre-prepared standard curve that uses histidine as a standard substance to observe singlet oxygen scavenging activity (antioxidant power). If the sample has high antioxidant power, more free radicals are scavenged and not replenished by the supplement, thus the signal becomes weaker.

[0102] Compared with uninoculated Komatsuna plants, the weight and antioxidant capacity of uninoculated Komatsuna plants were 3.15 g and the antioxidant capacity was 1560 μmol histidine / g, while the weight of inoculated Komatsuna plants was 4.07 g and the antioxidant capacity was 1890 μmol histidine / g. Therefore, a significant improvement in weight and antioxidant capacity was observed in inoculated Komatsuna plants.

[0103] 3.2 Promicromonospora citrea

[0104] For Komatsuna (Japanese amaranth), a cultivation experiment was conducted by inoculating the roots of Komatsuna seedlings isolated by the applicant with strain 27624-02-C06 (accession number NITE BP-03025). Specifically, inoculation was performed by soaking the roots of Komatsuna seedlings grown in vermiculite on the 10th day of cultivation in a suspension of the fungus for approximately 30 seconds. Sterilized water was used in the control area. Cultivation was carried out using soil obtained by mixing steam-sterilized farm soil with vermiculite in a 1:1 ratio, and liquid fertilizer was applied approximately once a week. Furthermore, cultivation was conducted in a plastic greenhouse.

[0105] The edible parts of the cultivated Komatsuna were used as analytical samples, cut into 1 cm cubes, mixed with 4 times their weight of water, crushed using a juicer, heated at 80°C for 30 minutes, cooled, and filtered to prepare a sample solution. 50 μL of the sample solution, 20 μL of 5.7 M 5,5-dimethyl-1-pyridine N-oxide (DMPO), and 90 μL of 2.5 mM hydrogen peroxide were mixed, and the mixture was irradiated with ultraviolet light for 30 seconds. The hydroxyl radical scavenging activity was then determined using an ESR method with the following conditions: Field = 335 ± 5 mT, Power = 3 mW, Modulation Width = 0.1 mT, Sweep Time = 1 minute, Time Constant = 0.1 seconds, and Amplify = 50, to evaluate the antioxidant capacity of Komatsuna.

[0106] The signal obtained from the above settings can be used to determine the strength of free radicals supplemented by DMPO as a supplement. The actual amount of substance can be estimated based on this strength from a standard curve prepared beforehand using DMSO as a standard substance for observing hydroxyl radical scavenging activity (antioxidant power). If the sample has high antioxidant power, more free radicals are scavenged and not supplemented by the supplement, thus the signal becomes weaker.

[0107] Compared with uninoculated Komatsuna plants, the weight and antioxidant capacity of uninoculated Komatsuna plants were 3.51 g and the antioxidant capacity was 1670 μmol DMSO / g, while the weight of inoculated Komatsuna plants was 4.29 g and the antioxidant capacity was 2350 μmol DMSO / g. Therefore, significant improvements in weight and antioxidant capacity were observed in inoculated Komatsuna plants.

[0108] 3.3 Olive-shaped fungal species (Olivibacter sp.)

[0109] For Komatsuna (Japanese amaranth), a cultivation experiment was conducted by inoculating the roots of Komatsuna seedlings isolated by the applicant with strain 27624-02-C07 (accession number NITE BP-03026). Specifically, inoculation was performed by soaking the roots of Komatsuna seedlings grown in vermiculite on the 10th day of cultivation in a suspension of the fungus for approximately 30 seconds. Sterilized water was used in the control area. Cultivation was carried out using soil obtained by mixing steam-sterilized farm soil with vermiculite in a 1:1 ratio, and liquid fertilizer was applied approximately once a week. Furthermore, cultivation was conducted in a plastic greenhouse.

[0110] The edible parts of the cultivated Komatsuna were used as the analytical sample, cut into 1 cm cubes, mixed with 4 times their weight of water, crushed using a juicer, heated at 80°C for 30 minutes, cooled, and filtered to prepare the sample solution. 50 μL of the sample solution, 30 μL of 8.55 M 5,5-dimethyl-1-pyridine N-oxide (DMPO), 50 μL of 1.25 mM hypoxanthine, 20 μL of 4.37 mM dimethyl sulfoxide (DMSO), and 50 μL of 0.1 U / ml xanthine oxidase were added and stirred for 60 seconds. The superoxide radical scavenging activity was then determined using the ESR method under the following conditions: Field = 335 ± 5 mT, Power = 3 mW, Modulation Width = 0.079 mT, Sweep Time = 1 minute, Time Constant = 0.1 seconds, and Amplify = 250, to evaluate the antioxidant capacity of Komatsuna.

[0111] The signal obtained from the above settings can be used to determine the strength of free radicals supplemented by DMPO as a supplement. The actual amount of substance can be estimated based on this strength using a pre-prepared standard curve that uses superoxide dismutase as a standard substance to observe superoxide radical scavenging activity (antioxidant power). If the sample has high antioxidant power, more free radicals are scavenged and not supplemented by the supplement, thus the signal becomes weaker.

[0112] Compared with uninoculated Komatsuna plants, the weight and antioxidant capacity of uninoculated Komatsuna plants were 3.51 g and the antioxidant capacity was 122 units of SOD / g, while the weight of inoculated Komatsuna plants was 4.47 g and the antioxidant capacity was 190 units of SOD / g. Therefore, a significant improvement in weight and antioxidant capacity was observed in inoculated Komatsuna plants.

[0113] The Patent Microbial Collection Center (Address: Room 122, 2-5-8 Kazusa-Kamazutsu, Kisarazu City, Chiba Prefecture 292-0818, Japan) of the National Institute for Technology Evaluation has deposited 2764-01-S16 with accession number NITE BP-02974 (original deposit date: June 20, 2019). Additionally, 27624-02-C06 has been deposited with accession number NITE BP-03025 (original deposit date: September 20, 2019), and 27624-02-C07 has been deposited with accession number NITE BP-03026 (original deposit date: September 20, 2019).

[0114] Collection Number

[0115] NITE BP-02974

[0116] NITE BP-03025

[0117] NITE BP-03026

Claims

1. A strain of Promicromonospora citrea, deposited at the Patent Microbial Collection Center with accession number NITE BP-03025.

2. Compost containing the strain of claim 1.

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