Method for cultivating plant, plant produced thereby, and composition for improving growing condition of plant

Abstract

Provided is a new plant cultivation method using microorganisms capable of promoting growth of plants and improving amounts of crop and biomass.　An actinomycete belonging to Streptomyces thermocarboxydus is applied to the plant or a breeding material thereof.

Description

Method for cultivating plants, plants produced thereby, and composition for improving plant growth conditions

[0001] The present invention relates to a method for cultivating plants and the plants produced thereby, as well as a composition for improving the growing conditions of plants.

[0002] In recent years, there has been a need for a stable supply of food to feed the growing world population and for the production of carbon dioxide (CO ２ Due to growing societal demand for greenhouse gas reduction through enhanced fixation capacity, there is a demand for increased yields and plant biomass production in crops, feed crops, trees, and other plants, and research and development in the field of plant science is being actively pursued. However, in Japan, the immediate implementation of plants developed using genetic engineering and genome editing technologies for outdoor use and food supply is difficult due to legal and social concerns, including concerns about the impact on the human body and ecosystem. Furthermore, growing interest in sustainable agriculture with reduced environmental impacts both domestically and internationally has led to a movement to reduce the use of chemical fertilizers, making highly productive plant cultivation even more difficult.

[0003] Therefore, plant cultivation processes utilizing microorganisms have recently attracted attention. A variety of microorganisms coexist and live symbiotically in the rhizosphere and on the surface and interior of leaves of outdoor plants, and are known to be closely related to plant productivity and growth. Therefore, there are growing expectations for cultivation methods that utilize the functions of these useful microorganisms. Examples of such useful microorganisms include various strains of actinomycetes belonging to the genus Streptomyces.

[0004] Patent Document 1 (JP 2011-188761 A) describes the actinomycete MBFA-172 strain (accession number NITE P-896) of the genus Streptomyces, which has a control effect against strawberry anthracnose, and a biological material for disease control containing the same.

[0005] Non-patent document 1 (Passari et al., PloS one, (2019), 14.7:e0219014) describes that when tomato (Solanum lycopersicum) seedlings were treated with the endophytic actinomycete Streptomyces thermocarboxydus isolate BPSAC147 and cultivated under greenhouse conditions, and the photochemical quantum yield and electron transport rate of photosystem II (PSII) were measured, the photosynthetic process was improved, and that this microorganism is therefore expected to be a new biological material for improving productivity by promoting photosynthesis, although this has not been proven.

[0006] Non-Patent Document 2 (Lasudee et al., Frontiers in Microbiology, (2018), 9:1247) describes that the Streptomyces thermocarboxydus S3 strain isolated from spores of the arbuscular mycorrhizal fungus Funneliformis mosseae had excellent phosphate solubilization ability, indole-3-acetic acid (IAA) production ability, siderophore production ability, etc., and that when mung beans (Vigna radiata) were treated with this microorganism and cultivated, the fresh weight, root length, and total length significantly increased. Furthermore, when rice (jasmine rice) was treated with this microorganism and cultivated in nutrient-poor soil under drought stress, growth was promoted in the early stages up to 45 days of cultivation.

[0007] The present inventors also reported in Non-Patent Document 3 (Kanno et al., Environ. Microbiol., 2016, 18[8]:2495-2506) the OS2C strain (accession number NITE ABP-03739), an actinomycete derived from Streptomyces thermocarboxydus, isolated in pure form from the stems of rice plants whose plant surfaces had been sterilized. The OS2C strain is a plant-symbiotic microorganism that possesses a unique enzyme gene capable of oxidizing dilute hydrogen at atmospheric concentrations and a high-affinity hydrogen oxidation ability. In this document, the present inventors reported that when rice seeds were treated with the OS2C strain and cultivated in sterile soil, its localization was observed within the roots and stems, suggesting that the OS2C strain is an endophyte. They also reported that after approximately four weeks of cultivation in sterile soil (approximately five weeks after germination), the length and dry mass of the above-ground parts and roots of the rice plants increased compared to untreated individuals.

[0008] JP 2011-188761 A

[0009] Passari et al.. PloS one, (2019), 14.7:e0219014Lasudee et al., Frontiers in Microbiology, (2018), 9:1247Kanno et al., Environ. Microbiol., 2016, 18[8]:2495-2506Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152Gravel et al., Soil Biol. Biochem., (2007), 39.8:1968-1977:Saito et al., Japanese Crop Science Journal, (2000), 69.3:385-390Wagner et al., Nat. Commun., (2016), 7:12151

[0010] However, all of the above-mentioned previous reports on Streptomyces thermocarboxydus, a species of the Streptomyces genus, were limited to reporting the effects on growth and stress tolerance during the vegetative growth phase when annual plants were treated with the strain and grown in soil. The effects of various actinomycete strains on the yield and biomass of annual plants grown to harvest time, as well as on perennial plants such as trees and cultivation systems from cuttings, were unknown.

[0011] The present invention has been made in view of the above-mentioned problems, and its object is to provide a new plant cultivation method using microorganisms that can promote plant growth and improve yield and biomass. A more specific object is to provide a new plant cultivation method that can improve yield and biomass when annual plants are cultivated until harvest time. Another specific object is to provide a new plant cultivation method that can promote plant growth and improve yield and biomass of agricultural crops in cultivation systems for perennial plants such as trees and cuttings.

[0012] As a result of extensive research, the present inventors have found that applying actinomycetes belonging to Streptomyces thermocarboxydus to plants or their propagation materials for cultivation can improve the plant's growth conditions, such as promoting plant growth and increasing the yield and biomass. In particular, they have found that the actinomycete can improve the yield and biomass of annual plants at the harvest time, and can promote the growth of perennial plants such as trees and in cultivation systems using cuttings, as well as suppress dormancy and growth cessation, leading to the present invention.

[0013] That is, the present invention encompasses the following: [Item 1] A method for cultivating a plant, the method comprising applying an actinomycete belonging to Streptomyces thermocarboxydus to the plant or its propagation material. [Item 2] The method of Item 1, wherein the plant is an annual plant and the method comprises applying the actinomycete to the plant at least six weeks after germination. [Item 3] The method of Item 1, wherein the plant is a perennial plant. [Item 4] The method of any one of Items 1 to 3, wherein the plant propagation material is a part or all of the plant. [Item 5] The method of any one of Items 1 to 4, wherein the plant part is selected from cells, tissues, organs, seeds, bulbs, cuttings, and seedlings. [Item 6] The method of any one of Items 1 to 5, wherein the actinomycete is the OS2C strain (Accession No. NITE ABP-03739) or a mutant strain thereof. [Item 7] The method according to any one of Items 1 to 6, wherein the actinomycete is applied by directly applying the actinomycete to the plant or its propagation material, or by indirectly applying the actinomycete to the environment surrounding the plant or its propagation material. [Item 8] The method according to any one of Items 1 to 7, wherein the growth condition of the plant is improved by applying the actinomycete. [Item 9] The method according to Item 8, wherein the improvement in the growth condition of the plant is an increase in crop yield, an increase in plant biomass, promotion of rooting, promotion of root, stem, and leaf development, suppression of plant dormancy, suppression of plant growth cessation, or a combination thereof. [Item 10] A plant or its propagation material produced by the method according to any one of Items 1 to 9. [Item 11] A composition for improving the growth condition of a plant, comprising an actinomycete belonging to Streptomyces thermocarboxydus. [Item 12] The composition according to Item 11, wherein the actinomycete is an OS2C strain (accession number NITE ABP-03739) or a mutant strain thereof. [Item 13] The method according to Item 11 or 12, wherein the improvement in plant growth conditions is an improvement in crop yield, an increase in plant biomass, promotion of rooting, promotion of root, stem, and leaf development, suppression of plant dormancy, suppression of plant growth cessation, or a combination thereof.[Item 14] The composition according to any one of Items 11 to 13, wherein the plant is an annual plant and the composition is applied to the plant at least 6 weeks after germination. [Item 15] The composition according to any one of Items 11 to 13, wherein the plant is a perennial plant.

[0014] According to the plant cultivation method using actinomycetes of the present invention, by applying the actinomycetes to plants or their propagation materials and cultivating them, it is possible to promote plant growth and improve the yield and biomass of the plants. In particular, the plant cultivation method of one aspect of the present invention can achieve effects such as improving the yield and biomass of annual plants at the harvest time. Furthermore, the plant cultivation method of one aspect of the present invention can achieve effects such as promoting the growth of perennial plants such as trees and in cultivation systems from cuttings, suppressing dormancy and growth cessation, and even improving the yield and biomass of agricultural crops.

[0015] Figures 1A and 1B are graphs showing the average length and dry mass (measurements of the aboveground and root portions of each individual plant) of rice plants grown in three soils with different nutrient concentrations 22 days after treatment with OS2C strain (29 days after germination) in the pot experiment of Example 2, compared with untreated rice. Figures 2A and 2B are photographs showing the overall appearance of rice plants 66 days after treatment with OS2C strain (71 days after germination; heading stage) and 136 days after treatment (141 days after germination; harvest stage), compared with untreated rice at the same time. Figure 3A is a graph showing the average leaf number of rice plants 51 days after treatment with OS2C strain (56 days after germination; immediately after heading), Figure 3B is the average tiller number, Figure 3C is the average aboveground dry mass excluding panicle dry mass (approximately 0.1 to 0.2 g each), and Figure 3D is the average root dry mass, all compared with untreated rice. Figures 4A and 4B are graphs showing the average dry mass of rice per cultivation pot and the average number of rice grains per cultivation pot harvested in the OS2C-treated plots, divided into imperfect rice and perfect rice, compared with untreated rice. Figure 5 is a photograph showing the overall appearance of pot-grown Erianthus 123 days after OS2C treatment (139 days after sowing) compared with untreated Erianthus. Figure 6A is a graph showing the average aboveground length per Erianthus plant 123 days after OS2C treatment (139 days after sowing) compared with untreated plants. Figure 6B is a graph showing the average aboveground dry mass of Erianthus treated with OS2C compared with untreated plants at the same time. Figure 6C is a graph showing the average root dry mass of Erianthus treated with OS2C compared with untreated plants at the same time. Figure 7A is a photograph showing the bottom view of an individual treated with OS2C strain 10 days after subculturing a portion of a sterilely cultured poplar plant, including a growing point, on a new medium, compared to an untreated individual. Figure 7B is a graph showing a comparison of the average root length of each individual in Figure 7A. Figure 8 is a photograph showing the overall appearance of an OS2C strain-treated poplar 88 days after the initial treatment (46 days after potting) compared to an untreated poplar. Figure 9A is a graph showing the average diameter of the stem base of an OS2C strain-treated poplar 88 days after the initial treatment (46 days after potting) compared to an untreated poplar.Figure 9B is a graph showing the average dry mass of aboveground stems of OS2C-treated poplars compared with untreated poplars during the same period. Figure 9C is a graph showing the average dry mass of roots of OS2C-treated poplars compared with untreated poplars during the same period. Figure 10 is a photograph showing the overall appearance of OS2C-treated poplars approximately 4 months after potting, compared with untreated poplars. The top of Figure 11A is a photograph showing the overall appearance of a eucalyptus individual 142 days after the initial OS2C treatment (152 days after sowing), and the bottom is a photograph showing the state of the roots in the pot, compared with an untreated individual. Figure 11B is a graph showing the average dry mass of roots per individual of OS2C-treated eucalyptus compared with an untreated individual during the same period. Figure 12 is a photograph showing the overall appearance of a potted eucalyptus 91 days after the initial OS2C treatment (105 days after sowing), compared with an untreated individual. FIG. 13A is a graph showing the average number of leaves per individual 91 days after the initial OS2C strain treatment (105 days after sowing) compared with untreated individuals. FIG. 13B is a graph showing the average aboveground dry mass per individual at the same time, compared with untreated individuals. FIG. 13C is a graph showing the average root dry mass per individual at the same time, compared with untreated individuals. FIG. 14 is a photograph showing an overall view of pot-grown eucalyptus 159 days after the initial OS2C strain treatment (180 days after sowing), compared with untreated individuals. FIG. 15A is a graph showing the average aboveground dry mass per eucalyptus individual 159 days after the initial OS2C strain treatment (180 days after sowing), compared with untreated individuals. FIG. 15B is a graph showing the average aboveground dry mass of stems excluding leaves per eucalyptus individual at the same time, compared with untreated individuals. Figure 15C is a graph showing the average root dry mass per Eucalyptus plant compared with untreated plants at the same time. Figure 15D is a graph showing the average stem diameter at 2 cm above ground level per Eucalyptus plant compared with untreated plants at the same time. Figures 16A and 16B are photographs showing the overall appearance of rice plants (Akitakomachi and Hokuriku 193) 19 days after treatment with OS2C strain (26 days after germination), compared with untreated rice at the same time.Figures 17A and 17B are photographs showing the overall growth of rice plants (Akitakomachi and Hokuriku 193) 78 days after OS2C treatment (59 days after transplanting) compared with untreated rice plants of the same period. Figures 17C and 17D are graphs showing the average number of tillers in OS2C-treated rice plants (Akitakomachi and Hokuriku 193) 78 days after OS2C treatment (59 days after transplanting) compared with untreated rice plants. Figures 18A and 18B are photographs showing rice plants (Akitakomachi and Hokuriku 193) harvested 155 days after OS2C treatment (136 days after transplanting) compared with untreated rice plants of the same period. Figure 19A is a graph showing the average aboveground fresh mass per plant of rice (Akitakomachi and Hokuriku 193) harvested 155 days after OS2C treatment (136 days after transplanting into paddy fields), compared with untreated plants. Figure 19B is a graph showing the average number of panicles per plant, and Figure 19C is a graph showing the average fresh mass of panicles per plant. Figure 20 is a photograph showing the root hairs of the primary seminal root 4 cm from the rice seed 3 days after OS2C treatment (6 days after sowing) compared with untreated rice. Figure 21A is a photograph showing the overall development of rice roots 7 days after OS2C treatment (10 days after sowing) compared with untreated rice. Figures 21B and 21C are graphs showing the total number and total length of lateral roots per rice plant 7 days after treatment with the OS2C strain (10 days after sowing), in comparison with untreated rice.

[0016] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments and can be embodied in any form without departing from the spirit of the present invention.

[0017] All patent documents (such as published patent applications and patent publications) and non-patent documents cited in the present invention are incorporated herein in their entirety for all purposes.

[0018] One aspect of the present invention relates to a method for cultivating a plant, the method comprising applying an actinomycete belonging to Streptomyces thermocarboxydus to the plant or its propagation material (appropriately referred to as the "method of the present invention").

[0019] As mentioned above, there have been several reports, including that by the present inventors, on treating plants with various actinomycete strains of the genus Streptomyces and cultivating them (see Patent Document 1 and Non-Patent Documents 1-3 mentioned above). However, all of these reports were limited to reporting the effects on growth and stress resistance during the vegetative growth phase when annual plants were treated with the strains and cultivated in soil. The effects of treating various actinomycete strains on the yield and biomass of annual plants cultivated to harvest time, as well as the effects of treating various actinomycete strains on perennial plants such as trees and cultivation systems from cuttings, were previously unknown.

[0020] The present inventors have found that when an actinomycete belonging to Streptomyces thermocarboxydus (OS2C strain described below) was treated on a monoennial plant (rice) and cultivated in soil indoors until harvest time, the yield and biomass amount were significantly improved (see Example 2 and Figures 1 to 4 described below). It has been suggested that the promotion of growth during the vegetative growth period of annual plants and the improvement of yield and biomass during the harvest period are not necessarily due to the same mechanism of action (see Non-Patent Document 4: Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152; Non-Patent Document 5: Gravel et al., Soil Biol. Biochem., (2007), 39.8:1968-1977; Non-Patent Document 6: Saito et al., Journal of the Crop Science Society of Japan, (2000), 69.3:385-390.). Therefore, the surprising finding that treating annual plants with an actinomycete belonging to Streptomyces thermocarboxydus and cultivating them improves their yield and biomass during the harvest period is not easily attainable from previous reports.

[0021] Next, the present inventors treated a herbaceous annual resource crop (Erianthus) with an actinomycete belonging to Streptomyces thermocarboxydus (OS2C strain, described below) and cultivated it in soil, and found that the biomass of the crop was significantly improved (see Example 3 and Figures 5 and 6, described below). It is a surprising finding that actinomycetes belonging to Streptomyces thermocarboxydus, isolated from agricultural crops, can also contribute to improving the biomass of resource crops.

[0022] The present inventors also treated seeds of a monoennial plant (rice) with an actinomycete belonging to Streptomyces thermocarboxydus (OS2C strain, described below) and cultivated them in soil. Furthermore, when stems containing the growing point and several leaves were excised from a perennial plant (poplar) in sterile culture and subcultured in new medium, the OS2C strain was treated and cultured, and the seeds were then potted and continued to be cultivated. The OS2C strain was also treated on seeds of a perennial plant (eucalyptus), which were then cultured on agar medium and then potted and continued to be cultivated. As a result, it was found that in both cases, effects such as growth promotion, suppression of dormancy and growth cessation, and even increased biomass could be obtained (see Example 4 and Figures 7 to 10, described below). It is known that the community structure of symbiotic microorganisms varies depending on the age of the plant (Non-Patent Document 7: Wagner et al., Nat. Commun., (2016), 7:12151). It is speculated that the mechanisms of growth promotion differ between annual and perennial plants. Furthermore, plant growth in soil and differentiation and development in non-soil cultivation systems, such as tissue culture and cuttings, generally involve completely different plant physiological mechanisms, and therefore are thought to be driven by different growth-promoting mechanisms. Therefore, when subcultivating a portion of a perennial plant (poplar) in sterile culture, treating it with an actinomycete belonging to the Streptomyces thermocarboxydus species and cultivating it results in subsequent growth promotion, suppression of dormancy and growth cessation, and even increased biomass. This surprising finding, which could not have been easily arrived at from previous reports, is a surprising finding.

[0023] Next, the present inventors found that when a perennial woody resource crop (eucalyptus) was treated with an actinomycete belonging to Streptomyces thermocarboxydus (OS2C strain described below) and cultivated in soil, the biomass of the crop was significantly improved (see Example 5 and Figures 11 to 15 described below). In light of the background described in the previous paragraph, the fact that an actinomycete belonging to Streptomyces thermocarboxydus also contributes to improving the biomass of woody resource crops is a surprising finding that could not have been easily arrived at from previous reports.

[0024] In the method of the present invention, examples of actinomycetes belonging to the genus Streptomyces thermocarboxydus include the OS2C strain and mutant strains thereof. The OS2C strain was isolated by the present inventors from the stems of paddy rice plants whose surfaces had been sterilized. It is a plant-symbiotic microorganism that possesses a unique enzyme gene capable of oxidizing dilute hydrogen at atmospheric concentrations and a high-affinity hydrogen oxidation ability. When the OS2C strain was applied to individual plants and cultured, its localization within the roots and stems was observed (Non-Patent Document 3), suggesting that the OS2C strain is an endophytic fungus (plant endophyte). According to sequence homology analysis of the 16S rRNA gene, a marker gene for bacterial phylogenetic classification, the sequence of the 16S rRNA gene of the OS2C strain (Accession no. AB894408) has been found to have 99.9% identity with the AT37 strain (Accession no. NR026072) of Streptomyces thermocarboxydus, a closely related strain.

[0025] The type of plant that is the target of the method of the present invention is not limited. For example, when classified according to their reproduction method, the plant may be a seed plant, or a seedless plant such as moss or a fern. In the case of a seed plant, it may be an angiosperm or a gymnosperm. In the case of angiosperms, it may be a monocotyledonous plant or a dicotyledonous plant. Furthermore, when classified according to their life cycle, the plant may be an annual plant or a perennial plant. Furthermore, the use of the plant is not limited, but useful plants such as crop plants such as agricultural crops and resource crops, and ornamental plants and horticultural plants are preferred.

[0026] In the method of the present invention, the form of the plant to which the actinomycete is applied is not limited. For example, it may be applied to a plant after germination or to plant propagation material before germination. Plant propagation material may be any part or all of a plant, such as cells, tissues, organs, seeds, bulbs, cuttings, and seedlings. Furthermore, plant cells, tissues, etc. may be in a differentiated state obtained from a plant, or may be in a dedifferentiated stem cell or callus state. Various techniques for dedifferentiating plant cells and tissues are known.

[0027] In the method of the present invention, the plant cultivation system is not particularly limited. Examples include soil culture systems using various types of soil and hydroponic systems using nutrient solutions instead of soil, but either may be used. In the case of soil culture systems, the soil may be natural soil or artificial soil. In the case of hydroponic systems, examples include solid culture systems using various solid media and liquid culture systems (hydroponic systems) using water or various liquid media, but either may be used. In either case, any fertilizer, such as natural fertilizer or artificial fertilizer, or various nutrients may be added. Furthermore, multiple cultivation systems may be used in combination, such as cultivating plants in a solid culture system or hydroponic system during the seed germination period or the vegetative growth period of seedlings, and then potting them up and transferring them to a soil culture system once they have grown to a certain extent.

[0028] In the method of the present invention, the timing and number of times of application of the actinomycete to plants or their propagation materials are not particularly limited. For example, when applied to plants after germination, the actinomycete may be applied at any one time (e.g., at any one time of the germination period, vegetative growth period, reproductive growth period, etc.) or at multiple different times (e.g., at any two or more times of the germination period, vegetative growth period, reproductive growth period, etc.). It may also be applied to propagation materials at any stage before germination or to a cultivation system such as soil used for plant cultivation. Furthermore, the actinomycete may be applied only once or two or more times at each time.

[0029] However, when targeting annual plants, it is preferable to apply the actinomycete at least from the vegetative growth stage onwards, specifically to plants six weeks or more after germination. As mentioned above, it has been suggested that the promotion of growth during the vegetative growth stage of annual plants and the improvement of yield and biomass during the harvest stage are not necessarily due to the same mechanism of action (see Non-Patent Document 4: Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152; Non-Patent Document 5: Gravel et al., Soil Biol. Biochem., (2007), 39.8:1968-1977; Non-Patent Document 6: Saito et al., Journal of the Crop Science Society of Japan, (2000), 69.3:385-390). By applying the actinomycetes to annual plants after the vegetative growth stage, it becomes easier to significantly achieve the effect of improving yield and biomass at the harvest stage, which was completely unknown in the prior art. However, when applying the actinomycetes to annual plants, they may be applied not only after the vegetative growth stage (i.e., after six weeks after germination) but also before the vegetative growth stage (i.e., up to five weeks after germination).

[0030] In the method of the present invention, the method of applying the actinomycete to plants or their propagation materials is not particularly limited, and examples thereof include direct application such as spraying or painting a culture solution of the actinomycete on the plants or their propagation materials, or immersing the plants or their propagation materials in the culture solution of the actinomycete, and indirect application such as mixing the culture solution of the actinomycete with the surrounding environment of the plants or their propagation materials (e.g., soil, medium, water, etc.) and absorbing it.

[0031] In the method of the present invention, the amount of actinomycete applied to plants or their propagation materials is not particularly limited, and may be appropriately selected depending on conditions such as the type of plant, application time, and application method. ６ ~10 １０ A culture solution adjusted to a concentration of CFU / mL is prepared. Then, in the case of direct application, approximately 0.1 to 10 mL of the culture solution is applied per plant. On the other hand, in the case of indirect application, the culture solution is mixed with soil, medium, or water to a concentration of 1 to 100 mL / kg (for soil or solid medium) or 0.01 to 50 mL / L (for liquid medium or water) and then applied. The actinomycetes are prepared in the form of spores or mycelium.

[0032] According to the method of the present invention, by applying the actinomycete to plants or their propagation materials, improvement of the growth condition (particularly improvement of the growth condition independent of the cultivation environment such as soil) is achieved. Therefore, in another aspect of the present invention, a composition for improving the growth condition of plants is provided, which comprises the actinomycete as an active ingredient. Examples of improvement of the growth condition include, but are not limited to, the following effects: - Improvement of the yield of agricultural crops (when the plant is an agricultural crop). - Increase in the biomass of resource crops (when the plant is a resource crop). - Promotion of rooting. - Promotion of development of roots, stems, and leaves. - Suppression of plant dormancy. - Suppression of plant growth cessation.

[0033] According to the method of the present invention, a plant can be produced that is symbiotic with the actinomycete and has one or more functions selected from the above. The plant produced by the method of the present invention and its propagation material are also within the scope of the present invention.

[0034] The present invention will be described in more detail below with reference to examples. However, these examples are merely examples shown for the convenience of explanation, and the present invention is not limited to these examples in any sense.

[0035] Example 1: Cultivation of actinomycete OS2C strain and preparation of bacterial suspension The actinomycete OS2C strain (accession number NITE ABP-03739) belonging to Streptomyces thermocarboxydus was aerobically cultured on an R2A agar medium at 30°C. The microorganism formed a gray spore mat on the agar medium after about 10 days of culture. An appropriate amount of sterilized water was poured onto the plate, the spore mat was scraped off, the collected bacterial suspension was centrifuged, and the supernatant was removed. An equal amount of sterilized water was added to the plate, and washing was repeated twice to prepare a spore suspension of the microorganism. The number of viable cells in the spore suspension was calculated by colony counting, and a bacterial cell concentration of 10 was determined for use in the treatment of rice in Examples 2, 6, and 7 described below, and in the treatment of Erianthus, poplar, and eucalyptus in Examples 3 to 5. ９ CFU / mL and 10 ８ The spore suspension was prepared so that the spore concentration was CFU / mL. The bacterial suspension was to be used for plant treatment on the same day and was stored on ice until then.

[0036] Example 2: Treatment of rice with actinomycete strain OS2C. Rice seeds (variety: Nipponbare) were selected by saltwater selection, dehulled, and then sterilized with 70% ethanol for 1 minute and 1% sodium hypochlorite for 30 minutes, followed by rinsing with water. Five days after sowing on 0.5% agar medium (containing no components other than agar), two immediately germinated rice plants were transplanted to pots containing 200 g of each of three types of soil, each previously wet-sterilized in an autoclave. The three types of soil, in descending order of nutrient concentration, were black soil, black soil plus nutrient solution (black soil with nutrient solution prepared by adding 200 g of black soil to the equivalent of 20 μL of liquid fertilizer (product name: Hyponex concentrate, Hyponex)), and synthetic potting soil (synthetic granular potting soil, product name: Bonsol No. 2, Sumitomo Chemical).

[0037] Furthermore, on the same day, the bacterial cell concentration of 10 ９One mL of a spore suspension of the OS2C strain (CFU / mL) was added to each rice plant in the vicinity of the transplant site. Rice plants in this example were cultivated in a growth chamber (28°C; photoperiod: 16 hours light / 8 hours dark; approximately 22,000 lux) and watered appropriately with sterilized water.

[0038] As shown in Figures 1A and 1B, the plant height (length) and dry mass (measured for the above-ground part and root part of each individual plant) of rice plants 22 days after treatment with the OS2C strain (29 days after germination) in the pot experiment of Example 2 were compared with those of untreated rice plants in three soils containing different nutrients (n = 2). The mean plant length (top and root combined) of the OS2C-treated rice plants increased by <41.8 cm to 46.6 cm> (+11.5%), <37.3 cm to 51.9 cm> (+39.1%), and <50.7 cm to 53.9 cm> (+6.3%) in the low-, medium-, and high-nutrient soils, respectively. The average dry mass increased from 0.063 g to 0.096 g (51.9%), from 0.075 g to 0.083 g (+10.5%), and from 0.120 g to 0.161 g (+34.2%), respectively. These results demonstrate that the symbiotic relationship between OS2C and rice plants promotes stable initial biomass production in soils with different nutrient contents.

[0039] Furthermore, after the fourth week of microbial treatment, the rice plants grown in the synthetic soil were placed in a growth chamber (30°C; humidity 70%; photoperiod: 11 hours light / 13 hours dark; CO ２ Cultivation was continued until the harvest time in a sterilized synthetic granular medium containing spores of the OS2C strain (approximately 2.0 × 10 per pot) at a concentration of 470 ppm. 31 and 51 days after the treatment, spores of the OS2C strain cultured in Example 1 (approximately 2.0 × 10 per pot) were added to the sterilized synthetic granular medium. ９ The pots were treated with additional microorganisms by adding soil mixed with microbial colonies (CFU).

[0040] As shown in Figures 2A and 2B, 66 days after treatment with OS2C (71 days after germination; heading stage) and 136 days after treatment (141 days after germination; harvest stage) (collective photographs of 10 rice plants, two plants per pot, five pots per test plot) were compared with untreated rice plants at the same time. These results demonstrate that treatment with OS2C significantly promoted rice development at both the heading and harvest stages.

[0041] As shown in Figure 3, plant biomass parameters (above-ground dry weight (DW) excluding number of leaves, number of tillers, and panicle mass (approximately 0.1-0.2 g each), and root dry weight (DW)) 51 days after treatment with OS2C strain (56 days after germination; immediately after the onset of heading) were compared with those of untreated rice. The total values ​​for two individuals per pot were averaged for eight pots (N = 8) in each test plot. These results demonstrate that treatment with OS2C strain significantly increased plant biomass in above-ground and root parts.

[0042] As shown in Figures 4A and 4B, the dry mass and kernel number of rice harvested 136 days after OS2C treatment (141 days after germination; harvest time) were compared between OS2C-treated and untreated plants, with imperfect and perfect rice varieties separated. The sums of the values ​​for two plants per pot were averaged across five pots (N = 5) in each experimental plot. These results demonstrate that OS2C treatment significantly reduced both the dry mass and kernel number of imperfect rice varieties, while significantly increased both the dry mass and kernel number of perfect rice varieties.

[0043] Furthermore, the period from heading to ripening was approximately two weeks shorter in the OS2C-treated plots than in the untreated plots. These results suggest that some kind of action may be promoting material allocation (nutrient translocation to the harvest) in rice plants treated with the OS2C strain.

[0044] Example 3: Treatment test of actinomycete OS2C strain on Erianthus Erianthus seeds (species name: Erianthus arundinaceus) were used. Sterile seeds were sterilized for 5 minutes using 1 / 20 diluted bleach and then washed with water. The seeds were sown on 1% agar medium containing 1% sucrose and 1 / 4 MS medium. The plates were then cultured upright in the dark at 22°C for 2 days, followed by light culture for 8 days under a 16-hour light / 8-hour dark photoperiod. The seeds were then transplanted into soil that had been sterilized in advance using an autoclave.

[0045] Furthermore, on the same day, the bacterial cell concentration was ８ One mL of a spore suspension of OS2C strain with a CFU / mL concentration was added to each Erianthus plant close to the transplant site. One month later, the same OS2C strain was added again, and thereafter, when transplanting the plants into larger pots, 1 mL of the OS2C strain spore suspension with the same bacterial cell concentration was added again to each Erianthus plant.

[0046] As shown in Figures 5 and 6A-C, Erianthus cultivation pots were prepared 123 days after the initial treatment with the OS2C strain (139 days after sowing), and the length, dry mass of the above-ground and below-ground parts per plant were compared with those of untreated plants. These increased by approximately 13%, 24%, and 13%, respectively. These results suggest that treatment with the OS2C strain may contribute to promoting the growth of both the above-ground and below-ground parts of the herbaceous plant Erianthus.

[0047] Example 4: Treatment test of actinomycete OS2C strain on poplar Hybrid poplar (Populus tremula x tremuloides) was used, which was cultured aseptically in 1 / 2 MS solid medium containing 0.3% gellan gum under a photoperiod of 16 hours light / 8 hours dark at 22°C. An internode located about 3 cm from the apex of the stem of an individual about one month after subculture was cut with a scalpel, and then about 1 cm from the cut surface was filled with the OS2C suspension (10 ７The fungus treatment was carried out by immersing the treated culture in a solution of 1000 cells / mL of 2% sucrose and leaving it to stand for 5 minutes. The stems of the treated cultures were then inserted into 1 / 2 MS solid medium containing 2% sucrose so that the plants could stand on their own, and the plants were again cultured under a 16-hour light / 8-hour dark photoperiod at 22°C. The effects of the fungus treatment on rooting and early growth were observed over time. As a control (untreated group), sterile water was used instead of the fungus suspension, and the cut cultures were immersed in sterile water for 5 minutes and then inserted into 1 / 2 MS solid medium also containing 2% sucrose for further observation.

[0048] As shown in Figure 7A, poplars treated with OS2C strain 10 days later were compared with untreated poplars. As shown in Figure 7B, root length of poplars treated with OS2C strain 10 days later was compared with that of untreated poplars. These results demonstrate that root length was significantly longer in the OS2C-treated group than in the untreated group, demonstrating that elongation growth was promoted.

[0049] Poplars were also potted from agar medium into soil pots and continued to be grown under artificial light. As shown in Figures 8 and 9A-C, poplar cultivation pots were prepared 88 days after the initial treatment with the OS2C strain (46 days after potting), and the stem base thickness, dry mass, and root dry mass per individual were compared with those of untreated individuals. Significant increases were confirmed compared to untreated individuals. These results suggest that treatment with the OS2C strain may promote the growth of poplar aboveground parts and roots.

[0050] As shown in Figure 10, the overall growth of OS2C-treated poplar trees approximately four months after potting was compared with that of untreated poplar trees. Untreated plants approximately four months after potting exhibited a transition to dormancy, with winter bud formation, growth cessation, and leaf yellowing. Meanwhile, OS2C-treated plants exhibited no such phenomena after about four months of potting; their leaves remained green and continued to grow and elongate at a good rate. These results suggest that the symbiosis of OS2C inhibits dormancy and growth cessation.

[0051] Example 5: Treatment test of actinomycete OS2C strain on eucalyptus Eucalyptus seeds (species name: Eucalyptus Globulus) were used. Sterile seeds were sterilized with 1 / 5 diluted Kitchen Bleachers for 30 minutes, 70% ethanol for 30 seconds, and then 1 / 5 diluted Kitchen Bleachers for 15 minutes, followed by rinsing with water. The seeds were sown on 1% agar medium containing 1% sucrose and 1 / 4 MS medium and cultured upright in the dark at 22°C for 2 days. After that, the seeds were cultured in the light for 8 days under a photoperiod of 16 hours light / 8 hours dark. They were then transplanted into soil that had been sterilized in advance by autoclaving.

[0052] Furthermore, on the same day, the bacterial cell concentration of 10 ８ One mL of a spore suspension of the OS2C strain (CFU / mL) was added to each Eucalyptus plant in the vicinity of the transplant site. After 43 days, an additional 1 mL of the OS2C strain at the same cell concentration was added to each Eucalyptus plant.

[0053] 11A and 11B, photographs were taken of the entire Eucalyptus plants 142 days after the initial treatment with the OS2C strain (152 days after sowing), and the dry weight of the roots per plant was compared with that of the untreated plants. The dry weight of the roots increased by approximately 54% compared to the untreated plants.

[0054] Eucalyptus plants were transplanted into larger pots than those used in the treatment tests in the previous two paragraphs, and four days after transplanting into soil, the fungus cells prepared in Example 1 at a concentration of 10 ８ One mL of a spore suspension of the OS2C strain (CFU / mL) was added to each eucalyptus plant in the vicinity of the transplant site. After 32 days, the plants were further treated with the same OS2C strain.

[0055] As shown in Figures 12 and 13A-C, eucalyptus cultivation pots were prepared 91 days after the initial treatment with the OS2C strain (105 days after sowing), and the leaf number and dry mass per individual were compared with untreated individuals. The leaf number, aboveground dry mass, and root dry mass increased by approximately 56%, 12%, and 58%, respectively. These results suggest that the symbiosis with the OS2C strain contributes to eucalyptus growth, particularly to improved aboveground biomass production through its effect of promoting root growth.

[0056] As shown in Figures 14 and 15A-C, eucalyptus cultivation pots were prepared 159 days after the initial OS2C treatment (180 days after sowing), and the dry mass of the aboveground parts, stems only, and underground parts per individual was compared with that of untreated individuals. The dry mass of the total aboveground parts, the dry mass of the stems only excluding leaves, and the dry mass of the underground parts increased by approximately 37%, 51%, and 2-fold, respectively. Furthermore, as shown in Figure 15D, the trunk diameter at 2 cm above ground level was compared between OS2C-treated and untreated individuals. The trunk diameter also tended to be larger in OS2C-treated individuals compared with untreated individuals. These results are similar to those shown in Figure 12, suggesting that symbiosis with OS2C is effective in increasing wood mass during long-term eucalyptus growth.

[0057] Example 6: Field test of rice seedlings treated with actinomycete strain OS2C. Rice seeds (varieties: Akitakomachi and Hokuriku 193) were selected by saltwater selection, sterilized with 70% ethanol for 1 minute and 1% sodium hypochlorite for 30 minutes, and then washed with water to obtain sterile seeds. The seeds were soaked in water for 3 days to induce germination, and the germinated seeds were sown in a cell tray containing synthetic soil (product name: Inaho granular soil), one seed per cell.

[0058] The series of cultivations were carried out in a growth chamber (28°C; photoperiod: 16 hours light / 8 hours dark; approximately 22,000 lux). ９ One mL of a spore suspension of the OS2C strain (CFU / mL) was added to the surface of each rice seed.

[0059] As shown in Figures 16A and 16B, rice seedlings of Akitakomachi and Hokuriku 193 were transplanted into paddy fields 19 days after treatment with OS2C (26 days after germination) in the field test of Example 6, and compared with untreated rice seedlings at the same time. These results demonstrate that treatment with OS2C promoted early seedling development. This early development promotion effect was particularly pronounced in Akitakomachi.

[0060] The seedlings were transplanted into paddy fields in Akita Prefecture in early June and continued to be grown outdoors until the harvest season in early October. The amount of fertilizer applied to the paddy fields before transplanting the seedlings and the spraying of herbicides before and after transplanting were all under normal cultivation conditions.

[0061] As shown in Figures 17A and 17B, photographs were taken from the paddy fields of Akitakomachi and Hokuriku 193 78 days after OS2C treatment (59 days after transplanting; vegetative growth stage), and compared with untreated rice from the same period. Furthermore, as shown in Figures 17C and 17D, the number of tillers in OS2C-treated Akitakomachi and Hokuriku 193 78 days after OS2C treatment (59 days after transplanting) was compared with that in untreated rice. The figures show the average values ​​for a total of 16 individuals in four plots. The number of tillers in the OS2C-treated plots was approximately 20% higher than in the untreated plots. These results demonstrate that treatment of seedlings with OS2C significantly increased the number of aboveground tillers in both varieties, even after approximately two months of cultivation in paddy fields.

[0062] As shown in Figures 18A and 18B, Akitakomachi and Hokuriku 193 (4-6 individuals) 155 days after treatment with OS2C (136 days after transplanting into paddy fields; harvest time) were compared with untreated rice plants at the same time. These results demonstrate that treatment with OS2C promoted rice development in both varieties, even at harvest time.

[0063] As shown in Figures 19A-C, the aboveground fresh weight (FW), a parameter of plant biomass, and the number and fresh weight (FW), a parameter of yield, were compared with untreated rice 155 days after OS2C treatment (136 days after transplanting into paddy fields; harvest time). For Akitakomachi, the values ​​are averaged over a total of 31-40 individuals (N = 31-40) across four plots. For Hokuriku 193, the values ​​are averaged over a total of 74 individuals (N = 74) across four plots. These results demonstrate that treatment of seedlings with OS2C significantly increased aboveground plant biomass and rice yield in both varieties.

[0064] Example 7: Comparative test of root hairs and lateral roots of rice treated with actinomycete strain OS2C. Rice seeds (variety: Nipponbare) were selected by saltwater selection, and the unhulled grains were removed. Sterile seeds were then used after sterilization with 70% ethanol for 1 minute and 1% sodium hypochlorite for 30 minutes and subsequent rinsing with water. Three days after sowing on 0.5% agar medium (containing no components other than agar), four individuals per dish were transplanted into sterilized No. 2 square Petri dishes containing 1 / 2 MS solid medium containing 0.5% gellan gum.

[0065] On the same day, before transplantation, the bacterial cells were cultured at a concentration of 10 ９ A spore suspension of the OS2C strain (CFU / mL) was applied to each petri dish at a volume of 150 μL. The cultivation was carried out in a growth chamber (28°C; photoperiod: 16 hours light / 8 hours dark; approximately 22,000 lux).

[0066] As shown in Figure 20, root hairs on the primary seminal roots at a distance of 4 cm from the seed 3 days after treatment with the OS2C strain (6 days after sowing) were compared with those of untreated rice plants. Root hairs in the OS2C strain-treated plot were significantly longer than those in the untreated plants, demonstrating that root hair differentiation and development were promoted within a short period after treatment.

[0067] As shown in Figure 21A, the overall root development of rice plants 7 days after OS2C treatment (10 days after sowing) was compared with that of untreated rice. As shown in Figures 21B and 21B, respectively, the total number and total length of lateral roots per plant 7 days after OS2C treatment (10 days after sowing) were compared with that of untreated rice. Average values ​​for eight plants are shown. In the OS2C-treated area, the length of lateral roots increased by approximately 14% compared with the untreated area. These results demonstrated that root length was significantly longer in the OS2C-treated area compared with the untreated area, and that elongation growth was promoted.

[0068] The present invention is extremely useful, particularly in the field of cultivation of crop plants such as agricultural crops and resource crops, and useful plants such as ornamental plants.

[0069] The OS2C strain was deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under accession number NITE P-03739 (original deposit date: September 6, 2022). Subsequently, a request for transfer of the international deposit under the Budapest Treaty was made for the microorganism on November 11, 2024 (accession number NITE ABP-03739).

Claims

1. A method for cultivating a plant, comprising applying to said plant or its propagation material an actinomycete belonging to the genus Streptomyces thermocarboxydus.

2. The method of claim 1, wherein the plant is an annual plant and the method comprises applying the actinomycete to the plant at or after 6 weeks of germination.

3. The method of claim 1, wherein the plant is a perennial plant.

4. The method according to any one of claims 1 to 3, wherein the plant propagation material is part or the whole of a plant.

5. The method according to any one of claims 1 to 4, wherein the plant part is selected from a cell, a tissue, an organ, a seed, a bulb, a cutting, and a seedling.

6. The method according to any one of claims 1 to 5, wherein the actinomycete is an OS2C strain (accession number NITE ABP-03739) or a mutant strain thereof.

7. The method according to any one of claims 1 to 6, wherein application of the actinomycete is carried out by direct application of the actinomycete to the plant or its propagation material, or indirect application by application of the actinomycete to the surrounding environment of the plant or its propagation material.

8. The method according to any one of claims 1 to 7, wherein application of the actinomycete improves the growth condition of the plant.

9. The method according to claim 8, wherein the improvement in the growth condition of a plant is an improvement in crop yield, an increase in plant biomass, promotion of root formation, promotion of development of roots, stems and leaves, inhibition of plant dormancy, inhibition of plant growth cessation, or a combination thereof.

10. A plant or its propagation material produced by the method according to any one of claims 1 to 9.

11. A composition comprising an actinomycete belonging to Streptomyces thermocarboxydus for improving the growth condition of a plant.

12. The composition according to claim 11, wherein the actinomycete is an OS2C strain (accession number NITE ABP-03739) or a mutant strain thereof.

13. The composition according to claim 11 or 12, wherein the improvement in the growth condition of a plant is an improvement in the yield of an agricultural crop, an increase in the amount of plant biomass, promotion of root formation, promotion of development of roots, stems and leaves, inhibition of dormancy of a plant, inhibition of cessation of growth of a plant, or a combination thereof.

14. The composition according to any one of claims 11 to 13, wherein the plant is an annual plant and the composition is applied to the plant 6 weeks or more after emergence.

15. The composition according to any one of claims 11 to 13, wherein the plant is a perennial plant.