Methods for cultivating plants and the resulting plants, as well as compositions for improving plant growth.

Abstract

This invention provides a novel plant cultivation method utilizing microorganisms that can promote plant growth and increase yield / biomass. An actinomycete belonging to *Streptomyces thermocarboxydus* is applied to the plant or its propagation material.

Description

Technical Field

[0001] The present invention relates to methods for cultivating plants and the resulting plants, as well as compositions for improving plant growth. Background Technology

[0002] In recent years, to ensure a stable food supply for the growing global population and to meet the expanding societal demand for reducing greenhouse gases by enhancing plants' ability to fix carbon dioxide (CO2), there is a need to further increase the yield and biomass of crops, forage crops, and trees. Research and development in plant science are actively underway. However, regarding the use of plants developed using gene recombination or genome editing technologies in the wild or for food supply, in Japan, concerns about their impact on human health and ecosystems hinder immediate social application at the legal and social levels. Furthermore, the increasing focus on sustainable agriculture that reduces environmental impact, both domestically and internationally, has led to trends towards controlling the use of chemical fertilizers, making high-yield plant cultivation even more challenging.

[0003] Therefore, the use of microorganisms in plant cultivation has recently attracted much attention. It is known that various microorganisms coexist and symbiotically exist in the rhizosphere, leaf surface, and interior of wild plants, and are closely related to plant productivity and growth. Expectations for cultivation methods utilizing the functions of these useful microorganisms are increasing. Examples of such useful microorganisms include various strains of actinomycetes belonging to the genus *Streptomyces*.

[0004] Patent document 1 (Japanese Patent Application Publication No. 2011-188761) discloses an actinomycete strain MBFA-172 (accession number NITE P-896) of the genus Streptomyces that is effective against strawberry anthracnose, and biological materials containing the strain for disease control.

[0005] Non-patent literature 1 (Passari et al., PloS one, (2019), 14.7: e0219014) describes the following: Tomato (Solanum lycopersicum) seedlings were treated with the endophytic actinomycete Streptomyces thermocarboxydus isolate BPSAC147 and cultivated under greenhouse conditions. The photochemical quantum yield and electron transport rate of photochemical system II (PSII) were measured. The results showed that the photosynthetic process was improved. Although it has not yet been confirmed, this microorganism is expected to serve as a new type of biomaterial for improving productivity by promoting photosynthesis.

[0006] Non-patent literature 2 (Lasudee et al., Frontiers in Microbiology, (2018), 9:1247) describes the following: The thermophilic Streptomyces thermocarboxydus S3 strain, isolated from spores of the arbuscular mycorrhizal fungus Funneliformis mosseae, exhibits excellent performance in terms of phosphate solubility, indole-3-acetic acid (IAA) production, and siderophore production. When this microorganism was used to treat mung beans (Vignaradiata) for cultivation, the results showed a significant increase in fresh weight, root length, and total length. Furthermore, when this microorganism was used to treat rice (jasmine rice) and cultivated under drought stress in low-nutrient soil, the results showed that growth was promoted in the initial stage within 45 days of cultivation.

[0007] The inventors also reported in Non-Patent Literature 3 (Kanno et al., Environ. Microbiol., 2016, 18[8]: 2495-2506) the OS2C strain of Streptomyces thermocarboxydus, purified and isolated from the surface-sterilized rice stems (Acceptance No. NITE ABP-03739). The OS2C strain is a plant symbiotic microorganism with specific enzyme genes for oxidizing dilute hydrogen at atmospheric concentrations and high affinity for hydroxylation. In this literature, the inventors reported that when rice seeds were treated with the OS2C strain for sterile soil cultivation, it was observed that the strain was locally present in the roots and stems, thus concluding that the OS2C strain is a plant endophyte. In addition, it was reported that after about 4 weeks (about 5 weeks after germination) of sterile soil cultivation, the length and dry weight of the aboveground parts and roots of rice increased compared with untreated individuals.

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: Japanese Patent Application Publication No. 2011-188761

[0011] Non-patent literature

[0012] Non-patent literature 1: Passari et al., PLoS one, (2019), 14.7: e0219014

[0013] Non-patent literature 2: Lasudee et al., Frontiers in Microbiology, (2018), 9: 1247

[0014] Non-patent literature 3: Kanno et al., Environ. Microbiol., 2016, 18[8]: 2495-2506

[0015] Non-patent literature 4: Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152

[0016] Non-patent literature 5: Gravel et al., Soil Biol. Biochem., (2007), 39.8: 1968-1977

[0017] Non-patent literature 6: Saito et al., Journal of the Japanese Society of Crop Science, (2000), 69.3: 385-390

[0018] Non-patent literature 7: Wagner et al., Nat. Commun., (2016), 7: 12151 Summary of the Invention

[0019] The problem that the invention aims to solve

[0020] However, previous reports on *Streptomyces*, a species of *Streptomyces*, have been limited to the effects of treating annual plants with the strain on growth rate or stress tolerance during the vegetative growth phase when cultivated in soil-based systems. The effects of various actinomycete strains on harvest yield or biomass when treating annual plants up to harvest time, and the effects of various actinomycete strains on perennial plants such as trees, or on cultivation systems such as those propagated by cuttings, remain unknown.

[0021] This invention was made in view of the aforementioned problems, and its object is to provide a novel plant cultivation method utilizing microorganisms that can promote plant growth and increase yield / biomass. More specifically, it aims to provide a novel plant cultivation method that can increase the yield / biomass of annual plants cultivated to harvest time. Another specific object is to provide a novel plant cultivation method that can promote plant growth and increase crop yield / biomass in cultivation systems such as trees or cuttings.

[0022] Methods for solving problems

[0023] Through dedicated research, the inventors discovered that applying actinomycetes belonging to the thermophilic Streptomyces thermocarboxydus species to the cultivation of plants or their propagation materials can improve plant growth, such as promoting plant growth and increasing yield / biomass. Specifically, they found that the following effects can be achieved: increasing the yield / biomass of annual plants during the harvest season; promoting growth and inhibiting dormancy / growth cessation in perennial plants such as trees or in cultivation systems such as cuttings; thus, the present invention was completed.

[0024] That is, the present invention includes the following.

[0025] [Item 1] A method of cultivating a plant, comprising applying an actinomycete belonging to Streptomyces thermocarboxydus to the plant or its propagation material.

[0026] [Item 2] The method of Item 1, wherein the plant is an annual plant, and includes applying the actinomycete to the plant after the 6th week following germination.

[0027] [Item 3] The method described in Item 1, wherein the plant is a perennial plant.

[0028] [Item 4] The method of any one of items 1 to 3, wherein the propagation material of the plant is part or all of the plant.

[0029] [Item 5] The method of any one of items 1 to 4, wherein a portion of the plant is selected from cells, tissues, organs, seeds, bulbs, cuttings, and seedlings.

[0030] [Item 6] The method of any one of items 1 to 5, wherein the actinomycete is OS2C strain (acceptance number NITEABP-03739) or its mutant strain.

[0031] [Item 7] The method of any one of items 1 to 6, wherein the application of the actinomycete is carried out by direct application of the actinomycete to the plant or its propagation material or by indirect application of the actinomycete to the surrounding environment of the plant or its propagation material.

[0032] [Item 8] The method of any one of items 1 to 7, which improves the growth status of the plant by applying the actinomycetes.

[0033] [Item 9] The method described in Item 8, wherein the improvement in plant growth status is an increase in crop yield, an increase in plant biomass, a promotion of root development, a promotion of root, stem and leaf growth, an inhibition of plant dormancy, an inhibition of plant growth cessation, or a combination thereof.

[0034] [Item 10] Plants or their propagation material produced by any one of items 1 to 9.

[0035] [Item 11] A composition for improving plant growth status, comprising an actinomycete belonging to Streptomyces thermocarboxydus.

[0036] [Item 12] The composition of Item 11, wherein the actinomycete is OS2C strain (acceptance number NITE ABP-03739) or a mutant strain thereof.

[0037] [Item 13] The method of Item 11 or 12, wherein the improvement in plant growth status is an increase in crop yield, an increase in plant biomass, a promotion of root development, a promotion of root, stem and leaf growth, an inhibition of plant dormancy, an inhibition of plant growth cessation, or a combination thereof.

[0038] [Item 14] The composition of any one of items 11 to 13, wherein the plant is an annual plant, and the composition is applied to the plant after the 6th week following germination.

[0039] [Item 15] The composition of any one of items 11 to 13, wherein the plant is a perennial plant.

[0040] Invention Effects

[0041] According to the plant cultivation method using the actinomycetes of the present invention, by applying them to plants or their propagation materials for cultivation, plant growth can be promoted and yield / biomass increased. In particular, the plant cultivation method according to an embodiment of the present invention can achieve effects such as increasing the yield / biomass of annual plants during the harvest period. Furthermore, the plant cultivation method according to an embodiment of the present invention can achieve effects such as promoting growth, inhibiting dormancy / inhibiting growth cessation in perennial plants such as trees or in cultivation systems such as cuttings, thereby increasing the yield / biomass of crops. Attached Figure Description

[0042] [ Figure 1 ] Figure 1 A and Figure 1Figure B represents a comparison of the average length and dry weight (measured values ​​of the aboveground parts and roots of each individual) of OS2C plants cultivated in three soils with different nutrient concentrations for 22 days after treatment (29 days after germination) in the pot experiment of Example 2 with untreated rice.

[0043] [ Figure 2 ] Figure 2 A and Figure 2 B are photographs showing the overall images of rice treated with OS2C strains 66 days after treatment (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.

[0044] [ Figure 3 ] Figure 3 Figure A shows a comparison of the average number of leaves in OS2C treated rice after 51 days (56 days after germination; just after heading) with that in untreated rice. Figure 3 Figure B is a comparison of the average number of tillers with that of untreated rice. Figure 3 C is a graph comparing the average dry weight of the aboveground parts (excluding panicle dry weight (approximately 0.1–0.2 g)) with that of untreated rice. Figure 3 Figure D is a graph comparing the average root dry weight with that of untreated rice.

[0045] [ Figure 4 ] Figure 4 A and Figure 4 Figure B represents the average dry weight of rice per pot and the average number of rice grains per pot harvested in the OS2C plant treatment area, divided into incomplete rice and complete rice, and compared with untreated rice.

[0046] [ Figure 5 ] Figure 5 A photograph showing an overall image of potted Erianthus plants after 123 days of OS2C treatment (139 days after sowing) compared to untreated Erianthus.

[0047] [ Figure 6 ] Figure 6 Figure A shows a comparison of the average length of the aboveground portion of each individual of the genus *Imperata* 123 days after treatment with OS2C strains (139 days after sowing) with that of untreated individuals. Figure 6 Figure B shows a comparison of the average dry weight of the aboveground parts of OS2C treated *Imperata* species with that of untreated individuals during the same period. Figure 6 Figure C represents a comparison of the average root dry weight of OS2C treated *Imperata* species with that of untreated individuals during the same period.

[0048] [ Figure 7 ] Figure 7 A represents a photograph taken from the bottom of a container comparing individuals treated with the OS2C strain for 10 days with untreated individuals when a portion of an organ containing a growth point from a sterile poplar tree was passaged into a new culture medium. Figure 7 B represents a comparison. Figure 7 A graph showing the average root length of each individual A.

[0049] [ Figure 8 ] Figure 8 A photograph showing an overall image of OS2C-treated poplar 88 days after the first treatment (46 days after potting) compared to an untreated poplar.

[0050] [ Figure 9 ] Figure 9 Figure A shows the average diameter of the stem base of treated poplars with OS2C strains 88 days after the first treatment (46 days after potting), compared with that of untreated poplars. Figure 9 Figure B shows a comparison of the average dry weight of the aboveground stems of OS2C-treated poplars with that of untreated poplars during the same period. Figure 9 Figure C represents a comparison of the average root dry weight of OS2C-treated poplars with that of untreated poplars during the same period.

[0051] [ Figure 10 ] Figure 10 A photograph showing an overall image of OS2C-treated poplar trees approximately 4 months after potting, compared to an untreated poplar tree.

[0052] [ Figure 11 ] Figure 11 The top image in A is a photograph comparing an OS2C strain 142 days after initial treatment (152 days after sowing) with an untreated strain. The bottom image is a photograph comparing the root condition in the pot with that of an untreated strain. Figure 11 Figure B shows a comparison of the average root dry weight of each individual OS2C-treated eucalyptus tree with that of the untreated individuals during the same period.

[0053] [ Figure 12 ] Figure 12 A photograph showing an overall image of potted eucalyptus trees 91 days after the initial treatment with OS2C strains (105 days after sowing) compared to an untreated individual.

[0054] [ Figure 13 ] Figure 13 Figure A shows a comparison of the average number of leaves per individual of OS2C strain 91 days after the first treatment (105 days after sowing) with that of untreated individuals. Figure 13 Figure B is a graph that compares the average aboveground dry weight of each individual during the same period with that of untreated individuals. Figure 13 C is a graph showing a comparison between the average root dry weight of each individual at the same time period and that of untreated individuals.

[0055] [ Figure 14 ] Figure 14 A photograph showing an overall image of potted eucalyptus trees 159 days after the first treatment with OS2C strains (180 days after sowing) compared to an untreated individual.

[0056] [ Figure 15 ] Figure 15 Figure A shows a comparison of the average aboveground dry weight of each individual eucalyptus tree 159 days after the first treatment with OS2C strains (180 days after sowing) with that of untreated individuals. Figure 15 Figure B represents a comparison of the average aboveground dry weight of the stem (excluding leaves) of each individual eucalyptus tree at the same period with that of an untreated individual. Figure 15 C is a graph showing the average root dry weight of each individual eucalyptus tree at the same period compared with that of untreated individuals. Figure 15 D is a graph that compares the average trunk diameter of each eucalyptus tree at a position 2 cm above the ground with that of an untreated individual at the same period.

[0057] [ Figure 16 ] Figure 16 A and Figure 16 B are images showing the overall appearance of rice (Akita Komachi and Hokuriku 193) treated with OS2C strains 19 days later (26 days after germination) compared with untreated rice at the same time.

[0058] [ Figure 17 ] Figure 17 A and Figure 17 B are photographs showing the overall growth of rice (Akita Komachi and Hokuriku 193) 78 days after OS2C treatment (59 days after transplanting into paddy fields), observed from above the paddy field, compared with untreated rice at the same time. Figure 17 C and Figure 17 Figures D and D represent the average number of tillers in OS2C-treated rice (Akita Komachi and Hokuriku 193) after 78 days of treatment (59 days after transplanting to paddy fields) compared to untreated rice.

[0059] [ Figure 18 ] Figure 18 A and Figure 18 Photographs B show a comparison between rice (Akita Komachi and Hokuriku 193) harvested 155 days after OS2C treatment (136 days after transplanting to paddy fields) and untreated rice at the same time.

[0060] [ Figure 19 ] Figure 19Figure A shows a comparison of the average aboveground fresh weight of each individual rice (Akita Komachi and Hokuriku 193) harvested 155 days after OS2C treatment (136 days after transplanting into paddy fields) with that of untreated individuals. Figure 19 B is a graph comparing the average number of ears per individual to that of untreated individuals. Figure 19 C is a graph comparing the average fresh ear weight of each individual with that of untreated individuals.

[0061] [ Figure 20 ] Figure 20 A photograph comparing the root hairs of the first seed root (4 cm from the seed) of rice treated with OS2C strains 3 days after (6 days after sowing) with those of untreated rice.

[0062] [ Figure 21 ] Figure 21 Image A shows an overall image of root development in rice treated with OS2C strains 7 days after treatment (10 days after sowing), compared with untreated rice. Figure 21 B and Figure 21 Figure C represents a comparison of the total number and length of lateral roots of each individual rice plant treated with OS2C strains 7 days after treatment (10 days after sowing) with untreated rice. Detailed Implementation

[0063] The present invention will now be described in detail with reference to specific embodiments. However, the present invention is not limited to the following embodiments and may be implemented in any form without departing from the spirit of the invention.

[0064] It should be noted that all patent documents (patent application publications and patent gazettes, etc.) and non-patent documents cited in this invention are incorporated herein in their entirety for all purposes.

[0065] One aspect of the invention is a method of cultivating plants, comprising applying an actinomycete belonging to the thermophilic Streptomyces thermocarboxydus to the plant or its propagation material (appropriately referred to as "the method of the invention").

[0066] As mentioned above, including the research of the inventors, there are several prior reports on the cultivation of plants treated with various actinomycete strains belonging to the genus *Streptomyces* (see Patent Document 1 and Non-Patent Documents 1-3 above). However, these prior reports are limited to the effects of treating annual plants with these strains and cultivating them in soil cultivation systems on growth rate or stress tolerance during the vegetative growth period. The effects of treating annual plants with various actinomycete strains until harvest time, and the effects of treating perennial plants such as trees or in cultivation systems such as cuttings, are previously unknown.

[0067] The inventors treated annual plants (rice) with an actinomycete belonging to *Streptomyces thermocarboxydus* (hereinafter referred to as OS2C strain) and cultivated them indoors in soil until harvest. The results showed a significant increase in yield and biomass (see Example 2 and [other examples described below]). Figures 1-4 Previous research suggests that growth enhancement during the vegetative growth phase of annual plants and increased harvest yield and biomass at harvest time are not necessarily based on the same mechanism of action (see Non-Patent Literature 4: Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152; Non-Patent Literature 5: Gravel et al., Soil Biol. Biochem., (2007), 39.8: 1968-1977; Non-Patent Literature 6: Saito et al., Chronicle of the Japanese Society of Crop Science, (2000), 69.3: 385-390). Therefore, the surprising insight that increasing harvest yield and biomass at harvest time by treating annual plants with actinomycetes belonging to the thermophilic Streptomyces thermocarboxydus is something that would not have been easily conceived from previous reports.

[0068] Next, the inventors treated an annual herbaceous resource crop (Streptomyces thermocarboxydus) with an actinomycete (OS2C strain described later) for soil cultivation, and found that its biomass was significantly increased (see Example 3 and below). Figure 5 , 6 Actinomycetes belonging to the thermocarboxydus bacterium, isolated from crops, also help increase the biomass of resource crops—a remarkable insight.

[0069] Furthermore, the inventors treated annual plant (rice) seeds with an actinomycete belonging to *Streptomyces thermocarboxydus* (hereinafter referred to as the OS2C strain) for soil cultivation. Additionally, when subculturing stems containing growth points and several leaves from aseptically cultured perennial plants (poplar) into new culture media, the OS2C strain was used for treatment, followed by potting and further cultivation. Furthermore, perennial plant (eucalyptus) seeds were treated with the OS2C strain and cultured on agar medium, followed by potting and further cultivation. The results showed that all methods achieved the following effects: promoted growth, inhibited dormancy / growth cessation, and thus increased biomass (see Example 4 and [example 5] described later). Figures 7-10 It is known that the community structure of symbiotic microorganisms varies with the age of the plant (Non-Patent Literature 7: Wagner et al., Nat. Commun., (2016), 7:12151), suggesting that the mechanisms of growth promotion in annual and perennial plants also differ. Furthermore, plant growth in soil cultivation systems and differentiation / development in non-soil cultivation systems such as tissue culture or cuttings are generally based on completely different physiological mechanisms, and are therefore believed to be based on different growth-promoting mechanisms. Therefore, the surprising insight that obtained subsequent growth promotion, dormancy / growth arrest inhibition, and increased biomass by treating and cultivating a portion of an organ from a sterile perennial plant (poplar) with an actinomycete belonging to *Streptomyces thermocarboxydus* during subculturing is something that would never have been easily conceived from previous reports.

[0070] Next, the inventors treated perennial resource crops (eucalyptus) belonging to the thermophilic Streptomyces thermocarboxydus (hereinafter referred to as OS2C strain) with soil cultivation, and found that their biomass was significantly increased (see Example 5 and below). Figures 11-15 Based on the background described above, actinomycetes belonging to the thermocarboxydus family also contribute to increasing the biomass of tree resource crops—a surprising insight that would never have been easily conceived from previous reports.

[0071] In the method of this invention, OS2C strain and its mutant strain are cited as examples of actinomycetes belonging to *Streptomyces thermocarboxydus*. OS2C strain is a microorganism purified and isolated by the inventors from the stems of rice plants used for surface sterilization. It is a plant symbiotic microorganism with a specific enzyme gene for oxidizing dilute hydrogen gas at atmospheric concentrations and a high affinity for hydrogen oxidization. When individual plants were treated with OS2C strain and cultured, it was observed to be locally present in the roots and stems (Non-Patent Document 3), thus OS2C strain is considered a plant endophyte. According to sequence homology analysis of the 16S rRNA gene, a marker gene for bacterial systematics, the sequence of the 16S rRNA gene of OS2C strain (serial number: AB894408) has 99.9% identity with the closest relative strain—*Streptomyces thermocarboxydus* AT37 strain (serial number: NR026072).

[0072] In the method of this invention, there is no limitation on the type of plant targeted. For example, when classified according to their mode of reproduction, the plant can be a seed plant or a non-seed-producing plant such as moss or ferns. If it is a seed plant, it can be an angiosperm or a gymnosperm. If it is an angiosperm, it can be a monocotyledonous plant or a dicotyledonous plant. Furthermore, when classifying the plant according to its life cycle, the plant can be an annual plant or a perennial plant. In addition, there is no limitation on the use of the plant, but useful plants such as agricultural crops, resource crops, foliage plants, and horticultural plants are preferred.

[0073] In the method of the present invention, there is no limitation on the plant morphology used as the target of the aforementioned actinomycetes. For example, it can be applied to plants after germination or to propagation material of plants before germination. Examples of plant propagation material include parts or all of a plant, such as cells, tissues, organs, seeds, bulbs, cuttings, and seedlings, and any of these. Furthermore, the plant cells and tissues can be substances derived from the differentiated state of the plant, or substances that have undergone dedifferentiation, such as stem cells or callus tissue. Various methods for dedifferentiating plant cells or tissues are known.

[0074] In the method of this invention, there are no particular limitations on the plant cultivation system. For example, soil cultivation systems using various types of soil and hydroponic cultivation systems using nutrient solutions instead of soil can be used; either type is acceptable. In the case of a soil cultivation system, the soil can be either natural or artificial soil. In the case of a hydroponic cultivation system, solid cultivation systems using various solid culture media and hydroponic cultivation systems using water or various liquid culture media can be used; either type is acceptable. Furthermore, in any case, any fertilizer, such as natural or artificial fertilizer, and various nutrients can be added. Additionally, multiple cultivation systems can be used in combination; for example, a solid cultivation system or a hydroponic system can be used during seed germination or seedling vegetative growth, and the plants can be potted and transferred to a soil cultivation system after they have grown to a certain size.

[0075] In the method of this invention, there are no particular limitations on the timing or frequency of applying the actinomycetes to plants or their propagation materials. For example, when applied to plants after germination, it can be applied at any time (e.g., any one of the germination period, vegetative growth period, reproductive growth period, etc.) or at multiple different times (e.g., any two or more of the germination period, vegetative growth period, reproductive growth period, etc.). Furthermore, it can be applied to propagation materials at any stage before germination, or to cultivation systems such as soil used for plant cultivation. Moreover, at each time point, the application of the actinomycetes can be performed only once or more than twice.

[0076] However, when applying the above-mentioned actinomycetes to annual plants, it is preferable to apply them at least after the vegetative growth stage, specifically after the 6th week post-germination. As mentioned earlier, previous studies have suggested that the growth promotion during the vegetative growth stage of annual plants and the increase in harvest yield and biomass at harvest time are not necessarily based on the same mechanism of action (see Non-Patent Literature 4: Isawa et al., Microbes and Environments, (2009), 1001180152-1001180152; Non-Patent Literature 5: Gravel et al., Soil Biol. Biochem., (2007), 39.8: 1968-1977; Non-Patent Literature 6: Saito et al., Chronicle of the Japanese Society of Crop Science, (2000), 69.3: 385-390). By applying the above-mentioned actinomycetes to annual plants after the vegetative growth stage, it is easy to obtain significant effects on increasing harvest yield and biomass at harvest time that were completely unknown in previous techniques. However, when applying the above-mentioned actinomycetes to annual plants, they can be applied not only after the vegetative growth period (i.e., after the 6th week of germination) but also before the vegetative growth period (i.e., after germination to the 5th week).

[0077] In the method of the present invention, there is no particular limitation on the manner in which the actinomycetes are applied to plants or their propagation materials. Examples include: direct application by spreading or coating the plants or their propagation materials with the culture solution of the actinomycetes; direct application by immersing the plants or their propagation materials in the culture solution of the actinomycetes; or indirect application by mixing the culture solution of the actinomycetes into the surrounding environment (e.g., soil, culture medium, water, etc.) of the plants or their propagation materials for absorption. Any of these methods may be used.

[0078] In the method of this invention, the amount of the actinomycetes used on the plant or its propagation material is not particularly limited, and can be appropriately selected according to the plant species, application period, application method, and other conditions. As an example, firstly, the actinomycetes are prepared to a concentration of approximately 10⁻⁶ cells / mL. 6 ~10 10 A culture medium containing CFU / mL is prepared. For direct application, approximately 0.1–10 mL of this culture medium is applied to each individual. Alternatively, for indirect application, the culture medium is mixed into soil, culture medium, or water at a concentration of 1–100 mL / kg (for soil or solid culture media) or 0.01–50 mL / L (for liquid culture media or water). It should be noted that the actinomycetes are prepared in the form of spores or mycelia.

[0079] According to the method of the present invention, by applying the above-mentioned actinomycetes to plants or their propagation material, the growth status can be improved (especially the growth status improvement independent of cultivation environments such as soil). Therefore, as another embodiment of the present invention, a composition for improving plant growth status is provided, which contains the above-mentioned actinomycetes as an active ingredient.

[0080] Improvements in growth status can be exemplified, for example, by the following (not limited to):

[0081] - Increased crop yields (in cases where the plant is an agricultural crop).

[0082] - Increase in the biomass of resource crops (when plants are resource crops).

[0083] - Promotes hair growth.

[0084] - Promotes the growth of roots, stems and leaves.

[0085] - Inhibition of plant dormancy.

[0086] - Inhibition of plant growth cessation.

[0087] According to the method of the present invention, plants exhibiting the above-described actinomycete symbiosis and possessing one or more of the aforementioned functions can be produced. Plants and their propagation materials produced by the method of the present invention are also the subject of this invention.

[0088] Example

[0089] The present invention will now be described in more detail with reference to embodiments, but these embodiments are merely examples for illustrative purposes and the present invention is not limited to these embodiments in any way.

[0090] [Example 1: Cultivation and preparation of the OS2C strain of actinomycete]

[0091] The actinomycete OS2C strain (application number NITE ABP-03739), belonging to *Streptomyces thermocarboxydus*, was cultured on R2A agar medium under aerobic conditions at 30°C. After approximately 10 days of culture, the microorganism formed gray spore pads on the agar medium. An appropriate amount of sterile water was added to the plate, the spore pads were scraped off, and the recovered bacterial solution was centrifuged, removing the supernatant. An equal volume of sterile water was added, and the mixture was washed twice to prepare a spore suspension of the microorganism. The number of viable cells in the spore suspension was counted using a colony counting method. For use in the treatment of rice in Examples 2, 6, and 7, and in the treatments of *Streptomyces*, poplar, and eucalyptus in Examples 3–5, bacterial suspensions with a concentration of 10 were prepared. 9 CFU / mL and 10 8 A spore suspension of CFU / mL. The bacterial suspension was used for plant treatment on the same day and stored on ice prior to application.

[0092] [Example 2: Treatment of rice with actinomycete OS2C strain]

[0093] Rice seeds (variety: Nipponbare) were selected using salt water, hulled, 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 sown on 0.5% agar medium (containing no components other than agar). Five days later, the newly germinated rice seedlings were transplanted into pots containing 200g each of three types of soil, pre-sterilized by wet autoclaving. Two seedlings were transplanted into each pot. The three types of soil, in order of increasing nutrient concentration, were: black soil, black soil + nutrient solution (black soil with nutrient solution added to 200g of black soil containing the equivalent of 20μL of liquid fertilizer (trade name: Hyponex stock solution), and synthetic potting mix (synthetic granular potting mix, trade name: Bonsol No. 2, Sumitomo Chemical).

[0094] Furthermore, on the same day, the bacterial cell concentration prepared in Example 1 was 10... 9 A spore suspension of OS2C strain at CFU / mL was added to the vicinity of the transplant site at a rate of 1 mL per rice plant. In this example, rice cultivation was carried out in a growth chamber (28°C; photoperiod: 16 hours light / 8 hours dark; approximately 22,000 lux), with sterile water supplied as appropriate.

[0095] like Figure 1 A and Figure 1 As shown in Figure B, the plant height (length) and dry weight (measured values ​​of the aboveground part and root of each individual) of rice plants treated with OS2C plants for 22 days (29 days after germination) in the pot experiment of Example 2 were compared with untreated rice plants in three soils with different nutrient contents (n=2). The average length of the aboveground part and root of the OS2C-treated rice plants increased from <41.8cm to 46.6cm> (+11.5%), from <37.3cm to 51.9cm> (+39.1%), and from <50.7cm to 53.9cm> (+6.3%) in low, medium, and high nutrient soils, respectively. Furthermore, the average dry weight increased from <0.063g to 0.096g> (51.9%), from <0.075g to 0.083g> (+10.5%), and from <0.120g to 0.161g> (+34.2%), respectively. These results indicate that even in soils with varying nutrient contents, symbiosis between OS2C plants and rice can stably promote the initial biomass production of rice.

[0096] Subsequently, rice cultivated with synthetic potting mix was uncovered after 4 weeks of microbial treatment and continued to be cultivated in a growth chamber (30°C; 70% humidity; photoperiod: 11 hours light / 13 hours dark; CO2 concentration 470 ppm) until harvest. 31 and 51 days after treatment, sterilized synthetic granular potting mix was topped with spores of the OS2C strain cultured in Example 1 (approximately 2.0 × 10⁶ spores per pot). 9 The soil (CFU) was subjected to additional microbial treatment.

[0097] like Figure 2 A and Figure 2 As shown in Figure B, composite photographs of OS2C plants treated for 66 days (71 days after germination; heading stage) and 136 days after treatment (141 days after germination; harvest stage) (2 individuals per pot, 5 pots per experimental area, totaling 10 individuals) were compared with untreated rice at the same time. These results indicate that OS2C treatment significantly promoted rice growth and development at any stage of heading and harvest.

[0098] like Figure 3As shown, the parameters of plant biomass (number of leaves, number of tillers, aboveground dry weight (DW) and root dry weight (DW)) of OS2C plants 51 days after treatment (56 days after germination; just after heading) were compared with those of untreated rice. The sum of the values ​​for two individuals in each pot was first calculated, and then the average of this sum was calculated for eight pots (N=8) in each experimental area. These results indicate that OS2C treatment significantly increased the aboveground and root biomass.

[0099] like Figure 4 A and Figure 4 As shown in Figure B, the dry weight and number of grains of rice harvested 136 days after OS2C treatment (141 days after germination; harvest time) were divided into incomplete rice and complete rice, and comparisons were made between OS2C-treated individuals and untreated individuals. First, the combined values ​​of two individuals in each pot were calculated, and then the average of these combined values ​​was calculated for five pots per experimental area (N=5). These results show that OS2C treatment significantly reduced the dry weight and number of grains in incomplete rice, while significantly increasing the dry weight and number of grains in complete rice.

[0100] Furthermore, compared to the untreated area, the OS2C treatment area showed an advancement of approximately two weeks from heading to maturity. This result suggests that, within the OS2C treated individuals, some mechanism facilitated nutrient allocation (nutrient translocation to the harvest).

[0101] [Example 3: Treatment experiment of the actinomycete OS2C strain against the genus *Sacchariformis*]

[0102] Seeds of the genus *Erianthus* (species name: *Erianthus arundinaceus*) were sterilized using a 1 / 20 dilution of kitchen bleach for 5 minutes and then rinsed with water. The seeds were sown on 1% agar medium containing 1% sucrose and 1 / 4 MS medium. The plates were placed upright and incubated in the dark at 22°C for 2 days, followed by 8 days of light incubation under a 16-hour light / 8-hour dark photoperiod. Finally, the seeds were transplanted into soil pre-sterilized by an autoclave.

[0103] Then, on the same day, the bacterial concentration was 10 8 A spore suspension of OS2C strain at CFU / mL was added to the vicinity of the transplanting site at a rate of 1 mL per *Cymbidium* plant. Further treatment with the same OS2C strain was administered one month later. Subsequently, when transplanting to larger pots, the same OS2C spore suspension at the same concentration was administered again at a rate of 1 mL per *Cymbidium* plant.

[0104] like Figure 5 and Figure 6As shown in A through C, pots containing *Imperata cylindrica* plants were prepared 123 days after the initial OS2C treatment (139 days after sowing). The length, aboveground and underground dry weight of each plant were compared with those of untreated plants. The increases were approximately 13%, 24%, and 13%, respectively. These results suggest that OS2C treatment may help promote the growth of both the aboveground and underground parts of the herbaceous *Imperata cylindrica*.

[0105] [Example 4: Treatment of poplar trees with the actinomycete OS2C strain]

[0106] Hybrid poplar (Populus tremula x tremuloides) individuals were aseptically cultured on 1 / 2 MS solid medium containing 0.3% gellan gum under a 16-hour light / 8-hour dark photoperiod and at 22°C. After approximately one month of subculture, the individuals were cut at an internode approximately 3 cm from the shoot tip using a scalpel. The cut surface, about 1 cm above the cut, was then immersed in the OS2C suspension prepared in Example 1 (10...). 7 The culture was treated with a bacterial culture medium (cells / mL) and allowed to stand for 5 minutes. Then, the stems of the treated culture were inserted into half MS solid medium containing 2% sucrose to allow the plants to stand upright. They were then cultured again under a 16-hour light / 8-hour dark photoperiod and at 22°C. The effects of the bacterial treatment on root development and initial growth were observed over time. As a control (untreated area), sterile water was used instead of the bacterial suspension. The cut culture sections were soaked in sterile water for 5 minutes and then inserted into half MS solid medium containing 2% sucrose for further observation.

[0107] like Figure 7 As shown in Figure A, poplar trees treated with OS2C strains for 10 days were compared with untreated poplar trees. Figure 7 As shown in Figure B, the root length of poplars treated with OS2C strains for 10 days was compared with that of untreated poplars. These results indicate that in the OS2C-treated area, root length significantly increased and elongation growth was promoted compared to untreated individuals.

[0108] In addition, the poplar trees were transferred from agar medium to soil pots and continued to be cultivated under artificial light. Figure 8 and Figure 9 As shown in A through C, poplar pots were prepared 88 days after the initial OS2C treatment (46 days after potting). The thickness and dry weight of the stem base of each individual plant, as well as the dry weight of the roots, were compared with those of untreated plants. Significant increases were confirmed compared to untreated plants. These results suggest that OS2C treatment may promote the growth of both the above-ground parts and roots of poplar.

[0109] like Figure 10As shown, the overall growth of OS2C-treated poplar trees approximately four months after potting was compared with that of untreated poplar trees. In the untreated individuals, approximately four months after potting, a transition to dormancy was observed, including winter bud formation, growth cessation, and leaf yellowing. However, these phenomena were not observed in the OS2C-treated individuals, whose leaves remained green and continued to elongate well. These results suggest that symbiosis with OS2C plants can effectively inhibit dormancy and growth cessation.

[0110] [Example 5: Treatment of Eucalyptus Trees with Actinomycete OS2C Strain]

[0111] Eucalyptus seeds (species name: *Eucalyptus Globulus*) were sterilized using a mixture of 1 / 5 diluted Kitchen Haiter for 30 minutes, 70% ethanol for 30 seconds, and then further sterilized with 1 / 5 diluted Kitchen Haiter for 15 minutes, followed by rinsing. The seeds were sown on 1% agar medium containing 1% sucrose and 1 / 4 MS medium. The plates were placed upright and incubated in the dark at 22°C for 2 days, followed by 8 days of light incubation under a 16-hour light / 8-hour dark photoperiod. Finally, the seeds were transplanted into pre-sterilized soil using an autoclave.

[0112] Furthermore, on the same day, the bacterial cell concentration prepared in Example 1 was 10... 8 A spore suspension of OS2C strain at CFU / mL was added to the vicinity of the transplant site at a rate of 1 mL per eucalyptus tree. Further, after 43 days, an additional 1 mL of OS2C strain at the same concentration was added to each eucalyptus tree.

[0113] like Figure 11 A and Figure 11 As shown in Figure B, whole-body images of eucalyptus individuals were taken 142 days after the initial treatment of OS2C plants (152 days after sowing), and the root dry weight of each individual was compared with that of untreated individuals. The root dry weight increased by approximately 54% compared to the untreated area.

[0114] For eucalyptus plants transplanted into pots larger than those used in the previous two treatment experiments, the bacterial cell concentration prepared in Example 1 was 10% after 4 days of transplanting into the soil. 8 A spore suspension of OS2C strain at CFU / mL was added to the vicinity of the transplant site at a rate of 1 mL per eucalyptus tree. Further treatment with the same OS2C strain was administered after 32 days.

[0115] like Figure 12 and Figure 13As shown in A through C, eucalyptus cultivation pots were prepared 91 days after the initial treatment with OS2C plants (105 days after sowing). The number of leaves and dry weight of each individual plant were compared with those of untreated plants. The number of leaves, aboveground dry weight, and root dry weight increased by approximately 56%, 12%, and 58%, respectively. These results suggest that the symbiosis of OS2C plants contributes to the growth of eucalyptus trees, particularly by enhancing aboveground biomass production through promoting root growth.

[0116] like Figure 14 and Figure 15 As shown in A through C, eucalyptus cultivation pots were prepared 159 days after the initial treatment of OS2C plants (180 days after sowing). The dry weight of the aboveground parts, stems only, and underground parts of each individual plant was compared with that of untreated plants. The total dry weight of the aboveground parts, the dry weight of the stems only (excluding leaves), and the dry weight of the underground parts increased by approximately 37%, 51%, and 2 times, respectively. Furthermore, as... Figure 15 As shown in Figure D, the thickness of the tree trunk at a position 2 cm above the ground was compared between OS2C treated individuals and untreated individuals. Regarding the trunk diameter, OS2C treated individuals showed a trend towards larger diameters than untreated individuals. These results are consistent with... Figure 12 The results shown are consistent, suggesting that symbiosis with OS2C strains is effective in increasing wood quality in the long-term cultivation of eucalyptus trees.

[0117] [Example 6: Field trial of rice seedlings treated with actinomycete OS2C strain]

[0118] The rice seeds (variety: Akita Komachi and Hokuriku 193) were obtained by sterilizing rice seeds selected through brine selection with 70% ethanol for 1 minute and 1% sodium hypochlorite for 30 minutes, followed by washing. The seeds were soaked in water to induce germination for 3 days, and the germinated seeds were sown into cell trays containing synthetic potting mix (trade name: Inaho granular potting mix), with one seed per cell.

[0119] A series of cultivations were carried out in a growth chamber (28°C; photoperiod: 16 hours light / 8 hours dark; approximately 22,000 lux). Seven days after germination, the bacterial cell concentration prepared in Example 1 was increased to 10⁻⁶. 9 A spore suspension of OS2C strain CFU / mL was added to the surface of each rice seed at a rate of 1 mL.

[0120] like Figure 16 A and Figure 16As shown in Figure B, in the field trial of Example 6, rice seedlings of Akita Komachi and Hokuriku 193, transplanted into paddy fields 19 days after treatment with OS2C plants (26 days after germination), were compared with untreated rice seedlings at the same time. These results indicate that OS2C treatment promotes early seedling growth. This early growth-promoting effect was particularly significant in Akita Komachi.

[0121] The seedlings were transplanted to paddy fields in Akita Prefecture in early June and continued to be cultivated in the wild until early October, the harvest season. The amount of fertilizer applied to the paddy fields before transplanting and the application of herbicides before and after transplanting were carried out according to general cultivation conditions.

[0122] like Figure 17 A and Figure 17 As shown in Figure B, photographs were taken above the paddy fields of Akita Komachi and Hokuriku 193 rice plants 78 days after OS2C treatment (59 days after transplanting to paddy fields; vegetative growth period), and compared with untreated rice at the same time. Further, as... Figure 17 C and Figure 17 As shown in Figure D, the number of tillers in the OS2C-treated individuals of Akita Komachi and Hokuriku 193 varieties after 78 days of OS2C treatment (59 days after transplanting to paddy fields) was compared with that in untreated rice. The average values ​​of a total of 16 individuals from each of the four divisions are shown. In the OS2C-treated areas, the number of tillers increased by approximately 20% compared to the untreated areas. These results indicate that treating seedlings with OS2C significantly increased the number of aboveground tillers in both varieties, even at a point approximately two months after transplanting to paddy fields.

[0123] like Figure 18 A and Figure 18 As shown in Figure B, Akita Komachi and Hokuriku 193 (4–6 individuals) treated with OS2C for 155 days (136 days after transplanting to paddy fields; harvest time) were compared with untreated rice at the same time. These results indicate that even at harvest time, OS2C treatment promoted rice growth and development in both varieties.

[0124] like Figure 19 As shown in A through C, the aboveground fresh weight (FW), a biomass parameter, and the number and fresh weight of panicles (FW), yield parameters, of OS2C plants after 155 days (136 days after transplanting to paddy fields; harvest time) were compared with untreated rice. For Akita Komachi, the average values ​​of 31–40 individuals (N=31–40) across four regions are shown; for Hokuriku 193, the average values ​​of 74 individuals (N=74) across four regions are shown. These results indicate that treatment with OS2C plants increased aboveground plant biomass and significantly increased rice yield in both varieties.

[0125] [Example 7: Comparative experiment on root hairs and lateral roots of rice treated with actinomycete OS2C strain]

[0126] Rice seeds (variety: Nipponbare) were selected using saline solution, hulled, sterilized with 70% ethanol for 1 minute and 1% sodium hypochlorite for 30 minutes, and then washed with water. The seeds were sown on 0.5% agar medium (containing no other components than agar). Three days later, four individuals per dish were transplanted into sterile No. 2 square Petri dishes containing 0.5% gellan gum on 1 / 2 MS solid medium.

[0127] Prior to the transplantation on that day, the bacterial cell concentration prepared in Example 1 was 10. 9 A spore suspension of OS2C strain at CFU / mL was spread at 150 μL per dish. A series of cultivations were carried out in a growth chamber (28°C; photoperiod: 16 h light / 8 h dark; approximately 22,000 lux).

[0128] like Figure 20 As shown, the root hairs of the first seed root (4 cm from the seed) of OS2C plants 3 days after treatment (6 days after sowing) were compared with those of untreated rice. In the OS2C plant treatment area, the root hairs were significantly longer than those of untreated individuals, indicating that root hair differentiation and development were promoted in the short term after treatment.

[0129] like Figure 21 As shown in Figure A, the overall root development of rice treated with OS2C plants for 7 days (10 days after sowing) is compared with that of untreated rice. Figure 21 B and Figure 21 As shown in Figures C, the total number of lateral roots and the total length of lateral roots for each individual of OS2C plants treated for 7 days (10 days after sowing) were compared with those of untreated rice. The average values ​​for each group of 8 individuals are shown. Compared to the untreated area, the lateral root length of rice in the OS2C-treated area increased by approximately 14%. These results indicate that in the OS2C-treated area, root length was significantly increased and elongation growth was promoted compared to untreated individuals.

[0130] Industrial practicality

[0131] This invention is particularly useful in the cultivation of crops, resource crops, and other useful plants such as ornamental plants.

[0132] Strain OS2C has been deposited at the Patent Microbial Collection Center of the Technical Base for Product Evaluation (address: Room 122, 2-5-8 Kamisamaashi, Kisarazu City, Chiba Prefecture 292-0818, Japan), with accession number NITE P-03739 (original deposit date: September 6, 2022). Subsequently, on November 11, 2024, a request was made for an international transfer of deposit of this microorganism in accordance with the Budapest Treaty (application number NITE ABP-03739). PCT / RO / 134 form

Claims

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

2. The method according to claim 1, wherein, The plant is an annual plant, and includes plants on which the actinomycetes have been applied for six weeks or more after germination.

3. The method according to claim 1, wherein, The plant in question is a perennial plant.

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

5. The method according to any one of claims 1 to 4, wherein, A portion of the plant is selected from cells, tissues, organs, seeds, bulbs, cuttings, and seedlings.

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

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

8. The method according to any one of claims 1 to 7, wherein the plant growth status is improved by applying the actinomycetes.

9. The method according to claim 8, wherein, The improvement in plant growth status is defined as increased crop yield, increased plant biomass, promoted root development, promoted root, stem and leaf growth, inhibited plant dormancy, inhibited 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 for improving plant growth status, comprising an actinomycete belonging to Streptomyces thermocarboxydus.

12. The composition according to claim 11, wherein, The actinomycete is the OS2C strain (application number NITEABP-03739) or its mutant strain.

13. The composition according to claim 11 or 12, wherein, The improvement in plant growth status is defined as increased crop yield, increased plant biomass, promoted root development, promoted root, stem and leaf growth, inhibited plant dormancy, inhibited plant growth cessation, 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 after the 6th week following germination.

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

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