New endospore-forming bacterium having plant-growth promoting action in dry environment, plant-growth promoting agent, and plant-cultivation method

Abstract

The present invention addresses the problem of providing a microbial material that can also be applied to agricultural crop cultivation in a dry area.　The present invention provides the following endospore-forming bacteria (A) to (C) having plant-growth promoting activity in a dry environment. (A) Either Bacillus thuringiensis TTREp1 strain identified by the accession number NITE BP-04159 or Bacillus altitudinis TCo-SSREp1 strain identified by the accession number NITE BP-04160, or a mutant strain thereof (B) Paenibacillus polysaccharolyticus TP-WSREp1 strain identified by the accession number NITE BP-04163, or a mutant strain thereof (C) either Priestia aryabhattai TTREn1 strain identified by the accession number NITE BP-04165 or Priestia megaterium TC-WSREn1 strain identified by the accession number NITE BP-04166, or a mutant strain thereof

Description

Novel spore-forming bacteria that promote plant growth in dry environments, plant growth promoter, and method for cultivating plants.

[0001] The present invention relates to a novel spore-forming bacterium that has an excellent growth-promoting effect on plants such as wheat in a dry environment, a plant growth promoter containing the spore-forming bacterium, and a method for cultivating plants using the spore-forming bacterium.

[0002] While many microbial materials have been developed worldwide, most of them use microorganisms that do not form spores, requiring storage and transportation at low temperatures, and having a short shelf life of only a few months, making them inconvenient for farmers to use.

[0003] On the other hand, spore-forming bacteria transform from vegetative cells into spores under unfavorable conditions such as drought, high temperatures, and nutrient deficiencies, making them resistant to such environments and thus suitable as microbial materials with high transportability and storage capabilities. To date, microbial materials using spore-forming bacteria that can be stored for long periods at room temperature have been developed. For example, it has been reported that a biofertilizer using Bacillus pumilus TUAT1 strain can maintain its bacterial count and effectiveness for more than six months at room temperature (around 25°C) (Patent Document 1). However, this biofertilizer using Bacillus pumilus TUAT1 strain was mainly developed as a microbial material for rice cultivation in water-rich environments, so it was unclear whether it could be adapted for field crop cultivation in areas with little rainfall. Furthermore, wheat, one of the world's three major grains along with rice and corn, requires cultivation conditions suitable for drought, unlike rice. In addition, recent global climate change has led to large-scale droughts and the expansion of arid areas such as deserts, which are major factors in reducing agricultural production. Therefore, until now, there has been no biofertilizer that can be used for crop cultivation in such arid regions and that can be transported and stored stably.

[0004] WO2015 / 093428 issue

[0005] Therefore, in view of the above-mentioned circumstances, the object of the present invention is to provide a microbial material that can also be applied to the cultivation of crops in arid regions.

[0006] The inventors diligently conducted research to solve the above problems. As a result, they isolated microorganisms from the rhizosphere of field crops, subjected them to high-temperature treatment, and isolated the surviving microorganisms as spore-forming bacteria. They then conducted inoculation tests on wheat using these spore-forming bacteria. While the Bacillus pumilus TUAT1 strain, which is used in existing microbial materials for rice cultivation, showed no growth-promoting effect under drought stress conditions, they found 11 strains among the isolated spore-forming bacteria that exhibited excellent growth-promoting effects even under drought stress conditions. Furthermore, they selected 5 strains that exhibited excellent growth-promoting effects even under high-temperature stress conditions, thus completing the present invention.

[0007] The present invention encompasses the following: (1) Spore-forming bacteria (A) to (C) below, which have plant growth promoting activity in a dry environment. (A) Either the Bacillus thuringiensis TTREp1 strain identified under accession number NITE BP-04159, or the Bacillus altitudinis TCo-SSREp1 strain identified under accession number NITE BP-04160, or a mutant thereof. (B) The Paenibacillus polysaccharolyticus TP-WSREp1 strain identified under accession number NITE BP-04163, or a mutant thereof. (C) The Priestia aryabhattai TTREn1 strain identified under accession number NITE BP-04165, or the Priestia megatherium identified under accession number NITE BP-04166. (1) A plant growth promoter containing any strain of megaterium TC-WSREn1 or a mutant thereof as an active ingredient. (2) A plant growth promoter containing the spore-forming bacteria described in (1) as an active ingredient. (3) A method for cultivating plants, comprising supplying the spore-forming bacteria described in (1) or the plant growth promoter described in (2) to the rhizosphere of a target plant or allowing it to act on a target plant. (4) The method according to (3), wherein the target plant is a plant selected from grasses and legumes. (5) The method according to (4), wherein the grass is wheat, rice, or maize. (6) The method according to (4), wherein the legume is soybean. (7) The spore-forming bacteria of (A) to (C) below.(A) Either the Bacillus thuringiensis TTREp1 strain identified under accession number NITE BP-04159, or the Bacillus altitudinis TCo-SSREp1 strain identified under accession number NITE BP-04160, or a mutant thereof. (B) The Paenibacillus polysaccharolyticus TP-WSREp1 strain identified under accession number NITE BP-04163, or a mutant thereof. (C) The Priestia aryabhattai TTREn1 strain identified under accession number NITE BP-04165, or the Priestia megatherium identified under accession number NITE BP-04166. Any strain of *Megalosa megaterium* TC-WSREn1, or a mutant thereof.

[0008] This application claims priority to Japanese Patent Application No. 2024-177139, filed on 9 October 2024, and encompasses the contents described in the specification of said patent application.

[0009] The present invention provides a novel spore-forming bacterium that promotes plant growth even in arid environments. Therefore, the spore-forming bacterium and the plant growth promoter containing the spore-forming bacterium according to the present invention can be applied to crop cultivation in arid regions and can suppress yield reductions. This will reduce drought damage to major grains worldwide (wheat, rice, corn, and soybeans) caused by the expansion of arid and semi-arid zones due to recent global warming, and enable a stable food supply.

[0010] Figure 2 shows the effects on the total biomass (dry weight) of wheat when wheat is cultivated under normal conditions (no stress), high-temperature stress conditions, and drought stress conditions after applying one known strain of B. pumilus TUAT and 13 newly isolated strains of spore-forming bacteria to the soil (asterisks indicate strains that showed a significant difference compared to the uninoculated area according to the Dunnette test). Figure 2 shows a photograph of wheat cultivated under drought stress conditions after inoculation with one known strain of B. pumilus TUAT and 13 newly isolated strains of spore-forming bacteria. Figure 3 shows the effects on the above-ground, underground, and above-ground + underground biomass (dry weight) of wheat when wheat is cultivated under high temperature + drought stress conditions after being treated with one known B. pumilus strain TUAT and six strains selected from 13 newly isolated spore-forming bacterial strains (one Bacillus strain TTREp, one TCo-SSREp, one Paenibacillus strain TC-CSREp, one TP-WSREp, and one Priestia strain TTREn and one TC-WSREn) in soil (asterisks indicate strains that showed a significant difference compared to the uninoculated area according to the Dunnette test). Figure 4 shows the effects on the above-ground and underground biomass (dry weight) of maize when inoculated with three strains selected from 13 newly isolated spore-forming bacterial strains (one Bacillus strain TCo-SSREp, one Paenibacillus silvae strain TC-CSREp, and one Priestia strain TTREn) under various conditions (normal conditions, high-temperature stress conditions, drought stress conditions, and high-temperature + drought stress conditions) (asterisks indicate strains that showed a significant difference compared to the uninoculated strain according to the Dunnette test). Figure 5 shows photographs of corn grown under various conditions (normal conditions, high-temperature stress conditions, drought stress conditions, and high-temperature + drought stress conditions) after applying three strains selected from 13 newly isolated spore-forming bacterial strains (one TCo-SSREp strain of the genus Bacillus, one TC-CSREp strain of the genus Paenibacillus silvae, and one TTREn strain of the genus Priestia) to the soil.Figure 6 shows the effects on the biomass (dry weight) of above-ground, underground, and above-ground + underground parts of wheat when inoculated with one of three strains: Bacillus genus TCo-SSREp1, Paenibacillus silvae genus TC-CSREp1, and Priestia genus TTREn1, either individually or in combination with two or three strains, under high temperature + drought stress conditions (asterisks indicate strains that showed a statistically significant difference compared to the uninoculated strain according to the Dunnette test).

[0011] The present invention will be described in detail below. 1. Novel spore-forming bacteria The spore-forming bacteria according to the present invention belong to one of the genera Bacillus, Paenibacillus, or Priestia, isolated by the inventors from the rhizosphere of field crops, and are novel spore-forming bacteria that can promote plant growth even in dry environments.

[0012] "Promoting plant growth" means increasing biomass. Here, "biomass" refers to the amount of plant material or its parts present in any given space at a given time, and is used to mean substances, food, materials, fuel, resources, etc. derived from such plant material or its parts. Specifically, increasing biomass means increasing the weight of the plant, enlargement of rhizomes (roots, corms, tubers, bulbs), above-ground stems, flower stalks, and vines, enlargement of seeds, promotion of elongation of stem height, plant height, culm height, and ear height, and increase in source organs such as leaves.

[0013] A "drought environment" refers to an environment in which a plant is exposed to moisture conditions lower than those under which it can normally grow, for any duration and any number of times, and experiences drought stress. Here, "drought stress" refers to the stress a plant experiences when exposed to a moisture environment in which the moisture content in the soil decreases due to a decrease in rainfall or irrigation, inhibiting water absorption and thus inhibiting plant growth. Specifically, although it may vary depending on the type of soil, examples include conditions where the soil moisture content in which the plant is cultivated is 15% by weight or less, strictly 10% by weight or less, or more strictly 7.5% by weight or less, or conditions where the pF value of the soil in which the plant is cultivated is 2.3 or higher, strictly 2.7 or higher, or more strictly 3.0 or higher.

[0014] Furthermore, "high-temperature stress" refers to stress caused by exposure of plants to temperatures higher than normal growing temperatures, such as 25°C or higher, more severely 30°C or higher, or even more severely 35°C or higher, and 50°C or lower. The daily duration of exposure to high temperatures varies depending on the temperature and is not particularly limited, but examples include 120 minutes or more, 180 minutes or more, 300 minutes or more, and 600 minutes or less.

[0015] The spore-forming bacteria belonging to the genus Bacillus mentioned above were named Bacillus amyloliquefaciens FRREp2, Bacillus subtilis TCoREn1, Bacillus thuringiensis TTREp1, Bacillus altitudinis TCo-SSREp1, and Bacillus velezensis TSEn-WSREn1. Of these, strain Bacillus thuringiensis TTREp1 was internationally deposited on September 19, 2024 (original deposit date) at the Patent Microbial Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) under the accession number NITE BP-04159 (identification: TTREp1) in accordance with the Budapest Convention. This deposited strain was transferred from domestic deposit (original deposit) to international deposit under the Budapest Convention on September 12, 2025. Bacillus altitudinis strain TCo-SSREp1 was internationally deposited on September 19, 2024 (original deposit date) at the Patent Microbial Depository Center (NPMD) of the National Institute of Technology and Evaluation (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) under the accession number NITE BP-04160 (identification: TCo-SSREp1) in accordance with the Budapest Convention. This deposited strain was transferred from domestic deposit (original deposit) to international deposit under the Budapest Convention on September 12, 2025.

[0016] The spore-forming bacteria belonging to the genus Paenibacillus mentioned above were named Paenibacillus silvae TC-CSREp1 strain and Paenibacillus polysaccharolyticus TP-WSREp1 strain. Of these, Paenibacillus polysaccharolyticus TP-WSREp1 strain was internationally deposited with the National Institute of Technology and Evaluation (NPMD) Patent Microbial Depository Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) on September 19, 2024 (original deposit date) under the Budapest Convention, with accession number NITE BP-04163 (identification mark: TP-WSREp1). These deposited shares were transferred from domestic deposit (original deposit) to international deposit under the Budapest Convention on September 12, 2025.

[0017] The spore-forming bacteria belonging to the genus Priestia mentioned above were named Priestia megaterium FTRen1, Priestia aryabhattai TTRen1, Priestia megaterium TC-WSREn1, and Priestia aryabhattai FP-WSREp1. Of these, the Priestia aryabhattai TTREn1 strain was internationally deposited on September 19, 2024 (original deposit date) at the Patent Microbial Depositary Center (NPMD) of the National Institute of Technology and Evaluation (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) under the Budapest Convention, with accession number NITE BP-04165 (identification mark: TTREn1). This deposited strain was transferred from domestic deposit (original deposit) to international deposit under the Budapest Convention on September 12, 2025. The Priestia megaterium TC-WSREn1 strain was internationally deposited on September 19, 2024 (original deposit date) at the National Institute of Technology and Evaluation (NPMD) Patent Microbial Depositary Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamatari, Kisarazu, Chiba, Japan 292-0818, Japan) under the designation NITE BP-04166 (identification mark: TC-WSREn1) in accordance with the Budapest Convention. This deposited strain was transferred from domestic deposit (original deposit) to international deposit under the Budapest Convention on September 12, 2025.

[0018] The spore-forming bacteria according to the present invention include bacteria belonging to the genus Bacillus that are classified as the same strains as the above-mentioned FRREp2 strain, TCoREn1 strain, TTREp1 strain, TCo-SSREp1 strain and TSEn-WSREn1 strain and have plant growth promoting activity in dry environments; bacteria belonging to the genus Paenibacillus that are classified as the same strains as the above-mentioned TC-CSREp1 and TP-WSREp1 strains and have plant growth promoting activity in dry environments; and bacteria belonging to the genus Priestia that are classified as the same strains as the above-mentioned FTRen1 strain, TTRen1 strain, TC-WSREn1 strain and FP-WSREp1 strain and have plant growth promoting activity in dry environments.

[0019] Furthermore, the bacteria belonging to the genus Bacillus, named FRREp2, TCoREn1, TTREp1, TCo-SSREp1, and TSEn-WSREn1, each possess 16S rDNA containing the nucleotide sequences shown in SEQ ID NOs. 1 to 5. Therefore, the spore-forming bacteria according to the present invention are bacteria belonging to the genus Bacillus that possess 16S rDNA containing a nucleotide sequence having 95% or more, preferably 98% or more, sequence identity with any of the nucleotide sequences shown in SEQ ID NOs. 1 to 5, and also include bacteria that have plant growth promoting activity in dry environments.

[0020] Furthermore, the bacteria belonging to the genus Paenibacillus, named TC-CSREp1 and TP-WSREp1, respectively, possess 16S rDNA containing the nucleotide sequences shown in SEQ ID NOs. 6 and 7. Therefore, the spore-forming bacteria according to the present invention are bacteria belonging to the genus Paenibacillus that possess 16S rDNA containing a nucleotide sequence having 95% or more, preferably 98% or more, and more preferably 99% or more sequence identity with either of the nucleotide sequences shown in SEQ ID NOs. 6 and 7, and also include bacteria that have plant growth promoting activity in dry environments.

[0021] Furthermore, the bacteria belonging to the genus Priestia, named FTRen1, TTRen1, TC-WSREn1, and FP-WSREp1, each possess 16S rDNA containing the nucleotide sequences shown in SEQ ID NOs. 8 to 11. Therefore, the spore-forming bacteria according to the present invention are bacteria belonging to the genus Priestia that possess 16S rDNA containing a nucleotide sequence having 95% or more, preferably 98% or more, and more preferably 99% or more sequence identity with any of the nucleotide sequences shown in SEQ ID NOs. 8 to 11, and also include bacteria that have plant growth promoting activity in dry environments.

[0022] Furthermore, the spore-forming bacteria according to the present invention may be mutant strains obtained by subjecting each of the above-mentioned strains FRREp2, TCoREn1, TTREp1, TCo-SSREp1, TSEn-WSREn1, TC-CSREp1, TP-WSREp1, FTRen1, TTRen1, TC-WSREn1, and FP-WSREp1 to mutagenic treatment, and may be bacteria that have plant growth promoting activity in a dry environment. Here, mutagenic treatment means inducing mutations in the genomic DNA of microorganisms, and examples include, but are not limited to, treatments in which compounds such as methylethylsulfonic acid (EMS), methylmethanesulfonic acid (MMS), alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and base analogs such as 5-bromouracil are brought into contact with the bacteria, or treatments in which ultraviolet light is irradiated onto the bacteria. Whether or not these mutagenic bacteria retain their plant growth-promoting activity even in a dry environment can be determined by growing wheat inoculated with the bacteria under drought stress conditions (reducing watering for one week from the third week of growth, and maintaining a pF level of 2.7-2.8 on a pF meter, indicating a dry state at the bottom of the box), and comparing the biomass amount with that of uninoculated plants, as described in the examples below.

[0023] 2. Plant Growth Promoter By utilizing the plant growth promoting effect of the spore-forming bacteria according to the present invention under dry conditions, it is possible to confer drought stress tolerance to the target plants. In other words, the spore-forming bacteria according to the present invention can be provided as a plant growth promoter that can be used in dry environments.

[0024] The spore-forming bacteria used as the active ingredient in the plant growth promoter according to the present invention are preferably spore-forming spore cells. Inoculation in the spore state maintains the survival of the spores for several months, increasing the stability of the inoculation effect. Methods for bacterial culture for spore formation (preferably mature spore formation) are well known, and such known methods can be used. For example, bacteria can be prepared by culturing them under appropriate conditions using a culture medium commonly used for culturing spore-forming bacteria. Specifically, a culture medium (natural or synthetic, liquid or solid) can be selected that contains a carbon source (lactose, glucose, sucrose, fructose, galactose, molasses, etc.), a nitrogen source (organic nitrogen-containing substances such as peptone, hydrolyzed casein, hydrolyzed whey protein, hydrolyzed soy protein), and inorganic salts (phosphates, sodium, potassium, magnesium, calcium, etc.) and allows for efficient cultivation of spore-forming bacteria. In addition, amino acids, vitamins, surfactants, etc. can be added to the culture medium if necessary. Suitable solid culture media for spore-forming bacteria include, for example, NFb medium (5.0 g malic acid, K2HPO4). 4 0.5g, MgSO4・7H2O 0.2g, NaCl 0.1g, CaCl 2 Examples of suitable media include 0.02g of spores, 0.002g of Na2MoO4・2H2O, 0.01g of MnSO4・H2O, 4.5g of KOH, 4.0ml of Fe EDTA (1.64% W / V%), and 0.1ml of Biotin, dissolved in 1L of distilled water, with the pH adjusted to 6.8 with NaOH or KOH; TS (Tryptocase Soy) agar; HI (Heart Infusion) agar; and spore-inducing media such as Difco Sporulation Medium (DSM).

[0025] Spore-forming bacteria can be cultured at 20°C to 70°C, preferably 30°C to 60°C, under aerobic or anaerobic conditions. Temperature conditions can be adjusted using a constant temperature bath, mantle heater, jacket, etc. Any culture method is acceptable, such as static culture, shaking culture, or tank culture. The culture time can be 12 hours to 20 days, preferably 12 hours to 10 days. It is preferable to maintain the pH of the culture medium at 5 to 9, preferably 6 to 8, at the start of culture. Those skilled in the art can select appropriate culture conditions depending on the type of spore-forming bacteria used. When using a liquid medium for static culture, spores tend to aggregate, so this can be prevented by adding a certain amount of an appropriate surfactant.

[0026] Bacterial culture solutions can be obtained by culturing bacteria in media and culture conditions conventionally used for spore-forming bacteria, as illustrated above.

[0027] Bacterial concentrates can be obtained, for example, by dehydrating the culture medium under reduced pressure at a temperature that does not kill the bacteria, or by concentrating the culture medium by filtering it using a cross-flow filter.

[0028] Bacterial dehydrates can be obtained, for example, by freeze-drying the culture medium.

[0029] Bacterial immobilized material can be obtained by adsorbing a bacterial culture solution onto one or more carriers, preferably porous carriers, such as calcium carbonate, rice bran, activated carbon, diatomaceous earth, talc, zeolite, vermiculite, peat moss, perlite, bentonite, or montmorillonite, and then drying the adsorbed material. Drying can be done by, for example, freeze-drying or vacuum drying. The dried material may be further pulverized to the extent that it does not destroy the bacteria.

[0030] When the spore-forming bacteria according to the present invention are used as a plant growth promoter, the spore-forming bacteria according to the present invention may be used alone, or they may be combined with other optional components to form a specific formulation. Examples of formulation forms include liquids, powders, granules, emulsions, oils, suspensions, wettable powders, water-soluble powders, pastes, capsules, and fumigants (aerosols).

[0031] Other optional formulations include, for example, carriers for supporting spore-forming bacteria such as liquid carriers and solid carriers, emulsifiers, dispersants, defoamers, diluents, excipients, binders, and bulking agents. Examples of liquid carriers include phosphate buffer, carbonate buffer, and physiological saline. Examples of solid carriers include natural mineral powders such as kaolin, clay, talc, bentonite, chalk, quartz, attapulgite, montmorillonite, white carbon, and diatomaceous earth, synthetic mineral powders such as silicic acid, alumina, and silicates, and high molecular weight natural products such as charcoal, crystalline cellulose, corn starch, gelatin, and alginic acid. In addition, inorganic substances such as vermiculite, silica sand, mica, pumice, gypsum, calcium carbonate, dolomite, magnesium, slaked lime, calcium phosphate, zeolite, and ammonium sulfate may be used as solid carriers. Furthermore, as solid carriers, plant-derived organic materials such as compost, peat, rice husks, rice bran, soybean flour, tobacco flour, walnut flour, wheat flour, wood flour, starch, and crystalline cellulose may be used. In addition, synthetic or natural polymer compounds such as coumarone resin, petroleum resin, alkyd resin, polyvinyl chloride, polyalkylene glycol, ketone resin, ester gum, copal gum, and dammar gum, as well as waxes such as carnauba wax and beeswax, and ureas may be used.

[0032] The plant growth promoter according to the present invention contains the above-mentioned spore-forming bacteria in the form of a bacterial culture solution, bacterial concentrate, bacterial dried product, or bacterial immobilized product. The content of spore-forming bacteria in the plant growth promoter according to the present invention is not particularly limited, but is 10 7 ~10 8 It can be expressed as cfu / ml.

[0033] 3. Method for cultivating plants The present invention further provides a method for cultivating plants, comprising supplying the above-mentioned spore-forming bacteria or plant growth promoter to the rhizosphere of a target plant or allowing it to act on a target plant.

[0034] In the present invention, the target plant may be either an annual or a perennial, and may be either a monocotyledon or a dicotyledon. The plant may be an ornamental flowering plant, an edible crop such as vegetables or fruits, or a plant used for the production of substances through genetic modification.

[0035] Examples of target plants include, but are not limited to, the following: Grasses: Corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), Ural wheat (Triticum urartu), winter wheat (Aegilops tauschii), Brachypodium distachyon, bamboo (Phyllostachys), sugarcane (Saccharum officinarum), Napier grass (Pennisetum pupureum), Erianthus ravenae, Japanese pampas grass (Miscanthus virgatum), sorghum (Sorghum bicolor), etc.Brassicaceae: Arabidopsis thaliana, Arabidopsis lyrata, Brassica rapa, Brassica napus, Brassica campestris, Cabbage (Brassica oleracea var. capitata), Chinese cabbage (Brassica rapa var. pekinensis), Bok choy (Brassica rapa var. chinensis), Turnip (Brassica rapa var. rapa), Broccoli (Brassica oleracea var. italica), Cauliflower (Brassica oleracea var. botrytis), Nozawana (Brassica rapa var. hakabura), Mizuna (Brassica rapa var. lanciniifolia), Komatsuna (Brassica rapa var. perviridis), Bok choy (Brassica rapa var. Examples include chinensis, radish (Raphanus sativus), wasabi (Wasabia japonica), rubella (Capsella rubella), arugula (ruca vesicaria subsp. sativa), watercress (Nasturtium officinale), and kale (Brassica oleracea var. acephala). Legumes: Soybeans (Glycine max), peas (Pisum sativum), broad beans (Vicia faba), wisteria (Wisteria floribunda), peanuts (Arachis hypogaea), lotus japonicus, kidney beans (Phaseolus vulgaris), adzuki beans (Vigna angularis), acacia (Acacia), meadow palm (Medicago truncatula), chickpeas (Cicer arietinum), cowpeas (Vigna unguiculata), mung beans (Vigna radiata), lentils (Lens culinaris), etc.Solanaceae family: Tobacco (Nicotiana tabacum), eggplant (Solanum melongena), potato (Solanum tuberosum), tomato (Solanum lycopersicum), chili peppers / bell peppers / paprika (Capsicum annuum), petunia (Petunia hybrida), etc. Malvaceae family: Okra (Abelmoschus esculentus), cotton (Gossypium arboreum L. var. obtusifolium (Roxb.) Roberty), etc. Apiaceae family: Carrot (Daucus carota subsp. sativus), Japanese parsley (Cryptotaenia japonica), parsley (Petroselinum crispum), celery (Apium graveolens var. dulce), etc. Lamiaceae: Perilla frutescens var. crispa, basil (Ocimum basilicum), thyme (Thymus vulgaris), sage (Salvia officinali), rosemary (Salvia rosmarinus), etc. Araceae: Taro (Colocasia esculenta), konjac (Amorphophallus konjac) Asteraceae: Chrysanthemum (Chrysanthemum morifolium), sunflower (Helianthus annuus), lettuce (Lactuca sativa), etc. Arecaceae: Oil palm (Elaeis guineensis, Elaeis oleifera), coconut palm (Cocos nucifera), date palm (Phoenix dactylifera), wax palm (Copernicia), etc. Anacardiaceae family: Includes Japanese wax tree (Rhus succedanea), cashew nut tree (Anacardium occidentale), lacquer tree (Toxicodendron vernicifluum), mango (Mangifera indica), pistachio (Pistacia vera), etc.Family Cucurbitaceae: Pumpkin (Cucurbita maxima, Cucurbita moschata, Cucurbita pepo), cucumber (Cucumis sativus), snake gourd (Trichosanthes cucumeroides), bottle gourd (Lagenaria siceraria var. gourda), melon (Cucumis melo), watermelon (Citrullus lanatus), etc. Rosaceae: Almond (Amygdalus communis), rose (Rosa), wild strawberry (Fragaria vesca), cherry (Prunus), apple (Malus pumila var. domestica), peach (Prunus persica), etc. Caryophyllaceae: Carnation (Dianthus caryophyllus), etc. Liliaceae: Tulip (Tulipa), lily (Lilium), etc. Amaryllidaceae: Onion (Allium cepa), welsh onion (Allium fistulosum L.), Chinese chive (Allium chinense), garlic (Allium sativum), Chinese leek (Allium tuberosum), etc. Salicaceae: Poplar (Populus trichocarpa, Populus nigra, Populus tremula), etc. Cannabaceae: Hemp (Cannabis sativa), hop (Humulus lupulus), etc. Chenopodiaceae: Spinach (Spinacia oleracea), etc. Convolvulaceae: Sweet potato (Ipomoea batatas), etc.

[0036] Among the above plants, Poaceae or Fabaceae plants, which are crops that require stable supply without being affected by drought stress, are preferred, Poaceae plants are more preferred, and among Poaceae plants, wheat, rice, and maize are more preferred.

[0037] As a method for supplying the spore-forming bacterium or the plant growth promoter to the rhizosphere of the target plant, there is no particular limitation, and for example, it can be carried out by methods such as spraying, mixing, embedding, chemical solution injection, chemical solution irrigation, etc. into the soil. When supplying to the soil, it may be carried out on a part of the soil in which the plant is cultivated, or on the entire surface. Specific examples of the place where the spore-forming bacterium or the plant growth promoter is applied include, for example, planting holes or the vicinity thereof, furrows or the vicinity thereof, between plants, the entire surface of the soil for cultivation, the entire surface of the soil, seedling boxes, seedling trays, seedling pots, seedbeds, etc.

[0038] The spore-forming bacterium or the plant growth promoter may be applied to the soil in advance before sowing or planting the target plant, or may be applied to the soil after sowing or planting the plant. The application time includes, for example, before sowing, at the time of sowing, the period from after sowing to before germination, the germination period, the breeding period, the time of transplanting seedlings, the time of cutting or budding, the growth period after planting (before flowering, during flowering, after flowering, immediately before heading or at the heading stage, etc.), the start period of fruit coloring, etc. At that time, it may be applied to the soil only once, or may be applied multiple times. From the viewpoint of sufficiently obtaining the plant growth promoting effect while minimizing the application amount, it is preferable to apply it at the initial growth stage of the plant (specifically, the period from germination to before flowering or heading) or earlier than that, and it is more preferable to apply it at the seedling raising stage or earlier than that.

[0039] In addition, as one embodiment of the method for allowing the spore-forming bacterium or the plant growth promoter to act on the target plant, for example, a method of attaching the spore-forming bacterium or the plant growth promoter to the surface of the seed of the target plant can be mentioned. Examples of seeds having the spore-forming bacterium or the plant growth promoter on the surface include seeds coated with the spore-forming bacterium or the plant growth promoter on the surface. As the method for producing the seeds, a method of preparing a spore suspension as the above plant growth promoter and applying the spore suspension to the seed surface, a method of spraying the spore suspension onto the seeds, a method of immersing the seeds in the spore suspension, etc. can be mentioned.

[0040] By sowing seeds having a spore-forming bacterium or a plant growth promoter on the surface, it is possible to obtain an effect of enhancing the drought stress tolerance of the target plant. By using seeds having a spore-forming bacterium or a plant growth promoter on the surface, as described above, the labor of applying the spore-forming bacterium or the plant growth promoter to the soil in advance before sowing or planting the target plant, or applying it to the soil after sowing or planting the plant can be saved. Even when seeds having a spore-forming bacterium or a plant growth promoter on the surface are used, as described above, the spore-forming bacterium or the plant growth promoter can be applied to the soil in advance before sowing or planting the target plant, or applied to the soil after sowing or planting the plant.

[0041] When cultivating a target plant using a spore-forming bacterium or a plant growth promoter, the concentration of the spore-forming bacterium in the soil during use is not limited, but for example, it can be 1×10 3 ~1×10 8 cells / g, preferably 1×10 5 ~1×10 8 cells / g, more preferably 1×10 7 ~1×10 8 cells / g.

[0042] Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited thereto.

[0043] (Example 1) Isolation of spore-forming bacterium (1) Collection of rhizosphere soil In October 2023, the roots and the surrounding rhizosphere soil of upland rice, taro, peanut, sorghum, corn, soybean, cotton cultivated in the black clay field of the Fuchu campus of Tokyo University of Agriculture and Technology, and taro, rice, peanut cultivated in the field of Fukushima Prefecture were collected.

[0044] (2) Isolation of epiphytes After removing the soil from the roots collected in (1) and washing with sterilized water, 9 mL of physiological saline was added to 1 g of fresh weight of roots, and the liquid obtained by shaking (1 hour, 180 rpm) was used as a liquid containing epiphytes (bacteria adhering to the surface of the roots).

[0045] (3) Isolation of endophytes After (2), the roots were surface-sterilized with a 3% sodium hypochlorite solution for 1 minute, and then washed 5 to 6 times with sterile water to remove the sodium hypochlorite. Then, sterile water was added to the roots and they were ground in a mortar and pestle to obtain an extract which was used as the solution containing endophytes (bacteria that lived inside the roots).

[0046] (4) Isolation of microorganisms contained in the rhizosphere soil Sterilized water was added to the soil removed in (2), and the soil was shaken at 25°C for 1 hour to extract the microorganisms. The microorganisms contained in the extract were inoculated onto sterilized soybean or wheat seeds along with the extract, and the seeds were cultivated in sterilized vermiculite for about 3 weeks. The seeds were sterilized by immersing them in 70% ethanol for 30 seconds and then in a 3% sodium hypochlorite solution for 3 minutes. After that, the roots were collected, and liquids containing epiphytes and endophytes were collected using the methods of (2) and (3), respectively.

[0047] (5) High-temperature treatment The solution containing epiphytes and endophytes obtained in (2) to (4) was subjected to a water bath at 65°C for 1 hour to kill the vegetative cells. Since spores can withstand this high-temperature treatment, the surviving microorganisms were designated as spore-forming microorganisms. The solution containing the microorganisms after high-temperature treatment was spread onto T-Soy agar plates. Subsequently, the plates were cultured in an incubator at 28°C under dark conditions for 2-3 days, and the resulting single colonies were taken and transferred to new agar plates. In this way, 243 strains were isolated, and after liquid culture, glycerol stocks were prepared and stored at -80°C.

[0048] (6) Observation of isolated spore-forming bacteria and stress tolerance tests The colony color and shape of the 243 isolated strains were observed. As a stress tolerance test, bacterial suspensions were applied to agar plates with pH adjusted to 5.0, 7.2, and 9.0 to check for growth under acidic and alkaline conditions, and bacterial suspensions were applied to agar plates with NaCl concentrations adjusted to 2% and 4% to check for growth under high salt concentrations. Based on these results, the 243 strains were narrowed down to 62 strains.

[0049] (7) Evaluation of plant growth promoting activity As evaluation items for the plant growth promoting activity of microorganisms, the plant hormone auxin (IAA), insoluble phosphorus solubility, insoluble potassium solubility, iron-dissolving siderophore production ability, and nitrogen fixation activity (ARA) were measured. Based on these results, 13 strains were selected from 62 strains and used in inoculation tests on wheat.

[0050] The 13 selected strains were sequenced using 16S rRNA, revealing that they belong to the genera Bacillus, Paenibacillus, and Priestia. Bacillus strains: 2 strains of Bacillus amyloliquefaciens (FRREp), 1 strain of Bacillus subtilis (TCoREn), 1 strain of Bacillus thuringiensis (TTREp), 1 strain of Bacillus altitudinis (TCo-SSREp), and 1 strain of Bacillus velezensis (TSEn-WSREn).

[0051] Paenibacillus genus / 4 strains: Paenibacillus silvae TC-CSREp1 strain, Paenibacillus xylanexedens TSEn-CSREp1 strain, Paenibacillus polysaccharolyticus TP-WSREp1 strain, Paenibacillus amylolyticus FR-WSREns1 strain

[0052] Priestia genus / 4 strains: 1 Priestia megaterium FTRen, 1 Priestia aryabhattai TTRen, 1 Priestia megaterium TC-WSREn, 1 Priestia aryabhattai FP-WSREp

[0053] (Example 2) Inoculation test on wheat (1) Method of inoculation test The 13 strains selected in Example 1 and the existing strain Bacillus pumilus TUAT1 (NITE BP-1356) were cultured in Difco Sporulation Medium (DSM) for 1 day, and then transferred to fresh DSM medium and cultured for 2 days to induce spore formation within the bacterial cells. After centrifugation, the cultures were washed three times with sterile water and then suspended in sterile water. Subsequent microscopic observation confirmed that all cultured cells were spores.

[0054] Wheat seeds were surface-sterilized in a 3% sodium hypochlorite solution, washed six times with sterile water, and then germinated on a damp paper towel for two days. Subsequently, the germinated seeds were sown four seeds per box in 300 mL plastic boxes filled with Shinano Soil (manufactured by Ohata Seed Co., Ltd.). After that, spore cells suspended in sterile water were raised to a cell density of 10 7 The solution was adjusted to a concentration of / mL, and 1mL was applied to each plant from above the sown area to begin wheat cultivation.

[0055] Wheat cultivation is performed at 25°C for 16 hours in light (300 μmol -1 m -2 s -1 The experiment was conducted for two weeks under conditions of 8 hours of darkness, and from the third week onward, it was conducted under the following conditions.

[0056] (High temperature stress conditions) Plants were exposed to 40°C for 6 hours a day for one week starting from the third week. (Drought stress conditions) Watering was reduced for one week starting from the third week, and the pF meter was maintained at pF 2.7-2.8, indicating a dry state at the bottom of the box. (Normal conditions) Plants were cultivated under the same conditions as up to the second week.

[0057] In this manner, the biomass (dry weight) of wheat cultivated for three weeks under each condition (normal conditions, high-temperature stress conditions, and drought stress conditions) was measured. The results are shown in Figure 1. A photograph of the wheat cultivated under drought stress conditions is shown in Figure 2.

[0058] Under drought stress conditions, the existing Bacillus pumilus TUAT1 strain showed no difference compared to uninoculated wheat. However, of the 13 newly isolated and selected strains, 11 strains—2 FRREp strains, 1 TCoREn strain, 1 TTRep strain, 1 TCo-SSREp strain, and 1 TSEn-WSREn strain from the genus Bacillus; 1 TC-CSREp strain, 1 TP-WSREp strain from the genus Paenibacillus; and 1 FTRen strain, 1 TTRen strain, 1 TC-WSREn strain, and 1 FP-WSREp strain from the genus Priestia—showed a significant difference in biomass (dry weight) compared to uninoculated wheat, according to Dunnett's test (Figure 1, asterisk). Furthermore, it was confirmed that wheat inoculated with these 11 strains grew larger than uninoculated wheat (Figure 2).

[0059] (Example 3) Inoculation test on wheat (high temperature stress + drought stress conditions) Six of the isolated strains (one Bacillus strain TTREp and one TCo-SSREp, one Paenibacillus strain TC-CSREp and one TP-WSREp, and one Priestia strain TTREn and one TC-WSREn) were inoculated into wheat.

[0060] Wheat cultivation was carried out in the same manner as in Example 2, except that conditions were used to apply both high-temperature stress and drought stress simultaneously. The biomass (dry weight) of the above-ground, underground, and above-ground + underground parts of the wheat was measured after 3 weeks of cultivation. The results are shown in Figure 3. There was no difference between the existing Bacillus pumilus TUAT1 strain and the uninoculated wheat, but the biomass (dry weight) of the above-ground, underground, and above-ground + underground parts of the wheat inoculated with the six strains mentioned above was significantly increased compared to the uninoculated wheat, according to the Dunnette test. Of the six strains, one strain of Bacillus thuringiensis TTREp, one strain of Bacillus altitudinis TCo-SSREp, one strain of Paenibacillus polysaccharolyticus TP-WSREp, one strain of Priestia aryabhattai TTREn, and one strain of Priestia megaterium TC-WSREn were internationally deposited after a comprehensive evaluation of their biomass increasing effect and bacterial growth characteristics.

[0061] (Example 4) Inoculation test on maize Three strains of bacteria were inoculated onto maize: Bacillus altitudinis TCo-SSREp1 strain, Paenibacillus silvae TC-CSREp1 strain, and Priestia aryabhattai TTREn1 strain.

[0062] Corn seeds were surface-sterilized in a 3% sodium hypochlorite solution, washed six times with sterile water, and then germinated on a damp paper towel for two days. Subsequently, the germinated seeds were sown four seeds per box in 300 mL plastic boxes filled with Shinano Soil (manufactured by Ohata Seed Co., Ltd.). After that, spore cells suspended in sterile water were raised to a cell density of 10 7 The solution was adjusted to a concentration of / mL, and 1mL was applied to each plant from above the sown area to begin corn cultivation.

[0063] Corn cultivation is performed at 25°C for 16 hours in light (300 μmol -1 m -2 s -1 The experiment was conducted for two weeks under conditions of 8 hours of darkness, and from the third week onward, it was conducted under the following conditions.

[0064] (High Temperature Stress Condition) Plants were exposed to 40°C for 6 hours a day for one week starting from the third week. (Drought Stress Condition) Watering was reduced for one week starting from the third week, and the pF meter was maintained at pF 2.7-2.8, indicating a dry state at the bottom of the box. (Normal Condition) Plants were cultivated under the same conditions as up to the second week. (High Temperature + Drought Stress Condition) Plants were cultivated under conditions where both the high temperature stress condition and the drought stress condition were applied simultaneously.

[0065] In this manner, the above-ground and underground biomass (dry weight) of corn was measured for three weeks under each of the following conditions (normal conditions, high-temperature stress conditions, drought stress conditions, and high-temperature + drought stress conditions). The results are shown in Figure 4. Photographs of the corn grown under each condition are shown in Figure 5.

[0066] Even under conditions where both high-temperature stress and drought stress were applied simultaneously, the biomass (dry weight) of corn inoculated with the three bacterial strains mentioned above showed a significant difference compared to uninoculated corn, according to Dunnett's test (Figure 4, asterisk). Furthermore, it was confirmed that corn inoculated with the three bacterial strains did not experience a decrease in biomass even under stress, and that the plant body grew larger than uninoculated corn under normal conditions (Figure 5).

[0067] (Example 4) Mixed inoculation test on wheat Inoculation tests were conducted on wheat by single inoculation of one of the three strains: Bacillus altitudinis TCo-SSREp1 strain, Paenibacillus silvae TC-CSREp1 strain, and Priestia aryabhattai TTREn1 strain, or by mixed inoculation of a combination of two or three strains.

[0068] Wheat cultivation was carried out in the same manner as in Example 2, except that it was done under conditions that simultaneously applied both high-temperature stress and drought stress. The biomass (dry weight) of the wheat cultivated for 3 weeks was measured. The results are shown in Figure 6.

[0069] As shown in Figure 6, it was confirmed that sufficient efficacy could be obtained with the above bacterial strains through single inoculation.

[0070] The present invention can be used in the field of manufacturing agricultural microbial materials. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. The following spore-forming bacteria (A) to (C) that have plant growth promoting activity in dry environments. (A) Either the Bacillus thuringiensis TTREp1 strain identified under accession number NITE BP-04159, or the Bacillus altitudinis TCo-SSREp1 strain identified under accession number NITE BP-04160, or a mutant thereof. (B) The Paenibacillus polysaccharolyticus TP-WSREp1 strain identified under accession number NITE BP-04163, or a mutant thereof. (C) The Priestia aryabhattai TTREn1 strain identified under accession number NITE BP-04165, or the Priestia megatherium identified under accession number NITE BP-04166. Any strain of *Megalosa megaterium* TC-WSREn1, or a mutant thereof.

2. A plant growth promoter containing the spore-forming bacteria described in claim 1 as an active ingredient.

3. A method for cultivating plants, comprising supplying the spore-forming bacteria described in claim 1 or the plant growth promoter described in claim 2 to the rhizosphere of a target plant or allowing it to act on a target plant.

4. The method according to claim 3, wherein the target plant is a plant selected from grasses and legumes.

5. The method according to claim 4, wherein the grass plant is wheat, rice, or maize.

6. The method according to claim 4, wherein the leguminous plant is soybean.

7. The following spore-forming bacteria (A) to (C). (A) Either the Bacillus thuringiensis TTREp1 strain identified under accession number NITE BP-04159, or the Bacillus altitudinis TCo-SSREp1 strain identified under accession number NITE BP-04160, or a mutant thereof. (B) The Paenibacillus polysaccharolyticus TP-WSREp1 strain identified under accession number NITE BP-04163, or a mutant thereof. (C) The Priestia aryabhattai TTREn1 strain identified under accession number NITE BP-04165, or the Priestia megatherium identified under accession number NITE BP-04166. Any strain of *Megalosa megaterium* TC-WSREn1, or a mutant thereof.

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