Material containing iron compound and filamentous fungal endophyte, method for promoting plant growth, and method for producing crop derived from plant

Abstract

To provide novel means for promoting plant growth.SOLUTION: The present invention provides a material containing an iron compound and a filamentous fungal endophyte, wherein the filamentous fungal endophyte includes at least one species selected from the group consisting of Fusarium sp. and Lecanicillium sp., and also provides a method for promoting plant growth using the material, and a method for producing a crop derived from a plant.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a material containing an iron compound and a filamentous fungal endophyte, a method for promoting plant growth, and a method for producing a crop obtained from a plant.

Background Art

[0002] Plants absorb inorganic nutrients from air and soil, and grow while converting them into organic substances using the energy of sunlight or using them in metabolic reactions and the like.

[0003] Among the nutrients (elements) essential for plant growth, iron (Fe) is classified as a micronutrient essential element because the required amount in plants is small. Iron is an element essential for nucleic acid synthesis such as DNA, energy production, and various enzyme reactions in organisms including plants. Therefore, iron is an essential element for maintaining life activities. However, since plants cannot move once they are rooted in the ground, a mechanism for preferentially taking up nutrients necessary for growth from the soil at the rooted location is required.

[0004] In neutral or alkaline soil, iron exists as trivalent iron (Fe 3+ ). Since trivalent iron (Fe 3+ ) is hardly soluble in water, the absorption of iron by plants is significantly inhibited in neutral or alkaline soil. Among the gramineous plants, there are those that secrete a molecule that strongly binds to trivalent iron (Fe 3+ ) called siderophore. Siderophore has a high affinity for trivalent iron (Fe 3+ ) and acts as a chelating agent that forms a complex with trivalent iron (Fe 3+ ). As a result of forming a complex with siderophore, trivalent iron (Fe 3+ ) is solubilized, absorbed from the roots, and taken up by plants.

[0005] Among microorganisms, there are those that produce siderophores, and those that symbiotically live with plant hosts as filamentous fungal endophytes have been reported (Non-Patent Document 1).

[0006] Filamentous fungal endophytes, also known as endophytes, refer to filamentous fungi that inhabit and symbiotically grow within the bodies of plant hosts, bringing benefits such as growth promotion and increased stress tolerance to the hosts (Patent Document 1). Among filamentous fungal endophytes, there are also reports of filamentous fungi having the ability to solubilize phosphate, which can convert insoluble phosphate and citrate-soluble phosphate (phosphate that dissolves under acidic conditions such as in citric acid) in the soil into water-soluble phosphate that can be absorbed by plant roots (Non-Patent Documents 2 and 3). In addition, there are reports of filamentous fungi that adsorb heavy metals (e.g., cadmium) present in the soil and that have an adverse effect on plant growth, thereby reducing their uptake by plants (Non-Patent Document 4).

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

[0009] An object of the present invention is to provide a novel means for solubilizing iron compounds in the rhizosphere of plants or their surrounding environment to promote plant growth. [Means for Solving the Problems]

[0010] As a result of intensive studies, the present inventors have found a new filamentous fungal endophyte that exhibits the action of solubilizing iron compounds in the rhizosphere of plants or their surrounding environment and promotes plant growth, and have completed the following invention.

[0011] [1] A material containing an iron compound and a filamentous fungal endophyte, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp.. [2] In the material according to [1], the iron compound is a trivalent iron compound. [3] In the material according to [1] or [2], the content of the iron compound is 10 to 50,000 ppm of the material. [4] In the material according to any one of [1] to [3], the filamentous fungal endophyte produces siderophores. [5] In the material according to any one of [1] to [4], the filamentous fungal endophyte produces indole-3-acetic acid. [6] In the material according to any one of [1] to [5], the material contains a raw material containing insoluble phosphate and / or citrate-soluble phosphate. [7] In the material according to any one of [1] to [6], the raw material is a calcined product of sludge. [8] In the material according to any one of [1] to [6], the filamentous fungal endophyte is a pulverized product of a dry solid culture. The material has a cumulative 50% particle size (D 50 ) of 100 to 900 μm in the volume particle size distribution. The material has a cumulative 90% particle size (D 90 ) of 1000 to 5000 μm. [9] In the material according to any one of [1] to [6], it is used for promoting plant growth.

[0012]

[10] A method for promoting plant growth, comprising contacting a filamentous fungal endophyte with an iron compound in the rhizosphere of a plant or its surrounding environment. In the above method, the filamentous fungal endophyte contains at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp..

[11] In the method according to

[10] , the iron compound is a trivalent iron compound.

[12] The method described in

[10] or

[11] is such that the content of the above iron compound is 10 to 50,000 ppm of the soil in which the above plant grows.

[13] The method described in any one of

[10] to

[12] includes contacting the above filamentous fungal endophyte with insoluble phosphate and / or soluble phosphate in the rhizosphere or the surrounding environment of the above plant.

[14] The method described in any one of

[10] to

[13] includes contacting the above filamentous fungal endophyte with the calcined product of sludge in the rhizosphere or the surrounding environment of the above plant.

[15] The method described in any one of

[10] to

[14] includes contacting the above filamentous fungal endophyte with heavy metals in the rhizosphere or the surrounding environment of the above plant.

[16] The method described in any one of

[10] to

[15] is such that the above heavy metals include cadmium and / or lead.

[0013]

[17] A method for producing a crop obtained from a plant, including contacting a filamentous fungal endophyte with an iron compound in the rhizosphere or the surrounding environment of the plant and growing the above plant. The above method includes harvesting the crop obtained from the above plant. In the above method, the above filamentous fungal endophyte includes at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp.

[18] In the method described in

[17] , the above iron compound is a trivalent iron compound.

[19] In the method described in

[17] or

[18] , the content of the above iron compound is 10 to 50,000 ppm of the soil in which the above plant grows.

[20] The method described in any one of

[17] to

[19] includes contacting the above filamentous fungal endophyte with insoluble phosphate and / or soluble phosphate in the rhizosphere or the surrounding environment of the above plant.

[21] The method described in any one of

[17] to

[20] includes contacting the above filamentous fungal endophyte with the calcined product of sludge in the rhizosphere or the surrounding environment of the above plant.

[22] The method according to any one of

[17] to

[21] includes contacting the above-mentioned filamentous fungal endophyte with heavy metals in the rhizosphere of the above-mentioned plant or its surrounding environment.

[23] In the method according to any one of

[17] to

[22] , the above-mentioned heavy metals include cadmium and / or lead. [Advantages of the Invention]

[0014] When a plant is grown by adopting the present invention, an iron compound in the rhizosphere of the plant or its surrounding environment is solubilized, and the growth of the plant is promoted. [Brief Description of the Drawings]

[0015] [Figure 1] Figure 1 shows the qualitative analysis results of siderophore production of strains by the CAS Agar Assay performed in Experiment 3-1. a: Example 5, b: Example 6. [Modes for Carrying Out the Invention]

[0016] Hereinafter, the present invention will be described in detail according to specific embodiments. However, the present invention is not limited to the following embodiments, and can be implemented in any form without departing from the gist of the present invention.

[0017] [Summary] The present inventors have found a novel filamentous fungal endophyte that exhibits an action of chelating an iron compound. Due to the factor that chelates the iron compound produced by these filamentous fungal endophytes, the iron compound is solubilized, and the growth of plants that take it up is promoted. The present invention is based on the discovery of such a new filamentous fungal endophyte.

[0018] One aspect of the present embodiment is a material containing an iron compound and a filamentous fungal endophyte, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp. The filamentous fungal endophyte produces a factor that chelates the iron compound. Thereby, the iron compound, particularly the insoluble iron compound, is solubilized, and the plant growth is promoted by taking in the water in which it is dissolved from the roots. In one aspect of the present embodiment, the material is a material used for promoting plant growth.

[0019] Also, one aspect of the present embodiment is a method for promoting plant growth, which includes contacting a filamentous fungal endophyte with an iron compound in the rhizosphere of a plant or its surrounding environment, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp.

[0020] Another aspect of the present embodiment is a method for producing crops obtained from plants, including contacting a filamentous fungal endophyte with an iron compound in the rhizosphere of the plant or its surrounding environment, growing the plant, and harvesting the crops obtained from the plant. The filamentous fungal endophyte includes at least one or more selected from the group consisting of Fusarium sp. and Lecanicillium sp. The crops grown and harvested by the method of one aspect of the present embodiment have an increased harvest weight. In addition, the crops grown and harvested by the method of one aspect of the present embodiment have reduced growth variability. In the present disclosure, "crops" is used in the sense of including both "the plant itself" and the "harvestable part" formed in the plant. The "harvestable part" refers to, for example, the edible part of the above-ground part of a plant such as salad greens. Also, for example, in the case of rice, it refers to its edible part, that is, "rice". Also, in the case of a plant that forms an edible part in the underground part such as an onion, it refers to the edible part of the underground part. Also, in the present disclosure, "contacting" includes physically directly contacting the filamentous fungal endophyte with a target substance (for example, an iron compound, insoluble phosphate, soluble phosphate, or heavy metal), or allowing a substance produced from the filamentous fungal endophyte (for example, a siderophore) to act on the target substance.

[0021] In the present disclosure, "filamentous fungal endophyte" refers to a filamentous fungus that can function as an endophyte. An endophyte, also called an endophytic bacterium, refers to a microorganism that inhabits and symbiotically lives within a plant host, bringing benefits such as growth promotion and increased stress tolerance to the host.

[0022] In one aspect of this embodiment, the filamentous fungal endophyte that can be used is Fusarium sp. An example of Fusarium is Fusarium solani. Further, the Fusarium solani that can be used in this embodiment can use, for example, the 15-B strain that was deposited with the National Institute of Technology and Evaluation (NITE) on October 16, 2023, and is available under the accession number NITE P-03991.

[0023] In one aspect of this embodiment, the filamentous fungal endophyte that can be used is Lecanicillium sp. The filamentous fungal endophyte of Lecanicillium that can be used in one aspect of this embodiment can use, for example, the 100-1 strain that was deposited with the National Institute of Technology and Evaluation (NITE) on September 15, 2023, and is available under the accession number NITE P-03984.

[0024] In the present disclosure, the iron compound is a compound containing iron element, and may be an insoluble or hardly soluble iron compound. When one aspect of this embodiment is applied, the insoluble or hardly soluble iron compound is solubilized and can be taken up from the roots of plants. In one aspect of this embodiment, the iron compound is an insoluble or hardly soluble iron compound under neutral or alkaline conditions, preferably a trivalent iron compound. Examples of the trivalent iron compound include iron(III) hydroxide, iron(III) oxide, iron(III) phosphate, etc. In one aspect of this embodiment, the iron compound may be 10 to 50,000 ppm, preferably 20 to 30,000 ppm, in the material or the soil to which it is applied.

[0025] In the present disclosure, a "siderophore" is a molecule having a high affinity for trivalent iron ions, and acts as a chelating agent that binds to trivalent iron ions to form an iron-siderophore complex. The siderophore forms an iron-siderophore complex with trivalent iron ions derived from an insoluble or sparingly soluble iron compound and is soluble in water. The iron-siderophore complex dissolved in water is taken up from the roots and makes iron available in plants.

[0026] The filamentous fungal endophyte that can be used in one aspect of the present embodiment produces a siderophore. The presence of the siderophore can be evaluated, for example, by an assay using CAS (Chrome azurol S). CAS is a dye compound that exhibits a characteristic color when bound to iron ions. By adding a compound having a high iron-binding ability such as a siderophore to a solution containing CAS, iron ions are taken away from the CAS-iron complex, and as a result, the characteristic color of the CAS-iron complex disappears. By utilizing this reaction, the presence of the siderophore can be determined.

[0027] As shown in the examples described below, the filamentous fungal endophyte has a significantly superior ability to chelate iron compounds as compared with the filamentous fungi described in Non-Patent Document 1.

[0028] In one aspect of the present embodiment, the material may include a raw material containing insoluble phosphate and / or citrate-soluble phosphate. The filamentous fungal endophyte used in one aspect of the present embodiment can solubilize insoluble phosphate and / or citrate-soluble phosphate and convert it into water-soluble phosphate.

[0029] In the present disclosure, "water-soluble phosphate" refers to phosphate that is soluble in water. An example of water-soluble phosphate is potassium dihydrogen phosphate. Water-soluble phosphate can be directly absorbed by plants from the roots.

[0030] In the present disclosure, "citrate-soluble phosphate" refers to phosphate that dissolves under acidic conditions such as in citric acid. An example of citrate-soluble phosphate is dicalcium phosphate (DCP). Although plants cannot absorb citrate-soluble phosphate as it is, they can secrete root acid from their roots to convert citrate-soluble phosphate into water-soluble phosphate and then absorb it. However, plants can utilize citrate-soluble phosphate in the soil only within a very narrow rhizosphere area where root acid can reach.

[0031] In the present disclosure, "insoluble phosphate" refers to phosphate that does not dissolve in water or under acidic conditions. Examples of insoluble phosphate include tricalcium phosphate (TCP) and aluminum phosphate (AP). Plants cannot absorb insoluble phosphate and do not have a means to make it absorbable.

[0032] In the present disclosure, "solubilization" of phosphate refers to the conversion of insoluble phosphate and / or citrate-soluble phosphate into water-soluble phosphate.

[0033] As shown in the examples described below, the filamentous fungal endophyte has a significantly superior ability to solubilize insoluble phosphate or an insoluble phosphate source compared to the bacteria described in Non-Patent Document 3. As described above, although plants can secrete root acid from their roots to solubilize citrate-soluble phosphate, they usually cannot utilize citrate-soluble phosphate in the soil except within a very narrow rhizosphere area where root acid can reach. However, the filamentous fungal endophyte symbiotically associates with plants, absorbs the sugars produced by plants through photosynthesis, and uses them for its own growth. The hyphae of the filamentous fungus are thinner than plant roots and can continuously grow into the tiny gaps (gas phase) in the soil. In this way, the filamentous fungus spreads its hyphae in a wider area of the soil than plant roots, decomposes (dissolves) the nutrients in the soil, and transports them to plant roots along the hyphae (Patent Document 1). Therefore, by using the filamentous fungal endophyte, the hyphae of the filamentous fungal endophyte extending from the roots can solubilize a wide range of citrate-soluble phosphate and insoluble phosphate that cannot be reached by root acid, making it possible for plants to absorb them.

[0034] In addition, as for the filamentous fungal endophyte, any one kind of fungus may be used alone, or two or more kinds of fungi may be used in combination at any combination and ratio. When two or more filamentous fungal endophytes are used in combination, two or more kinds of fungi belonging to the same genus may be combined, or one or two or more kinds of fungi may be selected from two or more different genera and combined. By using two or more filamentous fungal endophytes in combination, an improvement in the ability to solubilize phosphate due to a synergistic effect may be expected in some cases.

[0035] The raw materials used in the materials of this embodiment are not limited as long as they are raw materials used for promoting plant growth. As an example of the raw material, soil can be mentioned. As an example of the raw material, phosphate ore, steel slag and their pulverized products, and mixtures of these with crop residues can be mentioned. As an example of the crop residue, sorghum residue can be mentioned. As an example of the raw material, sludge containing phosphoric acid can be mentioned. Specifically, sewage sludge, animal manure (for example, cow dung, chicken manure) and sludge derived therefrom, meat and bone meal and sludge derived therefrom, mushroom fruiting beds and sludge derived therefrom, and calcined products thereof can be mentioned. The above calcined products may also be referred to as incinerated products. In addition, those obtained by subjecting these raw materials to concentration, dehydration, separation, digestion, drying or calcination alone or in a plurality of combinations are also included in the raw materials used in the materials of this embodiment. These raw materials may be used alone, or two or more kinds may be used in combination at any combination and ratio.

[0036] The raw materials used in the materials of this embodiment may contain at least insoluble phosphate and / or citrate-soluble phosphate. The raw materials used in this embodiment may contain only one of insoluble phosphate and citrate-soluble phosphate, or may contain both. In addition, the respective contents of insoluble phosphate and citrate-soluble phosphate in the raw materials used in this embodiment are not limited either.

[0037] However, among raw materials containing both insoluble phosphate and citrate-soluble phosphate, in the case of a raw material where {content of insoluble phosphate} > {content of citrate-soluble phosphate} (i.e., a raw material in which the content of insoluble phosphate is greater than the content of citrate-soluble phosphate), even if there are microorganisms that can solubilize only citrate-soluble phosphate, the amount of water-soluble phosphate necessary for plant growth may be insufficient. In an environment where {content of insoluble phosphate} > {content of citrate-soluble phosphate}, there is an advantage that sufficient phosphate for plant growth can be supplied by including filamentous fungal endophytes that can solubilize both citrate-soluble phosphate and insoluble phosphate. That is, according to one aspect, the raw material and the material contain both insoluble phosphate and citrate-soluble phosphate, and {content of insoluble phosphate} > {content of citrate-soluble phosphate}. Examples of raw materials where {content of insoluble phosphate} > {content of citrate-soluble phosphate} include phosphate ore and its pulverized products, and volcanic ash soil.

[0038] On the other hand, among raw materials containing both insoluble phosphate and citrate-soluble phosphate, in the case of a raw material where {content of insoluble phosphate} < {content of citrate-soluble phosphate} (i.e., a raw material in which the content of insoluble phosphate is less than the content of citrate-soluble phosphate), since the solubility of phosphate is higher than that of the above raw material where {content of insoluble phosphate} > {content of citrate-soluble phosphate}, it has an immediate effect on plant growth. That is, according to one aspect, the raw material and the material contain both insoluble phosphate and citrate-soluble phosphate, and {content of insoluble phosphate} < {content of citrate-soluble phosphate}. Examples of raw materials where {content of insoluble phosphate} < {content of citrate-soluble phosphate} include sewage sludge, animal manure (e.g., cow dung, chicken manure) and sludge derived therefrom, meat and bone meal, mushroom spawn beds, and calcined products (or incinerated products) thereof.

[0039] According to one aspect, the material of the present embodiment and / or the rhizosphere of the plant to which it is applied or its surrounding environment may contain heavy metals. The filamentous fungal endophyte that can be used in the present embodiment can adsorb heavy metals present in the material and / or the rhizosphere of the plant to which it is applied or its surrounding environment. Thereby, it is possible to suppress the uptake of heavy metals that can have an adverse effect on the plant targeted for growth promotion. Heavy metals that can be adsorbed by the filamentous fungal endophyte of the present embodiment include, for example, cadmium (Cd), nickel (Ni), chromium (Cr), and lead (Pb), preferably cadmium (Cd) or lead (Pb).

[0040] According to one aspect, the total content ratio of insoluble phosphoric acid and citric acid-soluble phosphoric acid in the material of the present embodiment and / or the raw materials used therein is preferably 0.5% by mass or more, particularly 3% by mass or more, and more preferably 5% by mass or more in both cases. By setting the total content of insoluble phosphoric acid and citric acid-soluble phosphoric acid in the material and / or the raw materials to be equal to or higher than the above lower limit, the solubilizing ability of the filamentous fungal endophyte can be reliably exerted, and it becomes possible to obtain a more effective plant growth promotion or soil improvement effect. On the other hand, the upper limit value of the total content is not particularly limited, but can be, for example, 95% by mass or less.

[0041] It should be noted that the content of insoluble phosphoric acid and the content of citric acid-soluble phosphoric acid in the raw materials and the material can both be measured by the quinoline mass method.

[0042] According to one aspect, the material of the present embodiment may further contain water-soluble phosphoric acid. The water-soluble phosphoric acid may be that contained in the raw materials. Also, the water-soluble phosphoric acid may be that generated by solubilizing the insoluble phosphoric acid and / or citric acid-soluble phosphoric acid contained in the raw materials by the filamentous fungal endophyte. When the material of the present embodiment contains water-soluble phosphoric acid, its content ratio is not restricted.

[0043] The filamentous fungal endophyte used in this embodiment may be used in combination with other microorganisms capable of solubilizing iron compounds and / or insoluble phosphoric acid. Other microorganisms capable of solubilizing iron compounds and / or insoluble phosphoric acid may be used alone, or two or more thereof may be used in combination in any combination and ratio. Examples of other microorganisms capable of solubilizing iron compounds include Aspergillus nidulans, Aspergillus sydowii, Aspergillus terreus, Azotobacter vinelandii, Bacillus subtilis, Burkholderia cepacia, Cymbidium aloifolium, Escherichia coli, Penicillium chrysogenum, Pseudomonas aeruginosa, Rhodotorula pilimanae, Streptomyces pilosus, Streptomyces coelicolor, and Ustilago sphaerogena.

[0044] Examples of other microorganisms capable of solubilizing insoluble phosphoric acid include Achromobacter marplatensis, Acinetobacter calcoaceticus, bacteria of the genus Advenella, Agromyces aruantiacus, Alcaligenes faecalis, bacteria of the genus Arthrobacter, Bacillus pumilus, Bacillus subtilis, Bordetella avium, Brevibacterium frigoritolerans, Burkholderia cenocepacia, Cronobacter sakazakii, bacteria of the genus Ensifer, Enterobacter ludwigii, bacteria of the genus Enterococcus, Flavobacterium ahuensis, Fusarium oxysporum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactococcus garvieae, Lactococcus lactis, bacteria of the genus Leifsonia, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium hydrocarbonoxydans, bacteria of the genus Nocardioides, Pantoea agglomerans, Pediococcus pentosaceus, Pediococcus stilesii, Pseudomonas aeruginosa, Rhizobium tropici, Serratia marcescens, Shinella kummerowiae, Sinorhizobium medicae, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptomyces roseofulvus, Trabulsiella guamensis, Trimyema compressum, Weissella confusa, and Weissella ghanensis.

[0045] The filamentous fungal endophyte that can be used in one aspect of the present embodiment produces indole-3-acetic acid (IAA), which is a kind of plant hormone called auxin. Indole-3-acetic acid is a plant growth regulator and exhibits effects such as promoting plant growth, promoting rooting, promoting cell elongation, and inducing the division of cambium cells. As shown in the examples described later, the filamentous fungal endophyte that can be used in one aspect of the present embodiment is significantly superior in the ability to produce indole-3-acetic acid compared to the bacteria described in Non-Patent Document 2.

[0046] The cell count of the filamentous fungal endophyte in the material of the present embodiment is not particularly limited. According to one aspect, when the material of the present embodiment is mainly a culture of the filamentous fungal endophyte, the cell count of the filamentous fungal endophyte, as the average colony count per 1 g of the material, can be, for example, 1×10 5 cfu / g or more, or 5×10 5 cfu / g or more, or 1×10 6 cfu / g or more, or 5×10 6 cfu / g or more, and can also be, for example, 1×10 11 cfu / g or less, or 5×10 10 cfu / g or less, or 1×10 10 cfu / g or less, or 5×10 9 cfu / g or less. In addition to the filamentous fungal endophyte, when other microorganisms capable of solubilizing iron compounds and / or insoluble phosphoric acid are used in combination, the cell count of such other microorganisms can also be in the same range as the cell count of the filamentous fungal endophyte. Further, when the material of the present embodiment contains a culture of the filamentous fungal endophyte and other raw materials, the cell count of the filamentous fungal endophyte in the culture, as the average colony count per 1 g of the material, can be 1×10 2 cfu / g or more, or 5×10 2 cfu / g or more, or 1×10 3 cfu / g or more, or 5×10 3 cfu / g or more, and can also be, for example, 1×10 11 cfu / g or less, or 5×10 10cfu / g or less, or 1×10 10 cfu / g or less, or 5×10 9 cfu / g or less.

[0047] The filamentous fungal endophyte used in this embodiment may be a culture of the filamentous fungal endophyte. Examples of the culture of the filamentous fungal endophyte include a solid culture and a liquid culture. The solid culture refers to a culture obtained as a result of solid culture, in which the filamentous fungal endophyte is cultured in a solid culture medium. The liquid culture refers to a culture obtained as a result of liquid culture, in which the filamentous fungal endophyte is cultured in a liquid culture medium. The preparation method of each culture will be described later.

[0048] According to one aspect, the filamentous fungal endophyte used in this embodiment is a solid culture of the filamentous fungal endophyte. The solid culture is preferably a dried solid culture, more preferably a pulverized product of the dried solid culture.

[0049] In addition to the filamentous fungal endophyte, when another microorganism capable of solubilizing an iron compound and / or insoluble phosphoric acid is used in combination, such another microorganism can also be used as a culture. Examples of the solid culture include a solid culture and a liquid culture. Among them, a solid culture is preferable, a dried solid culture is more preferable, and a pulverized product of the dried solid culture is particularly preferable.

[0050] The particle size of the pulverized product is not limited, but is, for example, as follows. According to one aspect, the cumulative 10% particle diameter (D 10 ) in the volume particle size distribution of the pulverized product is, for example, 50 μm or more, or 100 μm or more, or 150 μm or more, or 200 μm or more, and is, for example, 400 μm or less, or 350 μm or less, or 300 μm or less. According to one aspect, the cumulative 50% particle diameter (D 50) is, for example, 100 μm or more, or 200 μm or more, or 300 μm or more, or 400 μm or more, and is also, for example, 900 μm or less, or 850 μm or less, or 800 μm or less. According to one aspect, the cumulative 90% particle size (D 90 ) is, for example, 1000 μm or more, or 1100 μm or more, or 1200 μm or more, and is also, for example, 5000 μm or less, or 4000 μm or less, or 3000 μm or less, or 2000 μm or less. When using a material containing a pulverized product of a dry solid culture with uniform particle size, the growth of plants can be further promoted, and unevenness in the effect of promoting plant growth can also be suppressed. In addition, when other microorganisms capable of solubilizing iron compounds and / or insoluble phosphoric acid are used in combination as a pulverized product of the dry solid culture in addition to the filamentous fungal endophyte, the particle size of the pulverized product of such other microorganisms can also be in the same range as the particle size of the pulverized product of the filamentous fungal endophyte.

[0051] Note that the method for measuring the particle size of the pulverized product is not limited, and it can be measured by any method. Examples of such measurement methods include laser diffraction method, scattering method, imaging method, light transmission centrifugal sedimentation method, sedimentation method, electrical resistance method, specific surface area method, and sieve passing method. The equipment for measuring the particle size of the pulverized product is also not limited, and it can be measured by any commercially available particle size measuring instrument. Examples of such measuring instruments include LMS-2000e and LMS-3000 (Seishin Enterprise Co., Ltd.). Note that since the particle size of the above pulverized product is measured after drying the culture and then pulverizing, it is preferable to use a dry measurement method.

[0052] The material of the present embodiment may contain components other than the raw material, the filamentous fungal endophyte, and optionally other microorganisms capable of solubilizing iron compounds and / or insoluble phosphoric acid. Examples include nitrogen sources, potassium sources, and carbon sources. Examples of nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, urea, and Chile saltpeter. Examples of potassium sources include potassium chloride, potassium sulfate, potassium nitrate, and potassium silicate. Examples of carbon sources include glucose, sucrose, maltose, and lactose.

[0053] The materials of this embodiment are used for promoting plant growth or improving soil. For example, the materials of this embodiment may be applied alone or mixed with other materials such as soil to the rhizosphere of plants or the surrounding environment. Thereby, insoluble or poorly soluble iron compounds, and / or insoluble phosphoric acid and / or citrate-soluble phosphoric acid in the materials of this embodiment and the soil mixed therewith are solubilized by filamentous fungal endophytes, and the iron and / or water-soluble phosphoric acid that can be absorbed by plants increases. In this way, the iron and / or phosphoric acid availability of plants is improved, and the growth of plants is promoted, or the soil is improved. In the present disclosure, the "rhizosphere" means the soil space affected by plant root exudates and soil organisms.

[0054] The plants to which the materials of this embodiment are applied are not limited. For example, they may be annual plants or perennial plants, and may be angiosperms or gymnosperms. It is preferable to apply the materials of this embodiment to plants with relatively high requirements for iron or phosphoric acid, because the growth promoting effect of the materials of this embodiment can be obtained more significantly. Examples of crop plants with relatively high requirements for phosphoric acid include strawberries, cucumbers, tomatoes, cabbages, eggplants, lettuces, onions, peas, corns, Chinese cabbages, tobaccos, shishito peppers, radishes, carrots, potatoes, broccoli, and flowers.

[0055] [Method for manufacturing materials] One embodiment relates to a method for manufacturing a material for promoting plant growth or improving soil. The method includes mixing a raw material containing an insoluble or poorly soluble iron compound and / or an insoluble phosphoric acid and / or a citrate-soluble phosphoric acid with a filamentous fungal endophyte. In the manufacturing method of this embodiment, the details of the raw material and the filamentous fungal endophyte are as described above.

[0056] · Cultivation In the manufacturing method of this embodiment, as described above, it is preferable to prepare a culture obtained by previously culturing the filamentous fungal endophyte and mix it with raw materials. The culture conditions of the filamentous fungal endophyte are not limited. For example, liquid culture or solid culture may be used.

[0057] In the present disclosure, "liquid culture" refers to culturing a strain using a liquid medium. "Liquid culture" refers to a liquid culture obtained as a result of liquid culture. Subjecting the filamentous fungal endophyte to liquid culture means inoculating the filamentous fungus into a liquid medium and culturing it in the liquid medium. The filamentous fungal endophyte may be in the state of an inoculum preserved in a medium. Examples of the medium include agar. In order to avoid contamination by miscellaneous bacteria, it is preferable to perform inoculation under sterile conditions.

[0058] The liquid medium can be prepared, for example, by adding various additives to a liquid so as to have a composition suitable for culturing the filamentous fungal endophyte. Examples of the liquid include water. Examples of the additives added to the liquid medium include saccharides, minerals, nitrogen sources, vitamins, organic acids, inorganic acids, organic bases, and inorganic bases. Preferably, the liquid medium contains saccharides and a nitrogen source. Examples of the nitrogen source contained in the liquid medium include peptone.

[0059] Examples of saccharides include, but are not limited to, one or more selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol. The upper limit of the saccharides is not restricted, but as the total amount of saccharides in the liquid medium, for example, it can be usually 60 g / L or less, or 50 g / L or less, or 40 g / L or less, or 30 g / L or less. If there are too many nutrients such as saccharides, the hyphal concentration may become too high. On the other hand, the lower limit of the saccharides is not restricted, but as the total amount of saccharides in the liquid medium, for example, it can be 0.5 g / L or more, or 1 g / L or more, or 5 g / L or more, or 10 g / L or more. If there are too few nutrients such as saccharides, the hyphae may not elongate sufficiently. For example, in one embodiment, the saccharide is glucose and is added in an amount of about 20 g / L of the medium.

[0060] Examples of minerals include, but are not limited to, inorganic salts such as alkali and alkaline earth metal salts and salts of other metals. Such inorganic salts include, for example, one or more salts selected from sulfates, phosphates, carbonates, chlorides, alkali metal oxides, molybdates, selenites, and halides. One or more kinds of salts can be used. The upper limit of the salts is not restricted, but excluding the amount contained in yeast extract and peptone, the total amount of salts in the liquid medium can be, for example, 10.0 g / L or less, or 8.0 g / L or less, or 5.0 g / L or less, or 2.0 g / L or less. If there are too many salts as nutrients, the hyphal concentration may become too high. On the other hand, the lower limit of the salts is not restricted, but excluding the amount contained in yeast extract, the total amount of salts in the liquid medium can be, for example, 0.01 g / L or more, or 0.05 g / L or more, or 0.1 g / L or more, or 0.25 g / L or more. If there are too few minerals such as salts, the hyphae may not elongate sufficiently. For example, in one embodiment, as salts, magnesium sulfate is added in an amount of about 0.5 g / L of the medium, and potassium dihydrogen phosphate is added in an amount of about 0.1 g / L of the medium.

[0061] Examples of the nitrogen source include one or more nitrogen sources selected from yeast extract, protein hydrolysate, and protein. Examples of the protein hydrolysate include peptone. When yeast extract and / or peptone is added to the liquid medium, nutrients including protein hydrolysates such as amino acids and peptides, proteins, and salts can be added. The amount of the nitrogen source for the liquid medium is not limited, but the upper limit of the nitrogen source can be, for example, 30.0 g / L or less, or 20.0 g / L or less, or 15.0 g / L or less, or 10.0 g / L or less as the total amount of nitrogen sources such as yeast extract and peptone. The lower limit of the nitrogen source can be, for example, 0.05 g / L or more, or 0.1 g / L or more, or 0.5 g / L or more, or 1.0 g / L or more as the total amount of nitrogen sources such as yeast extract and peptone. The amount of the yeast extract for the liquid medium is not limited, but the upper limit of the yeast extract can be, for example, 20.0 g / L or less, or 15.0 g / L or less, or 10.0 g / L or less, or 5.0 g / L or less. The lower limit of the yeast extract can be, for example, 0.01 g / L or more, or 0.05 g / L or more, or 0.1 g / L or more, or 0.5 g / L or more. The amount of the peptone for the liquid medium is not limited, but the upper limit of the peptone can be, for example, 10.0 g / L or less, or 8.0 g / L or less, or 5.0 g / L or less, or 2.0 g / L or less. The lower limit of the peptone can be, for example, 0.01 g / L or more, or 0.05 g / L or more, or 0.1 g / L or more, or 0.5 g / L or more. Similar to the above, if there are too many nutrients, the hyphal concentration may become too high. On the other hand, if the nitrogen source is too small, the hyphae may not elongate sufficiently. For example, in one embodiment, yeast extract is added in an amount of about 2.0 g / L of the medium, and peptone is added in an amount of about 1.0 g / L of the medium. Further, if necessary, the pH may be appropriately adjusted by adding an appropriate acid or base.

[0062] Liquid culture can be carried out by appropriate culture means. For example, it can be cultured while stirring with a stirring device such as a stirrer using a container such as an Erlenmeyer flask. Also, it is preferable that the culture conditions of liquid culture are adjusted by a temperature control device or the like. For liquid culture, one or more devices selected from a vibration device, a humidity measurement device, a pH adjustment device, a turbidity measurement device, a light control device, a specific gas concentration measurement device, and a pressure measurement device may be used as necessary. The specific gas concentration measurement device may be able to measure, for example, O2 and CO2 as specific gases. For liquid culture, a silicone stopper may be arbitrarily used from the viewpoint of preventing contamination. Appropriately, for liquid culture, aeration agitation culture, shaking culture, or static culture may be performed. For example, in a certain embodiment, a liquid medium and a stirrer are put into an Erlenmeyer flask, the mouth of the flask is sealed with a silicone stopper, and stirring culture with ensured air permeability is performed.

[0063] The period of liquid culture is not limited, but it can be completed when the bacterial cells reach a desired concentration. Specifically, although not limited, as the average number of colonies per liter of the liquid culture of the bacterial cells, for example, 1×10 3 cfu / culture medium L or more, or 1×10 4 cfu / culture medium L or more, or 1×10 5 cfu / culture medium L or more, or 1×10 6 cfu / culture medium L or more, or about 5×10 6 When cfu / culture medium L is reached, the culture can be completed. For example, the upper limit of the period of liquid culture can be, for example, 8 days or less, or 6 days or less, or 5 days or less, or 4 days or less. On the other hand, the lower limit of the period of liquid culture can usually be 6 hours or more, or 12 hours or more, or 18 hours or more, or 1 day or more. If the period of liquid culture is too long, the mycelium concentration may become too high. On the other hand, if the period of liquid culture is too short, the mycelium may not elongate sufficiently. For example, in a certain embodiment, if the period of liquid culture is 2 to 4 days, for example, 3 days, a liquid culture of an appropriate concentration for performing solid culture following the liquid culture can be obtained.

[0064] On the one hand, in this specification, "solid culture" refers to culturing a strain using a solid culture medium and a solid culture medium containing water. "Solid culture product" refers to a culture product obtained as a result of solid culture, in which the strain is cultured in the solid culture medium. Subjecting the filamentous fungal endophyte to solid culture means inoculating a liquid culture of the filamentous fungus into the solid culture medium and performing the culture in the solid culture medium.

[0065] As the solid medium, for example, bran, okara, bamboo powder, sawdust, pine nuts, bagasse, cellulose powder, cellobiose, coffee grounds, and starch can be used as the medium. The water used for solid culture may, for example, be added with various additives that contribute to the growth of the strain. Examples of the additives include antibiotics that can suppress the growth of bacteria other than the filamentous fungal endophyte.

[0066] The solid culture medium can be prepared by adding water to the solid medium and mixing them. The water content of the solid culture medium is not limited, but for example, the upper limit of the water content is preferably usually 85% by mass or less, particularly 80% by mass or less, more preferably 75% by mass or less, and especially 70% by mass or less. The lower limit of the water content is preferably usually 30% by mass or more, particularly 40% by mass or more, more preferably 45% by mass or more, and especially 50% by mass or more. If the water content of the solid culture medium is too high, there will be too much moisture and sufficient gaps will not be formed in the medium, so the growth will not be promoted. On the other hand, if the water content of the solid culture medium is too low, sufficient moisture for the culture cannot be ensured. For example, in a certain embodiment, when the water content of the solid culture medium is 50 to 70% by mass, for example, 60% by mass, a solid culture product having an appropriate mycelium concentration as an endophyte material can be obtained.

[0067] Inoculation of the filamentous fungal endophyte into the medium for solid culture is not restricted. For example, it can be inoculated using a micropipette or other instruments, or by at least one of the methods such as directly inoculating after decanting and sterilizing by measures like heating the mouth of the container containing the strain with a burner. Whichever method is used, inoculating the liquid culture into the medium for solid culture enables the liquid culture to spread throughout the medium for solid culture. As a result, the mycelium propagates so as to extend more uniformly throughout the medium for solid culture, and a more uniform culture can be obtained, and it is considered that particles of the culture with more uniform particle size can be obtained.

[0068] Solid culture can be carried out by appropriate culturing means. For example, solid culture can be performed by using a container such as a bag made of plastic or the like and allowing the container to stand still. Also, for solid culture, it is preferable to adjust the culture conditions by a temperature control device, a humidity measuring device, etc. If necessary, one or more devices selected from a stirring device, a vibration device, a pH adjusting device, a turbidity measuring device, a light control device, a specific gas concentration measuring device, and a pressure measuring device may be used. The specific gas concentration measuring device only needs to be able to measure, for example, O2 and CO2 as specific gases. From the viewpoint of preventing contamination, it is preferable to use disposable containers for solid culture, and measures such as optionally closing the mouth of the bag may be taken. Appropriate stirring culture, shaking culture, or static culture, etc. may be carried out. For example, in a certain embodiment, a disposable bag equipped with an air filter is used, and the mouth of the bag is closed and static culture is performed.

[0069] The period of solid culture is not restricted, but it can be completed when the cell mass reaches the desired concentration. Specifically, although not restricted, as the average colony number per 1 g of the solid culture, for example, 1×10 3 ~1×10 11 cfu / g, or 5×10 3 ~5×10 10 cfu / g, or 1×10 4 ~1×10 10 cfu / g, or 5×10 4 ~5×10 9cfu / g, or about 1×10 9 The culture can be completed when it reaches cfu / g. The upper limit of the solid culture period is preferably, for example, usually 20 days or less, particularly 18 days or less, more preferably 15 days or less, and especially 12 days or less. The lower limit of the solid culture period is preferably usually 5 days or more, particularly 6 days or more, more preferably 7 days or more, and especially 8 days or more. If the solid culture period is too long, the risk of contamination by miscellaneous bacteria may increase. Also, if the solid culture period is too long, it may prevent the efficient production of endophyte materials. On the other hand, if the solid culture period is too short, the mycelia may not grow sufficiently. For example, in one embodiment, if the solid culture is carried out for 8 to 12 days, for example, 10 days, a solid culture having an appropriate mycelia concentration as an endophyte material can be obtained.

[0070] The temperature during culture is not particularly limited in either liquid culture or solid culture, and can be any temperature within a range that does not prevent the growth of the filamentous fungal endophyte. Specifically, the upper limit of the temperature during culture can be, for example, 40°C or less, or 35°C or less, or 30°C or less, or 25°C or less. On the other hand, the lower limit of the temperature during culture can be, for example, 5°C or more, or 10°C or more, or 15°C or more, or 20°C or more. In one embodiment, the culture can be carried out at 20 - 25°C, for example, 25°C ± 1°C.

[0071] The humidity during culture is not particularly limited in either liquid culture or solid culture, and can be any humidity within a range that does not prevent the growth of the filamentous fungal endophyte. Specifically, the upper limit of the humidity during culture can be, for example, 100%RH or less, or 95%RH or less, or 90%RH or less. On the other hand, the lower limit of the humidity during culture can be, for example, 55%RH or more, or 60%RH or more, or 65%RH or more. In one embodiment, the culture can be carried out at a humidity of 60 - 80%RH, for example, about 70%RH.

[0072] Also, from the perspective of pollution prevention, the liquid medium, solid medium, and / or solid culture medium are preferably sterilized by any known means such as filtration sterilization, autoclave sterilization, boiling sterilization, radiation sterilization, sodium hypochlorite, and ozone treatment. Each operation such as inoculation is preferably carried out in a sterile atmosphere. For example, in the case of a solid culture medium, it can be sterilized after adding appropriate moisture to the solid medium.

[0073] · Post-treatment When the filamentous fungal endophyte is solid-cultured, the solid culture may be dried. Drying can be carried out using general-purpose equipment such as an air conditioner and a dehumidifier. For example, it can be carried out by leaving it in an environment of 25°C and 50% RH for 3 days.

[0074] Also, after drying the solid culture of the filamentous fungal endophyte, it may be pulverized into a pulverized product. The pulverization of the dried solid culture can be carried out using general-purpose pulverizers such as a hood processor, a coffee mill, and a pepper mill. According to one aspect, the pulverization of the dried solid culture of the filamentous fungal endophyte is such that the cumulative 10% particle size (D 10 ), cumulative 50% particle size (D 50 ), and / or cumulative 90% particle size (D 90 ) in the volume particle size distribution of the pulverized product is preferably adjusted within the aforementioned range.

[0075] · Mixing In the production method of this embodiment, the mixing ratio of the raw material and the culture of the filamentous fungal endophyte is not limited. According to one aspect, the ratio of the amount of the culture of the filamentous fungal endophyte to the total amount of the raw material and the culture of the filamentous fungal endophyte can be, for example, 10% by mass or more, or 20% by mass or more, or 30% by mass or more, or 40% by mass or more, and can also be, for example, 95% by mass or less, or 90% by mass or less, or 80% by mass or less, or 70% by mass or less.

[0076] In the production method of the present embodiment, the mixing conditions and mixing method of the raw material and the filamentous fungal endophyte are not limited as long as the environment is such that the filamentous fungal endophyte does not die. However, it is preferably an environment that does not hinder the growth of the filamentous fungal endophyte, and more preferably an environment in which the filamentous fungal endophyte can be cultured. Specifically, it is as follows.

[0077] In the production method of the present embodiment, the upper limit of the temperature during mixing of the raw material and the culture of the filamentous fungal endophyte can be, for example, 40°C or lower, or 35°C or lower, or 30°C or lower, or 25°C or lower. On the other hand, the lower limit of the temperature during mixing can be, for example, 5°C or higher, or 10°C or higher, or 15°C or higher, or 20°C or higher. In one embodiment, the mixing can be carried out at 20 - 25°C, for example, 25°C ± 1°C.

[0078] In the production method of the present embodiment, the upper limit of the humidity during mixing of the raw material and the culture of the filamentous fungal endophyte can be, for example, 100%RH or lower, or 95%RH or lower, or 90%RH or lower. On the other hand, the lower limit of the humidity during mixing can be, for example, 55%RH or higher, or 60%RH or higher, or 65%RH or higher. In one embodiment, the mixing can be carried out at a humidity of 60 - 80%RH, for example, about 70%RH.

[0079] [Method for promoting plant growth or improving soil] One embodiment relates to a method for promoting plant growth. The method of the present embodiment includes bringing the filamentous fungal endophyte and / or a material containing the same into contact with an iron compound and / or insoluble phosphate and / or soluble phosphate in the rhizosphere of a plant or its surrounding environment. Specifically, by applying the culture of the filamentous fungal endophyte to the soil or material present in the rhizosphere of a plant or its surrounding environment, the iron compound and / or insoluble phosphate and / or soluble phosphate in the rhizosphere of the plant or its surrounding environment can be solubilized. Details of the filamentous fungal endophyte used in the method of the present embodiment and the plants to which it is applied are as described above.

[0080] One embodiment relates to a method for improving soil. The method of this embodiment includes contacting soil containing an iron compound and / or insoluble phosphoric acid and / or citric acid-soluble phosphoric acid with a filamentous fungal endophyte and / or a material containing the same. Thereby, the iron compound and / or insoluble phosphoric acid and / or citric acid-soluble phosphoric acid in the soil is solubilized by the filamentous fungal endophyte, and the soil is improved to become soil suitable for plant growth. Details of the filamentous fungal endophyte used in the method of this embodiment and the target soil are as described above.

Example

[0081] Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are merely examples shown for convenience of explanation, and the present invention is not limited to these examples in any sense.

[0082] In addition, the experimental equipment and reagents used in each experiment described below are as follows.

Table 1

[0083] Also, the strains used in the following experiments are as follows. Fusarium solani: Strain 15 - B (NITE P - 03991) Lecanicillium sp.: Strain 100 - 1 (NITE P - 03984)

[0084] [Experiment 1] Production of indole-3-acetic acid (IAA)

[0085] <Example 1> (1) A DYGS medium having the following composition was prepared and autoclaved at 121°C for 20 minutes.

Table 2

Table 3

[0086] <Example 2> An experiment was conducted in the same manner as <Example 1>, except that 3 pieces of inoculum agar of Lecanicillium sp. were added instead of F. solani.

[0087] <Comparative Example 1> An experiment was conducted in the same manner as <Example 1>, except that no inoculum agar was added.

[0088] [Results of Experiment 1] As a result of the above experiment, the production amount of indole-3-acetic acid (IAA) after culture was as follows.

Table 4

[0089] Since the IAA amounts in <Example 1> and <Example 2> were greater than that in <Comparative Example 1>, it was suggested that F. solani and Lecanicillium sp. are strains that produce IAA. The IAA production abilities of the strains were 23 μg / ml and 15 μg / ml, respectively, showing higher values than those of the Bacillus endophytes isolated in Non-Patent Document 2.

[0090] [Experiment 2] Solubilization of insoluble phosphate

[0091] <Example 3> (1) An NBRIP medium having the following composition was prepared.

Table 5

[0092] <Comparative Example 2> An experiment was conducted in the same manner as in <Example 3>, except that the inoculum agar of F. solani was not added.

[0093] <Example 4> An experiment was conducted in the same manner as in <Example 3>, except that Lecanicillium sp. was added instead of the inoculum agar of F. solani.

[0094] <Comparative Example 3> An experiment was conducted in the same manner as in <Example 3>, except that the inoculum agar of F. solani was not added, and it was conducted simultaneously with <Example 4>.

[0095] [Results of Experiment 2] As a result of the above experiments, the solubilized amounts of water - soluble phosphoric acid after culturing were as follows.

Table 6

[0096] <Example 3> and <Example 4> each contained more water - soluble phosphoric acid than <Comparative Example 2> and <Comparative Example 3>, indicating that the insoluble phosphoric acid source in the medium was solubilized by the strains. Also, these values were higher than those in Non - Patent Document 3.

[0097] [Experiment 3] Production of siderophore

[0098] [Experiment 3-1] Qualitative analysis of siderophore production <Example 5> (1) Solution I, Solution II, and Solution III were each prepared. ·Solution I

Table 7

[0099] ·Solution II (i) 10 μL of HCl (12 mol / L) was added to 110 μL of pure water to obtain 1 mol / L HCl. (ii) 100 μL of 1 mol / L HCl was added to 9.9 mL of pure water to obtain 10 mM HCl. (iii) 0.003 g of FeCl3·6H2O was added to 10 mM HCl.

[0100] ·Solution III

Table 8

[0101] (2) 9 mL of Solution II was added to 50 mL of Solution I and mixed, and then 40 mL of Solution III was further added. Thereafter, the mixture was autoclaved at 121 °C for 20 minutes. After sterilization, the mixture was transferred to a PP bottle in a sterile environment.

[0102] (3) The following reagents were each prepared. ·Minimal Media 9 (MM9) Salt Solution

Table 9

Table 10

Table 11

Table 12

[0103] (4) Dilute 20 ml of MM9 Salt Solution with 150 ml of pure water, and add NaOH solution to adjust the pH to 6.0. (5) While gradually adding 6.44 g of PIPES to the solution obtained in (4), dissolve it and prepare three types of solutions with pH values of 6.0, 6.3, and 6.55 using NaOH solution. (6) Add 3 g of agar for bacteria to the solution obtained in (5), and sterilize it by autoclaving at 121 °C for 20 minutes. (7) Add 6 ml of casamino acid solution and 2 ml of 20% glucose solution to the solution obtained in (6). (8) Slowly add 20 ml of Blue Dye Solution to the solution obtained in (7) while wetting the wall of the container. (9) Shake the solution obtained in (8) gently without foaming, and dispense it into 10 petri dishes to obtain CAS Agar medium. (10) Place a seed agar block of about 5 mm square filled with mycelium of F. solani on the CAS Agar medium, and let it stand at 25 °C and 70% RH for 5 days.

[0104] <Example 6> An experiment was conducted in the same manner as <Example 5>, except that a seed agar block of about 5 mm square filled with mycelium of Lecanicillium sp. was placed instead of F. solani.

[0105] [Experiment 3-2] Quantitative analysis of siderophore production amount <Example 7> (1) Prepare a liquid medium having the following composition and sterilize it by autoclaving at 121 °C for 20 minutes.

Table 13

[0106] <Example 8> An experiment was conducted in the same manner as <Example 7>, except that Lecanicillium sp. was added instead of F. solani.

[0107] <Comparative Example 4> An experiment was conducted in the same manner as <Example 7>, except that the inoculum agar of F. solani was not added.

[0108] [Results of Experiment 3]

[0109] When the CAS agar assay was performed, in <Example 5> and <Example 6>, a red Halo was formed around the strain, suggesting that F. solani and Lecanicillium sp. produce siderophores (Figure 1).

[0110] Therefore, the production amount of siderophore was quantitatively analyzed by CAS liquid assay. For F. solani and Lecanicillium sp., mycelia were sufficiently grown in the liquid medium at the third day of culture. In <Example 7> and <Example 8>, the production amounts of siderophore did not vary significantly, chelating 62.78% and 63.36% of iron, respectively (Table 14). These values are higher than those in Non-Patent Document 1, a previous study, and it can be expected that more iron can be chelated. [Table 14]

[0111] [Experiment 4] Heavy metal adsorption (1)

[0112] <Example 9> (1) 30 ml of an aqueous solution of 5 mg / L cadmium nitrate tetrahydrate was added to a tube. (2) 50 mg of mycelial powder of F. solani was added to (1). (3) The solution obtained in (2) was shaken at room temperature at 100 rpm for 60 minutes. (4) The solution obtained in (3) was filtered through a syringe filter with a pore size of 0.22 μm. (5) For the solution obtained in (4), the concentration of cadmium (Cd) was measured by ICP.

[0113] <Example 10> An experiment was conducted in the same manner as in <Example 9>, except that mycelial powder of Lecanicillium sp. was added instead of F. solani.

[0114] <Comparative Example 5> An experiment was conducted in the same manner as in <Example 9>, except that no mycelial powder was added.

[0115] [Results of Experiment 4] Since the amount of cadmium contained in <Example 9> and <Example 10> was less than that in <Comparative Example 5>, it was suggested that cadmium was adsorbed onto the mycelia (Table 15). In addition, Lecanicillium sp. showed a higher adsorption capacity than F. solani. This value is comparable to that in Non-Patent Document 4, a prior study, and it is expected to adsorb an equivalent amount of cadmium.

Table 15

[0116] [Experiment 5] Heavy metal adsorption (2)

[0117] <Example 11> (1) 30 ml of a 10 mg / L lead nitrate aqueous solution was added to a tube. (2) 10 mg of mycelial powder of F. solani was added to (1). (3) The solution obtained in (2) was shaken at room temperature at 100 rpm for 60 minutes. (4) The solution obtained in (3) was filtered through a syringe filter with a pore size of 0.22 μm. (5) For the solution obtained in (4), the concentration of lead (Pb) was measured by ICP.

[0118] <Example 12> An experiment was conducted in the same manner as in <Example 11>, except that mycelial powder of Lecanicillium sp. was added instead of F. solani.

[0119] <Comparative Example 6> An experiment was conducted in the same manner as in <Example 11>, except that no mycelial powder was added.

[0120] [Results of Experiment 5] Since the amount of lead contained in <Example 11> and <Example 12> was less than that in <Comparative Example 6>, it was suggested that lead was adsorbed onto the mycelia (Table 15). In addition, F. solani showed a higher adsorption capacity than Lecanicillium sp.

Table 16

[0121] [Experiment 6] Preparation method of materials and measurement of colony number

[0122] <Example 13> (1) 100 ml of pure water was added to 2.85 g of glucose peptone medium. (2) The solution of (1) was sterilized in an autoclave at 121 °C for 20 minutes. (3) Three pieces of inoculum agar, about 5 mm square, filled with mycelia of F. solani were added to the solution of (2), and cultured with stirring at 25 °C for 3 days at 700 rpm. (4) 500 ml of pure water was added to 500 g of bran and mixed well, and then transferred to a filter-equipped mushroom bed bag. (5) (4) was sterilized in an autoclave at 121 °C for 60 minutes. (6) 100 ml of the culture solution of F. solani obtained in (3) was added to the mushroom bed bag sterilized in (5) and sealed. (7) It was allowed to stand at 25 °C and 70% RH for 5 days. (8) The mushroom bed was taken out of the bag, crushed, and dried at 25 °C for 3 days. (9) The dried product was pulverized with a hood processor and sieved through a sieve with a diameter of 2 mm. This was designated as "Fusarium material". (10) 1 g of Fusarium material was suspended in 10 ml of pure water to obtain a 10-fold dilution. (11) 1 ml was collected from the 10-fold dilution obtained in (10), and 9 ml of pure water was added to obtain a 10 2 -fold dilution. Thereafter, 10 5 -fold dilutions were prepared in the same procedure. (12) 500 ml of pure water was added to 15.75 g of DRBC medium and sterilized in an autoclave at 121 °C for 20 minutes. (13) It was poured into a plastic petri dish and allowed to stand to solidify. (14) 1 ml of the 10 3 ~10 5 -fold dilutions prepared in (11) were spread on the DRBC solid medium. (15) It was allowed to stand at 25 °C and 70% RH for 2 days. (16) The number of formed colonies was measured.

[0123] <Example 14> An inoculum agar piece of about 5 mm square filled with mycelia of Lecanicillium sp. was added instead of F. solani, and it was allowed to stand for 3 days in (15). The experiment was conducted in the same manner as <Example 13> except that the completed material was designated as "Lecanicillium material".

[0124] [Results of Experiment 6] From <Example 13> and <Example 14>, the number of colonies contained in 1 g of the material was about 10 - 17 x 10 8 individuals and 2 - 5 x 10 6 individuals, respectively (Table 17).

Table 17

[0125] [Experiment 7] Growth experiment of lettuce

[0126] <Example 15> (1) Sterile culture soil and vermiculite were mixed at a volume ratio of 1:1 to prepare a cultivation soil. (2) To 1000 ml of the cultivation soil prepared in (1), 270 mg of ammonium sulfate as a nitrogen source, 450 mg of superphosphate as a phosphate source, 270 mg of potassium chloride as a potassium source, and 2 g of the Fusarium material prepared in <Example 13> were added and mixed well. (3) 100 ml of the cultivation soil prepared in (2) was distributed into each cultivation pot with a diameter of 9 cm, and salad greens (Okayama salad greens) were sown. (4) Cultivation was carried out in the pot for 60 days from sowing.

[0127] <Example 16> An experiment was conducted in the same manner as <Example 15> except that the Lecanicillium material prepared in <Example 14> was added instead of the Fusarium material.

[0128] <Comparative Example 7> An experiment was conducted in the same manner as in <Example 15>, except that neither Fusarium material nor Lecanicillium material was added.

[0129] [Results of Experiment 7] In <Example 15>, the harvested weight of the edible part increased by about 1.21% compared to <Comparative Example 7>. Furthermore, the variation in growth was suppressed and the standard deviation became smaller. In <Example 16>, the harvested weight of the edible part increased by 64.46% compared to <Comparative Example 7>.

Table 18

[0130] [Summary] The present invention proposes the use of filamentous fungal endophytes that promote plant growth, and contributes to the healthy growth of agricultural crops and an increase in yield. Patent Document 1 can be cited as a similar prior study, but in the present invention, the mechanism of action of plant growth promotion by fungi has been analyzed in more detail, and more functions are expected.

Industrial Applicability

[0131] The present invention can be widely applied in fields such as agriculture and horticulture, and its utility value is extremely large. For example, it becomes possible to cultivate plants even in soils where it is difficult to supply iron, such as alkaline soils, the cultivable arable land area is expanded, and it becomes possible to solve future food and resource problems.

Deposit Number

[0132] NITE P-03991 NITE P-03984

Claims

1. A material containing an iron compound and a filamentous fungal endophyte, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of the genus Fusarium (Fusarium sp.) and the genus Lecanicillium (Lecanicillium sp.).

2. The material according to claim 1, wherein the iron compound is a trivalent iron compound.

3. The material according to claim 1, wherein the content of the iron compound is 10 to 50,000 ppm of the material.

4. The material according to claim 1, wherein the filamentous fungal endophyte produces siderophore.

5. The material according to claim 1, wherein the filamentous fungal endophyte produces indole-3-acetic acid.

6. The material according to claim 1, further comprising a raw material containing insoluble phosphate and / or soluble phosphate.

7. The material according to claim 6, wherein the raw material is a fired product of sludge.

8. The filamentous fungal endophyte is a pulverized product of a dry solid culture, and the cumulative 50% particle size (D ５０ ) in the volume particle size distribution is 100 to 900 μm, and the cumulative 90% particle size (D ９０ ) is 1000 to 5000 μm. The material according to claim 1.

9. A method for promoting plant growth, comprising: contacting a filamentous fungal endophyte with an iron compound in the rhizosphere of a plant or its surrounding environment, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of the genus Fusarium (Fusarium sp.) and the genus Lecanicillium (Lecanicillium sp.).

10. The method according to claim 9, further comprising contacting the filamentous fungal endophyte with insoluble phosphate and / or soluble phosphate in the rhizosphere of the plant or its surrounding environment.

11. The method according to claim 9, further comprising contacting the filamentous fungal endophyte with a fired product of sludge in the rhizosphere of the plant or its surrounding environment.

12. The method according to claim 9, further comprising contacting the filamentous fungal endophyte with heavy metals in the rhizosphere of the plant or its surrounding environment.

13. A method for producing a crop obtained from a plant, comprising: contacting a filamentous fungal endophyte with an iron compound in the rhizosphere of a plant or its surrounding environment, growing the plant, and harvesting the crop obtained from the plant, wherein the filamentous fungal endophyte contains at least one or more selected from the group consisting of the genus Fusarium (Fusarium sp.) and the genus Lecanicillium (Lecanicillium sp.).

14.

15.

16. The method according to claim 13, comprising contacting the filamentous fungal endophyte with insoluble phosphate and / or citrate-soluble phosphate in the rhizosphere of the plant or its surrounding environment.

15. The method according to claim 13, comprising contacting the filamentous fungal endophyte with the fired product of sludge in the rhizosphere of the plant or its surrounding environment.

16. The method according to claim 13, comprising contacting the filamentous fungal endophyte with heavy metals in the rhizosphere of the plant or its surrounding environment.

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