JP7840922B2 - Materials containing iron compounds and filamentous fungal endophytes, a method for promoting plant growth, and a method for producing crops obtained from plants. - Google Patents

Description

NITE NITE P-03984 NITE NITE P-03991

This 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 crops obtained from plants.

Plants absorb inorganic nutrients from the air and soil, and grow by converting them into organic matter using sunlight energy or utilizing them in metabolic reactions.

Among the nutrients (elements) essential for plant growth, iron (Fe) is classified as a trace essential element because plants require only a small amount. Iron is essential for nucleic acid synthesis (such as DNA), energy production, and various enzymatic reactions in living organisms, including plants. Therefore, iron is an indispensable element for maintaining life activities. However, since plants cannot move once they take root in the soil, they need a mechanism to preferentially absorb the nutrients necessary for growth from the soil where they are rooted.

In neutral or alkaline soils, iron exists as trivalent iron ( ^Fe³⁺ ). Because trivalent iron ( ^Fe³⁺ ) is poorly soluble in water, iron absorption by plants is significantly inhibited in neutral or alkaline soils. Some grasses secrete molecules called siderophores that strongly bind to trivalent iron ( ^Fe³⁺ ). Siderophores have a high affinity for trivalent iron ( ^Fe³⁺ ) and act as chelating agents, forming complexes with it. As a result of forming complexes with siderophores, trivalent iron ( ^Fe³⁺ ) becomes solubilized and is absorbed by the roots ^and taken up by the plant.

Some microorganisms produce siderophores, and some have been reported to live in symbiosis with plant hosts as filamentous fungal endophytes (Non-Patent Literature 1).

Endophytes, also known as endophytes, are filamentous fungi that live symbiotically within plant hosts, providing benefits to the host such as increased growth and stress tolerance (Patent Document 1). Among filamentous endophytes, some have been reported to possess phosphate-solubilizing ability, converting insoluble phosphate and citrate-soluble phosphate (phosphate that dissolves under acidic conditions such as in citrate) in soil into water-soluble phosphate that can be absorbed by plant roots (Non-Patent Documents 2 and 3). Furthermore, some filamentous fungi have been reported to reduce the uptake of heavy metals (e.g., cadmium) by adsorbing them in the soil, thereby negatively impacting plant growth (Non-Patent Document 4).

Japanese Patent Publication No. 2018-174708

Chowdappa, S. et al., Detection and characterization of antibacterial siderophores secreted by endophytic fungi from Cymbidium aloifolium. Biomolecules. (2020), 10(10):1412. Milani R., et al. Bacillus subtilis isolates with different abilities to promote plant growth in maize, cotton and soybean crops isolation and characterization of bacterial strains. Asian Jr. of Microbiol. Biotech. Env. Sc.(2019), Vol.21, No.(4): 827-836 Kumer, P. et al., Seed bio-priming with tri-species consortia of phosphate solubilizing rhizobacteria (PSR) and its effect on plant growth promotion. Heliyon.(2020), Volume 6, issue 12, e05701. Joner, E. J., et al., (2000) Metal-binding capacity of arbuscular mycorrhizal mycelium. (2000) Plant and Soil volume 226:227-234

The object of this invention is to provide a novel means for solubilizing iron compounds in the rhizosphere or surrounding environment of plants to promote plant growth.

As a result of diligent research, the inventors have discovered a novel filamentous fungal endophyte that solubilizes iron compounds in the plant rhizosphere or its surrounding environment, thereby promoting plant growth, and have completed the following invention.

[1] A material containing an iron compound and a filamentous fungal endophyte,
The material is one in which the filamentous fungal endophyte contains at least one species selected from the group consisting of the genera Fusarium and Lecanicillium.
[2] The material described in [1] is a trivalent iron compound.
[3] The materials described in [1] or [2] have an iron compound content of 10 to 50,000 ppm of the above material.
[4] The materials described in any one of items [1] to [3] produce siderophores from the filamentous fungal endophytes.
[5] The materials described in any one of items [1] to [4] are those in which the filamentous fungal endophytes produce indole-3-acetic acid.
[6] The materials described in any one of items [1] to [5] include raw materials containing insoluble phosphoric acid and/or citrate-soluble phosphoric acid.
[7] The materials described in any one of items [1] to [6] are those in which the raw material is calcined sludge.
[8] The material described in any one of items [1] to [6] is a pulverized product of a dried solid culture of the above-mentioned filamentous fungal endophytes. The above material has a cumulative 50% particle size (D ₅₀ ) in the volume particle size distribution of 100 to 900 μm. The above material has a cumulative 90% particle size (D ₉₀ ) of 1000 to 5000 μm.
[9] Any material described in any one of items [1] to [6] is used to promote plant growth.

[10] A method for promoting the growth of plants,
The method involves contacting a filamentous fungal endophyte with iron compounds in the rhizosphere of a plant or its surrounding environment. The method includes at least one species of filamentous fungal endophyte selected from the group consisting of Fusarium sp. and Lecanicillium sp.
[11] The method according to [10] is wherein the iron compound is a trivalent iron compound.
[12] The method according to [10] or [11] is characterized in that the content of the iron compound is 10 to 50,000 ppm of the soil in which the plants are grown.
[13] The method described in any one of [10] to [12] comprises contacting the filamentous fungal endophyte with insoluble phosphate and/or citrate phosphate in the rhizosphere of the plant or the surrounding environment.
[14] The method described in any one of items [10] to [13] includes contacting the filamentous fungal endophyte with a calcined product of sludge from the rhizosphere of the above-mentioned plant or the surrounding environment.
[15] The method described in any one of the items [10] to [14] includes contacting the filamentous fungal endophyte with heavy metals in the rhizosphere of the plant or the surrounding environment.
[16] The method described in any one of items [10] to [15] includes, in which the heavy metals include cadmium and/or lead.

[17] A method for producing crops obtained from plants,
The method includes contacting a filamentous fungal endophyte with iron compounds in the rhizosphere or surrounding environment of a plant, and growing the plant. The method also includes harvesting a crop obtained from the plant. In the method, the filamentous fungal endophyte includes at least one species selected from the group consisting of Fusarium sp. and Lecanicillium sp.
[18] The method described in [17] is a trivalent iron compound.
[19] The method according to [17] or [18] is such that the iron compound content is 10 to 50,000 ppm in the soil in which the plants are grown.
[20] The method described in any one of [17] to [19] comprises contacting the filamentous fungal endophyte with insoluble phosphate and/or citrate phosphate in the rhizosphere of the plant or the surrounding environment.
[21] The method described in any one of items [17] to [20] includes contacting the filamentous fungal endophyte with a calcined product of sludge from the rhizosphere of the plant or the surrounding environment.
[22] The method described in any one of the items [17] to [21] includes contacting the filamentous fungal endophyte with heavy metals in the rhizosphere of the plant or the surrounding environment.
[23] The method described in any one of the items [17] to [22], wherein the heavy metal includes cadmium and/or lead.

When plants are grown using this invention, iron compounds in the plant's rhizosphere or surrounding environment are solubilized, thereby promoting plant growth.

Figure 1 shows the qualitative analysis results of siderophore production of the bacterial strains using CAS Agar Assay, conducted in Experiment 3-1. a: Example 5, b: Example 6.

The present invention will be described in detail below with reference 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 spirit of the invention.

[overview]
The inventors have discovered novel filamentous fungal endophytes that exhibit a chelating effect on iron compounds. The iron-chelating factors produced by these filamentous fungal endophytes solubilize the iron compounds, promoting the growth of plants that take them up. This invention is based on the discovery of such novel filamentous fungal endophytes.

One aspect of this embodiment relates to 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. The filamentous fungal endophyte produces a factor that chelates the iron compound. This solubilizes the iron compound, particularly insoluble iron compounds, and the plant's growth is promoted by the absorption of the water containing the dissolved iron compounds through the roots. In one aspect of this embodiment, the material is used to promote plant growth.

Furthermore, one aspect of this embodiment relates to a method for promoting plant growth, comprising contacting filamentous fungal endophytes with iron compounds in the plant's rhizosphere or surrounding environment, wherein the filamentous fungal endophytes include at least one species selected from the group consisting of Fusarium sp. and Lecanicillium sp.

Furthermore, one aspect of this embodiment relates to a method for producing crops obtained from plants, comprising contacting filamentous fungal endophytes with iron compounds in the plant's rhizosphere or surrounding environment, growing the plants, and harvesting crops obtained from the plants, wherein the filamentous fungal endophytes include at least one species selected from the group consisting of Fusarium sp. and Lecanicillium sp. Crops grown and harvested by the method of one aspect of this embodiment have increased harvest weight. In addition, crops grown and harvested by the method of one aspect of this embodiment have reduced growth variability. In this disclosure, "crop" is used to mean both "the plant itself" and "the part of the plant that is to be harvested." "The part that is to be harvested" refers to the edible part above ground in the case of plants that form edible parts above ground, such as lettuce. In the case of rice, for example, it refers to its edible part, i.e., "rice." Furthermore, in the case of plants that form edible parts underground, such as onions, this refers to the edible underground parts. Also, in this disclosure, "contact" includes both physically bringing filamentous fungal endophytes into direct contact with a target substance (e.g., iron compounds, insoluble phosphates, citrate-soluble phosphates, or heavy metals) and allowing substances produced from filamentous fungal endophytes (e.g., siderophores) to act on the target substance.

In this disclosure, "filamentous fungal endophytes" refers to filamentous fungi capable of functioning as endophytes. Endophytes, also known as endophytes, are microorganisms that live symbiotically within plant hosts, providing benefits to the host such as increased growth and stress tolerance.

One example of a filamentous fungal endophyte that can be used in this embodiment is from the genus Fusarium. An example of a Fusarium is Fusarium solani. Furthermore, the Fusarium solani that can be used in this embodiment is, for example, strain 15-B, which was deposited with the National Institute of Technology and Evaluation (NITE) on October 16, 2023, and is available under accession number NITE P-03991.

One example of a filamentous fungal endophyte that can be used in one embodiment of this embodiment is that of the genus Lecanicillium sp. For example, strain 100-1, which was deposited with the National Institute of Technology and Evaluation (NITE) on September 15, 2023, and is available under accession number NITE P-03984, can be used as a filamentous fungal endophyte of the genus Lecanicillium that can be used in one embodiment of this embodiment.

In this disclosure, an iron compound is a compound containing the element iron, and may be an insoluble or sparingly soluble iron compound. When one aspect of this embodiment is applied, an insoluble or sparingly soluble iron compound is solubilized and can be absorbed by plant roots. In one aspect of this embodiment, the iron compound is an insoluble or sparingly soluble iron compound under neutral or alkaline conditions, and is preferably a trivalent iron compound. Examples of trivalent iron compounds include iron(III) hydroxide, iron(III) oxide, and iron(III) phosphate. In one aspect of this embodiment, the iron compound may be present in the material or applied soil at a concentration of 10 to 50,000 ppm, preferably 20 to 30,000 ppm.

In this disclosure, "siderophore" refers to a molecule with high affinity for trivalent iron ions, acting as a chelating agent to bind to trivalent iron ions and form iron-siderophore complexes. Siderophore forms iron-siderophore complexes with trivalent iron ions derived from insoluble or sparingly soluble iron compounds, and these complexes become soluble in water. The water-soluble iron-siderophore complexes are absorbed by the roots, making iron available to plants.

In one embodiment of this model, a filamentous fungal endophyte may be used to produce siderophores. The presence of siderophores can be evaluated, for example, by an assay using CAS (Chrome azurol S). CAS is a pigment compound that exhibits a characteristic coloration when bound to iron ions. By adding a compound with high iron-binding ability, such as a siderophore, to a solution containing CAS, iron ions are removed from the CAS-iron complex, resulting in the disappearance of the characteristic coloration of the CAS-iron complex. This reaction can be used to determine the presence of siderophores.

As shown in the examples described below, the above-mentioned filamentous fungal endophyte exhibits significantly superior ability to chelate iron compounds compared to the filamentous fungus described in Non-Patent Document 1.

In one embodiment of this invention, the material may include raw materials containing insoluble phosphate and/or citrate-soluble phosphate. The filamentous fungal endophyte used in one embodiment of this invention can solubilize insoluble phosphate and/or citrate-soluble phosphate, converting it into water-soluble phosphate.

In this disclosure, "water-soluble phosphate" refers to phosphate that dissolves in water. An example of water-soluble phosphate is potassium dihydrogen phosphate. Water-soluble phosphate can be absorbed by plants through their roots in its original state.

In this disclosure, "citric acid-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). Plants cannot directly absorb citrate-soluble phosphate, but they can absorb it by secreting root acids from their roots, which convert citrate-soluble phosphate into water-soluble phosphate. However, plants can only utilize citrate-soluble phosphate in the soil within a very narrow range of the rhizosphere, where root acids can reach.

In this 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 have no means to make it absorbable.

In this disclosure, "solubilization" of phosphoric acid refers to the conversion of insoluble phosphoric acid and/or citrate-soluble phosphoric acid into water-soluble phosphoric acid.

As shown in the examples described later, filamentous fungal endophytes exhibit significantly superior ability to solubilize insoluble phosphate or insoluble phosphate sources compared to the bacteria described in Non-Patent Document 3. As mentioned above, although plants can solubilize citrate-soluble phosphate by secreting root acids from their roots, they cannot normally utilize citrate-soluble phosphate in the soil except in a very narrow range of the rhizosphere that root acids can reach. However, filamentous fungal endophytes live in symbiosis with plants and absorb sugars produced by plants through photosynthesis for their own growth. The hyphae of filamentous fungi are thinner than plant roots and can extend rapidly into even the smallest gaps (gas phase) in the soil. In this way, filamentous fungi spread their hyphae throughout the soil, which is wider than the area of plant roots, and decompose (dissolve) nutrients in the soil, sending them to the plant roots via the hyphae (Patent Document 1). Therefore, by using filamentous fungal endophytes, it is possible to solubilize a wide range of citrate-soluble and insoluble phosphates that are not reached by root acids, through the hyphae of filamentous fungal endophytes extending from the roots, making them absorbable by plants.

Furthermore, filamentous fungal endophytes may be used individually, or two or more species may be used in any combination and ratio. When using two or more filamentous fungal endophytes, two or more species from the same genus may be combined, or one or more species from two or more different genera may be selected and combined. Using two or more filamentous fungal endophytes in combination may be expected to improve phosphate solubilization ability through a synergistic effect.

The raw materials used in the materials of this embodiment are not limited as long as they are used to promote plant growth. Examples of raw materials include soil. Other examples include phosphate rock, steel slag and their crushed products, as well as mixtures of these with crop residues. An example of crop residue is sorghum residue. Another example of raw materials is sludge containing phosphoric acid. Specifically, this includes sewage sludge, animal manure (e.g., cow dung, chicken manure) and sludge derived therefrom, meat and bone meal and sludge derived therefrom, mushroom excretion substrate and sludge derived therefrom, and calcined products thereof. The calcined products may also be referred to as incinerated materials. Furthermore, materials subjected to concentration, dehydration, separation, digestion, drying, or calcination, either individually or in combination, are also included as raw materials used in the materials of this embodiment. These raw materials may be used individually, or two or more may be used in any combination and ratio.

The raw materials used in this embodiment may contain at least insoluble phosphoric acid and/or citrate-soluble phosphoric acid. The raw materials used in this embodiment may contain only one of insoluble phosphoric acid and citrate-soluble phosphoric acid, or both. Furthermore, the respective content ratios of insoluble phosphoric acid and citrate-soluble phosphoric acid in the raw materials used in this embodiment are not limited.

However, in raw materials containing both insoluble and citrate-soluble phosphate, if the amount of insoluble phosphate is greater than the amount of citrate-soluble phosphate (i.e., raw materials where the amount of insoluble phosphate is greater than the amount of citrate-soluble phosphate), even if microorganisms capable of solubilizing only citrate-soluble phosphate are present, the amount of water-soluble phosphate necessary for plant growth may be insufficient. In such environments where the amount of insoluble phosphate is greater than the amount of citrate-soluble phosphate, the inclusion of filamentous fungal endophytes capable of solubilizing both citrate-soluble and insoluble phosphate offers the advantage of supplying sufficient phosphate for plant growth. In other words, according to one embodiment, the raw materials and materials contain both insoluble and citrate-soluble phosphate, and the amount of insoluble phosphate is greater than the amount of citrate-soluble phosphate. Examples of raw materials where the amount of insoluble phosphate is greater than the amount of citrate-soluble phosphate include phosphate rock and its crushed material, and volcanic ash soil.

On the other hand, among raw materials containing both insoluble and citrate-soluble phosphate, raw materials where {content of insoluble phosphate} < {content of citrate-soluble phosphate} (i.e., raw materials where the content of insoluble phosphate is less than the content of citrate-soluble phosphate) have higher phosphate solubility compared to raw materials where {content of insoluble phosphate} > {content of citrate-soluble phosphate}, thus having a more immediate effect on plant growth. In other words, according to one embodiment, the raw materials and materials contain both insoluble 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 manure, chicken manure) and sludge derived therefrom, meat and bone meal, mushroom substrate, and their calcined (or incinerated) products.

According to one embodiment, the material of this embodiment and/or the rhizosphere or surrounding environment of the plant to which it is applied may contain heavy metals. The filamentous fungal endophytes used in this embodiment can adsorb heavy metals present in the material and/or the rhizosphere or surrounding environment of the plant to which it is applied. This suppresses the uptake of heavy metals that could adversely affect the plant whose growth is being promoted. Examples of heavy metals that the filamentous fungal endophytes of this embodiment can adsorb include cadmium (Cd), nickel (Ni), chromium (Cr), and lead (Pb), but cadmium (Cd) or lead (Pb) are preferred.

According to one embodiment, the total content of insoluble phosphate and citrate-soluble phosphate in the material and/or raw materials used therein is preferably 0.5% by mass or more, more preferably 3% by mass or more, and more preferably 5% by mass or more. By setting the total content of insoluble phosphate and citrate-soluble phosphate in the material and/or raw materials to be above the aforementioned lower limit, the solubilizing ability of filamentous fungal endophytes can be reliably exerted, making it possible to more effectively promote plant growth or improve soil quality. On the other hand, the upper limit of the total content is not particularly limited, but for example, it can be 95% by mass or less.

Furthermore, the insoluble phosphate and citrate-soluble phosphate content of raw materials and supplies can both be measured by the quinoline mass method.

According to one embodiment, the material of this embodiment may further contain water-soluble phosphate. This water-soluble phosphate may be present in the raw materials. Alternatively, this water-soluble phosphate may be produced when insoluble and/or citrate-soluble phosphate present in the raw materials is solubilized by filamentous fungal endophytes. If the material of this embodiment contains water-soluble phosphate, its content is not limited.

The filamentous fungal endophytes used in this embodiment may be used in combination with other microorganisms capable of solubilizing iron compounds and/or insoluble phosphates. These other microorganisms capable of solubilizing iron compounds and/or insoluble phosphates may be used individually or in combination of two or more in any combination and ratio. Other examples of microorganisms that can solubilize iron compounds include Aspergillus nidulans, Aspergillus sydowii, Aspergillus terreus, Azotobacter vinelandii, Bacillus subtilis, Burkholderia cepacia, Cymbidium aloifolium, Escherichia coli, Penicillium chrysogenum, Pseudomonas aeruginosa, Rhodotomorula pilimanae, Streptomyces pilosus, and Streptomyces. Examples include *coelicor* and *Ustilago sphaerogenea*.

Other examples of microorganisms that can solubilize insoluble phosphate include Achromobacter marplatensis, Acinetobacter calcoaceticus, bacteria of the genus Advenella, Agromyces aruantiacus, Alcaligenes faecalis, bacteria of the genus Arthrobacter, Bacillus pumila, Bacillus subtilis, Bordetella avium, Brevibacterium frigoritolerans, Burkholderia cenocepacia, and Cronobacter. sakazakii, Ensifer genus bacteria, Enterobacter ludwigii, Enterococcus genus bacteria, Flavobacterium ahuensis, Fusarium oxysporum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactococcus garvieae, Lactococcus lactis, Leifsonia bacteria, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Microbacterium hydrocarbonoxydans, Nocardioides bacteria, 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.

The filamentous fungal endophyte used in one embodiment of this model produces indole-3-acetic acid (IAA), a type of plant hormone called auxin. Indole-3-acetic acid is a plant growth regulator, exhibiting effects such as promoting plant growth, root development, cell elongation, and cambium cell division. As shown in the examples described later, the filamentous fungal endophyte used in one embodiment of this model exhibits significantly superior indole-3-acetic acid production compared to the bacteria described in Non-Patent Document 2.

The number of filamentous fungal endophytes in the material of this embodiment is not particularly limited. In one embodiment, if the material of this embodiment is mainly a culture of filamentous fungal endophytes, the number of filamentous fungal endophytes can be, for example, 1 × ^10⁵ cfu/g or more, or 5 × ^10⁵ cfu/g or more, or 1 × 10⁶ cfu/g or more, or 5 × ^10⁶ cfu/g or more, as an average number of colonies ^per gram of material, or it can be, for example, 1 × ^10¹¹ cfu/g or less, or 5 × ^10¹⁰ cfu/g or less, or 1 × ^10¹⁰ cfu/g or less, or 5 × ^10⁹ cfu/g or less. Furthermore, if other microorganisms capable of solubilizing iron compounds and/or insoluble phosphates are used in combination with filamentous fungal endophytes, the number of cells of such other microorganisms can be within the same range as the number of filamentous fungal endophytes. Furthermore, if the material of this embodiment includes a culture of filamentous fungal endophytes and other raw materials, the number of fungal cells of the cultured filamentous fungal endophytes can be 1 × ^10² cfu/g or more, or 5 × ^10² cfu/g or more, or 1 × 10³ cfu/g or more, or 5 × ^10³ cfu/g or more, as an average number ^{of colonies per gram of material. Alternatively, it can be, for example, 1 × 10¹¹ cfu} /g or less, or 5 × ^10¹⁰ cfu/g or less, or 1 × ^10¹⁰ cfu/g or less, or 5 × ^10⁹ cfu/g or less.

The filamentous fungal endophytes used in this embodiment may be cultures of filamentous fungal endophytes. Examples of such cultures include solid cultures and liquid cultures. A solid culture refers to a culture obtained as a result of solid culture, in which filamentous fungal endophytes are cultured in a solid culture medium. A liquid culture refers to a culture obtained as a result of liquid culture, in which filamentous fungal endophytes are cultured in a liquid culture medium. The preparation methods for each culture will be described later.

According to one embodiment, 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, and more preferably a pulverized dried solid culture.

Furthermore, when other microorganisms capable of solubilizing iron compounds and/or insoluble phosphates are used in combination with filamentous fungal endophytes, these other microorganisms can also be used as cultures. Examples of the above-mentioned solid cultures include solid cultures and liquid cultures. Among these, solid cultures are preferred, dried solid cultures are more preferred, and pulverized dried solid cultures are particularly preferred.

The particle size of the above-mentioned pulverized material is not limited, but is as follows for example. In one embodiment, the cumulative 10% particle size (D ₁₀ ) in the volume particle size distribution of the above-mentioned pulverized material is, for example, 50 μm or more, or 100 μm or more, or 150 μm or more, or 200 μm or more, and also, for example, 400 μm or less, or 350 μm or less, or 300 μm or less. In one embodiment, the cumulative 50% particle size (D ₅₀ ) in the volume particle size distribution of the above-mentioned pulverized material is, for example, 100 μm or more, or 200 μm or more, or 300 μm or more, or 400 μm or more, and also, for example, 900 μm or less, or 850 μm or less, or 800 μm or less. According to one embodiment, the cumulative 90% particle size (D ₉₀ ) in the volume particle size distribution of the pulverized material is, for example, 1000 μm or more, or 1100 μm or more, or 1200 μm or more, and also, for example, 5000 μm or less, or 4000 μm or less, or 3000 μm or less, or 2000 μm or less. Using a material containing pulverized dried solid culture with uniform particle size can further promote plant growth and suppress unevenness in the effect of promoting plant growth. Furthermore, when other microorganisms capable of solubilizing iron compounds and/or insoluble phosphate are used in combination as pulverized dried solid culture in addition to filamentous fungal endophytes, the particle size of the pulverized material of such other microorganisms can be within the same range as the particle size of the pulverized filamentous fungal endophytes.

Furthermore, the method for measuring the particle size of the pulverized material is not limited and can be measured by any method. Examples of such measurement methods include laser diffraction, scattering, imaging, light transmission centrifugal sedimentation, sedimentation, electrical resistance, specific surface area, and sieve passage methods. The equipment used to measure the particle size of the pulverized material is also not limited and can be any commercially available particle size analyzer. Examples of such analyzers include the LMS-2000e and LMS-3000 (Seishin Corporation). Since the pulverized material is prepared by drying the culture before pulverization, it is preferable to use a dry measurement method.

The materials of this embodiment may contain raw materials and filamentous fungal endophytes, as well as other components other than other microorganisms capable of solubilizing iron compounds and/or insoluble phosphates, which may be used optionally. Examples include nitrogen sources, potassium sources, and carbon sources. Examples of nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, urea, and Chilean nitrate. Examples of potassium sources include potassium chloride, potassium sulfate, potassium nitrate, and potassium silicate. Examples of carbon sources include glucose, sucrose, maltose, and lactose.

The material of this embodiment is used to promote plant growth or improve soil. For example, the material of this embodiment can be applied alone or mixed with other materials such as soil to the plant's rhizosphere or surrounding environment. This solubilizes insoluble or sparingly soluble iron compounds, and/or insoluble and/or citrate phosphate, in the material of this embodiment or the soil mixed with it, by filamentous fungal endophytes, increasing the amount of iron and/or water-soluble phosphate available to plants. Thus, the plant's utilization of iron and/or phosphate improves, promoting plant growth or improving soil. In this disclosure, "rhizosphere" refers to the soil space influenced by plant root secretions and soil organisms.

The plants to which the material of this embodiment can be applied are not limited. For example, it may be an annual or perennial plant, and may be angiosperms or gymnosperms. Applying the material of this embodiment to plants with relatively high iron or phosphorus requirements is preferable because the growth-promoting effect of the material of this embodiment is more pronounced. Examples of crop plants with relatively high phosphorus requirements include strawberries, cucumbers, tomatoes, cabbage, eggplants, lettuce, onions, peas, corn, Chinese cabbage, tobacco, shishito peppers, radishes, carrots, potatoes, broccoli, and flowering plants.

[Material manufacturing method]
One embodiment relates to a method for producing materials for promoting plant growth or improving soil. The above method includes mixing raw materials containing an insoluble or sparingly soluble iron compound and/or insoluble phosphate and/or citrate-soluble phosphate with filamentous fungal endophytes. In the production method of this embodiment, the details of the raw materials and filamentous fungal endophytes are as described above.

• In the manufacturing method of this embodiment, as described above, it is preferable to prepare a culture in which filamentous fungal endophytes have been cultured in advance and mix it with the raw materials. The culture conditions for filamentous fungal endophytes are not limited. For example, it may be a liquid culture or a solid culture.

In this disclosure, "liquid culture" refers to culturing a strain using a liquid medium. "Liquid culture" refers to the liquid culture obtained as a result of liquid culture. Subjecting filamentous fungal endophytes to liquid culture means inoculating the filamentous fungi into a liquid medium and culturing them in the liquid medium. The filamentous fungal endophytes may be in the form of inoculum carried in a medium. Agar is an example of such a medium. To avoid contamination by other bacteria, inoculation is preferably performed under sterile conditions.

Liquid culture media can be prepared, for example, by adding various additives to a liquid to create a composition suitable for culturing filamentous fungal endophytes. Examples of liquids include water. Additives to the liquid culture medium include, for example, sugars, minerals, nitrogen sources, vitamins, organic acids, inorganic acids, organic bases, and inorganic bases. Preferably, the liquid culture medium contains sugars and a nitrogen source. An example of a nitrogen source in the liquid culture medium is peptone.

The sugars used are not limited to those listed above, but examples include one or more selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol. While there is no upper limit to the amount of sugars, the total amount of sugars relative to the liquid medium can be, for example, typically 60 g/L or less, or 50 g/L or less, or 40 g/L or less, or 30 g/L or less. Too much sugar or other nutrients may result in excessively high mycelial concentration. Conversely, while there is no lower limit to the amount of sugars, the total amount of sugars relative to the liquid medium can be, for example, 0.5 g/L or more, or 1 g/L or more, or 5 g/L or more, or 10 g/L or more. Too little sugar or other nutrients may prevent sufficient mycelial growth. For example, in one embodiment, the sugar is glucose, added at an amount of approximately 20 g/L.

The minerals may be inorganic salts, such as alkali and alkaline earth metal salts, or other metal salts, although they are not limited to these. Examples of such inorganic salts include one or more salts selected from sulfates, phosphates, carbonates, chlorides, alkali metal oxides, molybdates, selenites, and halides. One or more types of salts may be used. There is no upper limit to the amount of salt, but the total amount of salts in the liquid medium, excluding the amount contained in the yeast extract and peptone, can be, for example, 10.0 g/L of medium or less, or 8.0 g/L of medium or less, or 5.0 g/L of medium or less, or 2.0 g/L of medium or less. If there are too many salts as nutrients, the mycelial concentration may become too high. On the other hand, while there is no lower limit to the amount of salts, the total amount of salts in the liquid medium, excluding the amount contained in the yeast extract, can be, for example, 0.01 g/L of medium or more, or 0.05 g/L of medium or more, or 0.1 g/L of medium or more, or 0.25 g/L of medium or more. If there are too few minerals such as salts, the mycelium may not grow sufficiently. For example, in one embodiment, magnesium sulfate is added at an amount of approximately 0.5 g/L of medium, and potassium dihydrogen phosphate is added at an amount of approximately 0.1 g/L of medium.

Examples of nitrogen sources include one or more nitrogen sources selected from yeast extract, protein hydrolysates, and proteins. An example of a protein hydrolysate is peptone. Adding yeast extract and/or peptone to the liquid culture medium adds nutrients including protein hydrolysates such as amino acids and peptides, proteins, and salts. While there are no restrictions on the amount of nitrogen source relative to the liquid culture medium, the upper limit of the nitrogen source can be set as the total amount of nitrogen sources such as yeast extract and peptone to, for example, 30.0 g/L or less, 20.0 g/L or less, 15.0 g/L or less, or 10.0 g/L or less. The lower limit of the nitrogen source can be set as the total amount of nitrogen sources such as yeast extract and peptone to, for example, 0.05 g/L or more, 0.1 g/L or more, 0.5 g/L or more, or 1.0 g/L or more. While there are no restrictions on the amount of yeast extract in the liquid medium, the upper limit of yeast extract can be, for example, 20.0 g/L or less, 15.0 g/L or less, 10.0 g/L or less, or 5.0 g/L or less. The lower limit of yeast extract can be, for example, 0.01 g/L or more, 0.05 g/L or more, 0.1 g/L or more, or 0.5 g/L or more. While there are no restrictions on the amount of peptone in the liquid medium, the upper limit of peptone can be, for example, 10.0 g/L or less, 8.0 g/L or less, 5.0 g/L or less, or 2.0 g/L or less. The lower limit of peptone can be, for example, 0.01 g/L or more, 0.05 g/L or more, 0.1 g/L or more, or 0.5 g/L or more. As described above, too much nutrient can lead to excessively high mycelial concentration. Conversely, too little nitrogen can prevent sufficient mycelial growth. For example, in one embodiment, approximately 2.0 g of yeast extract per liter of culture medium is added, along with approximately 1.0 g of peptone per liter of culture medium. Furthermore, the pH may be adjusted as needed by adding an appropriate acid or base.

Liquid culture can be carried out using appropriate culture methods. For example, it can be cultured using a container such as an Erlenmeyer flask and stirring with a stirring device such as a stirrer. Furthermore, it is preferable to adjust the culture conditions in liquid culture using a temperature control device or the like. For liquid culture, one or more devices selected from a vibrator, humidity measuring device, pH adjusting device, turbidity measuring device, light control device, specific gas concentration measuring device, and pressure measuring device may be used as needed. For the specific gas concentration measuring device, for example, it is sufficient if it can measure _O₂ and _CO₂ as specific gases. A silicone stopper may be optionally used in liquid culture from the viewpoint of preventing contamination. Depending on the circumstances, aeration stirring culture, shaking culture, or static culture may be performed in liquid culture. For example, in one embodiment, liquid culture medium and a stirrer are placed in an Erlenmeyer flask, the mouth of the flask is sealed with a silicone stopper, and stirring culture is performed while ensuring aeration.

While there are no restrictions on the duration of liquid culture, it can be considered complete when the fungal cells reach the desired concentration. Specifically, although there are no restrictions, the culture can be completed when the average number of colonies per liter of liquid culture reaches, for example, 1 × ^10³ cfu/L or more, or 1 × ^10⁴ cfu/L or more, or 1 × ^10⁵ cfu/L or more, or 1 × ^10⁶ cfu/L or more, or approximately 5 × ^10⁶ cfu/L. For example, the upper limit of the liquid culture period 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 liquid culture period can usually be 6 hours or more, or 12 hours or more, or 18 hours or more, or 1 day or more. If the liquid culture period is too long, the hyphal concentration may become too high. On the other hand, if the liquid culture period is too short, the hyphal growth may not be sufficient. For example, in one embodiment, if the liquid culture period is 2 to 4 days, for example 3 days, a liquid culture of a suitable concentration for subsequent solid culture can be obtained.

On the other hand, in this specification, "solid culture" refers to culturing a strain using a solid culture medium containing a solid culture medium and water. "Solid culture product" refers to the culture obtained as a result of solid culture, in which the strain has been cultured in a solid culture medium. Subjecting a filamentous fungal endophyte to solid culture means inoculating a liquid culture of the filamentous fungus into a solid culture medium and culturing it in the solid culture medium.

Solid culture media can be made from, for example, wheat bran, okara (soy pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, or starch. The water used for solid culture may contain various additives that contribute to the growth of the bacterial strain. Examples of additives include antibiotics that can inhibit the growth of bacteria other than filamentous endophytes.

Solid culture media can be prepared by adding water to a solid medium and mixing. While the water content of the solid culture medium is not limited, for example, the upper limit is usually 85% by mass or less, more preferably 80% by mass or less, even more preferably 75% by mass or less, and especially preferably 70% by mass or less. The lower limit of the water content is usually 30% by mass or more, more preferably 40% by mass or more, even more preferably 45% by mass or more, and especially preferably 50% by mass or more. If the water content of the solid culture medium is too high, it becomes excessively watery, preventing sufficient pores from forming in the medium, thus hindering growth. On the other hand, if the water content of the solid culture medium is too low, it cannot provide sufficient moisture for cultivation. For example, in one embodiment, if the water content of the solid culture medium is 50-70% by mass, for example 60% by mass, a solid culture with an appropriate mycelial concentration as an endophyte material can be obtained.

The inoculation of filamentous fungal endophytes into solid culture media is not restricted, but methods such as inoculation using a micropipette or other instrument, or direct inoculation after sterilization by decanting the container opening with a burner, can be employed. Regardless of the method used, inoculating the liquid culture into the solid culture medium allows the liquid culture to spread throughout the entire medium. This results in more uniform growth of the hyphae throughout the solid culture medium, leading to a more uniform culture and, consequently, a culture with more consistent particle size.

Solid-state culture can be carried out using appropriate culture methods. For example, solid-state culture can be performed by using a container such as a plastic bag and leaving the container undisturbed. It is also preferable to adjust the culture conditions for solid-state culture using a temperature control device, a humidity measuring device, etc. If necessary, one or more devices selected from a stirring device, a vibrating device, a pH adjuster, a turbidity measuring device, a light control device, a specific gas concentration measuring device, and a pressure measuring device may be used. For example, the specific gas concentration measuring device should be capable of measuring _O₂ and _CO₂ as specific gases. For solid-state culture, it is preferable to use disposable containers from the viewpoint of preventing contamination, and measures such as closing the opening of the bag may be taken as needed. Stirring culture, shaking culture, or static culture may be performed as appropriate. For example, in one embodiment, a disposable bag equipped with a ventilation filter is used, and static culture is performed with the opening of the bag closed.

The duration of solid culture is not limited, but it can be completed when the bacterial cells reach the desired concentration. Specifically, although not limited, the culture can be completed when the bacterial cells reach an average number of colonies per gram of solid culture, for example, 1 × ^10³ to 1 × ^10¹¹ cfu/g, or 5 × ^10³ to 5 × ^10¹⁰ cfu/g, or 1 × ^10⁴ to 1 × ^10¹⁰ cfu/g, or 5 × ^10⁴ to 5 × ^10⁹ cfu/g, or approximately 1 × ^10⁹ cfu/g. The upper limit of the solid culture period is preferably 20 days or less, more preferably 18 days or less, even more preferably 15 days or less, and especially preferably 12 days or less. The lower limit of the solid culture period is preferably 5 days or more, more preferably 6 days or more, even more preferably 7 days or more, and especially preferably 8 days or more. If the solid culture period is too long, the risk of contamination by unwanted bacteria may increase. Furthermore, if the solid culture period is too long, it may hinder the efficient production of endophyte materials. On the other hand, if the solid culture period is too short, the mycelium may not grow sufficiently. For example, in one embodiment, if solid culture is carried out for 8 to 12 days, for example, 10 days, a solid culture with an appropriate mycelial concentration for use as endophyte material can be obtained.

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

The humidity during cultivation is not particularly limited in either liquid or solid culture, and can be any humidity as long as it does not hinder the growth of filamentous fungal endophytes. Specifically, the upper limit of the humidity during cultivation can be, for example, 100% RH or less, 95% RH or less, or 90% RH or less. On the other hand, the lower limit of the humidity during cultivation can be, for example, 55% RH or more, 60% RH or more, or 65% RH or more. In one embodiment, cultivation can be carried out at a humidity of 60-80% RH, for example, about 70% RH.

Furthermore, from the viewpoint of preventing contamination, liquid culture media, solid culture media, and/or solid culture media are preferably sterilized by any known means, such as filtration sterilization, autoclave sterilization, boiling sterilization, radiation sterilization, sodium hypochlorite treatment, or ozone treatment. Each operation, such as inoculation, is preferably performed under a sterile atmosphere. For example, in the case of solid culture media, an appropriate amount of water can be added to the solid media before sterilization.

- Post-treatment: If filamentous fungal endophytes are cultured in solid form, the solid culture may be dried. Drying can be done using general-purpose equipment such as an air conditioner or dehumidifier. For example, it can be done by leaving it in an environment of 25°C and 50% RH for 3 days.

Alternatively, the solid culture of filamentous fungal endophytes may be dried and then pulverized to obtain a pulverized product. The dried solid culture can be pulverized using a general-purpose pulverizer such as a food processor, coffee mill, or pepper mill. In one embodiment, it is preferable that the pulverization of the dried solid culture of filamentous fungal endophytes is adjusted so that the cumulative 10% particle size (D ₁₀ ), the cumulative 50% particle size (D ₅₀ ), and/or the cumulative 90% particle size (D ₉₀ ) in the volume particle size distribution of the pulverized product are within the aforementioned ranges.

- Mixing In the manufacturing method of this embodiment, the mixing ratio of the raw materials and the culture of filamentous fungal endophytes is not limited. According to one embodiment, the ratio of the amount of filamentous fungal endophyte culture to the total amount of raw materials and filamentous fungal endophyte culture can be, for example, 10% by mass or more, 20% by mass or more, 30% by mass or more, or 40% by mass or more, or 95% by mass or less, 90% by mass or less, 80% by mass or less, or 70% by mass or less.

In the manufacturing method of this embodiment, the mixing conditions and method of the raw materials and filamentous fungal endophytes are not limited as long as the environment does not kill the filamentous fungal endophytes. However, it is preferable that the environment does not hinder the growth of the filamentous fungal endophytes, and more preferably that the environment is suitable for culturing the filamentous fungal endophytes. Specifically, the following applies:

In the manufacturing method of this embodiment, the upper limit of the temperature during mixing of the raw materials and the filamentous fungal endophyte culture can be, for example, 40°C or lower, 35°C or lower, 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, 10°C or higher, 15°C or higher, or 20°C or higher. In one embodiment, mixing can be carried out at 20-25°C, for example, 25°C ± 1°C.

In the manufacturing method of this embodiment, the upper limit of humidity during mixing of the raw materials and the filamentous fungal endophyte culture can be, for example, 100% RH or less, 95% RH or less, or 90% RH or less. On the other hand, the lower limit of humidity during mixing can be, for example, 55% RH or more, 60% RH or more, or 65% RH or more. In one embodiment, mixing can be carried out at a humidity of 60-80% RH, for example, about 70% RH.

[Methods for promoting plant growth or improving soil]
One embodiment relates to a method for promoting plant growth. The method of this embodiment involves contacting iron compounds and/or insoluble phosphate and/or citrate phosphate in the plant's rhizosphere or surrounding environment with filamentous fungal endophytes and/or materials containing them. Specifically, the iron compounds and/or insoluble phosphate and/or citrate phosphate in the plant's rhizosphere or surrounding environment can be solubilized by applying a culture of filamentous fungal endophytes to soil or materials present in the plant's rhizosphere or surrounding environment. Details of the filamentous fungal endophytes used in the method of this embodiment and the plants to which it is applied are as described above.

One embodiment relates to a method for improving soil. The method of this embodiment involves contacting soil containing iron compounds and/or insoluble phosphate and/or citrate-soluble phosphate with filamentous fungal endophytes and/or materials containing them. This solubilizes the iron compounds and/or insoluble phosphate and/or citrate-soluble phosphate in the soil, improving the soil to a state suitable for plant growth. Details of the filamentous fungal endophytes used in the method of this embodiment and the target soil are as described above.

The present invention will be described in further detail below with reference to examples. However, these examples are merely illustrative examples for explanatory purposes, and the present invention is not limited in any sense to these examples.

The experimental equipment and reagents used in each of the experiments described below are as follows.

Furthermore, the bacterial strains used in the following experiments are as follows:
Fusarium solani: 15-B strain (NITE P-03991)
Lecanicillium sp.: 100-1 plant (NITE P-03984)

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

<Example 1>
(1) Prepare a DYGS medium consisting of the following composition and autoclave it at 121°C for 20 minutes.
(2) Three pieces of inoculum agar, each approximately 5 mm square and filled with mycelium of F. solani, were added to DYGS medium.
(3) The culture was kept at 25°C in the dark for 4 days while being stirred with a stirrer.
(4) The culture medium from (3) above was collected and filtered through a φ0.22 μm syringe filter to obtain the filtrate.
(5) A 50 × Salkowski's reagent was prepared with the following composition.
(6) The above 50x Salkowski's reagent was diluted 25 times with pure water to prepare 2x Salkowski's reagent.
(7) Add 2 ml of 2 × Salkowski's reagent to 2 ml of the filtrate of the culture medium from (4) above, cover with aluminum foil to protect from light, and let stand at room temperature for 30 minutes.
(8) The absorbance at a wavelength of 526 nm was measured using a spectrophotometer for the mixture after carrying out the above (7). A reference solution was prepared by adding 2 ml of 2 × Salkowski's reagent to 2 mL of pure water and letting it stand at room temperature for 30 minutes. A standard solution was also prepared by adding 2 ml of 2 × Salkowski's reagent to 2 mL of 100 μg/ml IAA and letting it stand at room temperature for 30 minutes. A calibration curve was created using the reference solution and the standard solution.

<Example 2>
The experiment was carried out in the same manner as in Example 1, except that three pieces of Lecanicillium sp. starter culture agar were added instead of F. solani.

<Comparative Example 1>
The experiment was conducted in the same manner as in Example 1, except that starter culture agar was not added.

[Results of Experiment 1]
Based on the results of the above experiment, the amount of indole-3-acetic acid (IAA) produced after culturing was as follows.

The IAA levels in Examples 1 and 2 were higher than those in Comparative Example 1, suggesting that F. solani and Lecanicillium sp. are IAA-producing strains. The IAA production levels of the strains were 23 μg/ml and 15 μg/ml, respectively, which are higher than those of the Bacillus endophytes isolated in Non-Patent Document 2.

[Experiment 2] Solubilization of insoluble phosphoric acid

<Example 3>
(1) An NBRIP medium was prepared with the following composition.
(2) As a source of phosphate, 0.1 g each of one of the following was added to three polypropylene (PP) bottles: tricalcium phosphate (TCP), aluminum phosphate (AP), dicalcium phosphate (DCP), phosphate rock powder (from Morocco), and calcined sewage sludge ash.
(3) NBRIP medium was dispensed into PP bottles in 20 ml portions.
(4) The PP bottles from (3) were autoclaved at 121°C for 20 minutes.
(5) After cooling sufficiently, one piece of approximately 5 mm square seed agar filled with F. solani mycelium was added to each of three PP bottles for each type of phosphate source.
The PP bottles from (6) and (5) were cultured at 25°C with shaking at 100 rpm.
(7) The TCP, AP, and DCP samples were stopped shaking on the fourth day, and the phosphate rock and sewage sludge calcined ash samples were stopped shaking on the fifth day. The culture solutions were then filtered and collected.
(8) 7.6 ml of pure water and 400 μL of culture medium were added to a test tube. 100 μg/ml _{KH₂₂PO₄} was _used as the standard solution, and pure water was used as the blank.
(9) Add 1 ml/sample of Truog chromogenic reagent A and 0.085 g/sample of Truog chromogenic reagent B, and dissolve completely to prepare the chromogenic reagent.
(10) Add 1 ml of the colorimetric reagent to the test tube, mix by tapping, and then let stand for 10 minutes.
(11) The amount of phosphorus was measured using a soil analyzer.

<Comparative Example 2>
The experiment was conducted in the same manner as in Example 3, except that F. solani starter culture agar was not added.

<Example 4>
The experiment was conducted in the same manner as in Example 3, except that Lecanicillium sp. was added instead of F. solani bacterial culture agar.

<Comparative Example 3>
The experiment was conducted in the same manner as in Example 3, except that F. solani starter culture agar was not added, and was carried out simultaneously with Example 4.

[Results of Experiment 2]
Based on the results of the above experiment, the amount of water-soluble phosphate solubilized after culturing was as follows.

Examples 3 and 4 contained more water-soluble phosphate than Comparative Examples 2 and 3, respectively, indicating that the insoluble phosphate source in the culture medium was solubilized by the bacterial strain. Furthermore, these values were higher than those reported in Non-Patent Document 3.

[Experiment 3] Production of siderophores

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

・Solution II
(i) Add 10 μL of HCl (12 mol/L) to 110 μL of pure water to make a 1 mol/L HCl solution. (ii) Add 100 μL of 1 mol/L HCl to 9.9 ml of pure water to make a 10 mM HCl solution.
(iii) 0.003 g of _FeCl3 · _6H2O was added to 10 mM HCl.

・Solution III

(2) Add 9 ml of Solution II to 50 ml of Solution I and mix. Then, add another 40 ml of Solution III. The mixture was then autoclaved at 121°C for 20 minutes. After sterilization, the mixture was transferred to a PP bottle in a sterile environment.

(3) The following reagents were prepared.
・Minimal Media 9 (MM9) Salt Solution
It was prepared by slowly adding NaOH to 90 ml of pure water.
After complete dissolution, it was autoclaved at 121°C for 20 minutes.
After complete dissolution, the solution was filtered through a φ0.22 μm syringe filter and sterilized.

(4) 20 ml of MM9 Salt Solution was diluted with 150 ml of pure water, and NaOH solution was added to adjust the pH to 6.0.
(5) PIPES 6.44 g was gradually added to the solution obtained in (4) and dissolved, and three solutions with pH values of 6.0, 6.3, and 6.55 were prepared using NaOH solution.
(6) Add 3 g of bacterial agar to the solution obtained in (5) and autoclave sterilize at 121°C for 20 minutes.
(7) To the solution obtained in (6), 6 ml of casamino acid solution and 2 ml of 20% glucose solution were added.
(8) 20 ml of Blue Dye Solution was slowly added to the solution obtained in (7), letting it run down the side of the container.
(9) The solutions obtained in (8) were shaken gently to avoid creating bubbles, and then dispensed into 10 petri dishes to form CAS Agar medium.
(10) A seed agar of about 5 mm square filled with mycelium of F. solani was placed on CAS Agar medium and left to stand at 25°C and 70% RH for 5 days.

<Example 6>
The experiment was conducted in the same manner as in Example 5, except that a roughly 5 mm square seed agar filled with the mycelium of Lecanicillium sp. was placed on top instead of F. solani.

[Experiment 3-2] Quantitative analysis of siderophore production <Example 7>
(1) A liquid culture medium having the following composition was prepared and autoclaved at 121°C for 20 minutes.
(2) Three pieces of inoculum agar, approximately 5 mm square and filled with F. solani mycelium, were added to the liquid culture medium obtained in (1).
(3) The liquid culture medium from (2) was stirred and cultured with a stirrer at 25°C and 700 rpm for 3 days.
(4) The liquid culture medium prepared in (3) was collected and filtered using a φ0.22 μm syringe filter.
(5) 2 ml of Blue Dye Solution was diluted fivefold by adding 8 ml of pure water.
(6) Two ml of the filtrate from (4) and two ml of a five-fold diluted Blue Dye Solution were mixed in a test tube.
(7) 10 μL of 0.2 M 5-sulfosalicylic acid was added to the solution of (6) and allowed to stand at room temperature for 30 minutes.
(8) The absorbance of the solutions from (7) at a wavelength of 626 nm was measured using a spectrophotometer.
(9) The amount of siderophore produced was calculated using the following formula.
% siderophore units = (Ar - As) / Ar x 100
Ar = Absorbance of culture medium; As = Absorbance of sample

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

<Comparative Example 4>
The experiment was conducted in the same manner as in Example 7, except that F. solani starter culture agar was not added.

[Results of Experiment 3]

CAS agar assay revealed that in Examples 5 and 6, red halos formed around the bacterial strains, suggesting that F. solani and Lecanicillium sp. produce siderophores (Figure 1).

Therefore, the amount of siderophore production was quantitatively analyzed using CAS liquid assay. F. solani and Lecanicillium sp. allowed sufficient mycelial growth in liquid medium on the third day of culture. In Example 7 and Example 8, the amount of siderophore production did not differ significantly, and 62.78% and 63.36% of iron were chelated, respectively (Table 14). This value is higher than the value in Non-Patent Literature 1, a prior study, suggesting that more iron can be chelated.

[Experiment 4] Heavy Metal Adsorption (1)

<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 F. solani mycelium powder 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 using a syringe filter with a diameter of φ0.22 μm.
(5) The concentration of cadmium (Cd) in the solution obtained in (4) was measured by ICP.

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

<Comparative Example 5>
The experiment was conducted in the same manner as in Example 9, except that mycelial powder was not added.

[Results of Experiment 4]
The amount of cadmium in Examples 9 and 10 was less than in Comparative Example 5, suggesting that cadmium was adsorbed by the mycelium (Table 15). Furthermore, Lecanicillium sp. showed higher adsorption capacity than F. solani. This value is comparable to that of Non-Patent Document 4, a prior study, suggesting that it can adsorb a similar amount of cadmium.

[Experiment 5] Heavy Metal Adsorption (2)

<Example 11>
(1) 30 ml of 10 mg/L lead nitrate aqueous solution was added to the tube.
(2) 10 mg of F. solani mycelium powder 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 using a syringe filter with a diameter of φ0.22 μm.
(5) The concentration of lead (Pb) in the solution obtained in (4) was measured by ICP.

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

<Comparative Example 6>
The experiment was conducted in the same manner as in Example 11, except that mycelial powder was not added.

[Results of Experiment 5]
The lower lead content in Examples 11 and 12 compared to Comparative Example 6 suggests that lead was adsorbed by the mycelium (Table 15). Furthermore, F. solani showed higher adsorption capacity than Lecanicillium sp.

[Experiment 6] Method of preparing materials and measurement of colony count

<Example 13>
(1) 2.85 g of glucose peptone medium was mixed with 100 ml of pure water.
(2) The solution from (1) was sterilized in an autoclave at 121°C for 20 minutes.
(3) Three pieces of inoculum agar, approximately 5 mm square and filled with F. solani mycelium, were added to the solution from (2), and the mixture was cultured at 25°C for 3 days with stirring using a 700 rpm stirrer.
(4) Add 500 ml of purified water to 500 g of wheat bran and mix well, then transfer to a mushroom substrate bag with a filter.
(5) and (4) were 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 sterile mushroom bed bag in (5) and sealed.
(7) The samples were left standing at 25°C and 70% RH for 5 days.
(8) Remove the mushroom substrate from the bag, crush it, and dry it at 25°C for 3 days.
(9) The dried material was crushed in a food processor and sieved through a φ2 mm sieve. This was designated as "Fusarium material".
(10) 1 g of Fusarium material was suspended in 10 ml of pure water to make a 10-fold dilution.
(11) 1 ml was collected from the 10-fold diluted solution obtained in (10) and 9 ml of pure water was added to make a ^102- fold diluted solution. The same procedure was then used to prepare dilutions up to ^105- fold.
(12) 15.75 g of DRBC medium was mixed with 500 ml of pure water and sterilized in an autoclave at 121°C for 20 minutes.
(13) Pour the mixture into a plastic petri dish and let it stand to solidify.
(14) One ml of the ^10³ to ^10⁵- fold diluted solution prepared in (11) was spread onto DRBC solid medium.
(15) The sample was left standing at 25°C and 70% RH for two days.
(16) The number of colonies formed was measured.

<Example 14>
The experiment was conducted in the same manner as in Example 13, except that a starter culture agar, approximately 5 mm square and filled with the mycelium of Lecanicillium sp., was added instead of F. solani, and the mixture was left to stand for 3 days in (15), and the completed material was designated as "Lecanicillium material".

[Results of Experiment 6]
From Examples 13 and 14, the number of colonies contained in 1 g of material was approximately 10 to ¹⁷ x 10⁸ and 2 to 5 x ^10⁶ , respectively (Table 17).

[Experiment 7] Experiment on the growth of lettuce

<Example 15>
(1) A growing medium was prepared by mixing nutrient-free growing soil and vermiculite in a volume ratio of 1:1.
(2) To 1000 ml of the cultivation soil from (1), add 270 mg of ammonium sulfate as a nitrogen source, 450 mg of superphosphate as a phosphorus source, 270 mg of potassium as a potassium source, and 2 g of Fusarium material prepared in <Example 13>, and mix well.
(3) Distribute 100 ml of the cultivation soil prepared in (2) into 9 cm diameter cultivation pots and sow lettuce (Okayama lettuce).
(4) The plants were grown in pots for 60 days after sowing.

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

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

[Results of Experiment 7]
In Example 15, the harvested weight of the edible portion increased by approximately 1.21% compared to Comparative Example 7. Furthermore, the variation in growth was suppressed, and the standard deviation was reduced. In Example 16, the harvested weight of the edible portion increased by 64.46% compared to Comparative Example 7.

[summary]
This invention proposes the use of filamentous fungal endophytes to promote plant growth, contributing to the healthy growth of crops and increased yields. While similar prior research can be found in Patent Document 1, this invention provides a more detailed analysis of the mechanism of action of fungi in promoting plant growth, and is expected to offer a wider range of functionalities.

This invention has broad applicability to fields such as agriculture and horticulture, and its utility is extremely high. For example, it enables plant cultivation even in soils where iron supply is difficult, such as alkaline soils, expanding the area of arable land and potentially solving future food and resource problems.

NITE P-03991
NITE P-03984

Claims (16)

A material containing iron compounds and filamentous fungal endophytes,
The aforementioned filamentous fungal endophytes include those of the genus Lecanicillium sp. The material according to claim 1, wherein the iron compound is a trivalent iron compound. The material according to claim 1, wherein the content of the iron compound is 10 to 50,000 ppm of the material. The material according to claim 1, wherein the filamentous fungal endophyte produces siderophores. The material according to claim 1, wherein the filamentous fungal endophyte produces indole-3-acetic acid. Furthermore, the material according to claim 1, comprising raw materials containing insoluble phosphoric acid and/or citrate-soluble phosphoric acid. The material according to claim 6, wherein the raw material is a calcined product of sludge. The material according to claim 1, wherein the filamentous fungal endophytes are pulverized from a dried 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. A method for promoting plant growth,
This involves contacting filamentous fungal endophytes with iron compounds in the plant's rhizosphere or surrounding environment.
A method wherein the filamentous fungal endophyte contains the genus Lecanicillium sp. The method according to claim 9, comprising contacting the filamentous fungal endophyte with insoluble phosphate and/or citrate-soluble phosphate in the rhizosphere of the plant or its surrounding environment. The method according to claim 9, comprising contacting the filamentous fungal endophytes with a calcined product of sludge from the rhizosphere of the plant or its surrounding environment. The method according to claim 9, comprising contacting the filamentous fungal endophyte with heavy metals in the rhizosphere or surrounding environment of the plant. A method for producing crops obtained from plants,
This includes contacting filamentous fungal endophytes with iron compounds in the rhizosphere or surrounding environment of a plant, growing the plant, and harvesting the crop obtained from the plant.
A method wherein the filamentous fungal endophyte contains the genus Lecanicillium sp. 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. The method according to claim 13, comprising contacting the filamentous fungal endophytes with a calcined product of sludge from the rhizosphere or surrounding environment of the plant. 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.