WO2024070193A1

WO2024070193A1 - Endophyte material, and method for cultivating endophyte

Info

Publication number: WO2024070193A1
Application number: PCT/JP2023/028021
Authority: WO
Inventors: 良恵井上; 創太井上
Original assignee: セトラスホールディングス株式会社
Priority date: 2022-09-30
Filing date: 2023-07-31
Publication date: 2024-04-04

Abstract

The present invention provides a material that can suppress the occurrence of unevenness in a growth-promoting effect of plants, and that includes a culture medium of xsd08001 strain of the Cephaliophora sp. genus, with a cumulative 50% particle size (D₅₀) in the volume particle size distribution of 100-900 μm and a cumulative 90% particle size (D₉₀) of 1,000-5,000 μm.

Description

Endophyte materials and endophyte cultivation method

The present invention relates to an endophyte material and a method for producing the same, as well as a method for culturing an endophyte, more specifically, to a material containing a culture of Cephaliophora sp. xsd08001 strain, a method for producing the same, and a method for culturing the strain, which includes subjecting the strain to liquid culture.

Endophytes, also known as endophytic fungi, are microorganisms that live and symbiotically within the body of a plant host, thereby providing the host with benefits such as promoting growth and increasing stress resistance. One such endophyte that has been reported as a useful microorganism is the Cephaliophora sp. strain xsd08001 (hereinafter sometimes referred to as xsd08001 strain) (Patent Document 1).

A culture of the xsd08001 strain is mixed into soil and used to increase the content of highly functional components in vegetables and other plants (Patent Document 2). However, it was not known what characteristics of endophyte materials affect the quality of plants.

JP 2018-174708 A Patent No. 6898637 Japanese Patent Application Laid-Open No. 62-171618 Japanese Patent Application Laid-Open No. 62-269624

The object of the present invention is to provide a material that can suppress unevenness in the effect of promoting plant growth. Another object of the present invention is to provide a novel culture method that can culture the xsd08001 strain more efficiently.

As a result of intensive research, the inventors have succeeded in making the grain size of the culture of the strain uniform by using a specific culture method. They have also discovered that the culture period can be shortened by combining culture in a liquid medium with culture in a conventional solid medium alone, and have completed the following invention.
[1] A material containing a culture of Cephaliophora sp. xsd08001 strain. The material has a cumulative 50% particle size (D ₅₀ ) in a volumetric particle size distribution of 100 to 900 μm. The material has a cumulative 90% particle size (D ₉₀ ) of 1000 to 5000 μm.
[2] The average number of colonies per 1 g of the dried solid culture material in the material described in [1] is 1×10 ⁵ to 1×10 ⁹ cfu (Colony Forming Unit)/g.
[3] The material according to [1] or [2] is intended for promoting plant growth.
[4] A method for producing a material containing a dried solid culture of Cephaliophora sp. xsd08001 strain, the method comprising a culturing step of obtaining the dried solid culture of the strain, and an adjusting step of adjusting the cumulative 50% particle size (D ₅₀ ) and cumulative 90% particle size (D ₉₀ ) in the volumetric particle size distribution of the dried solid culture to 100 to 900 μm and 1000 to 5000 μm, respectively.
[5] In the production method according to [4], the culturing step includes a liquid culturing step of subjecting the strain to liquid culture. The culturing step includes a solid culturing step. In the solid culturing step, the strain cultured in the liquid culturing step is cultured in solid culture. The culturing step includes a drying step of drying the solid culture obtained in the solid culturing step to produce the dried solid culture. The preparation step includes a grinding step of grinding the dried solid culture dried in the drying step.
[6] The solid culture step is carried out using a solid culture medium containing a solid medium and water in the production method described in [5].
[7] In the production method according to [6], the solid culture medium has a water content of 50 to 70%.
[8] In the production method according to [6] or [7], the solid culture medium contains bran.
[9] A method for culturing a Cephaliophora sp. xsd08001 strain. The method comprises subjecting the Cephaliophora sp. xsd08001 strain to liquid culture to obtain a liquid culture. The method comprises subjecting the obtained liquid culture to solid culture to obtain a solid culture.
[10] The solid culture is carried out using a medium for solid culture in the culture method according to [9]. The medium for solid culture contains a solid medium and water.
[11] In the culture method according to [9] or [10], the solid culture medium has a water content of 50 to 70%.
[12] In the culture method according to any one of [9] to [11], the solid medium contains wheat bran.
[13] The liquid culture is carried out for 2 to 4 days in the culture method according to any one of [9] to [12].
[14] The solid culture is carried out for 7 to 12 days in the culture method according to any one of [9] to [13].
[15] At least one of the liquid culture and the solid culture is carried out at a room humidity of 60 to 90% RH in the culture method according to any one of [9] to [14].
[16] A culture obtained by the culture method according to any one of [9] to [15].

By using the material of the present invention, it is possible to prevent unevenness in the effect of promoting plant growth using the material. In addition, by using the cultivation method of the present invention, the xsd08001 strain can be cultivated more efficiently.

Fig. 1 shows lettuce on the 25th day after sowing in Experiment 2 of Example Group I. In Fig. 1, "a" shows the state of lettuce in Example 1. In Fig. 1, "b" shows the state of lettuce in Comparative Example 1. In Fig. 1, "c" shows the state of lettuce in Comparative Example 2. Figure 2 is an electrophoretic photograph showing the PCR detection results of the ITS region of Cephaliophora sp. in Experiment 3 of Example Group I. In Figure 2, lane M shows TAKARA 100 bp DNA Ladder Marker (for the base length of each DNA band, see the manufacturer's website below: https://catalog.takara-bio.co.jp/product/basic_info.php?unitid=U100006433). In Figure 2, lane 1 shows Comparative Example 3-2. In Figure 2, lane 2 shows Example 3-2. Fig. 3 shows the solid culture medium of the Example and Comparative Example of Example Group II on the 10th day after the start of solid culture. In Fig. 3, a shows the side of the solid culture medium of the Comparative Example. In Fig. 3, b shows the side of the solid culture medium of the Example. In Fig. 3, c shows the back of the solid culture medium of the Comparative Example. In Fig. 3, d shows the back of the solid culture medium of the Example. Fig. 4 shows the solid medium of Comparative Example of Example Group II on the 25th day from the start of solid culture. In Fig. 4, a, c, d, and e show the side of the solid medium of Comparative Example. In Fig. 4, b shows the back of the solid medium of Comparative Example. Fig. 5 shows the state of mycelium elongation of xsd08001 strain in a solid culture medium having a moisture content of 50 to 70% by mass in Reference Experiment 1 of Example Group II. In Fig. 5, the moisture content of 50% by mass corresponds to Reference Example 1. In Fig. 4, the moisture content of 60% by mass corresponds to Reference Example 2. In Fig. 5, the moisture content of 70% by mass corresponds to Reference Example 3. In Fig. 5, a shows the state of the front surface of the petri dish. In Fig. 5, b shows the state of the back surface of the petri dish. Fig. 6 shows the xsd08001 strain in a solid culture medium with a water content of 80% by mass in Reference Experiment 1 of Example Group II. Fig. 6 corresponds to Comparative Reference Example 3 with a water content of 80% by mass. Fig. 6 shows the appearance of the surface of the petri dish. Fig. 7 shows the xsd08001 strain in solid culture media with a moisture content of 50% by mass and 60% by mass at an environmental humidity of 70% RH in Reference Experiment 2 of Example Group II. In Fig. 7, a, b, and c correspond to Reference Example 4 with a moisture content of 50% by mass. In Fig. 7, d, e, and f correspond to Reference Example 5 with a moisture content of 60% by mass. All Fig. 7 shows the surface appearance of the petri dish.

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 present invention.

I. Endophyte Materials
One embodiment relates to an endophyte material. Adjusting the particle size of a microbial material is important for improving ease of handling, stabilizing effects, promoting plant growth, and the like. However, since filamentous fungi grow by extending strong hyphae, there is a large variation in the density of the hyphae in the medium, making it difficult to adjust the particle size of the culture. Despite this technical common knowledge, the present inventors have discovered that by using the xsd08001 strain, the hyphae can be uniformly extended and a culture with an adjusted particle size can be obtained.

Furthermore, as described in Patent Document 1, since the xsd08001 strain was isolated from soil, it is believed that an environment similar to that of soil is suitable, and thus the xsd08001 strain is usually cultivated by solid culture. In addition, liquid culture of filamentous fungi is difficult because the mycelium adheres and aggregates to form mycelium clumps (Non-Patent Document 1). Despite these circumstances, the inventors were surprised to succeed in liquid culture of the xsd08001 strain. Although a culture with adjusted particle size can be obtained even in the case of solid culture, when liquid culture becomes possible, a culture with an even more adjusted particle size can be obtained. This is thought to be because inoculating the liquid culture allows the seed fungus to spread throughout the entire medium, enabling the mycelium to spread evenly in the medium, resulting in a uniform concentration of fungal cells per unit mass.

[Materials]
Thus, the present embodiment provides a culture of the xsd08001 strain having a cumulative 50% particle size (D ₅₀ ) of 100 to 900 μm and a cumulative 90% particle size (D ₉₀ ) of 1000 to 5000 μm in volumetric particle size distribution, and a material containing the same.

[KK]
The Cephaliophora sp. xsd08001 strain used in this embodiment has been deposited at the National Institute of Technology and Evaluation (NITE), an independent administrative institution, and is available under the accession number NITE P-02438.

[Grain size]
The particle size of the culture of the strain used in this embodiment is not limited, but is as follows. The cumulative 10% particle size (D10) in the volumetric particle size distribution of the culture of the strain used in this embodiment is, for example, 50 to 400 μm, among which 100 to 350 μm, more preferably 150 to 350 μm, and even more preferably 200 to 300 μm. The cumulative 50% particle size (D ₅₀ ) of the culture of the strain used in this embodiment is, for example, 100 to 1000 μm, among which 200 to 900 μm, more preferably 300 to 850 μm, and even more preferably 400 to 800 μm. The cumulative 90% particle size (D ₉₀ ) of the culture of the strain used in this embodiment is, for example, 1000 to 5000 μm, among which 1100 to 4000 μm, more preferably 1200 to 3000 μm, and even more preferably 1200 to 2000 μm. For example, in one embodiment, the cumulative 50% particle size (D ₅₀ ) is 200-800 μm, and the cumulative 90% particle size (D ₉₀ ) is 1000-3000 μm. The particle size can be measured by any method, such as a laser diffraction method, a scattering method, an imaging method, a light transmission centrifugal sedimentation method, a sedimentation method, an electrical resistance method, a specific surface area method, or a sieve passing method. The particle size can be measured using an appropriate particle size measuring instrument, for example, a commercially available instrument such as LMS-2000e or LMS-3000 (Seishin Enterprise Co., Ltd.). Since the culture is dried and then pulverized, it is preferable to use a dry measurement method for the particle size.

In one embodiment, the culture of the xsd08001 strain is obtained by subjecting the xsd08001 strain to solid culture. In another embodiment, the culture of the xsd08001 strain is obtained by subjecting the xsd08001 strain to liquid culture and then to solid culture. In these embodiments, the particles of the culture of the xsd08001 strain may be obtained by a process including drying the solid culture obtained by solid culture and grinding the dried solid culture.

[Liquid culture]
In this specification, "liquid culture" refers to culturing a strain using a liquid medium. The process of performing such liquid culture may be referred to as a "liquid culture process." Subjecting the xsd08001 strain to liquid culture refers to inoculating the strain into a liquid medium and culturing it in the liquid medium. The strain may be in the form of a seed culture medium carried by a medium. An example of the medium is agar. In order to avoid contamination by various bacteria, inoculation is preferably performed under sterile conditions.

In this specification, the term "liquid culture" refers to a liquid culture obtained as a result of liquid culture. A liquid medium can be prepared, for example, by adding various additives to the liquid to adjust the composition to be suitable for culture. An example of the liquid is water. Examples of additives added to the liquid medium include sugars, minerals, nitrogen sources, vitamins, organic acids, inorganic acids, organic bases, and inorganic bases. Preferably, the liquid medium contains sugars and a nitrogen source. An example of the nitrogen source contained in the liquid medium is peptone.

The sugars are not limited, but may be one or more selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol. The upper limit of the sugars is not limited, but the total amount of sugars in the liquid medium is usually 60 g/L or less, more preferably 50 g/L or less, even more preferably 40 g/L or less, and especially preferably 30 g/L or less. This is because, as described in Patent Document 3, if the liquid medium contains too many nutrients such as sugars, the mycelium concentration becomes too high. On the other hand, the lower limit of the sugars is not limited, but the total amount of sugars in the liquid medium is usually 0.5 g/L or more, more preferably 1 g/L or more, even more preferably 5 g/L or more, and especially preferably 10 g/L or more. This is because if the nutrients such as sugars are too few, the mycelium does not grow sufficiently. For example, in one embodiment, the sugar is glucose, and is added in an amount of 20 g/L.

The minerals may be inorganic salts, such as, but not limited to, alkali and alkaline earth metal salts, and salts of other metals. 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 salts may be used. The upper limit of the salts is not limited, but the total amount of salts in the liquid medium, excluding the amount contained in yeast extract and peptone, is usually 10.0 g/L or less, preferably 8.0 g/L or less, more preferably 5.0 g/L or less, and particularly preferably 2.0 g/L or less. This is because, as described above, if the liquid medium contains too many salts as nutrients, the mycelium concentration will become too high. On the other hand, the lower limit of the salts is not limited, but the total amount of salts in the liquid medium, excluding the amount contained in the yeast extract, is usually 0.01 g/L or more, preferably 0.05 g/L or more, more preferably 0.1 g/L or more, and particularly preferably 0.25 g/L or more. This is because if the liquid medium contains too few minerals such as salts, the mycelium will not grow sufficiently. For example, in one embodiment, magnesium sulfate is added as salts in an amount of 0.5 g/L of medium, and potassium dihydrogen phosphate is added in an amount of 0.1 g/L of medium.

The nitrogen source may be 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 a liquid medium can add nutrients including protein hydrolysates such as amino acids and peptides, proteins, and salts. The amount of nitrogen source relative to the liquid medium is not limited, but for example, the upper limit of the nitrogen source, as the total amount of nitrogen sources such as yeast extract and peptone, is usually 30.0 g/L or less, particularly 20.0 g/L or less, even 15.0 g/L or less, and particularly 10.0 g/L or less. The lower limit of the nitrogen source, as the total amount of nitrogen sources such as yeast extract and peptone, is usually 0.05 g/L or more, particularly 0.1 g/L or more, even 0.5 g/L or more, and particularly 1.0 g/L or more. For example, the amount of yeast extract relative to the liquid medium is not limited, but the upper limit of yeast extract is usually 20.0 g/L or less, particularly 15.0 g/L or less, further 10.0 g/L or less, and particularly 5.0 g/L or less. The lower limit of yeast extract is usually 0.01 g/L or more, particularly 0.05 g/L or more, further 0.1 g/L or more, and particularly 0.5 g/L or more. The amount of peptone relative to the liquid medium is not limited, but the upper limit of peptone is usually 10.0 g/L or less, particularly 8.0 g/L or less, further 5.0 g/L or less, and particularly 2.0 g/L or less. The lower limit of peptone is usually 0.01 g/L or more, particularly 0.05 g/L or more, further 0.1 g/L or more, and particularly 0.5 g/L or more. As above, if there are too many nutrients, the mycelium concentration will be too high, while if there is too little nitrogen source, the mycelium will not grow sufficiently. For example, in one embodiment, yeast extract is added in an amount of 2.0 g/L of medium, and peptone is added in an amount of 1.0 g/L of medium. Furthermore, if necessary, the pH may be adjusted appropriately by adding an appropriate acid or base.

The liquid culture can be carried out by a suitable culture means. For example, a container such as an Erlenmeyer flask can be used, and the culture can be carried out while stirring with a stirring device such as a stirrer. In addition, it is preferable that the culture conditions of the liquid culture are adjusted by a temperature control device or the like. For the liquid culture, one or more devices selected from a vibration device, a humidity measuring 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 as necessary. For example, the specific gas concentration measuring device may be capable of measuring O ₂ and CO ₂ as specific gases. For the liquid culture, a silicon plug may be used optionally from the viewpoint of preventing contamination. For the liquid culture, aeration stirring culture, shaking culture, stationary culture, or the like may be appropriately carried out. For example, in one embodiment, a liquid medium and a stirrer are placed in an Erlenmeyer flask, the mouth of the flask is sealed with a silicon plug, and stirring culture with breathability is carried out.

The period of liquid culture is not limited, but the culture can be completed when the desired concentration, for example, the average number of colonies per 1 L of liquid culture is usually 1 x 10 ³ cfu/L or more, particularly 1 x 10 ⁴ cfu/L ^or more, even more preferably 1 x 10 ⁵ cfu/L or more, for example, about 5 x 10 ⁶ cfu/L or more, is reached. For example, the upper limit of the liquid culture period is usually 8 days or less, particularly 6 days or less, even 5 days or less, and particularly 4 days or less. The lower limit of the liquid culture period is usually 6 hours or more, particularly 12 hours or more, even 18 hours or more, and particularly 1 day or more. In liquid culture, if the culture period is too long, the mycelium concentration becomes too high. On the other hand, if the culture period is too short, the mycelium does not grow sufficiently. For example, in one embodiment, the liquid culture period is 2 to 4 days, for example 3 days, which is sufficient to obtain a liquid culture of a suitable concentration for subsequent solid culture.

[Solid culture]
In this specification, "solid culture" refers to culturing a strain using a solid culture medium containing a solid medium and water. The process of performing such solid culture may be referred to as a "solid culture process". A solid culture refers to a culture obtained as a result of solid culture in which a strain is cultured in a solid culture medium. For example, bran, soybean pulp, bamboo powder, sawdust, rice husk, bagasse, cellulose powder, cellobiose, coffee grounds, and starch can be used as the solid culture medium. The water used for solid culture may contain, for example, various additives that contribute to the growth of the strain. Examples of additives include antibiotics that can suppress the growth of bacteria other than the Cephaliophora sp. xsd08001 strain used in this embodiment.

The solid culture medium can be prepared by adding water to the solid culture medium and mixing. The moisture content of the solid culture medium is not limited, but for example, the upper limit of the moisture content is usually 85% by mass or less, particularly 80% by mass or less, further 75% by mass or less, and particularly 70% by mass or less is preferable. The lower limit of the moisture content is usually 30% by mass or more, particularly 40% by mass or more, further 45% by mass or more, and particularly 50% by mass or more is preferable. If the moisture content of the solid culture medium is too high, it becomes too watery and sufficient gaps are not formed in the medium, so growth is not promoted. On the other hand, if the moisture content of the solid culture medium is too low, sufficient moisture for culture cannot be secured. For example, in one embodiment, when the moisture content of the solid culture medium is 50 to 70% by mass, for example 60% by mass, a solid culture having a mycelium concentration appropriate as an endophyte material can be obtained.

In this specification, subjecting a liquid culture to solid culture refers to inoculating a solid culture medium with the liquid culture and carrying out solid culture. For inoculation, for example, a micropipette or other tool can be used to inoculate, or at least one of the following methods can be used: heating the mouth of the container containing the strain with a burner, followed by sterilization, followed by decanting and direct inoculation. Regardless of which method is used, inoculating a liquid culture into a solid culture medium makes it possible to spread the liquid culture throughout the solid culture medium. This allows the mycelium to grow more uniformly across the entire solid culture medium, resulting in a more uniform culture, which in turn is believed to result in culture particles with a more uniform particle size.

Solid culture can be carried out by a suitable culture means. For example, solid culture can be carried out by using a container such as a plastic bag and allowing the container to stand. In addition, for solid culture, it is preferable to adjust the culture conditions using 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 may be capable of measuring, for example, O ₂ and CO ₂ as specific gases. For solid culture, it is preferable to use a disposable container from the viewpoint of preventing contamination, and measures such as closing the mouth of the bag may be taken as appropriate. Stirring culture, shaking culture, static culture, etc. may be carried out as appropriate. For example, in one embodiment, a disposable bag equipped with a ventilation filter is used, and static culture is carried out by closing the mouth of the bag.

The period of solid culture is not limited, but the culture can be completed when the desired concentration, for example, the average number of colonies per 1 g of solid culture, is usually 1×10 ³ to 1×10 ⁷ cfu/g, preferably 5×10 ³ to 5×10 ⁶ cfu/g, more preferably 1×10 ⁴ to 1×10 ⁶ cfu/g, particularly preferably 5×10 ⁴ to 5×10 ⁵ cfu/g, for example, about 1×10 ⁵ cfu/g. For example, the upper limit of the period of solid culture is usually 20 days or less, more preferably 18 days or less, more preferably 15 days or less, and particularly preferably 12 days or less. The lower limit of the period of solid culture is usually 5 days or more, more preferably 6 days or more, more preferably 7 days or more, and particularly preferably 8 days or more. If the solid culture period is too long, the risk of contamination by various bacteria increases. Furthermore, if the solid culture period is too long, it will hinder efficient production of the endophyte material. On the other hand, in solid culture, if the culture period is too short, the mycelium does not grow sufficiently. For example, in one embodiment, solid culture for 8 to 12 days, e.g., 10 days, can provide a solid culture having a mycelium concentration suitable for use as an endophyte material.

[Humidity]
The humidity during liquid culture is not particularly limited and can be any humidity. On the other hand, the humidity during solid culture is not limited, but the upper limit of the humidity for solid culture is usually 100% RH or less, particularly 95% RH or less, more preferably 90% RH or less. The lower limit of the humidity for solid culture is usually 55% RH or more, particularly 60% RH or more, more preferably 65% RH or more. In some embodiments, the culture can be carried out at a humidity of 60 to 80% RH, for example, 70% RH.

[temperature]
In both liquid culture and solid culture, the upper limit of the temperature is usually 40° C. or lower, preferably 35° C. or lower, more preferably 30° C. or lower, and particularly preferably 25° C. or lower. The lower limit of the temperature in liquid culture and solid culture is usually 5° C. or higher, preferably 10° C. or higher, more preferably 15° C. or higher, and particularly preferably 20° C. or higher. In one embodiment, the culture can be carried out at 20 to 25° C., for example, 25° C.±1° C.

[Sterilization]
From the viewpoint of preventing contamination, it is preferable to sterilize the liquid medium, solid medium, and/or solid culture medium by any known means, such as filtration sterilization, autoclave sterilization, boiling sterilization, radiation sterilization, sodium hypochlorite, or ozone treatment. Each operation, such as inoculation, is preferably performed in a sterile atmosphere. For example, in the case of a solid culture medium, an appropriate amount of moisture can be added to the solid culture medium before sterilization.

[Cultures and materials]
The present embodiment provides a material containing a culture of the xsd08001 strain. The ratio of the culture of the xsd08001 strain in the present material is not limited. For example, the upper limit of the ratio of the culture of the xsd08001 strain in the present material is 100 mass% or less, 95 mass% or less, 90 mass% or less, or 80 mass% or less. The lower limit of the ratio of the culture of the xsd08001 strain in the present material is 10 mass% or more, 20 mass% or more, or 30 mass% or more. The present material may be composed of a culture of the xsd08001 strain. The present embodiment also provides a method for producing a material containing a culture of the xsd08001 strain. The production method includes obtaining a culture of the xsd08001 strain. In one aspect, obtaining a culture of the xsd08001 strain includes subjecting the xsd08001 strain to liquid culture and then to solid culture. In one embodiment of the present production method, obtaining a culture of the xsd08001 strain includes drying the solid culture obtained by solid culture and pulverizing the dried solid culture. In addition, it may also include classifying the pulverized product as necessary. For example, a sieve can be used for classification. In this specification, drying the solid culture obtained by the solid culture step to produce a dried solid culture is sometimes referred to as a "drying step". In addition, pulverizing the dried solid culture is sometimes referred to as a "pulverizing step".

Drying can be carried out using general-purpose equipment such as an air conditioner or dehumidifier to achieve the following culture concentrations. For example, drying can be carried out by leaving it in an environment of 25°C and 50% RH for 3 days. Grinding can be carried out using general-purpose grinders such as a food processor, coffee mill, or pepper mill.

In this production method, since the mycelium extends uniformly within the culture, the culture particles can be adjusted to a desired particle size simply by drying and pulverizing the solid culture, but in one embodiment, the method may further include adjusting the cumulative 50% particle size (D ₅₀ ) in the volumetric particle size distribution to 100-900 μm and the cumulative 90% particle size (D ₉₀ ) to 1000-5000 μm. In this specification, the adjustment of the cumulative 50% particle size (D ₅₀ ) in the volumetric particle size distribution of the dried solid culture to 100-900 μm and the cumulative 90% particle size (D ₉₀ ) to 1000-5000 μm is sometimes referred to as an "adjustment step". The adjustment step includes a pulverization step.

This material has a consistent particle size and the bacteria are uniformly distributed, so it can stably function as an endophyte on plants, which is preferable from the standpoint of plant growth and quality improvement. In addition, the consistent particle size makes it easy to handle, and is advantageous for tableting, granulation, packaging, transportation, etc. By using the strain of this embodiment, a material with uniformly distributed bacteria can be preferably obtained even with solid culture alone. Furthermore, when liquid culture is used in combination, the mycelium extends throughout the medium, making the concentration of the bacteria even more uniform.

The concentration of the solid culture obtained in this embodiment is, for example, usually 1 x ¹⁰ to 1 x 10 cfu/g, preferably 5 x ¹⁰ to 5 x ¹⁰ cfu/g, more preferably 1 x ¹⁰ to 1 x ¹⁰ cfu/g, and particularly preferably 5 x ¹⁰ to ^{5 x 10} ^cfu /g, for example, about 1 x ¹⁰ cfu/g, as the average number of colonies per gram of solid culture. The concentration of the culture in the material of this embodiment is, for example, ¹ x 10 to 1 x ¹⁰ cfu/g, preferably 5 x ¹⁰ to 5 x ¹⁰ cfu/g, more preferably 1 x ¹⁰ to 1 x ¹⁰ cfu/g, as the average number of colonies ^per gram ^of dry solid culture. The average number of colonies per gram of dry solid culture product does not change before and after the crushing step.

[II. Method for culturing endophytes]
One embodiment relates to a method for culturing an endophyte. As described in Patent Document 2, since the xsd08001 strain was isolated from soil, it is considered that an environment similar to that of soil is suitable for the xsd08001 strain, and the xsd08001 strain is usually cultured by solid culture. However, details of how to efficiently culture the xsd08001 strain are not known.

In addition, if the time from inoculation of the seed fungus to obtaining a culture is long, this hinders the efficient production of endophyte products that contain a sufficient amount of fungal bodies for the growth of the intended symbiotic plant. Furthermore, as described in Patent Document 3, a long culture period increases the risk of the endophyte product being contaminated by various bacteria. If an endophyte contaminated by various bacteria is used, the intended symbiotic plant will also be contaminated, causing disease and hindering cultivation, so it is important that endophyte products are as little contaminated as possible.

The use of liquid culture for fungi such as mushrooms has been reported to shorten the culture period and prevent contamination (Patent Document 3). However, in the case of filamentous fungi, mycelium adheres and aggregates to form mycelial masses, making liquid culture difficult and hindering industrial cultivation. For this reason, it was thought that special procedures such as genetic manipulation were necessary to apply liquid culture to filamentous fungi. For example, it has been reported that a gene was knocked out in the filamentous fungus Aspergillus nidulans to create an α-1,3-glucan-deficient strain in order to facilitate liquid culture (Non-Patent Document 1). The inventors discovered that the same difficulties existed with the xsd08001 strain of the genus Cephaliophora, which belongs to filamentous fungi.

In addition, the xsd08001 strain does not grow uniformly in the culture medium, and if its effect as an endophyte is unstable, there is a risk that the quality of plants cultivated using it will not be satisfactory.

In light of the above problems, the inventors conducted extensive research and were surprised to discover that it was possible to culture the xsd08001 strain in liquid form without the need for special procedures such as gene knockout, and that by optimizing the conditions for solid culture to culture it efficiently, they were able to shorten the time required for culture and commercialization and reduce the risk of contamination.

[Culture method]
The present embodiment provides a culture method comprising subjecting the xsd08001 strain to liquid culture. One aspect of the culture method relates to a method for culturing the Cephaliophora sp. xsd08001 strain, the culture method comprising subjecting the strain to liquid culture to obtain a liquid culture, and subjecting the obtained liquid culture to solid culture to obtain a solid culture.

[KK]
The Cephaliophora sp. xsd08001 strain used in this embodiment has been deposited at the National Institute of Technology and Evaluation (NITE) as described above, and is available under the accession number NITE P-02438.

[Liquid culture]
As used herein, "liquid culture" refers to culturing a strain using a liquid medium, as described above. Subjecting the xsd08001 strain to liquid culture refers to inoculating the strain into a liquid medium and culturing it in the liquid medium. The strain may be in the form of a seed culture medium carried by a medium. Examples of the medium include agar. In order to avoid contamination, it is preferable to take at least one of the following measures: adding the bacteria directly to a culture tank by decantation from a container containing the bacteria, using a holding tool such as sterilized tweezers, or performing the inoculation under a sterile atmosphere in a clean bench.

As used herein, the term "liquid culture" refers to a liquid culture obtained as a result of liquid culture, as described above. A liquid medium can be prepared, for example, by adding various additives to the liquid to adjust the composition to be suitable for culture. An example of the liquid is water. Examples of the additives include sugars, minerals, nitrogen sources, vitamins, organic acids, inorganic acids, organic bases, and inorganic bases. Preferably, the liquid medium contains sugars and a nitrogen source. An example of the sugars is glucose. An example of the nitrogen source is peptone. Details of these components are as described in detail in [I. Endophyte materials] .

[Solid culture]
In one embodiment of the culture method, a liquid culture of the xsd08001 strain obtained by liquid culture is subjected to solid culture to obtain a solid culture. As described above, "solid culture" in this specification refers to culturing a strain using a solid culture medium containing a solid medium and water. A solid culture refers to a culture obtained as a result of solid culture in which a strain is cultured in a solid culture medium. For example, bran, bean pulp, bamboo powder, sawdust, rice husk, bagasse, cellulose powder, cellobiose, coffee grounds, and starch can be used as the solid culture medium.

The solid culture medium can be prepared by adding water to the solid culture medium and mixing. The moisture content of the solid culture medium is not limited, but for example, the upper limit of the moisture content is usually 85% by mass or less, particularly 80% by mass or less, further 75% by mass or less, and particularly 70% by mass or less. The lower limit of the moisture content is usually 30% by mass or more, particularly 40% by mass or more, further 45% by mass or more, and particularly 50% by mass or more. If the moisture content of the solid culture medium is too high, it becomes too watery and there are not enough gaps in the medium, so growth is not promoted, while if the moisture content of the solid culture medium is too low, sufficient moisture for culture cannot be secured. For example, in one embodiment, when the moisture content of the solid culture medium is 50 to 70% by mass, for example 60% by mass, a solid culture having a mycelium concentration appropriate as an endophyte material can be obtained.

In this specification, subjecting a liquid culture to solid culture refers to inoculating a liquid culture into a solid culture medium and carrying out solid culture. For inoculation, for example, a method of inoculating using a micropipette or other tool, or at least one of measures of heating the mouth of a container containing the strain with a burner, followed by decanting and direct inoculation can be adopted. Regardless of the method used, by inoculating the liquid culture, it is possible to apply it so that it spreads throughout the entire solid culture medium, and the mycelium can extend and grow more quickly throughout the entire solid culture medium. In addition, in the conventional technology, as described in Patent Document 2, inoculation was performed by placing culture pieces of the seed strain on the medium, but it was necessary to mix the culture pieces during inoculation so that the microorganisms spread throughout the medium. Such work must be performed in a sterile atmosphere to avoid the risk of contamination, and even if it is performed in a sterile atmosphere, the risk of contamination cannot be completely eliminated. However, in this culture method, such stirring work itself is not necessary, or the number of stirring operations can be reduced compared to inoculation by placing culture pieces of the seed strain on the medium. Therefore, this culture method can reduce the risk of contamination associated with stirring, and can also save time and labor required for the work. In addition, as described in Patent Document 2, special equipment is used in mushroom cultivation to inoculate the culture liquid and allow it to penetrate into the mushroom bed. However, in the case of the strains used in this culture method, even with the use of a simple tool such as a micropipette, the liquid culture is able to permeate the entire solid culture medium, resulting in a uniform culture.

Other details regarding the culture method and culture period for solid culture are as described in detail in [I. Endophyte Materials] .

[Humidity]
The humidity during liquid culture is not particularly limited and can be any humidity. The humidity during solid culture is not limited, but the upper humidity limit for solid culture is usually 100% RH or less, particularly 95% RH or less, more preferably 90% RH or less. The lower humidity limit for solid culture is usually 55% RH or more, particularly 60% RH or more, more preferably 65% RH or more. In some embodiments, it can be performed at a humidity of 60 to 80% RH, for example, 70% RH. In the case of normal mushroom culture, for example, oyster mushroom is cultured at a high humidity of 95% RH and slimy oyster mushroom is cultured at 85% RH (Non-Patent Documents 2 and 3), but the culture method of the present application can be performed at a relatively low humidity of, for example, 70% RH. If the humidity of solid culture is low, it is advantageous in terms of the time, labor, electricity, water, cost, etc. required for installation of the equipment.

[Sterilization]
From the viewpoint of preventing contamination, it is preferable that the liquid medium, solid medium, and/or solid culture medium be sterilized by any known means, such as filtration sterilization, autoclave sterilization, boiling sterilization, radiation sterilization, sodium hypochlorite, or ozone treatment, and that each operation, such as inoculation, be carried out in a sterile atmosphere. For example, in the case of a solid culture medium, an appropriate amount of moisture can be added to the solid culture medium before sterilization.

[Total period required for culture]
The total period required for liquid culture and solid culture is not limited, but for example, the upper limit of the total culture period from inoculation to completion of culture is usually 30 days or less, particularly 25 days or less, further 20 days or less, and particularly 15 days or less. The lower limit of the total culture period is usually 13 days or more, particularly 12 days or more, further 11 days or more, and particularly 10 days or more. For example, in one embodiment, a solid culture having a mycelium concentration appropriate for an endophyte material can be obtained by performing the culture for 10 to 15 days, for example, 13 days. As described above, if the total culture period is too long, the mycelium concentration becomes too high and the risk of contamination increases. In addition, if the solid culture period is too long, the xsd08001 strain cannot be cultured efficiently. On the other hand, if the total culture period is too short, the mycelium does not extend sufficiently. When the main culture method including liquid culture is used, the period required to obtain the same amount of fungal mass can be shortened compared to the case of culturing only by solid culture. The present culture method is advantageous not only in that the total culture period can be shortened, thereby reducing the time, labor, cost, etc. required for culture, but also in that the spread of contamination during growth can be prevented.

[Cultures and materials]
Hereinafter, a culture obtained by the present culture method and a material containing the culture are provided. Furthermore, this embodiment is described as providing a method for producing a material, which includes recovering a culture obtained by the present culture method including liquid culture, and drying and pulverizing the recovered culture. Since the present culture employs liquid culture, it has the advantage that contamination is low and the mycelium extends throughout the medium, resulting in a uniform concentration of the fungus. Therefore, the use of the present material using such a culture reduces the risk of contamination of plants using endophytes, and since the fungus is uniformly distributed, it is preferable from the viewpoint of plant growth, since it can stably exert its function as an endophyte on plants.

The concentration of the culture obtained by this culture method is, for example, usually 1 x ¹⁰ to 1 x 10 cfu/g, preferably 5 x ¹⁰ to 5 x ¹⁰ cfu/g, more preferably 1 x ¹⁰ to 1 x ¹⁰ cfu/g, and particularly preferably 5 x ¹⁰ to 5 x ¹⁰ cfu/g, for example, about 1 x 10 cfu/g, as the average number of colonies per gram of solid culture. The concentration of the present material is usually 1 x 10 to ¹ x ¹⁰ cfu/g, preferably 5 x ¹⁰ to ⁵ x ¹⁰ cfu/g, more preferably 1 x ¹⁰ to 1 x ¹⁰ cfu/g, and particularly preferably 5 x ¹⁰ to ⁵ x ¹⁰ cfu/g, as the average number of colonies per gram of material.

The present invention will now be described in more detail with reference to examples. Note that the present invention is not limited to these examples.

[Example Group I] Examples related to endophyte materials
Experiment 1: Cultivation of xsd08001 strain The strain used was Cephaliophora sp. xsd08001, deposited at the National Institute of Technology and Evaluation (NITE) under the accession number NITE P-02438. The strain was attached to an agar medium and stored at -80°C and used as the seed culture.

In addition, a liquid medium was prepared by adding the following reagents to a 500 mL Erlenmeyer flask.
Glucose 4.0g
Yeast extract 0.4g
Magnesium sulfate 0.1g
Peptone 0.2g
Potassium dihydrogen phosphate 0.2g
Ion-exchanged water 200mL

A stirrer was added to the flask, the mouth of the flask was sealed with a silicone stopper, and the flask was sterilized in an autoclave at 121°C for 20 minutes. After cooling, the seed bacteria attached to the agar medium melted from -80°C in a clean bench was added directly to the flask by decantation from the test tube to inoculate the liquid medium. The mouth of the flask was sealed with a silicone stopper, and the mixture was stirred and cultured at 25°C using a stirrer (700 rpm). The culture was completed when the average number of colonies per liter reached approximately 3.0 x ¹⁰⁶ cfu/L of medium, and it took 3 days to complete the culture.

The solid culture medium was prepared by adding 1500 mL of ion-exchanged water to 1000 g of bran as a solid culture medium in a plastic tub and mixing well to a moisture content of 60%. The solid culture medium was added to a disposable bag equipped with a ventilation filter, and the mouth of the bag was taped and sterilized in an autoclave at 121°C for 60 minutes. After the medium was cooled to room temperature together with the bag, the bag was opened in a clean bench, and 80 mL of liquid culture cultured for 3 days was inoculated so as to spread throughout the solid culture medium by using a sterile micropipette or by heating the mouth of the flask with a burner and then decanting and adding directly. After inoculation, the mouth of the bag was sealed and solid culture was performed by leaving it in a thermo-hygrostat for 10 days under an environment of 25°C and 70% RH. The culture was completed when it was confirmed that the average number of colonies per 1 g reached about 1 x 10 ⁵ cfu/g. It took another 10 days after the start of liquid culture to complete the culture. After the solid culture, the solid culture was taken out of the bag and dried by leaving it to stand for 3 days in an environment of 25° C. and 50% RH using an air conditioner or a dehumidifier.

In the obtained solid culture, the mycelium spread was uniform, and no visible contamination was confirmed. Furthermore, when the concentration of the dried solid culture was measured, the number of colonies per gram was about 5 x ¹⁰⁶ to 4 x ¹⁰⁷ cfu/g. Furthermore, this dried solid culture was crushed using a crusher (Capsule Cutter Bonne, manufactured by Winners Co., Ltd.) and used as the material of Example 1, and the uncrushed dried product was used as the material of Comparative Example 1. Thereafter, the particle size of the material was measured using an LMS-2000e (Seishin Enterprise Co., Ltd.).

The results of particle size measurement are shown in Table 1. From Table 1, it can be seen that the material of Example 1 has a cumulative 50% particle size (D ₅₀ ) in the range of 200 to 800 μm and a cumulative 90% particle size (D ₉₀ ) in the range of 1000 to 5000 μm for all refractive indices, and thus has uniform particle size and small quality variation.

The material in Comparative Example 1 had a particle size of 5000 μm or more, which exceeded the upper measurement limit of the LMS-2000e (Seishin Enterprise Co., Ltd.) used for particle size measurement, and therefore could not be measured.

The material of Example 2 was produced by culturing, drying, and pulverizing the same method as in Example 1, except that the same strain was used and the seed culture was directly inoculated into the solid culture medium without using liquid culture, and cultured only in solid culture. Although the culture took longer in Example 2 than in Example 1, a material with sufficiently uniform particle size was obtained.

Experiment 2: Evaluation of plant growth due to differences in particle size
Example 1
The cultivation soil was a mixture of horticultural soil and vermiculite at a volume ratio of 1:1. The materials of Example 1 prepared in Experiment 1 were added to the cultivation soil at a mass ratio of 1.0%, dry mixed, and dispensed at 135 g per pot into eight 9 cm pots. Three lettuce seeds were sown per pot, watered, and the germinated lettuce was thinned to one plant per pot. After counting the number of true leaves for several days, on the 25th day after sowing when the lettuce reached a diameter of 20 cm, which is the harvest time, the soil was removed from the roots, dried, and the mass was measured.

(Comparative Example 1)
The growth of lettuce was evaluated in the same manner as in Example 1, except that the material of Comparative Example 1 prepared in Experiment 1 was used.

(Comparative Example 2)
Lettuce growth evaluation was carried out in the same manner as in Example 1, except that the material containing the culture of the xsd08001 strain was not mixed into the cultivation soil.

The results are shown in FIG.

From Tables 2 and 3, it can be seen that in Example 1 and Comparative Example 1, in which a culture of the xsd08001 strain was used, the growth of lettuce was promoted compared to Comparative Example 2, in which this culture was not used. Furthermore, in the case of Example 1, in which the particle size of the culture of the xsd08001 strain was uniform, the dry mass of the edible above-ground part increased by 39.8% and the dry mass of the root part also increased by 16.7% compared to Comparative Example 1, in which the particle size was uneven. Furthermore, regardless of the number of days, Example 1 showed less variation in the number of true leaves compared to Comparative Example 1, and it was possible to suppress unevenness in the effect of promoting plant growth. From the above, it can be seen that materials containing a culture of the xsd08001 strain have the effect of promoting plant growth, and this effect is further increased by uniforming the particle size of the culture.

The above results show that the use of the present invention makes it possible to obtain a material containing a culture of the xsd08001 strain with a more uniform particle size than materials produced using conventional technology. The use of such a material can further promote plant growth and also suppress unevenness in the effects of promoting plant growth.

Experiment 3: Cultivation of strawberries with the addition of endophytic materials

Example 3-1 and Comparative Example 3-1 (endophyte material added at planting)
Strawberry (variety name: Kaorino) was used as the test plant. The harvest period was from November 2021 to May 2022.

(Example 3-1)
2520 strawberry seedlings were grown by conventional farming methods. The grown strawberry seedlings were planted in a planting hole with 2 g of endophyte per seedling added, and cultivated by conventional farming methods. The yield of strawberry fruit was measured by the number of packs.

Here, "number of packs" refers to a pack containing 250g of fruit, which is the shipping standard for strawberries. Since the weight of each fruit varies, the weight per pack can go up to a maximum of 255g, but it will never go below 250g.

(Comparative Example 3-1)
Cultivation was carried out in the same manner as in Example 3-1, except that no endophyte material was added to the planting hole.

(Test results)
The results are shown in Table 4. As is clear from these results, the fruit yield was greater in Example 3-1 than in Comparative Example 3-1 in all months. The total number of packs throughout the year was increased by about 19.64% in Example 3-1 compared to Comparative Example 3-1.

Example 3-2 and Comparative Example 3-2 (endophyte material added during seedling raising)
Strawberry (variety name: Kaorino) was used as the test plant. The harvest period was from November 2022 to May 2023.

(Example 3-2)
2520 strawberry plants were grown in a conventional manner by adding 2 g of endophyte powder per plant to the soil for growing. The grown strawberry plants were planted and cultivated in a conventional manner. The yield of strawberry fruits was measured by the number of packs.

After harvesting, the roots were collected and nucleic acid was extracted, after which the ITS region (approximately 330 bp) of Cephaliophora sp. xsd08001 strain was detected by PCR. The primer sequences used are shown in Table 5.

(Comparative Example 3-2)
Cultivation and detection of the microorganism were carried out in the same manner as in Example 3-2, except that no endophyte material was added to the seedling soil.

(Test results)
The results are shown in FIG. 2 and Table 6. As is clear from these results, the fruit yield was higher in Example 3-2 than in Comparative Example 3-2 in all months. The total number of packs throughout the year was increased by about 25.63 % in Example 3-2 compared to Comparative Example 3-2 . In addition, as shown in FIG. 2, the ITS region (about 330 bp) of Cephaliophora sp. xsd08001 strain was detected from the root of Example 3-2, whereas it was not detected from the root of Comparative Example 3-2.

[Example Group II] Examples related to the cultivation method of endophytes
(Comparative Example: Cultivation of xsd08001 strain by solid culture only)
The strain used was the Cephaliophora sp. xsd08001 strain deposited at the National Institute of Technology and Evaluation (NITE) under the accession number NITE P-02438. The strain was attached to an agar medium and stored at -80°C and used as the seed culture.

The solid culture medium was prepared by adding 1,000 g of bran to 1,500 mL of ion-exchanged water in a plastic tub and mixing well to give a moisture content of 60%.

The solid culture was carried out by the following method. The solid culture medium was added to a bag with a filter, the mouth of the bag was taped, and sterilized in an autoclave at 121 ° C. for 60 minutes. After cooling the medium together with the bag to room temperature, the bag was opened in a clean bench, the above-mentioned inoculation bacteria were placed on the medium, and the solid culture was carried out by leaving it in a thermostatic chamber under an environment of 25 ° C. and 70% RH. After 7 days of culture, the bag was opened in a clean bench, the solid culture was inoculated into a new solid culture medium, and the solid culture was carried out by leaving it in a thermostatic chamber under an environment of 25 ° C. and 70% RH. Thereafter, the bag was opened on a tray, the solid culture was crushed and mixed well, and the number of colonies per 1 g was measured. When it was confirmed that the number of colonies reached about 1 × 10 ⁵ cfu / g, the culture was completed, and it took 25 days to complete the culture. In addition, the solid culture was observed on the 10th and 25th days after inoculation into the new solid culture medium, and the state of contamination and the degree of mycelium extension were confirmed. After the cultivation, the solid culture was dried using an air conditioner or a dehumidifier, and the dried material was ground in a food processor to prepare a material.

The results are shown in Figures 3 and 4. At the 10th day, in the comparative example, as shown in Figure 3, the white hyphae had not fully extended in the solid culture, and the hyphae were not uniformly spread in the solid culture. In addition, it took another 25 days from inoculation into the new solid culture medium to obtain the desired concentration. In other words, it took a total of 32 days from inoculation of the seed fungus to obtaining the final culture, and the culture took a very long time. In addition, at the 25th day from inoculation into the new solid culture medium, the hyphae extended to the bottom of the solid culture, but did not reach the bottom (Figures 4a, b), and furthermore, contamination by black mold (Figure 4c) and blue mold (Figure 4d) was confirmed everywhere. Furthermore, bacterial contamination was confirmed in the parts where the hyphae had not extended (Figure 4e), and a unique putrid odor and stickiness were generated. Furthermore, when the concentration of the material was measured, it was found to be 5×10 ⁶ cfu/g on average, but it was confirmed that there were many colonies that were not of the xsd08001 strain.

Example: Cultivation of strain xsd08001, including liquid and solid cultures
Liquid culture was carried out as follows.
A liquid medium was prepared by adding the following reagents to a 500 mL Erlenmeyer flask:
Glucose 4.0g
Yeast extract 0.4g
Magnesium sulfate 0.1g
Peptone 0.2g
Potassium dihydrogen phosphate 0.2g
Ion-exchanged water 200mL

A stirrer was added to the flask, the mouth of the flask was sealed with a silicone stopper, and the flask was sterilized in an autoclave at 121°C for 20 minutes. After cooling, the seed bacteria attached to the agar medium melted from -80°C in a clean bench was added directly to the flask by decantation from the test tube to inoculate the liquid medium. This seed bacteria was the same as the seed bacteria used in the comparative example. The mouth of the flask was sealed with a silicone stopper, and the mixture was stirred and cultured at 25°C and 700 rpm using a stirrer. The culture was completed when the average number of colonies per liter reached about 3.0 x ¹⁰⁶ cfu/L of medium, and it took 3 days to complete the culture.

Solid culture was carried out by the following method. The solid culture medium was prepared, bagged, and sterilized in the same manner as in the comparative example. After 3 days of liquid culture, a disposable bag equipped with an aeration filter containing the sterilized solid culture medium was opened in a clean bench, and 80 mL of liquid culture was inoculated so as to spread throughout the solid culture medium by using a sterile micropipette or by decanting and directly adding the liquid culture medium after heating the mouth of the flask with a burner. After inoculation, the mouth of the bag was sealed with a sealer, and solid culture was carried out under the same environment as in the comparative example. The culture was completed when it was confirmed that the average number of colonies per gram reached about 1 x ¹⁰⁵ cfu/g. It took another 10 days after the start of liquid culture until the culture was completed. After solid culture, the solid culture was dried in the same manner as in the comparative example, and the dried product was crushed in a food processor.

The results are shown in Figure 3. As shown in Figure 3, in the comparative example, the white mycelium had not yet fully extended on the fungal bed at the time of the 10th day, whereas in the example, the mycelium had extended to the back side, and the culture was completed in 10 days. In addition, the period from the inoculation of the seed fungus to obtaining the final culture was only 13 days in total, which was significantly shorter than that of the comparative example. Furthermore, the mycelium spread evenly in the solid culture, no visible contamination was confirmed, and there was no putrid odor or stickiness. In addition, the concentration of the material was confirmed to be 5 x 10 ⁶ to 4 x 10 ⁷ cfu/g in terms of the number of colonies per 1 g of material, and furthermore, the particle size was uniform and there was little variation in quality.

Reference experiment 1: Effect of water content in solid culture medium
Reference Experiment 1-1:
The optimal water content in the solid culture medium was determined by the following method.

(Reference Example 1)
Ion-exchanged water was added to 10 g of bran to a moisture content of 50% by mass, and this was used as a medium for solid culture. This medium was placed in a glass petri dish and sterilized in an autoclave at 121°C for 60 minutes, and then inoculated with an inoculum of xsd08001 strain aseptically in a clean bench. The medium was left to stand at 25°C and 90% RH, and the shortest length passing through the center of the area where the elongated mycelium could be confirmed on the back of the glass petri dish was measured, and this was taken as the diameter of the colony of the elongated mycelium.

(Reference Example 2)
The diameter of colonies of elongated hyphae was measured in the same manner as in Reference Example 1, except that the amount of ion-exchanged water was adjusted to give a water content of 60% by mass.

(Comparative Reference Example 1)
The diameter of colonies of elongated hyphae was measured in the same manner as in Reference Example 1, except that the amount of ion-exchanged water was adjusted to give a water content of 40% by mass.

(Comparative Reference Example 2)
The diameter of colonies of elongated hyphae was measured in the same manner as in Reference Example 1, except that the amount of ion-exchanged water was adjusted to give a water content of 45% by mass.

The results are shown in Table 7. In the case of Reference Examples 1 and 2 and Comparative Reference Examples 1 and 2, which have a moisture content of 60% by mass or less, as shown in Table 7, it was confirmed that the higher the moisture content, the larger the diameter of the colonies of elongated hyphae.

Reference Experiment 1-2:
The results of Reference Experiment 1-1 suggested that a higher moisture content would result in higher culture efficiency. Therefore, solid culture media with a moisture content of 70% by mass and 80% by mass were prepared as follows by further increasing the moisture content, and experiments were conducted to compare the media with those of Reference Examples 1 and 2 prepared in the same manner as in Reference Experiment 1-1.

(Reference Example 3)
The diameter of colonies of elongated hyphae was measured in the same manner as in Reference Example 1, except that the amount of ion-exchanged water was adjusted to give a water content of 70% by mass.

(Comparative Reference Example 3)
Cultivation was carried out in the same manner as in Reference Example 1, except that the amount of ion-exchanged water was adjusted to a water content of 80% by mass.

The results are shown in Table 8 and Figures 5 and 6. When the moisture content was 70% by mass or less, the higher the moisture content, the larger the diameter of the colony of elongated hyphae, as shown in Table 8, and the longer the mycelium, as shown in Figure 5, the higher the mycelium density, and the white mycelium was clearly visible. Therefore, when the moisture content was 70% by mass or less, it is suggested that the higher the moisture content of the solid culture medium, the higher the culture efficiency. However, when the moisture content of the solid culture medium was 80% by mass as shown in Comparative Reference Example 3, as shown in Figure 6, the excess moisture did not create gaps in the medium, preventing the mycelium from elongating and sufficient growth. This is thought to be due to the fact that Cephaliophora sp., to which the xsd08001 strain belongs, is an aerobic filamentous fungus.

These results suggest that the moisture content in solid culture media should preferably be greater than 45% by mass and less than 80% by mass, for example, 50% by mass or more and 75% by mass or less, or 50% by mass or more and 70% by mass or less.

Reference Experiment 2: Effect of humidity on solid culture The effect of humidity on solid culture was investigated using the moisture contents of 50% and 60% by mass, which are the solid culture media that showed good results in Reference Experiment 1. Since Reference Experiment 1 confirmed that the growth was sufficiently good at a humidity of 90% RH, in order to investigate the lower limit, culture at a humidity of 70% RH was attempted as follows.

(Reference Example 4)
The diameter of colonies of elongated hyphae was measured in a solid culture medium having a moisture content of 50% by mass in the same manner as in Reference Example 1, except that the humidity was set to 70% RH.

(Reference Example 5)
The diameter of colonies of elongated hyphae was measured in a solid culture medium having a moisture content of 60% by mass in the same manner as in Reference Example 2, except that the humidity was set to 70% RH.

The results on the sixth day of cultivation are shown in Figure 7. As shown in Figure 7, even when the humidity was lowered to 70% RH, good cultivation was possible at both 50% and 60% by mass. Therefore, it is preferable for the humidity in solid cultivation to be in the range of 70% to 90% RH, and it is suggested that good cultivation can be performed in a wide range of humidity, for example, 60% to 100% RH, or even at a relatively low humidity, for example, 60 to 80% RH.

The above results show that the use of the present invention can shorten the culture time compared to solid culture alone, reduces contamination, and produces high-quality cultures in which mycelia grow uniformly throughout the medium.

Claims

A material comprising a dry solid culture of Cephaliophora sp. xsd08001 strain,
The dried solid culture material has a cumulative 50% particle size (D 50 ) of 100 to 900 μm and a cumulative 90% particle size (D 90 ) of 1000 to 5000 μm in a volumetric particle size distribution.
2. The material according to claim 1, wherein the average number of colonies per gram of the dry solid culture is at a concentration of 1×10 5 to 1×10 9 cfu (Colony Forming Unit)/g.
A material as described in claim 1 or claim 2 for promoting plant growth.
A method for producing a material containing a dried solid culture of Cephaliophora sp. xsd08001 strain, comprising:
a culturing step to obtain said dry solid culture of said strain;
and adjusting the cumulative 50% particle size (D 50 ) in the volumetric particle size distribution of the dried solid culture product to 100 to 900 μm and the cumulative 90% particle size (D 90 ) to 1000 to 5000 μm.
The culture step comprises:
A liquid culture step of subjecting the strain to liquid culture;
A solid culture step of culturing the strain cultured in the liquid culture step in solid culture;
A drying step of drying the solid culture obtained by the solid culture step to produce the dried solid culture,
The method according to claim 4 , wherein the adjusting step includes a grinding step of grinding the dried solid culture product dried in the drying step.
The method according to claim 5, wherein the solid culture step is carried out using a solid culture medium containing a solid medium and water.
The method of claim 6, wherein the solid culture medium has a water content of 50 to 70%.
The material according to claim 6 or claim 7, wherein the solid culture medium contains bran.
A method for culturing Cephaliophora sp. xsd08001 strain, comprising:
Obtaining a liquid culture by subjecting the strain to liquid culture;
The culture method includes subjecting the obtained liquid culture to solid culture to obtain a solid culture.
The culture method of claim 9, wherein the solid culture is carried out using a solid culture medium containing a solid medium and water.
The culture method of claim 9, wherein the moisture content of the solid culture medium is 50 to 70%.
The culture method of claim 9, wherein the solid medium contains bran.
The culture method of claim 9, wherein the liquid culture is carried out for 2 to 4 days.
The culture method of claim 9, wherein the solid culture is carried out for 7 to 12 days.
The culture method of claim 9, wherein the liquid culture and/or solid culture is carried out at an indoor humidity of 60 to 90% RH.
A culture obtained by the culture method according to any one of claims 9 to 15.