Endophyte materials and methods for culturing endophytes

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

Technical Field

[0001] The present invention relates to endophyte materials, a method for producing the same, and a method for culturing endophytes. More specifically, it relates to a material containing a culture of Cephaliophora sp. xsd08001 strain, a method for producing the same, and a method for culturing the strain including subjecting the strain to liquid culture.

Background Art

[0002] Endophytes, also called endophytic bacteria, refer to microorganisms that inhabit and symbiotically grow inside plant hosts, bringing benefits such as growth promotion and increased stress tolerance to the hosts. As such an endophyte, Cephaliophora sp. xsd08001 strain (hereinafter sometimes referred to as the xsd08001 strain) has been reported as a useful microorganism (Patent Document 1).

[0003] The culture of the xsd08001 strain has been used to increase the content of highly functional components in plants such as vegetables when mixed with soil (Patent Document 2). By the way, regarding endophyte materials, it has not been known what characteristics of the materials affect the quality of plants.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0005]

Non-Patent Document 1

[0006] The objective of this invention is to provide a material that can suppress unevenness in the effect of promoting plant growth. Another objective of this invention is to provide a novel cultivation method that can cultivate the xsd08001 strain more efficiently. [Means for solving the problem]

[0007] As a result of diligent research, the inventors succeeded in standardizing the particle size of the culture of the bacterial strain by using a predetermined culture method. Furthermore, they discovered that combining culture with liquid medium, rather than solely with solid medium as in conventional methods, shortened the culture period, thus completing the following invention. [1] The material contains a culture of Cephaliophora sp. xsd08001 strain. The above material has a cumulative 50% particle size (D) in its volume particle size distribution. 50 The particle size is 100-900 μm. The above material has a cumulative 90% particle size (D 90 The size is 1000-5000 μm. [2] The average number of colonies per gram of dried solid culture is 1 × 10 for the materials described in [1]. 5 ~1 × 10 9 This is the concentration in cfu (Colony Forming Unit) / g. The materials described in [3] [1] or [2] are intended to promote plant growth. [4] A method for producing a material containing a dried solid culture of the genus Cephaliophora sp. xsd08001 strain. The method includes a culture step to obtain the dried solid culture of the strain. The method includes a cumulative 50% particle size (D) in the volume particle size distribution of the dried solid culture. 50 ) with a particle size of 100-900 μm and a cumulative 90% particle size (D 90 This includes an adjustment step to adjust the particle size to 1000-5000 μm. [5] The culture step described above includes a liquid culture step in the manufacturing method described in [4], in which the strain is subjected to liquid culture. The culture step includes a solid culture step. The solid culture step involves culturing the strain cultured in the liquid culture step in solid culture. The culture step includes a drying step of drying the solid culture obtained in the solid culture step to produce a dried solid culture. The preparation step includes a grinding step of grinding the dried solid culture dried in the drying step. [6] The above solid culture step is carried out using a solid culture medium containing a solid culture medium and water in the manufacturing method described in [5]. [7] The above solid culture medium has a water content of 50-70% in the manufacturing method described in [6]. [8] The above solid culture medium includes wheat bran in the manufacturing method described in [6] or [7]. [9] A culture method for culturing the genus Cephaliophora sp. xsd08001 strain. The culture method involves subjecting the genus Cephaliophora sp. xsd08001 strain to liquid culture to obtain a liquid culture. The culture method involves subjecting the obtained liquid culture to solid culture to obtain a solid culture.

[10] The above solid culture is carried out using a solid culture medium in the culture method described in [9]. The above solid culture medium contains a solid medium and water.

[11] The above solid culture medium has a water content of 50-70% in the culture method described in [9] or

[10] .

[12] The above solid culture medium includes wheat bran in the culture method described in any one of [9] to

[11] .

[13] The liquid culture described above is carried out for 2 to 4 days in the culture method described in any one of the items [9] to

[12] .

[14] The above solid culture is carried out for 7 to 12 days using the culture method described in any one of the items [9] to

[13] .

[15] At least one of the above liquid culture or the above solid culture is carried out in the culture method described in any one of items [9] to

[14] at room humidity of 60 to 90% RH.

[16] The culture is obtained by the culture method described in any one of the items [9] to

[15] . [Effects of the Invention]

[0008] By using the materials of the present invention, it is possible to suppress inconsistencies in the effect of promoting plant growth using these materials. Furthermore, by adopting the cultivation method of the present invention, the xsd08001 strain can be cultivated more efficiently. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the lettuce on day 25 of sowing in Experiment 2 of Example Group I. In Figure 1, a shows the lettuce of Example 1. In Figure 1, b shows the lettuce of Comparative Example 1. In Figure 1, c shows the lettuce of Comparative Example 2. [Figure 2] Figure 2 is an electrophoresis image 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 the TAKARA 100bp DNA Ladder Marker (for the base lengths of each DNA band, please refer to 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. [Figure 3]Figure 3 shows the solid culture media of the examples and comparative examples of Example Group II 10 days after the start of solid culture. In Figure 3, a shows a side view of the solid culture media of the comparative example. In Figure 3, b shows a side view of the solid culture media of the example. In Figure 3, c shows a back view of the solid culture media of the comparative example. In Figure 3, d shows a back view of the solid culture media of the example. [Figure 4] Figure 4 shows the solid culture medium of the comparative example in Example Group II 25 days after the start of solid culture. In Figure 4, a, c, d, and e show the side view of the solid culture medium of the comparative example. In Figure 4, b shows the back view of the solid culture medium of the comparative example. [Figure 5] Figure 5 shows the mycelial growth of the xsd08001 strain in solid culture medium with a moisture content of 50-70% by mass in Reference Experiment 1 of Example Group II. In Figure 5, the 50% by mass moisture content corresponds to Reference Example 1. In Figure 4, the 60% by mass moisture content corresponds to Reference Example 2. In Figure 5, the 70% by mass moisture content corresponds to Reference Example 3. In Figure 5, a shows the surface of the petri dish. In Figure 5, b shows the underside of the petri dish. [Figure 6] Figure 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. Figure 6 corresponds to Comparative Reference Example 3 with a water content of 80% by mass. Figure 6 shows the surface of the petri dish. [Figure 7] Figure 7 shows the xsd08001 strain in solid culture media with a moisture content of 50% by mass and 60% by mass at an ambient humidity of 70% RH in Reference Experiment 2 of Example Group II. In Figure 7, a, b, and c correspond to Reference Example 4 with a moisture content of 50% by mass. In Figure 7, d, e, and f correspond to Reference Example 5 with a moisture content of 60% by mass. All images in Figure 7 show the surface of the petri dish. [Modes for carrying out the invention]

[0010] 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.

[0011] [I. Endofight Materials] One embodiment relates to an endophyte material. Adjusting the particle size of microbial materials is important for improving handling, ensuring stable effectiveness, and promoting plant growth. However, because filamentous fungi grow by extending strong hyphae, there is considerable variation in the density of hyphae in the culture medium, making it difficult to adjust the particle size of the culture. Despite this common technical knowledge, the inventors have discovered that using strain xsd08001 allows for uniform hyphae extension, resulting in a culture with adjusted particle size.

[0012] Furthermore, as described in Patent Document 1, since strain xsd08001 was isolated from soil, it is thought that an environment similar to soil is suitable, and cultivation of strain xsd08001 is usually performed using solid culture. In addition, liquid culture of filamentous fungi is difficult for reasons such as the adhesion and aggregation of hyphae to form hyphae (Non-Patent Document 1). Despite these circumstances, the inventors surprisingly succeeded in liquid culture of strain xsd08001. Although a culture with adjusted particle size can be obtained even with solid culture, liquid culture allows for the acquisition of a culture with even more adjusted particle size. This is thought to be because inoculating with liquid culture allows the inoculum to spread throughout the entire culture medium, enabling the hyphae to spread uniformly within the medium, resulting in a uniform concentration of fungal cells per unit mass.

[0013] [Materials] Therefore, this embodiment shows the cumulative 50% particle size (D) in the volume particle size distribution. 50 ) is 100-900 μm and has a cumulative 90% particle size (D 90 The present invention provides a culture of the xsd08001 strain having a size of 1000 to 5000 μm, and materials containing the same.

[0014] [KK] The Cephaliophora sp. xsd08001 strain used in this embodiment is deposited with the National Institute of Technology and Evaluation (NITE) and is available under NITE accession number P-02438.

[0015] [Particle size] Although the particle size of the culture of the strain used in this embodiment is not limited, it is as follows. The cumulative 10% particle diameter (D10) in the volume particle size distribution of the culture of the strain used in this embodiment is, for example, 50 to 400 μm, particularly 100 to 350 μm, more preferably 150 to 350 μm, and still more preferably 200 to 300 μm. The cumulative 50% particle diameter (D 50 ) of the culture of the strain used in this embodiment is, for example, 100 to 1000 μm, particularly 200 to 900 μm, more preferably 300 to 850 μm, and still more preferably 400 to 800 μm. The cumulative 90% particle diameter (D 90 ) of the culture of the strain used in this embodiment is, for example, 1000 to 5000 μm, particularly 1100 to 4000 μm, more preferably 1200 to 3000 μm, and still more preferably 1200 to 2000 μm. For example, in one embodiment, the cumulative 50% particle diameter (D 50 ) is 200 to 800 μm, and the cumulative 90% particle diameter (D 90 ) is 1000 to 3000 μm. The particle size can be measured by any method such as laser diffraction method, scattering method, imaging method, light transmission centrifugal sedimentation method, sedimentation method, electrical resistance method, specific surface area method, sieve passing method, etc. The particle size can be measured using a suitable particle size measuring instrument, for example, commercially available equipment such as LMS-2000e, LMS-3000 (Seishin Enterprise Co., Ltd.). Since the culture is dried and then pulverized, it is preferable to use a dry measurement method.

[0016] Also, 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 pulverizing the dried solid culture.

[0017] [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 the "liquid culture process." Subjecting the xsd08001 strain to liquid culture means 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 carried in a medium. Agar is an example of a medium. To avoid contamination by other bacteria, inoculation is preferably performed under sterile conditions.

[0018] In this specification, "liquid culture" refers to a liquid culture obtained as a result of liquid culture. A liquid culture medium can be prepared, for example, by adding various additives to a liquid to adjust its composition to one suitable for culture. Examples of liquids include water. Examples of additives to be added to a liquid culture medium include 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 contained in a liquid culture medium is peptone.

[0019] The sugars used are not limited to, but examples include one or more selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and glycerol. The upper limit of the sugars is not limited, but it is generally preferable that the total amount of sugars in the liquid medium is 60 g / L of medium or less, more preferably 50 g / L of medium or less, even more preferably 40 g / L of medium or less, and especially preferably 30 g / L of medium or less. This is because if the liquid medium contains too many nutrients such as sugars, the hyphal concentration will become too high, as described in Patent Document 3. On the other hand, the lower limit of the sugars is not limited, but it is generally preferable that the total amount of sugars in the liquid medium is 0.5 g / L of medium or more, more preferably 1 g / L of medium or more, even more preferably 5 g / L of medium or more, and especially preferably 10 g / L of medium or more. This is because if there are too few nutrients such as sugars, the hyphal growth will not be sufficient. For example, in one embodiment, the sugar is glucose, and it is added in an amount of 20 g / L of medium.

[0020] The minerals may be inorganic salts, such as alkali and alkaline earth metal salts, or other metal salts, although this is not limited to them. 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 salts, but excluding the amounts contained in the yeast extract and peptone, the total amount of salts in the liquid medium is usually 10.0 g / L of medium or less, more preferably 8.0 g / L of medium or less, even more preferably 5.0 g / L of medium or less, and especially preferably 2.0 g / L of medium or less. This is because if the liquid medium contains too many salts as nutrients as described above, the concentration of mycelium will become too high. On the other hand, while there is no lower limit to the amount of salts, it is preferable that the total amount of salts in the liquid medium, excluding the amount contained in the yeast extract, is usually 0.01 g / L of medium or more, more preferably 0.05 g / L of medium or more, even more preferably 0.1 g / L of medium or more, and especially preferably 0.25 g / L of medium 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 at an amount of 0.5 g / L of medium and potassium dihydrogen phosphate is added at an amount of 0.1 g / L of medium as salts.

[0021] Examples of nitrogen sources include one or more nitrogen sources selected from yeast extract, protein hydrolysates, and proteins. Examples of protein hydrolysates include 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. The amount of nitrogen source relative to the liquid culture medium is not limited, but for example, the upper limit of the nitrogen source is usually 30.0 g / L of medium or less, more preferably 20.0 g / L of medium or less, even more preferably 15.0 g / L of medium or less, and especially preferably 10.0 g / L of medium or less, as a total amount of nitrogen sources such as yeast extract and peptone. The lower limit of the nitrogen source is usually 0.05 g / L of medium or more, more preferably 0.1 g / L of medium or more, even more preferably 0.5 g / L of medium or more, and especially preferably 1.0 g / L of medium or more, as a total amount of nitrogen sources such as yeast extract and peptone. For example, while there are no restrictions on the amount of yeast extract in a liquid medium, the upper limit of yeast extract is usually 20.0 g / L of medium or less, more preferably 15.0 g / L of medium or less, even more preferably 10.0 g / L of medium or less, and especially preferably 5.0 g / L of medium or less. The lower limit of yeast extract is usually 0.01 g / L of medium or more, more preferably 0.05 g / L of medium or more, even more preferably 0.1 g / L of medium or more, and especially preferably 0.5 g / L of medium or more. While there are no restrictions on the amount of peptone in a liquid medium, the upper limit of peptone is usually 10.0 g / L of medium or less, more preferably 8.0 g / L of medium or less, even more preferably 5.0 g / L of medium or less, and especially preferably 2.0 g / L of medium or less. The lower limit of peptone is usually 0.01 g / L of medium or more, more preferably 0.05 g / L of medium or more, even more preferably 0.1 g / L of medium or more, and especially preferably 0.5 g / L of medium or more. As described above, too much nutrition leads to an excessively high concentration of mycelium, while too little nitrogen prevents sufficient mycelial growth. For example, in one embodiment, yeast extract is added at a rate of 2.0 g / L of medium, and peptone is added at a rate of 1.0 g / L of medium. Furthermore, if necessary, the pH may be adjusted as appropriate by adding a suitable acid or base.

[0022] 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 adjuster, 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.

[0023] The duration of liquid culture is not limited, but it depends on the desired concentration, for example, the average number of colonies per liter of liquid culture is usually 1 × 10⁶. 3 cfu / medium L or more, especially 1 × 10⁶ 4 cfu / L or more, and even 1 × 10 5 cfu / medium L or more, particularly preferably 1 × 10 6 cfu / medium L or more, for example, about 5 × 10 6 The culture can be completed when the cfu / medium L level is reached. For example, the upper limit of the liquid culture period is usually 8 days or less, preferably 6 days or less, even more preferably 5 days or less, and especially preferably 4 days or less. The lower limit of the liquid culture period is usually 6 hours or more, preferably 12 hours or more, even more preferably 18 hours or more, and especially preferably 1 day or more. If the liquid culture period is too long, the hyphal concentration becomes too high. On the other hand, if the liquid culture period is too short, the hyphal growth is insufficient. For example, in one embodiment, if the liquid culture period is 2 to 4 days, for example 3 days, a liquid culture with a suitable concentration for subsequent solid culture can be obtained.

[0024] [Solid culture] In this specification, "solid culture" refers to culturing a strain using a solid culture medium containing a solid culture medium and water. The process of performing such solid culture may be referred to as the "solid culture process." A solid culture refers to a culture obtained as a result of solid culture, in which the strain has been cultured in a solid culture medium. As for the solid culture medium, for example, wheat bran, okara (soy pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, and starch can be used as the culture medium. The water used in solid culture may have various additives added to it that contribute to the growth of the bacterial strain. Examples of additives include antibiotics that can suppress the growth of bacteria other than the Cephaliophora sp. xsd08001 strain used in this embodiment.

[0025] Solid culture media can be prepared by adding water to a solid medium and mixing. The water content of the solid culture medium is not limited, but for example, the upper limit of the water content 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, there will be excess moisture and insufficient space will be created in the medium, so growth will not be promoted. On the other hand, if the water content of the solid culture medium is too low, it will not be possible to secure sufficient moisture for cultivation. For example, in one embodiment, if the water content of the solid culture medium is 50 to 70% by mass, for example 60% by mass, a solid culture with an appropriate mycelial concentration as an endophyte material can be obtained.

[0026] In this specification, "submitting a liquid culture to a solid culture" refers to inoculating a liquid culture into a solid culture medium to perform solid culture. For inoculation, for example, inoculation using a micropipette or other instrument, or sterilization and decantation by heating the mouth of the container containing the strain with a burner, and direct inoculation can be employed. Regardless of the method used, inoculating a liquid culture into a solid culture medium allows the liquid culture to spread throughout the solid culture medium. As a result, the mycelium proliferates more uniformly throughout the solid culture medium, leading to a more uniform culture and, consequently, a culture with more uniform particle size.

[0027] 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. 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.

[0028] The duration of solid culture is not limited, but it depends on the desired concentration, for example, the average number of colonies per gram of solid culture, which is usually 1 × 10⁶. 3 ~1 × 10 7 cfu / g, especially 5×10 3 ~5×10 6 cfu / g, and even 1 × 10⁻⁶ 4 ~1 × 10 6 cfu / g, particularly preferably 5 × 10 4 ~5×10 5cfu / g, for example, approximately 1 × 10⁻⁶ 5 Culturing can be completed when the cfu / g is reached. For example, the upper limit of the solid culture period is usually 20 days or less, 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 usually 5 days or more, preferably 6 days or more, even more preferably 7 days or more, and especially preferably 8 days or more. If the culture period is too long in solid culture, the risk of contamination by unwanted bacteria increases. Also, if the culture period is too long in solid culture, it hinders the efficient production of endophyte material. On the other hand, if the culture period is too short in solid culture, the mycelium will not grow sufficiently. For example, in one embodiment, if solid culture is performed for 8 to 12 days, for example 10 days, a solid culture with an appropriate mycelial concentration as endophyte material can be obtained.

[0029] [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 for solid culture humidity is usually 100%RH or less, more preferably 95%RH or less, and more preferably 90%RH or less. The lower limit for solid culture humidity is usually 55%RH or higher, more preferably 60%RH or higher, and more preferably 65%RH or higher. In one embodiment, it can be carried out at a humidity of 60-80%RH, for example, 70%RH.

[0030] [temperature] In both liquid culture and solid culture, the upper temperature limit is usually 40°C or lower, more preferably 35°C or lower, more preferably 30°C or lower, and especially preferably 25°C or lower. The lower temperature limit for both liquid and solid culture is usually 5°C or higher, more preferably 10°C or higher, more preferably 15°C or higher, and especially preferably 20°C or higher. In one embodiment, the process can be carried out at 20-25°C, for example, 25°C ± 1°C.

[0031] [Sterilization] 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.

[0032] [Cultures and materials] This embodiment provides a material containing a culture of strain xsd08001. The proportion of strain xsd08001 culture in this material is not limited. For example, the upper limit of the proportion of strain xsd08001 culture in this material is 100% by mass or less, 95% by mass or less, 90% by mass or less, or 80% by mass or less. The lower limit of the proportion of strain xsd08001 culture in this material is 10% by mass or more, 20% by mass or more, or 30% by mass or more. This material may consist of a culture of strain xsd08001. This embodiment also provides a method for producing a material containing a culture of strain xsd08001. This production method includes obtaining a culture of strain xsd08001. In one embodiment, obtaining a culture of strain xsd08001 includes subjecting strain xsd08001 to liquid culture followed by solid culture. In one embodiment of this manufacturing method, obtaining a culture of strain xsd08001 includes drying the solid culture obtained by solid culture and grinding the dried solid culture. The process may also include classifying the ground material as needed. For example, a sieve can be used for classification. In this specification, the process of drying the solid culture obtained by the solid culture process to produce a dried solid culture may be referred to as the "drying process." Furthermore, the process of grinding the dried solid culture may be referred to as the "grinding process."

[0033] Drying can be done using general-purpose equipment such as air conditioners and dehumidifiers, to achieve the following culture concentrations. For example, it can be done by leaving it in an environment of 25°C and 50% RH for 3 days. Grinding can be done using general-purpose grinders such as food processors, coffee grinders, and pepper mills.

[0034] In this manufacturing method, since the mycelium grows uniformly within the culture, it is possible to obtain the desired particle size of the culture simply by drying and pulverizing the solid culture. However, in one embodiment, the cumulative 50% particle size (D) in the volume particle size distribution is further reduced. 50 ) is 100-900 μm and has a cumulative 90% particle size (D 90 This may include adjusting the cumulative 50% particle size (D) in the volume particle size distribution of the dried solid culture. 50 ) is 100-900 μm and has a cumulative 90% particle size (D 90 The process of adjusting the particle size to 1000-5000 μm is sometimes referred to as the "adjustment process." The adjustment process includes the grinding process.

[0035] This material has a consistent particle size and uniformly distributed fungi, allowing it to function stably as an endophyte for plants, which is desirable from the perspective of plant growth and quality improvement. Furthermore, the consistent particle size makes it easy to handle, which is advantageous for tableting, granulation, packaging, and transportation. Using the strain of this embodiment, a material with uniformly distributed fungi can be preferably obtained even with solid culture alone. Moreover, by combining it with liquid culture, the mycelium can extend throughout the culture medium, further homogenizing the concentration of fungi.

[0036] The concentration of the solid culture obtained in this embodiment is typically 1 × 10¹⁶ colonies per gram of solid culture, for example. 3 ~1 × 10 7 cfu / g, especially 5×10 3 ~5×10 6 cfu / g, and even 1 × 10⁻⁶ 4 ~1 × 10 6 cfu / g, particularly preferably 5 × 10 4 ~5×10 5 cfu / g, for example, approximately 1 × 10⁻⁶ 5 The concentration is cfu / g. The culture concentration in the material of this embodiment is, for example, 1 × 10¹⁶ colonies per gram of dry solid culture. 5 ~1 × 10 9cfu / g, especially 5×10 5 ~5×10 8 cfu / g, and even 1 × 10⁻⁶ 6 ~1 × 10 8 cfu / g, particularly preferably 5 × 10 6 ~5×10 7 The value is cfu / g. Note that the average number of colonies per gram of dried solid culture does not change before or after the grinding process.

[0037] [II. Endophyte Culture Methods] One embodiment relates to a method for culturing endophytes. As described in Patent Document 2, since the xsd08001 strain was isolated from soil, it is thought that an environment similar to soil is suitable, and the xsd08001 strain is usually cultured in solid form. However, the details of how to efficiently culture the xsd08001 strain are not known.

[0038] Furthermore, a long time between inoculation of the spawn and obtaining the culture hinders the efficient production of endophyte products containing a sufficient amount of microbial cells for the growth of the symbiotic plant. In addition, as described in Patent Document 3, a longer culture period increases the risk of endophyte products being contaminated with other bacteria. Using endophytes contaminated with other bacteria can contaminate the symbiotic plant, causing diseases and hindering cultivation; therefore, it is important that endophyte products are minimally contaminated.

[0039] For fungi such as mushrooms, the use of liquid culture has been reported to shorten the culture period and prevent contamination (Patent Document 3). However, in the case of filamentous fungi, liquid culture is difficult because the hyphae adhere and aggregate to form hyphae masses, which is an obstacle to industrial cultivation. Therefore, it was thought that special operations such as genetic manipulation were necessary to apply liquid culture to filamentous fungi. For example, in order to facilitate liquid culture, it has been reported that a gene knockout was performed on the filamentous fungus Aspergillus nidulans to create an α-1,3-glucan-deficient strain (Non-Patent Document 1). The inventors have found that the xsd08001 strain of the genus Cephaliophora, which belongs to the filamentous fungus, also faces similar difficulties.

[0040] Furthermore, in the case of strain xsd08001, if its endophyte effect is not stable due to uneven growth in the culture medium, there is a risk that the quality of plants cultivated using it may not be sufficient.

[0041] In light of the above challenges, the inventors have diligently conducted research and, as a result, have surprisingly succeeded in enabling liquid culture of the xsd08001 strain without requiring special operations such as gene knockout. Furthermore, by optimizing the conditions for solid culture and culturing efficiently, they have succeeded in shortening the period from cultivation to commercialization and reducing the risk of contamination.

[0042] [Culture method] This embodiment provides a culture method that includes subjecting the xsd08001 strain to liquid culture. One aspect of the culture method is a method for culturing the genus Cephaliophora (Cephaliophora sp.) xsd08001 strain, which includes obtaining a liquid culture by subjecting the strain to liquid culture, and obtaining a solid culture by subjecting the obtained liquid culture to solid culture.

[0043] [KK] As mentioned above, the Cephaliophora sp. xsd08001 strain used in this embodiment is deposited with the National Institute of Technology and Evaluation (NITE) and is available under accession number NITE P-02438.

[0044] [Liquid culture] In this specification, "liquid culture" refers to culturing a strain using a liquid medium, as described above. Subjecting the xsd08001 strain to liquid culture means 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 carried in a medium. Agar is an example of a medium. To avoid contamination, it is preferable to take at least one of the following measures when inoculating: for example, adding the strain directly to the culture tank by decantation from a container containing the bacterial cells, using a sterilized gripping instrument such as tweezers, or performing the inoculation under a sterile atmosphere in a clean bench.

[0045] In this specification, "liquid culture" refers to the liquid culture obtained as a result of liquid culture, as described above. A liquid culture medium can be prepared, for example, by adding various additives to a liquid to adjust its composition to one suitable for culture. Examples of liquids include water. Examples of additives include 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. Examples of sugars include glucose. Examples of nitrogen sources include peptone. Details of these components are as follows: [I. Endofight Materials] As detailed elsewhere.

[0046] [Solid culture] One embodiment of the culture method includes obtaining a solid culture by subjecting the liquid culture of the xsd08001 strain obtained by liquid culture to solid culture. In this specification, "solid culture" refers to culturing the strain using a solid culture medium containing a solid culture medium and water, as described above. A solid culture refers to a culture obtained as a result of solid culture, in which the strain has been cultured in a solid culture medium. As a solid culture medium, for example, wheat bran, okara (soy pulp), bamboo powder, sawdust, rice hulls, bagasse, cellulose powder, cellobiose, coffee grounds, and starch can be used.

[0047] Solid culture media can be prepared by adding water to a solid medium and mixing. The water content of the solid culture medium is not limited, but for example, the upper limit of the water content 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, there will be excess moisture and insufficient pores will not form in the medium, so growth will not be promoted. On the other hand, if the water content of the solid culture medium is too low, it will not be possible to secure sufficient moisture for cultivation. For example, in one embodiment, if the water content of the solid culture medium is 50 to 70% by mass, for example 60% by mass, a solid culture with an appropriate mycelial concentration as an endophyte material can be obtained.

[0048] In this specification, "substituting a liquid culture for solid culture" refers to inoculating a liquid culture into a solid culture medium to perform solid culture. For inoculation, for example, a method can be employed in which the liquid culture is inoculated using a micropipette or other instrument, or a method of sterilization followed by decanting by heating the mouth of the container containing the strain with a burner and then direct inoculation. In either method, inoculating the liquid culture allows it to spread throughout the solid culture medium, enabling the mycelium to grow and proliferate more quickly throughout the solid culture medium. Furthermore, in the conventional technique, as described in Patent Document 2, inoculation was performed by placing culture pieces of the strain on the medium, but it was necessary to mix the culture pieces during inoculation to ensure that the microorganisms spread throughout. Such work had to be performed under a sterile atmosphere to avoid the risk of contamination, and even under a sterile atmosphere, the risk of contamination could not be completely eliminated. However, in this culture method, such mixing work itself is unnecessary, or the number of mixing operations can be reduced compared to inoculation by placing culture pieces of the strain on the medium. Therefore, this cultivation method can reduce the risk of contamination associated with stirring and save time and labor. Furthermore, as described in Patent Document 2, special equipment is used in mushroom cultivation to inoculate the culture solution and allow it to permeate the substrate. However, with the strains of this cultivation method, even with the use of simple equipment such as a micropipette, the liquid culture can spread throughout the solid culture medium, resulting in a uniform culture.

[0049] For further details on solid-state culture methods and culture periods, please see below. [I. Endofight Materials] As detailed elsewhere.

[0050] [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 limit for solid culture humidity is usually 100%RH or less, more preferably 95%RH or less, and more preferably 90%RH or less. The lower limit for solid culture humidity is usually 55%RH or higher, more preferably 60%RH or higher, and more preferably 65%RH or higher. In one embodiment, it can be carried out at a humidity of 60-80%RH, for example, 70%RH. Normally, when cultivating mushrooms, for example, oyster mushrooms are cultured at high humidity levels such as 95%RH and false oyster mushrooms at 85%RH (Non-patent documents 2 and 3), but the cultivation method of the present invention can be carried out at relatively low humidity levels, such as 70%RH. Lower humidity for solid culture is advantageous in terms of the time, labor, electricity, water, and cost required for equipment installation.

[0051] [temperature] In both liquid culture and solid culture, the upper temperature limit is usually 40°C or lower, more preferably 35°C or lower, more preferably 30°C or lower, and especially preferably 25°C or lower. The lower temperature limit for both liquid and solid culture is usually 5°C or higher, more preferably 10°C or higher, more preferably 15°C or higher, and especially preferably 20°C or higher. In one embodiment, the process can be carried out at 20-25°C, for example, 25°C ± 1°C.

[0052] [Sterilization] Furthermore, from the viewpoint of preventing contamination, it is preferable that liquid culture media, solid culture media, and / or solid culture media be sterilized by any known means such as filtration sterilization, autoclave sterilization, boiling sterilization, and radiation sterilization, sodium hypochlorite, or ozone treatment, and that each operation such as inoculation be carried out 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.

[0053] [Total period required for culturing] 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, more preferably 25 days or less, even more preferably 20 days or less, and especially preferably 15 days or less. The lower limit of the total culture period is usually 13 days or more, more preferably 12 days or more, even more preferably 11 days or more, and especially preferably 10 days or more. For example, in one embodiment, a solid culture with an appropriate mycelial concentration for endophyte material can be obtained by culturing for 10 to 15 days, for example, 13 days. As mentioned above, if the total culture period is too long, the mycelial concentration becomes too high and the risk of contamination increases. Also, in solid culture, if the 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 will not grow sufficiently. By using this culture method which includes liquid culture, the period required to obtain the same amount of fungal cells can be shortened compared to culturing with solid culture alone. This culture method offers advantages not only in shortening the total culture period and reducing the time, effort, and cost involved in culturing, but also in preventing the spread of contamination during the growth process.

[0054] [Cultures and materials] The following describes the provision of a culture obtained by this culture method and a material containing said culture. Furthermore, this embodiment describes a method for producing the material, which includes recovering the culture obtained by this culture method, which includes liquid culture, and drying and pulverizing the recovered culture. Because this culture employs liquid culture, it has the advantage of less contamination and the mycelium extends throughout the culture medium, resulting in a uniform concentration of fungi. Therefore, using this material with such a culture reduces the risk of contamination of plants using endophytes, and because the fungi are uniformly distributed, they can stably function as endophytes for plants, which is preferable from the viewpoint of plant growth.

[0055] The concentration of the culture obtained by this culture method is typically 1 × 10¹⁶ colonies per gram of solid culture, for example. 3 ~1 × 10 7 cfu / g, especially 5×103 ~5×10 6 cfu / g, and even 1 × 10⁻⁶ 4 ~1 × 10 6 cfu / g, particularly preferably 5 × 10 4 ~5×10 5 cfu / g, for example, approximately 1 × 10⁻⁶ 5 The concentration is cfu / g. The concentration of this material is typically 1 × 10¹⁶ colonies per gram of material. 5 ~1 × 10 9 cfu / g, especially 5×10 5 ~5×10 8 cfu / g, and even 1 × 10⁻⁶ 6 ~1 × 10 8 cfu / g, particularly preferably 5 × 10 6 ~5×10 7 It is cfu / g. [Examples]

[0056] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited thereto.

[0057] [Example Group I] Examples relating to endophyte materials Experiment 1: Culturing of strain xsd08001 The bacterial strain used was Cephaliophora sp. xsd08001, deposited with the National Institute of Technology and Evaluation (NITE) under accession number NITE P-02438. This strain was used as a starter culture, stored at -80°C while attached to an agar plate.

[0058] Furthermore, a liquid culture 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 200mL of deionized water

[0059] 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 inoculum attached to the agar medium, which had been melted from -80°C in a clean bench, was decanted directly from the test tube into the flask and inoculated into the liquid medium. The mouth of the flask was sealed with a silicone stopper, and the flask was cultured with a stirrer at 25°C (700 rpm). The average number of colonies per liter was approximately 3.0 × 10⁶. 6 When the culture was completed when the cfu / medium L level was reached, it took 3 days to complete the culture.

[0060] The solid culture medium was prepared by adding 1500 mL of deionized water to 1000 g of wheat bran in a plastic container and mixing thoroughly to achieve a moisture content of 60%. The solid culture medium was added to a disposable bag equipped with a breathable filter, the bag was sealed with tape, and sterilized in an autoclave at 121°C for 60 minutes. After the medium in the bag was cooled to room temperature, the bag was opened in a clean bench, and 80 mL of liquid culture, which had been liquid cultured for 3 days, was inoculated into the solid culture medium by using a sterile micropipette or by decanting the flask opening after heating it with a burner and adding it directly, ensuring that it covered the entire solid culture medium. After inoculation, the bag was sealed with a sealing machine, and solid culture was performed by standing it in a constant temperature and humidity chamber at 25°C and 70% RH for 10 days, with an average colony count of approximately 1 × 10⁶ per gram. 5 The culture was considered complete when the cfu / g level was confirmed. It took an additional 10 days from the start of liquid culture until the culture was complete. After solid culture, the solid culture was removed from the bag and dried by standing for 3 days in an environment of 25°C and 50% RH using an air conditioner or dehumidifier.

[0061] The obtained solid culture showed uniform hyphae distribution, and no visible contamination was observed. Furthermore, when the concentration of the dried solid culture was measured, the number of colonies per gram was approximately 5 × 10⁶. 6 ~4×10 7The concentration was cfu / g. Furthermore, this dried solid culture was pulverized using a pulverizer (Capsule Cutter Bonne, manufactured by Winners Co., Ltd.) and used as the material for Example 1, while the unpulverized dried material was used as the material for Comparative Example 1. Subsequently, the particle size of the materials was measured using an LMS-2000e (Seishin Corporation).

[0062] The particle size measurement results are shown in Table 1. From Table 1, the material of Example 1 has a cumulative 50% particle size (D) for all refractive indices. 50 ) is 200-800 μm, cumulative 90% particle size (D 90 The particle size is within the range of 1000 to 5000 μm, indicating uniform particle size and low variation in quality.

[0063] [Table 1]

[0064] Furthermore, the material in Comparative Example 1 had a particle size of 5000 μm or more, which exceeded the measurement limit of the LMS-2000e (Seishin Corporation) used for particle size measurement, and therefore could not be measured.

[0065] Furthermore, using the same strain, the material for Example 2 was prepared by culturing, drying, and grinding in the same manner as in Example 1, except that liquid culture was not employed and the inoculum was directly inoculated into a solid culture medium for cultivation using only solid culture. In Example 2, although cultivation took longer compared to Example 1, a material with sufficiently uniform particle size was obtained.

[0066] Experiment 2: Evaluation of plant growth based on differences in particle size (Example 1) The growing medium was a mixture of horticultural soil and vermiculite in a 1:1 volume ratio. The materials prepared in Example 1 in Experiment 1 were added to the growing medium at a mass ratio of 1.0% and dry-mixed, then dispensed into eight 9cm pots, 135g each. Three lettuce seeds were sown per pot, watered, and the germinated lettuce plants were thinned to one plant per pot. After counting the true leaves for several days, on the 25th day after sowing, when the plants reached a diameter of 20cm (the harvest time for lettuce), the soil was removed from the roots, dried, and the mass was measured.

[0067] (Comparative Example 1) The growth of lettuce was evaluated using the same method as in Example 1, except that the materials of Comparative Example 1, which were prepared in Experiment 1, were used.

[0068] (Comparative Example 2) The growth of lettuce was evaluated using the same method as in Example 1, except that the cultivation soil was not mixed with materials containing the culture of strain xsd08001.

[0069] The results are shown in Figure 1 and Tables 2 and 3. [Table 2]

[0070] [Table 3]

[0071] Tables 2 and 3 show that in Example 1 and Comparative Example 1, which used the culture of strain xsd08001, the growth of lettuce was promoted compared to Comparative Example 2, which did not use this culture. Furthermore, in Example 1, where the particle size of the culture of strain xsd08001 was uniform, the dry mass of the edible above-ground portion increased by 39.8% and the dry mass of the root portion increased by 16.7% compared to Comparative Example 1, where the particle size was uneven. In addition, in all time periods, the variation in the number of true leaves was smaller in Example 1 compared to Comparative Example 1, and unevenness in the effect of promoting plant growth was suppressed. From the above, it can be seen that materials containing the culture of strain xsd08001 have the effect of promoting plant growth, and this effect is further increased by uniformizing the particle size of the culture.

[0072] Based on the above results, 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 by conventional techniques. Using such a material can further promote plant growth and suppress unevenness in the effect of promoting plant growth.

[0073] Experiment 3: Cultivating strawberries with the addition of endophyte materials

[0074] Example 3-1 and Comparative Example 3-1 (Endophyte material added at planting time) The test plant used was strawberry (variety name: Kaorino). The harvest period was from November 2021 to May 2022.

[0075] (Example 3-1) 2520 strawberry seedlings were grown using conventional farming methods. The seedlings were planted in holes containing 2g of endophyte material per plant, and then cultivated using conventional farming methods. The yield of strawberries was measured by the number of packs.

[0076] Here, "number of packs" refers to packs containing 250g of strawberries, which is the standard shipping size for strawberries. Note that because the mass of individual strawberries varies, the weight per pack can exceed 255g, but it will never fall below 250g.

[0077] (Comparative Example 3-1) Cultivation was carried out in the same manner as in Example 3-1, except that endophyte material was not added to the planting holes.

[0078] (Test results) The results are shown in Table 4. As is clear from these results, Example 3-1 yielded a higher fruit harvest than Comparative Example 3-1 in all months. The total number of packs throughout the year increased by approximately 19.64% in Example 3-1 compared to Comparative Example 3-1.

[0079] [Table 4]

[0080] Example 3-2 and Comparative Example 3-2 (Endophyte material added during seedling cultivation) The test plant used was strawberry (variety name: Kaorino). The harvest period was from November 2022 to May 2023.

[0081] (Example 3-2) 2520 strawberry seedlings were grown using conventional farming methods, with 2g of endophyte powder per plant added to the seedling soil. After transplanting the seedlings, cultivation was carried out using conventional farming methods. The yield of strawberries was measured by the number of packs.

[0082] Furthermore, after harvesting, the roots were collected and nucleic acid extraction was performed, followed by PCR detection of the ITS region (approximately 330 bp) of the Cephaliophora sp. xsd08001 strain. The primer sequences used are shown in Table 5.

[0083] (Comparative Example 3-2) Cultivation and detection of the microorganism were carried out in the same manner as in Example 3-2, except that endophyte materials were not added to the seedling growing medium.

[0084] [Table 5]

[0085] (Test results) The results are shown in Figure 2 and Table 6. As is clear from these results, in all months, Example 3-2 yielded a larger fruit harvest than Comparative Example 3-2. The total number of packs throughout the year was as follows: 3- In 2, the comparative example 3- Compared to example 2, the amount increased by approximately 25.63%. Furthermore, as shown in Figure 2, the ITS region (approximately 330 bp) of the Cephaliophora sp. xsd08001 strain was detected in the root of example 3-2, whereas it was not detected in the root of comparative example 3-2.

[0086] [Table 6]

[0087] [Example Group II] Examples of methods for culturing endophytes (Comparative example: Culturing of strain xsd08001 by solid culture only) The bacterial strain used was Cephaliophora sp. xsd08001, deposited with the National Institute of Technology and Evaluation (NITE) under accession number NITE P-02438. This strain was used as a starter culture, stored at -80°C while attached to an agar plate.

[0088] The solid culture medium was prepared by adding 1000g of wheat bran to 1500mL of deionized water in a plastic container and mixing thoroughly until the water content reached 60%.

[0089] Solid culture was performed using the following method: Solid culture medium was added to a filter-equipped bag, the bag was sealed with tape, and sterilized in an autoclave at 121°C for 60 minutes. After the medium in the bag was cooled to room temperature, the bag was opened in a clean bench, the inoculum was placed on the medium, and solid culture was performed in a constant temperature and humidity chamber at 25°C and 70% RH. After 7 days of culture, the bag was opened in a clean bench, the solid culture was inoculated into new solid culture medium, and solid culture was performed in a constant temperature and humidity chamber at 25°C and 70% RH. After that, the bag was opened on a tray, the solid culture was crushed and thoroughly mixed, and the number of colonies per gram was measured to be approximately 1 × 10⁶. 5 The culture was considered complete when the cfu / g level was confirmed, and it took 25 days to complete the culture. In addition, the solid culture was observed 10 and 25 days after inoculation into a new solid culture medium to check the level of contamination and the degree of mycelial growth. After culturing, the solid culture was dried using an air conditioner or dehumidifier, and the dried material was crushed in a food processor to prepare the material.

[0090] The results are shown in Figures 3 and 4. At day 10, as shown in Figure 3, the white mycelium in the comparative example had not fully extended within the solid culture, and the spread of mycelium within the solid culture was uneven. Furthermore, it took another 25 days from inoculation into a new solid culture medium to obtain the desired concentration. In other words, the total period from inoculation with the starter culture to obtaining the final culture was 32 days, indicating a very long cultivation time. Also, at day 25 from inoculation into a new solid culture medium, although the mycelium had extended to the bottom of the solid culture, it had not reached the bottom surface (Figures 4a, b), and contamination by black mold (Figure 4c) and blue mold (Figure 4d) was observed in various places. Furthermore, bacterial contamination was observed in areas where the mycelium had not extended (Figure 4e), resulting in a sticky texture accompanied by a distinctive putrid odor. In addition, when the concentration of the material was measured, the average was 5 × 10⁶ 6 Although the cfu / g result was observed, it was confirmed that there were numerous colonies that did not belong to the xsd08001 strain.

[0091] (Example: Culture of strain xsd08001 including liquid culture and solid culture) Liquid culture was performed using the following method. A liquid culture 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 200mL of deionized water

[0092] 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 inoculum attached to the agar medium, which had been melted from -80°C in a clean bench, was decanted directly from the test tube into the flask and inoculated into the liquid medium. This inoculum is the same as the inoculum used in the comparative example. The mouth of the flask was sealed with a silicone stopper, and the flask was cultured by stirring at 25°C and 700 rpm using a stirrer. The average number of colonies per liter was approximately 3.0 × 10⁶. 6 When the culture was completed when the cfu / medium L level was reached, it took 3 days to complete the culture.

[0093] Solid culture was performed using the following method. Solid culture media were prepared, packaged, and sterilized in the same manner as in the comparative example. After 3 days of liquid culture, a disposable bag containing sterile solid culture media and equipped with a breathable filter was opened in a clean bench. 80 mL of liquid culture was inoculated into the solid culture media by using a sterile micropipette or by decanting the flask opening with a burner and adding it directly, ensuring even distribution. After inoculation, the bag opening was sealed with a sealing machine, and solid culture was performed under the same conditions as in the comparative example, with an average colony count of approximately 1 × 10⁶ per gram. 5 The culture was considered complete when it was confirmed that the cfu / g level had been reached. It took an additional 10 days from the start of liquid culture until the culture was complete. After solid culture, the solid culture was dried using the same method as in the comparative example, and the dried material was pulverized in a food processor.

[0094] The results are shown in Figure 3. As shown in Figure 3, in the comparative example, the white mycelium had not fully extended onto the substrate by the 10th day, whereas in the example, the mycelium had extended to the underside, and cultivation was completed in 10 days. Furthermore, the total period from inoculation with the spawn to obtaining the final culture was only 13 days, which was significantly shorter than in the comparative example. In addition, the spread of mycelium in the solid culture was uniform, no visible contamination was observed, and there was no putrid odor or stickiness. The concentration of the material was such that the number of colonies per gram of material was 5 × 10⁶. 6 ~4×10 7 The cfu / g concentration was confirmed, and furthermore, the particle size was uniform and the variation in quality was small.

[0095] Reference Experiment 1: Effect of water content in solid culture media Reference Experiment 1-1: The optimal water content for solid culture media was determined using the following method.

[0096] (Reference example 1) 10 g of wheat bran was mixed with deionized water to achieve a moisture content of 50% by mass, and this was used as a solid culture medium. This medium was placed in a glass petri dish and autoclaved at 121°C for 60 minutes. Then, the xsd08001 strain was aseptically inoculated in a clean bench. The culture was left to stand at 25°C and 90% RH, and the shortest length passing through the center of the area where elongated mycelium was observed on the underside of the glass petri dish was measured and defined as the diameter of the colony of elongated hyphae.

[0097] (Reference example 2) Except for adjusting the amount of deionized water to achieve a water content of 60% by mass, the diameter of the elongating hyphae colonies was measured in the same manner as in Reference Example 1.

[0098] (Comparison Example 1) Except for adjusting the amount of deionized water to achieve a water content of 40% by mass, the diameter of the elongated hyphae colonies was measured in the same manner as in Reference Example 1.

[0099] (Comparison Example 2) Except for adjusting the amount of deionized water to achieve a water content of 45% by mass, the diameter of the elongated hyphae colonies was measured in the same manner as in Reference Example 1.

[0100] The results are shown in Table 7. In the case of Reference Examples 1 and 2 with a moisture content of 60% by mass or less, and Comparative Reference Examples 1 and 2, as shown in Table 7, it was confirmed that the diameter of the elongating hyphae colonies tended to increase with increasing moisture content.

[0101] [Table 7]

[0102] Reference Experiment 1-2: Based on the results of Reference Experiment 1-1, it was suggested that higher water content leads to higher culture efficiency. Therefore, we further increased the water content and prepared solid culture media with 70% and 80% by mass as follows, and conducted experiments to compare them with the solid culture media of Reference Examples 1 and 2, which were prepared using the same method as in Reference Experiment 1-1.

[0103] (Reference example 3) Except for adjusting the amount of deionized water to achieve a water content of 70% by mass, the diameter of the elongated hyphae colonies was measured in the same manner as in Reference Example 1.

[0104] (Comparison Example 3) The culture was carried out in the same manner as in Reference Example 1, except that the amount of deionized water was adjusted to achieve a water content of 80% by mass.

[0105] The results are shown in Table 8 and Figures 5 and 6. When the moisture content was 70% by mass or less, as shown in Table 8, the diameter of the elongating hyphae colonies increased with higher moisture content, and as shown in Figure 5, the length of the mycelium increased, and the density of the mycelium tended to increase, with white hyphae clearly visible. Therefore, when the moisture content is 70% by mass or less, it is suggested that the culture efficiency of the solid culture medium increases with higher moisture content. 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, excessive moisture prevented gaps from forming in the medium, preventing the hyphae from elongating and resulting in insufficient growth. This is thought to be due to the fact that Cephaliophora sp., to which strain xsd08001 belongs, is an aerobic filamentous fungus.

[0106] [Table 8]

[0107] Based on these results, it is suggested that the water content of the solid culture medium should preferably be greater than 45% by mass and less than 80% by mass, for example, between 50% by mass and 75% by mass, or between 50% by mass and 70% by mass.

[0108] Reference Experiment 2: Effect of Humidity on Solid-State Culture We adopted the water content of 50% and 60% by mass of the solid culture medium that showed good results in Reference Experiment 1, and investigated the effect of humidity on solid culture. As Reference Experiment 1 showed that sufficiently good growth was confirmed at 90% RH humidity, we attempted cultivation at 70% RH humidity as follows to determine the lower limit.

[0109] (Reference example 4) Except for setting the humidity to 70% RH, the diameter of the elongating hyphae colonies was measured in a solid culture medium with a moisture content of 50% by mass, in the same manner as in Reference Example 1.

[0110] (Reference example 5) Except for setting the humidity to 70% RH, the diameter of the elongating hyphae colonies was measured in a solid culture medium with a water content of 60% by mass, in the same manner as in Reference Example 2.

[0111] Figure 7 shows the results after 6 days of culture. As shown in Figure 7, even when the humidity was reduced to 70% RH, good culture was possible in both the 50% by mass and 60% by mass cases. Therefore, it is suggested that the humidity in solid culture is preferably in the range of 70% RH to 90% RH, and that good culture is possible even in a wide humidity range such as 60% RH to 100% RH, or even in relatively low humidity such as 60% to 80% RH.

[0112] Based on the above results, the present invention allows for a shorter culture time compared to solid-state culture methods, reduces contamination, and produces a high-quality culture in which the mycelium extends uniformly throughout the culture medium.

Claims

1. A material for promoting plant growth, containing a dried solid culture of strain xsd08001, The aforementioned dried solid culture has a cumulative 50% particle size (D) in its volume particle size distribution. ５０ ) is 100-900 μm and the cumulative 90% particle size (D ９０ A material whose particle size is between 1000 and 5000 μm.

2. The average number of colonies per gram of the aforementioned dried solid culture is 1 × 10⁻⁶ ５ ~1 x 10 ９ The material according to claim 1, having a concentration of cfu (Colony Forming Unit) / g.

3. A method for producing a material for promoting plant growth, which includes a dried solid culture of strain xsd08001, A culture step to obtain the dried solid culture of the strain, The cumulative 50% particle size (D) in the volume particle size distribution of the aforementioned dried solid culture. ５０ ) with a particle size of 100-900 μm and a cumulative 90% particle size (D ９０ This includes an adjustment step to adjust the particle size to 1000 to 5000 μm, The aforementioned culture step is, A liquid culture step in which the aforementioned strain is subjected to liquid culture, A solid culture step is performed in which the strain cultured in the liquid culture step is cultured in solid culture, A manufacturing method comprising a drying step of drying the solid culture obtained in the solid culture step to produce the dried solid culture.

4. The manufacturing method according to claim 3, wherein the adjustment step includes a grinding step of grinding the dried solid culture that was dried in the drying step.

5. The manufacturing method according to claim 4, wherein the solid culture step is carried out using a solid culture medium containing a solid culture medium and water.

6. The manufacturing method according to claim 5, wherein the solid culture medium has a water content of 50 to 70%.

7. The method for producing the solid culture medium according to claim 5 or claim 6, wherein the solid culture medium includes wheat bran.

8. A method for culturing strain xsd08001, A liquid culture is obtained by subjecting the aforementioned strain to liquid culture, This includes obtaining a solid culture by subjecting the obtained liquid culture to solid culture, A culture method wherein the obtained liquid culture is subjected to solid culture by inoculating the liquid culture so that it is distributed throughout the solid culture medium.

9. The culture method according to claim 8, wherein the solid culture is carried out using the solid culture medium containing a solid culture medium and water.

10. The culture method according to claim 9, wherein the water content of the solid culture medium is 50 to 70%.

11. The culture method according to claim 9, wherein the solid culture medium includes wheat bran.

12. The culture method according to claim 8, wherein the liquid culture is carried out for 2 to 4 days.

13. The culture method according to claim 8, wherein the solid culture is carried out for 7 to 12 days.

14. The culture method according to claim 8, wherein the liquid culture and / or solid culture is carried out at room humidity of 60 to 90% RH.

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