Materials for plant cultivation, methods for manufacturing the same, and methods for cultivating plants

Abstract

This invention provides a plant cultivation material in which microorganisms are supported on charcoal, and which is less prone to contamination. [Solution] In a plant cultivation material in which microorganisms are supported on charcoal, the microorganisms are cellulose-assimilating fungi, and cellulose compounds are attached to the charcoal, and the total weight of low-molecular-weight sugars attached to the charcoal is 50% or less of the weight of cellulose compounds attached to the charcoal. This makes it possible to suppress the growth of microorganisms that cannot assimilate cellulose compounds, and thus makes contamination less likely to occur.

Description

[Technical Field]

[0001] This invention relates to a plant cultivation material comprising charcoal on which microorganisms are supported, and a method for producing the same. The invention also relates to a method for cultivating plants using this plant cultivation material. [Background technology]

[0002] In recent years, due to growing environmental awareness, there has been increasing interest in using plant cultivation materials that utilize microorganisms such as fungi and bacteria (so-called "biostimulants") as an alternative to chemical fertilizers and pesticides in fields such as agriculture and forestry. However, simply applying specific beneficial microorganisms to farmland or forests can sometimes fail to produce the desired effects because they lose out in competition with indigenous microorganisms already present in the land, or because the soil and other environmental conditions are not suitable for them, preventing the beneficial microorganisms from establishing themselves.

[0003] Therefore, beneficial microorganisms are being supported on porous carriers such as charcoal. For example, Patent Document 1 discloses a greening material in which various mycorrhizal fungi (VA mycorrhizal fungi, ectomycorrhizal fungi, or ericoid mycorrhizal fungi) are supported on heat-treated woody materials (e.g., charcoal, bamboo charcoal, etc.). This is said to enhance the effectiveness of beneficial microorganisms and make the material less susceptible to the influence of indigenous microorganisms. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2007-074986 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Patent Document 1 describes the following method for manufacturing greening materials: Specifically, after adding potato dextrose liquid medium (PDB medium) to heat-treated woody material and autoclaving it, mycorrhizal fungi are cultured in advance on potato dextrose agar medium (PDA medium), the required amount is inoculated onto the heat-treated woody material, and cultured at 25°C to 28°C to allow the mycelium to grow sufficiently, thereby manufacturing the greening material described in the document.

[0006] However, because PDB medium is a suitable growing medium for many microorganisms, the greening materials described in the same document, which were impregnated with PDB medium, carried a high risk of contamination (i.e., the proliferation of microorganisms other than the target beneficial microorganisms) occurring during manufacturing or storage. Using contaminated greening materials not only reduces the effectiveness of the beneficial microorganisms but may also adversely affect the growth of the plants being cultivated. For this reason, the greening materials described in the same document required maintaining a sterile environment during manufacturing and storage, which often resulted in extra effort and cost.

[0007] This invention was made to solve the above problems and provides a plant cultivation material in which microorganisms are supported on charcoal, and which is less prone to contamination. Another object of this invention is to provide a method for manufacturing this plant cultivation material. Furthermore, another object of this invention is to provide a method for cultivating plants using this plant cultivation material. [Means for solving the problem]

[0008] The above issues are, A plant cultivation material in which microorganisms are supported on charcoal, The aforementioned microorganisms include cellulose-utilizing fungi, The aforementioned charcoal has a cellulosic compound attached to it. The total weight of the low-molecular-weight sugars attached to the charcoal is 50% or less of the weight of the cellulosic compounds attached to the charcoal. Materials for plant cultivation This is solved by providing [a solution].

[0009] Here, "cellulose-assimilating fungi" refers to fungi that possess the ability to produce cellulase. The cellulase of cellulose-assimilating fungi may be produced constitutively or by induction by an inducer (e.g., a water-soluble low-molecular-weight sugar). Furthermore, "cellulose-based compounds" refers to cellulose or derivatives of cellulose.

[0010] In the microorganisms supported on charcoal, the weight ratio of cellulose-assimilating fungi is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. There is no upper limit to the weight ratio of cellulose-assimilating fungi, but it is usually 100% or less.

[0011] In this specification, "low molecular weight sugars" refers to monosaccharides, disaccharides, trisaccharides, and tetrasaccharides, as well as their derivatives (e.g., sugar alcohols). A monosaccharide is a sugar that cannot be further hydrolyzed, while disaccharides, trisaccharides, and tetrasaccharides are sugars in which two, three, and four monosaccharide molecules are linked by glycosidic bonds, respectively.

[0012] "Low molecular weight sugars" may also be fructose, glucose, sucrose, maltose, lactose, galactose, mannose, arabinose, xylose, rhamnose, ribose, fucose, trehalose, erythritol, xylitol, sorbitol, maltitol, mannitol, palatinite, maltotriitol, maltotetraitol, 4'-galactosyl lactose, raffinose, stachyose, lactosucrose, 1-kestose, nystose, fructofuranosylnistose, isomaltose, panose, isomalttriose, isomalttetraose, isomaltopentaose, maltotriose, maltotetraose, maltopentaose, inositol, cellobiose, palatinose, lactulose, galacturonic acid, glucuronic acid, xylobiose, xylotriose, glucosamine, N-acetylglucosamine, or sucralose. These low-molecular-weight sugars can be quantitatively detected by HPLC (high-performance liquid chromatography). Carboxyalkyl-modified glucose, hydroxyalkyl-modified glucose, and their salts may be excluded.

[0013] In the plant cultivation material according to the present invention, cellulose-assimilating fungi capable of assimilating cellulose compounds are supported on charcoal to which cellulose compounds are attached. In addition, the ratio of the total weight of low-molecular-weight sugars attached to the charcoal to the total weight of cellulose compounds attached to the charcoal (hereinafter referred to as the "low-molecular-weight sugar ratio") is kept to 50% or less. While almost all microorganisms can grow using low-molecular-weight sugars as a carbon source, the types of microorganisms that can grow using cellulose compounds as a carbon source are limited. For this reason, contamination is less likely to occur in the plant cultivation material according to the present invention, even without maintaining a strict sterilization state. Therefore, the effort and cost related to managing the manufacturing and storage environment can be reduced. Furthermore, the plant cultivation material according to the present invention is suitable for long-term storage and is easy to distribute to the market.

[0014] The low molecular weight sugar ratio may be 40% or less, 20% or less, 10% or less, 5% or less, or 2% or less. The lower the low molecular weight sugar ratio, the less likely contamination is to occur. There is no lower limit to the low molecular weight sugar ratio, but it is usually considered to be 0% or higher (above the detection limit).

[0015] The low-molecular-weight sugar ratio in the above-mentioned plant cultivation material can be determined, for example, by measuring the amount of low-molecular-weight sugar and the amount of cellulosic compound contained in a certain amount of the above-mentioned plant cultivation material, and then dividing the amount of low-molecular-weight sugar by the amount of cellulosic compound.

[0016] The amount of low molecular weight sugars can be determined as follows. First, the plant cultivation material is immersed in a 50 vol% ethanol solution (a solution with a volume ratio of water to ethanol of 1) to a sufficient extent, and the low molecular weight sugars are extracted by ultrasonic extraction. The intensity of the ultrasound and the temperature of the 50 vol% ethanol solution (20-60 degrees Celsius) are appropriately selected to maximize the extraction amount. The application time of ultrasound is, for example, 1 hour, but the application time should be extended by another hour until the change in the extraction amount is 5% or less. Next, the 50 vol% ethanol solution in which the plant cultivation material was immersed after ultrasonic extraction is filtered through filter paper, and the resulting filtrate is adjusted to an appropriate concentration by evaporation, concentration, and hydrolysis to prepare the filtrate for HPLC. Next, the above HPLC filtrate is subjected to HPLC (High Performance Liquid Chromatography) to detect various low molecular weight sugars, such as fructose and glucose. By comparing the detection values ​​from HPLC with those of a reference solution (a solution of specified concentration), the concentrations of various low-molecular-weight sugars in the HPLC filtrate can be determined. The concentrations of various low-molecular-weight sugars in the filtrate can be calculated from the degree of concentration and dilution when preparing the HPLC filtrate from the HPLC filtrate. Furthermore, the amount of low-molecular-weight sugars adhering to the plant cultivation material can be calculated from the weight of the plant cultivation material and the weight of the filtrate obtained by filtration. The HPLC detector may be a trend detector. For example, a Shimadzu LC-40D can be used for HPLC.

[0017] The amount of cellulosic compounds can be determined as follows: First, the plant cultivation material is immersed in an amount of water, DMF, DMAc, or a mixture thereof (extraction solvent) sufficient to fully immerse it, and the cellulosic compounds are extracted by ultrasonic extraction. The extraction solvent is selected to yield the highest amount of cellulosic compounds adhering to the plant cultivation material. The ultrasonic intensity and the temperature of the extraction solvent (20-60 degrees Celsius) are appropriately selected to maximize the extraction amount. The ultrasonic application time is, for example, 1 hour, but the application time should be extended by another hour to a point where the change in the extraction amount is 5% or less. Next, the extraction solvent in which the plant cultivation material was immersed after ultrasonic extraction is filtered through filter paper, and the resulting filtrate is adjusted to an appropriate concentration by evaporation, concentration, and dilution with the extraction solvent to obtain the filtrate for GPC. For the above dilution, any solvent that dissolves the extract can be used, which is commonly used in GPC (Gel Permeation Chromatography). For example, if the cellulosic compound is carboxymethylcellulose, 0.5M Na2SO4 water may be used. Next, the GPC filtrate is subjected to a GPC to detect the cellulosic compound. By comparing the GPC detection value with the detection value of the reference solution (a solution of specified concentration), the concentration of the cellulosic compound contained in the GPC filtrate can be determined. The concentration of various cellulosic compounds in the filtrate can be calculated from the degree of concentration and dilution when preparing the GPC filtrate from the filtrate. Furthermore, the cellulosic compound contained in the plant cultivation material can be calculated from the weight of the plant cultivation material and the weight of the filtrate obtained by filtration. The GPC detector may be a differential refractometer (RI).

[0018] In the materials for plant cultivation according to the present invention, as long as the carbon is a porous body mainly composed of carbon, its type and raw material are not particularly limited. As the carbon, it is preferable to adopt a solid substance produced by heating the raw material under an oxygen concentration controlled at a non-combustible level. The heating temperature of the raw material of the carbon is preferably 250°C or higher and 600°C or lower, and more preferably 300°C or higher and 500°C or lower. The carbon may be, for example, so-called biochar obtained by processing plants at a low oxygen concentration and high temperature. The biochar may be rice husk charcoal, rice straw charcoal, bamboo charcoal, sweet potato vine charcoal, sugarcane pulp charcoal, corn charcoal, bark charcoal, charcoal, etc. Only one type of carbon can be used, or two or more types can be used in combination.

[0019] The materials for plant cultivation according to the present invention is a method for manufacturing materials for plant cultivation in which microorganisms are carried on carbon, the microorganism is a cellulose-assimilating fungus, a liquid culture step of obtaining a culture solution of the cellulose-assimilating fungus by culturing the cellulose-assimilating fungus in a liquid medium containing a cellulose-based compound, a loading step of loading the cellulose-assimilating fungus on the carbon by bringing the culture solution obtained in the liquid culture step into contact with the carbon and includes the total weight of the low molecular weight sugars contained in the liquid medium is 50% or less of the weight of the cellulose-based compound contained in the liquid medium Method for manufacturing materials for plant cultivation can be manufactured by.

[0020] In the above manufacturing method, the cellulose-assimilating fungus is cultured in a liquid medium containing a cellulose-based compound, and the total weight of the low molecular weight sugars contained in the liquid medium is set to 50% or less of the weight of the cellulose-based compound contained in the liquid medium. Thereby, not only the materials for plant cultivation themselves but also the liquid medium and the culture solution used for their production can be made less likely to cause contamination. For this reason, the labor and cost involved in the production of the materials for plant cultivation can be suppressed.

[0021] In particular, as with the greening material in the aforementioned reference 1, conventional microbial materials require sterilization of the porous carrier before the microorganisms are placed on it to prevent contamination. However, with the plant cultivation material according to the present invention, contamination is less likely to occur even without this sterilization process. Since sterilization is usually performed under high pressure and high temperature using an autoclave or the like, eliminating the need for this process reduces the equipment costs and heating costs associated with sterilization.

[0022] In the plant cultivation material according to the present invention, it is preferable that the cellulosic compound is water-soluble. This is because if a cellulosic compound that is insoluble or sparingly soluble in water is used, it may become difficult to adhere the cellulosic compound to the charcoal. Furthermore, in the liquid culture medium used in the manufacture of the plant cultivation material, the cellulosic compound may not dissolve and disperse sufficiently, such as by forming clumps, making it difficult to ensure many opportunities for contact between the cellulosic compound and cellulose-assimilating fungi, and thus making it difficult to efficiently grow the cellulose-assimilating fungi. An example of a water-soluble cellulosic compound is carboxymethylcellulose (CMC).

[0023] In the plant cultivation material according to the present invention, it is preferable that the cellulose-assimilating fungus is a non-mycorrhizal filamentous fungus. Unlike mycorrhizal fungi, non-mycorrhizal filamentous fungi do not require a symbiotic relationship with a host for their proliferation. In other words, they can be propagated in an isolated state as clones. Therefore, by using a non-mycorrhizal filamentous fungus as the cellulose-assimilating fungus, it is possible to propagate the cellulose-assimilating fungus with simple processes and equipment, thereby reducing the effort and cost of manufacturing plant cultivation material. Examples of such cellulose-assimilating fungi include non-mycorrhizal filamentous fungi belonging to the genus Tricoderma.

[0024] When the cellulose-utilizing fungus is a non-mycorrhizal filamentous fungus, it is preferable that the cellulose-utilizing fungus is supported on the charcoal in the form of mycelium. Conventionally, in plant cultivation materials using non-mycorrhizal filamentous fungi, conidia or sporophytes have been used for purposes such as improving preservation. However, since conidia and sporophytes are dormant in non-mycorrhizal filamentous fungi, it is difficult to obtain the desired effect as is. For this reason, in plant cultivation materials using conidia or sporophytes, it was necessary to germinate the conidia or sporophytes and grow the mycelium after applying them to, for example, soil, but this was time-consuming, and under certain conditions, the mycelium may not grow well. In contrast, in plant cultivation materials in which cellulose-utilizing fungi are supported on charcoal in the form of mycelium, the mycelium can grow smoothly upon application, making it easier to obtain the desired effect quickly.

[0025] The plant cultivation material according to the present invention can be used in a plant cultivation method that involves growing plants on a vegetation substrate containing the material. [Effects of the Invention]

[0026] As described above, the present invention makes it possible to provide a plant cultivation material in which microorganisms are supported on charcoal, and which is less prone to contamination. Furthermore, it is also possible to provide a method for manufacturing this plant cultivation material. Moreover, it is also possible to provide a method for cultivating plants using this plant cultivation material. [Modes for carrying out the invention]

[0027] Preferred embodiments of the present invention will be described in more detail.

[0028] 1. Overview The plant cultivation material of this embodiment is a material used when cultivating plants that are artificially grown (hereinafter sometimes referred to as "cultivated plants"). This plant cultivation material consists of charcoal (hereinafter sometimes referred to as "supporting charcoal") on which a specific type of microorganism (hereinafter sometimes referred to as "supported microorganism") is supported. The supporting charcoal has a substance attached to it that the supported microorganism can use as a carbon source. By supporting the supported microorganism on supporting charcoal to which the carbon source is attached, it becomes easier to maintain the supported microorganism in an activated state, and the long-term storage of the plant cultivation material can be improved.

[0029] However, if a substance that is easily assimilated by other microorganisms is used as a carbon source, other (unintended) microorganisms besides the supported microorganisms may proliferate on the plant cultivation material, potentially leading to contamination. For this reason, the plant cultivation material of this embodiment includes cellulose-assimilating fungi (fungi that can proliferate using cellulosic compounds as a carbon source) as the supported microorganisms, and employs cellulosic compounds as the carbon source attached to the supporting charcoal. In addition, the ratio of the total weight of low-molecular-weight sugars (monosaccharides, disaccharides, trisaccharides, and tetrasaccharides and their derivatives (e.g., sugar alcohols, etc.)) attached to the supporting charcoal to the total weight of cellulosic compounds attached to the supporting charcoal (hereinafter referred to as the "low-molecular-weight sugar ratio") is set to 50% or less. Since the number of microorganisms that can assimilate cellulosic compounds is overwhelmingly smaller than the number of microorganisms that can assimilate low-molecular-weight sugars, adopting the above configuration makes contamination of the plant cultivation material less likely to occur.

[0030] Furthermore, the definition of "low molecular weight sugars" can exclude monosaccharides, disaccharides, trisaccharides, or tetrasaccharides that have undergone chemical modification of their hydroxyl groups (for example, those in which an alkylcarboxyl group or alkylhydroxyl group is ether-bonded to the hydroxyl group). This is because the number of microorganisms capable of assimilating hydroxyl-modified monosaccharides, disaccharides, trisaccharides, or tetrasaccharides is smaller than the number of microorganisms capable of assimilating monosaccharides, disaccharides, trisaccharides, and tetrasaccharides themselves, or sugar alcohols derived from them.

[0031] 2. Manufacturing method The plant cultivation material of this embodiment can be manufactured by going through a liquid culture process, a dispersion process, a loading process, and a drying process. Each of these processes will be described in detail below.

[0032] 2-1.Liquid culture process The liquid culture process involves culturing cellulose-assimilating fungi in a liquid medium containing cellulosic compounds to obtain a culture solution of cellulose-assimilating fungi. The liquid medium used is primarily water (base material) and contains a carbon source. Examples of carbon sources include cellulosic compounds and low-molecular-weight sugars, and the liquid medium used in the liquid culture process in this embodiment must contain cellulosic compounds.

[0033] On the other hand, the total weight of low-molecular-weight sugars in the liquid medium should be 50% or less of the weight of cellulosic compounds in the liquid medium. This not only makes it less likely for the plant cultivation materials after production to be contaminated, but also makes it less likely for the liquid medium itself and the culture solution during the liquid culture process to be contaminated. Preferably, the total weight of low-molecular-weight sugars in the liquid medium should be 40% or less of the total weight of cellulosic compounds in the liquid medium. The total weight of low-molecular-weight sugars in the liquid medium can also be 20% or less, 10% or less, 5% or less, or 2% or less of the total weight of cellulosic compounds in the liquid medium. There is no lower limit to the total weight of low-molecular-weight sugars in the liquid medium, but it is usually 0% by weight or more.

[0034] Generally, when culturing cloned microorganisms in liquid culture, it is common practice to sterilize the liquid culture medium before use (e.g., autoclave sterilization or filter sterilization) to prevent contamination. However, in this embodiment, by using only cellulosic compounds as the carbon source in the liquid culture medium, contamination of the liquid culture medium and culture solution can be reduced without sterilization of the liquid culture medium. This reduces costs and labor.

[0035] The total amount of carbon source added to the liquid culture medium (total amount of cellulosic compounds and low molecular weight sugars; the same applies hereinafter) is not limited, but if it is too little, it may be difficult to increase the growth efficiency of cellulose-assimilating fungi, and if it is too much, the viscosity of the liquid culture medium tends to increase, which may make it difficult for the culture solution of cellulose-assimilating fungi to permeate the carbon used for support in the subsequent support process. For this reason, the total amount of carbon source added to the liquid culture medium is preferably 0.2 parts by weight or more and 10 parts by weight or less, more preferably 0.5 parts by weight or more and 5 parts by weight or less, and more preferably 1 part by weight or more and 3 parts by weight or less, per 100 parts by weight of water, which is the base material.

[0036] The liquid culture medium may contain a nitrogen source. Examples of nitrogen sources include inorganic nitrogen (ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium sulfate, etc.) and organic nitrogen (various amino acids, proteins). The liquid culture medium may contain only one type of nitrogen source, or two or more types. The amount of nitrogen source added is not limited and varies depending on the type of nitrogen source, but is preferably around 0.001 to 1 part by weight, and more preferably around 0.01 to 0.5 parts by weight, per 100 parts by weight of water (the base material).

[0037] The liquid culture medium may contain organic salts and inorganic salts. Examples of organic and inorganic salts include, but are not limited to, phosphates, sulfates, chlorides, acetates, and carbonates of magnesium, calcium, sodium, potassium, iron, manganese, cobalt, zinc, etc. Vitamins such as thiamine may also be added to the liquid culture medium. The liquid culture medium may contain only one type of organic salt, inorganic salt, or vitamin, or two or more types. The amount of organic salt, inorganic salt, or vitamin added to the liquid culture medium varies depending on the type and is not limited, but is preferably about 0.001 to 1 part by weight, and more preferably about 0.01 to 0.5 parts by weight, per 100 parts by weight of water, which is the base material.

[0038] In the liquid culture process, cellulose-assimilating fungi are typically cultured under aerobic conditions. That is, cellulose-assimilating fungi isolated and stored as clones on solid culture media are either scraped off with a platinum loop or the like and inoculated into liquid culture media, or the solid culture media is cut out and placed in liquid culture media, followed by shaking culture or stirring culture. Shaking culture can be performed, for example, using a shaker and a container to be shaken, such as a test tube or flask. Stirring culture can be performed, for example, using a spinner flask and a stirrer, a jar fermenter, or a culture tank. The culture temperature in the liquid culture process is not limited. In some embodiments, the culture temperature is 10°C to 35°C, and in some embodiments, it is 15°C to 30°C.

[0039] In this embodiment, during the liquid culture process, only one type of cellulose-assimilating fungus is added to one batch of liquid culture medium, and liquid culture is performed. This can sometimes efficiently grow the desired cellulose-assimilating fungus. In another embodiment, two or more types of cellulose-assimilating fungi can be added to one batch of liquid culture medium. That is, two or more types of cellulose-assimilating fungi can be co-cultured in one batch of liquid culture medium.

[0040] The culture time in the liquid culture process is not limited. In some embodiments, the culture time is between 1 day (24 hours) and 10 days, and in some embodiments, it is between 2 days and 5 days. The culture time in the liquid culture process can also be determined by the turbidity and absorbance of the culture medium. For example, the culture can be continued until the turbidity of the culture medium or clumps of mycelium can be clearly observed visually. The culture time can also be determined by the absorbance of the culture medium. That is, for example, a portion of the culture medium during cultivation can be sampled and dispersed in the same manner as in the dispersion process described later to prepare a mycelial suspension, and the culture can be terminated when the absorbance of the mycelial suspension at a predetermined wavelength (e.g., 600 nm, 660 nm, 700 nm, etc.) reaches a predetermined value (e.g., 0.5, 1.0, 2.0, 3.0, etc.) or higher.

[0041] 2-2.Dispersion process At the end of the liquid culture process, most of the cellulose-assimilating fungi are in the form of mycelium. The dispersion process involves cutting and dispersing (suspending) the mycelium of the cellulose-assimilating fungi contained in the culture medium obtained in the liquid culture process. Depending on the culture conditions in the liquid culture process and the type of cellulose-assimilating fungi, the mycelium may become entangled with each other, resembling a ball of fur or a marimo moss ball, making it difficult to load onto the charcoal in the subsequent loading process. Dispersing the mycelium through the dispersion process makes it easier to load the mycelium onto the charcoal.

[0042] The method of cutting the mycelium (dispersion method) in the dispersion step is not limited. The dispersion step can be carried out, for example, by passing the culture solution obtained in the liquid culture step through a mixer, blender, mill, homogenizer, etc. Note that in other embodiments, this dispersion step can be omitted.

[0043] 2-3. Loading process The loading process involves loading cellulose-assimilating fungi onto the loading charcoal by contacting the culture solution obtained in the liquid culture process with the loading charcoal. This process not only loads the cellulose-assimilating fungi onto the loading charcoal but also allows cellulose-based compounds contained in the culture solution to adhere to the loading charcoal. The method of contacting the loading charcoal with the culture solution is not particularly limited. The loading process can be carried out, for example, by immersing the loading charcoal in the culture solution, or by pouring, coating, or spraying the culture solution onto the loading charcoal.

[0044] As mentioned earlier, in conventional microbial materials, including the greening materials described in Reference 1, sterilization of the porous carrier before microorganisms are placed on it was performed to prevent contamination. However, in the manufacturing method of the plant cultivation material of this embodiment, the charcoal used for support is not sterilized before microorganism placement. This is because, in this embodiment, cellulosic compounds are used as the main carbon source in the liquid culture medium (culture solution), and the content of low-molecular-weight sugars is kept low, making contamination less likely even without sterilization. This reduces the effort and cost of manufacturing the plant cultivation material.

[0045] 2-4.Drying process The drying process involves removing the supported charcoal, which has undergone the loading process, from the culture medium of the cellulose-assimilating fungus and drying it. The supported charcoal removed from the culture medium can be dried as is, or it can be washed with a washing solution (for example, a liquid culture medium with the same composition as that used in the liquid culture process) before drying. The drying method is not limited, and methods such as air drying or static drying can be employed. The drying location is also not limited, and can be used in a drying oven, indoors, or in the shade outdoors. In this embodiment, for the same reasons already mentioned, contamination can be minimized in the drying process even without strictly sterilizing the drying environment. This reduces equipment costs and labor.

[0046] The drying process is usually carried out at room temperature (around 20-30°C). The humidity during the drying process is not limited, but if it is too low, the cellulose-assimilating fungi supported on the charcoal may weaken, and if it is too high, the drying process may take too long. For this reason, the humidity during the drying process is preferably around 40-80%, and more preferably around 50-70%. Once the drying process is complete, the production of the plant cultivation material in this embodiment is complete.

[0047] 3. Cellulose compounds The specific type of cellulosic compound is not limited to cellulose or a derivative of cellulose. However, if a cellulosic compound that is insoluble or poorly soluble in water is used, it may be difficult to adhere the cellulosic compound to the charcoal. Furthermore, in the liquid culture medium used in the aforementioned method for producing plant growth materials, the cellulosic compound may not dissolve and disperse sufficiently, such as by forming clumps, making it difficult to ensure sufficient contact opportunities between the cellulosic compound and cellulose-assimilating fungi, and thus making it difficult to efficiently grow the cellulose-assimilating fungi.

[0048] Therefore, it is preferable to use a water-soluble cellulosic compound. More specifically, it is preferable to use a cellulosic compound with a solubility (solubility in 100g of pure water at 1 atmosphere and 20°C; the same applies hereinafter) of 0.1g / 100gH2O or higher, more preferably 0.5g / 100gH2O or higher, and even more preferably 1g / 100gH2O or higher.

[0049] When using a water-soluble cellulosic compound, the viscosity of the cellulosic compound when dissolved in water is not limited. However, if the viscosity of the aqueous solution of the cellulosic compound is too high, the culture solution may not permeate the charcoal used for support in the loading process of the plant cultivation material manufacturing method described above, and the cellulosic-utilizing fungi may not be able to be loaded deep into the charcoal. For this reason, the viscosity of a 1% by weight aqueous solution of the cellulosic compound (viscosity measured at 25°C using a rotational viscometer in accordance with JIS Z8803; the same applies hereinafter) is preferably 2000 mPa·s or less, more preferably 1000 mPa·s or less, and even more preferably 500 mPa·s or less. The lower limit of the viscosity of a 1% by weight aqueous solution of the cellulosic compound is not limited, but is usually 10 mPa·s or more.

[0050] Examples of water-soluble cellulosic compounds include cellulose ethers. Examples of cellulose ethers include carboxymethylcellulose (CMC), carboxyethylcellulose (CEC), carboxymethylhydroxyethylcellulose (CMHEC), methylcellulose (MC), ethylcellulose (EC), ethylmethylcellulose (EMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxyethylmethylcellulose (HEMC), and hydroxypropylmethylcellulose (HPMC). Cellulosic compounds can be used individually or in combination of two or more. In this embodiment, carboxymethylcellulose (CMC) is used as the cellulosic compound.

[0051] When CMC is used as the cellulosic compound, the degree of substitution (degree of etherification) of CMC is not limited, but if it is too low, it becomes difficult to increase the water solubility of CMC. For this reason, the degree of substitution of CMC is preferably 0.5 or higher, and more preferably 0.6 or higher. On the other hand, if the degree of substitution of CMC is too high, CMC becomes difficult to decompose by cellulase, making it difficult to utilize as a carbon source for cellulose-assimilating fungi. For this reason, the degree of substitution of CMC is preferably 1.5 or lower, and more preferably 1.0 or lower.

[0052] 4. Cellulose-assimilating fungi Cellulose-assimilating fungi are not limited to any specific type of fungus as long as they possess the ability to produce cellulase. Another definition of cellulose-assimilating fungi is, for example, "fungi that show statistically significant growth when cultured for one week with shaking in a sterile flask at a temperature of 25°C under late-stage conditions using a sterile liquid medium containing only CMC as a carbon source."

[0053] As cellulose-assimilating fungi, it is preferable to use non-mycorrhizal filamentous fungi. This is because, as already mentioned, unlike mycorrhizal fungi, non-mycorrhizal filamentous fungi can be grown (cultured) independently as clones, even without a symbiotic relationship with a host. Examples of such cellulose-assimilating fungi include non-mycorrhizal filamentous fungi belonging to the genus Tricoderma.

[0054] As cellulose-assimilating fungi, one type of fungus may be used, or a combination of two or more types of fungi may be used. When two or more types of fungi are included as cellulose-assimilating fungi, these fungi may belong to the same order or to two or more different orders. Furthermore, the fungi may belong to the same family or to two or more different families. In addition, the fungi may belong to the same genus or to two or more different genera.

[0055] When a non-mycorrhizal filamentous fungus is employed as the cellulose-utilizing fungus, it is preferable that the cellulose-utilizing fungus is supported on the carrier carbon in the form of mycelia. More specifically, among the cellulose-utilizing fungi supported on the carrier carbon, the proportion occupied by the mycelia (hereinafter sometimes expressed as "mycelium ratio R sc ").) is preferably 80% or more. Thereby, as already described, when applying the plant-growing material, the mycelia of the cellulose-utilizing fungus can be easily grown smoothly, and the desired effect by the cellulose-utilizing fungus can be easily obtained at an early stage. The mycelium ratio R m is more preferably 9% or more, and even more preferably 95% or more. The upper limit of the mycelium ratio R m is not limited, but is usually 100% or less.

[0056] That the cellulose-utilizing fungus supported on the carrier carbon is mycelia can be specified by observing the plant-growing material with an optical microscope. Also, the mycelium ratio R m is obtained by photographing the plant-growing material through an optical microscope, and the area (number of pixels) S m occupied by the mycelia and the area (number of pixels) S sc occupied by the sporophyte and / or conidiophore (hereinafter sometimes referred to as "sporophyte etc.") in the photographed field of view, and can be calculated by the following formula. R m =S m / (S m +S sc )×100

[0057] As cellulose-assimilating fungi, fungi that can exert beneficial effects on the growth of cultivated plants are typically employed. Examples of "beneficial effects on the growth of cultivated plants" include, for example, growth-promoting effects that can accelerate the growth of cultivated plants, control effects that can control disease-causing fungi and / or pests of cultivated plants, diversity-enhancing effects that can increase the diversity of the biome in the rhizosphere of cultivated plants, resistance-enhancing effects that can increase the resistance of cultivated plants to abiotic stress (examples include, but are not limited to, drought, heat damage, salt damage, cold damage, frost damage, damage from reactive oxygen species, hail damage, wind damage, and phytotoxicity from pesticides, etc.), and biome-modifying effects that can alter the biome in the rhizosphere of cultivated plants.

[0058] The growth-promoting effect of cellulose-assimilating fungi on specific cultivated plants can be determined as follows: Specifically, when a culture solution of cellulose-assimilating fungi cultured in a liquid medium containing only cellulosic compounds as a carbon source is applied to cultivated plants after a predetermined period, and a control group is formed by applying a liquid medium without cellulose-assimilating fungi and allowing a predetermined period to pass, if the weight or length of all or part of the plant body in the application group is statistically significantly larger than that of the control group, then the cellulose-assimilating fungi are considered to have a growth-promoting effect on those cultivated plants. This growth-promoting effect can be the promotion of growth of the entire plant body or the promotion of growth of only a part of the plant body. Examples of growth-promoting effects include a root-enhancing effect that promotes rooting and increases root mass, an above-ground growth-promoting effect that promotes the growth of the above-ground parts of the plant, and an effect that suppresses pests and diseases by altering the composition of the biome within the rhizosphere of the plant.

[0059] The effectiveness of cellulose-assimilating fungi in controlling specific pathogenic fungi can be determined as follows (hereinafter, the fungi to be controlled may be referred to as "target pathogenic fungi"). Specifically, when cellulose-assimilating fungi and target pathogenic fungi are cultured side-by-side on a solid medium, if the growth and hyphal elongation of the target pathogenic fungi are inhibited, and / or the target pathogenic fungi are killed, then the cellulose-assimilating fungi are considered to have an effective control effect against that pathogenic fungi. Alternatively, a culture solution prepared by culturing cellulose-assimilating fungi in a liquid medium containing only cellulosic compounds as a carbon source is applied to soil containing the target pathogen (for example, about 30-80 ml of culture solution per 200 ml of soil containing the target pathogen; in some embodiments, 50 ml). The microbial flora in the soil is then analyzed using amplicon sequencing before application and after a predetermined period (for example, about 1-3 weeks; in some embodiments, about 2 weeks). If the relative amount of DNA of the target pathogen is significantly reduced, the cellulose-assimilating fungi can be considered to have a control effect against that target pathogen.

[0060] The target pathogen is not limited to any particular type, as long as it can cause disease in at least one type of plant. The target pathogen can be a fungus (e.g., filamentous fungi), a bacterium, a virus, or an archaea. Examples of fungal target pathogens include, but are not limited to, those that cause diseases such as Fusarium wilt, Verticillium wilt, Late blight, Black spot disease, Sclerotinia rot, Rust, Damping-off, Anthracnose, Fusarium wilt, Blight, Clubroot, White spot disease, Verticillium wilt, Downy mildew, Root rot, Dry rot, Black rot Sclerotinia rot, and Basin rot (e.g., Sweet potato basal rot). Examples of fungal target pathogens include Fusarium, Sclerotium cepivorum, Verticillium, Rhizoctonia, Pythium, Plasmodiophora brassicae, Phytophthora, and Pectobacterium. Examples include, but are not limited to, *carotovorum*, *Alternaria*, *Pseudoperonospora*, *Peronospora*, *Plasmopara*, *Bremia*, *Albugo*, *Puccinia*, *Botrytis*, *Colletotrichum*, *Glomerella*, *Cercospora*, *Cercosporella*, *Mycovellosiella*, and *Diaporthe* (e.g., *Diaporthe destruens*). Examples of bacterial target pathogens include, but are not limited to, the causative agents of diseases such as bacterial wilt, fusarium wilt, black rot, soft rot, and decay. Examples of viral target pathogens include, but are not limited to, the causative viruses of diseases such as viral mosaic, yellowing necrosis, rice stripe blight, and dwarf disease.

[0061] The effectiveness of cellulose-assimilating fungi in controlling specific pests can be determined as follows (hereinafter, the insects to be controlled may be referred to as "target pests"). Specifically, live target pests are immersed for about 15 seconds in a culture medium in which cellulose-assimilating fungi are cultured using only cellulosic compounds as a carbon source (this group is called the fungal contact group), and those immersed for the same amount of time in a liquid medium without cellulose-assimilating fungi are called the control group. After rearing each group of target pests for a period of time (about 1-2 weeks) after immersion, if the mortality rate of the fungal contact group is statistically significantly higher than that of the control group, then the cellulose-assimilating fungi can be considered to have an effective control effect against that target pest.

[0062] The target pest is not limited to any particular type of plant, as long as it can cause damage to at least one type of plant (e.g., feeding, sap-sucking, disease, etc.). The target pest may be, for example, an organism belonging to the phylum Nematoda, phylum Mollusca, or phylum Arthropoda, but is not limited to these. The target pest may be, for example, an organism belonging to the order Thysanoptera, Hemiptera, Coleoptera, Acari, Lepidoptera, Diptera, Orthoptera, Hymenoptera, Porcelliophora, Stichodactyla, Isopoda, etc.

[0063] The biodiversity-enhancing effect of cellulose-assimilating fungi can be identified as follows: A culture solution in which cellulose-assimilating fungi are cultured in a liquid medium containing only cellulosic compounds as a carbon source is applied to test soil (for example, soil in a planting site for cultivated plants; the same applies hereinafter). (For example, about 30-80 ml of culture solution per 200 ml of test soil; in some embodiments, 50 ml.) The microbial community in the test soil is analyzed using amplicon sequencing before application and after a predetermined period (for example, about 1-3 weeks; in some embodiments, about 2 weeks). If the diversity of the microbial community in the test soil (e.g., number of genera) is significantly increased, then the cellulose-assimilating fungi are considered to have a biodiversity-enhancing effect.

[0064] The effectiveness of cellulose-assimilating fungi in enhancing the resistance of specific cultivated plants can be determined as follows: Cultivated plants that have been cultured in a liquid medium containing only cellulosic compounds as a carbon source and have been treated for a predetermined period are designated as the treatment group, and cultivated plants that have been treated in a liquid medium without cellulose-assimilating fungi and have been treated for a predetermined period are designated as the control group. When both the treatment group and the control group are subjected to approximately the same abiotic stress for approximately the same period, if the proportion of healthy individuals (not withered, diseased, or weakened) in the treatment group is statistically significantly higher than the proportion of healthy individuals in the control group, then the cellulose-assimilating fungi are considered to have an effective resistance-enhancing effect on those cultivated plants.

[0065] The biocommunity-modifying effect of cellulose-assimilating fungi can be identified as follows: A culture solution of cellulose-assimilating fungi cultured in a liquid medium containing only cellulosic compounds as a carbon source is applied to test soil (for example, about 30-80 ml of culture solution per 200 ml of test soil; 50 ml in some embodiments). The microbial community in the test soil is then analyzed using amplicon sequencing before and after a predetermined period (for example, about 1-3 weeks; about 2 weeks in some embodiments). If the difference in fungal community composition at the genus level before and after application exceeds a predetermined threshold value (hereinafter sometimes referred to as the "biocommunity modification threshold value") based on the Bray-Curtis index beta diversity, then the cellulose-assimilating fungi are considered to have a biocommunity-modifying effect. Primers targeting at least a portion of the fungal ITS (Internal Transcribed Spacer) region can be used for amplicon sequencing analysis. For example, 0.3, 0.35, 0.4, 0.45, etc., can be adopted as standard values ​​for biome modification.

[0066] 5.Applications Plant cultivation materials can be used in plant cultivation methods that involve growing plants on a vegetation substrate containing these materials. The vegetation substrate is not limited to any specific type as long as it allows the plant to take root. Examples of vegetation substrates include soil (whether artificially mixed or naturally occurring soil), sand, mud, porous materials, gels, sols, and liquids.

[0067] There are no particular limitations on the timing of applying plant growth materials to cultivated plants. Plant growth materials can be applied, for example, by germinating the plants in soil containing the plant growth materials, adding the plant growth materials to pots in which seedlings are planted, planting seedlings in soil containing the plant growth materials, or adding the plant growth materials to soil in which plants are planted. [Examples]

[0068] To verify the susceptibility of plant cultivation materials to contamination, an experiment was conducted as follows.

[0069] First, an experimental liquid culture medium was prepared by adding 2 parts by weight of a carbon source to 100 parts by weight of water as the base material. As the carbon source, CMC (chemical reagent "carboxymethylcellulose sodium (093-01335)" manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and / or sucrose, a low molecular weight sugar, were used, and five types of experimental liquid culture media were prepared so that the ratio of low molecular weight sugars in the total carbon source (sucrose weight divided by CMC weight) was 0%, 10%, 20%, 50%, and 150%, respectively.

[0070] The five types of experimental liquid culture media described above were each placed in Erlenmeyer flasks, inoculated with cellulose-assimilating fungi belonging to the genus Trichoderma, and cultured with shaking at 25°C to prepare experimental culture solutions. The amount of experimental liquid culture medium was such that even if the inoculated cellulose-assimilating fungi were to grow sufficiently, a sufficient amount of CMC and low-molecular-weight sugars would remain. The five types of experimental culture solutions obtained were each mixed in a mixer to obtain mycelial suspensions. Unsterilized support charcoal (obtained by treating rice husks at 400°C, in a size that fits within a circle with a diameter of 6 mm to 8 mm, and stored in an open-type bag for one month) was immersed in each of the five obtained mycelial suspensions for one hour to support the cellulose-assimilating fungi on the charcoal. After that, the support charcoal was removed from the mycelial suspensions and air-dried at approximately 20°C and a relative humidity of about 50% to obtain five types of experimental plant cultivation materials. Subsequently, the experimental plant cultivation material was left to stand for two days in an open-type container (without a lid and open at the top) in a room at approximately 20°C and 50% relative humidity. No sterilization was performed during the process from preparing the experimental culture solution to obtaining the experimental cultivation material.

[0071] Five 1 / 4 PDA culture media (Accudia potato dextrose agar medium, Shimadzu Diagnostics Corporation) were prepared in a sterile state in petri dishes (5.5 cm in diameter). Four of the five types of experimental plant growth materials prepared as described above were placed approximately in the center of each petri dish, and the dishes were covered and left undisturbed for two weeks at a temperature of 30°C and a relative humidity of 70%. After that, the growth of mycelium from the experimental plant growth materials in the petri dishes was observed with the naked eye.

[0072] It was found that the lower the ratio of low-molecular-weight sugars in the experimental liquid culture medium, the higher the proportion of the area on the bottom surface of the petri dish that exhibited the characteristic growth appearance of the cellulose-utilizing bacteria (Trichoderma species), such as dark green color. When the low-molecular-weight sugar ratio was 150%, the cellulose-utilizing bacteria occupied about half of the bottom surface of the petri dish, but in the remaining half, bacteria of a different color, presumably caused by other bacteria, were prominently observed, indicating contamination. In contrast, when the low-molecular-weight sugar ratio was 50%, the cellulose-utilizing bacteria occupied almost the entire bottom surface of the petri dish, and only a small amount of bacteria of a different color, presumably caused by other bacteria, were observed, and were only recognizable as scattered dots. When the low-molecular-weight sugar ratio was 20%, the cellulose-utilizing bacteria occupied almost the entire bottom surface of the petri dish, and when the low-molecular-weight sugar ratio was 10%, the cellulose-utilizing bacteria occupied the entire area.

Claims

1. A plant cultivation material in which microorganisms are supported on charcoal, The aforementioned microorganisms include cellulose-utilizing fungi, The aforementioned charcoal has a cellulosic compound attached to it. The total weight of the low-molecular-weight sugars attached to the charcoal is 50% or less of the weight of the cellulosic compounds attached to the charcoal. Materials for plant cultivation.

2. The plant cultivation material according to claim 1, wherein the cellulose compound is water-soluble.

3. The plant cultivation material according to claim 2, wherein the cellulosic compound is carboxymethylcellulose.

4. The plant cultivation material according to claim 1, wherein the cellulose-utilizing fungus is a non-mycorrhizal filamentous fungus.

5. The plant cultivation material according to claim 4, wherein the cellulose-assimilating fungus is a non-mycorrhizal filamentous fungus belonging to the genus Trichoderma.

6. The plant cultivation material according to claim 4, wherein a cellulose-assimilating fungus is supported on the charcoal in the form of mycelium.

7. A method for manufacturing a plant cultivation material in which microorganisms are supported on charcoal, The aforementioned microorganism is a cellulose-utilizing fungus. A liquid culture step to obtain a culture solution of cellulose-assimilating fungi by culturing cellulose-assimilating fungi in a liquid medium containing cellulose compounds, A loading step is to bring the culture solution obtained in the liquid culture step into contact with the charcoal, thereby supporting the cellulose-utilizing fungi onto the charcoal. Includes, The total weight of low-molecular-weight sugars contained in the liquid culture medium is 50% or less of the weight of cellulosic compounds contained in the liquid culture medium. A method for manufacturing plant cultivation materials.

8. A method for growing plants, comprising growing plants on a vegetation substrate containing a plant cultivation material according to any one of claims 1 to 6 or a plant cultivation material manufactured by the manufacturing method according to claim 7.