Microbial preparations that improve the inoculation rate of matsutake mushroom mycelium and their applications

Abstract

The present invention provides a formulation that improves the inoculation rate of matsutake mushroom mycelium into trees, and a method for producing matsutake mushroom mycelium-inoculated seedlings. [Solution] A microbial preparation is provided that includes the Paenibacillus cineris strain and the Micrococcus sp. strain, which improves the inoculation rate of Matsutake mycelium into trees. [Effects] The microbial preparation or composition according to the present invention does not inhibit the growth of matsutake mycelium and has the effect of suppressing harmful bacteria that inhibit the growth of matsutake mycelium. Furthermore, it has the effect of significantly improving the survival rate of matsutake mycelium-inoculated seedlings. According to the method for producing matsutake mycelium-inoculated seedlings of the present invention, it is possible to produce a large quantity of inoculated seedlings with a high inoculation rate of matsutake mycelium and a high survival rate after inoculation, and to supply them to farmers in large quantities.

Description

Technical Field

[0001] The present invention relates to a microbial preparation for improving the inoculation rate of Tricholoma matsutake mycelium and its use. More specifically, it relates to a microbial preparation for improving the inoculation rate of Tricholoma matsutake mycelium against trees, a composition for improving the inoculation rate of Tricholoma matsutake mycelium for trees containing this microbial preparation, a method for improving the inoculation rate of Tricholoma matsutake mycelium on trees using the composition, and a method for producing Tricholoma matsutake mycelium-inoculated seedlings.

Background Art

[0002] Tricholoma matsutake is a fungus belonging to the family Tricholomataceae of the order Agaricales. It is collected around coniferous tree communities such as pine, Thuja koraiensis, Chamaecyparis obtusa, and Abies firma, and is known to occur in pine communities in Korea. Tricholoma matsutake is an edible mushroom favored by people in Korea and Japan, and is a high-income source especially for farmers in the eastern coastal region.

[0003] Tricholoma matsutake is a mushroom that forms ectomycorrhiza on the roots of pine trees. Generally, Tricholoma matsutake occurs in pine forests with a tree age of 20 to 60 years. The Tricholoma matsutake mycelium is a biotrophic parasite that attaches to the young roots of pine trees to form mycorrhiza and symbiosis. The spores of Tricholoma matsutake germinate in a suitable environment, grow as mycelium, and attach to the young roots of pine trees. The white or light yellow living young roots change to dark brown while forming mature mycorrhiza. The mycorrhiza grows and reproduces in a cushion shape underground while forming white patches (small groups), which spread circularly and are called mycorrhizal rings. When the underground temperature remains below 19°C for 5 to 7 days, the fully developed mycelium starts to produce fruiting bodies.

[0004] Because matsutake mushrooms are parasitic fungi, artificial formation of fruiting bodies (mushrooms) is difficult, and artificial cultivation is not practiced. Therefore, in order to increase matsutake production, methods have been employed such as cultivating matsutake mycelium and then scattering the mycelium in matsutake production areas to incorporate mycelial masses into the soil, collecting spores from matsutake fruiting bodies and scattering them in production areas, and transplanting soil in which mycelium is present to areas where matsutake does not grow. However, due to the characteristic of matsutake mycelium that its growth rate is slower than that of other bacteria and fungi, these methods resulted in a loss of vitality, and the mycelium was washed away without infecting the pine roots due to rainfall and soil conditions, so no substantial effect on increasing matsutake yield was obtained. For this reason, a method called the matsutake infection seedling method has been attempted, in which pine seedlings are planted near the mycelial ring to obtain infected seedlings for transplantation, but the success rate remains low, and the development of improved techniques is needed. [Prior art documents] [Patent Documents]

[0005] Korean Registered Patent No. 10-1127514 Japanese Registered Patent No. 3057676 Korean Registered Patent No. 10-0390048 [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, in order to meet the demands of the prior art, the inventors continued their research and confirmed that a microbial preparation containing Paenibacillus cineris and Micrococcus sp. significantly improves the inoculation rate of matsutake mycelium into pine trees and also improves the survival rate of pine inoculated seedlings, thus completing the present invention.

[0007] Therefore, the object of the present invention is to provide a microbial preparation containing Paenibacillus cineris and Micrococcus sp. that improves the inoculation rate of Matsutake mycelium onto trees.

[0008] Another object of the present invention is to provide a composition for improving the inoculation rate of matsutake mycelium into trees, comprising the above-mentioned microbial preparation as an active ingredient.

[0009] A further object of the present invention is to provide a method for improving the inoculation rate of matsutake mycelium onto trees using the above-mentioned microbial preparation or composition.

[0010] Another object of the present invention is to provide a method for producing inoculated seedlings of Matsutake mushroom mycelium using the above-mentioned microbial preparation or composition. [Means for solving the problem]

[0011] To achieve the above-mentioned objectives of the present invention, the present invention provides a microbial preparation comprising the Paenibacillus cineris strain and the Micrococcus sp. strain, which improves the inoculation rate of Matsutake mycelium onto trees.

[0012] In this invention, the trees are those on which matsutake mushrooms can coexist, such as pine, fir, cypress, and spruce, and in Korea, they mainly grow on pine trees.

[0013] In this invention, inoculation means directly infecting a tree with matsutake mycelium.

[0014] In this invention, the terms "matsutake mycelium" and "matsutake fungus" are used synonymously.

[0015] In the course of research to improve the inoculation of matsutake mycelium into pine trees, the inventors isolated three strains, NIFoS B_44(44), NIFoS B_48(48), and NIFoS B_49(49), from coniferous forest soil that are beneficial to matsutake mycelium (matsutake fungus), suppress bacteria harmful to the growth of matsutake mycelium (harmful bacteria), and have the activity to improve the inoculation rate of matsutake mycelium into pine trees (Figure 1b).

[0016]

[0016] To identify the three strains isolated in the present invention, the 16S-rRNA region was amplified and the base sequence was analyzed. As a result, it was confirmed that two strains belonged to Paenibacillus cineris and the remaining strain belonged to Micrococcus sp. Based on this, the three strains of the present invention were named Paenibacillus cineris 44 (NIFoS B_44), Paenibacillus cineris 49 (NIFoS B_49), and Micrococcus sp. 48 (NIFoS B_48), respectively (Figure 4).

[0017] According to one embodiment of the present invention, the two strains (three strains) described above significantly improved the inoculation rate of matsutake mycelium into pine trees (41.2%) (Figure 5, Table 4). This result suggests that the two strains discovered in the present invention can significantly improve the inoculation rate of matsutake mycelium into trees. The two strains of the present invention did not inhibit the growth of matsutake mycelium and showed the effect of suppressing harmful fungi that inhibit the growth of matsutake mycelium (Figure 3, Table 1). Furthermore, the two strains of the present invention also significantly improved the survival rate (85%) of inoculated seedlings after inoculation with matsutake mycelium (Table 5). Therefore, the two strains of the present invention can be used as microbial preparations for improving the inoculation rate of matsutake mycelium.

[0018] In the microbial formulation of the present invention, the above-mentioned bacterial strain may be included in any of the following forms: live bacteria, dead bacteria, dried bacteria, culture medium, or bacterial strain pellets.

[0019] The above-mentioned strain pellets can be obtained by centrifuging the medium in which the strain is cultured, removing the supernatant.

[0020] In the present invention, the microbial preparation can be formulated in the form of strain pellets, wettable powders, granules or encapsulation agents for the purpose of stable formulation of microorganisms.

[0021] In the microbial preparation of the present invention, the above two strains, namely Penibacillus chibensis strain and Micrococcus sp. strain, can be combined at a volume ratio of 2:1 to 1:2, preferably supplied in the state of being contained in the microbial preparation at a volume ratio of 2:1.

[0022] Also, the above two strains can be stored separately for long-term preservation and mixed immediately before use. When storing the above two strains for a long time, they can be stored in a glycerol preservation solution at -70°C or below, or freeze-dried and stored at -20 to -80°C for use.

[0023] The wettable powder of the present invention can be produced by drying and pulverizing the medium in which the microorganism is cultured, and adding and mixing a surfactant and a bulking agent / nutrient agent.

[0024] The granules of the present invention can be produced by drying and pulverizing the medium inoculated with the microorganism, and adding a surfactant, a bulking agent / nutrient agent and a disintegrant.

[0025] As described above, one or more surfactants selected from the group consisting of polycarboxylates, sodium lignosulfonates, calcium lignosulfonates, sodium dialkyl sulfosuccinates, sodium alkylallyl sulfonates, polyoxyethylene alkylphenyl ethers, sodium tripolyphosphates, polyoxyethylene alkylallyl phosphates, polyoxyethylene alkylallyl ethers, polyoxyethylene alkylallyl polymers, polyoxyalkylene alkylphenyl ethers, polyoxyethylene nonylphenyl ethers, sodium sulfonate naphthalene formaldehyde, Triton 100, and Twin 80 may be used. As fillers and nutrients, one or more surfactants selected from the group consisting of soy flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose, and starch may be used, and as disintegrants, one or more surfactants selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate may be used.

[0026] The granular formulation of the present invention can be manufactured by further adding one or more substances selected from the group consisting of surfactants, inert carriers, preservatives, wetting agents, supply promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, UV protectants, buffers, and fluidizers to microorganisms.

[0027] Another object of the present invention is to provide a composition for improving the inoculation rate of Matsutake mycelium into trees, comprising the above-mentioned microbial preparation as an active ingredient.

[0028] In the present invention, the composition can be commercialized as a plant nutrient composition, a soil conditioner composition, and the like.

[0029] Methods for applying the microbial preparation or composition of the present invention include soaking the tree roots in a mixture of matsutake mycelium and the microbial preparation or composition, spraying the mixture onto the tree roots, or pouring the mixture around planted trees. Other known microbial application methods can also be used.

[0030] Another object of the present invention is to provide a method for improving the inoculation rate of Matsutake mycelium onto trees using the above-mentioned microbial preparation or composition.

[0031] Another object of the present invention is to provide a method for producing matsutake mycelium-inoculated seedlings using the above-mentioned microbial preparation or composition.

[0032] The above method, i) A process of producing a matsutake mycelial culture solution by culturing matsutake mycelium in liquid and then homogenizing it using a homogenizer; ii) A step of culturing Paenibacillus cineris and Micrococcus sp., followed by centrifugation to produce a strain pellet; iii) The process of washing the roots of pine seedlings to shorten them; iv) Inoculating the roots of shortened pine seedlings by immersing them in a mixture of the matsutake mycelium culture solution from step i) and the strain pellets from step ii); and v) The process of planting inoculated pine seedlings in soil and growing them;

[0033] The above method may further include, if necessary, the step of vi) planting pine seedlings in the soil and then pouring a mixture of the matsutake mycelium culture solution from step i) and the strain pellets from step ii) around the roots for additional inoculation.

[0034] Process i) Production of Matsutake mycelium culture solution After culturing the matsutake mycelium in liquid, a homogenized matsutake mycelium culture solution is produced using a homogenizer.

[0035] Liquid culture of matsutake mycelium can be performed by culturing in TMB (Tricholoma matsutake broth; Glucose 20g / L, Yeast extract 1.5g / L, Soytone 1.5g / L, pH 5.2), a culture medium for matsutake mycelium, at 25°C for one month with shaking at 150 rpm.

[0036] Matsutake mycelium used in liquid culture is prepared by first storing matsutake mycelium on a slant medium of PDA (Potato Dextrose agar, Difco 00980; Potato starch 4g / L, Dextrose 20g / L, Agar 15g / L), a solid medium for matsutake mycelial growth. Then, it is cultured statically for one month at 25°C in TMM (Tricholoma matsutake media; Glucose 20g / L, Yeast extract 1.5g / L, Soytone 1.5g / L, Agar 20g / L, pH 5.2), another solid medium for matsutake mycelial growth. After this, the fully grown matsutake mycelium is shredded to 5mm or less using a sterile scalpel, and then processed in PDB (Potato Dextrose broth, Difco 254920; Potato starch 4g / L, Dextrose) liquid medium for matsutake mycelial growth. It can be obtained by transferring it to a 20g / L culture medium and culturing it at 25°C for 1 month with shaking at 150 rpm.

[0037] To improve contact between the pine roots and the matsutake mycelium, the matsutake mycelium is homogenized using a homogenizer and uniformly crushed to a size of approximately 1-2 mm.

[0038] Process (ii): Production of bacterial strain pellets After culturing Paenibacillus cineris and Micrococcus sp., the strains are centrifuged to produce a pellet. The above strains were isolated from preserved glycerol stocks and streaked in TSA (Tryptone soy agar; tryptone 17 g / L, soy peptone 3 g / L, NaCl 5 g / L, K2HPO4 2.5 g / L, dextrose 2.5 g / L, agar 20 g / L) medium. After static incubation at 26-30°C for 2-3 days, single colonies were selected and cultured with shaking in TSB (Tryptone soy broth; Pancreatic digest of casein 17 g / L, Pancreatic digest of soybean 3 g / L, dextrose 2.5 g / L, sodium chloride 5 g / L, dipotassium phosphate 2.5 g / L) for 3 days to determine the strain concentration OD. 600 After adjusting the ratio to 0.1, the strain culture solution is centrifuged at 3,000-4,000 rpm for 10 minutes, and the supernatant is removed to obtain a strain pellet.

[0039] Step iii) Shortening the roots of pine seedlings The roots of the pine saplings are washed and shortened to prepare them for planting.

[0040] Pine seedlings can be 1-2 year old (1-0 seedlings, 2-0 seedlings) grown in potting soil, but are not limited to these.

[0041] Root cleaning can be performed by removing the potting soil from the pine seedling and washing it with tap water at least three times.

[0042] After washing, the roots of the pine seedlings are shortened to 15-20 cm, preferably 16 cm, to facilitate inoculation with matsutake mycelium and strains, and are prepared by retaining sufficient moisture to prevent the root tips from drying out.

[0043] Step iv) Inoculation of pine seedlings The roots of pine seedlings with shortened roots are inoculated by immersing them in a mixture of the matsutake mycelium culture solution from step i) and the strain pellets from step ii).

[0044] Immersion should be carried out for 20 to 40 minutes, preferably 30 minutes.

[0045] Step v) Growth of the inoculated pine seedlings The inoculated pine seedlings are planted in the soil and allowed to grow.

[0046] The planted inoculated seedlings can be grown in an environment of 25-30°C and 50% humidity, but are not limited to these conditions.

[0047] Step (vi): Additional vaccination If necessary, after planting the inoculated pine seedlings in the soil, an additional inoculation is performed by pouring (pouring) a mixture of the matsutake mycelium culture solution from step i) and the strain pellets from step ii) around the roots.

[0048] Booster inoculations can be performed 2 to 3 times at 2-week intervals after planting the inoculated seedlings, but are not limited to this.

[0049] The amount of the mixture to be administered is 5 to 15 ml, preferably 10 ml.

[0050] According to the present invention's method for producing seedlings inoculated with matsutake mycelium, the inoculation rate of matsutake mycelium into pine trees is improved, and the survival rate of the inoculated seedlings is also significantly improved. [Effects of the Invention]

[0051] The microbial preparation or composition according to the present invention significantly improves the inoculation rate of matsutake mycelium into trees. Furthermore, the microbial preparation or composition according to the present invention does not inhibit the growth of matsutake mycelium and has the effect of suppressing harmful bacteria that inhibit the growth of matsutake mycelium. The microbial preparation or composition according to the present invention also has the effect of significantly improving the survival rate of matsutake mycelium-inoculated seedlings.

[0052] The present invention's method for producing matsutake mycelium-inoculated seedlings makes it possible to mass-produce inoculated seedlings with a high inoculation rate of matsutake mycelium and a high survival rate after inoculation, and to supply them to farmers in large quantities. [Brief explanation of the drawing]

[0053] [Figure 1] This photograph shows the results of counterculture of Penibacillus cineris 44 (NIFoS B_44), Penibacillus cineris 49 (NIFoS B_49), and Micrococcus sp. 48 (NIFoS B_48) with Matsutake mushroom fungus (NIFoS2001) according to the present invention. [Figure 2]

[0058] This is a photograph showing the confrontation culture process to confirm the interaction between beneficial and harmful bacteria with Matsutake fungus. [Figure 3] The experimental results for confirming the ability of Penibacillus cineris 44 (NIFoS B_44), Penibacillus cineris 49 (NIFoS B_49), and Micrococcus sp. 48 (NIFoS B_48) to suppress harmful bacteria against Matsutake mushrooms according to the present invention are shown. [Figure 4] The 16S rRNA gene sequences of Penibacillus cineris 44 (NIFoS B_44), Penibacillus cineris 49 (NIFoS B_49), and Micrococcus sp. 48 (NIFoS B_48) according to the present invention are shown. [Figure 5] This is a photograph showing pine seedlings inoculated according to the present invention being planted. [Figure 6] A schematic diagram showing the primer positions and PCR conditions used for evaluating pine inoculated seedlings is provided. [Figure 7] The PCR results for pine seedlings inoculated with pine are shown. [Modes for carrying out the invention]

[0054] The following describes the configuration and effects of the present invention in more detail through specific examples to aid in understanding the present invention. However, the following examples are merely illustrative to illustrate the present invention more clearly, and the scope of the present invention is not limited by these examples.

[0055] Example 1: Searching for candidate strains and selecting strains To search for strains that improve the inoculation rate of Matsutake mushrooms (Matsutake mycelium), microorganisms were isolated from mycologically circulating soils where Matsutake mushrooms grow and from the roots of pine trees growing naturally in water source areas of Korea.

[0056] Specifically, soil was collected from a pine forest after removing the matsutake mycelium and the leaf litter humus layer from the pine canopy area. The mycelium soil was diluted in a 50 ml Falcon tube with sterilized water in a volume ratio of 10:1, and the diluted sterilized soil was mixed with 1:10 -3 ~10 -8 After being gradually diluted in the specified ratio, the samples were spread onto PDA and TSA media. In addition, pine roots were sterilized with 2% sodium hypochlorite (NaOCl), and the surface-sterilized roots were shredded with a sterile scalpel and placed on PDA and TSA media to isolate a total of 436 strains. Of these, 412 strains that inhibited the growth of matsutake mushrooms and 24 strains that did not have harmful effects were isolated based on the presence or absence of matsutake fungal growth inhibitory activity.

[0057] The growth inhibitory activity of Matsutake mushrooms was evaluated using Matsutake mushrooms (NIFoS 2001). After culturing the mycelium in PDA medium at 24°C for 25 days, mycelial discs were collected using a 5 mm diameter cork borer. PDA medium was dispensed and placed in the center of a disposable Petri dish, and each isolated fungus was streaked and cultured in opposition at a distance of 1-2 mm from the disc (Figure 1).

[0058] To confirm the interaction between harmful bacteria that inhibit the growth of Matsutake mycelium by more than 20% and beneficial bacteria that do not inhibit the growth of Matsutake mycelium, confrontation cultures were performed. Harmful bacteria were inoculated onto TSA (Tryptone soybean agar; tryptone 17 g / L, soy peptone 3 g / L, NaCl 5 g / L, K2HPO4 2.5 g / L, dextrose 2.5 g / L, agar 20 g / L) medium, and then paper discs cultured with beneficial bacteria were placed on top of the inoculated medium to observe the interaction between the strains (Figure 2). When beneficial bacteria inhibit the growth of harmful bacteria, the growth inhibition zone (clear zone) is observed as a transparent ring-shaped morphology. As a result of these confrontation cultures, three strains were selected from the isolated strains that showed an antagonistic effect against harmful bacteria that inhibit the growth of Matsutake mycelium (Figure 3, Table 1). [Table 1]

[0059] Three selected strains were streaked onto TSA medium to obtain pure colonies. The obtained cultures were incubated in TSB (Tryptone soy broth; Pancreatic digest of casein 17 g / L, Papaic digest of soybean 3 g / L, dextrose 2.5 g / L, sodium chloride 5 g / L, dipotassium phosphate 2.5 g / L) at 25°C for 2 days in a 200 rpm shaking incubator. The resulting strain culture solution was then divided into 10 8 The solution was adjusted to CFU / mL (colony forming units), treated with 20% (v / v) glycerol for additional experiments, and stored frozen at -80°C.

[0060] Example 2: Identification of selected bacterial strains To identify the three bacterial strains (44, 48, and 49) selected in Example 1, Microgen Co., Ltd., Korea was commissioned to analyze the 16S rRNA gene sequences of the three strains using the primer combinations in Table 2 (27F [V1-V9 region], 785F [V5-V9 region], 907R [V1-V5 region], 1492R [V1-V9 region]) to include all V1-V9 regions of the 16S rDNA commonly used for bacterial molecular identification. The analyzed 16S rRNA gene sequences are shown in Figure 4 and Sequence IDs 1-3. [Table 2]

[0061] To confirm the taxonomic position of the above strains, we analyzed them based on the most widely used microbial 16S rRNA gene sequence comparison data. The results showed that the 16S rRNA gene sequences of strains 44 and 49 had a 99.55% and 99.81% agreement rate with the 16S rRNA gene sequence of Paenibacillus cineris, respectively, and the 16S rRNA gene sequence of strain 48 had a 99.93% agreement rate with the 16S rRNA gene sequence of Micrococcus sp.

[0062] Based on the above identification results, strains 44, 49, and 48 were named Penibacillus cineris 44 (NIFoS B_44), Penibacillus cineris 49 (NIFoS B_49), and Micrococcus sp. 48 (NIFoS B_48), respectively.

[0063] Manufacturing Example 1: Production of Matsutake mushroom culture and Matsutake mushroom inoculation (Matsutake mushroom culture solution) The matsutake mycelium strain NIFoS 2001, derived from wild matsutake mushrooms collected in Yeongwol County, Gangwon Province, South Korea, was used as the inoculum. Matsutake mycelium was isolated from matsutake fruiting bodies and registered in the NCBI Gene Bank (Gene Bank accession number GCA_026213095.1). The matsutake mycelium, stocked on PDA (Potato Dextrose agar, Difco 00980; Potato starch 4 g / L, Dextrose 20 g / L, Agar 15 g / L) slant medium, was subcultured and stabilized in TMM (Tricholoma matsutake media; Glucose 20 g / L, Yeast extract 1.5 g / L, Soytone 1.5 g / L, Agar 20 g / L, pH 5.2) medium at 25°C for 1 month. Fully grown matsutake mycelium was shredded to 5 mm or less using a sterile scalpel, and transferred to PDB (Potato Dextrose broth, Difco 254920; Potato starch 4 g / L, Dextrose 20 g / L) liquid medium using sterile tweezers. The culture was then performed at 25°C for 1 month with shaking at 150 rpm. To use as matsutake inoculum, the cultured matsutake mycelium was then cultured in fresh TMB (Tricholoma matsutake broth; Glucose 20 g / L, Yeast extract 1.5 g / L, Soytone 1.5 g / L, pH 5.2) medium at 25°C for 1 month with shaking at 150 rpm. Finally, the matsutake mycelium was homogenized to 1-2 mm using a homogenizer to prepare a matsutake mycelium culture solution. The wet weight of the matsutake mycelium in the used inoculum was 30-70 g per 500 ml.

[0064] Production Example 2: Production of strain culture and strain inoculation (strain pellets) according to the present invention The three strains isolated and identified according to the present invention were each isolated from stored glycerol stocks, streaked onto TSA (Tryptone soy agar; tryptone 17 g / L, soy peptone 3 g / L, NaCl 5 g / L, K2HPO4 2.5 g / L, dextrose 2.5 g / L, agar 20 g / L) medium, and incubated statically at 28°C for 2 days. After selecting single colonies, they were incubated with TSB (Tryptone soy broth; Pancreatic digest of casein 17 g / L, Papaic digest of soybean 3 g / L, dextrose 2.5 g / L, sodium chloride 5 g / L, dipotassium phosphate 2.5 g / L) for 3 days with shaking at 150 rpm to perform seed culture in 50 ml tubes. 600 The cells were cultured so that the ratio was 1.0.

[0065] To be used for inoculation into pine trees, each seed culture solution was prepared using Matsutake mycelial culture medium (TMB) and OD 600 The cultures were diluted to 0.1 and mixed in equal volume ratios (resulting in a volume ratio of Paenibacillus strains to Micrococcus strains of 2:1). 500-700 ml of the resulting culture solution was prepared.

[0066] Manufacturing Example 3: Production of Matsutake Mycelium and Strain Inoculum for Matsutake Mycelial Inoculation According to manufacturing example 2, OD 600 The seed culture solution of the isolated and identified strain of the present invention, diluted to 0.1, was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was removed to obtain a strain pellet. The obtained strain pellet was uniformly mixed with the Matsutake mycelium culture solution (500-700 ml) cultured in Production Example 1. The Matsutake mycelium and strain inoculum inoculated into pine seedlings contained 30-70 g of Matsutake mycelium (biomedium) and strain pellet (OD) per 500 ml. 600 We prepared the mixture by adding (=0.1).

[0067] Example 3: Inoculation test on pine seedlings (analysis of improvement in matsutake mycelium inoculation rate) Inoculation tests on pine seedlings were conducted in three groups: a treatment group (mixed treatment with matsutake mycelium and strain), a control group (treatment with matsutake mycelium only, without strain), and an untreated group. 100 one-year-old pine seedlings were prepared. The culture medium was removed from 80 of the seedlings, the roots were washed at least three times with tap water, and the roots were shortened to 16 cm to facilitate inoculation. Sufficient moisture was maintained to prevent the root tips from drying out. The remaining 20 seedlings were similarly shortened and kept moist, and used as the untreated group.

[0068] For the inoculum used to inoculate pine seedlings, the treatment group used an inoculum prepared by mixing the strain pellets produced in Production Example 3 with the matsutake mycelium culture solution, while the control group used only the matsutake mycelium cultured in Production Example 1.

[0069] In the treatment group, 60 pine seedlings with shortened roots were immersed in the inoculum from Production Example 3 for 30 minutes, ensuring that the pine roots were completely submerged. They were then planted in 20 culture pots filled with a culture medium consisting of a 4:1 volume ratio mixture of decomposed granite and perlite. Watering was performed twice a week, and additional inoculation was performed twice at two-week intervals by pouring 10 ml of the inoculum around the pine roots (Figure 5).

[0070] For comparison, in the control group, 20 pine seedlings with shortened roots were immersed for 30 minutes in the matsutake mycelium culture solution cultivated in Production Example 1, ensuring that the pine roots were submerged, and then planted in the same culture pots. These were also watered twice a week, and additional inoculation was performed twice at two-week intervals by pouring 10 ml of matsutake mycelium culture solution around the roots.

[0071]

[0081] Twenty untreated pine seedlings, neither inoculated with matsutake mycelium nor strain, were also planted in the same culture pots.

[0072] The planted pine seedlings were cultured in a constant temperature and humidity chamber in a glass greenhouse maintained at 25-30°C and 50% humidity. After 7 months, the pines were transplanted into larger pots (size: 115 mm × 115 mm × 160 mm), and the presence or absence of matsutake mycelium inoculation was confirmed using matsutake mycelium-specific primers shown in Table 3. [Table 3]

[0073] Specifically, a portion of the roots of inoculated pine seedlings that survived seven months after planting were excavated, and genomic DNA (gDNA) was extracted. gDNA extraction was performed using the Qiagen Plant DNeasy kit and the CTAB method manual. DTm3_2001 primers were designed based on the ITS sequence of the Matsutake mushroom (NIFoS 2001) genome (Table 3), and PCR was performed on each gDNA under the conditions shown in Figure 6 to amplify the corresponding region. To confirm the quality of the pine root gDNA, the translation initiation factor (Pd_EF1) gene region based on the pine genome was used as an internal control. The results are shown in Figure 7 and Table 4. [Table 4]

[0074] As shown in Table 4, the inoculation rate (inoculation success rate) of matsutake mycelium in the control group, which was inoculated with matsutake mycelium alone without using the strain of the present invention, was only 9.2±1.2% (8.0~10.4%) on average. However, in the pine seedlings of the test group inoculated with the strain of the present invention in combination with the matsutake mycelium inoculation rate was 41.2±4.0% (37.2~45.2%), which was confirmed to be an improvement of approximately 4.5 times (approximately 3.6~5.7 times) on average.

[0075] The number of surviving pine seedlings and their survival rate were calculated seven months after planting (inoculated seedlings), and the results are shown in Table 5. [Table 5]

[0076] As shown in Table 5, the survival rate of the control group inoculated with only Matsutake mycelium without using the strain of the present invention was 52.5 ± 3.5%, while the survival rate of the inoculated seedlings in the test group inoculated with the strain of the present invention in combination was 84.2 ± 1.2% (83-85.4%), confirming an improvement of approximately 1.5 to 1.7 times. [Industrial applicability]

[0077] Therefore, the microbial preparation or composition and the method for producing matsutake mycelium-inoculated seedlings according to the present invention do not cause environmental pollution in matsutake cultivation, are non-toxic to humans, and can significantly improve the inoculation of trees with matsutake mycelium. As a result, by supplying a large quantity of inoculated seedlings according to the present invention, efficient matsutake cultivation can be made possible for matsutake farmers in Japan, and a stable income can be guaranteed.

Claims

1. A microbial preparation containing Paenibacillus cineris strain and Micrococcus sp. strain that improves the inoculation rate of Matsutake mycelium into trees.

2. A microbial preparation according to claim 1, characterized in that the strain is included in any form selected from the group consisting of live bacteria, dead bacteria, dried bacteria, culture solution, and strain pellets, which improves the inoculation rate of Matsutake mycelium into trees.

3. A microbial preparation according to claim 1, characterized in that the Penibacillus cineris strain and the Micrococcus sp. strain are combined in a volume ratio of 2:1 to 1:2, thereby improving the inoculation rate of Matsutake mycelium into trees.

4. A microbial preparation according to claim 1, characterized in that the microbial preparation is formulated in the form of a wettable powder, granules, or encapsulated agent, which improves the inoculation rate of Matsutake mycelium into trees.

5. A microbial preparation according to claim 1, characterized in that the microbial preparation is manufactured by further adding at least one selected from the group consisting of surfactants, bulking agents, and nutrients, thereby improving the inoculation rate of Matsutake mycelium into trees.

6. A microbial preparation according to claim 1, characterized in that the microbial preparation is applied by one or more of the following methods: soaking the roots of the tree, spraying the roots of the tree, or injecting or pouring the preparation around the planted tree, thereby improving the inoculation rate of Matsutake mycelium onto the tree.

7. A microbial preparation according to claim 1, characterized in that the inoculation rate of matsutake mycelium on trees is 37.2 to 45.2%, and the survival rate of inoculated seedlings after inoculation is 84.2 ± 1.2%, thereby improving the inoculation rate of matsutake mycelium on trees.

8. A method for producing seedlings inoculated with matsutake mycelium, wherein the method i) A process of producing a matsutake mycelial culture solution by culturing matsutake mycelium in liquid and then homogenizing it using a homogenizer; ii) A process of culturing Paenibacillus cineris and Micrococcus sp., followed by centrifugation to produce a strain pellet; iii) The process of washing the roots of pine seedlings to shorten them; iv) Inoculating the roots of shortened pine seedlings by immersing them in a mixture of the matsutake mycelium culture solution from step i) and the strain pellets from step ii); and v) A method comprising the step of planting inoculated pine seedlings in soil and growing them.

9. The method according to claim 8, further vi) A method for producing matsutake mushroom mycelium-inoculated seedlings, characterized by including the step of planting pine seedlings in soil and then pouring a mixture of the matsutake mushroom mycelium culture solution from step i) and the strain pellets from step ii) around the roots to perform additional inoculation.

10. A method for producing matsutake mushroom mycelium-inoculated seedlings, characterized in that the inoculation rate of the inoculated seedlings with matsutake mycelium is 37.2 to 45.2%, and the survival rate of the inoculated seedlings after inoculation is 84.2 ± 1.2%.

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