Fungi used in the cultivation of ginseng

Abstract

The objective is to provide a cultivation method for Panax ginseng (also known as Korean ginseng or Panax japonicus) that prevents the severe continuous cropping problems unique to this species. Furthermore, the objective is to develop a cultivation method for Panax ginseng that mitigates the difficulties of cultivation caused by continuous cropping problems, inefficient land use, the use of chemically synthesized soil disinfectants, and the high barriers to entry for new producers. [Solution] We have identified root endophytes (endophytes) that have both the effect of preventing root rot disease in Panax ginseng and the effect of promoting growth, and we provide a cultivation method in which these endophytes coexist with the roots of Panax ginseng. Furthermore, we provide materials containing these endophytes, growing medium, and seedlings in which these endophytes coexist.

Description

Technical Field

[0001] The present invention relates to the cultivation of Panax ginseng C.A. Meyer Regarding fungi used in .

Background Art

[0002] Panax ginseng C.A. Meyer is a perennial rhizomatous herb belonging to the Araliaceae family and is a semi-shade-loving plant that grows in cool regions. Its native range is North Korea located between 39° and 47° north latitude, the northeastern part of China to the coastal areas of Russia. Since cultivation and processing have developed mainly in the Korean Peninsula, it is also called Korean ginseng or Koryo ginseng.

[0003] Cultivation in Japan dates back to the development of cultivation methods for the first time in Nikko by the order of Tokugawa Yoshimune during the Edo period. Currently, cultivation is carried out in the Aizu region of Fukushima Prefecture, the Toshinga region of Nagano Prefecture, and Oda Island of Shimane Prefecture.

[0004] The roots of Panax ginseng C.A. Meyer have been used in numerous prescriptions as crude drugs in traditional Chinese medicine since ancient times, such as for tonifying essence, strengthening the stomach and regulating the intestines, suppressing vomiting, and quenching thirst. With the development of analytical techniques, it has been found that the active ingredient is a saponin unique to Panax ginseng C.A. Meyer and is called ginsenoside. Dozens of types of ginsenosides have been confirmed to exist (Non-Patent Document 1). It is also widely used in health foods and cosmetics.

[0005] In recent years, research and development of highly safe bio-formulations derived from plants instead of chemically synthesized drugs have been active, and it is expected that the global demand for medicinal plants will increase in the future. In Japan, which depends on imports from China for more than 90% of Panax ginseng C.A. Meyer, domestic production has become an urgent issue in terms of improving the self-sufficiency rate of medicinal crops (Non-Patent Document 2).

[0006] However, the cultivation of Panax ginseng C.A. Meyer is difficult and few new producers appear. The current producers are also decreasing due to aging, and Panax ginseng C.A. Meyer cultivation is facing a crisis of extinction. One of the biggest reasons making Panax ginseng C.A. Meyer cultivation difficult is the strong continuous cropping obstacle peculiar to this plant.

[0007] Once a field has been planted, it cannot be replanted for a long period of time. In Shimane and Fukushima prefectures, it is said that it cannot be replanted for 10 to 15 years, and in Nagano prefecture, it is said that it is not good even after 30 years, or in extreme cases, 50 years (Non-Patent Literature 3). It is said that "a person can only harvest once or twice in their lifetime," and that "carrot fields are nomadic" because farmers move in search of new land (Non-Patent Literature 4). This is an obstacle to the efficient use of land both domestically and internationally. In particular, it is a major barrier to entry for new producers.

[0008] Research institutions in Fukushima and Nagano prefectures have revealed that the cause of continuous cropping problems is biological factors such as soilborne pathogens and nematodes. Among these, the soilborne pathogen Cylindrocarpon panacis. causes root rot (red rot) in all roots from the first to the sixth year of the plant, resulting in devastating damage to the harvest. Note that Cylindrocarpon panacis. is now called Ilyonectria mors-panacis, so in this invention, Ilyonectria mors-panacis will be used hereafter.

[0009] Soil disinfection with the soil fumigant chloropicrin (drochlor) is effective in controlling soilborne pathogens and nematodes (Non-Patent Document 3) and is still practiced today. However, these chemically synthesized soil fumigants are highly toxic, and producers must handle them with caution. In addition, considering consumers' preference for natural products and the negative impact on the environment, including natural ecosystems, there is a demand to discontinue their use.

[0010] As an alternative, a soil disinfection method using low-concentration ethanol for soil reduction (Non-Patent Document 5) has been developed. It is safe, has a low environmental impact, and has been confirmed to be effective in suppressing soilborne diseases. However, it has not yet been put into practical use due to challenges such as high cost, long processing time, and unpleasant odor.

[0011] For this reason, dedicated ginseng farmers seek out new land free from continuous cropping problems, cultivate green manure crops (such as sorghum and oats), and plow them into the soil, spending two to three years preparing the soil to reduce the amount of chloropicrin they use. After that, they install expensive shades in the fields, pre-germinate the seeds to grow seedlings, carefully transplant each one, and spend a lot of time and effort on pesticide application and weeding to protect against diseases and pests. However, as the years of cultivation increase, the damage from diseases and pests increases, and it becomes difficult to prevent root rot. In reality, they then have to seek out new land and start the soil preparation process all over again.

[0012] In order to solve or improve these various problems related to ginseng cultivation, new cultivation methods are being researched (Non-Patent Literature 2), but the following invention has been disclosed as the only method for preventing continuous cropping problems in ginseng.

[0013] Patent Document 1 discloses a method for preventing continuous cropping problems in Panax ginseng by planting seeds or 1-3 year old seedlings in a cultivation bed made of sand, gravel, and pebbles set up in a river in a mountainous area, and constantly supplying fresh flowing water.

[0014] The cultivation method described in Patent Document 1 is an effective method for preventing continuous cropping problems, but its implementation requires an environment where the water flow velocity is 3 cm / s or more, the water temperature is 10 to 25°C, and the average daily illuminance is 30,000 lux or less. There are not many mountainous areas where such conditions can be secured, so there are significant constraints on location selection. Therefore, it is expected to be difficult to realize this on an industrial scale.

[0015] Furthermore, there are concerns about the environmental impact on river ecosystems and other areas caused by the chemical and liquid fertilizers used in cultivation. In addition, sowing seeds and transplanting seedlings into cultivation beds made of sand, gravel, and pebbles is clearly more labor-intensive than in conventional fields.

[0016] This cultivation method requires clean, natural, non-recirculating water for supply, and the inclusion of mud can lead to disease outbreaks. In fact, one comparative example states that all the plants died due to mud contamination caused by a typhoon. Considering the frequency of natural disasters in recent years, the risks are significant, making this cultivation method unsuitable for current producers, especially new ones.

[0017] On the other hand, in recent years, the use of endophytes (root-derived fungi) has been proposed as a means of controlling soil-borne diseases, which are a major cause of continuous cropping problems. It is attracting attention as a biological disease control method that has minimal adverse effects on natural ecosystems.

[0018] For example, Patent Document 2 proposes a method for suppressing soil-borne diseases of Chinese cabbage by inoculating the roots of Chinese cabbage with Black Sterile Mycelia and Westerdichella multispora, which live symbiotically within the roots of Chinese cabbage in the soil, have the ability to suppress clubroot disease of Chinese cabbage, and whose colonies on the culture medium are black or brown, grow slowly, and do not form spores on the culture medium.

[0019] Furthermore, Patent Document 3 lists Heteroconium chaetospira as a root endophyte that has the ability to suppress soilborne diseases.

[0020] Furthermore, Patent Document 4 describes a method for preventing soil-borne diseases by endocrinating or impregnating the roots of fruit trees of the Rosiformes suborder with a microbial material containing at least one of the cells, spores, or products of a microorganism belonging to the genus Microsferopsis.

[0021] Furthermore, Patent Document 5 describes a cultivation method using the FERM AP-22158 strain of Phialocephala as a root endophyte that suppresses soilborne pathogens such as Fusarium species in asparagus seedlings and promotes their growth.

[0022] Such root endophytes that inhabit root cells are said to have good colonization and affinity for plants and low host specificity. However, it also varies depending on the combination of fungal and plant species, and the relationship between the two is very complex. It is known that there are specific useful fungi for each plant, with varying degrees of compatibility.

[0023] Furthermore, in order to ensure the effect of root endophytes, it is necessary to consider not only the plants but also the target diseases. Therefore, as seen in the above-mentioned patent documents, it is necessary to identify effective root endophytes for different host plant species and pathogen species.

[0024] However, effective root endophytes against Ilyonectria mors-panacis, the main pathogen causing continuous cropping problems in carrot, have not been identified.

Prior Art Documents

Patent Documents

[0025]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0026]

Non-Patent Document 1

Non-Patent Document 2

[0027] The problem that this invention aims to solve is to provide a method for cultivating Panax ginseng that prevents the severe continuous cropping problems unique to Panax ginseng.

[0028] Furthermore, the goal is to develop a cultivation method for Panax ginseng that mitigates difficulties in cultivation caused by continuous cropping problems, inefficient land use, the use of chemically synthesized soil disinfectants, and the high barriers to entry for new producers. [Means for solving the problem]

[0029] Therefore, the inventors diligently conducted research and discovered a root endophyte that possesses both excellent disease control and growth-promoting effects against the root rot pathogen, which is the main cause of continuous cropping problems in ginseng, thus completing the present invention. In other words, in order to solve the above-mentioned problems, the method for cultivating ginseng according to the present invention has the following configuration.

[0030] The first aspect of the present invention relates to a cultivation method for Panax ginseng that suppresses root rot pathogens and / or promotes the growth of Panax ginseng, and is a method for cultivating Panax ginseng in which endophytes coexist. Here, "coexisting in the culture medium" means growing in close proximity to the ginseng roots and the fungus so that they can come into contact.

[0031] By incorporating the above-mentioned root endophytes into the growing medium for Panax ginseng, the roots of the Panax ginseng can be reliably inoculated with the root endophytes, establishing a symbiotic relationship between the two. This improves disease resistance, such as that to root rot. The Panax ginseng planted in the growing medium can be either seeds or 1- to 3-year-old seedlings. If sowing seeds, germination can be increased by treating the seeds with a germination-promoting agent. The ideal time to introduce the endophytes to maximize symbiosis is from March to May, when root activity begins, but this is not necessarily limited to this period depending on the form of the endophyte-containing material.

[0032] The second configuration of the present invention relates to the method for cultivating ginseng according to the first configuration, characterized in that the endophytes are Leptodophora orchidicola, Dactylonectria pauciseptata, and Lasionectriopsis pteridii.

[0033] The third configuration of the present invention relates to a method for cultivating Panax ginseng according to the second configuration, characterized in that the endophytes are strain YS102 belonging to Leptodophora orchidicola deposited as accession number NITE P-04203, strain YS024 belonging to Dactylonectria pauciseptata deposited as NITE P-04202, and strain YS147 deposited as NITE P-04204. These three strains were received by the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) on November 14, 2024, and tests conducted on November 22, 2024, confirmed that the microorganisms were viable. On December 6, 2024, certificates of deposit and certificates of viability were issued for each of these three strains.

[0034] The fourth aspect of the present invention relates to a cultivation method for suppressing root rot pathogens of Panax ginseng and / or promoting the growth of Panax ginseng, the method comprising coexisting with at least one of Leptodophora orchidicola, Dactylonectria pauciseptata, or Lasionectriopsis pteridii.

[0035] The fifth configuration of the present invention relates to a fourth configuration method for cultivating Panax ginseng, characterized in that the Leptodophora orchidicola is strain YS102 deposited under accession number NITE P-04203, the Dactylonectria pauciseptata is strain YS024 deposited under accession number NITE P-04202, and the Lasionectriopsis pteridii is strain YS147 deposited under NITE P-04204. These three strains were received by the Patent Microorganism Depositary Center of the National Institute of Technology and Evaluation (NITE) on November 14, 2024, and tests conducted on November 22, 2024, confirmed that the microorganisms were viable. On December 6, 2024, certificates of deposit and certificates of viability were issued for each of these three strains.

[0036] The sixth component of the present invention is Leptodophora orchidicola, Dactylonectria pauciseptata, or Lasionectriopsis pteridii. This invention relates to fungi that have an inhibitory effect on the root rot pathogens of Panax ginseng and / or an effect on promoting the growth of Panax ginseng.

[0037] The seventh component of the present invention relates to the fungus according to the sixth component, characterized in that Leptodophora orchidicola is strain YS102 deposited under accession number NITE P-04203, Dactylonectria pauciseptata is strain YS024 deposited under accession number NITE P-04202, and Lasionectriopsis pteridii is strain YS147 deposited under NITE P-04204.

[0038] The eighth component of the present invention relates to a material for cultivating ginseng, characterized by containing at least one of the fungi described in the sixth or seventh component. This material is a liquid carrier prepared by culturing endophytes in a liquid culture medium with shaking, then washing them with sterile water, pulverizing them in a mixer, and diluting the resulting microbial material with sterile water. This is used by directly spreading it onto the growing medium. However, the liquid carrier is inconvenient for transport and storage. There is also a risk of contamination by other bacteria, so careful handling is required. Therefore, the endophytes cultured in the liquid culture medium can be dried and powdered by freeze-drying or other methods, and can also be used as a powder. Alternatively, it can be used in any form such as granules, emulsions, or wettable powders by using a suitable solid carrier or emulsifying dispersant. It can also be adsorbed onto zeolite, diatomaceous earth, vermiculite, charcoal, etc., and air-dried. Furthermore, it can be adsorbed and mixed into bark compost made from fermented cedar or cypress leaves and bark.

[0039] The ninth component of the present invention relates to a growing medium for cultivating Panax ginseng, characterized by containing the materials described in the eighth component. To facilitate and simplify soil preparation work for ginseng producers, the above-mentioned microbial material may be combined with organic fertilizer, inorganic fertilizer, herbicide, soil, etc., to create seedling growing medium or soil amendment material. In this case, the above material should contain a number of fungi according to the present invention that will exert a disease-suppressing effect or growth-promoting effect on ginseng.

[0040] The tenth component of the present invention relates to a ginseng root and / or seedling characterized in that at least one of the fungi described in the sixth or seventh component is symbiotic with the root. Ginseng seeds have immature embryos and slow development, requiring germination treatment to promote sprouting. Sprouting treatment involves burying the seeds in river sand in a large unglazed pot, placing it in a shady, well-ventilated location, and watering just enough to keep the surface from drying out. This should be done from August to mid-November. Afterwards, sow the seeds in soil mixed with the seven components of this invention or in a growing medium with the eighth component. The seedlings will germinate in April of the following year and be cultivated in the shade with a sunshade or similar covering. Water as needed during this time. In addition, while checking the symbiotic state with endophytes, spray with a liquid carrier material containing at least one fungus, either Leptodophora orchidicola, Dactylonectria pauciseptata, or Lasionectriopsis pteridii, as needed. Dig up the seedlings around mid-October. Inoculating seedlings with endophytes at an early developmental stage improves fungal colonization and enhances the inoculation effect, providing healthy seedlings resistant to root rot with symbiotic endophytes.

[0041] To obtain the above-mentioned means, the inventors conducted extensive research including long-term experimental cultivation, isolation, culture, and identification of fungi, antimicrobial activity tests, and growth tests. As a result, they succeeded in discovering a root endophyte that possesses both excellent root rot disease control and growth-promoting effects. Specifically, it is as follows:

[0042] Panax ginseng is a plant that originally grows wild in the forests of Changbai Mountain in the northern part of the Korean Peninsula. In addition, Panax japonicus, which belongs to the same genus as Panax ginseng, grows wild in Japan, and colonies where it has been reproducing for many generations have been observed without any problems from continuous cropping. The inventors of this invention hypothesized that this might be due to the action of rhizosphere microorganisms. They particularly focused on the biological control characteristics of root endophytes (Dark Septate Endophytic fungi: DSE).

[0043] To identify root endophytes, the first step was to sow Panax ginseng seeds or transplant one-year-old seedlings in various forests and conduct forest floor trials. Cultivation was carried out in a variety of locations, including forests with different tree species such as cedar, sawtooth oak, pine, Japanese white pine, and broadleaf forests, forests with different elevations, forests with different canopy coverage (illumination), forests with different slope directions, and forests where Japanese ginseng grows naturally on the forest floor, as well as forests where wild plants such as Cypripedium macranthos and Arisaema serratum grow (Figure 1). After nearly 10 years of trial cultivation, it was found that there were significant differences depending on the forest, with some locations where the seeds did not germinate, some locations where they germinated but disappeared after 2 to 3 years, and others where they survived and continued to grow for more than 8 years.

[0044] We collected Panax ginseng roots (Figure 2) and Panax japonicus roots from these forests, isolated and cultured the endophytes living symbiotically in each root using the following procedure, and identified the species. (1) The collected roots were washed with a surfactant and the surface was disinfected with mercury chloride. (2) Place the subdivided roots on a 24-well microplate containing CMA medium (Figure 3). (3) After culturing at 15°C in the dark for 1 week to 3 months, establish a pure culture strain from the grown mycelium. (4) Species identification based on morphological characteristics and the nucleotide sequence of the ITS region of rRNA.

[0045] As a result, Acremonium alternatum, Alternaria sp., Chaetomicaceae sp., Chaetothyriales sp., Cladophialophora chaetospira, Cladophialophora sp., Cladosporium cladosporioides, Cladosporium sp., Colletorichum sp., Curvularia protuberata, Dactylonectria ecuadoriensis, Dactylonectria pauciseptata, Dothideomycetes sp.1, Dothideomycetes sp.2, Fusarium nematophilum, Globisporangium intermedium, Globisporangium intermedium, Helotiales sp.1, Helotiales sp.2, Helotiales sp.3, Helotiales sp.4, Helotiales sp.5, Helotiales sp.6, Herpotrichiellaceae sp., Humicola olivacea, Humicola sp., Hyaloscypha variabilis, Hyphodiscaceae sp.4, Ilyonectria mors-panacis, Ilyonectria rubusta, Lasionectriopsis sp., Lasionectriopsis pteridii, Leotiomycetes sp.1, Leotiomycetes sp.2, Leptodophora cf. echinata, Leptodophora orchidicola, Mariannaea pinicola, Metapochonia bulbillosa, Oidiodendron maius, Penicillium saturniforme, Penicillium sp., Pezicula ericae, Pezicula radicicola, Phialocephala sp.1, Phialocephala sp.2, Plectosphaerella cucumerina, Pleosporales sp.A total of approximately 50 species of root endophytes were identified, including *Tetracladium sp.*, *Trichoderma asperellum*, *Trichoderma polysporum*, and *Umbelopsisi nana*.

[0046] Furthermore, the presence of Ilyonectria mors-panacis was confirmed in almost all roots. As mentioned earlier, this fungus is a pathogen that causes root rot in ginseng and is a major cause of continuous cropping problems. Nevertheless, for ginseng plants that have survived and continued to grow for more than 5 years without developing root rot, 15 species of root endophytes were selected: Alternaria sp., Cladophialophora sp., Cladosporium sp, Dactylonectria pauciseptata, Dothideomycetes sp.1, Helotiales sp.1, Humicola olivacea, Hyphodiscaceae sp.4, Ilyonectria sp.2, Lasionectriopsis pteridii, Leptodophora orchidicola, Oidiodendron maius, Penicillium sp., Pezicula ericae, and Philocephala fortinii. From this group, we conducted indoor experiments to select fungi that exhibited superior effects in suppressing root rot pathogens and promoting growth.

[0047] First, regarding the inhibitory effect on root rot pathogens, an antimicrobial test was conducted by pitting the plants against Ilyonectria mors-panacis on a culture medium. As a result, three strains of Leptodophora orchidicola, YS102, YS094, and YS58, showed a strong inhibitory effect. Subsequently, Dactylonectria pauciseptata strain YS024 and Lasionectriopsis pteridii strain YS147 were also found to have inhibitory effects.

[0048] Next, we examined the effects of each endophyte on the growth of ginseng. Even if a disease can be controlled, if it negatively affects growth or does not promote growth, it cannot be adopted at all.

[0049] Laboratory inoculation experiments were conducted with these three types of endophytes (Figure 4). Specifically, each endophyte was cultured and propagated in test tubes. A growing medium consisting of equal parts akadama soil and peat moss was placed on top of the propagated endophytes. Since both ginseng and endophytes prefer slightly acidic conditions, unadjusted pH peat moss was used. Ginseng seeds were buried in the growing medium, and germination was allowed to occur naturally without any environmental control such as temperature. Rooting and germination occurred in April, and the seeds grew well until the end of August. As a result, the seeds inoculated with any of the endophytes showed a higher growth-promoting effect compared to those not inoculated with endophytes.

[0050] As a result of these studies, the inventors have found that all strains of Leptodophora orchidicola, Dactylonectria pauciseptata, and Lasionectriopsis pteridii exhibit excellent effects in suppressing root rot pathogens and promoting growth, thus preventing continuous cropping problems in ginseng. [Effects of the Invention]

[0051] According to the first configuration of the present invention, endophytes obtained from the roots of Panax ginseng cultivated in forest floor trials contain endophytes that suppress root rot pathogens and / or promote the growth of Panax ginseng. By incorporating these endophytes into the growing medium for Panax ginseng cultivation, it becomes possible to prevent continuous cropping problems in Panax ginseng, which are a problem in field cultivation, and to promote its growth.

[0052] According to the second configuration of the present invention, it is possible to control Ilyonectria mors-panacis, a root rot pathogen that is the main cause of continuous cropping problems in ginseng. By mixing multiple fungal species to increase diversity, it is expected that even if mutant strains of the pathogen Ilyonectria mors-panacis appear, they can be controlled. This makes it possible to replant in the same field. This eliminates the need to search for new land with an environment and soil suitable for ginseng. Furthermore, it eliminates the need for the time and effort of preparing the soil from scratch in new land. There is also no need to relocate the shade equipment. This greatly reduces the burden on producers. Furthermore, it is not limited by the location constraint of mountainous rivers as described in Patent Document 1, and there is no environmental burden on the river ecosystem due to fertilizer runoff. In addition, there is no difficulty in sowing or transplanting due to cultivation beds made of sand, gravel, and pebbles, and it is not time-consuming. Moreover, there is no risk of complete destruction due to mud inflow during natural disasters. Eliminating the need for the soil fumigant chloropicrin reduces the workload for producers and ensures safety. Furthermore, not using chemically synthesized pesticides caters to consumers' preference for natural products, increasing market value. It also reduces the environmental impact on natural ecosystems. Furthermore, it can prevent root rot, a disease that begins to increase sharply after about four years of cultivation. This helps to secure a good harvest. Moreover, by extending the cultivation period, for example to six years or more, the market value increases, contributing to increased income for producers.

[0053] According to the third configuration of the present invention, by using the endophytes of Leptodophora orchidicola strain YS102, deposited as NITE P-04203, Dactylonectria pauciseptata strain YS024, deposited as NITE P-04202, and Lasionectriopsis pteridii strain YS147, deposited as NITE P-04204, it becomes possible to reliably obtain superior control of root rot pathogens and growth promotion effects.

[0054] According to the fourth configuration of the present invention, it is possible to control Ilyonectria mors-panacis, a root rot pathogen that is the main cause of continuous cropping problems in ginseng. At least one species is sufficient for effective control, but mixing multiple species increases diversity, and it is expected that even if mutant strains of the pathogen Ilyonectria mors-panacis appear, they will be controlled. This makes it possible to replant in the same field. This eliminates the need to search for new land with an environment and soil suitable for ginseng. Furthermore, it eliminates the need for the time and effort of preparing the soil from scratch in new land. There is also no need to relocate the shade equipment. This greatly reduces the burden on producers.

[0055] According to the fifth configuration of the present invention, by using strains in which Leptodophora orchidicola is strain YS102 deposited under accession number NITE P-04203, Dactylonectria pauciseptata is strain YS024 deposited under accession number NITE P-04202, and Lasionectriopsis pteridii is strain YS147 deposited under NITE P-04204, it becomes possible to reliably obtain superior control of root rot pathogens and growth promotion effects.

[0056] According to the sixth configuration of the present invention, fungi belonging to Leptodophora orchidicola, Dactylonectria pauciseptata, and Lasionectriopsis pteridii are endophytes that have an inhibitory effect or a growth-promoting effect against the root rot pathogen of Panax ginseng.

[0057] According to the seven components of the present invention, strains belonging to Leptodophora orchidicola, Dactylonectria pauciseptata, and Lasionectriopsis pteridii can be easily obtained. These fungi, isolated and cultured from the roots of Panax ginseng cultivated on the forest floor, are stored and managed at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation. The Leptodophora orchidicola strain is YS102, deposited with accession number NITE P-04203; the Dactylonectria pauciseptata strain is YS024, deposited with accession number NITE P-04202; and the Lasionectriopsis pteridii strain is YS147, deposited with accession number NITE P-04204. The inhibitory effect of each strain on root rot pathogens and the growth-promoting effect of each strain on Panax ginseng have been confirmed experimentally.

[0058] According to the eighth and ninth components of the present invention, the difficulty of cultivation can be greatly reduced. The biggest factor in the difficulty of cultivation is soil preparation. By using this material and growing medium, even new producers can easily prepare the soil, and the work that takes two to three years becomes unnecessary. Furthermore, by continuously spraying this fungus on the field, it is expected that this fungus will inhabit the field, similar to how yeast and koji mold inhabit sake breweries and soy sauce breweries. This makes it possible to continuously cultivate the same field, and since there is no need to prepare the soil from scratch in new land, the amount of work can be greatly reduced. It can also contribute to the efficient use of land.

[0059] According to the ten components of the present invention, the cultivation period can be shortened and the barriers to entry for new producers can be lowered. By providing roots and / or seedlings according to the present invention, farmers are freed from tasks requiring expertise, such as seed germination and raising young seedlings susceptible to pathogens like damping-off, thereby lowering the barrier to entry for new farmers. Furthermore, starting cultivation with one-year-old or two-year-old seedlings shortens the time to harvest, which is a significant advantage for farmers. This is especially beneficial for new farmers, as the long period without income from cultivation to harvest has been a major barrier to entry. Moreover, by using chain pots for seedling cultivation, it is possible to transplant seedlings into the field while maintaining a soil environment containing endophytes, thus suppressing infection from indigenous fungi until the seedlings are established in the field, and reducing the need for pesticide application. Additionally, using chain pots improves the efficiency and reduces the labor required for transplanting.

[0060] In recent years, attempts have been made to cultivate ginseng under solar power generation panels. This has attracted considerable attention as an effective use of shaded land, but ginseng cannot be planted again in the same spot. While it might be possible to cultivate other crops, the lack of sunlight under the panels limits the types of crops that can be grown. As a result, farming is often abandoned. However, the cultivation method of the present invention makes it possible to replant ginseng, enabling a sustainable business model. For new producers, it is also an excellent business model in which they can compensate for the lack of income until harvest with electricity revenue. Across Japan, solar power generation facilities with a capacity of 0.5 MW or more occupy a total area of ​​229.211 km2 (0.079% of Japan's land area), with 66.36% of that being medium-sized facilities ranging from 0.5 MW to 10 MW. Furthermore, many of these are installed in secondary forests, artificial forests, artificial grasslands, fields, and rice paddies. Utilizing the land beneath solar power panels for ginseng cultivation could contribute to improving land-use efficiency in Japan, a country with limited land area.

[0061] Furthermore, it would be possible to increase the self-sufficiency rate of ginseng. Domestic use of ginseng is 611 tons, of which 610 tons are imported and only 1 ton is domestically produced, meaning that almost the entire amount is dependent on imports. Of this, 609 tons are imported from China (Source: Japan Kampo and Crude Drug Preparations Association, Report on the Survey of Raw Material Crude Drug Usage, etc. (July 15, 2011)). The fact that almost the entire amount of this raw material, which is extremely important for Kampo medicines and health foods, is dependent on China represents a significant risk similar to the rare earth crisis of 2010. Many other crude drugs besides ginseng, such as licorice, peony, and angelica, are also imported from China, and this situation is also known as the rare plant risk. From the perspective of economic security, it is strongly desired that this situation be rectified.

[0062] Expanding the ginseng cultivation method according to the present invention to be used in conjunction with solar power generation facilities can significantly contribute to reducing this risk. [Brief explanation of the drawing]

[0063] [Figure 1] This is a photograph of the above-ground parts of Panax ginseng growing in a forest floor experimental cultivation. [Figure 2] This is a photograph of Panax ginseng roots grown in a forest floor experimental cultivation. [Figure 3] This is a photograph of subdivided roots being cultured in a 24-well microplate containing CMA medium. [Figure 4] This is a photograph of an endophyte inoculation experiment conducted in a laboratory. [Figure 5] This is a photograph of endophyte inoculation cultivation in a planter. [Figure 6] This is a photograph of the ginseng roots cultivated in Example 1. [Figure 7] This is a photograph of the ginseng roots cultivated in Example 6. [Figure 8] This is a photograph of the ginseng root cultivated in Comparative Example 1. [Figure 9] This is a comparison table of this example and a comparative example. [Modes for carrying out the invention]

[0064] The embodiments of the present invention will now be described in more detail, but the present invention is not limited in any way to these embodiments.

[0065] The ginseng seeds used in the examples and comparative examples were collected from 4-year-old plants in early summer of the sowing year. After carefully removing the pulp, germination treatment was performed, and only seeds whose shells opened and embryonic development was confirmed were used. For the germination treatment, the seeds were buried in river sand in a large unglazed pot, placed in a shady, well-ventilated area, and watered just enough to keep the surface from drying out. The period was from August to mid-November.

[0066] For the growing medium, a mixture of akadama soil, kanuma soil, and peat moss was used in a volume ratio of 6 parts, 1 part, and 3 parts. However, different growing media were used for Example 8 and Comparative Example 2. Details will be described in those sections. Growing medium that has been used to cultivate ginseng for more than one year is called continuous crop growing medium, and soil that has been used to cultivate ginseng for more than one year and then disinfected is called disinfected growing medium.

[0067] The fertilizer consisted of 3 liters of leaf mold per planter as organic fertilizer, and 90g of minerals (Nissan Chemical Highminecon), 90g of ancient seaweed clay (Yawata Mining Minegreen), and 120g of gypsum (Yoshino Gypsum Darwin 5000) as inorganic fertilizer. The soil pH was slightly acidic at 4.7-5.0, which is preferred by ginseng and endophytes. In the comparative example, this soil was used as is, while in the example, a growing medium was used in which a liquid carrier material for endophyte bacteria was applied to this soil. The timing of application of the endophyte material can be either before or after sowing, as noted in each example.

[0068] Soil disinfection was carried out using a soil reduction method utilizing low-concentration ethanol (Non-Patent Literature 5). Specifically, after drying the growing medium to a certain extent, it was placed in a transparent, thick plastic bag, filled with 2% ethanol (diluted Ecologiaru), and the air was removed before sealing. It was left in direct sunlight for at least two months from July to August. Disinfection was considered successful when a butyric acid odor was detected and the red color of photosynthetic bacteria could be seen. From mid-September, the moisture was allowed to evaporate to maintain a suitable humidity level.

[0069] I used an Iris Ohyama planter with a capacity of approximately 60 liters (90 cm long x 30 cm wide x 30 cm high). I placed the planter in a shady spot with a sunshade to prevent it from being exposed to direct sunlight.

[0070] Regarding the sowing method, the seeds were sown in rows, meaning that grooves approximately 3 cm deep and 1 cm wide were dug on the surface of the growing medium, and the seeds were sown at a density where they did not overlap, and then covered with soil. Approximately 200 seeds were sown per planter.

[0071] Endophyte inoculation was carried out by spraying each fungal solution material with a watering can after sowing in mid-November and again in mid-March of the following year when root emergence began. The fungal solution materials were prepared by culturing the fungal cells in a liquid medium with shaking, then washing them with sterile water, crushing them in a mixer, and diluting the resulting liquid carrier material with sterile water. The amount sprayed per planter was 1 liter. Each liter of fungal solution contained approximately 15 g (wet weight) of fungal cells. [Examples]

[0072] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of Leptodophora orchidicola strain YS102 was sprayed using a watering can. A second application of the same fungal solution was made in mid-March of the following year, when root development began.

[0073] The seedlings sprouted in mid-April and grew well, with the number of sprouting measured in mid-May. By the end of June, above-ground growth stopped and root growth began in earnest. In September, some of the above-ground parts began to wither. In mid-October, the roots were dug up and their number and weight were measured. The root survival rate was 100%. The survival rate was calculated by dividing the number of excavated roots by the number of sprouting. The average weight of the roots was 0.35g. The average weight of the top 20% of roots was 0.64g. [Examples]

[0074] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of a mycelial solution of Dactylonectria pauciseptata strain YS024 was sprayed using a watering can. A second application of the same mycelial solution was made in mid-March of the following year, when root development began.

[0075] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 98%, the average root weight was 0.33g, and the average weight of the top 20% of roots was 0.52g. [Examples]

[0076] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of a mycelial solution of Lasionectriopsis pteridii strain YS147 was sprayed using a watering can. A second application of the same mycelial solution was made in mid-March of the following year, when root development began.

[0077] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 87%, the average root weight was 0.26g, and the average weight of the top 20% of roots was 0.51g. [Examples]

[0078] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of Leptodophora orchidicola strain YS094 was sprayed using a watering can. A second application of the same fungal solution was made in mid-March of the following year, when root development began.

[0079] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 89%, the average root weight was 0.28g, and the average weight of the top 20% of roots was 0.52g. [Examples]

[0080] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of Leptodophora orchidicola strain YS058 was sprayed using a watering can. A second application of the same fungal solution was made in mid-March of the following year, when root development began.

[0081] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 91%, the average root weight was 0.30g, and the average weight of the top 20% of roots was 0.56g. [Examples]

[0082] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, a 1-liter mixture of 1 / 3 liter of Leptodophora orchidicola strain YS102, 1 / 3 liter of Dactylonectria pauciseptata strain YS024, and 1 / 3 liter of Lasionectriopsis pteridii strain YS147 was applied using a watering can. A second application of the same solution was made in mid-March of the following year, when root development began.

[0083] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 98%, the average root weight was 0.35g, and the average weight of the top 20% of roots was 0.63g. [Examples]

[0084] Fertilizer was added to the soil used for continuous cropping and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, a total of 1 liter of a solution was mixed with Leptodophora orchidicola strain YS102, Leptodophora orchidicola strain YS094, and Leptodophora orchidicola strain YS058, and the mixture was sprayed using a watering can. A second application of the same solution was made in mid-March of the following year, when root development began.

[0085] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 100%, the average weight of the roots was 0.36g, and the average weight of the top 20% of roots was 0.65g. [Examples]

[0086] The growing medium is Takii Cell growing medium. <tm-1>Fertilizer was added to the soil used for cultivating ginseng for over a year, mixed well, placed in planters, and sowed in mid-November. After sowing, 1 liter of Leptodophora orchidicola strain YS102 solution was sprayed using a watering can. A second application of the same solution was made in mid-March of the following year when root development began.

[0087] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 85%, the average root weight was 0.25g, and the average weight of the top 20% of roots was 0.50g. [Examples]

[0088] Fertilizer was added to the disinfected potting soil and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of Leptodophora orchidicola strain YS102 was sprayed using a watering can. A second application of the same fungal solution was made in mid-March of the following year, when root development began.

[0089] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. By the end of June, above-ground growth stopped and root growth began in earnest. In September, some of the above-ground parts began to wither. In mid-October, the roots were dug up and their number and weight were measured. The root survival rate was 100%. The survival rate was calculated by dividing the number of excavated roots by the number of sprouting. The average weight of the roots was 0.36g. The average weight of the top 20% of roots was 0.67g. [Examples]

[0090] Fertilizer was added to the disinfected potting soil and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of a mycelial solution of Dactylonectria pauciseptata strain YS024 was sprayed using a watering can. A second application of the same mycelial solution was made in mid-March of the following year, when root development began.

[0091] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 96%, the average root weight was 0.34g, and the average weight of the top 20% of roots was 0.62g. [Examples]

[0092] Fertilizer was added to the disinfected potting soil and mixed well. The mixture was then placed in planters, and seeds were sown in mid-November. After sowing, 1 liter of a mycelial solution of Lasionectriopsis pteridii strain YS147 was sprayed using a watering can. A second application of the same mycelial solution was made in mid-March of the following year, when root development began.

[0093] The seedlings sprouted in mid-April and grew well, with the number of sprouting counted in mid-May. The roots were dug up in mid-October, and their number and weight were measured. The root survival rate was 100%, the average root weight was 0.35g, and the average weight of the top 20% of roots was 0.63g. Comparative Example 1

[0094] Fertilizer was added to the soil used for continuous cropping and mixed well. It was placed in planters and seeds were sown in mid-November. The seedlings germinated in mid-April of the following year and grew well, with the number of germinated seedlings counted in mid-May. Around the end of June, the growth of the above-ground parts stopped and root growth began in earnest. In July, some of the above-ground parts began to wither. By the end of August, the above-ground parts had almost completely disappeared. In mid-October, the roots were dug up and the number and weight of the roots were measured. The root survival rate was 37%, the average weight of the roots was 0.11g, and the average weight of the top 20% of roots was 0.18g. Comparative Example 2

[0095] The growing medium is Takii Cell growing medium. <tm-1>In a soil mix that had been used for cultivating ginseng for over a year, fertilizer was added and thoroughly mixed, then placed in a planter, and seeds were sown in mid-November. Germination occurred in mid-April of the following year, and the plants grew well, with the number of germinated plants counted in mid-May. Around the end of June, above-ground growth stopped, and root growth began in earnest. In July, some above-ground parts began to wither. By the end of August, the above-ground parts had almost completely disappeared. In mid-October, the roots were dug up, and the number and weight of the roots were measured. The root survival rate was 13%, the average root weight was 0.10g, and the average weight of the top 20% of roots was 0.15g. Comparative Example 3

[0096] Fertilizer was added to disinfected potting soil and mixed well. The mixture was placed in planters and seeds were sown in mid-November. Germination occurred in mid-April, and the plants grew well. The number of germinated seedlings was counted in mid-May. The roots were dug up in mid-October, and the number and weight of the roots were measured. The root survival rate was 96%, the average weight of the roots was 0.34g, and the average weight of the top 20% of roots was 0.61g.

[0097] For Examples 1-11 and Comparative Examples 1-3, the growing medium, endophyte strain name, root survival rate, average root weight, and average weight of the top 20% of root weights are summarized in the table in Figure 9.

[0098] Examples 1-5 and Comparative Example 1 show that when endophytes are present in continuous cropping soil, the root survival rate is higher and the root weight is greater compared to when they are not present. Among these, Example 1 (YS102) showed the highest survival rate and weight. Furthermore, the difference in the number of surviving roots and growth is clear from the photographs of carrot roots in Figure 6 (Example 1) and Figure 8 (Comparative Example 1). In other words, it is clear that continuous cropping problems in continuous cropping soil can be controlled by the presence of these endophytes.

[0099] Example 6 involved the coexistence of three endophytes: Leptodophora orchidicola, Dactylonectria pauciseptata, and Lasionectriopsis pteridii. The survival rate and root weight were greater than in Comparative Example 1. Furthermore, as shown in Figure 7, there was less variation in root morphology compared to Figure 6. It is believed that the coexistence of multiple endophytes increased the diversity of the microbial community, thereby reducing variability in the effectiveness of controlling various soilborne pathogens and promoting growth. For Panax ginseng, low root morphology variation is a crucial factor in both production and commercial value.

[0100] In Example 7, three strains of Leptodophora orchidicola, namely YS102, YS094, and YS058, were coexisted, and the survival rate and root weight were greater than in Comparative Example 1. In Examples 1 (YS102), 4 (YS094), and 5 (YS058), where single strains of Leptodophora orchidicola were coexisted, the YS102 strain showed the highest survival rate and weight. However, when three strains were coexisted, further improvements were observed in both survival rate and weight, confirming that there were no adverse effects from mixing multiple strains of Leptodophora orchidicola and demonstrating a synergistic effect. In other words, it can be seen that Leptodophora orchidicola, regardless of the strain, has an effect of controlling continuous cropping problems and promoting growth in Panax ginseng.

[0101] Examples 1, 6, and 7, along with Comparative Example 3, demonstrate that coexisting endophytes in the growing medium has a greater effect on preventing continuous cropping problems than soil reductive disinfection, and furthermore, it also promotes root growth.

[0102] Example 8 uses commercially available growing medium, TakiiCell growing medium. <tm-1>Even when using [this], the survival rate and root weight were greater than in Comparative Example 2. This indicates that endophytes have a preventative effect against continuous cropping problems even when different types of growing media are used. However, the survival rate and root weight were lower compared to soil mainly composed of akadama soil. This is because of the Takii Cell growing media. <tm-1>The high concentration of nitrogen (85 mg / l), phosphorus (90 mg / l), and potassium (85 mg / l) likely suppressed the activity of endophytes.

[0103] Examples 9-11 and Comparative Example 3 show that when endophytes were inoculated into disinfected growing medium, the root survival rate was equal to or better than that of uninoculated medium, and the root weight was greater. Among these, Example 9, inoculated with strain YS102, showed a high survival rate and weight. In other words, by coexisting endophytes with disinfected growing medium, it is possible to prevent continuous cropping problems and achieve a growth-promoting effect that surpasses that of soil reductive disinfection. As can be seen from the comparison between Examples 1-3 and Examples 9-11, by using soil reductive disinfected growing medium and endophytes together, a higher reduction in continuous cropping problems and a growth-promoting effect can be obtained.

[0104] One-year-old roots and / or seedlings from Examples 1-11 and Comparative Examples 1-3, dug up in mid-October, were transplanted to the field in November. The field was prepared using conventional methods by producers engaged in general vegetable cultivation. Sprouting occurred in mid-April of the following year, and the number of sprouting plants was counted in mid-May. No pesticides were applied, and the roots were dug up in mid-October, with the number and weight of roots measured. The root survival rate of Examples 1-11, which were inoculated with endophytes, was 95-100%, and the average root weight was 2.1g. In contrast, the root survival rate of Comparative Examples 1-2, which were not inoculated with endophytes, was 50%, and the average root weight was 0.7g, while the root survival rate of Comparative Example 3 was 70%, and the average root weight was 1.4g. Roots and / or seedlings with endophytes in symbiosis showed superior survival rate and growth. Therefore, providing roots and / or seedlings with endophytes in symbiosis will facilitate entry for new entrants.

Claims

1. A fungus belonging to Leptodophora orchidicola, Dactylonectria pauciseptata, or Lasionectriopsis pteridii that has the effect of suppressing root rot pathogens and / or promoting growth in the cultivation of Panax ginseng.

2. The fungus according to claim 1, wherein Leptodophora orchidicola is strain YS102 deposited under accession number NITE P-04203, Dactylonectria pauciseptata is strain YS024 deposited under accession number NITE P-04202, and Lasionectriopsis pteridii is strain YS147 deposited under accession number NITE P-04204.

3. A material for cultivating Panax ginseng, characterized by containing at least one of the fungi described in claim 1 or 2.

4. A growing medium for cultivating Panax ginseng, characterized by containing at least one of the fungi described in claim 1 or 2.

5. A ginseng root and / or seedling characterized in that at least one of the fungi described in claim 1 or 2 is symbiotic with the root.

6. Potted seedlings of Panax ginseng grown in pots filled with growing medium containing at least one of the fungi described in claim 1 or 2.

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