Method for evaluating capability of soil microbial flora that imparts beneficial character to plant
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-02-21
- Publication Date
- 2026-03-04
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to a method for evaluating the ability of a soil microbiome to confer beneficial traits to a plant. The present invention also relates to a method for screening a soil microbiome that confer beneficial traits to a plant. The present invention also relates to a method for screening a soil microbiome that suppresses the occurrence of a soil-borne plant disease in a plant. The present invention further relates to a method for suppressing the occurrence of a soil-borne plant disease in a plant. [Background technology]
[0002] In recent years, there has been a strong demand both in Japan and overseas to reduce the use of chemical pesticides and fertilizers due to concerns about their impact on the human body and the environment, as well as resource depletion. In the agricultural field, research has been conducted focusing on plant growth promoting microorganisms (PGPM) that have the ability to promote plant growth and control diseases as a key to breaking away from agricultural production systems that are dependent on chemical pesticides and fertilizers, and many PGPM strains with high activity potential against various crops and diseases have been discovered.
[0003] However, it is difficult to establish these PGPM strains when they are introduced into soil alone, and they often do not fully demonstrate their capabilities. Therefore, there are very few examples of PGPM strains being put to practical use in soil environments. Given this background, there is currently a great deal of attention being paid to the agricultural use of microbiota, which are a collection of microorganisms.
[0004] In order to appropriately utilize microbiota in agricultural fields, a deep understanding of the microbiota that confers beneficial traits to plants is required. To this end, it is necessary to discover and precisely analyze many microbiota that confers beneficial traits to plants. So far, when soils that are less susceptible to disease or soils that grow well in plants have been discovered in agricultural fields, the composition and function of these soil microbiota have been analyzed (Non-Patent Documents 1 to 3). For example, in Patent Document 3, Fusarium oxysporum is inoculated into several types of soil, such as strawberry monoculture soil, and strawberry seedlings are planted to evaluate the soil for its ability to suppress the occurrence of strawberry wilt disease caused by the filamentous fungus.
[0005] However, because the discovery of disease-suppressing soil is accidental, the number of research subjects is insufficient. In addition to the microbiome, soil also contains physical components, which are solid particulate components composed of inorganic matter such as stones, sand, soil, silt, and clay, and physicochemical components, which are water-soluble components such as organic and inorganic components. However, conventional analytical methods evaluate the entire soil, which means that the ability of the soil microbiome itself cannot be directly evaluated. [Prior art documents] [Non-patent literature]
[0006] [Non-Patent Document 1] Okazaki, K. et al. (2021), Microbes and environments, 36(2), ME20137 [Non-Patent Document 2] Yin, C. et al. (2021), Microbiome, 9(1), 86.https: / / link.springer.com / article / 10.1186 / s40168-020-00997-5 [Non-Patent Document 3] Cha, JY et al. (2016), The ISME journal, 10(1), 119-129. https: / / www.nature.com / articles / ismej201595 Summary of the Invention [Problem to be solved by the invention]
[0007] Therefore, in order to obtain a microbial flora that confers beneficial traits to plants, such as suppressing the occurrence of plant diseases and promoting plant growth (hereinafter referred to as "useful microbial flora"), a method for directly evaluating the capabilities of the soil microbial flora itself was desired. [Means for solving the problem]
[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that it is possible to directly evaluate the soil microbial flora by separating one or more soils containing a microbial flora into a fraction containing only the microbial flora and a fraction not containing the microbial flora, and cultivating plants in artificial soil treated with each fraction, thereby completing the present invention. The inventors also discovered that it is possible to screen for useful microbial flora from multiple soil microbial flora using a similar technique to that described above using multiple soils containing microbial flora, and thus completed the present invention. The inventors also discovered that, using a plurality of soils containing microbial flora, it is possible to screen for soil microbial flora that suppress the occurrence of soil-borne plant diseases caused by microorganisms that cause soil-borne plant diseases by a method similar to that described above, and thus completed the present invention. Furthermore, the inventors have discovered that by mixing soil having a high ability to suppress the occurrence of soil-borne plant diseases, obtained by the above-mentioned method for screening soil microbial flora that suppresses the occurrence of soil-borne plant diseases, with soil having a low ability to suppress the occurrence of soil-borne plant diseases, the ability of the latter to suppress the occurrence of soil-borne plant diseases can be improved, and thus the present invention has been completed.
[0009] That is, the present invention provides the following. (1) A method for assessing the ability of a soil microbiome to confer beneficial traits to a plant, comprising: (i) preparing a soil suspension from soil containing a microbiota and separating said soil suspension into a fraction containing a microbiota and a fraction not containing a microbiota; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating a plant and monitoring the occurrence of disease in said plant and / or the growth of said plant; (v) evaluating the soil as having the ability to impart a beneficial trait to a plant if the soil suppresses the occurrence of a disease and / or promotes the growth of the plant; A method comprising: (2) A method for screening a soil microbiome that confers beneficial traits to plants, comprising: (i) preparing soil suspensions from a plurality of soils containing a microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating a plant and monitoring the occurrence of disease in said plant and / or the growth of said plant; (v) selecting from the plurality of soils a soil that suppresses the occurrence of the disease and / or promotes the growth of the plant as a soil microbiome that confers beneficial traits to the plant; A method comprising: (3) A method for screening a soil microflora that suppresses the occurrence of a soil-borne plant disease in a plant, comprising: (i) preparing soil suspensions from a plurality of soils containing a microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) inoculating each of the treated artificial soils with a microorganism causing a soil-borne plant disease, followed by sowing plant seeds; (iv) cultivating the plant and monitoring the plant for occurrence of said soil-borne plant disease; (v) selecting from the plurality of soils a soil microbiota in which a fraction containing the microbiota suppresses the occurrence of the soil-borne plant disease by more than 50% and a fraction not containing the microbiota suppresses the occurrence of the soil-borne plant disease by less than 20%; A method comprising: (4) The method according to any one of (1) to (3), wherein the step (i) of separating the soil suspension into a fraction containing a microbiota and a fraction not containing a microbiota comprises a step of filtering the soil suspension through a filter having a pore size of 10 to 50 μm, and then filtering the obtained filtrate through a filter having a pore size of 0.1 to 1.0 μm. (5) The method according to any one of (1) to (4), wherein the artificial soil is selected from the group consisting of vermiculite, zeolite, and perlite. (6) The method according to (5), wherein the artificial soil is vermiculite. (7) The method according to any one of (3) and (4) to (6) which cites (3), wherein the soil-borne plant disease is Fusarium disease. (8) The method according to any one of (3) and (4) to (7) which cites (3), wherein the microorganism causing the soil-borne plant disease is a fungus of the genus Fusarium. (9) The method according to (8), wherein the Fusarium fungus is Fusarium oxysporum. (10) A method for suppressing occurrence of a soil-borne plant disease in a plant, comprising the steps of: (i) (3) and a step of selecting a soil (a) containing an antagonistic microorganism group, in which a fraction containing the microorganism suppresses the occurrence of the soil-borne plant disease by more than 80% and a fraction not containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 20%, from a soil (b) in which a fraction containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 30% and a fraction not containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 20%; (ii) mixing the soil (a) with the soil (b); (iii) a step of improving the ability of the soil (b) to suppress the occurrence of the soil-borne plant disease by transferring and establishing the antagonistic microorganisms in the soil (a) to the soil (b); A method comprising: (11) The method according to (10), wherein the soil (a) and the soil (b) are soils originating from the same field or from different fields. (12) The method according to (10) or (11), wherein the soil (a) and the soil (b) are mixed in a ratio of 1:9 to 9:1. (13) The method according to any one of (10) to (12), wherein the antagonistic microorganisms are bacteria of the genus Pseudolabrys and / or Sphingomonas. Effect of the Invention
[0010] According to the present invention, the ability of the soil microflora itself to impart useful traits to plants can be directly evaluated, thereby enabling useful microbial flora to be obtained with high accuracy. [Brief description of the drawings]
[0011] [Figure 1]Fig. 1 shows the Fusarium disease suppression effect of the mixed soil microflora component solution. Comparison of the average disease intensity of three replicate tests of five plants in each treatment area shows that the suppression effect of the microflora of soil 1 was transferred to soils 12 and 23, but not to soils 28 and 40. [Diagram 2] 2 shows the Fusarium disease inhibitory effect of a soil microbiota component solution mixed with gamma-ray sterilized soil 1. Comparison of the average disease intensity of three replicate tests of five plants in each treatment plot shows that the inhibitory effect of the microbiota of gamma-ray sterilized soil 1 was not transferred to soil 12 and soil 23. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments and can be practiced with appropriate modifications.
[0013] A first embodiment of the invention is a method for assessing the ability of a soil microbiome to confer beneficial traits to plants. The evaluation method according to the first embodiment includes the following steps. (i) preparing a soil suspension from soil containing a microbiota and separating said soil suspension into a fraction containing a microbiota and a fraction not containing a microbiota; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating a plant and monitoring the occurrence of disease in said plant and / or the growth of said plant; (v) assessing the soil as having the ability to impart a beneficial trait to a plant if the soil suppresses the occurrence of a disease and / or promotes the growth of the plant.
[0014] In the present invention, the "beneficial trait" refers to, for example, suppression of the occurrence of plant diseases, promotion of plant growth, improvement of plant yield, ease of harvesting of plants, and improvement of the production and accumulation of functional components in plants.
[0015] A plant "disease" refers to an abnormality in a plant that occurs as a result of the continuous action of a certain cause, and when the cause of the disease is a microorganism (filamentous fungi, bacteria, viruses, viroids, phytoplasmas, nematodes), it is specifically called an infectious disease. Examples of "diseases" include bacterial diseases such as soft rot, rot, bacterial spot, bacterial leaf spot, canker, bacterial perforation, bacterial black spot, bacterial brown spot, bacterial stem necrosis, bacterial grain blight, bacterial seedling blight, bacterial wilt, white leaf blight, black rot, and fire blight; and fungal diseases such as clubroot, white root rot, gray mold, gray spot, sclerotinia rot, brown spot, black spot, leaf spot, black spot, summer blight, ring spot, leaf mold, sooty mold, scab, anthracnose, powdery mildew, rust, black spot, late blight, and downy mildew.
[0016] "Suppressing the occurrence of disease" means exerting an antagonistic effect against microorganisms that cause plant diseases. A microflora that imparts beneficial traits to a plant (useful microflora) exerts an antagonistic effect against microorganisms that cause plant diseases, thereby preventing or curing plant diseases caused by those microorganisms. In addition, "suppressing the occurrence of disease" means that the microflora improves the immunity of the plant, thereby indirectly suppressing the occurrence of plant diseases. "Preventing plant diseases" means that, in plants that are not infected with microorganisms that cause plant diseases or that do not show symptoms, a plant grown in the presence of useful microbial flora has a lower degree of disease than a plant grown under the same conditions except for being grown in the absence of useful microbial flora. "Curing a plant disease" means that in a plant that is infected with a pathogen causing a plant disease and is showing symptoms, the severity of the disease is less in a plant that is grown in the presence of a useful microflora than in a plant that is grown under the same conditions except for in the absence of a useful microflora. "Low degree of disease damage" means, for example, that the disease severity or disease incidence rate is low, specifically, that the control value represented by Formula 2 in Example 2 described below is greater than 0. A greater control value is preferable, with 30% or more being excellent, 50% or more being even better, 60% or more being particularly excellent, and 70% or more being even more particularly excellent.
[0017] "Promoting plant growth" refers to the fact that the beneficial microflora has an antagonistic effect against microorganisms that cause plant diseases, thereby suppressing the occurrence and proliferation of the microorganisms, resulting in stabilization and improvement of plant yields. Also, "promoting plant growth" refers to the fact that the beneficial microflora assists in the supply of nutrients, resulting in stabilization and improvement of plant yields.
[0018] "Plants" are not particularly limited, but examples thereof include grains (e.g., rice, barley, wheat, rye, oats, corn, etc.), beans (soybeans, adzuki beans, broad beans, peas, kidney beans, peanuts, etc.), fruit trees and fruits (apples, citrus fruits, pears, grapes, peaches, plums, cherries, walnuts, chestnuts, almonds, bananas, etc.), leafy vegetables (cabbage, tomato, spinach, broccoli, lettuce, onion, green onion (chives, scallions), bell peppers, eggplant, strawberries, peppers, okra, chives, etc.), root vegetables (carrots, potatoes, sweet potatoes, taro, daikon, turnips, Examples of such crops include: lotus root, burdock, garlic, and scallions, processed crops (cotton, hemp, beets, hops, sugar cane, sugar beets, olives, rubber, coffee, tobacco, and tea, etc.), melons (pumpkin, cucumber, watermelon, Sakhalin gourd, and melon, etc.), pasture grasses (orchard grass, sorghum, timothy, clover, and alfalfa, etc.), turfgrass (Korean grass and bentgrass, etc.), ornamental crops for fragrances and the like (lavender, rosemary, thyme, parsley, pepper, ginger, etc.), and flowers (chrysanthemums, roses, carnations, orchids, tulips, lilies, etc.).
[0019] "Soil" refers to a mixture of naturally occurring solid inorganic matter such as minerals, rocks, sand, etc.; organic matter such as biological products, excrement, corpses, and humus; natural liquids such as petroleum, petroleum precursors, petroleum spring water, mineral water, hot spring water, river water, lake water, and sea water; and living organisms (living organisms). It includes soil that exists on the ground as well as at the bottom of bodies of water such as rivers, lakes, and seas. The target "soil" may be one or more. When there are multiple soils, they may be obtained from the same field or from different fields. "Soil suspension" refers to soil suspended in water. The water that can be used includes sterilized water, distilled water, purified water, pure water, ultrapure water, RO water, ion-exchanged water, elixirs water, tap water, etc. Among these, sterilized water is preferred. The concentration is preferably 1.0 x 10 -7 % to 99%, and more preferably 0.01% to 10%.
[0020] "Microbiota" refers to a collection of microorganisms in an ecosystem, such as bacteria, fungi, archaea, viruses, and protists. The types of microorganisms that make up the collection vary depending on the environment in which the microbiota is established. "Soil microbiota" refers to the microorganisms that are established in the soil. The "fraction containing microbiota" refers to the filtrate obtained by filtering a soil suspension with a filter of a specific pore size capable of separating physical components such as rocks (hereinafter referred to as the "microbiota-containing filtrate"). The "fraction not containing microbiota" refers to the filtrate obtained by further filtering the microbiota-containing filtrate with a filter of a pore size capable of removing microorganisms. The "fraction not containing microbiota" refers to a filtrate that does not contain any microbiota at all, or, if it does contain any microbiota, it is at a level below the detection limit (e.g., 1×10 to 1×10 2 The filtrate contains microbial flora (< cfu / ml).
[0021] In step (i) of the first embodiment of the present invention, first, the microflora contained in the soil is separated from physical components, which are solid particulate components composed of particles such as rocks, stones, sand, and soil, and inorganic substances such as silt (fine sand), and clay. For example, the soil is suspended in sterilized water or the like to prepare a soil suspension, and the physical components are removed by filtering through a filter with a coarse pore size. The obtained filtrate contains physicochemical components, which are water-soluble components such as organic and inorganic components, in addition to the microflora (hereinafter, appropriately referred to as a "fraction containing the microflora"). Next, the filtrate is further filtered through a filter with a finer pore size to obtain a physicochemical component solution from which the microflora has been removed (hereinafter, appropriately referred to as a "fraction not containing the microflora"). To remove the physical components, for example, a filter with a pore size of about 10 to about 50 μm, preferably about 20 to about 45 μm, is used. To remove the microflora, for example, a filter having a pore size of about 0.1 to about 1.0 μm, preferably about 0.1 to about 0.3 μm, is used, and more preferably a filter having a pore size of 0.22 μm is used.
[0022] In step (ii), the "artificial soil derived from natural minerals" is not particularly limited as long as it has a low content of physicochemical components and uniform physicochemical properties. Examples include vermiculite, zeolite, and perlite. Vermiculite is particularly preferred because it is porous, very light, and has excellent water retention, breathability, and fertilizer retention properties. The artificial soil may also be mixed with beet moss, red soil, or other soil-improving soil. In that case, the artificial soil should account for 70% or more by weight, preferably 80% or more by weight, of the total soil. The artificial soil to be used is preferably sterilized, which can be carried out by a conventional method such as autoclaving.
[0023] In step (ii), the treatment of the artificial soil with the fraction containing the microbiota or the fraction not containing the microbiota can be carried out by adding or spraying the fraction containing the microbiota or the fraction not containing the microbiota, or a concentrate, paste, dried product, diluted product, etc. thereof, to the artificial soil. In addition, artificial fertilizer such as Hyponex may be added to the artificial soil.
[0024] In step (iv), after sowing the plant seeds, the state of the seedlings that have grown is monitored. For example, it is monitored to see whether the seedlings are healthy or diseased, and when root rot, root gall formation, wilting, yellowing, decay, spot formation, browning of xylem, yellowing and browning of hypocotyl, wilting, yellowing, decay, wilting of the entire plant, complete withering, or the like is observed, the plant is deemed to be diseased, and the disease severity of the plant is evaluated, for example, according to the relative disease severity described in Example 2 below.
[0025] "Monitoring" refers to obtaining information on the growth state of the entire plant and / or each tissue such as the fruit, roots, leaves, etc. Specifically, the photosynthesis rate, transpiration rate, water absorption rate, leaf area, leaf height, leaf number, plant weight, growth abnormalities, bacterial count, etc. related to the plant being cultivated are monitored. The monitoring is performed by a monitoring device such as a camera or a sensor such as a beam sensor or color sensor. For example, a monitoring device for monitoring the plants is provided in a plant culture room, and a display device is provided outside the culture room, and an output signal from the monitoring device is transmitted to the display device. By installing the monitoring device, the growth state can be monitored outside the culture room without having to take the cultivation container out each time. "Evaluation" refers to determining the degree of inhibition of plant disease occurrence based on the relative disease severity described in Example 2 below, or using well-known evaluation methods to determine whether plant growth is promoted compared to healthy plants, whether yield is increased or decreased, or the amount of accumulated functional components is increased or decreased.
[0026] A second embodiment of the present invention is a method for screening soil microbiomes that confer beneficial traits on plants. A second embodiment of the present invention includes the following steps. (i) preparing soil suspensions from a plurality of soils containing a microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating a plant and monitoring the occurrence of disease in said plant and / or the growth of said plant; (v) selecting from the plurality of soils a soil that suppresses the occurrence of the disease and / or promotes the growth of the plant as a soil microbiome that confers beneficial traits to the plant.
[0027] Steps (i) to (iv) in the screening method according to the second embodiment of the present invention are substantially the same as steps (i) to (iv) in the evaluation method according to the first embodiment, but are characterized in that step (i) includes a step (v) of preparing a plurality of soils and selecting useful microbial flora from the plurality of soils. Here, "multiple" is not particularly limited as long as it is 2 or more, but is preferably 5 or more, preferably 6 or more, and more preferably 7 or more. The multiple soils may be obtained from the same farm field or may be obtained from different farm fields.
[0028] A third embodiment of the present invention is a method for screening for a soil microflora that suppresses the occurrence of a soil-borne plant disease in a plant. A third embodiment of the present invention includes the following steps. (i) preparing soil suspensions from a plurality of soils containing a microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction; (ii) preparing an artificial soil derived from natural minerals and treating the artificial soil with each of a fraction containing the microbial flora and a fraction not containing the microbial flora; (iii) inoculating each of the treated artificial soils with a microorganism causing a soil-borne plant disease, followed by sowing plant seeds; (iv) cultivating the plant and monitoring the plant for occurrence of said soil-borne plant disease; (v) selecting from the plurality of soils a soil microbiota in which a fraction containing said microbiota suppresses the occurrence of said soil-borne plant disease by more than x% and a fraction not containing said microbiota suppresses the occurrence of said soil-borne plant disease by less than y%; A method comprising:
[0029] Steps (i), (ii) and (iv) in the third embodiment of the present invention are the same as those in the second embodiment.
[0030] The "soil-borne plant disease" in step (iii) is not particularly limited as long as it is transmitted to other fields through soil by agricultural machinery or work shoes. It also includes a disease in which the zoospores of a microorganism present in a certain soil swim in water and infect plants in other soils. The "microorganisms" that cause soil-borne plant diseases are, for example, bacteria, filamentous fungi, etc. Filamentous fungi include, for example, Oomycetes, Ascomycetes, Deuteromycetes, Basidiomycetes, Zygomycetes, etc.
[0031] Specific examples of soil-borne plant diseases and the microorganisms that cause them include Ralstonia solanacearum, Erwinia carotovora, Pythium ultimum, Phytophthora capsici, Verticillium dahliae, Fusarium oxysporum, Fusarium oxysporum, Plasmodiophora brassicae, Gaeumannomyces gramineum, Athelia rolfsii, Helicobasidium mompa, Rosellinia necatrix, Aphanomyces euteiches, Aphanomyces arbutifolia, and others. These include Sclerotium raphani, Sclerotium cepivorum, Spongospora subterranea, Streptomyces scabies, Agrobacterium tumefaciens, Cephalosporium gramineum, Cephalosporium gregatum, Thielaviopsis basicola, and Rhizoctonia solani.
[0032] Methods for inoculating artificial soil with microorganisms that cause soil-borne plant diseases include adding or spraying the microbial cells themselves, a suspension containing the cells, a culture solution containing the cells, or concentrates, pastes, dried products, dilutions, etc. of these.
[0033] The inoculation concentration of the microorganism is, for example, 1×10 in terms of bacterial cell concentration. 2 ~1×10 11 cfu / ml, preferably 1×10 4 ~1×10 9 cfu / ml range.
[0034] Step (v) of the third embodiment involves assessing the extent (or rate) of inhibition of the occurrence of a soil-borne plant disease in a plant by the fraction containing the microbiota and the extent (or rate) of inhibition of the occurrence of a soil-borne plant disease in a plant grown under the same conditions as for the treatment with the fraction containing the microbiota, but with the exception of the treatment with the fraction not containing the microbiota.
[0035] The greater the difference between the degree of inhibition of the occurrence of soil-borne plant disease by the fraction containing microbiota (x%) and the degree of inhibition of the occurrence of soil-borne plant disease by the fraction not containing microbiota (y%), the better, but for example, it may be about 20% or more. For example, the inhibition rate of the fraction containing microbiota may be 30% and the inhibition rate of the fraction not containing microbiota may be 10%, or the inhibition rate of the fraction containing microbiota may be 40% and the inhibition rate of the fraction not containing microbiota may be 20%, but generally the inhibition rate of the fraction containing microbiota is more than 50% and the inhibition rate of the fraction not containing microbiota is less than 20%. More preferably, the inhibition rate of the fraction containing the microbiota is more than 60% and the inhibition rate of the fraction not containing the microbiota is less than 20%, more preferably, the inhibition rate of the fraction containing the microbiota is more than 70% and the inhibition rate of the fraction not containing the microbiota is less than 20%, more preferably, the inhibition rate of the fraction containing the microbiota is more than 80% and the inhibition rate of the fraction not containing the microbiota is less than 20%, more preferably, the inhibition rate of the fraction containing the microbiota is more than 90% and the inhibition rate of the fraction not containing the microbiota is less than 20%, more preferably, the inhibition rate of the fraction containing the microbiota is more than 95% and the inhibition rate of the fraction not containing the microbiota is less than 20%, and more particularly preferably, the inhibition rate of the fraction containing the microbiota is more than 99% and the inhibition rate of the fraction not containing the microbiota is less than 20%.
[0036] A fourth embodiment of the present invention is a method for inhibiting the occurrence of a soil-borne plant disease in a plant. A fourth embodiment of the present invention includes the following steps. (i) in the screening method according to the third embodiment of the present invention, selecting a soil (a) containing an antagonistic microorganism group, in which a fraction containing the microorganism suppresses the occurrence of the soil-borne plant disease by more than 80% and a fraction not containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 20%, and a soil (b) in which a fraction containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 30% and a fraction not containing the microorganism suppresses the occurrence of the soil-borne plant disease by less than 20%; (ii) mixing the soil (a) with the soil (b); (iii) a step of improving the ability of the soil (b) to suppress the occurrence of the soil-borne plant disease by transferring and establishing the antagonistic microorganisms in the soil (a) to the soil (b).
[0037] The "antagonistic microorganisms" contained in the soil (a) are not particularly limited as long as they are microbial groups that have an antagonistic effect against microorganisms that cause plant diseases. Antagonism generally appears in various forms, such as parasitism, antibiotics, competition, predation, and bacteriolysis, and one of these may act, or a combination of two or more of these may act. Examples of antagonistic microorganisms include antagonistic bacteria and antagonistic filamentous fungi. Examples of antagonistic bacteria include the genus Bacillus, non-pathogenic Agrobacterium, Enterobacter, Pseudomonas, Xanthomonas, Streptomyces, non-pathogenic Erwinia, Pasteuria, Sphingomonas, and Pseudolabrys. Examples of antagonistic filamentous fungi include the genus Aspergillus, non-pathogenic genus Fusarium, genus Gliocladium, genus Penicillium, genus Pythium, genus Trichoderma, genus Phoma, and genus Talaromyces.
[0038] Examples of antagonistic microorganisms against Fusarium oxysporum include Pseudomonas, Pseudolabrys, and Sphingomonas. Examples of Pseudomonas include Pseudomonas fluorescens, Pseudolabrys taiwanensis, and Sphingomonas include Sphingomonas paucimobilis.
[0039] The soil (a) and the soil (b) may be obtained from the same field or from different fields.
[0040] In step (ii), the mixing ratio of soil (a) to soil (b) is sufficient if the mixture contains about 10% or more of soil (a), and is preferably about 1:9 to about 9:1, more preferably about 2:8 to about 8:2, more preferably about 3:7 to about 7:3, more preferably about 4:6 to about 6:4, and more preferably about 1:1.
[0041] By mixing soil (a) and soil (b), antagonistic microbial groups present in soil (a) are transferred to soil (b) and colonized by the root fungi of microorganisms in soil (b), thereby imparting antagonistic ability, i.e., the ability to suppress the occurrence of soil-borne plant diseases, to soil (b). According to the method of the present invention, it has been found that antagonistic microbial groups present in soil (a) are transferred to and colonized in soil (b) even when soil (a) and soil (b) are collected from fields that are geographically very far apart. "Inhibitory ability" refers to the ability to "inhibit the occurrence of disease damage" as described in the first embodiment of the present invention, and can be expressed, for example, as a control value. A control value of more than 80% is excellent, more than 85% is better, more than 90% is better, more than 95% is better, more than 98% is particularly excellent, and more than 99% is particularly excellent.
[0042] According to the method of the fourth embodiment, soil containing antagonistic microorganisms can be effectively and simply utilized for controlling soil-borne plant diseases. EXAMPLES
[0043] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples and can be carried out with appropriate modifications.
[0044] Example 1 Preparation of soil microbial flora component solution and physicochemical component solution A total of 40 types of soil were collected from 13 fields in 10 prefectures across Japan. 5 g of soil was added to 45 ml of sterilized water and vigorously shaken at room temperature for 5 minutes. The soil suspension was then filtered through a 41 μm pore size filter to remove large soil particles. 10 ml of the filtrate was mixed with 90 ml of sterilized water to obtain a liquid containing the microbiota components (fraction containing the microbiota). The microbiota components were then filtered through a 0.22 μm pore size filter, and the filtrate from which the microbiota components had been removed was used as a physicochemical components liquid (fraction not containing the microbiota).
[0045] Example 2 Screening of soil microflora with Fusarium disease suppression effect Cucumbers (variety: Tokiwajiha) were used to evaluate the disease suppression effect of each soil microflora against Fusarium wilt (a plant disease caused by Fusarium oxysporum in this specification). Approximately 2.8 g of sterilized vermiculite was packed into a plant culture test tube (30 mm × 120 mm). The sterilized vermiculite in the plant culture test tube was treated with 3 ml of each soil microflora component solution, physicochemical component solution, or sterilized water (control group) and allowed to stand at 25°C for 12 hours. Then, a suspension of Fusarium oxysporum f. sp. cucumerinum GUS77 strain (approximately 1.0 × 10 4 spores / ml) or 2 ml of sterile water were inoculated.
[0046] Next, about 2.8 g of sterilized vermiculite was layered, and one surface-sterilized germinated cucumber seed was sown and then covered with a small amount of sterilized vermiculite.Furthermore, 1 ml of 1000-fold diluted Hyponex (manufactured by Hyponex Japan Co., Ltd.) was applied as fertilizer, and the test tube was capped and cultivated in an artificial climate chamber (25°C, 12 hours of light) for about 3 weeks. After cultivation, the condition of the cucumber seedlings was monitored and the disease severity was evaluated using a four-level disease index (0: healthy; 1: wilting, yellowing, or browning of xylems; 2: yellow-brown discoloration of hypocotyls; 3: severe wilting or complete death of entire plant). The relative disease severity of each treatment group compared to the control group was calculated using the following formula 1, and the control value of each treatment group was calculated using the following formula 2.
[0047]
number
[0048]
number
[0049] Soil microflora that met the following two conditions were defined as having a Fusarium disease suppression effect: 1) the control value (A value) of the area treated with a microbial flora component solution was more than 80%, and 2) the control value (B value) of the area treated with a physicochemical component solution was less than 20%. This experiment was repeated three times with five seedlings per plot. The test results are shown in Tables 1-1 and 1-2.
[0050] [Table 1-1]
[0051] [Table 1-2]
[0052] Of the 40 types evaluated, 13 types of soil microbiota had a Fusarium disease suppression effect. In particular, three types of soil microbiota, Soil 1, Soil 28, and Soil 40, completely suppressed the occurrence of Fusarium disease (control value = 100%).
[0053] The above results show that the screening method of the present invention can screen for microbial flora that have a Fusarium disease-suppressing effect. On the other hand, two types of microbial flora (soil 12 and soil 23) with a very low Fusarium disease-suppressing effect (A value < 30% and B value < 20%) were also found.
[0054] Example 3: Verification of transferability of the Fusarium disease suppression effect of soil microflora By mixing soils 1, 28, and 40, which have soil microflora with an extremely strong Fusarium disease inhibitory effect, with soils 12 and 23, which have microflora with a very low Fusarium disease inhibitory effect, it was examined whether the Fusarium disease inhibitory effect would be transferred. Soils 1, 28, and 40 were mixed with soils 12 and 23 in a ratio of 1:9, respectively, and left to stand at 25°C for 7 days. Then, a microflora component solution of each soil was prepared by the same method as in Example 1. The Fusarium disease inhibitory effect of each microflora component solution was evaluated by the same method as in Example 2, and the disease incidence of each treatment area was calculated by Equation 3.
[0055]
number
[0056] This experiment was repeated three times with five seedlings per plot.
[0057] The test results are shown in Figure 1. The disease incidence in the sterilized water treatment area (control area) was 57.8%. In the areas treated with the microflora component solution of soil 1, soil 28, and soil 40, the occurrence of Fusarium disease was completely suppressed (disease incidence = 0%), whereas the disease incidence in the areas treated with the microflora component solution of soil 12 and soil 23 was 51.1% and 46.7%, respectively, and no significant difference was observed from the control area. Furthermore, in the areas treated with the microflora component solution when soil 1 was mixed with soil 12 or soil 23, the occurrence of Fusarium disease was significantly suppressed. On the other hand, in the areas treated with the microflora component solution when soil 28 and soil 40 were mixed with soil 12 or soil 23, no significant difference was observed in the disease incidence compared to the control area. These results indicate that the Fusarium disease suppressive effect of the microflora of soil 1 was transferred and established in other soils by the mixing of the soils.
[0058] Example 4: Verification of the role of microflora in the transfer of Fusarium disease suppression effect Soil 1 was irradiated with 60 kGy of gamma rays to produce sterilized soil 1. Sterilized soil 1 was mixed with soil 12 and soil 23 in a ratio of 1:9 and allowed to stand at 25°C for 7 days. Microbiota component solutions were then prepared for each soil using the same method as in Example 1. The Fusarium disease inhibitory effect of each microbiota component solution was evaluated using the same method as in Example 2, and the disease incidence rate for each treatment plot was calculated. This experiment was repeated three times with five seedlings per plot.
[0059] The test results are shown in Figure 2. The disease incidence in the sterilized water-treated area (control area) was 51.1%. In the area treated with the microflora component solution of soil 1, the occurrence of Fusarium disease was completely suppressed (disease incidence = 0%), whereas no Fusarium disease inhibitory effect was observed in the area treated with the microflora component solution of sterilized soil 1. Furthermore, when sterilized soil 1 was mixed with soil 12 or soil 23, no significant difference in disease incidence was observed compared to the control area. These indicate that the living microflora, rather than the physicochemical components of soil 1, plays an important role in the transfer and establishment of the Fusarium disease inhibitory effect by the mixing of soil 1.
[0060] Example 5 Identification of microorganisms transferred and colonized by soil mixing Microbiome analysis was used to identify microorganisms that were transferred and colonized in soil 12 or soil 23 due to the mixing of soil 1. Soil 1 was mixed with soil 12 or soil 23 in a ratio of 1:9, respectively, and DNA was extracted from the soils ("soil 1+12" and "soil 1+23") that had been left to stand for one week. Next, the V3-V4 region of each DNA sample was probed with specific primers: Forward primer: CCTACGGGNGGCWGCAG (SEQ ID NO: 1) Reverse primer: GACTACHVGGGTATCTAATCC (SEQ ID NO: 2) The DNA was amplified by and paired-end sequenced using Illumina MiSeq (trademark) (Illumina Inc.). Analysis of the sequence data was performed using QIIME2 (trademark) to determine the relative abundance of the bacterial groups in each soil. First, the bacterial groups specifically present in soil 1 (soil 1-specific bacterial groups) were defined as bacterial groups with significantly higher relative abundance in soil 1 compared to soil 12 and soil 23. Linear discriminant analysis effect size (LEfSe) test was used for significance testing. In addition, using the LEfSe test, the bacterial groups specifically present in "soil 1+12" and "soil 1+23" ("soil 1+12-specific bacterial groups" and "soil 1+23-specific bacterial groups") were defined as bacterial groups with significantly higher relative abundance compared to soil 12 and soil 23. Next, the bacterial groups common to the “soil 1-specific bacterial groups,” “soil 1+12-specific bacterial groups,” and “soil 1+23-specific bacterial groups” were defined as the bacterial groups that were commonly transferred to soil 12 and soil 23 due to the mixing of soil 1. As a result, it was suggested that two types of antagonistic microorganisms, Pseudolabrys and Sphingomonas, which have been reported to be involved in the biocontrol effect against Fusarium wilt (Xue, C. et al. (2015), Scientific Reports, 5, 11124;Shen, Z. et al. (2015), Applied Soil Ecology, 93, 111-119;Tan, L. et al. (2019), Sustainability, 11, 4428;Kim, Y. et al. (2020), International Journal of Molecular Sciences, 21(6), 2019), were present in soil 1, and that the antagonistic microorganisms were transferred and established in both soils 12 and 23. This suggests that the transfer and establishment of the antagonistic microorganisms may be involved in the transfer and establishment of the Fusarium wilt suppression effect. [Industrial Applicability]
[0061] The method of the present invention makes it possible to utilize useful microbial flora.
Claims
1. 1. A method for assessing the ability of a soil microbiome to confer beneficial traits to a plant, comprising: (i) preparing a soil suspension from soil containing a microbiota and separating the soil suspension into a fraction containing a microbiota and a fraction not containing a microbiota, wherein the fraction containing a microbiota refers to a microbiota-containing isolate obtained by separating the soil suspension, and the fraction not containing a microbiota refers to a microbiota-free isolate obtained after further separating the soil suspension; (ii) preparing an artificial soil derived from natural minerals, and treating the artificial soil with each of a fraction containing the microbiota, a fraction not containing the microbiota, and sterilized water; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating the plant and monitoring the occurrence of disease in the plant and / or the growth of the plant; (v) comparing the ability of the artificial soil treated with the fraction containing the microbiota, the artificial soil treated with the fraction not containing the microbiota, and the artificial soil treated with sterilized water alone to suppress the occurrence of disease in the plant and / or to affect the growth of the plant; (vi) evaluating the soil microbiota as having the ability to impart beneficial traits to plants when the artificial soil treated with the fraction containing the microbiota suppresses the occurrence of diseases in the plant and / or promotes the growth of the plant more than the artificial soil treated with the fraction not containing the microbiota and the artificial soil treated with sterilized water alone; A method comprising:
2. A method for screening soil microbiota that confers beneficial traits to plants, comprising: (i) preparing soil suspensions from a plurality of soils containing microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction, wherein the microbiota-containing fraction refers to a microbiota-containing isolate obtained by separating the soil suspension, and the microbiota-free fraction refers to a microbiota-free isolate obtained by further separating the soil suspension; (ii) preparing an artificial soil derived from natural minerals, and treating the artificial soil with fractions containing the microbiota derived from a plurality of soils, fractions not containing the microbiota derived from a plurality of soils, and sterilized water; (iii) sowing plant seeds in each of the treated artificial soils; (iv) cultivating the plant and monitoring the occurrence of disease in the plant and / or the growth of the plant; (v) comparing the ability of an artificial soil treated with fractions containing the microbiota derived from a plurality of soils, an artificial soil treated with fractions not containing the microbiota derived from a plurality of soils, and an artificial soil treated with sterilized water alone to suppress the occurrence of disease in the plant and / or to affect the growth of the plant; (vi) selecting, from among the plurality of soils, a soil microbiome that inhibits the occurrence of plant diseases and / or promotes the growth of the plant more effectively in an artificial soil treated with a fraction containing a microbiome derived from the plurality of soils than in an artificial soil treated with a fraction not containing a microbiome derived from the plurality of soils and than in an artificial soil treated with sterilized water alone, as a soil microbiome that confers beneficial traits to plants; A method comprising:
3. A method for screening a soil microbiota that suppresses the occurrence of a soil-borne plant disease in a plant, comprising: (i) preparing soil suspensions from a plurality of soils containing microbiota and separating each of the soil suspensions into a microbiota-containing fraction and a microbiota-free fraction, wherein the microbiota-containing fraction refers to a microbiota-containing isolate obtained by separating the soil suspension, and the microbiota-free fraction refers to a microbiota-free isolate obtained by further separating the soil suspension; (ii) preparing an artificial soil derived from natural minerals, and treating the artificial soil with each of a fraction containing the microbiota, a fraction not containing the microbiota, and sterilized water; (iii) inoculating each of the treated artificial soils with a microorganism causing a soil-borne plant disease, and then sowing plant seeds; (iv) cultivating the plants and monitoring the plants for the occurrence of the soil-borne plant disease; (v) comparing the ability of the artificial soil treated with the fraction containing the microbiota, the artificial soil treated with the fraction not containing the microbiota, and the artificial soil treated with sterilized water alone to suppress the occurrence of the soil-borne plant disease; (vi) selecting, from the plurality of soils, a soil microbiome that suppresses the occurrence of the soil-borne plant disease by more than 50% when treated with the fraction containing the microbiome and that suppresses the occurrence of the soil-borne plant disease by less than 20% when treated with the fraction not containing the microbiome, as a soil microbiome that suppresses the occurrence of the soil-borne plant disease; A method comprising:
4. The method according to any one of claims 1 to 3, wherein the step (i) of separating into a fraction containing microbiota and a fraction not containing microbiota comprises the steps of filtering the soil suspension through a filter having a pore size of 10 to 50 µm to obtain a fraction containing microbiota, and then further filtering the fraction containing microbiota through a filter having a pore size of 0.1 to 1.0 µm to obtain a fraction not containing microbiota.
5. The method according to any one of claims 1 to 3, wherein the artificial soil is selected from the group consisting of vermiculite, zeolite, and perlite.
6. The method of claim 5, wherein the artificial soil is vermiculite.
7. 4. The method of claim 3, wherein the soil-borne plant disease is a Fusarium disease.
8. The method according to claim 3, wherein the microorganism causing the soil-borne plant disease is a fungus of the genus Fusarium.
9. The method according to claim 8, wherein the Fusarium fungus is Fusarium oxysporum.
10. 1. A method for suppressing the occurrence of a soil-borne plant disease in a plant, comprising: (i) selecting a soil containing a soil microbiota selected by the screening method of claim 3 as soil (a) containing antagonistic microorganisms, and further selecting a soil (b) in which the artificial soil treated with a fraction containing the microbiota suppresses the occurrence of the soil-borne plant disease by less than 30% and the artificial soil treated with a fraction not containing the microbiota suppresses the occurrence of the soil-borne plant disease by less than 20%; (ii) mixing the soil (a) with the soil (b); (iii) improving the ability of the soil (b) to suppress the occurrence of the soil-borne plant disease by transferring and establishing the antagonistic microorganisms in the soil (a) to the soil (b); A method comprising:
11. The method according to claim 10, wherein the soil (a) and the soil (b) are soils derived from the same field or from different fields.
12. The method according to claim 10, wherein the soil (a) and the soil (b) are mixed in a ratio of 1:9 to 9:
1.
13. The method according to claim 10, wherein the antagonistic microorganisms are bacteria of the genus Pseudolabrys and / or Sphingomonas.