Microorganisms, plant growth supplements, and plant growth methods

Abstract

To provide microorganisms, plant growth supplements, and plant growth methods beneficial to plants growing in metal-containing soil.SOLUTION: The present invention provides: [1] a microorganism classified as Pezicula sp. that colonizes the roots of plants in metal-containing soil and aids in their growth; [2] a plant growth supplement comprising the microorganism described in [1] supported on a carrier; and [3] a plant growth method comprising administering the microorganism described in [1] or the plant growth supplement described in [2] to the roots or the vicinity of the roots of a plant growing in soil containing a metal that causes stress to the plant.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to microorganisms, plant growth aids, and plant growth methods that are beneficial to plants growing in soil containing metals that cause stress to plants (hereinafter referred to as metal-containing soil).

Background Art

[0002] Due to mine development, tunnel excavation, natural volcanic activity, etc., high concentrations of metals may be contained in the soil. When the metal concentration in the soil is high, there is a problem that the growth of plants is inhibited and the restoration of vegetation is hindered. For example, in Patent Document 1, it has been proposed to reduce the heavy metal content in edible plant bodies such as the Solanaceae, Gramineae, and Leguminosae families by administering bacteria isolated from general farmlands to the roots of plants, thereby reducing the oral intake of heavy metals.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, a technique for promoting the establishment of plants using microorganisms has not been established on land where forests have been devastated, such as mine sites. Compared to general soil such as farmland, metal-containing soil has resistance to excessive metals, and there are various microorganisms that are in a symbiotic relationship with native plants. Therefore, it is desired to provide microorganisms that enhance the metal tolerance of native plants such as trees and herbs, rather than the bacteria involved in the absorption of heavy metals in vegetables disclosed in Patent Document 1. The present invention provides microorganisms, plant growth aids, and plant growth methods that are beneficial to plants growing in metal-containing soil.

Means for Solving the Problems

[0005] [1] A microorganism that colonizes the roots of plants in metal-containing soil and aids in their growth, and is classified as Pezicula sp. [2] The microorganism according to [1], wherein the Pezicula sp. is strain JN89 (accession number: NITE P-03951). [3] A plant growth promoter, wherein the microorganism according to [1] or [2] is supported on a carrier. [4] The plant growth promoter according to [3], wherein the carrier is an alginate gel capsule. [5] A method for growing a plant, wherein the microorganism according to [1] or [2] or the plant growth promoter according to [3] or [4] is administered to the roots or the periphery of the roots of a plant growing in soil containing a metal that causes stress to the plant. [6] The method for growing a plant according to [5], wherein the plant is a plant of the Salicaceae family.

Effect of the Invention

[0006] According to the present invention, it is possible to provide a microorganism, a plant growth promoter, and a method for growing a plant that are useful for plants growing in metal-containing soil.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

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Figure 4

Mode for Carrying Out the Invention

[0008] ≪Microorganism≫ The first aspect of the present invention is a microorganism that colonizes the roots of plants in metal-containing soil and helps their growth, and is a microorganism classified as Pezicula sp.

[0009] The microorganism of this aspect is preferably a filamentous fungus and a bacterium (endophyte) that infects the plant body. The microorganism of this aspect is preferably capable of producing a compound having an action of chelating metal ions, so-called siderophore. Since the siderophore produced by the microorganism chelates metals harmful to the plant body, it is considered that the metal stress on the roots of the plant infected with the microorganism is reduced.

[0010] Pezicula sp. can colonize various plants and is suitable for colonizing the roots of plants in the Salicaceae family, especially suitable for colonizing the roots of Salix miyamae. Pezicula sp. is known to produce siderophores.

[0011] The Pezicula sp. used in the examples described below is the JN89 strain (deposit number: NITE P-03951) entrusted to the National Institute of Technology and Evaluation (NITE) on July 26, 2023. Capsules encapsulating the JN89 strain in alginate gel capsules were prepared and administered to the roots of Salix miyamae growing in soil with a high content concentration of metals (especially iron and aluminum). As a result, it was confirmed that the number of root tips clearly increased and the roots grew vigorously compared to the case where they were not administered. The increase in the number of root tips greatly contributes to the growth of Salix miyamae, which is a pioneer tree.

[0012] In this specification, "metal" refers to heavy metal elements with a specific gravity of 4 to 5 or more and metal and metalloid elements such as Al and As. Examples of metals that cause metal stress in plants include cadmium, copper, lead, chromium, nickel, zinc, mercury, manganese, cobalt, copper, iron, aluminum, etc. By administering the microorganism of this embodiment to a plant, the metal stress of the plant can be reduced, and thus its growth and development can be assisted or promoted.

[0013] It is more efficient to isolate the microorganism of this embodiment from the roots of plants growing in soil with a high content concentration of metals such as mine sites. The method for isolating endophytic bacteria in roots can be performed, for example, by the following method. The roots of the collected plants are sterilized with alcohol, hydrogen peroxide, etc., root sections are prepared in a sterile environment such as a clean bench, and placed on a medium to culture the endophytic bacteria in the roots, whereby the target microorganism can be obtained. Furthermore, colonies can be formed on the medium for each strain of the microorganism, and they can be isolated as a single strain. The microbiological classification of the obtained isolated strain is identified by DNA sequence analysis of the 16S rRNA region by conventional methods.

[0014] When isolating Pezicula sp. from Myrica adenophora collected from a mine site, which is soil rich in metals, it is preferable to cut out fine roots in which endophytes are considered to be established among the roots of Myrica adenophora and prepare sections.

[0015] ≪Plant growth promoter≫ A second aspect of the present invention is a plant growth promoter in which the microorganism of the first aspect is supported on a carrier. As the carrier, a carrier that can be used for supporting or immobilizing known microorganisms can be applied. For example, activated carbon, diatomaceous earth, zeolite, peat moss, perlite, bentonite, montmorillonite, vermiculite, alumina, silicate, crystalline cellulose, corn starch, gelatin, alginic acid, etc. can be mentioned. These carriers can adsorb or incorporate microorganisms on or inside their surfaces in a state where they can grow later. Specifically, for example, the form of a gel or a gel capsule can be mentioned.

[0016] When using Pezicula sp. as the microorganism of the first aspect, it can be encapsulated in an alginate gel capsule to obtain a plant growth promoter. When the plant growth promoter is administered near the roots of a plant, the microorganisms encapsulated in the alginate gel capsule are gradually released, move to the roots around the capsule, and can be established as endophytes. Examples of the average diameter of the capsule include 1 mm to 5 mm. The method of encapsulating microorganisms in an alginate gel capsule is not particularly limited. For example, a method of dropping a bacterial solution containing sodium alginate, microorganisms, and water into an aqueous gelling solution containing calcium chloride can be mentioned. At this time, the degree of cross-linking of the gel can be adjusted by adjusting the calcium chloride concentration. Examples of the calcium chloride concentration include 10 to 300 mM, and 100 to 200 mM is preferable. Also, the concentration of sodium alginate contained in the bacterial solution is preferably 1.0 to 2.0 wt%. The detailed method will be described later in the examples.

[0017] ≪Method for growing plants≫ The third aspect of the present invention is a method for growing a plant, which comprises administering the microorganism of the first aspect or the plant growth promoter of the second aspect to the root or the vicinity of the root of the plant.

[0018] An amount of the microorganism or the plant growth promoter required to establish the microorganism of the first aspect at a density higher than the natural state is administered to the root or the vicinity of the root of the plant. As a result, the microorganism can colonize the root of the plant, relieve the metal stress of the plant, and assist or promote the growth of the plant.

[0019] When using Pezicula sp., the plants targeted by the plant growth promoter are preferably plants of the Salicaceae family, and particularly preferably Salix gracilistyla. In addition, it can also be applied to plants of the genus Diospyros in the Ebenaceae family, plants of the genus Ilex in the Aquifoliaceae family, etc.

[0020] As a specific method of using the plant growth promoter of this aspect on the root or the vicinity of the root of the plant, the plant growth promoter may be directly added to the root, or added or mixed with the soil or hydroponic solution in the vicinity of the root. For example, the plant growth promoter can be mixed within 5 cm from the root so as to directly touch the root. Here, the soil refers not only to the metal-containing soil, but also to all the soils used for plant cultivation. For example, it includes the fertile soil used for sowing, the soil used for cultivation, the soil containing fertilizers, the culture soil, etc.

[0021] As a method of administering the plant growth promoter of this aspect, for example, a suspension in which the plant growth promoter is suspended in water is supplied to the soil in which the plant grows, or the root of the plant is immersed in the suspension. When administering the plant growth promoter of this aspect, the growth stage of the plant and the season of the growth environment are not particularly limited.

[0022] The plant growth promoter of this aspect may contain components useful for the growth of microorganisms, such as inorganic salts, nitrogen sources, other nutrient sources, sugars (glucose, sucrose, etc.), starches, organic materials (beef extract, yeast extract, etc.). Furthermore, it may contain components useful for the growth of plants, that is, various fertilizers, etc.

[0023] The soil in which the plant to which the plant growth promoter of this embodiment is administered grows may be a soil containing metal at a high concentration that affects plant growth, and the soil may be acidic.

Examples

[0024] [Isolation and Deposition of Pezicula sp.] A method for isolating the JN89 strain of Pezicula sp. from Salix myrsinifolia collected from a mine site (dump site) which is a soil rich in metals will be described. First, surface sterilization of the roots of the collected Salix myrsinifolia was performed as follows in a clean bench. It was immersed in 70% EtOH for 1 minute, immersed in 7.5% H2O2 water and sterilized with stirring for 5 minutes, immersed in 70% EtOH again for 1 minute, immersed in 7.5% H2O2 water and washed with stirring for 5 minutes, further immersed in sterilized water for 5 minutes, immersed in another sterilized water for 5 minutes and washed, and the sterilization treatment was completed. Next, after removing the excess moisture attached to the roots on the sterilized filter paper, 10 mm root sections were prepared using a sterilized scalpel, and the sections were placed on a 1% malt extract agar medium (MA). The endophytes contained in the placed root sections were cultured on MA at 23°C in the dark for 1 month, and from the emerged endophytes, the JN89 strain of Pezicula sp. was obtained, deposited with NITE, and deposited under the above-mentioned accession number. The JN89 strain of Pezicula sp. may be simply referred to as "JN89" hereinafter.

[0025] [Method for Determining DNA Sequence] PrepMan TM Ultra Sample Preparation Reagent (Applied Biosystems TMGenomic DNA was extracted from the mycelium using []. The ITS regions of each isolate were amplified using the primers (ITS5 and ITS4) (White et al., 1990, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, M.A. Innes, D.H. Gelfand, J.J. Sninsky, et al. (Eds.), PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 315-322). In each case, 50 μL of a PCR mixture containing 1 μL of template DNA, 25 μL of GoTaq Green Master Mix (Promega, Madison, WI, USA), and 10 μL of each primer (10 pmol) was used. PCR was performed for 40 cycles with a cycle of 95°C for 4 minutes, 94°C for 30 seconds, 53°C for 30 seconds, 72°C for 50 seconds, and 72°C for 8 minutes. PCR was carried out using a thermal cycler T1000 (manufactured by Applied Biosystems). The amplicons were purified using Exosap-it, and the nucleotide sequences were determined using the BigDye Terminator kit v.3.1 (Applied Biosystems) on an ABI 3130XL genetic analyzer (Applied Biosystems TM ) (SEQ ID NO: 1).

[0026] [Identification of the biological classification of the JN89 strain] Observation was carried out using a stereomicroscope and an optical microscope. In addition, an alignment dataset was created by aligning the obtained DNA sequence with the sequences registered in the NCBI database using MUSCLE (Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797.). The genus name was determined from the phylogenetic position and the morphology of the conidia.

[0027] [Method for encapsulating microorganisms in alginate gel capsules] (1) Preparation of pregel solution As sodium alginate, 80 - 120 of sodium alginate (FUJIFILM Wako Pure Chemical Corporation) was used. The pregel solution for preparing alginate gel capsules encapsulating microorganisms was prepared by mixing an aqueous sodium alginate solution (2 wt%) filtered through a 0.45 μm filter or an autoclaved aqueous sodium alginate solution (2 wt%) and a bacterial suspension (Pezicula sp. bacterial solution) in a volume ratio of 9:1 using a stirrer. The pregel solution for preparing control capsules without bacteria was prepared using Millipore water instead of the bacterial suspension.

[0028] (2) Preparation of aqueous solution for gelation An aqueous solution containing calcium chloride (200 mM) and 0.1 wt% Tween 20 was prepared, autoclaved before gelation, and used as the aqueous solution for gelation.

[0029] (3) Preparation of bacterial solution The cells of Pezicula sp. JN89 were prepared according to the following procedures. The hyphal tips of Pezicula sp. JN89 grown on 1% MA were punched out into 5.5-mm disks. Five 300-mL Erlenmeyer flasks containing 100 mL of 1% malt extract (BD Difco) were prepared, and 10 disks were inoculated into each Erlenmeyer flask. The flasks were statically cultured at 23°C in the dark for 20 days. After culturing, the culture solution was filtered using a sterilized tea strainer in a clean bench to obtain the cells of JN89. The culture solution adhering to the obtained cells was thoroughly washed with sterilized water, and then the cells of JN89 were transferred into a 50-mL sterilized plastic tube. A small amount of sterilized water was added, and the hyphae of JN89 were disrupted with a sterilized mixer to obtain a cell-disrupted solution (i.e., a cell solution). A part (500 μL) of the cell solution was placed on a filter paper and dried at 80°C for 4 hours. The dry weight was measured, and the weight of the cell body was calculated by subtracting the weight of the filter paper. The measurement of the cell body weight was performed in triplicate, and the cell concentration was calculated from the average value. In addition, a small amount of the cell solution was cultured on 1% MA to confirm that the growth of the cells was good.

[0030] (4) Preparation of alginate gel capsules containing cells To 2 L of the aqueous gelation solution contained in a 3-L beaker stirred with a stirrer, 400 mL of a pregelled solution in which the cell solution of JN89 was suspended was dropped through a pipette tip for 200 μL connected with a silicon tube. The dropping rate was adjusted to 200 mL / hr with a peristaltic pump. Thirty minutes after the completion of dropping, stirring was stopped, and the capsules composed of alginate gel encapsulating microorganisms (i.e., JN89-encapsulating alginate gel capsules) formed in the beaker were washed with a large amount of Millipore water and then transferred into a container containing Millipore water and stored at 4°C. The average diameter of the obtained JN89-encapsulating alginate gel capsules was about 2.5 mm.

[0031] (5) Confirmation of the survival of cells encapsulated in alginate gel capsules The JN89-encapsulating alginate gel capsules were gently disrupted by immersing them in a phosphate buffer (pH 6) for about 10 minutes, and the growth and survival of the cells were confirmed by culturing JN89 contained in the disrupted product on 1% MA at 23°C in the dark. Twenty JN89-encapsulated alginate gel capsules were used in the above-mentioned survival test. After two weeks of culture, Pezicula sp. was re-isolated from all the capsules. From these results, it was found that the bacteria encapsulated in the alginate gel capsules survived and were suitable for subsequent growth.

[0032] When the calcium chloride concentration of the aqueous solution for gelation is lowered (10 mM, 50 mM, 100 mM), alginate gel capsules with low cross-linking strength can be prepared. Similar to the case of 200 mM above, JN89-encapsulated alginate gel capsules with low cross-linking strength were prepared and a survival test of the encapsulated bacteria was conducted. As a result, not only Pezicula sp. but also other bacteria were frequently isolated from the capsules with the lowest cross-linking strength (using an aqueous solution for gelation with a calcium chloride concentration of 10 mM). In addition, the growth inhibition of Pezicula sp. due to the appearance of other bacteria was significant. From these results, it was judged that the higher the cross-linking strength of the capsules, the more preferable it is to avoid the influence of other bacteria, and the use of an aqueous solution for gelation with a calcium chloride concentration of 200 mM is most preferable.

[0033] [Inoculation of Plants with JN89-Encapsulated Alginate Gel Capsules] Seeds were collected from Cryptomeria japonica var. sachalinensis growing in the local area (landfill) in 2020. Since the germination rate of Cryptomeria japonica var. sachalinensis seeds drops extremely after one week of collection, they were sown immediately after collection in sterilized Kanuma soil and germinated. After germination, Hyponex (2000-fold diluted solution; liquid fertilizer) was appropriately administered once a month as a guide, and one-year-old seedlings with stable number of true leaves were obtained. In June 2021, the one-year-old seedlings obtained above were transplanted to the landfill. At the time of transplantation, JN89-encapsulated alginate gel capsules (dry weight of JN89 cells: 5 mg / 4 mL), or non-encapsulated capsules without bacteria (control capsules: 4 mL) were added to the roots of the seedlings. Furthermore, a windbreak was installed according to the wind direction, and each seedling was provided with a numbering tape and a plate for snow protection for individual identification, and they were left in the natural environment for one year. The outline of the transplantation site is shown in Fig. 1. Con indicates the control capsule inoculation group (the transplantation group of uninoculated seedlings), and Ino indicates the JN89-encapsulated alginate gel capsule inoculation group (the transplantation group of inoculated seedlings). Three replicates were set for each inoculation group. 10 to 20 seedlings were transplanted into each group. In June 2022, similar to the above transplantation, JN89-encapsulated alginate gel capsules or control capsules were additionally inoculated.

[0034] [Observation and analysis of Miyamayagi seedlings grown in the field] In August 2022, 8 seedlings inoculated with JN89-encapsulated alginate gel capsules and 9 seedlings inoculated with control capsules were collected. The collected seedlings were placed in a cooler box at about 4 - 10 °C and taken back to the laboratory. After washing with tap water and Millipore water, they were separated by site and used for the following analysis.

[0035] 1) Growth evaluation, root morphology observation, and measurement using a root scanner Growth evaluation (seedling height, number of leaves, fresh weight (FW), dry weight (DW)) and measurement of root length, number of root tips, and surface area were performed using the device (WinRhizo version 2016a, Regent Instruments, Quebec, Canada). [Results] Table 1 shows the growth (seedling height, number of leaves, roots) of two-year-old seedlings. In the table, Con indicates the control capsule inoculation group, and Ino indicates the JN89-encapsulated alginate gel capsule inoculation group. It is shown as the mean ± SE (Con; n = 9, Ino; n = 8). It was tested by Student's t-test (*P < 0.05). Fig. 2 shows the root conditions of each inoculation group. The number of root tips in the JN89-encapsulated alginate gel capsule inoculation group was significantly increased compared with that in the control capsule inoculation group.

[0036] [Table 1]

[0037] For the leaves and roots taken from each seedling, 2) elemental analysis was performed. For the roots taken from each seedling, 3) re-isolation of endophytes and 4) observation of the infection structure of endophytes were carried out. The specific methods for 2) to 4) are described below.

[0038] 2) Elemental analysis After washing each seedling with tap water and Millipore water, it was divided into each part and dried in an oven at 80 °C for 2 days. The dried plant body was pulverized, its weight was measured, and then it was transferred to a test tube. 1 mL of nitric acid for measuring harmful metals was added, a glass bead was placed on it, and it was left overnight. Then, the organic matter was thermally decomposed in an aluminum block thermostat set at around 130 °C. After confirming that the decomposition solution became transparent and the organic matter was completely decomposed, the glass bead was removed, concentrated, and allowed to cool. After cooling, Millipore water was added, made up to 5 mL, and then filtered through a PTFE filter with a pore size of 0.2 μm. The obtained decomposition solution was subjected to inductively coupled plasma optical emission spectrometry (ICP-OES) for analysis of the elemental concentrations (Fe, Al, Mn, Cd, Cr, Cu, Pb, Zn, K, Na, Ca, Mg, P, S) in the leaves and roots. For C and N, analysis was performed using a CN coder. Statistical analysis was carried out by Student's t-test (significance level 5%). <Results> The elemental concentrations of the seedlings are shown in Figure 3. In the inoculation group with JN89-encapsulated alginate gel capsules, an increase in the P concentration in the leaves (Figure 3b) and a tendency for an increase in the Mg concentration in the roots (P = 0.056) (Figure 3d) were confirmed. From the above, it is considered that Pezicula sp. has an effect of promoting the absorption of the nutrient elements P and Mg, and it was suggested that the increase in the number of root tips by Pezicula sp. (Table 1) influenced the present results. Also, in the leaves of the inoculation group with JN89-encapsulated alginate gel capsules, a tendency for an increase in Al and Fe was confirmed (Al; P = 0.179, Fe; P = 0.097) (Figure 3a). Although no difference was confirmed in the Al and Fe concentrations in the roots among the treatments, the concentrations were significantly higher than the maximum values of Al and Fe concentrations contained in general plants (Larcher, 2004, Plant Ecophysiology. Toshiro Saeki, Masaki Tateno, translated by Springer-Verlag, Tokyo.) (Figure 3c).

[0039] 3) Re-isolation of endophytes Sterilization of the root surface taken from each seedling was carried out in a clean bench as follows, and endophytes were re-isolated from the roots. The roots were immersed in 70% EtOH for 1 minute, then immersed in 7.5% H2O2 solution and sterilized with stirring for 5 minutes, immersed in 70% EtOH again for 1 minute, immersed in 7.5% H2O2 solution and washed with stirring for 5 minutes, further immersed in sterilized water for 5 minutes, immersed in another sterilized water for 5 minutes and washed, and the sterilization treatment was completed. Next, after removing the excess moisture on the roots on sterilized filter paper, 10-mm root sections were prepared using a sterilized scalpel, and endophytes were separated by placing 5 sections on each MA. 50 root sections were prepared per individual seedling, and the endophytes contained in the placed root sections were cultured on MA at 23 °C in the dark for 1 month, and the emergence rate of endophytes was calculated. The morphology of the endophytes that emerged by culturing was observed with an optical microscope. The emergence rate (%) was defined as the ratio of the number of endophytes confirmed in all 50 sections, and finally, the average emergence rate ± SE of 5 individuals was calculated. Statistical analysis was performed using Student's t-test (significance level 5%) on the values after arcsine transformation. The results are shown in Figure 4.

[0040] 4) Observation of the infection structure of endophytes Following the method proposed by Oba et al. in 2006 (Oba, K., Saito, K., Fujiyoshi, M. (2006) Soil and Microorganisms 60, 57-61), the washed roots were stained with trypan blue as follows: First, the washed roots were cut to an appropriate length, placed in Millipore water, and autoclaved (121°C, 30 min) to soften the root tissue. Next, the roots were immersed in 10% KOH solution and decolorized by autoclaving (121°C, 30 min). After further treatment with 5% HCl solution for 5 min, the roots were transferred to Millipore water and left at room temperature overnight. Next, the Millipore water was discarded, and the roots were placed in trypan blue staining solution and boiled for 10 min for staining. Then, the roots were placed in lactglycerin and boiled for 5 min. After cooling, the roots were placed in a plastic tube covered with aluminum foil and stored at room temperature in the dark until use. Finally, the five stained roots were arranged perpendicular to the short side of a slide glass, sealed with a cover glass, and a preparation was prepared and observed under an optical microscope. <Result> The re-isolation of Pezicula sp. was shown by calculating the isolation rate from the number of occurrences in the total number of root pieces (Figure 4a). As a result, significant infection in the roots was confirmed in the JN89-encapsulated alginate gel capsule inoculation area compared to the control capsule inoculation area. In addition, trypan blue staining of the roots in both areas confirmed microsclerotia, which are the infection structure of Pezicula sp. (Figure 4b). From the above, it was confirmed that inoculation of JN89-encapsulated alginate gel capsules promotes Pezicula sp. infection in seedlings. Pezicula sp. is an indigenous bacterium of the deposition site (Ito, 2020, Master's thesis, Department of Environmental Science, Graduate School of Life and Environmental Sciences, University of Tsukuba), and the infection confirmed in the control capsule inoculation area was thought to be due to natural infection.

[0041] [Soil analysis of the pile-up site] In October 2020, the soil of the deposition site collected from a depth of 10 cm from the part excluding the surface layer (collected in 10×10×10 cm from 6 locations) was air-dried, passed through a 2-mm sieve, and used for the following analysis. The analysis items were: 1) measurement of soil pH (H2O), 2) analysis of contained elements by nitric acid-perchloric acid decomposition, 3) exchangeable Al analysis, and 4) exchangeable Fe analysis. 1) and 2) were carried out according to the Soil Environmental Analysis Method Editorial Committee (1997) Soil Environmental Analysis Method. Hakuyusha. Tokyo. 3) and 4) were carried out according to the Soil Nutrient Measurement Method Committee (1970) Soil Nutrient Analysis Method. Youkendo. Tokyo. For 4), an elution method using a 1M sodium acetate solution (pH 4.8) was selected. <Results> The soil pH (H2O) showed weak acidity at 5.17±0.14 (mean±SE; n = 6). The Fe and Al concentrations eluted by nitric acid-perchloric acid decomposition in the soil at each site were 40765.3±758.8 mg / kg and 31949.1±3740.0 mg / kg (mean±SE; n = 6), respectively, which were within the normal concentration range (Larcher, 2004, Plant Ecophysiology. Toshiro Saeki, Masaki Tateno, translators, Springer-Verlag, Tokyo). Other metal elements were not detected. The exchangeable Al concentration was 716.4±71.8 mg / kg (mean±SE; n = 6), which was higher than the average value of the exchangeable Al concentration in the brown forest soil across the country (340 mg / kg) (Environmental Agency, 1999, Third Report on the Summary of Acid Rain Countermeasure Investigations, Acid Rain Countermeasure Study Group of the Environmental Agency). Since the soil pH (H2O) at each site was weakly acidic and the exchangeable Al concentration was high, it was considered that the situation was such that plants could easily absorb and accumulate Al in the soil, and the influence of Al on plant growth was inferred. It has been confirmed that Miyama willow in the deposition site accumulates a high concentration of Al in its roots (Ito, 2020, Master's Thesis, Environmental Science, Graduate School of Life and Environmental Sciences, University of Tsukuba). The result that the exchangeable Al concentration was high in the soil this time can be said to support the Al accumulation property of Miyama willow. The exchangeable Fe concentration was 825.0 ± 134.3 mg / kg (mean ± SE; n = 6). Compared with the concentration of exchangeable Fe (8 - 10 mg / kg), which is used as an index of healthy soil for crops, the concentration was high at the deposition site, and it was considered that there was a possibility of phytotoxicity to plants. It has been found that the willow in the deposition site accumulates a high concentration of Fe in its roots (Ito, 2020, Master's thesis of the Department of Environmental Science, Graduate School of Life and Environmental Sciences, University of Tsukuba). It was considered that the concentration of exchangeable Fe in a form that is easily absorbed by plants affected this.

Industrial Applicability

[0042] The present invention can be widely used in the field of environmental conservation for the purpose of promoting ecological conservation-type greening with minimal impact on the ecosystem on soils such as mine sites in environments where plant growth is difficult due to metal stress.

Claims

1. A microorganism that colonizes the roots of plants in metal-containing soil and aids in their growth, a microorganism classified as Pezicula sp.

2. The microorganism according to Claim 1, wherein the Pezicula sp. is strain JN89 (Accession No.: NITE P-03951).

3. A plant growth promoter, wherein the microorganism according to Claim 2 is supported on a carrier.

4. The plant growth promoter according to Claim 3, wherein the carrier is an alginate gel capsule.

5. A method for growing a plant, comprising administering the microorganism according to Claim 1 or 2 or the plant growth promoter according to Claim 3 or 4 to the roots or the vicinity of the roots of a plant growing in soil containing a metal that causes stress to the plant.

6. The method for growing a plant according to Claim 5, wherein the plant is a plant of the Salicaceae family.

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