JP2007210981A - Plant-rooting stabilizer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant-rooting stabilizer useful for the growth of plants. <P>SOLUTION: This method for producing the plant-rooting stabilizer is characterized by immersing a ceramic in an actinomyces suspension and proliferating the actinomyces on the ceramic to fix the actinomyces to the ceramic. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a plant survival stabilizer containing actinomycetes useful for plant growth, for example, fixed on ceramics.

  Conventionally, a method of treating a seedling with a plant hormone (eg, indole butyric acid) before planting in soil in order to promote the rooting of tissue culture seedlings, seedlings or seedlings of plants, and to promote the establishment of soil. Has been. However, this method using plant hormones may require several months until rooting. In particular, when the method is applied to a tissue culture seedling that promotes rooting in an excessively humid state, it takes a long time, and the excessively humid state tends to cause growth failure of the seedling including disease and insect damage. Therefore, when promoting rooting of tissue culture seedlings and promoting survival on soil, a method capable of shortening the retention period in an overhumid state is desired.

  On the other hand, it is known that endogenous actinomycetes of plants are isolated from defoliation, litter and roots. Alternatively, endogenous actinomycetes are also isolated from the roots, leaves or stems of living plants that are growing naturally.

  When a strain belonging to the genus Streptomyces isolated from rhododendrons and rhododendrons of rhododendron plants is treated with rhododendron and carmia tissue culture seedlings or soil transplanted seedlings and allowed to grow endogenously, the disease resistance and It is known that drought resistance is enhanced. As a mechanism of disease resistance enhancement, it is considered that wall apposition and phytoalexin are induced in plant cells to which actinomycetes are attached (Patent Document 1). On the other hand, the mechanism of drought resistance is thought to be due to the increase in cell osmotic pressure of plants in which actinomycetes are endogenous, and the strengthening of cell walls due to increased hemicellulose and lignin.

  Conventionally, when introducing useful microorganisms such as actinomycetes, bacteria, filamentous fungi into soil, a method of directly mixing cells cultured in a suspension or solid medium into soil, or growing the bacteria in organic matter, A method of adhering grown bacteria to a carrier and mixing it with soil has been used. Examples of conventionally used organic solid carriers include naturally occurring polymer substances such as gelatin, cellulose, alginic acid and laminarin. Examples of inorganic solid carriers include clay, talc, quartz, and diatomaceous earth. Furthermore, examples of the liquid carrier include a buffer solution, physiological saline, and various liquid media. Alternatively, a surfactant (for example, polyoxyethylene-fatty acid ester, alkyl sulfonate, etc.) or an auxiliary agent (for example, starch, lactose, skim milk, etc.) is used to maintain the adhesion between the carrier and the microorganism and the activity of the microorganism. (Patent Document 2).

  On the other hand, at the stage of producing ceramics mainly composed of clay, feldspar, silica stone, etc., it is known that useful microorganisms such as actinomycetes and bacteria or their extracts are mixed and fired to form ceramics.

  For example, Patent Document 3 discloses a ceramic containing a useful microorganism group extract produced by kneading a clay containing a useful microorganism group extract and a calcium aluminate-based compound such as calcium oxide or aluminum oxide and firing at high temperature. It is disclosed. Patent Document 4 discloses carbonized ceramics produced by mixing Kibushi clay organic matter and useful microorganisms into raw materials and firing them at a high temperature at the stage of producing ceramics mainly composed of clay, feldspar, silica, and the like. Is disclosed. Furthermore, in Patent Document 5, the organic material is produced, clay containing one or more useful microorganisms such as photosynthetic bacteria, lactic acid bacteria, yeasts, actinomycetes, and ceramic raw materials not containing these useful microorganisms are fired at high temperature. Ceramics baked with useful microorganisms are disclosed.

  However, the use of ceramics as a carrier for useful microorganisms has not been known before.

Japanese Patent No. 3629212 JP 2004-143102 A JP 2000-186270 A JP 11-79864 A Japanese Patent Laid-Open No. 10-212156

  In view of the above-described circumstances, an object of the present invention is to provide a plant survival stabilizer containing actinomycetes useful for plant growth, for example, fixed on ceramics.

  As a result of diligent research to solve the above-mentioned problems, the inventors have found that actinomycetes that promote plant rooting and root elongation are isolated and fix the actinomycetes to ceramics, thereby completing the present invention.

The present invention includes the following.
(1) A plant survival stabilizer containing actinomycetes fixed on ceramics.
(2) The plant survival stabilizer according to (1), wherein the actinomycetes have a function of promoting plant root elongation, seed rooting, seedling rooting or seedling survival.
(3) The plant survival stabilizer according to (1), wherein the actinomycetes are actinomycetes belonging to the genus Streptomyces or Microbispora.
(4) The plant survival stabilizer according to (1), wherein the actinomycetes are microorganisms identified by the accession number NITE P-169.
(5) The above plant is azalea, orchid, gramineous, taro, solanaceae, legume, araceae, chrysanthemum, perilla, camellia, red crustaceae, cruciferous, cucurbitaceae, ginger, lily , Aceraceae, Teddyaceae, Rose, Lily, Convolvulaceae, Iridaceae, Sphagnum, Ranunculaceae, Oleander, Poppy, Pepper, Papaveraceae, Prunusaceae, Violet, Pleurotus, Tulipaceae, Thripsaceae In the family, urchinaceae, pineapple family, cypress family, diatomaceae family, gentian family, palm family, saxifrage family, mallow family, cedar family, euphorbiaceae family, cypress family, cypress family, cypress family, pine family, pine family, and oleaceae family The plant survival stabilizer according to (1), which is a plant selected from the group consisting of plants to which it belongs.
(6) A method for promoting plant survival, comprising a step of applying the plant activation stabilizer according to any one of (1) to (5) to soil and a step of growing a plant in the soil.
(7) A plant survival stabilizer according to any one of (1) to (5), wherein the plant survival stabilizer is stored under conditions of a humidity of 70% or less and a temperature of 25 ° C to 40 ° C. Preservation method.
(8) A method for producing a plant survival stabilizer comprising a step of immersing ceramics in an actinomycete suspension and a step of growing the actinomycetes on the ceramics.
(9) The plant activation stabilizer according to (8), wherein the actinomycetes have a function of promoting plant root elongation, seed rooting, seedling rooting or seedling survival. Production method.
(10) The method for producing a plant survival stabilizer according to (8), wherein the actinomycetes are actinomycetes belonging to the genus Streptomyces or Microbispora.
(11) The method for producing a plant survival stabilizer according to (8), wherein the actinomycetes are microorganisms identified by the accession number NITE P-169.

  According to the present invention, there is provided a plant survival stabilizer that stably and efficiently promotes plant seed rooting or seedling rooting and / or planting by the activity of actinomycetes.

Hereinafter, the present invention will be described in detail.
The plant survival stabilizer according to the present invention contains actinomycetes fixed on ceramics. Here, engraftment means a state in which the plant is rooted and absorbs nutrients. The plant survival stabilizer according to the present invention has an effect of promoting plant seed rooting or seedling rooting and / or planting.

  As ceramics used in the present invention, for example, commercially available ceramics such as FFC (trademark) ceramics (Atom Japan Co., Ltd.) can be used. For example, bead-shaped FFC ceramics (diameter 0.5-1.2 cm) (hereinafter abbreviated as “FFC ceramic beads”) pulverize the sedimentary rocks of ancient seafloor deposits and knead the powder with water. And then molded into a spherical shape having a diameter of 0.5 to 1.2 cm and fired at 200 to 850 ° C.

  On the other hand, the actinomycetes used in the present invention are actinomycetes belonging to any genus and species as long as they have a function of promoting plant root elongation, seed rooting, seedling rooting or seedling survival. For example, Streptomyces, Microbispora, Nocardia, Micromonospora, Actinomadura, Streptosporandium ( Streptosporangium), Actinoplanes genus (Actinoplanes) and Thermomonospora (Termomonospora) actinomycetes. Actinomycetes belonging to the genus Streptomyces or Microvispora are particularly preferred.

  Furthermore, for example, plant actinomycetes that can be used in the present invention can be isolated and purified as follows.

  Herbaceous and woody plants growing in the field are used as a source of actinomycetes. For example, actinomycetes used in the present invention can be separated from a plant by the following method (Patent Documents 1 and 2). That is, a part of an organ (for example, root, stem, leaf, flower) of a plant growing in the field is cut out, and after the surface is washed with tap water or the like, sodium hypochlorite and then ethanol (for example, Soak in 70% ethanol to sterilize the surface. After surface sterilization, this plant sample fragment is thoroughly washed with water, air-dried, placed on a solid actinomycete separation medium, and cultured at 25 to 40 ° C. for 10 to 60 days. As an actinomycete isolation medium, for example, IMA-2 medium (glucose 50 g, soluble starch 5.0 g, meat extract 1.0 g, NZ-casein 2.0 g, salt 2.0 g, calcium carbonate 1.0 g, agar 15.0 g, distilled water 1 L) Is used. Further, if necessary, an antibacterial agent or an antifungal agent may be added to the actinomycete separation medium.

  The mycelium of actinomycetes extended from the plant sample fragments placed on the medium is transplanted to a freshly prepared solid actinomycete isolation medium (for example, IMA-2 medium) and cultured to form mycelium colonies. After colony formation, a part of the colony is cut out and transplanted onto a membrane filter (pore size 0.2 μm) placed on the surface of a freshly prepared solid actinomycete isolation medium (for example, IMA-2 medium). The colonies transplanted on the membrane filter are cultured at 30 ° C. for about 7 days, and the mycelium of actinomycetes extended under the membrane filter is prepared from a newly prepared solid or liquid actinomycete isolation medium (for example, IMA-2 medium). The actinomycetes can be selectively purified by transplanting them into (3) and extending the mycelium.

  The isolated and purified spores of actinomycetes can be suspended, for example, in a sterilized glycerol solution (for example, 10% glycerol + 10% dimethyl sulfoxide, etc.) and stored frozen as a spore suspension.

  The actinomycetes that have been isolated, purified or known as described above, for example, the effect of actinomycetes themselves or metabolites derived from actinomycetes on plant root elongation significantly (e.g., 1.2. In the case of promoting (-1.3 times or more), it can be determined that the actinomycetes can be used in the present invention. The root elongation can be determined based on, for example, the main root length of the plant, the root dry weight, the number of side roots, the number of indefinite roots, and the like. Alternatively, when root growth is promoted, the growth of the above-ground part is also promoted. Therefore, the effects of isolated, purified, or known actinomycetes on plants include stem length and thickness, leaf area and number of leaves. By the increase, it can be determined whether or not the actinomycetes can be used in the present invention.

  Among the actinomycetes specifically isolated and purified by the present inventors as described above, those that can be used in the present invention include, for example, the MBR-52 strain belonging to the genus Streptomyces isolated from field-grown rhododendron (Streptomyces sp.). MBR-52 was established in the National Institute for Product Evaluation Technology Patent Microorganisms Depositary Center (NPMD) (Nite Biotechnology Headquarters, Patent Microorganisms Deposit Center, 2-5-8 Kazusa Kamashizu, Chiba Prefecture) January 27, 2006 Deposited by date, the deposit number is NITE P-169.

  The MBR-52 strain is a microorganism belonging to the genus Streptomyces by analyzing the colony shape, spore morphology with a scanning electron microscope, sugar availability, saccharides in the cell, amino acid composition of the cell wall, and the base sequence of 16S rDNA. Identified.

  Actinomycetes used in the present invention can be cultured in a large amount, for example, as follows. For example, in the case of actinomycetes belonging to the genus Streptomyces, spore suspension is prepared by, for example, IMA-2 liquid medium, A-3M liquid medium (glucose 5.0 g, glycerin 20.0 g, soluble starch 20.0 g, Pharmamedia 15.0 g Yeast extract 3.0 g, Dianin HP-20 1.0 g, distilled water 1 L) or other liquid medium for culturing actinomycetes, and shaking culture or stationary culture at 25-30 ° C. for about 1 day. In this case, a spore suspension that has been cryopreserved can be used. If necessary, actinomycetes may be further transplanted into these newly prepared media to increase the amount of cells.

  Using the ceramics and actinomycetes described above, the plant survival stabilizer according to the present invention can be produced. First, the ceramic is immersed in the actinomycete suspension. The amount of actinomycetes relative to ceramics can be 3-6 cfu / ml, preferably 3-4 cfu / ml. After soaking, it can be appropriately selected according to the actinomycetes, for example, cultured at 15-40 ° C. (preferably 28-32 ° C.) for 1-5 days (preferably 1-2 days), It grows and settles on the ceramic surface. Here, colonization means that actinomycetes extend mycelium on the ceramic surface to form spores. In culturing, after actinomycetes have settled on the ceramic surface, the ceramic may be taken out, washed, and cultured again.

  Hereinafter, for example, a method for producing a plant survival stabilizer according to the present invention when FFC ceramic beads and MBR-52 strain as actinomycetes are used will be described in detail.

  First, about 50-100g of FFC ceramic beads sterilized by autoclaving is immersed in the cell suspension of MBR-52 strain cultured in IMA-2 liquid medium in a 100-200mL glass flask, and at about 25-40 ° C. Incubate statically. Next, 7 to 10 days after the start of stationary culture, the FFC ceramic beads are aseptically removed in a clean bench, and the surface is lightly rinsed with sterile water. Air-dry the FFC ceramic beads in a clean bench until the surface is dry. The FFC ceramic beads after air drying are placed in, for example, an autoclave-sterilized glass beaker or plastic box, the glass beaker or plastic box is covered with an autoclave-sterilized aluminum foil, and cultured in a thermostat at 25 to 40 ° C. The glass beaker or plastic box is vibrated about every 3 days so that actinomycetes grow on the surface of each FFC ceramic bead. In about 3 to 10 days, actinomycetes grow to cover the surface of FFC ceramic beads. In this way, FFC ceramic beads having MBR-52 strain established on the surface can be used as the plant survival stabilizer according to the present invention.

  The plant survival stabilizer according to the present invention can contain nutrients such as a culture solution for maintaining the growth of actinomycetes for a long period of time, in addition to actinomycetes fixed on ceramics.

  The plant survival stabilizer according to the present invention produced as described above has a humidity of 70% when placed in a paper bag or box or a plastic bag or box together with a desiccant (for example, silica gel). Store for at least 6 months under the following conditions (preferably 60% or less, particularly preferably 50% or less) and at a temperature of 5 ° C to 40 ° C (preferably 25 ° C to 40 ° C, particularly preferably 25 ° C to 30 ° C). can do.

  On the other hand, plant survival can be promoted by applying the plant survival stabilizer according to the present invention to the soil where the plant grows. Examples of target plants include azalea, orchidaceae, gramineous, taroaceae, eggplant, legume, arachaceae, chrysanthemum, persimmonaceae, camellia, redwood, cruciferous, cucurbitaceae, ginger , Water lily family, cereal family, aceae family, rose family, liliaceae family, convolvulaceae family, iridaceae family, cinnamon family, buttercup family, oleander family, poppy family, pepper family, genus magnolia family, primrose family, violet family, red family family, cypress family Family, Thripsaceae, Pinusceae, Pineapple, Pleuroaceae, Bentulaceae, Glycaceae, Palm, Yukinosidae, Mallowaceae, Cryptomeriaceae, Euphorbiaceae, Cyprinidae, Papaveraceae, Cypressaceae, Pinaceae, Pinaceae Examples include plants belonging to the family Asteraceae. In particular, plants belonging to the azalea family such as rhododendron and carmia are preferable.

  The plant survival stabilizer according to the present invention can be applied, for example, by being mixed with cell culture trays for raising seedlings, plastic pots for raising seedlings, and soil for nursery beds. The type of soil to be mixed is not particularly limited, and can be used in combination with compost and chemical fertilizers used for normal cultivation. For example, when used in a cell tray for raising seedlings, for example, one FFC ceramic bead with a diameter of about 5 to 10 mm in which useful actinomycetes are fixed as a plant survival stabilizer according to the present invention is mixed in 1 cell (about 25 cc) of soil. To do. The amount of the plant survival stabilizer according to the present invention to be mixed with the soil can be appropriately increased or decreased depending on the soil capacity of the plastic pot for raising seedlings or the nursery bed to be used. Plant growth is promoted by growing the plant in soil mixed with the plant survival stabilizer according to the present invention. For example, plant survival in soil containing the plant survival stabilizer according to the present invention is significantly (for example, 1.5 to 2 times or more) compared to plant survival in soil not containing the plant survival stabilizer according to the present invention. In the case of promoting, the plant survival stabilizer according to the present invention can be determined to be useful. In addition, you may determine the plant survival by root elongation, such as the main root length of a plant, root part dry weight, the number of side roots, and an indefinite root number. Alternatively, plant survival can be determined based on a decrease in the occurrence of wilt seedlings, an increase in stem length, the number of leaves and leaf area, drought tolerance of seedlings, and the like.

  According to the plant survival stabilizer according to the present invention described above, it is possible to stably promote plant seed rooting or seedling rooting and / or planting by the activity of actinomycetes without polluting the environment. Can do. The plant survival stabilizer according to the present invention is useful as, for example, tissue culture seedlings, seedlings, transplanted seedlings, microbial materials that promote the survival of transplanted seedlings, or agricultural materials used for plant production. . In addition, since the plant survival stabilizer according to the present invention promotes rooting of seedlings, the time can be shortened compared to rooting of seedlings by the treatment of plant hormones conventionally used (for example, 1/2 Reduced to time). By using the plant survival stabilizer according to the present invention for the rooting of tissue culture seedlings and young seedlings that promote rooting in an excessively humid state, growth failure can be reduced. Furthermore, in the production of the plant survival stabilizer according to the present invention, it is not necessary to use a surfactant or an auxiliary agent that has been conventionally used to maintain the adhesion between the carrier and the microorganism or the activity of the microorganism.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

[Example 1] Isolation and purification of actinomycetes A portion of rhododendron roots growing in the field was cut out, and the surface was washed with tap water, followed by sodium hypochlorite and then ethanol (for example, 70% ethanol). ) To sterilize the surface. After surface sterilization, this plant sample fragment was sufficiently washed with water, air-dried, placed on a medium for solid actinomycete separation, and cultured at 25 to 40 ° C. for 10 to 60 days. As an actinomycete isolation medium, IMA-2 medium (glucose 5.0 g, soluble starch 5.0 g, meat extract 1.0 g, NZ-casein 2.0 g, salt 2.0 g, calcium carbonate 1.0 g, agar 15.0 g, distilled water 1 L) Using.

  Subsequently, the mycelium of actinomycetes extended from the plant sample fragment placed on the medium was transplanted to a freshly prepared solid IMA-2 medium and cultured to form mycelium colonies. After colony formation, a part of the colony was cut out and transplanted onto a membrane filter (pore size 0.2 μm) placed on the surface of a newly prepared solid IMA-2 medium. The colony transplanted on the membrane filter is cultured at 30 ° C for about 7 days, and the mycelium of actinomycetes extended under the membrane filter is transplanted into the newly prepared solid IMA-2 medium, and the mycelium is extended, The actinomycetes were selectively purified.

The isolated and purified actinomycete spores were suspended in a sterilized glycerol solution (for example, 10% glycerol) and stored frozen as a spore suspension.
The strain thus obtained is referred to as MBR-52 strain.

[Example 2] Taxonomic properties of MBR-52 strain Colony shape of isolated strain MBR-52 strain, observation of spore form by scanning electron microscope, sugar utilization, etc., intracellular sugar, amino acid composition of cell wall and 16S Analysis of the rDNA base sequence was performed as follows.

(1) Examination of colony shape and sugar availability
The MBR-52 strain cultured on IMA-2 solid medium at 30 ° C. for 2 weeks was transplanted to the solid medium of International Streptomyces Project (hereinafter referred to as “ISP”) medium No. 2-5, and 3-4 at 30 ° C. Cultured for a week. The colony properties on the medium were visually judged. The results are shown in Table 1. ISP medium No. 2 is yeast / malt agar medium, ISP medium No. 3 is oatmeal agar medium, ISP medium No. 4 is starch / inorganic salt agar medium, ISP medium No. 5 is glycerin / asparagine agar medium . The color numbers in Table 1 are based on the new color name encyclopedia (edited by Nippon Color Research Institute, published by Nippon Color Research Co., Ltd.) 4th edition (1998).

  As shown in Table 1, the basic mycelium of the MBR-52 strain grew well in any ISP medium, and formed many bluish beige to gray spores at maturity. The color of the basic mycelium colony and the color of the back surface of the colony differed depending on the medium.

  In addition, the MBR-52 strain cultured on IMA-2 solid medium at 30 ° C. for 2 weeks was transplanted to the solid medium of ISP medium No. 6 and 7, and cultured at 30 ° C. for 3 to 4 weeks. The presence or absence of melanin pigment diffusing around the colony was visually determined. The results are shown in Table 2. ISP medium No. 6 is a peptone / yeast / iron agar medium, and ISP medium No. 7 is a tyrosine agar medium.

  As shown in Table 2, melanin-like pigments were observed on ISP medium No. 6, but not on ISP medium No. 7.

  Furthermore, the MBR-52 strain cultured on IMA-2 solid medium at 30 ° C for 2 weeks was transplanted to a solid medium of ISP medium No. 9 to which various monosaccharides were added (no addition to the control group), and 30 ° C For 3-4 weeks. The extent of colony growth on the medium was determined with the naked eye, and the availability of each sugar by the MBR-52 strain was examined. The results are shown in Table 3. The result is the degree of colony elongation (“+”, “++” indicates the activity of colony elongation) compared to the control group without addition of sugar. ISP medium No. 9 is a pridoham goat-living agar medium.

  On the ISP medium No. 9 containing no sugar, the basic hyphae grew only slightly, but as shown in Table 3, on the ISP medium No. 9 to which D-glucose was added, The live mycelium grew actively. The basic mycelium grew more actively on ISP medium No. 9 supplemented with inositol, D-xylose or sucrose than on the medium supplemented with D-glucose. Such effects were not observed in the medium added with other sugars.

(2) Observation of spore morphology with a scanning electron microscope The MBR-52 strain that had been cryopreserved was suspended in sterile water. 50 μl of the cell suspension was applied to the surface of 100 ml of ISP medium No. 3 solid medium and cultured at 30 ° C. for 1 week. After culturing, the colony pieces were cut out together with the agar medium, and steam-fixed with osmium tetroxide in a sealed container. Samples were frozen in liquid nitrogen and then dried overnight in a lyophilizer. The sample was gold-deposited and the spore morphology of MBR-52 was observed with a scanning electron microscope. The results are shown in FIG. FIG. 1 shows a scanning electron microscope image of the spore chain of the MBR-52 strain. In FIG. 1, the scale bar is 3 μm.

  As shown in FIG. 1, the aerial mycelium had a smooth surface, a width of 0.5 to 0.7 μm, and the spore pattern was branched from some places to form slightly curved spore chains. The spores were saddle-shaped and the average size was 0.6-1.2 μm.

(3) Analysis of intracellular sugar
1 ml of 1N sulfuric acid was added to 50 mg of frozen cells of MBR-52 strain and heated at 120 ° C. for 15 minutes in a sealed glass tube. After heating, the supernatant of the liquid was collected by centrifugation and adjusted to pH 5.5 with barium hydroxide. Again, the supernatant was collected with a centrifuge, a small amount of ethanol was added, and the mixture was dried with a rotary evaporator. The dried sample was dissolved in 300 μl of distilled water and developed by thin layer chromatography together with a standard sugar sample solution. For the development of the thin layer, a solvent of n-butanol: distilled water: pyridine: toluene = 10: 6: 6: 1 was used. The spots were developed by spraying aniline phthalate.
As a result, the MBR-52 strain was found to have galactose as the main sugar in the cell.

(4) Analysis of cell wall amino acid composition
1 ml of 6N hydrochloric acid was added to 50 mg of frozen cells of MBR-52 strain and heated at 100 ° C. overnight in a sealed glass tube. After heating, the mixture was completely dried at 45 ° C. An additional 2 ml of distilled water was added and dried again. The dried sample was dissolved in 0.2 ml of distilled water and developed by thin layer chromatography together with diaminopimelic acid and glycine as standard sample solutions. For the development of the thin layer, a solvent of distilled water: 6N hydrochloric acid: pyridine = 80: 264: 10 was used. Ninhydrin was sprayed and heated at 120 ° C. to develop spots. The results are shown in FIG. FIG. 2 is a thin layer chromatography showing the cell wall amino acid composition of MBR-52 strain. In FIG. 2, “A ₂ pm” indicates a diaminopimelic acid standard sample, “MBR-52” indicates a MBR-52 strain sample, and “Glysine” indicates a glycine standard sample. “LL type A ₂ pm” indicated by an arrow indicates the position of LL type diaminopimelic acid. Similarly, “Meso type A ₂ pm” and “Glysine” indicated by arrows indicate the positions of meso-type diaminopimelic acid and glycine, respectively.
As shown in FIG. 2, the cell wall of the MBR-52 strain was found to contain LL type diaminopimelic acid and glycine.

(5) Base sequence analysis of 16S rDNA
DNA was extracted from MBR-52 strain cells cultured in IMA-2 medium at 30 ° C. for 10 days. Using PCR, 16S rDNA was amplified using the extracted DNA as a template, and the nucleotide sequence was analyzed with a DNA sequencer. Next, based on the base sequence of the 16S rDNA obtained, homology search and systematic analysis were performed by BLAST compared with the DNA base sequence database. The results of the homology search are shown in Table 4. In Table 4, the accession number for the 16S rDNA base sequence of each bacterial species is a number registered in GenBank. The homology value was calculated based on the DNA base sequence database (GenBank / DDBJ / EMBL) using BLAST.

As a result of the homology search, all of the top 20 strains having high homology with the MBR-52 strain were occupied by Streptomyces. Table 4 shows the top five species of bacteria with high homology and their homology values (%). As shown in Table 4, the MBR-52 strain showed the highest homology of 99.6% with S. ciscaucasicus, but the possibility of a strain that is physiologically different from this strain cannot be ruled out. Therefore, the MBR-52 strain was judged to be appropriate as a Streptomyces sp.
From the above results, the MBR-52 strain was identified as belonging to the genus Streptomyces.

[Example 3] Root elongation promoting effect of MBR-52 strain The test method and results of the MBR-52 strain root elongation promoting effect on the growth of cucumber seeds are described in this example.

(1) Root elongation promoting effect test method of actinomycetes on cucumber seeds In Example 1, the spore suspension of MBR-52 strain isolated, purified and cryopreserved was respectively used as antibiotic extraction medium A-3M medium (Glucose 5.0 g, Glycerin 20.0 g, Soluble starch 20.0 g, Pharmamedia 15.0 g, Yeast extract 3.0 g, Dianin HP-20 1.0 g, Distilled water 1 L) and cultured for 3 days at 30 ° C. before shaking. 1 mL of this culture solution was placed in a newly prepared A-3M medium, and further cultured for 6 days under the same conditions.

  In order to extract the metabolite of MBR-52 strain, the same amount of acetone as the culture solution after the main culture was added to the culture solution, followed by shaking culture at 30 ° C. for 24 hours.

  After shaking culture, the shaking culture was centrifuged and the supernatant was collected. The collected supernatant was subjected to a rotary evaporator to evaporate acetone, and then the metabolite extract was collected. Further, the metabolite extract was sterilized by filtration using a Millipore filter (pore size 0.2 μm), and the filtrate was used as an actinomycete metabolite solution.

  On the other hand, cucumber seeds were surface sterilized with 1% sodium hypochlorite and thoroughly washed with sterilized water. A sterilized filter paper soaked with sterilized water was placed in a sterilized Petri dish, and sterilized cucumber seeds were placed thereon, and germinated in the dark at 28 ° C. for 2 days.

  Next, two sterilized filter papers were placed in a glass tube (10 cm long, 2.5 cm caliber) sterilized by autoclaving, and 1 ml of a 10, 100, or 1000-fold diluted solution of the above-mentioned actinomycete metabolite solution was added. Furthermore, the above-mentioned germinated cucumber seeds were placed in these glass tubes, covered with autoclaved aluminum foil, and cultured at 28 ° C. in the dark for 7 days.

(2) Test result of root elongation promoting effect of actinomycetes on cucumber seeds The main root length and root (underground) dry weight of the roots grown on the cucumber seeds cultured as described in (1) above were measured. The results are shown in Table 5. The control group was cultured in the absence of actinomycete metabolite solution. On the other hand, the negative control (“A-3M medium” in Table 5) was obtained by adding 10, 100, or 1000-fold diluted solution of A-3M medium and culturing in the same manner.

  As shown in Table 5, it became clear that the MBR-52 strain belonging to the genus Streptomyces isolated from field-grown rhododendron promotes root growth of cucumber seeds.

[Example 4] Establishment of MBR-52 strain on rhododendron tissue culture seedlings In this example, MBR-52 strain was established on rhododendron tissue culture seedlings, and the influence on the number of adventitious roots of the same seedlings was examined.
(1) Test method of colonization of Streptomyces MBR-52 in tissue culture seedlings of rhododendron Rhododendron tissue culture seedlings (no roots) (variety Sakigake and Bouquet) grown for 3 weeks in tissue culture growth medium were used for the assay.

  0.5 mL of the spore suspension of MBR-52 strain that had been cryopreserved was added to 200 mL of GY broth (10 g glucose, 10 g yeast extract, 1 L ion exchange water) sterilized by autoclaving, and cultured with shaking at 30 ° C. for 12 hours. . Next, about 2 mL of the obtained MBR-52 strain culture solution was applied to the surface of the growth medium in the flask in which rhododendron tissue culture seedlings were growing, and cultured at 25 ° C. for 10 days.

  After autoclaving a plastic plant box (6.5 cm x 6.5 cm x 10 cm) containing 20 g of culture soil (volume ratio peat moss: vermiculite: perlite: saturite = 67: 16: 16: 1) and 100 mL of ion-exchanged water, Five strains of tissue culture seedlings of the cultivar Sakigake cultured as described above were transplanted into 15 plant boxes in a clean bench. On the other hand, tissue culture seedlings of the variety bouquet cultured as described above were transplanted into another 15 plant boxes. In addition, a group in which rhododendron tissue culture seedlings were transplanted into another 15 plant boxes from the MBR-52 strain-untreated flask was used as a control group. To prevent the inside of the plant box from becoming highly humid, open five holes with a diameter of 6 mm in the lid of the plant box, attach a filter (trade name Milli-Seal, manufactured by Millipore), circulate air, and mix airborne microorganisms I tried to prevent it. Each plant box was placed in an artificial weather apparatus (25 ° C) with illumination of 4000 lux (irradiation time 12 hours / day), and seedlings were cultured.

  One week after the start of culturing, three seedlings were extracted from each plant box in a clean bench, and each seedling was aseptically cut into small pieces and subterranean parts corresponding to two segments. Place these small pieces on IMA-2 solid medium (glucose 5.0g, soluble starch 5.0g, meat extract 1.0g, NZ-casein 2.0g, salt 2.0g, calcium carbonate 1.0g, agar 15.0g, distilled water 1L) Then, the cells were cultured at 30 ° C. for 1 week, and the presence or absence of the hyphae of the MBR-52 strain extended from the seedling pieces was confirmed with a stereomicroscope.

(2) Test results of colonization of Streptomyces MBR-52 strains in rhododendron tissue culture seedlings As shown in (1) above, by examining the presence or absence of MBR-52 strain mycelia extended from seedling pieces, hyphae extension The number of seedling pieces in which the sera were observed was counted. The results are shown in Table 6.

  As shown in Table 6, in the cultivar Sakigake, the re-separation rate from the underground equivalent part and from the ground to the upper first and second segments was 100%, and from the ground to the upper third segment was 93%. It was. On the other hand, in the varietal bouquet, the re-separation rate from the subsurface equivalent part and from the top first and second segments was 100%, and from the top third segment was 86%. In all varieties, MBR-52 strain was established from the border to the top.

(3) Effect of MBR-52 strain on the number of adventitious roots of rhododendron tissue culture seedlings By the same method as (1) above, rhododendron tissue culture seedlings (no-root) inoculated with MBR-52 strain in a flask Bouquet) was cultured in a plant box. Every week after the start of culture, 15 seedlings were extracted from the plant box in each section in a clean bench, and adventitious roots generated from the tissue culture seedlings were counted with a stereomicroscope. The results are shown in FIG. FIG. 3 shows the state of adventitious root elongation (transplanted in sterilized soil in a sterilized plastic box) of tissue culture seedlings of rhododendron (variety Sakigake and Bouquet) inoculated with MBR-52 strain. In FIG. 3, A to D show photographs of the following tissue culture seedlings. A: Variety Sakigake (no inoculation of MBR-52 strain), B: Variety Sakigake (inoculation of MBR-52 strain), C: Variety bouquet (no inoculation of MBR-52 strain), D: Variety bouquet (inoculation of MBR-52 strain), A and B: 3 weeks after transplantation, C and D: 5 weeks after transplantation.

  As shown in Fig. 3 and Table 7, the average number of adventitious roots up to 3 weeks after transplanting was not more than 1 in the cultivar Sakigake that was transplanted in the soil uninoculated with MBR-52, but the soil inoculated with MBR-52 The average number of adventitious roots from seedlings of the same cultivar transplanted to 1.1 was 1.1 in the second week after transplantation and 2.7 in the third week. On the 5th week, the average number of adventitious roots was 4-5 regardless of inoculated or uninoculated soil.

  On the other hand, the occurrence of adventitious roots was slower in the variety bouquet than in the variety Sakigake for about two weeks. The average number of adventitious roots from seedlings transplanted in soil uninoculated with MBR-52 was 0.6 in the 4th week after transplanting and 1.7 in 5th week, but the same variety transplanted in the soil inoculated with MBR-52 The average number of adventitious roots from the seedlings was 1.9 and 3.4 at 4 and 5 weeks after transplanting, respectively.

  As described above, it was confirmed that the occurrence of adventitious roots from tissue culture seedlings was accelerated in the soil inoculated with MBR-52 strain, regardless of which cultivar was used.

[Example 5] Production of plant survival stabilizer according to the present invention
As the carrier of the MBR-52 strain cells, FFC (trademark) ceramic beads (diameter 0.5 to 1.2 cm) (manufactured by Atom Japan Co., Ltd.) were used.

  On the other hand, the spore suspension of Streptomyces sp. MBR-52 strain that has been cryopreserved is inoculated into the IMA-2 liquid medium in a 100-200 mL glass flask, and cultured with shaking at 25-30 ° C. for about 1 day. A large amount of cell culture solution (cell suspension) was obtained.

  About 50 to 100 g of autoclaved FFC ceramic beads were immersed in the resulting cell suspension of the MBR-52 strain, and statically cultured at about 25 to 40 ° C. for 7 to 10 days. The FFC ceramic beads were aseptically removed in a clean bench and the surface was lightly rinsed with sterile water. Next, the FFC ceramic beads were air-dried in a clean bench until the surface was dry.

  The FFC ceramic beads after air drying were placed in an autoclave-sterilized glass beaker or plastic box, the glass beaker or plastic box was covered with an autoclave-sterilized aluminum foil, and cultured in a thermostatic chamber at 25 to 40 ° C. The glass beaker or the plastic box was vibrated about every 3 days so that actinomycetes were uniformly grown on the surface of each FFC ceramic bead.

  FIG. 4 shows the growth status of the MBR-52 strain cells grown on the surface of the FFC ceramic beads as described above. 4A to 4D, the following photographs are shown. A: MBR-52 strain growing on the surface of FFC ceramic beads (white cells), B: White colony of MBR-52 strain on the surface of FFC ceramic beads (stereomicroscopic image), C: Extending the surface of FFC ceramic beads MBR-52 strain mycelium (scanning electron microscope image), D: Spores formed from MBR-52 strain mycelia on the surface of FFC ceramic beads (scanning electron microscope image).

  As shown in FIG. 4, each MBR-52 strain grew to cover the surface of the FFC ceramic beads in about 3 to 10 days.

[Example 6] Preservation of plant survival stabilizer according to the present invention In this example, MBR-52 strain produced in Example 5 was grown on the surface and fixed on FFC ceramic beads (plant according to the present invention). (Stabilizer) was stored under different temperature conditions, and the survival of actinomycetes on the FFC ceramic beads was confirmed by the following method at regular intervals.

  FFC ceramic beads with MBR-52 strain produced in Example 5 fixed on the surface are placed in a glass beaker or plastic box covered with autoclaved aluminum foil and stored at 25 ° C, 5 ° C and -20 ° C, respectively. did. At regular intervals, a part of the FFC ceramic beads was taken out in a clean bench and placed on an IMA-2 agar medium in a newly prepared Petri dish. This petri dish was kept in a thermostat at 25 to 40 ° C. and cultured for about 7 to 180 days. When the mycelium of MBR-52 strain was extended on the medium from the FFC ceramic beads, it was determined that the MBR-52 strain was alive on the FFC ceramic beads. That is, when the mycelium of MBR-52 strain was extended on the medium from the FFC ceramic beads, it was determined that the MBR-52 strain was re-separated from the FFC ceramic beads. Table 8 shows the results of examining the change in the reseparation rate of MBR-52 strain with respect to the storage conditions / storage period of FFC ceramic beads.

  As shown in Table 8, the re-separation rate of MBR-52 strain from FFC ceramic beads stored at 25 ° C. was 100% during 7 to 180 days from the start of storage. The re-separation rate from the same beads stored at 5 ° C. was 67% on the 7th day after the start of storage, but 100% between 14 and 180 days. The reason why the re-separation rate on the 7th day was slightly lower than that at 25 ° C. was thought to be because the growth of the MBR-52 strain on the FFC ceramic beads was slightly delayed due to the low temperature.

  On the other hand, the re-separation rate of MBR-52 strain from FFC ceramic beads stored at −20 ° C. was 25-42% during 7-180 days after the start of storage. This indicates that the MBR-52 strain cannot grow in the frozen state, and the cells attached to the FFC ceramic beads remain alive during the fixing process. These re-separation rates were considered to indicate the rate at which the cells attached to the FFC ceramic beads grew in the re-separation medium.

[Example 7] Examination of the effect of the plant adhesion stabilizer according to the present invention on the rooting of a Kalmia tissue culture seedling The tissue culture seedling of the azalea plant Kalmia (Kalmia latifolia L.) is a culture seedling that is difficult to root. It does not root in the growth / planting medium for tissue culture in the flask. The tissue culture seedling transplanted from the flask to the soil in the cell tray is rooted in 2 to 3 months when placed in a high temperature and high humidity conditioned vinyl tunnel, and grows into a pot transplanted seedling. In this example, the effect of the plant survival stabilizer according to the present invention on rooting was tested using such a carmia tissue culture seedling that is difficult to root.

(1) Raising Kalmia tissue culture seedlings For the purpose of growing Kalmia tissue culture seedlings, firstly, a new tree of Kalmia was collected, and the surface was sterilized by dipping in ethanol and sodium hypochlorite. Next, in a clean bench, this new tree was cut to about 1 cm from the tip and planted as an explant in a culture medium for carmia growth shown in Table 9.

  By culturing the explants aseptically described above in an incubation room at 25 ° C under artificial light irradiation (approximately 4000-5000 lux, 12 hours / day) for 1-2 months, A shoot was formed. By further culturing for 1-2 months, young plants with stems and leaves were formed. Next, the formed seedlings are taken out from the container in a clean bench, and each seedling is aseptically divided, planted in a fresh Kalmia growth medium (Table 9), and further subjected to artificial light irradiation (about 4000 to 5000 lux, The seedlings for propagation were obtained by culturing aseptically in a culture chamber at 25 ° C. for 12 hours / day. By dividing the seedlings and subcultured, the desired number of seedlings could be obtained.

  Next, in order to grow the seedlings obtained in the culture medium for Kalmia growth into tissue culture seedlings that can be transplanted to the cell tray, they were planted in a Kalmia planting medium to which activated carbon was added as shown in Table 9 without hormones. These seedlings can be transplanted from flasks to cell trays after 2 to 3 months by aseptically cultivating them in an incubation room at 25 ° C under artificial light irradiation (approximately 4000 to 5000 lux, 12 hours / day). Grown on tissue culture seedlings.

(2) Preparation of tissue culture seedling transplantation soil containing the plant survival stabilizer according to the present invention Nonsterilized seedling culture soil (capacity ratio peat moss: vermiculite: perlite: saturite = 67: 16: 16: 1), cell tray 1 cell (about 25 cm ³ ) was filled. Next, FFC ceramic beads (diameter 3 to 10 mm) (the plant survival stabilizer according to the present invention) in which the MBR-52 strain produced in Example 5 which had been stored at room temperature was fixed on the surface were used as the culture soil of each cell Embedded one by one. This was made into the plant survival stabilizer treatment area. Neither FFC ceramic beads having MBR-52 strain produced in Example 5 fixed on the surface nor actinomycetes-untreated FFC ceramic beads were embedded in the ceramic untreated section. In addition, for comparison purposes, a ceramic test zone was prepared by embedding one MBC-52 strain and other FFC ceramic beads in which no other actinomycetes were established in each cell. Tested.

(3) Transplanting Kalmia tissue culture seedlings to transplanting soil containing the plant survival stabilizer according to the present invention Avoid drying after removing the Kalmia tissue culture seedlings in the flasks grown as described in (1) above from the flasks For this reason, they were separated one by one in water. Next, the tissue culture seedlings were inserted one by one with tweezers into each cell of the cell tray in which the culture soil for raising seedlings of (2) was placed, and transplanted. These cell trays were placed in a sealed vinyl tunnel in a glass greenhouse and replenished with automatic mist. Appropriate amount of liquid fertilizer was given every 10-15 days, and seedlings were grown for 2 months.

  FIG. 5 shows the state of root elongation from the tissue culture seedlings in each group 2 months after transplantation of the tissue culture seedlings, and Table 10 shows the dry weight of the roots in each group. In FIG. 5, AC shows the photograph of each following ward. A: Ceramic untreated section, B: Ceramics test section, C: Plant survival stabilizer treatment section.

  As shown in FIG. 5, many long roots are generated from seedlings grown in the soil treated with ceramics (ceramics test zone) compared to the zone grown for 2 months in the ceramic-untreated soil (ceramics untreated zone). In addition, many longer roots were generated in the soil treated with the plant survival stabilizer (plant survival treatment area). As shown in Table 10, assuming that the root dry weight of 60 seedlings in the ceramic-untreated group was 1, the ceramic test group was about twice, and the plant survival stabilizer-treated group was about three times. From these results, it is clear that the growth of roots from cultured Kalmia tissue culture seedlings is promoted by treating the ceramics themselves with soil, but the growth of roots is further promoted by treating the plant survival stabilizer with soil. It was.

FIG. 1 shows a scanning electron microscope image of the spore chain of the MBR-52 strain. FIG. 2 shows thin layer chromatography showing the cell wall amino acid composition of MBR-52 strain. FIG. 3 shows adventitious root elongation of tissue culture seedlings of rhododendron (variety Sakigake and Bouquet) inoculated with MBR-52 strain. FIG. 4 shows the state of growth of MBR-52 strain cells grown on the surface of FFC ceramic beads. FIG. 5 shows the state of root extension from the tissue culture seedlings in each section 2 months after transplantation of the Kalmia tissue culture seedlings.

Claims (11)

  A plant survival stabilizer containing actinomycetes fixed on ceramics.   2. The plant survival stabilizer according to claim 1, wherein the actinomycetes have a function of promoting plant root elongation, seed rooting, seedling rooting or seedling survival.   The plant survival stabilizer according to claim 1, wherein the actinomycetes are actinomycetes belonging to the genus Streptomyces or Microbispora.   The plant survival stabilizer according to claim 1, wherein the actinomycete is a microorganism specified by a deposit number NITE P-169.   The above plants are Azalea, Orchidaceae, Gramineae, Araceae, Eggplant, Legume, Araceae, Asteraceae, Perillaceae, Camellia, Azaza, Brassicaceae, Cucurbitaceae, Ginger, Waterlily , Taceaceae, Rose, Lily, Convolvulaceae, Iridaceae, Siwa tobacco, Buttercup, Oleander, Poppy, Pepper, Prunus, Prunus, Violet, Pleurotus, Thripsaceae, Prunus From plants belonging to the family belonging to the family, pineapple family, auropodae, diatomaceae, gentian family, palm family, saxifrage family, mallow family, cedar family, euphorbiaceae family, cyperaceae family, genus genus family, cypress family, pine family, aceae family, and oleaceae family The plant survival stabilizer according to claim 1, wherein the plant survival stabilizer is a plant selected from the group consisting of: Applying the plant survival stabilizer according to any one of claims 1 to 5 to soil;
Growing a plant in the soil;
A method for promoting plant survival, comprising:   A method for storing a plant survival stabilizer, comprising storing the plant survival stabilizer according to any one of claims 1 to 5 under conditions of a humidity of 70% or less and a temperature of 25 ° C to 40 ° C. Immersing ceramics in actinomycete suspension;
Growing the actinomycetes on the ceramic;
A method for producing a plant survival stabilizer comprising:   9. The method for producing a plant survival stabilizer according to claim 8, wherein the actinomycetes have a function of promoting plant root elongation, seed rooting, seedling rooting or seedling survival.   9. The method for producing a plant survival stabilizer according to claim 8, wherein the actinomycetes are actinomycetes belonging to the genus Streptomyces or Microbispora.   The method for producing a plant survival stabilizer according to claim 8, wherein the actinomycetes are microorganisms identified by the accession number NITE P-169.

Images