JP6267073B2 - New agricultural use of Escherichia bacteria - Google Patents

Description

  The present invention relates to a method for artificially infecting a plant with Escherichia genus bacteria, which is an endophyte, to impart pest resistance to the plant, a method for promoting the growth of the plant, and a method for increasing the yield of the plant.

  So far, pest control technology centered on chemical pesticides has contributed to efficient food security. However, in recent years, not only the efficiency of cultivation, but also environmentally friendly agriculture using pesticide-free and reduced pesticides, including areas such as safety and security, has been desired, and pest control technology (for example, microbial pesticides) and productivity improvement technology suitable for it. Is needed.

  In the agricultural field, chemical fertilizers are still used to promote the growth of crops, and chemically synthesized pesticides are used for disease and pest damage. In addition, microbial pesticides using microorganisms and substances produced by microorganisms are also known in part. However, there are problems that a large amount of energy is input and a large burden on the environment when the raw material of agricultural chemicals is produced by chemical synthesis. On the other hand, microbial pesticides that are currently used with a low environmental load are expensive, and a stable control effect cannot be obtained.

  Under such circumstances, among endophytes that are microorganisms symbiotic to plants, microorganisms imparting beneficial characteristics to plants such as pest control and yield increase have been reported (Patent Documents 1 to 9). . However, since there are few known types of endophytes, the search and application of microorganisms with high possibility of practical use are required for practical use in this field.

JP 2013-042695 A JP 2011-051902 A JP2009-232721 JP 2009-067717 JP 2009-051771 A JP 2009-050206 A JP 2003-300805 JP JP2003-274779 JP 2002-223747

The present invention aims to impart beneficial properties to agriculturally useful plants by microbiological means.
In particular, a technique for increasing the yield by promoting growth by fixing the Escherichia genus bacteria related to the present invention to a plant such as the grass family, the legume family, and the lily family has been unknown.

The present invention includes the following features.
(1) An agricultural process comprising the step of artificially infecting a plant with a bacterium belonging to the genus Escherichia and having the ability to symbiotically grow in an agriculturally useful plant and impart disease resistance to the plant. A method for imparting pest resistance to useful plants.
(2) An agriculturally useful method comprising the step of artificially infecting a plant with a bacterium belonging to the genus Escherichia and having the ability to symbiotically grow in an agriculturally useful plant and increase the yield of the plant. A method to increase plant yield.
(3) An agriculturally useful method comprising the step of artificially infecting a plant with a bacterium belonging to the genus Escherichia and having the ability to symbiotically grow in the agriculturally useful plant body and promote the growth of the plant. A method of promoting plant growth.
(4) Agriculturally useful plants are plants selected from leguminous plants, gramineous plants, liliaceae plants, cruciferous plants, asteraceae plants, solanaceous plants and celeryaceae plants, (1) to ( The method according to any one of 3).
(5) The ability of the bacterium to establish symbiosis in Escherichia sp. MYK100 (Accession No. NITE BP-267) or an agriculturally useful plant to impart disease resistance to the plant, and to increase the yield of the plant And / or the method according to any one of (1) to (4), which is a mutant strain having the ability to promote the growth of the plant.

(6) Ability to belong to the genus Escherichia and to live in agriculturally useful plants to impart disease resistance to the plants, to increase the yield of the plants, or to promote the growth of the plants An agriculturally useful microbial preparation for plants which contains bacteria having an active ingredient as an active ingredient.
(7) The agriculturally useful plant is a plant selected from leguminous plants, gramineous plants, liliaceae plants, cruciferous plants, asteraceae plants, solanaceous plants, and sericaceae plants, Microbial preparations.
(8) The microorganism preparation according to (6) or (7), wherein the bacterium is Escherichia sp. MYK100 (Accession No. NITE BP-267) or a mutant thereof.

(9) Escherichia sp. MYK100 (Accession No. NITE BP-267), or the ability to symbiotically grow in an agriculturally useful plant body to impart disease resistance to the plant, the ability to increase the yield of the plant, and / or Or a mutant thereof having the ability to promote the growth of the plant.

  As used herein, “Escherichia genus bacteria” refers to the ability to symbiotically grow in plants useful in agriculture to impart disease resistance to plants, the ability to increase plant yield, or the ability to promote plant growth Refers to endophytic bacteria. In general, the term “endophyte” refers to a microorganism symbiotic to a plant.

  According to the present invention, by the microbiological means, that is, Escherichia bacterium (for example, NITE BP-267 strain) which can be used as an endophyte, the present invention is not limited to the following, but legumes, grasses, lilies For plants selected from the family plants, solanaceous plants, cruciferous plants, asteraceae plants, and antaceae plants, imparting pest resistance to the plants, promoting the growth of the plants, and / or It becomes possible to increase the yield of the plant. For example, it is possible not only to impart disease and insect resistance to crops belonging to legumes, gramineous plants, liliaceae plants, etc., but also to promote plant growth and increase plant yield. As illustrated in the examples described later, the extent of damage in rice caused by drought beetles, stink bugs, etc. (decrease in the number of leaf damage caused by drought beetles was about 20%, reduction in spotted rice caused by stink bugs was about 55%, browning Panicle reduction is about 65%.) In rice, soybeans, and onions, growth is promoted and yield is increased (3-5% for gramineae, 11% for legumes, and about 5% for lilies). Is). Regarding other plants (for example, Komatsuna, lettuce, leek), weight increase or growth promotion of 10 to 20% was observed. Regarding disease, for example, the incidence of clubroot decreased by 23% for cabbage and 45% for komatsuna.

FIG. 1 is a diagram showing an evaluation method for the degree of disease of clubroot. Based on the disease severity shown in the figure, the severity of clubroot disease was evaluated on a scale from 0 to 4.

1. Bacteria Bacteria that can be used in the present invention belong to the genus Escherichia and are useful in agriculture, such as legumes, grasses, liliaceae, cruciferous plants, chrysanthemum, chrysanthemum. Ability to symbiotize in the body of a plant selected from the family of plants, solanaceous and celery, to impart pest resistance to the plant, to promote the growth of the plant, and / or the yield of the plant Any bacteria can be used as long as it has the ability to increase

  In this specification, the yield of a plant refers to the yield of plant components such as seeds, leaves (including bulbs), berries, stems, roots, and flowers.

  As used herein, a bacterium belonging to the genus Escherichia is an endophyte bacterium that can impart any one, two, or three of the above-mentioned abilities to a plant, and only a bacterium isolated from the natural world. In addition, mutants produced by subjecting such bacteria to mutation treatment are also included.

  As a specific example of the bacterium, Escherichia sp. MYK100 (accession number NITE BP-267; hereinafter, simply referred to as “NITE BP-267 strain”), which is a novel Escherichia bacterium. And mutants thereof having any one or more of the above-mentioned capabilities.

  The above Escherichia sp. MYK100 is a strain that has been selected and isolated from among the strains isolated from rice cultivated in a field by a selection test for growth, yield or pest resistance. Escherichia sp. MYK100 is an international depositary organization under the Budapest Treaty with an international deposit date of October 5, 2006, the National Institute for Product Evaluation Technology and the Patent Microorganism Depositary Center (Kisarazu City, Chiba Prefecture 292-0818, Japan) Deposited by the present applicant in Kazusa Kama feet 2-5-8) and assigned the deposit number NITE BP-267.

This NITE BP-267 strain is a partial sequence of 16S rDNA gene (in this case, Escherichia sp. MYK100 (Accession No. NITE BP-267) strain corresponding partial bases of various bacterial genera and species. As a result of homology search using the sequence (SEQ ID NO: 1), Escherichia hermannii and 99% (1506bp / 1512bp), Escherichia senegalensis and 98% (1517bp / 1533bp) ), And 97% (1497bp / 1536bp) high sequence identity with Escherichia fergusonii, which proved to be a novel bacterium belonging to the genus Escherichia.
The NITE BP-267 strain has the following substrate assimilation properties.

[Substrate to be utilized]
Dextrin, N-acetyl-D-glucosamine, L-arabinose, cellobiose, D-fructose, L-fucose, D-galactose, gentiobiose, α-D-glucose, α-D-lactose, maltose, D-mannitol, D- mannose, D-melibiose, β-methyl D-glucoside, D-raffinose, L-rhamnose, D-sorbitol, D-trehalose, methyl pyruvate, mono-methyl succinate, cis-aconitic acid, citric acid, formic acid, D- galactonic acid lactone, D-galacturonic acid, D-gluconic acid, D-glucuronic acid, D, L-lactic acid, D-saccharic acid, succinic acid, bromo succinic acid, glucuronamide, D-alanine, L-alanine, L- asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, inosine, thymidine, glycerol, D, L-α-glycerol phosphate, glucose-1-phosphate, glucose-6-phosphate

[Substrate not assimilated]
α-cyclodextrin, glycogen, tween 40, tween 80, N-acetyl-D-galactosamine, adonitol, D-arabitol, i-erythritol, m-inositol, lactolose, D-psicose, sucrose, turanose, xylitol, acetic acid, D -glucosaminic acid, α-hydroxybutyric acid, β-hydroxybutyric acid, γ-hydroxybutyric acid, p-hydroxy phenylacetic acid, itaconic acid, α-keto butyric acid, α-keto glutaric acid, α-keto valeric acid, malonic acid, propinic acid, quinic acid, sebacic acid, succinamic acid, alaninamide, L-alanyl-glycine, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L-histidine, hydroxy L-proline, L-leucine, L-ornithine, L-phenylalanine, L-pyroglutamic acid, D-serine, L-thrreonine, D, L-carnitine, γ-amino butyric acid, urocanic acid, uridine, phenyl ethylamine, putrescine, 2-amino ethanol, 2,3-butanediol

  As a bacterium that can be used in the present invention, a bacterium having the above-mentioned ability equivalent to the NITE BP-267 strain, which is a novel Escherichia bacterium, for example, belonging to the genus Escherichia, having any one or more of the above-mentioned abilities, And the same bacterium having the substrate assimilation property as the NITE BP-267 strain, or belonging to the genus Escherichia, having any one or more of the above-mentioned properties, and the nucleotide sequence shown in SEQ ID NO: 1 Examples include, but are not limited to, bacteria having 16S rDNA in at least a portion thereof. Furthermore, the NITE BP-267 strain is a mutant strain produced by artificial mutagenesis, and is useful in agriculture, such as legumes, grasses, liliaceae, cruciferous plants. A plant selected from the family Asteraceae, Solanum plant, and Apiaceae, capable of imparting pest resistance to the plant, promoting the growth of the plant, and / or the plant Mutants having the ability to increase the yield of can also be used in the present invention.

When separating Escherichia genus bacteria that can be used in the present invention from the natural world, bacteria that are symbiotic to the plant are isolated from the constituent parts of the plant body such as roots, stems, and leaves of agriculturally useful plants, and the above-mentioned ability A selection test can be performed for any of the above and / or for the sequence identity with the base sequence shown in SEQ ID NO: 1 and / or the substrate assimilation property.
When mutagenesis is performed, mutation can be performed using any appropriate mutagen for NITE BP-267 strain.

  Here, the term “mutagen” has a broad meaning, and includes not only a drug having a mutagenic action but also a treatment having a mutagenic action such as irradiation with high energy rays such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, gamma irradiation, N-methyl-N′-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, Any other effective mutagen can also be used.

  Alternatively, another means for introducing a mutation into a bacterium is a method using a gene recombination method. In particular, in the case of countermeasures against insect damage, introduction of a gene or cDNA capable of producing a repellent substance into a bacterial genome, introduction of a gene or cDNA encoding an insecticidal protein such as Bt toxin and the like can be mentioned.

  The bacterium used in the present invention can be cultured under the conditions normally used for Escherichia bacteria by a conventional culture method such as shaking culture. As a medium for culturing, saccharides such as glucose, sucrose, starch and dextrin as carbon sources, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate as nitrogen sources, inorganic nitrogen sources such as nitrates, yeast extract, corn Organic nitrogen sources such as steep leaker, meat extract, wheat germ, polypeptone, sugar cane squeezed (bacus), beer casks, soy flour, rice bran, fish meal, etc., and inorganic salts such as monopotassium phosphate, magnesium sulfate, manganese sulfate, sulfuric acid Examples thereof include synthetic or natural media containing salts containing phosphorus, potassium, manganese, magnesium, iron, etc., such as monoiron. The culture temperature is usually 20 to 37 ° C., preferably 27 to 32 ° C., and can be performed under aerobic conditions for 12 to 48 hours.

  In the method of the present invention, a bacterial culture solution can be used as it is, but a high bacterial concentration obtained by separating the bacterial culture solution by a method such as membrane separation, centrifugation, or filtration separation can also be used. .

  In the method of the present invention, a dried bacterial culture solution can also be used. Moreover, what dried by making it adsorb | suck to porous adsorbents, such as activated carbon, diatomaceous earth, talc, a zeolite, peat moss, perlite, bentonite, montmorillonite, vermiculite, can be used. One kind of porous adsorbent may be used, or a plurality of carriers may be used in combination. The drying method may be a normal method, for example, freeze drying or vacuum drying. These dried products may be further pulverized by a pulverizing means such as a ball mill after drying.

  Bacteria can be used for the purposes of the present invention alone as the above culture solution, high-concentration product, or dry product, but in the same form as normal microbial preparations (for example, powders) in combination with other optional components. , Wettable powders, granules, emulsions, solutions, suspensions, flowables, coatings, etc.). Examples of optional components that can be used in combination include materials that can be applied to plants such as solid carriers and adjuvants.

2. Agriculturally useful plants The target plants that can be used in the method of the present invention are not limited to the following. For example, legumes, gramineous plants, liliaceae plants, cruciferous plants, asteraceae plants, solanaceous plants and Agriculturally useful plants selected from the Apiaceae plants can be mentioned.
As used herein, “agriculturally useful plants” refers to plants to be produced in agriculture such as crops such as vegetables and cereal plants, and fruit trees.

  Examples of the grass family include cereals such as rice, wheat, barley, rye, triticale, pearl barley, sorghum, oat, corn, sugarcane, millet, and millet. Examples of grasses include feeds and grasses such as buckwheat, buffalo grass, bermuda grass, weeping grass, centipede grass, carpet grass, dalice grass, chrysanthemum grass and St. Augustine grass.

  Examples of legumes include soybean, azuki bean, peanut, kidney bean, pea, pea bean, broad bean, cowpea, chickpea, mung bean, lentil, lentil and bambara bean.

  Examples of the lily family include onions, leeks, raccoons, garlic, leek, chives, lilies, asparagus, shallots, and bamboo shoots.

  Examples of the Brassicaceae plants include: Brassica, Turnip, Chingensai, Nozawana, Mustard, Takana, Kobutana, Mizuna, Kohl Rabbi, Arugula, Watercress, Taasai, Cauliflower, Cabbage, Kale, Chinese cabbage, Komatsuna, Daikon, Hatsukadaikon, Broccoli, Mekabetsu, Wasabi , Horseradish, and Arabidopsis thaliana.

  Examples of the Asteraceae plants include lettuce, sunny lettuce, garlic, chrysanthemum and the like.

  Examples of solanaceous plants include eggplant, tomato, pepper, shishito, chili pepper, potato, wolfberry, paprika, jalapeno, habanero and the like.

  Examples of celery family plants include carrot, honeybee, parsley, celery, celery, ashitaba, soup celery, charbell, fennel and the like.

3. Pests and pests The bacterium of the present invention imparts pest resistance by providing symbiosis in the above-mentioned agriculturally useful plants, but the target plant diseases and pests are not limited to the following, but examples include the following: It is done.

Examples of plant diseases are as follows.
Examples of plant diseases of the grass family include, for example, Sheath brown rot of rice, rice blast, white leaf blight, seedling blight, coat blight, and idiot seedling.
Examples of plant diseases of leguminous plants include gray mold disease, rust disease, powdery mildew.
Examples of plant diseases of the liliaceae include soft rot, downy mildew, shrinkage disease, dry rot, rust disease, ginkgo biloba, stem blight disease, and spot disease.
Examples of cruciferous plant diseases include soft rot, black rot, bacterial seed blight, downy mildew, yellowish disease, mosaic disease, root-knot disease, white rot, and rot rot.
Examples of plant diseases of Asteraceae plants include mosaic disease, soft rot, rot, powdery mildew, downy mildew and the like.
Plant diseases of solanaceous plants include, for example, mosaic disease, yellowing wilt (TSWV), bacterial wilt, scab, brown root rot, seedling wilt, powdery mildew, half body rot, leaf mold, etc. Is mentioned.
Examples of plant diseases of celery family include mosaic disease, soft rot, black leaf blight, spotted disease, powdery mildew, mycorrhizal disease and the like.

Examples of pests are as follows. Insect damage includes feeding, sucking, and virus transmission.
The pests of the grass family include, Feeding by gale nematodes, other nematodes, rice weevil, click beetles, scarab beetles, grasshoppers, scallops, white-winged beetles, gangambo, scallops, white-spotted plant, leafhopper, scallop, leafhopper, leafhopper, leafhopper And thrips.

  Leguminous pests include, for example, white-headed beetles, beetles, turmeric moths, azuki bean moths, larvae, bean moths, bean squirrels, blue-green wings, figs and cinnamon moths Examples include nashikenmon, honey monk wabas, long-tailed squirrel moth, black butterfly, brown squirrel turtle, white-bellied helicopter, and white beetle.

  Examples of the pests of the liliaceae plant include Negia thrips, Negihamaburie, Thrips, and weevil.

  Examples of the cruciferous pests include diamondback moth, white butterfly, giant white butterfly, red-tailed moth, cabbage moth, red snapper, weevil, leafhopper, cabbage beetle, hornbill beetle, cabbage weevil, aphid, thrips, and the like.

Examples of the pests of the Asteraceae plants include Shiroshitayoto, Figukinuwaba, Tamanaginuwaba, Hosobasedakamokume, Nashikenmon, Artemisia, Ootobis Diadasaku, Nagame and the like.
Examples of the pests of the solanaceous plant include giant cigarette moth, giant moth beetle, eggplant beetle, azuki bean moth, fig cinnamon moth, and tofu tent beetle.
Examples of the pests of the Apiaceae plants include cucumber moth, yellow swallowtail, and honeymooner.
Other examples include snails and nematodes.

4). A microbiological method for imparting pest resistance, increasing yield, and promoting growth The method of the present invention belongs to the genus Escherichia, and is symbiotic in the body of the above-mentioned plant useful in agriculture to give pest resistance to the plant. Artificially infecting the plant with bacteria having the ability to confer, the ability to increase the yield of the plant, and / or the ability to promote the growth of the plant.

  Giving pest resistance to a plant leads to suppressing or preventing the plant's influence by the pest, for example, feeding, sucking, pathogenic fungal disease, viral disease, and the like. Although not wishing to be bound by theory, it is thought that the bacterium according to the present invention does not act directly on the pathogen but imparts pest resistance to the plant by activating the defense function of the plant itself. Reactions caused by resistance induction include, but are not limited to, hypersensitive reactions, production of antibacterial and anti-insect pest proteins, formation of papillas, and cell wall hardening. In general, it is known that if plant resistance is induced, it is resistant to a wide range of pests.

  Increasing the yield of a plant is, for example, for vegetables, for example, leaves (including bulbs), roots, rhizomes, seeds, flowers, etc., for cereals, for seeds, for fruit trees, It means to increase the yield of fruits.

  Promoting plant growth means that the growth of the plant is accelerated compared to a control that is not inoculated with the bacteria of the present invention.

  Examples of methods for applying to plants include seed coats, irrigation, application, or spraying treatments on young plants. In particular, a method of artificially scratching seeds or plants and spraying and applying the bacterial solution is preferable. As other application conditions, it is desirable to apply before planting in the field, such as at the time of sowing and the seedling season. Furthermore, high expression of the effect can be expected by spraying the plant, and in some cases, the soil around the root of the plant during field cultivation.

  As an example, in order to artificially infect a plant of the bacterium of the present invention, a bacterial solution is sprayed on young seedlings (for example, seedlings at the time when 1 to 4 true leaves appear) before being planted in the field. be able to. About 3 to 15 days after spraying, seedlings can be planted in the field. It is thought that the bacteria that invaded the plant body will eventually coexist with the plant.

The concentration of the bacterial solution is 1 × 10 ⁵ to 1 × 10 ⁹ cells / ml, but is not limited to these concentration ranges. The medium for suspending bacteria is preferably water or a medium. Usually, a high concentration bacterial solution can be used by diluting to a predetermined concentration with water or a medium.

Specifically, the method of the present invention comprises the following first to third embodiments.
According to the first aspect of the method of the present invention, a bacterium belonging to the genus Escherichia and having the ability to symbiotically grow in an agriculturally useful plant body and impart disease resistance to the plant is artificially given to the plant. There is provided a method for imparting pest damage resistance to an agriculturally useful plant, comprising a step of infecting a plant.

  According to the second aspect, the method of the present invention artificially infects a plant belonging to the genus Escherichia and has the ability to symbiotically grow in an agriculturally useful plant body and increase the yield of the plant. And a method for increasing the yield of agriculturally useful plants.

  According to the third aspect, the method of the present invention artificially infects the plant with a bacterium belonging to the genus Escherichia and having the ability to coexist in an agriculturally useful plant body and promote the growth of the plant. And a method for promoting the growth of an agriculturally useful plant.

5. Microbial preparation The present invention further belongs to the genus Escherichia, and is capable of symbiosis in an agriculturally useful plant to impart disease resistance to the plant, the ability to increase the yield of the plant, and / or the Provided is an agriculturally useful plant microbial preparation containing, as an active ingredient, a bacterium having the ability to promote plant growth.

Examples of agriculturally useful plants are the plants specifically exemplified above, selected from legumes, gramineous plants, liliaceae plants, solanaceous plants, cruciferous plants, asteraceae plants and sericaceae plants. is there.
A preferred bacterium is Escherichia sp. MYK100 (Accession No. NITE BP-267), or a mutant thereof.

  As the microbial preparation, a high concentration of bacteria, a dried culture solution of bacteria, or the like can be used. Moreover, what dried by making it adsorb | suck to porous adsorbents, such as activated carbon, diatomaceous earth, talc, a zeolite, peat moss, perlite, bentonite, montmorillonite, vermiculite, can be used. One kind of porous adsorbent may be used, or a plurality of carriers may be used in combination. The drying method may be a normal method, for example, freeze drying or vacuum drying. These dried products may be further pulverized by a pulverizing means such as a ball mill after drying.

  Bacteria can be used for the purposes of the present invention alone as the above culture solution, high-concentration product, or dry product, but in the same form as normal microbial preparations (for example, powders) in combination with other optional components. , Wettable powders, granules, emulsions, solutions, suspensions, flowables, coatings, etc.). Examples of optional components that can be used in combination include materials that can be applied to plants such as solid carriers and adjuvants.

  The present invention further includes Escherichia sp. MYK100 (Accession No. NITE BP-267), or an ability to symbiotically grow in an agriculturally useful plant to impart disease resistance to the plant, an ability to increase the yield of the plant, And / or a mutant thereof having the ability to promote the growth of the plant.

  The bacterium of the present invention can be cultured under the above-mentioned conditions as commonly used for Escherichia bacteria by a conventional culture method such as shaking culture.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited by these examples.

[Example 1]
<Isolation of Escherichia sp. MYK100 (Accession Number NITE BP-267)>
Rice cultivated in the field was collected and its stem was cut. Surface sterilization was performed by immersing the cut stems in 70% ethanol for 30 seconds and in 2.5% sodium hypochlorite solution for 5 minutes. Thereafter, the plant body was transferred to a mortar and ground while adding 1 ml of sterilized physiological saline and an appropriate amount of sea sand. 100 μl of the ground supernatant was applied to NA (Nutrient Agar) medium, and after culturing at 30 ° C. for several days, a single colony was isolated. The isolated strain was inoculated into cultivated rice, and the title NITE BP-267 strain was selected and isolated by evaluating insect damage resistance against the corn borer.

[Example 2]
<Rice resistance by NITE BP-267 strain>
1) Purpose By establishing a new Escherichia bacterium isolated from rice (hereinafter referred to as NITE BP-267) on crops, the damage of diseases and pests will be reduced, and growth will be increased by promoting growth even with reduced or non-pesticide cultivation. I confirmed the possibility. Furthermore, in order to confirm the effect in the field, rice inoculated with NITE BP-267 was cultivated in the field and the inoculation effect was examined.

1.1) Dromedary damage, test method rice varieties: Nanatsuboshi, Kirara 397
Endophyte strain: NITE BP-267 strain inoculation date: Nanatsuboshi May 21, 2006, Kirara 397 May 23, 2006 Inoculation method: 500 ml of 1 x 10 ⁸ / ml bacterial suspension / Seedling box irrigation treatment The cell suspension was adjusted with water so that the number of viable bacteria suspension was 10 ⁸ per ml. Rice seedlings grown from 1.5 leaves to 3.5 leaves were treated by irrigating 500 ml of bacterial suspension per seedling box with rain dew.
Date of planting: Nanatsuboshi May 24, 2006, Kirara 397 May 26, 2006 Field: Biei, Hokkaido Trial scale: 76.8m ² , 4 seedling boxes (8 rows planted, about 1790)

1.2) Damage to stink bugs, test methods Rice varieties: Nanatsuboshi, Kirara 397
Endophyte strain: NITE BP-267 strain inoculation date: Nanatsuboshi May 21, 2006, Kirara 397 May 23, 2006 Inoculation method: 500 ml of 1 x 10 ⁸ / ml bacterial suspension / Seedling box irrigation treatment The cell suspension was adjusted with water so that the number of viable bacteria suspension was 10 ⁸ per ml. Rice seedlings grown from 1.5 leaves to 3.5 leaves were treated by irrigating 500 ml of bacterial suspension per seedling box with rain dew.
Date of planting: Nanatsuboshi May 24, 2006, Kirara 397 May 26, 2006 Field: Biei, Hokkaido Trial scale: 76.8m ² , 4 seedling boxes (8 rows planted, about 1790)

1.3) Damage to brown ear (formal name: Sheath brown rot of rice), test method Rice varieties: Nanatsuboshi, Fuchimenko endophyte strain: NITE BP-267 inoculation date: 2007 May 18 inoculation method: 500 ml / seedling box irrigation treatment of 1 × 10 ⁸ cells / ml of bacterial suspension The cells were adjusted with water so that the number of viable bacteria suspension was 10 ⁸ per ml. Rice seedlings grown from 1.5 leaves to 3.5 leaves were treated by irrigating 500 ml of bacterial suspension per seedling box with rain dew.
Date of planting: May 21, 2007 Field: Biei City, Hokkaido Trial scale: 230.4m ² , 12 nursery boxes (8 rows, about 5370 plants)

2) Results and discussion

  The number of leaf damage caused by Drosophila and the number of strains were decreased in the inoculated area of NITE BP-267 (Tables 1 and 2). The spotted rice rate and coloring damage rate due to stink bugs also decreased in the inoculated area of NITE BP-267 strain (Tables 3 and 4). Furthermore, the browning damage rate also decreased in the inoculated area of NITE BP-267 strain (Tables 5 and 6).

[Example 3]
<NITE BP-267 strain promotes rice growth and increases yield>
(1) Field cultivation test of rice inoculated with bacterial endophyte
1) Method Rice varieties: Nanatsuboshi Fukkenko Endophyte strain: NITE BP-267 Inoculation date: Nanatsuboshi May 21, 2006 Fukunkenko May 18, 2007 Inoculation method: 1 × 10 ⁸ cells / ml fungus suspension 500 ml / nursing box irrigation treatment The cell suspension was adjusted with water so that the number of viable cells was 10 ⁸ per ml. Rice seedlings grown from 1.5 leaves to 3.5 leaves were treated by irrigating 500 ml of bacterial suspension per seedling box with rain dew.
Date of planting: Nanatsuboshi May 24, 2006 Fukuchinko May 21, 2007 Field: Biei City, Hokkaido
Test scale: 76.8m ² 4 nursery boxes (8 rows, about 1790 plants)
The non-inoculation section was performed in 2 sections, and the average value was calculated.

2) Results and discussion

  In both Nanatsubo and Fumiko, the splitting was promoted and the number of stems increased in the inoculated area of NITE BP-267 (Tables 7 and 8). The yield also increased in the inoculation area of NITE BP-267, both in Nanatsubo and Fluffy (Tables 9 and 10).

[Example 4]
<Acceleration of soybean growth and increase in yield by NITE BP-267>
1) Purpose To investigate the growth promotion and yield increase of new Escherichia bacteria isolated from cultivated rice (hereinafter referred to as NITE BP-267) in soybean, cultivate soybean inoculated with NITE BP-267 in the field The inoculation effect was examined.

2) Method Soybean varieties: Toyohoma renofite strain: NITE BP-267 strain Inoculation date: June 8, 2006 Inoculation method: 1 x 10 ⁸ / ml bacterial suspension 10ml / strain spray treatment The solution was adjusted with water so that the number of viable bacteria was 10 ⁸ per ml. The soybean seedlings from the first leaf development to the third leaf development were treated by spraying 10 ml of the bacterial suspension per soybean strain.
Farm: Biei City, Hokkaido Test scale: Total area 9.9m ² 4.95m ² (1.32m × 3.75m) × 2 stations 200 shares

3) Results

  In the inoculation area of NITE BP-267, the stem length and the number of branches tended to increase, and the number of arrivals increased (Table 11). Also, the yield increased by 11% (Table 12).

[Example 5]
Onion growth promotion and yield increase
1) Purpose Endophite Escherichia spp.Bacteria isolated from rice (NITE BP-267 strain) are established in the onion to promote onion growth and increase the yield by increasing the size of the onion. There was a possibility. Therefore, NITE BP-267 inoculated onion was cultivated in the field and the inoculation effect was examined.

2) Method Onion variety: Okhotsk 222
Endophyte strain: NITE BP-267 (rice isolate)
Inoculation date: May 2, 2006 Inoculation method: 500 ml of seedling suspension of 1 × 10 ⁸ cells / ml / nursing seedling irrigation with water so that the number of viable bacteria suspension is 10 ⁸ per ml It was adjusted. The seedlings at the 1st to 3rd leaf stages were treated by irrigating the fungus suspension with 500 ml of rain dew per seedling box.
Date of planting: May 7, 2006 Field: Furano, Hokkaido Experiment size: 448 (224 shares x 2)

3) Results and discussion

  In the growth survey, 12 strains were counted and the average of 10 strains excluding the highest and lowest values was calculated. There was a tendency for the plant height to be slightly larger in the NITE BP-267 strain inoculation zone (Table 13). In addition, the yield increased by 5% in the inoculated area of NITE BP-267, due to the increase in the number of bulbs and the number of bulbs within the specification (Table 14).

[Example 6]
<Promoting growth of Komatsuna by NITE BP-267>
1) Materials and methods Komatsuna varieties: Summer Rakuten sowing date and inoculation date: July 31, 2013 Harvest date: August 23, 2013 Test scale: Cell tray 10-hole test method: Cell tray filled with culture soil, Two seeds were sown and inoculated with 100 μl of a bacterial solution (viable cell concentration 1 × 10 ⁸ to 10 ⁹ cells / ml) cultured for 24 hours. From the next day, the plants were watered daily and cultivated in a glass house. After cultivation for 23 days, the above-ground part was cut off, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results The dry matter weights of the 10 stocks in each treatment area were measured and converted to the weight of 1 stock. The results are shown in Table 15. The numbers in parentheses are relative values when the untreated section is 100. As a result, an increase of 16.7% in weight was observed in the NITE BP-267 treated group compared to the untreated group.

[Example 7]
<Promoting radish growth with NITE BP-267>
1) Method Daikon varieties: Kotaro sowing date and inoculation date: September 20, 2013 Harvest date: October 11, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Fill the cell tray with culture soil, Two seeds were sown and inoculated with 100 μl of a bacterial solution (viable cell concentration 1 × 10 ⁸ to 10 ⁹ cells / ml) cultured for 24 hours. From the next day, the plants were watered daily and cultivated in a glass house. After cultivation for 21 days, the above-ground part was cut, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 16 shows the results of measuring the dry matter weight of each of the 10 treatment groups and converting them to the weight of one strain and averaging the values in the three columns. The numbers in parentheses are relative values when the untreated section is 100. As a result, an increase of 11.1% in weight was observed in the NITE BP-267 treated group compared to the untreated group.

[Example 8]
<Promoting growth of lettuce with NITE BP-267>
1) Method lettuce variety: Green wave sowing date and inoculation date: July 31, 2013 Harvest date: August 23, 2013
Test scale: Cell tray 10-hole Test method: Cell tray is filled with culture soil, seeds are sown 2 seeds at a time, and the bacterial solution cultured for 24 hours (viable cell concentration 1 x 10 ⁸ to 10 ⁹ cells / ml) 100 μl each was inoculated and covered with soil. From the next day, the plants were watered daily and cultivated in a glass house. After cultivation for 23 days, the above-ground part was cut off, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 17 shows the results of measuring the weight of dry matter collected from 10 stocks in each treatment area and converting them to the weight of 1 stock. The numbers in parentheses are relative values when the untreated section is 100. As a result, an increase of 22.1% in weight was observed in the NITE BP-267 treated group compared to the untreated group.

[Example 9]
<Promotion of tomato growth by NITE BP-267>
1) Method tomato varieties: Hokin Fuju Tomato sowing date and inoculation date: July 31, 2013 Harvest date: August 23, 2013 Test scale: Cell tray 10-hole Test method: Fill the cell tray with culture soil, Two seeds were sown and inoculated with 100 μl of a bacterial solution (viable cell concentration 1 × 10 ⁸ to 10 ⁹ cells / ml) cultured for 24 hours. From the next day, the plants were watered daily and cultivated in a glass house. After cultivation for 23 days, the above-ground part was cut off, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 18 shows the results of measuring the dry matter weight of the 10 stocks in each treatment area and converting them to the weight of one stock. The numbers in parentheses are relative values when the untreated section is 100. As a result, an increase of 6.8% in weight was observed in the NITE BP-267 treated group compared to the untreated group.

[Example 10]
<Acceleration of leek growth by NITE BP-267>
1) Method Leek variety: All-purpose small leek sowing date and inoculation date: September 20, 2013 Harvest date: November 1, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Filling cell tray with culture soil Then, seeds were seeded two by two, and 100 μl each of the bacterial solution (viable cell concentration 1 × 10 ⁸ to 10 ⁹ cells / ml) cultured for 24 hours was inoculated and covered with soil. From the next day, the plants were watered daily and cultivated in a glass house. After 42 days of cultivation, the above-ground part was cut, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 19 shows the results of measuring the dry matter weight of each of the 10 treatment groups and converting them to the weight of one strain, and averaging the values in the three columns. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE BP-267 treatment group showed an increase of 10.3% in weight compared to the non-treatment group.

[Example 11]
<Acceleration of carrot growth by NITE BP-267>
1) Method Carrot varieties: 5 inch ginseng sowing date and inoculation date: September 20, 2013 Harvest date: November 1, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Fill the cell tray with culture soil Then, seeds were seeded two by two, and 100 μl each of the bacterial solution (viable cell concentration 1 × 10 ⁸ to 10 ⁹ cells / ml) cultured for 24 hours was inoculated and covered with soil. From the next day, the plants were watered daily and cultivated in a glass house. After 42 days of cultivation, the above-ground part was cut, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 20 shows the results of measuring the dry matter weight of each of the 10 treatment groups and converting them to the weight of one strain and averaging the values in the three columns. The numbers in parentheses are relative values when the untreated section is 100. As a result, an increase of 6.8% in weight was observed in the NITE BP-267 treated group compared to the untreated group.

[Example 12]
<Promoting soybean growth with NITE BP-267>
1) Method Soybean varieties: Early seedling date and inoculation date: September 20, 2013 Harvest date: October 11, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Fill the cell tray with culture soil Then, seeds were sown two by two, and 100 μl each of the bacterial solution cultured for 24 hours (viable cell count concentration of 1 × 10 ⁸ to 10 ⁹ cells / ml) was inoculated and covered with soil. From the next day, the plants were watered daily and cultivated in a glass house. After cultivation for 21 days, the above-ground part was cut, 10 strains were put together in a bag, dried at 80 ° C. for 2 days or more, and then weighed.

2) Results Table 21 shows the results of measuring the dry matter weight of each of the 10 treatment groups and converting them to the weight of one strain and averaging the values in the three columns. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE BP-267 treated area showed a 9.0% weight increase compared to the untreated area.

[Example 13]
<Disease resistance test against clubroot of cruciferous plants 1: Field cultivation test of cabbage inoculated with NITE BP-267 in root-knot nematode contaminated soil>
1) Materials and methods Cabbage variety: Shinran
Sowing date: July 27, 2013 Inoculation date: August 10, 2013 Survey date: December 12, 2014 Inoculation method: Bacterial suspension with a viable cell concentration of 1 × 10 ⁸ cells / ml 10ml / strain spray treatment test scale: 4 seedling boxes for about 500 strains Measurement item: NITE BP-267 stock treatment area, untreated treatment area 100 each cabbage in five stages of visual inspection Evaluation was made (see FIG. 1).

2) Results Table 22 shows the average degree of damage that occurred in 100 treatment areas. As a result, the damage decreased in the NITE BP-267 strain inoculation area. The numbers in parentheses are relative values when the non-inoculation section is 100.

[Example 14]
<Drug resistance test against clubroot of cruciferous plants 2: Pot seedling cultivation test of Komatsuna inoculated with NITE BP-267>
1) Materials and methods Komatsuna varieties: Summer Rakuten sowing date: March 11, 2014 Inoculation method: NITE BP-267 strain cultured at seeding for 24 hours (viable cell count concentration 1 x 10 ⁸ to 10 ⁹ cells / ml) 100μl / cell inoculation test scale: 10 strains in 1 treatment area, 3 repeated experiments Method: Prepare root-knot nematode contaminated soil so that root-knot nematode density per cell is 50 individuals, sow 3 komatsuna seeds per cell, NITE BP-267 cultured for 24 hours was inoculated at 100 μl / cell and covered with soil. Cultivated in a glass greenhouse, and when the true leaves began to appear, the plants were thinned to one per cell, and grown until 5 or more true leaves were developed. Thereafter, the presence or absence of diseased clubroot was observed visually.

2) Results Three repeated tests were performed for each treatment group. In the NITE BP-267 vaccination group, the incidence of clubroot was reduced by 45% (Table 23).

  The present invention artificially infects agriculturally useful plants such as gramineous plants, legumes, and liliaceae with Escherichia genus bacteria, thereby suppressing disease and pest damage of these plants, promoting growth, or Agriculturally useful because it increases the yield.

Claims (7)

Escherichia sp. MYK100 (Accession No. NITE BP-267), and comprising the step of artificially infected with plant, imparting pest resistance to agriculturally useful plants. Escherichia sp. MYK100 (Accession No. NITE BP-267), and comprising the step of artificially infected with plant, a method for increasing the yield of agriculturally useful plants. Escherichia sp. MYK100 (Accession No. NITE BP-267), and comprising the step of artificially infected with plant, a method for promoting the growth of agriculturally useful plants.   The agriculturally useful plant is a plant selected from legumes, gramineous plants, liliaceae plants, cruciferous plants, asteraceae plants, solanaceous plants, and sericaceae plants. 2. The method according to item 1. An agriculturally useful microbial preparation for plants containing Escherichia sp. MYK100 (Accession No. NITE BP-267 ) as an active ingredient.   The microbial preparation according to claim 5, wherein the agriculturally useful plant is a plant selected from legumes, gramineous plants, liliaceae plants, cruciferous plants, asteraceae plants, solanaceous plants and sericaceae plants. . Escherichia sp. MYK100 (Accession Number NITE BP-267 ).

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