JP6267064B2 - New agricultural use of Stenotrophomonas bacteria - Google Patents

Description

  The present invention relates to a method for artificially infecting a plant with a genus Stenotrophomonas, 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.

The present invention includes the following features.
(1) It includes a step of artificially infecting a plant with a bacterium belonging to the genus Stenotrophomonas 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 agriculturally useful plants.
(2) Agriculture comprising a step of artificially infecting a plant with a bacterium belonging to the genus Stenotrophomonas and having an ability to increase the yield of the plant symbiotically in a plant useful in agriculture A method to increase the yield of top useful plants.
(3) Agriculture, comprising a step of artificially infecting a plant with a bacterium that belongs to the genus Stenotrophomonas and has the ability to symbiotically grow in an agriculturally useful plant body and promote the growth of the plant A method for promoting the growth of a useful plant.
(4) The agriculturally useful plant is a plant selected from the Brassicaceae plant, Asteraceae plant, Legume plant, Gramineae plant, Solanum plant, Lily family plant, and Apiaceae plant, (1) to ( The method according to any one of 3).
(5) The method according to any one of (1) to (3), wherein the agriculturally useful plant is a cruciferous plant.
(6) Stenotrophomonas sp. MYK101 (Accession No. NITE P-492), Ability to symbiosis in agriculturally useful plants and impart disease resistance to the plants, Ability to increase the yield of the plants And / or the method according to any one of (1) to (5), which is a mutant strain having the ability to promote the growth of the plant.
(7) It belongs to the genus Stenotrophomonas and has 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, or the growth of the plant. An agriculturally useful microbial preparation for plants containing bacteria having an ability to promote as an active ingredient.
(8) The agriculturally useful plant is a plant selected from the family Brassicaceae, Asteraceae, Legume, Gramineae, Solanum, Lily and Apiaceae, Microbial preparations.
(9) The microorganism preparation according to (7) or (8), wherein the bacterium is Stenotrophomonas sp. MYK101 (Accession No. NITE P-492) or a mutant thereof.
(10) Stenotrophomonas sp. MYK101 (Accession No. NITE P-492), or the ability to symbiosis in agriculturally useful plants to impart disease resistance to the plants, the ability to increase the yield of the plants, and / or Or a mutant thereof having the ability to promote the growth of the plant.

  As used herein, the term “Stenotrophomonas genus bacteria” refers to the ability of symbiosis in agriculturally useful plants to impart disease resistance to plants, the ability to increase plant yield, or plant growth. Refers to endophyte bacteria that have the ability to promote. In general, the term “endophyte” refers to a microorganism symbiotic to a plant.

  According to the present invention, by the microbiological means, that is, the genus Stenotrophomonas which can be used as an endophyte (for example, NITE P-492 strain), Brassicaceae, Asteraceae, Legume, Gramineae, Solanum, It becomes possible to impart pest resistance to a plant selected from the liliaceae plant and the sericaceae plant, to promote the growth of the plant, and / or to increase the yield of the plant. . For example, it is possible not only to impart disease resistance to cruciferous crops, but also to promote plant growth and increase the yield of plant components such as leaves. As illustrated in the examples below, the degree of insect damage in kale, for example, diamondback moth, was reduced to 73.5%, and the survival rate for damage caused by, for example, snail death was 144.5%. Regarding diseases, for example, the incidence of clubroot decreased by 23% for cabbage and 17% for komatsuna. Moreover, the yield increased to 166.7% due to the growth promoting effect. In Komatsuna, the yield increased to 112.9%. For other plants, a yield increase of several to tens of percent was observed. By using the bacterium of the present invention in the agricultural field, the yield can be increased and the yield can be increased even in pesticide-free and pesticide-free cultivation.

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 Stenotrophomonas and are useful in agriculture, such as cruciferous plants, asteraceae plants, legumes, and gramineous families. The ability to coexist in the body of a plant selected from a plant, solanaceous plant, liliaceae and sericaceae to impart disease resistance to the plant, the ability to promote the growth of the plant, and / or the The bacterium is not particularly limited as long as it has the ability to increase plant yield.

  As used herein, a Stenotrophomonas genus bacterium is an endophyte bacterium that can impart any one, two, or three of the above-mentioned abilities to plants, and has been isolated from the natural world. It includes not only bacteria but also mutants produced by subjecting such bacteria to mutation treatment.

  Specific examples of the bacterium include Stenotrophomonas sp. MYK101 (accession number NITE P-492; hereinafter, simply referred to as “NITE P-492 strain”), which is a novel Stenotrophomonas bacterium, or any of the above Or mutants thereof having one or more abilities.

  The above Stenotrophomonas sp. MYK101 is an isolated strain selected from a strain isolated from kale grown in a field by a selection test for growth, yield or pest resistance. On March 5, 2008, Stenotrophomonas sp. MYK101, a domestic depository institution, the National Institute for Product Evaluation Technology and the Patent Microorganism Deposit Center (Kazusa-Kamazu 2-5-8, Kisarazu City, Chiba Prefecture 292-0818) ) And is assigned the deposit number NITE P-492.

  This NITE P-492 strain is a partial sequence of the 16S rDNA gene (in this case, Stenotrophomonas sp. MYK101 (accession number NITE P-492) strain corresponding to the genus and species of various bacteria). As a result of homology search using comparison with SEQ ID NO: 1), Stenotrophomonas humi and 99% (725bp / 728bp), Stenotrophomonas terrae and 98% (717bp / 728bp) As a result, a high sequence identity of 98% (716 bp / 728 bp) with Stenotrophomonas maltophilia was confirmed, and it was found to be a novel bacterium belonging to the genus Stenotrophomonas.

  The NITE P-492 strain has substrate assimilation properties shown in Tables 1 and 2 below.

  As a bacterium that can be used in the present invention, a bacterium having the above-mentioned ability equivalent to the novel Stenotrophomonas genus NITE P-492 strain, for example, belonging to the genus Stenotrophomonas, having any one or more of the above-mentioned ability, And the same substrate-utilizing bacterium as NITE P-492 strain, belonging to the genus Stenotrophomonas, having any one or more of the above-mentioned ability, 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 P-492 strain is a mutant strain produced by artificial mutagenesis treatment, and is an agriculturally useful plant such as a cruciferous plant, asteraceae plant, a legume plant, a gramineous plant. A plant selected from the group consisting of solanaceous plants, liliaceae plants, and sericaceae plants, 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 isolating Stenotrophomonas 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 plant constituent parts such as roots, stems, leaves, etc. of agriculturally useful plants by 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 P-492 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 Stenotrophomonas bacteria by a conventional culture method such as shaking culture. As a medium for culturing, sugars such as glucose, sucrose, starch and dextrin are used as a carbon source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate are used 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, but examples include cruciferous plants, asteraceae plants, legumes, gramineous plants, solanaceous plants, liliaceous 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 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 grass family include cereals such as rice, wheat, barley, rye, triticale, pearl barley, sorghum, oat, corn, sugarcane, millet, and millet. Further, examples of grasses include feed or grass 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 Asteraceae plants include lettuce, sunny lettuce, garlic, chrysanthemum and the like.

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

  Examples of solanaceous plants include eggplant, tomato, pepper, shishito, chili pepper, potato, wolfberry, publica, 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 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 Gramineae plants include rice blast disease, white leaf blight, seedling blight, coat blight, and idiot disease.

  Examples of plant diseases of leguminous plants include gray mold disease, rust disease, powdery mildew.

  Examples of plant diseases of Asteraceae plants include mosaic disease, soft rot, rot, powdery mildew, downy mildew and the like.

  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.

  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.

  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.

  The pests of the grass family include, , Other nematodes, rice weevil, click beetles, scarab beetles, grasshoppers, scallop apples, white-winged beetles, ganganbo, tamabetsu, feeding on white-spotted plant, leafhopper, leafhopper, other planthopper, leafhopper, aphid This includes thrips and stink bugs.

  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 Asteraceae plants include Shiroshitayoto, Figukinuwaba, Tamanaginuwaba, Hosobasedakamokume, Nashikenmon, Artemisia, Ootobis Diadasaku, Nagame and the like.

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

  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). Microbiological method for imparting pest resistance, increasing yield, and promoting growth 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, increasing the yield of edible parts such as leaves, roots, rhizomes, and seeds for vegetables, seeds for cereals, and fruits for fruit trees. Means that.

  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 the application method to the plant include a seed coat, irrigation, application or spraying treatment to a young plant. 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, the method of the present invention relates to a bacterium belonging to the genus Stenotrophomonas and having the ability to symbiotically grow in an agriculturally useful plant body and impart disease resistance to the plant. The present invention provides a method for imparting pest resistance to agriculturally useful plants, including the step of artificially infecting plants.

  According to the second aspect of the method of the present invention, a bacterium belonging to the genus Stenotrophomonas and having the ability to symbiotically grow in an agriculturally useful plant and increase the yield of the plant is artificially introduced into the plant. A method is provided for increasing the yield of agriculturally useful plants, including the step of infecting.

  According to the third aspect of the method of the present invention, a bacterium belonging to the genus Stenotrophomonas and having the ability to coexist in an agriculturally useful plant body and promote the growth of the plant is artificially added to the plant. A method is provided for promoting the growth of agriculturally useful plants, including the step of infecting them.

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

  Examples of agriculturally useful plants are the plants specifically selected above, selected from the family Brassicaceae, Asteraceae, Legume, Gramineae, Solanum, Lily and Apiaceae. is there.

  A preferred bacterium is Stenotrophomonas sp. MYK101 (accession number NITE P-492), 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 Stenotrophomonas sp. MYK101 (Accession No. NITE P-492) 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 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 Stenotrophomonas 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 Stenotrophomonas sp. MYK101 (Accession Number NITE P-492)>
The kale grown in the field was collected, and after cutting, surface sterilization was performed by immersing 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, ground with 1 ml of sterilized physiological saline and an appropriate amount of sea sand, and transferred to a 1.5 ml tube. 100 μl of the supernatant was applied to Nutrient Agar medium and cultured at 30 ° C. for several days. By this operation, a single colony was isolated on the medium. The isolated strain was grown in the field and subjected to a pest resistance selection test to select and isolate the title strain.

[Example 2]
<Kale growth promotion or yield increase test>
Field cultivation test of kale inoculated with bacterial endophyte 1) Method Kale variety: Portuguese kale endophyte strain: NITE P-492 Inoculation date: May 25, 2007 Inoculation method: 1 × 10 ⁸ cells / ml suspension 500 ml of liquid / nursing box irrigation treatment
Date of planting of 4th leaf: Date of planting and method: June 6, 2007 Scale of hand-planting trial: 3 treatments per treatment area (1 square: 42 shares) About 126 shares in each treatment area No treatment area and no treatment Measurement item: NITE P-492 treatment group placed between the plots: Leaf size and yield Leaf size: The vertical and horizontal sizes of the leaf were measured and multiplied by these values to calculate the value.
Yield: Leaves were harvested until they exceeded 10 kg, and the harvest weight (fresh weight) per strain was calculated.

2) Results and discussion The results are shown in Tables 3 and 4. For the leaf size, 20 strains in each treatment area were measured. The numbers in parentheses are relative numbers when the non-inoculated section is 100.

  Regarding the growth of kale, the leaves were enlarged in the NITE P-492 inoculated area, and the growth was promoted. Regarding the yield, the harvest weight per strain increased in the NITE P-492 inoculated area (Tables 3 and 4).

[Example 3]
<Kale insect damage resistance test (1)>
Examination of inoculation effect in field cultivation of kale
1) Method Kale variety: Portuguese kale endophyte strain: NITE P-492 Inoculation date: May 25, 2007 Inoculation method: 500ml / seedling box irrigation treatment of 1 x 10 ⁸ cells / ml
Date of planting of 4th leaf: Date of planting and method: June 6, 2007 Scale of hand-planting trial: 3 treatments per treatment area (1 square: 42 shares) About 126 shares in each treatment area No treatment area and no treatment Measurement item with NITE P-492 treated treatment area between wards: Evaluation of the degree of damage caused by konaga Damage degree: 20 strains of each treated area were measured Visually evaluated the degree of damage to leaves per strain in five stages ( 0: Eating rate 0%, 1: Eating rate 1-20%, 2: Eating rate 21-40%, 3: Eating rate 41-60%, 4: Eating rate 61-80%, 5: Eating rate 81-100 %)

2) Results and discussion 20 strains in each treatment area were measured. The results are shown in Tables 5 and 6. The numbers in parentheses are relative numbers when the non-inoculated section is 100.

  As of July 26th, diamondback moths were present, but the degree of food damage decreased in the NITE P-492 strain inoculation zone (Tables 5 and 6).

[Example 4]
<Kale insect damage resistance test (2)>
Examination of inoculation effect in field cultivation of kale (snail)
1) Method Kale variety: Portuguese kale endophyte strain: NITE P-492 strain Seeding date: June 1, 2009 Inoculation date: June 13, 2009 Inoculation method: 1 x 10 ⁸ cells / ml The suspension was inoculated with 10ml / strain spray
Treatment planting date and method when the 4th leaf is deployed: June 15, 2009, hand-planting test scale: 1 treatment area 1 畝 each (1 畝: 17 shares) each treatment area about 17 shares Untreated and untreated Measurement items with NITE P-492 stock treatment ward placed between wards:
Survival strain rate: Kale measured the number of strains that remained without being killed because the plants were killed by snail damage.

2) Results and discussion The results are shown in Tables 7 and 8. The numbers in parentheses are relative numbers when the non-inoculated section is 100.

  At 55 days after sowing, there were many strains that were invaded by snails and were damaged by the snail, but the survival rate was high in the NITE P-492 inoculated area (Tables 7 and 8).

[Example 5]
<Komatsuna yield increase test>
Pot seedling cultivation test of Komatsuna inoculated with bacterial endophyte 1) Method Komatsuna variety: Summer Rakuten endophyte strain: NITE P-492 Inoculation method: Bacterial suspension cultured at seeding for 24 hours (viable cell count concentration 1 × 10 ⁸ 〜 10 ⁹ cells / ml) 100 μl / cell inoculation test scale: 10 strains in 1 treatment area, 3 replicates

2) Experimental method Culture soil and vermiculite mixed at 50:50 were filled in a cell tray, seeded with 3 Komatsuna seeds per cell, and inoculated with 100 µl / cell of NITE-P492 strain cultured for 24 hours. Cultivation was carried out in a glass greenhouse, and when the true leaves began to appear, one plant was thinned out per cell, and cultivation was continued until 4 or more true leaves were developed. Then, after cutting from the border and removing the foliage, dry weight was measured.

3) Results and discussion Three treatments were repeated in each treatment group. The results are shown in Table 9. The numbers in parentheses are relative numbers when the non-inoculated section is 100. Regarding the yield, dry matter weight increased by 14.4% in the NITE-P492 inoculated area (Table 9).

[Example 6]
<Examination of inoculation effect in field cultivation of Komatsuna>
1) Method prototype: Komatsuna
Endophyte strain: NITE P-492 inoculation date: December 28, 2012 (1 × 10 ⁸ cells / ml of microbial suspension)
Processing cultivation method: House cultivation test scale: 90cm × 200cm / treatment area Measurement item: Yield (fresh weight) measured

2) Results and discussion Table 10 shows the results. The numbers in parentheses are relative numbers when the non-inoculated section is 100.

  As a result of harvesting on March 4, 2013 and calculating the yield, the weight increased by 12.9% in the NITE P-492 inoculated area (Table 10).

[Example 7]
<Pot seedling cultivation test of radish inoculated with bacterium endophyte>
1) Materials and methods 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: 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 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 and Discussion Table 11 shows the results of measuring the dry matter weight of 10 stocks in each treatment area, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed an increase of 6.4% in weight compared to the untreated area.

[Example 8]
<Pot seedling cultivation test of bacterial endophyte inoculated lettuce>
1) Materials and methods Lettuce varieties: Green wave sowing date and inoculation date: July 31, 2013 Harvest date: August 23, 2013 Test scale: Cell tray 10-hole test method: Filling 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 25 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 and Discussion Table 12 shows the results of measuring the dry matter weight of each of the 10 treatment plants and converting them to the weight of one strain. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed a 5.2% weight increase compared to the untreated area.

[Example 9]
<Pot seedling cultivation test of bacterial endophyte inoculated rice>
1) Materials and methods Rice varieties: Japan seeding date and inoculation date: September 20, 2013 Harvest date: November 1, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Filling culture soil into cell tray 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 and Discussion Table 13 shows the results of measuring the dry matter weight of 10 stocks in each treatment group, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed a 14.3% weight increase compared to the untreated area.

[Example 10]
<Pot seedling cultivation test of bacterial endophyte inoculated tomato>
1) Materials and methods Tomato varieties: Hokin Fukuju Tomato sowing date and inoculation date: July 31, 2013 Harvest date: August 23, 2013 Test scale: Cell tray 10-hole Test method: Filling culture soil into cell tray 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 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 and Discussion Table 14 shows the results of measuring the dry matter weight of 10 stocks in each treatment area, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed an increase of 6.8% in weight compared to the untreated area.

[Example 11]
<Pot seedling cultivation test of green onion inoculated leek>
1) Materials and methods 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: Culture tray with cell tray The seeds were seeded in two seeds, and 100 μl of the bacterial solution (viable cell count concentration of 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 and discussion Table 15 shows the results of measuring the dry matter weight of 10 stocks in each treatment area, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, 7.0% weight increase was observed in the NITE P-492 treated area compared to the untreated area.

[Example 12]
<Pot seedling cultivation test of carrot inoculated bacterial carrot>
1) Materials and methods 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: Culture tray with cell tray The seeds were seeded in two seeds, and 100 μl of the bacterial solution (viable cell count concentration of 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 and Discussion Table 16 shows the results of measuring the dry matter weight of 10 stocks in each treatment area, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed a 4.7% weight increase compared to the untreated area.

[Example 13]
<Pot seedling cultivation test of soybean endophyte inoculated soybean>
1) Materials and methods Soybean varieties: Early edamame seeding date and inoculation date: September 20, 2013 Harvest date: October 11, 2013 Test scale: Cell tray 10 holes x 3 rows Test method: Culture soil into cell tray After filling, seeds were seeded two by two, and 100 μl each of the bacterial solution (viable cell count 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 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 and discussion Table 17 shows the results of measuring the dry matter weight of 10 stocks in each treatment area, converting them to the weight of 1 stock, and averaging the values in 3 rows. The numbers in parentheses are relative values when the untreated section is 100. As a result, the NITE P-492 treated area showed an increase of 11.2% in weight compared to the untreated area.

[Example 14]
<Disease resistance test for clubroot of cruciferous plants 1: Field cultivation test of cabbage inoculated with bacterial endophyte in soil contaminated with root-knot nematodes>
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 P-492 stock treatment area, untreated treatment area 100 each cabbage in five stages by visual inspection Evaluation was made (see FIG. 1).
2) Results and discussion Table 18 shows the average degree of damage in each treatment area. As a result, the damage decreased in the NITE P-492 inoculation area. The numbers in parentheses are relative values when the non-inoculation section is 100.

[Example 15]
<Disease resistance test against clubroot of cruciferous plant 2: Pot seedling cultivation test of Komatsuna inoculated with bacterial endophyte>
1) Materials and methods Komatsuna variety: Summer Rakuten sowing date: March 11, 2014 Inoculation method: NITE P-492 strain cultured for 24 hours at the time of sowing (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-P492 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 and discussion Three treatments were repeated in each treatment group. In the NITE-P492 inoculated area, the incidence of clubroot was reduced by 17% (Table 19).

  The present invention artificially infects agriculturally useful plants such as cruciferous plants with Stenotrophomonas genus bacteria to suppress the disease and pest damage of these plants, promote their growth, or increase leaf yield. It is useful above.

Claims (8)

Stenotrophomonas sp. MYK101 (accession number NITE P-492), and comprising the step of artificially infected with plant, imparting pest resistance to agriculturally useful plants. Stenotrophomonas sp. MYK101 (accession number NITE P-492), and comprising the step of artificially infected with plant, a method for increasing the yield of agriculturally useful plants. Stenotrophomonas sp. MYK101 (accession number NITE P-492), 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 cruciferous plants, asteraceae plants, legumes, gramineous plants, solanaceous plants, liliaceae plants and sericaceae plants. 2. The method according to item 1.   The method according to any one of claims 1 to 3, wherein the agriculturally useful plant is a cruciferous plant. An agriculturally useful plant microbial preparation containing Stenotrophomonas sp. MYK101 (Accession Number NITE P-492 ) as an active ingredient.   The microbial preparation according to claim 6, wherein the agriculturally useful plant is a plant selected from the family Brassicaceae, Asteraceae, Legume, Gramineae, Solanum, Lily and Apiaceae. . Stenotrophomonas sp. MYK101 (accession number NITE P-492 ).

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