JP5198015B2 - New uses of Herbaspirillum bacteria - Google Patents

Description

  The present invention relates to a method for artificially infecting a plant with Herbaspirillum 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 About.

  So far, pest control technology centered on chemical pesticides has contributed to efficient food security. However, in recent years, environmentally friendly agriculture with pesticide-free and reduced pesticides, including not only the efficiency of cultivation but also safe and secure areas, is desired, and pest control technology (for example, microbial pesticides) suitable for it is required. .

  The object of the present invention is to impart various favorable properties to agriculturally useful plants by biological means.

The present invention includes the following inventions.
(1) Insect resistance to cruciferous plants, including the step of artificially infecting cruciferous plants with bacteria that belong to the genus Herbaspirillum and have the ability to symbiotize in cruciferous plants and impart insect resistance to the plants A method of imparting sex.
(2) including the step of artificially infecting the cruciferous plant with a bacterium belonging to the genus Herbaspirillum and having the ability to symbiotically in the cruciferous plant and increase the yield of the leaf of the plant, How to increase yield.
(3) Insect resistance to gramineous plants, including the step of artificially infecting gramineous plants with bacteria that belong to the genus Herbaspirillum and have the ability to symbiotically exist in gramineous plants and impart pest resistance to the plants A method of imparting sex.
(4) Promote the growth of gramineous plants, including the step of artificially infecting gramineous plants with bacteria belonging to the genus Herbaspirillum and having the ability to symbiotically grow in the gramineous plants and promote the growth of the plants. Method.
(5) Grain yield in Gramineae plants, including the step of artificially infecting Gramineae plants with bacteria belonging to the genus Herbaspirillum and having the ability to symbiotically in Gramineae plants and increase grain yield in the plants. How to increase.
(6) Promote the growth of legumes, including the step of artificially infecting legumes with bacteria that belong to the genus Herbaspirillum and have the ability to symbiotically grow in the legumes and promote the growth of the plants Method.
(7) Leguminous plant implantation, including the step of artificially infecting leguminous plants with bacteria belonging to the genus Herbaspirillum and having the ability to symbiotically in the leguminous plant body and increase the number of plants of the plant How to increase the number.
(8) The number of root nodules in legumes, which includes the step of artificially infecting legumes with bacteria belonging to the genus Herbaspirillum and having the ability to symbiotically in the legumes and increase the number of nodules in the plants, How to increase.
(9) It belongs to the genus Herbaspirillum and includes a step of artificially infecting a lily family plant having a bulb with a bacterium that has the ability to symbioticly in the lily family plant having a bulb and increasing the yield of the bulb in the plant. A method for increasing the bulb yield in a lily family.
(10) The method according to any one of (1) to (9), wherein the bacterium is a novel genus Herbaspirillum (accession number NITE BP-193) or a mutant thereof.

  As used herein, “a novel bacterium belonging to the genus Herbaspirillum” refers to a bacterium belonging to the genus Herbaspirillum, which is disclosed in International Application PCT / JP2007 / 054624 and assigned accession number NITE BP-193.

  According to the present invention, by biological means, imparting pest resistance to cruciferous plants, imparting pest resistance to gramineous plants, promoting the growth of gramineous plants, grains in gramineous plants Increase yield, promote leguminous plant growth, increase the number of legumes, increase the number of nodules in legumes, increase the yield of cruciferous leaves Or it becomes possible to increase the yield of the bulb in a lily family plant.

1. Bacteria The bacterium that can be used in the present invention is a genus Herbaspirillum, the ability to symbiotically in the cruciferous plant and impart pest resistance to the plant, and the symbiotic plant in the gramineous plant. Ability to impart pest resistance to plants, Ability to symbiotically grow in grasses, Ability to symbiotic in grasses and increase grain yield in plants, Symbiosis in legumes The ability to promote the growth of the plant, the ability to grow symbiotically in the leguminous plant body, increase the number of buds of the plant, the ability to live in the legume plant body and increase the number of nodules in the plant, At least one selected from the group consisting of the ability to symbiotic in the plant and increase the yield of leaves of the plant, and the ability to symbiotic in the lily family having a bulb and increase the yield of bulbs in the plant Horn It not particularly limited as long as bacteria with a force. As a specific example, a novel bacterium belonging to the genus Herbaspirillum (Accession No. NITE BP-193) may be mentioned.

  A novel bacterium belonging to the genus Herbaspirillum (Accession No. NITE BP-193) is a novel bacterium isolated from rice by the present inventors and disclosed in International Application PCT / JP2007 / 054624. A new bacterium belonging to the genus Herbaspirillum has the substrate utilization ability shown in the following table. In addition to L-rhamnose, this strain has the ability to assimilate sodium ketoglutarate, m-erythritol, and diammonium sebacate. In addition to the substrates shown in the table, the same strain could not assimilate potassium gluconate, n-capric acid, adipic acid, DL-malic acid, sodium citrate, phenyl acetate and sucrose.

A partial base sequence of 16S rDNA of a novel bacterium of the genus Herbaspirillum is shown in SEQ ID NO: 1 in the sequence listing.
Further, the bacterium that can be used in the present invention is a bacterium having the same ability as that of a novel genus Herbaspirillum (accession number NITE BP-193), for example, a bacterium belonging to the genus Herbaspirillum and having the same carbon source as the above-mentioned novel genus Herbaspirillum. Examples include, but are not limited to, bacteria having assimilation ability and bacteria having 16S rDNA belonging to the genus Herbaspirillum and having at least a portion of the base sequence shown in SEQ ID NO: 1. Furthermore, a mutant strain produced by mutagenesis treatment of a new genus Herbaspirillum bacterium (Accession No. NITE BP-193), which is capable of symbiotic in the cruciferous plant body and imparting insect resistance to the plant, Ability to symbiotically grow in plants and impart pest resistance to plants, Ability to symbiotic in grasses and promote growth, Grain yields in grasses increase in plants Ability to symbioticly in the leguminous plant and promote the growth of the plant, ability to symbiotic in the leguminous plant and increase the number of arrivals of the plant, The ability to increase the number of nodules, the ability to symbiotic in the Brassicaceae plant and increase the yield of the leaves of the plant, and the ability to symbiotic to the Lilyaceae plant having a bulb and increase the yield of bulbs in the plant, From the group of Mutants having at least one capability is-option can also be suitably used in the present invention. Among such mutant strains, it belongs to the genus Herbaspirillum and has the same carbon source assimilation ability as described above, or a bacterium belonging to the genus Herbaspirillum, and at least a part of the base sequence shown in SEQ ID NO: 1. Bacteria having 16S rDNA contained in are preferred. The mutagenesis treatment can be performed using any suitable mutagen. Here, the term “mutagen” has a broad meaning and should be understood to include not only a drug having a mutagenic effect but also a treatment having a mutagenic effect such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other effect A typical mutagen can also be used.

  The bacteria used in the present invention can be cultured under normal conditions by a normal 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., inorganic salt, potassium 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.

  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. Alternatively, a bacterial culture solution adsorbed on a porous adsorbent such as activated carbon powder, diatomaceous earth, or talc and dried can be used. 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 alone for the use of the present invention as the above-mentioned culture medium, high-concentration product or dried product, but in the same form as a normal microbial preparation (for example, powder, (Forms of wettable powders, emulsions, liquids, flowables, coatings, etc.) can also be used. 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. Giving pest resistance to cruciferous plants, increasing leaf yield of cruciferous plants One form of the present invention belongs to the genus Herbaspirillum, and symbioticly exists in cruciferous plants to impart pest resistance to the plants. The present invention relates to a method for imparting insect resistance to a cruciferous plant, which comprises the step of artificially infecting the cruciferous plant with a bacterium having the ability to act.

  Another aspect of the present invention includes the step of artificially infecting a cruciferous plant with a bacterium belonging to the genus Herbaspirillum and having the ability to symbiotically in the cruciferous plant and increase the yield of the leaves of the plant, It relates to a method for increasing the yield of cruciferous leaves.

  In these forms, the cruciferous plants include rape, turnip, chingensai, nozawana, mustard, Takana, Kobutana, mizuna, callaby, arugula, watercress, taasai, cauliflower, cabbage, kale, Chinese cabbage, komatsuna, Japanese radish, Japanese radish, broccoli, and medicinal cabbage. , Horseradish, horseradish, and Arabidopsis thaliana.

  Examples of insect damage include feeding by a diamondback moth, a white butterfly, a white-tailed butterfly, a cabbage moth, a cabbage moth, a red-tailed moth, a weevil, a leafhopper, a hornbill beetle, a hornbill beetle, a weevil, aphids, and a thrips.

  Examples of the method of applying the bacterium or the composition containing the same to these cruciferous plants in these embodiments include a seed coat, a method of irrigating, applying, or spraying 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 sowing in the field, such as at the time of sowing and seedling raising. Moreover, high expression of the effect can be expected by spraying the plants during field cultivation.

3. Giving pest resistance to grasses, promoting growth, increasing grain yield One form of the present invention belongs to the genus Herbaspirillum and is the ability to symbioticly exist in grasses to impart pest resistance to the plants. The present invention relates to a method for imparting pest resistance to gramineous plants, including the step of artificially infecting gramineous plants with bacteria having

  Another aspect of the present invention includes a step of artificially infecting a gramineous plant with a bacterium belonging to the genus Herbaspirillum and having the ability to coexist in the gramineous plant and promote the growth of the plant. The present invention relates to a method for promoting plant growth.

  Another aspect of the present invention includes a step of artificially infecting a gramineous plant with a bacterium belonging to the genus Herbaspirillum and having the ability to symbiotically grow in the gramineous plant and increase grain yield in the plant. The present invention relates to a method for increasing grain yield in a family plant.

  Grains such as rice, wheat, barley, rye, triticale, pearl barley, sorghum, oats, corn, sugar cane, millet, millet, etc. can be mentioned as the gramineous plant in these forms. The grasses further include feed or grass such as buckwheat, buffalo grass, Bermuda grass, weeping grass, centipede grass, carpet grass, dalice grass, Kikuyu grass, and St. Augustine grass. Most preferred is rice.

  Insect damages in grasses include Drosophila, Nikaameiga, Ichimongiseseri, Knotomoiga, Ineyotou, Awayoto, Sujiriyotou, Futobiboyaga, Inebobiso hamushi, other potato beetles, rice flies, rice leaf moths, rice leaf moths, Other nematodes, rice weevil, click beetles, scarab beetles, grasshoppers, scallop apples, white-winged beetles, gangambo, scallops, feeding on white planthoppers, leafhoppers, leafhoppers, other planthoppers, leafhoppers, leafworms, beetles And soup by stink bugs.

  Examples of the method for applying the bacterium or the composition containing the same to the grass family in this embodiment include a seed coat, a method of irrigating, applying, or spraying the seedling. 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 sowing in the field, such as at the time of sowing and seedling raising. Moreover, high expression of the effect can be expected by spraying the plants during field cultivation.

4. Growth promotion of leguminous plants, increase in the number of arrivals, increase in the number of root nodules One form of the present invention is a bacterium belonging to the genus Herbaspirillum and having the ability to promote symbiosis within the legumes. The present invention relates to a method for promoting the growth of legumes, which comprises the step of artificially infecting legumes.

  Another aspect of the present invention includes the step of artificially infecting leguminous plants with bacteria belonging to the genus Herbaspirillum and having the ability to symbiotically grow in the legumes and increase the number of arrivals of the plants. The present invention relates to a method for increasing the number of arrivals of legumes.

  Another aspect of the present invention includes the step of artificially infecting legumes with a bacterium belonging to the genus Herbaspirillum and having the ability to symbiotically in the legumes and increase the number of nodules in the plants. The present invention relates to a method for increasing the number of nodules in a family plant.

  In these forms, examples of legumes include soybean, azuki bean, groundnut, kidney bean, pea, hana bean, broad bean, cowpea, chickpea, mung bean, lentil, lentil, and banbara bean. Most preferred is soybean.

  Examples of the method for applying the bacterium or the composition containing the same to the leguminous plant in this embodiment include a seed coat, a method of irrigating, applying, or spraying the seedling. 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 sowing in the field, such as at the time of sowing and seedling raising. Moreover, high expression of the effect can be expected by spraying the plants during field cultivation.

5. Increasing the yield of bulbs in lily family One form of the present invention is a bacterium belonging to the genus Herbaspirillum and having the ability to live in lily family plants having bulbs and increase the yield of bulbs in the plant. It relates to a method for increasing the yield of bulbs in a lily family plant, comprising the step of artificially infecting a lily family plant having

  In this form, the lily family plants having bulbs include onion, rakkyo, garlic, leek, chives and lily. Most preferred is onion.

  Examples of the method of applying the bacterium or the composition containing the bacterium in this embodiment to a lily family include a seed coat, a method of irrigating, applying, or spraying 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 sowing in the field, such as at the time of sowing and seedling raising. Moreover, high expression of the effect can be expected by spraying the plants during field cultivation.

Golden moth feeding test
1． To elucidate the mechanism of insect resistance induction induced by the target endophyte, the resistance of Herbaspirillum sp. NITE BP-193 is clarified using Arabidopsis, a model plant of disease resistance.

2． Method <br/> Test strain: NITE BP-193 strain
Inoculation concentration: 5 × 10 ⁶ cell / seed (converted to 1 × 10 ⁸ cell / ml) Control group: Non-inoculated plant: Arabidopsis Col-0
Target pests: Golden moth feeding time: 4 hours Number of samples processed: 15 samples in each treatment area

3． Experimental Method Arabidopsis seeds were sterilized by treatment with 70% ethanol for 20 seconds and 1% aqueous hypochlorous acid for 5 minutes, and then washed three times with sterilized distilled water for 20 minutes. About 20 seeds of sterilized seeds are sown in a plastic container (5 x 5 x 5 cm) containing horticultural soil (Kureha) that has been sterilized by autoclaving (121 ° C, 40 minutes). Cultivated under conditions of 60%, 16 hours light / 8 hours dark. Thereafter, the test strain was inoculated at 1 × 10 ⁸ cell / ml on the 10th day, and the cut leaves of the plant body were fed to the 3rd instar larvae 20 days later, and the feeding rate was measured.

  Moreover, the endophytic infection rate of the plant body at this time was measured. Feeding tests were performed on individuals with confirmed infection.

Four. result

There was an individual whose infection was not confirmed in the endfight treatment section (table above).
From this study, it was clarified that inoculation of Arabidopsis thaliana with Herbaspirillum sp.

About rice insect resistance
Examination of inoculation effect in field cultivation of rice
1.1) Drosophila damage, test method <br/> Rice varieties: Hoshinoyume endophyte strain: NITE BP-193 Inoculation date: May 26, 2005 Fixed planting date: May 28, 2005 Field: Pesticide-free cultivation Test scale: 57.7 m ² , 3 seedling boxes (6 plants, about 1,340 plants)

1.2) Stink bug damage, test method <br/> Rice varieties: Nanatsuboshi, Kirara 397
Endophyte strain: NITE BP-193 Strain inoculation date: Nanatsuboshi May 21, 2006, Kirara 397 May 23, 2006 (1 x 10 ⁸ cells / ml of bacterial suspension 500 ml (Box irrigation treatment)
Date of planting: Nanatsuboshi May 24, 2006, Kirara 397 May 26, 2006 Field: Pesticide-free cultivation Test scale: 76.8 m ^{2 for} each treatment area (8 plants, about 1,790 plants) )

2) Results and discussion

The number of leafworms damaged in the duck beetle decreased in the NITE BP-193 test plot.
The number of grains damaged by stink bugs, the percentage of speckled rice, and the percentage of speckled (small) showed a decreasing trend in the NITE BP-193 test area.

About rice growth promotion, yield increase
1. Field cultivation test of rice inoculated with bacteria
1) Method <br/> Rice variety: Kirara 397
Endophyte strain: NITE BP-193 Inoculation date: May 23, 2006 (1 x 10 ⁸ cells / ml of suspension in 500 ml / nursing box irrigation)
Planting date: May 30, 2006 Field: Organic fertilizer, pesticide-free cultivation Test scale: Approximately 60 plants in each treatment area

2) Results and discussion 20 strains in each treatment area of the test rice were measured. The numbers in parentheses are relative numbers when the non-inoculated section is 100.

  Regarding the growth of rice, the plant height and the number of stems were increasing during the NITE BP-193 inoculation, and the growth was promoted. Regarding harvesting, drought increased in NITE BP-193 inoculation area.

2. Examination of the inoculation effect in the field cultivation of endfight inoculated rice
1) Method <br/> Rice varieties: Nanatsuboshi, Kirara 397
Endophyte strain: NITE BP-193 Strain inoculation date: Nanatsuboshi May 21, 2006, Kirara 397 May 23, 2006 (1 x 10 ⁸ cells / ml of bacterial suspension 500 ml (Box irrigation treatment)
Date of planting: Nanatsuboshi May 24, 2006, Kirara 397 May 26, 2006 Field: Pesticide-free cultivation Test scale: 76.8 m ^{2 for} each treatment area (8 plants, about 1,790 plants) )
Two uninoculated wards were prepared and the average was calculated.

2) Results and discussion

The initial growth on the 10th day after transplantation showed a tendency for the plant height to be slightly lower in Kirara 397 inoculated with Endfite NITE BP-193. After that, the number of stems during growth tended to be higher in the NITE BP-193 inoculation group than in the non-inoculation group, and it was confirmed that the tillering was promoted.
The yield increased for both Nanatsubo and Kirara 397 in the NITE BP-193 inoculation group.

Promoting soybean growth, increasing yield, and increasing nodulation
1. The purpose of this study was to investigate the effects of NITE BP-193, a new bacterium belonging to the genus Herbasprillum, isolated from target- grown rice on growth promotion and yield increase in soybean.

2. Experimental method
2-1. Prototype product <br/> Soybean variety: Toyohomare 200 treatment areas
2-2. Farm field <br/> Farm field first year
2-3. Test bacteria
NITE BP-193 (Herbasprillum genus)
2-4. Test Method Soybeans were sown in the field and cultivated in alleys. When there were about 1 to 3 true leaves, the bacterial solution prepared to 10 ⁸ CFU / ml was sprayed using a sprayer. Under normal management, growth and yield surveys were conducted during cultivation (57 days after sowing) and during the harvest period (135 days after sowing). The growth survey items were the number of leaves, stem length, and the number of established plants. In the yield survey, the number of arrivals was measured. The number of nodules was also measured.

3. Results
3-1. Growth Survey Results Table 11 shows the results of growth studies on the 57th day and 135th day after sowing. From the results on the 57th day, the leaf number and stem length of the NITE BP-193 inoculated group showed growth promotion over the control group. In addition, in the harvest period survey results on day 135, the NITE BP-193 treated group showed growth promotion over the control group in all items.

  In addition, Table 12 shows the number of root nodules and the number of landings. NITE BP-193 treatment group had more pods and nodules than control group.

About increase in kale harvest
1. Examination of inoculation effect in field cultivation of endfight inoculated kale
1) Method <br/> Breeding: Autologous breeding endophyte strain: NITE BP-193 Inoculation date: May 18, 2006 (1 x 10 ⁸ cells / ml of suspension in 500ml / nursery box irrigation processing)
Planting date: June 15, 2006, Hand-planted field: Sand, organic fertilizer, pesticide-free cultivation Test scale: More than 100 strains in each treatment area

NITE BP-193接種区で無接種区と比較してより生育が促進され、収量が増加した。 2) Results and discussion

In the NITE BP-193 inoculation group, the growth was further promoted and the yield increased compared to the non-inoculation group.

Onion yield increase
1. Examination of inoculation effect in field cultivation of endfight inoculated onion
1, Method <br/> Onion variety: North maple 2000
Endophyte strain: NITE BP-193 Inoculation date: May 2, 2006 (1 x 10 ⁸ cells / ml of bacterial suspension 500 ml / nursing box irrigation treatment)
Planting date: May 4-7, 2006 Field: Conventional cultivation test scale: 448 strains (224 strains x 2) in each treatment area

2) Results and discussion

In Northern Maple 2000, the leaf sheath diameter tended to increase in the NITE BP-193 inoculation group. Regarding the plant height, a slightly large tendency was seen in the NITE BP-193 inoculation area, but the level was almost the same.
As for the harvest, the size of the balls increased in the NITE BP-193 inoculation zone, and the yield increased.

Claims (9)

A method for imparting pest resistance to a cruciferous plant, comprising a step of artificially infecting the cruciferous plant with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for increasing the yield of a cruciferous plant leaf, comprising a step of artificially infecting the cruciferous plant with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for imparting pest resistance to a gramineous plant, comprising a step of artificially infecting the gramineous plant with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for promoting the growth of gramineous plants, including the step of artificially infecting gramineous plants with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for increasing grain yield in gramineous plants comprising the step of artificially infecting gramineous plants with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for promoting the growth of legumes, comprising a step of artificially infecting legumes with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for increasing the number of arrivals of legumes, comprising a step of artificially infecting legumes with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for increasing the number of nodules in a leguminous plant, comprising a step of artificially infecting the leguminous plant with a novel genus Herbaspirillum (accession number NITE BP-193) . A method for increasing the yield of bulbs in a lily family plant, comprising artificially infecting a lily family plant having a bulb bulb with a novel genus Herbaspirillum (accession number NITE BP-193) .