JP5074866B2 - Methods to inhibit legume feeding by pests - Google Patents

Description

  The present invention relates to a novel bacterial endophyte, a method using this to inhibit the feeding of legumes by pests, and a plant produced by this method.

  In today's crop cultivation, chemically synthesized pesticides that exhibit excellent pest control effects play an important role in efficient food production, enabling high-quality crops to be harvested in high yields. In recent years, however, environmental problems on a global scale have become serious, and there is a demand for a shift to environmentally friendly agriculture by reducing the use of chemically synthesized pesticides. Therefore, development of a new control technology (for example, microbial pesticide) that enables safe and safe crop supply and environmental conservation agriculture is desired. Plants are originally known to induce immune systemic resistance against various diseases and pests. As one of them, inductive resistance is known. The present invention is a technique for artificially inducing the inherent ability of such a plant and utilizing it for agriculture.

  The present invention provides an agriculturally useful legume plant with pest feeding inhibition, promotes growth, increases the number of buds, increases the number of nodules, and provides means for increasing the yield. With the goal.

The present invention includes the following inventions.
(1) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to give pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of arrivals of the plant is increased. Confer legume feeding inhibition on legumes, including the step of artificially infecting legumes with the ability, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant how to.
(2) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to impart pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of plants that grow A method of promoting leguminous plant growth comprising the step of artificially infecting legumes with bacteria having the ability, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant.

(3) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to impart pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of plants that grow A method for increasing the number of legumes growing, comprising the step of artificially infecting legumes with a bacterium having the ability, an ability to increase the number of nodules in the plant, and an ability to increase the yield of the plant .
(4) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to give pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of plants that grow A method for increasing the number of nodules in a legume, comprising the step of artificially infecting a legume with a bacterium having the ability, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant.

(5) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to impart pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of plants that grow A method of increasing legume yield, comprising the step of artificially infecting legumes with bacteria having the ability, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant.
(6) The method according to any one of (1) to (5), wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or a mutant strain thereof.
(7) The method according to any one of (1) to (5), wherein the bacterium is Methylobacterium sp. M143R1 (Accession No. NITE P-96) or a mutant strain thereof.

(8) It belongs to the genus Variovorax or Methylobacterium, and the ability to symbiotically exist in the leguminous plant body to impart pest feeding inhibition to the plant, the ability to promote the growth of the plant, and the number of plants that grow A microbial preparation for legumes, comprising as an active ingredient a bacterium having an ability, an ability to increase the number of nodules in the plant, and an ability to increase the yield of the plant.
(9) The microorganism preparation according to (8), wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or a mutant strain thereof.
(10) The microorganism preparation according to (9), wherein the bacterium is Methylobacterium sp. M143R1 (Accession No. NITE P-96) or a mutant strain thereof.
(12) (11) Ability of Variovorax sp. M30P3 (Accession No.NITE P-95) or a mutant thereof to coexist in the leguminous plant body and impart pest feeding inhibition to the plant, growth of the plant A mutant having the ability to promote the growth of plants, the ability to increase the number of plants settled, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant.

(12) Methylobacterium sp. M143R1 (Accession No. NITE P-96) or a mutant thereof, symbiotic to the body of legumes and imparting pest feeding inhibition to the plant, promoting the growth of the plant A mutant having the ability, the ability to increase the number of plants settled, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant.
(13) Variovorax sp. M30P3 (Accession No. NITE P-95), Methylobacterium sp. M143R1 (Accession No. NITE P-96), and mutants thereof, which are symbiotic in the leguminous plant and are harmful to the plant. Has the ability to impart feeding inhibition, the ability to promote the growth of the plant, the ability to increase the number of plants that grow, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant A leguminous plant in which at least one selected from the group consisting of mutants has been artificially infected.

  According to the present invention, the ability to symbiotically exist in the leguminous plant body to impart pest feeding inhibition to the plant, the ability to promote the growth of the plant, the ability to increase the number of plants growing, the number of nodules in the plant Having the ability to increase the yield of the plant and the ability to increase the yield of the plant, a method for imparting pest feeding inhibition to legumes using the bacterium, a method for promoting the growth of legumes, legume Provided are a method for increasing the number of plant settlements, a method for increasing the number of nodules in legumes, a method for increasing the yield of legumes, and legumes produced by the method.

  As a leguminous plant to which resistance to feeding by pests is imparted by the infection of the present invention, growth is promoted, the number of buds is increased, the number of root nodules is increased, and the yield is increased, for example , Soybean, azuki bean, vine azuki bean, broad bean, green beans, peas, cowpea, peanut, mung bean, lotus root, alfalfa, clovers, vetches. In particular, soybean is preferable.

  The present invention also relates to a leguminous plant that has been artificially infected with the bacterium of the present invention, has high insect pest feeding inhibition, is easy to grow, has a large number of buds, has a large number of nodules, and has a high yield.

  The pests that can inhibit the intake of legumes according to the present invention include lepidoptera, aphids, leafhoppers, stink bugs, crickets, leaf beetles, weevil, weevil, bark beetles, scarabs, stag beetles, Examples include insects belonging to planthoppers, grasshoppers, locusts, flying beetles, thrips, and the like. Inhibition of feeding according to the present invention is particularly effective against insects belonging to the order Lepidoptera, particularly larvae thereof. For example, Sedokukuga), Higgariidae (for example, Obihitori, Kiharagomadarahitori, Shilohitori) or Yaga (for example, Hammonyotou, Ezochairoyotou, Shiroshitayoto, Futaobikiyotou, Tampokiyotou, Kushirokiyotou, Inagitoyo, Insects, Insect It is effective against this.

The bacterium that can be used in the present invention belongs to the genus Variovorax or Methylobacterium, is capable of symbiotic in the leguminous plant body and imparts pest feeding inhibition to the plant, the ability to promote the growth of the plant, It is not particularly limited as long as it is a bacterium having the ability to increase the number of plants settled, the ability to increase the number of nodules in the plant, and the ability to increase the yield of the plant. Specific examples include Variovorax sp. M30P3 (Accession number NITE P-95), and Methylobacterium sp.
M143R1 (Accession number NITE P-96). In addition, a bacterium having the same ability as Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96), for example, belonging to the genus Variovorax and shown in Example 1 Bacteria having the same ability to assimilate carbon sources as M30P3, bacteria belonging to the genus Variovorax, having 16S rDNA containing at least a part of the base sequence shown in SEQ ID NO: 1, and belonging to the genus Methylobacterium, shown in Example 2 Examples include, but are not limited to, bacteria having the same ability to assimilate carbon sources as Methylobacterium sp. M143R1, and bacteria belonging to the genus Methylobacterium and having 16S rDNA containing at least a portion of the base sequence shown in SEQ ID NO: 2. Variovorax sp.M30P3 (Accession No.NITE P-95) or Methylobacterium sp. M143R1 (Accession No.NITE P-96) is a mutant strain produced by mutagenesis treatment, Ability to impart pest feeding inhibition to a plant, ability to promote the growth of the plant, ability to increase the number of plants planted, ability to increase the number of nodules in the plant, and increase the yield of the plant A mutant having an ability can also be preferably used in the present invention. 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. Examples thereof include synthetic or natural media containing salts containing phosphorus, potassium, manganese, magnesium, iron, etc., such as monoiron.

  The present invention also contains a bacterium of the present invention as an active ingredient, imparts pest feeding inhibition to leguminous plants, promotes growth, increases the number of arrivals, increases the number of root nodules, and yield The present invention relates to a microbial preparation for legumes to increase For the production of a microbial preparation, the bacterial culture solution of the present invention can be used as it is, but preferably the culture solution is subjected to membrane separation, centrifugation, filtration separation, etc. for the purpose of enhancing the effect. Separate high concentrations can be used.

  As the microbial preparation of the present invention, a dried culture solution of the bacterium of the present invention can also be used. Moreover, what dried the culture medium of this invention by making it adsorb | suck to porous adsorbents, such as activated carbon powder, diatomaceous earth, and a talc, 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.

  The bacterium of the present invention can be used alone in the present invention as the above-mentioned culture solution, high-concentration product, or dried product, but in the same form as a normal microbial preparation in combination with other optional components (for example, a powder agent) , Wettable powders, emulsions, liquids, flowables, coatings, etc.). Examples of optional components that can be used in combination include a solid carrier and an adjuvant.

  Infection of the bacterium of the present invention with a plant is preferably carried out at the seed, seedling stage, or vegetative growth stage of the plant.

  The method of applying the bacterium of the present invention to a plant includes spraying, irrigation, pickling, application to the plant, contact with artificially wounds, injection with a syringe, mixing with soil, hydroponics Mixing into sand, etc., and spraying like sand blasting can be considered. In addition, tissue culture plants can be mixed into the medium.

Selection of Variovorax sp. M30P3 (Material)
Legumes and cruciferous plants were collected around the Asagiri plateau in Nagano Prefecture, Shizuoka Prefecture, and around Ishigaki Island in Okinawa Prefecture. Of the obtained plants, insect resistance evaluation tests and endophytic separation were performed using leguminous 52 strains and 47 cruciferous plants collected around Nagano Prefecture and Asagiri Plateau in Shizuoka Prefecture. Lepidoptera (Spodoptera litura) were used as the lepidopteran to evaluate feeding inhibition.

(Method)
Evaluation of the feeding inhibition effect was performed by a leaf disk test. The leaf of the plant body was cut into a diameter of 12 mm with a cork borer to make a leaf disk. For those that are difficult to cut out, such as small leaves, several treatments were used to make the area of the same level. Lotus japonicus used the 1st instar larvae from the first day to the fourth day of hatching. 250 pieces of Spodoptera litura were added to 16 leaf discs and fed for about 2 days in the dark at 25 ° C. In the evaluation, those with a food intake level of less than 5% were regarded as intensity inhibition, and those with less than 70% as moderate inhibition. This was the primary screening. Next, only plants that showed moderate or higher inhibition in the primary screening were collected, and the secondary screening was performed in the same manner.

  Isolation of endophytes from legumes and cruciferous plants, which were selected after secondary screening and showed an antifeeding effect on Spodoptera litura, was attempted. Plants were surface sterilized using 70% ethanol and 2.5% hypochlorous acid and isolated on NA medium and PDA medium.

(result)
From the primary screening, 5 strains showing strong inhibition in 52 legumes and 13 strains showing moderate inhibition were selected. Furthermore, when secondary screening was carried out using only the selected strains, 3 strains showed strong inhibition and 6 strains showed moderate inhibition (Table 1).

  The Brassicaceae was also selected in the same manner. In the primary screening, 1 strain with strong inhibition and 8 strains with moderate inhibition were selected from 47 strains, and 3 strains with moderate inhibition were selected in the secondary screening (Table 2).

  In addition, bacteria and filamentous fungi were isolated as endophytes from 7 selected plants in the legume family and from 1 plant in the Brassicaceae family.

(Verification test 1)
(the purpose)
From the endophytes separated as described above, those that can coexist with soybean and give host soybean resistance to feeding by pests are selected.

(Materials and methods)
Bacteria (M30N2, M30P1, M30P2, M30P3, M30P5, M71N1, and M72N4) isolated from leguminous and cruciferous native plant lines in which feeding inhibition was observed were inoculated into soybean cultivar Enrei. The inoculation method is as follows. In other words, soybean seed sterilized with 70% EtOH for 20 seconds and 2.5% sodium hypochlorite aqueous solution for 5 minutes was inoculated with 1 × 10 ⁸ cells / seed of the bacterial suspension, and the seeds after inoculation were cultivated. The individual was obtained. Three individuals were prepared for each fungus. As a control, a non-inoculated bacterium group was also prepared and treated in the same manner.

  Cultivation of the inoculated individual was carried out by aseptically seeding and cultivating the above inoculated seeds in a magenta box filled with 200 mL of soil sterilized at 121 ° C. for 30 minutes, then opening the above-ground part, It was grown in an incubator with sterile water for 10 days, finally transplanted into a pot, and further cultivated in a pot.

  10 individuals / soybean individuals of Spodoptera litura were inoculated into individuals on the 30th day of pot cultivation, and the degree of feeding damage was measured 20 days after the inoculation. During the feeding test, the cultivation pots were placed next to each other so that the Japanese clover can move freely between plants. As a follow-up test, we tried to inoculate M30P1, M30P2, and M30P3 with a non-woven fabric so that the larvae would not escape to the outside, and inoculated 20 Spodoptera litura larvae / 20 soybean individuals.

  The eating damage level was measured according to the following procedure. That is, the visual index (1: feeding area 10% or less; 2: feeding area 10-30%; 3: feeding area 30-50%; 4: feeding area 50-70%; 5: feeding area (70% or more) was counted, and the index was multiplied by the number of diseased leaves, and the total was calculated as the degree of eating damage.

(result)
When the level of each eating damage was tested by the least significant difference method from the level of eating damage of the non-inoculated individuals, the degree of eating damage was significantly reduced at the 1% level for M30P1, M30P2 and M30P3 inoculated individuals. Results were obtained (Table 3 and Figure 1). Since there are 24 plants in the entire test area, a total of 240 Spodoptera litura were released, but the number of larvae that had inhabited on the plant at the end of the feeding test (day 20) began. It decreased to 4.1% (10 individuals) of the time. There were no larvae in the three inoculated individuals of M30P1, M30P2 and M30P3 whose feeding was significantly suppressed. In a test where the larvae were covered with a non-woven fabric to prevent them from escaping, the M30P2-inoculated individuals were the least affected by feeding, but no significant difference was obtained.

(Verification test 2)
(the purpose)
The supplementary test was conducted for the three strains M30P1, M30P2 and M30P3 selected above.

(Materials and methods)
Endfight: M30P1 strain, M30P2 strain and M30P3 strain Control group: Non-inoculated individual soybean variety: Enrei insect: Lotus japonicus 1-year-old insect cultivation: Pot cultivation, 4 individuals / pot / treatment test: Lotus field 1-year-old on the 60th day of pot cultivation Ten insects / soybean individuals were inoculated and fed in a greenhouse.
Measurement method: Evaluated as damage index.
Damage index: Index (1: Feeding area 10% or less; 2: Feeding area 10-30%; 3: Feeding area 30-50%; 4: Feeding area 50-70%; 5: Feeding area 70 % Or more) was visually counted, and the damage index was calculated by the following formula.
Damage index = 100 x [1 x (number of leaves of damage index 1) + 2 x (number of leaves of damage index 2) + 3 x (number of leaves of damage index 3) + 4 x (number of leaves of damage index 4) + 5 x (damage Number of leaves with index 5)] / [5 × (total number of leaves)]

(result)
As a result of the verification test, it was proved that soybean individuals inoculated with the M30P3 strain among the three strains significantly suppressed the degree of damage (Table 4).

  At this time, the whole test area was damaged by spider mites, but the soybean individuals inoculated with M30P3 suffered less damage.

(Identification of M30P3 strain)
The M30P3 strain was identified using a biolog system (microbe identification system). As a result, Variovorax paradoxus (Gram negative, non-enteric bacteria) showed the highest homology.

  Table 5 summarizes the carbon sources confirmed to be utilized in the M30P3 strain and Variovorax paradoxus ATCC 17713 strain. As shown in Table 5, the carbon sources that can be assimilated are different.

The base sequence of 16S rDNA of M30P3 strain is shown in SEQ ID NO: 1.
The strain was cultured in Nutrient Broth, and genomic DNA was isolated from the cells. Using the isolated DNA as a template, the base sequence of about 500 bp in the first half of the 16S rDNA region was determined by the dye primer method (SEQ ID NO: 1). Using the homology search program FASTA, the determined base sequence and DDBJ / EMBL / GenBank
A homology search with an international nucleotide sequence database was performed.

  As a result of homology search, it was revealed that the base sequence of 16S rDNA of M30P3 strain showed high homology with the base sequence of 16S rDNA of Variovorax paradoxus ATCC17713 strain (same strain as standard strain DSM66). FIG. 2 shows the results of comparison of the 16S rDNA base sequence between the M30P3 strain and the Variovorax paradoxus DSM66 strain using the base sequence multiple alignment program CLASTAL W. The base sequences that can be compared were 487 bp, of which 7 bases were different. That is, the homology was about 98.6%.

  From the above results, it was concluded that the M30P3 strain is a new strain belonging to the genus Variovorax. The M30P3 strain has been deposited as a Variovorax sp. M30P3 at the Patent Microorganism Depositary Center for Product Evaluation and Technology on March 31, 2005 (Accession Number NITE P-95).

Selection of Methylobacterium sp. M143R1 (Materials and Methods)
In order to search for endfights that are highly resistant to vegetable pests, legumes and rapes were found in 6 locations in Ishigaki Island, 6 locations in Yonaguni Island, and 4 locations in Iriomote Island from March 4 to 7, 2003. We collected plant native plants.

  A plant having resistance to Lepidoptera was selected from the collected plants. In the same manner as in Example 1, plants having resistance to feeding were screened in two stages using Spodoptera litura.

  Next, we tried to isolate endophyte from plants resistant to feeding by Spodoptera litura. As possible, stems and leaves were used for the plants that could not use the ground due to the fact that there was only one plant. At the time of isolation, we tried to isolate various strains with different assimilation ability using three kinds of agar media, NA, PDA and Reny. Surface sterilization was performed by immersing in 70% ethanol for 10 seconds and then in 2.5% hypochlorous acid for 8 to 10 minutes. Then, it grind | pulverized in the mortar, and applied the grinding liquid to the agar medium. Single colonies were further isolated from the grown colonies. Thereafter, the same strain was selected and removed by RAPD PCR.

(result)
As a result of plant collection in Okinawa Prefecture, 75 plants estimated to be leguminous from the form and 18 plants estimated to be cruciferous were collected. As a result of the feeding test using the first instar larvae, the strains showing resistance with a feeding rate of 5% or less show moderate resistance from legumes and cruciferous plants to 1 plant each, about 50% Four plants were selected from the legume family.

  As a result of attempts to isolate endophyte, 47 bacteria were isolated from 5 legumes and 1 filamentous fungus was isolated from 1 cruciferous plant.

(Verification test 1)
(the purpose)
From the endophytes separated as described above, those that can coexist with soybean and give host soybean resistance to feeding by pests are selected.

(Method)
The endophyte isolated as described above was inoculated into soybean (Glycine max cv. Enrei), and the establishment of the colony was evaluated. Bacterial endophytes were inoculated one by one into sterilized soybean seeds. As a rule, soybeans in each inoculation area are 6 individuals (some of which 6 individuals could not be tested), and on the 40th to 50th day of seeding, 25 individuals were released immediately after hatching. The intake of soybean leaves was observed. The feeding test was conducted in a state where the cultivation pot was placed adjacent to each other and the Japanese sardine was able to move freely between plants. The evaluation of the results was carried out by calculating the damage index in the same manner as the damage verification evaluation of the three strains M30P1, M30P2 and M30P3.
Damage index: Index (1: Feeding area 10% or less; 2: Feeding area 10-30%; 3: Feeding area 30-50%; 4: Feeding area 50-70%; 5: Feeding area 70 % Or more) was visually counted, and the damage index was calculated by the following formula.
Damage index = 100 x [1 x (number of leaves of damage index 1) + 2 x (number of leaves of damage index 2) + 3 x (number of leaves of damage index 3) + 4 x (number of leaves of damage index 4) + 5 x (damage Number of leaves with index 5)] / [5 × (total number of leaves)]

(result)
Table 7 shows the results of the feeding test for 13 types of strains isolated from the plant M143. As a result, the M143R1 strain suppressed the feeding of soybean, and M143R1 was selected as a bacterial endophyte that imparts a feeding-inhibiting action to the host soybean. The other 12 strains isolated from plant M143 were not significantly different from the non-inoculated area.

(Identification of M143R1 strain)
Table 8 shows the carbon sources that were confirmed to be capable of assimilating the M143R1 strain.

The nucleotide sequence of 16S rDNA of M143R1 strain is shown in SEQ ID NO: 2.
The strain was cultured in Nutrient Broth, and genomic DNA was isolated from the cells. Using the isolated DNA as a template, the base sequence of about 500 bp in the first half of the 16S rDNA region was determined by the dye primer method (SEQ ID NO: 2). Using the homology search program FASTA, a homology search between the determined nucleotide sequence and the DDBJ / EMBL / GenBank international nucleotide sequence database was performed.

  As a result of homology search, the base sequence of 16S rDNA of M143R1 strain is given by Methylobacterium sp. (Accession No. AY468371) (Accession No.) when each base sequence is registered in the DNA data bank. It was clarified that it shows high homology with the base sequence of 16S rDNA. FIG. 3 shows a comparison result of comparing the base sequence of 16S rDNA between M143R1 strain and Methylobacterium sp. (AY468371). The homology was 99.362%.

  From the above results, it was concluded that the M143R1 strain is a new strain belonging to the genus Methylobacterium. The M143R1 strain has been deposited as Methylobacterium sp. M143R1 with the Patent Microorganism Depositary Center for Product Evaluation and Technology on March 31, 2005 (Accession Number NITE P-96).

Promoting soybean growth, increasing yield, and increasing nodulation
1. the purpose
The purpose of this study was to investigate the effects of Variovorax sp. M30P3 (NITE P-95) and Methylobacterium sp. M143R1 (NITE P-96) on the growth promotion and yield increase of soybean.

2. experimental method
2-1. Prototype <br/> Soybean variety: Toyohomale 200 treatment areas
2-2. Field <br/> First year of paddy field conversion

2-3. Test bacteria
Variovorax sp. M30P3 (NITE P-95)
Methylobacterium sp. M143R1 (NITE P-96)

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 (large ≧ 8 mm, medium 8-7 mm, scrap 7 mm ≦) and grain weight were measured.
In the analysis, the total yield was calculated from the above yield survey results.

3. result
3-1. Growth Survey Results Table 9 shows the results of growth survey on the 57th day and 135th day after sowing. From the results on the 57th day, regarding the number of leaves and the stem length, growth promotion was slightly observed in the endophyte inoculated group of the present invention compared to the control group. Further, in the harvest period survey results on the 135th day, the growth promotion was observed in the endophyte inoculation group of the present invention over the control group in all items.

  Table 10 shows the results of the harvest survey and the total yield. The number of arrivals was higher in the endophyte inoculation group of the present invention than in the control group. Moreover, regarding the total seed weight, the endophyte inoculation group of the present invention was heavier than the control group. In addition, regarding the number of nodules, the endophyte inoculation group of the present invention was more than the control group (Table 11).

  The final total yield results showed an increase in yield in both of the two endophytic inoculation zones of the present invention.

It is a figure which shows the relationship between the symbiosis of each bacterial endophyte in Example 1, and the feeding inhibition degree of host soybean. It is a figure which shows the comparison result which compared the base sequence of 16S rDNA of M30P3 stock | strain and Variovorax paradoxus ATCC17713 strain | stump | stock (same as standard microbe DSM66). It is a figure which shows the comparison result which compared the base sequence of 16S rDNA of M30P3 stock | strain and Variovorax paradoxus ATCC17713 strain | stump | stock (same as standard microbe DSM66). It is a figure which shows the comparison result which compared the base sequence of 16S rDNA of M143R1 strain | stump | stock and Methylobacterium sp. Strain (Accession No. AY468371).

Claims (9)

A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, ability to increase the number of nodules in plants, and a bacterium having the ability to increase the yield of the plant, comprising the step of artificially infected with legumes, in a manner to impart pest feeding inhibitory legumes And the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, A method of promoting leguminous plant growth comprising the step of artificially infecting legumes with a bacterium having the ability to increase the number of nodules in the plant and the ability to increase the yield of the plant, The above method, wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, ability to increase the number of nodules in plants, and a bacterium having the ability to increase the yield of the plant, comprising the step of artificially infected with legumes, a method of increasing the Chakusaya number legumes The method as described above, wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, A method for increasing the number of nodules in a legume, comprising the step of artificially infecting a legume with an ability to increase the number of nodules in the plant and the ability to increase the yield of the plant , The above method, wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, A method of increasing legume yield, comprising the step of artificially infecting legumes with a bacterium having the ability to increase the number of nodules in the plant and the ability to increase the yield of the plant, The above method, wherein the bacterium is Variovorax sp. M30P3 (Accession No. NITE P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . A genus belonging to the genus Variovorax or Methylobacterium, capable of giving symbiosis in legumes and imparting pest feeding inhibition to the plant, an ability to promote the growth of the plant, an ability to increase the number of arrivals of the plant, A microbial preparation for legumes, which contains a bacterium having an ability to increase the number of nodules in a plant and an ability to increase the yield of the plant as an active ingredient , wherein the bacterium is Variovorax sp. M30P3 (Accession No. P-95) or Methylobacterium sp. M143R1 (Accession No. NITE P-96) . Variovorax sp. M30P3 (Accession number NITE P-95 ). Methylobacterium sp. M143R1 (Accession number NITE P-96 ). Variovorax sp. M30P3 (accession number NITE P-95), and Methylobacterium sp. M143R1 (accession number NITE P-96) or Ranaru least one artificially infected legume is selected from the group.

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